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Effect of using a physical assistance device for movements involving trunk bending Exoskeletons are a solution to physically relieve workers while allowing them to control the execution of their tasks and assist them (Baltrusch et al.2018).

The study investigated in the search for elements able to quantify the action of a physical assistance device (PAD) on the human body for movements responsible for pathologies recognized as occupational diseases.

The evaluation of a PAD allows to determine in which framework the exoskeleton can be useful for the realization of the movement. It is specified that a relevant way to insert exoskeletons in a company by always implementing a study or analysis beforehand to ensure its good integration.

The objective of the study is to perform several tasks with and without the Posture Harness (HAPO) in order to analyze the kinematics of the movements and the electrical activity of erector muscles of the spine involved in carrying a load at work to understand its effects on the human body.

sentences: Complexity-stability relationship in empirical microbial ecosystems Mays stability theory [1, 2], which holds that large ecosystems can be stable up to a critical level of complexity, a product of the number of resident species and the intensity of their interactions, has been a central paradigm in theoretical ecology [3-7]. So far, however, empirically demonstrating this theory in real ecological systems has been a long-standing challenge, with inconsistent results [8]. Especially, it is unknown whether this theory is pertinent in the rich and complex communities of natural microbiomes, mainly due to the challenge of reliably reconstructing such large ecological interaction networks [9-11]. Here, we introduce a novel computational framework for estimating an ecosystems complexity without relying on a priori knowledge of its underlying interaction network. By applying this method to human-associated microbial communities from different body sites [12] and sponge-associated microbial communities from different geographical locations [13], we found that in both cases the communities display a pronounced trade-off between the number of species and their effective connectance. These results suggest that natural microbiomes are shaped by stability constraints, which limit their complexity.

sentences: Leveraging eDNA metabarcoding to characterize nearshore fish communities in Southeast Alaska: Do habitat and tide matter? Nearshore marine habitats are critical for a variety of commercially important fish species, but assessing fish communities in these habitats is costly and time-intensive. Here, we leverage eDNA metabarcoding to characterize nearshore fish communities near Juneau, Alaska, USA, a high-latitude environment with large tidal swings, strong currents, and significant freshwater input. We investigated whether species richness and community composition differed across three habitat types (sand beaches, eelgrass beds, and rocky shorelines) and between high and low tides. Additionally, we tested whether replication of field samples and PCR reactions influenced either species richness or composition. We amplified a 12S mitochondrial locus in our samples and identified 188 fish amplicon sequence variants (ASVs), corresponding to 21 unique taxa, with approximately half of these resolved to single species. Species richness and composition inferred from eDNA differed substantially among habitats, with rock habitats containing fewer taxa and fewer overall detections than sand and eelgrass habitats. The effect of tide was more subtle and suggested a habitat-tide interaction, with differences in taxa between tides largely isolated to sand habitats. Power analyses indicated that additional field sampling is useful to detect subtle changes in species richness such as those due to tide. PCR replicates typically identified a small number of additional taxa. The most notable result from our study was that shore morphology appeared to substantially influence community structure. Rocky shorelines sloped quickly into deep water, while sand and eelgrass habitats descended much more gradually. We hypothesize that differences in taxa observed among habitats were largely due to lack of mixing between bottom and surface water, providing further evidence that eDNA transport is minimal and that many marine eDNA detections are derived from highly localized sampling locations. We suggest that future studies could explore the extent to which habitat and nearshore physical processes influence eDNA detections.

sentences: Two types of hand withdraw movement to place food in the mouth mediated by somatosensation in 22-species of strepsirrhines The evolution of visual control of the hand to assist feeding by primates is uncertain but in anthropoid primates vision contributes not only to reaching for food and grasping it but also to the withdraw movement that brings food to the mouth. The strepsirrhines are a relatively large monophyletic group of Euarchontoglires near the base of the primate cladogram that are described as using vision to reach for food, but it is not known whether they use vision to assist the withdraw movement. The present study answere this question in 22 species of captive strepsirrhines from 6 of the seven strepsirrhine families, Daubentoniidae, Cheirogaleidae, Indriidae, Lemuridae, Lorisidae and Galagidae. Animals were videorecorded as they ate their normal food provisions. Dependent measures for analyses were ground withdraw movements, bringing grasped food to the mouth, and inhand withdraw movements, brining food held in the hand to the mouth, as well as the posture and head movements associated with each type of withdraw. Frame-by-frame scores from the video record showed that there were large differences between and within strepsirrhine families in these movements. Nevertheless, for all species, the withdraw movement was mediated by somatosensation, with mouth reaching and perioral contact with food determining how food was eventually eaten. Nonvisual behavior also contributed to food grasping as many species sniffed food before or during grasping. Even amongst species that made most use of the hand for their withdraws, the insectivores Loris lydekkerianus and Galago senegalensis, and herbivores, Hapalemur simus and Eulemur flavifrons, perioral contact was used to orient food for biting. The use of somatosensation and the absence of vision in mediating getting food in strepsirrhines suggests that visual mediation of the withdraw is an anthropoid innovation.

sentences: The compound of baicalein, wogonin, and oroxylin-A inhibits EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC.

sentences: No need to choose: independent regulation of cognitive stability and flexibility challenges the stability-flexibility tradeoff Adaptive behavior requires the ability to focus on a current task and protect it from distraction (cognitive stability) as well as the ability to rapidly switch to another task in light of changing circumstances (cognitive flexibility). Cognitive stability and flexibility have been conceptualized as opposite endpoints on a stability-flexibility tradeoff continuum, implying an obligatory reciprocity between the two: greater flexibility necessitates less stability, and vice versa. Surprisingly, rigorous empirical tests of this critical assumption are lacking. Here, we acquired simultaneous measurements of cognitive stability (congruency effects) and flexibility (switch costs) on the same stimuli within the same task, while independently varying contextual demands on these functions with block-wise manipulations of the proportion of incongruent trials and task switches, respectively. If cognitive stability and flexibility are reciprocal, increases in flexibility in response to higher switch rates should lead to commensurate decreases in stability, and increases in stability in response to more frequent incongruent trials should result in decreased flexibility. Across three experiments, using classic cued task switching (Experiments 1 and 3) and attentional set shifting (Experiment 2) protocols, we found robust evidence against an obligatory stability-flexibility tradeoff. Although we observed the expected contextual adaptation of stability and flexibility to changing demands, strategic adjustments in stability had little influence on flexibility, and vice versa. These results refute the long-held assumption of a stability-flexibility tradeoff, documenting instead that the cognitive processes mediating these functions can be regulated independently - it is possible to be both stable and flexible at the same time.

sentences: Effect of Pre-harvest Calcium Chloride and Salicylic Acid Spray on Morphological and Biochemical Traits of Guava (Psidium guajava) Guava is a small, tropical fruit tree grown in various tropical and subtropical regions. Salicylic acid (SA) is a phenolic compound that enhances disease resistance and delays the fruit ripening process. Calcium is an essential cell component that delays ripening, particularly softening of the fruit. The effect of foliar spray of CaCl2, and SA, on the physical and biochemical traits of guava was investigated in the present investigation. The application of CaCl2 2% + SA 2mM was more effective as compared with both when applied alone. The data were recorded on fruit set (%), fruit weight (g), fruit length (cm), fruit diameter (cm), fruit yield (kg), ripening period (days), TSS, acidity, total sugar, ascorbic acid, nitrogen, phosphorus, potassium. CaCl2 2% + SA 2mM was showed better performance in all cases, followed by SA 2mM and CaCl2 2%. Overall, this work determines the influence on guava's essential traits by pre-harvest calcium chloride and salicylic acid.

sentences: Efficient Estimation of Indirect Effects in Case-Control Studies Using a Unified Likelihood Framework Mediation models are a set of statistical techniques that investigate the mechanisms that produce an observed relationship between an exposure variable and an outcome variable in order to deduce the extent to which the relationship is influenced by intermediate mediator variables. For a case-control study, the most common mediation analysis strategy employs a counterfactual framework that permits estimation of indirect and direct effects on the odds ratio scale for dichotomous outcomes, assuming either binary or continuous mediators. While this framework has become an important tool for mediation analysis, we demonstrate that we can embed this approach in a unified likelihood framework for mediation analysis in case-control studies that leverages more features of the data (in particular, the relationship between exposure and mediator) to improve efficiency of indirect effect estimates. One important feature of our likelihood approach is that it naturally incorporates cases within the exposure-mediator model to improve efficiency. Our approach does not require knowledge of disease prevalence and can model confounders and exposure-mediator interactions, and is straightforward to implement in standard statistical software. We illustrate our approach using both simulated data and real data from a case-control genetic study of lung cancer.

sentences: Indication-specific tumor evolution and its impact on neoantigen targeting and biomarkers for individualized cancer immunotherapies Individualized neoantigen specific immunotherapy (iNeST) requires robustly expressed clonal neoantigens for efficacy, but tumor mutational heterogeneity, loss of neoantigen expression, and variable tissue sampling present challenges. To characterize these potential obstacles, we combined multi-region sequencing (MR-seq) analysis of five untreated, synchronously sampled metastatic solid tumors with re-analysis of published MR-seq data from 103 patients. Branching evolution in colorectal cancer and renal cell carcinoma led to fewer clonal neoantigens and to clade-specific neoantigens (those shared across a subset of tumor regions but not fully clonal), with the latter not being readily distinguishable in single tumor samples. Prioritizing mutations with higher purity- and ploidy-adjusted variant allele frequency enriched for globally clonal neoantigens (those found in all tumor regions), whereas estimated cancer cell fraction derived from clustering-based tools, surprisingly, did not. Neoantigen quality was associated with loss of neoantigen expression in the bladder cancer case, and HLA-allele loss was observed in the renal and non-small cell lung cancer cases. Our results show that indication type, multi-lesion sampling, neoantigen expression, and HLA allele retention are important factors for iNeST targeting and patient selection.

sentences: Syrbactin-class dual constitutive- and immuno-proteasome inhibitor TIR-199 impedes myeloma-mediated bone degeneration in vivo. Proteasome-addicted neoplastic malignancies present a considerable refractory and relapsed phenotype with patients exhibiting drug-resistance and high mortality rates. To counter this global problem, novel proteasome-based therapies are being developed. In the current study we extensively characterize TIR-199, a syrbactin-class proteasome inhibitor derived from a plant virulence factor of bacterium Pseudomonas syringae pv syringae. We report that TIR-199 is a potent constitutive and immunoproteasome inhibitor, capable of inducing cell death in multiple myeloma, triple-negative breast cancer and non-small cell lung cancer lines, effectively inhibit proteasome in primary myeloma cells of refractory patients, and bypass the PSMB5 A49T+A50V bortezomib-resistant mutant. TIR-199 treatment leads to accumulation of canonical proteasome substrates in cells, it is specific, and does not inhibit 50 other enzymes tested in vitro. The drug exhibits synergistic cytotoxicity in combination with proteasome-activating-kinase DYRK2 inhibitor LDN192960. Furthermore, low dose TIR-199 exhibits in vivo activity in delaying myeloma-mediated bone degeneration in a mouse xenograft model. Together, our data indicates that proteasome inhibitor TIR-199 could indeed be a next generation drug within the repertoire of proteasome-based therapeutics with a potential to target relapsed and refractory proteasome-addicted neoplasia.

sentences: Directed Evolution of Aptamer Discovery Technologies ConspectusAlthough antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our lab to accelerate the discovery of high quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multi-parametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrices like serum. The third method is a click chemistry-based particle display (click-PD) that enables the generation and high-throughput screening of "non-nattural" aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Lastly, we describe the non-natural aptamer array (N2A2) platform, in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in a massively parallel fashion, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases, and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

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Perception affects the brain's metabolic response to sensory stimulation Processing of incoming sensory stimulation triggers an increase of cerebral perfusion and blood oxygenation (neurovascular response) as well as an alteration of the metabolic neurochemical profile (neurometabolic response). Here we show that perceived and unperceived isoluminant chromatic flickering stimuli designed to have similar neurovascular responses as measured by blood oxygenation level dependent functional MRI (BOLD-fMRI) in primary visual cortex (V1) have markedly different neurometabolic responses as measured by functional MRS. In particular, a significant regional buildup of lactate, an index of aerobic glycolysis, and glutamate, an index of malate-aspartate shuttle, occurred in V1 only when the flickering is perceived, without any relation with behavioral or physiological variables. Wheras the BOLD-fMRI signal in V1, a proxy for input to V1, was insensitive to flickering perception by design, the BOLD-fMRI signal in secondary visual areas was larger during perceived than unperceived flickering indicating increased output from V1. These results indicate that the upregulation of energy metabolism induced by visual stimulation depends on the type of information processing taking place in V1, and that 1H-fMRS provides unique information about local input/output balance that is not measured by BOLD-fMRI.

Significance statementVisual perception has a measurable metabolic effect in the primary visual cortex (V1). Here we show that the upregulation of energy metabolism induced by isoluminant chromatic flickering depends on subjective visual perception. Within V1, perceived and unperceived stimuli that are contrast-matched to elicit similar blood-oxygenation level-dependent fMRI response are associated with clearly distinct neurochemical profiles. Specifically, regional accumulations of lactate (an index of aerobic glycolysis) and glutamate (an index of malate-aspartate shuttle) only develop during perceived stimuli, together with a larger activation of secondary visual areas. Our results imply a dissociation between metabolic and functional response, and indicate that that the upregulation of energy metabolism induced by visual stimulation depends on the type of information processing taking place in V1.

sentences: Learning enhances behaviorally relevant representations in apical dendrites Learning alters cortical representations and improves perception. Apical tuft dendrites in Layer 1, which are unique in their connectivity and biophysical properties, may be a key site of learning-induced plasticity. We used both two-photon and SCAPE microscopy to longitudinally track tuft-wide calcium spikes in apical dendrites of Layer 5 pyramidal neurons as mice learned a tactile behavior. Mice were trained to discriminate two orthogonal directions of whisker stimulation. Reinforcement learning, but not repeated stimulus exposure, enhanced tuft selectivity for both directions equally, even though only one was associated with reward. Selective tufts emerged from initially unresponsive or low-selectivity populations. Animal movement and choice did not account for changes in stimulus selectivity. Enhanced selectivity persisted even after rewards were removed and animals ceased performing the task. We conclude that learning produces long-lasting realignment of apical dendrite tuft responses to behaviorally relevant dimensions of a task.

sentences: Single-cell foundations of live social gaze interaction in the prefrontal cortex and amygdala Social gaze interaction powerfully shapes interpersonal communication in humans and other primates. However, little is known about the neural underpinnings of these social behavioral exchanges. Here, we studied neural responses associated with naturalistic, face-to-face, social gaze interactions between pairs of macaques. We examined spiking activity in a large number of neurons spanning four different brain regions involved in social behaviors - the amygdala, orbitofrontal cortex, anterior cingulate cortex, and dorsomedial prefrontal cortex. We observed widespread single-cell representations of social gaze interaction functionalities in these brain regions - social discriminability, social gaze monitoring, and mutual eye contact selectivity. Many of these neurons discriminated looking at social versus non-social stimuli with rich temporal heterogeneity, or parametrically tracked the gaze positions of oneself or the conspecific. Furthermore, many neurons displayed selectivity for mutual eye contact as a function of the initiator or follower of mutual gaze events. Crucially, a significant proportion of neurons coded for more than one of these three signatures of social gaze interaction, supporting the recruitment of partially overlapping neuronal ensembles. Our findings emphasize integrated contributions of the amygdala and prefrontal circuits within the social interaction networks in processing real-life social interactions.

sentences: Mice immunized with the vaccine candidate HexaPro spike produce neutralizing antibodies against SARS-CoV-2 Updated and revised versions of COVID-19 vaccines are vital due to genetic variations of the SARS-CoV-2 spike antigen. Furthermore, vaccines that are safe, cost-effective, and logistically friendly are critically needed for global equity, especially for middle to low income countries. Recombinant protein-based subunit vaccines against SARS-CoV-2 have been reported with the use of the receptor binding domain (RBD) and the prefusion spike trimers (S-2P). Recently, a new version of prefusion spike trimers, so called "HexaPro", has been shown for its physical property to possess two RBD in the "up" conformation, as opposed to just one exposed RBD found in S-2P. Importantly, this HexaPro spike antigen is more stable than S-2P, raising its feasibility for global logistics and supply chain. Here, we report that the spike protein HexaPro offers a promising candidate for SARS-CoV-2 vaccine. Mice immunized by the recombinant HexaPro adjuvanted with aluminium hydroxide using a prime-boost regimen produced high-titer neutralizing antibodies for up to 56 days after initial immunization against live SARS-CoV-2 infection. In addition, the level of neutralization activity is comparable to that of convalescence sera. Our results indicate that the HexaPro subunit vaccine confers neutralization activity in sera collected from mice receiving the prime-boost regimen.

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Functional amyloids in the microbiomes of a rat Parkinson's disease model and wild-type rats Cross-seeding between amyloidogenic proteins in the gut is receiving increasing attention as a possible mechanism for initiation or acceleration of amyloid formation by aggregation-prone proteins such as SN, which is central in the development of Parkinsons disease. This is particularly pertinent in view of the growing number of functional (i.e. benign and useful) amyloid proteins discovered in bacteria. Here we identify two functional amyloid proteins, Pr12 and Pr17, in fecal matter from Parkinsons disease transgenic rats and their wild type counterparts, based on their stability against dissolution by formic acid. Both proteins show robust aggregation into ThT-positive aggregates that contain higher-order {beta}-sheets and have a fibrillar morphology, indicative of amyloid proteins. In addition, Pr17 aggregates formed in vitro showed significant resistance against formic acid, suggesting an ability to form highly stable amyloid. Treatment with proteinase K revealed a protected core of approx. 9 kDa. Neither Pr12 nor Pr17, however, affected SN aggregation in vitro. Thus, amyloidogenicity does not per se lead to an ability to cross-seed fibrillation of SN. Our results support the use of proteomics and formic acid to identify amyloid protein in complex mixtures and indicates the existence of numerous functional amyloid proteins in microbiomes.

IMPORTANCEThe bacterial microbiome in the gastrointestinal tract is increasingly seen as important for human health and disease. One area of particular interest is that of neurodegenerative diseases such as Parkinsons which involve pathological aggregation into amyloid of human proteins such as - synuclein (SN). Bacteria are known to form benign or functional amyloid, some of which may initiate unwanted aggregation of e.g. SN in the enteric nervous system through cross-seeding via contact with the microbiome. Here we show that the rat microbiome contains several proteins which form this type of amyloid aggregate both in vivo and in vitro. Although the two proteins we investigate in depth do not directly promote SN aggregation, our work shows that the microbiome potentially harbors a significant number of bacterial amyloid which could play a role in human physiology at various levels.

sentences: Live visualization of cytosolic and endoplasmic reticulum redox potential in zebrafish embryos reveals region-specific differences Redox homeostasis is an integral part of many cellular processes and its perturbation is associated with conditions such as diabetes, aging, and neurodegenerative disorders. Redox homeostasis or redox potential in organelles is maintained within a particular range to facilitate the organelle specific cellular redox reactions. Previous studies using yeast, cell systems, and nematodes have demonstrated the Endoplasmic Reticulum (ER) has a more oxidizing environment while the cytosol exhibits a reducing redox potential. However, we know very little about redox state of ER in the vertebrate systems. We created transgenic zebrafish (Danio rerio) lines with roGFP sensors targeted to the cytosol and ER for studying physiological redox potential at the systems level. In the process, we characterized the ER-targeting signal sequence in D. rerio for the first time. Measurements of the redox state in live embryos found that the ER and cytosol exhibit deviations from the expected range in different regions of the developing embryos. The ER is more reducing than expected in certain tissues, and cytosol has oxidizing regions at par with the ER in other parts of the embryo. This was particularly true for the brain which showed the heterogeneity of redox state in both the ER and the cytosol. These differences became even more evident in a transgenic line expressing the more sensitive roGFPiE variant. Thus, live monitoring of redox potential across the developing zebrafish embryos revealed interesting regions of redox heterogeneity at the organism level.

sentences: The C-terminal domain of SEC-10 is fundamental for exocyst function, Spitzenkorper organization and cell morphogenesis in Neurospora crassa. The exocyst is a conserved multimeric complex that participates in the final steps of the secretion of vesicles. In the filamentous fungus Neurospora crassa, the exocyst is crucial for polar growth, morphology, and the organization of the Spitzenkorper (Spk), the apical body where secretory vesicles accumulate before being delivered to the plasma membrane. In the highly polarized cells of N. crassa, the exocyst subunits SEC-3, SEC-5, SEC-6, SEC-8, and SEC-15 were previously found localized at the plasma membrane of the cells apices, while EXO-70 and EXO-84 occupied the frontal outer layer of the Spk, occupied by vesicles. The localization of SEC-10 had remained so far elusive. In this work, SEC-10 was tagged with the green fluorescent protein (GFP) either at its N- or C-terminus and found localized at the plasma membrane of growing hyphal tips, similar to what was previously observed for some exocyst subunits. While expression of an N-terminally tagged version of SEC-10 at its native locus was fully viable, expression of a C-terminally tagged version at its native locus resulted in severe hyphal growth and polarity defects. Additionally, a sec-10 knockout mutant in a heterokaryotic state (with genetically different nuclei) was viable but showed a strongly aberrant phenotype, confirming that this subunit is essential to maintain hyphal morphogenesis. Transmission electron microscopy analysis revealed the lack of a Spk in the SEC-10-GFP strain, suggesting a critical role of the exocyst in the vesicular organization at the Spk. Mass spectrometry analysis revealed fewer peptides of exocyst subunits interacting with SEC-10-GFP than with GFP-SEC-10, suggesting an essential role of the C-terminus of SEC-10 in exocyst assembly and/or stability. Altogether, our data suggest that an unobstructed C-terminus of SEC-10 is indispensable for the exocyst complex function and that a GFP tag could be blocking important subunit-subunit interactions.

sentences: Cell Painting predicts impact of lung cancer variants Most variants in most genes across most organisms have an unknown impact on the function of the corresponding gene. This gap in knowledge is especially acute in cancer, where clinical sequencing of tumors now routinely reveals patient-specific variants whose functional impact on the corresponding gene is unknown, impeding clinical utility. Transcriptional profiling was able to systematically distinguish these variants of unknown significance (VUS) as impactful vs. neutral in an approach called expression-based variant-impact phenotyping (eVIP). We profiled a set of lung adenocarcinoma-associated somatic variants using Cell Painting, a morphological profiling assay that captures features of cells based on microscopy using six stains of cell and organelle components. Using deep-learning-extracted features from each cells image, we found that cell morphological profiling (cmVIP) can predict variants functional impact and, particularly at the single-cell level, reveals biological insights into variants which can be explored in our public online portal. Given its low cost, convenient implementation, and single-cell resolution, cmVIP profiling therefore seems promising as an avenue for using non-gene-specific assays to systematically assess the impact of variants, including disease-associated alleles, on gene function.

sentences: Cell-intrinsic functions of the transcription factor Bhlhe40 in activated B cells and T follicular helper cells restrain the germinal center reaction and prevent lymphomagenesis The generation of high-affinity antibodies against pathogens and vaccines requires the germinal center (GC) reaction - a process that relies on a complex interplay between specialized effector subsets of B and CD4 T lymphocytes - GC B cells and T follicular helper (TFH) cells. Intriguingly, several key positive regulators of the GC reaction are common for both cell types. Here, we report that the transcription factor Bhlhe40 is a crucial cell-intrinsic negative regulator affecting both the B and T cell sides of the GC reaction. In activated CD4 T cells, Bhlhe40 was required to restrain proliferation thus limiting the number of TFH cells. In B cells, Bhlhe40 executed its function in the first days after immunization by selectively restricting the generation of the earliest GC B cells but not of early memory B cells or plasmablasts. Conditional Bhlhe40 inactivation confirmed cell-autonomous functions of Bhlhe40 in both GC B and TFH cells, while the GC phenotype was further enhanced upon loss of Bhlhe40 in both cell types. This negative regulation of the GC reaction by Bhlhe40 was of crucial importance, as Bhlhe40-deficient mice with progressing age succumbed to a B cell lymphoma characterized by accumulation of monoclonal GC B-like cells and polyclonal TFH cells in various tissues.

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Mental Workload Classification of Oceanauts from EEG Data Using Multiclass Support Vector Machines BackgroundIn the actual operation task, the workload of the oceanaut is mainly mental workload. For the oceanaut, too high or too low mental workload will significantly reduce work efficiency and even lead to major safety accidents. Classification of mental workload of the oceanaut in operational task research is one of the key problems in operational task research. However, the traditional mechanism modeling method is complex, computational complexity and low accuracy.

MethodIn this paper, machine learning method got used to the model. Based-on Electroencephalograph (EEG) data collected from the simulation experiment of the operating manipulator, multiclass support vector machine (MSVM) was used to train the samples, and the optimal training sample size was selected. The parameters of the model were optimized by grid search mixed with particle-swarm optimization algorithm (GSPSO) to obtain the optimal classification of oceanauts mental workload.

ResultsAs the research results showed that the method could accurately classify the mental workload of oceanauts. Furthermore, GSPSO-MSVM in the classification of oceanauts mental workload had the advantage over K-nearest neighbors (KNN), BP neural network (BP), random forest (RF) and support vector machines (SVM).

ConclusionGSPSO-MSVM in the classification of oceanauts mental workload had the advantage over K-nearest neighbors (KNN), BP neural network (BP), random forest (RF) and support vector machines (SVM).

Key points: The significance of the studyO_LIClassification of mental workload of the oceanaut in operational task research is one of the key problems in operational task research.
C_LIO_LIBased-on Electroencephalograph (EEG) data collected from the simulation experiment of the operating manipulator, multiclass support vector machine (MSVM) was used to train the samples, and the optimal training sample size was selected.
C_LIO_LIThe parameters of the model were optimized by grid search mixed with particle-swarm optimization algorithm (GSPSO) to obtain the optimal classification of oceanauts mental workload.
C_LIO_LIGSPSO-MSVM in the classification of oceanauts mental workload had the advantage over K-nearest neighbors (KNN), BP neural network (BP), random forest (RF) and support vector machines (SVM).
C_LI

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A Novel Family of RNA-Binding Proteins Regulate Polysaccharide Metabolism in Bacteroides thetaiotaomicron Human gut microbiome composition is constantly changing, and diet is a major driver of these changes. Gut microbial species that persist in mammalian hosts for long periods of time must possess mechanisms for sensing and adapting to nutrient shifts to avoid being outcompeted. Global regulatory mechanisms mediated by RNA-binding proteins (RBPs) that govern responses to nutrient shifts have been characterized in Proteobacteria and Firmicutes but remain undiscovered in the Bacteroidetes. Here we report the identification of RBPs that are broadly distributed across the Bacteroidetes, with many genomes encoding multiple copies. Genes encoding these RBPs are highly expressed in many Bacteroides species. A purified RBP, RbpB, from Bacteroides thetaiotaomicron binds to single-stranded RNA in vitro with an affinity similar to other characterized regulatory RBPs. B. thetaiotaomicron mutants lacking RBPs show dramatic shifts in expression of polysaccharide utilization and capsular polysaccharide loci, suggesting that these RBPs may act as global regulators of polysaccharide metabolism. A B. thetaiotaomicron {Delta}rbpB mutant shows a growth defect on dietary sugars belonging to the raffinose family of oligosaccharides (RFOs). The {Delta}rbpB mutant had reduced expression of BT1871, encoding a predicted RFO-degrading melibiase, compared to the wild-type strain. Mutation of BT1871 confirmed that the enzyme it encodes is essential for growth on melibiose and promotes growth on the RFOs raffinose and stachyose. Our data reveal that RbpB is required for optimal expression of BT1871 and other polysaccharide-related genes, suggesting that we have identified an important new family of global regulatory proteins in the Bacteroidetes.

ImportanceThe human colon houses hundreds of bacterial species, including many belonging to the genus Bacteroides, that aid in breaking down our food to keep us healthy. Bacteroides have many genes responsible for breaking down different dietary carbohydrates and complex regulatory mechanisms ensure that specific genes are only expressed when the right carbohydrates are available. In this study, we discovered that Bacteroides use a family of RNA-binding proteins as global regulators to coordinate expression of carbohydrate utilization genes. The ability to turn different carbohydrate utilization genes on and off in response to changing nutrient conditions is critical for Bacteroides to live successfully in the gut, and thus the new regulators we have identified may be important for life in the host.

sentences: Innate Immune sensing of Influenza A viral RNA through IFI16 promotes pyroptotic cell death Programmed cell death pathways are triggered by various stresses or stimuli, including viral infections. The mechanism underlying the regulation of these pathways upon Influenza A virus IAV infection is not well characterized. We report that a cytosolic DNA sensor IFI16 is essential for the activation of programmed cell death pathways in IAV infected cells. We have identified that IFI16 functions as an RNA sensor for influenza A virus by binding to genomic RNA. The activation of IFI16 triggers the production of type I, III interferons, and also other pro-inflammatory cytokines via the STING-TBK1 and Pro-caspase-1 signaling axis, thereby promoting cell death (apoptosis and pyroptosis in IAV infected cells). Whereas, IFI16 knockdown cells showed reduced inflammatory responses and also prevented cell mortality during IAV infection. These results demonstrate the pivotal role of IFI16-mediated IAV sensing and its essential role in activating programmed cell death pathways.

sentences: Synchrony of mind and body are distinct in mother-child dyads Hyperscanning studies have begun to unravel the brain mechanisms underlying social interaction, indicating a functional role for interpersonal neural synchronization (INS), yet the mechanisms that drive INS are poorly understood. While interpersonal synchrony is considered a multimodal phenomenon, it is not clear how different biological and behavioral synchrony markers are related to each other. The current study, thus, addresses whether INS is functionally-distinct from synchrony in other systems - specifically the autonomic nervous system (ANS) and motor behavior. To test this, we used a novel methodological approach, based on concurrent functional near-infrared spectroscopy-electrocardiography, recorded while N = 34 mother-child and stranger-child dyads (child mean age 14 years) engaged in cooperative and competitive tasks. Results showed a marked differentiation between neural, ANS and behavioral synchrony. Importantly, only in the neural domain was higher synchrony for mother-child compared to stranger-child dyads observed. Further, ANS and neural synchrony were positively related during competition but not during cooperation. These results suggest that synchrony in different behavioral and biological systems may reflect distinct processes. Mother-child INS may arise due to neural processes related to social affiliation, which go beyond shared arousal and similarities in behavior.

sentences: Impaired ERK MAPK activation in mature osteoblasts enhances bone formation via the mTOR pathway Emerging evidence supports that osteogenic differentiation of skeletal stem cells (SSCs) is a key determinant of overall bone formation and bone mass. Despite extensive studies showing mitogen-activated protein kinase (MAPK) function in osteoblast differentiation, none of these studies properly show in vivo evidence of impacting post-lineage commitment and subsequent maturation. Here, we describe how the extracellular signal-regulated kinase (ERK) pathway in osteoblasts controls bone formation by suppressing the mechanistic target of rapamycin (mTOR) pathway. We also show that, while ERK inhibition blocks the differentiation of osteogenic precursors when initiated at an early stage, ERK inhibition surprisingly promotes the later stages of osteoblast differentiation. Accordingly, inhibition of the ERK pathway using a small compound inhibitor or conditional deletion of the MAP2Ks Mek1 and Mek2, in mature osteoblasts and osteocytes (Mek1/2Dmp1), markedly increased bone formation due to augmented osteoblast differentiation. Mice with inducible deletion of the ERK pathway in mature osteoblasts (Mek1/2Ocn-Ert) also displayed similar phenotypes, demonstrating that this phenotype reflects continuous postnatal inhibition of late-stage osteoblast maturation. Mechanistically, ERK inhibition increases mitochondrial function and SGK1 phosphorylation via mTOR2 activation, which leads to osteoblast differentiation and production of angiogenic and osteogenic factors to promote bone formation. This phenotype was partly reversed by inhibiting mTOR. Our study uncovers a surprising dichotomy of ERK pathway functions in osteoblasts, whereby ERK activation promotes the early differentiation of osteoblast precursors, but inhibits the subsequent differentiation of committed osteoblasts via mTOR-mediated regulation of mitochondrial function and SGK1.

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A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients by phage display is binding to the ACE2-RBD interface and is tolerant to most known recently emerging RBD mutations The novel betacoranavirus SARS-CoV-2 causes a form of severe pneumonia disease, termed COVID-19 (coronavirus disease 2019). Recombinant human antibodies are proven potent neutralizers of viruses and can block the interaction of viral surface proteins with their host receptors. To develop neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor binding domain (RBD) of the S1 subunit of the viral spike (S) protein were selected by phage display. The selected antibodies were produced in the scFv-Fc format and 30 showed more than 80% inhibition of spike (S1-S2) binding to cells expressing ACE2, assessed by flow cytometry screening assay. The majority of these inhibiting antibodies are derived from the VH3-66 V-gene. The antibody STE90-C11 showed a sub nM IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody was demonstrated in the Syrian hamster and in the hACE2 mice model using a silenced human IgG1 Fc part. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD was solved at 2.0 [A] resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibtion of STE90-C11 is not blocked by many known RBD mutations including N439K, L452R, E484K or L452R+E484Q (emerging B.1.617). STE90-C11 derived human IgG1 with Fc{gamma}R silenced Fc (COR-101) is currently undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.

In BriefHuman antibodies were selected from convalescent COVID-19 patients using antibody phage display. The antibody STE90-C11 is neutralizing authentic SARS-CoV-2 virus in vitro and in vivo and the crystal structure of STE90-C11 in complex with SARS-CoV-2-RBD revealed that this antibody is binding in the RBD-ACE2 interface. S1 binding of STE90-C11 and inhibition of ACE2 binding is not blocked by many known RBD mutations.

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Listeria spp. Isolated from Soil Samples Collected in the Great Smoky Mountains Listeria monocytogenes, a foodborne pathogen, and other Listeria spp. are present in natural environments. Isolating and characterizing strains from natural reservoirs can provide insight into the prevalence and diversity of Listeria spp. in these environments, elucidate their contribution to contamination of agricultural and food processing environments and food products, and lead to the discovery of novel species. In this study, we evaluated the diversity of Listeria spp. isolated from soil samples in a small region of the Great Smoky Mountains National Park (GSMNP), which is the most biodiverse national park in the United States National Park system. Of the 17 Listeria isolates that were recovered, whole-genome sequencing revealed that 14 were unique strains. The unique strains were shown to represent a diversity of Listeria spp., including L. monocytogenes (n=9), L. cossartiae subsp. cossartiae (n=1), L. marthii (n=1), L. booriae (n=1), and a novel Listeria sp. (n=2). The Listeria isolated in this study were collected from high elevation sites near a creek that drains into a series of rivers ultimately leading to the Mississippi River; thus, the Listeria present in this natural environment could potentially travel downstream to a large region that includes portions of nine southeastern and midwestern states in the U.S. The Listeria spp. isolated and described in this study provide insight into the diversity of Listeria spp. found in the Great Smoky Mountains and indicate that this environment is a reservoir of novel Listeria spp.

IMPORTANCEListeria monocytogenes is a foodborne pathogen that can cause serious systemic illness that, although rare, usually results in hospitalization and has a relatively high mortality rate compared to other foodborne pathogens. Identification of novel and diverse Listeria spp. provides insight into the genomic evolution, ecology, and evolution and variance of pathogenicity of this genus, especially in natural environments. Comparing L. monocytogenes and Listeria spp. isolates from natural environments, such as those recovered in this study, to contamination and/or outbreak strains may provide more information about the original natural sources of these strains and the pathways and mechanisms that lead to contamination of food products and agricultural or food processing environments.

sentences: Stenoparib, an inhibitor of cellular poly (ADP-ribose) polymerases (PARPs), blocks in vitro replication of SARS-CoV-2 variants We recently published a preliminary assessment of the activity of a poly (ADP-ribose) polymerase (PARP) inhibitor, stenoparib, also known as 2X-121, which inhibits viral replication by affecting pathways of the host. Here we show that stenoparib effectively inhibits a SARS-CoV-2 wt (BavPat1/2020) strain and four additional variant strains; alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and gamma (P.1) in vitro, with 50% effective concentration (EC50) estimates of 4.1 M, 8.5 M, 24.1 M, 8.2 M and 13.6 M, respectively. A separate experiment focusing on a combination of 10 M stenoparib and 0.5 M remdesivir, an antiviral drug, resulted in over 80% inhibition of the alpha (B.1.1.7) variant, which is substantially greater than the effect achieved with either drug alone, suggesting at least additive effects from combining the different mechanisms of activity of stenoparib and remdesivir.

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High Throughput Intact Protein Analysis Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry Mass spectrometry (MS) is the primary analytical tool used to characterize proteins within the biopharmaceutical industry. Electrospray ionization (ESI) coupled to liquid chromatography is the gold standard technique for intact protein analysis. However, speed limitations prevent analysis of large sample numbers (>1000) in a day. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MS, an ambient ionization MS technology, has recently been established as a platform for high throughput small molecule analysis. Here we report the application of such system for intact protein analysis. Up to 150 kDa proteins are detectable on a high throughput IR-MALDESI-MS system and we have evaluated how matrix affects signal. The system can analyze up to 22 protein samples in a second. Protein autophosphorylation, compound binding kinetics and compound modifications to a probe protein are demonstrated as applications. Top-down protein sequencing was conducted to identify a cysteine modification site. Two plate-based high throughput sample cleanup methods were coupled to IR-MALDESI-MS to enable analysis of samples containing high amounts of salts and buffers without compromising speed.

Graphical Abstract

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Oral subunit SARS-CoV-2 vaccine induces systemic neutralizing IgG, IgA and cellular immune responses and can boost neutralizing antibody responses primed by an injected vaccine The rapid spread of the COVID-19 pandemic, with its devastating medical and economic impacts, triggered an unprecedented race toward development of effective vaccines. The commercialized vaccines are parenterally administered, which poses logistic challenges, while adequate protection at the mucosal sites of virus entry is questionable. Furthermore, essentially all vaccine candidates target the viral spike (S) protein, a surface protein that undergoes significant antigenic drift. This work aimed to develop an oral multi-antigen SARS-CoV-2 vaccine comprised of the receptor binding domain (RBD) of the viral S protein, two domains of the viral nucleocapsid protein (N), and heat-labile enterotoxin B (LTB), a potent mucosal adjuvant. The humoral, mucosal and cell-mediated immune responses of both a three-dose vaccination schedule and a heterologous subcutaneous prime and oral booster regimen were assessed in mice and rats, respectively. Mice receiving the oral vaccine compared to control mice showed significantly enhanced post-dose-3 virus-neutralizing antibody, anti-S IgG and IgA production and N-protein-stimulated IFN-{gamma} and IL-2 secretion by T cells. When administered as a booster to rats following parenteral priming with the viral S1 protein, the oral vaccine elicited markedly higher neutralizing antibody titres than did oral placebo booster. A single oral booster following two subcutaneous priming doses elicited serum IgG and mucosal IgA levels similar to those raised by three subcutaneous doses. In conclusion, the oral LTB-adjuvanted multi-epitope SARS-CoV-2 vaccine triggered versatile humoral, cellular and mucosal immune responses, which are likely to provide protection, while also minimizing technical hurdles presently limiting global vaccination, whether by priming or booster programs.

HighlightsO_LIMigVax-101 is a multi-epitope oral vaccine for SARS-CoV-2.
C_LIO_LIMigVax-101 elicits neutralizing IgG and IgA production and cellular responses in mice
C_LIO_LIMigVax-101 serves as an effective booster in rats to a parenteral anti-S1 vaccine.
C_LI

sentences: Temporal integration of rod signals in luminance and chromatic pathways We assessed how rod excitation (R) affects luminance (L+M+S) and chromatic [L/(L+M)] reaction times (RTs). A four-primary display based on the overlapped images of two spectrally-modified monitors, which allowed specific or combined [L+M+S+R, L/(L+M) + R] photoreceptor stimulation, was used to present a C-target stimulus differing from the background only by the selected stimulation. For the luminance pathway, rod input increased RTs, suggesting a suppressive rod-cone interaction. The responses of the chromatic pathway were faster when rods were involved, suggesting a major role of rods in mesopic color perception.

sentences: Unravelling the puzzle of anthranoids metabolism in living plant cells using spectral imaging coupled to mass spectrometry Vismione H (VH) is a fluorescent prenylated anthranoid produced by plants from the Hypericaceae family, with antiprotozoal activities against malaria and leishmaniosis. Little is known about its biosynthesis and metabolism in plants or its mode of action against parasites. When VH is isolated from Psorospermum glaberrimum, it is rapidly converted into madagascine anthrone and anthraquinone, which are characterized by markedly different fluorescent properties. To locate the fluorescence of VH in living plant cells and discriminate it from that of the other metabolites, an original strategy combining spectral imaging (SImaging), confocal microscopy and non-targeted metabolomics using mass spectrometry, was developed. Besides VH, structurally related molecules including madagascine, emodin, quinizarin as well as lapachol and fraxetin were analyzed. This strategy readily allowed a spatiotemporal characterization and discrimination of spectral fingerprints from anthranoids-derived metabolites and related complexes with cations and proteins. In addition, our study validates the capability of plant cells to metabolize VH into madagascine anthrone, anthraquinones and unexpected metabolites, leading to new hypotheses on the metabolism of anthranoids in plants.

sentences: Personalized genome assembly for accurate cancer somatic mutation discovery using cancer-normal paired reference samples The use of personalized genome assembly as a reference for detecting the full spectrum of somatic events from cancers has long been advocated but never been systematically investigated. Here we address the critical need of assessing the accuracy of somatic mutation detection using personalized genome assembly versus the standard human reference assembly (i.e. GRCh38). We first obtained massive whole genome sequencing data using multiple sequencing technologies, and then performed de novo assembly of the first tumor-normal paired genomes, both nuclear and mitochondrial, derived from the same donor with triple negative breast cancer. Compared to standard human reference assembly, the haplotype phased chromosomal-scale personalized genome was best demonstrated with individual specific haplotypes for some complex regions and medical relevant genes. We then used this well-assembled personalized genome as a reference for read mapping and somatic variant discovery. We showed that the personalized genome assembly results in better alignments of sequencing reads and more accurate somatic mutation calls. Direct comparison of mitochondrial genomes led to discovery of unreported nonsynonymous somatic mutations. Our findings provided a unique resource and proved the necessity of personalized genome assembly as a reference in improving somatic mutation detection at personal genome level not only for breast cancer reference samples, but also potentially for other cancers.

sentences: Pan-ErbB inhibition protects from SARS-CoV-2 replication, inflammation, and injury Effective therapies are needed to combat emerging viruses. Seventeen candidates that rescue cells from SARS-CoV-2-induced lethality and target diverse functions emerged in a screen of 4,413 compounds. Among the hits was lapatinib, an approved inhibitor of the ErbB family of receptor tyrosine kinases. Lapatinib and other pan-ErbB inhibitors suppress replication of SARS-CoV-2 and unrelated viruses with a high barrier to resistance. ErbB4, but not lapatinibs cancer targets ErbB1 and ErbB2, is required for SARS-CoV-2 entry and encephalitis alphavirus infection and is a molecular target mediating lapatinibs antiviral effect. In human lung organoids, lapatinib protects from SARS-CoV-2-induced activation of pathways implicated in non-infectious acute lung injury and fibrosis downstream of ErbBs (p38-MAPK, MEK/ERK, and AKT/mTOR), pro-inflammatory cytokine production, and epithelial barrier injury. These findings reveal regulation of viral infection, inflammation, and lung injury via ErbBs and propose approved candidates to counteract these effects with implications for pandemic coronaviruses and unrelated viruses.

sentences: Tunable human myocardium derived decellularized extracellular matrix for 3D bioprinting and cardiac tissue engineering The generation of 3D tissue constructs with multiple cell types and matching mechanical properties remains a challenge in cardiac tissue engineering. Recently, 3D bioprinting has become a powerful tool to achieve these goals. Decellularized extracellular matrix (dECM) is a common scaffold material due to providing a native biochemical environment. Unfortunately, dECMs low mechanical stability prevents usage for bioprinting applications alone. In this study, we developed bioinks composed of decellularized human heart ECM (dhECM) with either gelatin methacryloyl (GelMA) or GelMA- methacrylated hyaluronic acid (MeHA) hydrogels dual crosslinked with UV light and microbial Transglutaminase (mTGase). We characterized the bioinks mechanical, rheological, swelling, printability and biocompatibility properties. Composite GelMA-MeHA-dhECM (GME) hydrogels demonstrated improved mechanical properties by an order of magnitude, compared to GelMA-dhECM (GE) hydrogels. All hydrogels were extrudable and compatible with human induced pluripotent stem cells derived cardiomyocytes (iCMs) and human cardiac fibroblasts (hCFs). Tissue-like beating of the printed constructs with striated sarcomeric alpha-actinin and Connexin 43 expression was observed. The order of magnitude difference between the elastic modulus of these hydrogel composites offers applications in in vitro modelling of the myocardial infarct boundary. Here, as a proof of concept, we created an infarct boundary region with control over mechanical properties along with cellular and macromolecular content through printing iCMs with GE bioink and hCFs with GME bioink.

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DRDOCK: A Drug Repurposing platform integrating automated docking, simulations and a log-odds-based drug ranking scheme MotivationDrug repurposing, where drugs originally approved to treat a disease are reused to treat other diseases, has received escalating attention especially in pandemic years. Structure-based drug design, integrating small molecular docking, molecular dynamic (MD) simulations and AI, has demonstrated its evidenced importance in streamlining new drug development as well as drug repurposing. To perform a sophisticated and fully automated drug screening using all the FDA drugs, intricate programming, accurate drug ranking methods and friendly user interface are very much needed.

ResultsHere we introduce a new web server, DRDOCK, Drug Repurposing DOcking with Conformation-sampling and pose re-ranKing - refined by MD and statistical models, which integrates small molecular docking and molecular dynamic (MD) simulations for automatic drug screening of 2016 FDA-approved drugs over a user-submitted single-chained target protein. The drugs are ranked by a novel drug-ranking scheme using log-odds (LOD) scores, derived from feature distributions of true binders and decoys. Users can submit a selection of LOD-ranked poses for further MD-based binding affinity evaluation. We demonstrated that our platform can indeed recover one of the substrates for nsp16, a cap ribose 2'-O methyltransferase, and recommends that fluralaner, tegaserod and fenoterol could be repurposed for the COVID19 treatment with the latter two being confirmed in SARS-CoV2 suppression experiments. All the sampled docking poses and trajectories can be 3D-viewed and played via our web interface. This platform shall be easy-to-use for general scientists and medicinal researchers to carry out drug repurposing within a couple of days which should add value to our timely responses to, particularly, emergent disease outbreaks.

Availability and implementationDRDOCK can be freely accessed from https://dyn.life.nthu.edu.tw/drdock/. (Due to the hardware upgrade, the service is NOT available before 7/18, 2021)

sentences: Empirical Transmit Field Bias Correction of T1w/T2w Myelin Maps T1-weighted divided by T2-weighted (T1w/T2w) myelin maps were initially developed for neuroanatomical analyses such as identifying cortical areas, but they are increasingly used in statistical comparisons across individuals and groups with other variables of interest. Existing T1w/T2w myelin maps contain radiofrequency transmit field (B1+) biases, which may be correlated with these variables of interest, leading to potentially spurious results. Here we propose two empirical methods for correcting these transmit field biases using either explicit measures of the transmit field or alternatively a 'pseudo-transmit' approach that is highly correlated with the transmit field at 3T. We find that the resulting corrected T1w/T2w myelin maps are both better neuroanatomical measures (e.g., for use in cross-species comparisons), and more appropriate for statistical comparisons of relative T1w/T2w differences across individuals and groups (e.g., sex, age, or body-mass-index) within a consistently acquired study at 3T. We recommend that investigators who use the T1w/T2w approach for mapping cortical myelin use these B1+ transmit field corrected myelin maps going forward.

sentences: Noncontact and high-precision sensing system for piano keys identified fingerprints of virtuosity Dexterous tool use is typically characterized by fast and precise motions performed by multiple fingers. One representative task is piano playing, which involves fast performance of a sequence of complex motions with high spatiotemporal precision. However, for several decades, a lack of contactless sensing technologies that are capable of precision measurement of piano key motions has been a bottleneck for unveiling how such an outstanding skill is cultivated. Here, we developed a novel sensing system that can record the vertical position of all piano keys with a time resolution of 1 ms and a spatial resolution of 0.01 mm in a noncontact manner. Using this system, we recoded the piano key motions while 49 pianists played a complex sequence of tones that required both individuated and coordinated finger movements to be performed as fast and accurately as possible. Penalized regression using various feature variables of the key motions identified distinct characteristics of the key-depressing and key-releasing motions in relation to the speed and accuracy of the performance. For the maximum rate of the keystrokes, individual differences across the pianists were associated with the peak key descending velocity, the key depression duration, and key-lift timing. For the timing error of the keystrokes, the interindividual differences were associated with the peak ascending velocity of the key and the interstrike variability of both the peak key descending velocity and the key depression duration. These results highlight the importance of dexterous control of the vertical motions of the keys for fast and accurate piano performance.

sentences: Pupillometry and electroencephalography in the digit span task This dataset consists of raw 64-channel EEG, cardiovascular (electrocardiography and photoplethysmography), and pupillometry data from 86 human participants recorded during 4 minutes of eyes-closed resting and during performance of a classic working memory task - digit span task with serial recall. The participants either memorized or just listened to sequences of 5, 9, or 13 digits presented auditorily every 2 seconds. The dataset can be used for (1) developing algorithms for cognitive load discrimination and detection of cognitive overload; (2) studying neural (event-related potentials and brain oscillations) and peripheral (electrocardiography, photoplethysmography, and pupillometry) physiological signals during encoding and maintenance of each sequentially presented memory item; (3) correlating cognitive load and individual differences in working memory to neural and peripheral physiology, and studying the relationship between the physiological signals; (4) integration of the physiological findings with the vast knowledge coming from behavioral studies of verbal working memory in simple span paradigms. The data are shared in Brain Imaging Data Structure (BIDS) format and freely available on OpenNeuro (https://openneuro.org/datasets/ds003838).

sentences: A Biologically Plausible Model for Continual Learning using Synaptic Weight Attractors The human brain readily learns tasks in sequence without forgetting previous ones. Artificial neural networks (ANNs), on the other hand, need to be modified to achieve similar performance. While effective, many algorithms that accomplish this are based on weight importance methods that do not correspond to biological mechanisms. Here we introduce a simple, biologically plausible, method for enabling effective continual learning in ANNs. We show that it is possible to learn a weight-dependent plasticity function that prevents catastrophic forgetting over multiple tasks. We highlight the effectiveness of our method by evaluating it on a set of MNIST classification tasks. We further find that the use of our method promotes synaptic multi-modality, similar to that seen in biology.

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Nucleolar targeting in an early-branching eukaryote suggests a general physicochemical mechanism for ribosome protein sorting The eukaryotic cell targets proteins to the organelles in which they function, both membrane-bound (like the nucleus) and non-membrane-bound (like the nucleolus). Nucleolar targeting relies on positively charged localisation signals, and has received rejuvenated interest since the widespread recognition of liquid-liquid phase separation (LLPS) as a mechanism contributing to nucleolus formation. Here, we exploit a new genome-wide analysis of protein localisation in an early-branching eukaryote, Trypanosoma brucei, to analyse general nucleolar protein properties. T. brucei nucleolar proteins have similar properties to those in common model eukaryotes, specifically basic amino acids. Using protein truncations and addition of candidate targeting sequences to proteins, we show both homopolymer runs and distributed basic amino acids give nucleolar partition, further aided by a nuclear localisation signal (NLS). These findings are consistent with phase separation models of nucleolar formation and protein physical properties being a major contributing mechanism for eukaryotic nucleolar targeting, conserved from the last eukaryotic common ancestor. Importantly, cytoplasmic ribosome proteins in comparison to mitochondrial ribosome proteins followed the same pattern - pointing to adaptation of physicochemical properties to assist segregation.

Summary StatementWe show protein targeting to the nucleolus is mediated by positive charge, likely across eukaryotes, and contributes to sorting of mitochondrial from cytoplasmic ribosome proteins.

sentences: Mapping mechanical stress in curved epithelia of designed size and shape The function of organs such as lungs, kidneys and mammary glands relies on the three-dimensional geometry of their epithelium. To adopt shapes such as spheres, tubes and ellipsoids, epithelia generate mechanical stresses that are generally unknown. Here we engineered curved epithelial monolayers of controlled size and shape and mapped their state of stress. We designed pressurized epithelia with circular, rectangular and ellipsoidal footprints. We developed a computational method to map the stress tensor in these epithelia. This method establishes a direct correspondence between epithelial shape and mechanical stress without assumptions of material properties. In epithelia with spherical geometry spanning more than one order of magnitude in radius, we show that stress weakly increases with areal strain in a size-independent manner. In epithelia with rectangular and ellipsoidal cross-section we found pronounced stress anisotropies consistent with the asymmetric distribution of tractions measured at the cell-substrate contact line. In these anisotropic profiles, cell shape tended to align with the direction of maximum principal stress but this alignment was non-universal and depended on epithelial geometry. Besides interrogating the fundamental mechanics of epithelia over a broad range of sizes and shapes, our approach will enable a systematic study of how geometry and stress influence epithelial fate and function in three-dimensions.

sentences: Caspase-mediated nuclear pore complex trimming in cell differentiation and endoplasmic reticulum stress Introductory ParagraphDuring apoptosis, caspases degrade 7 out of [~]30 nucleoporins (Nups) to irreversibly demolish the nuclear pore complex (NPC)1,2. However, for poorly understood reasons, caspases are also activated during differentiation at sublethal levels3,4. Here, we describe reversible, caspase-mediated NPC "trimming" during myogenesis. We find that sublethal levels of caspases selectively proteolyze 4 peripheral Nups, Nup358, Nup214, Nup153, and Tpr, resulting in the transient block of nuclear export pathways. Consequently, several nuclear export signal (NES)-containing focal adhesion proteins accumulate in the nucleus where they function as transcription cofactors5. One such protein, FAK (focal adhesion kinase), drives a global reconfiguration of MBD2 (methyl CpG binding domain protein 2)-mediated genome regulation. Caspase-mediated NPC trimming was also observed during neurogenesis and endoplasmic reticulum (ER) stress. Our results illustrate that NPC composition can be proteolytically modulated in response to non-apoptotic cues, and call for a reassessment of the death-centric view of caspases.

sentences: Discovery of diverse human BH3-only and non-native peptide binders of pro-apoptotic BAK indicate that activators and inhibitors use asimilar binding mode and are not distinguished by binding affinity or kinetics Apoptosis is a programmed form of cell death important for the development and maintenance of tissue homeostasis. The BCL-2 protein family controls key steps in apoptosis, dysregulation of which can lead to a wide range of human diseases. BCL-2 proteins comprise three groups: anti-apoptotic proteins, pro-apoptotic proteins, and BH3-only proteins. BAK is one of two pro-apoptotic proteins, and previous work has shown that binding of certain BH3-only proteins such as truncated BID (tBID), BIM, or PUMA to BAK leads to mitochondrial outer membrane permeabilization, the release of cytochrome c, and ultimately cell death. This process, referred to as activation, involves the BH3-stimulated conversion of BAK from monomer to dimer and then to oligomers that promote membrane disruption. Crystal structures of putative intermediates in this pathway, crosslinking data, and in vitro functional tests have provided insights into the activation event, yet the sequence-function relationships that make some but not all BH3-only proteins function as activators remain largely unexamined. In this work, we used computational protein design, yeast surface-display screening of candidate BH3-like peptides, and structure-based energy scoring to identify ten new binders of BAK that span a large sequence space. Among the new binders are two peptides from human proteins BNIP5 and PXT1 that promote BAK activation in liposome assays and induce cytochrome-c release from mitochondria, expanding current views of how BAK-mediated cell death may be triggered in cells. High-resolution crystal structures and binding experiments revealed a high degree of similarity in binding geometry, affinity, and association kinetics between peptide activators and inhibitors, including peptides described previously and those identified in this work. We propose a model for BAK activation that is based on differential engagement of BAK monomers vs. the BAK activation transition state that integrates our observations with previous reports of BAK binders, activators, and inhibitors.

sentences: Characterization of TR-107, a Novel Chemical Activator of the Human Mitochondrial Protease ClpP We recently described the identification of a new class of small molecule activators of the mitochondrial protease ClpP. These compounds synthesized by Madera Therapeutics showed increased potency of cancer growth inhibition over the related compound ONC201. In this study, we describe chemical optimization and characterization of the next generation of highly potent and selective small molecule ClpP activators (TR compounds) and demonstrate their efficacy against breast cancer models in vitro and in vivo. One of these compounds (TR-107) with excellent potency, specificity and drug-like properties was selected for further evaluation. Examination of TR-107 effects in triple-negative breast cancer (TNBC) cell models showed growth inhibition in the low nanomolar range, equipotent to paclitaxel, in a ClpP-dependent manner. TR-107 reduced specific mitochondrial proteins including OXPHOS and TCA cycle components, in a time, dose and ClpP-dependent manner. Seahorse XF analysis and glucose deprivation experiments confirmed inactivation of OXPHOS and demonstrated an increased dependence on glycolysis following TR-107 exposure. The pharmacokinetic properties of TR-107 were compared to other known ClpP activators including ONC201 and ONC212. TR-107 displayed excellent exposure and serum t1/2 after oral administration. The antitumor response to TR-107 was investigated using human TNBC MDA-MB-231 cell line-induced xenograft tumors. Oral administration of TR-107 resulted in reduction in tumor volume and extension of survival in the treated compared with vehicle control mice. In summary, we describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.

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Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma Immune cell composition within the tumor microenvironment is regulated by tumor-derived factors. Cell Communication Network factor 4 (CCN4/WISP1) is a matricellular protein secreted by cancer cells that promotes metastasis by inducing the epithelial-mesenchymal transition. While metastatic dissemination limits patient survival, the absence of anti-tumor immunity also associates with poor patient out-comes with recent work suggesting these two clinical correlates are linked. Motivated by finding that CCN4 was associated with a dampened anti-tumor immune contexture in patients diagnosed with primary melanoma, we tested for a direct causal link by knocking out CCN4 (CCN4-KO) in the B16F0 and YUMM1.7 mouse models for melanoma. Tumor growth was significantly reduced when CCN4-KO melanoma cells were implanted subcutaneously in immunocompetent C57BL/6 mice but not in immunodeficient NSG mice. Correspondingly, the frequency of total CD45+ tumor-infiltrating leukocytes was significantly increased in CCN4-KO tumors, with increased natural killer (NK) and effector CD8+ T cells and reduced myeloid-derived suppressor cells (MDSC). Additionally, the absence of tumor-derived CCN4 was associated with an impaired splenic generation of suppressive granulocytic MDSC. Among mechanisms linked to local immunosuppression, we found CCN4 directly suppressed antigen-induced IFN{gamma} release by CD8+ T cells, promoted glycolysis and consequent lactate release by melanoma cells, and enhanced tumor secretion of MDSC-attracting chemokines like CCL2 and CXCL1. Finally, CCN4-KO in B16F0 and YUMM1.7 melanoma cells complemented the anti-tumor effect of immune checkpoint blockade (ICB) therapy. Overall, our results suggest that CCN4 promotes tumor-induced immunosuppression and is a potential target for therapeutic combinations with ICB.

Statement of SignificanceGiven emerging interest in understanding the interplay between functional plasticity and anti-tumor immunity, Cell Communication Network factor 4, a secreted matricellular protein linked to promoting metastasis in melanoma, also suppresses anti-tumor immunity.

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Belowground plant responses to root herbivory depend on the composition and structure of their root-colonising arbuscular mycorrhizal fungi O_LIThere is growing interest in managing arbuscular mycorrhizal (AM) fungi in agriculture to support plant production. These fungi can support crop growth and nutrient uptake but also affect plant-herbivore interactions. Our knowledge of how native AM fungal diversity and community composition influence these interactions is limited, while our understanding of this in relation to root-herbivory is lacking altogether.
C_LIO_LITo begin to address these knowledge gaps, plants were grown with no AM fungi or were inoculated with native fungal communities sourced from either a crop field (field community), a sclerophyll forest (forest community), or a crop field in fallow (fallow community). We then explored how the composition and structure (species richness and relative abundance) of root-colonising AM fungal communities was associated with the growth and belowground nutrient responses of a major crop (Sorghum bicolor) to attack from a root-feeding insect (Dermolepida albohirtum).
C_LIO_LIDNA metabarcoding revealed plants associated with three distinct root-colonising AM fungal communities. Fungal taxon richness in roots was highest in the field community and lowest in the fallow community. Both the field and fallow communities were dominated by the putatively ruderal genera Glomus and Claroideoglomus, while the forest-derived community contained greater proportions of Paraglomus and Ambispora.
C_LIO_LIIn response to root herbivory, plants without AM fungi and plants colonised by the forest community exhibited root biomass losses of 61% and 44%, respectively. Similarly, these two groups also had reductions of 59% and 65% in their root phosphorus content, respectively, when subjected to the root herbivore. In contrast, plants associating with communities harbouring greater proportions of Glomus and Claroideoglomus (the field and fallow communities) did not exhibit reductions in root biomass or nutrient content.
C_LIO_LIOur results show that plant responses to root-herbivory vary with root-colonising AM fungal community composition and structure. In a community context, our findings suggest that stronger associations with the genera Claroideoglomus and Glomus may potentially support crop tolerance-associated responses belowground. There is an urgent need for more exploration of how natural assemblages of AM fungi differentially mediate plant-herbivore interactions if we are to effectively manage soil fungi in sustainable agricultural systems.
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sentences: ATR-I774Yfs*5 promotes genomic instability through micronuclei formation Although mismatch repair (MMR) defects are associated with high risk of malignancy, the specific oncogenic drivers pertinent to MMR-affected cancers are poorly characterized. The heterozygous ATR-I774Yfs*5 mutation, the result of strand slippage in a poly-A tract of the Ataxia Telangiectasia and Rad3 related (ATR) gene, is overexpressed in MMR-defective malignancies including colorectal carcinoma (CRC) and is the most common ATR mutation in cancer. Here, we explore the contribution of ATR-I774Yfs*5 to genomic integrity. Using heterozygous ATR-I774Yfs*5 HCT-116 cells to mimic the native mutation, we found this mutation reduced ATR activity as measured by damage-induced Chk1 phosphorylation at S317 and ATR autophosphorylation ATR at T1989. ATR-I774Yfs*5 expression impaired genomic stability as visualized by the appearance of micronuclei in two stable expression models as well as in cell lines transfected with ATR-I774Yfs*5. Micronucleus development was dependent on replication and independent of ATR copy number. ATR-I774Yfs*5 expression did not alter cellular viability, cell cycle progression, or replicative rate, suggesting this mutation is well-tolerated despite its destabilizing effect on the genome. Taken together, these data suggest that the ATR-I774Yfs*5, whose development is favored in the context of MMR deficiency, may represent an important driver of a mutator phenotype by promoting genomic instability.

sentences: Improved post-stroke spontaneous recovery by astrocytic extracellular vesicles Spontaneous recovery after a stroke accounts for a major part of the neurological recovery in patients. However limited, the spontaneous recovery is mechanistically driven by axonal restorative processes for which several molecular cues have been previously described. We report the acceleration of spontaneous recovery in a preclinical model of ischemia/reperfusion in rats via a single intracerebroventricular administration of extracellular vesicles released from primary cortical astrocytes. We used MRI, confocal and multiphoton microscopy to correlate the structural remodeling of the corpus callosum and striatocortical circuits with neurological performance over 21 days. We also evaluated the functionality of the corpus callosum by repetitive recordings of compound action potentials to show that the recovery facilitated by astrocytic extracellular vesicles was both anatomical and functional. Our data provide compelling evidence that astrocytes can hasten the basal recovery that naturally occurs post-stroke through the release of cellular mediators contained in extracellular vesicles.

sentences: Green Fluorescent Carbon Dots as Targeting Probes for LED-Dependent Bacterial Killing The emergence of antimicrobial resistance represents a significant health and economic challenge worldwide. The slow pace of antibacterial discovery necessitates strategies for optimal use of existing agents, including effective diagnostics able to drive informed prescribing; and development of alternative therapeutic strategies that go beyond traditional small-molecule approaches. Thus, the development of novel probes able to target bacteria for detection and killing, and that can pave the way to effective theranostic strategies, is of great importance. Here we demonstrate that metal-free green-emitting fluorescent carbon dots (FCDs) synthesized from glucosamine HCl and m-phenylenediamine, and featuring 2,5-deoxyfructosazine on a robust amorphous core, can label both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) bacterial pathogens within 10 minutes of exposure. Moreover, effective killing of Gram-positive and -negative bacteria can be induced by combining FCD treatment with irradiation by LED light in the visible range. Cell-based, electron microscopy and Tandem Mass Tag (TMT) proteomic experiments indicate that FCD administration in combination with LED exposure gives rise to local heating, ROS production, and membrane- and DNA-damage, suggesting multiple routes to FCD-mediated bacterial killing. Our data identify FCDs as materials that combine facile synthesis from low-cost precursors with labelling and light-dependent killing of clinically important bacterial species, and that thus warrant further exploration as the potential bases for novel theranostics.

sentences: Intrabody induced cell death by targeting the T. brucei cytoskeletal protein TbBILBO1 Trypanosoma brucei belongs to a genus of protists that cause life-threatening and economically important diseases of human and animal populations in Sub-Saharan Africa. T. brucei cells are covered in surface glycoproteins some of which are used to escape the host immune system. Exo-/endocytotic trafficking of these and other molecules occurs via a single copy organelle called the flagellar pocket (FP). The FP is maintained and enclosed around the flagellum by the flagellar pocket collar (FPC). To date, the most important cytoskeletal component of the FPC is an essential, calcium-binding, polymer-forming protein called TbBILBO1. In searching for novel immune-tools to study this protein, we raised nanobodies against TbBILBO1. Nanobodies (Nb) that were selected according to their binding properties to TbBILBO1, were tested as immunofluorescence tools, and expressed as intrabodies (INb). One of them, Nb48, proved to be the most robust nanobody and intrabody. We further demonstrate that inducible, cytoplasmic expression of INb48 was lethal to these parasites, producing abnormal phenotypes resembling those of TbBILBO1 RNAi knockdown. Our results validate the feasibility of generating functional single-domain antibody derived intrabodies to target trypanosome cytoskeleton proteins.

sentences: Substitution Rate Variation in a Robust Procellariiform Seabird Phylogeny is not Solely Explained by Body Mass, Flight Efficiency, Population Size or Life History Traits Substitution rate variation among branches can lead to inaccurate reconstructions of evolutionary relationships and obscure the true phylogeny of affected clades. Body mass is often assumed to have a major influence on substitution rate, though other factors such as population size, life history traits, and flight demands are also thought to have an influence. Birds of the order Procellariiformes--which encompasses petrels, storm-petrels and albatrosses--show a striking 900-fold difference in body mass between the smallest and largest members, divergent life history traits, and substantial heterogeneity in mitochondrial substitution rates. Here, we used genome-scale nuclear DNA sequence data from 4365 ultraconserved element loci (UCEs) in 51 procellariiform species to examine whether phylogenetic reconstruction using genome-wide datasets is robust to the presence of rate heterogeneity, and to identify predictors of substitution rate variation. Our results provide a backbone phylogeny for procellariiform seabirds and resolve several controversies about the evolutionary history of the order, demonstrating that albatrosses are basal, storm-petrels are paraphyletic and diving petrels nestled within the Procellariidae. We find evidence of rate variation; however, all phylogenetic analyses using both concatenation and multispecies coalescent approaches recovered the same branching topology, including analyses implementing different clock models, and analyses of the most and least clock-like loci. Overall, we find that rate heterogeneity is little impacted by body mass, population size, age at first breeding, and longevity but moderately correlated with hand-wing index, a proxy for wing shape and flight efficiency. Given our results and the context of the broader literature perhaps it is time that we begin to question the prevailing paradigm that one or a few traits largely explain rate variation and accept instead that substitution rate may be the product of weak interactions among many, potentially taxon-specific, variables.

sentences: Oxytocin promotes social grooming in bonobos: testing the biobehavioural feedback loop hypothesis Oxytocin has attracted research attention due to its role in promoting social bonding. One notable recent hypothesis is the biobehavioral feedback loop, which posits that the oxytocin system has evolved to support the formation and maintenance of social bonds through a positive feedback loop, where oxytocin promotes social behaviours which then cause oxytocin release themselves. In the two Pan species, humans closest relatives, oxytocin is known to be released following key behaviours related to social bonding, such as social grooming in chimpanzees and female-female sexual behaviour in bonobos. However, no experimental evidence has demonstrated that oxytocin promotes such socio-positive behaviours. To test this, we administered nebulized oxytocin or saline placebo to a group of female bonobos and subsequently observed the change in their gross behavior during free interaction. We found that bonobos groomed other group members significantly more frequently in the oxytocin compared to placebo condition. Other behavioural measures did not largely differ between conditions, except for a nonsignificant trend for reduction in abnormal regurgitation/reingestion behaviour. Overall, we found that oxytocin promoted socio-positive interaction in bonobos, providing support for the biobehavioural feedback loop hypothesis of oxytocin in bonobo social evolution.

sentences: Structural architecture of a dimeric paramyxovirus polymerase complex Human parainfluenza virus type 3 (hPIV3), a member of non-segmented, negative-strand RNA viruses (nsNSVs), is the second most common cause of severe respiratory disease in pediatrics. The transcription and replication processes of nsNSVs are catalyzed by a multi-functional RNA-dependent RNA polymerase (RdRp) complex composed of the large protein (L) and the phosphoprotein (P). Previous studies have shown that the polymerase can adopt a dimeric form, however, the structure of the dimer and how it functions are not understood. Here we determined the cryo-EM structure of hPIV3 L-P complex at 2.7 [A] with substantial structural details. A putative catalytic magnesium ion could be built in our structure, and structural comparison revealed atomic features conserved with other RNA viruses. Interactions identified between the two priming and intrusion loops and the connector domain potentially trigger the spatial movement of three C-terminal L domains for different steps of transcription and replication. Structural comparison with other nsNSV RdRps suggests common features of L-P binding. Furthermore, we report for the first time the structural basis of the L-L interaction in the partially modelled dimeric L-P structure, in which the connector domain of one L is positioned at the putative RNA template entry of the other L. Based on these findings, we propose a model by which L dimerization promotes efficient conversion of nascent RNA into a template.

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Ultrasound-mediated blood-brain barrier disruption improves anti-pyroglutamate3 Aβ antibody efficacy and enhances phagocyte infiltration into brain in aged Alzheimer's disease-like mice Pyroglutamate-3 amyloid-{beta} (pGlu3 A{beta}) is an N-terminally modified, toxic form of amyloid-{beta} that is present in cerebral amyloid plaques and vascular deposits. Using the Fc-competent murine anti-pGlu3 A{beta} monoclonal antibody (mAb), 07/2a, we present here a nonpharmacological approach using focused ultrasound (FUS) with intravenous (i.v.) injection of microbubbles (MB) to facilitate i.v. delivery of the 07/2a mAb across the blood brain barrier (BBB) in order to improve A{beta} removal and restore memory in aged APP/PS1 mice, an Alzheimers disease (AD)-like model of amyloidogenesis.

Compared to sham-treated controls, aged APP/PS1 mice treated with 07/2a immediately prior to FUS-mediated BBB disruption (mAb + FUS-BBBD combination treatment) showed significantly better spatial learning and memory in the Water T Maze. FUS-BBBD treatment alone improved contextual fear learning and memory in aged WT and APP/PS1 mice, respectively. APP/PS1 mice given the combination treatment had reduced A{beta}42 and pGlu3 A{beta} hippocampal plaque burden compared to PBS-treated APP/PS1 mice.

Hippocampal synaptic puncta density and synaptosomal synaptic protein levels were also higher in APP/PS1 mice treated with 07/2a just prior to BBB disruption. Increased Iba-1+ microglia were observed in the hippocampi of AD mice treated with 07/2a with and without FUS-BBBD, and APP/PS1 mice that received hippocampal BBB disruption and 07/2a showed increased Ly6G+ monocytes in hippocampal CA3. FUS-induced BBB disruption did not increase the incidence of microhemorrhage in mice with or without 07/2a mAb treatment.

Our findings suggest that FUS is useful tool that may enhance delivery of an anti-pGlu3 A{beta} mAb for immunotherapy. FUS-mediated BBB disruption in combination with the 07/2a mAb also appears to facilitate monocyte infiltration in this AD model. Overall, these effects resulted in greater sparing of synapses and improved cognitive function without causing overt damage, suggesting the possibility of FUS as a noninvasive method to increase the therapeutic efficacy in AD patients.

sentences: The NUCKS1-SKP2-p21/p27 axis controls S phase entry Efficient entry into S phase of the cell cycle is necessary for embryonic development and tissue homeostasis. However, unscheduled S phase entry triggers DNA damage and promotes oncogenesis, underlining the requirement for strict control. Here, we identify the NUCKS1-SKP2-p21/p27 axis as a checkpoint pathway for the G1/S transition. In response to mitogenic stimulation, NUCKS1, a transcription factor, is recruited to chromatin to activate expression of SKP2, the F-box component of the SCFSKP2 ubiquitin ligase, leading to degradation of p21 and p27 and promoting progression into S phase. In contrast, DNA damage induces p53-dependent transcriptional repression of NUCKS1, leading to SKP2 downregulation, p21/p27 upregulation, and cell cycle arrest. We propose that the NUCKS1-SKP2-p21/p27 axis integrates mitogenic and DNA damage signalling to control S phase entry. TCGA data reveal that this mechanism is hijacked in many cancers, potentially allowing cancer cells to sustain uncontrolled proliferation.

sentences: Transcriptional targets of senataxin and E2 promoter binding factors are associated with neuro-degenerative pathways during increased autophagic flux Autophagy is an intracellular recycling process that degrades harmful molecules, maintains optimal composition of cellular organelles and enables survival during starvation. Previous studies have demonstrated how transcription factors (TFs) can increase autophagy with therapeutic potential (impaired autophagy in the ageing brain, in particular, may be an important risk factor for dementia). To investigate the transcriptional regulation of autophagy from a systems perspective, we induced autophagy by amino acid starvation and mTOR inhibition in HeLa, HEK 293 and SH-SY5Y cells and used RNA-seq to measure gene expression at three time points. We observed 453 differentially expressed (DE) genes due to starvation and 284 genes due to mTOR inhibition (PFDR < 0.05 in every cell line). Pathway analyses confirmed enrichment of genes implicated in Alzheimers (PFDR < 0.001 in SH-SY5Y and HeLa) and Parkinsons (PFDR [≤] 0.024 in SH-SY5Y and HeLa) diseases and amyotrophic lateral sclerosis (ALS, PFDR < 0.05 in 4 of 6 experiments). We then integrated Signaling Pathway Impact Analysis and TF target enrichment testing to predict which TF target genes were contributing to pathway perturbation. Differential expression of the Senataxin (SETX) target gene set was predicted to activate multiple neurodegenerative pathways (PFDR [≤] 0.04). Notably, SETX is a causal gene for a rare form of ALS. In the SH-SY5Y cells of neuronal origin, the E2F transcription family was predicted to activate Alzheimers disease pathway (PFDR [≤] 0.0065). SETX and E2F may be important mediators of transcriptional regulation of autophagy and may provide new therapeutic opportunities for neuro-degenerative conditions.

sentences: Human orbitofrontal cortex signals decision outcomes to sensory cortex during behavioural adaptations The ability to respond adaptively to an ever-changing environment relies on the orbitofrontal cortex (OFC). The OFC sends neuroanatomical projections to the primary sensory cortex - yet the contribution of this top-down feedback projection to behavioural flexibility in humans is unknown. Inspired from recent rodent studies, we here combined a probabilistic Go/No-Go tactile reversal learning task with functional magnetic resonance imaging (fMRI) in human participants to investigate how OFC interacts with the primary somatosensory cortex (S1) to promote flexible decision-making. We show a distinct task-dependent engagement of S1 and lateral OFC: while the lateral OFC responds saliently and transiently to rule-switches, activity in S1 reflects initial task learning and persistent engagement after each rule-switch event. Unlike the contralateral S1, which represents the sensory input, activity in ipsilateral S1 mirrors the outcome value during re-learning. Importantly, the implementation of this outcome-selectivity in ipsilateral S1 is dependent on top-down  teaching signals from lateral OFC. Overall, as in mice, we show comparable physiological and computational signatures of how dynamic interactions between OFC and sensory cortex support flexible decision-making in humans.

sentences: Speed-related but not detrended gait variability increases with more sensitive self-paced treadmill controllers at multiple slopes Self-paced treadmills are being used more frequently to study humans walking with their self-selected gaits on a range of slopes. There are multiple options to purchase a treadmill with a built-in controller, or implement a custom written self-paced controller, which raises questions about how self-paced controller affect treadmill speed and gait biomechanics on multiple slopes. This study investigated how different self-paced treadmill controller sensitivities affected gait parameters and variability on decline, level, and incline slopes. We hypothesized that increasing self-paced controller sensitivity would increase gait variability on each slope. We also hypothesized that detrended variability could help mitigate differences in variability that arise from differences in speed fluctuations created by the self-paced controllers. Ten young adults walked on a self-paced treadmill using three controller sensitivities (low, medium, and high) and fixed speeds at three slopes (decline, -10{degrees}; level, 0{degrees}; incline, +10{degrees}). Within each slope, average walking speeds and spatiotemporal gait parameters were similar regardless of self-paced controller sensitivity. With higher controller sensitivities on each slope, speed fluctuations, speed variance, and step length variance increased whereas step frequency variance and step width variance were unaffected. Detrended variance was not affected by controller sensitivity suggesting that detrending variability helps mitigate differences associated with treadmill speed fluctuations. Speed-trend step length variances, however, increased with more sensitive controllers. Further, detrended step length variances were similar for self-paced and fixed speed walking, whereas self-paced walking included substantial speed-trend step length variance not present in fixed speed walking. In addition, regardless of the self-paced controller, subjects walked fastest on the level slope with the longest steps, narrowest steps, and least variance. Overall, our findings suggest that separating gait variability into speed-trend and detrended variability could be beneficial for interpreting gait variability among multiple self-paced treadmill studies and when comparing self-paced walking with fixed speed walking.

sentences: Voluntary motor commands are preferentially released during restricted sensorimotor beta rhythm phases Voluntary movement requires motor commands to be released from motor cortex (M1) and transmitted to spinal motoneurons and effector muscles. M1 activity oscillates between brief excitatory and inhibitory states that correlate with single neuron spiking rates. Here, we asked if the motor commands needed to produce voluntary, self-paced finger movements are preferentially released from M1 during restricted phases of this ongoing sensorimotor oscillatory activity. 21 healthy adults performed a self-paced finger movement task while EEG and EMG signals were recorded. For each finger movement, we identified the individual sensorimotor mu (8-12 Hz) and beta (13-35 Hz) oscillatory phase at the estimated time of motor command release from M1 by subtracting individually-defined MEP latencies from EMG-determined movement onset times. We report that motor commands were preferentially released at ~120{degrees} along the beta cycle but were released uniformly across the mu cycle. These results suggest that motor commands are preferentially released from M1 near optimal peak phases of endogenous beta rhythms.

sentences: Low affinity integrin states have faster binding kinetics than the high affinity state Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins 4{beta}1 and 5{beta}1 and show that the low-affinity states bind substantially faster than the high-affinity state. On and off-rate measurements are similar for integrins on cell surfaces and ectodomain fragments. Although the extended-open conformations on-rate is [~]20-fold slower, its off-rate is [~]25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. These kinetic measurements, together with previous equilibrium measurements of integrin conformational state affinity and relative free energy on intact cells, are key to a definitive understanding of the mechanism of integrin activation.

sentences: High-speed large-scale 4D activities mapping of moving C. elegans by deep-learning-enabled light-field microscopy on a chip We report a novel fusion of microfluidics and light-field microscopy, to achieve high-speed 4D (space + time) imaging of moving C. elegans on a chip. Our approach combines automatic chip-based worm loading / compartmentalization / flushing / reloading with instantaneous deep-learning light-field imaging of moving worm. Taken together, we realized intoto image-based screening of wild-type and uncoordinated-type worms at a volume rate of 33 Hz, with sustained observation of 1 minute per worm, and overall throughput of 42 worms per hour. With quickly yielding over 80000 image volumes that four-dimensionally visualize the dynamics of all the worms, we can quantitatively analyse their behaviours as well as the neural activities, and correlate the phenotypes with the neuron functions. The different types of worms can be readily identified as a result of the high-throughput activity mapping. Our approach shows great potential for various lab-on-a-chip biological studies, such as embryo sorting and cell growth assays.

sentences: A marine fungus efficiently degrades polyethylene Plastics pollution has been a global concern. Huge quantities of polyethylene (PE), the most abundant and refractory plastic in the world, have been accumulating in the environment causing serious ecological problems. However, the paucity of microorganisms and enzymes that efficiently degrading PE seriously impedes the development of bio-products to eliminate this environmental pollution. Here, by screening hundreds of plastic waste-associated samples, we isolated a fungus (named Alternaria sp. FB1) that possessing a prominent capability of colonizing, degrading and utilizing PE. Strikingly, the molecular weight of PE film decreased 95% after the fungal treatment. Using GC-MS, we further clarified that a four-carbon product (named Diglycolamine) accounted for 93.28% of all degradation products after the treatment by strain FB1. We defined potential enzymes that involved in the degradation of PE through a transcriptomic method. The degradation capabilities of two representative enzymes including a laccase and a peroxidase were verified. Lastly, a complete biodegradation process of PE is proposed. Our study provides a compelling candidate for further investigation of degradation mechanisms and development of biodegradation products of PE.

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Brainprints: identifying individuals from magnetoencephalograms Magnetoencephalography (MEG) is used to study a wide variety of cognitive processes. Increasingly, researchers are adopting principles of open science and releasing their MEG data. While essential for reproducibility, sharing MEG data has unforeseen privacy risks. Individual differences may make a participant identifiable from their anonymized recordings. However, our ability to identify individuals based on these individual differences has not yet been assessed. Here, we propose interpretable MEG features to characterize individual difference. We term these features brainprints (brain fingerprints). We show through several datasets that brainprints accurately identify individuals across days, tasks, and even between MEG and Electroencephalography (EEG). Furthermore, we identify consistent brainprint components that are important for identification. We study the dependence of identifiability on the amount of data available. We also relate identifiability to the level of preprocessing, the experimental task. Our findings reveal specific aspects of individual variability in MEG. They also raise concerns about unregulated sharing of brain data, even if anonymized.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/159913v3_fig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@50c58corg.highwire.dtl.DTLVardef@d99cc1org.highwire.dtl.DTLVardef@c255cdorg.highwire.dtl.DTLVardef@8f2c13_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFigure 1:C_FLOATNO Graphical abstract.

Identifying which subject a segment of MEG data belongs to is strikingly easy when other data from the same session is available for every subject. We propose three types of interpretable features that can also be used to identify individuals across sessions with high accuracy. Identifiability of individuals is influenced by factors such as resting state vs. task state, components of each feature, the sample size and the level of preprocessing. Our results reveal aspects of individual variability in MEG signals and highlight privacy risks associated with MEG data sharing.

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sentences: Physiological and life history changes associated with seasonal adaptation in the cactophilic Drosophila mojavensis Many insects inhabiting temperate climates are faced with changing environmental conditions throughout the year. Depending on the species, these environmental fluctuations can be experienced within a single generation or across multiple generations. Strategies for dealing with these seasonal changes vary across populations. Drosophila mojavensis is a cactophilic Drosophila species endemic to the Sonoran Desert. The Sonoran Desert regularly reaches temperatures of 50{degrees}C in the summer months. As individuals of this population are rare to collect in the summer months, we therefore simulated in a temperature and light controlled chamber the cycling temperatures experienced by D. mojavensis in the Sonoran Desert from April to July (four generations) to understand the physiological and life history changes that allow this population to withstand these conditions. In contrast to our hypothesis of a summer aestivation, we found that D. mojavensis continue to reproduce during the summer months, albeit with lower viability, but the longevity of the population is highly reduced during this period. As expected, stress resistance increased during the summer months in both the adult and the larval stages. This study examines several strategies for withstanding the Sonoran Desert summer conditions which may be informative in the study of other desert endemic species.

sentences: In Silico Molecular-Based Rationale for SARS-CoV-2 Spike Circulating Mutations Able to Escape Bamlanivimab and Etesevimab Monoclonal Antibodies The purpose of this work was to provide an in silico molecular rationale of the role eventually played by currently circulating S-RBDCoV-2 mutations in evading the immune surveillance effects elicited by the two Eli Lilly LY-CoV555/bamlanivimab and LY-CoV016/etesevimab monoclonal antibodies. The main findings from this study and shows that, compared to the wild-type SARS-CoV-2 spike protein, mutations E484A/G/K/Q/R/V, Q493K/L/R, S494A/P/R, L452R and F490S are predicted to be markedly resistant to neutralization by LY-CoV555, while mutations K417E/N/T, D420A/G/N, N460I/K/S/T, T415P, and Y489C/S are predicted to confer LY-CoV016 escaping advantage to the viral protein. A challenge of our global in silico results against the relevant experimental data resulted in an overall 90% agreement. This achievement not only constitutes a further, robust validation of our computer-based approach but also yields a molecular-based rationale for all relative experimental findings, and leads us to conclude that the current circulating SARS-CoV-2 and all possible emergent variants carrying these mutations in the spike protein can present new challenges for mAb-based therapies and ultimately threaten the fully-protective efficacy of currently available vaccines.

sentences: Immune elicitation by sensing the conserved signature from phytocytokines and microbes via the Arabidopsis MIK2 receptor Sessile plants encode a large number of small peptides and cell surface-resident receptor kinases, most of which have unknown functions. Here, we report that the Arabidopsis receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) recognizes the conserved signature motif of SERINE RICH ENDOGENOUS PEPTIDEs (SCOOPs) from plants as well as proteins present in fungal Fusarium spp. and bacterial Comamonadaceae, and elicits potent immune responses. SCOOP signature peptides trigger diverse immune and physiological responses in a MIK2-dependent manner with a sub-nanomolar sensitivity and directly bind to the extracellular leucine rich-repeat domain of MIK2 in vivo and in vitro, indicating that MIK2 is the receptor of SCOOP peptides. Perception of SCOOP peptides induces the association of MIK2 and the coreceptors SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (SERK3) and SERK4 and relays the signaling through the cytosolic receptor-like kinases BOTRYTIS-INDUCED KINASE 1 (BIK1) and AVRPPHB SUSCEPTIBLE1 (PBS1)-LIKE 1 (PBL1). Our study identified a unique plant receptor that bears a dual recognition capability sensing the conserved peptide motif from phytocytokines and microbial proteins via a convergent signaling relay to ensure a robust immune response.

sentences: DUF581-9 (At2g44670; FLZ3) negatively regulates SnRK1 activity by interference with T-loop phosphorylation In plants, SUCROSE NON-FERMENTING RELATED KINASE 1 (SnRK1) is a key energy-sensor that orchestrates large-scale transcriptional reprogramming to maintain cellular homeostasis under energy deficit. SnRK1 activity is under tight negative control, although the exact mechanisms leading to its activation are far from being understood. We show that the Arabidopsis DOMAIN OF UNKNOWN FUNCTION (DUF581) protein DUF581-9/FLZ3 binds to the catalytic SnRK1 subunit KIN10 to inhibit its activation by GRIK-dependent T-loop phosphorylation. Overexpression of DUF581-9 in Arabidopsis dampens SnRK1 signalling and interferes with adaptation to dark-induced starvation. The presence of DUF581-9 significantly reduced SnRK1 activity in protoplasts and in vitro. This was accompanied by a reduction in T175 T-loop phosphorylation and also diminished KIN10 auto-phosphorylation. Furthermore, DUF581-9 reduced binding of the up-stream activating kinase GRIK2 to KIN10, explaining the reduced KIN10 T-loop phosphorylation. Ectopically expressed DUF581-9 protein was rapidly turned over by the proteasome when Arabidopsis plants were subjected to starvation treatment, likely releasing its inhibitory activity on the SnRK1 complex. Taken together, our results support a model in which DUF581-9 negatively regulates SnRK1 activity under energy sufficient conditions. Turnover over of the protein provides a rapid way for SnRK1 activation under energy deficit without the need of de-novo protein synthesis.

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Accelerometer-measured physical activity and cognitive functioning: A Mendelian Randomization study Physical activity and cognitive functioning are strongly intertwined. However, the causal relationships underlying this association are still unclear. Physical activity can enhance brain functions, but healthy cognition may also promote engagement in physical activity. Here, we assessed the bidirectional relationships between physical activity and general cognitive functioning using Latent Heritable Confounder Mendelian Randomization (LHC-MR). Association data were drawn from two large-scale genome-wide association studies (UK Biobank and COGENT) on accelerometer-measured moderate, vigorous, and average physical activity (N = 91,084) and cognitive functioning (N = 257,841). After Bonferroni correction, we observed significant LHC-MR associations suggesting that increased fraction of both moderate (b = 0.32, CI95% = [0.17,0.47], P = 2.89e-05) and vigorous physical activity (b = 0.22, CI95% = [0.06,0.37], P = 0.007) lead to increased cognitive functioning. In contrast, we found no evidence of a causal effect of average physical activity on cognitive functioning, and no evidence of a reverse causal effect (cognitive functioning on any physical activity measures). These findings provide new evidence supporting a beneficial role of moderate and vigorous physical activity (MVPA) on cognitive functioning.

Significance StatementWhether the observed correlation between physical activity and cognitive functioning reflects causal relationships in either direction is still unclear. To investigate the potential causal relationships underlying this association, we conducted recently-improved genetically-informed analyses with two large-scale datasets including accelerometer-measured physical activity. In line with theoretical models and previous experimental work explaining the mechanisms underlying the association between physical activity and cognitive functioning, our results revealed a one-way association: Higher levels of moderate and vigorous physical activity can potentially cause higher cognitive functioning. These findings suggest that physical activity plays a fundamental role in general cognitive functioning, and that health policies and interventions promoting moderate or vigorous physical activity are relevant to improve cognitive performance and to delay its decline.

sentences: Single-cell genomics reveals region-specific developmental trajectories underlying neuronal diversity in the prenatal human hypothalamus The hypothalamus is critically important for regulating most autonomic, metabolic, and behavioral functions essential for life and species propagation, yet a comprehensive understanding of neuronal subtypes and their development in the human brain is lacking. Here, we characterized the prenatal human hypothalamus by sequencing the transcriptomes of 45,574 single-cells from 12 embryos, spanning gestational weeks 4 through 25. These cells describe a temporal trajectory from proliferative stem cell populations to maturing neurons and glia, including 38 distinct excitatory and inhibitory neuronal subtypes. Merging these data with paired samples from the cortex and ganglionic eminences (GE) revealed two distinct neurogenesis pathways, one shared between GE and hypothalamus and a second unique to cortex. Gene regulatory network modeling predicted that these distinct maturation trajectories involve the activation of region- and cell type-specific transcription factor networks. These results provide the first comprehensive transcriptomic view of human hypothalamus development at cellular resolution.

sentences: Novel regulators of PrPC biosynthesis revealed by genome-wide RNA interference The cellular prion protein PrPC is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels of PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. We cultured human U251-MG glioblastoma cells in the presence of 64752 unique siRNAs targeting 21584 annotated human genes, and measured PrPC using a one-pot fluorescence-resonance energy transfer immunoassay in 51128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies.

sentences: Flavonols and dihydroflavonols inhibit the main protease activity of SARS-CoV-2 and the replication of human coronavirus 229E Since December 2019, the deadly novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the current COVID-19 pandemic. To date, vaccines are available in the developed countries to prevent the infection of this virus, however, medicines are necessary to help control COVID-19. Human coronavirus 229E (HCoV-229E) causes the common cold. The main protease (Mpro) is an essential enzyme required for the multiplication of these two viruses in the host cells, and thus is an appropriate candidate to screen potential medicinal compounds. Flavonols and dihydroflavonols are two groups of plant flavonoids. In this study, we report docking simulation with two Mpro enzymes and five flavonols and three dihydroflavonols, in vitro inhibition of the SARS-CoV-2 Mpro, and in vitro inhibition of the HCoV 229E replication. The docking simulation results predicted that (+)-dihydrokaempferol, (+)-dihydroquercetin, (+)-dihydromyricetin, kaempferol, quercetin, myricentin, isoquercetin, and rutin could bind to at least two subsites (S1, S1, S2, and S4) in the binding pocket and inhibit the activity of SARS-CoV-2 Mpro. Their affinity scores ranged from -8.8 to -7.4. Likewise, these compounds were predicted to bind and inhibit the HCoV-229E Mpro activity with affinity scores ranging from -7.1 to -7.8. In vitro inhibition assays showed that seven available compounds effectively inhibited the SARS-CoV-2 Mpro activity and their IC50 values ranged from 0.125 to 12.9 {micro}M. Five compounds inhibited the replication of HCoV-229E in Huh-7 cells. These findings indicate that these antioxidative flavonols and dihydroflavonols are promising candidates for curbing the two viruses.

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Engineering CAR-NK cells to secrete IL15 sustains their anti-AML functionality, but is associated with systemic toxicities BackgroundThe prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. NK cells can elicit an anti-leukemic response via a repertoire of activating receptors that bind AML surface ligands. NK cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity, and interleukin-15 (IL15) to enhance their persistence.

MethodsWe characterized in detail NK cell populations expressing a panel of AML (CD123)-specific CARs and/or IL15 in vitro and in AML xenograft models.

ResultsCARs with 2B4.{zeta} or 4-1BB.{zeta} signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.{zeta} CAR-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.{zeta} CAR-NK cells. However, the benefit was transient due to limited CAR-NK cell persistence. Transgenic expression of secretory (s)IL15 in 2B4.{zeta} CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK cell subsets. 2B4.{zeta}/sIL15 CAR and sIL15 NK cells maintained an overall activated NK cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK cell groups. In vivo, 2B4.{zeta}/sIL15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.{zeta}/sIL15 CAR and sIL15 NK cells induced lethal toxicity in a second model.

ConclusionTransgenic expression of CD123-CARs and sIL15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early phase clinical testing.

O_FIG O_LINKSMALLFIG WIDTH=161 HEIGHT=200 SRC="FIGDIR/small/461509v1_ufig1.gif" ALT="Figure 1">
View larger version (42K):
org.highwire.dtl.DTLVardef@4d09eaorg.highwire.dtl.DTLVardef@1c61411org.highwire.dtl.DTLVardef@14f8097org.highwire.dtl.DTLVardef@8375ac_HPS_FORMAT_FIGEXP  M_FIG C_FIG In BriefSecretion of IL15 from anti-CD123.2B4.CD3{zeta} CAR-NK cells enhances their activation and killing potential against Acute Myelogenous Leukemia, but carries the risk of severe systemic toxicity.

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Relevance of Auditory Errors Decreases When Errors Are Introduced Suddenly PurposeWhen the speech motor system encounters errors, it generates adaptive responses to compensate for the errors. We previously showed that adaptive responses to task-irrelevant errors are significantly smaller than responses to task-relevant errors when errors are introduced gradually. The current study aimed to examine responses to task-irrelevant and task-relevant errors when errors are introduced suddenly.

MethodWe used an adaptation paradigm in which participants experienced task-relevant errors (induced by formant-shift perturbations) and task-irrelevant errors (induced by formant-clamp perturbations). For one group of participants (N = 30), we applied the perturbations gradually. The second group of participants (N = 30) received the perturbations suddenly. We designed the perturbations based on participant-specific vowel configurations such that a participants first and second formants of /{varepsilon}/ were perturbed toward their /ae/. To estimate adaptive responses, we measured formant changes (within 0-100 ms of the vowel onset) in response to the formant perturbations.

ResultsWe found that (1) the difference between adaptive responses to formant-shift and formant-clamp perturbations was the smallest for the suddenly introduced perturbations, and (2) responses to formant-shift perturbations positively correlated with responses to formant-clamp perturbations for the suddenly (but not gradually) introduced perturbations.

ConclusionsThese results showed that the speech motor system responds to task-relevant errors and task-irrelevant errors more differently when errors are introduced gradually than suddenly. Overall, the speech motor system evaluates the relevance of errors and uses its evaluation to modulate its adaptive responses to errors.

sentences: Pvr and downstream signaling factors are required for spreading of Drosophila hemocytes at larval wound sites Tissue injury is typically accompanied by inflammation. In Drosophila melanogaster, wound-induced inflammation involves adhesive capture of hemocytes at the wound surface followed by hemocyte spreading to assume a flat, lamellar morphology. The factors that mediate this cell spreading at the wound site are not known. Here, we discover a role for the Platelet-derived growth factor (PDGF)/ Vascular endothelial growth factor (VEGF)-related receptor (Pvr) and its ligand, Pvf1, in blood cell spreading at the wound site. Pvr and Pvf1 are required for spreading in vivo and in an in vitro spreading assay where spreading can be directly induced by Pvf1 application or by constitutive Pvr activation. In an effort to identify factors that act downstream of Pvr, we performed a genetic screen in which select candidates were tested to determine if they could suppress the lethality of Pvr overexpression in the larval epidermis. Some of the suppressors identified are required for epidermal wound closure, another Pvr-mediated wound response, some are required for hemocyte spreading in vitro, and some are required for both. One of the downstream factors, Mask, is also required for efficient wound-induced hemocyte spreading in vivo. Our data reveals that Pvr signaling is required for wound responses in hemocytes (cell spreading) and defines distinct downstream signaling factors that are required for either epidermal wound closure or hemocyte spreading.

sentences: Human Lung-resident Mucosal-Associated Invariant T cells are Abundant, Express Antimicrobial Proteins, and are Cytokine Responsive Mucosal-associated Invariant T (MAIT) cells are an innate-like T cell subset that recognize a broad array of microbial pathogens, including respiratory pathogens. Here we investigate the transcriptional profile of MAIT cells localized to the human lung, and postulate that MAIT cells may play a role in maintaining homeostasis at this mucosal barrier. Using the MR1/5-OP-RU tetramer, we identified MAIT cells and non-MAIT CD8+ T cells in lung tissue not suitable for transplant from human donors. We used RNA-sequencing of MAIT cells compared to non-MAIT CD8+ T cells to define the transcriptome of MAIT cells in the human lung. We show that, as a population, lung MAIT cells are polycytotoxic, secrete the directly antimicrobial molecule IL-26, express genes associated with persistence, and selectively express cytokine and chemokine-related molecules distinct from other lung-resident CD8+ T cells, such as interferon-{gamma}- and IL-12-receptors. These data highlight MAIT cells predisposition to rapid pro-inflammatory cytokine responsiveness and antimicrobial mechanisms in human lung tissue, concordant with findings of blood-derived counterparts, and support a function for MAIT cells as early sensors in the defense of respiratory barrier function.

sentences: Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis Synucleinopathies including Parkinsons disease and dementia with Lewy bodies are neurodegenerative diseases characterized by the intracellular accumulation of the protein alpha-synuclein called "Lewy pathology." Alpha-synuclein within Lewy pathology is aggregated into protease resistant filamentous structures and is predominantly phosphorylated at serine 129 (PSER129). Lewy pathology has been hypothesized to spread throughout the nervous system as the disease progresses. Cross-sectional studies have shown the olfactory bulb and olfactory tract consistently bare LP for common synucleinopathies, making these structures likely starting points for the spreading process, and thus disease. Here we examined the distribution of PSER129 in non-diseased brain. To do this we used a sensitive tyramide signal amplification (TSA) technique to detect low abundance endogenous PSER129 under ideal antibody binding conditions. In wild-type non-diseased mice, PSER129 was detected in the olfactory bulb and several brain regions of the olfactory cortex across the neuroaxis (i.e., olfactory bulb to brain stem). PSER129 was particularly apparent in the mitral cell layer and the outer plexiform layer of the olfactory bulb where it was observed as cytosolic/nuclear puncta or fibers, respectively. PSER129 immunoreactivity in the healthy olfactory bulb was abolished by pretreatment of the tissue with proteinase K, pre-absorption of the primary antibody against the purified PSER129 peptide fragment, or the omission of the PSER129 antibody. Furthermore, PSER129 immunoreactivity was not observed in any brain region of alpha-synuclein knockout mice. Dual labeling for the PSER129 and the mitral cell marker TBX21 showed that PSER129 positive structures of the healthy OB were found in mitral cells. We found evidence of the same PSER129 positive structures in the olfactory bulb of non-diseased rats, non-human primates, healthy humans, but not individuals diagnosed with PD. Results suggest biological pathways responsible for alpha-synuclein phosphorylation are constitutively active in OB mitral cells and alpha-synuclein in these cells may be predisposed to pathological aggregation. Pathological seeds originating in mitral cells may act as a source for alpha-synuclein spread competent assemblies that spreads throughout the brain via fibers of the olfactory tract. Future studies should investigate the normal function of alpha-synuclein in the mitral cells of the olfactory bulb, which may give insight into synucleinopathy disease origins.

sentences: An Immune Cell Atlas Reveals Dynamic COVID-19 Specific Neutrophil Programming Amendable to Dexamethasone Therapy SARS-CoV-2 is a novel coronavirus that causes acute respiratory distress syndrome (ARDS), death and long-term sequelae. Innate immune cells are critical for host defense but are also the primary drivers of ARDS. The relationships between innate cellular responses in ARDS resulting from COVID-19 compared to other causes of ARDS, such as bacterial sepsis is unclear. Moreover, the beneficial effects of dexamethasone therapy during severe COVID-19 remain speculative, but understanding the mechanistic effects could improve evidence-based therapeutic interventions. To interrogate these relationships, we developed an scRNA-Seq and plasma proteomics atlas (biernaskielab.ca/COVID_neutrophil). We discovered that compared to bacterial ARDS, COVID-19 was associated with distinct neutrophil polarization characterized by either interferon (IFN) or prostaglandin (PG) active states. Neutrophils from bacterial ARDS had higher expression of antibacterial molecules such as PLAC8 and CD83. Dexamethasone therapy in COVID patients rapidly altered the IFNactive state, downregulated interferon responsive genes, and activated IL1R2+ve neutrophils. Dexamethasone also induced the emergence of immature neutrophils expressing immunosuppressive molecules ARG1 and ANXA1, which were not present in healthy controls. Moreover, dexamethasone remodeled global cellular interactions by changing neutrophils from information receivers into information providers. Importantly, male patients had higher proportions of IFNactive neutrophils, a greater degree of steroid-induced immature neutrophil expansion, and increased mortality benefit compared to females in the dexamethasone era. Indeed, the highest proportion of IFNactive neutrophils was associated with mortality. These results define neutrophil states unique to COVID-19 when contextualized to other life-threatening infections, thereby enhancing the relevance of our findings at the bedside. Furthermore, the molecular benefits of dexamethasone therapy are also defined, and the identified pathways and plasma proteins can now be targeted to develop improved therapeutics.

sentences: Extracellular cues accelerate neurogenesis of induced pluripotent stem cell derived neurons Neurogenesis is a complex process encompassing neuronal progenitor cell expansion/proliferation and differentiation, followed by neuron maturation. In vivo models are most commonly used to study neurogenesis; however, human induced pluripotent stem cell-derived (iPSC) neurons are increasingly used to establish cellular models of human neurological processes. Unfortunately, the differentiation and maturation of iPSC-derived neurons varies in methodology, is asynchronous, and has restricted experimental utility because of extended differentiation/maturation times. To accelerate and standardize iPS neuronal maturation, we differentiated and matured feeder layer-free iPSC-derived neuronal cultures under physiological oxygen levels (5%), and modified the underlying extracellular matrix and medium composition. Our results demonstrate that calretinin gene expression occurred earlier under our optimized iPS conditions and the corresponding "neurogenesis burst" associated with proliferative expansion occurred more synchronously, reliably emerging two and three weeks after differentiation. As expected, the expression of mature neuronal markers (i.e., NeuN+/Calbindin+) started at 4-weeks post-differentiation. qPCR microarray, western blot and single cell analyses using high content imaging indicated that 4-week iPS neuronal cultures were non-cycling with decreased expression of cyclin D1 and Ki67. Our data demonstrate that extracellular cues influence the kinetics of neurogenesis models and that feeder layer-free iPSC-derived neurogenesis can be reproducibly miniaturized.

sentences: Python-Microscope: A new open source Python library for the control of microscopes Custom built microscopes often require control of multiple hardware devices and precise hardware coordination. It is also desirable to have a solution that is scalable to complex systems and translatable between components from different manufacturers. Here we report Python-Microscope, a free and open source Python library for high performance control of arbitrarily complex and scalable custom microscope systems. Python-Microscope offers simple to use Python-based tools, abstracting differences between physical devices by providing a defined interface for different device types. Concrete implementations are provided for a range of specific hardware and a framework exists for further expansion. Python-Microscope supports the distribution of devices over multiple computers while maintaining synchronisation via highly precise hardware triggers. We discuss the architecture choices of Python-Microscope that overcome the performance problems often raised against Python and demonstrate the different use cases that drove its design: its integration in user facing projects, namely in the Microscope-Cockpit project; in controlling complex microscopes at high speed while using the Python programming language; and as a microscope simulation tool for software development.

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Modelling the distribution of rare invertebrates by correcting class imbalance and spatial bias AimSoil arthropods are important decomposers and nutrient cyclers, but are poorly represented on national and international conservation Red Lists. Opportunistic biological records for soil invertebrates are often sparse, and contain few observations of rare species but a relatively large number of non-detection observations (a problem known as class imbalance). Robinson et al. (2018) proposed a method for sub-sampling non-detection data using a spatial grid to improve class balance and spatial bias in bird data. For taxa that are less intensively sampled, datasets are smaller, which poses a challenge because under-sampling data removes information. We tested whether spatial under-sampling improved prediction performance of species distribution models for millipedes, for which large datasets are not available. We also tested whether using environmental predictor variables provided additional information beyond what is captured by spatial position for predicting species distributions.

LocationIsland of Ireland.

MethodsWe tested the spatial under-sampling method of Robinson et al. (2018) by using biological records to train species distribution models of rare millipedes.

ResultsUsing spatially under-sampled training data improved species distribution model sensitivity (true positive rate) but decreased model specificity (true negative rate). The decrease in specificity was minimal for rarer species and was accompanied by substantial increases in sensitivity. For common species, specificity decreased more, and sensitivity increased less, making spatial under-sampling most useful for rare species. Geographic coordinates were as good as or better than environmental variables for predicting distributions of two out of six species.

Main ConclusionsSpatial under-sampling improved prediction performance of species distribution models for rare soil arthropod species. Spatial under-sampling was most effective for rarer species. The good prediction performance of models using geographic coordinates is promising for modeling distributions of poorly studied species for which little is known about ecological or physiological determinants of occurrence.

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Hippocampal Neuropeptide Y2 receptor blockade improves spatial memory retrieval and modulates limbic brain metabolism IntroductionThe neuropeptide Y (NPY) is broadly distributed in the central nervous system (CNS), and it has been related to neuroprotective functions. NPY seems to be an important component to counteract brain damage and cognitive impairment mediated by drugs of abuse and neurodegenerative diseases, and both NPY and its Y2 receptor (Y2R) are highly expressed in the hippocampus, critical for learning and memory. We have recently demonstrated its influence on cognitive functions; however, the specific mechanism and involved brain regions where NPY modulates spatial memory by acting on Y2R remain unclear.

MethodsHere, we examined the involvement of the hippocampal NPY Y2R in spatial memory and associated changes in brain metabolism by bilateral administration of the selective antagonist BIIE0246 into the rat dorsal hippocampus. To further evaluate the relationship between memory functions and neuronal activity, we analysed the regional expression of the mitochondrial enzyme cytochrome c oxidase (CCO) as an index of oxidative metabolic capacity in limbic and non-limbic brain regions.

ResultsThe acute blockade of NPY Y2R significantly improved spatial memory recall in rats trained in the Morris water maze that matched metabolic activity changes in spatial memory processing regions. Specifically, CCO activity changes were found in the dentate gyrus of the dorsal hippocampus and CA1 subfield of the ventral hippocampus, the infralimbic region of the PFC and the mammillary bodies.

ConclusionsThese findings suggest that the NPY hippocampal system, through its Y2R receptor, influences spatial memory recall (retrieval) and exerts control over patterns of brain activation that are relevant for associative learning, probably mediated by Y2R modulation of long-term potentiation and long-term depression.

HighlightsO_LIUnder hippocampal Y2R antagonism, place preference memory retrieval is enhanced
C_LIO_LISpatial retrieval enhancement under Y2R blockade is correlated with changes in regional brain energy metabolism
C_LIO_LIEnhanced retrieval associated CCO activity increases in the dorsal DG, while decreasing in the ventral CA1, IL cortex and mammillary bodies
C_LIO_LIY2R exert control over patterns of brain activation that are relevant for spatial memory expression
C_LI

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From the origin and molecular diversity of the amastins, to the origin and diversity of intracellular parasitism from human Trypanosomatids The large families of amastins from Leishmania donovani, L. infantum, L. major, L. braziliensis and Trypanosoma cruzi are strongly associated with the evolution of intracellular parasitism of rich cells in human MHC.1 molecules such as the macrophages, dendritic cells, and Langerhans cells by these parasites, recognize the MHC-1 molecules as host receptor. The internalization and transport of the paraste in the cytoplas of infected cell is facilitated by the MHC-1 recycle and endosome formation drag and transport the parasite in the cytoplasm of infected cell. The microbody amastins participate as coreceptor potency the infection, the tropism of L. major and L. braziliensis by the cells from the skin is facilitated by two molecular interactions, the first molecular interaction is faclitated by the amastins interact the human MHC-1 molecules, and the second molecular interaction is facilitated by the numerous microbody amastins; which also participate in the biogenesis of the small prasitophorous vcuole from L. major, and large parasitophorous vacuole from L. braziliensis.

All amastins from these parasites developed deactivation domains, in different grade L. donovani develop an amastin surface coat specialized in deactivation of infected macrophages heavily glycosylated developed 38 amastins with 38 glycosylation Asp. N-Glycosylation sites and 45 N-glucosamina glycosylation sites, whereas L. infantum, L. major and L. braziliensis developed one half of glycosylated amastins in asparagine N-glycosylation sites, and T. cruzi did not developed none glycosylated amastin.

The amastins surface coat from L. donovani is rich in phosphorylation sites, developed 45 amastins with 45 casein kinase II phosphorylations sites, and 48 amastins with 48 protein kinase phosphorylation sites. L. infantum, L. braziliensis, and T. cruzi developed 32, 42, and 8 amastins, with 94, 114, 21 casein kinase II phosphorylation sites; in similar way developed 35,38, 11 amastins with 89,78, and 22 protein kinase phosphorylation sites. The family of amastins from L. donovani develop 137 phosphoserines. and 128 phosphothreonine, L. major developed 14 phosphoserine and 4 phosphothreonine; L. infantum 1 phophoserine and 7 phosphothreonine; L. braziliensis did not developed phosphoserine and phosphothreonine and T. cruzi 4 phosphoserine and 4 phosphothreonine. The results show that amastin surface coat is equiped with numerous phosphorylations sites atractive for phosphohrylases from the infected host contribute with the dephosphorylation and deactivation of infectetd host cells.

The amastins from L. major develop a membrane amastin with laminin G domain, which can interact with the collagen and heparin sulfate proteoglycan sites from the extracellular matrix of the skin tissue. Furthermore develop 14 amastins with tyrosine sulfation site, evade the activation of receptor of chemokines and the activation of the immune response by chemokines.

There is an alternative mechanism of polarization of the immune response from protective TH1 to non protective TH2.

The parasite nutrition is mediated by amastins that dissimilate the MHC-1 molecules and other subsets of proteins, the dissimilation products can be translocated through of the parasite cell membrane and employed as nutrient source.

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Subnuclear localisation is associated with gene activation not repression or parental origin at the imprinted Dlk1-Dio3 locus At interphase, de-condensed chromosomes have a non-random three-dimensional architecture within the nucleus, however, little is known about the extent to which nuclear organisation might influence expression or vice versa. Here, using imprinting as a model, we use 3D RNA- and DNA-fluorescence-in-situ-hybridisation in normal and mutant mouse embryonic stem cells to assess the relationship between imprinting control, gene expression and allelic distance from the nuclear periphery. We compared the two parentally inherited imprinted domains at the Dlk1-Dio3 domain and find a small but reproducible trend for the maternally inherited domain to be further away from the periphery if the maternally expressed gene Gtl2/Meg3 is active compared to when it is silenced. Using Zfp57KO ES cells, which harbour a paternal to maternal epigenotype switch, we observe active alleles significantly further away from the nuclear periphery with the distance from the periphery being proportional to the number of alleles active within the cell. This distribution of alleles suggests an activating effect of the nuclear interior rather than a repressive association with the nuclear periphery. Although we see a trend for the paternally inherited copy of the locus to be closer to the nuclear periphery, this appears to be linked to stochastic gene expression differences rather than parental origin. Our results suggest that transcriptional activity, rather than transcriptional repression or parental origin, defines sub-nuclear localisation at an endogenous imprinted domain.

Author summaryGenomic imprinting is an epigenetically regulated process that results in the preferential expression of a subset of developmentally regulated genes from maternally or paternally inherited chromosomes. We have used imprinted genes as a model system to investigate the relationship between the localisation of genes within the cell nucleus and their active expression while at the same time distinguishing gene repression by genomic imprinting, and gene repression by other mechanisms that act on the active allele. We find that there is a significant correlation between transcription and distance to the edge of the nucleus for the Gtl2/Meg3 gene in the imprinted Dlk1-Dio3 region. However, this correlation has a very small effect size and the nuclear envelope, which is commonly thought to act as a repressive environment for gene expression, does not appear to play a major role. We show that position effects, which have been shown for artificially lamina-targeted genes, also exist for endogenous loci and consider the possible biological relevance of the observed small effect.

sentences: Is single nucleus ATAC-seq accessibility a qualitative or quantitative measurement? Single nucleus ATAC-seq is a key assay for gene regulation analysis. Existing approaches to scoring feature matrices from sequencing reads are inconsistent with each other, creating differences in downstream analysis, and displaying artifacts. We show that even with sparse single cell data, quantitative counts are informative for estimating a cells regulatory state, which calls for consistent treatment. We propose Paired-Insertion-Counting (PIC) as a uniform method for snATAC-seq feature characterization.

sentences: Populus ERF85 balances xylem cell expansion and secondary cell wall formation in hybrid aspen Secondary growth relies on precise and specialized transcriptional networks that determine cell division, differentiation, and maturation of xylem cells. We identify a novel role for the ethylene induced Populus ETHYLENE RESPONSE FACTOR ERF85 (Potri.015G023200) in balancing xylem cell expansion and secondary cell wall (SCW) formation in hybrid aspen (Populus tremula x tremuloides). Expression of ERF85 is high in phloem and cambium cells and during expansion of xylem cells, while it is low in maturing xylem tissue. Extending ERF85 expression into SCW forming zones of woody tissues through ectopic expression reduced wood density and SCW thickness of xylem fibers but increased fiber diameter. Xylem transcriptomes from the transgenic trees revealed transcriptional induction of genes involved in cell expansion, translation and growth. Expression of genes associated with plant vascular development and biosynthesis of SCW chemical components such as xylan and lignin, was downregulated in the transgenic trees. Our results suggest that ERF85 activates genes related with xylem cell expansion, while preventing transcriptional activation of genes related to SCW formation. The importance of precise spatial expression of ERF85 during wood development together with the observed phenotypes in response to ectopic ERF85 expression suggests that ERF85 functions as a switch between different phases of xylem differentiation during wood formation.

sentences: Filamentous Bacteriophage Delay Healing of Pseudomonas-Infected Wounds We have identified a novel role for filamentous bacteriophage in the delayed healing associated with chronic Pseudomonas aeruginosa (Pa) wound infections. Pf phage delays wound re-epithelialization in the absence of live Pa, indicative that Pf effects on wound healing are independent of Pa pathogenesis. Pf phage directly inhibits autocrine signaling of CXCL1 (KC) to impede keratinocyte migration and wound re-epithelization. In agreement with these studies, a prospective cohort study of 36 human patients with chronic Pa wound infections revealed that wounds infected with Pf positive strains of Pa took longer to heal and were more likely to increase in size compared to wounds infected with Pf negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian target cells. We propose that Pf phage may have potential as a biomarker and therapeutic target in delayed wound healing.

sentences: A muscle-related contractile tissue specified by MRTF-activity in Porifera Muscle-based movement is a hallmark of animal biology, but the evolutionary origins of myocytes - the cells that comprise muscle tissues - are unknown. Sponges (Porifera) provide an opportunity to reconstruct the earliest periods of myocyte evolution. Although sponges are believed to lack muscle, they are capable of coordinated whole-body contractions that purge debris from internal water canals. This behavior has been observed for decades, but their contractile tissues remain uncharacterized; it is an open question whether they have affinity to muscle or non-muscle contractile tissues in other animals. Here, we characterize the endothelial-like lining of water canals (the endopinacoderm) as a primary contractile tissue in the sponge Ephydatia muelleri. We find tissue-wide organization of contractile actin-bundles that contain striated-muscle myosin II and transgelin, and that contractions are regulated by the release of internal Ca2+ stores upstream of the myosin-light-chain-kinase (MLCK) pathway. Further, we show that the endopinacoderm is developmentally specified by myocardin-related transcription factor (MRTF) as part of an environmentally-inducible transcriptional complex that otherwise is known to function in muscle development, plasticity, and regeneration in other animals. We conclude that both muscle tissues and the endopinacoderm evolved from a common, multifunctional contractile tissue in the animal stem-lineage. Furthermore, as an actin-regulated force-sensor, MRTF-activity offers a mechanism for how water canals dynamically remodel in response to flow and can re-form normally from stem-cells in the absence of the intrinsic positional cues characteristic of embryogenesis in other animals.

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Three PilZ domain proteins, PlpA, PixA and PixB, have distinct functions in regulation of motility and development in Myxococcus xanthus In bacteria, the nucleotide-based second messenger bis-(3-5)-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pili-dependent motility and the starvation-induced developmental program that results in the formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP receptors. We confirm that PlpA, a stand-alone PilZ-domain protein, is specifically important for motility and that Pkn1, which is composed of a Ser/Thr domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ-domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pili-dependent and gliding motility upstream of the Frz chemosensory system as well as development. The acetyltransferase domain is required and sufficient for function during growth while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility downstream of the Frz chemosensory system by setting up the polarity of the two motility systems. Our results support a model whereby the three proteins PlpA, PixA and PixB act in parallel pathways and have distinct functions to regulation of motility.

Importancec-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts upstream of the Frz chemosensory system to regulate motility via the acetyltransferase domain while the intact protein and c-di-GMP binding are essential for PixB to support development. By contrast, PixA acts downstream of the Frz system to regulate motility. Together with previous observations, we conclude that PilZ-domain proteins and c-di-GMP act in multiple parallel pathways to regulate motility and development in M. xanthus.

sentences: Discovery and Biosynthesis of Persiathiacins: Unusual Polyglycosylated Thiopeptides Active against Multi-drug resistant Tuberculosis Thiopeptides are ribosomally biosynthesized and post-translationally modified peptides (RiPPs) that potently inhibit the growth of Gram-positive bacteria by targeting multiple steps in protein biosynthesis. The poor pharmacological properties of thiopeptides, in particular their low aqueous solubility, has hindered their development into clinically useful antibiotics. Antimicrobial activity screens of a library of Actinobacterial extracts led to discovery of the novel polyglycosylated thiopeptides persiathiacins A and B from Actinokineospora sp. UTMC 2475 and Actinokineospora sp. UTMC 2448. Persiathiacin A is active against methicillin-resistant Staphylococcus aureus (MRSA) and several Mycobacterium tuberculosis strains, including drug-resistant and multidrug-resistant clinical isolates, and does not significantly affect the growth of ovarian cancer cells at concentrations up to 400 M. In vitro translation assays showed that, like other thiopeptide antibiotics, persiathiacin A targets protein biosynthesis. Polyglycosylated thiopeptides are extremely rare and nothing is known about their biosynthesis. Sequencing and analysis of the Actinokineospora sp. UTMC 2448 genome enabled identification of the putative persiathiacin biosynthetic gene cluster. A cytochrome P450 encoded by this gene cluster catalyses the hydroxylation of nosiheptide in vitro and in vivo, consistent with the proposal that the cluster directs persiathiacin biosynthesis. Several genes in the cluster encode homologues of enzymes known to catalyse the assembly and attachment of deoxysugars during the biosynthesis of other classes of glycosylated natural products. The discovery of the persiathiacins and their biosynthetic gene cluster thus provides the basis for the development of biosynthetic engineering approaches to the creation of novel (poly)glycosylated thiopeptide derivatives with enhanced pharmacological properties.

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NamZ1 and NamZ2 from the oral pathogen Tannerella forsythia are peptidoglycan processing exo-β-N-acetylmuramidases with distinct substrate specificity The Gram-negative periodontal pathogen Tannerella forsythia is inherently auxotrophic for N-acetylmuramic acid (MurNAc), which is an essential carbohydrate constituent of the peptidoglycan (PGN) of the bacterial cell wall. Thus, to build up its cell wall, T. forsythia strictly depends on the salvage of exogenous MurNAc or sources of MurNAc, such as polymeric or fragmentary PGN, derived from cohabiting bacteria within the oral microbiome. In our effort to elucidate how T. forsythia satisfies its demand for MurNAc, we recognized that the organism possesses three putative orthologs of the exo-{beta}-N-acetylmuramidase BsNamZ from Bacillus subtilis, which cleaves non-reducing end, terminal MurNAc entities from the artificial substrate pNP-MurNAc and the naturally-occurring disaccharide substrate MurNAc-{beta}-1,4-N-acetylglucosamine (GlcNAc). TfNamZ1 and TfNamZ2 were successfully purified as soluble, pure recombinant His6-fusions and characterized as exo-lytic {beta}-N-acetylmuramidases with distinct substrate specificities. The activity of TfNamZ1 was considerably lower compared to TfNamZ2 and BsNamZ, in the cleavage of pNP-MurNAc and MurNAc-GlcNAc. When peptide-free PGN glycans were used as substrates, we revealed striking differences in the specificity and mode of action of these enzymes, as analyzed by mass spectrometry. TfNamZ1, but not TfNamZ2 or BsNamZ, released GlcNAc-MurNAc disaccharides from these glycans. In addition, glucosamine (GlcN)-MurNAc disaccharides were generated when partially N-deacetylated PGN glycans from B. subtilis 168 were applied. This characterizes TfNamZ1 as a unique disaccharide-forming exo-lytic {beta}-N-acetylmuramidase (exo-disaccharidase), and, TfNamZ2 and BsNamZ as sole MurNAc monosaccharide-lytic exo-{beta}-N-acetylmuramidases.

IMPORTANCETwo exo-{beta}-N-acetylmuramidases from T. forsythia belonging to glycosidase family GH171 (www.cazy.org) were shown to differ in their activities, thus revealing a functional diversity within this family: NamZ1 releases disaccharides (GlcNAc-MurNAc/GlcN-MurNAc) from the non-reducing ends of PGN glycans, whereas NamZ2 releases terminal MurNAc monosaccharides. This work provides a better understanding of how T. forsythia may acquire the essential growth factor MurNAc by the salvage of PGN from cohabiting bacteria in the oral microbiome, which may pave avenues for the development of anti-periodontal drugs. On a broad scale, our study indicates that the utilization of PGN as a nutrient source, involving exo-lytic N-acetylmuramidases with different modes of action, appears to be a general feature of bacteria, particularly among the phylum Bacteroidetes.

sentences: Comprehensive profiling of genomic and transcriptomic  differences between risk groups of lung adenocarcinoma and lung squamous cell carcinoma Lung cancer is the second frequently diagnosed cancer type and responsible for the highest number of cancer deaths worldwide. Lung adenocarcinoma and lung squamous cell carcinoma are subtypes of non-small cell lung cancer which has the highest frequency of lung cancer cases. We aimed to analyze genomic and transcriptomic variations including simple nucleotide variations (SNVs), copy number variations (CNVs) and differential expressed genes (DEGs) in order to find key genes and pathways for diagnostic and prognostic prediction for lung adenocarcinoma and lung squamous cell carcinoma. We performed univariate cox model and then lasso regularized cox model with leave-one-out cross-validation using TCGA gene expression data in tumor samples. We generated a 35-gene signature and a 33-gene signature for prognostic risk prediction based on the overall survival time of the patients with LUAD and LUSC, respectively. When we clustered patients into high-risk and low-risk groups, the survival analysis showed highly significant results with high prediction power for both training and test datasets. Then we characterized the differences including significant SNVs, CNVs, DEGs, active subnetworks, and the pathways. We described the results for the risk groups and cancer subtypes separately to identify specific genomic alterations between both high-risk groups and cancer subtypes. Both LUAD and LUSC high-risk groups have more down-regulated immune pathways and upregulated metabolic pathways. On the other hand, low-risk groups have both upregulated and downregulated genes on cancer-related pathways. Both LUAD and LUSC have important gene alterations such as CDKN2A and CDKN2B deletions with different frequencies. SOX2 amplification occurs in LUSC and PSMD4 amplification in LUAD. EGFR and KRAS mutations are mutually exclusive in LUAD samples. EGFR, MGA, SMARCA4, ATM, RBM10, and KDM5C genes are mutated only in LUAD but not in LUSC. CDKN2A, PTEN, and HRAS genes are mutated only in LUSC samples. Low-risk groups of both LUAD and LUSC, tend to have a higher number of SNVs, CNVs, and DEGs. The signature genes and altered genes have the potential to be used as diagnostic and prognostic biomarkers for personalized oncology.

sentences: SARS-CoV-2 in the Republic of Guinea: Fragment and Whole-Genome Sequencing, Phylogenetic Analysis Genetic diversity of SARS-CoV-2 isolates circulating in the Republic of Guinea in May and June 2020, as well as in March 2021, has been demonstrated using fragment (S gene) and whole genome sequencing of 14 strains. Analysis of nucleotide sequences and phylogenetic constructs make it possible to divide the studied strains into 3 groups. Comparison of the obtained data with the already available epidemiological data proves the initial importation of COVID-19 from Western European countries, and also demonstrates four independent import routes in two time periods (March 2020 and no later than March 2021).

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Tracking the circulating SARS-CoV-2 variants in Turkey: Complete genome sequencing and molecular characterization of 1000 SARS-CoV-2 samples Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus and has caused a pandemic of acute respiratory disease, named  coronavirus disease 2019 (COVID-19). COVID-19 has a deep impact on public health as one of the most serious pandemics in the last century. Tracking SARS-CoV-2 is important for monitoring and assessing its evolution. This is only possible by detecting all mutations in the viral genome through genomic sequencing. Moreover, accurate detection of SARS-CoV-2 and tracking its mutations is also required for its correct diagnosis. Potential effects of mutations on the prognosis of the disease can be observed. Assignment of epidemiological lineages in an emerging pandemic requires efforts. To address this, we collected 1000 SARS-CoV-2 samples from different geographical regions in Turkey and analyze their genome comprehensively. To track the virus across Turkey we focus on 10 distinct cities in different geographic regions. Each SARS-CoV-2 genome was analyzed and named according to the nomenclature system of Nextclade and Pangolin Lineage. Furthermore, the frequency of the variations observed in 10 months was also determined by region. In this way, we have observed how the virus mutations and what kind of transmission mechanism it has. The effects of age and disease severity on lineage distribution were other considered parameters. The temporal rates of SARS-CoV-2 variants by time in Turkey were close to the global trend. This study is one of the most comprehensive whole genome analyses of SARS-CoV-2 that represents a general picture of the distribution of SARS-CoV-2 variations in Turkey in 2021.

Author SummarySince the outbreak of the COVID-19 pandemic in 2019, the viral genome of SARS-CoV-2 was analysed intensively all over the world both to detect its zoonotic origin and the emerging variants worldwide together with the variants effect on the prognosis and treatment, respectively, of the infection. Remarkable COVID-19 studies were also made in Turkey as it was in the rest of the world. To date, indeed, almost all studies on COVID-19 in Turkey either sequenced only a small number of the viral genome or analysed the viral genome which was obtained from online databases. In respect thereof, our study constitutes a milestone regarding both the huge sample size consisting of 1000 viral genomes and the widespread geographic origin of the viral genome samples. Our study provides new insights both into the SARS-CoV-2 landscape of Turkey and the transmission of the emerging viral pathogen and its interaction with its vertebrate host.

sentences: Neuromodulation-induced burst firing in parvalbumin interneurons of the basolateral amygdala mediates transition between fear-associated network and behavioral states Patterned coordination of network activity in the basolateral amygdala (BLA) is important for fear expression. Neuromodulatory systems play an essential role in regulating changes between behavioral states, however the mechanisms underlying the neuromodulatory control of BLA circuits that mediates transitions between brain and behavioral states remain largely unknown. We examined the role of neuromodulation of parvalbumin (PV)-expressing interneurons in the BLA in coordinating network and behavioral states using combined chemogenetics, ex vivo patch clamp recordings, and in vivo and ex vivo local field potential recordings. We show that Gq signaling, whether by the designer receptor, hM3D, 1A adrenoreceptors, or 5-HT2a serotonergic receptors, induces a previously undescribed, highly stereotyped bursting pattern of activity in BLA PV interneurons that generates synchronous bursts of inhibitory postsynaptic currents and phasic firing in the BLA principal neurons. The Gq activation in PV interneurons induced a transition from tonic to phasic firing in the BLA PV neurons and principal neurons and suppressed BLA gamma oscillations in slices and suppressed BLA gamma and potentiated theta power in vivo. Gq activation in BLA PV interneurons also facilitated fear memory recall, consistent with previous reports of BLA gamma suppression and theta potentiation during conditioned fear expression. Thus, our data reveal a BLA parvalbumin neuron-specific Gq neuromodulatory mechanism that mediates the transition to a fear-associated network and behavioral state.

sentences: Functional characterization of SARS-CoV-2 vaccine elicited antibodies in immunologically naive and pre-immune humans As the COVID-19 pandemic continues, the authorization of vaccines for emergency use has been crucial in slowing down the rate of infection and transmission of the SARS-CoV-2 virus that causes COVID-19. In order to investigate the longitudinal serological responses to SARS-CoV-2 natural infection and vaccination, a large-scale, multi-year serosurveillance program entitled SPARTA (SARS SeroPrevalence and Respiratory Tract Assessment) was initiated at 4 locations in the U.S. The serological assay presented here measuring IgG binding to the SARS-CoV-2 receptor binding domain (RBD) detected antibodies elicited by SARS-CoV-2 infection or vaccination with a 95.5% sensitivity and a 95.9% specificity. We used this assay to screen more than 3100 participants and selected 20 previously infected pre-immune and 32 immunologically naive participants to analyze their antibody binding to RBD and viral neutralization (VN) responses following vaccination with two doses of either the Pfizer-BioNTech BNT162b2 or the Moderna mRNA-1273 vaccine. Vaccination not only elicited a more robust immune reaction than natural infection, but the level of neutralizing and anti-RBD antibody binding after vaccination is also significantly higher in pre-immune participants compared to immunologically naive participants (p<0.0033). Furthermore, the administration of the second vaccination did not further increase the neutralizing or binding antibody levels in pre-immune participants (p=0.69). However, ~46% of the immunologically naive participants required both vaccinations to seroconvert.

sentences: A tractable bottom-up model of the yeast polarity genotype-phenotype map for evolutionary relevant predictions Accurate phenotype prediction based on genetic information has numerous societal applications, such as crop design or cellular factories. Epistasis, when biological components interact, complicates modelling phenotypes from genotypes. Here we show an approach to mitigate this complication for polarity establishment in budding yeast, where mechanistic information is abundant. We coarse-grain molecular interactions into a so-called mesotype, which we combine with gene expression noise into a physical cell cycle model. Firstly, we show with computer simulations that the mesotype allows validation of the most current biochemical polarity models by quantitatively matching doubling times. Secondly, the mesotype elucidates epistasis emergence as exemplified by evaluating the predicted mutational effect of key polarity protein Bem1p when combined with known interactors or under different growth conditions. This example also illustrates how unlikely evolutionary trajectories can become more accessible. The tractability of our biophysically justifiable approach inspires a road-map towards bottom-up phenotype modelling beyond statistical inferences.

sentences: Dissociable mechanisms of reward learning co-mature during human adolescence as predicted by macaque lesion models Reward-guided learning and decision-making is a fundamental adaptive ability and depends on a number of component processes. We investigate how such component processes mature during human adolescence. Our approach was guided by analyses of the effects of lateral orbitofrontal lesions in macaque monkeys, as this part of the brain shows clear developmental maturation in humans during adolescence. Using matched tasks and analyses in humans (n=388, 11-35yrs), we observe developmental changes in two key learning mechanisms as predicted from the monkey data. First, choice-reward credit assignment - the ability to link a specific outcome to a specific choice - is reduced in adolescents. Second, the effects of the global reward state - how good the environment is overall recently - exerts a distinctive pattern of influence on learning in humans compared to other primates and across adolescence this pattern becomes more pronounced. Both mechanisms were correlated across participants suggesting that associative learning of correct reward assignments and GRS based learning constitute two complementary mechanisms of reward-learning that co-mature during adolescence.

sentences: Epigenomic translocation of H3K4me3 broad domains over oncogenes following hijacking of super-enhancers Chromosomal translocations are important drivers of haematological malignancies whereby proto-oncogenes are activated by juxtaposition with enhancers, often called enhancer hijacking. We analysed the epigenomic consequences of rearrangements between the super-enhancers of the immunoglobulin heavy locus (IGH) and proto-oncogene CCND1 that are common in B-cell malignancies. By integrating BLUEPRINT epigenomic data with DNA breakpoint detection, we characterised the normal chromatin landscape of the human IGH locus and its dynamics after pathological genomic rearrangement. We detected an H3K4me3 broad domain (BD) within the IGH locus of healthy B cells that was absent in samples with IGH-CCND1 translocations. The appearance of H3K4me3-BD over CCND1 in the latter was associated with overexpression and extensive chromatin accessibility of its gene body. We observed similar cancer-specific H3K4me3-BDs associated with hijacking of super-enhancers of other common oncogenes in B-cell (MAF, MYC and FGFR3/NSD2) and T-cell malignancies (LMO2, TLX3 and TAL1). Our analysis suggests that H3K4me3-BDs can be created by super-enhancers and supports the new concept of epigenomic translocation, where the relocation of H3K4me3-BDs from cell identity genes to oncogenes accompanies the translocation of super-enhancers.

sentences: Reorganization of thalamocortical connections in congenitally blind humans Evidence of cross-modal plasticity in blind individuals has been reported over the past decades showing that non-visual information is carried and processed by  visual brain structures. This feature of the blind brain makes it a pivotal model to explore the limits and mechanisms of brain plasticity. However, despite multiple efforts, the structural underpinnings of cross-modal plasticity in congenitally blind individuals remain unclear. Using advanced neuroimaging techniques, we mapped thalamocortical connectivity and assessed cortical thickness and integrity of white matter of ten congenitally blind individuals and ten sighted controls. We hypothesized an aberrant thalamocortical pattern of connectivity taking place in the absence of visual stimuli from birth as a potential mechanism of cross-modal plasticity. In addition to the increased cortical thickness of the primary visual cortex and reduced integrity of visual white matter bundles, we observed structural connectivity changes between the thalamus and both occipital and temporal cortices. Specifically, the thalamic territory dedicated to connections with the occipital cortex was found to be smaller and displayed weaker connectivity in congenitally blind individuals, whereas the one that connects with the temporal cortex showed greater volume and stronger connectivity when compared to sighted controls. The abnormal pattern of thalamocortical connectivity included the lateral and medial geniculate nuclei and the pulvinar nucleus. For the first time in humans, a remapping of structural thalamocortical connections involving both unimodal and multimodal thalamic nuclei has been demonstrated, shedding light on the possible mechanisms of cross-modal plasticity in humans. Future studies should employ neurophysiologic approaches to explore the functional relevance of present findings.

sentences: Spontaneous thalamic activity modulates the cortical innervation of the primary visual nucleus of the thalamus Sensory processing relies on the correct development of thalamocortical loops. Visual corticothalamic axons (CTAs) invade the dorsolateral geniculate nucleus (dLGN) of the thalamus in early postnatal mice according to a regulated program that includes activity-dependent mechanisms. Spontaneous retinal activity influences the thalamic incursion of CTAs, yet the perinatal thalamus also generates intrinsic patterns of spontaneous activity whose role in modulating afferent connectivity remains unknown. Here, we found that patterned spontaneous activity in the dLGN contributes to proper spatial and temporal innervation of CTAs. Disrupting patterned spontaneous activity in the dLGN delays corticogeniculate innervation under normal conditions and upon eye enucleation. The delayed innervation was evident throughout the first two postnatal weeks but resumes after eye-opening, suggesting that visual experience is necessary for the homeostatic recovery of corticogeniculate innervation.

sentences: Perturbation of ACE2 structural ensembles by SARS-CoV-2 spike protein binding The human ACE2 enzyme serves as a critical first recognition point of coronaviruses, including SARS-CoV-2. In particular, the extracellular domain of ACE2 interacts directly with the S1 tailspike protein of the SARS-CoV-2 virion through a broad protein-protein interface. Although this interaction has been characterized by X-ray crystallography and Cryo-EM, these structures do not reveal significant differences in ACE2 structure upon S1 protein binding. In this work, using several all-atom molecular dynamics simulations, we show persistent differences in ACE2 structure upon binding. These differences are determined with the Linear Discriminant Analysis (LDA) machine learning method and validated using independent training and testing datasets, including long trajectories generated by D. E. Shaw Research on the Anton 2 supercomputer. In addition, long trajectories for 78 potent ACE2-binding compounds, also generated by D. E. Shaw Research, were projected onto the LDA classification vector in order to determine whether the ligand-bound ACE2 structures were compatible with S1 protein binding. This allows us to predict which compounds are "apo-like" vs "complex-like", as well as to pinpoint long-range ligand-induced allosteric changes of ACE2 structure.

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Functional Redundancy in Ocean Microbiomes Controls Trait Stability Theory predicts that functional redundancy in microbial communities increases trait stability, meaning that traits or functions are less likely to be lost from the community when species go extinct. However, few experiments have empirically tested this prediction, especially in the context of microbial communities and at the landscape scale. In part, the lack of metrics for functional redundancy in microbial ecosystems has prevented addressing this question. In a companion manuscript we proposed a quantitative metric for functional redundancy called Contribution Evenness (CE) that is optimized to reflect trait stability. Here, we use CE to predict the stability of marine microbial functions to species and transcription loss. Using transcriptomes deposited in the Ocean Microbial Reference Gene Catalog (OM-RGC.v2), a catalog of genes and transcripts sequenced by the TARA Ocean expedition, we quantified the functional redundancy for 4,314 KEGG Orthologs (KOs) across 124 marine sites. Functional redundancy was highly correlated with a latent variable consisting of four ocean physiochemical parameters: oxygen and chlorophyll a concentrations, depth, and salinity. Functional redundancy was higher at the poles than in non-polar regions. Simultaneously, regional {beta}-diversity for individual functions was higher for functions with higher functional redundancy. These observations provide evidence that higher functional redundancy indicates increased stability of microbial ecosystem functions on spatiotemporal scales consistent with surface ocean mixing. We suggest that future changes in ocean physiochemistry could likely influence this stability for functions with lower functional redundancy.

ImportanceFunctional redundancy describes the state of multiple species performing the same function. Theory suggests functional redundancy stabilizes microbial community functions from disturbances leading to species loss or other changes to the microbiome. Previous work suggests that functional redundancy is common in ocean microbiomes which implies traits should be more stable among metacommunities. Some laboratory experiments demonstrate this idea, but it is difficult to test in the natural world. In a companion manuscript, we proposed a functional redundancy metric sensitive to trait stability. Here, we used this metric to show that functional redundancy varied substantially among ocean microbiomes and that regions with higher functional redundancy had higher regional trait stability. Last, we noted that variations in functional redundancy strongly correlated to ocean physiochemistry. Thus, changes in ocean physiochemistry via climate change may alter community traits to become more or less resistant to disturbance relative to contemporary conditions.

sentences: Microglia and perivascular macrophages act as antigen presenting cells to promote CD8 T cell infiltration of the brain CD8 T cell infiltration of the central nervous system (CNS) is necessary for host protection but contributes to neuropathology. Antigen presenting cells (APCs) situated at CNS borders are thought to mediate T cell entry into the parenchyma during neuroinflammation. The identity of the CNS-resident APC that presents antigen via major histocompatibility complex (MHC) class I to CD8 T cells is unknown. Herein, we characterize MHC class I expression in the naive and virally infected brain and identify microglia and macrophages (CNS-myeloid cells) as APCs that upregulate H-2Kb and H-2Db upon infection. Conditional ablation of H-2Kb and H-2Db from CNS-myeloid cells allowed us to determine that antigen presentation via H-2Db, but not H-2Kb, was required for CNS immune infiltration during Theilers murine encephalomyelitis virus (TMEV) infection and drives brain atrophy as a consequence of infection. These results demonstrate that CNS-myeloid cells are key APCs mediating CD8 T cell brain infiltration.

sentences: Rapid quantification of tissue perfusion properties with a two-stage look-up table: a simulation study Tissue perfusion properties reveal crucial information pertinent to clinical diagnosis and treatment. Multispectral spatial frequency domain imaging (SFDI) is an emerging imaging technique that has been widely used to quantify tissue perfusion properties. However, slow processing speed limits its usefulness in real-time imaging applications. In this study, we present a two-stage look-up table (LUT) approach that accurately and rapidly quantifies optical (absorption and reduced scattering maps) and perfusion (total hemoglobin and oxygen saturation maps) properties using stage-1 and stage-2 LUTs, respectively, based on reflectance images at 660nm and 850nm. The two-stage LUT can be implemented on both CPU and GPU computing platforms. Quantifying tissue perfusion properties using the simulated diffuse reflectance images, we achieved a quantification speed of 266, 174, and 74 frames per second for three image sizes 512x512, 1024x1024, and 2048x2048 pixels, respectively. Quantification of tissue perfusion properties was highly accurate with only 3.5% and 2.5% error for total hemoglobin and oxygen saturation quantification, respectively. The two-stage LUT has the potential to be adopted in existing SFDI applications to enable real-time imaging capability of tissue hemodynamics.

sentences: Welfare concerns for mounted load carrying by working donkeys in Pakistan Working donkeys (Equus asinus) are vital to peoples livelihoods. They are essential for carrying goods, however globally, overloading is one of the primary welfare concerns of working donkeys. We studied mounted load carrying by donkeys and associated factors in Pakistan. A cross-sectional study of donkey owners (n = 332) was conducted, and interviews were undertaken based on a questionnaire. Owners estimated that the median weight of their donkeys was 110kg (interquartile range (IQR) 100-120kg), and that they carried a median mounted load of 81.5kg (IQR 63-99kg). We found that 87.4% of donkeys carried a load above 50% of their bodyweight ratio (BWR), the median BWR carried was 77.1% (IQR 54.5-90.7%), and 25.3% of donkeys carried above 90% BWR. Donkeys that were loaded at more than 50% BWR were more likely to sit, compared to donkeys loaded with less weight (p=0.01). Donkeys working in peri-urban and urban areas were more likely to carry a greater BWR than donkeys working in rural areas (P<0.001), as were those carrying construction materials or bricks, compared to agricultural materials (p=0.004). Age (p=0.03) and breed (p=0.01) were also associated with carrying a higher weight. Overloading based on current recommendations (50% BWR) was common, with the majority (87.4%) of donkeys reported to carry more than the recommended 50% limit. This survey provides evidence of on-the-ground working practices and factors associated with mounted load carrying, which is critical for developing evidence-based recommendations for loading, in order to improve the welfare of working donkeys.

sentences: Attentional Modulations of Alpha Power Are Sensitive to the Task-relevance of Auditory Spatial Information The topographical distribution of oscillatory power in the alpha band is known to vary depending on the current focus of spatial attention. Here, we investigated to what extend univariate and multivariate measures of post-stimulus alpha power are sensitive to the required spatial specificity of a task. To this end, we varied the perceptual load and the spatial demand in an auditory search paradigm. A centrally presented sound at the beginning of each trial indicated the to-be-localized target sound. This spatially unspecific pre-cue was followed by a sound array, containing either two (low perceptual load) or four (high perceptual load) simultaneously presented lateralized sound stimuli. In separate task blocks, participants were instructed either to report whether the target was located on the left or the right side of the sound array (low spatial demand) or to indicate the exact target location (high spatial demand). Univariate alpha lateralization magnitude was neither affected by perceptual load nor by spatial demand. However, an analysis of onset latencies revealed that alpha lateralization emerged earlier in low (vs. high) perceptual load trials as well as in low (vs. high) spatial demand trials. Finally, we trained a classifier to decode the specific target location based on the multivariate alpha power scalp topography. A comparison of decoding accuracy in the low and high spatial demand conditions suggests that the amount of spatial information present in the scalp distribution of alpha-band power increases as the task demands a higher degree of spatial specificity. Altogether, the results offer new insights into how the dynamic adaption of alpha-band oscillations in response to changing task demands is associated with post-stimulus attentional processing.

sentences: Modulating neutrophils leads to changes in CNS output It has become increasingly evident that immune cells play a role in modulating brain function. Neutrophils are the most abundant innate immune cell in mammals. Until recently, our understanding of the physiological role of these cells was limited to infection and cancer. Recently, it has emerged that neutrophils play critical roles in modulating tissue health and physiology. There has been evidence that neutrophils populate the rodent meninges. As such, we hypothesized that like other meningeal immune cells, neutrophils play a neuromodulatory role. Using a depletion paradigm, anti-Ly6G antibody injection, we demonstrate that the loss of neutrophils leads to changes in cognitive flexibility and social interactions in mice. Furthermore, the depletion paradigm led to a 50% decrease in the number of neutrophils in the meninges, which in turn led to the proliferation of resident immature neutrophils within the meninges. Finally, our data show that this immature population reenters the cell cycle only in response to specific physiological stimuli (neutrophil depletion, and stress). In conclusion, this study demonstrates that there are resident neutrophils within the meninges and that elucidating their function in the nervous system will deepen our understanding of immune regulation of brain function.

sentences: Rattus Economicus: A neural model for rational regulation of water-seeking effort in rodents In the laboratory, animals motivation to work tends to be positively correlated with reward magnitude. But in nature, rewards earned by work are essential to survival (e.g., working to find water), and the payoff of that work can vary on long timescales (e.g., seasonally). Under these constraints, the strategy of working less when rewards are small could be fatal. We found that instead, rats in a closed economy did more work for water rewards when the rewards were stably smaller, a phenomenon also observed in human labor supply curves. Like human consumers, rats also showed elasticity of demand, consuming far more water per day when its price in effort was lower. The neural mechanisms underlying such "rational" market behaviors remain largely unexplored. We propose a dynamic utility maximization model that can account for the dependence of rat labor supply (trials/day) on the wage rate (ml/trial), and also predict the temporal dynamics of when rats work. Based on data from mice, we hypothesize that SFOGLUT neurons in lamina terminalis continuously compute the instantaneous marginal utility of voluntary work for water reward, and causally determine the amount and timing of work.

sentences: Scalable Fabrication of 3D Structured Microparticles Using Induced Phase Separation Microparticles with defined shapes and spatial chemical modification can enable new opportunities to interface with cells and tissues at the cellular scale. However, conventional methods to fabricate shaped microparticles have trade-offs between the throughput of manufacture and precision of particle shape and chemical functionalization. Here, we achieved scalable production of hydrogel microparticles at rates of greater than 40 million/hour with localized surface chemistry using a parallelized step emulsification device and temperature-induced phase-separation. The approach harnesses a polymerizable polyethylene glycol (PEG) and gelatin aqueous-two phase system (ATPS) which conditionally phase separates within microfluidically-generated droplets. Following droplet formation, phase separation is induced and phase separated droplets are subsequently crosslinked to form uniform crescent and hollow shell particles with gelatin functionalization on the boundary of the cavity. The gelatin localization enabled deterministic cell loading in sub nanoliter-size crescent-shaped particles, which we refer to as nanovials, with cavity dimensions tuned to the size of cells. Loading on nanovials also imparted improved cell viability during analysis and sorting using standard fluorescence activated cell sorters, presumably by protecting cells from shear stress. This localization effect was further exploited to selectively functionalize capture antibodies to nanovial cavities enabling single-cell secretion assays with reduced cross-talk in a simplified format.

sentences: Fetal Electrocardiogram Extraction from the Mother's Abdominal Signal Using the Ensemble Kalman Filter Fetal electrocardiogram (fECG) assessment is essential throughout pregnancy to monitor the wellbeing and development of the fetus, and to possibly diagnose potential congenital heart defects. Due to the high noise incorporated in the abdominal ECG (aECG) signals, the extraction of fECG has been challenging. And it is even a lot more difficult for fECG extraction if only one channel of aECG is provided, i.e. in a compact patch device. In this paper, we propose a novel algorithm based on the Ensemble Kalman Filter (EnKF) for non-invasive fECG extraction from a single-channel aECG signal. To assess the performance of the proposed algorithm, we used our own clinical data, obtained from a pilot study with 10 subjects each of 20 min recording, and data from the PhysioNet 2013 Challenge bank with labeled QRS complex annotations. The proposed methodology shows the average positive predictive value (PPV) of 97.59%, sensitivity (SE) of 96.91%, and F1-score of 97.25% from the PhysioNet 2013 Challenge bank. Our results also indicate that the proposed algorithm is reliable and effective, and it outperforms the recently proposed Extended Kalman Filter (EKF) based algorithm.

sentences: Doctoring Direct-to-Consumer Genetic Tests with DNA Spike-Ins Direct-to-consumer (DTC) genetic testing companies have provided personal genotyping services to millions of customers. Customers mail saliva samples to DTC service providers to have their genotypes analyzed and receive back their raw genetic data. Both consumers and the DTC companies use the results to perform ancestry analyses, relative matching, trait prediction, and estimate predisposition to disease, often relying on genetic databases composed of the data from millions of other DTC-genotyped individuals. While the digital integrity risks to this type of data have been explored, we considered whether data integrity issues could manifest upstream of data generation through physical manipulation of DNA samples themselves, for example by adding synthetic DNA to a saliva sample ("spiked samples") prior to sample processing by a DTC company. Here, we investigated the feasibility of this scenario within the standard DTC genetic testing pipeline. Starting with the purchase of off-the-shelf DTC genetic testing kits, we found that synthetic DNA can be used to precisely manipulate the results of saliva samples genotyped by a popular DTC genetic testing service and that this method can be used to modify arbitrary single nucleotide polymorphisms (SNPs) in multiplex to create customized doctored genetic profiles. This capability has implications for the use of DTC-generated results and the outcomes of their downstream analyses.

sentences: Cultural diffusion dynamics depend on behavioural production rules Culture is an outcome of the acquisition of knowledge about behaviour through social transmission, and its subsequent production. Transmission and production are often discussed interchangeably or modeled separately, yet to date, no study has accounted for both processes and explored their interaction. We present a generative model that integrates the two in order to explore how variation in either might shape cultural diffusion dynamics. Agents make behavioural choices that change as they learn from their behavioural productions. Their repertoires also change over time, and the social transmission of behaviours depends on their frequency. We diffuse a novel behaviour through social networks across a large parameter space to demonstrate how accounting for both transmission and production reveals dependencies between individual-level behavioural production rules and population-level diffusion dynamics. We then investigate how such dependencies might affect the performance of two commonly used inferential models for social learning; Network-based Diffusion Analysis (NBDA), and Experienced Weighted Attraction models (EWA). By clarifying the distinction between acquisition and usage, we illuminate often-overlooked theoretical differences between social learning and social influence. These distinctions yield consequences and new considerations for how inferential methods are applied to empirical studies of culture.

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Exosome TDP-43 profile in canine model of ALS: A preliminary study in developing surrogate biomarker ObjectiveBlood-based biomarkers provide a crucial information in progress of neurodegenerative diseases with minimally invasive sampling method. Validated blood-based biomarker application in people with amyotrophic lateral sclerosis would derive numerous benefits. Canine degenerative myelopathy is a naturally occurring animal disease model to study the biology of human amyotrophic lateral sclerosis. Serum derived exosomes are potential carriers for cell-specific cargoes making them ideal venue to study biomarkers for a variety of diseases and biological processes. This study assessed the exosomal proteins that may be assigned as surrogate biomarker that may reflect biochemical changes in central nervous system.

MethodsExosomes were isolated from canine serum using commercial exosome isolation reagents. Exosomes target proteins contents were analysed by Western blotting method.

ResultsThe profiles of potential biomarker candidates in spinal cord homogenate and that of serum-derived exosomes were found elevated in dogs with degenerative myelopathy as compare to control subjects.

ConclusionsSerum-derived exosomal biomolecules can serve as surrogate biomarkers in neuro degenerative diseases.

Key MessagesO_LIA canine with degenerative myelopathy can serve as a model animal to study human amyotrophic lateral sclerosis.
C_LIO_LISerum-derived exosomes contains Transactive Response DNA Binding Protein 43 (TDP-43), potential biomarker candidate.
C_LIO_LIThe levels of spinal cord TDP-43 proteins and that of serum-derived exosomes exhibited a similar profiling. Therefore, serum derived exosomes may be used as a venue for establishing blood-based biomarkers for neurodegenerative diseases.
C_LI

sentences: A new protocol for multiple muscle mapping using nTMS The representation of muscles in the cortex can be mapped using navigated transcranial magnetic stimulation. The mapping can be quantified via measures like the centre of gravity or the size of the region of excitability. Determining these measures typically relies only on stimulation points that yield motor-evoked potentials (MEPs); stimulations that do not elicit an MEP, i.e., non-MEP points, are ignored entirely. In this study, we show how incorporating non-MEP points may affect the estimates of the size and centroid of the excitable area in eight hand and forearm muscles after mono-phasic single-pulse TMS. We performed test-retest assessments in twenty participants and estimated the reliability of centroids and sizes of the corresponding areas using inter-class correlation coefficients. For most muscles, the reliability turned out good, if not excellent. As expected, removing the non-MEP points significantly decreased area sizes and area weights, suggesting that conventional approaches that do not account for non-MEP points are likely to overestimate the regions of excitability.

sentences: Using EEG Microstates to Examine Post-Encoding Quiet Rest and Subsequent Word-Pair Memory Evidence suggests that the brain preferentially consolidates memories during "offline" periods, in which an individual is not performing a task and their attention is otherwise undirected, including spans of quiet, resting wakefulness. Moreover, research has demonstrated that factors such as the initial encoding strength of information influence which memories receive the greatest benefit. Recent studies have begun to investigate these periods of post-learning quiet rest using EEG microstate analysis to observe the electrical dynamics of the brain during these stretches of memory consolidation, specifically finding an increase in the amount of the canonical microstate D during a post-encoding rest period. Here, we implement an exploratory analysis to probe the activity of EEG microstates during a post-encoding session of quiet rest in order to scrutinize the impact of learning on microstate dynamics and to further understand the role these microstates play in the consolidation of memories. We examined 54 subjects (41 female) as they completed a word-pair memory task designed to use repetition to vary the initial encoding strength of the word-pair memories. In this study, we were able to replicate previous research in which there was a significant increase (p < .05) in the amount of microstate D (often associated with the dorsal attention network) during post-encoding rest. This change was accompanied by a significant decrease (p < .05) in the amount of microstate C (often associated with the default mode network). We also found preliminary evidence indicating a positive relationship between the amount of microstate D and improved memory for weakly encoded memories, which merits further exploration.

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SRSF1 as a novel interacting partner for IFITM1/3 unravels the emergent role of IFITM1/3 mediating protein expression IFITM proteins play a role in cancer cell progression through undefined mechanisms. Here, we propose an emergent role of IFITM1/3 regulating protein synthesis. SBP-tagged IFITM1 protein was used to identify an association of IFITM1 protein with the cytosolic isoform of SRSF1 that transports mRNA to the ribosome. This cytosolic association was confirmed in situ using proximity ligation assays for SRSF1 and IFITM1/3, suggesting a role associated with translation. Accordingly, IFITM1/3 were shown to interact with HLA-B mRNA in response to IFN{gamma} stimulation using RNA-protein proximity ligation assays. In addition, shotgun RNA sequencing in IFITM1/IFITM3 null cells and wt-SiHa cells indicated that reduced HLA-B gene expression does not account for lowered HLA-B protein synthesis in response to IFN{gamma}. Furthermore, ribosome profiling using sucrose gradient sedimentation identified a reduction in 80S ribosomal fraction an IFITM1/IFITM3 null cells compared to their wild-type counterpart, partially reverted by IFITM1/3 complementation. Our data all together link the binding of IFITM1/3 proteins to HLA-B mRNA and SRSF1 as a mechanism to catalyze the synthesis of target proteins, suggesting an RNA chaperonin role for IFITM1/3 proteins.

SignificanceIFITMs are widely studied for their role in inhibiting viruses, and multiple studies have associated IFITMs with cancer progression. However, mechanistic insights are not well understood. Our study proposes that IFITMs have a role regulating protein synthesis, a pivotal function highly relevant for viral infection and cancer progression. Our results suggest that IFITM1/3 is present in the ribosomal fraction and regulates particular protein expression; among them, we identified HLA-B. Changes in HLA-B expression could impact the presentation and recognition of oncogenic antigens on the cell surface by cytotoxic T cells and, ultimately, limit tumor cell eradication. In addition, the role of IFITMs mediating protein translation is relevant, as has the potential of regulating the expression of viral and oncogenic proteins.

sentences: CNest: A Novel Copy Number Association Discovery Method Uncovers 862 New Associations from 200,629 Whole Exome Sequence Datasets in the UK Biobank Copy number variation (CNV) has long been known to influence human traits having a rich history of research into common and rare genetic disease and although CNV is accepted as an important class of genomic variation, progress on copy number (CN) phenotype associations from Next Generation Sequencing data (NGS) has been limited, in part, due to the relative difficulty in CNV detection and an enrichment for large numbers of false positives. To date most successful CN genome wide association studies (CN-GWAS) have focused on using predictive measures of dosage intolerance or gene burden tests to gain sufficient power for detecting CN effects. Here we present a novel method for large scale CN analysis from NGS data generating robust CN estimates and allowing CN-GWAS to be performed genome wide in discovery mode. We provide a detailed analysis in the large scale UK BioBank resource and a specifically designed software package for deriving CN estimates from NGS data that are robust enough to be used for CN-GWAS. We use these methods to perform genome wide CN-GWAS analysis across 78 human traits discovering 862 genetic associations that are likely to contribute strongly to trait distributions based solely on their CN or by acting in concert with other genetic variation. Finally, we undertake an analysis comparing CNV and SNP association signals across the same traits and samples, defining specific CNV association classes based on whether they could be detected using standard SNP-GWAS in the UK Biobank.

sentences: Fast Numerical Optimization for Genome Sequencing Data in Population Biobanks We develop two efficient solvers for optimization problems arising from large-scale regularized regressions on millions of genetic variants sequenced from hundreds of thousands of individuals. These genetic variants are encoded by the values in the set {0, 1, 2, NA}. We take advantage of this fact and use two bits to represent each entry in a genetic matrix, which reduces memory requirement by a factor of 32 compared to a double precision floating point representation. Using this representation, we implemented an iteratively reweighted least square algorithm to solve Lasso regressions on genetic matrices, which we name snpnet-2.0. When the dataset contains many rare variants, the predictors can be encoded in a sparse matrix. We utilize the sparsity in the predictor matrix to further reduce memory requirement and computational speed. Our sparse genetic matrix implementation uses both the compact 2-bit representation and a simplified version of compressed sparse block format so that matrix-vector multiplications can be effectively parallelized on multiple CPU cores. To demonstrate the effectiveness of this representation, we implement an accelerated proximal gradient method to solve group Lasso on these sparse genetic matrices. This solver is named sparse-snpnet, and will also be included as part of snpnet R package. Our implementation is able to solve group Lasso problems on sparse genetic matrices with more than 1, 000, 000 columns and almost 100, 000 rows within 10 minutes and using less than 32GB of memory.

sentences: Stem cell heterogeneity and reiteration of developmental signaling underlie melanocyte regeneration in zebrafish Tissue-resident stem cells are present in many adult organs, where they are important for organ homeostasis and repair in response to injury. However, the signals that activate these cells and the mechanisms governing how these cells self-renew or differentiate are highly context-dependent and incompletely understood, particularly in non-hematopoietic tissues. In the skin, melanocyte stem cells (McSCs) are responsible for replenishing mature pigmented melanocytes. In mammals, these cells reside in the hair follicle bulge and bulb niches where they are activated during homeostatic hair follicle turnover and following melanocyte destruction, as occurs in vitiligo and other skin hypopigmentation disorders. Recently, we identified adult McSCs in the zebrafish. To elucidate mechanisms governing McSC self-renewal and differentiation we analyzed individual transcriptomes from thousands of melanocyte lineage cells during the regeneration process. We identified transcriptional signatures for McSCs, deciphered transcriptional changes and intermediate cell states during regeneration, and analyzed cell-cell signaling changes to discover mechanisms governing melanocyte regeneration. We identified KIT signaling via the RAS/MAPK pathway as a regulator of McSC direct differentiation. Analysis of the scRNAseq dataset also revealed a population of mitfa/aox5 co-expressing cells that divides following melanocyte destruction, likely corresponding to cells that undergo self-renewal. Our findings show how activation of different subpopulations of mitfa-positive cells underlies self-renewal and differentiation to properly reconstitute the melanocyte pigmentary system following injury.

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DNA methylation in human gastric epithelial cells allows cell type-related plasticity and defines regional identity BackgroundEpigenetic modifications in mammalian DNA are commonly manifested by DNA methylation. In the stomach, altered DNA methylation patterns have been observed following chronic Helicobacter pylori infections and in gastric cancer. In the context of epigenetic regulation, the regional nature of the stomach has been rarely considered in detail.

ResultsHere, we describe the DNA methylation landscape across the phenotypically different regions of the healthy human stomach (i.e., antrum, corpus, fundus) together with the corresponding transcriptomes. We show that stable regional DNA methylation differences translate to a limited extent into regulation of the transcriptomic phenotype, indicating a largely permissive epigenetic regulation. We identify a small number of transcription factors with novel region-specific activity and likely epigenetic impact in the stomach, including GATA4, IRX5, IRX2, PDX1, and CDX2. Detailed analysis of the Wnt pathway reveals differential regulation along the craniocaudal axis, which involves non-canonical Wnt signaling in determining cell fate in the proximal stomach.

ConclusionsBy extending our analysis to pre-neoplastic lesions and gastric cancers, we conclude that epigenetic dysregulation already characterizes intestinal metaplasia as a founding basis for functional changes in gastric cancer. Finally, our study provides a well-defined resource of regional stomach transcription and epigenetics as a starting point for further studies.

sentences: Hybrid metachronal rowing augments swimming speed and acceleration via increased stroke amplitude Numerous aquatic invertebrates use drag-based metachronal rowing for swimming, in which closely spaced appendages are oscillated starting from the posterior, with each appendage phase-shifted in time relative to its neighbor. Continuously swimming species such as Antarctic krill generally use "pure metachronal rowing" consisting of a metachronal power stroke and a metachronal recovery stroke, while burst swimming species such as many copepods and mantis shrimp typically use "hybrid metachronal rowing" consisting of a metachronal power stroke followed by a synchronous or nearly synchronous recovery stroke. Burst swimming organisms need to rapidly accelerate in order to capture prey and/or escape predation, and it is unknown whether hybrid metachronal rowing can augment acceleration and swimming speed compared to pure metachronal rowing. Simulations of rigid paddles undergoing simple harmonic motion showed that collisions between adjacent paddles restrict the maximum stroke amplitude for pure metachronal rowing. Hybrid metachronal rowing similar to that observed in mantis shrimp (Neogonodactylus bredini) permits oscillation at larger stroke amplitude while avoiding these collisions. We comparatively examined swimming speed, acceleration, and wake structure of pure and hybrid metachronal rowing strategies by using a self-propelling robot. Both swimming speed and peak acceleration of the robot increased with increasing stroke amplitude. Hybrid metachronal rowing permitted operation at larger stroke amplitude without collision of adjacent paddles on the robot, augmenting swimming speed and peak acceleration. Hybrid metachronal rowing generated a dispersed wake unlike narrower, downward-angled jets generated by pure metachronal rowing. Our findings suggest that burst swimming animals with small appendage spacing, such as copepods and mantis shrimp, can use hybrid metachronal rowing to generate large accelerations via increasing stroke amplitude without concern of appendage collision.

sentences: The parallel origins of vertebrate venoms Evolution can occur with surprising predictability when organisms face similar ecological challenges. How and why traits arise repeatedly remains a central question in evolutionary biology, but the complexity of most traits makes it challenging to answer. Reptiles and mammals independently evolved oral venoms consisting of proteinaceous cocktails that allow mapping between genotype and phenotype. Although biochemically similar toxins can occur as major venom components across many taxa, whether these toxins evolved via convergent or parallel processes remains unknown. Most notable among them are kallikrein-like serine proteases that form the core of most vertebrate oral venoms. We used a combination of comparative genomics and phylogenetics to investigate whether serine protease recruitment into the venom occurred independently or in parallel in mammalian and reptilian lineages. Using syntenic relationships between genes flanking known toxins, we traced the origin of kallikreins to a single locus containing one or more nearby paralogous kallikrein-like clusters. Independently, phylogenetic analysis of vertebrate serine proteases revealed that kallikrein-like toxins in mammals and reptiles are homologous. Given the shared regulatory and genetic machinery, these findings suggest a unified model underlying vertebrate venom evolution. Namely, the common tetrapod ancestor possessed salivary genes that were biochemically suitable for envenomation. We term such genes  toxipotent - in the case of salivary kallikreins they already had potent vasodilatory activity that was weaponized by venomous lineages. Furthermore, the ubiquitous distribution of kallikreins across vertebrates suggests that the evolution of envenomation may be more common than previously recognized, blurring the line between venomous and non-venomous animals.

sentences: SARS-CoV-2 Omicron (BA.1 and BA.2) specific novel CD8+ and CD4+ T cell epitopes targeting spike protein. The Omicron (BA.1/B.1.1.529) variant of SARS-CoV-2 harbors an alarming 37 mutations on its spike protein, reducing the efficacy of current COVID-19 vaccines. This study identified CD8+ and CD4+ T cell epitopes from SARS-CoV-2 S protein mutants. To identify the highest quality CD8 and CD4 epitopes from the Omicron variant, we selected epitopes with a high binding affinity towards both MHC I and MHC II molecules and applied other clinical checkpoint predictors including immunogenicity, antigenicity, allergenicity, instability, and toxicity. Subsequently, we found eight Omicron (BA.1/B.1.1.529) specific CD8+ and eleven CD4+ T cell epitopes with a world population coverage of 76.16% and 97.46%, respectively. Additionally, we identified common epitopes across Omicron BA.1 and BA.2 lineages that target mutations critical to SARS-CoV-2 virulence. Further, we identified common epitopes across B.1.1.529 and other circulating SARS-CoV-2 variants, such as B.1.617.2 (Delta). We predicted CD8 epitopes binding affinity to murine MHC alleles to test the vaccine candidates in preclinical models. The CD8 epitopes were further validated using our previously developed software tool PCOptim. We then modeled the three-dimensional structures of our top CD8 epitopes to investigate the binding interaction between peptide-MHC and peptide-MHC-TCR complexes. Importantly, our identified epitopes are targeting the mutations on the RNA-binding domain and the fusion sites of S protein. This could potentially eliminate viral infections and form long-term immune responses compared to rather short-lived mRNA vaccines and maximize the efficacy of vaccine candidates against the current pandemic and potential future variants.

sentences: In vivo identification and validation of novel potential predictors for human cardiovascular diseases Genetics crucially contributes to cardiovascular diseases (CVDs), the global leading cause of death. Since the majority of CVDs can be prevented by early intervention there is a high demand for predictive markers. While genome wide association studies (GWAS) correlate genes and CVDs after diagnosis and provide a valuable resource for such markers, preferentially those with previously known or suspected function are addressed further. To tackle the unaddressed blind spot of understudied genes, we particularly focused on the validation of heart GWAS candidates with little or no apparent connection to cardiac function. Building on the high conservation of basic heart function and underlying genetics from fish to human we combined CRISPR/Cas9 genome editing of the orthologs of human GWAS candidates in isogenic medaka with automated high-throughput heart rate analysis. Our functional analyses of understudied human candidates uncovered a prominent fraction of heart rate associated genes from adult human patients displaying a heart rate effect in embryonic medaka already in the injected generation. Following this pipeline, we identified 16 GWAS candidates with potential diagnostic and predictive power for human CVDs.

sentences: Simultaneous in vivo Pharmacokinetics and Pharmacodynamics of TDF Intravaginal Ring during High-dose Vaginal SHIV Challenge in Pigtail Macaques The demonstration of complete protection of macaques in a repeated low dose virus challenge by a tenofovir disoproxil fumarate (TDF) intravaginal ring (IVR) and the success of the dapivirine IVR in clinical trials highlighted the potential of IVRs as pre- exposure prophylaxis against HIV. Efficacy of TDF ring was not investigated in sexually active women. Our understanding of the mechanisms of protection is limited. To address this knowledge gap, we performed simultaneous pharmacokinetic and pharmacodynamic analysis of a TDF-IVR at the site of SIV challenge in pigtail macaques at the anatomical and cellular level. Specifically, we challenged TDF-IVR administered pigtail macaques with a single high dose of a non-replicative SIV-based vector containing a dual reporter system that helped us to identify the earliest targets of SIV infection within the mucosa. Two and three days after challenge, the macaques were euthanized and tenofovir (TFV) concentrations were measured in the female reproductive tract (FRT) by HPLC-MS/MS to correlate drug concentrations and SIV-vector transduction efficiency. TFV formed a gradient through the mucosal tissue, with the highest concentrations near the ring, in the upper vagina and endocervix. Despite this, several transduction events were identified with the most common sites being in the ovaries. Moreover, proviral DNA was detected in the cervix and vagina. Thus, our studies demonstrate an uneven distribution of TFV in the FRT of macaques after release from a TDF-IVR that leads to incomplete FRT protection from high viral dose challenge.

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Magnetic DNA random access memory with nanopore readouts and exponentially-scaled combinatorial addressing The storage of data in DNA typically involves encoding and synthesizing data into short oligonucleotides, followed by reading with a sequencing instrument. Major challenges include the molecular consumption of synthesized DNA, issues with basecalling errors, and limitations with scaling up read access operations for individual data elements. Addressing these challenges, we describe a DNA storage system called MDRAM (Magnetic DNA-based Random Access Memory) that enables repetitive and efficient readouts of targeted files with nanopore-based sequencing. Through conjugation of synthesized DNA to magnetic beads, we enabled repeated readouts of data while preserving the original DNA analyte and maintaining data readout quality. MDRAM also utilizes an efficient convolutional coding scheme that leverages soft information in raw nanopore sequencing signals to achieve information reading costs comparable to Illumina sequencing despite substantially higher error rates. Finally, we demonstrate a proof-of-concept DNA-based proto-filesystem that enables an exponentially-scalable data address space using only small numbers of targeting primers for assembly and readout.

ONE-SENTENCE SUMMARYWe demonstrate a novel DNA data storage system that leverages conjugation of DNA onto magnetic beads, new computational advances in data encoding, and exponentially scalable access of individual data elements.

sentences: Simplifying the development of portable, scalable, and reproducible workflows Command-line software plays a critical role in biology research. However, processes for installing and executing software differ widely. The Common Workflow Language (CWL) is a community standard that addresses this problem. Using CWL, tool developers can formally describe a tools inputs, outputs, and other execution details in a manner that fosters use of shared computational methods and reproducibility of complex analyses. CWL documents can include instructions for executing tools inside software containers--isolated, operating-system environments. Accordingly, CWL tools are portable--they can be executed on diverse computers--including personal workstations, high-performance clusters, or the cloud. This portability enables easier adoption of bioinformatics pipelines. CWL supports workflows, which describe dependencies among tools and using outputs from one tool as inputs to others. To date, CWL has been used primarily for batch processing of large datasets, especially in genomics. But it can also be used for analytical steps of a study. This article explains key concepts about CWL and software containers and provides examples for using CWL in biology research. CWL documents are text-based, so they can be created manually, without computer programming. However, ensuring that these documents confirm to the CWL specification may prevent some users from adopting it. To address this gap, we created ToolJig, a Web application that enables researchers to create CWL documents interactively. ToolJig validates information provided by the user to ensure it is complete and valid. After creating a CWL tool or workflow, the user can create "input-object" files, which store values for a particular invocation of a tool or workflow. In addition, ToolJig provides examples of how to execute the tool or workflow via a workflow engine.

sentences: Low abundance members of the gut microbiome are potent drivers of immune cell education. One of the main goals of microbiome research is to identify bacterial members that significantly affect host phenotypes and understand their contributions to disease pathogenesis. Studies identifying bacterial members that dictate host phenotype have focused mainly on the dominant members, and the role of low abundance microbes in determining host phenotypes and pathogenesis of diseases remains unexplored. In this study, we compared the gut bacterial community of mice with wide-ranging microbial exposure to determine if low abundance bacteria vary based on microbial exposure or remain consistent. We noted that similar to the high abundance bacterial community, a core community of low abundance bacteria made up a significant portion of the gut microbiome irrespective of microbial exposure. To determine the effect of low abundance bacteria on community structure and host gene expression, we devised a microbiome dilution strategy to "delete" out low abundance bacteria and engrafted the diluted microbiomes into germ-free mice. Our approach successfully excluded low abundance bacteria from small and large intestinal bacterial communities and induced global changes in microbial community structure and composition in the large intestine. Gene expression analysis of intestinal tissue revealed that loss of low abundance bacteria resulted in a drastic reduction in expression of multiple genes involved MHC class II antigen presentation pathway and T-cell cytokine production in the small intestine. The effect of low abundance bacteria on MHC class II expression was found specific to the intestinal epithelium at an early timepoint post-colonization and correlated with bacteria belonging to the family Erysipelotrichaceae. We conclude that low abundance bacteria have a significantly higher immuno-stimulatory effect compared to dominant bacteria and are thus potent drivers of early immune education in the gut. Therefore, characterizing immune interaction of low abundance bacteria with the host will offer greater insight into the intestinal immune landscape and disease pathogenesis.

sentences: Automated strain separation in low-complexity metagenomes using long reads High-throughput short-read metagenomics has enabled large-scale species-level analysis and functional characterization of microbial communities. Microbiomes often contain multiple strains of the same species, and different strains have been shown to have important differences in their functional roles. Despite this, strain-level resolution from metagenomic sequencing remains challenging. Recent advances on long-read based methods enabled accurate assembly of bacterial genomes from complex microbiomes and an as-yet-unrealized opportunity to resolve strains. Here we present Strainberry, a metagenome assembly method that performs strain separation in single-sample low-complexity metagenomes and that relies uniquely on long-read data. We benchmarked Strainberry on mock communities and showed it consistently produces strain-resolved assemblies with near-complete reference coverage and 99.9% base accuracy. We also applied Strainberry on real datasets for which it improved assemblies generating 20-118% additional genomic material than conventional metagenome assemblies on individual strain genomes. Our results hence demonstrate that strain separation is possible in low-complexity microbiomes using a single regular long read dataset. We show that Strainberry is also able to refine microbial diversity in a complex microbiome, with complete separation of strain genomes. We anticipate this work to be a starting point for further methodological improvements aiming to provide better strain-resolved metagenome assemblies in environments of higher complexities.

sentences: Epigenetic reprogramming by TET enzymes impacts co-transcriptional R-loops DNA oxidation by ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming. The conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) initiates developmental and cell-type-specific transcriptional programs through mechanisms that include changes in the chromatin structure. Here, we show that the presence of 5hmC in the transcribed DNA promotes the annealing of the nascent RNA to its template DNA strand, leading to the formation of an R-loop. The genome-wide distribution of 5hmC and R-loops show a positive correlation in mouse and human embryonic stem cells and overlap in half of all active genes. Moreover, R-loop resolution leads to differential expression of a subset of genes that are involved in crucial events during stem cell proliferation. Altogether, our data reveal that epigenetic reprogramming via TET activity promotes co-transcriptional R-loop formation, and disclose novel links between R-loops and the regulation of gene expression programs in stem cells.

sentences: Twenty years of change in benthic communities across the Belizean Barrier Reef Disease, ocean warming, and pollution have caused catastrophic declines in the cover of living coral on reefs across the Caribbean. Subsequently, reef-building corals have been replaced by invertebrates and macroalgae, leading to changes in ecological functioning. We describe changes in benthic community composition and cover at 15 sites across the Belizean Barrier Reef (BBR) following numerous major disturbances--bleaching, storms, and disease outbreaks--over the 20-year period 1997-2016. We tested the role of potential drivers of change on coral reefs, including local human impacts and ocean temperature. From 1997 to 2016, mean coral cover significantly declined from 26.3% to 10.7%, while macroalgal cover significantly increased from 12.9% to 39.7%. We documented a significant decline over time of the reef-building corals Orbicella spp. and described a major shift in benthic composition between early sampling years (1997-2005) and later years (2009-2016). The covers of hard-coral taxa, including Acropora spp., M. cavernosa, Orbicella spp., and Porites spp., were negatively related to marine heatwave frequency. Only gorgonian cover was related, negatively, to our metric of the magnitude of local impacts (the Human Influence Index). Changes in benthic composition and cover were not associated with local protection or fishing. This result is concordant with studies throughout the Caribbean that have documented living coral decline and shifts in reef-community composition following disturbances, regardless of local fisheries restrictions. Our results suggest that benthic communities along the BBR have experienced disturbances that are beyond the capacity of the current management structure to mitigate. We recommend that managers devote greater resources and capacity to enforce and expand existing marine protected areas and that government, industry, and the public act to reduce global carbon emissions.

sentences: A nitrite-oxidizing bacterium constitutively consumes atmospheric hydrogen Chemolithoautotrophic nitrite-oxidizing bacteria (NOB) of the genus Nitrospira contribute to nitrification in diverse natural environments and engineered systems. Nitrospira are thought to be well-adapted to substrate limitation owing to their high affinity for nitrite and capacity to use alternative energy sources. Here, we demonstrate that the canonical nitrite oxidizer Nitrospira moscoviensis oxidizes hydrogen (H2) below atmospheric levels using a high-affinity group 2a nickel-iron hydrogenase [Km(app) = 32 nM]. Atmospheric H2 oxidation occurred under both nitrite-replete and nitrite-deplete conditions, suggesting low-potential electrons derived from H2 oxidation promote nitrite-dependent growth and enable survival during nitrite limitation. Proteomic analyses confirmed the hydrogenase was abundant under both conditions and indicated extensive metabolic changes occur to reduce energy expenditure and growth under nitrite-deplete conditions. Respirometry analysis indicates the hydrogenase and nitrite oxidoreductase are bona fide components of the aerobic respiratory chain of N. moscoviensis, though they transfer electrons to distinct electron carriers in accord with the contrasting redox potentials of their substrates. Collectively, this study suggests atmospheric H2 oxidation enhances the growth and survival of NOB in amid variability of nitrite supply. These findings also extend the phenomenon of atmospheric H2 oxidation to a seventh phylum (Nitrospirota) and reveal unexpected new links between the global hydrogen and nitrogen cycles.

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Prefrontally Modulated Vagal Tone Inhibits Inflammatory Responses to Prevent Telomere Damage in Healthy Participants BackgroundAccumulated senescent cells are proposed to be one of the main drivers of age-related pathology through disruption of tissue structure and function. We recently proposed the Neuro-Immuno-Senescence Integrative Model (NISIM; Ask et al., 2018) which relates prefrontally modulated vagal tone and subsequent balance between vagal and sympathetic input to the spleen to inflammatory responses leading to generation of reactive oxygen species and oxidative telomere damage. The NISIM is based on converging evidence and argues for the existence of a prefrontal cortex-autonomic nervous system-spleen (PAS) axis, suggesting that the inflammation that induces reactive oxygen species-generation is downstream of this axis.

AimIn this study, we aim to assess inflammation as a mediator in the relationship between prefrontally modulated vagal tone and leukocyte telomere length to test the hypothesis that PAS axis dysregulation accelerates cellular aging. We also assess the relationship between a recently proposed index of vagal immunomodulation (vagal tone/inflammation ratio; NIM index; Gidron et al., 2018) and telomere length, and compare results between the NIM index and vagal tone as predictors of telomere length.

MethodsThis study uses participant data from a large nationally representative longitudinal study since 1974 with a total of 45,000 Norwegian residents so far. A sub-sample of 1372 participants from which vagal tone, C Reactive Protein, and leukocyte telomere length could be obtained were included in the study. Relationships were analyzed with hierarchical multiple linear regression using either vagal tone and C Reactive Protein or the NIM index to predict telomere length. Sleeping problems, tobacco use status, alcohol use status, time since last meal, and symptoms of depression were included as control variables.

ResultsIn the mediation analysis, vagal tone was a significant positive predictor of telomere length, while C Reactive Protein was a significant negative predictor of telomere length. This relationship remained significant when individually controlling for some but not all confounding variables. The NIM index was a significant positive predictor of telomere length. This relationship remained significant when controlling for all confounding variables except one. In a reduced dataset excluding all participants where confounders were present, the NIM index remained a significant predictor of telomere length.

ConclusionThis is the first study suggesting that PAS axis activity is associated with telomere length thus supporting the NISIM. Results indicate that the NIM index is a more sensitive indicator of PAS axis activity than vagal tone and C Reactive Protein in isolation. Clinical relevance and suggestions for future research are discussed.

sentences: Prevention and therapy of SARS-CoV-2 and the B.1.351 variant in mice Improving the standard of clinical care for individuals infected with SARS-CoV-2 variants is a global health priority. Small molecule antivirals like remdesivir (RDV) and biologics such as human monoclonal antibodies (mAb) have demonstrated therapeutic efficacy against SARS-CoV-2, the causative agent of COVID-19. However, it is not known if combination RDV/mAb will improve outcomes over single agent therapies or whether antibody therapies will remain efficacious against variants. In kinetic studies in a mouse-adapted model of ancestral SARS-CoV-2 pathogenesis, we show that a combination of two mAbs in clinical trials, C144 and C135, have potent antiviral effects against even when initiated 48 hours after infection. The same antibody combination was also effective in prevention and therapy against the B.1.351 variant of concern (VOC). Combining RDV and antibodies provided a modest improvement in outcomes compared to single agents. These data support the continued use of RDV to treat SARS-CoV-2 infections and support the continued clinical development of the C144 and C135 antibody combination to treat patients infected with SARS-CoV-2 variants.

sentences: A UHM - ULM interface contributes to U2AF2 and SF3B1 association for pre-mRNA splicing In the early stages of spliceosome assembly, the 3 splice site is recognized by sequential complexes of U2AF2 with SF1 followed by the SF3B1 subunit of the U2 small nuclear ribonucleoprotein particle. The U2AF2 - SF1 interface comprises a U2AF homology motif (UHM) of U2AF2 and a well-characterized U2AF ligand motif (ULM)/coiled coil region of SF1. However, the structure of the U2AF2 - SF3B1 interface and its importance for pre-mRNA splicing is unknown. To address this knowledge gap, we determined the crystal structure of the U2AF2 UHM bound to a SF3B1 ULM site at 1.8 [A] resolution. The trajectory of the SF3B1 ULM across the U2AF2 UHM surface differed from prior UHM/ULM structures. This distinctive structure is expected to modulate the orientations of the fulllength proteins. Using isothermal titration calorimetry, we established similar binding affinities of a minimal U2AF2 UHM - SF3B1 ULM complex and a nearly full-length U2AF2 protein binding the N-terminal SF3B1 region, with or without an auxiliary SF3B6 subunit. We showed that key residues at the U2AF2 UHM - SF3B1 ULM interface are required for high affinity association and co-immunoprecipitation of the splicing factors. Moreover, disrupting the U2AF2 - SF3B1 interface altered splicing of representative human transcripts. Further analysis of these transcripts and genome-wide data sets indicated that the subset of splice sites co-regulated by U2AF2 and SF3B1 are largely distinct from those co-regulated by U2AF2 and SF1. Altogether, these findings support distinct structural and functional roles for the sequential SF1 and SF3B1 complexes with U2AF2 during the pre-mRNA splicing process.

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"Holey" niche! Finding holes in niche hypervolumesusing persistence homology O_LIHutchinsons niche hypervolume concept has enabled significant progress in our understanding of species ecological needs and distributions across environmental gradients. Nevertheless, the properties of Hutchinsons n-dimensional hypervolumes can be challenging to calculate and several methods have been proposed to extract meaningful measurements of hypervolumes properties (e.g., volume).
C_LIO_LIOne key property of hypervolumes are holes, which provide important information about the ecological occupancy of species. However, to date, current methods rely on volume estimates and set operations to identify holes in hypervolumes. Yet, this approach can be problematic because in high-dimensions, the volume of region enclosing a hole tends to zero.
C_LIO_LIHere, we propose the use of the topological concept of persistence homology (PH) to identify holes in hypervolumes and in ecological datasets more generally. PH allows for the estimates of topological properties in n-dimensional niche hyper-volumes and is independent of the volume estimates of the hypervolume. We demonstrate the application of PH to canonical datasets and to the identification of holes in the hypervolumes of five vertebrate species with diverse niches, highlighting the potential benefits of this approach to gain further insights into animal ecology.
C_LIO_LIOverall, our approach enables the study of an yet unexplored property of Hutchinsons hypervolumes (i.e., holes), and thus, have important implications to our understanding of animal ecology.
C_LI

sentences: ANCHOR, a technical approach to monitor single-copy locus localization in planta RESUMEGene expression is governed by several layers of regulation which in addition to genome organization, local chromatin structure, gene accessibility and the presence of transcription factors also includes gene positioning. Although basic mechanisms are expected to be conserved in Eukaryotes, surprisingly little information on the role of gene positioning is available in plant cells, mainly due to the lack of a highly resolutive approach. In this manuscript, we adapted the use of the ANCHOR system to perform real-time single-locus detection in planta. ANCHOR is a DNA-labelling tool derived from the partitioning system. We demonstrate its suitability to monitor a single-locus in planta and used this approach to track chromatin mobility during cell differentiation in Arabidopsis root epidermal cells. Finally, we discuss the potential of this approach to investigate the role of gene positioning during transcription and DNA repair in plants.

sentences: A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for high-throughput screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified four molecules, including the broad-spectrum antiviral merimepodib/VX-497, which show anti-nsp5 activity and inhibit SARS-CoV-2 replication in A549-ACE2 cells with IC50 values in the 4-21 {micro}M range. The here described assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.

sentences: A monotone single index model for missing-at-random longitudinal proportion data Beta distributions are commonly used to model proportion valued response variables, commonly encountered in longitudinal studies. In this article, we develop semi-parametric Beta regression models for proportion valued responses, where the aggregate covariate effect is summarized and flexibly modeled, using a interpretable monotone time-varying single index transform of a linear combination of the potential covariates. We utilize the potential of single index models, which are effective dimension reduction tools and accommodate link function misspecification in generalized linear mixed models. Our Bayesian methodology incorporates the missing-at-random feature of the proportion response, and utilize Hamiltonian Monte Carlo sampling to conduct inference. We explore finite-sample frequentist properties of our estimates, and assess the robustness via detailed simulation studies. Finally, we illustrate our methodology via application to a motivating longitudinal dataset on obesity research recording proportion body fat.

sentences: Human-in-the-loop optimization of visual prosthetic stimulation AO_SCPLOWBSTRACTC_SCPLOWRetinal prostheses are a promising strategy to restore sight to patients with retinal degenerative diseases. These devices compensate for the loss of photoreceptors by electrically stimulating neurons in the retina. Currently, the visual function that can be recovered with such devices is very limited. This is due, in part, to current spread, unintended axonal activation, and the limited resolution of existing devices. Here we show, using a recent model of prosthetic vision, that optimizing how visual stimuli are encoded by the device can help overcome some of these limitations, leading to dramatic improvements in visual perception. We propose a strategy to do this in practice, using patients feedback in a visual task. The main challenge of our approach comes from the fact that, typically, one only has access to a limited number of noisy responses from patients. We propose two ways to deal with this: first, we use a model of prosthetic vision to constrain and simplify the optimization; second, we use preferential Bayesian optimization to efficiently learn the encoder using minimal trials. To test our approach, we presented healthy subjects with visual stimuli generated by a recent model of prosthetic vision, to replicate the perceptual experience of patients fitted with an implant. Our optimization procedure led to significant and robust improvements in perceived image quality, that transferred to increased performance in other tasks. Importantly, our strategy is agnostic to the type of prosthesis and thus could readily be implemented in existing implants.

sentences: Profiling sensory neuron microenvironment after peripheral and central axon injury reveals key pathways for neural repair Sensory neurons with cell bodies in dorsal root ganglia (DRG) represent a useful model to study axon regeneration. Whereas regeneration and functional recovery occurs after peripheral nerve injury, spinal cord injury or dorsal root injury is not followed by regenerative outcomes. Regeneration of sensory axons in peripheral nerves is not entirely cell autonomous. Whether the DRG microenvironment influences the different regenerative capacities after injury to peripheral or central axons remains largely unknown. To answer this question, we performed a single cell transcriptional profiling of mouse DRG in response to peripheral (sciatic nerve crush) and central axon injuries (dorsal root crush and spinal cord injury). Each cell type responded differently to the three types of injuries. All injuries increased the proportion of a cell type that shares features of both immune cells and glial cells. A distinct subset of satellite glial cells (SGC) appeared specifically in response to peripheral nerve injury. Activation of the PPAR signaling pathway in SGC, which promotes axon regeneration after peripheral nerve injury, failed to occur after central axon injuries. Treatment with the FDA-approved PPAR agonist fenofibrate increased axon regeneration after dorsal root injury. This study provides a map of the distinct DRG microenvironment responses to peripheral and central injuries at the single cell level and highlights that manipulating non-neuronal cells could lead to avenues to promote functional recovery after CNS injuries or disease.

sentences: Allele-resolved single-cell multi-omics uncovers the dynamics and transcriptional kinetics of X-chromosome upregulation X-chromosome inactivation (XCI) and upregulation (XCU) are the major opposing chromosome-wide modes of gene regulation that collectively achieve dosage compensation in mammals, but the regulatory link between the two remains elusive. Here, we use allele-resolved single-cell RNA-seq combined with chromatin accessibility profiling to finely dissect the separate effects of XCI and XCU on RNA levels during mouse development. We uncover that balanced X dosage is flexibly attained through expression tuning by XCU in a sex- and lineage-specific manner along varying degrees of XCI and across developmental and cellular states. Male blastomeres achieve XCU upon zygotic genome activation while females experience two distinct waves of XCU, upon imprinted- and random XCI, and ablation of Xist impedes female XCU. Contrary to widely established models of mammalian dosage compensation, naive female embryonic cells carrying two active X chromosomes do not exhibit upregulation but express both alleles at basal level, yet collectively exceeding the RNA output of a single hyperactive allele. We show, in vivo and in vitro, that XCU is kinetically driven by X-specific modulation of transcriptional burst frequency, coinciding with increased compartmentalization of the hyperactive allele. Altogether, our data provide unprecedented insights into the dynamics of mammalian XCU, prompting a revised model of the chain in events of allelic regulation by XCU and XCI in unitedly achieving stable cellular levels of X-chromosome transcripts.

sentences: Svep1 stabilizes developmental vascular anastomosis in reduced flow conditions. We report the discovery that flow and Svep1 are modulator of vessel anastomosis during developmental angiogenesis in zebrafish embryos. We show that loss of Svep1 and blood flow reduction both contribute to defective anastomosis of intersegmental vessels. We show that this defect in primary angiogenic sprouts is associated with an expansion of Apelin-positive tip cells and with reduced formation and lumenisation of the dorsal longitudinal anastomotic vessel. Mechanistically, our results suggest that flow and Svep1 act synergistically to modulate vascular network formation in the zebrafish trunk.

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Sulfur limitation increases duckweed starch accumulation without compromising growth Duckweeds contain relatively high levels of starch and are a potential biomass feedstock for biofuel production. Here, the biomass and starch yield of duckweed under three different nutrient-limited conditions were analyzed to investigate possible ways of further increasing the efficiency of starch production. The results showed that sulfur limitation resulted in the highest starch yield, which was 42% and 73% higher than in nitrogen or phosphorus limitation, respectively. The high yield of sulfur-limited duckweed is largely due to the combinations of little effects on biomass and high accumulations of starch. Although nitrogen limitation led to higher starch content (67.4%), it severely reduced biomass production. The photosynthetic performance indicator Fv/Fm was a simple and sensitive indicator of starch content in nutrient-limited duckweed. Taken together, this study demonstrates that sulfur limitation is a simple and efficient way to increase starch yield, highlighting the great potential of duckweed for biofuel production. We report that sulfur limitation is a practical approach to increase starch yields in duckweed without affecting growth or biomass.

HighlightsO_LISulfur limitation induces starch production in a duckweed specie.
C_LIO_LINitrogen limitation triggers the highest starch content, but limits growth.
C_LIO_LISulfur limitation results in the highest starch yield.
C_LIO_LIFv/Fm is a rapid and robust proxy of starch content in nutrient-limited duckweed.
C_LI

sentences: SH3-domain mutations selectively disrupt Csk homodimerization or PTPN22 binding The kinase Csk is the primary negative regulator of the Src-family kinases (SFKs, e.g., Lck, Fyn, Lyn, Hck, Fgr, Blk, Yes), phosphorylating a tyrosine on the SFK C-terminal tail that mediates autoinhibition. Csk also binds phosphatases, including PTPN12 (PTP-PEST) and immune-cell PTPN22 (LYP/Pep), which dephosphorylate the SFK activation loop to promote autoinhibition. Csk-binding proteins (e.g., CBP/PAG1) oligomerize within membrane microdomains, and high local concentration promotes Csk function. Purified Csk homodimerizes in solution through an interface that overlaps the phosphatase binding footprint. Here we demonstrate that Csk can homodimerize in Jurkat T cells, in competition with PTPN22 binding. We designed SH3-domain mutations in Csk that selectively impair homodimerization (H21I) or PTPN22 binding (K43D) and verified their kinase activity in solution. Disruption of either interaction, however, decreased the negative-regulatory function of Csk in cells. Csk W47A, a substitution previously reported to block PTPN22 binding, also impairs homodimerization. Csk H21I and K43D will be useful tools for dissecting the protein-specific drivers of autoimmunity mediated by the human polymorphism PTPN22 R620W, which impairs interaction with Csk and with the E3 ubiquitin ligase TRAF3. Future investigations of Csk homodimer activity and phosphatase interactions may reveal new facets of SFK regulation in hematopoietic and non-hematopoietic cells.

sentences: Recombination marks the evolutionary dynamics of a recently endogenized retrovirus All vertebrate genomes have been colonized by retroviruses along their evolutionary trajectory. While endogenous retroviruses (ERVs) can contribute important physiological functions to contemporary hosts, such benefits are attributed to long-term co-evolution of ERV and host because germline infections are rare and expansion is slow, because the host effectively silences them. The genomes of several outbred species including mule deer (Odocoileus hemionus) are currently being colonized by ERVs, which provides an opportunity to study ERV dynamics at a time when few are fixed. Because we have locus-specific data on the distribution of cervid endogenous retrovirus (CrERV) in populations of mule deer, in this study we determine the molecular evolutionary processes acting on CrERV at each locus in the context of phylogenetic origin, genome location, and population prevalence. A mule deer genome was de novo assembled from short and long insert mate pair reads and CrERV sequence generated at each locus. CrERV composition and diversity have recently measurably increased by horizontal acquisition of a new retrovirus lineage. This new lineage has further expanded CrERV burden and CrERV genomic diversity by activating and recombining with existing CrERV. Resulting inter-lineage recombinants endogenized and subsequently retrotransposed. CrERV loci are significantly closer to genes than expected if integration were random and gene proximity might explain the recent expansion by retrotransposition of one recombinant CrERV lineage. Thus, in mule deer, retroviral colonization is a dynamic period in the molecular evolution of CrERV that also provides a burst of genomic diversity to the host population.

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CARDIOVASCULAR AUTONOMIC NEUROPATHY IN TYPE 1 DIABETES IS ASSOCIATED WITH SEVERAL METABOLIC PATHWAYS: New Risk Markers on the horizon ObjectiveCardiovascular autonomic neuropathy (CAN) in diabetes is associated with increased mortality and morbidity and is a non-treatable complication. We investigated associations between circulating metabolites and presence of CAN in persons with type 1 diabetes (T1D).

MethodsCAN was assessed by cardiovascular reflex tests (CARTs) in 302 persons with T1D as heart rate response to: deep breathing; lying-to-standing test; and the Valsalva manoeuvre. More than 1 pathological CART defined the CAN diagnosis.

Serum metabolomics and lipidomics profiles were analysed with two complementary non-targeted massspectrometry methods. Cross-sectional associations between single metabolites and CAN were assessed by linear regression. Models were fitted with and without adjustments for relevant confounders and multiple testing.

ResultsParticipants were mean (IQR) aged 55(49, 63) years, 50% males, with diabetes duration 39(32, 47) years, HbA1c 63(55,69) mmol/mol and 34% had the CAN diagnosis.

A total of 75 metabolites and 106 lipids were examined. In crude models, CAN diagnosis was associated with higher levels of hydroxy fatty acids (2,4- and 3,4-dihydroxybutanoic acids, 4-deoxytetronic acid), creatinine, sugar derivates (ribitol, ribonic acid, myo-inositol), citric acid, glycerol, phenols, phosphatidylcholines and lower levels of free fatty acids and amino acid methionine (p<0.05). Upon adjustment, positive associations with CAN were retained with hydroxy fatty acids, tricarboxylic acid (TCA) cycle-based sugar derivates, and citric acid and phenols (Padjusted<0.05).

ConclusionsMetabolic pathways, including the TCA cycle, hydroxy fatty acids, phosphatidylcholines and sugar derivatives, were associated with CAN in T1D. These novel metabolic pathways associated with CAN could prove to be future modifiable risk factors.

sentences: Region-specific maladaptive gray matter myelination is associated with differential susceptibility to stress-induced behavior in male rodents and humans Individual reactions to traumatic stress vary dramatically, yet the biological basis of this variation remains poorly understood. Recent studies demonstrate the surprising plasticity of oligodendrocytes and myelin with stress and experience, providing a potential mechanism by which trauma induces aberrant structural and functional changes in the adult brain. In this study, we utilized a translational approach to test the hypothesis that gray matter myelin contributes to traumatic-stress-induced behavioral variation in both rats and humans. We exposed adult, male rats to a single, severe stressor and used a multimodal approach to characterize avoidance, startle, and fear-learning behavior, as well as oligodendrocyte and myelin content in multiple brain areas. We found that oligodendrocyte cell density and myelin content were correlated with behavioral outcomes in a region-specific manner. Specifically, stress-induced avoidance positively correlated with hippocampal dentate gyrus oligodendrocytes and myelin. Viral overexpression of the oligodendrogenic factor Olig1 in the dentate gyrus was sufficient to induce an anxiety-like behavioral phenotype. In contrast, contextual fear learning positively correlated with myelin in the amygdala and spatial processing regions of the hippocampus. In a group of trauma-exposed US veterans, T1-/T2-weighted magnetic resonance imaging estimates of hippocampal and amygdala myelin associated with symptom profiles in a region-specific manner that mirrored the findings in rats. These results demonstrate a species- independent relationship between region-specific, gray matter oligodendrocytes and myelin and differential behavioral phenotypes following traumatic stress exposure. This study suggests a novel mechanism for brain plasticity that underlies individual variance in sensitivity to traumatic stress.

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Year-round dynamics of amplicon sequence variant communities differ among eukaryotes, Mimiviridae, and prokaryotes in a coastal ecosystem Coastal seawater is the habitat of different microbial communities. These communities are affected by seasonal environmental changes and fluctuating nutrient availability, as well as competitive and cooperative interspecific interactions. In this work, we investigated the seasonal dynamics of communities of eukaryotes, a major group of double-stranded DNA viruses infecting eukaryotes (i.e. Mimiviridae), as well as prokaryotes in the Uranouchi Inlet, Kochi, Japan. We performed metabarcoding using ribosomal RNA genes and the Mimiviridae polB gene as marker genes in 43 seawater samples collected during 20 months. Communities characterized by the compositions of amplicon sequence variants (ASVs) showed synchronic seasonal cycles for eukaryotes, Mimiviridae, and prokaryotes. However, the community dynamics showed intriguing differences in several aspects such as the recovery rate after a year. We further show that the differences in the community dynamics can be explained by differences in the recurrence/persistence levels of individual ASVs among eukaryotes, Mimiviridae, and prokaryotes. Mimiviridae ASVs were less persistent than eukaryotic ASVs, and prokaryotic ASVs were the most persistent. We argue that the differences in the specificity of interactions (i.e. virus-eukaryote vs prokaryote-eukaryote) as well as the survival strategies are at the origin of the distinct community dynamics among eukaryotes, Mimiviridae, and prokaryotes.

One sentence summaryA one year observation of coastal microbial communities revealed similar but different community dynamics for eukaryotes, a group of large viruses, and prokaryotes.

sentences: How much pollen do solitary bee larvae consume? Or establishing realistic exposure estimates of solitary bee larvae via pollen for the use in risk assessment Bees foraging in agricultural habitats can be exposed to plant protection products. In order to limit the risk of adverse events to occur a robust risk assessment is needed, which requires reliable estimates for the expected exposure. Especially the exposure pathways to developing solitary bees are not well described and in the currently proposed form rely on limited information. To address this topic, we used a published data set on the volume of pollen solitary bees provide for their larvae to build two scaling models predicting the amount of protein and pollen developing solitary bees need based on adult body weight. We test our models using both literature and experimental data, which both support the validity of the presented models. Using scaling models in the bee risk assessment could complement existing risk assessment approaches, facilitate the further development of accurate risk characterization for solitary bees and ultimately will help to protect them during their foraging activity in agricultural settings.

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Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells BackgroundNonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge.

ResultsTo identify and analyze endogenous targets of NMD, we applied cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identified, most derive from alternative exon usage. The isoform-aware analysis revealed many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3'UTR and, for those mRNAs with a termination codon in the last exon, the length of the 3'UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, though the main function of NMD seems to be ridding the transcriptome of isoforms resulting from spurious splicing events.

ConclusionsLong-read sequencing enabled the identification of many novel NMD-sensitive mRNAs and revealed both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications.

sentences: Peeling Back the Layers: Complex Dynamics Shaping the Evolution of the Ledebouriinae (Scilloideae, Asparagaceae) The Ledebouriinae (Scilloideae, Asparagaceae) are a widespread group of bulbous geophytes found predominantly throughout seasonal climates in sub-Saharan Africa, with a handful of taxa in Madagascar, the Middle East, India, and Sri Lanka. Phylogenetic relationships within the group have been historically difficult to elucidate. Here, we provide the first phylogenomic perspective into the Ledebouriinae. Using the Angiosperms353 targeted enrichment probe set, we consistently recovered four major clades (i.e., two Ledebouria clades, Drimiopsis, and Resnova). The two Ledebouria clades closely align with geography, either consisting almost entirely of sub-Saharan African taxa (Ledebouria Clade A), or East African and non-African taxa (Ledebouria Clade B). Our results suggest that the Ledebouriinae likely underwent a rapid radiation leading to rampant incomplete lineage sorting. We additionally find evidence for potential historical hybridization between Drimiopsis and a subclade within Ledebouria Clade A.

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The anterior Hox gene ceh-13 and elt-1/GATA activate the posterior Hox genes nob-1 and php-3 to specify posterior lineages in the C. elegans embryo Hox transcription factors play a conserved role in specifying positional identity during animal development, with posterior Hox genes typically repressing the expression of more anterior Hox genes. Here, we dissect the regulation of the posterior Hox genes nob-1 and php-3 in the nematode C. elegans. We show that nob-1 and php-3 are co-expressed in gastrulation-stage embryos in cells that previously expressed the anterior Hox gene ceh-13. This expression is controlled by several partially redundant transcriptional enhancers. These enhancers require ceh-13 for expression, providing a striking example of an anterior Hox gene positively regulating a posterior Hox gene. Several other regulators also act positively through nob-1/php-3 enhancers, including elt-1/GATA, ceh-20/ceh-40/Pbx, unc-62/Meis, pop-1/TCF, ceh-36/Otx and unc-30/Pitx. We identified defects in both cell position and cell division patterns in ceh-13 and nob-1;php-3 mutants, suggesting that these factors regulate lineage identity in addition to positional identity. Together, our results highlight the complexity and flexibility of Hox gene regulation and function and the ability of developmental transcription factors to regulate different targets in different stages of development.

Author SummaryHox genes are critical for head-to-tail patterning during embryonic development in all animals. Here we examine the factors that are necessary to turn on two posterior Hox genes, nob-1 and php-3, in the nematode worm, C. elegans. We identified six new transcription factors and three enhancer regions of DNA that can activate expression of nob-1/php-3. Unexpectedly, these activating transcription factors included ceh-13, an anterior Hox gene, and elt-1, a regulator of skin development that is briefly expressed in many cells that do not adopt skin fates, including the cells that express nob-1. Furthermore, the cellular defects we observed in ceh-13 and nob- 1;php-3 mutant embryos indicate that the early embryonic functions of these Hox genes help determine the identity of cells as well as their position within the embryo. Our findings identify new roles for Hox genes in C. elegans and emphasize the ability of transcription factors to contribute to the diversification of cell types and the adoption of specific cell types at different phases of embryonic development.

sentences: A mutational hotspot that determines highly repeatable evolution can be built and broken by silent genetic changes Mutational hotspots can determine evolutionary outcomes and make evolution repeatable. Hotspots are products of multiple evolutionary forces including mutation rate heterogeneity, but this variable is often hard to identify. In this work we reveal that a powerfully deterministic genetic hotspot can be built and broken by a handful of silent mutations. We observed this when studying homologous immotile variants of the bacteria Pseudomonas fluorescens, AR2 and Pf0-2x. AR2 resurrects motility through highly repeatable de novo mutation of the same nucleotide in >95% lines in minimal media (ntrB A289C). Pf0-2x, however, evolves via a number of mutations meaning the two strains diverge significantly during adaptation. We determined that this evolutionary disparity was owed to just 6 synonymous variations within the ntrB locus, which we demonstrated by swapping the sites and observing that we were able to both break (>95% to 0% in AR2) and build (0% to 80% in Pf0-2x) a powerfully deterministic mutational hotspot. Our work reveals a fundamental role for silent genetic variation in determining adaptive outcomes.

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Biocatalytic Formation of Novel Polyesters with para-Hydroxyphenyl groups in the Backbone - Engineering Cupriavidus necator for production of high-performance materials from CO2 and electricity Synthetic materials are integral components of consumables and durable goods and indispensable in our modern world. Polyesters are the most versatile bulk- and specialty-polymers, but their production is not sustainable, and their fate at end-of-life of great concern. Bioplastics are highly regarded alternatives but have shortcomings in material properties and commercial competitiveness with conventional synthetic plastics. These constraints have limited the success in global markets. Enabling bio-production of advanced bioplastics with superior properties from waste-derived feedstocks could change this.

We have created microbial cell factories that can produce a range of aliphatic and aromatic polyesters. A {Delta}phaC1 mutant of Cupriavidus necator H16 was complemented with hydroxyacyl-CoA transferases from either Clostridium propionicum (pct540) or Clostridium difficile (hadA), respectively. These were combined with a mutant PHA synthase (phaC1437) from Pseudomonas sp. MBEL 6-19, which rescued the PHA- phenotype of the knock-out mutant and allowed polymerization of various hydroxy carboxylates, including phloretic acid. This is the first-time, incorporation of an aromatic ring in the backbone of a biological polyester was achieved. Polymers contain para-hydroxyphenyl subunits are structurally analogous to synthetic aromatic polyesters like PET and high-strength polyarylates.

In a further advance, the transgenic strain was cultivated in a bio-electrochemical system under autotrophic conditions, enabling synthesis of aromatic bio-polyesters from H2 and O2 generated in situ, while assimilating CO2. Follow-up elementary flux-mode analysis established the feasibility of de novo production of twenty different polyesters from five different carbon- and energy-sources. This comprehensive study opens the door to sustainable bio-production of high-performance thermoplastics and thermosets.

Significance statementNew biomaterials can facilitate transition to a carbon-neutral chemical industry and a circular economy while at the same time preventing accumulation of plastic wastes in the environment. This can be accomplished by developing "drop-in" replacements for existing fossil carbon-based plastics. To that end, this work demonstrates for the first time that biocatalytic polymerization can incorporate an aromatic para-hydroxyphenyl carbonic acid into the backbone of a bio-polyester. This was accomplished using a genetically engineered microbial cell factory that assimilates carbon dioxide using hydrogen that is produced electrochemically in situ. A bio-electrochemical system eliminates the need for external supply of oxyhydrogen and avoids explosive mixtures, opening the door for sustainable production of biomaterials analogous to aromatic bulk-polyesters such as PET and high-performance "liquid-crystal polymers".

Highlights- Biocatalytic formation of aromatic polyesters with structural analogy to polyarylates
- Production of novel PHAs from CO2 and H2+O2 produced in situ in a bio-electrochemical system
- Expression-level of PHA synthase and molecular weight of polyesters are inversely correlated
- In silico design and analysis of pathways towards novel PHAs from different carbon-sources

sentences: Factors regulating translation coupled mRNA degradation in yeast Codon optimality is a major factor governing translation speed and efficiency, mRNA expression, and stability. Presence of optimal codons maintains a steady translation time, while non-optimal codons can lead to slow peptidyl transfer, prolonged translation time and frequent halt. Owing to this, there are co-translational surveillance protein complexes which monitor this process meticulously and can trigger the release of the mRNA transcript after prolonged halt. The surveillance protein complexes are closely associated with various degradation enzymes which can break down these released transcripts. Degradation of the released transcripts will be ensured if they contain amenable regions for enzyme binding and action. Here, we have used sequence data to estimate the relative abundance of non-optimal codons within a transcript. We found that transcripts with high relative abundance of non-optimal codon show reduced stability and half-life compared to transcripts with lower relative abundance of non-optimal codon. We have further integrated the relative abundance of non-optimal codons with internal unstructured segments for the transcripts, to show that the former is responsible for transcript release and the latter provides susceptible platform for endonuclease cleavage, which together is capable of destabilising and reducing the transcripts half-life. Since this is a translational system, sequestration of mRNA into a dense forest of ribosomes is seen to prolong the transcripts half-life even in the presence of destabilising factors.

sentences: High-speed widefield handheld swept-source OCT angiography with a VCSEL light source Optical coherence tomography (OCT) and OCT angiography (OCTA) enable noninvasive structural and angiographic imaging of the eye. Portable handheld OCT/OCTA systems are required for imaging patients in the supine position. Examples include infants in the neonatal intensive care unit (NICU) and operating room (OR). The speed of image acquisition plays a pivotal role in acquiring high quality OCT/OCTA images, particularly with the handheld system, since both the operator hand tremor and subject motion can cause significant motion artifacts. In addition, having a large field of view and the ability of real-time data visualization are critical elements in rapid disease screening, reducing imaging time, and detecting peripheral retinal pathologies. The arrangement of optical components is less flexible in the handheld system due to the limitation of size and weight. In this paper, we introduce a 400-kHz, 55-degree field of view handheld OCT/OCTA system that has overcome many technical challenges as a portable OCT system as well as a high-speed OCTA system. We demonstrate imaging premature infants with retinopathy of prematurity (ROP) in the NICU, and patients with incontinentia pigmenti (IP) in the OR using our handheld OCT system. Our design may have potential for improving the diagnosis of retinal diseases and help provide a practical guideline for designing a flexible and portable OCT system.

sentences: NanoDam identifies novel temporal transcription factors conserved between the Drosophila central brain and visual system Temporal patterning of neural progenitors is an evolutionarily conserved strategy for generating neuronal diversity. Type II neural stem cells in the Drosophila central brain produce transit-amplifying intermediate neural progenitors (INPs) that exhibit temporal patterning. However, the known temporal factors cannot account for the neuronal diversity in the adult brain. To search for new temporal factors, we developed NanoDam, which enables rapid genome-wide profiling of endogenously-tagged proteins in vivo with a single genetic cross. Mapping the targets of known temporal transcription factors with NanoDam identified Homeobrain and Scarecrow (ARX and NKX2.1 orthologues) as novel temporal factors. We show that Homeobrain and Scarecrow define middle-aged and late INP temporal windows and play a role in cellular longevity. Strikingly, Homeobrain and Scarecrow have conserved functions as temporal factors in the developing visual system. NanoDam enables rapid cell type-specific genome-wide profiling with temporal resolution and can be easily adapted for use in higher organisms.

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Insights into the mutation T1117I in the spike and the lineage B.1.1.389 of SARS-CoV-2 circulating in Costa Rica Emerging mutations and genotypes of the SARS-CoV-2 virus, responsible for the COVID-19 pandemic, have been reported globally. In Costa Rica during the year 2020, a predominant genotype carrying the mutation T1117I in the spike (S:T1117I) was previously identified. To investigate the possible effects of this mutation on the function of the spike, i.e. the biology of the virus, different bioinformatic pipelines based on phylogeny, natural selection and co-evolutionary models, molecular docking and epitopes prediction were implemented.

Results of the phylogeny of sequences carrying the S:T1117I worldwide showed a polyphyletic group, with the emergency of local lineages. In Costa Rica, the mutation is found in the lineage B.1.1.389 and it is suggested to be a product of positive/adaptive selection. Different changes in the function of the spike protein and more stable interaction with a ligand (nelfinavir drug) were found. Only one epitope out 742 in the spike was affected by the mutation, with some different properties, but suggesting scarce changes in the immune response and no influence on the vaccine effectiveness.

Jointly, these results suggest a partial benefit of the mutation for the spread of the virus with this genotype during the year 2020 in Costa Rica, although possibly not strong enough with the introduction of new lineages during early 2021 which became predominant later. In addition, the bioinformatics pipeline offers an integrative and exhaustive in silico strategy to eventually study other mutations of interest for the SARS-CoV-2 virus and other pathogens.

HighlightsO_LIIn Costa Rica during the year 2020, a predominant SARS-CoV-2 genotype carrying the mutation T1117I in the spike (S:T1117I) was identified.
C_LIO_LIThe S:T1117I was assessed for possible effects of this mutation on the function of the spike with a in silico approach.
C_LIO_LIPhylogeny revealed that sequences carrying the S:T1117I worldwide define a polyphyletic group, with the emergency of local lineages, including the lineage B.1.1.389 in Costa Rica.
C_LIO_LIA positive/adaptive selection was identified for S:T1117I, with different changes in the function of the spike protein, more stable interaction with ligands and scarce changes in the immune response.
C_LIO_LIThe bioinformatics pipeline can be eventually used to study other mutations of the SARS-CoV-2 virus and other pathogens.
C_LI

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A meta-analysis of gRNA library screens enables an improved understanding of the impact of gRNA folding and structural stability on CRISPR-Cas9 activity. CRISPR systems are known to be inhibited by unwanted secondary structures that form within the guide RNA (gRNA). The minimum free energy of predicted secondary structures has been used in prediction algorithms. However, the types of structures as well as the degree to which a predicted structure can inhibit Cas9/gRNA activity is not well characterized. Here we perform a meta-analysis of published CRISPR-Cas9 datasets to better understand the role of secondary structures in inhibiting gRNA activity. We identify two inhibitory structures and provide estimated free energy cutoffs at which they become impactful. Further, we identify the prevalence of these structures in existing datasets. The cutoffs provided help to explain conflicting impacts of free energy values in different datasets as well as providing a guideline for future gRNA designs.

HighlightsO_LIClearly define two secondary structures that inhibit CRISPR-Cas9 activity
C_LIO_LIProvide free energy calculations and cutoffs at which each structure begins to inhibit activity
C_LIO_LIEvaluate impact of these structures in published datasets
C_LI

sentences: Higher-order olfactory neurons in the lateral horn supports odor valence and odor identity coding in Drosophila Understanding neuronal representations of odor-evoked activities and their progressive transformation from the sensory level to higher brain centers features one of the major aims in olfactory neuroscience. Here, we investigated how odor information is transformed and represented in higher-order neurons of the lateral horn, one of the higher olfactory centers implicated in determining innate behavior, using Drosophila melanogaster. We focused on a subset of third-order glutamatergic lateral horn neurons (LHNs) and characterized their odor coding properties in relation to their presynaptic partner neurons, the projection neurons (PNs) by two-photon functional imaging. We found that odors evoke reproducible, stereotypic and odor-specific response patterns in LHNs. Notably, odor-evoked responses in these neurons are valence-specific in a way that their response amplitude is positively correlated with innate odor preferences. We postulate that this valence-specific activity is the result of integrating inputs from multiple olfactory channels through second-order neurons. GRASP and micro-lesioning experiments provide evidence that glutamatergic LHNs obtain their major excitatory input from uniglomerular PNs, while they receive an odor-specific inhibition through inhibitory multiglomerular PNs. In summary, our study indicates that odor representations in glutamatergic LHNs encode hedonic valence and odor identity and primarily retain the odor coding properties of second-order neurons.

sentences: Ultrafast, alignment-free detection of repeat expansions in next-generation DNA and RNA sequencing data Short tandem repeat expansions are an established cause of diseases such as Huntingtons disease. Bioinformatic methods for detecting repeat expansions in short-read sequencing have revealed new repeat expansions in humans. Current bioinformatic methods to detect repeat expansions require alignment information to identify repetitive motif enrichment at genomic locations. We present superSTR, an ultrafast method that does not require alignment. We demonstrate superSTRs ability to efficiently process both whole-genome and whole-exome sequencing data. Using superSTR we perform the first analysis of the UK Biobank to efficiently screen the exomes of 49,953 biobank participants for repeat expansions. We identify known mutations, as well as diseases not previously associated with REs. We further demonstrate the first bioinformatic screening of RNA sequencing data to detect repeat expansions in patients with spinocerebellar ataxia and Fuchs endothelial corneal dystrophy, and mouse models of myotonic dystrophy. superSTR is a highly computationally-efficient repeat expansion tool screening and detection tool for genomewide novel repeat expansion analysis, significantly outperforming existing methods. superSTR is available from https://github.com/bahlolab/superSTR.

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Selection of software and database for metagenomics sequence analysis impacts the outcome of microbial profiling and pathogen detection AimShotgun metagenomic sequencing analysis is widely used for microbial profiling of biological specimens and pathogen detection. However, very little is known about the technical biases caused by the choice of analysis software and databases. In this study, we evaluated shotgun metagenomics taxonomical profiling software to characterize the microbial compositions of biological samples collected from wild rodents.

Method and ResultsUsing nine of the most widely used metagenomics software and four different databases, we analyzed shotgun metagenomic sequence data from three sets of wild rodent tissue samples. We demonstrated the discrepancies in results when different databases and software were used, which cause significant variation in microbial community characterizations. Our analysis also showed that these software differed in their ability to detect the presence of Leptospira, a major zoonotic pathogen of one health importance.

ConclusionsSignificant differences in compositional profiles for the same dataset while using different databases and software combinations can result in confounding biological conclusions in microbial profiling.

Significance and Impact of StudyThis study cautions that the selection of software and databases to analyze metagenomics data can influence the outcome and biological interpretation.

sentences: The efficiency of neutral honey as a tissue fixative in histopathology In the routine laboratory, 10% neutral buffered formalin (NBF) is the fixative of choice. However, formalin is a human carcinogen. To the best of our knowledge, neutral honey, not natural or artificial honey, has not been tested to fix histological tissues. This study aimed to examine the efficiency of neutral buffered honey and other types of honey fixatives to fix histological tissues. The most two natural common Omani honey were used as fixatives, namely Sumar and date. We tested samples of rat liver, kidney, and stomach. Nine types of fixatives were used. All tissues were treated equally. The evaluation was performed blindly by three senior biomedical scientists who work in a histopathology laboratory. Hematoxylin and eosin showed adequate staining in all groups when compared to 10% NBF. The intensity and specificity of Jones Methenamine silver stain in 10% Sumer and Date honey and 10% alcoholic Sumer honey showed similar findings of 10% NBF. The specificity and intensity of all groups for Periodic acid-Schiff were comparable with 10% neutral buffered formalin accepts for 10% Sumer honey and 10% Alcoholic Date honey. However, all honey groups showed weak staining for the reticulin fibers using Gordon and Sweets method. Vimentin showed comparable findings with 10% NBF as there were no significant differences. The findings of this study are promising. Further in depth research on honey as a possible safe substitute fixative for formalin should be conducted.

sentences: Plasmid co-infection: linking biological mechanisms to ecological and evolutionary dynamics As infectious agents of bacteria and vehicles of horizontal gene transfer, plasmids play a key role in bacterial ecology and evolution. Plasmid dynamics are shaped not only by plasmid-host interactions, but also by ecological interactions between plasmid variants. These interactions are complex: plasmids can co-infect the same host cell and the consequences for the co-resident plasmid can be either beneficial or detrimental. Many of the biological processes that govern plasmid co-infection-from systems to exclude infection by other plasmids to interactions in the regulation of plasmid copy number per cell-are well characterised at a mechanistic level. Modelling plays a central role in translating such mechanistic insights into predictions about plasmid dynamics, and in turn, the impact of these dynamics on bacterial evolution. Theoretical work in evolutionary epidemiology has shown that formulating models of co-infection is not trivial, as some modelling choices can introduce unintended ecological assumptions. Here, we review how the biological processes that govern co-infection can be represented in a mathematical model, discuss potential modelling pitfalls, and analyse this model to provide general insights into how co-infection impacts eco-evolutionary outcomes. In particular, we demonstrate how beneficial and detrimental effects of co-infection give rise to frequency-dependent selection.

sentences: Large-scale biophysically detailed model of somatosensory thalamocortical circuits in NetPyNE The primary somatosensory cortex (S1) of mammals is critically important in the perception of touch and related sensorimotor behaviors. In 2015 the Blue Brain Project developed a groundbreaking rat S1 microcircuit simulation with over 31,000 neurons with 207 morpho-electrical neuron types, and 37 million synapses, incorporating anatomical and physiological information from a wide range of experimental studies. We have implemented this highly-detailed and complex model S1 model in NetPyNE, using the data available in the Neocortical Microcircuit Collaboration Portal. NetPyNE provides a Python high-level interface to NEURON and allows defining complicated multiscale models using an intuitive declarative standardized language. It also facilitates running parallel simulations, automates the optimization and exploration of parameters using supercomputers, and provides a wide range of built-in analysis functions. This will make the S1 model more accessible and simpler to scale, modify and extend in order to explore research questions or interconnect to other existing models. Despite some implementation differences, the NetPyNE model closely reproduced the original cell morphologies, electrophysiological responses, spatial distribution for all 207 cell types; and the connectivity properties of the 1941 pathways, including synaptic dynamics and short-term plasticity (STP). The NetPyNE S1 simulations produced reasonable physiological rates and activity patterns across all populations, and, when STP was included, produced a 1 Hz oscillation comparable to that of the original model. We also extended the model by adding thalamic circuits, including 6 distinct thalamic populations with intrathalamic, thalamocortical and corticothalamic connectivity derived from experimental data. Overall, our work provides a widely accessible, data-driven and biophysically-detailed model of the somatosensory thalamocortical circuits that can be utilized as a community tool for researchers to study neural dynamics, function and disease.

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Neural oscillation coupling selectively predicts speech reception in young children with Autism Spectrum Disorder Communication difficulties in autism spectrum disorder (ASD) involve a speech reception deficit, whose biological causes are not yet identified. This deficit could denote atypical neuronal ensemble activity, as reflected by neural oscillations. Atypical cross-frequency oscillation coupling in particular could disrupt the possibility to jointly track and predict dynamic acoustic stimuli, a dual process that is essential for speech comprehension. Whether such oscillation anomalies can already be found in very young children with ASD, and with what specificity they relate to individual language reception capacity is unknown. In this study, neural activity was collected using EEG in 64 very young children with and without ASD (mean age 3) while they were exposed to naturalistic-continuous speech via an age-appropriate cartoon. EEG power typically associated with phrase-level chunking (delta, 1-3Hz), phonemic encoding (low-gamma, 25-35Hz) and top-down control (beta, 12-20Hz) was markedly reduced in ASD relative to typically developing (TD) children. Speech neural-tracking by delta and theta oscillations was also weaker in ASD than TD children. Critically, children with ASD exhibited slightly atypical theta/gamma coupling (PAC) involving a higher-than-normal gamma frequency, and markedly atypical beta/gamma PAC. Even though many oscillation features were atypical in our sample of 31 very young children with ASD, the beta/gamma coupling anomaly was the single best predictor of individual speech reception difficulties. These findings suggest that early interventions targeting the normalization of low-gamma and low-beta activity, might help young children with ASD to engage more in oral interactions.

HighlightsVery young children diagnosed with autism spectrum disorder already present marked alterations of neural oscillatory activity in response to natural speech.

The hierarchical processing of phonemic- and syllabic-range information (theta/gamma coupling) is atypical in young ASD children.

Speech reception deficit in children with ASD is indexed by abnormal bottom-up (low-gamma) and top-down (low-beta) coordination.

sentences: Phylogenetic context using phylogenetic outlines AO_SCPLOWBSTRACTC_SCPLOWMicrobial studies typically involve the sequencing and assembly of draft genomes for individual microbes or whole microbiomes. Given a draft genome, one first task is to determine its phylogenetic context, that is, to place it relative to the set of related reference genomes. We provide a new interactive graphical tool that addresses this task using Mash sketches to compare against all bacterial and archaeal representative genomes in the GTDB taxonomy, all within the framework of SplitsTree5. The phylogenetic context of the query sequences is then displayed as a phylogenetic outline, a new type of phylogenetic network that is more general that a phylogenetic tree, but significantly less complex than other types of phylogenetic networks. We propose to use such networks, rather than trees, to represent phylogenetic context, because they can express uncertainty in the placement of taxa, whereas a tree must always commit to a specific branching pattern. We illustrate the new method using a number of draft genomes of different assembly quality.

sentences: Pericyte-to-endothelial cell signaling via vitronectin-integrin regulates blood-CNS barrier Endothelial cells of blood vessels of the central nervous system (CNS) constitute blood-CNS barriers. Barrier properties are not intrinsic to these cells; rather they are induced and maintained by CNS microenvironment. Notably, the abluminal surface of CNS capillaries are ensheathed by pericytes and astrocytes. However, extrinsic factors from these perivascular cells that regulate barrier integrity are largely unknown. Here, we establish vitronectin, an extracellular-matrix protein secreted by CNS pericytes, as a regulator of blood-CNS barrier function via interactions with its integrin receptor, 5 in endothelial cells. Genetic ablation of vitronectin or mutating vitronectin to prevent integrin binding as well as endothelial-specific deletion of integrin 5 causes barrier leakage. Furthermore, vitronectin-integrin 5 signaling maintains barrier integrity by actively inhibiting transcytosis in endothelial cells. These results demonstrate that signaling from perivascular cells to endothelial cells via ligand-receptor interactions is a key mechanism to regulate barrier permeability.

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A Novel Model of Estrogen Receptor-Positive Breast Cancer Bone Metastasis with Antiestrogen Responsiveness Most women diagnosed with breast cancer (BC) have estrogen receptor alpha positive (ER+) disease. ER+ BC preferentially metastasizes to bone; at which time it is considered incurable. Treatments for bone metastasis have not advanced in decades, in part due to a lack of appropriate ER+ BC bone metastasis models. We developed an immunocompetent ER+ BC murine model with spontaneous bone metastasis and antiestrogen responsiveness. To do this, we transduced triple-negative (TN) bone-tropic murine BC cell lines 4T1.2 and E0771/Bone to express ER. These cells were then injected into the mammary fat pads of Balb/c (n=21) or C57Bl/6 (n=27), respectively. Once tumors established, mice were treated with either the selective estrogen receptor modulator (SERM) tamoxifen (TAM), the selective estrogen receptor degrader (SERD) ICI 182,780 (ICI, Faslodex, fulvestrant), or vehicle control for 21 days. Tumor volumes and weights significantly decreased in the ER+ groups treated with TAM and ICI compared with ER+ vehicle-treated groups. Staining for immune profiles and total RNA sequencing demonstrated modified immune cell infiltration between TN and ER-derived tumors. Approximately 25% of the mice with ER+ 4T1.2 tumors developed metastases to long bones while none of the mice with TN tumors developed metastases. This immunocompetent ER+ 4T1.2 BC model may allow for further exploration of ER+ BC bone metastasis mechanisms and for the development of new therapeutics for women diagnosed with bone metastasis from ER+ BC.

Simple SummaryEstrogen receptor alpha positive (ER+) breast cancer is the most common subtype of breast cancer. When it metastasizes to bone, it becomes incurable. Little advancement has occurred in the treatment of bone metastasis from ER+ breast cancer, partly due to the lack of animal models. To establish an animal model of ER+ BC, we genetically modified two triple-negative breast cancer cell lines to express ER and injected the cell lines into murine mammary glands. Mice were treated with standard antiestrogen therapies, the selective estrogen receptor modulator tamoxifen or the selective estrogen receptor degrader ICI 182,780. We found that compared to mice with triple-negative breast cancer, mice with ER+ breast cancer developed bone metastases and were responsive to antiestrogen therapy. This model allows for further exploration of bone metastasis mechanisms and for the development of new therapeutics, translating into improved clinical outcomes for women with bone metastasis from ER+ breast cancer.

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Recruitment of Peroxin14 to lipid droplets affects triglyceride storage in Drosophila The activity of multiple organelles must be coordinated to ensure cellular lipid homeostasis. This includes the peroxisomes which metabolise certain lipids and lipid droplets which act as neutral lipid storage centres. Direct organellar contact between peroxisomes and lipid droplets has been observed, and interaction between proteins associated with the membranes of these organelles has been shown, but the functional role of these interactions is not clear. In Drosophila cells, we identified a novel localization of a subset of three transmembrane Peroxin proteins (Peroxin3, Peroxin13, and Peroxin14), normally required for peroxisome biogenesis, to newly formed lipid droplets. This event was not linked to significant changes in peroxisome size or number, nor was recruitment of other Peroxin proteins or mature peroxisomes observed. The presence of these Peroxin proteins at lipid droplets influences their function as changes in the relative levels of Peroxin14 associated with the lipid droplet surface directly affected the presence of regulatory perilipin and lipases with corresponding effects on triglyceride storage.

Summary statementInteractions between peroxisomes and lipid droplets is thought to help coordinate management of cellular lipids. Peroxin proteins are required for peroxisome biogenesis. A spectrum of effects on triacylglyceride storage was seen when each of the 12 conserved Peroxins are knocked down in the Drosophila fat body with Peroxin14 knockdown having the largest effect. When Drosophila S2 cells were cultured in excess oleic acid, Peroxin3, Peroxin13, and Peroxin14, but not other Peroxins were localized to lipid droplets independently of other peroxisome markers. The presence of Peroxin14 at the lipid droplet surface altered recruitment of perilipin and lipase proteins.

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Anaerobic Benzene Biodegradation Linked to Growth of Highly Specific Bacterial Clades Reliance on bioremediation to remove benzene from anoxic environments has proven risky for decades but for unknown reasons. Years of research have revealed a strong link between anaerobic benzene biodegradation and the enrichment of highly specific microbes, namely Thermincola in the family Peptococcaceae and the deltaproteobacterial Candidate Sva0485 clade. Using aquifer material from Canadian Forces Base Borden, we compared five bioremediation approaches in batch microcosms. Under conditions simulating natural attenuation or sulfate biostimulation, benzene was not degraded after 1-2 years of incubation and no enrichment of known benzene-degrading microbes occurred. In contrast, nitrate-amended microcosms reported benzene biodegradation coincident with significant growth of Thermincola spp., along with a functional gene presumed to catalyze anaerobic benzene carboxylation (abcA). Inoculation with 2.5% of a methanogenic benzene-degrading consortium containing Sva0485 (Deltaproteobacteria ORM2) resulted in benzene biodegradation in the presence of sulfate or under methanogenic conditions. The presence of other hydrocarbon co-contaminants decreased rates of benzene degradation by a factor of 2-4. Tracking the abundance of the abcA gene and 16S rRNA genes specific for benzene-degrading Thermincola and Sva0485 is recommended to monitor benzene bioremediation in anoxic groundwater systems to further uncover growth rate limiting conditions for these two intriguing phylotypes.

SYNOPSISAnaerobic benzene biodegradation was accelerated by biostimulation with nitrate or by bioaugmentation under methanogenic or sulfate-reducing conditions.

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High Transcriptional Activity and Diverse Functional Repertoires of Hundreds of Giant Viruses in a Coastal Marine System Viruses belonging to the Nucleocytoviricota phylum are globally-distributed and include members with notably large genomes and complex functional repertoires. Recent studies have shown that these viruses are particularly diverse and abundant in marine systems, but the magnitude of actively replicating Nucleocytoviricota present in ocean habitats remains unclear. In this study, we compiled a curated database of 2,431 Nucleocytoviricota genomes and used it to examine the gene expression of these viruses in a 2.5-day metatranscriptomic time-series from surface waters of the California Current. We identified 145 viral genomes with high levels of gene expression, including 90 Imitervirales and 49 Algavirales viruses. In addition to recovering high expression of core genes involved in information processing that are commonly expressed during viral infection, we also identified transcripts of diverse viral metabolic genes from pathways such as glycolysis, the TCA cycle, and the pentose phosphate pathway, suggesting that virus-mediated reprogramming of central carbon metabolism is common in oceanic surface waters. Surprisingly, we also identified viral transcripts with homology to actin, myosin, and kinesin domains, suggesting that viruses may use them to manipulate host cytoskeletal dynamics during infection. We performed phylogenetic analysis on the virus-encoded myosin and kinesin proteins, which demonstrated that most belong to deep-branching viral clades, but that others appear to have been acquired from eukaryotes more recently. Our results highlight a remarkable diversity of active Nucleocytoviricota in a coastal marine system and underscore the complex functional repertoires expressed by these viruses during infection.

ImportanceThe discovery of giant viruses has transformed our understanding of viral complexity. Although viruses have traditionally been viewed as filterable infectious agents that lack metabolism, giant viruses can reach sizes rivalling cellular lineages and possess genomes encoding central metabolic processes. Recent studies have shown that giant viruses are widespread in aquatic systems, but the activity of these viruses and the extent to which they reprogram host physiology in situ remains unclear. Here we show that numerous giant viruses consistently express central metabolic enzymes in a coastal marine system, including components of glycolysis, the TCA cycle, and other pathways involved in nutrient homeostasis. Moreover, we found expression of several viral-encoded actin, myosin, and kinesin genes, indicating viral manipulation of the host cytoskeleton during infection. Our study reveals a high activity of giant viruses in a coastal marine system and indicates they are a diverse and underappreciated component of microbial diversity in the ocean.

sentences: Implicit Laplacian of Enhanced Edge (ILEE): An unguided quantitative cytoskeletal imaging algorithm The eukaryotic cytoskeleton plays essential roles in cell signaling and trafficking, which is broadly associated with immunity and diseases of human and plants. To date, most analyses aiming at defining the temporal and spatial dynamics of the cytoskeleton have relied on qualitative and quantitative analysis of fluorescence images to describe cytoskeletal function. While state-of-the-art, these approaches have limitations: the diverse shape and brightness of the cytoskeleton cause considerable inaccuracy in both human-driven and automated approaches, and the widely adopted image projection process (3D to 2D) leads to bias and information loss. Here, we describe the development and application of Implicit Laplacian of Enhanced Edge (ILEE), an unguided approach that uses a 2D/3D-compatible local thresholding algorithm for the quantitative evaluation of cytoskeletal status and organization at high performance. Using ILEE, we constructed a Python library to enable automated cytoskeletal image analysis, providing numerous biologically-interpretable indices measuring the density, bundling, severing, branching, and directionality of the cytoskeleton. The data presented herein demonstrate that ILEE resolves the defects of classic cytoskeleton analysis approaches, enables the measurement of novel cytoskeletal features, and yields quantitatively descriptive data with superior accuracy, stability, and robustness. We released the ILEE algorithm as an open-source library and further developed a Google Colab interface as a community resource.

sentences: HP1 oligomerization compensates for low-affinity H3K9me recognition and provides a tunable mechanism for heterochromatin-specific localization HP1 proteins bind with low affinity but high specificity to histone H3 lysine 9 methylation (H3K9me), forming transcriptionally inactive genomic compartments referred to as heterochromatin. How HP1 proteins traverse a complex and crowded chromatin landscape on the millisecond timescale and yet recognize H3K9me with high specificity remains paradoxical. Here, we visualize the single-molecule dynamics of an HP1 homolog, the fission yeast Swi6, in its native chromatin environment. By analyzing the motions of individual Swi6 molecules, we identify mobility states that map to discrete biochemical intermediates. Using mutants that perturb Swi6 H3K9me recognition, oligomerization, or nucleic acid binding, we parse the mechanism by which each biochemical property affects protein dynamics. We find that rather than enhancing chromatin binding, nucleic acid interactions, compete with and titrates Swi6 away from heterochromatin. However, as few as four tandem Swi6 chromodomains are necessary and sufficient to restore H3K9me-dependent localization. Our studies propose propose that HP1 oligomerization stabilizes higher-order protein configurations of a defined stoichiometry that facilitates high-specificity H3K9me recognition and outcompetes the inhibitory effects of nucleic acid-binding.

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Evidence for complex interplay between quorum sensing and antibiotic resistance in Pseudomonas aeruginosa Quorum sensing (QS) is a cell-density-dependent, intercellular communication system mediated by small diffusible signaling molecules. QS regulates a range of bacterial behaviors, including biofilm formation, virulence, drug resistance mechanisms, and antibiotic tolerance. Enzymes capable of degrading signaling molecules can interfere in QS - a process termed quorum quenching (QQ). Remarkably, previous work reported some cases where enzymatic interference in QS was synergistic to antibiotics against Pseudomonas aeruginosa. The premise of combination therapy is attractive to fight against multidrug-resistant bacteria, yet comprehensive studies are lacking. Here we evaluate the effects of QS signal disruption on the antibiotic resistance profile of P. aeruginosa by testing 222 antibiotics and antibacterial compounds from 15 different classes. We found compelling evidence that QS signal disruption does indeed affect antibiotic resistance (40% of all tested compounds; 89/222), albeit not always synergistically (not synergistic for 48% of compounds with an effect (43/89)). For some tested antibiotics, like sulfathiazole and trimethoprim, we were able to relate the changes in resistance caused by QS signal disruption to the modulation of the expression of key genes of the folate biosynthetic pathway. Moreover, using a P. aeruginosa-based Caenorhabditis elegans killing model, we confirm that enzymatic QQ modulates the effects of antibiotics on P. aeruginosas pathogenicity in vivo. Altogether, these results show that signal disruption has profound and complex effects on the antibiotic resistance profile of P. aeruginosa. This work suggests that combination therapy including QQ and antibiotics should not be discussed globally but rather in case-by-case studies.

IMPORTANCEInterference in bacterial Quorum Sensing (QS) is a promising approach to control microbial behavior. Of particular interest is the potential of this strategy to reduce biofilms and virulence of antibiotic resistant strains. Interestingly, several studies report synergistic interactions between antibiotic treatments and interference in QS. However, it is unclear whether this is a generality, let alone the molecular mechanisms underlying the observed synergies. Here, we provide a comprehensive description of combination treatment in the model organism, opportunistic human pathogen P. aeruginosa. Screening > 200 antimicrobials, and combining them to QS signals and disruption strategies, we show that there is no systematic synergy between these approaches in vitro, as well as in vivo, in a C. elegans infection model. Altogether, this work show that QS has complex connections to the antibiotic resistance profile of P. aeruginosa, and that combination treatment should not be discussed globally, but rather in case-by-case studies.

sentences: Production of recombinant human acid α-glucosidase with mannosidic N-glycans in α-mannosidase I mutant rice cell suspension culture High-mannose glycans, containing 6-9 mannose residues, are favorable for in vitro mannose phosphorylation to produce Mannose-6-phosphate (M6P) residues. These are important for lysosomal enzyme targeting and essential for uptake. Golgi -mannosidase-I mediates mannose trimming in the N-glycosylation pathway. In this study, I used mutant rice with a T-DNA insertion in the Os04g0606400 ({Delta}-manI) gene to produce recombinant human acid -glucosidase (rhGAA) for enzyme replacement therapy to treat Pompe disease. After characterization of {Delta}-manI mutant rice, the gene encoding rhGAA was introduced into mutant calli by Agrobacterium-mediated transformation. Integration of the target sequence into the rice genome was detected by genomic DNA PCR. Expression of rhGAA from {Delta}-manI mutant ({Delta}-manI-GAA) and its secretion into culture media were confirmed by western blot analysis. The N-glycosylation pattern of purified {Delta}-manI-GAA was analysed by MALDI-TOF mass spectrometry. This showed that the high-mannose type N-glycan with man8 (63.8%) was the most abundant form, followed by Man7 (22.5%), GnGnXF3 (6.7%), Man6 (5.4%), and Man5 (1.6%). The results suggest the successful production of rhGAA with N-glycan containing 6-8 mannose residue in the {Delta}-manI mutant rice cell suspension culture.

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A Retrotranslocation Assay that Predicts Defective VCP/p97-Mediated Trafficking of a Retroviral Signal Peptide Studies of viral replication have provided critical insights into host processes, including protein trafficking and turnover. Mouse mammary tumor virus (MMTV) is a betaretrovirus that encodes a functional 98-amino acid signal peptide (SP). MMTV SP is generated from both Rem and envelope precursor proteins by signal peptidase cleavage in the endoplasmic reticulum (ER) membrane. We previously showed that SP functions as an HIV-1 Rev-like protein that is dependent on the AAA ATPase VCP/p97 to subvert ER-associated degradation (ERAD). SP contains a nuclear/nucleolar localization sequence (NLS/NoLS) within the N-terminal 45 amino acids. To directly determine the SP regions needed for membrane extraction and trafficking, we developed a quantitative retrotranslocation assay with biotin acceptor peptide (BAP)-tagged SP proteins. Use of alanine substitution mutants of BAP-tagged MMTV SP in retrotranslocation assays revealed that mutation of amino acids 57 and 58 (M57-58) interfered with ER membrane extraction, whereas adjacent mutations did not. The M57-58 mutant also showed reduced interaction with VCP/p97 in co-immunoprecipitation experiments. Using transfection and reporter assays to measure activity of BAP-tagged proteins, both M57-58 and an adjacent mutant (M59-61) were functionally defective compared to wild-type SP. Confocal microscopy revealed defects in SP nuclear trafficking and abnormal localization of both M57-58 and M59-61. Furthermore, purified GST-tagged M57-58 and M59-61 demonstrated reduced ability to oligomerize compared to tagged wild-type SP. These experiments suggest that SP amino acids 57-58 are critical for VCP/p97 interaction and retrotranslocation, whereas residues 57-61 are critical for oligomerization and nuclear trafficking independent of the NLS/NoLS. Our results emphasize the complex host interactions with long signal peptides.

IMPORTANCEEndoplasmic reticulum-associated degradation (ERAD) is a form of cellular protein quality control that is manipulated by viruses, including the betaretrovirus, mouse mammary tumor virus (MMTV). MMTV-encoded signal peptide (SP) has been shown to interact with an essential ERAD factor, VCP/p97 ATPase, to mediate its extraction from the ER membrane, also known as retrotranslocation, for RNA-binding and nuclear function. In this manuscript, we developed a quantitative retrotranslocation assay that identified an SP substitution mutant, which is defective for VCP interaction as well as nuclear trafficking, oligomer formation, and function. An adjacent SP mutant was competent for retrotranslocation and VCP interaction, but shared the other defects. Our results revealed the requirement for VCP during SP trafficking and the complex cellular pathways used by long signal peptides.

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Photoprotection is regulated by light-independent CO2 availability Photosynthetic algae cope with suboptimal levels of light and CO2. In low CO2 and excess light, the green alga Chlamydomonas reinhardtii activates a CO2 Concentrating Mechanism (CCM) and photoprotection; the latter is mediated by LHCSR1/3 and PSBS. How light and CO2 signals converge to regulate photoprotective responses remains unclear. Here we show that excess light activates expression of photoprotection- and CCM-related genes and that depletion of CO2 drives these responses, even in total darkness. High CO2 levels, derived from respiration or impaired photosynthetic fixation, repress LHCSR3 and CCM genes while stabilizing the LHCSR1 protein. We also show that CIA5, which controls CCM genes, is a major regulator of photoprotection, elevating LHCSR3 and PSBS transcript accumulation while inhibiting LHCSR1 accumulation. Our work emphasizes the importance of CO2 in regulating photoprotection and the CCM, demonstrating that the impact of light on photoprotection is often indirect and reflects intracellular CO2 levels.

TeaserPhotoprotection- and CCM-related genes can be activated by changes in CO2 availability even in the absence of light.

sentences: PGP1 personal genome assembly - a hybrid assembly dataset using ONT's PromethION and PacBio's HiFi sequencing PGP1 is the first participant of Personal Genome Project. We present the PGP1s chromosome-scale genome assembly. It was constructed using 255 Gb ultra-long PromethION reads and 97 Gb short paired-end reads. For reducing base calling errors, we corrected PromethION reads using 72 Gb PacBio HiFi reads. 327 Gb Hi-C chromosomal mapping data were utilized to maximize the assemblys contiguity. PGP1s contig assembly was 3.01 Gb in length comprising of 4,234 contigs with an N50 value of 33.8 Mb. After scaffolding with Hi-C data and extensive manual curation, we obtained a chromosome-scale assembly that represents 3,880 scaffolds with an N50 value of 142 Mb. From the Merqury assessment, PGP1 assembly achieved a high QV score of Q45.45. For a gene annotation, we predicted 106,789 genes with a liftover from the Gencode 38 and an assembly of transcriptome data.

sentences: Repertoire of naturally acquired maternal antibodies transferred to infants for protection against shigellosis Shigella is the second leading cause of diarrheal diseases, accounting for >200,000 infections and >50,000 deaths in children under 5 years of age annually worldwide. The incidence of Shigella-induced diarrhea is relatively low during the first year of life and increases substantially, reaching its peak between 11 to 24 months of age. This epidemiological trend hints at an early protective immunity of maternal origin and an increase in disease incidence when maternally acquired immunity wanes. The magnitude, type, antigenic diversity, and antimicrobial activity of maternal antibodies transferred via placenta that can prevent shigellosis during early infancy are not known. To address this knowledge gap, Shigella-specific antibodies directed against the lipopolysaccharide (LPS) and virulence factors (IpaB, IpaC, IpaD, IpaH, and VirG), and antibody-mediated serum bactericidal (SBA) and opsonophagocytic killing antibody (OPKA) activity were measured in maternal and cord blood sera from a longitudinal cohort of mother-infant pairs living in rural Malawi. Protein-specific (very high levels) and Shigella LPS IgG were detected in maternal and cord blood sera; efficiency of placental transfer was 100% and 60%, respectively, and had preferential IgG subclass distribution (protein-specific IgG1 > LPS-specific IgG2). In contrast, SBA and OPKA activity in cord blood was substantially lower as compared to maternal serum and varied among Shigella serotypes. LPS was identified as the primary target of SBA and OPKA activity. Maternal sera had remarkably elevated Shigella flexneri 2a LPS IgM, indicative of recent exposure. Our study revealed a broad repertoire of maternally acquired antibodies in infants living in a Shigella-endemic region and highlights the abundance of protein-specific antibodies and their likely contribution to disease prevention during the first months of life. These results contribute new knowledge on maternal infant immunity and target antigens that can inform the development of vaccines or therapeutics that can extend protection after maternally transferred immunity wanes.

sentences: First record of Temnothorax convexus (Forel, 1894) in Portugal (Hymenoptera: Formicidae) with an updated checklist of ants from the country The arboreal ant Temnothorax convexus (Forel, 1894) is reported for the first time in Portugal. A single colony was found nesting inside an abandoned gall near Lisbon. A full morphometric evaluation of the specimens is provided, including a small sample from the recently discovered population in South Iberia. High-definition photographs of the worker caste are given, together with a comparison with the most similar species in Iberia and an update of the Portuguese ant checklist.

sentences: A Meta-analysis of the known Global Distribution and Host Range of the Ralstonia Species Complex The Ralstonia species complex is a genetically diverse group of plant wilt pathogens. Ralstonia strains are classified by a "phylotype-sequevar" phylogenetic system. Since the development of the phylotype-sequevar system, over one hundred papers have described the genetic diversity of Ralstonia strains isolated from agriculturally important crops, ornamental plants, and plants in natural ecosystems. Our goal is to create a database that contains the reported global distribution and host range of Ralstonia sequevars. In this third release, we have catalogued information from 93 manuscripts that report one or more Ralstonia strain isolated from 86 geographic regions. The database is hosted as a GitHub repository (https://github.com/lowepowerlab/Ralstonia_Global_Diversity) that will be updated regularly.

sentences: Competition and environmental gradients structure assemblages of Canidae across the planet The phylogenetic information of assemblages carries the signature of ecological and evolutionary processes that assembled these communities. Identifying the mechanisms that shape communities is not simple, as they can vary spatially. Here, we investigated how the phylogenetic structure of Canidae across the globe is affected by the environment and competition. We first identified phylogenetically clustered and overdispersed assemblages of canids over the planet taking into account regional pools of species in South and North America, Eurasia, and Africa. Then, we applied Structural Equation Models in these communities in order to identify the effect of temperature, vegetation cover, human impact, and body size dissimilarity on the spatial distribution of the phylogenetic information of canids. Only southern South America and the Middle East had strong phylogenetic clustered patterns, while the rest of the planet predominantly showed overdispersed assemblages. Body size dissimilarity and vegetation cover were the most important variables to explain the spatial patterns of phylogenetic structure in clustered and overdispersed communities, respectively. Canidae community composition across the world presents significant patterns of clustering and overdispersion, which vary following mainly the environmental gradient, suggesting habitat filtering as the main force acting on Canidae assemblages.

sentences: Structural basis for the peptidoglycan editing activity of YfiH Bacterial cells are encased in peptidoglycan (PG), a polymer of disaccharide N-acetyl-glucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) cross-linked by peptide stems. PG is synthesized in the cytoplasm as UDP-MurNAc-peptide precursors, of which the amino-acid composition of the peptide is unique, with L-Ala added at the first position in most bacteria but L-Ser or Gly in some bacteria. YfiH is a PG-editing factor whose absence causes misincorporation of L-Ser instead of L-Ala into peptide stems; but its mechanistic function is unknown. Here we report the crystal structures of substrate-bound and product-bound YfiH, showing that YfiH is a cytoplasmic amidase that controls the incorporation of the correct amino acid to the nucleotide precursors by preferentially cleaving the nucleotide precursor byproduct UDP-MurNAc-L-Ser. This work reveals an editing mechanism in the cytoplasmic steps of peptidoglycan biosynthesis.

sentences: Computational modeling of posteroanterior lumbar traction by an automated massage bed: predicting intervertebral disc stresses and deformation Spinal traction is a physical intervention that provides constant or intermittent stretching axial force to the lumbar vertebrae to gradually distract spinal tissues into better alignment, reduce intervertebral disc (IVD) pressure, and manage lower back pain (LBP). However, such axial traction may change the normal lordotic curvature, and result in unwanted side effects and/or inefficient reduction of the IVD pressure. An alternative to axial traction has been recently tested, consisting of posteroanterior (PA) traction in supine posture, which was recently shown effective to increase the intervertebral space and lordotic angle using MRI. PA traction aims to maintain the lumbar lordosis curvature throughout the spinal traction therapy while reducing the intradiscal pressure. In this study, we developed finite element simulations of mechanical therapy produced by a commercial thermo-mechanical massage bed capable of spinal PA traction. The stress relief produced on the lumbar discs by the posteroanterior traction system was investigated on human subject models with different BMI (normal, overweight, moderate obese and extreme obese BMI cases). We predict typical traction levels lead to significant distraction stresses in the lumbar discs, thus producing a stress relief by reducing the compression stresses normally experienced by these tissues. Also, the stress relief experienced by the lumbar discs was effective in all BMI models, and it was found maximal in the normal BMI model. These results are consistent with prior observations of therapeutic benefits derived from spinal AP traction.

sentences: Integrated Systems-Level Proteomics and Metabolomics Reveals the Diel Molecular Landscape of Diverse Kale Cultivars The diverse cultivars of Brassica oleracea and Brassica napus constitute an important global food source. Of those, the Brassica oleracea convar. acephala cultivar group containing var. sabellica and var. palmifolia and Brassica napus var. pabularia, collectively known as kale, are nutritious leafy greens consumed for their abundance of vitamins and micronutrients. Typified by their curly, serrated or wavy leaves, kale varieties have been primarily defined based on their leaf morphology and geographic origin, despite having complex genetic backgrounds. Kale is a very promising crop for vertical farming due to its high nutritional content. However, systematic analysis of kale growth and molecular composition under different light regimes suitable for vertical farming has been lacking. Hence, we selected nine diverse, publicly available and commercially grown kale cultivars for growth under light emitting diode (LED)-replicated sunlight-like conditions. We then analyzed their growth and nutrition using a combination of phenomics, proteomics and metabolomics. As plant growth and development are driven by the diel molecular activities of plants, we harvested kale leaf tissue at both end-of-day (ED) and end-of-night (EN) time-points for all molecular analyses. Our results reveal that diel metabolome and proteome signatures sub-divide the selected kale cultivars into two distinct groups, defined by their amino acid and sugar content, along with significant proteome differences involving carbon and nitrogen metabolism, mRNA splicing, protein translation and light harvesting. Together, our analysis has generated robust quantitative insights into the diel growth and development landscape of kale, significantly advancing our fundamental understanding of this nutritious leafy green for next-generation breeding and biotechnology.

sentences: Population genomics of Pocillopora corals: insights from RAD-sequencing Scleractinian corals are of great ecological interest as ecosystem engineer species. Accordingly, there is a wealth of studies on their adaptive abilities facing climate change. Such studies should rely on precise species and population delimitation. Nevertheless species delimitation in corals can be hindered by the lack of adequate genetic markers, by hybridization, and by morphological plasticity. Here we applied RAD sequencing to the study of species delimitation and genetic structure in populations of Pocillopora spp. from Oman and French Polynesia with the objectives to test primary species hypotheses based on mitochondrial DNA sequencing, and to study the genetic structure among sampling sites inside species. Regarding the varying levels of missing data observed among samples we tested different filtering strategy. The main genetic differentiation was observed between samples from Oman and French Polynesia, which also corresponded to different mitochondrial lineages and species hypotheses. In Oman, we did not observe any clear differentiation according to the main mitochondrial lineages considered here, nor between sampling sites. In French Polynesia where a single mitochondrial lineage was studied, we did not evidence any differentiation according to sampling sites. These results provide an additional example of the importance of using independent nuclear markers for the study of species delimitation. Our analyses also allowed the identification of clonal lineages in our samples, and to take them into account in our interpretations. We used simulations to study the impact of clonal reproduction on the distribution of statistics of genetic diversity and genetic structure among loci.

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Artificial intelligence reveals nuclear pore complexity Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport. Their intricate 120 MDa architecture remains incompletely understood. Here, we report a near-complete structural model of the human NPC scaffold with explicit membrane and in multiple conformational states. We combined AI-based structure prediction with in situ and in cellulo cryo-electron tomography and integrative modeling. We show that linker Nups spatially organize the scaffold within and across subcomplexes to establish the higher-order structure. Microsecond-long molecular dynamics simulations suggest that the scaffold is not required to stabilize the inner and outer nuclear membrane fusion, but rather widens the central pore. Our work exemplifies how AI-based modeling can be integrated with in situ structural biology to understand subcellular architecture across spatial organization levels.

One sentence summaryAn AI-based, dynamic model of the human nuclear pore complex reveals how the protein scaffold and the nuclear envelope are coupled inside cells.

sentences: Dissecting CD8+ T cell pathology of severe SARS-CoV-2 infection by single-cell epitope mapping The current COVID-19 pandemic represents a global challenge. A better understanding of the immune response against SARS-CoV-2 is key to unveil the differences in disease severity and to develop future vaccines targeting novel SARS-CoV-2 variants. Feature barcode technology combined with CITE-seq antibodies and DNA-barcoded peptide-MHC I Dextramer reagents enabled us to identify relevant SARS-CoV-2-derived epitopes and compare epitope-specific CD8+ T cell populations between mild and severe COVID-19. We identified a strong CD8+ T cell response against an S protein-derived epitope. CD8+ effector cells in severe COVID-19 displayed hyperactivation, T cell exhaustion and were missing characteristics of long-lived memory T cells. We identify A*0101 WTAGAAAYY as an immunogenic CD8+ T cell epitope with the ability to drive clonal expansion. We provide an in-depth characterization of the CD8+ T cell-mediated response to SARS-CoV-2 infection which will be relevant for the development of molecular and targeted therapies and potential adjustments of vaccination strategies.

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The dynamics of cell-free DNA from urine and blood after a full marathon PurposeCell-free DNA (cfDNA) has been investigated as a minimally invasive biomarker for many diseases, particularly cancer. An increase in cfDNA has been observed during exercise. Neutrophil extracellular traps (NETs) may be the origin of cfDNA in response to acute exercise, but the mechanisms of generation of cfDNA during exercise remain unclear. In this study we investigated the dynamics of serum and urinary cfDNA levels and determined the relevance of other biomarkers to serum and urinary cfDNA levels and fragment size after a full marathon.

MethodsSamples were collected from 23 healthy male subjects. Blood and urine samples were collected before and immediately, two hours, and one day after the full marathon. The measurements included serum and urinary cfDNA, creatine kinase, myoglobin, creatinine, white blood cells, platelets, and lactoferrin from blood, and amylase, albumin, and creatinine from urine.

ResultsSerum and urinary cfDNA levels increased after a full marathon. Creatine kinase, myoglobin, and creatinine in blood, and albumin and creatinine in urine also increased significantly after a full marathon. Serum cfDNA showed peak values about 180 bp after the full marathon. Values over 1000 bp were present at two hours post-marathon. Urinary cfDNA showed peak values from 35 bp to 50 bp after the full marathon. Values over 1000 bp appeared at Immediately and two hours post marathon.

ConclusionThis study revealed that both serum and urinary cfDNA levels transiently increased after a full marathon. In addition, these cfDNA fragment varied in size.

sentences: In vivo single-cell profiling of lncRNAs during Ebola virus infection Long non-coding RNAs (lncRNAs) are pivotal mediators of systemic immune response to viral infection, yet most studies concerning their expression and functions upon immune stimulation are limited to in vitro bulk cell populations. This strongly constrains our understanding of how lncRNA expression varies at single-cell resolution, and how their cell-type specific immune regulatory roles may differ compared to protein-coding genes. Here, we perform the first in-depth characterization of lncRNA expression variation at single-cell resolution during Ebola virus (EBOV) infection in vivo. Using bulk RNA-sequencing from 119 samples and 12 tissue types, we significantly expand the current macaque lncRNA annotation. We then profile lncRNA expression variation in immune circulating single-cells during EBOV infection and find that lncRNAs expression in fewer cells is a major differentiating factor from their protein-coding gene counterparts. Upon EBOV infection, lncRNAs present dynamic and mostly cell-type specific changes in their expression profiles especially in monocytes, the main cell type targeted by EBOV. Such changes are associated with gene regulatory modules related to important innate immune responses such as interferon response and purine metabolism. Within infected cells, several lncRNAs have positively and negatively correlated expression with viral load, suggesting that expression of some of these lncRNAs might be directly hijacked by EBOV to attack host cells. This study provides novel insights into the roles that lncRNAs play in the host response to acute viral infection and paves the way for future lncRNA studies at single-cell resolution.

sentences: The CRISPR-Cas System differentially regulates surface-attached and pellicle-biofilm in Salmonella enterica serovar Typhimurium CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) system has been studied for its role in biofilm regulation and expression of outer membrane proteins in Salmonella. We investigated the CRISPR-Cas mediated biofilm regulation in Salmonella enterica serovar Typhimurium by deleting CRISPR-Cas components,{Delta} crisprI,{Delta} crisprII, {Delta}{Delta}crisprI crisprII, and{Delta} cas op. We determined that the system positively regulates surface-biofilm while inhibiting pellicle-biofilm. In knockout strains, flagellar (fliC, flgK) and curli (csgA) genes were repressed, causing reduced surface-biofilm. Conversely, they displayed altered pellicle-biofilm architecture possessing bacterial multilayers and a denser ECM with enhanced cellulose and lesser Curli, ergo weaker pellicle. The intracellular cellulose concentration was less in the knockout strains due to upregulation of bcsC, necessary for cellulose export. We hypothesized that exported cellulose integrates into the pellicle. We determined that crp is upregulated in the knockout strains, thereby inhibiting the expression of csgD, hence csgA and bcsA. The conflicting upregulation of bcsC, the last gene of bcsABZC operon, could be independently regulated by the CRISPR-Cas system owing to a partial match between the CRISPR-spacers and bcsC gene. The CRP-mediated regulation of the flagellar genes in the knockout strains was probably circumvented through the regulation of Yddx governing the availability of {sigma}28 factor that further regulates class3 flagellar genes (fliC, flgK). Additionally, the variations in the LPS profile and expression of LPS-related genes (rfaC, rfbG, rfbI) in the knockout strains could also contribute to the altered pellicle architecture. Collectively, we establish that the CRISPR-Cas system differentially regulates the formation of surface-attached and pellicle-biofilm.

sentences: Identification of 370 genetic loci for age at first sex and birth linked to externalising behaviour Age at first sexual intercourse (AFS) and age at first birth (AFB) have implications for health and evolutionary fitness. In the largest genome-wide association study to date (AFS, N=387,338; AFB, N=542,901), we identify 370 independent signals, 11 sex-specific, with a 5-6% polygenic score (PGS) prediction. Heritability of AFB shifted from 9% [CI=4-14] for women born in 1940 to 22% [CI=19-25] in 1965. Signals are driven by the genetics of reproductive biology and externalising behaviour, with key genes related to follicle stimulating hormone (FSHB), implantation (ESR1), infertility, and spermatid differentiation. Polycystic Ovarian Syndrome leads to later AFB, linking with infertility. Late AFB is protective against later-life disease and associated with parental longevity. Higher childhood socioeconomic circumstances and those in the highest PGS decile (90%+) experience markedly later reproductive onset. Results are relevant for improving teenage and late-life health, for understanding longevity, and guiding experimentation into mechanisms of infertility.

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Intrinsic Network Activity Reflects the Fluctuating Experience of Tonic Pain Although we know sensation is continuous, research on long-lasting and continuously changing stimuli is scarce and the dynamic nature of ongoing cortical processing is largely neglected.

In a longitudinal study with 152 fMRI sessions, participants were asked to continuously rate the intensity of applied tonic heat pain for 20 minutes. Using group independent component analysis and dual-regression, we extracted the subjects time courses of intrinsic network activity. The relationship between the dynamic fluctuation of network activity with the varying time courses of three pain processing entities was computed: pain intensity, the direction of pain intensity changes and temperature.

We were able to dissociate the spatio-temporal patterns of objective (temperature) and subjective (pain intensity/changes of pain intensity) aspects of pain processing in the human brain. We found two somatosensory networks with distinct functions: one network which encodes the small fluctuations in temperature and consists mainly of bilateral SI. A second right-lateralised network that encodes the intensity of the subjective experience of pain consists of SI, SII, the PCC, and the thalamus.

We revealed the somatosensory dynamics that build up towards a current subjective percept of pain. The timing suggests a cascade of subsequent processing steps towards the current pain percept.

sentences: Validation and Application of a Protocol for the Extraction and Quantitative Analysis of Sphingomyelin in Erythrocyte Membranes of Patients with NAFLD A set of constituents of the erythrocyte membrane lipidome has been proposed to serve as biomarkers for liver disease and acute coronary syndrome. In erythrocytes, sphingomyelin hydrolysis provides ceramide, a signaling lipid necessary for phosphatidylserine exposure and eryptosis. Phosphatidylserine exposure further amplifies hepatic inflammation and fibrosis during non-alcoholic fatty liver disease (NAFLD). In this study, we developed and applied a quantitative TLC for erythrocyte membrane sphingomyelin of NAFLD patients. We also compared 10 extraction methods for the isolation of sphingomyelin from erythrocytes. For quantitative TLC, lipids were separated in Silica gel 60 F254 using a mixture of chloroform/methanol/acetic acid/water (60/50/1/4) (v/v/v/v). The separated lipids were stained in a chamber containing iodine, and the intensity of each of the primary colors (red, green, blue) and the sum of the Red plus Green colors (R+G) was analyzed. The method was linear over a wide range of concentrations, presented acceptable precision (inter-day CV(%) 0.34, 0.006 and 0.44 for 2.5, 5.0 and 10 g, respectively), good accuracy (recovery range 85.2-97.1%), and excellent limit of detection (0.137 g/spot) and limit of quantification (0.41 g/spot). Using this quantitation method, we compared various lipid extraction methods and found that lipid extraction with methanol led to higher yield of erythrocyte sphingomyelin (135.35{+/-}1.04% recovery, compared to the Folch method). Application of these methods showed that erythrocytes from NAFLD patients (9 men, 15 women, 57.95{+/-}11.11 years old) contained statistically significantly less sphingomyelin (829.82{+/-}511.60 vs 1892.08{+/-}606.25 g/ml of packed erythrocytes) compared to healthy controls (4 men, 6 women, 39.3{+/-}15.55 years old).

sentences: System Environmental Metrics Collector for EM facilities Recent developments in cryo-electron microscopy (cryoEM) have led to the routine determination of structures at near atomic resolution and greatly increased the number of biomedical researchers wanting access to high-end cryoEM instrumentation. The high costs and long wait times for gaining access encourages facilities to maximize instrument uptime for data collection. To support these goals, we developed a System Environmental Metrics Collector for facilities (SEMCf) that serves as a laboratory performance and management tool. SEMCf consists of an architecture of automated and robust sensors that track, organize and report key facility metrics. The individual sensors are connected to Raspberry Pi (RPi) single board computers installed in close proximity to the input metrics being measured. The system is controlled by a central server that may be installed on a RPi or existing microscope support PC. Tracking the system and the environment provides feedback of imminent issues, suggestions for interventions that are needed to optimize data production, and indications as to when preventative maintenance should be scheduled. The sensor components are relatively inexpensive and widely commercially available, and the open-source design and software enables straightforward implementation, customization, and optimization by any facility that would benefit from real time environmental monitoring and reporting.

sentences: Identifying developing interneurons as a potential target for multiple genetic autism risk factors in human and rodent forebrain. Autism spectrum condition or  autism is associated with numerous monogenic and polygenic genetic risk factors including the polygenic 16p11.2 microdeletion. A central question is what neural cells are affected. To systematically investigate we analysed single cell transcriptomes from gestational week (GW) 8-26 human foetal prefrontal cortex and identified a subset of interneurons (INs) first appearing at GW23 with enriched expression of a disproportionately large fraction of risk factor transcripts. This suggests the hypothesis that these INs are disproportionately vulnerable to mutations causing autism. We investigated this in a rat model of the 16p11.2 microdeletion. We found no change in the numbers or position of either excitatory or inhibitory neurons in the somatosensory cortex or CA1 of 16p11.2+/- rats but found that CA1 Sst INs were hyperexcitable with an enlarged axon initial segment, which was not the case for CA1 pyramidal cells. This study prompts deeper investigation of IN development as a convergent target for autism genetic risk factors.

sentences: Distinct natural syllable-selective neuronal ensembles in the primary auditory cortex of awake marmosets Marmosets are highly social non-human primates living in families. They exhibit rich vocalization, but neural basis underlying complex vocal communication is largely unknown. Here we report the existence of specific neuronal ensembles in marmoset A1 that respond selectively to distinct monosyllable or disyllable calls made by conspecific marmosets. These neurons were spatially dispersed within A1 but distinct from those responsive to pure tones. Syllable-selective responses were markedly diminished when individual domains of the syllable were deleted or the domain sequence was altered, indicating the importance of global rather than local spectral-temporal properties of the sound. Disyllable-selective responses also disappeared when the sequence of the two monosyllable components was reversed or their interval was extended beyond 1 second. Light anesthesia largely abolished disyllable-selective responses. Our findings demonstrate extensive inhibitory and facilitory interactions among syllable-evoked responses, and provide the basis for further study of circuit mechanisms underlying vocal communication in awake non-human primates.

sentences: Investigating the antioxidant effects of Aloe vera: Possible role in regulating lipid profile and liver function in high fat and fructose diet (HFFD) fed mice. Fat rich diets are believed to induce obesity and contributes to the development of diabetes and cardiovascular disease while high fructose diet was reported to increase gut surface area and enhance nutrient uptake resulting in weight gain. The study investigate the role of Aloe vera supplementation on lipid profiles, oxidative stress as well as liver and hear histology in high fat and fructose diet fed mice. Twenty mice were distributed into four groups (n=5). The groups received regular diet, high fat and fructose died (HFFD), HFFD plus 10% Aloe vera (HFFD+AV1) and HFFD plus 20% Aloe vera (HFFD+AV2) respectively for 10 weeks. The cholesterol level of HFFD+AV treated mice were significantly lower compared to HFFD treated mice. The ALT level was significantly increased in HFFD treated mice relative to the control. Aloe vera significantly improve albumin level as well as Catalase and superoxide dismutase activities of HFFD treated mice. The liver tissues of control and HFFD+AV2 treated mice showed normal hepatocytes. The study suggest that Aloe vera supplementation could protect against HFFD induced oxidative stress and hyperlipidemia. These findings might be used for further research on food supplementation for the control of metabolic disorders.

sentences: Molecular basis of unidirectional information transmission in two-component systems: lessons from the DesK-DesR thermosensor Cellular signaling systems transmit information over long distances using allosteric transitions and/or post-translational modifications. In two-component systems the sensor histidine kinase and response regulator are wired through phosphoryl-transfer reactions, using either a uni- or bi-directional transmission mode, allowing to build rich regulatory networks. Using the thermosensor DesK-DesR two-component system from Bacillus subtilis and combining crystal structures, QM/MM calculations and integrative kinetic modeling, we uncover that: i) longer or shorter distances between the phosphoryl-acceptor and -donor residues can shift the phosphoryl-transfer equilibrium; ii) the phosphorylation-dependent dimerization of the regulator acts as a sequestering mechanism by preventing the interaction with the histidine kinase; and iii) the kinases intrinsic conformational equilibrium makes the phosphotransferase state unlikely in the absence of histidine phosphorylation, minimizing backwards transmission. These mechanisms allow the system to control the direction of signal transmission in a very efficient way, showcasing the key role that structure-encoded allostery plays in signaling proteins to store and transmit information.

sentences: Molecular evolution and structural analyses of the spike glycoprotein from Brazilian SARS-CoV-2 genomes: the impact of the fixation of selected mutations The COVID-19 pandemic caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached by July 2021 almost 200 million cases and more than 4 million deaths worldwide since its beginning in late 2019, leading to enhanced concern in the scientific community and the general population. One of the most important pieces of this host-pathogen interaction is the spike protein, which binds to the human Angiotensin-converting enzyme 2 (hACE2) cell receptor, mediates the membrane fusion and is the major target of neutralizing antibodies against SARS-CoV-2. The multiple amino acid substitutions observed in this region, specially in the Receptor Binding Domain (RBD), mainly after almost one year of its emergence (late 2020), have enhanced the hACE2 binding affinity and led to several modifications in the mechanisms of SARS-CoV-2 pathogenesis, improving the viral fitness and/or promoting immune evasion, with potential impact in the vaccine development. In this way, the present work aimed to evaluate the effect of positively selected mutations fixed in the Brazilian SARS-CoV-2 lineages and to check for mutational evidence of coevolution. Additionally, we evaluated the impact of selected mutations identified in some of the VOC and VOI lineages (C.37, B.1.1.7, P.1, and P.2) of Brazilian samples on the structural stability of the spike protein, as well as their possible association with more aggressive infection profiles by estimating the binding affinity in the RBD-hACE2 complex. We identified 48 sites under selective pressure in Brazilian spike sequences, 17 of them with the strongest evidence by the HyPhy tests, including VOC related mutation sites 138, 142, 222, 262, 484, 681, and 845, among others. The coevolutionary analysis identified a number of 28 coevolving sites that were found not to be conditionally independent, such as the couple E484K - N501Y from P.1 and B.1.351 lineages. Finally, the molecular dynamics and free energy estimates showed the structural stabilizing effect and the higher impact of E484K for the improvement of the binding affinity between the spike RBD and the hACE2 in P.1 and P.2 lineages, as well as the stabilizing and destabilizing effects for the positively selected sites.

sentences: Incertohypothalamic A13 dopaminergic neurons are involved in fine forelimb movements but not reward Tyrosine hydroxylase (TH)-containing neurons of the dopamine (DA) cell group A13 are well-positioned to impact known dopamine-related functions since their descending projections innervate target regions that regulate vigilance, sensorimotor integration and execution. Despite this known connectivity little is known regarding the functionality of A13-DA circuits. Using TH-specific loss-of-function methodology and techniques to monitor population activity in transgenic rats in vivo we investigated the contribution of A13-DA neurons in reward and movement-related actions. Our work demonstrates a role for A13-DA neurons in grasping and handling of objects that is independent from reward. A13-DA neurons respond strongly when animals grab and manipulate food items while their inactivation or degeneration prevents animals from successfully doing so - a deficit partially attributed to a reduction in grip strength. In contrast, there was no relation between A13-DA activity and food-seeking behavior when animals were tested on a reward-based task that did not include a reaching/grasping response. Moreover, motivation for food was unaffected as goal-directed behavior for food items was in general intact following A13 neuronal inactivation/degeneration. These results demonstrate a functional role for A13-DA neurons in prehensile actions that are uncoupled from reward and as such position A13-DA neurons into the functional framework regarding centrally-located DA populations and their ability to coordinate movement.

sentences: The role of the first transmembrane helix in bacterial YidC: Insights from the crystal structure and molecular dynamics simulations The evolutionary conserved YidC is a unique dual-function protein that adopts insertase and chaperone conformations. The TM1 helix of Escherichia coli YidC mediates the interaction between the YidC chaperone and the Sec translocon. Here, we report the first crystal structure of Thermotoga maritima YidC (TmYidC) including the TM1 helix (PDB ID: 6Y86). The TM1 helix lies on the periplasmic side of the bilayer forming an angle of about 15 degrees with the membrane surface. Our functional studies suggest a role of TM1 for the species-specific interaction with the Sec translocon. The reconstitution data and the superimposition of TmYidC with known YidC structures suggest an active insertase conformation for YidC. Molecular dynamics (MD) simulations of TmYidC provide evidence that the TM1 helix acts as a membrane anchor for the YidC insertase and highlight the flexibility of the C1 region underlining its ability to switch between insertase and chaperone conformations. A structure-based model is proposed to rationalize how YidC performs the insertase and chaperone functions by re-positioning of the TM1 helix and the other structural elements.

sentences: Transcriptome-wide Association Study and eQTL colocalization identify potentially causal genes responsible for bone mineral density GWAS associations Genome-wide association studies (GWASs) for bone mineral density (BMD) have identified over 1,100 associations to date. However, identifying causal genes implicated by such studies has been challenging. Recent advances in the development of transcriptome reference datasets and computational approaches such as transcriptome-wide association studies (TWASs) and expression quantitative trait loci (eQTL) colocalization have proven to be informative in identifying putatively causal genes underlying GWAS associations. Here, we used TWAS/eQTL colocalization in conjunction with transcriptomic data from the Genotype-Tissue Expression (GTEx) project to identify potentially causal genes for the largest BMD GWAS performed to date. Using this approach, we identified 512 genes as significant (Bonferroni <= 0.05) using both TWAS and eQTL colocalization. This set of genes was enriched for regulators of BMD and members of bone relevant biological processes. To investigate the significance of our findings, we selected PPP6R3, the gene with the strongest support from our analysis which was not previously implicated in the regulation of BMD, for further investigation. We observed that Ppp6r3 deletion in mice decreased BMD. In this work, we provide an updated resource of putatively causal BMD genes and demonstrate that PPP6R3 is a putatively causal BMD GWAS gene. These data increase our understanding of the genetics of BMD and provide further evidence for the utility of combined TWAS/colocalization approaches in untangling the genetics of complex traits.

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Proteostatic regulation of caveolins avoids premature oligomerisation and preserves ER homeostasis Caveolin-1 (CAV1) and CAV3 are membrane sculpting proteins driving formation of plasma membrane caveolae. Caveola formation is unique as it requires oligomerisation of newly synthesised caveolins through the biosynthetic-secretory pathway. Here, we combine structural, biochemical, and microscopy analyses to examine the early proteostasis of caveolin family members and mutants. We describe striking trafficking differences between newly synthesised caveolins, with CAV1 rapidly exported to the Golgi but CAV3 showing ER retention and targeting to lipid droplets. Only monomeric/low oligomeric caveolins are efficiently exported from the ER, with oligomers assembling beyond the cis-Golgi and disease-causing mutations leading to detrimental non-functional complexes. Caveolins in the ER are maintained at low levels by active proteasomal degradation, avoiding premature oligomerisation and ER stress. Increasing lipid availability, cholesterol for CAV1 and fatty acids for CAV3, enhances trafficking and reduces proteasomal degradation. In conclusion, we identify proteostatic mechanisms that modulate stability and trafficking of newly synthesised caveolins, protecting cells against ER stress but perturbed in caveolin-related disease.

SummaryUnderstanding the unique proteostasis of caveolins has important implications for cell biology and physiopathology. Combining structural, microscopy, and biochemical analyses, we uncover new insights into the mechanisms that differentiate the early biosynthetic steps of caveolin family members, isoforms, and pathogenic mutants.

sentences: The CPEB3 ribozyme modulates hippocampal-dependent memory A self-cleaving ribozyme mapping to an intron of the cytoplasmic polyadenylation element binding protein 3 (CPEB3) gene has been suggested to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. The ribozymes self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the CPEB3 mRNA. Here we report that the murine ribozyme modulates its own host mRNA maturation in both cultured cortical neurons and the hippocampus. Inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory. These findings reveal a previously unknown role for self-cleaving ribozyme activity in regulating experience-induced co-transcriptional and local translational processes required for learning and memory.

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Sabatier principle for rationalizing enzymatic hydrolysis of a synthetic polyester Interfacial enzyme reactions are common in nature and in industrial settings, including the enzymatic deconstruction of poly(ethylene terephthalate) (PET) waste. Kinetic descriptions of PET hydrolases are necessary for both comparative analyses, discussions of structure-function relations and rational optimization of technical processes. We investigated whether the Sabatier principle could be used for this purpose. Specifically, we compared the kinetics of two well-known PET hydrolases, leaf-branch compost cutinase (LCC) and a cutinase from the bacterium T. fusca (TfC) when adding different concentrations of the surfactant cetyltrimethylammonium bromide (CTAB). We found that CTAB consistently lowered the strength of enzyme-PET interactions, while its effect on enzymatic turnover was strongly biphasic. Thus, at gradually increasing CTAB concentrations, turnover was initially promoted and subsequently suppressed. This correlation with maximal turnover at an intermediate binding strength is in accordance with the Sabatier principle. One consequence of these results is that both enzymes had too strong intrinsic interaction with PET for optimal turnover, especially TfC, which showed a 20-fold improvement of kcat at the maximum. LCC on the other hand had an intrinsic substrate affinity closer to the Sabatier optimum and the turnover rate was 5-fold improved at weakened substrate binding. Our results show that the Sabatier principle may indeed rationalize enzymatic PET degradation and support process optimization. Finally, we suggest that future discovery efforts should consider enzymes with weakened substrate binding, since strong adsorption seems to limit their catalytic performance.

ToC graphics O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: Structural Basis for Mis18 Complex Assembly: Implications for Centromere Maintenance The centromere, defined by the enrichment of CENP-A (a Histone H3 variant)-containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. During each round of the cell cycle, CENP-A levels undergo DNA replication-mediated dilution. To maintain centromere identity, CENP-A levels must be restored. A central player mediating this process is the Mis18 complex (composed of Mis18, Mis18{beta} and Mis18BP1), which recruits the CENP-A specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multipronged approach we provide the structural basis for the assembly of the Mis18 complex. We show that the Mis18/{beta} hetero-trimer (2 Mis18:1 Mis18{beta}) is assembled by the formation of a triple helical bundle with a Mis18/{beta}Yippee hetero-dimer and Mis18Yippee monomer on opposite ends. Two such Mis18/{beta} hetero-trimers, each bound to a Mis18BP1, assemble as a hetero-octamer via Mis18Yippee homo-dimerisation. Evaluation of structure-guided separation of function mutants in cells reveal structural determinants essential for Mis18 complex assembly and function.

sentences: Automated assessment reveals extinction risk of reptiles is widely underestimated across space and phylogeny The Red List of Threatened Species, published by the International Union for Conservation of Nature (IUCN), is a crucial tool for conservation decision making. However, despite substantial effort, numerous species remain unassessed, or have insufficient data available to be assigned a Red List threat category. Moreover, the Red Listing process is subject to various sources of uncertainty and bias. The development of robust automated assessment methods could serve as an efficient and highly useful tool to accelerate the assessment process and offer provisional assessments. Here we aimed to: 1) present a machine learning based automated threat assessment method that can be used on less known species; 2) offer provisional assessments for all reptiles - the only major tetrapod group without a comprehensive Red List assessment; and 3) evaluate potential effects of human decision biases on the outcome of assessments. We use the method presented here to assess 4,369 reptile species that are currently unassessed or classified as Data Deficient by the IUCN. Our models range in accuracy from 88% to 93% for classifying species as threatened/non-threatened, and from 82% to 87% for predicting specific threat categories. Unassessed and Data Deficient reptiles were more likely to be threatened than assessed species, adding to mounting evidence that they should be considered threatened by default. The overall proportion of threatened species greatly increased when we included our provisional assessments. Assessor identities strongly affected prediction outcomes, suggesting that assessor effects need to be carefully considered in extinction risk assessments. Regions and taxa we identified as likely to be more threatened should be given increased attention in new assessments and conservation planning. Lastly, the method we present here can be easily implemented to help bridge the assessment gap on other less known taxa.

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Navigating the amino acid sequence space between functional proteins using a deep learning framework MotivationShedding light on the relationships between protein se-quences and functions is a challenging task with many implications in protein evolution, diseases understanding, and protein design. Protein sequence / function space is however hard to comprehend due to its com-plexity. Generative models help to decipher complex systems thanks to their abilities to learn and recreate data specificity. Applied to protein sequences, they can point out relationships between protein positions and functions capture the sequence patterns associated with functions or navigate through uncharted area of molecular evolution.

ResultsIn this study, an unsupervised generative approach based on adversarial auto-encoder (AAE) is proposed to generate and explore new sequences with respect to their functions thanks to the prior distribution allowing a continuous exploration of the latent space. AAEs are tested on three protein families known for their multiple functions. Clustering re-sults on the encoded sequences from the latent space computed by AAEs display high level of homogeneity regarding the protein sequence func-tions. The study also reports and analyzes for the first time two sampling strategies based on latent space interpolation and latent space arithmetic to generate intermediate protein sequences sharing sequential and functional properties of original sequences issued from different families and functions. Generated sequences by interpolation between latent space data points demonstrate the ability of the AAE to generalize and to pro-duce meaningful biological sequences from an evolutionary uncharted area of the biological sequence space. Finally, 3D structure models generated by comparative modelling between different combinations of structures of different sub-families and of generated sequences from latent space or sub-family sequences point out to the ability of the latent space arithmetic to successfully transfer functional properties between sub-families. All in all this study confirms the ability of deep learning frameworks to model biological complexity and bring new tools to explore amino acid sequence and functional spaces.

AvailabilityCode and data used for this study are freely available at https://github.com/T-B-F/aae4seq.

Contacttristan@bitardfeildel.fr

Supplementary informationSupplementary data are available at online.

sentences: Cannabinoid tetrad effects of oral Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in male and female rats: sex, dose-effects and time course evaluations Rationale The legalization of medicinal use of Cannabis sativa in most US states and the removal of hemp from the Drug Enforcement Agency (DEA) controlled substances act has resulted in a proliferation of products containing {Delta}9-tetrahydrocannabinol (THC) and cannabidiol (CBD) for oral consumption (e.g., edibles, oils and tinctures) that are being used for recreational and medicinal purposes. Objective This study examined the effects of cannabinoids THC and CBD when administered orally on measures of pain sensitivity, body temperature, locomotor activity, and catalepsy (i.e., cannabinoid tetrad) in male and female Sprague Dawley rats. Methods Rats (N=24, 6 per sex/drug group) were administered THC (1-20 mg/kg), CBD (3-30 mg/kg), or sesame oil via oral gavage. Thermal and mechanical pain sensitivity (tail flick assay, von Frey test), rectal measurements for body temperature, locomotor activity, and the bar-test of catalepsy were completed. A separate group of rats (N=8/4 per sex) were administered morphine (5-20 mg/kg; intraperitoneal, IP) and evaluated for pain sensitivity as a positive control. Results We observed classic tetrad effects of antinociception, hypothermia, hyper- and hypolocomotion, and catalepsy after oral administration of THC that were long lasting (>7 hours). CBD modestly increased mechanical pain sensitivity and produced sex-dependent effects on body temperature and locomotor activity. Conclusions Oral THC and CBD produced long lasting effects, that differed in magnitude and time course when compared with other routes of administration. Examination of cannabinoid effects administered via different routes of administration, species, and in both males and females is critical to enhance translation.

sentences: Transposable elements continuously remodel the regulatory landscape, transcriptome, and function of decidual stromal cells Gene expression evolution underlies the origin, divergence, and conservation of biological characters including cell-types, tissues, and organ systems. Previously we showed that large-scale gene expression changes in decidual stromal cells contributed to the origins of pregnancy in eutherians and the divergence of pregnancy traits in primates (Marini[c] et al., 2021; Mika et al., 2021a, 2021b), and that transposable elements likely contributed to these gene expression changes (Mika et al., 2021a). Here we show that two large waves of TEs remodeled the transcriptome and regulatory landscape of decidual stromal cells, including a major wave in primates. Genes nearby TE-derived regulatory elements are among the most progesterone responsive in the genome and play essential roles in orchestrating progesterone responsiveness and the core function of decidual cells by donating progesterone receptor binding sites to the genome. We tested the regulatory abilities of 89 TE consensus sequences and found nearly all acted as repressors in mammalian cells but that treatment with histone deacetylase inhibitors unmasked latent enhancer functions. These data indicate TEs have played an important role in the development, evolution, and function of primate decidual stromal cells and suggest a two-step model in which latent enhancer functions of TEs are unmasked after they lose primary repressors functions.

sentences: Single-cell analysis reveals cellular heterogeneity and molecular determinants of hypothalamic leptin-receptor cells Hypothalamic nuclei which regulate homeostatic functions express leptin receptor (LepR), the primary target of the satiety hormone leptin. Single-cell RNA sequencing (scRNA-seq) has facilitated the discovery of a variety of hypothalamic cell types. However, low abundance of LepR transcripts prevented further characterization of LepR cells. Therefore, we perform scRNA-seq on isolated LepR cells and identify eight neuronal clusters, including three uncharacterized Trh-expressing populations as well as 17 non-neuronal populations including tanycytes, oligodendrocytes and endothelial cells. Food restriction had a major impact on Agrp neurons and changed the expression of obesity-associated genes. Multiple cell clusters were enriched for GWAS signals of obesity. We further explored changes in the gene regulatory landscape of LepR cell types. We thus reveal the molecular signature of distinct populations with diverse neurochemical profiles, which will aid efforts to illuminate the multi-functional nature of leptins action in the hypothalamus.

sentences: Maize brace root mechanics vary by whorl, genotype, and reproductive stage Root lodging is responsible for significant crop losses world-wide. During root lodging, roots fail by breaking, buckling, or pulling out of the ground. In maize, above-ground roots, called brace roots, have been shown to reduce root lodging susceptibility. However, the underlying structural-functional properties of brace roots that prevent root lodging are poorly defined. In this study, we quantified structural mechanical properties, geometry, and bending moduli for brace roots from different whorls, genotypes, and reproductive stages. Using 3-point bend tests, we show that brace root mechanics are variable by whorl, genotype, and reproductive stage. Generally, we find that within each genotype and reproductive stage, the brace roots from the whorl closest to the ground had higher structural mechanical properties and a lower bending modulus than brace roots from the second whorl. There was additional variation between genotypes and reproductive stages. Specifically, genotypes with higher structural mechanical properties also had a higher bending modulus, and senesced brace roots had lower structural mechanical properties than hydrated brace roots. Collectively these results highlight the importance of considering whorl-of-origin, genotype, and reproductive stage for quantification of brace root mechanics, which is important for mitigating crop loss due to root mechanical failure.

sentences: Human genetic variations reveal Chromosomal Instability aiding Variants (CIVa) in kinetochore-microtubule associated proteins The vast majority of Chromosomal Instability (CIN) promoting mutations remain unknown. We assess the prevalence of Chromosomal Instability aiding Variants (CIVa) by collating Loss-of-Function (LoF) variants predicted in 135 chromosome segregation genes from over 150,000 humans, including consanguineous individuals. Surprisingly, we observe heterozygous and homozygous CIVa in Astrin and SKA3 genes that encode evolutionarily conserved microtubule-associated proteins essential for chromosome segregation. By combining high-resolution microscopy and controlled protein expression, we show the naturally occurring Astrin variant, p.Q1012*, as potentially harmful because it fails to localise normally, delays anaphase onset, induces chromosome misalignment and promotes chromosome missegregation. We show that N-terminal frameshift variants in Astrin and SKA3 are likely to generate shorter isoforms that do not compromise chromosome segregation revealing resilient mechanisms to cope with harmful variants. This study provides a framework to predict and stratify naturally occurring CIVa, an important step towards precision medicine for CIN syndromes.

sentences: Genomic analyses identify biological processes and functions between M1 and M2 macrophages Macrophages can be induced by a variety of factors to change their phenotype and functions. M1 and M2 macrophages play converse roles during inflammation or other diseases. However, the mechanism and function of M1 and M2 macrophages are still not clear. This study aims to identify the different molecules and functions by analyzing the RNA-seq data. The GSE189354 was created by the Illumina HiSeq 4000 (Mus musculus). The KEGG and GO analyses indicated the Phagosome and Hematopoietic cell lineage are the major biological processes between M1 and M2 macrophages. Moreover, we further identified ten molecules including Il6, Il1b, Tlr2, Myc, Fn1, Itgax, Cxcl10, Ccl5, Cxcr4, and Pparg. Therefore, our study may provide novel knowledge of innate immunity.

sentences: Multivariate genome-wide association study identifies 780 unique genetic loci associated with cortical morphology. Brain morphology has been shown to be highly heritable, yet only a small portion of the heritability is explained by the genetic variants discovered so far. Here we exploit the distributed nature of genetic effects across the brain and apply the Multivariate Omnibus Statistical Test (MOSTest) to genome-wide association studies (GWAS) of vertex-wise structural magnetic resonance imaging (MRI) cortical measures from N=35,657 participants in the UK Biobank. We identified 695 loci for cortical surface area and 539 for cortical thickness, in total 780 unique genetic loci associated with cortical morphology. This reflects an approximate 10-fold increase compared to the commonly applied univariate GWAS methods. Power analysis indicates that applying MOSTest to vertex-wise structural MRI data triples the effective sample size compared to conventional univariate GWAS approaches. Functional follow up including gene-based analyses implicate 10% of all protein-coding genes and point towards pathways involved in neurogenesis and cell differentiation.

sentences:
DiSCERN - Deep Single Cell Expression ReconstructioN for improved cell clustering and cell subtype and state detection Single cell sequencing provides detailed insights into biological processes including cell differentiation and identity. While providing deep cell-specific information, the method suffers from technical constraints, most notably a limited number of expressed genes per cell, which leads to suboptimal clustering and cell type identification. Here we present DISCERN, a novel deep generative network that reconstructs missing single cell gene expression using a reference dataset. DISCERN outperforms competing algorithms in expression inference resulting in greatly improved cell clustering, cell type and activity detection, and insights into the cellular regulation of disease. We used DISCERN to detect two novel COVID-19-associated T cell types, cytotoxic CD4+ and CD8+ Tc2 T helper cells, with a potential role in adverse disease outcome. We utilized T cell fraction information of patient blood to classify mild or severe COVID-19 with an AUROC of 81 % that can serve as a biomarker of disease stage. DISCERN can be easily integrated into existing single cell sequencing workflows and readily adapted to enhance various other biomedical data types.

2010 MSC: 00-01, 99-00

sentences: Anti-inflammatory and neuroprotective activity of Viphyllin a standardized extract of β-caryophyllene from black pepper (Piper Nigrum L) and its associated mechanisms in mouse macrophage cells and Human Neuroblastoma SH-SY5Y cells Oxidative stress breeds various chronic lifestyle ailments including inflammatory conditions and neurodegenerative diseases. {beta}-caryophyllene natural bicyclic sesquiterpene, obtained from various plants sources found to be effective against inflammation and neuroprotection. In this study, we have evaluated the protective effect of Viphyllin, a standardized extract of {beta}-caryophyllene from black pepper against inflammation induced by lipopolysaccharide in RAW264.7 macrophage cells and mechanisms involved in hydrogen peroxide (H2O2)-challenged oxidative stress in human neuroblastoma SH-SY5Y cells. Viphyllin demonstrated the anti-inflammatory activity by subsiding the release of the pro-inflammatory intermediaries like NO, cytokines, interleukins, and protein expression levels of cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS). In addition, Viphyllin suppressed the extracellular signal-regulated kinase (ERK). c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. On the other hand, Viphyllin showed neuroprotective effect against neuronal oxidative damage caused by H2O2. Viphyllin lessened the expression B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-2-associated X protein (BAX), cleaved caspase-9, and PARP-1 proteins associated with apoptosis. Our results indicate that Viphyllin ameliorated LPS-mediated inflammation in macrophages by regulating inflammation and Viphyllin exerted remarkable anti apoptotic effect against neuronal damage challenged by H2O2. Altogether, Viphyllin could be potential functional food ingredient for inflammation and neurodegenerative diseases.

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Sequence conservation, domain architectures, and phylogenetic distribution of the HD-GYP type c-di-GMP phosphodiesterases The HD-GYP domain, named after two of its conserved sequence motifs, was first described in 1999 as a specialized version of the widespread HD phosphohydrolase domain that had additional highly conserved amino acid residues. Domain associations of HD-GYP indicated its involvement in bacterial signal transduction and distribution patterns of this domain suggested that it could serve as a hydrolase of the bacterial second messenger c-di-GMP, in addition to or instead of the EAL domain. Subsequent studies confirmed the ability of various HD-GYP domains to hydrolyze c-di-GMP to linear pGpG and/or GMP. Certain HD-GYP-containing proteins hydrolyze another second messenger, cGAMP, and some HD-GYP domains participate in regulatory protein-protein interactions. The recently solved structures of HD-GYP domains from four distinct organisms clarified the mechanisms of c-di-GMP binding and metal-assisted hydrolysis. However, the HD-GYP domain is poorly represented in public domain databases, which causes certain confusion about its phylogenic distribution, functions, and domain architectures. Here, we present a refined sequence model for the HD-GYP domain and describe the roles of its most conserved residues in metal and/or substrate binding. We also calculate the numbers of HD-GYPs encoded in various genomes and list the most common domain combinations involving HD-GYP, such as the RpfG (REC-HD-GYP), Bd1817 (DUF3391- HD-GYP), and PmGH (GAF-HD-GYP) protein families. We also provide the descriptions of six HD-GYP-associated domains, including four novel integral membrane sensor domains. This work is expected to stimulate studies of diverse HD-GYP-containing proteins, their N-terminal sensor domains and the signals to which they respond.

IMPORTANCEThe HD-GYP domain forms class II of c-di-GMP phosphodiesterases that control the cellular levels of the universal bacterial second messenger c-di-GMP and therefore affect flagellar and/or twitching motility, cell development, biofilm formation, and, often, virulence. Despite more than 20 years of research, HD-GYP domains are insufficiently characterized; they are often confused with  classical HD domains that are involved in various housekeeping activities and may participate in signaling, hydrolyzing (p)ppGpp and c-di-AMP. This work provides an updated description of the HD-GYP domain, including its sequence conservation, phylogenetic distribution, domain architectures, and the most widespread HD-GYP-containing protein families. This work shows that HD-GYP domains are widespread in many environmental bacteria and are predominant c-di-GMP hydrolases in many lineages, including clostridia and deltaproteobacteria.

sentences: Dual role of an essential HtrA2/Omi protease in the human malaria parasite: maintenance of mitochondrial homeostasis and induction of apoptosis like cell death under cellular stress HtrA family of serine protease is known to play role in mitochondrial homeostasis as well as in the programmed cell death. Mitochondrial homeostasis and metabolism are crucial for survival and propagation of malaria parasite within the host. Here we have functionally characterized a Plasmodium falciparum HtrA2 (PfHtrA2) protein, which harbours trypsin like protease activity that can be inhibited by its specific inhibitor, ucf-101. Transgenic parasite line was generated, using HA-glmS C-terminal tagging approach, for localization as well as for inducible knockdown of PfHtrA2. The PfHtrA2 was localized in the parasite mitochondrion during asexual life cycle. Genetic ablation of PfHtrA2 caused significant parasite growth inhibition, decreased replication of mtDNA, increased mitochondrial ROS production, caused mitochondrial fission/fragmentation, and hindered parasite development. However, ucf-101 treatment had no effect on the parasite growth, suggesting non-protease/chaperone role of PfHtrA2 in the parasite. Under cellular stress conditions, inhibition of PfHtrA2 by ucf-101 reduced activation of the caspase like protease as well as parasite cell death, suggesting involvement of protease activity of PfHtrA2 in apoptosis like cell death in the parasite. Under these cellular stress conditions, the PfHtrA2 gets processed but remains localized in the mitochondrion, suggesting that it acts within the mitochondrion by cleaving intra-mitochondrial substrate(s). This was further supported by trans-expression of PfHtrA2 protease domain in the parasite cytosol, which was unable to induce any cell death in the parasite. Overall, we show specific role of PfHtrA2 in maintaining mitochondrial homeostasis as well as in regulating the stress induced cell death.

sentences: On Improving an Already Competitive Segmentation Algorithm for the Cell Tracking Challenge - Lessons Learned The virtually error-free segmentation and tracking of densely packed cells and cell nuclei is still a challenging task. Especially in low-resolution and low signal-to-noise-ratio microscopy images erroneously merged and missing cells are common segmentation errors making the subsequent cell tracking even more difficult. In 2020, we successfully participated as team KIT-Sch-GE (1) in the 5th edition of the ISBI Cell Tracking Challenge. With our deep learning-based distance map regression segmentation and our graph-based cell tracking, we achieved multiple top 3 rankings on the diverse data sets. In this manuscript, we show how our approach can be further improved by using another optimizer and by fine-tuning training data augmentation parameters, learning rate schedules, and the training data representation. The fine-tuned segmentation in combination with an improved tracking enabled to further improve our performance in the 6th edition of the Cell Tracking Challenge 2021 as team KIT-Sch-GE (2).

sentences: Ensemble-function relationships to evaluate catalysis in the ketosteroid isomerase oxyanion hole Following decades of insights from structure-function studies, there is now a need to progress from a static to dynamic view of enzymes. Comparison of prior cryo X-ray structures suggested that deleterious effects from ketosteroid isomerase (KSI) mutants arise from misalignment of the oxyanion hole catalytic residue, Y16. However, multi-conformer models from room temperature X-ray diffraction revealed an ensemble of Y16 conformers indistinguishable from WT for Y32F/Y57F KSI and a distinct, non-native ensemble for Y16 in Y57F KSI. Functional analyses suggested rate effects arise from weakened hydrogen bonding, due to disruption of the Y16/Y57/Y32 hydrogen bond network, and repositioning of the general base. In general, catalytic changes can be deconvoluted into effects on the probability of occupying a state (P-effects) and the reactivity of each state (k-effects). Our results underscore the need for ensemble-function analysis to decipher enzyme function and ultimately manipulate their extraordinary capabilities.

sentences: Chronic Alcohol Consumption Alters Home-Cage Behaviors and Responses to Ethologically Relevant Predator Tasks in Mice Alcohol use disorders (AUD) are the most prevalent substance use disorders worldwide. Considering recent reports indicating an increase in alcohol use particularly in females, it is vital to understand how alcohol history impacts behavior. Animal model research on withdrawal-associated affective states tends to focus on males, forced alcohol paradigms, and a few traditional anxiety/stress tests. While this has been essential, heavy alcohol use triggers adverse withdrawal-related affective states that can influence how people respond to a large variety of life events and stressors. To this end, we show that behaviors in the home-cage, open field, looming disc, and robogator predator threat task, which vary in task demand and intensity, are altered in mice with a history of voluntary alcohol consumption. In alcohol-exposed males, behaviors in the home cage, a low anxiety baseline environment, suggest increased vigilance/exploration. However, in the open field and robogator task, which induce heightened arousal and task demands, a more hesitant/avoidant phenotype is seen. Female alcohol mice show no behavioral alterations in the home cage and open field test, however, in the looming disc task, which mimics an overhead advancing predator and forces a behavioral choice, we see greater escape responses compared to water controls, indicative of active stress coping behaviors. This suggests females may begin to show alcohol-induced alterations as task demands increase. To date, few drugs have advanced past clinical trials for the treatment of AUD, and those that have are predominately used in life-threatening situations only. No treatments exist for ameliorating negative withdrawal related states, which could aid in harm reduction related to heavy alcohol use. Understanding how withdrawal alters a variety of behavioral responses that are linked to stress coping can widen our understanding of alcohol abuse and lead us closer to better therapeutics to help individuals with AUD.

sentences: Snail maintains the stem/progenitor state of skin epithelial cells and carcinomas through the autocrine effect of the matricellular protein Mindin Intratumoral heterogeneity poses a major challenge in designing effective anti-cancer strategies. Accumulating evidence suggests that this heterogeneity arises from cancer stem cells (CSCs) that also drives tumor aggressiveness and drug resistance. The stemness of CSCs are preserved by an ill-defined combination of intrinsic and external factors and is particularly intriguing since they exist within a sea of similar cells at various degrees of differentiation. In models of cutaneous squamous cell carcinoma (cSCC), we discovered a non-EMT function for the transcription factor Snail in maintaining stemness of keratinocytes. This is accomplished by the secretion of the matricellular protein Mindin from Snail expressing cells, which creates a protective niche that impedes differentiation. In an autocrine fashion, extracellular Mindin activates a Src -STAT3 pathway to reinforce the stem/progenitor phenotype and disruption of this signalling module in human cSCC attenuates tumorigenesis. The expression of Mindin in multiple carcinomas, and its critical role in cancer progression suggests that it would be a promising target for therapeutic intervention.

sentences: Uncertainty Quantification in Variable Selection for Genetic Fine-Mapping using Bayesian Neural Networks In this paper, we propose a new approach for variable selection using a collection of Bayesian neural networks with a focus on quantifying uncertainty over which variables are selected. Motivated by fine-mapping applications in statistical genetics, we refer to our framework as an "ensemble of single-effect neural networks" (ESNN) which generalizes the "sum of single-effects" regression framework by both accounting for nonlinear structure in genotypic data (e.g., dominance effects) and having the capability to model discrete phenotypes (e.g., case-control studies). Through extensive simulations, we demonstrate our methods ability to produce calibrated posterior summaries such as credible sets and posterior inclusion probabilities, particularly for traits with genetic architectures that have significant proportions of non-additive variation driven by correlated variants. Lastly, we use real data to demonstrate that the ESNN framework improves upon the state-of-the-art for identifying true effect variables underlying various complex traits.

sentences: Readers move their eyes mindlessly using midbrain visuo-motor principles Saccadic eye movements rapidly shift our gaze over 100,000 times daily, enabling countless tasks ranging from driving to reading. Long regarded as a window to the mind1 and human information processing2, they are thought to be cortically/cognitively controlled movements aimed at objects/words of interest3-10. Saccades however involve a complex cerebral network11-13 wherein the contribution of phylogenetically older sensory-motor pathways14-15 remains unclear. Here we show using a neuro-computational approach16 that mindless visuo-motor computations, akin to reflexive orienting responses17 in neonates18-19 and vertebrates with little neocortex15,20, guide humans eye movements in a quintessentially cognitive task, reading. These computations occur in the superior colliculus, an ancestral midbrain structure15, that integrates retinal and (sub)cortical afferent signals13 over retinotopically organized, and size-invariant, neuronal populations21. Simply considering retinal and primary-visual-cortex afferents, which convey the distribution of luminance contrast over sentences (visual-saliency map22), we find that collicular population-averaging principles capture readers prototypical word-based oculomotor behavior2, leaving essentially rereading behavior unexplained. These principles reveal that inter-word spacing is unnecessary23-24, explaining metadata across languages and writing systems using only print size as a predictor25-26. Our findings demonstrate that saccades, rather than being a window into cognitive/linguistic processes, primarily reflect rudimentary visuo-motor mechanisms in the midbrain that survived brain-evolution pressure27.

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A high-throughput neutralization assay for yellow fever serodiagnostics Quick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks, for surveillance and to evaluate vaccine efficacy in population-wide studies. All this requires serological assays that can process a large number of samples in a highly standardized format. Albeit being laborious, time-consuming and limited in throughput, classical plaque reduction neutralization test (PRNT) is still considered gold standard for the detection and quantification of nAbs due to its sensitivity and specificity. Here we report the development of an alternative fluorescence-based serological assay (SNTFLUO) with an equally high sensitivity and specificity that is fit for high-throughput testing with the potential for automation. Finally, our novel SNTFLUO was cross-validated in several reference laboratories and against international WHO standards showing its potential to be implemented in clinical use. SNTFLUO assays with similar performance are available for the Japanese encephalitis, Zika and dengue viruses amenable for differential diagnostics.

IMPORTANCEFast and accurate detection of neutralizing antibodies (nAbs) against yellow fever virus (YFV) is key in yellow fever serodiagnosis, outbreak surveillance and monitoring of vaccine efficacy. Although classical PRNT still remains gold standard for measuring YFV nAbs, this methodology suffers from inherent limitations such as a low throughput and an overall high labor intensity. We present a novel fluorescence-based serum neutralization test (SNTFLUO) with equally high sensitivity and specificity that is fit for processing large number of samples in a highly standardized manner and has the potential to be implemented in clinical use. In addition, we present SNTFLUO assays with similar performance for Japanese encephalitis, Zika and dengue viruses opening new avenues for differential diagnostics.

sentences: Identifying transcription factor-DNA interactions using machine learning Machine learning approaches have been applied to identify transcription factor (TF)-DNA interaction important for gene regulation and expression. However, due to the enormous search space of the genome, it is challenging to build models capable of surveying entire reference genomes, especially in species where models were not trained. In this study, we surveyed a variety of methods for classification of epigenomics data in an attempt to improve the detection for 12 members of the Auxin Response Factor (ARF) binding DNAs from maize and soybean as assessed by DNA Affinity Purification and sequencing (DAP-seq). We used the classification for prediction by minimizing the genome search space by only surveying unmethylated regions (UMRs). For identification of DAP-seq binding events within the UMRs, we achieved 93.54% accuracy, 6.2% false positive, and a 43.29% false negative rate across 12 members of ARFs of maize on average by encoding DNA with count vectorization for k-mer with a logistic regression classifier with up-sampling and feature selection. Importantly, feature selection helps to uncover known and potentially novel ARF binding motifs. This demonstrates an independent method for identification of transcription factor binding sites. Finally, we tested the model built with maize DAP-seq data and applied it directly to the soybean genome and found unacceptably high false positive rates, which accounted for more than 40% across the ARF TFs tested. The findings in this study suggest the potential use of various methods to predict TF-DNA interactions within and between species with varying degrees of success.

sentences: Methods for in situ quantitative root biology When dealing with plant roots, a multi-scale description of the functional root structure is needed. Since the beginning of XXI century, new devices like laser confocal microscopes have been accessible for coarse root structure measurements, including 3D reconstruction. Most researchers are familiar with using simple 2D geometry visualization that does not allow quantitatively determination of key morphological features from an organ-like perspective. We provide here a detailed description of the quantitative methods available for three-dimensional (3D) analysis of root features at single cell resolution, including root asymmetry, lateral root analysis, xylem and phloem structure, cell cycle kinetics, and chromatin determination. Quantitative maps of the distal and proximal root meristems are shown for different species, including Arabidopsis thaliana, Nicotiana tabacum and Medicago sativa. A 3D analysis of the primary root tip showed divergence in chromatin organization and cell volume distribution between cell types and precisely mapped root zonation for each cell file. Detailed protocols are also provided. Possible pitfalls in the usage of the marker lines are discussed. Therefore, researchers who need to improve their quantitative root biology portfolio can use them as a reference.

sentences: Versatile mixotrophs employ CO2 fixation as fill-up function to dominate in modern groundwater The current understanding of organic carbon inputs into ecosystems lacking photosynthetic primary production is predicated on data and inferences derived almost entirely from metagenomic analyses. The elevated abundances of putative chemolithoautotrophs in groundwaters suggest that dark CO2 fixation is an integral component of subsurface trophic webs. To understand the impact of autotrophically-fixed carbon, the flux of CO2-derived carbon through various subpopulations of subsurface microbiota must first be resolved, both quantitatively and temporally. Here, we implement novel stable isotope cluster analysis to render a time-resolved and quantitative evaluation of 13CO-derived carbon flow through a groundwater microbiome stimulated with reduced sulfur compounds. We demonstrate that mixotrophs, not obligate chemolithoautotrophs, were the most abundant active organisms in groundwater microcosms. Species of Hydrogenophaga, Polaromonas, Dechloromonas, and other metabolically versatile mixotrophs drove the recycling of organic carbon and, when chance afforded, supplemented their carbon requirements via chemolithoautotrophy and uptake of available organic compounds. Mixotrophic activity facilitated the replacement of 43 and 80% of total microbial carbon stores with 13C in just 21 and 70 days, respectively. This opportunistic "utilize whatever pathways net the greatest advantage in fitness" strategy may explain the great abundances of mixotrophs in other oligotrophic habitats, like the upper ocean and boreal lakes.

sentences: Immune characterization and profiles of SARS-CoV-2 infected patients reveals potential host therapeutic targets and SARS-CoV-2 oncogenesis mechanism The spread of SARS-CoV-2 and the increasing mortality rates of COVID-19 create an urgent need for treatments, which are currently lacking. Although vaccines have been approved by the FDA for emergency use in the U.S., patients will continue to require pharmacologic intervention to reduce morbidity and mortality as vaccine availability remains limited. The rise of new variants makes the development of therapeutic strategies even more crucial to combat the current pandemic and future outbreaks. Evidence from several studies suggests the host immune response to SARS-CoV-2 infection plays a critical role in disease pathogenesis. Consequently, host immune factors are becoming more recognized as potential biomarkers and therapeutic targets for COVID-19. To develop therapeutic strategies to combat current and future coronavirus outbreaks, understanding how the coronavirus hijacks the host immune system during and after the infection is crucial. In this study, we investigated immunological patterns or characteristics of the host immune response to SARS-CoV-2 infection that may contribute to the disease severity of COVID-19 patients. We analyzed large bulk RNASeq and single cell RNAseq data from COVID-19 patient samples to immunoprofile differentially expressed gene sets and analyzed pathways to identify human host protein targets. We observed an immunological profile of severe COVID-19 patients characterized by upregulated cytokines, interferon-induced proteins, and pronounced T cell lymphopenia, supporting findings by previous studies. We identified a number of host immune targets including PERK, PKR, TNF, NF-kB, and other key genes that modulate the significant pathways and genes identified in COVID-19 patients. Finally, we identified genes modulated by COVID-19 infection that are implicated in oncogenesis, including E2F transcription factors and RB1, suggesting a mechanism by which SARS-CoV-2 infection may contribute to oncogenesis. Further clinical investigation of these targets may lead to bonafide therapeutic strategies to treat the current COVID-19 pandemic and protect against future outbreaks and viral escape variants.

sentences: Regenerating vascular mural cells in zebrafish fin blood vessels are not derived from pre-existing ones and differentially require pdgfrb signaling for their development Vascular networks are comprised of endothelial cells and mural cells, which include pericytes and smooth muscle cells. It is well established that new endothelial cells are derived from pre-existing ones during the angiogenic phase of blood vessel growth. By contrast, mural cell ontogeny is less clear with an ongoing debate whether mural cells possess mesenchymal stem cell properties. To elucidate the mechanisms controlling mural cell recruitment during development and tissue regeneration, we studied the formation of zebrafish caudal fin arteries. Mural cells showed morphological heterogeneity: cells colonizing arteries proximal to the body wrapped around them, while those in more distal regions extended protrusions along the proximo-distal vascular axis. Despite these differences, both cell populations expressed platelet-derived growth factor receptor beta (Pdgfrb) and the smooth muscle cell marker myosin heavy chain 11a (Myh11a). Loss of Pdgfrb signalling during development or tissue regeneration resulted in a substantial decrease in mural cells at the vascular front, while those proximal to the body were less affected. Using lineage tracing, we demonstrate that precursor cells located in periarterial regions of the caudal fin and expressing Pgdfrb can give rise to mural cells, while in regeneration newly formed mural cells were not derived from pre-existing ones. Together, our findings reveal conserved roles for pdgfrb signalling in development and regeneration, while at the same time illustrating a limited capacity of mural cells to self-renew or contribute to other cell types during tissue regeneration.

sentences: The cannabinoid agonist CB-13 produces peripherally mediated analgesia in mice but elicits tolerance and signs of CNS activity with repeated dosing Activation of cannabinoid receptor type 1 (CB1) produces analgesia in a variety of preclinical models of pain; however, engagement of central CB1 receptors is accompanied by unwanted side effects, such as tolerance and dependence. Efforts to develop novel analgesics have focused on targeting peripheral CB1 receptors to circumvent central CB1-related side effects. In the present study, we evaluated the effects of acute and repeated dosing with the peripherally selective CB1-preferring agonist CB-13 on nociception and central CB1-related phenotypes in an inflammatory model of pain in mice. We also evaluated cellular mechanisms underlying CB-13-induced antinociception in vitro using cultured mouse dorsal root ganglion (DRG) neurons. CB-13 reduced inflammation-induced mechanical allodynia in a peripheral CB1 receptor-dependent manner and relieved inflammatory thermal hyperalgesia. In cultured mouse DRG neurons, CB-13 reduced TRPV1 sensitization and neuronal hyperexcitability induced by the inflammatory mediator prostaglandin E2, providing potential mechanistic explanations for the analgesic actions of peripheral CB1 receptor activation. With acute dosing, phenotypes associated with central CB1 receptor activation occurred only at a dose of CB-13 approximately 10-fold the ED50 for reducing allodynia. Strikingly, repeated dosing resulted in both analgesic tolerance and CB1 receptor dependence, even at a dose that did not produce central CB1 receptor-mediated phenotypes on acute dosing. This suggests repeated CB-13 dosing leads to increased CNS exposure and unwanted engagement of central CB1 receptors. Thus, caution is warranted regarding therapeutic use of CB-13 with the goal of avoiding CNS side effects. Nonetheless, the clear analgesic effect of acute peripheral CB1 receptor activation suggests that peripherally restricted cannabinoids are a viable target for novel analgesic development.

sentences: A mitotic screen in Indian muntjac cells unveils how Augmin drives kinetochore fiber maturation Chromosome segregation in mammals relies on the maturation of a thick bundle of kinetochore-attached microtubules known as k-fibers. How k-fibers mature from initial kinetochore-microtubule attachments remains a fundamental question. By combining functional perturbations in Indian muntjac, a placental mammal with the lowest known chromosome number (n=3) and distinctively large kinetochores, with fixed- and subsecond live-cell super-resolution CH-STED nanoscopy, we uncovered the mechanism by which Augmin mediates k-fiber maturation. Augmin promoted kinetochore microtubule turnover by sustaining microtubule formation from kinetochores and poleward flux, regardless of pioneer centrosomal microtubules. Tracking of microtubule growth events within individual k-fibers revealed a wide angular dispersion, consistent with Augmin-mediated branched microtubule nucleation. Augmin depletion reduced the frequency of microtubule growth events within k-fibers and hampered normal repair after acute k-fiber injury by laser microsurgery. Our work underscores the contribution of microtubule formation from kinetochores and Augmin-mediated microtubule amplification for k-fiber maturation and spindle assembly in mammals.

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Rapid appetitive transitions are sculpted by amygdala to accumbens pathways Foraging, pursuit, and predation rapidly transition into behavioral quiescence during reward capture and consumption. While appetitive-consummatory dissociations are embedded at both psychological and neural levels, the mechanisms controlling switches or transitions between appetitive seeking and consummatory behaviors remain poorly understood. Here we identify the BLA[->]AcbSh pathway as critical to these transitions by showing that this pathway inhibits the appetitive seeking response in the presence of consummatory demands. Using an appetitive cue-discrimination task in male rats, we show that reward delivery is a significant driver of seeking inhibition and that a BLA[->]AcbSh pathway mediates this inhibition. This role in suppressing seeking responses during periods of consumption was not due to a general suppression of behavior because responding to other cues during the same test was unaffected. Moreover, it was specific to the BLA[->]AcbSh pathway, because the contribution of the BLA[->]AcbC pathway to appetitive switching was distinct and modest. State-dependent silencing of BLA[->]AcbSh revealed that the modulation of seeking before and after reward delivery are co-dependent. Finally, we found that BLA terminals in AcbSh have functional connectivity to LH-projecting AcbSh neurons, thereby identifying a BLA[->]AcbSh[->]LH pathway as a putative route for the rapid regulation of appetitive behaviors. Taken together, these findings suggest that the BLA[->]AcbSh pathway is a core component of an appetitive switching system, recruited under conditions requiring rapid or dynamic shifts in appetitive behavior, and that this pathway enables these shifts by actively inhibiting seeking.

Significance statementForaging, pursuit, and predation quickly transition into behavioral quiescence during reward capture and consumption. These transitions are critical for flexible and responsive sequences of behavior. Here we show that behavioral transitions are actively controlled at the limbic-striatal interface. We identify reward receipt as a proximal trigger for transition between reward seeking and taking, we identify active inhibition as the functional operation of this transition, and we identify the basolateral amygdala[->]accumbens shell pathway as critical to this functional operation.

sentences: SiCoDEA: a simple, fast and complete app for analyzing the effect of individual drugs and their combinations The administration of combinations of drugs is a method widely used in the treatment of different pathologies as it can lead to an increase in the therapeutic effect and a reduction in the dose compared to the administration of the single drugs. For these reasons, it is of interest to study combinations of drugs and in particular to determine whether a specific combination has a synergistic, antagonistic or additive effect, i.e greater, less than or equal to the effect expected by the sum of the individual drugs. For this purpose, various mathematical models have been developed, which use different methods to evaluate the synergy of a combination of drugs. Most of these methods are based on the Loewe Additivity Principle (Loewe et al., 1953), which has its key step in choosing the model used for predicting the effect of individual drugs. Creating a model for this purpose and calculating its parameters, however, requires a certain level of mathematical and programming knowledge or the use of commercial software. For this purpose, therefore, we have developed an open access and easy to use app that allows to explore different models and to choose the most fitting for the specific experimental data: SiCoDEA (Single and Combined Drug Effect Analysis, https://sicodea.shinyapps.io/shiny/). The data used to test SiCoDEA comes from cell line samples treated with different drug combinations and analyzed through metabolic or viability assays. SiCoDEA is developed through a Shiny interactive and easy-to-use interface (R based). There are five models taken into consideration for the analysis of single drugs and the calculation of combination index. The first is one of the most used, that of the median-effect; while the others are different forms of the log-logistic equation, with two, three and four parameters. The purpose of SiCoDEA is, on the one hand, to provide an easy-to-use tool for analyzing drug combination data and, on the other hand, also to have a view of the various steps and to offer different results based on the model chosen. An important prerequisite in analyzing drug combinations is in fact the dose-response curve calculated for individual drugs. For this purpose, SiCoDEA allows you to view the plots of the individual drugs both to evaluate the distribution of the calculated points and therefore identify any outliers, and to view the curve of the different models taken into consideration and evaluate which one best fits the data. A table showing all the R2 values for the five different models is created with the curve plot. In addition to the type of model, it is also possible to choose between two different normalization methods, one based on the maximum or minimum value and the other on the value calculated at drug concentrations equal to zero. For the chosen options, a plot is then created that shows the trend of the combination index for the different drug combinations and consequently whether it is synergy, antagonism or additivity. Finally, it is possible to export the results in single png files or in a summary report in pdf. SiCoDEA is an open-source app among the most complete and offers more functions even than the famous CompuSyn (Chou et al., 2010), as it allows you to analyze drug curves with different models, rather than just one, and it also allows the analysis of single drug curves.

sentences: Uncovering hidden assembly artifacts: when unitigs are not safe and bidirected graphs are not helpful Recent assemblies by the T2T and VGP consortia have achieved significant accuracy but required a tremendous amount of effort and resources. More typical assembly efforts, on the other hand, still suffer both from mis-assemblies (joining sequences that should not be adjacent) and from under-assemblies (not joining sequences that should be adjacent). To better understand the common algorithm-driven causes of these limitations, we investigated the unitig algorithm, which is a core algorithm at the heart of most assemblers. We prove that, contrary to popular belief, even when there are no sequencing errors, unitigs are not always safe (i.e. they are not guaranteed to be substrings of the sequenced genome). We also prove that the unitigs of a bidirected de Bruijn graph are different from those of a doubled de Bruijn graph and, contrary to our expectations, result in under-assembly. Using experimental simulations, we then confirm that these two artifacts exist not only in theory but also in the output of widely used assemblers. In particular, when coverage is low then even error-free data results in unsafe unitigs; also, unitigs may unnecessarily split palindromes in half if special care is not taken. To the best of our knowledge, this paper is the first to theoretically predict the existence of these assembler artifacts and confirm and measure the extent of their occurrence in practice.

sentences: Transmembrane Signaling Regulates the Remodeling of the Actin Cytoskeleton:Roles of PKC and Adducin We tested the hypothesis that Pkc53E regulates adducin to orchestrate the remodeling of the membrane skeleton following the transmembrane GPCR-Gq signaling. Adducin is a known substrate of PKC and is critical for the assembly of the membrane skeleton by cross-linking actin filaments with the spectrin network. In Drosophila photoreceptors, loss of function in pkc53E leads to retinal degeneration while Pkc53E-RNAi negatively impacts the actin cytoskeleton of the visual organelle rhabdomeres. Unexpectedly, Pkc53E-RNAi enhances the degeneration caused by the loss of PLC{beta}4 (norpAP24). We show that when PLC{beta}4 is absent Plc21C may be activated instead for activating Pkc53E. We investigate whether Pkc53E phosphorylates adducin in vivo and observed that levels of phosphorylated adducin at the conserved PKC site were greatly reduced in a null allele of pkc53E. We show Pkc53E-RNAi did not modify adducin-RNAi, which exerts a more severe effect on the actin cytoskeleton. Moreover, overexpression of the mCherry-tagged adducin that appears to act in a dominant-negative manner interferes with the spectrin interaction leading to the apical expansion of rhabdomeres similar to that of {beta}-spectrin-RNAi. We performed epistasis analysis and show that double mutants of the tagged adducin and Pkc53E-RNAi display the expansion phenotype at the eclosion, but progress to severe degeneration in adult photoreceptors. Together, most of our findings support that adducin is likely regulated by Pkc53E in Drosophila photoreceptors.

sentences: Toward a first principles understanding of the activation and deactivation mechanisms of class A G-protein coupled receptors and voltage-gated cation channels We previously reported a first principles multi-scale theory called Biodynamics that attributes cellular functions to sets of coupled molecular and ionic fluxes operating in the non-equilibrium/non-linear dynamic regime. Fluxes build and decay over time and undergo dynamic non-covalent intra- and intermolecular state transitions powered principally by the storage and release of free energy to/from the H-bond networks of external and internal solvation (that we refer to as solvation dynamics) at rates governed by the desolvation and resolvation costs incurred during their entry and exit, respectively. We have thus far examined the functional state transitions of cereblon and COVID Mpro in this context, and now turn to the agonist-induced activating and deactivating state transitions of class A G-protein coupled receptors (GPCRs) and membrane potential-/dipole potential-induced activating and deactivating state transitions of voltage-gated cation channels (VGCCs). We analyzed crystal structures of the activated and deactivated forms of the human {beta}2 adrenergic receptor ({beta}2AR) and cryo-EM structures of the activated and deactivated forms of Nav1.7 channels. We postulate that activation and deactivation of the {beta}2AR is conveyed by switchable changes in transmembrane helix (TMH) orientations relative to extracellular loop 2 (ECL2) and curvature of TMH6 and TMH7, all of which are powered by solvation free energy and kickstarted by agonist binding. The known activation and deactivation mechanisms of Nav1.7 consist of S4 translations toward and away from the extracellular membrane surface, respectively, resulting in S4-S5 linker repositioning, followed by rearrangements of the S5 and S6 helices. The latter TMH conveys channel opening and closing by respectively curving away from and toward the central pore axis. We postulate that all of these rearrangements are likewise powered by solvation free energy and kickstarted by changes in the membrane and dipole potentials. The results of our study may facilitate structure-based design of GPCR agonists/antagonists and mitigation of drug-induced ion channel blockade.

sentences: Evaluation of a high-throughput deorphanization strategy to identify cytochrome p450s important for odor degradation in Drosophila Members of the cytochrome p450 (CYP) enzyme family are abundantly expressed in insect olfactory tissues, where they are thought to act as Odorant Degrading Enzymes (ODEs). However, their contribution to olfactory signaling in vivo is poorly understood. This is due in part to the challenge of identifying which of the dozens of antennal-expressed CYPs might inactivate a given odorant. Here, we tested a high-throughput deorphanization strategy in Drosophila to identify CYPs that are transcriptionally induced by exposure to a plant volatile. We discovered three CYPs selectively upregulated by the odorant using transcriptional profiling. Although these CYPs are shown to be broadly expressed in the antenna in non-neuronal cells, electrophysiological recordings from CYP mutants did not reveal any changes in olfactory neuron responses to the odorant. Neurons were desensitized by pre-exposing flies to the odorant, but this effect was similar in CYP mutants. Together, our data suggest that this transcriptomic approach may not be useful for identifying CYPs that contribute to olfactory signaling. We go on to show that some CYPs have highly restricted expression patterns in the antenna, and suggest that such CYPs may be useful candidates for further studies on olfactory CYP function.

sentences: Gastric cancer derived exosomes induce peritoneal mesothelial cell EMT through TGF-β1/Smads pathway to promote peritoneal metastasis Epithelial-mesenchymal transition (EMT) plays an important role in peritoneal metastasis of Gastric cancer (GC). Tumor exosomes can mediate tumor directed metastasis, and TGF-{beta}1 is an important factor in inducing tumor Epithelial mesenchymal transition. However, it is not clear whether GC derived exosomes can induce peritoneal mesothelial cells through the TGF-{beta}1/ Smads pathway and the effect of injured peritoneal mesothelial cells on the biological characteristics of GC cells. In this study, we demonstrated that GC-derived exosomes can activate the TGF-{beta}1/Smads pathway in peritoneal mesothelial cells and induce the corresponding EMT process, and that the injured peritoneal mesothelial cells can improve the migration and adhesion of GC cells. Taken together, these data further support the critical role of exosomes in the remodeling of the pre-metastatic microenvironment.

sentences: Extensive germline-somatic interplay drives prostate cancer through HNF1B co-option of TMPRSS2-ERG Genome-wide association studies have identified 270 loci conferring risk for prostate cancer (PCa), yet the underlying biology and clinical impact remain to be investigated. Here we observe an enrichment of transcription factor genes including HNF1B within PCa risk-associated regions. While focused on the 17q12/HNF1B locus, we find a strong eQTL for HNF1B and multiple potential causal variants involved in the regulation of HNF1B expression in PCa. An unbiased genome-wide co-expression analysis reveals PCa-specific somatic TMPRSS2-ERG fusion as a transcriptional mediator of this locus and the HNF1B eQTL signal is ERG fusion status dependent. We investigate the role of HNF1B and find its involvement in several pathways related to cell cycle progression and PCa severity. Furthermore, HNF1B interacts with TMPRSS2-ERG to co-occupy large proportion of genomic regions with a remarkable enrichment of additional PCa risk alleles. We finally show that HNF1B co-opts ERG fusion to mediate mechanistic and biological effects of the PCa risk-associated locus 17p13.3/VPS53/FAM57A/GEMIN4. Taken together, we report an extensive germline-somatic interaction between TMPRSS2-ERG fusion and genetic variations underpinning PCa risk association and progression.

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Determination of Slow-binding HDAC Inhibitor Potency and Subclass Selectivity Histone deacetylases (HDACs) 1-3 regulate chromatin structure and gene expression. These three enzymes are targets for cancer chemotherapy and are studied for the treatment of immune disorders and neurodegeneration, but there is a lack of selective pharmacological tool compounds to unravel their individual roles. Potent inhibitors of HDACs 1-3 often display slow-binding kinetics, which causes a delay in inhibitor-enzyme equilibration and may affect assay readout. Here, we compare the potency and selectivity of slow-binding inhibitors measured by discontinuous and continuous assays. We find that entinostat, a clinical candidate, inhibits HDACs 1-3 by a two-step, slow-binding mechanism with lower potencies than previously reported. In addition, we show that RGFP966, commercialized as HDAC3-selective probe, is a slow-binding inhibitor with inhibitor constants of 57 nM, 31 nM, and 13 nM against HDACs 1-3, respectively. These data highlight a need for thorough kinetic investigation in the development of selective HDAC probes.

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sentences: Virus transmission by ultrasonic scaler and its prevention by antiviral agent During an ultrasonic scaler (USS) operation, droplets and aerosol are generated that may contribute to the transmission of viruses contained in saliva and gingival crevicular fluid. The purpose of this research was to develop an experimental model for testing the spread of viruses during USS instrumentation and to examining the prevention of spreading by replacing the coolant with an antiviral agent. In a virus transmission tunnel, USS operation with saline coolant and delivery of a viral suspension to the vicinity of USS tip generated droplets and aerosol containing Equine Arteritis Virus (EAV). Evaluation of droplet transmission was evaluated with adherent 48h cell culture monolayer RK13 cell lines in standard 48-well-plates positioned at a distance from 30 to 55 cm. The aerosol was collected by a cyclone aero-sampler flow of 100l/min. Antiviral activity of 0.25% sodium hypochlorite or electrolyzed water (EOW) was tested by suspension test. The two tested antiviral agents transmission prevention ability was evaluated by repeating the same experiment as with saline coolant. All experiments were repeated twice. With saline coolant, the cytopathic effect on cells was found in cells up to the distance of 45 cm, with the number of infected wells decreasing with distance. Viral particles were detected in only one AS in a very low concentration ([≤]4.2 TCID50/ml). In suspension test of 0.25% NaOCl and EOW, the TCID50/ml was below detection limit after 5s. With both antiviral agents, no cytopathic effect was found. However, the cytotoxic effect of 0.25% NaOCl was evident up to the distance of 35 cm. By USS activity, EAV could be transmitted by droplets up to a distance of 45 cm. Both antiviral agents could prevent virus droplet transmission. The transmission of EAV by aerosol yielded inconclusive results.

sentences: Decreased synthesis and variable gene transcripts of oxytocin in a domesticated avian species The Bengalese finch was domesticated more than 250 years ago from the wild white-rumped munia. Similar to other domesticated species, Bengalese finches show a reduced fear response and have lower corticosterone levels, compared to white-rumped munias. Bengalese finches and munias also have different song types. Since oxytocin (OT) has been found to be involved in stress coping and auditory processing, we tested whether the OT sequence and brain expression pattern and content differ in wild munias and domesticated Bengalese finches. We identified intra-strain variability in the untranslated regions of the OT sequence in Bengalese finches in comparison to the munia OT. Several of these changes fall in specific transcription factor binding sites, which show either a conserved or a relaxed evolutionary trend in the avian lineage, and in vertebrates in general. Although in situ hybridization in several hypothalamic nuclei did not reveal significant differences in the number of cells expressing OT between the two strains, real-time quantitative PCR showed significantly lower OT mRNA expression in the diencephalon of the Bengalese finches relative to munias. Our study thus points to a decreased OT synthesis in the domestic strain compared with the wild strain in birds. This is an opposite pattern from that found in some domesticated mammals, suggesting that different processes of OT function might have occurred in mammals and birds under domestication.

sentences: Make Interactive Complex Heatmaps in R Heatmap is a powerful visualization method on two-dimensional data to reveal patterns shared by subsets of rows and columns. In R, there are many packages that make heatmaps. Among them, ComplexHeatmap provides rich tools for constructing highly customizable heatmaps. It can easily establish connections between information from multiple sources by automatically concatenating and adjusting multiple heatmaps as well as complex annotations, which makes it widely applied in data analysis in various fields, especially in Bioinformatics. Nevertheless, the limit of ComplexHeatmap still exists. It only generates static plots which restricts deeper inspections on complex heatmaps, e.g., to look into a subset of rows and columns when a specific pattern of interest is observed from the heatmap. In this work, we described a new R/Bioconductor package InteractiveComplexHeatmap that brings interactivity to ComplexHeatmap. InteractiveComplexHeatmap is designed with an easy-to-use interface where static complex heatmaps can be directly exported to an interactive Shiny web application only with one extra line of code. The interactive application contains comprehensive tools for manipulating heatmaps. Besides common tools as supported in other interactive heatmap packages, InteractiveComplexHeatmap additionally supports, e.g., selecting over multiple heatmaps and searching heatmaps via row or column labels. Also, InteractiveComplexHeatmap provides methods for exporting static heatmaps from other popular heatmap functions, e.g., heatmap.2() or pheatmap(), to interactive heatmap applications. Finally, InteractiveComplexHeatmap provides flexible functionalities for integrating interactive heatmap widgets into other Shiny applications. InteractiveComplexHeatmap provides a user interface for self-defining response to the selection events on heatmaps, which helps to implement more complex Shiny web applications.

sentences: Differential Interaction between RAC/ROP-GTPases and RIC-Effectors: A Network Hub for Broader Signal Transduction in Pollen Tubes To date knowledge about plant RAC/ROP-GTPase effectors and downstream targets is still limited. This work aims on elucidation of related signal transduction networks involved in pollen tube growth. Yeast two-hybrid and Pull Down methodology were used to identify and characterize hitherto unknown components of RAC-related protein complexes from Nicotiana tabacum. Nt-RIC11pt specifically interacts with diverse active tobacco RAC-GTPases, and it is particularly significant, that their binding affinity is differential, thus implicating a multifaceted role in an interconnected RIC-RAC network. Moreover, Y2H-screening for Nt-RIC11pt targets identified Nt-CAR4, which is phylogenetically assigned to a multifaceted family of novel unusual GTPase activating proteins (GAP). It is argued that scaffold Nt-RIC11pt connects active Nt-RAC3 with membrane-bound Nt-CAR4, thus relaying GAP-activity. Quantitative RT-PCR demonstrates Nt-RIC11pt is primarily expressed in pollen and YFP-fusion proteins show homogeneous cytoplasmic localization in growing tubes, what builds the prerequisite for a proposed role in broader signal transduction. By synoptically integrating experimental data, bioinformatic sequence comparison, phylogenetic analyses, and detailed literature review, this study hypothesizes a concept in which RIC-effectors collectively constitute a multifaceted network hub linking diverse GTPase-dependent processes.

sentences: Comparative Morphometric Analysis Of Captive Jaguars Vs. Wild Jaguars (Panthera onca) In Venezuela In Venezuela the oldest, although anecdotical, record known of a captive jaguar is a male in the city of Maracay between 1918 and 1935. Body measurements were made on 22 jaguars kept in Venezuelan zoos between 1996 and 2009 to provide data on external morphometrics and compare with the measurements of 25 free-living animals, published by Hoogesteijn and Mondolfi (1992) and determine whether there are significant differences in the morphology between sexes of captive and free-living animals. The evaluation of morphometric variation was focused on three body measurements and body weight: Head-and-body length (HBL) and Tail length (Tl). Total body length (TBL) was calculated by adding HBL and Tl. Body weight (BW) was determined using a clock scale to the nearest gramme/kilogram. To determine the values all animals were anesthetized. The sexes were analyzed separately. All data were used for the calculation of descriptive statistics. Student t-tests were used with a significance level  0.05 to establish if there is a significant difference between the measurements and weights of the sexes. The TBL and HBL in captive males were observed to be 8.78% and 12.13%, respectively, less than that of free-living males. A principal components analysis made it possible to assess the variability between HBL, TBL, and BW between sexes and between free-living/wild and captive animals. This study showed no significant differences in the BW of captives and wild females. Some captive males jaguars in this study may be thinner than normal, because of their diet and others may be fatter because of the lack of physical activity imposed by captivity. Body mass index (BMI) was used to contrast the difference in the average mass of each sex in both captive and free-living animals. Simple linear regressions and correlation coefficients were used to determine the relationship between body mass and male/female size. According to correlation coefficients, the change in weight that can be attributed to the ratio of weight to body length is 8.55 % for males and 9.83 % for females.

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Coverage-preserving sparsification of overlap graphs for long-read assembly Read-overlap-based graph data structures play a central role in computing de novo genome assembly using long reads. Many assembly tools use the string graph model [Myers, Bioinformatics 2005] to sparsify overlap graphs. Graph sparsification improves accuracy by removing spurious and redundant connections. However, a graph model must be coverage-preserving, i.e., it must ensure that each chromosome can be spelled as a walk in the graph, given sufficient sequencing coverage. This property becomes even more important for diploid genomes, polyploid genomes and metagenomes where there is a risk of losing haplotype-specific information.

We develop a novel theoretical framework under which the coverage-preserving properties of a graph model can be analysed. We first prove that de Bruijn graph and overlap graph models are guaranteed to be coverage-preserving. We also prove that the standard string graph model lacks this guarantee. The latter result is consistent with the observation made in [Hui et al., 2016; Nurk et al., 2021] that removal of contained reads during string graph construction can lead to coverage gaps. To remedy this, we propose practical heuristics that are well-supported by our theoretical results to sparsify overlap graphs. In our experiments using simulated long reads from HG002 human diploid genome, we find that 98 and 5 coverage gaps are introduced by ignoring contained reads from nanopore and HiFi long-read datasets respectively. We test the proposed heuristics for deciding which contained reads should be retained to avoid the coverage gaps. The proposed method retains a small fraction of contained reads (1 - 2%) and closes all coverage gaps. This compares favorably to other known solutions for this problem.

sentences: Resource Profile and User Guide of the Polygenic Index Repository Polygenic indexes (PGIs) are DNA-based predictors. Their value for research in many scientific disciplines is rapidly growing. As a resource for researchers, we used a consistent methodology to construct PGIs for 47 phenotypes in 11 datasets. To maximize the PGIs prediction accuracies, we constructed them using genome-wide association studies--some of which are novel--from multiple data sources, including 23andMe and UK Biobank. We present a theoretical framework to help interpret analyses involving PGIs. A key insight is that a PGI can be understood as an unbiased but noisy measure of a latent variable we call the "additive SNP factor." Regressions in which the true regressor is the additive SNP factor but the PGI is used as its proxy therefore suffer from errors-in-variables bias. We derive an estimator that corrects for the bias, illustrate the correction, and make a Python tool for implementing it publicly available.

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AGO1 regulates major satellite transcripts and H3K9me3 distribution at pericentromeric regions in mESCs Argonaute proteins (AGOs), that play an essential role in cytosolic post-transcriptional gene silencing, have been also reported to function in nuclear processes like transcriptional activation or repression, alternative splicing and, chromatin organization. As most of these studies have been conducted in human cancer cell lines, the relevance of AGOs nuclear functions in the context of mouse early embryonic development remains uninvestigated. Here, we examined a possible role of the AGO1 protein on the distribution of constitutive heterochromatin in mouse Embryonic Stem Cells (mESCs). We observed a specific redistribution of the repressive histone mark H3K9me3 and the heterochromatin protein HP1, away from pericentromeric regions upon Ago1 depletion. Furthermore, we demonstrated that major satellite transcripts are strongly upregulated in Ago1_KO mESCs and that their levels are partially restored upon AGO1 rescue. We also observed a similar redistribution of H3K9me3 and HP1 in Drosha_KO mESCs, suggesting a role for microRNAs (miRNAs) in the regulation of heterochromatin distribution in mESCs. Finally, we showed that specific miRNAs with complementarity to major satellites can partially regulate the expression of these transcripts.

Summary blurbDepletion of AGO1 in mESCs leads to a redistribution of H3K9me3 and HP1 away from pericentromeric regions and is accompanied by an upregulation of major satellites transcripts.

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Fatty acid metabolic reprogramming promotes C. elegans development Acetylcholine signaling has been reported to play essential roles in animal metabolic regulation and disease affected by diets. However, the underlying mechanisms that how diets regulate animal physiology and health are not well understood. Here we found that the acetylcholine receptor gene eat-2 was expressed in most of the pharyngeal muscles, which is in accordance to our previous report that EAT-2 received synaptic signals not only from pharyngeal MC neurons. The expression of fatty acid synthesis genes was significantly increased in both eat-2 and tmc-1 fast-growth mutants on CeMM food environment, compared to the wild-type. Excitingly, dietary fatty acids such as 15-methyl-hexadecanoic acid (C17ISO), palmitic acid (PA, C16:0) and stearic acid (SA, C18:0) supplementation, significantly accelerated wild-type worm development on CeMM, indicating that the fatty acid synthesis reprogramming is an essential strategy for C. elegans to regulate its development and growth on CeMM diet. Furthermore, we found that fatty acid elongase gene elo-6 knock-out significantly attenuated eat-2 mutant fast growth, while overexpression of elo-6 could rescue the eat-2; elo-6 double mutant slow development, which suggested that elo-6 played a major role in the above metabolic remodeling. Taken together, our report indicates that diets regulate neuromuscular circuit and modulate C. elegans development via fatty acid metabolic reprogramming. As most of the key genes and metabolites found in this study are conserved in both invertebrate and vertebrate animals, we believed that our results might provide essential clues to the molecular mechanisms underlying interactions among animal nutrition sensation, metabolism reprogramming and developmental regulation.

Significance StatementDiets and nutritional composition affect animal development and human health, however the underlying mechanisms remain elusive. We demonstrate that the acetylcholine receptor gene eat-2 is expressed in most of pharyngeal muscles, and the expression of fatty acid synthesis genes is significantly increased in both eat-2 and tmc-1 fast-growth mutants on the synthetic chemical defined CeMM food environment. Dietary supplementation of several fatty acids significantly speed up animal development. Furthermore, we demonstrate that the fatty acid elongase gene elo-6 knock-out attenuates eat-2 mutant fast growth, and overexpression of wild-type elo-6 promotes the eat-2; elo-6 double mutant slow development. Our findings describe that acetylcholine signaling coordinate nutrition sensation and developmental regulation through fatty acid metabolic remodeling.

sentences: AC2P20 selectively kills M. tuberculosis  at acidic pH by depleting free thiols Mycobacterium tuberculosis (Mtb) senses and adapts to host immune cues as part of its pathogenesis. One environmental cue sensed by Mtb is the acidic pH of its host niche in the macrophage phagosome. Disrupting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. Previously, a high throughput screen of a [~]220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb growth at acidic pH. The screen discovered chemical probes that kill Mtb at pH 5.7 but are inactive at pH 7.0. In this study, AC2P20 was prioritized for continued study to test the hypothesis that it was targeting Mtb pathways associated with pH-driven adaptation. RNAseq transcriptional profiling studies showed AC2P20 modulates expression of genes associated with redox homeostasis. Gene enrichment analysis revealed that the AC2P20 transcriptional profile had significant overlap with a previously characterized pH-selective inhibitor, AC2P36. Like AC2P36, we show that AC2P20 kills Mtb by selectively depleting free thiols at acidic pH. Mass spectrometry studies show the formation of a disulfide bond between AC2P20 and reduced glutathione, supporting a mechanism where AC2P20 is able to deplete intracellular thiols and dysregulate redox homeostasis. The observation of two independent molecules targeting free thiols to kill Mtb at acidic pH further supports that Mtb has restricted redox homeostasis and sensitivity to thiol-oxidative stress at acidic pH.

sentences: Do examinations prepare students for higher education? A lesson from the Covid-19 lockdown. The COVID-19 pandemic caused severe disruption to education in the UK in 2020, with most of the school teaching moving online and national school examinations being cancelled. This was particularly disruptive for those taking end of school examinations in preparation for higher education. Biological science courses require students to absorb a lot of new vocabulary and concepts, with examinations traditionally focusing on content recall rather than reasoning. Students who had entered university in September 2019 were compared with those arriving in September 2020 with respect to their knowledge of bioscience vocabulary and understanding of key concepts. Results showed no significant difference between those who had gone through the examination process in 2019 relative to those who had not, in 2020. This suggests the cramming of information for examinations has no detectable effect on the knowledge and understanding of biology that students take with them to university.

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An atypical ABC transporter is involved in antifungal resistance and host interactions in the pathogenic fungus Cryptococcus neoformans ATP-binding cassette (ABC) transporters represent one of the largest protein superfamilies. Functionally diverse, ABC transporters have been implicated in many aspects of microbial physiology. The genome of the human fungal pathogen Cryptococcus neoformans encodes 54 putative ABC transporters and the majority of them remain uncharacterized. In a previous genetic screen for fungal regulators of phagocytosis, we identified an uncharacterized gene, CNAG_06909, that modulates host interactions. This gene encodes a half-size ABC transporter of the PDR-type, and phenotypic studies of a strain missing this gene revealed an altered antifungal profile, including hypersensitivity to fluconazole (FLC). This gene, which we have named PDR6, localizes to the endoplasmic reticulum (ER) and plasma membrane (PM), and when absent, less ergosterol was observed in the PM. Additionally, we observed that the pdr6{Delta} strain displays a reduction in secreted polysaccharide capsular material. These changes to the cellular surface may explain the observed increased uptake by macrophages and the reduced intracellular survival. Finally, studies in mice demonstrate that PDR6 function is required for normal progression of cryptococcal infection. Taken together, this study demonstrates a novel dual role for PDR transporters in C. neoformans, which could represent a potential target for antifungal therapeutics. Furthermore, the atypical half-size transporter encoded by PDR6 is conserved in many fungal pathogens, but absent in non-pathogenic fungal models. Hence, this study provides for the first time, a function for this unique group of fungal half-size PDR transporters that, although conserved, remain largely understudied.

IMPORTANCEConserved across all kingdoms of life, ABC transporters comprise one of the largest protein families. They are associated with multidrug resistance, affecting aspects such as resistance to antimicrobials or cancer treatments. Despite their importance, they are understudied in fungal pathogens. In the environmental fungus Cryptococcus neoformans, a leading cause of fungal infections, only few ABC transporters have been studied. Here we characterize an atypical, half-size, ABC transporter of the PDR-type, that affects both antifungal resistance and host-pathogen interactions. PDR-type transporters are only present in fungi and plants, and this subgroup of half-size transporters is conserved in fungal pathogens, yet their function was completely unknown. Because the current treatments for cryptococcal infection are suboptimal, understanding the mechanisms of antifungal resistance and the host interactions that drive the infection is critical to improve the management of this disease. Here we provide insights into these important aspects of cryptococcal pathogenesis.

sentences: Lattice micropatterning for cryo-electron tomography studies of cell-cell contacts Cryo-electron tomography is the highest resolution tool available for structural analysis of macromolecular complexes within their native cellular environment. At present, data acquisition suffers from low throughput, in part due to the low probability of positioning a cell such that the subcellular structure of interest is on a region of the electron microscopy (EM) grid that is suitable for imaging. Here, we photo-micropatterned EM grids to optimally position endothelial cells so as to enable high-throughput imaging of cell-cell contacts. Lattice micropatterned grids increased the average distance between intercellular contacts and the thicker cell nuclei such that the regions of interest were sufficiently thin for direct imaging. We observed a diverse array of membranous and cytoskeletal structures at intercellular contacts, demonstrating the utility of this technique in enhancing the rate of data acquisition for cellular cryo-electron tomography studies.

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Representation learning for neural population activity with Neural Data Transformers Neural population activity is theorized to reflect an underlying dynamical structure. This structure can be accurately captured using state space models with explicit dynamics, such as those based on recurrent neural networks (RNNs). However, using recurrence to explicitly model dynamics necessitates sequential processing of data, slowing real-time applications such as brain-computer interfaces. Here we introduce the Neural Data Transformer (NDT), a non-recurrent alternative. We test the NDTs ability to capture autonomous dynamical systems by applying it to synthetic datasets with known dynamics and data from monkey motor cortex during a reaching task well-modeled by RNNs. The NDT models these datasets as well as state-of-the-art recurrent models. Further, its non-recurrence enables 3.9ms inference, well within the loop time of real-time applications and more than 6 times faster than recurrent baselines on the monkey reaching dataset. These results suggest that an explicit dynamics model is not necessary to model autonomous neural population dynamics.

Codegithub.com/snel-repo/neural-data-transformers.

sentences: DNA methylation meta-analysis confirms the division of the genome into two functional groups Based on a meta-analysis of human genome methylation data, we tested a theoretical model in which aging is explained by the redistribution of limited resources in cells between two main tasks of the organism: its self-sustenance based on the function of the housekeeping gene group (HG) and functional differentiation, provided by the (IntG) integrative gene group. A meta-analysis of methylation of 100 genes, 50 in the HG group and 50 in IntG, showed significant differences (p<0.0001) between our groups in the level of absolute methylation values of genes bodies and its promoters. We showed a reliable decrease of absolute methylation values in IntG with rising age in contrast to HG, where this level remained constant. The one-sided decrease in methylation in the IntG group is indirectly confirmed by the dispersion data analysis, which also decreased in the genes of this group. The imbalance between HG and IntG in methylation levels suggests that this IntG-shift is a side effect of the ontogenesis grownup program and the main cause of aging. The theoretical model of functional genome division also suggests the leading role of slow dividing and post mitotic cells in triggering and implementing the aging process.

sentences: An efficient algorithm for estimating population history from genetic data The Legofit statistical package uses genetic data to estimate parameters describing population history. Previous versions used computer simulations to estimate probabilities, an approach that limited both speed and accuracy. This article describes a new deterministic algorithm, which makes Legofit faster and more accurate. The speed of this algorithm declines as model complexity increases. With very complex models, the deterministic algorithm is slower than the stochastic one. In an application to simulated data sets, the estimates produced by the deterministic and stochastic algorithms were essentially identical. Reanalysis of a human data set replicated the findings of a previous study and provided increased support for the hypotheses that (a) early modern humans contributed genes to Neanderthals, and (b) a "superarchaic" population (which separated from all other humans early in the Pleistocene) was either large or deeply subdivided.

sentences: Commensal Pseudomonas fluorescens protect Arabidopsis from closely-related Pseudomonas pathogens in a colonization-dependent manner Plants form commensal associations with soil microorganisms, creating a root microbiome that provides benefits to the host including protection against pathogens. While bacteria can inhibit pathogens through production of antimicrobial compounds in vitro, it is largely unknown how microbiota contribute to pathogen protection in planta. We developed a gnotobiotic model system consisting of Arabidopsis thaliana, and an opportunistic pathogen Pseudomonas sp. N2C3, to identify mechanisms that determine the outcome of plant-pathogen-microbiome interactions in the rhizosphere. We screened 25 phylogenetically diverse Pseudomonas strains for their ability to protect against N2C3 and found that commensal strains closely related to N2C3 were more likely to protect against pathogenesis. We used a comparative genomics approach to identify unique genes in the protective strains that revealed no genes that correlate with protection, suggesting that variable regulation of components of the core Pseudomonas genome may contribute to pathogen protection. We found that commensal colonization level was highly predictive of protection and so tested deletions in genes previously shown to be required for Arabidopsis rhizosphere colonization. We identified a response regulator colR that is required for Pseudomonas protection from N2C3 and fitness in competition with N2C3 indicating that competitive exclusion may contribute to pathogen protection. We found that Pseudomonas WCS365 also protects against the agricultural pathogen Pseudomonas fuscovaginae SE-1, the causal agent of bacterial sheath brown rot of rice. This work establishes a gnotobiotic model to uncover mechanisms by which members of the microbiome can protect hosts from pathogens and informs our understanding of the use of beneficial strains for microbiome engineering in dysbiotic soil systems.

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Differential regulation of degradation and immune pathways underlies adaptation of the ectosymbiotic nematode Laxus oneistus to oxic-anoxic interfaces Eukaryotes may experience oxygen deprivation under both physiological and pathological conditions. Because oxygen shortage leads to a reduction in cellular energy production, all eukaryotes studied so far conserve energy by suppressing their metabolism. However, the molecular physiology of animals that naturally and repeatedly experience anoxia is underexplored. One such animal is the marine nematode Laxus oneistus. It thrives, invariably coated by its sulfur-oxidizing symbiont Candidatus Thiosymbion oneisti, in anoxic sulfidic or hypoxic sand. Here, transcriptomics and proteomics showed that, whether in anoxia or not, L. oneistus mostly expressed genes involved in ubiquitination, energy generation, oxidative stress response, immune response, development, and translation. Importantly, ubiquitination genes were also upregulated when the nematode was subjected to anoxic sulfidic conditions, together with genes involved in autophagy, detoxification and ribosome biogenesis. We hypothesize that these degradation pathways were induced to recycle damaged cellular components (mitochondria) and misfolded proteins into nutrients. Remarkably, when L. oneistus was subjected to anoxic sulfidic conditions, lectin and mucin genes were also upregulated, potentially to promote the attachment of its thiotrophic symbiont. Furthermore, the nematode appeared to survive oxygen deprivation by using an alternative electron carrier (rhodoquinone) and acceptor (fumarate), to rewire the electron transfer chain. On the other hand, under hypoxia, genes involved in costly processes (e.g., amino acid biosynthesis, development, feeding, mating) were upregulated, together with the worms Toll- like innate immunity pathway and several immune effectors (e.g., Bacterial Permeability Increasing proteins, fungicides).

In conclusion, we hypothesize that, in anoxic sulfidic sand, L. oneistus upregulates degradation processes, rewires oxidative phosphorylation and by reinforces its coat of bacterial sulfur-oxidizers. In upper sand layers, instead, it appears to produce broad-range antimicrobials and to exploit oxygen for biosynthesis and development.

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MRI-derived brain age as a biomarker of ageing in rats: validation using a healthy lifestyle intervention MRI data can be used as input to machine learning models to accurately predict brain age in healthy human subjects. A large difference between predicted and chronological brain age (the so-called BrainAGE score) has been associated with disease and neurodegeneration, indicating the potential utility of neuroimaging-based ageing biomarkers. So far, most brain age prediction studies have been carried out on humans. However, it is important for such a biomarker to be validated on laboratory animals too, in order to better account for specific environmental or genetic factors within a more controlled laboratory framework.

In this work, we developed a new algorithm for rat brain age prediction based on the combination of Gaussian process regression and a logistic regression classifier. The algorithm was trained on a cohort of 31 normal rats. High prediction accuracy was achieved using leave-one-out cross-validation (mean absolute error = 4.87 weeks, correlation between predicted and chronological age r = 0.92), supporting the validity and potential of the method.

Furthermore, the trained model was tested on two independent groups of 24 rats each: a new normal control group and a "healthy lifestyle" group that underwent long-term environmental enrichment and dietary restriction (EEDR) between 3 and 17 months of age. After fitting a linear mixed-effects model, the BrainAGE values were found to increase more slowly with chronological age in the EEDR group than in the controls (slope = 0.52 vs. 0.61; p = 0.015 for the interaction term). When survival analysis was performed with a Cox regression model, the BrainAGE score at 5 months of age had a significant prediction power (p = 0.03).

Our results demonstrate that BrainAGE, as computed by the proposed approach, is significantly modulated by EEDR intervention, hence it is a sensitive marker of biological ageing. These findings also support the potential of lifestyle-related prevention approaches to slow down the brain ageing process. Moreover, the results of the survival analysis further demonstrate that BrainAGE is indeed a predictor of ageing outcome.

sentences: Senataxin and RNase H2 act redundantly to suppress genome instability during class switch recombination Class switch recombination generates antibody distinct isotypes critical to a robust adaptive immune system and defects are associated with auto-immune disorders and lymphomagenesis. Transcription is required during class switch to recruit the cytidine deaminase AID--an essential step for the formation of DNA double-strand breaks--and strongly induces the formation of R loops within the immunoglobulin heavy chain locus. However, the impact of R loops on double-strand break formation and repair during class switch recombination remains unclear. Here we report that cells lacking two enzymes involved in R loop removal-- Senataxin and RNase H2--exhibit increased R loop formation and genome instability at the immunoglobulin heavy chain locus without impacting class switch recombination efficiency, transcriptional activity, or AID recruitment. Senataxin and RNase H2-deficient cells also exhibit increased insertion mutations at switch junctions, a hallmark of alternative end joining. Importantly, these phenotypes were not observed in cells lacking Senataxin or RNase H2B alone. We propose that Senataxin acts redundantly with RNase H2 to mediate timely R loop removal, promoting efficient repair while suppressing AID-dependent genome instability and insertional mutagenesis.

sentences: A quantile integral linear model to quantify genetic effects on phenotypic variability Detecting genetic variants associated with the variance of complex traits, i.e. variance quantitative trait loci (vQTL), can provide crucial insights into the interplay between genes and environments and how they jointly shape human phenotypes in the population. We propose a quantile integral linear model (QUAIL) to estimate genetic effects on trait variability. Through extensive simulations and analyses of real data, we demonstrate that QUAIL provides computationally efficient and statistically powerful vQTL mapping that is robust to non-Gaussian phenotypes and confounding effects on phenotypic variability. Applied to UK Biobank (N=375,791), QUAIL identified 11 novel vQTL for body mass index (BMI). Top vQTL findings showed substantial enrichment for interactions with physical activities and sedentary behavior. Further, variance polygenic scores (vPGS) based on QUAIL effect estimates showed superior predictive performance on both population-level and within-individual BMI variability compared to existing approaches. Overall, QUAIL is a unified framework to quantify genetic effects on the phenotypic variability at both single-variant and vPGS levels. It addresses critical limitations in existing approaches and may have broad applications in future gene-environment interaction studies.

sentences: The human nose organoid respiratory virus model: an ex-vivo human challenge model to study RSV and SARS-CoV-2 pathogenesis and evaluate therapeutics There is an unmet need for pre-clinical models to understand the pathogenesis of human respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a non-invasive technique to generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made air liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation) while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration dependent manner. Thus, the HNO-ALI model can serve as an alternate to lung organoids to study respiratory viruses and testing therapeutics.

sentences: Dendrites use mechanosensitive channels to proofread ligand-mediated guidance during morphogenesis. Ligand-receptor interactions guide axon navigation and dendrite arborization. Mechanical forces also influence guidance choices. However, the nature of such mechanical stimulations, the mechanosensors identity and how they interact with guidance receptors are unknown. Here, we demonstrate that mechanosensitive DEG/ENaC channels are required for dendritic arbor morphogenesis in Caenorhabditis elegans. Inhibition of DEG/ENaC channels causes reduced dendritic outgrowth and branching in vivo, a phenotype that is alleviated by overexpression of the mechanosensitive channels PEZO-1/Piezo or YVC1/TrpY1. DEG/ENaCs trigger local Ca2+ transients in growing dendritic filopodia via activation of L-type voltage-gated Ca2+ channels. Anchoring of filopodia by guidance ligand/receptor is required for the mechanical activation of DEG/ENaC channels. Therefore, mechanosensitive channels serve as a checkpoint for appropriate chemoaffinity by activating Ca2+ transients required for neurite growth.

sentences: Structures of the active HER2/HER3 receptor complex reveal dynamics at the dimerization interface induced by binding of a single ligand The Human Epidermal Growth Factor Receptor 2 (HER2) and HER3 form a potent pro-oncogenic heterocomplex upon binding of growth factor neuregulin-1{beta} (NRG1{beta})1-3. The mechanism by which HER2 and HER3 interact remains unknown in the absence of any structures of the complex. We isolated the NRG1{beta}-bound near full-length HER2/HER3 dimer and obtained a 2.9[A] cryo-electron microscopy (cryo-EM) reconstruction of the extracellular domain module which reveals unexpected dynamics at the HER2/HER3 dimerization interface. We show that the dimerization arm of NRG1{beta}-bound HER3 is unresolved likely because the apo HER2 monomer fails to undergo a ligand-induced conformational change needed to establish a HER3 dimerization arm binding pocket. In a second structure of an oncogenic extracellular domain mutant of HER2, S310F, we observe a compensatory interaction with the HER3 dimerization arm that stabilizes the dimerization interface. We show that both HER2/HER3 and HER2-S310F/HER3 retain the capacity to bind to the HER2-directed therapeutic antibody, trastuzumab, but the mutant complex does not bind to pertuzumab. Our 3.5[A] structure of the HER2-S310F/HER3/NRG1{beta}/trastuzumab Fragment antigen binding (Fab) complex shows that the receptor dimer undergoes a conformational change to accommodate trastuzumab. Thus, like oncogenic mutations, therapeutics exploit the intrinsic dynamics of the HER2/HER3 heterodimer. The unique features of a singly liganded HER2/HER3 heterodimer underscore the allosteric sensing of the ligand occupancy by the dimerization interface and explain why extracellular domains of HER2 do not homo-associate via canonical active dimer interface.

sentences: Incomplete bunyavirus particles contribute to within-host spread and between-host transmission Bunyaviruses lack a specific mechanism to ensure the incorporation of a complete set of genome segments into each virion, explaining the generation of incomplete virus particles lacking one or more genome segments. Such incomplete virus particles, which may represent the majority of particles produced, are generally considered to interfere with virus infection and spread. Using the three-segmented Rift Valley fever virus as a model bunyavirus, we here show that two distinct incomplete virus particle populations that are unable to spread autonomously, are able to efficiently complement each other in both mammalian and insect cells following co-infection. We further show that incomplete virus particles are capable of co-infecting mosquitoes, resulting in the rescue of infectious virus that is able to disseminate to the mosquito salivary glands. Our findings reveal a significant role of incomplete particles in within-host spread and between-host transmission, reminiscent of the life cycle of multipartite viruses.

sentences: Mechanically active integrins direct cytotoxic secretion at the immune synapse The secretory output of cell-cell interfaces must be tightly controlled in space and time to ensure functional efficacy. This is particularly true for the cytotoxic immune synapse (IS), the stereotyped junction formed between a cytotoxic lymphocyte and the infected or transformed target cell it aims to destroy1. Cytotoxic lymphocytes kill their targets by channeling a mixture of granzyme proteases and the pore forming protein perforin directly into the IS2, 3. The synaptic secretion of these toxic molecules constrains their deleterious effects to the target cell alone, thereby protecting innocent bystander cells in the surrounding tissue from collateral damage. Despite the importance of this process for immune specificity, the molecular and cellular mechanisms that establish secretory sites within the IS remain poorly understood. Here, we identified an essential role for integrin mechanotransduction in cytotoxic secretion using a combination of single cell biophysical measurements, ligand micropatterning, and functional assays. Upon ligand-binding, the L{beta}2 integrin LFA-1 functioned as a spatial cue, attracting lytic granules containing perforin and granzyme and inducing their fusion at closely adjacent sites within the synaptic membrane. LFA-1 molecules were subjected to pulling forces within these secretory domains, and genetic or pharmacological suppression of these forces abrogated cytotoxicity. We conclude that lymphocytes employ an integrin-dependent mechanical checkpoint to enhance both the potency and the security of their cytotoxic output.

sentences: Computational Prediction of Heteromeric Protein Complex Disassembly Order with Hybrid Monte Carlo/Molecular Dynamics Simulation The physicochemical entity of biological phenomenon in the cell is a network of biochemical reactions and the activity of such a network is regulated by multimeric protein complexes. Mass spectroscopy (MS) experiments and multimeric protein docking simulations based on structural bioinformatics techniques have revealed the molecular-level stoichiometry and static configuration of subcomplexes in their bound forms, then revealing the subcomplex populations and formation orders. Meanwhile, these methodologies are not designed to straightforwardly examine temporal dynamics of multimeric protein assembly and disassembly, essential physicochemical properties to understand functional expression mechanisms of proteins in the biological environment. To address the problem, we had developed an atomistic simulation in the framework of the hybrid Monte Carlo/Molecular Dynamics (hMC/MD) method and succeeded in observing disassembly of homomeric pentamer of the serum amyloid P component protein in experimentally consistent order. In this study, we improved the hMC/MD method to examine disassembly processes of the tryptophan synthase tetramer, a paradigmatic heteromeric protein complex in MS studies. We employed the likelihood-based selection scheme to determine a dissociation-prone subunit pair at each hMC/MD simulation cycle and achieved highly reliable predictions of the disassembly orders with the success rate over 0.9 without a priori knowledge of the MS experiments and structural bioinformatics simulations. We similarly succeeded in reliable predictions for the other three tetrameric protein complexes. These achievements indicate the potential availability of our hMC/MD approach as the general purpose methodology to obtain microscopic and physicochemical insights into multimeric protein complex formation.

sentences: Mutational effects of ubiquitously present gamma radiation on Arabidopsis thaliana: insight into radiosensitivity in the reproductive stage Earth has always been exposed to ionizing radiation from natural sources, and man-made sources have added to this radiation. In order to assess mutational effects of ubiquitously present radiation on plants, we performed a whole-genome resequencing analysis of mutations induced by chronic irradiation throughout the life-cycle of Arabidopsis thaliana under controlled conditions. Resequencing data from 12 M1 lines and 36 M2 progeny derived under gamma-irradiation conditions ranging from 0.0 to 2.0 Gy/d were obtained to identify de novo mutations, including single base substitutions (SBSs) and small insertions/deletions (INDELs). The relationship between de novo mutation frequency and a low-to-middling dose of radiation was assessed by statistical modeling. The increasing of de novo mutations in response to doses of irradiation fit the negative binomial model, accounting for the high variability of mutation frequency observed. Among the different types of mutations, SBSs were more prevalent than INDELs, with deletions being more frequent than insertions. Furthermore, we observed that the mutational effects of chronic radiation are more intensive during the reproductive stage. These outcomes could provide valuable insights into practical strategies for environmental radioprotection of plants on Earth and in space.

sentences: A Genome-Scale Metabolic Model of Marine Heterotroph Vibrio splendidus sp. 1A01 While the Vibrio splendidus species is best known as an opportunistic pathogen in oysters, the Vibrio splendidus sp. 1A01 strain was first identified as an early colonizer of synthetic chitin particles incubated in seawater. To gain a better understanding of its metabolism, a genome-scale metabolic model (GSMM) of V. splendidus sp. 1A01 was reconstructed. GSMMs enable us to simulate all metabolic reactions in a bacterial cell using Flux Balance Analysis. A draft model was built using an automated pipeline from BioCyc. Manual curation was then performed based on experimental data, in part by gap-filling metabolic pathways and tailoring the models biomass reaction to V. splendidus sp. 1A01. The challenges of building a metabolic model for a marine microorganism like V. splendidus sp. 1A01 are described.

sentences: PSGL-1 directs early TCR signaling to repress metabolism and promote T cell exhaustion by modulating the TCF-1/TOX axis in CD8+ T cells. Summary/AbstractWe previously identified the adhesion molecule PSGL-1 as a T cell intrinsic immune checkpoint regulator of T cell exhaustion. Here we show that the ability of PSGL-1 to restrain TCR sginaling correlates with decreased expression of the Zap70 inhibitor Ubash3b (Sts-1) in PSGL-1-deficient T cells. PSGL-1-deficency in T cells supports antitumor responses to a PD-1 blockade resistant melanoma wherein tumor-specific CD8+ T cells sustain an enhanced metabolic state, with an elevated metabolic gene signature that promotes increased glycolysis and glucose uptake to support effector functions. In models of chronic virus infection and cancer, this outcome was associated with CD8+ T cell stemness, as PSGL-1 deficient CD8+ T cells displayed increased TCF-1 and decreased TOX expression, a phenotype shown to be crucial for responsiveness to checkpoint inhibition. Further, we demonstrate that PSGL-1 signaling promotes development of exhaustion in human CD8+ T cells. Finally, pharmacologic blockade of PSGL-1 was sufficient to curtail T cell exhaustion and enhance functionality both with melanoma tumors and chronic LCMV infection, demonstrating that PSGL-1 represents a therapeutic target for immunotherapy for PD-1/PD-L1 resistant tumors.

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Selective neural coding of object, feature, and geometry spatial cues in humans Orienting in space requires the processing and encoding of visual spatial cues. The dominant hypothesis about the brain structures mediating the coding of spatial cues stipulates the existence of a hippocampal-dependent system for the representation of geometry and a striatal-dependent system for the representation of landmarks. However, this dual-system hypothesis is based on paradigms that presented spatial cues conveying either conflicting or ambiguous spatial information and that amalgamated the concept of landmark into both discrete 3D objects and wall features. These confounded designs introduce difficulties in interpreting the spatial learning process. Here, we test the hypothesis of a complex interaction between the hippocampus and the striatum during landmark and geometry visual coding in humans. We also postulate that object-based and feature-based navigation are not equivalent instances of landmark-based navigation as currently considered in human spatial cognition. We examined the neural networks associated with geometry-, object-, and feature-based spatial navigation in an unbiased, two-choice behavioral paradigm using fMRI. We showed evidence of a synergistic interaction between hippocampal and striatal coding underlying flexible navigation behavior. The hippocampus was involved in all three types of cue-based navigation, whereas the striatum was more strongly recruited in the presence of geometric cues than object or feature cues. We also found that unique, specific neural signatures were associated with each spatial cue. Critically, object-based navigation elicited a widespread pattern of activity in temporal and occipital regions relative to feature-based navigation. These findings challenge and extend the current view of a dual, juxtaposed hippocampal-striatal system for visual spatial coding in humans. They also provide novel insights into the neural networks mediating object vs. feature spatial coding, suggesting a need to distinguish these two types of landmarks in the context of human navigation.

Highlights O_LIComplex hippocampal-striatal interaction during visual spatial coding for flexible human navigation behavior.
C_LIO_LIDistinct neural signatures associated with object-, feature-, and geometry-based navigation.
C_LIO_LIObject- and feature-based navigation are not equivalent instances of landmark-based navigation.
C_LI

sentences: IsoSolve: an integrative framework to improve isotopic coverage and consolidate isotopic measurements by MS and/or NMR Stable-isotope labeling experiments are widely used to investigate the topology and functioning of metabolic networks. Label incorporation into metabolites can be quantified using a broad range of mass spectrometry (MS)and nuclear magnetic resonance (NMR)spectroscopy methods, but in general, no single approach can completely cover isotopic space, even for small metabolites. The number of quantifiable isotopic species could be increased, and the coverage of isotopic space improved, by integrating measurements obtained by different methods; however, this approach has remained largely unexplored because no framework able to deal with partial, heterogeneous isotopic measurements has yet been developed. Here, we present a generic computational framework based on symbolic calculus that can integrate any isotopic dataset by connecting measurements to the chemical structure of the molecules. As a test case, we apply this framework to isotopic analyses of amino acids, which are ubiquitous to life, central to many biological questions, and can be analyzed by a broad range of MS and NMR methods. We demonstrate how this integrative framework helps to i) clarify and improve the coverage of isotopic space, ii) evaluate the complementarity and redundancy of different techniques, iii) consolidate isotopic datasets, iv) design experiments, and v) guide future analytical developments. This framework, which can be applied to any labeled element, isotopic tracer, metabolite, and analytical platform, has been implemented in IsoSolve (available at https://github.com/MetaSysLISBP/IsoSolve and https://pypi.org/project/IsoSolve), an open source software that can be readily integrated into data analysis pipelines.

sentences: The miR-430 locus with extreme promoter density is a transcription body organizer, which facilitates long range regulation in zygotic genome activation In anamniote embryos the major wave of zygotic genome activation (ZGA) starts during the mid-blastula transition. This major wave of ZGA is facilitated by several mechanisms, including dilution of repressive maternal factors and accumulation of activating transcription factors during the fast cell division cycles preceding the mid-blastula transition. However, a set of genes escape global genome repression and are activated substantially earlier, during what is called, the minor wave of genome activation. While the mechanisms underlying the major wave of genome activation have been studied extensively, the minor wave of genome activation is little understood. In zebrafish the earliest expressed RNA polymerase II (Pol II) transcribed genes are activated in a pair of large transcription bodies depleted of chromatin, abundant in elongating Pol II and nascent RNAs (Hadzhiev et al., 2019; Hilbert et al., 2021). This transcription body includes the miR-430 gene cluster required for maternal mRNA clearance. Here we explored the genomic, chromatin organisation and cis-regulatory mechanisms of the minor wave of genome activation occurring in the transcription body. By long read genome sequencing we identified a remarkable cluster of miR-430 genes with over 300 promoters and spanning 0.6 Mb, which represent the highest promoter density of the genome. We demonstrate that the miR-430 gene cluster is required for the formation of the transcription body and acts as a transcription organiser for minor wave activation of a set of zinc finger genes scattered on the same chromosome arm, which share promoter features with the miR-430 cluster. These promoter features are shared among minor wave genes overall and include the TATA-box and sharp transcription start site profile. Single copy miR-430 promoter transgene reporter experiments indicate the importance of promoter-autonomous mechanisms regulating escape from global repression of the early embryo. These results together suggest that formation of the transcription body in the early embryo is the result of high promoter density coupled to a minor wave-specific core promoter code for transcribing key minor wave ZGA genes, which are required for the overhaul of the transcriptome during early embryonic development.

sentences: Maternal SMCHD1 controls both imprinted Xist expression and imprinted X chromosome inactivation Embryonic development is dependent on the maternal supply of proteins through the oocyte, including factors setting up the adequate epigenetic patterning of the zygotic genome. We previously reported that one such factor is the epigenetic repressor SMCHD1, whose maternal supply controls autosomal imprinted expression in mouse preimplantation embryos and mid-gestation placenta. In mouse preimplantation embryos, X chromosome inactivation is also an imprinted process. Combining genomics and imaging, we show that maternal SMCHD1 is required not only for the imprinted expression of Xist in preimplantation embryos, but also for the efficient silencing of the inactive X in both the preimplantation embryo and mid-gestation placenta. These results expand the role of SMCHD1 in enforcing the silencing of Polycomb targets. The inability of zygotic SMCHD1 to fully restore imprinted X inactivation further points to maternal SMCHD1s role in setting up the appropriate chromatin environment during preimplantation development, a critical window of epigenetic remodelling.

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Training neural networks to recognize speech increased their correspondence to the human auditory pathway but did not yield a shared hierarchy of acoustic features The correspondence between the activity of artificial neurons in convolutional neural networks (CNNs) trained to recognize objects in images and neural activity collected throughout the primate visual system has been well documented. Shallower layers of CNNs are typically more similar to early visual areas and deeper layers tend to be more similar to later visual areas, providing evidence for a shared representational hierarchy. This phenomenon has not been thoroughly studied in the auditory domain. Here, we compared the representations of CNNs trained to recognize speech (triphone recognition) to 7-Tesla fMRI activity collected throughout the human auditory pathway, including subcortical and cortical regions, while participants listened to speech. We found no evidence for a shared representational hierarchy of acoustic speech features. Instead, all auditory regions of interest were most similar to a single layer of the CNNs: the first fully-connected layer. This layer sits at the boundary between the relatively task-general intermediate layers and the highly task-specific final layers. This suggests that alternative architectural designs and/or training objectives may be needed to achieve fine-grained layer-wise correspondence with the human auditory pathway.

HighlightsO_LITrained CNNs more similar to auditory fMRI activity than untrained
C_LIO_LINo evidence of a shared representational hierarchy for acoustic features
C_LIO_LIAll ROIs were most similar to the first fully-connected layer
C_LIO_LICNN performance on speech recognition task positively associated with fmri similarity
C_LI

sentences: Evolution and systematics of the Aculeata and kin (Hymenoptera),with emphasis on the ants (Formicoidea: {dagger}@@@idae fam. nov., Formicidae). Fossils provide unique opportunity to understand the tempo and mode of evolution and are essential for modeling the history of lineage diversification. Here, we interrogate the Mesozoic fossil record of the Aculeata, with emphasis on the ants (Formicidae), and conduct an extended series of ancestral state estimation exercises on distributions of tip-dated combined-evidence phylogenies. We developed and illustrated from ground-up a series of 576 morphological characters which we scored for 144 extant and 431 fossil taxa, including all families of Aculeata, Trigonaloidea, Evanioidea, and {dagger}Ephialtitoidea. We used average posterior probability support to guide composition of a target matrix of 303 taxa, for which we integrated strongly filtered ultraconserved element (UCE) data for 115 living species. We also implemented reversible jump MCMC (rjMCMC) and hidden state methods to model complex behavioral characters to test hypotheses about the pathway to obligate eusociality. In addition to revising the higher classification of all sampled groups to family or subfamily level using estimated character polarities to diagnose nodes across the phylogeny, we find that the mid-Cretaceous genera {dagger}Camelomecia and {dagger}Camelosphecia form a clade which is robustly supported as sister to all living and fossil Formicidae. For this reason, we name this extinct clade as {dagger}@@@idae fam. nov. and provide a definition for the expanded Formicoidea. Based on our results, we recognize three major phases in the early evolution of the ants: (1) origin of Formicoidea as ground-adapted huntresses during the Late Jurassic in the "stinging aggressor" guild (Aculeata) among various lineages of "sneaking parasitoids" (non-aculeate Vespina); (2) the first formicoid radiation during the Early Cretaceous, by the end of which all major extant linages originated; and (3) turnover of the Formicoidea at the end-Cretaceous leading to the second formicoid radiation. We conclude with a concentrated series of considerations for future directions of study with this dataset and beyond.

sentences: Permissive aggregative group formation favors coexistence in yeast In Saccharomyces cerevisiae, the FLO1 gene encodes flocculins that lead to formation of multicellular flocs, that offer protection to the constituent cells. Flo1p was found to preferentially bind to fellow cooperators compared to defectors lacking FLO1 expression, resulting in enrichment of cooperators within the flocs. Given this dual function in cooperation and kin recognition, FLO1 has been termed a  green beard gene. Because of the heterophilic nature of Flo1p binding however, we hypothesize that kin recognition is permissive and depends on the relative stability of FLO1+/flo1- versus FLO1+/FLO1+ bonds, which itself can be dependent on environmental conditions and intrinsic cell properties. We combine single cell measurements of adhesion strengths, individual cell-based simulations of cluster formation and evolution, and in vitro flocculation experiments to study the impact of relative bond stability on defector exclusion as well as benefit and stability of cooperation. We hereto vary the relative bond stability by changing the shear flow rate and the inherent bond strength. We identify a marked trade-off between both aspects of the green beard mechanism, with reduced relative bond stability leading to increased kin recognition, but at the expense of decreased cluster sizes and benefit of cooperation. Most notably, we show that the selection of FLO1 cooperators is negative-frequency dependent, which we directly attribute to the permissive character of the Flo1p bond. Taking into account the costs associated to FLO1 expression, this asymmetric selection results in a broad range of ecological conditions where coexistence between cooperators and defectors is stable. Although the kin recognition aspect of the FLO1  green beard gene is thus limited and condition dependent, the negative-frequency dependency of selection can conserve the diversity of flocculent and non-flocculent phenotypes ensuring flexibility towards variable selective pressures.

sentences: Cortical adaptation to sound reverberation In almost every natural environment, sounds are reflected by nearby objects, producing many delayed and distorted copies of the original sound, known as reverberation. Our brains usually cope well with reverberation, allowing us to recognize sound sources regardless of their environments. In contrast, reverberation can cause severe difficulties for speech recognition algorithms and hearing-impaired people. The present study examines how the auditory system copes with reverberation. We trained a linear model to recover a rich set of natural, anechoic sounds from their simulated reverberant counterparts. The model neurons achieved this by extending the inhibitory component of their receptive filters for more reverberant spaces, and did so in a frequency-dependent manner. These predicted effects were observed in the responses of auditory cortical neurons of ferrets in the same simulated reverberant environments. Together, these results suggest that auditory cortical neurons adapt to reverberation by adjusting their filtering properties in a manner consistent with dereverberation.

sentences: Evaluation of lamin A/C mechanotransduction under different surface topography in LMNA related muscular dystrophy. Most of the single point mutations of the LMNA gene are associated with distinct muscular dystrophies, marked by heterogenous phenotypes but primarily the loss and symmetric weakness of skeletal muscle tissue. The molecular mechanism and phenotype-genotype relationships in these muscular dystrophies are poorly understood. An effort has been here to delineating the adaptation of mechanical inputs into biological response by mutant cells of lamin A associated muscular dystrophy. In this study we implement engineered smooth and pattern surfaces of particular young modulus to mimic muscle physiological range. Using fluorescence and atomic force microscopy we present distinct architecture of the actin filament along with abnormally distorted cell and nuclear shape in mutants, which showed a tendency to deviate from wild type cells. Topographic features of pattern surface antagonizes the binding of the cell with it. Correspondingly, from the analysis of genome wide expression data in wild type and mutant cells, we report differential expression of the gene products of the structural components of cell adhesion as well as LINC (linkers of nucleoskeleton and cytoskeleton) protein complexes. This study also reveals mis expressed downstream signaling processes in mutant cells, which could potentially lead to onset of the disease upon the application of engineered materials to substitute the role of conventional cues in instilling cellular behaviors in muscular dystrophies. Collectively, these data support the notion that lamin A is essential for proper cellular mechanotransduction from extracellular environment to the genome and impairment of the muscle cell differentiation in the pathogenic mechanism for lamin A associated muscular dystrophy.

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SkewDB: A comprehensive database of GC and a 10 other skews for over 28,000 chromosomes and plasmids GC skew denotes the relative excess of G nucleotides over C nucleotides on the leading versus the lagging replication strand of eubacteria. While the effect is small, typically around 2.5%, it is robust and pervasive. GC skew and the analogous TA skew are a localized deviation from Chargaffs second parity rule, which states that G and C, and T and A occur with (mostly) equal frequency even within a strand.

Most bacteria also show the analogous TA skew. Different phyla show different kinds of skew and differing relations between TA and GC skew.

This article introduces an open access database (https://skewdb.org) of GC and 10 other skews for over 28,000 chromosomes and plasmids. Further details like codon bias, strand bias, strand lengths and taxonomic data are also included. The SkewDB database can be used to generate or verify hypotheses. Since the origins of both the second parity rule, as well as GC skew itself, are not yet satisfactorily explained, such a database may enhance our understanding of microbial DNA.

sentences: Structures of SARS-CoV-2 B.1.351 neutralizing antibodies provide insights into cocktail design against concerning variants The spread of the SARS-CoV-2 variants could seriously dampen the global effort to tackle the COVID-19 pandemic. Recently, we investigated the humoral antibody responses of SARS-CoV-2 convalescent patients and vaccinees towards circulating variants, and identified a panel of monoclonal antibodies (mAbs) that could efficiently neutralize the B.1.351 (Beta) variant. Here we investigate how these mAbs target the B.1.351 spike protein using cryo-electron microscopy. In particular, we show that two superpotent mAbs, BD-812 and BD-836, have non-overlapping epitopes on the receptor-binding domain (RBD) of spike. Both block the interaction between RBD and the ACE2 receptor; and importantly, both remain fully efficacious towards the B.1.617.1 (Kappa) and B.1.617.2 (Delta) variants. The BD-812/BD-836 pair could thus serve as an ideal antibody cocktail against the SARS-CoV-2 VOCs.

sentences: The Detection of Bergeyella zoohelcum in Young Children after Cat Bite Bergeyella zoohelcum is an uncommon zoonotic pathogen typically associated with cat or dog bites. Previously, only 14 cases of B. zoohelcum infection have been reported. We isolated the bacteria from the face of a 2-year-old girl who was bitten by a cat. The organism was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequence, and phylogenetic analysise supported that this isolate was belong to B. zoohelcum. Due to the contradicts of culture characteristics of B. zoohelcum which described in reported literatures, we used different media to culture the bacteria. After 24 h incubation, Colombia blood agar (CBA), Mueller-Hinton agar plate with 5% sheep blood and chocolate agar (CA) grew well, but blood agar (BA) grew well until 72 h. The strain did not grow on McConkey agar (MAC), Mueller-Hinton agar (MHA) and chocolate agar (containing vancomycin). The low detection rate of this strain was related to its harsh growth conditions and the limitations of traditional identification techniques. With the popularization of MALDI-TOF MS and 16S rRNA gene sequence and our further understanding of the fastidious bacteria, we will quickly and accurately identify the B. zoohelcum, meanwhile the detection rate of this bacterium will also be significantly improved. So that clinicians can achieve precise anti-infective treatment according to antimicrobial susceptibility testing.

sentences: Nuclear inhibitor of protein phosphatase 1 (NIPP1) regulates CNS tau phosphorylation and myelination during development Nuclear inhibitor of protein phosphatase 1 (NIPP1) is a known regulator of gene expression and has been shown to play roles in many physiological or pathological processes such as stem cell proliferation and skin inflammation. While NIPP1 has many regulatory roles in proliferating cells, its function in the central nervous system (CNS) has not been directly investigated. In the present study, we examined NIPP1 CNS function using a conditional knockout (cKO) mouse model, in which the nipp1 gene is excised from neural precursor cells. These mice demonstrate severe developmental impairments that lead to premature lethality within the first few postnatal weeks. To delineate some of the neurological changes occurring in these animals, we first assessed microtubule associated protein tau, a known target of NIPP1 activity. Furthermore, observed tremors prompted exploration of myelin integrity, an integral structure for CNS function, whose disruption is associated with various neurological disorders and neurodegenerative diseases. First, immunoblotting demonstrated increased phospho-tau and altered AKT and PP1 activity in NIPP1 cKO mice, suggesting increased tau phosphorylation likely results from a shift in kinase/phosphatase activity. Second, immunoblots, electron microscopy, and electrophysiology demonstrated a myelin deficit within the brain and optic nerve. Our study suggests that NIPP1 in neural precursors regulates phosphorylation of tau and CNS myelination and may represent a novel therapeutic target for neurodegenerative diseases.

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T cell derived HB-EGF prevents Th17 cell differentiation in an autocrine way. CD4 T cells critically contribute to host immunity against infections, but can also contribute to the development of autoimmune diseases. The underlying mechanisms that govern differentiation of naive CD4 T cells into different effector populations remain poorly understood. Here, we show that the expression of the Epidermal Growth Factor (EGF)-like growth factor HB-EGF by CD4 T cells sustained their expression of Interleukin (IL)-2 and reduced their capacity to differentiate into T Helper 17 (Th17) cells. Concordantly, mice with a T cell specific deficiency of HB-EGF showed an enhanced differentiation of naive CD4 T cells into Th17 cells and a more rapid onset of experimental autoimmune encephalomyelitis (EAE). Furthermore, transfer of naive HB-EGF-deficient CD4 T cells into Rag1-/- mice led to the rapid induction of multi-organ inflammation in recipient mice. Together, our data reveal a novel mechanism by which an HB-EGF-mediated constrain on Th17 differentiation prevents the development of autoimmune diseases.

SUMMARYCD4 T cell activation induces the expression of the EGFR and its high-affinity ligand HB-EGF. HB-EGF sustains IL-2 expression in an autocrine manner, preventing the differentiation of Th17 cells and the subsequent induction of Th17 cell-mediated autoimmune diseases.

sentences: Large-scale systematic feasibility study on the pan-cancer predictability of multi-omic biomarkers from whole slide images with deep learning We assessed the pan-cancer predictability of multi-omic biomarkers from haematoxylin and eosin (H&E)-stained whole slide images (WSI) using deep learning (DL) throughout a systematic study. A total of 13,443 DL models predicting 4,481 multi-omic biomarkers across 32 cancer types were trained and validated. The investigated biomarkers included a broad range of genetic, transcriptomic, proteomic, and metabolic alterations. Furthermore, established markers relevant for prognosis, molecular subtypes and clinical outcomes were included. Overall, we established the general feasibility of predicting multi-omic markers directly from routine histology images with DL across solid cancer types, where 50% of the models could perform at an area under the curve (AUC) of more than 0.633 (with 25% of the models having an AUC larger than 0.711). A wide range of biomarkers were detectable from routine histology images across all investigated cancer types, with a mean AUC of at least 0.62 in almost all malignancies. Strikingly, we observed that biomarker predictability was mostly consistent and not dependent on sample size and class ratio, suggesting a degree of true predictability inherent in histomorphology. Together, the results of our study show the potential of DL to predict a multitude of biomarkers across the omics spectrum using only routine slides. This paves the way for accelerating diagnosis and developing more precise treatments for cancer patients.

sentences: Spontaneous neural synchrony links intrinsic spinal sensory and motor networks during unconsciousness Non-random functional connectivity during unconsciousness is a defining feature of supraspinal networks. However, its generalizability to intrinsic spinal networks remains incompletely understood. Previously, Barry et al. (2014) used fMRI to reveal bilateral resting state functional connectivity within sensory-dominant and, separately, motor-dominant regions of the spinal cord. Here, we record spike trains from large populations of spinal interneurons in vivo in rats and demonstrate that spontaneous functional connectivity also links sensory- and motor-dominant regions during unconsciousness. The spatiotemporal patterns of connectivity could not be explained by latent afferent activity or by populations of interconnected neurons spiking randomly. We also document connection latencies compatible with mono- and di-synaptic interactions and putative excitatory and inhibitory connections. The observed activity is consistent with the hypothesis that salient, experience-dependent patterns of neural transmission introduced during behavior or by injury/disease are reactivated during unconsciousness. Such a spinal replay mechanism could shape circuit-level connectivity and ultimately behavior.

sentences: Pancreas resident macrophage-induced fibrosis has divergent roles in pancreas inflammatory injury and PDAC Tissue-resident macrophages (TRMs) are long-lived cells that maintain locally and can be phenotypically distinct from monocyte-derived macrophages (MDMs). However, whether TRMs and MDMs have functional distinction under differing pathologies is not understood. Here, we show a significant portion of macrophages that accumulated during pancreatitis and pancreatic cancer were expanded from TRMs. We further established that pancreas TRMs have a distinct extracellular matrix remodeling phenotype that was critical for maintaining tissue homeostasis during inflammation. Loss of TRMs led to exacerbation of severe pancreatitis and animal death, due to impaired acinar cell survival and recovery. In pancreatitis, TRMs elicited protective effects by triggering the accumulation and activation of fibroblasts, which was necessary for initiating fibrosis as a wound healing response. The same TRM-driven fibrosis, however, drove pancreas cancer pathogenesis and progression. Together, these findings indicate that TRMs play divergent roles in the pathogenesis of pancreatitis and cancer through regulation of stromagenesis.

sentences: A masculinizing supergene underlies an exaggerated male reproductive morph in a spider. In many species, individuals can develop into strikingly different morphs, which are determined by a simple Mendelian locus. How selection shapes loci that control complex phenotypic differences remains poorly understood. In the spider Oedothorax gibbosus, males either develop into a  hunched morph with conspicuous head structures or as a fast developing  flat morph with a female-like appearance. We show that the hunched-differs from the flat-determining allele by a hunch-specific genomic fragment of approximately 3 megabases. This fragment comprises dozens of genes that duplicated from genes found at different chromosomes. All functional duplicates, including doublesex - a key sexual differentiation regulatory gene, show male-specific expression, which illustrates their combined role as a masculinizing supergene. Our findings demonstrate how extensive indel polymorphisms and duplications of regulatory genes may contribute to the evolution of co-adapted gene clusters, sex-limited reproductive morphs, and the enigmatic evolution of exaggerated sexual traits in general.

sentences: Structural basis for receptor selectivity and inverse agonism in S1P5 receptors The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PR) - S1P1-5. S1P5 is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects. In this article, we describe a 2.2 [A] resolution room temperature crystal structure of the human S1P5 receptor in complex with a selective inverse agonist determined by serial femtosecond crystallography (SFX) at the Pohang Accelerator Laboratory X-Ray Free Electron Laser (PAL-XFEL) and analyze its structure-activity relationship data. The structure demonstrates a unique ligand-binding mode, involving an allosteric subpocket, which clarifies the receptor subtype selectivity and provides a template for structure-based drug design. Together with previously published S1PR structures in complex with antagonists and agonists, the new S1P5-inverse agonist structure sheds light on the activation mechanism and reveals structural determinants of the inverse agonism in the S1PR-family.

sentences: Single neuron activity predicts behavioral performance of individual animals during memory retention In 1972 Rescorla and Wagner formulated their model of classical Pavlovian conditioning postulating that the associative strength of a stimulus is expressed directly in the behavior it elicits1. Many biologists and psychologists were inspired by this model, and numerous experiments thereafter were interpreted assuming that the magnitude of the conditioned response (CR) reflects an associative effect at the physiological level. However, a correlation between neural activity and the expression of the CR in individual animals has not yet been reported. Here we show that, following differential odor conditioning, the change in activity of single mushroom body output neurons (MBON) of the honeybee predicts the behavioral performance of the individual during memory retention. The encoding of the stimulus-reward association at the mushroom body output occurs about 600 ms before the initiation of the CR. We conclude that the MB provides a stable representation of the stimulus-reward associative strength, and that this representation is required for behavioral decision-making during memory retention.

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Orientation dependent transverse relaxation in human brain white matter: The magic angle effect on a cylindrical helix PurposeTo overcome some limitations of prior orientation-dependent proton transverse relaxation formalisms in white matter (WM) with a novel framework based on the generalized magic angle effect function.

MethodsA cylindrical helix model was developed embracing anisotropic rotational and translational diffusion of restricted molecules in human brain WM, with the former characterized by an axially symmetric system. Transverse relaxation rates R2 and [Formula] were divided into isotropic [Formula] and anisotropic parts, [Formula], with  denoting an open angle and{varepsilon} 0 an orientation ({Phi}) offset from DTI-derived primary diffusivity direction. The proposed framework (Fit A) was compared with prior models without{varepsilon} 0 on previously published water and methylene proton transverse relaxation rates from developing, healthy, and pathological WM at 3T. Goodness of fit was represented by root-mean-square error (RMSE). F-test and linear correlation were used with statistical significance set to P [≤] 0.05.

ResultsFit A significantly (P<0.01) outperformed prior models as demonstrated by reduced RMSEs, e.g., 0.349 vs. 0.724 in myelin water. Fitted{varepsilon} 0 was in good agreement with calculated{varepsilon} 0 from directional diffusivities. Compared with those from healthy adult, the fitted [Formula], and  from neonates were substantially reduced but{varepsilon} 0 increased, consistent with incomplete myelination. Significant positive [Formula] and negative ( and [Formula]) correlations were found with aging (demyelination) in elderly.

ConclusionThe developed framework can better characterize orientation dependences from a wide range of proton transverse relaxation measurements in human brain WM, shedding new light on myelin microstructural alterations at the molecular level.

sentences: Wnt and BMP signaling enhance melanocyte regeneration and suppress melanoma cell migration Tissue regeneration and cancer share remarkable features including activation of cell proliferation and migration. Yet, tumors considerably differ from the regenerating tissue with respect to abnormal proliferation, invasive growth and metastasis. Thus, it is likely that cancer resembles early stages of regeneration with increased proliferation, but separates from the later stages with reduced proliferation and enhanced differentiation. Here, by exploiting the zebrafish melanocytes that can efficiently regenerate and be induced to undergo malignant melanoma, we unravel the transcriptome profiles of the regenerating melanocytes during early and late regeneration, and the melanocytic nevi and malignant melanoma. Our global comparison of the gene expression profiles of melanocyte regeneration and nevi/melanoma uncovers opposite regulation of Wnt and BMP signaling pathways between regeneration and cancer. Functional inhibition of canonical Wnt or BMP pathways during melanocyte regeneration impeded the regenerative capacity of the melanocytes. In contrast, activation of either pathway significantly promoted melanocyte regeneration and potently suppressed migration of human melanoma cells. Therefore, mechanisms of regeneration can be exploited to correct dysregulated molecular pathways and malignant transformation in cancer.

sentences: The Impact of Multivesicular Release on the Transmission of Sensory Information by Ribbon Synapses The statistics of vesicle release determine how information is transferred at the synapse but the classical Poisson model of independent fusion does not hold at the first stages of vision and hearing. There, ribbon synapses encoding analogue signals can coordinate the release of two or more vesicles as a single event. The implications of such multivesicular release (MVR) for spike generation are not known. Here we investigate the hypothesis that MVR might provide advantages over Poisson synapses. We used leaky integrate-and-fire models incorporating the statistics of release measured experimentally from glutamatergic synapses of retinal bipolar cells and compared these with models assuming Poisson inputs constrained to operate at the same average rates. We find that MVR can increase the number of spikes generated per vesicle while reducing interspike intervals and latency to first spike. The combined effect was to increase the efficiency of information transfer (bits per spike) over a range of conditions mimicing retinal ganglion cells of different size. MVR was most advantageous in neurons with short time-constants and reliable synaptic inputs, when a lower degree of convergence was required to trigger spikes. In the special case of a single input driving a neuron with short time-constant, as occurs at the first synapse in hearing, MVR increased information transfer whenever spike generation required more than one vesicle. This study demonstrates how presynaptic integration of vesicles by MVR has the potential to compensate for less effective summation post-synaptically to increase the efficiency with which sensory information is transmitted.

sentences: Identification of leptospiral protein antigens recognized by WC1+ γδ T cell subsets as target for development of recombinant vaccines Pathogenic Leptospira species cause leptospirosis, a neglected zoonotic disease recognized as a global public health problem. It is also the cause of the most common cattle infection that results in major economic losses due to reproductive problems. {gamma}{delta} T cells play a role in the protective immune response in livestock species against Leptospira while human {gamma}{delta} T cells also respond to Leptospira. Thus, activation of {gamma}{delta} T cells has emerged as a potential component for optimization of vaccine strategies. Bovine {gamma}{delta} T cells proliferate and produce IFN-{gamma} in response to vaccination with inactivated leptospires and this response is mediated by a specific subpopulation of the WC1-bearing {gamma}{delta} T cells. WC1 molecules are members of the group B scavenger receptor cysteine rich (SRCR) superfamily and are composed of multiple SRCR domains, of which particular extracellular domains act as ligands for Leptospira. Since WC1 molecules function as both pattern recognition receptors and {gamma}{delta} TCR coreceptors, the WC1 system has been proposed as a novel target to engage {gamma}{delta} T cells. Here, we demonstrate the involvement of leptospiral protein antigens in the activation of WC1+ {gamma}{delta} T cells and identified two leptospiral outer membrane proteins able to interact directly with them. Interestingly, we show that the protein-specific {gamma}{delta} T cell response is composed of WC1.1+ and WC1.2+ subsets, although a greater number of WC1.1+{gamma}{delta} T cells respond. Identification of protein antigens will enhance our understanding of the role {gamma}{delta} T cells play in the leptospiral immune response and in recombinant vaccine development.

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Patterns and mechanisms underlying ecoregion delineation in North American freshwater plants AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSAimC_ST_ABSBiogeographical regionalisations are actively studied in different ecosystems, because they increase our understanding on fundamental broad{square}scale patterns and can help us in the establishment of conservation areas. Thus, we studied how well existing freshwater ecoregions describe geographical delineation for inland water plants and which ecogeographical gradients explain them.

LocationNorth America, excluding Mexico and remote islands.

TaxonFreshwater vascular plants of all taxa and different functional groups.

MethodsUsing newly available fine-grained data on freshwater plant distributions, we calculated internal homogeneity and cross-boundary heterogeneity among neighbouring ecoregions. We further integrated measures of community dissimilarity to assess whether the degree of within-ecoregion homogeneity and distinctness are driven by their relationships to species replacements and richness differences, and explored how a complex suite of ecogeographical mechanisms and plant life forms affect ecoregion delineation using spatially explicit regression routines.

ResultsWe found a clear geographical patterning of ecoregion robustness for North American freshwater plants, with their communities being more internally homogeneous and more similar to one another in polar and subtropical inland waters. Surprisingly, the degree of internal homogeneity and ecoregion distinctness were almost equally driven by species replacements and richness differences. Considering different life forms, ecoregion delineation performed best for emergent and floating-leaved plants. Finally, within-ecoregion homogeneity and distinctness were best explained by annual mean temperature and terrain ruggedness, respectively, with mean water alkalinity, ecoregion area and Late Quaternary Ice Age legacies having supplementary effects.

Main conclusionsOur findings emphasise that geographical regionalisations founded on a particular organismal group are not applicable for all taxa. Our study is a promising starting point for further investigations of geographical delineations for different freshwater taxa. These updated regionalisations can then be used for conserving different biotas in freshwaters, which are currently among the most threatened ecosystems in the world.

SO_SCPLOWTATEMENTC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWOFC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWSIGNIFICANCEC_SCPLOWIn biogeographical regionalisation biota is categorized to meaningful geographical units, such as ecoregions. However, ecoregions delineated for a particular group of organisms may not be applicable to another assemblages. We studied how ecoregions founded on fish are suitable for freshwater plants across North America. Our findings suggest that these ecoregions did not show consistent robustness for freshwater plants in North America. This study is a promising starting point for further investigations of geographical delineations for different freshwater taxa, having also value in conservation planning and management.

sentences: How many giraffes are there? A comparison of abundance estimators at Ongava Game Reserve, Namibia Camera-traps are a versatile and widely adopted tool for collecting biological data for wildlife conservation and management. While estimating population abundance from camera-trap data is the primarily goal of many projects, the question of which population estimator is suitable for analysing these data needs to be investigated. We took advantage of a 21 day camera-trap monitoring period of giraffes (Giraffa camelopardalis angolensis) on the Ongava Game Reserve (Namibia) to compare capture-recapture (CR), rarefaction curves and N-mixture estimators of population abundance. A marked variation in detection probability of giraffes was observed both in time and between individuals. Giraffes were also less likely to be detected after they were seen at a waterhole (mean daily visit frequency of f = 0.25). We estimated the population size to be 119 giraffes (Cv = 0.10) using the most robust reference estimator (CR). All other estimators deviated from the CR population size by ca. -20 to > +80%. This was due the fact that these models did not account for the temporal and individual variations in detection probability. We found that modelling choice was much less forgiving for N-mixture models than CR estimators because it leads to very variable and inconsistent estimations of abundance. Double counts were problematic for N-mixture models, challenging the use of raw counts (i.e. when individuals are not identified) at waterholes, to monitor the abundance of giraffe or of other species without idiosyncratic coat patterns.

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A simple, cost-effective and automation-friendly direct PCR approach for bacterial community analysis Bacterial communities in water, soil, and humans play an essential role in environmental ecology and human health. PCR-based amplicon analysis, such as 16S ribosomal RNA sequencing, is a fundamental tool for quantifying and studying microbial composition, dynamics, and interactions. However, given the complexity of microbial communities, a substantial number of samples becomes necessary to analyses that parse the factors that determine microbial composition. A common bottleneck in performing these kinds of experiments is genomic DNA (gDNA) extraction, which is time-consuming, expensive, and often biased on the types of species. Direct PCR methods are a potentially simpler and more accurate alternative to gDNA extraction methods that do not require the intervening purification step. In this study, we evaluated three variations of direct PCR methods using diverse heterogeneous bacterial cultures, ZymoBIOMICS Microbial Community Standards, and groundwater. By comparing direct PCR methods with DNeasy blood and tissue kits and DNeasy Powersoil kits, we found a specific variant of the direct PCR method exhibits a comparable overall efficiency to the conventional DNeasy Powersoil protocol. We also found the method showed higher efficiency for extracting gDNA from the gram negative strains compared to DNeasy blood and tissue protocol. This direct PCR method is 1600 times cheaper ($0.34 for 96 samples), 10 times simpler (15 min hands-on time for 96 samples) than DNeasy Powersoil protocol. The direct PCR method can also be fully automated, and is compatible with small volume samples, thereby permitting scaling of samples and replicates needed to support high-throughput large-scale bacterial community analysis.

IMPORTANCEUnderstanding bacterial interaction and assembling in complex microbial communities using 16S ribosomal RNA sequencing normally requires a large experimental load. However, the current DNA extraction methods including cell disruption and genomic DNA purification are normally biased, costly, time and labor consuming, and not amenable to miniaturization by droplets or 1536 well plates due to the significant DNA loss during purification step for tiny volume and low cell density samples. Direct PCR method could potentially solve these problems. In this study, we demonstrate a direct PCR method which exhibits similar efficiency as the widely used method - DNeasy Powersoil protocol, while 1600 times cheaper and 10 times faster to execute. This simple, cost-effective, and automation friendly direct PCR based 16S ribosomal RNA sequencing method allows us to study the dynamics, microbial interaction and assembly of varying microbial communities in a high throughput fashion.

sentences: Density-dependent attributes of schooling in small pelagic fishes Many small pelagic fishes obligately form schools; some of these schools attain a remarkable size. Although the school is a fundamental and important ecological unit and is the site of biological interactions such as competition and predation, information on schooling processes in the field remains scarce. Here, we examined the quantitative relationships between population density and school size, the number of schools, and other school characteristics (i.e., packing density, volume, and cross-sectional area) in three species of small pelagic fishes: chub mackerel Scomber japonicus, Japanese sardine Sardinops melanostictus, and Japanese anchovy Engraulis japonicus. We found that school size increased almost linearly with population density, whereas the number of schools and other characteristics increased non-linearly with population density, whereby the rate of increase slowed radically as population density increased. These results indicate that, at low population densities, an increase in density causes an increase in both school size and the number of schools, whereas at higher population densities, an increase in density triggers the formation of larger schools rather than more schools. Furthermore, we found that the shapes of the relationships of all school characteristics with population density differed among species. Our results indicate that the schooling behaviour of small pelagic fishes is density-dependent, and responses to changes in density are species-specific. Our results provide insight into of how biological interactions such as intra- and inter-specific competition and predator-prey interactions mediate the density-dependent processes that underlie the population dynamics and community structure of small pelagic fishes in marine ecosystems.

sentences: A ubiquitously expressed UDP-glucosyltransferase, UGT74J1, controls basal salicylic acid levels in rice Salicylic acid (SA) is a phytohormone that regulates a variety of physiological and developmental processes, including disease resistance. SA is a key signaling component in the immune response of many plant species. However, the mechanism underlying SA-mediated immunity is obscure in rice (Oryza sativa). Prior analysis revealed a correlation between basal SA level and blast resistance in a range of rice varieties. This suggested that resistance might be improved by increasing basal SA level. Here, we identified a novel UDP-glucosyltransferase gene, UGT74J1, which is expressed ubiquitously throughout plant development. Mutants of UGT74J1 generated by genome editing accumulated high levels of SA under non-stressed conditions, indicating that UGT74J1 is a key enzyme for SA homeostasis in rice. Microarray analysis revealed that the ugt74j1 mutants constitutively overexpressed a set of pathogenesis-related (PR) genes. An inoculation assay demonstrated that these mutants had increased resistance against rice blast, but they also exhibited stunted growth phenotypes. To our knowledge, this is the first report of a rice mutant displaying SA overaccumulation.

sentences: Involvement of mechanical cues in the migration of Cajal-Retzius cells in the marginal zone during neocortical development Emerging evidence points to coordinated action of chemical and mechanical cues during brain development. At early stages of neocortical development, angiogenic factors and chemokines such as CXCL12, ephrins, and semaphorins assume crucial roles in orchestrating neuronal migration and axon elongation of postmitotic neurons. Here we explore the intrinsic mechanical properties of the developing marginal zone of the pallium in the migratory pathways and brain distribution of the pioneer Cajal-Retzius cells. These pioneer neurons are generated in several proliferative regions in the developing brain (e.g., the cortical hem and the pallial subpallial boundary) and migrate tangentially in the preplate/marginal zone covering the upper portion of the neocortex. These cells play crucial roles in correct neocortical layer formation by secreting several molecules such as Reelin. Our results indicate that the motogenic properties of Cajal-Retzius cells and their perinatal distribution in the marginal zone are also modulated by both chemical and mechanical factors, by the specific mechanical properties of Cajal-Retzius cells, and by the differential stiffness of the migratory routes. Indeed, cells originating in the cortical hem display higher migratory capacities than those generated in the pallial subpallial boundary which may be involved in the differential distribution of these cells in the dorsal-lateral axis in the developing marginal zone.

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LACTATE IS A MAJOR ENERGY SUBSTRATE FOR CORTICAL NEURONS AND ENHANCES THEIR FIRING ACTIVITY Glucose is the mandatory fuel for the brain, yet the relative contribution of glucose and lactate for neuronal energy metabolism is unclear. We found that increased lactate, but not glucose concentration, enhances the spiking activity of neurons of the cerebral cortex. Enhanced spiking was dependent on ATP-sensitive potassium (KATP) channels formed with Kir6.2 and SUR1 subunits, which we show are functionally expressed in most neocortical neuronal types. We also demonstrate the ability of cortical neurons to take-up and metabolize lactate. We further reveal that ATP is produced by cortical neurons largely via oxidative phosphorylation and only modestly by glycolysis. Our data demonstrate that in active neurons, lactate is preferred to glucose as an energy substrate, and that lactate metabolism shapes neuronal activity in the neocortex through KATP channels. Our results highlight the importance of metabolic crosstalk between neurons and astrocytes for brain function.

HighlightsO_LIMost cortical neurons subtypes express pancreatic beta-cell like KATP channels.
C_LIO_LILactate enhances spiking activity via its uptake and closure of KATP channels.
C_LIO_LICortical neurons take up and oxidize lactate.
C_LIO_LICortical neurons produce ATP mainly by oxidative phosphorylation.
C_LI

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DeepTMB: An uncertainty-aware deep calibration of tumor mutational burden with a synthetic tumor-only dataset BackgroundTumor mutational burden (TMB) is commonly investigated as a biomarker for immune checkpoint blockade (ICB) therapy, but lack of uniform standards in its calculation may be preventing consensus recommendations. Gene panels have mostly supplanted exome sequencing for calculating TMB, but these panels have a strong tendency to overestimate TMB. With TMB cutoffs being developed with panel-based estimates and suggestions to use The Cancer Genome Atlas (TCGA) exome data to model panel data, there is a need to understand how to properly compare values from different assays.

DesignOur approach to TMB calibration allows for a nonlinear fit and a heteroscedastic error distribution, utilizes a per-batch read depth augmentation to upsample the exomic data to what would be seen at panel depth, and is trained on a novel tumor-only synthetic dataset generated by utilizing germline as well as somatic calls.

ResultsWe were able to accurately model the distribution of tumor-normal data. Removing hotspots and including synonymous mutations resulted in better regression metrics, as did including NCIt label. Correcting for read depth resulted in a slight increase in the spread of the data but did not affect the model fit. When applying a model trained on tumor-normal data to synthetic tumor-only input there was a dramatic drop in the positive predictive value (PPV) at a TMB of 10. This was largely mitigated by training with synthetic tumor-only input (up to over a 200% improvement in PPV).

ConclusionsA deep learning model is capable of accurately modeling the tumor-normal TMB distribution. Using a synthetic tumor-only input for training appears to be a viable strategy for calibrating tumor-only panels.

sentences: De novo design of peptides that form transmembrane barrel pores killing antibiotic resistant bacteria De novo design of peptides that self-assemble into transmembrane barrel pores is challenging due to the complexity of several competing interactions involving peptides, lipids, water, and ions. Here, we develop a computational approach for the de novo design of -helical peptides that self-assemble into stable transmembrane barrel pores with a central functional channel. We formulate the previously missing design guidelines and report 52 sequence patterns that can be tuned for specific applications using the identified role of each residue. Atomic force microscopy and fluorescent dye leakage experiments confirm that the designed peptides form leaky membrane pores in vitro. Customized designed peptides act as antimicrobial agents able to kill even antibiotic-resistant ESKAPE bacteria at micromolar concentrations, while exhibiting low toxicity to human cells. The peptides can be similarly fine-tuned for other medical and biotechnological applications.

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SPaRTAN, a computational framework for linking cell-surface receptors to transcriptional regulators The identity and functions of specialized cell types are dependent on the complex interplay between signaling and transcriptional networks. We present SPaRTAN (Single-cell Proteomic and RNA based Transcription factor Activity Network), a computational method to link cell-surface receptors to transcription factors (TFs) by exploiting cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) datasets with cis-regulatory information. SPaRTAN is applied to immune cell types in the blood to predict the coupling of signaling receptors with cell context-specific TFs. The predictions are validated by prior knowledge and flow cytometry analyses. SPaRTAN is then used to predict the signaling coupled TF states of tumor infiltrating CD8+ T cells in malignant peritoneal and pleural mesotheliomas. SPaRTAN greatly enhances the utility of CITE-seq datasets to uncover TF and cell-surface receptor relationships in diverse cellular states.

SignificanceRecently single-cell technologies such as CITE-seq have been developed that enable simultaneous quantitative analysis of cell-surface receptor expression with transcriptional states. To date, these datasets have not been used to systematically develop cell-context-specific maps of the interface between signaling and transcriptional regulators orchestrating cellular identity and function. We developed a computational framework, SPaRTAN (Single-cell Proteomic and RNA based Transcription factor Activity Network) that integrates single-cell proteomic and transcriptomic data based on CITE-seq with cis-regulatory information. We applied our method to publicly available (PBMCs) and new CITE-seq datasets (mesothelioma). Our predicted TF activity and cell-surface receptor relationships are validated by prior knowledge as well as experimental testing. SPaRTAN reveals many previously unidentified surface-receptor associated TFs.

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In vitro reconstituted quorum sensing pathways enable rapid evaluation of quorum sensing inhibitors Quorum sensing, as inner- or inter-species microbial communication process orchestrated by diffusible autoinducers, typically results in collective pathogenic behaviours, being recognized as a promising druggable target for anti-virulence. Here, we reconstituted las and rhl quorum sensing pathways of Pseudomonas aeruginosa, mediated by acyl-homoserine lactones (AHLs) and LuxI/LuxR-family proteins, with fluorescence output in Escherichia coli cell-free expression system, offering a platform to rapidly evaluate quorum sensing inhibitors (QSIs) in vitro. Previously reported small-molecule quorum sensing inhibitors for interfering with P. aeruginosa quorum sensing systems were tested and showed mild to high on-target inhibition as well as off-target toxicity. Of note, quercetin displayed potent on-target inhibition to quorum sensing pathways as well as acceptable off-target toxicity to cell-free expression machinery. Upon our work, cell-free platform is anticipated to further facilitate automated and high-throughput drug screening, bridge in silico and in vivo drug-screening methods, and accelerate the upgrading of antimicrobial arsenal.

GRAPHICAL ABSTRACT

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sentences: USP28 deletion and small molecule inhibition destabilises c-Myc and elicits regression of squamous cell lung carcinoma Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient 5-year survival rate is less than 5%. The ubiquitin specific protease 28 (USP28) has been implicated in tumorigenesis through its stabilization of the oncoprotein c-MYC. Here, we show that genetic inactivation of Usp28 induced regression of established murine LSCC lung tumors. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-Myc proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumors and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.

sentences: SUV39 SET domains mediate crosstalk of heterochromatic histone marks The SUV39 class of methyltransferase enzymes deposits histone H3 lysine 9 di- and trimethylation (H3K9me2/3), the hallmark of constitutive heterochromatin. How these enzymes are regulated to mark specific genomic regions as heterochromatic is poorly understood. Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast S. pombe and recent evidence suggests that ubiquitination of lysine 14 on histone H3 (H3K14ub) plays a key role in H3K9 methylation. However, the molecular mechanism of this regulation and its role in heterochromatin formation remains to be determined. Our structure-function approach shows that the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby stimulates the enzyme by over 250-fold. Mutations that disrupt this mechanism lead to a loss of H3K9me2/3 and abolish heterochromatin silencing similar to clr4 deletion. Comparison with mammalian SET domain proteins suggests that the Clr4 SET domain harbours a conserved sensor for H3K14ub, which mediates licensing of heterochromatin formation.

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Genetic mapping of flowering time and plant height in a maize Stiff Stalk MAGIC population The Stiff Stalk heterotic pool is a foundation of US maize seed parent germplasm and has been heavily utilized by both public and private maize breeders since its inception in the 1930s. Flowering time and plant height are critical characteristics for both inbred parents and their test crossed hybrid progeny. To study these traits, a six parent multiparent advanced generation intercross (MAGIC) population was developed including maize inbred lines B73, B84, PHB47 (B37 type), LH145 (B14 type), PHJ40 (novel early Stiff Stalk), and NKH8431 (B73/B14 type). A set of 779 doubled haploid lines were evaluated for flowering time and plant height in two field replicates in 2016 and 2017, and a subset of 689 and 561 doubled haploid lines were crossed to two testers, respectively, and evaluated as hybrids in two locations in 2018 and 2019 using an incomplete block design. Markers were derived from a Practical Haplotype Graph built from the founder whole genome assemblies and genotype-by-sequencing and exome capture-based sequencing of the population. Genetic mapping utilizing an update to R/qtl2 revealed differing profiles of significant loci for both traits between 636 of the DH lines and two sets of 571 and 472 derived hybrids. Genomic prediction was used to test the feasibility of predicting hybrid phenotypes based on the per se data. Predictive abilities were highest on direct models trained using the data they would predict (0.55 to 0.63), and indirect models trained using per se data to predict hybrid traits had slightly lower predictive abilities (0.49 to 0.55). Overall, this finding is consistent with the overlapping and non-overlapping significant QTL found within the per se and hybrid populations and suggests that selections for phenology traits can be made effectively on doubled haploid lines before hybrid data is available.

Core IdeasA multi-parent advanced generation intercross (MAGIC) mapping population was developed from six founder Stiff Stalk maize inbreds with commercial relevance. Genetic mapping utilizing an update to R/qtl2 was demonstrated for flowering and plant height traits.

Genetic mapping using maize inbred and hybrid information was compared and provided insight into trait expression in inbreds relative to heterotic testcross hybrids.

sentences: Genetic variation among wMel strains of Wolbachia pipientis differentially rescues a bag of marbles partial loss of function mutant in Drosophila melanogaster In Drosophila melanogaster, the maternally inherited endosymbiont Wolbachia pipientis interacts with germline stem cell genes during oogenesis. One such gene, bag of marbles (bam) is the key switch for differentiation and also shows signals of adaptive evolution for protein diversification. These observations have led us to hypothesize that W. pipientis could be driving the adaptive evolution of bam for control of oogenesis. To test this hypothesis, we must understand the specificity of the genetic interaction between bam and W. pipientis. Previously, we documented that the W. pipientis variant, wMel, rescued the fertility of the bamBW hypomorphic mutant as a transheterozygote over a bam null. However, bamBW was generated more than 20 years ago in an uncontrolled genetic background and maintained over a balancer chromosome. Consequently, the chromosome carrying bamBW accumulated mutations that have prevented controlled experiments to further assess the interaction. Here, we used CRISPR/Cas9 to engineer the same single amino acid bam hypomorphic mutation (bamL255F) and a new bam null disruption mutation into the w1118 isogenic background. We assess the fertility of wildtype bam, bamL255F/bamnull hypomorphic, and bamL255F/ bamL255F mutant females, each infected individually with ten W. pipientis wMel variants representing three phylogenetic clades. Overall, we find that all of the W. pipientis variants tested here rescue bam hypomorphic fertility defects with wMelCS-like variants exhibiting the strongest rescue effects. Additionally, these variants did not increase wildtype bam female fertility. Therefore, both bam and W. pipientis interact in genotype-specific ways to modulate female fertility, a critical fitness phenotype.

sentences: Combination of the Systemin peptide with the beneficial fungus Trichoderma afroharzianum T22 improves plant defense responses against pests and diseases Trichoderma spp. are among the most widely used plant beneficial fungi in agriculture. A novel approach to enhance their effectiveness in plant defense is to use the fungi in combination with bioactive molecules including plant-derived compounds. Here, we show that plant treatment with Trichoderma afroharzianum (strain T22) and Systemin (Sys), a tomato plant peptide active in triggering plant defense, confers protection against the fungal pathogens Fusarium oxysporum, Botrytis cinerea and the insect pest Tuta absoluta. The observed defensive response was associated with increased accumulation of metabolites and transcripts involved in the Jasmonic acid (JA) pathway. Our findings suggest that the innovative combination of T. afroharzianum T22 and Sys can result in a more effective and robust control of different biotic stress agents.

sentences: LMO7-dependent apical constriction requires the binding of the Myosin heavy chain Apical constriction, or reduction of the apical domain, underlies many morphogenetic events during development, such as furrow or tube formation. Actomyosin complexes play an essential role in apical constriction, however the detailed analysis of molecular mechanisms is still pending. Here we show that Lim domain only protein 7 (Lmo7), a multidomain adaptor at apical junctions, promotes apical constriction in the Xenopus superficial ectoderm, whereas apical domain size increases in Lmo7-depleted cells. Lmo7 is localized to and promotes the formation of the circumferential actomyosin belt adjacent to apical junctions. We find that Lmo7-dependent apical constriction requires the RhoA-ROCK-Non-muscle myosin II (NMII) pathway. Strikingly, Lmo7 binds and recruits NMII heavy chains to apical junctions. Lmo7 overexpression altered the subcellular distribution of Wtip, a sensor of mechanical tension. Our findings suggest that Lmo7 serves as a scaffold organizing the actomyosin network to regulate contractility at apical junctions.

sentences: Fighting over defence chemicals disrupts mating behaviour Studies on intraspecific contest behaviour predominantly focus on contests between individuals of the same sex, however contest behaviour is also expected to occur between individuals of the opposite sex including possible mates. Here we investigate potential trade-offs between mating and fighting behaviour in the turnip sawfly (Athalia rosae). Adults of this species collect chemical defence compounds (clerodanoids) directly from plants but also indirectly by nibbling on conspecifics that have already obtained clerodanoids themselves, a highly aggressive behavioural interaction. An A. rosae individual without clerodanoids may therefore be the potential mate or attacker of an individual of the opposite sex that has gained clerodanoids. We paired males and females with or without clerodanoid access and manipulated body mass differences between the sexes via the early life starvation of females. We show that asymmetrical clerodanoid acquisition between male-female pairs causes an increase in agonistic nibbling behaviour, irrespective of sex. Moreover, fighting over clerodanoids disrupted mating behaviour, and the frequency of aggressive nibbling behaviour in these pairs was determined by the comparative body mass of the attacking individual. Our study highlights the vital importance of investigating agonistic intersex interactions not only over mating but also over resources.

sentences: Real-Time Brain-Machine Interface Achieves High-Velocity Prosthetic Finger Movements using a Biologically-Inspired Neural Network Decoder Despite the rapid progress and interest in brain-machine interfaces that restore motor function, the performance of prosthetic fingers and limbs has yet to mimic native function. The algorithm that converts brain signals to a control signal for the prosthetic device is one of the limitations in achieving rapid and realistic finger movements. To achieve more realistic finger movements, we developed a shallow feed-forward neural network, loosely inspired by the biological neural pathway, to decode real-time two-degree-of-freedom finger movements. Using a two-step training method, a recalibrated feedback intention-trained (ReFIT) neural network achieved a higher throughput with higher finger velocities and more natural appearing finger movements than the ReFIT Kalman filter, which represents the current standard. The neural network decoders introduced herein are the first to demonstrate real-time decoding of continuous movements at a level superior to the current state-of-the-art and could provide a starting point to using neural networks for the development of more naturalistic brain-controlled prostheses.

sentences: The Pebble/Rho1/Anillin pathway controls polyploidization and axonal wrapping activity in the glial cells of the Drosophila eye During development glial cell are crucially important for the establishment of neuronal networks. Proliferation and migration of glial cells can be modulated by neurons, and in turn glial cells can differentiate to assume key roles such as axonal wrapping and targeting. To explore the roles of actin cytoskeletal rearrangements in glial cells, we studied the function of Rho1 in Drosophila developing visual system. We show that the Pebble (RhoGEF)/Rho1/Anillin pathway is required for glia proliferation and to prevent the formation of large polyploid perineurial glial cells, which can still migrate into the eye disc if generated. Surprisingly, this Rho1 pathway is not necessary to establish the total glial membrane area or for the differentiation of the polyploid perineurial cells. The resulting polyploid wrapping glial cells are able to initiate wrapping of axons in the basal eye disc, however the arrangement and density of glia nuclei and membrane processes in the optic stalk are altered and the ensheathing of the photoreceptor axonal fascicles is reduced.

sentences: Modeling of Aryl Hydrocarbon Receptor Pathway Intrinsic Immunometabolic Role using Glioblastoma Stem Cells and Patient-Derived Organoids The intrinsic genetic program of glioblastoma (GBM) stem cells is critical for tumor evolution and recurrence. We recently identified intrinsic phenotypes and immune-like genetic programs of GBM organoids (GBMO)1 from patient derived glioblastoma stem cells (GSCs), replicating genomic, metabolic, and cellular aspects of GBM in vivo. Aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, is a key regulator of infiltrating immune cells in gliomas2, 3 and associated with poor prognosis, but its role in GSC biology is unknown2. Here, we show that AHR is a patient-specific regulator of the glioma intrinsic gene program in GSCs and GSC-derived GBMO that are enriched for AHR. We find that AHR is required for GSC self-renewal, GBMO expansion, radial glia-like cell proliferation, and expression of immune mediators seen in the mesenchymal subtype. CRISPR-Cas9 genetic ablation and pharmacological inhibition revealed that AHR regulates genes linked to intrinsic immunity, proliferation, and migration in GBMO. Genomic analysis of GBMO treated with AHR inhibitors identified expression signatures and candidate markers associated with survival of gliomas. Our work defines the glioma intrinsic function of AHR in a model of early GBM formation, offering a rationale for clinical exploration of a potential  two-hit target of both GBM cells and infiltrating immune cells in patients with GBM expressing high levels of AHR.

sentences: Segmenting biological specimens from photos to understand the evolution of UV plumage in passerine birds Ultraviolet (UV) colouration is thought to be an important signalling mechanism in many bird species, yet broad insights regarding the prevalence of UV plumage colouration and the factors promoting its evolution are currently lacking. Here, we develop a novel image segmentation pipeline based on deep learning that considerably outperforms classical (i.e. non-deep learning) segmentation methods, and use this to extract accurate information on whole-body plumage colouration from photographs of >24,000 museum specimens covering >4,500 species of passerine birds. Our results demonstrate that UV reflectance, particularly as a component of other colours, is widespread across the passerine radiation but is strongly phylogenetically conserved. We also find clear evidence in support of the role of light environment in promoting the evolution of UV plumage colouration, and a weak trend towards higher UV plumage reflectance among bird species with ultraviolet rather than violet-sensitive visual systems. Overall, our study provides important broad-scale insight into an enigmatic component of avian colouration, as well as demonstrating that deep learning has considerable promise for allowing new data to be bought to bear on long-standing questions in ecology and evolution.

sentences: Automated and parallelized spike collision tests to identify spike signal projections The spike collision test is a highly reliable technique to identify axonal projection of a neuron recorded electrophysiologically for investigating functional spike information among brain areas. It is potentially applicable to more neuronal projections by combining multi-channel recording with optogenetic stimulation. Yet, it remains inefficient and laborious because an experimenter must visually select spikes in every channel and manually repeat spike collision tests for each neuron serially. Here, we established a novel technique to automatically perform spike collision tests for all channels in parallel (Multi-Linc analysis), employing two distinct protocols implemented in a multi-channel real-time processing system. The rat cortical neurons identified with this technique displayed physiological spike features consistent with excitatory projection neurons. Their antidromic spikes were similar in shape but slightly larger in amplitude compared with spontaneous spikes. In addition, we demonstrated simultaneous identification of reciprocal or bifurcating projections among cortical areas. Thus, our Multi-Linc analysis will be a powerful research approach to elucidate interareal spike communication.

sentences: A role of Lck annular lipids in the steady upkeep of active Lck in T cells Theoretical work suggests that collective spatiotemporal behaviour of integral membrane proteins (IMPs) can be modulated by annular lipids sheathing their hydrophobic moiety. Here, we present evidence for this prediction in a natural membrane by investigating the mechanism that maintains steady amount of active isoform of Lck kinase (LckA) by Lck trans-autophosphorylation offset by the phosphatase CD45. We gauged experimental suitability by quantitation of CD45 and LckA subcellular localisation, LckA generation as a function of Lck and pharmacological perturbation. Steady LckA was challenged by swapping Lck membrane anchor with structurally divergent ones expected to substantially modify Lck annular lipids, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45, respectively. The data showed only small alteration of LckA, except for CD45 hydrophobic anchor that thwarted LckA, due to excessive lateral proximity to CD45. The data are best explained by annular lipids facilitating or penalising IMPs lateral proximity, hence modulating IMPs protein-protein functional interactions. Our findings can contribute to improve the understanding of biomembranes organisation.

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Comparative analysis of molecular representations in prediction of drug combination effects AO_SCPLOWBSTRACTC_SCPLOWApplication of machine and deep learning methods in drug discovery and cancer research has gained a considerable amount of attention in the past years. As the field grows, it becomes crucial to systematically evaluate the performance of novel computational solutions in relation to established techniques. To this end we compare rule-based and data-driven molecular representations in prediction of drug combination sensitivity and drug synergy scores using standardized results of 14 throughput screening studies, comprising 64 200 unique combinations of 4 153 molecules tested in 112 cancer cell lines. We evaluate the clustering performance of molecular representations and quantify their similarity by adapting the Centered Kernel Alignment metric. Our work demonstrates that to identify an optimal molecular representation type it is necessary to supplement quantitative benchmark results with qualitative considerations, such as model interpretability and robustness, which may vary between and throughout preclinical drug development projects.

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sentences: Temporal specificity of abnormal neural oscillations during phonatory events in Laryngeal Dystonia Laryngeal Dystonia is a debilitating disorder of voicing in which the laryngeal muscles are intermittently in spasm resulting in involuntary interruptions during speech. The central pathophysiology of laryngeal dystonia, underlying computational impairments in vocal motor control, remains poorly understood. Although prior imaging studies have found aberrant activity in the central nervous system during phonation in patients with laryngeal dystonia, it is not known at what timepoints during phonation these abnormalities emerge and what function may be impaired. To investigate this question, we recruited 22 adductor laryngeal dystonia patients (15 female, age range = 28.83-72.46 years) and 18 controls (8 female, age range = 27.40-71.34 years). We leveraged the fine temporal resolution of magnetoencephalography to monitor neural activity around glottal movement onset, subsequent voice onset and after the onset of pitch feedback perturbations. We examined event-related beta-band (12-30 Hz) and high-gamma band (65-150 Hz) neural oscillations. Prior to glottal movement onset, we observed abnormal frontoparietal motor preparatory activity. After glottal movement onset, we observed abnormal activity in somatosensory cortex persisting through voice onset. Prior to voice onset and continuing after, we also observed abnormal activity in the auditory cortex and the cerebellum. After pitch feedback perturbation onset, we observed no differences between controls and patients in their behavioural responses to the perturbation. But in patients, we did find abnormal activity in brain regions thought to be involved in the auditory feedback control of vocal pitch (premotor, motor, somatosensory and auditory cortices). Our study results confirm the abnormal processing of somatosensory feedback that has been seen in other studies. However, there were several remarkable findings in our study. First, patients have impaired vocal motor activity even before glottal movement onset, suggesting abnormal movement preparation. These results are significant because: (i) they occur before movement onset, abnormalities in patients cannot be ascribed to deficits in vocal performance, and (ii) they show that neural abnormalities in laryngeal dystonia are more than just abnormal responses to sensory feedback during phonation as has been hypothesised in some previous studies. Second, abnormal auditory cortical activity in patients begins even before voice onset, suggesting abnormalities in setting up auditory predictions before the arrival of auditory feedback at voice onset. Generally, activation abnormalities identified in key brain regions within the speech motor network around various phonation events not only provide temporal specificity to neuroimaging phenotypes in laryngeal dystonia but also may serve as potential therapeutic targets for neuromodulation.

sentences: Synthetic periphyton as a model system to understand species dynamics in complex microbial freshwater communities Phototrophic biofilms, also known as periphyton, are microbial freshwater communities that drive crucial ecological processes in streams and lakes. Gaining a deep mechanistic understanding of the biological processes occurring in natural periphyton remains challenging due to the high complexity and variability of such communities. To address this challenge, we rationally developed a workflow to construct a synthetic community by co-culturing 26 phototrophic species (i.e., diatoms, green algae and cyanobacteria) that were inoculated in a successional sequence to create a periphytic biofilm on glass slides. We show that this community is diverse, stable and highly reproducible in terms of microbial composition, function and 3D spatial structure of the biofilm. We also demonstrate the ability to monitor microbial dynamics at the single species level during periphyton development and how their abundances are impacted by stressors such as increased temperature and a herbicide, singly and in combination. Overall, such a synthetic periphyton, grown under controlled conditions, can be used as a model system for theory testing through targeted manipulation.

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Sex differences and similarities in the neuroimmune response to central administration of poly I:C BackgroundThe neuroimmune system is required for normal neural processes, including modulation of cognition, emotion, and adaptive behaviors. Aberrant neuroimmune activation is associated with dysregulation of memory and emotion, though the precise mechanisms at play are complex and highly context dependent. Sex differences neuroimmune activation and function further complicate our understanding of its roles in cognitive and affective regulation.

MethodsHere, we characterized the physiological sickness and inflammatory response of the hippocampus following intracerebroventricular (ICV) administration of a synthetic viral mimic, polyinosinic:polycytidylic acid (poly I:C), in both male and female C57Bl/6 mice.

ResultsWe observed that poly I:C induced weight loss, fever, and elevations of cytokine and chemokines in the hippocampus of both sexes. Specifically, we found transient increases in gene expression and protein levels of IL-1, IL-1{beta}, IL-4, IL-6, TNF, CCL2, and CXCL10, where males showed a greater magnitude of response compared with females. Only males showed increased IFN and IFN{gamma} in response to poly I:C, whereas both males and females exhibited elevations of IFN{beta}, demonstrating a specific sex difference in the anti-viral response in the hippocampus.

ConclusionOur data suggest that type I interferons are one potential node mediating sex-specific cytokine responses and neuroimmune effects on cognition. Together, these findings highlight the importance of using both males and females and analyzing a broad set of inflammatory markers in order to identify the precise, sex-specific roles for neuroimmune dysregulation in neurological diseases and disorders.

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Social Touch is associated with Neural but not Physiological Synchrony in Naturalistic Mother-Infant Interactions Caregiver touch plays a vital role in infants growth and development, but its role as a communicative signal in human parent-infant interactions is surprisingly poorly understood. Here, we assessed whether touch and proximity in caregiver-infant dyads are related to neural and physiological synchrony. We simultaneously measured brain activity and respiratory sinus arrhythmia of 4- to 6-month-old infants and their mothers (N=69 dyads) in distal and proximal joint watching conditions as well as in an interactive face-to-face condition. Neural synchrony was higher during the proximal than during the distal joint watching conditions, and even higher during the face-to-face interaction. Physiological synchrony was highest during the face-to-face interaction and lower in both joint watching conditions, irrespective of proximity. Maternal affectionate touch during the face-to-face interaction was positively related to neural but not physiological synchrony. This is the first evidence that touch mediates mutual attunement of brain activities, but not cardio-respiratory rhythms in caregiver-infant dyads during naturalistic interactions. Our results also suggest that neural synchrony serves as a biological pathway of how social touch plays into infant development and how this pathway could be utilized to support infant learning and social bonding.

HighlightsO_LIMother-infant neural and physiological synchrony arise during mutual engagement.
C_LIO_LIBehavioral correlates of neural and physiological synchrony diverge.
C_LIO_LIInterpersonal neural synchrony is related to proximity and affective touch.
C_LIO_LIInterpersonal physiological synchrony is related to infant negative affect.
C_LI

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Comprehensive characterization of internal and cuticle surface microbiota of laboratory-reared F1 Anopheles albimanus originating from different sites BackgroundResearch on mosquito-microbe interactions may lead to new tools for mosquito and mosquito-borne disease control. To date, such research has largely utilized laboratory-reared mosquitoes that typically lack the microbial diversity of wild populations. A logical progression in this area involves working under controlled settings using field-collected mosquitoes or, in most cases, their progeny. Thus, an understanding of how laboratory colonization affects the assemblage of mosquito microbiota would aid in advancing mosquito microbiome studies and their applications beyond laboratory settings.

MethodsUsing high throughput 16S rRNA amplicon sequencing, we characterized the internal and cuticle surface microbiota of F1 progeny of wild-caught adult Anopheles albimanus from four locations in Guatemala. A total of 132 late instar larvae and 135 2-5day old, non-blood-fed virgin adult females that were reared under identical laboratory conditions, were pooled (3 individuals/pool) and analyzed.

ResultsResults showed geographical heterogeneity in both F1 larval internal (p=0.001; pseudo-F = 9.53) and cuticle surface (p=0.001; pseudo-F = 8.51) microbiota, and only F1 adult cuticle surface (p=0.001; pseudo-F = 4.5) microbiota, with a more homogenous adult internal microbiota (p=0.12; pseudo-F = 1.6) across collection sites. Overall, ASVs assigned to Leucobacter, Thorsellia, Chryseobacterium and uncharacterized Enterobacteriaceae, dominated F1 larval internal microbiota, while Acidovorax, Paucibacter, and uncharacterized Comamonadaceae, dominated the larval cuticle surface. F1 adults comprised a less diverse microbiota compared to larvae, with ASVs assigned to the genus Asaia dominating both internal and cuticle surface microbiota, and constituting at least 70% of taxa in each microbial niche.

ConclusionsThese results suggest that location-specific heterogeneity in filed mosquito microbiota can be transferred to F1 progeny under normal laboratory conditions, but this may not last beyond the F1 larval stage without adjustments to maintain field-derived microbiota. Our findings provide the first comprehensive characterization of laboratory-colonized F1 An. albimanus progeny from field-derived mothers. This provides a background for studying how parentage and environmental conditions differentially or concomitantly affect mosquito microbiome composition, and how this can be exploited in advancing mosquito microbiome studies and their applications beyond laboratory settings.

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Total number and ratio of GABAergic neuron types in the mouse lateral and basal amygdala GABAergic neurons are key circuit elements in cortical networks. In spite of growing evidence showing that inhibitory cells play a critical role in the lateral (LA) and basal (BA) amygdala functions, neither the number of GABAergic neurons nor the ratio of their distinct types have been determined in these amygdalar nuclei. Using unbiased stereology, we found that the ratio of GABAergic neurons in the BA (22 %) is significantly higher than in the LA (16 %) in both male and female mice. No difference was observed between the right and left hemispheres in either sexes. In addition, we assessed the ratio of the major inhibitory cell types in both amygdalar nuclei. Using transgenic mice and a viral strategy for visualizing inhibitory cells combined with immunocytochemistry, we estimated that the following cell types together compose the vast majority of GABAergic cells in the LA and BA: axo-axonic cells (5.5-6 %), basket cells expressing parvalbumin (17-20 %) or cholecystokinin (7-9 %), dendrite-targeting inhibitory cells expressing somatostatin (10-16 %), NPY-containing neurogliaform cells (14-15 %), VIP and/or calretinin-expressing interneuron-selective interneurons (29-38 %) and GABAergic projection neurons expressing somatostatin and neuronal nitric oxide synthase (nNOS, 5.5-8 %). Our results show that these amygdalar nuclei contain all major GABAergic neuron types as found in other cortical regions. Furthermore, our data offer an essential reference for future studies aiming to reveal changes in GABAergic cell number and in inhibitory cell types typically observed under different pathological conditions, and to model functioning amygdalar networks in health and disease.

Significance statementGABAergic cells in cortical structures, like in the lateral and basal nucleus of the amygdala, have a determinant role in controlling circuit operation. In this study, we provide the first estimate for the total number of inhibitory cells in these two amygdalar nuclei. In addition, our study is the first to define the ratio of the major GABAergic cell types present in these cortical networks. Taking into account that hyper-excitability in the amygdala, arising from the imbalance between excitation and inhibition typifies many altered brain functions including anxiety, posttraumatic stress disorder, schizophrenia and autism, uncovering the number and ratio of distinct amygdalar inhibitory cell types offers a solid base for comparing the changes in inhibition in pathological brain states.

sentences: The impact of AlphaFold on experimental structure solution AlphaFold2 is a machine-learning based program that predicts a protein structure based on the amino acid sequence. In this article, we report on the current usages of this new tool and give examples from our work in the Coronavirus Structural Task Force. With its unprecedented accuracy, it can be utilized for the design of expression constructs, de novo protein design and the interpretation of Cryo-EM data with an atomic model. However, these methods are limited by their training data and are of limited use to predict conformational variability and fold flexibility; they also lack co-factors, posttranslational modifications and multimeric complexes with oligonucleotides. They also are not always perfect in terms of chemical geometry. Nevertheless, machine learning based fold prediction are a game changer for structural bioinformatics and experimentalists alike, with exciting developments ahead.

sentences: Harnessing genetic diversity in the USDA pea (Pisum sativum L.) germplasm collection through genomic prediction Phenotypic evaluation and efficient utilization of germplasm collections can be time-intensive, laborious, and expensive. However, with the plummeting costs of next-generation sequencing and the addition of genomic selection to the plant breeders toolbox, we now can more efficiently tap the genetic diversity within large germplasm collections. In this study, we applied and evaluated genomic selections potential to a set of 482 pea accessions - genotyped with 30,600 single nucleotide polymorphic (SNP) markers and phenotyped for seed yield and yield-related components - for enhancing selection of accessions from the USDA Pea Germplasm Collection. Genomic prediction models and several factors affecting predictive ability were evaluated in a series of cross-validation schemes across complex traits. Different genomic prediction models gave similar results, with predictive ability across traits ranging from 0.23 to 0.60, with no model working best across all traits. Increasing the training population size improved the predictive ability of most traits, including seed yield. Predictive abilities increased and reached a plateau with increasing number of markers presumably due to extensive linkage disequilibrium in the pea genome. Accounting for population structure effects did not significantly boost predictive ability, but we observed a slight improvement in seed yield. By applying the best genomic prediction model (e.g., RR-BLUP), we then examined the distribution of genotyped but nonphenotyped accessions and the reliability of genomic estimated breeding values (GEBV). The distribution of GEBV suggested that none of the nonphenotyped accessions were expected to perform outside the range of the phenotyped accessions. Desirable breeding values with higher reliability can be used to identify and screen favorable germplasm accessions. Expanding the training set and incorporating additional orthogonal information (e.g., transcriptomics, proteomics, metabolomics, physiological traits, etc.) into the genomic prediction framework could enhance prediction accuracy.

sentences: Estimations of the weather effects on brain functions using functional MRI: a cautionary note The influences of environmental factors such as weather on the human brain are still largely unknown. A few neuroimaging studies have demonstrated seasonal effects, but were limited by their cross-sectional design or sample sizes. Most importantly, the stability of the MRI scanner hasnt been taken into account, which may also be affected by environments. In the current study, we analyzed longitudinal resting-state functional MRI (fMRI) data from eight individuals, where the participants were scanned over months to years. We applied machine learning regression to use different resting-state parameters, including the amplitude of low-frequency fluctuations (ALFF), regional homogeneity (ReHo), and functional connectivity matrix, to predict different weather and environmental parameters. For careful control, the raw EPI and the anatomical images were also used for predictions. We first found that daylight length and air temperatures could be reliably predicted with cross-validation using the resting-state parameters. However, similar prediction accuracies could also be achieved by using one frame of EPI image, and even higher accuracies could be achieved by using segmented or raw anatomical images. Finally, the signals outside of the brain in the anatomical images and signals in phantom scans could also achieve higher prediction accuracies, suggesting that the predictability may be due to the baseline signals of the MRI scanner. After all, we did not identify detectable influences of weather on brain functions other than the influences on the baseline signals of MRI scanners. The results highlight the difficulty of studying long-term effects using MRI.

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Automatic Identification and Avoidance of Axon Bundle Activation for Epiretinal Prosthesis Retinal prostheses must be able to activate cells in a selective way in order to restore high-fidelity vision. However, inadvertent activation of far-away retinal ganglion cells (RGCs) through electrical stimulation of axon bundles can produce irregular and poorly controlled percepts, limiting artificial vision. Therefore, the problem of axon bundle activation can be defined as the axonal stimulation of RGCs with unknown soma and receptive field locations, typically outside the electrode array. Here, a new algorithm is presented that utilizes electrical recordings to determine the stimulation current amplitudes above which bundle activation occurs. The method exploits several spatiotemporal characteristics of electrically-evoked spikes to overcome the challenge of detecting small axonal spikes in extracellular recordings. The algorithm was validated using large-scale ex vivo stimulation and recording experiments in macaque retina, by comparing algorithmically and manually identified bundle activation thresholds. The algorithm could be used in a closed-loop manner by a future epiretinal prosthesis to reduce poorly controlled visual percepts associated with bundle activation. The method may also be applicable to other types of retinal implants and to cortical implants.

ContributionsPT developed the algorithm and analyzed the data, with input from SMi and EJC. NB and NS helped with the analysis. SMa and LG performed dissections and collected the data. PT and VFH performed manual identification. PH, AS and AML developed and supported recording hardware and software. PT, EJC and SMi wrote the manuscript. NS and SMa edited it. EJC and SMi supervised the project.

sentences: Signalling speed along a key white matter tract influences memory recall ability in humans Conduction velocity is the speed at which electrical signals travel along axons and is a crucial determinant of neural communication. Inferences about conduction velocity can now be made in vivo in humans using magnetic resonance (MR) imaging and a measure called the MR g-ratio. Here, in the first application to cognition, we found that increased MR g-ratio, and by inference faster conduction velocity, specifically in the parahippocampal cingulum bundle, which connects the hippocampus with a range of other brain areas, was associated with better memory recall ability in 217 healthy adults. Moreover, two tract features seemed to favour better memory retrieval - large inner axon diameters and coherently organised neurites, both of which facilitate speedy signalling. These results offer a new perspective on drivers of individual differences in memory recall ability. More broadly, they show that MR g-ratio can provide novel insights into how the human brain processes and integrates information.

sentences: Developing an empirical model for spillover and emergence: Orsay virus host range in Caenorhabditis A lack of tractable experimental systems in which to test hypotheses about the ecological and evolutionary drivers of disease spillover and emergence has limited our understanding of these processes. Here we introduce a promising system: Caenorhabditis hosts and Orsay virus, a positive-sense single-stranded RNA virus that naturally infects C. elegans. We assayed the susceptibility of species across the Caenorhabditis tree and found 21 of 84 wild strains belonging to 14 of 44 species to be susceptible to Orsay virus. Confirming patterns documented in other systems, we detected effects of host phylogeny on susceptibility. We then tested whether susceptible strains were capable of transmitting Orsay virus by transplanting exposed hosts and determining whether they transmitted infection to conspecifics during serial passage. We found no evidence of transmission in 10 strains (virus undetectable after passaging), evidence of low-level transmission in 5 strains (virus lost between passage 1 and 5), and evidence of sustained transmission in 6 strains (including all 3 experimental C. elegans strains). Transmission was associated with host phylogeny and with viral amplification in exposed populations. Variation in Orsay virus susceptibility and transmission among Caenorhabditis species suggests that the system could be powerful for studying spillover and emergence.

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Alternative electron pathways of photosynthesis drive the algal CO2 concentrating mechanism Global photosynthesis consumes ten times more CO2 than net anthropogenic emissions, and microalgae account for nearly half of this consumption1. The great efficiency of algal photosynthesis relies on a mechanism concentrating CO2 (CCM) at the catalytic site of the carboxylating enzyme RuBisCO, thus enhancing CO2 fixation2. While many cellular components involved in the transport and sequestration of inorganic carbon (Ci) have been uncovered3,4, the way microalgae supply energy to concentrate CO2 against a thermodynamic gradient remains elusive4-6. Here, by monitoring dissolved CO2 consumption, unidirectional O2 exchange and the chlorophyll fluorescence parameter NPQ in the green alga Chlamydomonas, we show that the complementary effects of cyclic electron flow and O2 photoreduction, respectively mediated by PGRL1 and flavodiiron proteins, generate the proton motive force (pmf) required by Ci transport across thylakoid membranes. We demonstrate that the trans-thylakoid pmf is used by bestrophin-like Ci transporters and further establish that a chloroplast-to-mitochondria electron flow contributes to energize non-thylakoid Ci transporters, most likely by supplying ATP. We propose an integrated view of the CCM energy supply network, describing how algal cells distribute photosynthesis energy to power different Ci transporters, thus paving the way to the transfer of a functional algal CCM in plants towards improving crop productivity.

One sentence summaryPhotosynthetic alternative electron flows and mitochondrial respiration drive the algal CO2 concentrating mechanism

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Nanopore adaptive sampling: a tool for enrichment of low abundance species in metagenomic samples BackgroundAdaptive sampling is a method of software-controlled enrichment unique to nanopore sequencing platforms recently implemented in Oxford Nanopores own control software. By examining the first few hundred bases of a DNA molecule as it passes through a pore, software can determine if the molecule is sufficiently interesting to sequence in its entirety. If not, the molecule is ejected from the pore by reversing the voltage across it, freeing the pore for a new molecule. User supplied sequences define the targets to be sequenced or ejected. Here we explore the potential of using adaptive sampling for enrichment of rarer species within metagenomic samples.

ResultsWe created a synthetic mock community consisting of seven bacterial species at different proportions ranging from 1.2% to 47% and used this as the basis for a series of enrichment and depletion experiments. To investigate the effect of DNA length on adaptive sampling efficiency, we created sequencing libraries with mean read lengths of 1.7 kbp, 4.7 kbp, 10.6 kbp, and 12.8 kbp and enriched or depleted for individual and multiple species over a series of sequencing runs. Across all experiments enrichment ranged from 1.67-fold for the most abundant species with the shortest read length to 13.87-fold for the least abundant species with the longest read length. Factoring in the reduction to sequence output associated with repeatedly rejecting molecules reduces the calculated efficiency of this enrichment to between 0.96-fold and 4.93-fold. We note that reducing ejections due to false negatives (approximately 36%) would significantly increase efficiency. We used the relationship between abundance, molecule length and enrichment factor to produce a mathematical model of enrichment based on molecule length and relative abundance, whose predictions correlated strongly with experimental data. A web application is provided to allow researchers to explore model predictions in advance of performing their own experiments.

ConclusionsOur data clearly demonstrates the benefit for enriching low abundant species in adaptive sampling metagenomic experiments, especially with longer molecules, and our mathematical model can be used to determine whether a given experimental DNA sample is suitable for adaptive sampling. Notably, repeated voltage reversals have no effect on pore stability.

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Matrix-Bound Growth Factors are Released upon Cartilage Compression by an Aggrecan-Dependent Sodium Flux that is Lost in Osteoarthritis Articular cartilage is a dense extracellular matrix-rich tissue that degrades following chronic mechanical stress, resulting in osteoarthritis (OA). The tissue has low intrinsic repair especially in aged and osteoarthritic joints. Here we describe three pro-regenerative factors; fibroblast growth factor 2 (FGF2), connective tissue growth factor, bound to transforming growth factor-beta (CTGF-TGF{beta}), and hepatoma-derived growth factor (HDGF), that are rapidly released from the pericellular matrix (PCM) of articular cartilage upon mechanical injury. All three growth factors bound heparan sulfate, and were displaced by exogenous NaCl. We hypothesised that sodium, sequestered within the aggrecan-rich matrix, was freed by injurious compression, thereby enhancing the bioavailability of pericellular growth factors. Indeed, growth factor release was abrogated when cartilage aggrecan was depleted by IL-1 treatment, and in severely damaged human osteoarthritic cartilage. A flux in free matrix sodium upon mechanical compression of cartilage was visualised by 23Na magnetic resonance imaging (MRI) just below the articular surface. This corresponded to a region of reduced tissue stiffness, measured by scanning acoustic microscopy and second harmonic generation microscopy, and where Smad2/3 was phosphorylated upon cyclic compression. Our results describe a novel intrinsic repair mechanism, controlled by matrix stiffness and mediated by the free sodium concentration, in which heparan sulfate-bound growth factors are released from cartilage upon injurious load. They identify aggrecan as a depot for sequestered sodium, explaining why osteoarthritic tissue loses its ability to repair. Treatments that restore matrix sodium to allow appropriate release of growth factors upon load are predicted to enable intrinsic cartilage repair in osteoarthritis.

Significance StatementOsteoarthritis is the most prevalent musculoskeletal disease, affecting 250 million people worldwide 1. We identify a novel intrinsic repair response in cartilage, mediated by aggrecan-dependent sodium flux, and dependent upon matrix stiffness, which results in the release of a cocktail of pro-regenerative growth factors after injury. Loss of aggrecan in late-stage osteoarthritis prevents growth factor release and likely contributes to disease progression. Treatments that restore matrix sodium in osteoarthritis may recover the intrinsic repair response to improve disease outcome.

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Electrobehavioral phenotype and seizure pharmacosensitivity in a novel mouse model of patient-derived SLC6A1 S295L mutation-associated neurodevelopmental epilepsy Solute carrier family 6 member 1 (SLC6A1) gene encodes GAT-1, a GABA transporter expressed on glia and presynaptic terminals of inhibitory neurons. Mutations in SLC6A1 are associated with myoclonic atonic epilepsy, absence epilepsy, autism, and intellectual disability. However, the mechanisms leading to these defects are unknown. Here, we used a novel mouse model harboring a point mutation (S295L) recently identified in the human SLC6A1 gene that results in impaired membrane trafficking of the GAT-1 protein. We performed chronic wireless telemetry recordings of heterozygous (GAT-1S295L/+) mice, and of mice lacking one or both copies of the gene (GAT-1+/- and GAT-1-/-). We assessed their behaviors and pharmacosensitivity, and investigated the relationship between seizure burden and behavioral performance. GAT-1S295L/+ mice exhibited frequent spikewave discharges (SWDs) associated with behavioral arrest, and there was a dose-effect relationship between GAT-1 gene copy number and the severity of electrocorticogram (ECoG) abnormalities. Seizure burden was inversely correlated with behavioral performance. Forelimb grip strength was reduced in female mice. Acute administration of GAT-1 antagonist NO-711 induced SWDs in wildtype mice, exacerbated the phenotype in GAT-1S295L/+ and GAT-1+/- mice, and had no effect on GAT-1-/- mice lacking the drug target. By contrast, ethosuximide normalized the ECoG in GAT-1S295L/+ and GAT-1+/- mice. In conclusion, GAT-1S295L/+ mice show haploinsufficiency with evidence of GAT-1 hypofunction. This mouse model reconstitutes major aspects of human disease and thus provides a useful preclinical model for drug screening and gene therapy.

Significance StatementThe SLC6A1 gene encodes for GAT-1, a major GABA transporter. Mutations in SLC6A1 lead to a spectrum of neurodevelopmental disorders and epilepsies collectively referred to as SLC6A1-related disorders (SRD). A critical contributor to disability in SRD patients is the burden of seizures and their sequelae. There is an urgent need to understand the mechanisms of SRD as they will inform therapeutic interventions. Mouse models can provide critical information allowing both the assessment of candidate therapies and the design of next generations therapies. Here we used behavioral assessments and wireless electrophysiology in a new mouse model of SRD to understand the disease pathogenesis and the association between seizure burden and behavioral deficits.

sentences: Single-Cell Transcriptomic Analyses of Tumor Ecosystems and Spatial Architectures in Human Small Cell Lung Cancers Small cell lung cancer (SCLC) is a highly heterogenous disease characterized by aggressive phenotypes and poor prognosis. In order to dissect the cellular components and their spatial distribution in SCLC ecosystems, here we performed single-cell RNA sequencing and spatial transcriptomics analyses of 12 resected human primary SCLCs. Our analysis revealed extensive heterogeneity of both tumor and non-tumor cells, and a predominant immunosuppressive microenvironment. Importantly, multiple hybrid tumor cell states were unmasked, including hybrid SCLC and lung adenocarcinoma cells as well as hybrid tumor and immune cells, indicating high phenotypic plasticity of tumor cells. Moreover, we revealed the cellular origin and evolution of these hybrid tumor cells, and critical roles of tumor-stromal cellular crosstalk in driving the development of hybrid cells. In conclusion, this study deepens our understanding of human SCLC ecosystems and provides rationales to target the hybrid tumor cells and immunosuppressive microenvironment for SCLC therapies.

sentences: Identifying targetable metabolic dependencies across colorectal cancer progression Colorectal cancer (CRC) is a multi-stage process initiated through the formation of a benign adenoma, progressing to an invasive carcinoma and finally metastatic spread. Tumour cells must adapt their metabolism to support the energetic and biosynthetic demands associated with disease progression. As such, targeting cancer cell metabolism is a promising therapeutic avenue in CRC. However, to identify tractable nodes of metabolic vulnerability specific to CRC stage, we must understand how metabolism changes during CRC development. Here, we use a unique model system - comprising human early adenoma to late adenocarcinoma. We show that adenoma cells transition to elevated glycolysis at the early stages of tumour progression but maintain oxidative metabolism. Progressed adenocarcinoma cells rely more on glutamine-derived carbon to fuel the TCA cycle, whereas glycolysis and TCA cycle activity remain tightly coupled in early adenoma cells. Adenocarcinoma cells are more flexible with respect to fuel source, enabling them to proliferate in nutrient-poor environments. Despite this plasticity, we identify asparagine (ASN) synthesis as a node of metabolic vulnerability in late-stage adenocarcinoma cells. We show that loss of asparagine synthetase (ASNS) blocks their proliferation, whereas early adenoma cells are largely resistant to ASN deprivation. Mechanistically, we show that late-stage adenocarcinoma cells are dependent on ASNS to support mTORC1 signalling and maximal glycolytic and oxidative capacity. Resistance to ASNS loss in early adenoma cells is likely due to a feedback loop, absent in late-stage cells, allowing them to sense and regulate ASN levels and supplement ASN by autophagy. Together, our study defines metabolic changes during CRC development and highlights ASN synthesis as a targetable metabolic vulnerability in later stage disease.

sentences: A new role of low barrier hydrogen bond in mediating protein stability by small molecules Low barrier hydrogen bond (LBHB) is a special type of hydrogen bond which occurs where two heteroatoms with similar pKa values share a single proton resulting in an unusually strong and short hydrogen bond. LBHBs in protein play important roles in enzyme catalysis and maintaining protein structural integrity but its other biochemical roles are unknown. Here we report a novel function of LBHB in selectively inducing tubulin protein degradation. A tubulin inhibitor, 3-(3-Phenoxybenzyl) amino-{beta}-carboline (PAC), promotes selective degradation of {beta}-tubulin heterodimers by binding to the colchicine site of {beta}-tubulin. Biochemical studies have revealed that PAC specifically destabilizes tubulin, making it prone to aggregation that then predisposes it to ubiquitinylation and then degradation. Structural activity analyses have indicated that the destabilization is mediated by a single hydrogen bond formed between the pyridine nitrogen of PAC and {beta}Glu198, which is identified as a LBHB. In contrast, another two tubulin inhibitors only forming normal hydrogen bonds with {beta}Glu198 exhibit no degradation effect. Thus, the LBHB accounts for the degradation. Most importantly, we screened for compounds capable of forming LBHB with {beta}Glu198 and demonstrated that BML284, a Wnt signaling activator, also promotes tubulin heterodimers degradation in a PAC-like manner as expected. Our study has identified a novel approach for designing tubulin degraders, providing a unique example of LBHB function and suggests that designing small molecules to form LBHBs with protein residues resulting in the highly specific degradation of a target protein could be a new strategy for drug development.

sentences: Antibiotic-induced recombination in Gram-negative bacteria requires the formation of double-strand breaks Recombination is an essential process in bacterial drug resistance evolution. Fluoroquinolones, an important class of antibiotics, are known to stimulate recombination in Escherichia coli. On the other hand, when bacteria are exposed to sublethal concentrations of different antibiotic families, including strong inducers of the SOS system, which upregulates recombination pathways, there is no detectable change in the recombination rate. Here we explore why fluoroquinolones, but not any other tested antimicrobials, increase recombination rates. One feature that makes the fluoroquinolones different from other antibiotics is the generation of DNA double-strand breaks (DSBs). We tested whether other antimicrobials known for their ability to cause double-strand breaks, such as mitomycin C and bleomycin, are also affecting bacterial recombination rate. We found that both drugs indeed affected recombination rates in E. coli. Furthermore, there was a positive correlation between the number of DNA double-strand breaks and the recombination frequency. The manipulation of the level of DSBs directly impacted the recombination frequency. These results imply that recombination, at least in Gram-negative bacteria, is conservative, and even if multiple copies of a DNA sequence are available, the basal recombination rate is low. Our results highlight that homologous recombination is intended more for DNA damage repair than generating diversity, or at least that they occur at a very different scale. We also show that only antibiotics that cause DNA double-strand breaks can increase genetic diversity via recombination. The stimulation of recombination by DSBs-causing antimicrobials is an additional factor leading to the risk of antibiotic resistance evolution.

sentences: Expression and subcellular localization of USH1C/harmonin in the human retina provides insights into pathophysiology and therapy of Usher syndrome Usher syndrome (USH) is the most common form of hereditary deafness-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retina uncovered harmonin a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Muller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones, and outer segments of rods as well as at adhesive junctions of MGCs and photoreceptors in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in MGCs and photoreceptors and rhodopsin in photoreceptors. Subcellular expression and colocalization of harmonin correlates with the clinical phenotype observed in USH1C patients. In addition, primary cilia defects in USH1C patient-derived fibroblasts could be reverted by delivery of harmonin a1 transcript isoform. Our data provide novel insights into photoreceptor cell biology, USH1C pathophysiology and for developing gene therapy treatment.

sentences: A single synonymous nucleotide change impacts the male-killing phenotype of prophage WO gene wmk Wolbachia are the most widespread bacterial endosymbionts in animals. Within arthropods, these maternally-transmitted bacteria can selfishly hijack host reproductive processes to increase the relative fitness of their transmitting females. One such form of reproductive parasitism called male killing, or the selective killing of infected males, is recapitulated to degrees by transgenic expression of the WO-mediated killing (wmk) gene. Here, we characterize the genotype-phenotype landscape of wmk-induced male killing in D. melanogaster using transgenic expression. While phylogenetically distant wmk homologs induce no sex-ratio bias, closely-related homologs exhibit complex phenotypes spanning no death, male death, or death of all hosts. We demonstrate that alternative start codons, synonymous codons, and notably a single synonymous nucleotide in wmk can ablate killing. These findings reveal previously unrecognized features of transgenic wmk-induced killing and establish new hypotheses for the impacts of post-transcriptional processes in male killing variation. We conclude that synonymous sequence changes are not necessarily silent in nested endosymbiotic interactions with life-or-death consequences.

sentences: Proteome Microarray Screening Identifies Human Polyphosphate-Binding Proteins in the Phosphatidylinositol Signaling Pathway Polyphosphates are linear chains of orthophosphate residues that are present in all living cells. Polyphosphates are released from platelet d-granules and are also produced in bacteria. Polyphosphates are procoagulant in mammalian species and in bacteria are required for energy and phosphate storage, stress resistance, chelation of metal ions and escaping host immunity. Despite these pleiotropic effects, sparse information is available on molecular binding partners of polyphosphates. Here, we used a slide-based human proteome microarray screen for the search of polyphosphate-binding proteins. This approach suggested several novel proteins with relation to the phosphatidylinositol signaling pathway. The highest signals were obtained for Disabled-1 (DAB1) and phosphatidylinositol-5-phosphate 4-kinase 2B (PIP4K2B). Isothermal titration calorimetry was used for confirmation of DAB1 interactions with long-chain polyphosphates. These results offer new rationale to further investigate the interference of polyphosphates with intracellular signaling pathways.

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Spatial transcriptomic characterization of COVID-19 pneumonitis identifies immune circuits related to tissue injury Severe lung damage in COVID-19 involves complex interactions between diverse populations of immune and stromal cells. In this study, we used a spatial transcriptomics approach to delineate the cells, pathways and genes present across the spectrum of histopathological damage in COVID-19 lung tissue. We applied correlation network-based approaches to deconvolve gene expression data from areas of interest within well preserved post-mortem lung samples from three patients. Despite substantial inter-patient heterogeneity we discovered evidence for a common immune cell signaling circuit in areas of severe tissue that involves crosstalk between cytotoxic lymphocytes and pro-inflammatory macrophages. Expression of IFNG by cytotoxic lymphocytes was associated with induction of chemokines including CXCL9, CXCL10 and CXCL11 which are known to promote the recruitment of CXCR3+ immune cells. The tumour necrosis factor (TNF) superfamily members BAFF (TNFSF13B) and TRAIL (TNFSF10) were found to be consistently upregulated in the areas with severe tissue damage. We used published spatial and single cell SARS-CoV-2 datasets to confirm our findings in the lung tissue from additional cohorts of COVID-19 patients. The resulting model of severe COVID-19 immune-mediated tissue pathology may inform future therapeutic strategies.

One Sentence SummarySpatial analysis identifies IFN{gamma} response signatures as focal to severe alveolar damage in COVID-19 pneumonitis.

sentences: Optimizing transcranial direct current stimulation (tDCS) electrode position, size, and distance doubles the on-target cortical electric field: Evidence from 3000 Human Connectome Project models Transcranial direct current stimulation (tDCS) is a widely used noninvasive brain stimulation technique with mixed results and no FDA-approved therapeutic indication to date. So far, thousands of published tDCS studies have placed large scalp electrodes directly over the intended brain target and delivered the same stimulation intensity to each person. Inconsistent therapeutic results may be due to insufficient cortical activation in some individuals and the inability to determine an optimal dose. Here, we computed 3000 MRI-based electric field models in 200 Human Connectome Project (HCP) participants, finding that the largely unexamined variables of electrode position, size, and between-electrode distance significantly impact the delivered cortical electric field magnitude. At the same scalp stimulation intensity, smaller electrodes surrounding the neural target deliver more than double the on-target cortical electric field while stimulating only a fraction of the off-target brain regions. This new optimized tDCS method can ensure sufficient cortical activation in each person and could produce larger and more consistent behavioral effects in every prospective research and transdiagnostic clinical application of tDCS.

sentences: Effective generation of tumor-infiltrating lymphocyte products from metastatic non-small-cell lung cancer (NSCLC) lesions irrespective of location and previous treatments. Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Because current treatment regimens show limited success rates, alternative therapeutic approaches are needed. We recently showed that treatment-naive, stage I/II primary NSCLC tumors contain a high percentage of tumor-reactive T cells, and that these tumor-reactive T cells can be effectively expanded and used for the generation of autologous TIL therapy. Whether these promising findings also hold true for metastatic lesions is unknown yet critical for the translation into the clinic. We here studied the lymphocytic composition and the feasibility to generate tumor-responsive TIL products from metastatic NSCLC lesions that were derived from different locations and from patients who had previously received different treatment regimens, or who were treatment naive. The overall numbers and composition of lymphocyte infiltrates from this various metastatic lesions was by and large comparable to that of early-stage primary NSCLC tumors. With the clinically approved TIL expansion protocol, we effectively expanded TILs from metastatic NSCLC lesions to numbers that were compatible with TIL transfusion, irrespective of the location of the metastasis and of the previous treatment. Importantly, 16 of 21 (76%) tested TIL products displayed anti-tumoral activity, as determined by their production of the key pro-inflammatory cytokines IFN-{gamma}, TNF, IL-2 and of CD137 expression upon incubation with autologous tumor digests by CD8+ and CD4+ T cells. In summary, metastatic NSCLC lesions constitute a viable source for the generation of tumor-reactive TIL products for therapeutic purposes irrespective of their location and the pretreatment regimens.

sentences: Structure and function of a Notch-like proteolytic switch domain in the extracellular matrix receptor dystroglycan The adhesion receptor dystroglycan provides a critical mechanical link between the extracellular matrix (ECM) and the actin cytoskeleton to help muscle cells withstand contraction and neural cells maintain the blood brain barrier. Disrupting the link is associated cancer and muscular dystrophy. Proteolysis of dystroglycan by matrix metalloproteinases (MMPs) provides a mechanism to break the mechanical anchor and is amplified in several pathogenic states, yet how MMP cleavage is regulated is largely unexplored. We solved the crystal structure of the extracellular membrane-adjacent domains of dystroglycan containing the MMP site and find the tandem Ig-like and SEA-like domains structurally interact in an L-shape to facilitate a permissive conformation for /{beta} auto-catalytic processing. The MMP cleavage site is located in a structural motif C-terminal to the SEA-like domain that folds back and nestles into a pocket within the SEA-like domain to protect the cleavage site from MMPs. We then show while the intact proteolytic switch domain is resistant to MMP cleavage, structurally disruptive muscular dystrophy-related mutations sensitize dystroglycan to proteolysis in secreted constructs and the full-length receptor. Intriguingly, previously uncharacterized cancer-associated mutations that map to the proteolytic switch domain similarly lead to increases in proteolysis. Taken together, this study demonstrates dystroglycan proteolysis is conformationally regulated and mutations in the domain lead to enhanced proteolysis.

sentences: Viscous effects and complex local flow behaviors dominate hemolymph circulation in the living wings of locusts An insects living systems - circulation, respiration, and a branching nervous system - extend from the body into the wing.1,2. Hemolypmh circulation in the wing is critical for hydrating tissues, such as the highly elastic resilin3 that enhances wing flexibility, and for supplying nutrients to living systems, including sensory organs such as scent-producing patches, sound-receiving tympana, and wind-sensing sensilla distributed across the wing.4-7During flight, the presence of hemolymph in the wings reduces aerodynamic instabilities like flutter8,9, and faster hemolymph flows are induced by flapping.10 Despite the critical role of hemolymph circulation in maintaining healthy wing function, wings are often considered "lifeless" cuticle, and most measurements remain qualitative or employ coarse, bulk-flow techniques. While pioneering work in the 1960s mapped hemolymph flow direction in 100 insect species,11 half a century later we still only have quantitative measurements of flow within the wings of a few insects. Here, we focused on the North American locust Schistocerca americana, a well-studied agricultural pest species, and performed a detailed, quantitative study of global and local hemolymph flows in the densely venated fore and hind wings, along with key regions in the body and pumping organs. Through high-speed fluorescent microscopy, we measured 800 individual trajectories of neutrally buoyant fluorescent particles that move in sync with hemolymph, in the wings and body of 8 live, resting locusts. Our data show that overall flow within the wings is circuitous, but local flow behavior is highly complex, with three distinct types of flow (pulsatile, continuous, and "leaky") occurring in various combinations in different areas of the wing. We provide the first quantitative measurements of "leaky" flow into wing regions that act as sinuses, where hemolymph flows out of tubular veins and pools within thin membranous regions. We also calculate Peclet, Reynolds, and Womersley numbers, and find that viscous effects dominate flow regimes throughout the wing. Pumping organs and wing regions closest to the body display significantly faster flows and higher Reynolds numbers, but remain within the viscous flow regime. Given the central role of wings in sustaining ecologically important insect behaviors such as pollination, migration, and mating, along with the vast diversity of insect wings seen in nature, this first detailed, quantitative map of hemolymph flows across a wing provides a template for future studies investigating the dynamics of hemolymph flows critical to sustaining wing health among insects.

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ifCNV: a novel isolation-forest-based package to detect copy number variations from NGS datasets Copy number variations (CNVs) are an essential component of genetic variation distributed across large parts of the human genome. CNV detection from next-generation sequencing data and artificial intelligence algorithms has progressed in recent years. However, only a few tools have taken advantage of machine learning algorithms for CNV detection, and none propose using artificial intelligence to automatically detect probable CNV-positive samples. Furthermore, in general, most CNV software that is developed for specific data types has sub-optimal reliability for routine practice. In addition, the most developed approach is to use a reference or normal dataset to compare with the samples of interest, and it is well known that selecting appropriate normal samples represents a challenging task which dramatically influences the precision of results in all CNV-detecting tools. With careful consideration of these issues, we propose here ifCNV, a new software based on isolation forests that creates its own reference, available in R and python with customisable parameters. ifCNV combines artificial intelligence using two isolation forests and a comprehensive scoring method to faithfully detect CNVs among various samples. It was validated using datasets from diverse origins (capture and amplicon, germline and somatic), and it exhibits high sensitivity, specificity and accuracy. ifCNV is a publicly available open-source software that allows the detection of CNVs in many clinical situations.

Key pointsO_LICopy number variation detection
C_LIO_LIMachine learning
C_LIO_LILocalisation scoring
C_LIO_LIBenchmark on various clinical situations and on various datasets
C_LIO_LIEasy-to-use R and Python open-source Package
C_LI

sentences: Bone marrow haematopoietic stem cells influence liver homeostatic networks and cancer development after dietary intervention In fatty liver disease, systemic homeostasis is perturbed. While pre-clinical models are used to understand its pathogenesis, translating this knowledge to patients is difficult. However, by focusing on the most preserved homeostasis systems between species and models, novel disease dimensions can be unearthed. We interrogated core liver gene co-expression networks in a mouse model of liver cancer following dietary challenge. Immunometabolic modules showed temporal changes under the influence of diet duration and aging. The behaviour of immune network in tumours mirrored their counterparts in non-tumour liver. A high immune response network was associated with a lower tumour burden in mice and humans. This module in mice was enriched for genes related to haematopoietic cell differentiation. Consistently, the bone marrow haematopoietic stem and progenitor cells response was reflective of the liver immune response. Linking haematopoiesis to hepatic homeostasis uncovers a hitherto unexplored dimension of tissue crosstalk that can inform pathogenesis.

sentences: Rethinking Statistical Learning as a Dynamic Stochastic Process, from The Motor Systems Perspective The brain integrates streams of sensory input and builds accurate predictions, while arriving at stable percepts under disparate time scales. This stochastic process bears different dynamics for different people, yet statistical learning (SL) currently averages out, as noise, individual fluctuations in data streams registered from the brain as the person learns. We here adopt the motor systems perspective to reframe SL. Specifically, we rethink this problem using the demands that the persons brain faces to predict, and control variations in biorhythmic activity akin to those present in bodily motions. This new approach harnesses gross data as the important signals, to reassess how individuals learn predictive information in stable and unstable environments. We find two types of learners: narrow-variance learners, who retain explicit knowledge of the regularity embedded in the stimuli -the goal. They seem to use an error-correction strategy steadily present in both stable and unstable cases. In contrast, broad-variance learners emerge only in the unstable environment. They undergo an initial period of memoryless learning characterized by a gamma process that starts out exponentially distributed but converges to Gaussian. We coin this mode exploratory, preceding the more general error-correction mode characterized by skewed-to-symmetric distributions and higher signal content from the start. Our work demonstrates that statistical learning is a highly dynamic and stochastic process, unfolding at different time scales, and evolving distinct learning strategies on demand.

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GSA: An Independent Development Algorithm for Calling Copy Number and Detecting Homologous Recombination Deficiency (HRD) from Target Capture Sequencing BackgroundThe gain or loss of large chromosomal regions or even whole chromosomes is termed as genomic scarring and can be observed as copy number variations resulting from the failure of DNA damage repair.

ResultsIn this study, a new algorithm called Genomic Scar Analysis (GSA) has developed and validated to calculate homologous recombination deficiency (HRD) score. The two critical submodules were tree recursion (TR) segmentation and filtering, and the estimation and correction of the tumor purity and ploidy. Then, this study evaluated the rationality of segmentation and genotype identification by the GSA algorithm and compared with other two algorithms, PureCN and ASCAT, found that the segmentation result of GSA algorithm was more logical. In addition, the results indicated that the GSA algorithm had an excellent predictive effect on tumor purity and ploidy, if the tumor purity was more than 20%. Furtherly, this study evaluated the HRD scores and BRCA1/2 deficiency status of 195 clinical samples, and the results indicated that the accuracy was 0.98 (comparing with Affymetrix OncoScan assay) and the sensitivity was 95.2% (comparing with BRCA1/2 deficiency status), both were well-behaved. Finally, HRD scores and 16 genes mutations (TP53 and 15 HRR pathway genes) were analyzed in 17 cell lines, the results showed that there was higher frequency in HRR pathway genes in high HRD score samples.

ConclusionsThis new algorithm, named as GSA, could effectively and accurately calculate the purity and ploidy of tumor samples through NGS data, and then reflect the degree of genomic instability and large-scale copy number variations of tumor samples.

sentences: Comprehensive profiling of mutations to influenza virus PB2 that confer resistance to the cap-binding inhibitor pimodivir Antivirals are used not only in current treatment of influenza, but are also stockpiled as a first line of defense against novel influenza strains for which vaccines have yet to be developed. Identifying drug resistance mutations can guide clinical deployment of the antiviral, and additionally define the mechanisms of drug action and drug resistance. Pimodivir is a first-in-class inhibitor of the polymerase basic protein 2 (PB2) subunit of the influenza A virus polymerase complex. A number of resistance mutations have previously been identified in treated patients or cell culture. Here, we generate a complete map of the effect of all single-amino-acid mutations to an avian PB2 on resistance to pimodivir. We identified both known and novel resistance mutations not only in the previously implicated cap-binding and mid-link domains, but also in the N-terminal domain. Our complete map of pimodivir resistance thus enables the evaluation of whether new viral strains contain mutations that will confer pimodivir resistance.

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T cell receptor (TCR) repertoire analysis reveals a highly conserved TCR repertoire in a bilateral tumor mouse model Temporal analysis of the T cell receptor (TCR) repertoire has been used to monitor treatment-induced changes in antigen-specific T cells in patients with cancer. However, the lack of experimental models that allow temporal analysis of the TCR repertoire in the same individual in a homogeneous population limits the understanding of the causal relationship between changes in TCR repertoire and antitumor responses. A bilateral tumor model, where tumor cells were inoculated bilaterally into the backs of mice, could be used for temporal analysis of the TCR repertoire. This study examined the prerequisite for this strategy: the TCR repertoire is conserved between bilateral tumors with the same growth rate. Bilateral tumors with equivalent size and draining lymph nodes (dLNs) were collected 13 days after tumor inoculation to analyze the TCR repertoire of CD4+ and CD8+ T cells. The tumor-infiltrating T cell clones were highly conserved between the bilateral tumors, and the extent of clonal expansion was equivalent. In addition, the similarity between the bilateral tumors was equivalent to heterogeneity on one side of the tumor. The similarity of the TCR repertoire in the bilateral dLNs was markedly lower than that in the tumor, suggesting that tumor-reactive T cell clones induced independently in each dLN integrated during recirculation and then infiltrated the tumor. These findings suggest that our bilateral tumor model is suitable for temporal monitoring of the TCR repertoire to evaluate temporal and treatment-induced changes in tumor-reactive T cell clones.

Significance StatementThe bilateral subcutaneous tumor model, where tumor cells were inoculated bilaterally into the backs of mice, is a promising model for temporal analysis of the antitumor response in cancer immunotherapy. This study demonstrated a highly conserved TCR repertoire in bilateral tumors and provided the basis for using a bilateral tumor model for evaluating temporal and treatment-induced changes in tumor-responsive T cell clones in individual mice. In humans, accurate statistical analysis is hampered by various background factors, including cancer type and stage and history of treatment. Moreover, temporal tumor biopsy in patients is highly invasive. Our bilateral tumor model overcomes these clinical issues and is expected to be a valuable tool for the development of novel immune monitoring and therapeutic strategies.

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Postnatal lymph node expansion of stromal progenitors towards reticular and CD34+ stromal cell subsets is determined by distinct transcriptional programs Gut-draining mesenteric lymph nodes (mLN) provide the framework and microenvironment to shape intestinal adaptive immune responses. We previously delineated transcriptional signatures in LN stromal cells (SC), pointing to tissue-specific variability in composition and immuno-modulatory function of SCs.

Here, we dissect the tissue-specific epigenomic DNA accessibility and CpG methylation landscape of LN non-endothelial SCs and identify a microbiota-independent core epigenomic signature of LN SCs. By combined analysis of transcription factor (TF) binding sites together with the gene expression profiles of non-endothelial SCs, we delineated TFs poising skin-draining peripheral LN (pLN) SCs for pro-inflammatory responses. Furthermore, using scRNA-seq, we dissected the developmental trajectory of mLN SCs derived from postnatal to aged mice, identifying two distinct putative progenitors, namely CD34+ SC and fibroblastic reticular stromal cell (FRC) progenitors, which both feed the rapid postnatal LN expansion. Finally, we identified Irf3 as a key differentiation TF inferred from the epigenomic signature of mLN SCs that is dynamically expressed along the differentiation trajectories of FRCs, and validated Irf3 as a regulator of Cxcl9+ FRC differentiation.

Together, our data constitute a comprehensive transcriptional and epigenomic map of mLN development and dissect location-specific, microbiota-independent properties of mLN non-endothelial SCs. As such, our findings represent a valuable resource to identify core transcriptional regulators that impinge on the developing mLN early in life, thereby shaping long-lasting intestinal adaptive immune responses.

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Evolutionary coupling range varies widely among enzymes depending on selection pressure Recent studies proposed that enzyme active sites induce evolutionary constraints at long distances. The physical origin of such long-range evolutionary coupling is unknown. Here, I use a recent biophysical model of evolution to study the relationship between physical and evolutionary couplings on a diverse data set of monomeric enzymes. I show that evolutionary coupling is not universally long-range. Rather, range varies widely among enzymes, from 2[A] to 20[A]. Furthermore, the evolutionary coupling range of an enzyme does not inform on the underlying physical coupling, which is short-range for all enzymes. Rather, evolutionary coupling range is determined by functional selection pressure.

SIGNIFICANCEUntil recently, only residues near enzyme active sites were thought to be evolutionarily constrained. However, recent studies proposed that active sites induce long-range evolutionary constraints. This seems to conflict with the common finding that physical couplings in proteins are short-range. This raises the question of how short-range physical couplings may cause long-range evolutionary couplings. Here, I show that the function that maps physical coupling into evolutionary coupling depends on functional selection pressure. Under weak selection, both couplings are similarly short-range; under strong selection, short-range physical coupling is non-linearly turned into long-range evolutionary coupling. Thus, due to a huge variation of selection pressure, evolutionary coupling range varies widely among enzymes, from very short (2 [A]) to very long (20 [A]).

sentences: Predictive link between systemic metabolism and immune signaling in the brain of APOE4 mice The {varepsilon}4 variant of apolipoprotein E (APOE) is the strongest and most common genetic risk factor for Alzheimers disease (AD). While the mechanism of conveyed risk is incompletely understood, promotion of inflammation, dysregulated metabolism, and protein misfolding and aggregation have been implicated as potential contributions to accelerating disease. Here we determined the concurrent effects of systemic metabolic changes and brain inflammation in young (3-month-old) and aged (18-month-old) female mice carrying the human APOE4 gene. Using functional metabolic assays alongside multivariate modeling of hippocampal cytokine signaling, we found that immune signaling patterns in the brain are predictive of systemic metabolic outcomes, independent of AD proteinopathies. The relationship between cytokine signaling networks in the hippocampus and systemic glucose tolerance and adiposity differ by APOE genotype, where APOE4 mice produce lower levels of neuroprotective cytokines as they age and as their metabolic phenotype declines. To our knowledge, ours is the first study to identify a quantitative and predictive link between systemic metabolism and specific pathological immune signaling signatures in the brain. Our results highlight the AD-relevant effects of APOE4 beyond the brain and highlight the potential for bidirectional systemic influences of risk factors in the brain and periphery.

sentences: Two new threatened species of Rinorea (Violaceae), forest trees from East and South Regions, Cameroon Two tree species are described as new to science: Rinorea spongiocarpa Achound. sp. nov (placed in Rinorea [unranked] Brachypetalae) and Rinorea dimakoensis Achound. sp. nov.( placed in Rinorea [unranked] Ilicifolieae). Both species are endemic to Cameroon, occurring south of the Sanaga river, the first from South and East Regions, occurring in evergreen forest from Ebolowa to Dja, while the second occurs in the northern part of East Region in semi-deciduous forest towards the interface with woodland habitats in the Dimako-Bertoua area. The two species are illustrated, and their affinities and conservation status according to the 2012 IUCN categories and criteria are discussed. Both species are threatened with extinction due to habitat destruction, the first is assessed as Vulnerable, the second Endangered.

sentences: Nrf2 modulates the hybrid epithelial/mesenchymal phenotype and Notch signaling during collective cancer migration Hybrid epithelial/mesenchymal cells (E/M) are key players in aggressive cancer metastasis. It remains a challenge to understand how these cell states, which are mostly non-existent in healthy tissue, become stable phenotypes participating in collective cancer migration. The transcription factor Nrf2, which is associated with tumor progression and resistance to therapy, appears to be central to this process. Here, using a combination of single cell biosensors, immunocytochemistry, and computational modeling, we show that Nrf2 functions as a phenotypic stability factor for hybrid E/M cells by inhibiting a complete epithelial-mesenchymal transition (EMT) during collective cancer migration. We also demonstrate that Nrf2 and EMT signaling are spatially coordinated near the migrating front. In particular, computational analysis of an Nrf2-EMT-Notch network and experimental modulation of Nrf2 by pharmacological treatment or CRISPR/Cas9 gene editing reveal that Nrf2 stabilizes a hybrid E/M phenotype which is maximally observed in the interior region immediately behind the leading edge. We further demonstrate that the Nrf2-EMT-Notch network enhances Dll4 and Jagged1 expression near the leading edge, which correlates with the formation of leader cells and protruding tips. Altogether, our results provide direct evidence that Nrf2 acts as a phenotypic stability factor in restricting complete EMT and plays an important role in coordinating collective cancer migration.

sentences: L-threonine mediated DAF-16/HSF-1 activation inhibits ferroptosis and increases healthspan The pathways that impact longevity in the wake of dietary restriction (DR) pathway remain still ill-defined. Most studies have focused on nutrient limitation and perturbations of energy metabolism to explain the beneficial effects of DR. We showed that the essential amino acid L-threonine was elevated in Caenorhabditis elegans under DR, and that L-threonine supplementation increased its healthspan. To elucidate the underlying mechanism, we conducted metabolic and transcriptomic profiling using LC-MS/MS and RNA-seq analyses in worms that were fed with RNAi to induce loss of function of key candidate mediators and evaluated healthspan. L-threonine supplementation and loss-of-threonine dehydrogenase, which govern L-threonine metabolism, increased the healthspan of C. elegans by attenuating ferroptosis in a ferritin-dependent manner. Tran-scriptomic analysis of C. elegans supplemented with L-threonine showed that FTN-1 encoding ferritin was elevated, implying FTN-1 is an essential mediator of longevity promotion through L-threonine. Organismal ferritin levels were positively correlated with chronological aging and L-thre-onine supplementation further increased ferritin levels, which protected against age-associated ferroptosis through the DAF-16 and HSF-1 pathways. Our investigation uncovered the role of a distinct and universal metabolite, L-threonine, in DR-mediated improvement in organismal healthspan, suggesting it could be an effective intervention for preventing senescence progression and age-induced ferroptosis.

sentences: Testing the adaptive walk model of gene evolution Understanding the dynamics of species adaptation to their environments has long been a central focus of the study of evolution. Theories of adaptation propose that populations evolve by "walking" in a fitness landscape. This "adaptive walk" is characterised by a pattern of diminishing returns, where populations further away from their fitness optimum take larger steps than those closer to their optimal conditions. Hence, we expect young genes to evolve faster and experience mutations with stronger fitness effects than older genes because they are further away from their fitness optimum. Testing this hypothesis, however, constitutes an arduous task. Young genes are small, encode proteins with a higher degree of intrinsic disorder, are expressed at lower levels, and are involved in species-specific adaptations. Since all these factors lead to increased protein evolutionary rates, they could be masking the effect of gene age. While controlling for these factors, we fitted models of the distribution of fitness effects to population genomic datasets of Arabidopsis and Drosophila. We found that a genes evolutionary age significantly impacts the molecular rate of adaptation. Moreover, we observed that substitutions in young genes tend to have larger fitness effects. Our study, therefore, provides strong evidence that molecular evolution follows an adaptive walk model across large evolutionary timescale.

sentences: Processing of oxidatively damaged DNA dirty ends by APE1 Reactive oxygen species attack the structure of DNA, thus altering its base-pairing properties. Consequently, oxidative stress-associated DNA lesions are a major source of the mutation load that gives rise to cancer and other diseases. Base excision repair (BER) is the pathway primarily tasked with repairing DNA base damage, with apurinic/apyrimidinic endonuclease (APE1) having both AP-endonuclease and 3 to 5 exonuclease (exo) DNA cleavage functions. The lesion 8-oxo-7,8-dihydroguanine (8-oxoG) can enter the genome as either a product of direct damage to the DNA, or through polymerase insertion at the 3-end of a DNA strand during replication or repair. Importantly, 3-8-oxoG impairs the ligation step of BER and therefore must be removed by the exo activity of a surrogate enzyme to prevent double stranded breaks and cell death. In the present study, we characterize the exo activity of APE1 on 3-8-oxoG substrates. These structures demonstrate that APE1 uses a unified mechanism for its exo activities that differs from its more canonical AP-endonuclease activity. In addition, through complementation of the structural data with enzyme kinetics and binding studies employing both wild-type and rationally designed APE1 mutants, we were able to identify and characterize unique protein:DNA contacts that specifically mediate 8-oxoG removal by APE1.

sentences: Initial efficacy determination and resistance profile of anti-Acinetobacter antibiotics, turnercyclamycins Drug-resistant Acinetobacter is a challenging, deadly pathogen of increasing prevalence in the US healthcare system. Recently, we described a series of lipopeptides, the turnercyclamycins, which retain potency against Acinetobacter strains that are resistant to the last-line antibiotic, colistin. To further evaluate the potential of turnercyclamycins, we completed mouse efficacy, pharmacokinetics, and toxicity studies. These demonstrate that turnercyclamycin A has a pharmacological profile with similarity to other lipopeptides that are in clinical use. Turnercyclamycin A was well tolerated in mice up to 25 mg/kg, and exhibited >99% and >98% reduction in bacterial load compared to vehicle control in a thigh infection model at 25 and 12.5 mg/kg, respectively. This result closely reflected the anticipated effectiveness based upon in vitro activity and was similar to the colistin control. Acinetobacter strains resistant to colistin often harbor the mcr-1 resistance gene. Here, we show that the effectiveness of turnercyclamycins against Escherichia coli is not greatly altered by mcr-1 (0- to 2-fold) whereas there is a 16-fold increase in the colistin minimal inhibitory concentration when mcr-1 is present. These data suggest that turnercyclamycins are suitable for further investigation and optimization as anti-Acinetobacter lead compounds.

sentences: A large-scale systematic survey of SARS-CoV-2 antibodies reveals recurring molecular features In the past two years, the global research in combating COVID-19 pandemic has led to isolation and characterization of numerous human antibodies to the SARS-CoV-2 spike. This enormous collection of antibodies provides an unprecedented opportunity to study the antibody response to a single antigen. From mining information derived from 88 research publications and 13 patents, we have assembled a dataset of [~]8,000 human antibodies to the SARS-CoV-2 spike from >200 donors. Analysis of antibody targeting of different domains of the spike protein reveals a number of common (public) responses to SARS-CoV-2, exemplified via recurring IGHV/IGK(L)V pairs, CDR H3 sequences, IGHD usage, and somatic hypermutation. We further present a proof-of-concept for prediction of antigen specificity using deep learning to differentiate sequences of antibodies to SARS-CoV-2 spike and to influenza hemagglutinin. Overall, this study not only provides an informative resource for antibody and vaccine research, but fundamentally advances our molecular understanding of public antibody responses to a viral pathogen.

sentences: NMR structures and functional roles of two related chitin-binding domains of a lytic polysaccharide monooxygenase from Cellvibrio japonicus Among the extensive repertoire of carbohydrate-active enzymes, lytic polysaccharide monooxygenases (LPMOs) have a key role in recalcitrant biomass degradation. LPMOs are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides such as cellulose and chitin. Several LPMOs contain carbohydrate-binding modules (CBMs) that are known to promote LPMO efficiency. Still, structural and functional properties of some of these CBMs remain unknown and it is not clear why some LPMOs, like CjLPMO10A from Cellvibrio japonicus, have two CBMs (CjCBM5 and CjCBM73). Here, we studied substrate binding by these two CBMs to shine light on the functional variation, and determined the solution structures of both by NMR, which includes the first structure of a member of the CBM73 family. Chitin-binding experiments and molecular dynamics simulations showed that, while both CBMs bind crystalline chitin with Kd values in the {micro}M range, CjCBM73 has higher affinity than CjCBM5. Furthermore, NMR titration experiments showed that CjCBM5 binds soluble chitohexaose, whereas no binding to soluble chitin was detected for CjCBM73. These functional differences correlated with distinctly different architectures of the substrate-binding surfaces of the two CBMs. Taken together, these results provide insight into natural variation among related chitin-binding CBMs and the possible functional implications of such variation.

sentences: Transcriptomic analysis of resistant and susceptible responses in a new model root-knot nematode infection system using Solanum torvum and Meloidogyne arenaria Root-knot nematodes (RKNs) are among the most devastating pests in agriculture. Solanum torvum Sw. (turkey berry) has been used as a rootstock for eggplant (aubergine) cultivation because of its resistance to RKNs, including Meloidogyne incognita and M. arenaria. We previously found that a pathotype of M. arenaria, A2-J, is able to infect and propagate in S. torvum. In vitro infection assays showed that S. torvum induces the accumulation of brown pigments during avirulent pathotype A2-O infection, but not during virulent A2-J infection. This experimental system is advantageous because resistant and susceptible responses can be distinguished within a few days, and because a single plant genome can yield information about both resistant and susceptible responses. Comparative RNA-sequencing analysis of S. torvum inoculated with A2-J and A2-O at early stages of infection was used to parse the specific resistance and susceptible responses. Infection with A2-J did not induce statistically significant changes in gene expression within one day post-inoculation (DPI), but afterward, A2-J specifically induced the expression of chalcone synthase, spermidine synthase, and genes related to cell wall modification and transmembrane transport. Infection with A2-O rapidly induced the expression of genes encoding class III peroxidases, sesquiterpene synthases, and fatty acid desaturases at 1 DPI, followed by genes involved in defense, hormone signaling, and the biosynthesis of lignin at 3 DPI. Both isolates induced the expression of suberin biosynthetic genes, which may be triggered by wounding during nematode infection. Histochemical analysis revealed that A2-O, but not A2-J, induced lignin accumulation at the root tip, suggesting that physical reinforcement of cell walls with lignin is an important defense response against nematodes. The S. torvum-RKN system can provide a molecular basis for understanding plant-nematode interactions.

sentences: Microwell array-based opto-electrochemical detections revealing co-adaptation of rheological properties and oxygen metabolism in budding yeast Microdevices composed of microwell arrays integrating nanoelectrodes (OptoElecWell) were developed to achieve dual high-resolution optical and electrochemical detections on single Saccharomyces cerevisiae budding yeast cells. Each array consists in 1.6 x 105 microwells of 8 {micro}m diameter and 5 {micro}m height, with a platinum nanoring electrode for in-situ electrochemistry, all integrated on a transparent thin wafer for further high-resolution live-cell imaging. After optimizing the filling rate, 32% of cells were effectively trapped within microwells. This allowed to analyse S. cerevisiae metabolisms associated with basal respiration while simultaneously measuring optically other cellular parameters. In this study, we focused on the impact of glucose concentration on respiration and intracellular rheology. We found that while oxygen uptake rate decreased with increasing glucose concentration, diffusion of tracer nanoparticles increased. Our OptoElecWell based respiration methodology provided similar results compared to the commercial gold-standard Seahorse XF analyser, while using 20 times lesser biological samples, paving the way to achieve single cell metabolomics. In addition, it facilitates an optical route to monitor the contents within single cells. The proposed device, in combination with the dual detection analysis, opens up new avenues for measuring cellular metabolism, and relating it to various cellular physiological and rheological indicators at single cell level.

sentences: Autophagy as a mechanism for adaptive prediction-mediated emergence of drug resistance Drug resistance is a major problem in treatment of microbial infections and cancers. There is growing evidence that a transient drug tolerant state may precede and potentiate the emergence of drug resistance. Therefore, understanding the mechanisms leading to tolerance is critical for combating drug resistance and for the development of effective therapeutic strategy. Through laboratory evolution of yeast, we recently demonstrated that adaptive prediction (AP), a strategy employed by organisms to anticipate and prepare for a future stressful environment, can emerge within 100 generations by linking the response triggered by a neutral cue (caffeine) to a mechanism of protection against a lethal agent (5-FOA). Here, we demonstrate that mutations selected across multiple laboratory evolved lines had linked the neutral cue response to core genes of autophagy. Across these evolved lines, conditional activation of autophagy through AP conferred tolerance, and potentiated subsequent selection of mutations in genes specific to overcoming the toxicity of 5-FOA. We propose a model to explain how extensive genome-wide genetic interactions of autophagy facilitates emergence of AP over short evolutionary timescales to potentiate selection of resistance-conferring mutations.

sentences: Quantitative fragmentomics allow affinity mapping of interactomes Human protein networks have been widely explored but most binding affinities remain unknown, hindering quantitative interactome-function studies. Yet interactomes rely on minimal interacting fragments displaying quantifiable affinities. Here we measured the affinities of 65,000 interactions involving PDZ domains and their target PDZ-binding motifs (PBM) within a human interactome region particularly relevant for viral infection and cancer. We calculate interactomic distances, identify hot spots for viral interference, generate binding profiles and specificity logos, and explain selected cases by crystallographic studies. Mass spectrometry experiments on cell extracts and literature surveys show that quantitative fragmentomics effectively complement protein interactomics by providing affinities and completeness of coverage, putting a full human interactome affinity survey within realistic reach. Finally, we show that interactome hijacking by the viral PBM of human papillomavirus (HPV) E6 oncoprotein deeply impacts the host cell proteome way beyond immediate E6 binders, illustrating the complex system-wide relationship between interactome and function.

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Design and Computational Analysis of a Chimeric Avian Influenza Antigen: A Yeast-displayed, Universal and Cross-protective Vaccine Candidate BackgroundA cross-protective avian influenza vaccine candidate can be designed by using a preserved antigen against mutation in various subtypes of influenza. M2e peptide sequence has remained remarkably unchanged in influenza type A isolated since 1918.

MethodsA consensus sequence of M2e peptide was obtained from 31 sequences of H5N8, H5N1, H9N2 and H7N9 subtypes of avian influenza virus isolated from 7 avian species in 5 Asian countries. A partial sequence of flagellin was considered as an adjuvant. Subsequently, two chimeric antigens were designed to be virtually cloned and expressed using PYD1 vector and EBY100 yeast strain. The stability and conformational features of these two antigens were assessed through molecular dynamic (MD) simulations. The detectability of vaccine candidates by a specific monoclonal antibody (MAb148) were estimated through docking studies.

ResultsIn spite of significant compactness and stability of the first candidate in comparison with the second design, it was less detectable by MAb148. Contrary to the first chimeric antigen, Van der Waals, electrostatic and binding energies of the interaction of the second antigen with MAb148 were significantly closer to the positive control. It is shown that epitopes of the second chimeric antigen could be correctly located in the specific pocket of CDR region of MAb148.

ConclusionThe second chimeric antigen could be considered as a yeast-displayed avian influenza vaccine candidate due to the capability of provoking humoral immunity and innate immune system by M2e and flagellin respectively.

sentences: The native - exotic plant choice in green roof design: using a multicriteria decision framework to foster urban biodiversity Green roofs are considered key elements of the urban green infrastructure since they offer several environmental benefits, including habitat provision for arthropods. To achieve these benefits and ensure green roof success, an appropriate plant selection is an important step in the design of these infrastructures, especially where green roof technology is emerging like in South American cities. So far, decisions of using native or exotic plant species in green roofs had never been evaluated taking into account the plant potential to foster beneficial arthropods. By applying an integrative multicriteria decision framework that combined the habitat template hypothesis with the potential of plants to attract floral visitors and natural enemies, we obtained a ranked set of candidate native and exotic plant species. Among the best-ranked candidate species, we further compared the performance of six native and six exotic species in 30 experimental green roofs installed in Cordoba city, Argentina. To evaluate plant success, the occurrence and cover of each species were recorded one year after establishment under two management conditions: regular watering and weeding of spontaneous plants, and no management (15 roofs each). All selected species increased their vegetative cover one year after establishment. More interestingly, native plants had an advantage over exotic plant species as they exhibited a significantly higher occurrence and a slightly higher cover with no management than exotics. Native annuals were able to reseed the following season even in the absence of management, thus highlighting the relative importance of lifespan as a useful plant trait for future studies in green roof design. Given that green roofs are one of the possible solutions to ameliorate the negative effects of urban habitat loss on arthropod diversity, the development of an integrative multicriteria decision framework that takes into account the potential of native and exotic plant species for promoting beneficial arthropods would give a new twist in plant selection processes for green roofs.

sentences: Fusobacterium nucleatum induces pancreatic cancer cell proliferation and migration by regulating host autocrine and paracrine signaling Pancreatic ductal adenocarcinoma (PDAC) harbors a complex tumor microbiome that has been implicated in cancer progression and resistance to chemotherapy. Recent clinical investigations uncovered a correlation between high loads of intratumor Fusobacterium nucleatum and decreased patient survival. Here we show that healthy and cancerous pancreatic cell lines harboring intracellular F. nucleatum secrete increased levels of cancer-associated cytokines including GM-CSF, CXCL1, IL-8, and MIP-3. We report that GM-CSF (granulocyte-macrophage colony stimulating factor) secretion directly increases the proliferation and migration of pancreatic cancer cells via an autocrine mechanism, notably in the absence of immune cell participation. Furthermore, we show that non-cancerous pancreatic epithelial cells do not exhibit increased proliferation or migration in response to these cytokines, but nevertheless, their secreted cytokines stimulate these responses in cancerous cell lines through paracrine signaling. Our results provide evidence that intratumor F. nucleatum in the pancreas elicits an infection-specific cytokine secretion profile from both normal and cancerous cells that adversely contributes to cancer progression through autocrine and paracrine mechanisms. Therefore, these results support the importance of investigating the contributions of both microbiome and host driven processes in pancreatic cancer to guide future therapeutic interventions.

sentences: High-throughput proteomics of nanogram-scale samples with Zeno SWATH DIA The ability to conduct high-quality proteomic experiments in high throughput has opened new avenues in clinical research, drug discovery, and systems biology. Next to an increase in quantitative precision, recent developments in high-throughput proteomics have also gained proteomic depth, to the extent that earlier gaps between classic and high-throughput experiments have significantly narrowed. Here we introduce and benchmark Zeno SWATH, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) in order to increase proteomic depth and dynamic range in proteomic experiments. Combined with the high acquisition speed, these gains in sensitivity are particularly attractive for conducting high-throughput proteomics experiments with high chromatographic flow rates and fast gradients. We demonstrate that when combined with either micro-flow- or analytical-flow-rate chromatography, Zeno SWATH increases protein identification in complex samples 5- to 10-fold when compared to current SWATH acquisition methods on the same instrument. Using 20-min micro-flow chromatography, Zeno SWATH identified > 6,000 proteins from a 62.5 ng load of human cell lysate with more than 5,000 proteins consistently quantified in triplicate injections with a median CV of 6%. Using 5-min analytical-flow-rate chromatography (800 {micro}l/min), Zeno SWATH identified 4,907 proteins from a triplicate injection of 2 {micro}g of a human cell lysate; or more than 3,000 proteins from 250 ng tryptic digest. Zeno SWATH hence facilitates precise proteomic experiments with small sample amounts using a fast and robust high flow-rate chromatographic method, broadening the application space that requires precise proteomic experiments on a large scale.

sentences: Multi-omics investigation of Clostridioides difficile-colonized patients reveals protective commensal carbohydrate metabolism Clostridioides difficile infection (CDI) imposes a substantial burden on the health care system in the United States. Understanding the biological basis for the spectrum of C. difficile-related disease manifestations is imperative to improving treatment and prevention of CDI. Here, we investigate the correlates of asymptomatic C. difficile colonization using a multi-omics approach, comparing the fecal microbiome and metabolome profiles of patients with CDI versus asymptomatically-colonized patients. We find that microbiomes of asymptomatic patients are significantly enriched for species in the class Clostridia relative to those of symptomatic patients. Asymptomatic patient microbiomes were enriched with fucose, rhamnose, and sucrose degradation pathways relative to CDI patient microbiomes. Fecal metabolomics corroborates this result: we identify carbohydrate compounds enriched in asymptomatic patients relative to CDI patients, and correlated with a number of commensal Clostridia. Further, we reveal that across C. difficile isolates, the carbohydrates rhamnose and lactulose do not serve as robust growth substrates in vitro, corroborating their enriched detection in our metagenomic and metabolite profiling of asymptomatic individuals. We conclude that in asymptomatically-colonized individuals, carbohydrate metabolism by other commensal Clostridia may prevent CDI by inhibiting C. difficile proliferation. These insights into C. difficile colonization and putative commensal competition suggest novel avenues to develop probiotic or prebiotic therapeutics against CDI.

sentences: A novel enhancer that regulates Bdnf expression in developing neurons Brain derived neurotrophic factor (BDNF) is a critical secreted peptide that promotes neuronal differentiation and survival, and its downregulation is implicated in many neurological disorders. Here, we investigated the regulation of the mouse Bdnf gene in cortical neurons and identified a novel enhancer that promotes the expression of many Bdnf transcript variants during differentiation, increasing total Bdnf mRNA levels. Enhancer activity contributes to Bdnf-mediated effects on neuronal clustering and activity-dependent dendritogenesis. During Bdnf activation, enhancer-promoter contacts increase, and the region moves away from the repressive nuclear periphery. Our findings suggest that changes in nuclear structure may contribute to the expression of essential growth factors during neuronal development.

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Gene-gene relationships in an Escherichia coli accessory genome are linked to function and mobility The pangenome contains all genes encoded by a species, with the core genome present in all strains and the accessory genome in only a subset. Coincident gene relationships are expected within the accessory genome, where the presence or absence of one gene is influenced by the presence or absence of another. Here, we analysed the accessory genome of an Escherichia coli pangenome consisting of 400 genomes from 20 sequence types to identify genes that display significant co-occurrence or avoidance patterns with one another. We present a complex network of genes that are either found together or that avoid one another more often than would be expected by chance, and show that these relationships vary by lineage. We demonstrate that genes co-occur by function, and that several highly connected gene relationships are linked to mobile genetic elements. We find that genes are more likely to co-occur with, rather than avoid, another gene, suggesting that cooperation is more common than conflict in the accessory genome. This work furthers our understanding of the dynamic nature of prokaryote pangenomes and implicates both function and mobility as drivers of gene relationships.

Data summaryAll Supplementary Data files and the Python scripts used in the analyses are available at doi.org/10.17639/nott.7103.

Impact statementThe pangenome of a species encompasses the core genes encoded by all genomes, as well as the accessory genes found in only a subset. Much remains to be understood about the relationships and interactions between accessory genes; in particular, what drives pairs of genes to appear together in the same genome, or what prevents them from being in the same genome together, more often than expected by chance. How these co-occurrence and avoidance relationships develop, and what effect they have on the dynamics and evolution of the pangenome as a whole, is largely unknown. Here, we present a springboard for understanding prokaryote pangenome evolution by uncovering significant gene relationships in a model Escherichia coli pangenome. We identify mobile genetic elements and the sharing of common function as possible driving forces behind the co-occurrence of accessory genes. Furthermore, this work offers an extensive dataset from which gene relationships could be identified for any gene of interest in this E. coli accessory genome, providing a rich resource for the community.

sentences: Injectable hydrogels with phase-separated structures that can encapsulate live cells Injectable hydrogels are biomaterials that can be administered minimally invasively in liquid form, and are considered promising artificial extracellular matrix (ECM) materials. However, ordinary injectable hydrogels are synthesized from water-soluble molecules to ensure injectability, resulting in nonphase-separated structures, making them structurally different from natural ECMs with phase-separated insoluble structural proteins, such as collagen and elastin. Here, we propose a simple material design approach to impart phase-separated structures to injectable hydrogels by adding inorganic salts. Injecting a gelling solution of mutually crosslinkable tetra-arm poly(ethylene glycol)s with potassium sulfate at optimal concentrations results in the formation of a hydrogel with phase-separated structures in situ. These phase-separated structures provide up to an 8-fold increase in fracture toughness while allowing the encapsulation of live mouse chondrogenic cells without compromising their proliferative activity. Our findings highlight that the concentration of inorganic salts is an important design parameter in injectable hydrogels for artificial ECMs.

sentences: PISCES: A pipeline for the Systematic, Protein Activity-based Analysis of Single Cell RNA Sequencing Data While single-cell RNA sequencing provides a new window on physiologic and pathologic tissue biology and heterogeneity, it suffers from low signal-to-noise ratio and a high dropout rate at the individual gene level, thus challenging quantitative analyses. To address this problem, we introduce PISCES (Protein-activity Inference for Single Cell Studies), an integrated analytical framework for the protein activity-based analysis of single cell subpopulations. PISCES leverages the assembly of lineage-specific gene regulatory networks, to accurately measure activity of each protein based on the expression its transcriptional targets (regulon), using the ARACNe and metaVIPER algorithms, respectively. It implements novel analytical and visualization functions, including activity-based cluster analysis, identification of cell state repertoires, and elucidation of master regulators of cell state and cell state transitions, with full interoperability with Seurats single-cell data format. Accuracy and reproducibility assessment, via technical and biological validation assays and by assessing concordance with antibody and CITE-Seq-based measurements, show dramatic improvement in the ability to identify rare subpopulations and to assess activity of key lineage markers, compared to gene expression analysis.

sentences: Non-lesional and Lesional Lupus Skin Share Inflammatory Phenotypes that Drive Activation of CD16+ Dendritic Cells Cutaneous lupus erythematosus (CLE) is a disfiguring and poorly understood condition frequently associated with systemic lupus. Studies to date suggest that non-lesional keratinocytes play a role in disease predisposition, but this has not been investigated in a comprehensive manner or in the context of other cell populations. To investigate CLE immunopathogenesis, normal-appearing skin, lesional skin, and circulating immune cells from lupus patients were analyzed via integrated single-cell RNA-sequencing and spatial-seq. We demonstrate that normal-appearing skin of lupus patients represents a type I interferon-rich,  prelesional environment that skews gene transcription in all major skin cell types and dramatically distorts cell-cell communication. Further, we show that lupus-enriched CD16+ dendritic cells undergo robust interferon education in the skin, thereby gaining pro-inflammatory phenotypes. Together, our data provide a comprehensive characterization of lesional and non-lesional skin in lupus and identify a role for skin education of CD16+ dendritic cells in CLE pathogenesis.

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4DPhenoMVS: A Low-Cost 3D Tomato Phenotyping Pipeline Using a 3D Reconstruction Point Cloud Based on Multiview Images Manual phenotyping of tomato plants is time consuming and labor intensive. Due to the lack of low-cost and open-access 3D phenotyping tools, the dynamic 3D growth of tomato plants during all growth stages has not been fully explored. In this study, based on the 3D structural data points generated by employing structures from motion algorithms on multiple-view images, we proposed a dynamic 3D phenotyping pipeline, 4DPhenoMVS, to calculate and analyze 14 phenotypic traits of tomato plants covering the whole life cycle. The results showed that the R2 values between the phenotypic traits and the manual measurements stem length, plant height, and internode length were more than 0.8. In addition, to investigate the environmental influence on tomato plant growth and yield in the greenhouse, eight tomato plants were chosen and phenotyped during 7 growth stages according to different light intensities, temperatures, and humidities. The results showed that stronger light intensity and moderate temperature and humidity contribute to a higher growth rate and higher yield. In conclusion, we developed a low-cost and open-access 3D phenotyping pipeline for tomato plants, which will benefit tomato breeding, cultivation research, and functional genomics in the future.

HighlightsBased on the 3D structural data points generated by employing structures from motion algorithms on multiple-view images, we developed a low-cost and open-access 3D phenotyping tool for tomato plants during all growth stages.

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Structural heterogeneity of the microtubule lattice Microtubules are polymers assembled from {beta}-tubulin heterodimers. They typically display lateral - and {beta}-{beta} homotypic interactions, except at one region, called the seam, where heterotypic -{beta} and {beta}- interactions occur. Here, we decorated microtubules assembled in vitro or in cytoplasmic Xenopus egg extracts with kinesin-motor domains, and analyzed their lattice organization using dual axis cryo-electron tomography followed by segmented sub-tomogram averaging. In both conditions, microtubules incorporated variable protofilament and/or tubulin subunit helix start numbers. While microtubules assembled in vitro displayed variable numbers of seams, those assembled in extracts displayed preferentially one seam. The seam location varied within individual microtubules implying the presence of lattice holes. Thus, the formation of discontinuous microtubule lattices is an intrinsic property of tubulin assembly, a process that is controlled in cells.

One-Sentence SummaryMicrotubules assembled from purified tubulin form discontinuous lattices, an intrinsic property strictly controlled in cytoplasmic Xenopus egg extracts.

sentences: The Inherent Flexibility of Receptor Binding Domains in SARS-CoV-2 Spike Protein Spike (S) protein is the primary antigenic target for neutralization and vaccine development for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It decorates the virus surface and undergoes large conformational changes of its receptor binding domain (RBD) to enter the host cell, as the abundant structural studies suggest. Here, we observe Down, one-Up, one-Open, and two-Up-like structures in enhanced molecular dynamics simulations without pre-defined reaction coordinates. The RBDA transition from Down to one-Up is supported by transient salt-bridges between RBDA and RBDC and by the glycan at N343B. Reduced interactions between RBDA and RBDB induce the RBDB motions toward two-Up. Glycan shielding for neutralizing antibodies is the weakest in one-Open. Cryptic pockets are revealed at the RBD interfaces in intermediate structures between Down and one-Up. The inherent flexibility in S-protein is, thus, essential for the structure transition and shall be considered for antiviral drug rational design or vaccine development.

sentences: Capture of Viruses and Microorganisms in Aerosols Using A Newly Designed Collection System: A Proof-of-concept Study Aerosols contain human pathogens that cause public health disasters such as tuberculosis (TB) and the ongoing COVID-19 pandemic. The current technologies for the collection of viruses and microorganisms in aerosols face critical limitations, necessitating the development of a new type of sampling system to advance the capture technology. Herein, we presented a new type of collection system, which exploits the affinity between carbon chains and organic molecules on the surfaces of viruses and microorganisms. We demonstrated that the physical capture efficiency of the collection system was over 99% for particle sizes from 0.3 to 10 {micro}m. We further evaluated the biochemical capture efficiency of the collection system using mass spectrometry approaches and showed that the biochemical information of viruses and microorganisms was well preserved. Coupled with well-established molecular technologies, this new type of capture technology can be used for the investigation of aerosol-related disease transmission models, as well as the development of innovative screening and monitoring tools for human diseases and public health issues.

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Sex differences in microRNA expression in first and third trimester human placenta Maternal and fetal pregnancy outcomes related to placental function vary based on fetal sex, which may be the result of sexually dimorphic epigenetic regulation of RNA expression. We identified sexually dimorphic miRNA expression throughout gestation in human placentae. Next-generation sequencing was used to identify miRNA expression profiles in first and third trimester uncomplicated pregnancies using tissue obtained at chorionic villous sampling (n=113) and parturition (n=47). Sequencing and differential expression (DE) analysis identified 432 mature miRNAs expressed in the first trimester female, 425 in the first trimester male, 400 in the third trimester female, and 508 in the third trimester male placenta (baseMean >10). Of these, 11 sexually dimorphic (FDR<0.05, baseMean >10) miRNAs were identified in the first and 4 miRNAs were identified in the third trimester, including miR-361-5p, significant in both trimesters, all upregulated in females. Across gestation, 207 miRNAs were DE across gestation, common to both females and males, miR-4483, the most DE across gestation. There were twice as many female-specific differences across gestation as male-specific (44 miRNAs vs 21 miRNAs), indicating that miRNA abundance across human gestation is sexually dimorphic. Pathway enrichment analysis identified significant pathways that were differentially regulated in first and third trimester as well as across gestation. This work provides the normative sex dimorphic miRNA atlas in first and third trimester, as well as the sex independent and sex specific placenta miRNA atlas across gestation which may be used to identify biomarkers of placental function and direct functional studies investigating placental sex differences.

Summary SentenceSex dimorphism in miRNA expression is more pronounced in first compared to third trimester placenta, and there are twice as many female-specific gestational differences, indicating miRNA abundance across human gestation is also sexually dimorphic.

sentences: A Drosophila Su(H) Model of Adams-Oliver Syndrome Reveals Notch Cofactor Titration as a Mechanism Underlying Developmental Defects Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that Drosophila with a single copy of the AOS-like Su(H) allele behave in an opposing manner as flies with a Su(H) null allele due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants decrease DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby a single RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.

sentences: Neurocomputational mechanisms of affected beliefs The feedback people receive on their behavior shapes the process of belief formation and self-efficacy in mastering a given task. However, the neural and computational mechanisms of how the subjective value of these beliefs, and the corresponding affect, bias the learning process remain unclear. We investigated these mechanisms during the learning of self-efficacy beliefs using fMRI, pupillometry, and computational modeling, and by analyzing individual differences in affective experience. Biases in the formation of self-efficacy beliefs were associated with affect, pupil dilation, and neural activity within the anterior insula, amygdala, ventral tegmental area/ substantia nigra, and mPFC. Specifically, neural and pupil responses map the valence of the prediction errors in correspondence with individuals experienced affect and learning bias during belief formation. Together with the functional connectivity dynamics of the anterior insula within this network, our results hint at neural and computational mechanisms that integrate affect into the process of belief formation.

sentences: Integrated Metabolomics to Reveal the Impacts of Common Antibiotics based on Drug Resistance Prediction of Gut Microbiota Due to the close interaction between the host and the gut microbiota, the alterations in gut microbiota metabolism may therefore contribute to various diseases. How to use antibiotics more wisely in clinical practice is a promising task in the field of pathophysiology related to gut microbiota. The hope fueling this research is that the alteration of gut microbial communities are paralleled by their capacity on metabolomic from the combined perspective of microbiome and metabolomics. In order to reveal the impacts of antibiotics on microbiota-associated host metabolomic phenotypes, a feasible methodology should be well developed to assess the pervasive effects of antibiotics on the population structure of gut microbial communities. Our attempt starts from predicting specific resistance phenotypes of the individuals in isolation from the rest of the gut microbiota community, according to their resistant genotypes. Once resistance phenotypes of microbiome is determined, we integrated metabolomics with machine learning by applying various analysis algorithms to explore the relationship between the predicted resistance and metabolites, including what the microbial community is after medication, which microbes produce metabolites, and how these metabolites enrich.

sentences: An Efficient Approach for SARS-CoV-2 Monoclonal Antibody Production via Modified mRNA-LNP Immunization Throughout the COVID-19 pandemic, many prophylactic and therapeutic drugs have been evaluated and introduced. Among these treatments, monoclonal antibodies (mAbs) that bind to and neutralize SARS-CoV-2 virus have been applied as complementary and alternative treatments to vaccines. Although different methodologies have been utilized to produce mAbs, traditional hybridoma fusion technology is still commonly used for this purpose due to its unmatched performance record. In this study, we coupled the hybridoma fusion strategy with mRNA-lipid nanoparticle (LNP) immunization. This time-saving approach can circumvent biological and technical hurdles, such as difficult to express membrane proteins, antigen instability, and the lack of posttranslational modifications on recombinant antigens. We used mRNA-LNP immunization and hybridoma fusion technology to generate mAbs against the receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein. Compared with traditional protein-based immunization approaches, inoculation of mice with RBD mRNA-LNP induced higher titers of serum antibodies. In addition, the mAbs we obtained can bind to SARS-CoV-2 RBDs from several variants. Notably, RBD-mAb-3 displayed particularly high binding affinities and neutralizing potencies against both Alpha and Delta variants. In addition to introducing specific mAbs against SARS-CoV-2, our data generally demonstrate that mRNA-LNP immunization may be useful to quickly generate highly functional mAbs against emerging infectious diseases.

sentences: The roles of NADPH oxidases during adult zebrafish fin regeneration Sustained elevated levels of reactive oxygen species (ROS) have been shown to be essential for whole body, appendage and organ regeneration in various organisms, including planarians, Hydra, zebrafish, axolotl, Xenopus, geckos and mice. In the majority of cases these roles have been shown via the use of NADPH oxidase pharmacological inhibitors, which generally target all NAPDH oxidases (NOXes). To identify the specific NOX or NOXes essential for ROS production during adult fin regeneration in zebrafish, we generated nox mutants for duox, nox5 and cyba (a key subunit of NOXes 1-4). We also crossed these mutant lines to a transgenic line ubiquitously expressing HyPer, which permits the measurement of ROS levels in adult zebrafish fins. Using this approach, we found that homozygous duox mutants have significantly attenuated ROS levels following fin amputation, and this correlated with a significantly diminished rate of fin regeneration. While the other nox homozygous mutants (nox5 and cyba) showed less of an effect on ROS levels or adult fin regeneration, duox/cyba double mutants showed a more diminished rate of fin regeneration than duox mutants alone, suggesting that Nox1-4 do play a role during regeneration, but one that is secondary to that of Duox. This work also serendipitously found that ROS levels in amputated adult zebrafish fins oscillate during the day with a circadian rhythm.

sentences: Hda1C restricts the transcription initiation frequency to limit divergent non-coding RNA transcription Nucleosome-depleted regions (NDRs) at gene promoters support initiation of RNA Polymerase II transcription. Interestingly, transcription often initiates in both directions, resulting in an mRNA, and a divergent non-coding (DNC) transcript with an unclear purpose. Here, we characterized the genetic architecture and molecular mechanism of DNC transcription in budding yeast. We identified the Hda1 histone deacetylase complex (Hda1C) as a repressor of DNC in high-throughput reverse genetic screens based on quantitative single-cell fluorescence measurements. Nascent transcription profiling showed a genome-wide role of Hda1C in DNC repression. Live-cell imaging of transcription revealed that Hda1C reduced the frequency of DNC transcription. Hda1C contributed to decreased acetylation of histone H3 in DNC regions, supporting DNC repression by histone deacetylation. Our data support the interpretation that DNC results as a consequence of the NDR-based architecture of eukaryotic promoters, but that it is governed by locus-specific repression to maintain genome fidelity.

sentences: Reduced sera neutralization to Omicron SARS-CoV-2 by bothinactivated and protein subunit vaccines and the convalescents Omicron variant continues to spread all over the world. There are lots of scientific questions remaining to be answered for such a devastating variant. There are a dozen of vaccines already in clinical use. The very urgent scientific question would be whether or not these vaccines can protect Omicron variant. Here, we tested the sera from both convalescents and vaccine recipients receiving either inactivated or protein subunits vaccines (CoronaVac from Sinovac, or BBIBP-CoV from Sinopharm, or ZF2001 from Zhifei longcom) for the binding antibody titers (ELISA) and neutralization antibodies titers (pseudovirus neutralization assay). We showed that Omicron do have severe immune escape in convalescents, with 15 of 16 were negative in neutralization. By contrast, in vaccinees who received three jabs of inactivated or protein subunit vaccine, the neutralizing activity was much better preserved. Especially in the ZF2001 group with an extended period of the second and third jab (4-6 months) remains 100% positive in Omicron neutralization, with only 3.1-folds reduction in neutralizing antibody (NAb) titer. In this case, we proposed that, the multi-boost strategy with an extended interval between the second and third jab for immune maturation would be beneficial for NAb against devastating variants such as Omicron.

sentences: RECIPROCALLY INHIBITORY CIRCUITS OPERATING WITH DISTINCT MECHANISMS ARE DIFFERENTLY ROBUST TO PERTURBATION AND MODULATION What features are important for circuit robustness? Reciprocal inhibition is a building block in many circuits. We used dynamic clamp to create reciprocally inhibitory circuits from pharmacologically isolated neurons of the crab stomatogastric ganglion by injecting artificial synaptic (ISyn) and hyperpolarization-activated inward (IH) currents. There are two mechanisms of antiphase oscillations in these circuits: "escape" and "release". In release, the active neuron primarily controls the off/on transitions. In escape, the inhibited neuron controls the transitions. We characterized the robustness of escape and release circuits to alterations in circuit parameters, temperature, and neuromodulation. We found that escape circuits rely on tight correlations between synaptic and H conductances to generate bursting but are resilient to temperature increase. Release circuits are robust to variations in synaptic and H conductances but fragile to temperature increase. The modulatory current (IMI) restores oscillations in release circuits but has little effect in escape circuits. Thus, the same perturbation can have dramatically different effects depending on the circuits mechanism of operation that may not be observable from basal circuit activity.

sentences: Repetition Probability Effects for Words Depend on Language Knowledge The magnitude of repetition suppression (RS), measured by fMRI, is modulated by the probability of repetitions (P(rep)) for various sensory stimulus categories. It has been suggested that for visually presented simple letters this P(rep) effect depends on the prior practices of the participants with the stimuli. Here we tested further if previous experiences affect the neural mechanisms of RS, leading to the modulatory effects of stimulus P(rep), for more complex lexical stimuli as well. We measured the BOLD signal in the Visual Word Form Area (VWFA) of native Chinese and German participants and estimated the P(rep) effects for Chinese characters and German words. The results showed a significant P(rep) effect for stimuli of the mother tongue in both participant groups. Interestingly, Chinese participants, learning German as a second language, also showed a significant P(rep) modulation of RS for German words while the German participants who had no prior experiences with the Chinese characters showed no such effects. Our findings suggest that P(rep) effects on RS are manifest for visual word processing as well, but only for words of a language with which participants are highly familiar. These results support further the idea that predictive processes, estimated by P(rep) modulations of RS, require prior experiences.

sentences: Progressive alterations in polysomal architecture in a mouse model of Huntington's disease Given their highly polarized morphology and functional singularity, neurons require precise spatial and temporal control of protein synthesis. Alterations in protein translation have been implicated in the development and progression of a wide range of neurological and neurodegenerative disorders, including Huntingtons disease (HD). We examined the architecture of polysomes in their native brain context by performing 3D electron tomography of striatal tissue from the zQ175 knock-in mouse model of HD. Results revealed progressive remodelling towards a more compacted polysomal architecture, an effect that coincided with the emergence and progression of HD related symptoms. This aberrant architecture is compatible with ribosome stalling phenomena. In fact, we also detected an increase in the striatal expression of the stalling relief factor EIF5A2. Polysomal sedimentation gradients showed a significant increase in the accumulation of free 40S ribosomal subunits in zQ175 striatal samples. Together, these findings indicate that changes in the architecture of the protein synthesis machinery may underlie translational alterations associated with HD, thus opening new avenues for understanding the progression of this disease.

sentences: Hidden Talents: Poly (I:C)-induced maternal immune activation improves mouse visual discrimination performance and reversal learning in a sex-dependent manner. While there is a strong focus on the negative consequences of maternal immune activation (MIA) on developing brains, very little attention is directed towards potential advantages of early life challenges. In this study we utilized a polyinosine-polycytidylic acid (poly(I:C)) MIA model to test visual discrimination (VD) and reversal learning (RL) in mice using touchscreen technology. Significant sex differences emerged in that MIA reduced the latency for males to make a correct choice in the VD task while females reached criterion sooner, made fewer errors, and utilized fewer correction trials in RL compared to saline controls. These surprising improvements were accompanied by the sex-specific upregulation of several genes critical to cognitive functioning, indicative of compensatory plasticity in response to MIA. In contrast, when exposed to a  two-hit stress model (MIA + loss of the social component of environmental enrichment (EE)), mice did not display anhedonia but required an increased number of PD and RL correction trials. These animals also had significant reductions of CamK2a mRNA in the prefrontal cortex. Appropriate functioning of synaptic plasticity, via mediators such as this protein kinase and others, are critical for behavioral flexibility. Although EE has been implicated in delaying the appearance of symptoms associated with certain brain disorders, these findings are in line with evidence that it also makes individuals more vulnerable to its loss. Overall, with the right  dose, early life stress exposure can confer at least some functional advantages, which are lost when the number or magnitude of these exposures become too great.

sentences: Effect of plant tissues on DNA quantity and quality of barley (Hordeum vulgare) validating through PCR technique The use of molecular techniques to deal with plant molecular breeding requires the extraction of genomic DNA in good quantity and quality, which can be influenced by method of extraction and source of DNA (plant species, plant part or tissues). However, this research focuses on plant tissue source and tried to describe the quality and quantity of DNA isolated from different tissues of barley crop. CTAB protocol was used for the isolation of DNA and both the quality and quantity of this DNA was validated through gel electrophoresis, Qubit quantification and PCR amplifications. The result showed that DNA could be successfully extracted from all tissues of plants and the yield of DNA obtained was variable ranging from 179ng l-1 in stem to 750ng l-1 in young leave. Band intensity of genomic and PCR amplified DNA was good for DNA isolated from young and matured leave. Faint band was observed in the PCR amplification for DNA isolated from stem but no to unreliable amplification was obtained for seeds and roots, respectively. Thus, for any molecular technique in barley crop research, the best tissue for DNA isolation using modified CTAB, is young or matured leave and alternatively stem can be used as DNA source.

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Quantitative 3D microscopy reveals a genetic network predicting the local activity of anti-Aβ compounds Genetic and biochemical evidence implicates amyloid-{beta} (A{beta}) in Alzheimers disease, yet many anti-A{beta} treatments are clinically ineffective. Regional heterogeneity of efficacy may contribute to these disappointing results. Here we mapped the regiospecificity of various anti-A{beta} treatments by high-resolution light-sheet imaging of amyloid plaques in electrophoretically clarified brains of Thy1-APP mice overexpressing A{beta}. We found that A{beta} plaques in whole brains progressed from 1.2*106 to 2.5*106 (standard deviation {+/-} 1.6*105 and {+/-} 4.3*105 respectively) over a 9-month period. We then assessed the regiospecific plaque clearance in mice subjected to {beta}-secretase inhibition, amyloid intercalation by polythiophenes, or anti-A{beta} antibodies. Each treatment showed unique spatiotemporal A{beta} clearance signatures, with polythiophenes emerging as potent anti-A{beta} compounds with promising pharmacokinetic properties. We then interrogated genes matching regiospecific A{beta} clearance by aligning voxels that showed drug effectiveness to spatial-transcriptomics atlases. Bace1 and Thy1 expression matched the regiospecific efficacy of BACE inhibition, confirming the validity of our analyses. Voxels cleared by polythiophenes correlated with transcripts encoding synaptic proteins, whereas voxels cleared by BACE inhibition correlated with oligodendrocyte-specific genes. The restricted regional susceptibility of A{beta} plaques to specific treatments may contribute to the clinical failure of anti-A{beta} therapies. The striking regiospecificity of the treatments studied suggests that combinatorial regimens may improve functional outcomes.

One sentence summary3D microscopy of A{beta} plaques reveals that Alzheimers disease therapeutics have regiospecific effects that can be mapped to distinct genetic networks.

sentences: Persistence of Plasmodium cynomolgi hypnozoites in cynomolgus monkey iPS-derived hepatocytes Plasmodium cynomolgi (Pc) is one of the few parasite species that forms quiescent liver stage parasites known as hypnozoites and is therefore a suitable model for Plasmodium vivax. Very little is known about liver stage dormancy, which hampers the search for compounds with anti-hypnozoite activity. Here, we present the development of a Pc in vitro infection model using stem cell-derived hepatocytes from Macaca fascicularis. IPS cells were established on feeder free condition and differentiated into hepatocytes via inducible overexpression of key transcription factors. The generated hepatocytes were infected with Pc sporozoites and hypnozoite formation as well as schizont development were confirmed by immunofluorescence. This system is a promising tool to study the mechanisms underlying liver stage dormancy and facilitate drug discovery against hypnozoites.

sentences: Distribution of the National Science Foundation's Advancing Informal STEM Learning Awards (AISL) between 2006-21 The COVID-19 pandemic tested many fundamental connections between science and society. A growing field working to strengthen those connections exists within the informal STEM learning (ISL) community which provides diverse learning and engagement environments outside the formal classroom. One of the largest funders of ISL initiatives is the National Science Foundation (NSF) which runs the Advanced Informal STEM Learning (AISL) program in the United States. The AISL program supports initiatives through six categories that include pilots and feasibility studies, research in service to practice, innovations in development, broad implementation, literature reviews, syntheses, or meta-analyses, and conferences. However, a number of questions remain unanswered with respect to the distribution and ultimately the broad impact of the awards. In this study, we analyzed publicly available awardee information across a 15-year period (2006-2021) to provide a preliminary analysis of how the AISL grants are distributed across individual states, organizations, and the principal investigators. Several states, organizations, and principal investigators stood out as prolific awardees. Massachusetts and California represented the largest share of awards at 14% and 13% respectively during that time. WGBH Educational Foundation located in Massachusetts and the Exploratorium located in California, received the largest number of awards during the 15 year period. Notably, 67% of the AISL awards list at least one co-principal investigator. Our report brings to light a number of new questions and charts new paths of exploration for future studies.

sentences: Characterization and structural prediction of the putative ORF10 protein in SARS-CoV-2 Upstream of the 3-untranslated region in the SARS-CoV-2 genome is ORF10 which has been proposed to encode for the ORF10 protein. Current research is still unclear on whether this protein is synthesized, but further investigations are still warranted. Herein, this study uses multiple bioinformatic tools to biochemically and functionally characterize the ORF10 protein, along with predicting its tertiary structure. Results indicate a highly ordered, hydrophobic, and thermally stable protein that contains at least one transmembrane region. This protein also possesses high residue protein-binding propensity, primarily in the N-terminal half. An assessment of forty-one missense mutations reveal slight changes in residue flexibility, mainly in the C-terminal half. However, these same mutations do not inflict significant changes on protein stability and other biochemical features. The predicted model suggests the ORF10 protein contains a {beta}--{beta} motif with a {beta}-molecular recognition feature occurring in the first {beta}-strand. Functionally, the ORF10 protein could be a membrane protein. A single pocket was identified in this protein but found to possess low druggability. The ORF10 itself consists of two distinct lineages: the SARS-CoV lineage and the SARS-CoV-2 lineage. Evidence of strong positive selection (dN/dS = 4.01) and purifying selection (dN/dS = 0.713) were found within the SARS-CoV-2 lineage and SARS-CoV lineage, respectively. Collectively, these results continue to assess the biological relevance of ORF10 and its putatively encoded protein, thereby aiding in diagnostic and possibly vaccine development.

sentences: Learning and motor-impulse control in crows and domestic chickens Cognitive abilities allow animals to navigate through complex, fluctuating environments. In the present study, we tested the performance of a captive group of eight crows, Corvus corone, and ten domestic chickens, Gallus gallus domesticus, in the cylinder task, as a test of motor inhibitory control, and reversal learning as a measure of learning ability and behavioural flexibility. Four crows and nine chickens completed the cylinder task, eight crows and six chickens completed the reversal learning experiment. Crows performed better in the cylinder task compared to chickens. In the reversal learning experiment, species did not significantly differ in the number of trials until learning criterion was reached. Performance in the reversal learning experiment did not correlate with performance in the cylinder task in chickens. Our results suggest crows to possess better motor inhibitory control compared to chickens. In contrast, learning performance in a reversal learning task did not differ between the species, indicating similar levels of behavioural flexibility. Interestingly, we describe notable individual differences in performance. We stress the importance not only to compare cognitive performance between species but also between individuals of the same species when investigating the evolution of cognitive skills.

sentences: Development of antibiotic resistance reveals diverse evolutionary pathways to face the complex and dynamic environment of a long-term treated patient Antibiotic resistance development has been studied using approaches that range from laboratory experimental evolution, surveillance and epidemiology, to clinical isolate sequencing. However, evolutionary trajectories depend on the environment in which selection takes place, compelling to address evolutionary analyses in antibiotic-treated patients, to embrace the whole inherent environmental complexities as well as their dynamics over time. Herein, we address the complexity of the bacterial adaptive response to changing antibiotic selective pressures by studying the long-term in-patient evolution of a broad diversity of {beta}-lactam resistant Pseudomonas aeruginosa clones. By using mutational and ultra-deep amplicon sequencing, we analyzed multiple generations of a P. aeruginosa hypermutator strain persisting for more than 26 years of chronic infection in the airways of a cystic fibrosis (CF) patient. We identified the accumulation of multiple alterations targeting the chromosomally encoded class C {beta}-lactamase (blaPDC), providing structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrate selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. Surprisingly, "a gain of function" of collateral resistance towards ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed and the biochemical basis of this cross-resistance phenomenon was elucidated. This work unveils the diversity of evolutionary trajectories driven by bacteria in the natural CF environmental setting, towards a multidrug resistant phenotype after years of antibiotic treatment against a formidable pathogen.

sentences: Reversal of lineage plasticity in RB1/TP53-deleted prostate cancer through FGFR and Janus kinase inhibition The inherent plasticity of tumor cells provides a mechanism of resistance to many molecularly targeted therapies, exemplified by adeno-to-neuroendocrine lineage transitions seen in prostate and lung cancer. Here we investigate the root cause of this lineage plasticity in a primary murine prostate organoid model that mirrors the lineage transition seen in patients. These cells lose luminal identity within weeks following deletion of Trp53 and Rb1, ultimately acquiring an Ar-negative, Syp+ phenotype after orthotopic in vivo transplantation. Single-cell transcriptomic analysis revealed progressive mixing of luminal-basal lineage features after tumor suppressor gene deletion, accompanied by activation of Jak/Stat and Fgfr pathway signaling and interferon-a and -g gene expression programs prior to any morphologic changes. Genetic or pharmacologic inhibition of Jak1/2 in combination with FGFR blockade restored luminal differentiation and sensitivity to antiandrogen therapy in models with residual AR expression. Collectively, we show lineage plasticity initiates quickly as a largely cell-autonomous process and, through newly developed computational approaches, identify a pharmacological strategy that restores lineage identity using clinical grade inhibitors.

sentences: Spermidine reduces neuroinflammation and soluble amyloid beta in an Alzheimer's disease mouse model Deposition of amyloid beta (A{beta}) along with glia cell-mediated neuroinflammation are prominent pathogenic hallmarks of Alzheimers disease (AD). In recent years, impairment of autophagy has been found to be another important feature, contributing to AD progression and aging. Therefore, we assessed the effect of the autophagy activator Spermidine, a small body-endogenous polyamine often used as dietary supplement and known to promote longevity, on glia cell-mediated neuroinflammation. Spermidine reduced TLR3- and TLR4- mediated inflammatory processes in microglia and astrocytes by decreasing cytotoxicity, inflammasome activity and NF-{kappa}B signaling. In line with these anti-inflammatory effects, oral treatment of the amyloid prone AD-like APPPS1 mice with Spermidine reduced neuroinflammation and neurotoxic soluble A{beta}. Mechanistically, single nuclei sequencing revealed microglia as one of the main targets of Spermidine treatment, with increased expression of genes implicated in cell motility and phagocytosis. Thus, Spermidine provides a promising therapeutic potential to target glia cells in AD progression.

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Mutations at the Alphavirus E2/E1 inter-dimer interface have host-specific phenotypes Alphaviruses are enveloped viruses transmitted by arthropod vectors to vertebrate hosts. The surface of the virion contains 80 glycoprotein spikes embedded in the membrane and these spikes mediate attachment to the host cell and initiate viral fusion. Each spike consists of a trimer of E2-E1 heterodimers. These heterodimers interact at two interfaces: (1) the intra-dimer interactions between E2 and E1 of the same heterodimer, and (2) the inter-dimer interactions between E2 of one heterodimer and E1 of the adjacent heterodimer (E1). We hypothesized that the inter-dimer interactions are essential for trimerization of the E2-E1 heterodimers into a functional spike. In this work, we made a mutant virus (CPB) where we replaced six inter-dimeric residues in the E2 protein of Sindbis virus (WT SINV) with those from the E2 protein from chikungunya virus, and studied its effect in both mammalian and mosquito cell lines. CPB produced fewer infectious particles in mammalian cells than in mosquito cells, relative to WT SINV. When CPB virus was purified from mammalian cells, particles showed reduced amounts of glycoproteins relative to capsid protein, and contained defects in particle morphology compared to virus derived from mosquito cells. Using cryo-EM, we determined that the spikes of CPB had a different conformation than WT SINV. Last, we identified two revertants, E2-H333N and E1-S247L, that restored particle growth and assembly to different degrees. We conclude the inter-dimer interface is critical for spike trimerization and is a novel target for potential antiviral drug design.

IMPORTANCEAlphaviruses, which can cause disease when spread to humans by mosquitoes, have been classified as an emerging pathogen, with infections occurring worldwide. The spikes on the surface of the alphavirus particle are absolutely required for the virus to enter a new host cell and initiate an infection. Using a structure-guided approach, we made a mutant virus that alters spike assembly in mammalian cells but not mosquito cells. This is important because it identifies a region in the spike that could be a target for antiviral drug design.

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Allosteric effect of nanobody binding on ligand-specific active states of the β2-Adrenergic Receptor Nanobody binding stabilizes the active state of G-protein-coupled receptor (GPCR) and modulates its affinity for bound ligands. However, the atomic level basis for this allosteric regulation remains elusive. Here, we investigate the conformational changes induced by the binding of a nanobody (Nb80) on the active-like {beta}2 adrenergic receptor ({beta}2AR) via enhanced sampling molecular dynamics simulations. Dimensionality reduction analysis shows that Nb80 stabilizes a highly active state of the {beta}2AR with a ~14 [A] outward movement of transmembrane helix 6 and close proximity of transmembrane (TM) helices 5 and 7. This is further supported by the residues located at hotspots located on TMs 5, 6 and 7, as shown by supervised machine learning methods. Besides, ligand-specific subtle differences in the conformations assumed by intercellular loop 2 and extracellular loop 2 are captured from the trajectories of various ligand-bound receptors in the presence of Nb80. Dynamic network analysis further reveals that Nb80 binding can enhance the communication between the binding sites of Nb80 and of the ligand. We identify unique allosteric signal transmission mechanisms between the Nb80-binding site and the extracellular domains in presence of full agonist and G-protein biased partial agonist, respectively. Altogether, our results provide insights into the effect of intracellular binding partners on the GPCR activation mechanism, which could be useful for structure-based drug discovery.

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Gymnasts' ability to modulate bioelectric sensorimotor rhythms during kinesthetic motor imagery of sports non-specific movements Previous psychological studies using questionnaires have consistently reported that athletes have superior motor imagery ability, both for sports-specific and sports non-specific movements. However, regarding motor imagery of sports non-specific movements, no physiological studies have demonstrated differences in neural activity between athletes and non-athletes. The purpose of the present study was to examine differences in bioelectric sensorimotor rhythms during kinesthetic motor imagery (KMI) of sports non-specific movements between gymnasts and non-gymnasts. We selected gymnasts as an example population because they are likely to have particularly superior motor imagery ability due to frequent usage of motor imagery including KMI as part of daily practice. Healthy young participants (16 gymnasts and 16 non-gymnasts) performed repeated motor execution and KMI of sports non-specific movements (wrist dorsiflexion and shoulder abduction of the dominant hand). Scalp electroencephalogram (EEG) was recorded over the contralateral sensorimotor cortex. During motor execution and KMI, sensorimotor EEG power is known to decrease in the - (8-15 Hz) and {beta}-bands (16-35 Hz), referred to as event-related desynchronization (ERD). We calculated the maximal peak of ERD both in the - (ERDmax) and {beta}-bands ({beta}ERDmax) as a measure of changes in corticospinal excitability. ERDmax was significantly greater in gymnasts, who subjectively evaluated their KMI as being more vivid, for both KMI tasks. On the other hand, {beta}ERDmax was greater in gymnasts only for shoulder abduction KMI. These findings suggest gymnasts signature of flexibly modulating sensorimotor rhythm with no movements, which may be the basis of their superior ability of KMI for sports non-specific movements.

New & NoteworthyKinesthetic motor imagery of sports non-specific movements was compared between gymnasts and non-gymnasts (i.e., healthy controls) from both physiological and psychological approaches. The EEG sensorimotor rhythms during kinesthetic motor imagery were more desynchronized in gymnasts who subjectively imaged their own movements as being more vivid. The work reveals novel ability in gymnasts to flexibly control their sensorimotor rhythms with no actual movements, which would be the basis of their superior ability of motor imagery.

sentences: Your pleasure is mine; when people share amusical emotional experience during a livemusic performance in a concert hall. Over the years, several publications have proposed that musical sound could be an ancestral emotional way of communication, thus positing an ancestral biological function for music. Understanding how musical emotions, and the pleasure derived from music regardless of the musical valence, can be shared between individuals is a fascinating question, and investigating it can shed light on the function of musical reward. Is the pleasure felt at the individual level transmitted on a collective level? And if so, how? We investigated these questions in a natural setting during an international competition for orchestra conductors. Participants (n=15) used a dedicated smartphone app to report their subjective emotional experiences in real time during a concert. We recorded participants electrodermal activity (EDA) and cerebral activity with electroencephalography (EEG). The overall behavioral real time ratings suggest a possible social influence on the reported and felt pleasure. The physically closer the participants, the more similar their reported pleasure. We estimated the inter-individual cerebral coherence, which indicates the degree of mutual cerebral information between pairs of participants in the frequency domain. The results show that when people simultaneously reported either high or low pleasure, their cerebral activities were closer than for simultaneous neutral pleasure reports. Participants skin conductance levels were also more coupled when reporting higher emotional degrees simultaneously. More importantly, the participants who were physically closer had higher cerebral coherence, but only when they simultaneously reported intense pleasure. We propose that mechanisms of emotional contagion and/or emotional resonance could explain why a form of  emotional connecting force could arise between people.

sentences: Antiviral activity of bacterial TIR domains via signaling molecules that trigger cell death The Toll/interleukin-1 receptor (TIR) domain is a canonical component of animal and plant immune systems. In plants, intracellular pathogen sensing by immune receptors triggers their TIR domains to generate a molecule which is a variant of cyclic ADP-ribose (v-cADPR). This molecule is hypothesized to activate plant cell death via a yet unresolved pathway. TIR domains were recently also shown to be involved in a bacterial anti-phage defense system called Thoeris, but the mechanism of Thoeris defense remained unknown. In this study we report that phage infection triggers Thoeris TIR-domain proteins to produce an isomer of cyclic ADP-ribose. This molecular signal activates a second protein, ThsA, which then depletes the cell of the essential molecule nicotinamide adenine dinucleotide (NAD) and leads to abortive infection and cell death. We further show that similar to eukaryotic innate immune systems, bacterial TIR-domain proteins determine the immunological specificity to the invading pathogen. Our results describe a new antiviral signaling pathway in bacteria, and suggest that generation of intracellular signaling molecules is an ancient immunological function of TIR domains conserved in both plant and bacterial immunity.

sentences: Single-domain antibodies efficiently neutralize SARS-CoV-2 variants of concern, including Omicron variant Virus-neutralizing antibodies are one of the few treatment options for COVID-19. The evolution of SARS-CoV-2 virus has led to the emergence of virus variants with reduced sensitivity to some antibody-based therapies. The development of potent antibodies with a broad spectrum of neutralizing activity is urgently needed. Here we isolated a panel of single-domain antibodies that specifically bind to the receptor-binding domain of SARS-CoV-2 S glycoprotein. Three of the selected antibodies exhibiting most robust neutralization potency were used to generate dimeric molecules. We observed that these modifications resulted in up to a 200-fold increase in neutralizing activity. The most potent heterodimeric molecule efficiently neutralized each of SARS-CoV-2 variant of concern, including Alpha, Beta, Gamma, Delta and Omicron variants. This heterodimeric molecule could be a promising drug candidate for a treatment for COVID-19 caused by virus variants of concern.

sentences: Control strategies for heterogeneous plant pathogens: evolutionary and agricultural consequences Optimising the use of chemical pesticide is required in order to reduce the inevitable environmental and economic costs related to it. The consequences of chemical control are particularly tricky to foresee in the presence of pathogens, displaying heterogeneous traits involved in their life cycle, because its effect will likely differ across the population. In this work, we investigate the effects of trait-dependent pesticide on heterogeneous plant pathogens, by means of a minimal model connecting evolutionary and agricultural states of the system. We model a pathogen population displaying continuous levels of virulence and transmission. Control strategies are modelled by the quantity of pesticide released and its degree of correlation with the pathogens heterogeneous traits. We show that the pathogen population can adapt towards opposite evolutionary states, that may be reversed by chemical control due to its heterogeneous selective pressure. This dual behaviour triggers saturating effects in yield production, with respect to pesticide use. As a consequence, we show that maximising yield production and minimising pesticide application are conflicting objectives. We identify Pareto-efficient solutions, where the optimal pesticide type depends on the applied quantity. Our results provide a theoretical framework to explore how to harness heterogeneity in pathogen populations to our advantage.

sentences: What are the reference strains of Acinetobacter baumannii referring to? We assembled the whole genome sequence (WGS) of a collection of 43 non-redundant modern clinical isolates and four broadly used reference strains of Acinetobacter baumannii. Comparison of these isolates and their WGS confirmed the high heterogeneity in capsule loci, sequence types, the presence of virulence and antibiotic resistance genes. However, a significant portion of clinical isolates strongly differ when compared to several reference strains in the light of colony morphology, cellular density, capsule production, natural transformability and in vivo virulence. These genetic and phenotypic differences between current circulating strains of A. baumannii and established reference strains could hamper the study of A. baumannii as an entity. The broadly used reference strains led to the current state of the art of the A. baumannii field, however, we propose that established reference strains in the A. baumannii field should be carefully used, because of the high genetic and phenotypic heterogeneities. In this study, we generated a collection of high-quality nucleotide sequences of 43 modern clinical isolates with the corresponding multi-level phenotypic characterizations. Beside the contribution of novel fundamental observations generated in this study, the phenotypic and genetic data, along with the bacterial strains themselves, will be further accessible using the first open access online platform called "Acinetobase". Therefore, a rational choice of modern strains will be possible to select the ones that suit the needs of specific biological questions.

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Neurophysiological Measures Of Auditory Sensory Processing Are Associated With Adaptive Behavior In Children With Autism Spectrum Disorder BackgroundAtypical auditory cortical processing is consistently found in scalp electrophysiological and magnetoencephalographic studies of Autism Spectrum Disorder (ASD), and may provide a marker of neuropathological brain development. However, the relationship between atypical cortical processing of auditory information and adaptive behavior in ASD is not yet well understood.

MethodsWe sought to test the hypothesis that early auditory processing in ASD is related to everyday adaptive behavior through the examination of auditory event-related potentials (AEPs) in response to simple tones and Vineland Adaptive Behavior Scales in a large cohort of children with ASD (N=89), aged 6-17, and in age- and IQ-matched neurotypically (NT) developing controls (N=120).

ResultsStatistical analyses revealed significant group differences in early AEPs over temporal scalp regions. Whereas the expected rightward lateralization of the AEP to tonal stimuli occurred in both groups, lateralization of the AEP was only significantly associated with adaptive functioning, in the domains of communication and daily living, in the ASD group.

ConclusionsThese results lend support to the hypothesis that atypical processing of sensory information is related to everyday adaptive behavior in autism.

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Mechanisms for Electron Uptake by Methanosarcina acetivorans During Direct Interspecies Electron Transfer Direct interspecies electron transfer (DIET) between bacteria and methanogenic archaea appears to be an important syntrophy in both natural and engineered methanogenic environments. However, the electrical connections on the outer surface of methanogens and the subsequent processing of electrons for carbon dioxide reduction to methane are poorly understood. Here we report that the genetically tractable methanogen Methanosarcina acetivorans can grow via DIET in co-culture with Geobacter metallireducens serving as the electron-donating partner. Comparison of gene expression patterns in M. acetivorans grown in co-culture versus pure culture growth on acetate revealed that transcripts for the outer-surface, multi-heme, c-type cytochrome MmcA were higher during DIET-based growth. Deletion of mmcA inhibited DIET. The high aromatic amino acid content of M. acetivorans archaellins suggests that they might assemble into electrically conductive archaella. A mutant that could not express archaella was deficient in DIET. However, this mutant grew in DIET-based co-culture as well as the archaella-expressing parental strain in the presence of granular activated carbon, which was previously shown to serve as a substitute for electrically conductive pili as a conduit for long-range interspecies electron transfer in other DIET-based co-cultures. Transcriptomic data suggesting that the membrane-bound Rnf, Fpo, and HdrED complexes also play a role in DIET were incorporated into a charge-balanced model illustrating how electrons entering the cell through MmcA can yield energy to support growth from carbon dioxide reduction. The results are the first genetics-based functional demonstration of likely outer-surface electrical contacts for DIET in a methanogen.

ImportanceThe conversion of organic matter to methane plays an important role in the global carbon cycle and is an effective strategy for converting wastes to a useful biofuel. The reduction of carbon dioxide to methane accounts for approximately a third of the methane produced in anaerobic soils and sediments as well as waste digesters. Potential electron donors for carbon dioxide reduction are H2 or electrons derived from direct interspecies electron transfer (DIET) between bacteria and methanogens. Elucidating the relative importance of these electron donors has been difficult due to a lack of information on the electrical connects on the outer surface of methanogens and how they process the electrons received from DIET. Transcriptomic patterns and gene deletion phenotypes reported here provide insight into how a group of Methanosarcina that play an important role in methane production in soils and sediments participate in DIET.

sentences: Maf/ham1-like pyrophosphatases of non-canonical nucleotides are host-specific partners of viral RNA-dependent RNA polymerases Cassava brown streak disease (CBSD), dubbed the "Ebola of plants", is a serious threat to food security in Africa caused by two viruses of the family Potyviridae: cassava brown streak virus (CBSV) and Ugandan (U)CBSV. Intriguingly, U/CBSV, along with another member of this family and one secoviridae, are the only known RNA viruses encoding a protein of the Maf/ham1-like family, a group of widespread pyrophosphatase of non-canonical nucleotides (ITPase) expressed by all living organisms. Despite the socio-economic impact of CDSD, the relevance and role of this atypical viral factor has not been yet established. Here, using an infectious cDNA clone and reverse genetics, we demonstrate that UCBSV requires the ITPase activity for infectivity in cassava, but not in the model plant Nicotiana benthamiana. HPLC MS/MS experiments showed that, quite likely, this host-specific constraint is due to an unexpected high concentration of non-canonical nucleotides in cassava. Finally, protein analyses and experimental evolution of mutant viruses indicated that keeping a fraction of the yielded UCBSV ITPase covalently bound to the viral RNA-dependent RNA polymerase (RdRP) optimizes viral fitness, and this seems to be a feature shared by the other members of the Potyviridae family expressing Maf/ham1-like proteins. All in all, our work (i) reveals that the over-accumulation of non-canonical nucleotides in the host might have a key role in antiviral defense, and (ii) provides the first example of an RdRP-ITPase partnership, reinforcing the idea that RNA viruses are incredibly versatile at adaptation to different host setups.

sentences: A comparison of methodological approaches to the study of young sex chromosomes: A case study in Poecilia Studies of sex chromosome systems at early stages of divergence are key to understanding the initial process and underlying causes of recombination suppression. However, identifying signatures of divergence in homomorphic sex chromosomes can be challenging due to high levels of sequence similarity between the X and the Y. Variations in methodological precision and underlying data can make all the difference between detecting subtle divergence patterns or missing them entirely. Recent efforts to test for X-Y sequence differentiation in the guppy have led to contradictory results. Here we apply different analytical methodologies to the same dataset to test for the accuracy of different approaches in identifying patterns of sex chromosome divergence in the guppy. Our comparative analysis reveals that the most substantial source of variation in the results of the different analyses lies in the reference genome used. Analyses using custom-made de novo genome assemblies for the focal species successfully recover a signal of divergence across different methodological approaches. By contrast, using the distantly related Xiphophorus reference genome results in variable patterns, due to both sequence evolution and structural variations on the sex chromosomes between the guppy and Xiphophorus. Changes in mapping and filtering parameters can additionally introduce noise and obscure the signal. Our results illustrate how analytical differences can alter perceived results and we highlight best practices for the study of nascent sex chromosomes.

sentences: Involvement of Brn3a-positive spinal dorsal horn neurons in the transmission of visceral pain in inflammatory bowel disease model mice Spinal dorsal horn plays crucial roles in the transmission and processing of somatosensory information. Although spinal neural circuits which process several distinct types of cutaneous sensation have been extensively studied, those responsible for visceral pain transmission remain poorly understood. In the present study, we analyzed the dextran sodium sulfate (DSS)-induced inflammatory bowel disease (IBD) model mice to characterize the spinal dorsal horn neurons involved in visceral pain transmission. DSS-treated mice exhibited increased abdominal licking behavior, suggestive of experiencing visceral pain. Immunostaining of c-fos, a marker indicating neuronal activity, demonstrated that numerous c-fos-positive cells were found bilaterally in the lumbosacral spinal dorsal horn, and their distribution was particularly abundant in the shallow dorsal horn. Neurochemical characterization of these neurons revealed that the percentage of the POU transcription factor Brn3a-positive neurons among the c-fos-positive neurons in the shallow dorsal horn was 30-40% in DSS-treated mice, which was significantly higher than that in the somatic pain model mice. We further demonstrated by neuronal tracing that within the shallow dorsal horn, Brn3a-positive neurons are represented more highly in spino-solitary projection neurons than in spino-parabrachial projection ones. These results raised the possibility that Brn3a-positive spinal dorsal horn neurons make a large contribution to visceral pain transmission, and part of which was mediated through spino-solitary pathway.

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CK2 alpha prime and alpha-synuclein pathogenic functional interaction mediates synaptic dysregulation in Huntington's disease BackgroundHuntingtons Disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the HTT gene for which no therapies are available. This mutation causes HTT protein misfolding and aggregation, preferentially affecting medium spiny neurons (MSNs) of the basal ganglia. Transcriptional perturbations in synaptic genes and neuroinflammation are key processes that precede MSN dysfunction and motor symptom onset. Understanding the interplay between these processes is crucial to develop effective therapeutic strategies to treat HD. We investigated whether protein kinase CK2, a kinase upregulated in MSNs in HD and previously associated with Parkinsons disease (PD), participates in the regulation of neuroinflammation and synaptic function during HD progression.

MethodsWe used the heterozygous knock-in zQ175 HD mouse model and compared that to zQ175 mice lacking one allele of CK2. We performed neuropathological analyses using immunohistochemistry, cytokine proteome profiling, RNA-seq analyses in the striatum, electrophysiological recordings, and behavioral analyses. We also used the murine immortalized striatal cell lines STHdhQ7 and STHdhQ111 and studied the expression of various synaptic genes dysregulated by CK2.

ResultsWe showed that CK2 haploinsufficiency in zQ175 mice ameliorated neuroinflammation, HTT aggregation, transcriptional alterations, excitatory synaptic transmission, and motor coordination deficits. RNA-seq analyses also revealed a connection between -syn, a protein associated with PD, and the transcriptional perturbations mediated by CK2 in HD. We also found increased -syn serine 129 phosphorylation (pS129--syn), a post-translational modification linked to -synucleinopathy, in the nuclei of MSNs in zQ175 mice and in patients with HD. Levels of pS129--syn were ameliorated in zQ175 lacking one allele of CK2.

ConclusionsOur data demonstrated that CK2 contributes to transcriptional dysregulation of synaptic genes and neuroinflammation in zQ175 mice and its depletion improved several HD-like phenotypes in this mouse model. These effects were related to increased phosphorylation of S129--syn in the striatum of HD mice, suggesting that CK2 contributes to worsening HD by mediating synucleinopathy. Our study highlights a possible convergent mechanism of neurodegeneration between HD and PD and suggests targeting CK2 as a potential therapeutic strategy to ameliorate synaptic dysfunction in HD as well as other neurodegenerative diseases.

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Adapting to extremes: reconstructing evolution in response to changing climate over time and space in the diverse Australian plant genus Acacia AimMacroevolutionary analysis is increasingly being used to study biodiversity responses to climate change, for example by using phylogenetic node ages to infer periods of diversification, or phylogenetic reconstruction of traits to infer adaptation to particular stresses. Here we apply a recently developed macroevolutionary method to investigate the responses of a diverse plant genus, Acacia, to increasing aridity and salinity in Australia from the Miocene to the present. We ask whether increase in tolerance of aridity and salinity coincided with periods of aridification, and if it allowed the radiation of Acacia into a wide range of niches.

TaxonAcacia

LocationAustralia

MethodsWe applied the Niche Evolution Model (NEMo), which combines Environmental (or Ecological) Niche Modelling (ENM) with phylogenetic comparative methods (PCM) in a single statistical framework, to a large database of Acacia presence-only records and presence-absence survey sites in order to infer current environmental tolerances of Australia Acacia species and reconstruct the evolution of environmental tolerance to increasing aridity and salinity.

ResultsWe find that patterns in evolution of Acacia, over time and across different habitat types, are consistent with the aridification history of Australia and suggests substantial ability to adapt to high aridity and salinity.

Main conclusionsOur results suggest that many Acacia lineages have been able to exploit new environments created during the aridification of Australia through evolution of environmental tolerance, resulting in their current dominance of many habitats across the continent. This study demonstrates that phylogenetic studies of the evolution of responses to changing environment can move beyond application of simple trait-based models, allowing the underlying processes of speciation, adaptation and dispersal to be explicitly modelled in a macroecological and macroevolutionary context.

Statement of significanceAcacia species are found throughout Australia, from rainforests to deserts, and are striking in their environmental adaptability, so they are a perfect case study for understanding evolution of tolerance to environmental extremes in a changing climate. We use the largest database of spatial distribution records yet assembled, using both surveys and atlas data, and a new analytical method that combines the strengths of environmental niche modelling with phylogenetic comparative methods, to demonstrate rapid evolution in aridity and salinity tolerance in response to aridification of the Australian continent during the Neogene and Quaternary.

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BBB damage in aging causes brain iron deposits via astrocyte-neuron crosstalk and Hepc/Fpn1 pathway During aging, iron accumulates in brains regions vulnerable to neurodegeneration: the cerebral cortex and the hippocampus. However, the mechanism of iron regulation in the brain remains scarce. Here, we demonstrated for the first time the involvement of the Hepcidin/Ferroportin1 pathway in brain iron metabolism during aging.

We demonstrated the alteration of BBB integrity, that leads to increased iron permeability and deregulation of iron homeostasis during aging. We found that brain iron overload drives Hepcidin upregulation and, consequently, the inhibition of the iron exporter Ferroportin1, neuroinflammation and oxidative stress. Moreover, both in the cerebral cortex and hippocampus Ferroportin1 colocalizes with astrocytes, while the iron storage protein ferritin light-chain with neurons. This differential distribution suggests that astrocytes mediate iron shuttling and neurons are unable to metabolize it. Furthermore, we observed NCOA4-dependent ferritinophagy of ferritin heavy-chain isoforms determining the increase of light-chain enriched ferritin heteropolymers that are more efficient as iron chelators. Altogether, these data highlight the involvement of the Hepcidin/Ferroportin1 axis and NCOA4 during mice aging as a response to a higher iron influx to the brain.

sentences: Improved AlphaFold modeling with implicit experimental information Machine learning prediction algorithms such as AlphaFold1 and RoseTTAFold2 can create remarkably accurate protein models, but these models usually have some regions that are predicted with low confidence or poor accuracy3-6. We hypothesized that by implicitly including experimental information, a greater portion of a model could be predicted accurately, and that this might synergistically improve parts of the model that were not fully addressed by either machine learning or experiment alone. An iterative procedure was developed in which AlphaFold models are automatically rebuilt based on experimental density maps and the rebuilt models are used as templates in new AlphaFold predictions. We find that including experimental information improves prediction beyond the improvement obtained with simple rebuilding guided by the experimental data. This procedure for AlphaFold modeling with density has been incorporated into an automated procedure for crystallographic and electron cryo-microscopy map interpretation.

sentences: RNA nucleotide repeats induce mitochondrial dysfunction and the ribosome associated quality control Nucleotide repeat sequences are prevalent in the genome and expansion of these sequences is associated with more than 40 neuromuscular disorders. To understand the pathogenic mechanisms underlying RNA-repeat toxicity, we performed a genetic screen in a Caenorhabditis elegans model expressing an expanded CUG repeat specifically in the muscle. Here, we show that expression of this RNA repeat impairs motility by mitochondrial dysfunction, disrupting mitochondrial morphology and respiration. The phenotype is dependent on the RNA-binding factor MBL-1 and requires factors from the ribosome-associated protein quality control complex. Furthermore, Coenzyme Q supplementation rescued the motility impairment and all of the mitochondrial phenotypes. Together, our data reveal the importance of mitochondrial dysfunction in the molecular pathogenesis of RNA repeat expansion disorders.

sentences: PspA adopts an ESCRT-III-like fold and remodels bacterial membranes PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 [A] resolution cryo-EM structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generate positive membrane curvature. Using cryo-EM we visualized how PspA remodels membrane vesicles into m-sized structures and how it mediates the formation of internalized vesicular structures. Hot spots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

sentences: DRP1 regulates endoplasmic reticulum sheets to control mitochondrial DNA replication and segregation Mitochondria are multi-faceted organelles crucial for cellular homeostasis that contain their own genome. Mitochondrial DNA (mtDNA) codes for several essential components of the electron transport chain, and mtDNA maintenance defects lead to mitochondrial diseases. mtDNA replication occurs at endoplasmic reticulum (ER)-mitochondria contact sites and is regulated by mitochondrial dynamics. Specifically, mitochondrial fusion is essential for mtDNA maintenance. In contrast, while loss of mitochondrial fission causes the aggregation of nucleoids (mtDNA-protein complexes), its role in nucleoid distribution remains unclear. Here, we show that the mitochondrial fission protein DRP1 regulates nucleoid segregation by altering ER sheets, the ER structure associated with protein synthesis. Specifically, DRP1 loss or mutation leads to altered ER sheets that enhance physical interaction with mitobulbs, mitochondrial structures containing aggregated nucleoids. Importantly, nucleoid distribution and mtDNA replication were rescued by expressing the ER sheet protein CLIMP63. Thus, our work identifies a novel fission-independent mechanism by which DRP1 regulates mtDNA replication and distribution.

sentences: A cadherin mutation in Celsr3 linked to Tourette Disorder affects dendritic patterning and excitability of cholinergic interneurons CELSR3 encodes an atypical protocadherin cell adhesion receptor that was recently identified as a high-risk gene for Tourette disorder. A putative damaging de novo variant was inserted into the mouse genome to generate an amino acid substitution within the fifth cadherin repeat. By contrast to Celsr3 constitutive null animals, mice homozygous for the R774H amino acid substitution are viable and have grossly normal forebrain development. The density of cortical and striatal interneuron subpopulations is normal, but 3D geometric analysis of cortical pyramidal neurons and striatal cholinergic interneurons revealed changes to dendritic patterning and types and distributions of spines. Furthermore, patch clamp recordings in cholinergic interneurons located within the sensorimotor striatum uncovered mild intrinsic hyperexcitability. Despite these changes, Celsr3R774H homozygous mice do not show obvious  tic-like stereotypies at baseline nor motor learning impairments, but females exhibited perseverative digging behavior. Our findings show that a human mutation in CELSR3 linked to Tourette disorder is sufficient to alter dendritic patterning in the cortex and striatum and also the intrinsic excitability of cholinergic interneurons.

sentences: SIRT5 is a proviral factor that interacts with SARS-CoV-2 Nsp14 protein SARS-CoV-2 non-structural protein Nsp14 is a highly conserved enzyme necessary for viral replication. Nsp14 forms a stable complex with non-structural protein Nsp10 and exhibits exoribonuclease and N7-methyltransferase activities. Protein-interactome studies identified human sirtuin 5 (SIRT5) as a putative binding partner of Nsp14. SIRT5 is an NAD-dependent protein deacylase critical for cellular metabolism that removes succinyl and malonyl groups from lysine residues. Here we investigated the nature of this interaction and the role of SIRT5 during SARS-CoV-2 infection. We showed that SIRT5 stably interacts with Nsp14, but not with Nsp10, suggesting that SIRT5 and Nsp10 are parts of separate complexes. We found that SIRT5 catalytic domain is necessary for the interaction with Nsp14, but that Nsp14 does not appear to be directly deacylated by SIRT5. Furthermore, knock-out of SIRT5 or treatment with specific SIRT5 inhibitors reduced SARS-CoV-2 viral levels in cell-culture experiments. SIRT5 knock-out cells expressed higher basal levels of innate immunity markers and mounted a stronger antiviral response. Our results indicate that SIRT5 is a proviral factor necessary for efficient viral replication, which opens novel avenues for therapeutic interventions.

sentences: Fiber density and matrix stiffness modulate distinct cell migration modes in a 3D stroma mimetic composite hydrogel The peritumoral stroma is a complex 3D tissue that provides cells with myriad biophysical and biochemical cues. Histologic observations suggest that during metastatic spread of carcinomas, these cues influence transformed epithelial cells, prompting a diversity of migration modes spanning single cell and multicellular phenotypes. Purported consequences of these variations in tumor escape strategies include differential metastatic capability and therapy resistance. Therefore, understanding how cues from the peritumoral stromal microenvironment regulate migration mode phenotypes has prognostic and therapeutic value. Here, we utilize a synthetic stromal mimetic in which matrix fiber density and bulk hydrogel stiffness can be orthogonally tuned to investigate the contribution of these two key matrix attributes on MCF10A migration mode phenotypes, epithelial-mesenchymal transition (EMT), and invasive potential. We developed an automated computational image analysis framework to extract migratory phenotypes from fluorescent images and determine 3D migration metrics relevant to metastatic spread. Using this analysis, we find that matrix fiber density and bulk hydrogel stiffness distinctly contribute to a variety of MCF10A migration modes including amoeboid, single mesenchymal, multicellular clusters, and collective strands. Taking advantage of the tunability of this material platform, we identify a combination of physical and soluble cues that induces distinct heterogeneous migration modes originating from the same MCF10A spheroid and use this setting to examine a functional consequence of migration mode - apoptotic resistance. We find that cells migrating as part of collective strands are more resistant to staurosporine-induced apoptosis than either disconnected multicellular clusters or individual invading cells. Improved models of the peritumoral stromal microenvironment that help elucidate relationships between matrix attributes and cell migration mode can contribute to ongoing efforts to identify efficacious cancer therapeutics that address migration plasticity-based therapy resistances.

sentences: Using top-down modulation to optimally balance shared versus separated task representations Human adaptive behavior requires continually learning and performing a wide variety of tasks, often with very little practice. To accomplish this, it is crucial to separate neural representations of different tasks in order to avoid interference. At the same time, sharing neural representations supports generalization and allows faster learning. Therefore, a crucial challenge is to find an optimal balance between shared versus separated representations. Typically, models of human cognition employ top- down modulatory signals to separate task representations, but there exist surprisingly little systematic computational investigations of how such modulation is best implemented. We identify and systematically evaluate two crucial features of modulatory signals. First, top-down input can be processed in an additive or multiplicative manner. Second, the modulatory signals can be adaptive (learned) or non-adaptive (random). We cross these two features, resulting in four modulation networks which are tested on a variety of input datasets and tasks with different degrees of stimulus-action mapping overlap. The multiplicative adaptive modulation network outperforms all other networks in terms of accuracy. Moreover, this network develops hidden units that optimally share representations between tasks. Specifically, different than the binary approach of currently popular latent state models, it exploits partial overlap between tasks.

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Computational simulations and Ca2+ imaging reveal that slow synaptic depolarizations (slow EPSPs) inhibit fast EPSP evoked action potentials for most of their time course in enteric neurons Transmission between neurons in the extensive enteric neural networks of the gut involves synaptic potentials with vastly different time courses and underlying conductances. Most enteric neurons exhibit fast excitatory post-synaptic potentials (EPSPs) lasting 20-50 ms, but many also exhibit slow EPSPs that last up to 100 s. When large enough, slow EPSPs excite action potentials at the start of the slow depolarization, but how they affect action potentials evoked by fast EPSPs is unknown. Furthermore, two other sources of synaptic depolarization probably occur in enteric circuits, activated via GABAA or GABAC receptors; how these interact with other synaptic depolarizations is also unclear. We built a compartmental model of enteric neurons incorporating realistic voltage-dependent ion channels, then simulated fast EPSPs, slow EPSPs and GABAA or GABAC ligand-gated Cl- channels to explore these interactions. Model predictions were tested by imaging Ca2+ transients in myenteric neurons ex vivo as an indicator of their activity during synaptic interactions. The model could mimic firing of myenteric neurons in mouse colon evoked by depolarizing current during intracellular recording and the fast and slow EPSPs in these neurons. Subthreshold fast EPSPs evoked spikes during the rising phase of a slow EPSP, but suprathreshold fast EPSPs could not evoke spikes later in a slow EPSP. This predicted inhibition was confirmed by Ca2+ imaging in which stimuli that evoke slow EPSPs suppressed activity evoked by fast EPSPs in many myenteric neurons. The model also predicted that synchronous activation of GABAA receptors and fast EPSPs potentiated firing evoked by the latter, while synchronous activation of GABAC receptors with fast EPSPs, potentiated firing and then suppressed it. The results reveal that so-called slow EPSPs have a biphasic effect being likely to suppress fast EPSP evoked firing over very long periods, perhaps accounting for prolonged quiescent periods seen in enteric motor patterns.

Author SummaryThe gastrointestinal tract is the only organ with an extensive semi-autonomous nervous system that generates complex contraction patterns independently. Communication between neurons in this "enteric" nervous system is via depolarizing synaptic events with dramatically different time courses including fast synaptic potentials lasting around 20-50 ms and slow depolarizing synaptic potentials lasting for 10 - 120 s. Most neurons have both. We explored how slow synaptic depolarizations affect generation of action potentials by fast synaptic potentials using computational simulation of small networks of neurons implemented as compartmental models with realistic membrane ion channels. We found that slow synaptic depolarizations have biphasic effects; they initially make fast synaptic potentials more likely to trigger action potentials, but then actually prevent action potential generation by fast synaptic potentials with the inhibition lasting several 10s of seconds. We confirmed the inhibitory effects of the slow synaptic depolarizations using live Ca imaging of enteric neurons from mouse colon in isolated tissue. Our results identify a novel form of synaptic inhibition in the enteric nervous system of the gut, which may account for the vastly differing time courses between signalling in individual gut neurons and rhythmic contractile patterns that often repeat at more than 60 s intervals.

sentences: A proteomic perspective and involvement of cytokines in SARS-CoV-2 infection Infection with the SARS-CoV-2 virus results in manifestation of several clinical observations from asymptomatic to multi-organ failure. Biochemically, the serious effects are due to what is described as cytokine storm. The initial infection region for COVID-19 is the nasopharyngeal/oropharyngeal region which is the site where samples are taken to examine the presence of virus. We have earlier shown that several defensin genes are down regulated in cells from this region in patients who tested positive in the RTPCR test. We have now carried out detailed proteomic analysis of the nasopharyngeal/oropharyngeal swab samples collected from normal individuals and those tested positive for SARS-CoV-2 by RTPCR, involving high throughput quantitative proteomics analysis. Several proteins like annexins, cytokines and histones were found differentially regulated in the host human cells following SARS-CoV-2 infection. Genes for these proteins were also observed to be differentially regulated when their expression was analyzed. Majority of the cytokine proteins were found to be up regulated in the infected individuals. Cell to Cell signaling interaction, Immune cell trafficking and inflammatory response pathways were found associated with the differentially regulated proteins based on network pathway analysis.

sentences: Pipeline for generating stable large genomic deletions in zebrafish, from small domains to whole gene excisions Here we describe a short feasibility study and methodological framework for the production of stable, CRISPR/Cas9-based, large genomic deletions in zebrafish, ranging from several base pairs (bp) to hundreds of kilobases (kb). Using a cocktail of four sgRNAs targeting a single genomic region mixed with a marker-sgRNA against the pigmentation gene tyrosinase (tyr), we demonstrate that one can easily and accurately excise genomic regions such as promoters, protein domains, specific exons or whole genes. We exemplify this technique with a complex gene family, neurexins, composed of three duplicated genes with multiple promoters and intricate splicing processes leading to thousands of isoforms. We precisely deleted small regions such as their transmembrane domains (150bp deletion in average) to their entire genomic locus (300kb deletion for nrxn1A for instance). We find that both the concentration and ratio of Cas9/sgRNAs are critical for the successful generation of these large deletions and, interestingly, that their transmission frequency does not decrease with increasing distance between sgRNA target sites. Considering the growing reports and debate about genetically compensated small indel mutants, the use of large-deletion approaches is likely to be widely adopted in studies of gene function. This strategy will also be key to the study of non-coding genomic regions.

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Regulation of blood-brain barrier integrity by microbiome-associated methylamines and cognition by trimethylamine N-oxide BackgroundCommunication between the gut microbiota and the brain is primarily mediated via soluble microbe-derived metabolites, but the details of this pathway remain poorly defined. Methylamines produced by microbial metabolism of dietary choline and L-carnitine have received attention due to their proposed association with vascular disease, but their effects upon the cerebrovascular circulation have hitherto not been studied.

ResultsHere we use an integrated in vitro/in vivo approach to show that physiologically relevant concentrations of the dietary methylamine trimethylamine N-oxide (TMAO) enhanced blood-brain barrier (BBB) integrity and protected it from inflammatory insult, acting through the tight junction regulator annexin A1. In contrast, the TMAO precursor trimethylamine (TMA) impaired BBB function and disrupted tight junction integrity. Moreover, we show that long-term exposure to TMAO protects murine cognitive function from inflammatory challenge, acting to limit astrocyte and microglial reactivity in a brain region-specific manner.

ConclusionOur findings demonstrate the mechanisms through which microbiome-associated methylamines directly interact with the mammalian BBB, with consequences for cerebrovascular and cognitive function.

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RCX - an R package adapting the Cytoscape Exchange format for biological networks MotivationThe Cytoscape Exchange (CX) format is a JSON-based data structure designed for the transmission of biological networks using standard web technologies. It was developed by the network data exchange (NDEx), which itself serves as online commons to share and collaborate on biological networks. Furthermore, the Cytoscape software for the analysis and visualization of biological networks contributes structure elements to capture the visual layout within the CX format. However, there is a fundamental difference between data handling in web standards and R. A manual conversion requires detailed knowledge of the CX format to reproduce and work with the networks.

ResultsHere we present a software package to create, handle, validate, visualize and convert networks in CX format to standard data types and objects within R. Networks in this format can serve as a source for biological knowledge, and also capture the results of the analysis of those while preserving the visual layout across all platforms. The RCX package connects the R environment for statistical computing with outside platforms for storage and collaboration, as well as further analysis and visualization of biological networks.

AvailabilityRCX is a free and open-source R package, available on Bioconductor from release 3.15 (https://bioconductor.org/packages/RCX) and via GitHub (https://github.com/frankkramer-lab/RCX).

Contactflorian.auer@informatik.uni-augsburg.de

Supplementary informationSupplementary data are available at Bioinformatics Advances online.

sentences: Internal and external features of wild birds after collisions without apparent trauma in Japan Wild birds often require rehabilitation after collisions, even with no apparent injury. Information about aftermath of collisions is still scares. Here, we investigated external characteristics and clinical features of the internal organs of wild birds that experienced collision and compared them with birds admitted to rehabilitation center for other reasons. Necropsy was performed on 55 bird carcasses from Passeriformes and Coraciiform. Five external characteristics were recorded before necropsy including; cause of admission, keel score, life stage, fat score, and number of days before death. The median survival time was calculated by Kaplan-Meier estimates. Data on external and internal features were compared using univariate and multivariate multinomial regressions. There was no significant difference in the median survival time among the causes of admission: 1 day for collision, 2 days for trauma, and 2 days for malnutrition. Kidney discoloration was more significantly associated with collision than with other trauma (p = 0.01). Although no apparent kidney abnormality (including enlargement) were observed, anterior lobe was significantly larger than posterior lobe with collision compared with malnutrition (p = 0.045). Birds that experienced collision exhibited a higher fat score than malnourished birds (p = 0.03). Our results suggested that wild birds with abundant fat were more likely to be admitted due to collision. The gross characteristics of collision included kidney discoloration and anterior lobe extension, which was possibly due to rupture of renal blood vessels by blunt external force. From these findings, it was considered that collision caused major axis of anterior lobe significantly larger than posterior lobe, even though no abnormal finding in renal size such as hypertrophy was recognized. This was the first study to evaluate the cause of admission, necropsy results, and external characteristics in wild birds admitted to rehabilitation centers. Absolute cage rest should be adhered to restore renal function for those birds admitted due to collision, and handling and treatment should be minimized to avoid excess movement of the birds.

sentences: Germline/soma distinction in Drosophila embryos requires regulators of zygotic genome activation In Drosophila melanogaster embryos, somatic versus germline identity is the first cell fate decision. Zygotic genome activation (ZGA) orchestrates regionalized gene expression, imparting specific identity on somatic cells. ZGA begins with a minor wave that commences at nuclear cycle (NC) 8 under the guidance of chromatin accessibility factors (Zelda, CLAMP, GAF), followed by the major wave during NC14. By contrast, primordial germ cell (PGC) specification requires maternally deposited and posteriorly anchored germline determinants. This is accomplished by a centrosome coordinated release and sequestration of germ plasm during the precocious cellularization of PGCs in NC10. Here we report a novel requirement for Zelda and CLAMP during the establishment of the germline/soma distinction. When their activity is compromised, PGC determinants are not properly sequestered, and specification is disrupted. Conversely, the spreading of PGC determinants from the posterior pole adversely influences transcription in the neighboring somatic nuclei. These reciprocal aberrations can be correlated with defects in centrosome duplication/separation that are known to induce inappropriate transmission of the germ plasm. Interestingly, consistent with the ability of BMP signaling to influence specification of embryonic PGCs, reduction in the transcript levels of a BMP family ligand, decapentaplegic (dpp), is exacerbated at the posterior pole.

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Reproducible Propagation of Species-Rich Soil Microbiomes Suggests Robust Underlying Deterministic Principles of Community Formation Microbiomes are typically characterised by high species diversity but it is poorly understood how such system-level complexity can be generated and propagated. Here, we used soils as a relevant model to study microbiome development. Despite inherent stochastic variation in manipulating species-rich communities, both laboratory-mixed medium complexity (21 soil bacterial isolates in equal proportions) and high-diversity natural top-soil communities followed highly reproducible succession paths, maintaining distinct soil microbiome signatures. Development trajectories and compositional states were different for communities propagated in soils than in liquid suspension. Microbiome states were maintained over multiple renewed growth cycles but could be diverged by short-term pollutant exposure. The different but robust trajectories demonstrated that deterministic taxa-inherent characteristics underlie reproducible development and self-organized complexity of soil microbiomes within their environmental boundary conditions. Our findings also have direct implications for potential strategies to achieve controlled restoration of desertified land.

TEASERSpecies-rich soil microbiomes grow and propagate reproducibly despite inherent stochastic complexity, paving the way for soil restoration.

sentences: Constitutive G protein coupling profiles of understudied orphan GPCRs A large number of GPCRs are potentially valuable drug targets but remain understudied. Many of these lack well-validated activating ligands and are considered "orphan" receptors, and G protein coupling profiles have not been defined for many orphan GPCRs. Here we asked if constitutive receptor activity can be used to determine G protein coupling profiles of orphan GPCRs. We monitored nucleotide-sensitive interactions between 48 understudied orphan GPCRs and five G proteins (240 combinations) using bioluminescence resonance energy transfer (BRET). No receptor ligands were used, but GDP was used as a common G protein ligand to disrupt receptor-G protein complexes. Constitutive BRET between the same receptors and {beta}-arrestins was also measured. We found sufficient GDP-sensitive BRET to generate G protein coupling profiles for 22 of the 48 receptors we studied. Altogether we identified 48 coupled receptor-G protein pairs, many of which have not been described previously. We conclude that receptor-G protein complexes that form spontaneously in the absence of guanine nucleotides can be used to profile G protein coupling of constitutively active GPCRs. This approach may prove useful for studying G protein coupling of other GPCRs for which activating ligands are not available.

sentences: The Influence of Phosphorus and Transplanting Date on Onion (Allium Cepa L.) Quality Parameters and yield of bulb Under Irrigation at Adami Tulu Jedo Kombolcha Woreda, East showa zone, Oromia Region. A field experiment was showed to decide the influence of phosphorus level and transplanting date on quality parameters of onion at Adami Tulu Jedo Kombolcha Woreda, during 2017/18 season. The treatments consisted of four phosphorus levels (0, 20, 40 and 60 kg P ha-1) and three transplanting date (42, 49 and 56 days). The experiment was laid out in randomized complete block design with four replications. The result showed that phosphorus level and transplanting date significantly influenced bulb dry matter content, marketable bulb yield, medium size bulb yield. Among these parameters, marketable bulb yield was also significantly influenced by the interaction of phosphorus level and transplanting date. On the other hand, small size bulb yield, large size bulb yield, over size bulb yield and under size bulb yield were only influenced by the effect of phosphorus levels. In this study, fertilized 60 kg P ha-1 with transplanting at 56 days of transplanting date recorded the highest marketable bulb yield, but no significant difference was showed with that obtained at 40 kg P ha-1 with the same transplanting date. Treatment combinations of no P (control) and transplanting date at 42 days produced the lowest amounts of marketable bulb yield. The economic analysis revealed that the highest net benefit with low cost of production was obtained in response to the application of 40 kg P ha-1 and the transplanting age of 56 days. The marginal rate of return for this treatment was 11983% which is found to be economically feasible for producing bulb in the districts.

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Mass photometric detection and quantification of nanoscale α-synuclein phase separation -Synuclein (-Syn) liquid-liquid phase separation (LLPS) leads to irreversible amyloid fibril formation associated with Parkinsons disease pathogenesis. Critical concentrations of -Syn LLPS are relatively high under physiological solution conditions. Moreover, -Syn exhibits delayed LLPS kinetics under certain conditions which deviates from the behaviour predicted by classical homogeneous nucleation theory. In the current body of work, using interferometric light scattering (iSCAT), also known as mass photometry, we experimentally probe that -Syn can form nanoscale phase separated assemblies/clusters, containing tens to hundreds of molecules-- both above and below the critical LLPS concentration down to physiologically relevant scales. The formation of these clusters is instantaneous, even under conditions where the formation of microscopically visible droplets takes several days. However, they account for a very small volume fraction below saturation concentration. The slow growth of the nanoclusters can be attributed to a kinetic barrier which can be overcome by increasing the solution temperature to just below the droplet melting point. We provide reasons for caution in quantifying dilute phase concentrations for -Syn LLPS samples containing nanoscale droplets--which can only be separated using ultracentrifugation. In addition, we also delineate that the presence of certain surfaces facilitates -Syn droplet nucleation under conditions of delayed kinetics but is not a mandatory prerequisite for nanocluster formation. Taken together, our findings reveal that phase separation of -Syn occurs at a wider range of solution conditions than predicted so far and provides an important step towards understanding -Syn LLPS within physiological scales.

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sentences: Exploring the effects of CO2 elevation on seedlings' growth of Acacia senegal in the era of changes Acacia senegal is a priority and important C3 tree species in drylands of Sudan and across the gum belt. Investigation of its seedlings response to elevated carbon dioxide (eCO2) is important as atmospheric ([CO2]) has increased and predicted to continue to rise. Many studies showed that eCO2 causes increased photosynthesis in plants, which leads to greater production of carbohydrates and biomass, and increased soil organic matter and carbon content. This study investigated the effects of eCO2 on A. senegal seedlings grown in sand and silt soils under irrigation intervals of every day and every two days. Seven days old seedlings were assigned to the treatments in Split - spilt plot design for 4 weeks. The main plot is eCO2 (600-800 ppm) and ambient ([≤]400 ppm) under Free Air CO2 Enrichment (FACE) system. Subplots are irrigation intervals and soil types. Seedling height and number of leaves were measured weekly, and seedlings were harvested after 4 weeks where growth parameters and soil properties were measured. The eCO2 showed no effect on the measured parameters except the significant increase in tap-root length. However, the irrigation every day showed significant increase than every two days in seedlings height, number of leaves, root length and seedlings dry weight but not seedlings and soil C% & N%. Soil treatment showed effects on stem height, leaf number, seedlings dry weight, leaves and root N% and soil C% but not root length, seedling C% and soil N%. The results indicate the importance of soil moisture, physical and chemical properties that reflects adaptation of the species to its dry land environment.

sentences: Predicting the zoonotic capacity of mammals to transmit SARS-CoV-2 Back and forth transmission of SARS-CoV-2 between humans and animals may lead to wild reservoirs of virus that can endanger efforts toward long-term control of COVID-19 in people, and protecting vulnerable animal populations that are particularly susceptible to lethal disease. Predicting high risk host species is key to targeting field surveillance and lab experiments that validate host zoonotic potential. A major bottleneck to predicting animal hosts is the small number of species with available molecular information about the structure of ACE2, a key cellular receptor required for viral cell entry. We overcome this bottleneck by combining species ecological and biological traits with 3D modeling of virus and host cell protein interactions using machine learning methods. This approach enables predictions about the zoonotic capacity of SARS-CoV-2 for over 5,000 mammals -- an order of magnitude more species than previously possible. The high accuracy predictions achieved by this approach are strongly corroborated by in vivo empirical studies. We identify numerous common mammal species whose predicted zoonotic capacity and close proximity to humans may further enhance the risk of spillover and spillback transmission of SARS-CoV-2. Our results reveal high priority areas of geographic overlap between global COVID-19 hotspots and potential new mammal hosts of SARS-CoV-2. With molecular sequence data available for only a small fraction of potential host species, predictive modeling integrating data across multiple biological scales offers a conceptual advance that may expand our predictive capacity for zoonotic viruses with similarly unknown and potentially broad host ranges.

sentences: Emergent Diversity and Persistent Turnover in Evolving Microbial Cross-Feeding Networks A distinguishing feature of many ecological networks in the microbial realm is the diversity of substrates that could potentially serve as energy sources for microbial consumers. The microorganisms are themselves the agents of compound diversification via metabolite excretion or overflow metabolism. It has been suggested that the emerging richness of different substrates is an important condition for the immense biological diversity in microbial ecosystems. In this work, we study how complex cross-feeding networks (CFN) of microbial species may develop from a simple initial community given some elemental evolutionary mechanisms of resource-dependent speciation and extinctions using a network flow model. We report results of several numerical experiments and report an in-depth analysis of the evolutionary dynamics. We find that even in stable environments, the system is subject to persisting turnover, indicating an ongoing co-evolution. Further, we compare the impact of different parameters, such as the ratio of mineralization, as well as the metabolic versatility and variability on the evolving community structure. The results imply that high microbial and molecular diversity is an emergent property of evolution in cross-feeding networks, which affects transformation and accumulation of substrates in natural systems, such as soils and oceans, with potential relevance to biotechnological applications.

sentences: Allosteric regulation of 3CL protease of SARS-CoV-2 and SARS-CoV observed in the crystal structure ensemble The 3C-like protease (3CLpro) of SARS-CoV-2 is a potential therapeutic target for COVID-19. Importantly, it has an abundance of structural information solved as a complex with various drug candidate compounds. Collecting these crystal structures (83 Protein Data Bank (PDB) entries) together with those of the highly homologous 3CLpro of SARS-CoV (101 PDB entries), we constructed the crystal structure ensemble of 3CLpro to analyze the dynamic regulation of its catalytic function. The structural dynamics of the 3CLpro dimer observed in the ensemble were characterized by the motions of four separate loops (the C-loop, E-loop, H-loop, and Linker) and the C-terminal domain III on the rigid core of the chymotrypsin fold. Among the four moving loops, the C-loop (also known as the oxyanion binding loop) causes the order (active)-disorder (collapsed) transition, which is regulated cooperatively by five hydrogen bonds made with the surrounding residues. The C-loop, E-loop, and Linker constitute the major ligand binding sites, which consist of a limited variety of binding residues including the substrate binding subsites. Ligand binding causes a ligand size dependent conformational change to the E-loop and Linker, which further stabilize the C-loop via the hydrogen bond between the C-loop and E-loop. The T285A mutation from SARS-CoV 3CLpro to SARS-CoV-2 3CLpro significantly closes the interface of the domain III dimer and allosterically stabilizes the active conformation of the C-loop via hydrogen bonds with Ser1 and Gly2; thus, SARS-CoV-2 3CLpro seems to have increased activity relative to that of SARS-CoV 3CLpro.

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PEERS - an open science "Platform for the Exchange of Experimental Research Standards" in biomedicine Laboratory workflows and preclinical models have become increasingly diverse and complex. Confronted with the dilemma of assessing a multitude of information with ambiguous relevance for their specific experiments, scientists run the risk of overlooking critical factors that can influence the planning, conduct and results of studies and that should have been considered a priori. Negligence of such crucial information may result in sub-optimal study design and study execution, bringing into question the validity of generated outcomes. As a corollary, a lot of resources are wasted on biomedical research that turns out to be irreproducible and not sufficiently robust for further project development.

To address this problem, we present  PEERS (Platform for the Exchange of Experimental Research Standards), an open-access online platform that is built to aid scientists in determining which experimental factors and variables are most likely to affect the outcome of a specific test, model or assay and therefore ought to be considered during the design, execution and reporting stages.

The PEERS database is categorized into in vivo and in vitro experiments and provides lists of factors derived from scientific literature that have been deemed critical for experimentation. Most importantly, the platform is based on a structured and transparent system for rating the strength of evidence related to each identified factor and its relevance for a specific method/model. In this context, the rating procedure will not solely be limited to the PEERS working group but will also allow for a community-based grading of evidence.

To generate a proof-of-concept that the PEERS approach is feasible, we focused on a set of in vitro and in vivo methods from the neuroscience field, which are presented in this article. On the basis of the Open Field paradigm in rodents, we describe the selection of factors specific to each experimental setup and the rating system, but also discuss the identification of additional general items that transcend categories and individual tests. Moreover, we present a working format of the PEERS prototype with its structured information framework for embedding data and critical back end/front end user functionalities. Here, PEERS not only offers users the possibility to search for information to facilitate experimental rigor, but also draws on the engagement of the scientific community to actively expand the information contained within the platform through a standardized approach to data curation and knowledge engineering.

As the database grows and benefits become more apparent, we will expand the scope of PEERS to any area of applied biomedical research.

Collectively, by helping scientists to search for specific factors relevant to their experiments, and to share experimental knowledge in a standardized manner, PEERS will serve as the ultimate exchange and analysis tool to enhance data validity and robustness as well as the reproducibility of preclinical research. PEERS offers a vetted, independent tool by which to judge the quality of information available on a certain test or model, identifies knowledge gaps and provides guidance on the key methodological considerations that should be prioritized to ensure that preclinical research is conducted to the highest standards and best practice.

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Positive Selection and Heat-Response Transcriptomes Reveal Adaptive Features of the Brassicaceae Desert Model, Anastatica hierochuntica O_LIPlant adaptation to a desert environment and its endemic heat stress is poorly understood at the molecular level. The naturally heat-tolerant Brassicaceae species Anastatica hierochuntica is an ideal extremophyte model to identify genetic adaptations that have evolved to allow plants to tolerate heat stress and thrive in deserts.
C_LIO_LIWe generated an A. hierochuntica reference transcriptome and pinpointed extremophyte adaptations by comparing Arabidopsis thaliana and A. hierochuntica transcriptome responses to heat and identifying positively selected genes in A. hierochuntica.
C_LIO_LIThe two species exhibit similar transcriptome adjustment in response to heat and the A. hierochuntica transcriptome does not exist in a constitutive heat "stress-ready" state. Furthermore, the A. hierochuntica global transcriptome as well as heat-responsive orthologs, display a lower basal and higher heat-induced expression than in A. thaliana. Genes positively selected in multiple extremophytes are associated with stomatal opening, nutrient acquisition, and UV-B induced DNA repair while those unique to A. hierochuntica are consistent with its photoperiod-insensitive, early-flowering phenotype.
C_LIO_LIWe suggest that evolution of a flexible transcriptome confers the ability to quickly react to extreme diurnal temperature fluctuations characteristic of a desert environment while positive selection of genes involved in stress tolerance and early flowering could facilitate an opportunistic desert lifestyle.
C_LI

sentences: The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate "handover" from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

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An in vivo model of echovirus-induced meningitis in neonates Echoviruses are amongst the most common causes of aseptic meningitis worldwide, which can cause long-term sequelae and death, particularly in neonates. However, the mechanisms by which these viruses induce meningeal inflammation is poorly understood, owing at least in part to the lack of in vivo models that recapitulate this aspect of echovirus pathogenesis. Here, we developed an in vivo neonatal mouse model that recapitulates key aspects of echovirus-induced meningitis. We found that expression of the human homologue of the neonatal Fc receptor (FcRn), the primary echovirus receptor, in neonatal mice was not sufficient for infection of the brain. However, ablation of type I, but not III, IFN signaling in mice expressing human FcRn permitted high levels of echovirus replication in the brain, with corresponding clinical symptoms including delayed motor skills and hind limb weakness. We also defined the immunological response of the brain to echovirus infections and identified key cytokines induced by this infection. Lastly, we found that echoviruses robustly replicate in the leptomeninges, where they induce profound inflammation and cell death. Together, this work establishes an in vivo model of aseptic meningitis associated with echovirus infections and defines the specificity of echoviral infections within the meninges.

Significance StatementEchoviruses are amongst the most common causes of aseptic meningitis worldwide, which can cause long-term sequelae or even death. The mechanisms by which echoviruses infect the brain are poorly understood, largely owing to the lack of robust in vivo models that recapitulate this aspect of echovirus pathogenesis. Here, we establish a neonatal mouse model of echovirus-induced aseptic meningitis and show that expression of the human homologue of the neonatal Fc receptor (FcRn), the primary receptor for echoviruses, and ablation of type I interferon (IFN) signaling are required to recapitulate echovirus-induced meningitis and clinical disease. These findings provide key insights into the host factors that control echovirus-induced meningitis and a model that could be used to test anti-echovirus therapeutics.

sentences: Evaluation of Commercially Available Glucagon Receptor Antibodies and Glucagon Receptor Expression Glucagon is a key regulator of numerous metabolic functions including glucose, protein and lipid metabolism, and glucagon-based therapies are explored for diabetes, fatty liver disease and obesity. Insight into tissue and cell specific expression of the glucagon receptor (GCGR) is important to understand the biology of glucagon as well as to differentiate between direct and indirect actions of glucagon. However, it has been challenging to accurately localize the GCGR in tissue due to low expression levels and lack of specific methodologies. Immunohistochemistry has frequently been used for GCGR localization, but G-protein-coupled receptors (GPCRs) targeting antibodies are notoriously unreliable. In this study, we systematically evaluated all commercially available GCGR antibodies. Initially, twelve GCGR antibodies were evaluated using HEK293 cells transfected with mouse or human GCGR cDNA. Of the twelve antibodies tested, eleven showed positive staining of GCGR protein from both species. Human liver tissue was investigated using the same GCGR antibodies. Five antibodies failed to stain human liver biopsies (despite explicit claims to the contrary from the vendors). Immunohistochemical (IHC) staining demonstrated positive staining of liver tissue from glucagon receptor knockout (Gcgr-/-) mice and their wild-type littermates (Gcgr+/+) with only one out of the twelve available GCGR antibodies. Three antibodies were selected for further evaluation by western blotting and bands corresponding to the predicted size of the GCGR (62 kDa) were identified using two of these. Finally, a single antibody (no. 11) was selected for specific GCGR localization studies in various tissues. In mouse tissue the most intense immunostainings were found in lever, kidney, ileum, heart, and pancreas. Western blotting, performed on liver tissue from Gcgr+/+ and Gcgr-/- mice, confirmed the specificity of antibody no. 11, detecting a band at high intensity in material from Gcgr+/+and no bands in liver tissue from Gcgr-/-mice. Staining of human kidney tissue, with antibody no. 11, showed GCGR localization to the distal tubules. Autoradiography was used as an antibody-independent approach to support the antibody-based findings, revealing specific binding in liver, pancreas, and kidney. As a final approach, RNA-sequencing and single-cell RNA (scRNA)-sequencing were implemented. RNA-sequencing confirmed GCGR presence within liver and kidney tissue. The GCGR was specifically found to be expressed in hepatocytes by scRNA-sequencing and potentially also in collecting and distal tubule cells in the kidney. Our results clearly indicate the liver and the kidneys as the primary targets of glucagon action.

sentences: The level of synovial human VEGFA, IL-8 and MIP-1α correlate with truncation of lubricin glycans in osteoarthritis Osteoarthrithis (OA) is an endemic disease due to the increase of the worlds elderly population. Previously thought to be a consequence of an imbalance between cartilage degradation and biosynthesis, it is now recognized as a disease also involving inflammation, hence influencing the level of inflammatory cytokines, growth factors and chemokines. Lubricin is a mucin type molecule where its OA induced glycosylation truncation propels a deteriorating lubrication of the articular cartilage. The objective of this study was to explore the OA driven truncation of O-linked glycosylation of synovial lubricin and its cross talk with systemic and local (synovial fluid, SF) inflammation. We compared the systemic level of cytokines/chemokine in OA patients and controls plasma with their local level in SF using a 44 plex screen. The level of 27 cytokines and chemokines was consistently measured in both plasma and SF. The data showed that the levels of cytokines and chemokines in OA plasma display limited correlation to their counterpart in SF. The level of synovial IL-8 and MIP-1 and VEGFA in OA patients, but not their plasma level, where the only cytokines that displayed a significant correlation to the observed lubricin O-linked glycosylation truncation. These cytokines were also shown to be upregulated exposing fibroblast like synoviocytes from healthy and OA patients to recombinant lubricin with truncated glycans mainly consisting of Tn-antigens, while lubricin with sialylated and non-sialylated T anigens did not have any effect. The data suggest that truncated glycans of lubricin, as found in OA, promotes the synovial cytokine production and exerebate the local synovial inflammation.

sentences: Systemic impact of the expression of the mitochondrial alternative oxidase on Drosophila development Despite the beneficial effects of xenotopically expressing the mitochondrial alternative oxidase AOX from Ciona intestinalis in mammalian and insect models, important detrimental outcomes have also been reported, raising concerns regarding its potential deployment as a therapeutic enzyme for human mitochondrial diseases. Because of its non-protonmotive terminal oxidase activity, AOX can bypass the cytochrome segment of the respiratory chain whilst not contributing to mitochondrial ATP synthesis. We have previously shown that pupal lethality occurs when AOX-expressing Drosophila larvae are cultured on a low-nutrient diet, indicating that AOX can perturb normal metabolism during development. Here, combined omics analyses revealed multiple correlates of this diet-dependent lethality, including a general alteration of larval amino acid and lipid metabolism, functional and morphological changes to the larval digestive tract, and a drastic decrease in larval biomass accumulation. Pupae at the pre-lethality stage presented a general downregulation of mitochondrial metabolism and a signature of starvation and deregulated signaling. AOX-induced lethality was partially rescued when the low-nutrient diet was supplemented with tryptophan and/or methionine, but not with proline and/or glutamate, strongly suggesting perturbation of one-carbon metabolism. The developmental dependence on tryptophan and/or methionine, associated with elevated levels of lactate dehydrogenase, 2-hydroxyglutarate, choline-containing metabolites and breakdown products of membrane phospholipids, indicates that AOX expression promotes tissue proliferation and larval growth, but this is ultimately limited by energy dissipation due to partial mitochondrial uncoupling. We speculate that the combination of dietary interventions and AOX expression might, nevertheless, be useful for the metabolic regulation of proliferative tissues, such as tumors.

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Evolution and antiviral activity of a human protein of retroviral origin Endogenous retroviruses are abundant components of mammalian genomes descended from ancient germline infections. In several mammals, the envelope proteins encoded by these elements protect against exogenous viruses, but this activity has not been documented in human. We report that our genome harbors a large pool of envelope-derived sequences with the potential to restrict retroviral infection. To further test this, we characterize in detail the envelope-derived protein, Suppressyn. We found that Suppressyn is expressed in preimplantation embryos and developing placenta using its ancestral retroviral promoter. Restriction assays in cell culture show that Suppressyn, and its hominoid orthologs, can restrict infection by extant mammalian type D retroviruses. Our data support a generalizable model of retroviral envelope cooption for host immunity and genome defense.

SummaryWe found that the human genome expresses a vast pool of envelope sequences of retroviral origin and provide proof of principle that such proteins can restrict zoonotic viruses.

sentences: Mechanical vibration patterns elicit behavioral transitions and habituation in crawling Drosophila larvae How animals respond to repeatedly applied stimuli, and how animals respond to mechanical stimuli in particular, are important questions in behavioral neuroscience. We study adaptation to repeated mechanical agitation using the Drosophila larva. Vertical vibration stimuli elicit a discrete set of responses in crawling larvae: continuation, pause, turn, and reversal. Through high-throughput larva tracking, we characterize how the likelihood of each response depends on vibration intensity and on the timing of repeated vibration pulses. By examining transitions between behavioral states at the population and individual levels, we investigate how the animals habituate to the stimulus patterns. We identify time constants associated with desensitization to prolonged vibration, with re-sensitization during removal of a stimulus, and additional layers of habituation that operate in the overall response. Known memory-deficient mutants exhibit distinct behavior profiles and habituation time constants. An analogous simple electrical circuit suggests possible neural and molecular processes behind adaptive behavior.

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Membraneless organelles formed by liquid-liquid phase separation increase bacterial fitness Liquid-liquid phase separation is emerging as a crucial phenomenon in several fundamental cell processes. A range of eukaryotic systems exhibit liquid condensates. However, their function in bacteria, which in general lack membrane-bound compartments, remains less clear. Here, we used high-resolution optical microscopy to observe single bacterial aggresomes, nanostructured intracellular assemblies of proteins, to undercover their role in cell stress. We find that proteins inside aggresomes are mobile and undergo dynamic turnover, consistent with a liquid state. Our observations are in quantitative agreement with phase-separated liquid droplet formation driven by interacting proteins under thermal equilibrium that nucleate following diffusive collisions in the cytoplasm. We have discovered aggresomes in multiple species of bacteria, and show that these emergent, metastable liquid-structured protein assemblies increase bacterial fitness by enabling cells to tolerate environmental stresses.

One Sentence SummaryBacteria use subcellular proteinaceous liquid droplets to survive stress

sentences: Revisiting a classic hybrid zone: rapid movement of the northern flicker hybrid zone in contemporary times Natural hybrid zones have provided important insights into the evolutionary process, and their geographic stability/instability over time can help to disentangle the underlying biological processes that maintain them. Here, we leverage replicated sampling of an identical transect across the hybrid zone between yellow-shafted and red-shafted flickers in the Great Plains to assess its stability over [~]60 years (1955-1957 to 2016-2018). We identify a [~]73 km westward shift in the hybrid zone center towards the range of the red-shafted flicker, but find no associated changes in width over our sampling period. In fact, the hybrid zone remains remarkably narrow, suggesting some kind of selective pressure maintains the zone. By comparing to previous work in the same geographic region, it appears likely that the movement in the hybrid zone has occurred rapidly in the years since the early 1980s. This recent, rapid movement may be related to changes in climate or land management practices that have allowed asymmetric westward movement of yellow-shafted flickers into the Great Plains.

sentences: Impact of maternal intermittent fasting during pregnancy on cardiovascular, metabolic and renal function in adult rat offspring Pregnant Muslim women are exempt from fasting during Ramadan; however a majority are reported to fast. The impact of this form of maternal intermittent fasting (IF) on fetal development and offspring health is not well defined. Using a rat model, we have shown previously that maternal IF results in fetal growth restriction accompanied by changes in placental nutrient transport function. The aim of this study was to assess cardiovascular, metabolic and renal function in adult offspring of IF-exposed dams. Food was withheld from Wistar rats from 17:00 to 09:00 daily throughout pregnancy; controls had ad libitum access to food. Birth weight was unaffected; however male IF pups grew more slowly up to 10 weeks of age (P < 0.01) whereas IF females matched their control counterparts. Systolic blood pressure (SBP), glucose tolerance and basal renal function at 14 weeks were not affected by IF exposure. When offered saline solutions (0.9-2.1%) to drink, females showed a greater salt preference than males (P < 0.01); however there were no differences between dietary groups. A separate group of pups was weaned onto a 4% NaCl diet. SBP increased in IF pups sooner, at 7 weeks (P < 0.01), than controls which became hypertensive from 10 weeks. Renal function did not appear to differ; however markers of renal injury were elevated in IF males (P < 0.05). Maternal IF does not affect resting cardiovascular, metabolic and renal function; but when challenged by dietary salt load male IF offspring are more prone to renal injury.

sentences: Multiscale model of the physiological control of myocardial perfusion to delineate putative metabolic feedback mechanisms Coronary blood flow is tightly regulated to ensure that myocardial oxygen delivery meets local metabolic demand via the concurrent action of myogenic, neural, and metabolic mechanisms. While several competing hypotheses exist, the specific nature of the local metabolic mechanism(s) remains poorly defined. To gain insights into the viability of putative metabolic feedback mechanisms and into the coordinated action of parallel regulatory mechanisms, we applied a multi-scale modeling framework to analyze experimental data on coronary pressure, flow, and myocardial oxygen delivery in the porcine heart in vivo. The modeling framework integrates a previously established lumped-parameter model of myocardial perfusion used to account for transmural hemodynamic variations and a simple vessel mechanics model used to simulate the vascular tone in each of three myocardial layers. Vascular tone in the resistance vessel mechanics model is governed by input stimuli from the myogenic, metabolic, and autonomic control mechanisms. Seven competing formulations of the metabolic feedback mechanism are implemented in the modeling framework, and associated model simulations are compared to experimental data on coronary pressures and flows under a range of experimental conditions designed to interrogate the governing control mechanisms. Analysis identifies a maximally likely metabolic mechanism among the seven tested models, in which production of a metabolic signaling factor is proportional to MVO2 and delivery proportional to flow. Finally, the identified model is validated based on comparisons of simulations to data on the myocardial perfusion response to conscious exercise that were not used for model identification.

sentences: Consequences of coupled barriers to gene flow for the build-up of genomic differentiation Theory predicts that when different barriers to gene flow become coincident, their joint effects enhance reproductive isolation and genomic divergence beyond their individual effects, but empirical tests of this  coupling hypothesis are rare. Here, we analyze patterns of gene exchange among populations of European corn borer moths that vary in the number of acting barriers, allowing for comparisons of genomic variation when barrier traits or loci are in coincident or independent states. We find that divergence is mainly restricted to barrier loci when populations differ by a single barrier, whereas the coincidence of temporal and behavioral barriers is associated with divergence of two chromosomes harboring barrier loci. Furthermore, differentiation at temporal barrier loci increases in the presence of behavioral divergence and differentiation at behavioral barrier loci increases in the presence of temporal divergence. Our results demonstrate how the joint action of coincident barrier effects leads to levels of genomic differentiation that far exceed those of single barriers acting alone, consistent with theory arguing that coupling allows indirect selection to combine with direct selection and thereby lead to a stronger overall barrier to gene flow. Thus, the state of barriers - independent or coupled - strongly influences the accumulation of genomic differentiation.

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Escitalopram Administration, Neuroplastic Effects and Relearning: A Diffusion Tensor Imaging Study in Healthy Individuals BackgroundNeuroplastic processes are influenced by selective serotonergic reuptake inhibitors, while learning in conjunction with the administration of serotonergic agents alters white matter microstructure in humans. The goal of this double-blind, placebo-controlled imaging study was to investigate the influence of escitalopram on white matter plasticity during (re)learning.

MethodsSeventy-one healthy individuals (age = 25.6{+/-}5.0, 43 females) underwent 3 diffusion magnetic resonance imaging sessions: at baseline, after 3-weeks of associative learning (emotional/non-emotional content) and after relearning shuffled associations for an additional 3 weeks. During the relearning phase, subjects received daily escitalopram 10 mg or placebo orally. Statistical analysis was performed with statistical parametric mapping (SPM) and using sandwich estimator.

ResultsA three-way and two-way rmANOVA was conducted to analyze the effects of escitalopram on AD, FA, MD and RD during the learning and relearning period. We found no significant three-way or two-way interactions for each DTI metrics (pFDR > 0.05), thus neither after 3 nor after 6 weeks we found significant changes in white matter microstructure.

ConclusionWe examined neither an effect of escitalopram nor learning (or relearning) interventions on different DTI metrics. The duration and intensity of study interventions (i.e. administration of escitalopram and learning as the relearning task) might have been chosen insufficiently to induce detectable alterations. Previous studies examining the effects of SSRIs on white matter tracts in humans are underrepresented, but do mainly yield towards non-significant findings. The results implicate that escitalopram does not impact white matter microstructures in healthy subjects.

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Intra and inter-annual climatic conditions have stronger effect than grazing intensity on root growth of permanent grasslands AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackground and AimsC_ST_ABSUnderstanding how direct and indirect changes in climatic conditions, management, and species composition affect root production and root traits is of prime importance for the delivery of carbon sequestration services of grasslands. This study considers the effects of climatic variability and gradients of herbage utilisation by grazing on root production over the course of two years. The root and leaf traits of the plant communities were determined to detect their capacity to predict above- and below-ground net primary production, ANPP and BNPP, respectively.

MethodsA long-term field experiment was used to compare the effects of abandonment and low (Ca-) and high (Ca+) grazing intensities (resulting in mean residual plant heights of 15.2 cm and 7.7 cm, respectively) induced by grazing rotations on upland fertile grasslands after 10 years of treatment application. Ingrowth cores and exclusion cages were used to measure, respectively, the root and shoot mass production several times each year and at an annual scale. The root and leaf traits of the communities were measured near the vegetations peak growing season.

ResultsWe observed strong seasonal root production across treatments in both a wet and a dry year, but the response to grazing intensity was hardly observable within growing seasons. In the abandonment treatment, the spring and autumn root growth peaks were delayed by approximately one month compared to the two cattle treatments, possibly due to a late plant canopy green-up induced by lower soil temperatures and an accumulation of litter. The BNPP was slightly lower in the abandonment treatment compared to the cattle treatments only during the dry year, whereas a decline of the ANPP in the abandonment treatment compared to the Ca+ treatment was observed during the wet year. In response to drought, which occurred during the second year, the root-to-shoot biomass ratio was stable in the cattle treatments but declined in the abandonment treatment. The higher allocation to root mass could benefit plant communities under drier conditions.

ConclusionsRotational grazing pressures and climatic condition variabilities had limited effects on root growth seasonality, although drought had stronger effects on the BNPP than on the ANPP. The stability of the root-to-shoot biomass ratio during the dry year evidenced a higher resistance to drought by grazed versus abandoned grassland communities.

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Multicellular Spatial Model of RNA Virus Replication and Interferon Responses Reveals Factors Controlling Plaque Growth Dynamics Respiratory viruses present major health challenges, as evidenced by the 2009 influenza pandemic and the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Severe RNA virus respiratory infections often correlate with high viral load and excessive inflammation. Understanding the dynamics of the innate immune response and its manifestation at the cell and tissue levels are vital to understanding the mechanisms of immunopathology and developing improved, strain independent treatments. Here, we present a novel spatialized multicellular spatial computational model of two principal components of tissue infection and response: RNA virus replication and type-I interferon mediated antiviral response to infection within lung epithelial cells. The model is parameterized using data from influenza virus infected cell cultures and, consistent with experimental observations, exhibits either linear radial growth of viral plaques or arrested plaque growth depending on the local concentration of type I interferons. Modulating the phosphorylation of STAT or altering the ratio of the diffusion constants of interferon and virus in the cell culture could lead to plaque growth arrest. The dependence of arrest on diffusion constants highlights the importance of developing validated spatial models of cytokine signaling and the need for in vitro experiments to measure these diffusion constants. Sensitivity analyses were performed under conditions creating both continuous plaque growth and arrested plaque growth. Findings suggest that plaque growth and cytokine assay measurements should be collected during arrested plaque growth, as the model parameters are significantly more sensitive and more likely to be identifiable. The models metrics replicate experimental immunostaining imaging and titer based sampling assays. The model is easy to extend to include SARS-CoV-2-specific mechanisms as they are discovered or to include as a component linking epithelial cell signaling to systemic immune models.

Author SummaryCOVID-19 is possibly the defining healthcare crisis of the current generation, with tens of millions of global cases and more than a million reported deaths. Respiratory lung infections form lesions in the lungs, whose number and size correlate with severity of illness. In some severe cases, the disease triggers a severe inflammatory condition known as cytokine storm. Given the complexity of the immune system, computational modeling is needed to link molecular signaling at the site of inflection to the signaling impact on the overall immune system, ultimately revealing how severe inflammatory conditions may emerge. Here, we created a computational model of the early stages of infection that simulates lung cells infected with RNA viruses, such those responsible for COVID-19 and influenza, to help explore how the disease forms viral plaques, an in vitro analog to lesion growth in the lung. Our model recapitulates in vitro observations that pretreatment of biological signaling molecules called with type-I interferons, which are currently being evaluated for treatment of COVID-19. Analyzing the model, we, can stop viral plaque growth. We found that enhancing certain aspects of the innate immune system, such as the JAK/STAT pathway, may be able to stop viral plaque growth, suggesting molecules involved in this pathway as possible drug candidates. Quantifying the parameters needed to model interferon signaling and viral replication, experiments should be performed under conditions that inhibit viral growth, such as pretreating cells with interferon. We present a computational framework that is essential to constructing larger models of respiratory infection induced immune responses, can be used to evaluate drugs and other medical interventions quickly, cheaply, and without the need for animal testing during the initial phase, and that defines experiments needed to improve our fundamental understanding of the mechanisms regulating the immune response.

sentences: Genetic basis of body shape variation along the benthic-pelagic axis in cichlid fishes Divergence along the benthic-pelagic axis is one of the most widespread and repeated patterns of morphological variation in fishes, producing body shape diversity associated with ecology and swimming mechanics. This ecological shift is also the first stage of the explosive adaptive radiation of cichlid fishes in the East African Rift Lakes. We use two hybrid crosses of cichlids (Metriaclima sp. x Aulonocara sp. and Labidochromis sp. x Labeotropheus sp., >975 animals total) along the benthic-pelagic ecomorphological axis to determine the genetic basis of body shape diversification. Using a series of both linear and geometric shape measurements, we identify 55 quantitative trait loci (QTL) that underlie various aspects of body shape variation associated with benthic-pelagic divergence. These QTL are spread throughout the genome, each explain 3.0-7.2% of phenotypic variation, and are largely modular. Further, QTL are distinct both between these two crosses of Lake Malawi cichlids and compared to previously identified QTL for body shape in fishes such as sticklebacks. We find that body shape is controlled by many genes of small effects. In all, we find that convergent benthic and pelagic body phenotypes commonly observed across fish clades are most likely due to distinct genetic and molecular mechanisms.

sentences: A new highly-specific Natural Killer cell-specific gene signature predicting recurrence in colorectal cancer patients. The protective role of Natural Killer (NK) cell tumour immunosurveillance has long been recognised in colorectal cancer (CRC). However, as most patients show limited intra-tumoral NK cell infiltration, improving our ability to identify those with high NK cell activity might aid in dissecting the molecular features which could trigger strong response to NK cell-mediated immune killing. Here, a novel CRC-specific NK cell gene signature capable of inferring NK cell load in primary tissue samples was derived and validated in multiple patient CRC cohorts. The specificity of the signature is substantiated in tumour-infiltrating NK cells from primary CRC tumours at the single cell level, and the expression profile of each constituent gene is explored in NK cells of different maturation states, activation status and anatomical origin. Thus, in contrast with other NK cell gene signatures that have several overlapping genes across different immune cell types, our NK cell signature has been extensively refined to be specific for CRC-infiltrating NK cells and includes genes which identify a broad spectrum of NK cell subtypes. Moreover, it was shown that this novel NK cell signature accurately discriminates murine NK cells, demonstrating the potential applicability of this signature when mining datasets generated from both clinical and animal studies. Differential gene expression analysis revealed tumour-intrinsic features associated with NK cell inclusion versus exclusion in CRC patients, with those tumours with predicted high NK activity showing strong evidence of enhanced chemotactic and cytotoxic transcriptional programs. Furthermore, survival modelling indicated that NK signature expression is associated with improved survival outcomes in two large cohorts of primary CRC patients. Thus, scoring CRC samples with this refined NK cell signature might aid in identifying patients with high NK cell activity who could be prime candidates for NK cell directed immunotherapies.

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SpaceX: Gene Co-expression Network Estimation for Spatial Transcriptomics MotivationThe analysis of spatially-resolved transcriptome enables the understanding of the spatial interactions between the cellular environment and transcriptional regulation. In particular, the characterization of the gene-gene co-expression at distinct spatial locations or cell types in the tissue enables delineation of spatial co-regulatory patterns as opposed to standard differential single gene analyses. To enhance the ability and potential of spatial transcriptomics technologies to drive biological discovery, we develop a statistical framework to detect gene co-expression patterns in a spatially structured tissue consisting of different clusters in the form of cell classes or tissue domains.

ResultsWe develop SpaceX (spatially dependent gene co-expression network), a Bayesian methodology to identify both shared and cluster-specific co-expression network across genes. SpaceX uses an over-dispersed spatial Poisson model coupled with a high-dimensional factor model which is based on a dimension reduction technique for computational efficiency. We show via simulations, accuracy gains in co-expression network estimation and structure by accounting for (increasing) spatial correlation and appropriate noise distributions. In-depth analysis of two spatial transcriptomics datasets in mouse hypothalamus and human breast cancer using SpaceX, detected multiple hub genes which are related to cognitive abilities for the hypothalamus data and multiple cancer genes (e.g. collagen family) from the tumor region for the breast cancer data.

Availability and implementationThe SpaceX R-package is available at github.com/bayesrx/SpaceX.

Contactxzhousph@umich.edu, veerab@umich.edu

Supplementary informationSupplementary data are available at bookdown.org/satwik91/SpaceX_supplementary/.

sentences: Placental mammals acquired the functional region and domain in NRK for regulating the CK2-PTEN-AKT pathway and placental cell proliferation The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologues across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: (1) mouse NRK is localised at the plasma membrane via the CNH domain, where the CIR inhibits CK2. (2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN, and (3) leads to the inhibition of AKT signalling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2-PTEN-AKT pathway and placental cell proliferation.

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Chemical ecology of Himalayan eggplant variety's antixenosis: Identification of geraniol as an oviposition deterrent against the eggplant shoot and fruit borer O_LIEggplant (Solanum melongena) suffers severe losses due to a multi-insecticide resistant lepidopteran pest, shoot and fruit borer (SFB, Leucinodes orbonalis). Heavy and combinatorial application of pesticides for SFB control renders eggplant risky for human consumption.
C_LIO_LIWe observed that 1) ovipositing SFB females can find even solitary plants of susceptible varieties and 2) they do not oviposit on Himalayan eggplant variety RC-RL-22 (RL22). We hypothesized that the olfactory cues influence ovipositing females host choice.
C_LIO_LITo find these cues, leaf volatile blends of seven eggplant varieties were profiled using GCMS. Seven compounds were present in >2.5-fold concentrations in RL22 than the other varieties. In choice assays, oviposition deterrence efficacies of these candidate compounds were independently tested by their foliar application on SFB-susceptible varieties. Complementation of geraniol, which was exclusively found in RL22, reduced oviposition (>90%). To validate geraniols role in RL22s SFB-deterrence, we silenced RL22s geraniol synthase gene using virus-induced gene silencing. Geraniol biosynthesis suppression rendered RL22 SFB-susceptible; foliar geraniol application on the geraniol synthase-silenced plants restored oviposition deterrence.
C_LIO_LIWe infer that geraniol is RL22s SFB oviposition deterrent. The use of natural compounds like geraniol, which influence the chemical ecology of oviposition can reduce the load of hazardous larvicidal pesticides.
C_LI

sentences: Mapping Human Pluripotent Stem Cell Derived Erythroid Differentiation by Single-Cell Transcriptome Analysis There is an imbalance between the supply and demand of functional red blood cells (RBCs) in clinical applications. This imbalance can be addressed by regenerating RBCs using several in vitro methods. Induced pluripotent stem cells (iPSCs) can handle the low supply of cord blood and the ethical issues in embryonic stem cell research and provide a promising strategy to eliminate immune rejection. However, no complete single-cell level differentiation pathway exists for the iPSC-derived RBC differentiation system. In this study, we used iPSC line BC1 to establish a RBCs regeneration system. The 10x genomics single-cell transcriptome platform was used to map the cell lineage and differentiation trajectories on day 14 of the regeneration system. We observed that iPSCs differentiation was not synchronized during embryoid body (EB) culture. The cells (day 14) mainly consisted of mesodermal and various blood cells, similar to the yolk sac hematopoiesis. We identified six cell classifications and characterized the regulatory transcription factors (TFs) networks and cell-cell contacts underlying the system. iPSCs undergo two transformations during the differentiation trajectory, accompanied by the dynamic expression of cell adhesion molecules and estrogen-responsive genes. We identified different stages of erythroid cells such as burst-forming unit erythroid (BFU-E) and orthochromatic erythroblasts (ortho-E) and found that the regulation of TFs (e.g., TFDP1 and FOXO3) is erythroid-stage specific. Immune erythroid cells were identified in our system. This study provides systematic theoretical guidance for optimizing the iPSCs-derived RBCs differentiation system, and this system is a useful model for simulating in vivo hematopoietic development and differentiation.

sentences: FAIRly big: A framework for computationally reproducible processing of large-scale data Large-scale datasets present unique opportunities to perform scientific investigations with un-precedented breadth. However, they also pose considerable challenges for the findability, accessibility, interoperability, and reusability (FAIR) of research outcomes due to infrastructure limitations, data usage constraints, or software license restrictions. Here we introduce a DataLad-based, domain-agnostic framework suitable for reproducible data processing in compliance with open science mandates. The framework attempts to minimize platform idiosyncrasies and performance-related complexities. It affords the capture of machine-actionable computational provenance records that can be used to retrace and verify the origins of research outcomes, as well as be re-executed independent of the original computing infrastructure. We demonstrate the frameworks performance using two showcases: one highlighting data sharing and transparency (using the studyforrest.org dataset) and another highlighting scalability (using the largest public brain imaging dataset available: the UK Biobank dataset).

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The heart rate discrimination task: a psychophysical method to estimate the accuracy and precision of interoceptive beliefs Interoception - the physiological sense of our inner bodies - has risen to the forefront of psychological and psychiatric research. Much of this research utilizes tasks that attempt to measure the ability to accurately detect cardiac signals. Unfortunately, these approaches are confounded by well-known issues limiting their validity and interpretation. At the core of this controversy is the role of subjective beliefs about the heart rate in confounding measures of interoceptive accuracy. Here, we recast these beliefs as an important part of the causal machinery of interoception, and offer a novel psychophysical "heart rate discrimination" method to estimate their accuracy and precision. By applying this task in 223 healthy participants, we demonstrate that cardiac interoceptive beliefs are more biased, less precise, and are associated with poorer metacognitive insight relative to an exteroceptive control condition. Our task, provided as an open-source python package, offers a robust approach to quantifying cardiac beliefs.

HighlightsO_LICurrent interoception tasks conflate cardiac beliefs with accuracy.
C_LIO_LIWe introduce a Bayesian method for estimating cardiac belief accuracy and precision.
C_LIO_LIIndividuals underestimate their heart rate by -7 BPM (95% CI [-8.6 -5.3]) on average.
C_LIO_LICardiac beliefs are associated with reduced precision and metacognitive insight.
C_LIO_LIThe task and modelling tools are provided in the Python Cardioception Package.
C_LI

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Neutrophil Peptidylarginine Deiminase 4 is Essential for Detrimental Age-related Cardiac Remodeling and Dysfunction in Mice AimsWe aimed to study the long-term effect of neutrophils on cardiac health during the process of natural aging. We hypothesized that neutrophil PAD4, via its role in neutrophil extracellular trap (NET) formation, is involved in myocardial remodeling and cardiac fibrosis development, resulting in turn in impaired cardiac function.

Methods and resultsWe generated mice with deletion of Padi4, a NET-essential gene, under the neutrophil-specific promoter S100A8 (PAD4fl/flMRP8Cre+). These mice and their littermate controls were aged for two years (coinciding with approximately 70 years of age in humans; the age at which HF is the number one cause of hospitalization), after which cardiac function and remodeling were evaluated. We performed a comprehensive echocardiography analysis including both structural and functional parameter measurements. Deletion of PAD4 in neutrophils resulted in a protection against both systolic, and diastolic dysfunction. Interestingly, these mice showed protection against age induced fibrosis, detected as through the absence of cardiac collagen deposition. To explore this further, cardiac gene expression and plasma cytokine levels were evaluated. Here we saw a clear impact of PAD4-deficiency on cardiac neutrophil recruitment, with both cardiac genes as well as plasma cytokines involved in neutrophil recruitment being downregulated in aged PAD4fl/flMRP8Cre+ animals in comparison to littermate PAD4fl/fl controls, including decreased plasma levels of C-X-C ligand 1 (CXCL1).

ConclusionOur data confirms neutrophil PAD4 involvement in heart failure progression by promoting cardiac remodeling, leading to cardiac dysfunction with old age. We saw that the deletion of PAD4 specifically in neutrophils had an influence on the CXCL1-CXCR2 axis, which is known to be involved in HF development.

Translational perspectiveIn the developed world, an estimated 2% of the population lives with heart failure (HF). HF can be viewed as an upcoming pandemic, which is only expected to increase due to the aging of the global population. Therefore, research in HF development and progression are needed to establish new avenues for treatment and improved therapies. In our study, we were able to show the contribution of neutrophil PAD4 to HF pathogenesis, providing new supporting evidence for the involvement of NETs in detrimental cardiac remodeling.

sentences: Impact of a native hemiparasitic plant on invasive and native hosts in the field. There is increasing evidence that native parasitic plants are showing potential as biocontrols for plant invasions which are a major global threat to biodiversity. However, for this potential to be realised, the range of invasive hosts that can be controlled needs to be identified while also evaluating any potential off-target effects the parasite may pose. To address this, we investigated the impact of the Australian native shoot hemiparasitic plant, Cassytha pubescens on the major invasive species Rubus anglocandicans and two native shrubs, Acacia pycnantha and Bursaria spinosa in naturally occurring populations in southern Australia. We measured foliar predawn and midday quantum yield, electron transport rate (ETR), stomatal conductance (gs), carbon isotope composition and foliar nitrogen concentration [N] of uninfected and infected plants and, apart from gs, also for C. pubescens. Infection significantly decreased predawn and midday quantum yield, ETR and gs of invasive R. anglocandicans. In contrast, infection had no effect on predawn and midday quantum yield, ETR or carbon isotope composition of the native hosts. However, C. pubescens had a significant negative effect on native host gs, and a positive effect on host [N]. Parasite stem [N] was significantly higher when infecting A. pycnantha than B. spinosa. These results strengthen evidence for native parasitic plants having greater impact on invasive hosts while having mild off-target effects on native hosts and thus, show potential to mitigate plant invasions and help protect biodiversity.

sentences: Analysis of non-human primate models for evaluating prion disease therapeutic efficacy Prion disease is a fatal neurodegenerative disease caused by the conformational corruption of the prion protein (PrP), encoded by the prion protein gene (PRNP). While no disease-modifying therapy is currently available, genetic and pharmacological proofs of concept support development of therapies that lower PrP levels in the brain. In light of proposals for clinical testing of such drugs in presymptomatic individuals at risk for genetic prion disease, extensive nonclinical data are likely to be required, with extra attention paid to choice of animal models. Uniquely, the entire prion disease process can be faithfully modeled through transmission of human prions to non-human primates (NHPs), raising the question of whether NHP models should be used to assess therapeutic efficacy. Here we systematically aggregate data from N=527 prion-inoculated animals spanning six decades of research studies. Using this dataset, we assess prion strain, route of administration, endpoint, and passage number to characterize the relationship of tested models to currently prevalent human subtypes of prion disease. We analyze the incubation times observed across diverse models and perform power calculations to assess the practicability of testing prion disease therapeutic efficacy in NHPs. We find that while some models may theoretically be able to support therapeutic efficacy studies, pilot studies would be required to confirm incubation time and attack rate before pivotal studies could be designed, cumulatively requiring several years. The models with the shortest and most tightly distributed incubation times are those with smaller brains and weaker homology to humans. Our findings indicate that it would be challenging to conduct efficacy studies in NHPs in a paradigm that honors the potential advantages of NHPs over other available models, on a timeframe that would not risk unduly delaying patient access to promising drug candidates.

sentences: Water levels primarily drive variation in photosynthesis and nutrient use of scrub Red Mangroves in the southeastern Florida Everglades We investigated how mangrove-island micro-elevation (i.e., habitat: center vs. edge) affects tree physiology in a scrub mangrove forest of the southeastern Everglades. We measured leaf gas exchange rates of scrub Rhizophora mangle L. trees monthly during 2019, hypothesizing that CO2 assimilation (Anet) and stomatal conductance (gsw) would decline with increasing water levels and salinity, expecting more-considerable differences at mangrove-island edges than centers, where physiological stress is greatest. Water levels varied between 0 and 60 cm from the soil surface, rising during the wet season (May-October) relative to the dry season (November-April). Porewater salinity ranged from 15 to 30 ppt, being higher at mangrove-island edges than centers. Anet maximized at 15.1 {micro}mol m-2 s-1, and gsw was typically <0.2 mol m-2 s-1, both of which were greater in the dry than the wet season and greater at island centers than edges, with seasonal variability being roughly equal to variation between habitats. After accounting for season and habitat, water level positively affected Anet in both seasons but did not affect gsw. Our findings suggest that inundation stress (i.e., water level) is the primary driver of variation in leaf gas exchange rates of scrub mangroves in the Florida Everglades, while also constraining Anet more than gsw. The interaction between inundation stress due to permanent flooding and habitat varies with season as physiological stress is alleviated at higher-elevation mangrove-island center habitats during the dry season. Freshwater inflows during the wet season, increase water levels and inundation stress at higher-elevation mangrove-island centers, but also potentially alleviate salt and sulfide stress in soils. Thus, habitat heterogeneity leads to differences in nutrient and water acquisition and use between trees growing in island centers versus edges, creating distinct physiological controls on photosynthesis, which likely affect carbon flux dynamics of scrub mangroves in the Everglades.

sentences: Nodal GABA facilitates axon spike transmission in the spinal cord. Movement and posture depend on sensory feedback that is regulated by specialized GABAergic neurons (GAD2+) that form axo-axonic contacts onto myelinated proprioceptive sensory axons and are thought to be inhibitory. However, we report here that activating GAD2+ neurons, directly with optogenetics or indirectly by cutaneous stimulation, facilitates sensory feedback to motoneurons in awake rodents and humans. GABAA receptors and GAD2+ innervation at or near nodes of Ranvier of sensory axons cause this facilitation, preventing spike propagation failure at the many axon branch points, which is otherwise common without GABA. In contrast, GABAA receptors are generally lacking from axon terminals (unlike GABAB) and do not presynaptically inhibit transmitter release onto motoneurons. GABAergic innervation near nodes and branch points allows individual branches to function autonomously, with GAD2+ neurons regulating which branches conduct, adding a computational layer to the neuronal networks generating movement and likely generalizing to other CNS axons.

sentences: Refractive-index matching enhanced polarization-sensitive optical coherence tomography quantification in human brain tissue The importance of polarization-sensitive optical coherence tomography (PS-OCT) has been increasingly recognized in human brain imaging. Despite the recent progress of PS-OCT in revealing white matter architecture and orientation, quantification of fine-scale fiber tracts in the human brain cortex has been a challenging problem, due to a low birefringence in the gray matter. In this study, we investigated the effect of refractive index matching by 2,2-thiodiethanol (TDE) immersion on the improvement of PS-OCT measurements in ex vivo human brain tissue. We obtain the cortical fiber orientation maps in the gray matter, which reveals the radial fibers in the gyrus, the U-fibers along the sulcus, as well as distinct layers of fiber axes exhibiting laminar organization. Further analysis shows that index matching reduces the noise in axis orientation measurements by 56% and 39%, in white and gray matter, respectively. Index matching also enables precise measurements of apparent birefringence, which was underestimated in the white matter by 82% but overestimated in the gray matter by 16% prior to TDE immersion. Mathematical simulations show that the improvements are primarily attributed to the reduction in the tissue scattering coefficient, leading to an enhanced signal-to-noise ratio in deeper tissue regions, which could not be achieved by conventional noise reduction methods.

sentences: TACSTD2 upregulation is an early reaction to lung infection TACSTD2 encodes a transmembrane glycoprotein Trop2 commonly overexpressed in carcinomas. While the Trop2 protein was discovered already in 1981 and first antibody-drug conjugate targeting Trop2 were recently approved cancer therapy, the physiological role of Trop2 is still not fully understood. In this article, we show that TACSTD2/Trop2 expression is evolutionarily conserved in lungs of various vertebrates. By analysis of publicly available transcriptomic data we demonstrate that TACSTD2 level consistently increases in lungs infected with miscellaneous pathogens. Single cell and subpopulation based transcriptomic data revealed that the major source of TACSTD2 transcript are lung epithelial cells and their progenitors and that TACSTD2 is induced directly in lung epithelial cells following infection. This increase may represent a mechanism to maintain/restore epithelial barrier function and contribute to regeneration process in infected/damaged lungs.

sentences: Supervised Phenotype Discovery from Multimodal Brain Imaging Data-driven discovery of image-derived phenotypes (IDPs) from large-scale multimodal brain imaging data has enormous potential for neuroscientific and clinical research by linking IDPs to subjects demographic, behavioural, clinical and cognitive measures (i.e., non-imaging derived phenotypes or nIDPs). However, current approaches are primarily based on unsupervised approaches, without use of information in nIDPs. In this paper, we proposed Supervised BigFLICA (SuperBigFLICA), a semi-supervised, multimodal, and multi-task fusion approach for IDP discovery, which simultaneously integrates information from multiple imaging modalities as well as multiple nIDPs. SuperBigFLICA is computationally efficient and largely bypasses the need for parameter tuning. Using the UK Biobank brain imaging dataset with around 40,000 subjects and 47 modalities, along with more than 17,000 nIDPs, we showed that SuperBigFLICA enhances the prediction power of nIDPs, bench-marked against IDPs derived by conventional expert-knowledge and unsupervised-learning approaches (with average nIDP prediction accuracy improvements of up to 46%). It also enables learning of generic imaging features that can predict new nIDPs. Further empirical analysis of the SuperBigFLICA algorithm demonstrates its robustness in different prediction tasks and the ability to derive biologically meaningful IDPs in predicting health outcomes and cognitive nIDPs, such as fluid intelligence and hypertension scores.

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The impact of genetic background and sex on the phenotype of IL-23 induced murine arthritis BackgroundOverexpression of IL-23 in adult mice by means of hydrodynamic tail vein injection of IL-23 minicircles has been reported to result in spondyloarthritis-like disease. The impact of genetic background and sex on the disease phenotype in this model has not been investigated.

MethodsWe compared male B10.RIII mice with male C57BL/6 mice, and male with female B10.RIII mice after hydrodynamic injection of IL-23 enhanced episomal vector (EEV) at 8-12 weeks of age. We monitored clinical arthritis scores, paw swelling, and body weight. Animals were euthanized after two weeks and tissues were harvested for histology, flow cytometry and gene expression analysis. Serum cytokine levels were determined by ELISA.

FindingsMale B10.RIII mice developed arthritis in the forepaws and feet within 6 days after IL-23 EEV injection; they also exhibited psoriasis-like skin disease, colitis, weight loss, and osteopenia. In contrast to previous reports, we did not observe spondylitis or uveitis. Male C57BL/6 mice injected with IL-23 EEV had serum IL-23 levels comparable with B10.RIII mice and developed skin inflammation, colitis, weight loss, and osteopenia but failed to develop arthritis. Female B10.RIII mice had more severe arthritis than male B10.RIII mice but did not lose weight.

ConclusionsSystemic IL-23 overexpression results in spondyloarthritis-like disease in B10.RIII mice. The development of extra-articular manifestations but absence of arthritis in C57BL/6 mice suggests organ-specific genetic control mechanisms of IL-23 driven inflammation. Discrepancies regarding the phenotype of IL-23 induced disease in different labs and the sexual dimorphism observed in this study warrant further exploration.

sentences: Human visual gamma for color stimuli: When LGN drive is equalized, red is not special Strong gamma-band oscillations in primate early visual cortex can be induced by spatially homogeneous, high-contrast stimuli, such as color surfaces. Compared to other hues, particularly strong gamma oscillations have been reported for red stimuli. However, precortical color processing and the resultant strength of input to V1 has often not been fully controlled for. This leaves the possibility that stronger responses to some hues were due to differences in V1 input strength. We presented stimuli that had equal luminance and color contrast levels in a color coordinate system based on color responses of the lateral geniculate nucleus, the main input source for area V1. With these stimuli, we recorded magnetoencephalography in 30 human subjects. We found narrowband color-induced gamma oscillations in early visual cortex, which, contrary to previous reports, did not differ between red and green stimuli of equal L-M cone contrast. Notably, blue stimuli with contrast exclusively on the S-cone axis induced very weak gamma responses, as well as smaller event-related fields and poorer change detection performance. The strength of human color gamma responses could be well explained by the strength of thalamic input induced by each hue and does not show a clear red bias when this input strength is properly equalized.

sentences: Mapping the human praxis network: an investigation of white matter disconnection in apraxia Left hemispheric cerebral stroke can cause apraxia, a motor-cognitive disorder characterised by deficits of higher-order motor skills such as the failure to accurately produce meaningful gestures. This disorder provides unique insights into the anatomical and cognitive architecture of the human praxis system. The present study aimed to map the structural brain network that is damaged in apraxia. We assessed the ability to perform meaningful gestures with the hand in 101 patients with chronic left hemisphere stroke. Structural white matter fibre damage was directly assessed by diffusion tensor imaging and fractional anisotropy mapping. We used multivariate topographical inference on tract-based fractional anisotropy topographies to identify white matter disconnection associated with apraxia. We found relevant pathological white matter alterations in a densely connected fronto-temporo-parietal network of short and long association fibres. Hence, the findings suggest that heterogeneous topographical results in previous lesion mapping studies might not only result from differences in study design, but also from the general methodological limitations of univariate topographical mapping in uncovering the structural praxis network. A striking role of middle and superior temporal lobe disconnection, including temporo-temporal short association fibres, was found, suggesting strong involvement of the temporal lobe in the praxis network. Further, the results stressed the importance of subcortical disconnections for the emergence of apractic symptoms. Our study provides a fine-grain view into the structural connectivity of the human praxis network and suggests a potential value of disconnection measures in the clinical prediction of behavioural post-stroke outcome.

sentences: The arthropod associates of 155 North American cynipid oak galls The identities of most arthropod associates of cynipid-induced oak galls in the western Palearctic are generally known. However, a comprehensive accounting of associates has been performed for only a small number of the galls induced by the estimated 700 species of cynipid gall wasp in the Nearctic. This gap in knowledge stymies many potential studies of diversity, coevolution, and community ecology, for which oak gall systems are otherwise ideal models. We report rearing records of insects and other arthropods from more than 527,306 individual galls representing 201 different oak gall types collected from 32 oak tree species in North America. Of the 201 gall types collected, 155 produced one or more animals. A total of 151,075 animals were found in association with these 155 gall types, and of these 61,044 (40.4%) were gall wasps while 90,031 (59.6%) were other arthropods. We identified all animals to superfamily, family, or, where possible, to genus. We provide raw numbers and summaries of collections, alongside notes on natural history, ecology, and previously published associations for each taxon. For eight common gall-associated genera (Synergus, Ceroptres, Euceroptres, Ormyrus, Torymus, Eurytoma, Sycophila, and Euderus), we also connect rearing records to gall wasp phylogeny, geography, and ecology - including host tree and gall location (host organ), and their co-occurrence with other insect genera. Though the diversity of gall wasps and the large size of these communities is such that many Nearctic oak gall-associated insects still remain undescribed, this large collection and identification effort should facilitate the testing of new and varied ecological and evolutionary hypotheses in Nearctic oak galls.

sentences: Multi-morph eco-evolutionary dynamics in structured populations Our understanding of the evolution of quantitative traits in nature is still limited by the challenge of including realistic trait distributions in the context of frequency-dependent selection and ecological feedbacks. We extend to class-structured populations a recently introduced "oligomorphic approximation" which bridges the gap between adaptive dynamics and quantitative genetics approaches and allows for the joint description of the dynamics of ecological variables and of the moments of multimodal trait distributions. Our theoretical framework allows us to analyse the dynamics of populations composed of several morphs and structured into distinct classes (e.g. age, size, habitats, infection status, species...). We also introduce a new approximation to simplify the eco-evolutionary dynamics using reproductive values. We illustrate the effectiveness of this approach by applying it to the important conceptual case of two-habitat migration-selection models. In particular, we show that our approach allows us to predict both the long-term evolutionary endpoints and the short-term transient dynamics of the eco-evolutionary process, including fast evolution regimes. We discuss the theoretical and practical implications of our results and sketch perspectives for future work.

sentences: Insights into the structure and function of the human organic anion transporter 1 in lipid bilayer membranes The human SLC22A6/OAT1 plays an important role in the elimination of a broad range of endogenous substances and xenobiotics thus attracting attention from the pharmacological community. Furthermore, OAT1 is also involved in key physiological events such as the remote inter-organ communication. Despite its significance, the knowledge about hOAT1 structure and the transport mechanism at the atomic level remains fragmented owing to the lack of resolved structures. By means of protein-threading modeling refined by s-scaled Molecular Dynamics simulations, the present study provides the first robust model of hOAT1 in outward-facing conformation. Taking advantage of the AlphaFold 2 predicted structure of hOAT1 in inward-facing conformation, we here provide the essential structural and functional features comparing both states. The intracellular motifs conserved among Major Facilitator Superfamily members create a so-called "charge-relay system" that works as molecular switches modulating the conformation. The principal element of the event points at interactions of charged residues that appear crucial for the transporter dynamics and function. Moreover, hOAT1 model was embedded in different lipid bilayer membranes highlighting the crucial structural dependence on lipid-protein interactions. MD simulations supported the pivotal role of phosphatidylethanolamine components to the protein conformation stability. The present model is made available to decipher the impact of any observed polymorphism and mutation on drug transport as well as to understand substrate binding modes.

sentences: CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 [A] resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.

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Competition is a determinant of multidrug resistant plasmid acquisition in Salmonella Fostering a balanced gut microbiome through the administration of beneficial microbes that can competitively exclude pathogens has gained a lot of attention and use in human and animal medicine. However, little is known about how microbes affect the horizontal gene transfer of antimicrobial resistance (AMR). To shed more light on this question, we challenged neonatal broiler chicks raised on reused broiler chicken litter - a complex environment made up of decomposing pine shavings, feces, uric acid, feathers, and feed, with Salmonella Heidelberg (S. Heidelberg), a model pathogen. Neonatal chicks challenged with S. Heidelberg and raised on reused litter were more resistant to S. Heidelberg cecal colonization than chicks grown on fresh litter. Furthermore, chicks grown on reused litter were at a lower risk of colonization with S. Heidelberg strains that encoded AMR on IncI1 plasmids. We used 16S rRNA gene sequencing and shotgun metagenomics to show that the major difference between chicks grown on fresh litter and reused litter was the microbiome harbored in the litter and ceca. The microbiome of reused litter samples was more uniform and enriched in functional pathways related to the biosynthesis of organic and antimicrobial molecules than fresh litter samples. We found that E. coli was the main reservoir of plasmids encoding AMR and that the IncI1 plasmid was maintained at a significantly lower copy per cell in reused litter compared to fresh litter. These findings support the notion that commensal bacteria play an integral role in the horizontal transfer of plasmids encoding AMR to pathogens like Salmonella.

Importance/SignificanceAntimicrobial resistance spread is a worldwide health challenge, stemming in large part, from the ability of microorganisms to share their genetic material through horizontal gene transfer. To address this issue, many countries and international organization have adopted a One health approach to curtail the proliferation of antimicrobial resistant bacteria. This includes the removal and reduction of antibiotics used in food animal production and the development of alternatives to antibiotics. However, there is still a significant knowledge gap in our understanding of how resistance spreads in the absence of antibiotic selection and the role commensal bacteria play in reducing antibiotic resistance transfer. In this study, we show that commensal bacteria play a key role in reducing the horizontal gene transfer of antibiotic resistance to Salmonella and provide the identity of the bacterial species that potentially perform this function in broiler chickens and also postulate the mechanism involved.

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A spiking neural network model of the Superior Colliculus that is robust to changes in the spatial-temporal input Previous studies have indicated that the location of a large neural population in the Superior Colliculus (SC) motor map specifies the amplitude and direction of the saccadic eye-movement vector, while the saccade trajectory and velocity profile are encoded by the population firing rates. We recently proposed a simple spiking neural network model of the SC motor map, based on linear summation of individual spike effects of each recruited neuron, which accounts for many of the observed properties of SC cells in relation to the ensuing eye movement. However, in the model, the cortical input was kept invariant across different saccades. Electrical microstimulation and reversible lesion studies have demonstrated that the saccade properties are quite robust against large changes in supra-threshold SC activation, but that saccade amplitude and peak eye-velocity systematically decrease at low input strengths. These features are not accounted for by the linear spike-vector summation model. Here we show that the models input projection strengths and intra-collicular lateral connections can be tuned to generate saccades that follow the experimental results.

Author statementThe midbrain SC generates fast saccadic eye movements through a large population of cells within a topographically organized motor map, in which the location, spike count and temporal firing patterns of recruited cells determine saccade metrics and kinematics. According to the dynamic ensemble-coding model, each recruited SC cell contributes to the saccade by linear vector summation of all its spike contributions. Our previous spiking neural network model used invariant cortical inputs to the SC cells for all saccades. We here improved the robustness of the model to large spatial-temporal variations in the input patterns, by tuning its top-down and lateral synaptic connections, to generate saccades with properties observed in electrophysiological experiments.

sentences: Permissive epigenetic landscape facilitates distinct transcriptional signatures of activating transcription factor 6 in the liver Proteostatic stress initiates a transcriptional response that is unique to the stress condition, yet the regulatory mechanisms underlying the distinct gene expression patterns observed in stressed cells remains unknown. Using a functional genomic approach, we investigated how activating transcription factor 6 (ATF6), a key transcription factor in the unfolded protein response (UPR), regulates target genes. We first designed a computational strategy to define Atf6 target genes based on the evolutionary conservation of predicted ATF6 binding in gene promoters, identifying 652 conserved putative Atf6 target (CPAT) genes. CPATs were overrepresented for genes functioning in the UPR, however, the majority functioned in cellular processes unrelated to proteostasis, including small molecule metabolism and development. Functional studies of stress-independent and toxicant based Atf6 activation in zebrafish livers showed that the pattern of CPAT expression in response to Atf6 overexpression, alcohol and arsenic was unique. Only 34 CPATs were differentially expressed in all conditions, indicating that Atf6 is sufficient to regulate a small subset of CPATs. Blocking Atf6 using Ceapins in zebrafish demonstrated that Atf6 is necessary for activation of these genes in response to arsenic. We investigated CPAT during physiologically mediated hepatocyte stress using liver regeneration in mice as a model. Over half of all CPATs were differentially expressed during this process. This was attributed to the permissive chromatin environment in quiescent livers on the promoters of these genes, characterized by the absence of H3K27me3 and enrichment of H3K4me3. Taken together, these data uncover a complex transcriptional response to Atf6 activation and implicate a permissive epigenome as a mechanism by which distinct transcriptional responses are regulated by Atf6.

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Alone in a crowd: Effect of a nonfunctional lateral line on expression of the social hormone parathyroid hormone 2. Parathyroid hormone 2 (Pth2) is a vertebrate-specific neuropeptide whose thalamic expression is upregulated by social contact with conspecifics. However, social interactions fail to stimulate pth2 expression in isolated zebrafish whose lateral line hair cells have been chemically ablated. These results suggest that modulation of pth2 by social context is acutely dependent on mechanosensory information from the lateral line. However, it is unclear how a congenital loss of lateral line function influences the ability of zebrafish to interpret their social environment. In this study, we measure pth2 levels in zebrafish mutants lacking hair cell function in either the lateral line only, or in both the inner ear and lateral line. Socially-raised lateral line mutants express lower levels of pth2 relative to wild type siblings, but there is no further reduction when all sensory hair cells are nonfunctional. However, social isolation of hair cell mutants causes a further reduction in pth2 expression, pointing to additional unidentified sensory cues that influence pth2 production. Lastly, we report that social context modulates fluorescent transgenes driven by the pth2 promoter. Altogether, these data suggest that lateral line mutants experience a chronic sense of isolation, even when raised in a social environment.

SUMMARY STATEMENTExpression of the pro-social neuropeptide pth2 is downregulated in larval zebrafish with a congenital loss of lateral line function. Thus, even in social environments, fish with compromised lateral lines may experience chronic loneliness.

sentences: Loss of Usp22 enhances histone H2B monoubiquitination and stimulates intracellular and systemic interferon immunity Interferons protect from virus infections by inducing hundreds of interferon-stimulated genes (ISG) which orchestrate anti-viral adaptive and innate immunity. Upon viral infection or type I interferon (IFN) stimulation of cell lines, a histone modification, monoubiquitinated histone 2B (H2Bub1), increases at ISG loci, raising the possibility that a specific chromatin state can broadly stimulate IFN immunity in vivo. Here we show that, in the absence of virus infection or elevated interferon levels, mice lacking the relevant deubiquitinase, Usp22, in immune cells have elevated H2Bub1 levels. Hypermonoubiquitinated H2B is physically associated with dozens of ISG loci, and expression of large numbers of ISG is upregulated. This epigenetic state promotes intracellular and systemic immune phenotypes akin to adaptive and innate interferon immunity, and thereby identifies Usp22 as a negative regulator of interferon immunity.

sentences: A-type lamins are critical for the recruitment of RPA and RAD51 to stalled replication forks to maintain fork stability Lamins provide a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. The mechanisms whereby lamins regulate genome stability remain poorly understood. Here, we demonstrate a crucial role for A-type lamins preserving the integrity of the replication fork (RF) during replication stress (RS). We find that lamins bind to nascent DNA strands, especially during RS, and ensure the recruitment of fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, play crucial roles in the stabilization, remodeling and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamins depletion elicits replication fork instability (RFI) depicted by MRE11 nuclease-mediated degradation of nascent DNA, RS-induced accumulation of DNA damage, and increased sensitivity to replication inhibitors. Importantly, in contrast to cells deficient in various homology recombination repair proteins, the RFI phenotype of lamins-depleted cells is not linked to RF reversal. This suggests that the point of entry of nucleases is not the reversed fork, but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamins-depleted cells is rescued by forced elevation of the heterotrimeric RPA complex or RAD51. These data unveil a clear involvement of structural nuclear proteins in the protection of ssDNA from the action of nucleases during RS by warranting proper recruitment of ssDNA binding proteins RPA and RAD51 to stalled RFs. In support of this model, we show physical interaction between RPA and lamins. Our study also suggests that RS is a major source of genomic instability in laminopathies and in lamins-depleted tumors.

sentences: A wipe-based stool collection and preservation kit for microbiome community profiling While a range of methods for stool collection exist, many require complicated, self-directed protocols and stool transfer. In this study, we introduce and validate a novel, wipe-based approach to fecal sample collection and stabilization for metagenomics analysis. A total of 72 samples were collected across four different preservation types: freezing at -20{degrees}C, room temperature storage, a commercial DNA preservation kit, and DESS (dimethyl sulfoxide, ethylenediaminetetraacetic acid, sodium chloride) solution. These samples were sequenced and analyzed for taxonomic abundance metrics, metabolic pathway classification, and diversity analysis. Overall, the DESS wipe results validated the use of a wipe-based capture method to collect stool samples for microbiome analysis, showing an R2 of 0.96 for species across all kingdoms, as well as exhibiting a maintenance of Shannon diversity (3.1-3.3) and species richness (151-159) compared to frozen samples. Moreover, DESS showed comparable performance to the commercially available preservation kit (R2 of 0.98), and samples consistently clustered by subject across each method. Future studies will be needed to further explore sample processing options and their applications in non-healthy subjects, particularly patients with irritable bowel syndrome, inflammatory bowel disease, and colorectal cancer, but these data suggest the DESS wipe method can be used for stable, room temperature collection and transport of human stool specimens.

sentences: The DNA sensors AIM2 and IFI16 are NET-binding SLE autoantigens Nucleic acid binding proteins are frequently targeted as autoantigens in systemic lupus erythematosus (SLE) and other interferon (IFN)-linked rheumatic diseases. The AIM-like receptors (ALRs) are IFN-inducible innate sensors that form supramolecular assemblies along double-stranded DNA of various origins. Here, we identify the ALR Absent in melanoma 2 (AIM2) as a novel autoantigen in SLE, with similar properties to the established ALR autoantigen interferon-inducible protein 16 (IFI16). Our SLE cohort revealed a frequent co-occurrence of anti-AIM2, anti-IFI16 and anti-DNA antibodies, and higher clinical measures of disease activity in patients positive for antibodies against these ALRs. We examined neutrophil extracellular traps (NETs) as DNA scaffolds on which these antigens might interact in a pro-immune context, finding that both ALRs bind NETs in vitro and in SLE renal tissues. We demonstrate that ALR binding causes NETs to resist degradation by DNase I, suggesting a mechanism whereby extracellular ALR-NET interactions may promote sustained IFN signaling. Our work suggests that extracellular ALRs bind NETs, leading to DNase resistant nucleoprotein fibers that are targeted as autoantigens in SLE.

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Early events in reovirus infection influence induction of innate immune response Mammalian orthoreovirus (reovirus) is a double-stranded (ds)RNA virus which encapsidates its 10 genome segments within a double-layered viral particle. Reovirus infection triggers an antiviral response in host cells which serves to limit viral replication. This antiviral response is initiated by recognition of the incoming viral genome by host sensors present in the cytoplasm. However, how host sensors gain access to the reovirus genome is unclear as this dsRNA is protected by the viral particle proteins throughout infection. To initiate infection, reovirus particles are endocytosed and the outer viral particle layer is disassembled through the action of host proteases. This disassembly event is required for viral escape into the cytoplasm to begin replication. We show that even after disassembly is complete, endosomal proteases are required to induce an immune response to reovirus. Additionally, counter to dogma, our data demonstrate at least some viral dsRNA genome is exposed and detectable during entry. We hypothesize that some proportion of reovirus particles remain trapped within endosomes, allowing for the breakdown of these particles and release of their genome. We show that rapidly uncoating mutants escape the endosome more rapidly and induce a diminished immune response. Further, we show that particles entering through dynamin-independent pathways evade detection by host sensors. Overall, our data provide new insight into how genomes from entering reovirus particles are detected by host cells.

IMPORTANCEViruses must infect host cells to replicate, often killing the host cell in the process. However, hosts can activate defenses to limit viral replication and protect the organism. To trigger these host defenses to viral infections, host cells must first recognize they are infected. Mammalian orthoreovirus (reovirus) is a model system used to study host-virus interactions. This study identifies aspects of host and virus biology which determine the capacity of host cells to detect infection. Notably, entry of reovirus into host cells plays a critical role in determining the magnitude of immune response triggered during infection. Mutants of reovirus which can enter cells more rapidly are better at avoiding detection by the host. Additionally, reovirus can enter cells through multiple routes. Entry through some of these routes also helps reovirus evade detection.

sentences: Scholar Metrics Scraper (SMS): automated retrieval of citation and author data Academic departments, research clusters and evaluators analyze author and citation data to measure research impact and to support strategic planning. We created a tool, Scholar Metrics Scraper (SMS), to automate the retrieval of this bibliometric data for our research team. The project contains Jupyter notebooks (publicly-shared here) that take a list of researchers as an input to export a CSV file of citation metrics from Google Scholar and figures to visualize the groups impact. SMS is a scalable, open and publicly-accessible solution for automating the retrieval of citation data over time for a group of researchers.

sentences: Mesoscale organization in the cell envelope of Deinococcus radiodurans S-layers are highly ordered coats of proteins localized on the cell surface of many bacterial species. In these structures, one or more proteins form elementary units that self-assemble into a crystalline monolayer tiling the entire cell surface. Here, the cell envelope of the radiation-resistant bacterium Deinococcus radiodurans was studied by high-resolution cryo-electron microscopy finding the crystalline regularity of the S-layer extended into the layers below. The cell envelope appears to be highly packed and resulting from a three-dimensional crystalline distribution of protein complexes organized in close continuity but allowing different degrees of voidness in the entire thickness. These insights grade S-layers to mesoscale hubs behaving as structural and functional architraves essential for the entire cell body.

sentences: First direct observation of 'elongated' conformational states in α-synuclein upon liquid-liquid phase separation -Synuclein (-syn) is an intrinsically disordered protein (IDP) that undergoes liquid-liquid phase separation (LLPS), fibrillation, and forms insoluble intracellular Lewys bodies in neurons, which are the hallmark of Parkinsons Disease (PD). Neurotoxicity precedes the formation of aggregates and is probably related to LLPS of -syn in the cell. The molecular mechanisms underlying the early stages of LLPS are still elusive. To obtain structural insights into -syn upon LLPS, we take advantage of cross-linking/mass spectrometry (XL-MS) and introduce an innovative approach, termed COMPASS (COMPetitive PAiring StatisticS). COMPASS unravels transient interactions between -syn molecules in liquid droplets. In this work, we show that the conformational ensemble of -syn shifts from a  hairpin-like structure towards more  elongated conformational states upon LLPS. We obtain insights into the critical initial stages of PD and establish a novel mass spectrometry-based approach that will aid to solve open questions in LLPS structural biology.

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An inexpensive setup for robust activity tracking in small animals: Portable Locomotion Activity Monitor (pLAM) O_LIAdvances in computer vision and deep learning have automated animal behaviour studies that previously required tedious manual input. However, tracking activity of small and fast flying animals remains a hurdle, especially in a field setting with variable light conditions. Commercial locomotor activity monitors (LAMs) can be expensive, closed source, and generally limited to laboratory settings.
C_LIO_LIHere, we present a portable locomotion activity monitor (pLAM), a mobile activity detector to quantify small animal circadian activity. Our setup uses inexpensive components, is based on open-source motion tracking software, and is easy to assemble and use in the field. It runs off-grid, supports low-light tracking with infrared lights, and can implement arbitrary light cycle colours and brightnesses with programmable LEDs. We provide a user-friendly guide to assembling pLAM hardware and accessing its pre-configured software and guidelines for using it in other systems.
C_LIO_LIWe benchmarked pLAM for insects under various lab and field conditions, then compared results to a commercial activity detector. They offer broadly similar activity measures, but our setup captures flight and bouts of motion that are often missed by beam-breaking activity detection.
C_LIO_LIpLAM will enable high-throughput quantification of small animal location and activity in a low-cost and accessible manner, crucial to studying behaviour that can help inform conservation and management decisions.
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sentences: Cerebral activity in monkeys Papio anubis during the perception of conspecific and heterospecific agonistic vocalizations: A functional Near Infrared Spectroscopy study Are conspecific emotional vocalizations special? Although often investigated in non-human primates using functional magnetic resonance imaging or positron emission tomography, it remains unclear whether the listening of conspecific vocal emotions leads to similar or different cerebral activations when compared to heterospecific calls (i.e. expressed by another primate species). Using a neuroimaging technique rarely employed in monkeys so far, functional Near Infrared Spectroscopy (fNIRS), the present study investigated cortical temporal activities during exposure to both conspecific and heterospecific calls in three female adult baboons (Papio anubis). The three subjects were lightly anesthetized and passively exposed to agonistic baboon and chimpanzee (Pan troglodytes) vocalizations, as well as energy matched white noises in order to control for this low-level acoustic feature. Despite inter-individual variabilities, permutation test analyses on the extracted OxyHemoglobin signal revealed for two subjects out of three significant differences between the passive listening of baboon versus chimpanzee stimuli. Additionally, in one subject, a modulation of the left temporal cortex activity was found for the perception of baboon calls contrasted to chimpanzee vocalizations as well as for the passive listening of baboon white noises compared to chimpanzee ones. Although the lack of generalization of those findings in all three subjects prevents us to drawn any conclusion and that more subjects would be needed, the hypothesis that baboons cortical temporal regions may be more sensitive to the processing of conspecific sounds compared to heterospecific stimuli is not excluded. Our study highlights that fNIRS may be a promising alternative to further investigate the auditory mechanisms at play in the right and left baboons temporal cortices for the processing of emotional vocalizations.

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Structure-driven effects on genomic DNA damage propensity at G-quadruplex sites Our genome contains about half a million sites capable of forming G-quadruplex (G4) structures. Such structural formations, often localised at important regulatory loci, have high capability of altering the predisposition of corresponding genomic spans to endogenous and exogenous DNA damage. In this work, we devised an approach to systematically enrich and zoom onto structure-driven effects on the propensity to undergo 9 types of DNA damage: ultraviolet radiation-induced pyrimidine-pyrimidone (6-4) photoproduct PP and cyclobutane pyrimidine dimer CPD couplings (two dyad-based subtypes in each), cisplatin-mediated G-G crosslinks, reactive oxygen species induced 8-oxoguanine damage, DNA fragmentation upon natural decay and fossilisation, breakages from artificial enzymatic cleavage and ultrasound sonication. Our results indicate that the structural effects on DNA damageability at G4 sites are not a simple combination of shielding (G4 strand) and de-shielding (opposite strand) against damaging factors, and the outcomes have different patterns and variation from one damage type to another, highly dependent on the G4 strength and relative strand localisation. The results are accompanied by electronic structure calculations, detailed structural parallels and considerations.

Graphical Abstract. An illustration of the considered damage factors acting on G-quadruplex sites, through strand-specific (two pointing thunder signs) and strand-invariant (a single pointing thunder sign) manner.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/471014v1_ufig1.gif" ALT="Figure 1">
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Modelling sexual violence in male rats: The sexual aggression test (SxAT) Sexual assault and rape are crimes that impact victims worldwide. Although the psychosocial and eco-evolutionary factors associated with this antisocial behavior have repeatedly been studied, the underlying neurobiological mechanisms are still unknown mainly due to the lack of an appropriate animal model of sexual aggression (SxA). Here, we established a novel paradigm to provoke and subsequently assess SxA in adult male Wistar rats: the sexual aggression test (SxAT). Briefly, male Wistar rats are sexually aroused by a receptive female, which is exchanged by a non-receptive female immediately after the first intromission. This protocol elicits forced mounting (FM) and aggressive behavior (AB) towards the non-receptive female to different degrees, which can be scored. In a series of experiments we can show that SxA behavior is a relatively stable trait in rats and correlates positively with sexual motivation. Rats with innate abnormal anxiety and aggressive behavior also show abnormal SxA behavior. In addition, central infusion of oxytocin moderately inhibits AB, but increases FM. Finally, we identified the agranular insular cortex to be specifically activated by SxA, and inhibition of this region mildly decreased AB during the SxAT.

Altogether, the SxAT is a paradigm that can be readily implemented in behavioral laboratories as a valuable tool to find answers regarding the biological mechanisms underlying SxA in humans, as well as social decision-making in general.

sentences: Complete loss of mitochondrial complex I genes in mistletoes (Viscaceae) and evidence of polyadenylated mitochondrial transcripts shown by whole transcriptome sequencing The profound absence of mitochondrial complex I (NADH-ubiquinone oxidoreductase) genes from the mitogenome of Viscum spp. and the rapid rates of molecular evolution characterizing most of their remaining mitochondrial genes raise questions regarding the possible transfer of the entire nad gene set to the nucleus, as well as for the functionality of the remaining highly divergent genes. Using whole transcriptome sequencing in three species of Viscaceae: V. album, V. crassulae, and Phoradendron leucarpum we were able to confirm transcription of all previously identified genes. However, we did not detect any nad gene transcripts, thus, providing further evidence of the complete loss of complex I in Viscaceae. The results from transcriptome sequencing also revealed that levels and patterns of RNA editing were not different from those found in autotrophic plant species. Hence, RNA editing is not a means of restoring conserved domains or folding sites of the proteins coded for by the divergent mitochondrial genes. Since we were able to recover mitochondrial genes transcripts following a sequencing protocol targeted towards polyadenylated mRNA molecules, it is suggested that mitochondrial genes undergo post-transcriptional polyadenylation in Viscaceae.

sentences: Algorithmic Reconstruction of GBM Network Complexity Glioblastoma (GBM) is a complex disease that is difficult to treat. Establishing the complex genetic interactions regulating cell fate decisions in GBM can help to shed light on disease aggressivity and improved treatments. Networks and data science offer alternative approaches to classical bioinformatics pipelines to study gene expression patterns from single-cell datasets, helping to distinguish genes associated with control of differentiation and thus aggressivity. Here, we applied a host of data theoretic techniques, including clustering algorithms, Waddington landscape reconstruction, trajectory inference algorithms, and network approaches, to compare gene expression patterns between pediatric and adult GBM, and those of adult glioma-derived stem cells (GSCs) to identify the key molecular regulators of the complex networks driving GBM/GSC and predict their cell fate dynamics. Using these tools, we identified critical genes and transcription factors coordinating cell state transitions from stem-like to mature GBM phenotypes, including eight transcription factors (OLIG1/2, TAZ, GATA2, FOXG1, SOX6, SATB2, YY1) and four signaling genes (ATL3, MTSS1, EMP1, and TPT1) as clinically targetable novel putative function interactions differentiating pediatric and adult GBMs from adult GSCs. Our study provides strong evidence of the applicability of complex systems approaches for reverse-engineering gene networks from patient-derived single-cell datasets and inferring their complex dynamics, bolstering the search for new clinically relevant targets in GBM.

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rTASSEL: an R interface to TASSEL for association mapping of complex traits SummaryThe need for efficient tools and applications for analyzing genomic diversity is essential for any genetics research program. One such tool, TASSEL (Trait Analysis by aSSociation, Evolution and Linkage), provides many core methods for genomic analyses. Despite its efficiency, TASSEL has limited means to use scripting languages for reproducible research and interacting with other analytical tools. Here we present an R package rTASSEL, a front-end to connect to a variety of highly used TASSEL methods and analytical tools. The goal of this package is to create a unified scripting workflow that exploits the analytical prowess of TASSEL in conjunction with Rs popular data handling and parsing capabilities without ever having the user to switch between these two environments. By implementing this workflow, we can achieve performances ranging from approximately 2 to 20 times faster than other widely used R packages for various functionalities.

Availability and implementationrTASSEL is implemented in R using core TASSEL methods written in Java. The source code for rTASSEL can be found at https://bitbucket.org/bucklerlab/rtassel/src/master/. The source code for TASSEL can be found at https://bitbucket.org/tasseladmin/tassel-5-source/src/master/.

Supplemental informationThe rTASSEL user manual and tutorials are freely available at https://maize-genetics.github.io/rTASSEL/. Supplemental material for this manuscript regarding performance metrics can be found in the attached file.

sentences: Anisotropic Rod-Shaped Particles Influence Injectable Granular Hydrogel Properties and Cell Invasion Granular hydrogels have emerged as a new class of injectable and porous biomaterials that improve integration with host tissue when compared to solid hydrogels. Granular hydrogels are typically prepared using spherical particles and this study considers whether particle shape (i.e., isotropic spheres versus anisotropic rods) influences granular hydrogel properties and cellular invasion. Simulations predict that anisotropic rods influence pore shape and interconnectivity, as well as bead transport through granular assemblies. Photocrosslinkable norbornene-modified hyaluronic acid is used to produce spherical and rod-shaped particles using microfluidic droplet generators and formed into shear-thinning and self-healing granular hydrogels at low and high particle packing. Rod-shaped particles form granular hydrogels that have anisotropic and interconnected pores, with pore number and size, storage moduli, and extrusion forces influenced by particle shape and packing. Robust in vitro sprouting of endothelial cells from embedded cellular spheroids is observed with rod-shaped particles, including higher sprouting densities and sprout lengths when compared to hydrogels with spherical particles. Cellular invasion into granular hydrogels when injected subcutaneously in vivo is significantly greater with rod-shaped particles, whereas a gradient of cellularity is observed with spherical particles. Overall, this work demonstrates potentially superior functional properties of granular hydrogels with rod-shaped particles for tissue repair.

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A Mendelian randomization study of glycemic and anthropometric traits and Parkinson's disease BackgroundImpaired glucose and obesity are frequently observed in patients with Parkinsons disease (PD), although it is unclear whether the impairment precedes or results from the neurodegeneration.

ObjectiveWe aimed to assess whether glycemic and anthropometric traits can influence the risk of PD in 33,674 cases and 449,056 healthy controls using the Mendelian randomization (MR) framework.

MethodsWe investigated causality with a two-sample MR approach in the European population to compute effect estimates with summary statistics from available discovery meta-analyses of genome-wide association studies (GWAS) on glycemic and anthropometric traits.

ResultsWe considered a threshold of p-value=0.0038 as significant after accounting for multiple testing, and p-value<0.05 was considered to be a suggestive evidence for a potential association. We observed a protective effect of waist-hip ratio (WHR) on PD (Inverse variance-weighted (IVW): OR IVW=0.735; 95%CI= 0.622-0.868; p-value=0.0003; I2 index=22.0%; MR-Egger intercept p-value=0.1508; Cochran Q test p-value=0.0003). The association was further retained after the exclusion of overlapping UK biobank (UKB) samples between the WHR and PD datasets (ORIVW=0.791; 95%CI=0.659-0.950; p-value=0.012; I2 index=13.0%; MR-Egger intercept p-value=0.733; Cochran Q test p-value=0.035). The sensitivity analysis provided suggestive evidence of an increased risk of PD on fasting glucose (FG) ({beta} IVW=0.0188; 95%CI=0.0062-0.0313, p-value=0.0055; I2 index=0.0%; MR-Egger intercept p-value=0.0957; Cochran Q test p-value=0.4555) and protective effect of PD on T2D (Weighted median effect: ORWME=0.946; 95%CI=0.9290.983; p-value=0.0051; Weighted mode effect: ORMBE=0.943; 95%CI=0.904-0.983; p-value=0.0116).

ConclusionsOur results showed that central or abdominal obesity may be protective against PD development, independent of glucose levels.

sentences: Methylation at CpG sites in genomes of aphid Acyrtosiphon pisum and its endosymbiont Buchnera Pea aphid Acyrtosiphon pisum, a sap-feeding insect, has established a mutualistic relationship with an endosymbiotic bacteria (Buchnera aphidicola) that constitutes an evolutionary successful symbiosis to synthetize complex chemical compounds from a nutrient deprived diet. In this study, led by the presence of DNMT1 and a putative DNMT3 methylase in the aphid genome, we investigated the distribution of the methyl groups on 5cytosine in CpG motifs on the whole genomes of host and endosymbiont, and looked into their correlation with gene expression. The DNA methylation turned to be present at low level in aphid (around 3% of total genomic cytosine) compared to mammals and plants, but increased to [~]9% in genes. Interestingly, the reduced genome of the endosymbiont Buchnera also shows global low level of methyl cytosine despite the fact that its genome does not shelter any DNA methylase. This finding argues for the import of DNA methylase from the host to the endosymbiont. The observed differences in methylation patterns between two clonal variants (host plus endosymbiont) are reported along with the differences in their transcriptome profiles. Our data allowed to decipher a dynamic combinatorial DNA methylation and epigenetic cross talk between host and symbiont in a clonality context that might count for the aphid adaptation to environment.

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Development of thalamus mediates paternal age effect on offspring reading: A preliminary investigation The importance of (inherited) genetic impact in reading development is well-established. De novo mutation is another important contributor that is recently gathering interest as a major liability of neurodevelopmental disorders, but has been neglected in reading research to date. Paternal age at childbirth (PatAGE) is known as the most prominent risk factor for de novo mutation, which has been repeatedly shown by molecular genetic studies. As one of the first efforts, we performed a preliminary investigation of the relationship between PatAGE, offsprings reading, and brain structure in a longitudinal neuroimaging study following 51 children from kindergarten through third grade. The results showed that greater PatAGE was associated significantly with worse reading, explaining an additional 9.5% of the variations after controlling for a number of confounds--including familial factors and cognitive-linguistic reading precursors. Moreover, this effect was mediated by volumetric maturation of the left posterior thalamus from ages 5 to 8. Complementary analyses indicated the PatAGE-related thalamic region was most likely located in the pulvinar nuclei and related to the dorsal attention network by using brain atlases, public datasets, and offsprings diffusion imaging data. Altogether, these findings provide novel insights into neurocognitive mechanisms underlying the PatAGE effect on reading acquisition during its earliest phase and suggest promising areas of future research.

HighlightsO_LIPaternal age at childbirth (PatAGE) is negatively correlated with offsprings reading abilities.
C_LIO_LIPatAGE is related to volumetric maturation of the left posterior thalamus.
C_LIO_LIThalamic development mediates the PatAGE effect on reading.
C_LIO_LIThe PatAGE-related thalamic area is more likely to connect with the dorsal attention network.
C_LI

sentences: Triazolopyrimidines target aerobic respiration in Mycobacterium tuberculosis We previously identified a series of triazolopyrimidines with anti-tubercular activity. We determined that Mycobacterium tuberculosis strains with mutations in a component of the cytochrome bc1 system (QcrB) were resistant to the series. A cytochrome bd oxidase deletion strain was also more sensitive to this series. We isolated resistant mutants, all of which had mutations in Rv1339. Compounds were active against intracellular bacteria but did not inhibit mitochondrial respiration in human HepG2 cells. These data are consistent with triazolopyrimidines acting via inhibition of M. tuberculosis QcrB.

sentences: Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies The SARS-CoV-2 B.1.1.529 variant (Omicron) contains 15 mutations on the receptor-binding domain (RBD). How Omicron would evade RBD neutralizing antibodies (NAbs) requires immediate investigation. Here, we used high-throughput yeast display screening1,2 to determine the RBD escaping mutation profiles for 247 human anti-RBD NAbs and showed that the NAbs could be unsupervised clustered into six epitope groups (A-F), which is highly concordant with knowledge-based structural classifications3-5. Strikingly, various single mutations of Omicron could impair NAbs of different epitope groups. Specifically, NAbs in Group A-D, whose epitope overlap with ACE2-binding motif, are largely escaped by K417N, G446S, E484A, and Q493R. Group E (S309 site)6 and F (CR3022 site)7 NAbs, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but still, a subset of NAbs are escaped by G339D, N440K, and S371L. Furthermore, Omicron pseudovirus neutralization showed that single mutation tolerating NAbs could also be escaped due to multiple synergetic mutations on their epitopes. In total, over 85% of the tested NAbs are escaped by Omicron. Regarding NAb drugs, the neutralization potency of LY-CoV016/LY-CoV555, REGN10933/REGN10987, AZD1061/AZD8895, and BRII-196 were greatly reduced by Omicron, while VIR-7831 and DXP-604 still function at reduced efficacy. Together, data suggest Omicron would cause significant humoral immune evasion, while NAbs targeting the sarbecovirus conserved region remain most effective. Our results offer instructions for developing NAb drugs and vaccines against Omicron and future variants.

sentences: Regulation by the pitcher plant Sarracenia purpurea of the structure of its inquiline food web AO_SCPLOWBSTRACTC_SCPLOWPhytotelmata, the water-filled habitats in pitcher plants, bromeliad tanks, and tree-holes, host multitrophic food webs that are model experimental systems for studying food-web structure and dynamics. However, the plant usually is considered simply as an inert container, not as an interacting part of the food web. We used a manipulative field experiment with a response-surface design to determine effects of nutrient enrichment (multiple levels of NH4NO3, PO4, and captured prey), top predator (removed or present), and the plant itself (with or without plastic tubes inserted into the pitchers to isolate the food web from the plant) on the macrobial food web within the modified leaves ("pitchers") of the carnivorous pitcher plant Sarracenia purpurea. Connection to the plant, addition of NH4NO3 and removal of the top predator significantly increased the food webs saturation, defined as its trophic depth and number of interactions. No effects on food-web saturation resulted from addition of PO4 or supplemental prey. Plants such as S. purpurea that create phytotelmata are more than inert containers and their inhabitants are more than commensal inquilines. Rather, both the plant and the inquilines are partners in a complex network of interactions.

sentences: Experimental Confirmation of Multiple Co-Existent DNA Secondary Structures using Low-Yield Bisulfite Sequencing The prediction of DNA secondary structures from DNA sequences using thermodynamic models is imperfect for many biological sequences, both due to insufficient experimental data for training and to the kinetics of folding that lead to metastable structures. Here, we developed low-yield bisulfite sequencing (LYB-seq) to query the secondary structure states of cytosine (C) nucleotides in thousands of different DNA oligonucleotides on a single-molecule level. We observed that the reaction kinetics between bisulfite and C nucleotides is highly dependent on the secondary structure state of the C nucleotides, with the most accessible C nucleotides (those in small hairpin loops) reacting 70-fold faster than those in stable duplexes. Next, we developed a statistical model to evaluate the likelihood of an NGS read being consistent with a particular proposed secondary structure. By analyzing thousands of NGS reads for each DNA species, we can infer the distribution of secondary structures adopted by each species in solution. We find that 84% of 1,057 human genome subsequences studied here adopt 2 or more stable secondary structures in solution.

sentences: An interactome landscape of SARS-CoV-2 virus-human protein-protein interactions by protein sequence-based multi-label classifiers The new coronavirus species, SARS-CoV-2, caused an unprecedented global pandemic of COVID-19 disease since late December 2019. A comprehensive characterization of protein-protein interactions (PPIs) between SARS-CoV-2 and human cells is a key to understanding the infection and preventing the disease. Here we present a novel approach to predict virus-host PPIs by multi-label machine learning classifiers of random forests and XGBoost using amino acid composition profiles of virus and human proteins. Our models harness a large-scale database of Viruses.STRING with >80,000 virus-host PPIs along with evidence scores for multi-level evidence prediction, which is distinct from predicting binary interactions in previous studies. Our multi-label classifiers are based on 5 evidence levels binned from evidence scores. Our best model of XGBoost achieves 74% AUC and 68% accuracy on average in 10-fold cross validation. The most important amino acids are cysteine and histidine. In addition, our model predicts experimental PPIs with higher accuracy than text mining-based PPIs by 4% despite their smaller data size by more than 6-fold. We then predict evidence levels of [~]2,000 SARS-CoV-2 virus-human PPIs from public experimental proteomics data. Interactions with SARS-CoV-2 Nsp7b show high evidence. We also predict evidence levels of all pairwise PPIs of [~]550,000 between the SARS-CoV-2 and human proteomes to provide a draft virus-host interactome landscape for SARS-CoV-2 infection in humans in a comprehensive and unbiased way in silico. Most human proteins from 140 highest evidence predictions interact with SARS-CoV-2 Nsp7, Nsp1, and ORF14, with significant enrichment in the top 2 pathways of vascular smooth muscle contraction (CALD1, NPR2, CALML3) and Myc targets (CBX3, PES1). Our prediction also suggests that histone H2A components are targeted by multiple SARS-CoV-2 proteins.

sentences: Cholinergic modulation of dentate gyrus processing through dynamic reconfiguration of inhibitory circuits The dentate gyrus (DG) of the hippocampus plays a key role in memory formation and it is known to be modulated by septal projections. By performing electrophysiology and optogenetics we evaluated the role of cholinergic modulation in the processing of afferent inputs in the DG. We showed that mature granule cells (GCs), but not adult-born immature neurons, have increased responses to afferent perforant path stimuli upon cholinergic modulation. This is due to a highly precise reconfiguration of inhibitory circuits, differentially affecting Parvalbumin and Somatostatin interneurons, resulting in a nicotinic-dependent perisomatic disinhibition of GCs. This circuit reorganization provides a mechanism by which mature GCs could escape the strong inhibition they receive, creating a window of opportunity for plasticity. Indeed, coincident activation of perforant path inputs with optogenetic release of acetylcholine produced a long-term potentiated response in GCs, essential for memory formation.

sentences: Expansion of Ventral Foregut Primes the Enhancer Landscape for Organ Specific Differentiation Cell proliferation is fundamental for almost all stages of development and differentiation that require an increase cell number. Although cell cycle phase has been associated with differentiation, the actual process of proliferation is not seen as having a specific role. Here we exploit human embryonic stem cell derived endodermal progenitors that we find are an in vitro model for the ventral foregut. These cells exhibit expansion dependent increases in differentiation efficiency to pancreatic progenitors that are linked to organ-specific enhancer priming at the level of chromatin accessibility and the decommissioning of lineage inappropriate enhancers. Our findings suggest that cell proliferation in embryonic development is about more than tissue expansion, it is required to ensure equilibration of gene regulatory networks allowing cells to become primed for future differentiation. The use of expansion of lineage specific intermediates may therefore be an important step in high fidelity in vitro differentiation.

sentences: The Polarization Dependence of 2D IR Cross-Peaks Distinguishes Parallel-Stranded and Antiparallel-Stranded DNA G-quadruplexes Guanine-rich nucleic acid sequences have a tendency to form four-stranded non-canonical motifs known as G-quadruplexes. These motifs may adopt a wide range of structures characterized by size, strand orientation, guanine base conformation, and fold topology. Using three K+-bound model systems, we show that vibrational coupling between guanine C6=O and ring modes varies between parallel-stranded and antiparallel-stranded G-quadruplexes, and that such structures can be distinguished by comparison of polarization dependent cross-peaks in their two-dimensional infrared (2D IR) spectra. Combined with previously defined vibrational frequency trends, this analysis reveals key features of a 30-nucleotide unimolecular variant of the Bcl-2 proximal promoter that are consistent with its reported structure. This study shows that 2D IR spectroscopy is a convenient method for analyzing G-quadruplex structures that can be applied to complex sequences where traditional high-resolution methods are limited by solubility and disorder.

sentences: Lonafarnib improves cardiovascular function and survival in a mouse model of Hutchinson-Gilford Progeria Syndrome Clinical trials have demonstrated that lonafarnib, a farnesyltransferase inhibitor, extends lifespan in patients afflicted by Hutchinson-Gilford progeria syndrome, a devastating condition that accelerates many characteristics of aging and results in premature death due to cardiovascular sequelae. The US Food and Drug Administration approved Zokinvy (lonafarnib) in November 2020 for treating these patients, yet a detailed examination of drug-associated effects on cardiovascular structure, properties, and function has remained wanting. In this paper, we report encouraging outcomes of daily post-weaning treatment with lonafarnib on the composition and biomechanical phenotype of elastic and muscular arteries as well as associated cardiac function in a well-accepted mouse model of progeria that exhibits severe end-stage cardiovascular disease. Lonafarnib resulted in 100% survival of the treated progeria mice to the study end-point (time of 50% survival of untreated mice), with associated improvements in arterial structure and function working together to significantly reduce pulse wave velocity and improve left ventricular diastolic function. By contrast, dual treatment with lonafarnib and rapamycin did not improve outcomes over that achieved with lonafarnib monotherapy.

sentences: High-resolution imaging and manipulation of endogenous AMPA receptor surface mobility during synaptic plasticity and learning Regulation of synaptic neurotransmitter receptor content is a fundamental mechanism for tuning synaptic efficacy during experience-dependent plasticity and behavioral adaptation. However, experimental approaches to track and modify receptor movements in integrated experimental systems are limited. Exploiting AMPA-type glutamate receptors (AMPAR) as a model, we generated a knock-in mouse expressing the biotin acceptor peptide (AP) tag on the GluA2 extracellular N-terminus. Cell-specific introduction of biotin ligase allows the use of monovalent or tetravalent avidin variants to respectively monitor or manipulate the surface mobility of endogenous AMPAR containing biotinylated AP-GluA2 in neuronal subsets. AMPAR immobilization precluded the expression of long-term potentiation and formation of contextual fear memory, allowing for target-specific control of the expression of synaptic plasticity and animal behavior. The AP tag knock-in model offers unprecedented access to resolve and control the spatiotemporal dynamics of endogenous receptors, and opens new avenues to study the molecular mechanisms of synaptic plasticity and learning.

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The role of mitochondrial energetics in the origin and diversification of eukaryotes The origin of eukaryotic cell size and complexity is thought by some to have required an energy excess provided by mitochondria, whereas others claim that mitochondria provide no energetic boost to eukaryotes. Recent observations show that energy demand scales continuously and linearly with cell volume across both prokaryotes and eukaryotes, and thus suggest that eukaryotes do not have an increased energetic capacity over prokaryotes. However, amounts of respiratory membranes and ATP synthases scale super-linearly with cell surface area. Furthermore, the energetic consequences of the contrasting genomic architectures between prokaryotes and eukaryotes have yet to be precisely quantified. Here, we investigated (1) potential factors that affect the cell volumes at which prokaryotes become surface area-constrained, (2) the amount of energy that is divested to increasing amounts of DNA due to the contrasting genomic architectures of prokaryotes and eukaryotes, and (3) the costs and benefits of respiring symbionts for a host cell. Our analyses suggest that prokaryotes are not necessarily constrained by their cell surfaces at cell volumes of 100-103 m3, and that the genomic architecture of eukaryotes is only slightly advantageous at genomes sizes of 106-107 bp. This suggests that eukaryotes may have first evolved without the need for mitochondria as these ranges hypothetically encompass the Last Eukaryote Common Ancestor and its proto-eukaryotic ancestors. At these slightly larger cell volumes, a host cell may also have derived a greater advantage (lower cost) from harboring a population of ATP-producing symbionts. However, our analyses also show that increasingly larger and fast-dividing prokaryotes would have a shortage of surface area devoted to respiration and would disproportionally divest more energy to DNA synthesis at larger genome sizes. We thus argue that, even though mitochondria may not have been required by the first eukaryotes, the successful diversification of eukaryotes into larger and faster-growing cells was ultimately contingent upon the origin of mitochondria.

SignificanceThere has been a lot of theorizing about the evolution of eukaryotes from prokaryotes, but no consensus seems to be on the horizon. Our quantitative analyses on the required amount of respiratory membrane, and the amount of energy diverted to DNA synthesis, by both prokaryotes and eukaryotes, suggest that mitochondria provided rather small advantages to the first eukaryotes, but were advantageous for the macro-evolutionary diversification of eukaryotes. This conclusion provides a middle road in the debate between those that claim that the origin of eukaryotes required a massive energy boost provided by mitochondria, and those that argue that the origin of mitochondria did not represent a quantum leap in energetic advantages to eukaryotes.

sentences: Lexical frequency and sentence context influence the brain's response to single words Typical adults read remarkably quickly. Such fast reading is facilitated by brain processes that are sensitive to both word frequency and contextual constraints. It is debated as to whether these attributes have additive or interactive effects on language processing in the brain. We investigated this issue by analysing existing magnetoencephalography data from 99 participants reading intact and scrambled sentences. Using a cross-validated model comparison scheme, we found that lexical frequency predicted the word-by-word elicited MEG signal in a widespread cortical network, irrespective of sentential context. In contrast, index (ordinal word position) was more strongly encoded in sentence words, in left front-temporal areas. This confirms that frequency influences word processing independently of predictability, and that contextual constraints affect word-by-word brain responses. With a conservative multiple comparisons correction, only the interaction between lexical frequency and surprisal survived, in anterior temporal and frontal cortex, and not between lexical frequency and entropy, nor between lexical frequency and index. However, interestingly, the uncorrected index*frequency interaction revealed an effect in left frontal and temporal cortex that reversed in time and space for intact compared to scrambled sentences. Finally, we provide evidence to suggest that, in sentences, lexical frequency and predictability may independently influence early (<150ms) and late stages of word processing, but also interact during late stages of word processing (>150-250ms), thus helping to converge previous contradictory eye-tracking and electrophysiological literature. Current neuro-cognitive models of reading would benefit from accounting for these differing effects of lexical frequency and predictability on different stages of word processing.

sentences: Shade-induced transcription of PIF-Direct-Target Genes precedes H3K4-trimethylation chromatin modifications The phytochrome (phy)-PIF (Phytochrome Interacting Factor) sensory module perceives and transduces light signals to Direct-Target Genes (DTGs), which then drive the adaptational responses in plant growth and development, appropriate to the prevailing environment. These signals include the first exposure of etiolated seedlings to sunlight upon emergence from subterranean darkness, and the change in color of the light that is filtered through, or reflected from, neighboring vegetation ( shade). Previously, we identified three broad categories of rapidly signal-responsive genes: those repressed by light and conversely induced by shade; those repressed by light, but subsequently unresponsive to shade; and those responsive to shade only. Here, we investigate the potential role of epigenetic chromatin modifications in regulating these contrasting patterns of phy-PIF module-induced expression of DTGs. Using RNA-seq and ChlP-seq, time-resolved profiling of transcript and histone 3 lysine 4 trimethylation (H3K4me3) levels, respectively, we show that, whereas the initial dark-to-light transition triggers a rapid, apparently temporally-coincident decline of both parameters, the light-to-shade transition induces similarly rapid increases in transcript levels that precede increases in H3K4me3 levels. Together with other recent findings, these data raise the possibility that, rather than being causal in the shade-induced expression changes, H3K4me3 may function to buffer the rapidly fluctuating shade/light switching that is intrinsic to vegetational canopies under natural sunlight conditions.

sentences: A conserved enzyme found in diverse human gut bacteria interferes with anticancer drug efficacy Pharmaceuticals are the top predictor of inter-individual variations in gut microbial community structure1, consistent with in vitro evidence that host-targeted drugs inhibit gut bacterial growth2 and are extensively metabolized by the gut microbiome3,4. In oncology, bacterial metabolism has been implicated in both drug efficacy5,6 and toxicity7,8; however, the degree to which bacterial drug sensitivity and metabolism can be driven by conserved pathways also found in mammalian cells remains poorly understood. Here, we show that anticancer fluoropyrimidine drugs inhibit the growth of diverse gut bacterial strains by disrupting pyrimidine metabolism, as in mammalian cells. Select bacteria metabolized 5-fluorouracil (5-FU) to its inactive metabolite dihydrofluorouracil (DHFU), mimicking the major host pathway for drug clearance. The preTA operon was necessary and sufficient for 5-FU inactivation in Escherichia coli, exhibited high catalytic efficiency for the reductive reaction, decreased the bioavailability and efficacy of oral fluoropyrimidine treatment in mice, and was prevalent in the gut microbiomes of colorectal cancer patients prior to and during treatment. The observed conservation of both the targets and pathways for metabolism of therapeutics across domains highlights the need to distinguish the relative contributions of human and microbial cells to drug disposition9, efficacy, and side effect profiles.

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A mutational gradient drives somatic mutation accumulation in mitochondrial DNA and influences germline polymorphisms and genome composition BackgroundMutations in the mitochondrial genome (mtDNA) can cause devastating maternally inherited diseases, while the accumulation of somatic mtDNA mutations is linked to common diseases of aging. Although mtDNA mutations impact human health, the process(es) that give rise to these mutations are unclear and are under considerable debate. We analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing to provide clues to the mechanism by which de novo mutations arise as well as how the genome is replicated.

ResultsWe observe two distinct mutational gradients in G[->]A and T[->]C transitions, but not their complements, that are delimited by the light-strand origin and the control region (CR). The gradients increase with age and are lost in the absence of DNA polymerase {gamma} proofreading activity. A nearly identical pattern is present in human mtDNA somatic mutations. The distribution of mtDNA single nucleotide polymorphisms (SNPs) in the human population and genome base composition across >3,000 vertebrate species mirror this gradient pattern, pointing to evolutionary conservation of this phenomenon. Lastly, high-resolution analysis of the mtDNA control region highlights mutational  hotspots and  cold-spots that strongly align with important regulatory regions.

ConclusionsCollectively, these patterns support an asymmetric strand-displacement mechanism with key regulatory structures in the CR and argue against alternative replication models. The mutational gradient is a fundamental consequence of mtDNA replication that drives somatic mutation accumulation and influences inherited polymorphisms and, over evolutionary timescales, genome composition.

sentences: Gut microbiome dysbiosis during COVID-19 is associated with increased risk for bacteremia and microbial translocation. The microbial populations in the gut microbiome have recently been associated with COVID-19 disease severity. However, a causal impact of the gut microbiome on COVID-19 patient health has not been established. Here we provide evidence that gut microbiome dysbiosis is associated with translocation of bacteria into the blood during COVID-19, causing life-threatening secondary infections. Antibiotics and other treatments during COVID-19 can potentially confound microbiome associations. We therefore first demonstrate in a mouse model that SARS-CoV-2 infection can induce gut microbiome dysbiosis, which correlated with alterations to Paneth cells and goblet cells, and markers of barrier permeability. Comparison with stool samples collected from 96 COVID-19 patients at two different clinical sites also revealed substantial gut microbiome dysbiosis, paralleling our observations in the animal model. Specifically, we observed blooms of opportunistic pathogenic bacterial genera known to include antimicrobial-resistant species in hospitalized COVID-19 patients. Analysis of blood culture results testing for secondary microbial bloodstream infections with paired microbiome data obtained from these patients indicates that bacteria may translocate from the gut into the systemic circulation of COVID-19 patients. These results are consistent with a direct role for gut microbiome dysbiosis in enabling dangerous secondary infections during COVID-19.

sentences: A benchmarking of human mitochondrial DNA haplogroup classifiers from whole-genome and whole-exome sequence data The mitochondrial genome (mtDNA) is of interest for a range of fields including evolutionary, forensic, and medical genetics. Human mitogenomes can be classified into evolutionary related haplogroups that provide ancestral information and pedigree relationships. Because of this and the advent of high-throughput sequencing (HTS) technology, there is a diversity of bioinformatic tools for haplogroup classification. We present a benchmarking of the 11 most salient tools for human mtDNA classification using empirical whole-genome (WGS) and whole-exome (WES) short-read sequencing data from 36 unrelated donors. Besides, because of its relevance, we also assess the best performing tool in third-generation long noisy read WGS data obtained with nanopore technology for a subset of the donors. We found that, for short-read WGS, most of the tools exhibit high accuracy for haplogroup classification irrespective of the input file used for the analysis. However, for short-read WES, Haplocheck and MixEmt were the most accurate tools. Based on the performance shown for WGS and WES, and the accompanying qualitative assessment, Haplocheck stands out as the most complete tool. For third-generation HTS data, we also showed that Haplocheck was able to accurately retrieve mtDNA haplogroups for all samples assessed, although only after following assembly-based approaches (either based on a referenced-based assembly or a hybrid de novo assembly). Taken together, our results provide guidance for researchers to select the most suitable tool to conduct the mtDNA analyses from HTS data.

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Reproducible Discovery of Cell-Binding Peptides "Lost" in Bulk Amplification via Emulsion Amplification in Phage Display Panning Many pharmaceutically-relevant cell surface receptors are functional only in the context of intact cells. Phage display, while being a powerful method for the discovery of ligands for purified proteins often fails to identify a diverse set of ligands to receptors on a cell membrane mosaic. To understand this deficiency, we examined growth bias in naive phage display libraries and observed that it fundamentally changes selection outcomes: The presence of growth-biased (parasite) phage clones in a phage library is detrimental to selection and cell-based panning of such biased libraries is poised to yield ligands from within a small parasite population. Importantly, amplification of phage libraries in water-oil emulsions suppressed the amplification of parasites and steered the selection of biased phage libraries away from parasite population. Attenuation of the growth bias through the use of emulsion amplification reproducibly discovers the ligands for cell-surface receptors that cannot be identified in screen that use conventional  bulk amplification.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/466683v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1ed14c5org.highwire.dtl.DTLVardef@1eec8deorg.highwire.dtl.DTLVardef@1c5741forg.highwire.dtl.DTLVardef@4aa542_HPS_FORMAT_FIGEXP  M_FIG Find Ligands in Droplets

Canonical phage display selection of ligands for breast cancer cells, which uses bulk amplification (BA) of phage library, reproducibly identified peptide ligands from a ~0.0001% sub-population of the library, which harbors fast-growing phage. Replacing BA by emulsion-amplification (EmA) altered the selection landscape and yielded cell-binding ligands not accessible to conventional phage-display select

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sentences: A field-wide assessment of differential high throughput sequencing reveals widespread bias Here we assess reproducibility and inferential quality in the field of differential HT-seq, based on analysis of datasets submitted 2008-2019 to the NCBI GEO data repository. Analysis of GEO submission file structures places an overall 56% upper limit to reproducibility without querying other sources. We further show that only 23% of experiments resulted in theoretically expected p value histogram shapes, although both reproducibility and p value distributions show marked improvement over time. Uniform p value histogram shapes, indicative of <100 true effects, were extremely few. Our calculations of {pi}0, the fraction of true nulls, showed that 36% of experiments have {pi}0 <0.5, meaning that in over a third of experiments most RNA-s were estimated to change their expression level upon experimental treatment. Both the fraction of different p value histogram types and {pi}0 values are strongly associated with the software used for calculating these p values by the original authors, indicating widespread bias.

sentences: Fc-independent neutralization of SARS-CoV-2 by recombinant human monoclonal antibodies The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in vivo neutralization of SARS-CoV-2 is not yet clear. Delineating the role this process plays in antibody-mediated protection will have a great impact on the design of such therapeutics. Here, the Fc of two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, targeting distinct domains of the spike, was engineered to abrogate their Fc-dependent functions. The protective activity of these antibodies was tested against lethal SARS-CoV-2 infections in K18-hACE2 transgenic mice, both before or two days post-exposure in comparison to their original, Fc-active antibodies. Antibody treatment with both Fc-variants similarly rescued the mice from death, reduced viral load and prevented signs of morbidity. In addition, surviving animals developed a significant endogenous immune response towards the virus. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in antibody-mediated protection, which should aid in future design of effective antibody-based therapies.

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Predicting coordinates of peptide features in raw timsTOF data with machine learning for targeted extraction reduces missing values in label-free DDA LC-MS/MS proteomics experiments 1The determination of relative protein abundance in label-free data dependant acquisition (DDA) LC-MS/MS proteomics experiments is hindered by the stochastic nature of peptide detection and identification. Peptides with an abundance near the limit of detection are particularly effected. The possible causes of missing values are numerous, including; sample preparation, variation in sample composition and the corresponding matrix effects, instrument and analysis software settings, instrument and LC variability, and the tolerances used for database searching.

There have been many approaches proposed to computationally address the missing values problem, predominantly based on transferring identifications from one run to another by data realignment, as in MaxQuants matching between runs (MBR) method, and/or statistical imputation. Imputation transfers identifications by statistical estimation of the likelihood the peptide is present based on its presence in other technical replicates but without probing the raw data for evidence.

Here we present a targeted extraction approach to resolving missing values without modifying or realigning the raw data. Our method, which forms part of an end-to-end timsTOF processing pipeline we developed called Targeted Feature Detection and Extraction (TFD/E), predicts the coordinates of peptides using machine learning models that learn the delta of each peptides coordinates from a reference library. The models learn the variability of a peptides location in 3D space from the variability of known peptide locations around it. Rather than realigning or altering the raw data, we create a run-specific  lens through which to observe the data, targeting a location for each peptide of interest and extracting it. By also creating a method for extracting decoys, we can estimate the false discovery rate (FDR). Our method outperforms MaxQuant and MSFragger by achieving substantially fewer missing values across an experiment of technical replicates. The software has been developed in Python using Numpy and Pandas and open sourced with an MIT license (DOI 10.5281/zenodo.6513126) to provide the opportunity for further improvement and experimentation by the community. Data are available via ProteomeXchange with identifier PXD030706.

2 Author SummaryMissed identifications of peptides in data-dependent acquisition (DDA) proteomics experiments are an obstacle to the precise determination of which proteins are present in a sample and their relative abundance. Efforts to address the problem in popular analysis workflows include realigning the raw data to transfer a peptide identification from one run to another. Another approach is statistically analysing peptide identifications across an experiment to impute peptide identifications in runs in which they were missing.

We propose a targeted extraction technique that uses machine learning models to construct a run-specific lens through which to examine the raw data and predict the coordinates of a peptide in a run. The models are trained on differences between observations of confidently identified peptides in a run and a reference library of peptide observations collated from multiple experiments. To minimise the risk of drawing unsound experimental conclusions based on an unknown rate of false discoveries, our method provides a mechanism for estimating the false discovery rate (FDR) based on the misclassification of decoys as target features. Our approach outperforms the popular analysis tool suites MaxQuant and MSFragger/IonQuant, and we believe it will be a valuable contribution to the proteomics toolbox for protein quantification.

sentences: STK25 inhibits PKA signaling by phosphorylating PRKAR1A In the heart, Protein Kinase A (PKA) is critical for activating calcium handling and sarcomeric proteins in response to beta adrenergic stimulation leading to increased myocardial contractility and performance. The catalytic activity of PKA is tightly regulated by regulatory subunits which inhibit the catalytic subunit until released by cAMP binding. Phosphorylation of Type II regulatory subunits promotes PKA activation, however the role of phosphorylation in Type I regulatory subunits remain uncertain. Here we utilized human induced pluripotent stem cell cardiomyocytes (iPSC-CM) to identify STK25 as a kinase of the Type I regulatory subunit PRKAR1A. Phosphorylation PRKAR1A led to inhibition of PKA kinase activity and increased binding to the catalytic subunit in the presence of cAMP. Stk25 knockout in mice diminished Prkar1a phosphorylation, increased Pka activity and augmented contractile response to beta adrenergic stimulation. Together, these data support STK25 as a negative regulator of PKA signaling through phosphorylation of PRKAR1A.

sentences: A Probabilistic Approach For Registration Of Multi-Modal Spatial Transcriptomics Data Observing the spatial characteristics of gene expression by image-based spatial transcriptomics technology allows studying gene activity across different cells and intracellular structures. We present a probabilistic approach for the registration and analysis of transcriptome images and immunostaining images. The method is based on particle filters and jointly exploits intensity information and image features. We applied our approach to synthetic data as well as real transcriptome images and immunostaining microscopy images of the mouse brain. It turns out that our approach accurately registers the multi-modal images and yields better results than a state-of-the-art method.

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Unraveling protein interactions between the temperate virus Bam35 and its Bacillus host using an integrative yeast two hybrid-high throughput sequencing approach AO_SCPLOWBSTRACTC_SCPLOWBacillus virus Bam35 is the model Betatectivirus and member of the Tectiviridae family, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. The interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein-protein interactions network for a tectivirus-host system by studying the Bam35-Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and Illumina high-throughput sequencing. We generated and thoroughly analyzed a genomic library of Bam35s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait-prey couples. In total, this screen resulted in the detection of over 4,000 potential interactions, of which 183 high-confidence interactions were defined as part of the core virus-host interactome. Overall, host metabolism proteins and peptidases are particularly enriched within the detected interactions, distinguishing this host-phage system from the other reported host-phage protein-protein interaction networks (PPIs). Our approach also suggests biological roles for several Bam35 proteins of unknown function, resulting in a better understanding of the Bam35-B. thuringiensis interaction at the molecular level.

AO_SCPLOWUTHORC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWSUMMARYC_SCPLOWMembers of the family Tectiviridae, composed of non-tailed icosahedral, membrane-containing bacteriophages, have been increasingly scrutinized in recent years for their possible role in the origin of dsDNA viruses. In particular, the genus Betatectivirus receives increased attention as these phages can infect clinical strains as well as industrially relevant members of the B. cereus group. However, little is known about the interactions between these temperate viruses and their hosts. Here, we present the first high-throughput study of tectivirus-host protein-protein interactions focusing on Bam35, model virus of betatectiviruses, and its host B. thuringiensis, an important entomopathogenic bacterium. We adapted the well-known technique yeast-two-hybrid and integrated high-throughput sequencing and bioinformatics for the downstream analysis of the results which enables large-scale analysis of protein-protein interactions. In total, 182 detected interactions show an enrichment in host metabolic proteins and peptidases, in contrast with the current knowledge on host-phage PPIs. Specific host-viral protein-protein interactions were also detected enabling us to propose functions for uncharacterized proteins.

sentences: Synthetic mammalian signaling circuits for robust cell population control In multicellular organisms, cells actively sense and control their own population density. Synthetic mammalian quorum sensing circuits could provide insight into principles of population control and extend cell therapies. However, a key challenge is reducing their inherent sensitivity to "cheater" mutations that evade control. Here, we repurposed the plant hormone auxin to enable orthogonal mammalian cell-cell communication and quorum sensing. We designed a paradoxical population control circuit, termed Paradaux, in which auxin stimulates and inhibits net cell growth at different concentrations. This circuit limited population size over extended timescales, of up to 42 days of continuous culture. By contrast, when operating in a non-paradoxical regime, the same cells limited population growth, but were more susceptible to mutational escape. These results establish auxin as a versatile "private" communication system, and demonstrate that paradoxical circuit architectures can provide robust population control.

sentences: Perphenazine-macrocycle conjugates rapidly sequester the Aβ42 monomer and inhibit amyloid formation Alzheimers disease is imposing a growing social and economic burden worldwide and effective therapies are required. Strategies aimed at the removal of fibrillar plaques formed by the amyloid-{beta} peptide have not proved therapeutic and the focus has shifted to approaches that target the cytotoxic oligomeric amyloid-{beta} species that are populated before fibrils are deposited. We have designed and synthesized perphenazine-cyclam conjugates that specifically and rapidly bind to the monomeric form of A{beta}42, reducing the production of both cytotoxic oligomers and amyloid fibrils. We have applied detailed kinetic analysis and NMR spectroscopy to show that the perphenazine-cyclam conjugates divert the A{beta}42 monomer into amorphous aggregates that are not toxic to differentiated SH-SY5Y cells in vitro. Unlike most other amyloid inhibitors studied to date, these conjugates inhibit oligomer and fibril assembly even in the presence of pre-formed fibrillar seeds, demonstrating that they act through a monomer sequestration mechanism. These modular, three-dimensional conjugates therefore effectively prevent monomer-dependent secondary nucleation, the autocatalytic process that generates the majority of toxic oligomers.

sentences: A parental transcriptional response to microsporidia infection induces inherited immunity in offspring Inherited immunity is an emerging field and describes how the transfer of immunity from parents to offspring can promote progeny survival in the face of infection. The mechanisms of how inherited immunity is induced are mostly unknown. The intracellular parasite Nematocida parisii is a natural microsporidian pathogen of Caenorhabditis elegans. Here, we show that N. parisii-infected worms produce primed offspring that are resistant to microsporidia infection. We find that immunity is induced in a dose dependent manner and lasts for a single generation. Intergenerational immunity prevents host cell invasion by N. parisii and also enhances survival to the bacterial pathogen Pseudomonas aeruginosa. Further, we show that inherited immunity is triggered by the host transcriptional response to infection, which can also be induced through maternal somatic depletion of negative regulators PALS-22 and the retinoblastoma protein ortholog LIN-35. We show that other biotic and abiotic stresses, such as viral infection and cadmium exposure, that induce a similar transcriptional response to microsporidia can also induce immunity in progeny. Our results demonstrate that distinct stimuli can induce inherited immunity to provide resistance against multiple classes of pathogens. These results show that activation of an innate immune response can provide protection against pathogens not only within a generation, but also in the next generation.

sentences: Centrosomal Enrichment and Proteasomal Degradation of SYS-1/-β-catenin Requires the Microtubule Motor Dynein The C. elegans Wnt/{beta}-catenin Asymmetry (W{beta}A) pathway utilizes asymmetric regulation of SYS- 1/{beta}-catenin and POP-1/TCF coactivators. W{beta}A differentially regulates gene expression during cell fate decisions, specifically by asymmetric localization of determinants in mother cells to produce daughters biased towards their appropriate cell fate. Despite the induction of asymmetry, {beta}-catenin localizes symmetrically to mitotic centrosomes in both mammals and C. elegans. Due to the mitosis-specific localization of SYS-1 to centrosomes and enrichment of SYS-1 at kinetochore microtubules when SYS-1 centrosomal loading is disrupted, we investigated active trafficking in SYS-1 centrosomal localization. Here, we demonstrate that trafficking by microtubule motor dynein is required to maintain SYS-1 centrosomal enrichment, by dynein RNAi-mediated decreases in SYS-1 centrosomal enrichment and by temperature-sensitive allele of the dynein heavy chain. Conversely, we observe depletion of microtubules by nocodazole treatment or RNAi of dynein-proteasome adapter ECPS-1 exhibits increased centrosomal enrichment of SYS-1. Moreover, disruptions to SYS-1 or negative regulator microtubule trafficking are sufficient to significantly exacerbate SYS-1 dependent cell fate misspecifications. We propose a model whereby retrograde microtubule-mediated trafficking enables SYS-1 enrichment at centrosomes, enhancing its eventual proteasomal degradation. These studies support the link between centrosomal localization and enhancement of proteasomal degradation, particularly for proteins not generally considered  centrosomal.

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Effective gene expression prediction from sequence by integrating long-range interactions The next phase of genome biology research requires understanding how DNA sequence encodes phenotypes, from the molecular to organismal levels. How noncoding DNA determines gene expression in different cell types is a major unsolved problem, and critical downstream applications in human genetics depend on improved solutions. Here, we report substantially improved gene expression prediction accuracy from DNA sequence through the use of a new deep learning architecture called Enformer that is able to integrate long-range interactions (up to 100 kb away) in the genome. This improvement yielded more accurate variant effect predictions on gene expression for both natural genetic variants and saturation mutagenesis measured by massively parallel reporter assays. Notably, Enformer outperformed the best team on the critical assessment of genome interpretation (CAGI5) challenge for noncoding variant interpretation with no additional training. Furthermore, Enformer learned to predict promoter-enhancer interactions directly from DNA sequence competitively with methods that take direct experimental data as input. We expect that these advances will enable more effective fine-mapping of growing human disease associations to cell-type-specific gene regulatory mechanisms and provide a framework to interpret cis-regulatory evolution. To foster these downstream applications, we have made the pre-trained Enformer model openly available, and provide pre-computed effect predictions for all common variants in the 1000 Genomes dataset.

One-sentence summaryImproved noncoding variant effect prediction and candidate enhancer prioritization from a more accurate sequence to expression model driven by extended long-range interaction modelling.

sentences: Efficient CRISPR/Cas-mediated Targeted Mutagenesis in Spring and Winter Wheat Varieties The CRISPR/Cas technology has recently become a molecular tool of choice for gene function studies in plants as well as crop improvement. Wheat is a globally important staple crop with a well annotated genome and there is plenty of scope for improving its agriculturally important traits using genome editing technologies, such as CRISPR/Cas. As part of this study we targeted three different genes in hexaploid wheat Triticum aestivum: TaBAK1-2 in the spring cultivar Cadenza as well as Ta-eIF4E and Ta-eIF(iso)4E in winter cultivars Cezanne, Goncourt and Prevert. The primary transgenic lines carrying CRISPR/Cas-induced indels were successfully generated for all targeted genes. As winter wheat varieties are generally less amenable to genetic transformation, the successful experimental methodology for transformation and genome editing in winter wheat presented in this study will be of interest to the research community working with this crop.

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GIGANTEA gene expression influence leaf senescence in Populus in two different ways GIGANTEA (GI) genes have a central role in plant development and influence several processes such as light signaling, circadian rhythm and abiotic stress tolerance. Hybrid aspen T89 (Populus tremula x tremuloides) trees with low GI expression through RNAi have a severely compromised growth. In order to study the effect of reduced GI expression on leaf traits with special emphasis on leaf senescence, we grafted GI-RNAi scions onto wild type (WT) rootstocks and managed to restore scions growth. The RNAi line had distorted leaf shape and reduced photosynthesis, probably caused by modulating phloem or stomatal function, increased starch accumulation, higher carbon-to-nitrogen (C/N) ratio and a reduced capacity to withstand moderate light stress. GI-RNAi also induced senescence under long day (LD) and moderate light conditions. Furthermore, the GI-RNAi lines were affected in their capacity to respond to "autumn environmental cues" inducing senescence, a type of leaf senescence with characteristics different from senescence induced directly by stress under LD conditions. Whereas Overexpression of GI delayed senescence. The two different effects on leaf senescence were not affected by the expression of FT (Flowering locus T), were "local" - they followed the genotype of the branch independent on the position in the tree - and trees with modified gene expression grown in the field were affected in a similar way as under controlled conditions. Taken together, GI plays a central role to sense the environmental changes during autumn and determine the appropriate timing for leaf senescence in Populus.

One sentence summaryLeaf senescence is a complex process that is not well understood, but this paper shows that changing the expression of one gene could influence leaf senescence in Populus trees in two separate ways.

sentences: Do we parse the background into separate streams in the cocktail party? AO_SCPLOWBSTRACTC_SCPLOWIn the cocktail party situation, people with normal hearing usually follow a single speaker among multiple concurrent ones. However, there is no agreement in the literature as to whether the background is segregated into multiple streams/speakers. The current study varied the number of concurrent speech streams and investigated target detection and memory for the contents of a target stream as well as the processing of distractors. A male-spoken target stream was either presented alone (single-speech), together with one male-spoken (one-distractor), or a male- and a female-spoken distractor (two-distractor). Behavioral measures of target detection and content tracking performance as well as target- and distractor detection related ERPs were assessed. We found that the detection sensitivity and the target N2b amplitude decreased whereas the P3b amplitude increased from the single-speech to the concurrent speech streams conditions. Importantly, the behavioral distractor effect differed between the conditions with one- vs. two-distractor (distraction by the female speaker was lower than that of the male speaker in either condition) and the target N2b elicited in the presence of two distractors was significantly smaller than that elicited in the presence of one distractor. Further, the voltage in the N2b time window significantly differed between the one- and two-distractor conditions for the same (M2) speaker. These results show that speech processing was different in the presence of one vs. two distractors, and thus, the current data suggest that the two background speech streams were segregated from each other.

sentences: Heat stress reveals the existence of a specialized variant of the pachytene checkpoint in meiosis of Arabidopsis thaliana Plant growth and fertility strongly depend on environmental conditions such as temperature. Remarkably, temperature also influences meiotic recombination and thus, the current climate change will affect the genetic make-up of plants. To further understand temperature effects on meiosis, we have followed male meiocytes of Arabidopsis thaliana by live cell imaging under three different temperature regimes, at 21{degrees}C and at heat shock conditions of 30{degrees}C and 34{degrees}C as well as after an acclimatization phase of one week at 30{degrees}C. This work led to a cytological framework of meiotic progression at elevated temperature. We found that an increase to 30{degrees}C, sped up meiotic progression with specific phases being more amenable to heat than others. An acclimatization phase often moderated this effect. A sudden increase to 34{degrees}C promoted a faster progression of meiosis in early prophase compared to 21{degrees}C. However, the phase in which cross-overs maturate was found to be prolonged at 34{degrees}C. Interestingly, mutants involved in the recombination pathway did not show the extension of this phase at 34{degrees}C demonstrating that the delay is recombination dependent. Further analysis revealed the involvement of the ATM kinase in this prolongation indicating the existence of a specialized variant of the pachytene checkpoint in plants.

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The role of epistasis in determining the fitness landscape of HIV proteins The rapid evolution of HIV is constrained by interactions between mutations which affect viral fitness. In this work, we explore the role of epistasis in determining the fitness landscape of HIV for multiple drug target proteins, including Protease, Reverse Transcriptase, and Integrase. Epistatic interactions between residues modulate the mutation patterns involved in drug resistance with unambiguous signatures of epistasis best seen in the comparison of a maximum entropy sequence co-variation (Potts) model predicted and experimental HIV sequence "prevalences" when expressed as higher-order marginals (beyond triplets) of the sequence probability distribution. In contrast, the evidence for epistasis based on experimental measures of fitness such as replicative capacity is weak; the correspondence with Potts model "prevalence"-based predictions is obscured by site conservation and limited precision. Double mutant cycles provide in principle one of the best ways to probe epistatic interactions experimentally without reference to a particular background, and we find they reveal that the most strongly interacting mutations in HIV involve correlated sets of drug-resistance-associated residues, however the analysis is complicated by the small dynamic range of measurements. The use of correlated models for the design of experiments to probe viral fitness can help identify the epistatic interactions involved in mutational escape, and lead to better inhibitor therapies.

Author summaryProtein covariation models provide an alternative to experimental measures for estimating the fitness of mutations in proteins from across a variety of organisms. Yet, for viral proteins, it has been shown that models including epistatic couplings between residues, or other machine learning models perform no better or even worse than a simpler independent model devoid of such epistatic couplings in estimating viral fitness measurements such as replicative capacities, providing weak or ambiguous evidence for epistasis. We show that the evidence for long-range epistasis is strong by the analysis of the high-order marginals of the MSA distribution (up to subsequences of length 14), which are accurately captured by a correlated Potts sequence-covariation model but not by an independent model. While double mutant cycles in principle provide well-established biophysical probes for epistatic interactions, we demonstrate that the analysis and comparison between model and experiment is difficult due to the much smaller dynamic range of the measurements, making them more susceptible to noise.

sentences: TNF-mediated neuroinflammation is linked to neuronal necroptosis in Alzheimer's disease hippocampus The pathogenetic mechanisms underlying neuronal death and dysfunction in Alzheimers disease (AD) remain unclear. However, chronic neuroinflammation has been implicated in stimulating or exacerbating neuronal damage. The tumor necrosis factor (TNF) superfamily of cytokines are involved in many systemic chronic inflammatory and degenerative conditions and are amongst the key mediators of neuroinflammation. TNF binds to the TNFR1 and TNFR2 receptors to activate diverse cellular responses that can be either neuroprotective or neurodegenerative. In particular, TNF can induce programmed necrosis or necroptosis in an inflammatory environment. Although activation of necroptosis has recently been demonstrated in the AD brain, its significance in AD neuron loss and the role of TNF signaling is unclear. We demonstrate an increase in expression of multiple proteins in the TNF/TNF receptor-1-mediated necroptosis pathway in the AD post-mortem brain, as indicated by the phosphorylation of RIPK3 and MLKL, predominantly observed in the CA1 pyramidal neurons. The density of phosphoRIPK3+ and phosphoMLKL+ neurons correlated inversely with total neuron density and showed significant sexual dimorphism within the AD cohort. In addition, apoptotic signaling was not significantly activated in the AD brain compared to the control brain. Exposure of human iPSC-derived glutamatergic neurons to TNF increased necroptotic cell death when apoptosis was inhibited, which was significantly reversed by small molecule inhibitors of RIPK1, RIPK3, and MLKL. In the post-mortem AD brain and in human iPSC neurons to TNF, we show evidence of altered expression of proteins of the ESCRT III complex, which has been recently suggested as an antagonist of necroptosis and a possible mechanism by which cells can survive after necroptosis has been triggered. Taken together, our results suggest that neuronal loss in AD is due to TNF-mediated necroptosis rather than apoptosis, which is amenable to therapeutic intervention at several points in the signaling pathway.

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A transcriptional mechanism involving R-loop, m6A modification and RNA abasic sites regulates an enhancer RNA of APOE. The DNA genetic code and the RNA regulatory code determine phenotypes from gene expression to disease susceptibility. DNA sequence variants lead to phenotypic differences among individuals, while within an individual, RNA dynamically confers cell identity and responds to cellular and environmental signals. To provide regulation for different cell types and conditions, the nucleotides of RNA are modified by hundreds of chemical reactions, and RNA folds into innumerable shapes. To decipher the RNA regulatory code is to understand how RNA sequence and structure respond to cellular needs. Here, we deciphered one part of the RNA code where RNA abasic sites in R-loops regulate transcription by pausing RNA polymerase II. We uncovered an enhancer RNA, AANCR, that regulates the transcription and expression of APOE. When AANCR is folded into an R-loop, which is then modified by N6-adenine methylation and N-glycosidic cleavage, it is a partially transcribed nonfunctional enhancer and APOE is not expressed. In contrast, in some cell types and under stress, AANCR does not form a stable R-loop as its sequence is not modified, so it is transcribed into a full-length enhancer that promotes APOE expression. By genetic analysis, we confirmed that AANCR regulates APOE expression. DNA sequence variants in AANCR are associated with APOE expression and also with Alzheimers disease. Our data show that DNA and RNA sequence and structure jointly regulate gene expression that influence disease risk.

HighlightsO_LIm6A, RNA abasic sites and R-loops jointly regulate transcription by pausing RNA Polymerase II.
C_LIO_LIAn enhancer RNA regulates APOE expression.
C_LIO_LIEnhancer RNA of APOE modifies susceptibility to Alzheimers disease.
C_LI

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Mutational analysis of DinG-family helicase YoaA: its expression and interaction with replication clamp-loader protein HolC in E. coli The XP-D/DinG family of DNA helicases contribute to genomic stability in all three domains of life. We investigate here the role of one of these proteins,YoaA, of Escherichia coli. In E. coli,YoaA aids tolerance to the nucleoside azidothymidine (AZT), a DNA replication inhibitor and physically interacts with a subunit of the DNA polymerase III holoenzyme, HolC. We map the residues of YoaA required for HolC interaction to its C-terminus by yeast two-hybrid analysis. We propose that this interaction competes with HolCs interaction with HolD and the rest of the replisome;YoaA indeed inhibits growth when overexpressed, dependent on this interaction region. By gene fusions we show YoaA is repressed by LexA and induced in response to DNA damage as part of the SOS response. Induction of YoaA by AZT is biphasic with an immediate response after treatment and a slower response that peaks in the late log phase of growth. This growth-phase dependent induction by AZT is not blocked by lexA3 (Ind-), which normally negates its self-cleavage, implying another means to induce the DNA damage response that responds to the nutritional state of the cell. We propose that YoaA helicase activity increases access to the 3 nascent strand during replication; consistent with this,YoaA appears to aid removal of potential A-to-T transversion mutations in ndk mutants, which are prone to nucleotide misincorporation. YoaA and its paralog DinG also may initiate template-switching that leads to deletions between tandem repeats in DNA.

IMPORTANCEMaintaining genomic stability is crucial for all living organisms. Replication of DNA frequently encounters barriers that must be removed to complete genome duplication. Balancing DNA synthesis with its repair is critical and not entirely understood at a mechanistic level.The YoaA protein, studied here, is required for certain types of DNA repair and interacts in an alternative manner with proteins that catalyze DNA replication. YoaA is part of the well-studied LexA-regulated response to DNA damage, the SOS response. We describe an unusual feature of its regulation that promotes induction after DNA damage as the culture begins to experience starvation. Replication fork repair integrates both DNA damage and nutritional signals. We also show that YoaA affects genomic stability.

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Depth-targeted intracortical microstroke by two-photon photothrombosis in rodent brain SignificancePhotothrombosis is a widely used model of ischemic stroke in rodent experiments. In the photothromboris model, the photosensitizer Rose Bengal is systemically introduced to the blood stream and activated by green light to induce aggregation of platelets that eventually cause vessel occlusion. Since the activation of Rose Bengal is a one-photon phenomenon and the molecules in the illuminated area (light path) are subject to excitation, targeting of thrombosis is unspecific especially in the depth dimension. We have developed a photothrombosis protocol that can target a single vessel in the cortical parenchyma by two-photon excitation.

AimWe aim to induce a thrombotic stroke in the cortical parenchyma by two-photon activation of Rose Bengal so that we confine photothrombosis within a vessel of a target depth.

ApproachFITC-dextran is injected into the blood stream to visualize the cerebral blood flow in anesthetized adult mice with a cranial window. After a target vessel is chosen by two-photon imaging (950 nm), Rose Bengal is injected into the blood stream. The scanning wavelength is changed to 720 nm and photothrombosis was induced by scanning the target vessel.

ResultsTwo-photon depth-targeted single vessel photothrombosis was achieved with a success rate of 84.9{+/-}1.7% within 80 s. Attempts without Rose Bengal (i.e., only with FITC) did not result in photothrombosis at the excitation wavelength of 720 nm.

ConclusionsWe described a protocol that achieves depth-targeted single vessel photothrombosis by two-photon excitation. Simultaneous imaging of blood flow in the targeted vessel using FITC dextran enabled the confirmation of vessel occlusion and prevention of excess irradiation that possibly induces unintended photodamage.

sentences: Reviewing the consequences of genetic purging on the success of rescue programs Genetic rescue is increasingly considered a promising and underused conservation strategy to reduce inbreeding depression and restore genetic diversity in endangered populations, but the empirical evidence supporting its application is limited to a few generations. Here we discuss on the light of theory the role of inbreeding depression arising from partially recessive deleterious mutations and of genetic purging as main determinants of the medium to long-term success of rescue programs. This role depends on two main predictions: (1) The inbreeding load hidden in populations with a long stable demography increases with the effective population size; and (2) After a population shrinks, purging tends to remove its (partially) recessive deleterious alleles, a process that is slower but more efficient for large populations than for small ones. We also carry out computer simulations to investigate the impact of genetic purging on the medium to long term success of genetic rescue programs. For some scenarios, it is found that hybrid vigor followed by purging will lead to sustained successful rescue. However, there may be specific situations where the recipient population is so small that it cannot purge the inbreeding load introduced by migrants, which would lead to increased fitness inbreeding depression and extinction risk in the medium to long term. In such cases, the risk is expected to be higher if migrants came from a large non-purged population with high inbreeding load, particularly after the accumulation of the stochastic effects ascribed to repeated occasional migration events. Therefore, under the specific deleterious recessive mutation model considered, we conclude that additional caution should be taken in rescue programs. Unless the endangered population harbors some distinctive genetic singularity whose conservation is a main concern, restoration by continuous stable gene flow should be considered, whenever feasible, as it reduces the extinction risk compared to repeated occasional migration and can also allow recolonization events.

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Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci. BackgroundThe discovery of the CRISPR-Cas9 system and its applicability in mammalian embryos has revolutionized the way we generate genetically engineered animal models. To date, models harbouring conditional alleles (i.e.: two loxP sites flanking an exon or a critical DNA sequence of interest) remain the most challenging to generate as they require simultaneous cleavage of the genome using two guides in order to properly integrate the repair template. In the current manuscript, we describe a modification of the sequential electroporation procedure described by Horii et al (2017). We demonstrate production of conditional allele mouse models for eight different genes via one of two alternative strategies: either by consecutive sequential electroporation (strategy A) or non-consecutive sequential electroporation (strategy B).

ResultsBy using strategy A, we demonstrated successful generation of conditional allele models for three different genes (Icam1, Lox, and Sar1b), with targeting efficiencies varying between 5 to 13%. By using strategy B, we generated five conditional allele models (Loxl1, Pard6a, Pard6g, Clcf1, and Mapkapk5), with targeting efficiencies varying between 3 to 25%.

ConclusionOur modified electroporation-based approach, involving one of the two alternative strategies, allowed the production of conditional allele models for eight different genes via two different possible paths. This reproducible method will serve as another reliable approach in addition to other well-established methodologies in the literature for conditional allele mouse model generation.

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Germinal center block exacerbates extrafollicular responses and accelerates autoimmune disease progression in a murine lupus model Systemic lupus erythematosus and numerous other autoimmune diseases are characterized by affinity-matured, class-switched autoantibodies to nuclear antigens. Such antibodies are generally thought to arise in germinal centers (GCs). Several strategies to block GC formation and progression are currently being explored clinically. However, recent studies have suggested a key role for extrafollicular responses in driving the early events in autoimmune development. To investigate the relative contribution of these two pathways in autoimmune disease development, we leveraged a lupus murine model, where we could genetically block the GC pathway. We find that a B cell intrinsic block in GC formation accelerates extrafollicular responses and exacerbates autoimmune progression. The manifestations included higher levels of circulating, class-switched autoantibodies, as well as antibody- and complement-deposition in the kidney glomeruli. GC B cell cultures in vitro showed that loss of the GC transcription factor Bcl-6 prevents cellular expansion and accelerates plasma cell differentiation. This suggests that the in vivo phenotype was a direct consequence of rewiring of B cell intrinsic transcriptional programming. In a competitive scenario in vivo, in autoreactive mixed bone marrow chimeras, B cells harboring the genetic GC block contributed disproportionately highly to the plasma cell output. Taken together, this emphasizes the extrafollicular pathway as a key contributor to autoimmune pathogenesis and suggests that strategies aimed at blocking GCs should simultaneously target this pathway to avoid rerouting the pathogenic response.

Highlights- Genetic GC block exacerbates autoimmune progression in a lupus model
- An intrinsic GC block drives B cell differentiation into terminally differentiated plasma cells in vitro
- B cells harboring a GC block competitively contribute to the plasma cell compartment in an autoreactive setting in vivo
- Lupus mice with a GC block display immune complex deposition in kidney glomeruli that is indistinguishable from their wild-type counterparts

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Analyzing the differences in olfactory bulb mitral cell spiking with ortho- and retronasal stimulation The majority of olfaction studies focus on orthonasal stimulation where odors enter via the front nasal cavity, while retronasal olfaction, where odors enter the rear of the nasal cavity during feeding, is understudied. The processing of retronasal odors via coordinated spiking of neurons in the olfactory bulb (OB) is largely unknown. To this end, we use multi-electrode array in vivo recordings of rat OB mitral cells (MC) in response to a food odor with both modes of stimulation, and find significant differences in evoked firing rates and spike count covariances (i.e., noise correlations). To better understand these differences, we develop a single-compartment biophysical OB model that is able to reproduce key properties of important OB cell types. Prior experiments in olfactory receptor neurons (ORN) showed retro stimulation yields slower and spatially smaller ORN inputs than with ortho, yet whether this is consequential for OB activity remains unknown. Indeed with these specifications for ORN inputs, our OB model captures the trends in our OB data. We also analyze how first and second order ORN input statistics dynamically transfer to MC spiking statistics with a phenomenological linear-nonlinear filter model, and find that retro inputs result in larger temporal filters than ortho inputs. Finally, our models show that the temporal profile of ORN is crucial for capturing our data and is thus a distinguishing feature between ortho and retro stimulation, even at the OB. Using data-driven modeling, we detail how ORN inputs result in differences in OB dynamics and MC spiking statistics. These differences may ultimately shape how ortho and retro odors are coded.

Author summaryOlfaction is a key sense for many cognitive and behavioral tasks, and is particularly unique because odors can naturally enter the nasal cavity from the front or rear, i.e., ortho- and retro-nasal, respectively. Yet little is known about the differences in coordinated spiking in the olfactory bulb with ortho versus retro stimulation, let alone how these different modes of olfaction may alter coding of odors. We simultaneously record many cells in rat olfactory bulb to assess the differences in spiking statistics, and develop a biophysical olfactory bulb network model to study the reasons for these differences. Using theoretical and computational methods, we find that the olfactory bulb transfers input statistics differently for retro stimulation relative to ortho stimulation. Furthermore, our models show that the temporal profile of inputs is crucial for capturing our data and is thus a distinguishing feature between ortho and retro stimulation, even at the olfactory bulb. Understanding the spiking dynamics of the olfactory bulb with both ortho and retro stimulation is a key step for ultimately understanding how the brain codes odors with different modes of olfaction.

sentences: The role of temporal coherence and temporal stability in the build-up of auditory grouping The cochlea decomposes sounds into separate frequency channels, from which the auditory brain must reconstruct the auditory scene. To do this the auditory system must make decisions about which frequency information should be grouped together, and which should remain distinct. Two key cues for grouping are temporal coherence, resulting from coherent changes in power across frequency, and temporal predictability, resulting from regular or predictable changes over time. To test how these cues contribute to the construction of a sound scene we present listeners with a range of precursor sounds, which act to prime the auditory system by providing information about each sounds structure, followed by a fixed masker in which participants were required to detect the presence of an embedded tone. By manipulating temporal coherence and/or temporal predictability in the precursor we assess how prior sound exposure influences subsequent auditory grouping. In Experiment 1, we measure the contribution of temporal predictability by presenting temporally regular or jittered precursors, and temporal coherence by using either narrow or broadband sounds, demonstrating that both independently contribute to masking/unmasking. In Experiment 2, we measure the relative impact of temporal coherence and temporal predictability and ask whether the influence of each in the precursor signifies an enhancement or disruption of unmasking. We observed that interfering precursors produced the largest changes to thresholds.

sentences: Distractibility and impulsivity neural states are distinct from attention states and modulate the implementation of attention In the context of visual attention, it has been classically assumed that missing the response to a target or erroneously selecting a distractor occurs as a consequence of the (miss)allocation of attention in space. In the present paper, we challenge this view and provide evidence that, in addition to encoding spatial attention, prefrontal neurons also encode a distractibility-to-impulsivity state. Using supervised dimensionality reduction techniques, we identify two partially overlapped neuronal subpopulations associated either with attention or overt behaviour. The degree of overlap accounts for the behavioural gain associated with the good allocation of attention. We further describe the neural variability accounting for distractibility-to-impulsivity behaviour by a two dimensional state associated with optimality in task and responsiveness. Overall, we thus show that behavioural performance arises from the integration of task-specific neuronal processes and pre-existing neuronal states describing task-independent behavioural states, shedding new light on attention disorders such as ADHD.

sentences: A Xeno-free Media for the In Vitro Expansion of Human Spermatogonial Stem Cells In vitro expansion of spermatogonial stem cells (SSCs) has been established using animal-derived fetal bovine serum (FBS) and bovine serum albumin (BSA). However, the use of animal components during cell culture introduces the risk of contaminating cells with pathogens, and leads to animal epitope expression, rendering them unsuitable for medical use. Therefore, this study set out to develop a xeno-free, fully defined media for the expansion of human SSCs. We show that the molecules Prostaglandin D2 (PGD-2) and Insulin-Like Growth Factor 1 (IGF-1) can replace FBS and BSA in cell culture media without loss of viability or expansion capability, and that Rho-Associated, Coiled-Coil Containing Protein Kinase (ROCK) inhibitor Y-27632 supplementation improves viability after cryopreservation. Long-term SSC cultures expanded in xeno-free, defined culture conditions shared identical protein expression profiles for well-known SSC markers, while gene expression analyses revealed a significant improvement in quiescent SSC and pan-germ markers. This xeno-free, defined formulation allows for standardized SSC culture free of animal pathogens.

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BMAL1 loss in oligodendroglial lineage cells dysregulates myelination and sleep Myelination depends on maintenance of oligodendrocytes that arise from oligodendrocyte precursor cells (OPCs). We show that the dynamic nature of oligodendroglia and myelination are regulated by the circadian transcription factor BMAL1. Bmal1 knockdown in OPCs during development - but not adulthood - decreases OPC proliferation, whereas BMAL1 regulates OPC morphology throughout life. OPC-specific Bmal1 deficiency impairs remyelination in an age-dependent manner, suggesting that age-associated decrements in circadian regulation of oligodendroglia may contribute to the deficient remyelination potential in demyelinating diseases like multiple sclerosis (MS). This oligodendroglial dysregulation and dysmyelination increase sleep fragmentation in OPC-specific Bmal1 knockout mice, and sleep fragmentation is causally associated with MS. These findings have broad mechanistic and therapeutic implications for numerous brain disorders that include both myelin and sleep phenotypes.

One-Sentence SummaryBMAL1 regulates the homeostatic maintenance of oligodendroglia and myelin, that subsequently controls sleep architecture.

sentences: BANKSY: A Spatial Omics Algorithm that Unifies Cell Type Clustering and Tissue Domain Segmentation Each cell type in a solid tissue has a characteristic transcriptome and spatial arrangement, both of which are observable using modern spatial omics assays. However, the common practice is still to ignore spatial information when clustering cells to identify cell types. In fact, spatial location is typically considered only when solving the related, but distinct, problem of demarcating tissue domains (which could include multiple cell types). We present BANKSY, an algorithm that unifies cell type clustering and domain segmentation by constructing a product space of cell and neighbourhood transcriptomes, representing cell state and microenvironment, respectively. BANKSYs spatial kernel-based feature augmentation strategy improves per-formance and scalability on both tasks when tested on FISH-based and sequencing-based spatial omics data. Uniquely, BANKSY identified hitherto undetected niche-dependent cell states in two mouse brain regions. Lastly, we show that quality control of spatial omics data can be formulated as a domain identification problem and solved using BANKSY. BANKSY represents a biologically motivated, scalable, and versatile framework for analyzing spatial omics data.

sentences: Informational masking vs. crowding - A mid-level trade-off between auditory and visual processing In noisy or cluttered environments, sensory cortical mechanisms help combine auditory or visual features into perceived objects. Knowing that individuals vary greatly in their ability to suppress unwanted sensory information, and knowing that the sizes of auditory and visual cortical regions are correlated, we wondered whether there might be a corresponding relation between an individuals ability to suppress auditory vs. visual interference. In auditory masking, background sound makes spoken words unrecognizable. When masking arises due to interference at central auditory processing stages, beyond the cochlea, it is called informational masking (IM). A strikingly similar phenomenon in vision, called visual crowding, occurs when nearby clutter makes a target object unrecognizable, despite being resolved at the retina. We here compare susceptibilities to auditory IM and visual crowding in the same participants. Surprisingly, across participants, we find a negative correlation (R = -0.7) between IM susceptibility and crowding susceptibility: Participants who have low susceptibility to IM tend to have high susceptibility to crowding, and vice versa. This reveals a mid-level trade-off between auditory and visual processing.

sentences: A Cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of genetic variants in vivo Human genetic studies have identified a large number of disease-associated de novo variants in presumptive regulatory regions of the genome that pose a challenge for interpretation of their effects: the impact of regulatory variants is highly dependent on the cellular context, and thus for psychiatric diseases these would ideally be studied in neurons in a living brain. Furthermore, for both common and rare variants, it is expected that only a subset fraction will affect gene expression. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of hundreds of predefined sequences in a single experiment. These assays have been used for functional screening of several different types of regulatory sequences in vitro. However, they have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. Here, using a test-case 3'UTR MPRA library with variants from ASD patients, we sought to develop a method to achieve reproducible measurements of variant effects in vivo in a cell type-specific manner. We implemented a Cre-dependent design to control expression of our library and first validated our system in vitro. Next, we measured the effect of >500 3'UTR variants in excitatory neurons in the mouse brain. Finally, we report >40 variants with significant effects on transcript abundance in the context of the brain. This new technique should enable robust, functional annotation of genetic variants in the cellular contexts most relevant to psychiatric disease.

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The Fox gene repertoire in the annelid Owenia fusiformis reveals multiple expansions of the foxQ2 class in Spiralia Fox genes are a large and conserved family of transcription factors involved in many key biological processes, including embryogenesis and body patterning. Although the role of Fox genes has been studied in an array of model systems, comprehensive comparative studies in Spiralia--a large clade of invertebrate animals including molluscs and annelids--are scarce but much needed to better understand the evolutionary history of this gene family. Here, we reconstruct and functionally characterise the Fox gene complement in the annelid Owenia fusiformis, a slow evolving species and member of the sister group to all remaining annelids. The genome of O. fusiformis contains at least a single ortholog of each of the 23 Fox gene classes that are ancestral to Bilateria, except for foxE and foxI. Temporal and spatial expression dynamics reveal a conserved role of Fox genes in gut formation (foxA), mesoderm patterning (foxF, foxL1, foxC, foxH) and cilia formation (foxJ1) in Annelida and Spiralia. Moreover, we uncover an ancestral expansion of foxQ2 genes in Spiralia, represented by 11 paralogs in O. fusiformis. Notably, although all foxQ2 copies have apical expression in O. fusiformis, they show variable spatial domains and staggered temporal activation, which suggest cooperation and sub-functionalisation among foxQ2 genes for the development of apical fates in this annelid. Altogether, our study informs the evolution and developmental roles of Fox genes in Annelida and Spiralia generally, providing the basis to explore how regulatory changes in Fox gene expression might have contributed to developmental and morphological diversification in Spiralia.

Significance statementThe role of Fox genes, a group of DNA-binding proteins required for the formation of many animal organs, is poorly understood in invertebrate groups such as molluscs and annelids. Here, by studying the genome and embryogenesis of the annelid Owenia fusiformis, we demonstrate that Fox genes are involved in the development of the gut, muscles, cilia, and nervous system. Importantly, we find that a group of Fox genes (referred to as foxQ2) expressed in the anterior end of most animals has more copies in annelids and molluscs than in other invertebrate groups like insects and sea stars. Together, our findings clarify the evolution of Fox genes and their contribution to the diversity of forms and organs found in marine invertebrates.

sentences: Peripheral neuropathy linked mRNA export factor GANP reshapes gene regulation in human motor neurons Loss-of-function of the mRNA export protein GANP (MCM3AP gene) cause early-onset sensorimotor neuropathy, characterised by axonal degeneration in long peripheral nerves. GANP functions as a scaffold at nuclear pore complexes, contributing to selective nuclear export of mRNAs. Here, we aimed to identify motor neuron specific transcripts that are regulated by GANP and may be limiting for local protein synthesis in motor neuron axons. We compared motor neurons with a gene edited mutation in the Sac3 mRNA binding domain of GANP to isogenic controls. We also examined patient-derived motor neurons. RNA sequencing of motor neurons as well as nuclear and axonal subcompartments showed that mutant GANP had a profound effect on motor neuron transcriptomes, with alterations in nearly 40 percent of all expressed genes and broad changes in splicing. Expression changes in multiple genes critical for neuronal functions, combined with compensatory upregulation of protein synthesis and early-stage metabolic stress genes, indicated that RNA metabolism was abnormal in GANP-deficient motor neurons. Surprisingly, limited evidence was found for large-scale nuclear retention of mRNA. This first study of neuropathy-linked GANP defects in human motor neurons shows that GANP has a wide gene regulatory role in a disease-relevant cell type that requires long-distance mRNA transport.

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Maternal age affects equine Day 8 embryo gene expression both in trophoblast and inner cell mass BackgroundBreeding a mare until she is not fertile or even until her death is common in equine industry but the fertility decreases as the mare age increases. Embryo loss due to reduced embryo quality is partly accountable for this observation. Here, the effect of mares age on blastocysts gene expression was explored. Day 8 post-ovulation embryos were collected from multiparous young (YM, 6-year-old, N = 5) and older (OM, > 10-year-old, N = 6) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure or inner cell mass (ICM) enriched trophoblast, obtained by embryo bisection, were RNA sequenced. Deconvolution algorithm was used to discriminate gene expression in the ICM from that in the trophoblast. Differential expression was analyzed with embryo sex and diameter as cofactors. Functional annotation and classification of differentially expressed genes and gene set enrichment analysis were also performed.

ResultsMaternal aging did not affect embryo recovery rate, embryo diameter nor total RNA quantity. In both compartments, the expression of genes involved in mitochondria and protein metabolism were disturbed by maternal age, although more genes were affected in the ICM. Mitosis, signaling and adhesion pathways and embryo development were decreased in the ICM of embryos from old mares. In trophoblast, ion movement pathways were affected.

ConclusionsThis is the first study showing that maternal age affects gene expression in the equine blastocyst, demonstrating significant effects as early as 10 years of age. These perturbations may affect further embryo development and contribute to decreased fertility due to aging.

sentences: Mother centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly Mitotic centrosomes are formed when centrioles start to recruit large amounts of pericentriolar material (PCM) around themselves in preparation for mitosis. This centrosome "maturation" requires the centrioles and also Polo/PLK1 protein kinase. The PCM comprises several hundred proteins and, in Drosophila, Polo cooperates with the conserved centrosome proteins Spd-2/CEP192 and Cnn/CDK5RAP2 to assemble a PCM scaffold around the mother centriole that then recruits other PCM client proteins. We show here that in Drosophila syncytial blastoderm embryos, centrosomal Polo levels rise and fall during the assembly process--peaking, and then starting to decline, even as levels of the PCM scaffold continue to rise and plateau. Experiments and mathematical modelling indicate that a centriolar pulse of Polo activity, potentially generated by the interaction between Polo and its centriole receptor Ana1 (CEP295 in humans), could explain these unexpected scaffold assembly dynamics. We propose that centrioles generate a local pulse of Polo activity prior to mitotic entry to initiate centrosome maturation, explaining why centrioles and Polo/PLK1 are normally essential for this process.

sentences: Pericyte-derived vitronectin regulates blood-CNS barrier function via integrin signaling Endothelial cells of blood vessels of the central nervous system (CNS) constitute blood-CNS barriers. Barrier properties are not intrinsic to these cells; rather they are induced and maintained by CNS microenvironment. Notably, the abluminal surface of CNS capillaries are ensheathed by pericytes and astrocytes. However, extrinsic factors from these perivascular cells that regulate barrier integrity are largely unknown. Here, we establish vitronectin, an extracellular-matrix protein secreted by CNS pericytes, as a regulator of blood-CNS barrier function via interactions with its integrin receptor, 5 in endothelial cells. Genetic ablation of vitronectin or mutating vitronectin to prevent integrin binding as well as endothelial-specific deletion of integrin 5 causes barrier leakage. Furthermore, vitronectin-integrin 5 signaling maintains barrier integrity by actively inhibiting transcytosis in endothelial cells. These results demonstrate that signaling from perivascular cells to endothelial cells via ligand-receptor interactions is a key mechanism to regulate barrier permeability.

sentences: Antibiotic Resistance Genes and Taxa Analysis from Mat and Planktonic Microbiomes of Antarctic Lake Fryxell and Lake Bonney, and a Mat-forming Culture of Rhodoferax antarcticus The Antarctic Taylor Valley Lakes Fryxell and Bonney harbor oligotrophic microbial communities that are separated geographically from other aquatic systems. Their microbiomes include planktonic as well as lift-off mat communities that float to the underside of the perennial ice cover and eventually emerge at the surface. We investigated the antibiotic resistance genes (ARGs) from metagenomes of lift-off mats emerging from ice, from filtered water samples of Lake Fryxell, and from filtered water samples of Lake Bonney. ARG sequence markers were designed by ShortBRED-Identify using the Comprehensive Antibiotic Resistance Database (CARD). The overall proportion of ARG hits in the metagenomes was found to be similar to that found in temperate-zone rural water bodies with moderate human inputs (0.0002-0.0007%). The specific ARGs found showed distinct distributions for the two lakes, and for mat versus planktonic sources. Metagenomic taxa distributions showed that mat phototrophs were mainly cyanobacteria or betaproteobacteria, whereas the water column phototrophs were mainly protists. An enrichment culture of the betaproteobacterium Rhodoferax antarcticus from a Lake Fryxell mat sample showed a mat-forming phenotype not previously reported for this species. Its genome showed no ARGs associated with betaproteobacteria, but had ARGs consistent with a minor Pseudomonas component. The Antarctic lake mats and water showed specific ARGs distinctive to the mat and water sources, but overall ARG levels were similar to those of temperate water bodies with minimal human inputs.

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Conserved patterns across ion channels correlate with variant pathogenicity and clinical phenotypes Clinically identified genetic variants in ion channels can be benign or cause disease by increasing or decreasing the protein function. Consequently, therapeutic decision-making is challenging without molecular testing of each variant. Our biophysical knowledge of ion channel structures and function is just emerging, and it is currently not well understood which amino acid residues cause disease when mutated.

We sought to systematically identify biological properties associated with variant pathogenicity across all major voltage and ligand-gated ion channel families. We collected and curated 3,049 pathogenic variants from hundreds of neurodevelopmental and other disorders and 12,546 population variants for 30 ion channel or channel subunits for which a high-quality protein structure was available. Using a wide range of bioinformatics approaches, we computed 163 structural features and tested them for pathogenic variant enrichment. We developed a novel 3D spatial distance scoring approach that enables comparisons of pathogenic and population variant distribution across protein structures.

We discovered and independently replicated that several pore residue properties and proximity to the pore axis were most significantly enriched for pathogenic variants compared to population variants. Using our novel 3D scoring approach, we showed that the strongest pathogenic variant enrichment was observed for pore-lining residues and alpha-helix residues within 5[A] distance from the pore axis center and not involved in gating. Within the subset of residues located at the pore, the hydrophobicity of the pore was the feature most strongly associated with variant pathogenicity. We also found an association between the identified properties and both clinical phenotypes and fucntional in vitro assays for voltage-gated sodium channels (SCN1A, SCN2A, SCN8A) and N-methyl-D-aspartate (NMDA) receptor (GRIN1, GRIN2A, GRIN2B) encoding genes. In an independent expert-curated dataset of 1,422 neurodevelopmental disorder pathogenic patient variants, and 679 electrophysiological experiments that pore axis distance is associated with seizure age of onset and cognitive performance as well as differential gain vs. loss-of-channel function.

In summary, we identified biological properties associated with ion-channel malfunction and show that these are correlated with in vitro functional read-outs and clinical phenotypes in patients with neurodevelopmental disorders. Our results suggest that clinical decision support algorithms that predict variant pathogenicity and function are feasible in the future.

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Arabidopsis ABIG1 Functions in Laminar Growth and Polarity Formation through Regulation by REVOLUTA and KANADI Leaf laminar growth and adaxial-abaxial boundary formation are fundamental outcomes of plant development. Boundary and laminar growth coordinate the further patterning and growth of the leaf, directing the differentiation of cell types within the top and bottom domains and promoting initiation of lateral organs along their adaxial/abaxial axis. Leaf adaxial-abaxial polarity specification and laminar out-growth are regulated by two transcription factors, REVOLUTA (REV) and KANADI (KAN). ABA INSENSITIVE TO GROWTH 1 (ABIG1) is a HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP) Class II transcription factor and is a direct target of the adaxial-abaxial regulators REV and KAN. To investigate the role of ABIG1 in the leaf development and establishment of polarity, we examined the phenotypes of both gain-of-function and loss-of-function mutants. Through genetic interaction analysis with REV and KAN mutants, we have determined that ABIG1 plays a role in leaf laminar-growth as well as in adaxial-abaxial polarity establishment. Genetic and physical interaction assays showed that ABIG1 interacts with the transcriptional corepressor TOPLESS (TPL). This study provides new evidence that another HD-ZIP II gene, ABIG1, facilitates growth through the corepressor TPL.

HighlightABIG1, a HD-ZIP Class II transcription factor, promotes laminar growth and adaxial-abaxial polarity through the regulation of REV and KAN.

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Deubiquitinase-Targeting Chimeras for Targeted Protein Stabilization Targeted protein degradation is a powerful therapeutic modality that uses heterobifunctional small-molecules to induce proximity between E3 ubiquitin ligases and target proteins to ubiquitinate and degrade specific proteins of interest. However, many proteins are ubiquitinated and degraded to drive disease pathology; in these cases targeted protein stabilization (TPS), rather than degradation, of the actively degraded target using a small-molecule would be therapeutically beneficial. Here, we present the Deubiquitinase-Targeting Chimera (DUBTAC) platform for TPS of specific proteins. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48 ubiquitin-specific deubiquitinase OTUB1. We then developed a heterobifunctional DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds {Delta}F508-CFTR. We demonstrated proof-of-concept of TPS by showing that this DUBTAC robustly stabilized {Delta}F508-CFTR in human cystic fibrosis bronchial epithelial cells in an OTUB1-dependent manner. Our study underscores the utility of chemoproteomics-enabled covalent ligand discovery approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.

Editorial summaryWe have developed the Deubiquitinase Targeting Chimera (DUBTAC) platform for targeted protein stabilization. We have discovered a covalent recruiter against the deubiquitinase OTUB1 that we have linked to the mutant {Delta}F508-CFTR targeting cystic fibrosis drug Lumacaftor to stabilize mutant CFTR protein in cells.

sentences: Curation, inference, and assessment of a globally reconstructed gene regulatory network for Streptomyces coelicolor Streptomyces coelicolor A3(2) is a model microorganism for the study of Streptomycetes, antibiotic production, and secondary metabolism in general. Even though S. coelicolor has an outstanding variety of regulators among bacteria, little effort to globally study its transcription has been made. We manually curated 29 years of literature and databases to assemble a meta-curated experimentally-validated gene regulatory network (GRN) with 5386 genes and 9707 regulatory interactions ([~]41% of the total expected interactions). This provides the most extensive and up-to-date reconstruction available for the regulatory circuitry of this organism. Only [~]6% (534/9707) are supported by experiments confirming the binding of the transcription factor to the upstream region of the target gene, the so-called "strong" evidence. While for the remaining interactions there is no confirmation of direct binding. To tackle network incompleteness, we performed network inference using several methods (including two proposed here) for motif identification in DNA sequences and GRN inference from transcriptomics. Further, we contrasted the structural properties and functional architecture of the networks to assess the reliability of the predictions, finding the inference from DNA sequence data to be the most trustworthy approach. Finally, we show two applications of the inferred and the curated networks. The inference allowed us to propose novel transcription factors for the key Streptomyces antibiotic regulatory proteins (SARPs). The curated network allowed us to study the conservation of the system-level components between S. coelicolor and Corynebacterium glutamicum. There we identified the basal machinery as the common signature between the two organisms. The curated networks were deposited in Abasy Atlas (https://abasy.ccg.unam.mx/) while the inferences are available as Supplementary Material.

sentences: Transgenerational impact of aberrant inflammation in rat pregnancy Children of women with pre-eclampsia have increased risk of cardiovascular (CV) and metabolic disease in adult life. Furthermore, the risk of pregnancy complications is higher in daughters born to women affected by pre-eclampsia than in daughters born after uncomplicated pregnancies. While aberrant inflammation contributes to the pathophysiology of pregnancy complications, including pre-eclampsia, the contribution of maternal inflammation to subsequent risk of CV and metabolic disease as well as pregnancy complications in the offspring remains unclear. Here we demonstrate that 24-week-old female rats (F1) born to dams (F0) exposed to lipopolysaccharide (LPS) during pregnancy (to induce inflammation) exhibited mild systolic dysfunction, increased cardiac growth-related gene expression, abnormal glucose tolerance and coagulopathy; whereas male F1 offspring exhibited abnormal glucose tolerance and increased visceral fat accumulation compared with F1 sex-matched offspring born to saline-treated dams. Both male and female F1 offspring born to LPS-treated dams had evidence of anemia. Fetuses (F2) from F1 females born to LPS-treated dams were growth restricted, and this reduction in fetal growth was associated with increased CD68 positivity and decreased expression of glucose transporter-1 in their utero-placental units. These results indicate that abnormal maternal inflammation can contribute to increased risk of CV and metabolic disease in offspring, and that the effects of inflammation may be transgenerational. This study provides evidence in support of early screening for CV and metabolic disease, as well as pregnancy complications in offspring affected by pre-eclampsia or other pregnancy complications associated with aberrant inflammation.

sentences: Evolution under juvenile malnutrition impacts adult metabolism and fitness Juveniles are often first to suffer from nutrient shortage, and juvenile undernutrition is likely an important force of natural selection shaping animal physiology, with consequences potentially extending into adulthood. We combined RNAseq, targeted metabolomics and genomics to study the consequences of experimental evolution under juvenile undernutrition for metabolism of reproductively active adult females of Drosophila melanogaster. Compared to six Control populations maintained on standard diet, six Selected populations evolved for over 230 generations on a nutrient-poor larval diet showed major changes in adult gene expression and metabolite abundance. In particular, Selected flies were relatively deficient in essential amino acids and purine nucleotides, but showed overabundance of several non-essential amino-acids involved in purine synthesis and overexpression of multiple enzymes catalyzing this pathway. Selected flies also accumulated medium-chain acylcarnitines suggestive of congestion in beta-oxidation, possibly linked to deficiency of electron transporters. Some aspects of the metabolic profile of Selected flies resembled that of flies subject to starvation. Furthermore, differences between Selected and Control populations in adult gene expression were in general positively correlated with differences in larval expression, consistent with pleiotropy in gene regulation between the life stages. Finally, Selected flies were less fit in terms of fecundity than Controls even when both were raised under the conditions under which the Selected populations evolved. These results suggest that evolutionary adaptation to juvenile undernutrition has large pleiotropic consequences for adult metabolism, and that they are costly rather than adaptive for adult fitness.

sentences: Resource selection by the Endangered Arabian tahr: identifying critical habitats for conservation and climate change adaptation. The Arabian tahr is an Endangered mountain ungulate endemic to the Hajar Mountains of Arabia. The Arabian tahr population is in decline and threats to tahr habitat are intensifying, in addition new potential challenges from climate change are emerging. Fundamental to future conservation planning is understanding tahr habitat selection patterns, so we can prioritise habitat protection, and understand how habitat may be used to provide thermal refuge and allow adaptation to climate change impacts. We used GPS collars and resource selection functions to characterise Arabian tahr habitat preferences in Wadi Sareen Nature Reserve, Oman. We found tahr habitat selection was dependent on scale, sex and season. Vegetation resources were only selected at the smallest scales of selection and avoided at other scales. Habitat providing low heat load and thermal refuge were intensely selected at small and medium scales, by both sexes and in both seasons, suggesting the importance of thermal refuges in facilitating thermoregulation. Higher elevations, steep slopes and rugged habitats were selected across all scales tested here, and in previous landscape-scale studies, indicating the fundamental importance of these habitats in supporting Arabian tahr populations. Our results identified critical habitats required to sustain Arabian tahr, and demonstrated the importance of thermal refuges to species living in the hot climates such as the Arabian Peninsula. Given the accessibility of habitat layers, and ease in which the identified habitats can be mapped using a geographical information system, understanding the habitat selection of tahr and other species is a crucial step to increasing conservation management capacity of threatened species. Given our uncertainty of how to conserve wildlife under future climate change, understanding the availability and distribution of wildlife habitat is an important baseline from where we can plan, connect and preserve the resources necessary for wildlife conservation.

sentences: STOmicsDB: a database of Spatial Transcriptomic data Recent technological development in spatial transcriptomics allows researchers to measure gene expression of cells and their spatial locations at the almost single-cell level, which generates detailed biological insight into biological processes. However, specialized spatial transcriptomics databases are rare. Here, we present the Spatial TranscriptOmics DataBase (STOmicsDB), a user-friendly database with multifunctions including search of relevant publications and tools, public dataset visualization, customized specialized databases, new data archive, and online analysis. The current version of STOmicsDB consists of 141 curated spatial transcript datasets covering 12 species, and includes 5,618 spatial multi-omics publications and 674 tools. STOmicsDB is freely accessible at https://db.cngb.org/stomics/.

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Developmental and environmental plasticity in opsin gene expression in Lake Victoria cichlid fish In many organisms, sensory abilities develop and evolve according to the changing demands of navigating, foraging and communication across different environments and life stages. Teleost fish inhabit heterogeneous light environments and exhibit a large diversity in visual system properties among species. Cichlids are a classic example of this diversity, generated by different tuning mechanisms that involve both genetic factors and phenotypic plasticity. Here, we document the developmental progression of visual pigment gene expression in Lake Victoria cichlids and test if these patterns are influenced by variation in light conditions. We reared two sister species of Pundamilia to adulthood in two distinct visual conditions that resemble the two light environments that they naturally inhabit in Lake Victoria. We also included interspecific first-generation hybrids. We then quantified (using RT-qPCR) the expression of the four Pundamilia opsins (SWS2B, SWS2A, RH2A and LWS) at 14 time points. We find that opsin expression profiles progress from shorter-wavelength sensitive opsins to longer-wavelength sensitive opsins with increasing age, in both species and their hybrids. The developmental trajectories of opsin expression also responded plastically to the visual conditions. Finally, we found subtle differences between reciprocal hybrids, possibly indicating parental effects and warranting further investigation. Developmental and environmental plasticity in opsin expression may provide an important stepping stone in the evolution of cichlid visual system diversity.

Research highlightsIn Lake Victoria cichlid fish, expression levels of opsin genes (encoding visual pigments) differ between developmental stages and between experimental light treatments. This plasticity may contribute to the evolution of cichlid visual system diversity.

sentences: Flavobacterium johnsoniae Tyrosine Ammonia Lyase (FjTAL) in-silico Structure Prediction and Molecular Docking to L-Tyrosine, p-Coumaric Acid (pHCA) and Caffeic Acid Responsible for the conversion of L-tyrosine to p-coumaric acid in Flavobacterium johnsoniae, FjTAL has drawn the attention of many biochemical engineers who wish to carry out a sustainable biosynthetic scheme for the production of aromatic compounds. In this study, with the aid of various computational tools, the secondary and tertiary structures of FjTAL have been predicted. The results suggest that FjTAL forms a homo-tetramer when active as a cytosolic enzyme and it is mostly consisted of alpha helices. With the aid of molecular docking, one can hypothesize that FjTAL is likely to bind to L-tyrosine, p-coumaric acid, and caffeic acid with a similar molecular mechanism and thus, p-coumaric acid and caffeic acid may exhibit a negative feedback response toward the enzyme and inhibit its activity competitively. Two distinct binding pockets have been discovered, one of which contains highly conserved residues among several species. The residues which form the prosthetic group 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) also emerge in the evolutionary conserved binding pocket. The other discovered cavity, could either be a second binding site for the ligands or simply an artifact of the molecular docking task.

sentences: Neural basis for adaptive motor behavior during car driving Car driving is supported by motor skills trained through continuous daily practice. One of the skills unique to expert drivers is the ability to detect abrupt changes in the driving environment and then quickly adapt their operation mode to the changes. Previous functional neuroimaging studies on motor control investigated the mechanisms underlying behaviors adaptive to changes in control properties of simple experimental devices such as a computer mouse or a joystick. The switching of multiple internal models mainly engages adaptive behaviors and underlies the interplay between the cerebellum and frontoparietal network (FPN) regions as the neural process. However, it remains unclear whether the neural mechanisms identified through an experimental paradigm using such simple devices also underlie practical driving behaviors. In the current study, we measure functional magnetic resonance imaging (fMRI) activities while participants control a realistic driving simulator inside the MRI scanner. Here, the accelerator sensitivity of a virtual car is abruptly changed, requiring participants to respond to this change as quickly as possible. We first compare brain activities before and after the sensitivity change. As a result, sensorimotor areas, including the left cerebellum, increase their activities after the sensitivity change. Moreover, after the change, activity significantly increases in the inferior parietal lobe and dorsolateral prefrontal cortex, parts of the FPN regions. By contrast, the posterior cingulate cortex, a part of the default mode network, deactivates after the sensitivity change. Our results suggest that the neural bases found in previous experiments using the simpler devices can serve as the foundation of adaptive car driving. At the same time, this study also highlights the unique contribution of non-motor-related regions to addressing the high cognitive demands of driving.

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Tuberculostearic acid (TSA)-containing phosphatidylinositols as reliable marker to determine Mycobacterium tuberculosis bacterial burden It is estimated that approximately one-fourth of the world's population is infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agents of tuberculosis (TB). In this study, we present rationally developed molecular markers for bacterial burden, which are derived from mycobacterial phospholipids. Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PI) are present in all clinically relevant MTBC lineages investigated. For the major abundant lipid PI 16:0_19:0 (TSA), a detection limit equivalent to 102 colony forming units (CFU) was determined for bacterial cultures and approximately 103 for cell culture systems. We further developed a mass spectrometry based targeted lipid assay, which - in contrast to bacterial quantification on solid medium - can be performed within several hours including sample preparation. Translation of this indirect and culture-free detection approach allowed the determination of pathogen loads in infected murine macrophages, human neutrophils and murine lung tissue. We show that marker lipids inferred from the mycobacterial PIs are increased in peripheral blood mononuclear cells (PBMCs) of TB patients beyond the lipid metabolic background in comparison to healthy controls. In a small cohort of drug-susceptible TB patients elevated levels of these marker molecules were detected at therapy start and declined following successful anti-tuberculosis treatment. The concentration of TSA-containing PIs can be used as correlate for reliable and rapid quantification of Mycobacterium tuberculosis (Mtb) burden in experimental in vitro model systems and may also provide a clinically relevant tool for monitoring TB therapy.

One Sentence SummaryTuberculostearic acid containing phosphatidylinositols represent a novel, fast to measure, reliable correlate of Mycobacterium tuberculosis bacterial burden in experimental model systems, which makes a future clinical application conceivable.

sentences: Molecular coevolution of nuclear and nucleolar localization signals inside basic domain of HIV-1 Tat During evolution, viruses had to adapt to an increasingly complex environment of eukaryotic cells. Viral proteins that need to enter the cell nucleus or associate with nucleoli possess nuclear localization signals (NLSs) and nucleolar localization signals (NoLSs) for nuclear and nucleolar accumulation, respectively. As viral proteins are relatively small, acquisition of novel sequences seems to be a more complicated task for viruses than for eukaryotes. Here, we carried out a comprehensive analysis of the basic domain (BD) of HIV-1 Tat to show how viral proteins might evolve with NLSs and NoLSs without an increase in protein size. The HIV-1 Tat BD is involved in several functions, the most important being the transactivation of viral transcription. The BD also functions as an NLS, although it is substantially longer than a typical NLS. It seems that different regions in the BD could function as NLSs due to its enrichment with positively charged amino acids. Additionally, the high positive net charge inevitably causes the BD to function as an NoLS through a charge-specific mechanism. The integration of NLSs and NoLSs into functional domains enriched with positively charged amino acids might be a mechanism that allows the condensation of different functional sequences in small protein regions and, as a result, to reduce protein size, influencing the origin and evolution of NLSs and NoLSs in viruses.

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Upregulated-flotillins and sphingosine kinase 2 derail vesicular trafic to stabilize AXL and promote epithelial-mesenchymal transition Altered endocytosis and vesicular trafficking are major players during tumorigenesis. Flotillin overexpression, a feature observed in many invasive tumors, and identified as a marker of poor prognosis, induces a deregulated endocytic and trafficking pathway called Upregulated Flotillin-Induced Trafficking (UFIT). Here, we found that, in non tumoral mammary epithelial cells, induction of the UFIT pathway promotes epithelial-to-mesenchymal transition (EMT) and accelerates the endocytosis of several transmembrane receptors, including AXL, in flotillin-positive late endosomes. AXL overexpression, frequently observed in cancer cells, is linked to EMT and metastasis formation. In flotillin-overexpressing non-tumoral mammary epithelial cells and in invasive breast carcinoma cells, we found that the UFIT-pathway-mediated AXL endocytosis allows its stabilization and depends on sphingosine-kinase 2, a lipid kinase recruited in flotillin-rich plasma membrane-domains and endosomes.

Thus, the deregulation of vesicular trafficking following flotillin upregulation, and through sphingosine kinase 2, emerges as a new mechanism of AXL overexpression and EMT-inducing signaling pathway activation.

sentences: EagleImp-Web: A Fast and Secure Genotype Phasing and Imputation Web Service using Field-Programmable Gate Arrays Imputation web servers have been developed that allow phasing and imputation of genome-wide data without the need for own computing resources. However, their terms of use, data sharing with third parties, the security architecture of the web service, and the algorithms and parameter settings used are only partially disclosed. We developed EagleImp-Web, a fast, secure and convenient web service for phasing and imputation of genome-wide data. The web service uses technical improvements in phasing and imputation algorithms and a field-programmable gate array (FPGA) accelerator design to reduce computation time without loss of phasing and imputation quality. Other key features include no exposure of user information and input/output data to third parties, high data security and fast secure download, user authentication through 2-factor authentication, full control in managing user accounts, and full transparency of algorithms and their settings. EagleImp-Web provides simple and convenient functionalities for monitoring running jobs and selecting parameter settings and output information. Due to the speed advantage over a purely CPU-based implementation, EagleImp-Web offers the user the ability to choose a more resource-intensive parameter setting in exchange for computation time to further improve phasing and imputation quality. EagleImp-Web is freely availabe at https://hybridcomputing.ikmb.uni-kiel.de.

sentences: Seasonal dynamics and repeatability of gastrointestinal parasite burdens in wild Soay sheep Seasonality is a ubiquitous feature of wildlife disease ecology, but is determined by a complex interplay of environmental, parasitological and host factors. Gastrointestinal parasites often exhibit strong seasonal dynamics in wild vertebrate populations due to, for example, environmental influences on free-living or vectored life stages, and variation in the physiological and immune status of hosts across their annual cycle. At the same time, wild populations are typically infected with multiple parasites. The seasonal dynamics of co-infecting parasites may differ depending on age and reproductive status, and associations among parasites may be driven by short-term within-individual changes or longer-term interactions that are consistent among hosts. Here, we used faecal samples and egg counts collected repeatedly from individually marked and monitored wild Soay sheep that were part of a long-term study to investigate seasonal dynamics of six gastrointestinal parasite groups (strongyle nematodes, coccidian protozoa, Capillaria, Strongyloides, Nematodirus, and Moniezia). Prevalence and abundance generally tended to be higher spring and summer, and burdens were higher in lambs than adults. Within the highly prevalent strongyle nematode group, we found differences in seasonality of egg counts depending on adult reproductive status. Reproductive ewes had increased counts in spring around the time of birth followed by a drop in abundance in summer, while barren ewes showed little evidence of seasonality. Males showed a sustained rise in egg counts through spring and summer, and sex differences were only strongly apparent in summer. In contrast, in similarly prevalent coccidia we found a peak in faecal oocyst counts in spring but no differences in seasonality among males, barren and pregnant ewes. Using multivariate mixed-effects models, we went on to show that both strongyle and coccidia counts are moderately repeatable across seasons among individuals. We further show that apparent positive correlation between strongyle and coccidia counts was driven by short-term within-individual changes in both parasite burdens rather than long-term among-individual covariation. Overall, our results demonstrate that seasonality varies across demographic and parasite groups and highlight the value of investigating fluctuating susceptibility and exposure over time for understanding epidemiology of a population.

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A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and catalytic activity is essential in the parasitic protozoan Leishmania Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite Leishmania. While GDP-Fucose synthesis is essential (Guo et al 2017), fucosylated glycoconjugates have not been reported in Leishmania major. Four predicted fucosyltransferases appear conventionally targeted to the secretory pathway; SCA1/2 play a role in side-chain modifications of lipophosphoglycan, while gene deletion studies here showed that FUT2 and SCAL were not essential. Unlike most eukaryotic glycosyltransferases, the predicted  1-2 fucosyltransferase encoded by FUT1 localized to the mitochondrion. A quantitative  plasmid segregation assay, expressing FUT1 from the multicopy episomal pXNG vector in a chromosomal null {Delta}fut1- background, established that FUT1 is essential. Similarly "plasmid shuffling" confirmed that both enzymatic activity and mitochondrial localization were required for viability, comparing import-blocked or catalytically inactive enzymes respectively. Enzymatic assays of tagged proteins expressed in vivo or of purified recombinant FUT1 showed it had a broad fucosyltransferase activity including glycan and peptide substrates. Unexpectedly a single rare {Delta}fut1-s segregant ({Delta}fut1s) was obtained in rich media, which showed severe growth defects accompanied by mitochondrial dysfunction and loss, all of which were restored upon FUT1 re-expression. Thus, FUT1 along with the similar Trypanosoma brucei enzyme TbFUT1 (Bandini et al 2021) joins the eukaryotic O-GlcNAc transferase isoform as one of the few glycosyltransferases acting within the mitochondrion. Trypanosomatid mitochondrial FUT1s may offer a facile system for probing mitochondrial glycosylation in a simple setting and their essentiality renders it an attractive target for chemotherapy of these serious human pathogens.

Significance StatementAbundant surface glycoconjugates play key roles in the infectious cycle of protozoan parasites including Leishmania. Through defining biosynthetic pathways we identified a fucosyltransferase FUT1 that was localized to the parasite mitochondrion, an atypical compartment for glycosyltransferases. FUT1 was essential for normal growth, requiring both mitochondrial localization and enzymatic activity. Loss of FUT1 in a unique segregant showed extensive mitochondrial defects. Enzymatic tests showed FUT1 could fucosylate glycan and peptide substrates in vitro, although as yet the native substrate is unknown. Trypanosomatid mitochondrial FUT1s may offer a facile system in the future for probing mitochondrial glycosylation in a setting uncomplicated by multiple isoforms targeted to diverse compartments, and its essentiality renders it an attractive target for chemotherapy of these deadly parasites.

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Effect of a low concentration in chloride, sodium and potassium on oocyte maturation, oocyte hydration, ovulation and egg quality in rainbow trout. Water salinity is an important environmental factor known to have detrimental effects on salmonid reproduction, mostly when migrating female broodfish are held in sea water. In contrast, data obtained in freshwater are scarce and the impact of low water salinity, and more specifically of low water concentrations in sodium, chloride and potassium, during reproduction in freshwater is currently unknown. For this reason, and because ion and water fluxes are critical for the final steps of the female gamete formation, including oocyte hydration and ovulation, the aim of the present study was to investigate the impact of low salinity water on final oocyte maturation, ovulation and, ultimately, on egg quality, using rainbow trout as a physiological model and relevant aquaculture species.

Fish from the same commercial strain were raised either in a site characterized by low concentrations of Na+, K+, and Cl- ions in the water or in a closely located control site exhibiting higher concentration in these elements. Egg quality and duration of final oocyte maturation were investigated using innovative phenotyping tools such as automatic assessment of egg viability using the VisEgg system and non-invasive echograph-based monitoring of final oocyte maturation duration, respectively. Oocyte hydration during final oocyte maturation and after ovulation was also investigated. Finally, molecular phenotyping was performed using real-time PCR-based monitoring of several key players of final oocyte maturation and ovulation associated with ion and water transport, inflammation, proteolytic activity, and coagulation. Oocyte hydration and gene expression data were analyzed in the light of the duration of final oocyte maturation.

Here we show that low water salinity (i.e., low water concentration in chloride, sodium and potassium) negatively influences final oocyte maturation, ovulation and, ultimately, egg quality. Low water salinity triggered delayed ovulation and lower oocyte viability. When investigating the impact of low water salinity on final oocyte maturation duration, individuals presenting the most severe phenotypes exhibited impaired oocyte hydration and abnormally reduced gene expression levels of several key players of the ovulatory process. While the under expression of water (i.e., aquaporins) and ion (i.e., solute carriers) transporters is consistent with impaired oocyte hydration, our observations also indicate that the overall ovulatory gene expression program is disrupted. Our results raise the question of the mechanisms underlying the negative influence of low salinity water on the dynamics of the preovulatory phase, on the control of the oocyte homeostasis, including hydration, and on the overall success of the maturation-ovulation process.

HighlightsO_LILow water salinity impairs final oocyte maturation and egg quality in rainbow trout
C_LIO_LILow water salinity induces delayed ovulation and impaired oocyte hydration
C_LIO_LILow water salinity induces a dysregulation of several key ovulatory genes
C_LIO_LIMonitoring of final oocyte maturation can be performed using ultrasound staging
C_LI

sentences: Fear in the mind's eye: Mental imagery can generate and regulate acquired differential fear conditioned reactivity Mental imagery is an important tool in the cognitive control of emotion. The present study tests the prediction that visual imagery can generate and regulate differential fear conditioning via the activation and prioritization of stimulus representations in early visual cortices. We combined differential fear conditioning with manipulations of viewing and imagining basic visual stimuli in humans. We discovered that mental imagery of a fear-conditioned stimulus compared to imagery of a safe conditioned stimulus generated a significantly greater conditioned response as measured by self-reported fear, the skin conductance response, and right anterior insula activity (experiment 1). Moreover, mental imagery effectively down- and up-regulated the fear conditioned responses (experiment 2). Multivariate classification using the functional magnetic resonance imaging data from retinotopically defined early visual regions revealed significant decoding of the imagined stimuli in V2 and V3 (experiment 1) but significantly reduced decoding in these regions during imagery-based regulation (experiment 2). Together, the present findings indicate that mental imagery can generate and regulate a differential fear conditioned response via mechanisms of the depictive theory of imagery and the biased-competition theory of attention. These findings also highlight the potential importance of mental imagery in the manifestation and treatment of psychological illnesses.

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Social hosts evade predation but have deadlier parasites Parasites exploit hosts to replicate and transmit, but overexploitation kills host and parasite (1): predators may shift this cost-benefit balance by consuming hosts (2-4) or changing host behavior, but the strength of these effects remains unclear. Modeling both, we find a primary, strong effect: hosts group to defend against predators (5), increasing parasite transmission, thus multiple infections, and therefore favoring more exploitative, virulent, parasites (6). Indeed, among 18 Trinidadian Gyrodactyus spp. parasite lines, those collected from high predation guppy populations were more virulent in common garden than those from low predation populations. Our model accurately predicted this result when parametrized with our experimentally demonstrated virulence-transmission trade-off, implicating the behavioral effects of predation. Broadly, our results indicate that reduced social contact selects against parasite virulence.

One-Sentence SummaryOur theory and data show predators cause increased host social grouping; the resulting transmission favors parasite virulence.

sentences: Structural basis of the P4B ATPase lipid flippase activity P4 ATPases are lipid flippases that are phylogenetically grouped into P4A, P4B and P4C clades. The P4A ATPases are heterodimers composed of a catalytic -subunit and accessory {beta}-subunit, and the structures of several heterodimeric flippases have been reported. The S. cerevisiae Neo1 and its orthologs represent the P4B ATPases, which function as monomeric flippases without a {beta}-subunit. It has been unclear whether monomeric flippases retain the architecture and transport mechanism of the dimeric flippases. Here we report the first structure of a P4B ATPase, Neo1, in its E1-ATP, E2P-transition, and E2P states. The structure reveals a conserved architecture as well as highly similar functional intermediate states relative to dimeric flippases. Consistently, structure-guided mutagenesis of residues in the proposed substrate translocation path disrupted Neo1s ability to establish membrane asymmetry. These observations indicate that evolutionarily distant P4 ATPases use a structurally conserved mechanism for substrate transport.

sentences: The natural sequence of events in larval settlement and metamorphosis of Hydroides elegans (Polychaeta; Serpulidae) The broadly distributed serpulid worm Hydroides elegans has become a model organism for studies of marine biofouling, development and the processes of larval settlement and metamorphosis induced by surface microbial films. Contrasting descriptions of the initial events of these recruitment processes, whether settlement is induced by (1) natural multi-species biofilms, (2) biofilms composed of single bacterial species known to induce settlement, or (3) a bacterial extract stimulated the research described here. We found that settlement induced by natural biofilms or biofilms formed by the bacterium Pseudoalteromonas luteoviolacea is invariably initiated by attachment and secretion of an adherent and larva-enveloping primary tube, followed by loss of motile cilia and ciliated cells and morphogenesis. The bacterial extract containing complex tailocin arrays derived from an assemblage of phage genes incorporated into the bacterial genome appears to induce settlement events by destruction of larval cilia and ciliated cells, followed by attachment and primary-tube formation. Similar destruction occurred when precompetent larvae and larvae of a nudibranch gastropod were exposed to the extract, neither of which metamorphosed. We further argue that larvae that lose their cilia before attachment would be swept away from the sites that stimulated settlement by the turbulent flow characteristic of most marine habitats.

sentences: The Dual Mechanisms of Cognitive Control dataset, a theoretically-guided within-subject task fMRI battery Cognitive control is a critical higher mental function, which is subject to considerable individual variation, and is impaired in a range of mental health disorders. We describe here the initial release of Dual Mechanisms of Cognitive Control (DMCC) project data, the DMCC55B dataset, with 55 healthy unrelated young adult participants. Each participant performed four well-established cognitive control tasks (AX-CPT, Cued Task-Switching, Sternberg Working Memory, and Stroop) while undergoing functional MRI scanning. The dataset includes a range of state and trait self-report questionnaires, as well as behavioural tasks assessing individual differences in cognitive ability. The DMCC project is on-going and features additional components (e.g., related participants, manipulations of cognitive control mode, resting state fMRI, longitudinal testing) that will be publicly released following study completion. This DMCC55B subset is released early with the aim of encouraging wider use and greater benefit to the scientific community. The DMCC55B dataset is suitable for benchmarking and methods exploration, as well as analyses of task performance and individual differences.

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Inferring inter- and intrachromosomal Dobzhansky-Muller incompatibilities                from imbalanced segregation of recombinant haplotypes in hybrid populations Dobzhansky-Muller incompatibilities (DMIs) are a major component of reproductive isolation between species. DMIs imply negative epistasis, and are exposed when two diverged populations hybridize. Mapping the locations of DMIs has largely relied on classical genetic mapping. Approaches to date are hampered by low power and the challenge of identifying DMI loci on the same chromosome, because strong initial linkage of parental haplotypes weakens statistical tests. Here, we propose new statistics to infer negative epistasis from haplotype frequencies in hybrid populations. When two divergent populations hybridize, the variance in heterozygosity at two loci decreases faster with time at DMI loci than at random pairs of loci. When two populations hybridize at near-even admixture proportions, the deviation of the observed variance from its expectation becomes negative for the DMI pair. This negative deviation enables us to detect intermediate to strong negative epistasis both within and between chromosomes. In practice, the detection window in hybrid populations depends on the demographic scenario, the recombination rate, and the strength of epistasis. When the initial proportion of the two parental populations is uneven, only strong DMIs can be detected with our method unless migration prevents parental haplotypes from being lost. We use the new statistics to infer candidate DMIs from three hybrid populations of swordtail fish. We identify numerous new DMI candidates, some of which are inferred to interact with several loci within and between chromosomes. Moreover, we discuss our results in the context of an expected enrichment in intrachromosomal over interchromosomal DMIs.

Author SummaryGenetic incompatibility in the form of (Bateson-)Dobzhansky-Muller incompatibilities (DMIs) is an important component of reproductive isolation between species. However, the evolutionary role of DMIs during the process of speciation is contentious. DMIs occur when two or more genetic variants interact to reduce their carriers fitness. Once recombination combines incompatible variants in hybrids, selection acts to remove these variants from the population. One step towards addressing the evolutionary role of DMIs is to quantify the prevalence of DMIs in incipient and hybridizing species. Here, we present statistics that are sensitive to the resulting recombinant imbalance and that can indicate the location of DMIs in hybrid genomes in various demographic scenarios. We use simulations to show that the time window during which a DMI is detectable depends on its genomic location, the severity of the DMI, and the populations demography. Importantly, our statistic distinguishes genetic associations arising due to physical linkage from those arising due to gene interactions, which allows for the inference of both inter- and intrachromosomal DMIs. Applying our statistics to three hybrid populations of swordtail fish, we confirm previously known DMIs and identify new candidate incompatibilities.

sentences: Active haptic training improves somatosensory-motor feedback control in the motor skills of experts A common problem experts experience is the challenge of overcoming the ceiling effects of highly trained sensorimotor skills. Recent studies have demonstrated that the amount of practice fails to explain interindividual variation in experts motor expertise, implicating hidden bottlenecks that prevent experts from overcoming this ceiling. Here, we show that somatosensory function in motion is one that defines further improvement of the sensorimotor skills of expert pianists. We found that even in expert pianists, specialized training that was designed to enhance somatosensory perception in motion improved sensorimotor skills in which feedback, but not feedforward, control plays a predominant role. Furthermore, training increased reliance on afferent information in motion relative to predictive information that could originate from internal models of movement. Together, these findings suggest that enhancement of somatosensory processing in motion improves the somatosensory-motor feedback loop and thereby enabling experts to surpass the ceiling that initially limited their motor expertise.

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Synthetic dysmobility screen unveils an integrated STK40-YAP-MAPK system driving cell migration Integrating signals is essential for cell survival, leading to the concept of synthetic lethality. However, how signaling is integrated to control cell migration remains unclear. By conducting a "two-hit" screen, we revealed the synergistic reduction of cell migration when serine-threonine kinase 40 (STK40) and mitogen-activated protein kinase (MAPK) were simultaneously suppressed. Single-cell analyses showed that STK40 knockdown reduced cell motility and coordination by strengthening focal adhesion (FA) complexes. Furthermore, STK40 knockdown reduced translocation of yes-associated protein (YAP) into the nucleus, while MAPK inhibition further weakened YAP activities in the nucleus to disturb FA remodeling. Altogether, we unveiled an integrated STK40-YAP-MAPK system regulating cell migration, and introduced "synthetic dysmobility" as a novel strategy to collaboratively control cell migration.

One Sentence SummaryBlocking collaborative pathways within the integrated signaling network synergistically disrupts the migration of cells.

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Eye-hand coordination during online reach corrections is task-dependent To produce accurate movements, the human motor system needs to deal with errors that can occur due to inherent noise, changes in the body, or disturbances in the environment. Here, we investigated the temporal coordination of rapid corrections of the eye and hand in response to a change in visual target location during the movement. In addition to a  classic double-step task in which the target stepped to a new position, participants performed a set of modified double-step tasks in which the change in movement goal was indicated by the appearance of an additional target, or by a spatial or symbolic cue. We found that both the absolute correction latencies of the eye and hand and the relative eye-hand correction latencies were dependent on the visual characteristics of the target change, with increasingly longer latencies in tasks that required more visual and cognitive processing. Typically, the hand started correcting slightly earlier than the eye, especially when the target change was indicated by a symbolic cue, and in conditions where visual feedback of the hand position was provided during the reach. Our results indicate that the oculomotor and limb-motor system can be differentially influenced by processing requirements of the task and emphasize that temporal eye-hand coordination is flexible rather than rigid.

New & NoteworthyEye movements support hand movements in many situations. Here we used variations of a double-step task to investigate temporal coupling of corrective hand and eye movements in response to target displacements. Correction latency coupling depended on the visual and cognitive processing demands of the task. The hand started correcting before the eye, especially when the task required decoding a symbolic cue. These findings highlight the flexibility and task-dependency of eye-hand coordination.

sentences: Analysis of diverse eukaryotes suggests the existence of an ancestral 1 mitochondrial apparatus derived from the bacterial type II secretion system The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.

sentences: The integrated role of resource memory and scent-mediated territoriality in the emergence of home-ranges Despite decades of animal movement research, an integrated understanding of the processes underlying the emergence of home-ranges remains inadequate. We explored the effects of integrating two key processes of home-ranging arising from individual movement decisions: (i) memory-based resource use; and (ii) scent-based territoriality. Both mechanisms together led to the formation of exclusive, resource- and population density-dependent home-ranges, which were responsive to perturbations in the conspecific environment (i.e., removing individuals). Home-range patterns (size, size skewness and overlap) were primarily influenced by memory and scent decay rate parameters, demonstrating that home-ranging is ultimately a balance between an animals inherent exploratory tendency and its desire to avoid conspecifics. Model application to a population of feral cats demonstrated that general space use patterns could be approximated through simulation, and replication of finer-scale space use patterns is plausible with further model development. Our modelling framework provides a foundation for sophisticated theoretical models of space use in interacting animals.

sentences: Simulating lesion-dependent functional recovery mechanisms Functional recovery after brain damage varies widely and depends on many factors, including lesion site and extent. When a neuronal system is damaged, recovery may occur by engaging residual (e.g., perilesional) components. When damage is extensive, recovery depends on the availability of other intact neural structures that can reproduce the same functional output (i.e., degeneracy). A systems response to damage may occur rapidly, require learning or both. Here, we simulate functional recovery from four different types of lesions, using a generative model of word repetition that comprised a default premorbid system and a less used alternative system. The synthetic lesions (i) completely disengaged the premorbid system, leaving the alternative system intact, (ii) partially damaged both premorbid and alternative systems, and (iii) limited the experience-dependent plasticity of both. The results, across 1000 trials, demonstrate that (i) a complete disconnection of the premorbid system naturally invoked the engagement of the other, (ii) incomplete damage to both systems had a much more devastating long-term effect on model performance and (iii) the effect of reducing learning capacity within each system. These findings contribute to formal frameworks for interpreting the effect of different types of lesions.

sentences: Regulatory dynamics distinguishing desiccation tolerance strategies within resurrection grasses Desiccation tolerance has evolved recurrently in grasses using two unique strategies to mitigate photooxidative damage under anhydrobiosis. The grass Oropetium thomaeum protects and retains chlorophyll, thylakoids, and the photosynthetic apparatus during desiccation (Homoiochlorophyly), while Eragrostis nindensis degrades and resynthesizes these components under desiccation and rehydration (Poikilochlorophyly). Here, we surveyed chromatin architecture and gene expression during desiccation in these two closely related species to identify regulatory dynamics underlying the distinct desiccation tolerance strategies in grasses. In both grasses, we observed a strong association between nearby chromatin accessibility and gene expression in desiccated tissues compared to well-watered, reflecting an unusual chromatin stability under anhydrobiosis. Integration of chromatin accessibility (ATACseq) and expression data (RNAseq) revealed a core desiccation response across these two grasses including many genes with binding sites for the core seed development transcription factor ABI5. O. thomaeum had a unique set of desiccation induced genes and regulatory elements associated with photoprotection, pigment biosynthesis, and response to high light, reflecting its adaptation of homoiochlorophyly. A tandem array of early light induced proteins (ELIPs) had massive shifts in gene expression and chromatin openness under desiccation in only O. thomaeum, and ELIPs acquired a novel desiccation related cis-regulatory motif, reflecting regulatory neofunctionalization during the evolution of desiccation tolerance. Together, our results highlight the complex regulatory and expression dynamics underlying desiccation tolerance in grasses.

sentences: A murine model of the human CREBRFR457Q obesity-risk variant does not influence energy or glucose homeostasis in response to nutritional stress Obesity and diabetes have strong heritable components, yet the genetic contributions to these diseases remain largely unexplained. In humans, a missense variant in Creb3 regulatory factor (CREBRF) [rs373863828 (p.Arg457Gln); CREBRFR457Q] is strongly associated with increased odds of obesity but decreased odds of diabetes. Although virtually nothing is known about CREBRFs mechanism of action, emerging evidence implicates it in the adaptive transcriptional response to nutritional stress downstream of TORC1. The objectives of this study were to generate a murine model with knockin of the orthologous variant in mice (CREBRFR458Q) and to test the hypothesis that this CREBRF variant promotes obesity and protects against diabetes by regulating energy and glucose homeostasis downstream of TORC1. To test this hypothesis, we performed extensive phenotypic analysis of CREBRFR458Q knockin mice at baseline and in response to acute (fasting/refeeding), chronic (low- and high-fat diet feeding), and extreme (prolonged fasting) nutritional stress as well as with pharmacological TORC1 inhibition. The results demonstrate that the murine CREBRFR458Q model of the human CREBRFR457Q variant does not influence energy/glucose homeostasis in response to these interventions. Alternative preclinical models and/or studies in humans will be required to decipher the mechanisms linking this variant to human health and disease.

sentences: Convergent loss of chemoreceptors across independent origins of slave-making in ants The evolution of an obligate parasitic lifestyle often leads to the reduction of morphological and physiological traits, which may be accompanied by loss of genes and functions. Slave-maker ants are social parasites that exploit the work force of closely related ant species for social behaviours such as brood care and foraging. Recent divergence between these social parasites and their hosts enables comparative studies of gene family evolution. We sequenced the genomes of eight ant species, representing three independent origins of ant slavery. During the evolution of eusociality, chemoreceptor genes multiplied due to the importance of chemical communication in societies. We investigated evolutionary patterns of chemoreceptors in relation to slave-making in ants. We found that slave-maker ant genomes harboured only half as many gustatory receptors as their hosts, potentially mirroring the outsourcing of foraging tasks to host workers. In addition, parasites had fewer odorant receptors and their loss shows patterns of convergence across origins of parasitism, representing a rare case of convergent molecular evolution. This convergent loss of specific odorant receptors suggests that selective deprivation of receptors is adaptive. The 9-exon odorant receptor subfamily, previously linked to social evolution in insects, was significantly enriched for convergent loss across the three origins of slavery in our study, indicating that the transition to social parasitism in ants is accompanied by the loss of receptors that are likely important for mediating eusocial behaviour. Overall, gene loss in slave-maker ants suggests that a switch to a parasitic lifestyle accompanies relaxed selection on chemical perception.

sentences: Circadian regulation of the transcriptome in a complex polyploid crop The circadian clock is a finely balanced time-keeping mechanism that coordinates programmes of gene expression. It is currently unknown how the clock regulates expression of homoeologous genes in polyploids. Here, we generate a high-resolution time-course dataset to investigate the circadian balance between sets of three homoeologous genes (triads) from hexaploid bread wheat. We find a large proportion of circadian triads exhibit imbalanced rhythmic expression patterns, with no specific sub-genome favoured. In wheat, period lengths of rhythmic transcripts are found to be longer and have a higher level of variance than in other plant species. Expression of transcripts associated with circadian controlled biological processes are largely conserved between wheat and Arabidopsis, however striking differences are seen in agriculturally critical processes such as starch metabolism. Together, this work highlights the ongoing selection for balance versus diversification in circadian homoeologs, and identifies clock-controlled pathways that might provide important targets for future wheat breeding.

sentences: Toxicology assessment of manganese oxide nanomaterials with enhanced electrochemical properties using human in vitro models representing different exposure routes In the present study, a comparative human toxicity assessment between newly developed Mn3O4 nanoparticles with enhanced electrochemical properties (GNA35) and their precursor material (Mn3O4) was performed, employing different in vitro cellular models representing alveolar, oral and dermal exposure routes, namely the human alveolar carcinoma epithelial cell line (A549), the human colorectal adenocarcinoma cell line (HT29), and the reconstructed 3D human epidermal model EpiDerm (RhE). The obtained results showed that Mn3O4 and GNA35 harbour similar morphological characteristics, while differences were observed in relation to their thermal stability and electrochemical properties. In regard to their toxicological properties, both nanomaterials induced oxidative stress in the A549 and HT29 cell lines that have demonstrated that manganese oxide nanoparticles reduce the cell viability in A549 cells while they do not produce any negative effect on that of HT29 cells. On the other hand, it was observed that Mn3O4 and GNA35 induce oxidative stress in both cell lines. Finally, it was noticed that none of the nanoparticles caused a reduction of the viability on the skin tissue so that they could not be classified as irritants. Our findings demonstrate that the evaluation of the toxicity of nanoparticles using different models is a critical aspect to increase the knowledge on their potential impact on human health.

sentences: DiSCs: Domains involving SETDB1 and Cohesin are critical regulators of genome topology and stem cell fate SETDB1 is a key regulator of lineage-specific genes and endogenous retroviral elements (ERVs) through its deposition of repressive H3K9me3 mark. Apart from its H3K9me3 regulatory role, SETDB1 has seldom been studied in terms of its other potential regulatory roles. To investigate this, a genomic survey of SETDB1 binding in mouse embryonic stem cells across multiple libraries was conducted, leading to the unexpected discovery of regions bereft of common repressive histone marks (H3K9me3, H3K27me3). These regions were enriched with the CTCF motif that is often associated with the topological regulator Cohesin. Further profiling of these non-H3K9me3 regions led to the discovery of a cluster of non-repeat loci that were co-bound by SETDB1 and Cohesin. These regions, which we named DiSCs (Domains involving SETDB1 and Cohesin) were seen to be proximal to the gene promoters involved in embryonic stem cell pluripotency and lineage development. Importantly, it was found that SETDB1-Cohesin co-regulate target gene expression and genome topology at these DiSCs. Depletion of SETDB1 led to localized dysregulation of Cohesin binding thereby locally disrupting topological structures. Dysregulated gene expression trends revealed the importance of this cluster in ES cell maintenance as well as at gene  islands that drive differentiation to other lineages. The  unearthing of the DiSCs thus unravels a unique topological and transcriptional axis of control regulated chiefly by SETDB1.

sentences: Imaging protein conformational space in liquid water Recent developments in electron-transparent materials have paved the way for liquid-phase electron microscopy (LPEM) leading to an unprecedented understanding of the structure and dynamics of specimens in their liquid environment. Image reconstruction in liquid-state poses several challenges, and most importantly, it undermines the single-particle analysis assumption that the three-dimensional objects captured on the image sensor are identical. We propose the combination of all-atom simulations with LPEM to complement structural studies with dynamic investigations. In this work, we exploited LPEM to image the dynamics of particles undergoing Brownian motion, using their natural rotation to access the particle structural landscape for reconstructing its architecture in 3D using tomographic techniques. We have selected two test cases for our approach according to prior data accessibility and physiological environment factors: apoferritin and archaeal RNA polymerase. We show that the adopted approach allows to achieve sub-nanometer spatial resolutions of protein structures, either imaging proteins one by one and assessing different conformational states or combining several proteins into one statistical conformational ensemble.

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Increased signal to noise ratios within experimental field trials by regressing spatially distributed soil properties as principal components. Environmental variability poses a major challenge to any field study. Researchers attempt to mitigate this challenge through replication. Thus, the ability to detect experimental signals is determined by the degree of replication and the amount of environmental variation, noise, within the experimental system. A major source of noise in field studies comes from the natural heterogeneity of soil properties which create micro-treatments throughout the field. To make matters worse, the variation within different soil properties is often non-randomly distributed across a field. We explore this challenge through a sorghum field trial dataset with accompanying plant, microbiome and soil property data. Diverse sorghum genotypes and two watering regimes were applied in a split-plot design. We describe a process of identifying, estimating, and controlling for the effects of spatially distributed soil properties on plant traits and microbial communities using minimal degrees of freedom. Importantly, this process provides a tool with which sources of environmental variation in field data can be identified and removed, improving our ability to resolve effects of interest and to quantify subtle phenotypes.

IMPORTANCEData from field experiments are notoriously noisy. Proper field designs with high replication aid in mitigating this challenge, yet true biological correlations are still often masked by environmental variability. This work identifies soil property composition as a spatially distributed source of variance to three types of characteristics: plant phenotype, microbiome composition, and leaf traits. We show that once identified, spatial principal component regression was able to account for these effects so that more precise estimates of experimental factors were obtained. This generalizable method is applicable to diverse field experiments.

sentences: A UAV-based high-throughput phenotyping approach to assess time-series nitrogen responses and identify traits associated genetic components in maize Advancements in the use of genome-wide markers have provided new opportunities for dissecting the genetic components that control phenotypic trait variation. However, cost-effectively characterizing agronomically important phenotypic traits on a large scale remains a bottleneck. Unmanned aerial vehicle (UAV)-based high-throughput phenotyping has recently become a prominent method, as it allows large numbers of plants to be analyzed in a time-series manner. In this experiment, 233 inbred lines from the maize diversity panel were grown in a replicated incomplete block under both nitrogen-limited conditions and following conventional agronomic practices. UAV images were collected during different plant developmental stages throughout the growing season. A pipeline for extracting plot-level images, filtering images to remove non-foliage elements, and calculating canopy coverage and greenness ratings based on vegetation indices (VIs) was developed. After applying the pipeline, about half a million plot-level image clips were obtained for 12 different time points. High correlations were detected between VIs and ground truth physiological and yield-related traits collected from the same plots, i.e., Vegetative Index (VEG) vs. leaf nitrogen levels (Pearson correlation coefficient, R = 0.73), Woebbecke index vs. leaf area (R = -0.52), and Visible Atmospherically Resistant Index (VARI) vs. 20 kernel weight - a yield component trait (R = 0.40). The genome-wide association study was performed using canopy coverage and each of the VIs at each date, resulting in N = 29 unique genomic regions associated with image extracted traits from three or more of the 12 total time points. A candidate gene Zm00001d031997, a maize homolog of the Arabidopsis HCF244 (high chlorophyll fluorescence 244), located underneath the leading SNPs of the canopy coverage associated signals that were repeatedly detected under both nitrogen conditions. The plot-level time-series phenotypic data and the trait-associated genes provide great opportunities to advance plant science and to facilitate plant breeding.

sentences: specificity	: an R package for generalized specificity analysis, with example analyses of Hawaiian foliar endophytic Fungi, human gut microbiome bacteria, and Antarctic glacier bacteria. Understanding the factors that influence microbes environmental distributions is important for determining drivers of microbial community composition. Species distributions are governed by parameters that influence their dispersal or survival. These include environmental variables like temperature and pH, and higher-dimensional variables like geographic distance and host species phylogenies. In microbial ecology, "specificity" is often described in the context of symbiotic or host parasitic interactions, but specificity can be more broadly used to describe the extent to which a species occupies a narrower range of an environmental variable than expected by chance. Using a standardization we describe here, Raos (1982, 2010) Quadratic Entropy can be conveniently applied to calculate specificity of a focal species to many different environmental variables, including 1-dimensional variables (vectors), dissimilarity matrices, phylogenies, and ontologies. We present our R package specificity for performing the above analyses, and apply it to four real-life microbial datasets to demonstrate its application: fungi living within the leaves of native Hawaiian plants, bacteria from the human gut microbiome, bacteria living within Antarctic glacier ice, and the Earth Microbiome Project data set. Finally, we present an interactive visualization and data exploration tool for these analyses, available as a companion R-package called specificity.shiny.

sentences: Resistance training rejuvenates the mitochondrial methylome in aged human skeletal muscle Resistance training (RT) alters skeletal muscle nuclear DNA methylation patterns (or the methylome). However, no study has examined if RT affects the mitochondrial DNA (mtDNA) methylome. Herein, ten older untrained males (65{+/-}7 years old) performed six weeks of full-body RT (twice weekly). Body composition and knee extensor torque were assessed prior to and 72 hours following the last RT session. Vastus lateralis (VL) biopsies were also obtained. VL DNA was subjected to reduced representation bisulfite sequencing providing excellent coverage across the ~16-kilobase mtDNA methylome (254 CpG sites). Various biochemical assays were also performed, and older male data were compared to younger trained males (22{+/-}2 years old, n=7). RT increased whole-body lean tissue mass (p=0.017), VL thickness (p=0.012), and knee extensor torque (p=0.029) in older males. RT also profoundly affected the mtDNA methylome in older males, as 63% (159/254) of the CpG sites demonstrated reduced methylation (p<0.05). Notably, several mtDNA sites presented a more "youthful" signature after RT in older males when comparisons were made to younger males. The 1.12 kilobase D-loop/control region on mtDNA, which regulates mtDNA replication and transcription, possessed enriched hypomethylation in older males following RT. Enhanced expression of mitochondrial H- and L-strand genes and increases in mitochondrial complex III and IV protein levels were also observed (p<0.05). This is the first study to show RT alters the mtDNA methylome in skeletal muscle. Observed methylome alterations may enhance mitochondrial transcription, and RT remarkably evokes mitochondrial methylome profiles to mimic a more youthful signature in older males.

sentences: The BNT162b2 mRNA vaccine induces polyfunctional T cell responses with features of longevity. Vaccination against SARS-CoV-2 infection has shown to be effective in preventing hospitalization for severe COVID-19. However, multiple reports of break-through infections and of waning antibody titers have raised concerns on the durability of the vaccine, and current discussions on vaccination strategies are centered on evaluating the opportunity of a third dose administration. Here, we monitored T cell responses to the Spike protein of SARS-CoV-2 in 71 healthy donors vaccinated with the Pfizer-BioNTech mRNA vaccine (BNT162b2) for up to 6 months after vaccination. We find that vaccination induces the development of a sustained anti-viral memory T cell response which includes both the CD4+ and the CD8+ lymphocyte subsets. These lymphocytes display markers of polyfunctionality, are fit for interaction with cognate cells, show features of memory stemness, and survive in significant numbers the physiological contraction of the immune response. Collectively, this data shows that vaccination with BNT162b2 elicits an immunologically competent and potentially long-lived SARS-CoV-2-specific T cell population. Understanding the immune responses to BNT162b2 provides insights on the immunological basis of the clinical efficacy of the current vaccination campaign and may instruct future vaccination strategies.

sentences: U-RISC: An Ultra-high Resolution EM Dataset Challenging Existing Deep Learning Algorithms Connectomics is a developing field aiming at reconstructing the connection of the neural system at nanometer scale. Computer vision technology, especially deep learning methods used in image processing, has promoted connectomic data analysis to a new era. However, the performance of the state-of-the-art methods still falls behind the demand of scientific research. Inspired by the success of ImageNet, we present the U-RISC, an annotated Ultra-high Resolution Image Segmentation dataset for Cell membrane, which is the largest cell membrane annotated Electron Microscopy (EM) dataset with a resolution of 2.18nm/pixel. Multiple iterative annotations ensured the quality of the dataset. Through an open competition, we reveal that the performance of current deep learning methods still has a considerable gap with human-level, different from ISBI 2012, on which the performance of deep learning is close to human. To explore the causes of this discrepancy, we analyze the neural networks with a visualization method, attribution analysis. We find that in U-RISC, it requires a larger area around a pixel to predict whether the pixel belongs to the cell membrane or not. Finally, we integrate currently available methods to provide a new benchmark (0.67, 10% higher than the leader of competition, 0.61) for cell membrane segmentation on U-RISC and propose some suggestions in developing deep learning algorithms. The U-RISC dataset and the deep learning codes used in this paper will be publicly available.

sentences: Detecting Cd adaptation footprint in C. riparius with a multi-genomic approach Evolutionary processes and acquired tolerance to toxicants are important factors governing how animals respond to chemical exposure. Evidence for increased tolerance to cadmium (Cd), a widely distributed toxic metal in aquatic environments, in Chironomus riparius is conflicting and still questioned if it happens through phenotypic plasticity or genetic adaptation. The present study considered the relevance of directional environmental changes by increasing contaminant concentration in a multigenerational selection experiment. Evaluation of measurable life-cycle traits, transcriptomic responses and quantitative genetics from an evolve and resequencing (E&R) experiment were integrated to assess the potential of C. riparius to adapt to Cd. Survival tests revealed some adaptation to Cd exposure. Genomic analyses showed a strong, genome-wide selective response in all replicates, emphasizing that even control laboratory conditions continually exert selective pressure. The integration of transcriptomic and genomic data could isolate the genes related to Cd acquired resistance. Those genes could be linked to an efflux of metals. Therefore, it is possible to conclude that C. riparius can endure long-term Cd exposure also through genetic adaptation.

sentences: Behaviorally emergent hippocampal place maps remain stable during memory recall The hippocampus is critical for the formation and recall of episodic memories1, 2 which store past experience of events ( what) occurring at particular locations ( where) in time ( when). Hippocampal place cells, pyramidal neurons which show location-specific modulation of firing rates during navigation3, 4, together form a spatial representation of the environment. It has long been hypothesized that place cells serve as the neural substrate for long-term episodic memory of space5, 6. However, recent studies call to question this tenet of the field by demonstrating unexpected levels of representational drift in hippocampal place cells with respect to the duration of episodic memories in mice7, 8. In the present study, we examined behaviorally driven long-term organization of the place map, to resolve the relationship between memory and place cells. Leveraging the stability of two-photon calcium imaging, we tracked activity of the same set of CA1 pyramidal neurons during learning and memory recall in an operant, head-fixed, odorcued spatial navigation task. We found that place cells are rapidly recruited into task-dependent spatial maps, resulting in emergence of orthogonal as well as overlapping representations of space. Further, task-selective place cells used a diverse set of remapping strategies to represent changing task demands that accompany learning. We found behavioral performance dependent divergence of spatial maps between trial types occurs during learning. Finally, imaging during remote recall spanning up to 30 days revealed increased stabilization of learnt place cell maps following memory consolidation. Our findings suggest that a subset of place cells is recruited by rule based spatial learning, actively reconfigured to represent task-relevant spatial relationships, and stabilized following successful learning and consolidation.

sentences: Disentangling the roles of neocortical alpha/beta and hippocampal theta/gamma oscillations in human episodic memory formation To form an episodic memory, we must first process a vast amount of sensory information about a to-be-encoded event and then bind these sensory representations together to form a coherent memory. While these two cognitive capabilities are thought to have two distinct neural origins, with neocortical alpha/beta oscillations supporting information representation and hippocampal theta-gamma phase-amplitude coupling supporting mnemonic binding, evidence for a dissociation between these two neural markers is conspicuously absent. To address this, seventeen human participants completed a sequence-learning task that first involved processing information about three stimuli, and then binding these stimuli together into a coherent memory trace, all the while undergoing MEG recordings. We found that decreases in neocortical alpha/beta power during sequence perception, but not mnemonic binding, correlated with enhanced memory performance. Hippocampal theta/gamma phase-amplitude coupling, however, showed the opposite pattern; increases during mnemonic binding (but not sequence perception) correlated with enhanced memory performance. These results demonstrate that memory-related decreases in neocortical alpha/beta power and memory-related increases in hippocampal theta/gamma phase-amplitude coupling arise at distinct stages of the memory formation process. We speculate that this temporal dissociation reflects a functional dissociation in which neocortical alpha/beta oscillations could support the processing of incoming information relevant to the memory, while hippocampal theta-gamma phase-amplitude coupling could support the binding of this information into a coherent memory trace.

sentences: Innate behavior sequence progression by peptide-mediated interorgan crosstalk Innate behaviors consist of a succession of genetically-hardwired motor and physiological subprograms that can be coupled to drastic morphogenetic changes. How these integrative responses are orchestrated is not completely understood. Here, we provide insight into these mechanisms by studying pupariation, a multi-step innate behavior of fly larvae that is critical for survival during metamorphosis. We find that the steroid-hormone ecdysone triggers parallel pupariation neuromotor and morphogenetic subprograms, which include the induction of the relaxin-peptide hormone, Dilp8, in the epidermis. Dilp8 acts on six Lgr3-positive thoracic interneurons to couple both subprograms in time and to instruct neuromotor subprogram switching during behavior. Our work reveals that interorgan feedback gates progression between subunits of an innate behavior and points to an ancestral neuromodulatory function of relaxin signaling.

sentences: Spontaneous parthenogenesis in the parasitoid wasp Cotesia typhae: low frequency anomaly or evolving process? Hymenopterans are haplodiploids and unlike most other Arthropods they do not possess sexual chromosomes. Sex determination typically happens via the ploidy of individuals: haploids become males and diploids become females. Arrhenotoky is believed to be the ancestral reproduction mode in Hymenopterans, with haploid males produced parthenogenetically, and diploid females produced sexually. However, a number of transitions towards thelytoky (diploid females produced parthenogenetically) have appeared in Hymenopterans, and in most cases populations or species are either totally arrhenotokous or totally thelytokous. Here we present the case of Cotesia typhae (Fernandez-Triana), a Braconidae that produces parthenogenetic females at a low frequency. The phenotyping of two laboratory strains and one natural population showed that this frequency is variable, and that this rare thelytokous phenomenon also happens in the wild. Moreover, mated females from one of the laboratory strains produce a few parthenogenetic daughters among a majority of sexual daughters. The analysis of daughters of heterozygous virgin females allowed us to show that a mechanism similar to automixis with central fusion is very likely at play in C. typhae. This mechanism allows some parts of the genome to remain heterozygous, especially at the chromosomes centromeres, which can be advantageous depending on the sex determination system involved. Lastly, in most species, the origin of thelytoky is either bacterial or genetic, and an antibiotic treatment as well as PCR experiments did not demonstrate a bacterial cause in C. typhae. The unusual case of low parthenogenetic frequency described in this species constitutes another example of the fascinating diversity of sex determination systems in Arthropods.

sentences: Metabolomics of aging in primary fibroblasts from small and large breed dogs Among several animal groups (eutherian mammals, birds, reptiles) lifespan positively correlates with body mass over several orders of magnitude. Contradicting this pattern are domesticated dogs, with small dog breeds exhibiting significantly longer lifespans than large dog breeds. The underlying mechanisms of differing aging rates across body masses are unclear, but it is generally agreed that metabolism is a significant regulator of the aging process. Herein, we performed a targeted metabolomics analysis on primary fibroblasts isolated from small and large breed young and old dogs. Regardless of size, older dogs exhibited lower glutathione and ATP, consistent with a role for oxidative stress and bioenergetic decline in aging. Furthermore, several size-specific metabolic patterns were observed with aging, including: (i) An apparent defect in the lower half of glycolysis in large old dogs at the level of pyruvate kinase. (ii) Increased glutamine anaplerosis into the TCA cycle in large old dogs. (iii) A potential defect in co-enzyme A biosynthesis in large old dogs. (iv) Low nucleotide levels in small young dogs that corrected with age. (v) An age dependent increase in carnitine in small dogs that was absent in large dogs. Overall, these data support the hypothesis that alterations in metabolism may underlie the different lifespans of small versus large breed dogs, and further work in this area may afford potential therapeutic strategies to improve the lifespan of large dogs.

sentences: Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants by Modular, Tetravalent, Bi-paratopic Antibodies Neutralizing antibodies (nAbs) that target the SARS-CoV-2 spike protein are approved for treatment of COVID-19. However, with the emergence of variants of concern, there is a need for new treatment options. We report a novel format that enables modular assembly of bi-paratopic, tetravalent nAbs with antigen-binding sites from two distinct nAbs. The tetravalent nAb was purified in high yield, and it exhibited biophysical characteristics that were comparable to those of approved therapeutic antibodies. The tetravalent nAb bound to the spike protein trimer at least 100-fold more tightly than bivalent IgGs (apparent KD < 1 pM), and it exhibited extremely high potencies against a broad array of pseudoviruses, chimeric viruses, and authentic virus variants. Together, these results establish the tetravalent diabody-Fc-Fab as a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that greatly exceed those of conventional bivalent IgGs.

sentences: A Bayesian Analysis of Steady-State Enzyme Data leads to Estimates of Rate Constants and Uncertainties in a Multi-Step Reaction The microscopic rate constants that govern an enzymatic reaction are only directly measured under certain experimental set-ups, such as stopped flow, quenched flow, or temperature-jump assays; the majority of enzymology proceeds from steady state conditions which leads to a set of more easily-observable parameters such as kcat, KM, and observed Kinetic Isotope Effects (Dkcat). This paper further develops a model from Toney (2013) to estimate microscopic rate constants from steady-state data for a set of reversible, four-step reactions. This paper uses the Bayesian modeling software Stan, and demonstrates the benefits of Bayesian data analysis in the estimation of these rate constants. In contrast to the optimization methods employed often in the estimation of kinetic constants, a Bayesian treatment is more equipped to estimate the uncertainties of each parameter; sampling from the posterior distribution using Hamiltonian Monte Carlo immediately gives parameter estimates as mean or median of the posterior, and also confidence intervals that express the uncertainty of each parameter.

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XENet: Using a new graph convolution to accelerate the timeline for protein design on quantum computers Graph representations are traditionally used to represent protein structures in sequence design protocols where the folding pattern is known. This infrequently extends to machine learning projects: existing graph convolution algorithms have shortcomings when representing protein environments. One reason for this is the lack of emphasis on edge attributes during massage-passing operations. Another reason is the traditionally shallow nature of graph neural network architectures. Here we introduce an improved message-passing operation that is better equipped to model local kinematics problems such as protein design. Our approach, XENet, pays special attention to both incoming and outgoing edge attributes.

We compare XENet against existing graph convolutions in an attempt to decrease rotamer sample counts in Rosettas rotamer substitution protocol. This use case is motivating because it allows larger protein design problems to fit onto near-term quantum computers. XENet outperformed competing models while also displaying a greater tolerance for deeper architectures. We found that XENet was able to decrease rotamer counts by 40% without loss in quality. This decreased the problem size of our use case by more than a factor of 3. Additionally, XENet displayed an ability to handle deeper architectures than competing convolutions.

Author summaryGraphs data structures are ubiquitous in the field of protein design and are at the core of the recent advances in artificial intelligence brought forth by graph neural networks (GNNs). GNNs have led to some impressive results in modeling protein interactions, but are not as common as other tensor representations.

Most GNN architectures tend to put little to no emphasis on the information stored on edges; however, protein modeling tools often use edges to represent vital geometric relationships about residue pair interactions. In this paper, we show that a more advanced processing of edge attributes can lead to considerable benefits when modeling chemical data.

We introduce XENet, a new member of the GNN family that is shown to have improved ability to model protein residue environments based on chemical and geometric data. We use XENet to intelligently simplify the optimization problem that is solved when designing proteins. This task is important to us and others because it allows larger proteins to be designed on near-term quantum computers. We show that XENet is able to train on our protein modeling data better than existing methods, successfully resulting in a dramatic decrease in protein design sample space with no loss in quality.

sentences: Structural covariance of amygdala subregions is associated with trait aggression and endogenous testosterone in healthy individuals Many studies point toward volume reductions in the amygdala as a potential neurostructural marker for trait aggression. However, most of these findings stem from clinical samples, rendering unclear whether the findings generalize to non-clinical populations. Furthermore, the notion of neural networks suggests that interregional correlations in grey matter volume (i.e., structural covariance) can explain individual differences in aggressive behavior beyond local univariate associations. Here, we tested whether structural covariance between amygdala subregions and the rest of the brain is associated with self-reported aggression in a large sample of healthy young students (n=263; 51% women). Salivary testosterone concentrations were measured for a subset of n=76 participants (45% women), allowing us to investigate the influence of endogenous testosterone on structural covariance. Aggressive individuals showed enhanced covariance between superficial amygdala (SFA) and dorsal anterior insula (dAI), but lower covariance between laterobasal amygdala (LBA) and dorsolateral prefrontal cortex (dlPFC). These structural patterns overlap with functional networks involved in the genesis and regulation of aggressive behavior, respectively. With increasing endogenous testosterone, we observed stronger structural covariance between centromedial amygdala (CMA) and medial prefrontal cortex in men and between CMA and orbitofrontal cortex in women. These results speak for structural covariance of amygdala subregions as a robust correlate of trait aggression in healthy individuals. Moreover, regions that showed structural covariance with the amygdala modulated by either testosterone or aggression did not overlap, speaking for a more complex role of testosterone in human social behavior rather than the simple assumption that testosterone only increases aggressiveness.

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Glucose-dependent miR-125b is a negative regulator of β-cell function Impaired pancreatic {beta}-cell function and insulin secretion are hallmarks of type 2 diabetes. MicroRNAs are short non-coding RNAs that silence gene expression, vital for the development and function of {beta}-cells. We have previously shown that {beta}-cell specific deletion of the important energy sensor AMP-activated protein kinase (AMPK) results in increased miR-125b-5p levels. Nevertheless, the function of this miRNA in {beta}-cells is unclear. We hypothesized that miR-125b-5p expression is regulated by glucose and that this miRNA mediates some of the deleterious effects of hyperglycaemia in {beta}-cells. Here we show that islet miR-125b-5p expression is up-regulated by glucose in an AMPK-dependent manner and that short-term miR-125b-5p overexpression impairs glucose stimulated insulin secretion (GSIS) in the mouse insulinoma MIN6 cells and in human islets. An unbiased high-throughput screen in MIN6 cells identified multiple miR-125b-5p targets, including the transporter of lysosomal hydrolases M6pr and the mitochondrial fission regulator Mtfp1. Inactivation of miR-125b-5p in the human {beta}-cell line EndoC{beta}-H1 shortened mitochondria and enhanced GSIS, whilst mice overexpressing miR-125b-5p selectively in {beta}-cells (MIR125B-Tg) were hyperglycaemic and glucose intolerant. MIR125B-Tg {beta}-cells contained enlarged lysosomal structures and showed reduced insulin content and secretion. Collectively, we identify miR-125b as a glucose-controlled regulator of organelle dynamics that modulates insulin secretion.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: RNA sensing via LGP2 is essential for the induction of a type I IFN response in ADAR1 deficiency RNA editing by the enzyme Adenosine Deaminase Acting on RNA 1 (ADAR1) is an important mechanism by which cells avoid innate immune responses to some endogenous RNAs. In ADAR1-deficient cells, unedited self RNAs can form base-paired structures that resemble viral RNAs and inadvertently activate antiviral innate immune pathways that lead to the induction of type I interferon (IFN). Rare mutations in ADAR1 cause Aicardi-Goutieres Syndrome (AGS), a severe childhood autoinflammatory syndrome that is characterized by chronic and excessive type I IFN production and developmental delay. Conversely, ADAR1 dysfunction and consequent type I IFN production helps restrict tumor growth and potentiates the activity of some chemotherapy drugs. Induction of type I IFN in ADAR1-deficient cells is thought to be due to triggering of the cytosolic RIG-I-like receptor (RLR), MDA5, by unedited self RNAs. Here, we show that another RLR, LGP2, also has an essential role. We demonstrate that ADAR1-deficient human cells fail to mount a type I IFN response in the absence of LGP2 and this involves the canonical function of LGP2 as an RNA sensor and facilitator of MDA5-dependent signaling. Further, we show that the sensitivity of tumor cells to ADAR1 loss requires the presence of LGP2. Finally, we find that type I IFN induction in tumor cells depleted of ADAR1 and treated with some chemotherapeutics is fully dependent on the expression of LGP2. These findings highlight a central role for LGP2 in self RNA sensing with important clinical implications for the treatment of AGS as well as for the potential application of ADAR1-directed anti-tumor therapy.

sentences: The structure of Vibrio cholerae FeoC reveals conservation of the helix-turn-helix motif but not the cluster-binding domain Most pathogenic bacteria require ferrous iron (Fe2+) in order to sustain infection within hosts. The ferrous iron transport (Feo) system is the most highly-conserved prokaryotic transporter of Fe2+, but its mechanism remains to be fully characterized. Most Feo systems are composed of two proteins: FeoA, a soluble SH3-like accessory protein and FeoB, a membrane protein that translocates Fe2+ across a lipid bilayer. Some bacterial feo operons encode FeoC, a third soluble, winged-helix protein that remains enigmatic in function. We previously demonstrated that select FeoC proteins bind O2-sensitive [4Fe-4S] clusters via Cys residues, leading to the proposal that some FeoCs could sense O2 to regulate Fe2+ transport. However, not all FeoCs conserve these Cys residues, and FeoC from the causative agent of cholera (Vibrio cholerae) notably lacks any Cys residues, precluding cluster binding. In this work, we determined the NMR structure of VcFeoC, which is monomeric and conserves the helix-turn-helix domain seen in other FeoCs. In contrast, however, the structure of VcFeoC reveals a truncated winged {beta}-sheet in which the cluster-binding domain is notably absent. To test the interactions of VcFeoC with VcFeoB, we used NMR to demonstrate that these proteins interact in a 1:1 stoichiometry with a Kd of ca. 25 M. Finally, using homology modeling, we predicted the structure of VcNFeoB and used docking to identify an interaction site with VcFeoC, which is confirmed by NMR spectroscopy. These findings provide the first atomic-level structure of VcFeoC and contribute to a better understanding of its role vis-a-vis FeoB.

sentences: Reliability of Mouse Behavioural Tests of Anxiety: a Systematic Review and Meta-Analysis on the Effects of Anxiolytics. Animal research on anxiety and anxiety disorders relies on valid animal models of anxiety. However, the validity of widely used rodent behavioural tests of anxiety has repeatedly been questioned, as they often fail to produce consistent results across independent replicate studies using different study populations or different anxiolytic compounds. In this study, we assessed the sensitivity of behavioural tests of anxiety in mice to detect anxiolytic effects of drugs prescribed to treat anxiety in humans. To this end, we conducted a pre-registered systematic review of studies reporting tests of anxiolytic compounds against a control treatment using common behavioural tests of anxiety in mice. PubMed and EMBASE were searched on August 21st 2019 for studies published in English and 814 papers were identified for inclusion. Risk of bias was assessed based on Syrcles risk of bias tool and the Camarades study quality checklist on a randomly selected subsample of 180 papers. Meta-analyses on effect sizes of treatments using standardized mean differences (Hedges g) showed that only two of 17 test measures reliably detected effects of anxiolytic compounds other than diazepam. Further, we report considerable variation in both direction and size of effects of most anxiolytics on most outcome variables, indicating poor replicability of test results. This was corroborated by high heterogeneity in most test measures. Finally, we found an overall high risk of bias. Our findings indicate a general lack of sensitivity of common behavioural tests of anxiety in mice to anxiolytic compounds and cast serious doubt on both construct and predictive validity of most of those tests. The use of animals to model human conditions can be justified only if the expected results are informative, reproducible, and translatable. In view of scientifically valid and ethically responsible research, we call for a revision of behavioural tests of anxiety in mice and the development of more predictive tests.

sentences: Grandparent inference from genetic data: The potential for parentage-based tagging programs to identify offspring of hatchery strays Fisheries managers routinely use hatcheries to increase angling opportunity. Many hatcheries operate as segregated programs where hatchery-origin fish are not intended to spawn with natural-origin conspecifics in order to prevent potential negative effects on the natural-origin population. Currently available techniques to monitor the frequency with which hatchery-origin strays successfully spawn in the wild rely on either genetic differentiation between the hatchery- and natural-origin fish or extensive sampling of fish on the spawning grounds. We present a method to infer grandparent-grandchild trios using only genotypes from two putative grandparents and one putative grandchild. We developed estimators of false positive and false negative error rates and showed that genetic panels containing 500 - 700 single nucleotide polymorphisms or 200 - 300 microhaplotypes are expected to allow application of this technique for monitoring segregated hatchery programs. We discuss the ease with which this technique can be implemented by pre-existing parentage-based tagging programs and provide an R package that applies the method.

sentences: Effects of land use and weather on the presence and abundance of mosquito-borne disease vectors in a urban and agricultural landscape in Eastern Ontario, Canada Weather and land use can significantly impact mosquito abundance and presence, and by consequence, mosquito-borne disease (MBD) dynamics. Knowledge of vector ecology and mosquito species response to these drivers will help us better predict risk from MBD. In this study, we evaluated and compared the independent and combined effects of weather and land use on mosquito species occurrence and abundance in Eastern Ontario, Canada. Data on occurrence and abundance (245,591 individuals) of 30 mosquito species were obtained from mosquito capture at 85 field sites in 2017 and 2018. Environmental variables were extracted from weather and land use datasets in a 1-km buffer around trapping sites. The relative importance of weather and land use on mosquito abundance (for common species) or occurrence (for all species) was evaluated using multivariate hierarchical statistical models. Models incorporating both weather and land use performed better than models that include weather only for approximately half of species (59% for occurrence model and 50% for abundance model). Mosquito occurrence was mainly associated with temperature whereas abundance was associated with precipitation and temperature combined. Land use was more often associated with abundance than occurrence. For most species, occurrence and abundance were positively associated with forest cover but for some there was a negative association. Occurrence and abundance of some species (47% for occurrence model and 88% for abundance model) were positively associated with wetlands, but negatively associated with urban (Culiseta melanura and Anopheles walkeri) and agriculture (An. quadrimaculatus, Cs. minnesotae and An. walkeri) environments. This study provides predictive relationships between weather, land use and mosquito occurrence and abundance for a wide range of species including those that are currently uncommon, yet known as arboviruses vectors. Elucidation of these relationships has the potential to contribute to better prediction of MBD risk, and thus more efficiently targeted prevention and control measures.

sentences: Gaze-Related Activity in Primate Frontal Cortex Predicts and Mitigates Spatial Uncertainty A remarkable feature of primate behavior is the ability to predict future events based on past experience and current sensory cues. To understand how the brain plans movements in the presence of unstable cues, we recorded gaze-related activity in the frontal cortex of two monkeys engaged in a quasi-predictable cue-conflict task. Animals were trained to look toward remembered visual targets in the presence of a landmark that shifted with fixed amplitude but randomized direction. As simulated by a probabilistic model based on known physiology/behavior, gaze end points assumed a circular distribution around the target, mirroring the possible directions of the landmark shift. This predictive strategy was reflected in frontal cortex activity (especially supplementary eye fields), which anticipated future gaze distributions before the actual landmark shift. In general, these results implicate prefrontal cortex in the predictive integration of environmental cues and their learned statistical properties to mitigate spatial uncertainty.

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Sugar beet cold-induced PMT5a and STP13 carriers are poised for taproot proton-driven plasma membrane sucrose and glucose import O_LIAs the major sugar-producing crop in the northern hemisphere, sugar beet taproots store sucrose at a concentration of about 20 %. While the vacuolar sucrose loader TST has already been identified in the taproot, sugar transporters mediating sucrose uptake across the plasma membrane of taproot parenchyma cells remained unknown.
C_LIO_LIWe electrophysiologically examined taproots for proton-coupled sugar uptake and identified potentially involved transporters by transcriptomic profiling. After cloning, the transporter features were studied in the heterologous Xenopus laevis oocyte expression system using the two-electrode voltage clamp technique. Insights into the structure were gained by 3D homology modeling.
C_LIO_LIAs with glucose, sucrose stimulation of taproot parenchyma cells caused inward H+-fluxes and plasma membrane depolarization, indicating a sugar/proton symport mechanism. As one potential candidate for sugar uploading, the BvPMT5a was characterized as a H+-driven low-affinity glucose transporter, which does not transport sucrose. BvSTP13 operated as a high-affinity H+/sugar symporter, transporting glucose and to some extent sucrose due to a binding cleft plasticity. Both transporter genes were upregulated upon cold exposure, with the transport capacity of BvSTP13 being more cold-resistant than BvPMT5a.
C_LIO_LIIdentification of BvPMT5a and BvSTP13 as taproot sugar transporters could improve breeding of cold-tolerant sugar beet to provide a sustainable energy crop.
C_LI

sentences: Molecular characterization and functional annotation of a hypothetical protein (TDB29877.1) from probiotic bacteria Lactobacillus acidophilus: an in-silico approach Lactobacillus acidophilus bacteria are widely used as probiotic and to produce various healthy fermented food products. The PNW3 strain of the bacteria has numerous proteins in its genome and some are considered as hypothetical proteins. The aim of the present study was to predict the structures and biological functions of the hypothetical protein (accession number: TDB29877.1) from L. acidophilus through an in-silico approach applying various bioinformatics tools. The sequence similarity was searched on the available biological databases using BLASTp program to find out the homologues proteins. Besides, determination of various properties like physicochemical characteristics, subcellular localization, phylogenetic analysis, functional annotation, protein-protein interaction, determination of secondary and tertiary structures, active site detection and further quality assessment analysis were done using appropriate computational methods of bioinformatics. In-silico analysis revealed that the hypothetical protein has contained TerB-N and TerB-C domains with the presence of YjbR-like superfamily. The Protein-protein interaction network analysis revealed that the protein highly interacted with various known and unknown proteins responsible for iron ion binding, DNA and RNA metabolisms and numerous repair mechanisms that maintain cellular integrity. It was also found that the protein has predominantly alpha-helices in its secondary structure and the three dimensional model has been found to be novel as it possessed expected quality while gone through various quality assessment tools. Thus, the present result indicated that the selected hypothetical protein which is cytoplasmic in nature with Belta-grasp fold, plays important role in responding during stress condition or phage defense mechanism.

sentences: Neural Basis of The Double Drift Illusion In the "double-drift" illusion, local motion within a window moving in the periphery alters the windows perceived path. The illusion is strong even when the eyes track a target whose motion matches the window so that the stimulus remains stable on the retina. This implies that the illusion involves the integration of retinal signals with non-retinal eye-movement signals. To identify where in the brain this integration occurs, we measured BOLD fMRI responses in visual cortex while subjects experienced the double drift illusion. We identified a number of cortical areas that responded more strongly during the illusion, but only in area hMT+ was it possible to decode the illusory trajectory. Our results provide evidence for a perceptual representation in human visual cortex that is not linked to retinal position.

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Augmenting Recombinant Antibody Production in HEK293E Cells: Optimising Transfection and Culture Parameters BackgroundOptimising recombinant antibody production is important for cost-effective therapeutics and diagnostics. With impact on commercialisation, higher productivity beyond laboratory scales is highly sought, where efficient production can also accelerate antibody characterisations and investigations.

MethodsInvestigating HEK293E cells for mammalian antibody production, various transfection and culture parameters were systematically analysed for antibody light chain production before evaluating them for whole antibody production. Transfection parameters investigated include seeding cell density, the concentration of the transfection reagent and DNA, complexation time, temperature, and volume, as well as culture parameters such as medium replacement, serum deprivation, use of cell maintenance antibiotic, incubation temperature, medium volume, post-transfection harvest day and common nutrient supplements.

ResultsUsing 2 mL adherent HEK293E cell culture transfections with 25 kDa linear Polyethylenimine in the most optimised parameters, we demonstrated a ~2-fold production increase for light chain alone and for whole antibody production reaching 536 and 49 g respectively in a cost-effective manner. With the addition of peptone, {kappa} light chain increased by ~4-fold to 1032 g while whole antibody increased to a lesser extent by ~2.5-fold to 51 g, with benefits potentially for antibodies limited by their light chains in production.

ConclusionsOur optimised findings show promise for a more efficient and convenient antibody production method through transfection and culture optimisations that can be incorporated to scale up processes and with potential transferability to other mammalian-based recombinant protein production using HEK293E cells.

Statement of SignificanceRecombinant antibody production is crucial for antibody research and development. Systematically investigating transfection and culture parameters such as PEI/DNA concentrations, complexation time, volume, and temperature, supplements, etc., we demonstrated a ~4-fold light chain alone production increase to 1032 g and a 2.5-fold whole antibody production increase to 51 g from 2 mL transfections.

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An exploratory study of EEG connectivity during the first year of life in preterm and full-term infants AimTo evaluate electroencephalography (EEG) connectivity during the first year of age in healthy full-term infants and preterm infants with prenatal and perinatal risk factors for perinatal brain damage.

MethodsThree groups of infants were studied: healthy full-term infants (n = 71), moderate/late-preterm infants (n = 54), and very preterm infants (n = 56). All preterm infants had perinatal and/or perinatal risk factors for brain damage. EEG was obtained during phase II of natural nonrapid eye movement (NREM) sleep. EEG analysis was performed in 24 2.56-s artifact-free segments. For the calculation of EEG sources, spectral structured sparse Bayesian learning was used. Connectivity was computed by the phase-lag index.

ResultsIn healthy full-term infants, EEG interhemispheric connectivity in the different frequency bands followed similar trends with age to those reported in each frequency band: delta connectivity decreased, theta increased at the end of the year, the alpha band showed different trends based on the region studied, and beta interhemispheric connectivity decreased with age. EEG connectivity in preterm infants showed differences from the results of the term group.

DiscussionImportant structural findings may explain the differences observed in EEG connectivity between the term and preterm groups.

ConclusionThe study of EEG connectivity during the first year of age provides essential information on normal and abnormal brain development.

sentences: Whole Genome and Exome Sequencing Reference Datasets from A Multi-center and Cross-platform Benchmark Study With the rapid advancement of sequencing technologies in the past decade, next generation sequencing (NGS) analysis has been widely applied in cancer genomics research. More recently, NGS has been adopted in clinical oncology to advance personalized medicine. Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from errors or artifacts introduced during NGS processes or data analysis. Therefore, there is an urgent need to develop best practices in cancer mutation detection using NGS and the need for standard reference data sets for systematically benchmarking sequencing platforms, library protocols, bioinformatics pipelines and for measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples, a human triple-negative breast cancer cell line and a matched normal cell line derived from B lymphocytes. We generated whole-genome (WGS) and whole-exome sequencing (WES) data using 16 NGS library preparation protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the paired reference samples to identify factors affecting detection reproducibility and accuracy in cancer genomes. These large cross-platform/site WGS and WES datasets using well-characterized reference samples will represent a powerful resource for benchmarking NGS technologies, bioinformatics pipelines, and for the cancer genomics studies.

sentences: Semantic scene-object consistency modulates N300/400 EEG components, but does not automatically facilitate object representations During natural vision, objects rarely appear in isolation, but often within a semantically related scene context. Previous studies reported that semantic consistency between objects and scenes facilitates object perception, and that scene-object consistency is reflected in changes in the N300 and N400 components in EEG recordings. Here, we investigate whether these N300/400 differences are indicative of changes in the cortical representation of objects. In two experiments, we recorded EEG signals while participants viewed semantically consistent or inconsistent objects within a scene; in Experiment 1, these objects were task-irrelevant, while in Experiment 2, they were directly relevant for behavior. In both experiments, we found reliable and comparable N300/400 differences between consistent and inconsistent scene-object combinations. To probe the quality of object representations, we performed multivariate classification analyses, in which we decoded the category of the objects contained in the scene. In Experiment 1, in which the objects were not task-relevant, object category could be decoded from around 100 ms after the object presentation, but no difference in decoding performance was found between consistent and inconsistent objects. By contrast, when the objects were task-relevant in Experiment 2, we found enhanced decoding of semantically consistent, compared to semantically inconsistent, objects. These results show that differences in N300/400 components related to scene-object consistency do not index changes in cortical object representations, but rather reflect a generic marker of semantic violations. Further, our findings suggest that facilitatory effects between objects and scenes are task-dependent rather than automatic.

sentences: Intact maternal buffering of stress response in infant rats despite altered responsivity towards maternal olfactory cues in the valproic acid model of autism-like behavior Disrupted processing of social cues and altered social behaviors are among the core symptoms of autism spectrum disorders (ASDs), and they emerge as early as the first year of life. These differences in sensory abilities may affect the ability of children with ASDs to securely attach to a caregiver and experience caregiver buffering of stress. Prenatal exposure to valproic acid (VPA) has been used to model some aspects of ASDs in rodents. Here, we asked whether prenatal VPA exposure altered infant rats behavioral responsivity to maternal olfactory cues in an odor preference test and affected maternal buffering of infants stress responsivity to shock. In the odor preference test, one-week old rats treated with VPA during pregnancy appeared to have impaired social recognition and/or may be less motivated to approach social odors in early infancy. These effects were particularly prominent in female pups. In two-week old rats, VPA-exposed pups and saline-exposed pups showed similar preferences for home cage bedding. Although VPA-exposed pups may initially have a deficit in this attachment-related behavior they do recover typical responses to home cage bedding in later infancy. Both control and VPA-exposed pups showed robust stress hormone responses to repeated shocks, an effect which was blocked when a calm mother was present during shock exposure. No sex differences in the effect of maternal presence on the stress response to shock and no interactions between sex and prenatal drug exposure were observed. Although VPA-exposed pups may show impaired responsivity to maternal cues in early infancy, maternal presence is still capable of regulating the stress response in VPA-exposed pups. In this study we demonstrate the importance of utilizing multiple batteries of tests in assessing behavior, dissecting the behavior on one test into different components. Our results inform about the underlying behavioral characteristics of some of the ASD phenotypes, including sex differences reported by clinical studies, and could shed light on potential opportunities for intervention.

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A new behavioral and neural marker of social vision Research on face perception has revealed highly specialized visual mechanisms such as configural processing, and provided markers of interindividual differences -including disease risks and alterations- in visuoperceptual abilities that traffic in social cognition. Is face perception unique in degree or kind of mechanisms, and in its relevance for social cognition? Combining functional MRI and behavioral methods, we address the processing of an uncharted class of socially relevant stimuli: minimal social scenes involving configurations of two bodies spatially close and face-to-face as if interacting (hereafter, facing dyads). We report category-specific activity for facing (vs. non-facing) two-body configurations in selective areas of the visual cortex. That activity shows face-like signatures of configural processing -i.e., stronger response, and greater susceptibility to stimulus inversion for facing (vs. non-facing) dyads-, and is predicted by performance-based measures of body-dyad perception (i.e., accuracy in a fast visual categorization task). Moreover, individual performance in body-dyad perception is reliable, stable-over-time and correlated with the individual social sensitivity, coarsely captured by the Autism-Spectrum Quotient. Further analyses clarify the relationship between single-body and body-dyad perception. We propose that facing dyads are processed through highly specialized mechanisms (and brain areas), analogously to other biologically/socially relevant stimuli such as faces. Like face perception, facing-dyad perception can reveal basic visual processes that lay the foundations for understanding others, their relationships and interactions.

Significance statementWith its specialization to faces and biological motion, vision houses the foundations of human social ability. Using a multimodal approach (meta-analysis, fMRI, visual-perception tasks and self-administered survey), we introduce a new class of visual stimuli -minimal social scenes with two face-to-face bodies-, whose processing highlights new behavioral and neural markers of visuoperceptual abilities that traffic in social cognition. Behavioral and neural effects of body-dyad perception reveal the recruitment of specialized configural processing, previously described for face perception. Furthermore, individual performance in body-dyad perception is stable over time, and predicts an individuals social sensitivity, measured in terms of autism-spectrum traits. Thus, body-dyad perception reveals uncharted aspects of visual functioning and specialization, which may critically contribute to human social life.

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Zinc as a non-hormonal contraceptive: a better alternative to the copper intrauterine device (IUD) Long-acting and reversible contraceptives (LARC) are the most widely used form of female contraception worldwide, however they have significant side-effects that often result in early removal. Most LARCs are hormonal, but the use of exogenous hormones is not suitable for all women and causes side-effects in many others. The copper IUD (CuIUD) is the only non-hormonal LARC, but a large proportion of users suffer severe side effects. This study proposes the use of zinc as a suitable alternative to the CuIUD.

A rat intrauterine device (IUD) model was established to test the efficacy of a zinc IUD (ZnIUD) against a CuIUD, and a control nylon IUD. The IUD was surgically implanted into one uterine horn while the other remained untreated. Both the ZnIUD and CuIUD resulted in significantly fewer implantation sites compared to untreated horns. There was no significant difference between treated and untreated horns in the control nylon IUD group. Histological assessment revealed damage and inflammation in the endometrium of CuIUD treated horns, but only minor epithelial damage in ZnIUD treated horns, closely resembling the normal appearance of the control horns. This suggests ZnIUDs may not share the side-effect profile of the CuIUD. To test the long-term efficacy of the ZnIUD, rats had a ZnIUD surgically implanted into both horns and cohoused with males for 3 months. These rats mated regularly but did not get pregnant, confirming the long-term effectiveness of the ZnIUD. Reversibility of the ZnIUD was also established, as removal of the ZnIUD after 3 months resulted in no significant difference in the number of implantation sites between treated and untreated horns.

This study demonstrated the contraceptive efficacy of zinc and its potential as a LARC. The ZnIUD had minimal histological impact on the endometrium compared to the current copper standard, indicating that IUDs containing zinc may offer highly effective contraception while causing fewer side effects.

sentences: Tacc3 modulates microtubule network dynamicity and focal adhesion remodeling to affect cranial neural crest cell migration in Xenopus laevis Coordinated cell migration is critical during embryogenesis, as cells must leave their point of origin, navigate a complex barrage of signals, and accurately position themselves to facilitate correct tissue and organ formation. The cell motility process relies on dynamic interactions of the F-actin and microtubule (MT) cytoskeletons. Our work focuses on how one MT plus-end regulator, Transforming Acidic Coiled-Coil 3 (Tacc3), can impact migration of cranial neural crest cells in Xenopus laevis. We previously demonstrated that tacc3 expression is expressed in cranial neural crest cells, and that Tacc3 can function as a MT plus-end tracking protein to regulate MT growth velocities. Here, we demonstrate that manipulation of Tacc3 protein levels is sufficient to alter cranial neural crest cell velocity in vitro. Tacc3 overexpression drives increased single-cell migration velocities, while Tacc3 KD results in reduced cell velocity and defective explant dispersion. We also show that Tacc3 can have spatially-enhanced effects on MT plus-end growth velocities as well as effects on focal adhesion remodeling. Together, we demonstrate that Tacc3 can facilitate neural crest cell motility through spatially-enhanced cytoskeletal remodeling, which may underlie the enhanced metastatic potential of Tacc3-overexpressing tumor cells.

sentences: A novel, ataxic mouse model of Ataxia Telangiectasia caused by a clinically relevant nonsense mutation Ataxia Telangiectasia (A-T) and Ataxia with Ocular Apraxia Type 1 (AOA1) are devastating neurological disorders caused by null mutations in the genome stability genes, A-T mutated (ATM) and Aprataxin (APTX), respectively. Our mechanistic understanding and therapeutic repertoire for treating these disorders is severely lacking, in large part due to the failure of prior animal models with similar null mutations to recapitulate the characteristic loss of motor coordination (i.e., ataxia) and associated cerebellar defects. By increasing genotoxic stress through the insertion of null mutations in both the Atm (nonsense) and Aptx (knockout) genes in the same animal, we have generated a novel mouse model that for the first time develops a progressively severe ataxic phenotype associated with atrophy of the cerebellar molecular layer. We find biophysical properties of cerebellar Purkinje neurons are significantly perturbed (e.g., reduced membrane capacitance, lower action potential thresholds, etc.), while properties of synaptic inputs remain largely unchanged. These perturbations significantly alter Purkinje neuron neural activity, including a progressive reduction in spontaneous action potential firing frequency that correlates with both cerebellar atrophy and ataxia over the animals first year of life. Double mutant mice also exhibit a high predisposition to developing cancer (thymomas) and immune abnormalities (impaired early thymocyte development and T-cell maturation), symptoms characteristic of A-T. Lastly, by inserting a clinically relevant nonsense-type null mutation in Atm, we demonstrate that Small Molecule Read-Through (SMRT) compounds can restore ATM production, indicating their potential as a future A-T therapeutic.

sentences: Unravelling the Graded Millisecond Allosteric Activation Mechanism of Imidazole Glycerol Phosphate Synthase Deciphering the molecular mechanisms of enzymatic allosteric regulation requires the structural characterization of key functional states and also their time evolution toward the formation of the allosterically activated ternary complex. The transient nature and usually slow millisecond timescale interconversion between these functional states hamper their detailed experimental and computational characterization. Here, we design a computational strategy tailored to reconstruct millisecond timescale events to describe the graded allosteric activation of imidazole glycerol phosphate synthase (IGPS) in the ternary complex. IGPS is a heterodimeric bienzyme complex responsible for the hydrolysis of glutamine to glutamate in the HisH subunit and delivering ammonia for the cyclase activity in HisF. Despite significant advances in understanding the underlying allosteric mechanism, essential molecular details of the long-range millisecond allosteric activation pathway of wild-type IGPS remain hidden. Without using a priori information of the active state, our simulations uncover how IGPS, with the allosteric effector bound in HisF, spontaneously captures glutamine in a catalytically inactive HisH conformation, subsequently attains a closed HisF:HisH interface, and finally forms the oxyanion hole in HisH for efficient glutamine hydrolysis. We show that effector binding in HisF dramatically decreases the conformational barrier associated with the oxyanion hole formation in HisH, in line with the experimentally observed 4500-fold activity increase in glutamine production. The formation of the allosterically active state is controlled by time-evolving dynamic communication networks connecting the effector and substrate binding sites. This computational strategy can be generalized to study other unrelated enzymes undergoing millisecond timescale allosteric transitions.

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Umibato: estimation of time-varying microbial interaction using continuous-time regression hidden Markov model MotivationAccumulating evidence has highlighted the importance of microbial interaction networks. Methods have been developed for estimating microbial interaction networks, of which the generalized Lotka-Volterra equation (gLVE)-based method can estimate a directed interaction network. The previous gLVE-based method for estimating microbial interaction networks did not consider time-varying interactions.

ResultsIn this study, we developed unsupervised learning based microbial interaction inference method using Bayesian estimation (Umibato), a method for estimating time-varying microbial interactions. The Umibato algorithm comprises Gaussian process regression (GPR) and a new Bayesian probabilistic model, the continuous-time regression hidden Markov model (CTRHMM). Growth rates are estimated by GPR, and interaction networks are estimated by CTRHMM. CTRHMM can estimate time-varying interaction networks using interaction states, which are defined as hidden variables. Umibato outperformed the existing methods on synthetic datasets. In addition, it yielded reasonable estimations in experiments on a mouse gut microbiota dataset, thus providing novel insights into the relationship between consumed diets and the gut microbiota.

AvailabilityThe C++ and python source codes of the Umibato software are available at https://github.com/shion-h/Umibato

Contactshion_hosoda@asagi.waseda.jp, mhamada@waseda.jp

sentences: A shared brain system forming confidence judgment across cognitive domains Confidence is typically defined as a subjective judgment about whether a decision is right. Decisions are based on sources of information that come from various cognitive domains and are processed in different brain systems. An unsettled question is whether the brain computes confidence in a similar manner whatever the domain or in a manner that would be idiosyncratic to each domain. To address this issue, human participants of both sexes performed two tasks probing confidence in decisions made about the same material (history and geography statements), but based on different cognitive processes: semantic memory for deciding whether the statement was true or false, and duration perception for deciding whether the statement display was long or short. At the behavioral level, we found that the same factors (difficulty, accuracy, response time and confidence in the preceding decision) predicted confidence judgments in both tasks. At the neural level, we observed using fMRI that confidence judgments in both tasks were associated to activity in the same brain regions: positively in the ventromedial prefrontal cortex and negatively in a prefronto-parietal network. Together, these findings suggest the existence of a shared brain system that generates confidence judgments in a similar manner across cognitive domains.

sentences: Health hazards posed by ionizing radiation to human space missions and newly developing methodologies to investigate them Ionizing radiation is one of the main threats to human space exploration beyond low Earth orbit (BLEO). It is thus of primary importance to determine safe career dose limits for astronauts involved in BLEO missions. In this work the average equivalent doses received by a hypothetical human crew are established using the data of several robotic missions to the Moon and to Mars. The probabilities of the occurrence of deterministic effects due to radiation that could impair the success of a mission or lower the life expectancy of astronauts are evaluated using statistical methods. The limits of the obtained results concerning deterministic and stochastic effects basing on statistical methods are discussed. The conclusion is that new strategies are necessary in order to predict the insurgence of stochastic effects. We argue that the use of the so-called 3D models or organoids could be a good candidate of such a strategy that will be complementary to in vitro 2D models and animal models. Organoids are in fact able to recapitulate several relevant features of the real human organs and their in vivo surroundings. Yet, the main problem of detecting the onset of cancer in the human body remains outstanding. To this purpose, we propose here to combine organoid techniques with those of precision oncology and molecular medicine. Exactly these disciplines have greatly contributed in the recent time to the understanding of the patterns of cancer formation in several cases of tumors. Precision oncology and molecular medicine provide powerful markers to track down the events leading to tumorigenesis following exposure to radiation in organoids.

sentences: Top-down control of hippocampal signal-to-noise by prefrontal long-range inhibition The prefrontal cortex (PFC) is postulated to exert  top-down control by modulating information processing throughout the brain to promote specific actions based on current goals. However, the pathways mediating top-down control remain poorly understood. In particular, knowledge about direct prefrontal connections that might facilitate top-down prefrontal control of information processing in the hippocampus remains sparse. Here we describe novel monosynaptic long-range GABAergic projections from PFC to hippocampus. These preferentially inhibit vasoactive intestinal polypeptide expressing interneurons, which are known to disinhibit hippocampal microcircuits. Indeed, stimulating prefrontal-hippocampal GABAergic projections increases hippocampal feedforward inhibition and reduces hippocampal activity in vivo. The net effect of these actions is to specifically enhance the signal-to-noise ratio for hippocampal representations of objects. Correspondingly, stimulation of PFC-to-hippocampus GABAergic projections promotes object exploration. Together, these results elucidate a novel top-down pathway in which long-range GABAergic projections target disinhibitory microcircuits, thereby enhancing signals and network dynamics underlying exploratory behavior.

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Continental generalization of an AI system for clinical seizure recognition BackgroundElectroencephalogram (EEG) monitoring and objective seizure identification is an essential clinical investigation for some patients with epilepsy. Accurate annotation is done through a time-consuming process by EEG specialists. Computer-assisted systems for seizure detection currently lack extensive clinical utility due to retrospective, patient-specific, and/or irreproducible studies that result in low sensitivity or high false positives in clinical tests. We aim to significantly reduce the time and resources on data annotation by demonstrating a continental generalization of seizure detection that balances sensitivity and specificity.

MethodsThis is a prospective inference test of artificial intelligence on nearly 14,590 hours of adult EEG data from patients with epilepsy between 2011 and 2019 in a hospital in Sydney, Australia. The inference set includes patients with different types and frequencies of seizures across a wide range of ages and EEG recording hours. The artificial intelligence (AI) is a convolutional long short-term memory network that is trained on a USA-based dataset. The Australian set is about 16 times larger than the US training dataset with very long interictal periods (between seizures), which is way more realistic than the training set and makes our false positives highly reliable. We validated our inference model in an AI-assisted mode with a human expert arbiter and a result review panel of expert neurologists and EEG specialists on 66 sessions to demonstrate achievement of the same performance with over an order-of-magnitude reduction in time.

FindingsOur inference on 1,006 EEG recording sessions on the Australian dataset achieved 76.68% with nearly 56 [0, 115] false alarms per 24 hours on average, against legacy ground-truth annotations by human experts, conducted independently over nine years. Our pilot test of 66 sessions with a human arbiter, and reviewed ground truth by a panel of experts, confirmed an identical human performance of 92.19% with an AI-assisted system, while the time requirements reduce significantly from 90 to 7.62 minutes on average.

InterpretationAccurate and objective seizure counting is an important factor in epilepsy. An AI-assisted system can help improve efficiency and accuracy alongside human experts, particularly in low and middle-income countries with limited expert human resources.

FundingsSOAR Fellowship from The University of Sydney, a Microsoft AI for Accessibility grant, and a Research Training Program (RTP) support provided by the Australian Government.

Research in contextO_ST_ABSEvidence before this studyC_ST_ABSDuring the development of our artificial intelligence (AI) system, we did a systematic review of the scientific literature with search via PubMed for research articles published on seizure detection with the following inclusion criteria: (1) Tests or inference evaluation is conducted on large-scale clinical EEG data; (2) Generalization is attempted or potentials for generalization is considered, e.g., in commercialized tools; (3) Seizure detection delay and real-time (aka. online) operation were not considered critical in this context as long as the test was conducted on raw EEG data. Note that ICU seizure detection or portable seizure alert systems are relying on detection delay and real-time needs. Our keywords include "prospective seizure detection", "automated seizure detection", "non-patient specific seizure detection", "seizure detection on continuous EEG", and "deep learning-based seizure detection" and "machine learning-based seizure detection". We found that the only two categories of works meet our criteria: two research papers published in 2020 and works published by commercial tools developers. We cited a recent review of 89 deep learning-based seizure detection, all of which are retrospective. One work from Stanford reported seizure detection on all ages (pediatric to adult ages) using post-acquisition EEG recordings and provided an avenue for independent evaluation by providing a test on a publicly available Temple University Hospital (TUH) EEG dataset. The other work pivoted on algorithmic-assisted real-time seizure risk monitoring in continuous EEG in neonatal intensive care unit (NICU) with 128 neonates (32 with seizures) showing about 20% improvement in seizure identification over 130 neonates (38 with seizures) with no algorithmic assistance.

Commercial tools we studied are Encevis (EpiScan), Besa, and Persyst. There is a recent comparative study on these tools on 81 patients. Encevis is reported as the best performing tool, and hence we provided a comparative study with Encevis ver. 1.9.2. Encevis is also the only tool that provided an avenue for comparative study on publicly available EEG data. The Stanford work, published in 2020, confirms many false positives with Persyst 13. We excluded our tests on Persyst 14 as it highly under-performed relative to Encevis. Only Stanfords work provides code availability. We compared our results with Stanfords work outcome and provided pilot test results with the Encevis (EpiScan) tool on the Australian dataset, which shows a considerably lower sensitivity.

Added value of this studyTo the best of our knowledge, the current study is the first continental generalization that demonstrates the potential to achieve an expert human-level seizure recognition rate in a clinical setting and in just a fraction of time. The two datasets used in this study are recorded with different infrastructure, which adds to the independence of inference from hardware types and improves clinical utility. This is particularly important as 80% of patients with epilepsy live in low and middle-income countries with limited resources, particularly EEG specialists and neurologists.

Implications of all the available evidenceOur results support the potential benefits of deep learning AI in clinical settings for seizure recognition and its contribution to significant sensitivity over available solutions. Our AI-assisted system achieves more than a ten-fold increase in time efficiency and reports identical performance to human experts for EEG interpretation with access to great neurophysiology support and auxiliary data. Our findings, particularly our tests on an available commercial tool, recommend that the evaluation, test, or inference in AI systems be performed on different datasets, with diverse infrastructures, and on large-scale and realistic sets with long interictal periods.

sentences: Elucidating design principles for engineering cell-derived vesicles to inhibit SARS-CoV-2 infection The ability of pathogens to develop drug resistance is a global health challenge. The SARS-CoV-2 virus presents an urgent need wherein several variants of concern resist neutralization by monoclonal antibody therapies and vaccine-induced sera. Decoy nanoparticles--cell-mimicking particles that bind and inhibit virions--are an emerging class of therapeutics that may overcome such drug resistance challenges. To date, we lack quantitative understanding as to how design features impact performance of these therapeutics. To address this gap, here we perform a systematic, comparative evaluation of various biologically-derived nanoscale vesicles, which may be particularly well-suited to sustained or repeated administration in the clinic due to low toxicity, and investigate their potential to inhibit multiple classes of model SARS-CoV-2 virions. A key finding is that such particles exhibit potent antiviral efficacy across multiple manufacturing methods, vesicle subclasses, and virus-decoy binding affinities. In addition, these cell-mimicking vesicles effectively inhibit model SARS-CoV-2 variants that evade monoclonal antibodies and recombinant protein-based decoy inhibitors. This study provides a foundation of knowledge that may guide the design of decoy nanoparticle inhibitors for SARS-CoV-2 and other viral infections.

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Metabolic function in aging retina and retinal pigment epithelium remains robust despite vision loss PurposeCharacterize how metabolic function in the murine retina and retinal pigment epithelium-choroid-sclera (eyecup) complex is impacted by natural aging.

MethodsWe examined scotopic and photopic visual function of young (3-6 months) and aged (23-26 months) C57Bl/6J mice using electroretinograms (ERGs). Metabolic changes in retina and eyecup explants were characterized by measuring uptake and usage of U-13C-glucose or U-13C-glutamine at different timepoints by gas chromatography-mass spectrometry (GC-MS), measuring oxygen consumption rate (OCR) using a perifusion apparatus, and determining ATP levels with a bioluminescence assay.

ResultsScotopic and photopic ERG responses declined in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected by the age of the mouse. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable.

ConclusionsThe ex vivo approach in our study to examine aging glucose and glutamine metabolism in retina and RPE showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The surprising metabolic stability of these tissues ex vivo suggests age-related metabolic alterations in these tissues may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or other structural changes influence ocular metabolism in vivo.

sentences: Computing Minimal Boolean Models of Gene Regulatory Networks Models of Gene Regulatory Networks (GRNs) capture the dynamics of the regulatory processes that occur within the cell as a means to understand the variability observed in gene expression between different conditions. Arguably the simplest mathematical construct used for modeling is the Boolean network, which dictates a set of logical rules for transition between states described as Boolean vectors. Due to the complexity of gene regulation and the limitations of experimental technologies, in most cases knowledge about regulatory interactions and Boolean states is partial. In addition, the logical rules themselves are not known a-priori. Our goal in this work is to present a methodology for inferring this information from the data, and to provide a measure for comparing network states under different biological conditions. We present a novel methodology for integrating experimental data and performing a search for the optimal consistent structure via optimization of a linear objective function under a set of linear constraints. We also present a statistical approach for testing the similarity of inferred network states under different conditions. Finally, we extend our methodology into a heuristic that can handle large datasets that are generated by single-cell RNA-Sequencing(scRNA-Seq). We demonstrate the effectiveness of these tools using a public scRNA-Seq dataset and the GRN that is associated with it. Our methodology will enable researchers to obtain a better understanding of the dynamics of gene regulatory networks and their biological role.

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Emergence of a Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae Co-harbouring a blaNDM-1-carrying Virulent Plasmid and a blaKPC-2-carrying Plasmid in an Egyptian Hospital The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and the analysis of the plasmids associated with CR-hypervirulent-KP (CR-HvKP) in Egypt are not presented. Therefore, we attempt to decipher the plasmids sequences, which are responsible for transferring the determinants of carbapenem-resistance, particularly the blaNDM-1 and blaKPC-2. Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383, and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed using Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as CR-HvKP strain, it harboured four plasmids, namely; pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes, blaNDM-1 and blaKPC-2, were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD, iutA). Thus, we set out this study to analyse in-depth the genetic basis of pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We reported for the first time a high-risk clone ST11 KL47 serotype of CR-HvKP strain isolated from the blood of a 60-year-old hospitalised female patient from the ICU in a tertiary-care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharbouring the blaNDM-1 and virulence genes, besides a blaKPC-2-carrying plasmid.

IMPORTANCECRKP had been registered in the critical priority tier by the World Health Organization and became a significant menace to public health. Therefore, we set out this study to analyse in-depth the genetic basis of pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. Herein, we reported for the first time (to the best of our knowledge) a high-risk clone ST11 KL47 serotype of CR-HvKP strain isolated from the blood of a 60-year-old hospitalised female patient in a tertiary-care hospital from the ICU in Egypt, which showed the cohabitation of a novel hybrid plasmid co-harbouring the blaNDM-1 and virulence genes, besides a blaKPC-2-carrying plasmid. Herein, the high rate of CRKP might be due to the continuous usage of carbapenems as empirical therapy, besides the failure to implement an antibiotic stewardship program in Egyptian hospitals. Thus, this study serves to alert the contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.

sentences: Is bigger always better? Neither body size nor aggressive behavior are good predictors to measure the degree of specialization of hummingbird interaction networks in rocky outcrops Interspecific competition can strongly influence community structure and shape niche breadth and overlap. One of the main factors that determine the hummingbird community structure is competition for food. Hummingbirds functional attributes, such as beak length and body mass, influence nectar acquisition in the flowers, shaping foraging niches according to hummingbird dominance and foraging strategy. This study evaluates how the hummingbirds functional and behavioral attributes are related to plants assemblage in rocky outcrops habitats. We tested the following hypothesis: H1) Functional traits (beak length and body mass) are related to the richness and frequency of pollen grain morphotypes carried by hummingbirds; H2) Dominant and territorial hummingbirds carry a lower richness and frequency of pollen types when compared to subordinate hummingbirds, and H3) Hummingbird species carry different types of pollen grains. We conducted the study between September 2018 and March 2019 in a Campo Rupestre (rocky outcrops) in Southeastern Brazil. Hummingbirds were captured with a trap built based on trapdoors. We recorded their beak size and body masses, marked with commercial bird rings and ink on parts of the body, and then released. Behavioral responses to artificial feeders were collected regarding each visits time and duration and the outcome of aggressive interactions. The pollen adhered to the body parts was collected and identified in the laboratory. Our results showed that neither body size nor aggressive behaviors influenced pollen richness and frequency in rocky outcrops. Beak length was the most important hummingbirds attribute that influenced pollen richness, but not pollen frequency. Short-billed hummingbirds carried the greatest richness of pollen grains. Pollen grain richness and frequency were not related to hummingbird body mass or aggressive behavior. The hummingbird-pollen grain interaction network has shown to be generalized in the pollen grain transport. We conclude that hummingbirds beak length is the central morphological variable to measure pollen grain transport. It has direct implications for the pollination of different plant species.

sentences: Bioerosion traces and borings in the Upper Devonian vertebrate remains from the Holy Cross Mountains, Poland Bioerosion in fish remains Among the hundreds of collected Devonian vertebrate macrofossils in the Holy Cross Mountains, placoderms dominate and provide data on their morphology, distribution and taphonomy. So far 17 out of more than 500 studied specimens have revealed bones with surfaces covered by sediment-filled trace fossils. The traces have been made on the vertebrate remains before their final burial. The borings, oval in cross-section, include dendroidal networks of shallow tunnels or short, straight or curved individual scratches and grooves, which frequently create groups on the both sides of the bones. ?Karethraichnus isp. from Kowala and ?Osteocallis isp. from Wietrznia are the oldest record of these ichnogenera. Sedimentological clues indicate a shallow water environment, probably from the slope below the storm wave base.

sentences: Minus one frameshifted puromycin N-acetyltransferase is targeted to the nucleolus: sporadic but reproducible chimera formation by a transfected Lc3b construct Map1Lc3b is a protein that has pivotal functions in cellular autophagy. At least three groups in the past decade have reported its presence in the nucleoli of cells, but its functions in that organelle remain unknown. We isolated a few clonal populations of cells stably expressing V5-tagged mouse Lc3b highly enriched in the nucleoli, but the frequency of occurrence of such clones was strikingly low. The phenomenon was readily reproducible, though the protein in the nucleolus puzzlingly had varying molecular masses in different clones but consistently displayed a very strong interaction with the mitochondrial protein C1qbp, which has well-documented functions in the nucleolus. We investigated further and discovered that, in at least one of the clones, Lc3b had formed a chimera with the puromycin resistance gene in the plasmid, plausibly by illegitimate recombination during or after integration of the construct into the cellular genomic DNA. The -1 shifted reading frame of puromycin N-acetyltransferase (pac) can encode a protein that is equally long as the one encoded by the complete pac ORF, but is targeted to the nucleoli due to a drastic shift in the isoelectric point (pI). Notably, this set of events again brings into focus the low threshold often reported for recombination events to occur in eukaryotic cells, the multiple factors influencing them, and calls for increased vigilance in experiments involving DNA transfection and gene targeting.

sentences: Chloride-dependent mechanisms of multimodal sensory discrimination and neuropathic sensitization in Drosophila Individual sensory neurons can be tuned to many stimuli, each driving unique, stimulus-relevant behaviors, and the ability of multimodal nociceptor neurons to discriminate between potentially harmful and innocuous stimuli is broadly important for organismal survival. Moreover, disruptions in the capacity to differentiate between noxious and innocuous stimuli can result in neuropathic pain. Drosophila larval Class III (CIII) neurons are peripheral noxious cold nociceptors and gentle touch mechanosensors; high levels of activation drive cold-evoked contraction (CT) behavior, while low levels of activation result in a suite of touch-associated behaviors. However, it is unknown what molecular factors underlie CIII multimodality. Here, we show that the TMEM16/anoctamins subdued and white walker (wwk; CG15270) are required for cold-evoked CT, but not for touch-associated behavior, indicating a conserved role for anoctamins in nociception. We also evidence that CIII neurons make use of atypical depolarizing chloride currents to encode cold, and that overexpression of ncc69--a fly homologue of NKCC1--results in phenotypes consistent with neuropathic sensitization, including behavioral hypersensitization and spontaneous nociceptor activity, making Drosophila CIII neurons a candidate system for future studies of the basic mechanisms underlying neuropathic pain.

sentences: Differentiating amyloid beta spread in autosomal dominant and sporadic Alzheimer's disease Amyloid-beta (A{beta}) deposition is one of the hallmark pathologies in both sporadic Alzheimers disease (sAD) and autosomal dominant Alzheimers disease (ADAD), the latter of which is caused by mutations in genes involved in A{beta} processing. Despite A{beta} deposition being a centerpiece to both sAD and ADAD, some differences between these AD subtypes have been observed with respect to the spatial pattern of A{beta}. Previous work has shown that the spatial pattern of A{beta} in individuals spanning the sAD spectrum can be reproduced with high accuracy using an epidemic spreading model (ESM), which simulates the diffusion of A{beta} across neuronal connections and is constrained by individual rates of A{beta} production and clearance. However, it has not been investigated whether A{beta} deposition in the rarer ADAD can be modeled in the same way, and if so, how congruent the spreading patterns of A{beta} across sAD and ADAD are. We leverage the ESM as a data-driven approach to probe individual-level variation in the spreading patterns of A{beta} across three different large-scale imaging datasets (2 SAD, 1 ADAD). We applied the ESM separately to the Alzheimers Disease Neuroimaging initiative (N=737), the Open Access Series of Imaging Studies (N=510), and the Dominantly Inherited Alzheimers Network (N=249), the latter two of which were processed using an identical pipeline. We assessed inter- and intra-individual model performance in each dataset separately, and further identified the most likely epicenter of A{beta} spread for each individual. Using epicenters defined in previous work in sAD, the ESM provided moderate prediction of the regional pattern of A{beta} deposition across all three datasets. We further find that, while the most likely epicenter for most A{beta}-positive subjects overlaps with the default mode network, 13% of ADAD individuals were best characterized by a striatal origin of A{beta} spread. These subjects were also distinguished by being younger than ADAD subjects with a DMN A{beta} origin, despite having a similar estimated age of symptom onset. Together, our results suggest that most ADAD patients express A{beta} spreading patters similar to those of sAD, but that there may be a subset of ADAD patients with a separate, striatal phenotype.

sentences: Loss of Rnf43 accelerates Kras-mediated neoplasia and remodels the tumor immune microenvironment in pancreatic adenocarcinoma RNF43 is an E3 ubiquitin ligase that is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC) and precursor cystic neoplasms of the pancreas. The impact of RNF43 mutations on PDAC is poorly understood and autochthonous models have not been sufficiently characterized. In this study we describe a genetically engineered mouse model (GEMM) of PDAC with conditional expression of oncogenic Kras and deletion of the catalytic domain of Rnf43 (KRC) in exocrine cells. We demonstrate that Rnf43 loss results in an increased incidence of high-grade cystic lesions of the pancreas and PDAC. Importantly, KRC mice have a significantly decreased survival compared to mice containing only an oncogenic Kras mutation. By use of single cell RNA sequencing we demonstrated that KRC tumor progression is accompanied by a decrease in macrophages, as well as an increase in T and B lymphocytes with evidence of increased immune checkpoint molecule expression and affinity maturation, respectively. This was in stark contrast to the tumor immune microenvironment observed in the Kras/Tp53 driven PDAC GEMM. Furthermore, expression of the chemokine, CXCL5, was found to be specifically decreased in KRC cancer cells by means of epigenetic regulation and emerged as a putative candidate for mediating the unique KRC immune landscape. This GEMM establishes RNF43 as a bona fide tumor suppressor gene in PDAC and puts forth a rationale for an immunotherapy approach in this subset of PDAC cases.

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Non-linear Dimensionality Reduction on Extracellular Waveforms Reveals Physiological, Functional, and Laminar Diversity in Premotor Cortex Cortical circuits are thought to contain a large number of cell types that coordinate to produce behavior. Current in vivo methods rely on clustering of specified features of extracellular waveforms to identify putative cell types, but these capture only a small amount of variation. Here, we develop a new method (WaveMAP) that combines non-linear dimensionality reduction with graph clustering to identify putative cell types. We apply WaveMAP to extracellular waveforms recorded from dorsal premotor cortex of macaque monkeys performing a decision-making task. Using WaveMAP, we robustly establish eight waveform clusters and show that these clusters recapitulate previously identified narrow- and broad-spiking types while revealing previously unknown diversity within these subtypes. The eight clusters exhibited distinct laminar distributions, characteristic firing rate patterns, and decision-related dynamics. Such insights were weaker when using feature-based approaches. WaveMAP therefore provides a more nuanced understanding of the dynamics of cell types in cortical circuits.

SignificanceHow different cell types sculpt activity patterns in brain areas associated with decision-making is a fundamentally unresolved problem in neuroscience. In monkeys, and other species where transgenic access is not yet possible, identifying physiological types in vivo relies on only a few discrete user-specified features of extracellular waveforms to identify cell types. Here, we show that non-linear dimensionality reduction with graph clustering applied to the entire extracellular waveform can delineate many different putative cell types and does so in an interpretable manner. We show that this method reveals previously undocumented physiological, functional, and laminar diversity in the dorsal premotor cortex of monkeys, a key brain area implicated in decision-making.

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Impact of immediate cryopreservation on the establishment of patient derived xenografts from head and neck cancer patients. BackgroundPatient-derived xenografts established from human cancers are important tools for investigating novel anti-cancer therapies. Establishing PDXs requires a significant investment and many PDXs may be used infrequently due to their similarity to existing models, their growth rate, or the lack of relevant mutations. We performed this study to determine whether we could efficiently establish PDXs after cryopreservation to allow molecular profiling to be completed prior to implanting the human cancer.

MethodsFresh tumor was split with half used to establish a PDX immediately and half cryopreserved for later implantation. Resulting tumors were assessed histologically and tumors established from fresh or cryopreserved tissues compared as to the growth rate, extent of tumor necrosis, mitotic activity, keratinization, and grade. All PDXs were subjected to short tandem repeat testing to confirm identity and assess similarity between methods.

ResultsTumor growth was seen in 70% of implanted cases. No growth in either condition was seen in 30% of tumors. One developed a SCC from the immediate implant but a lymphoproliferative mass without SCC from the cryopreserved specimen. No difference in growth rate was seen. No difference between histologic parameters was seen between the two approaches.

ConclusionsFresh human cancer tissue can be immediately cryopreserved and later thawed and implanted to establish PDXs. This resource saving approach allows for tumor profiling prior to implantation into animals thus maximizing the probability that the tumor will be utilized for future research.

sentences: Gut microbiota transplantation drives the adoptive transfer of colonic genotype-phenotype characteristics between mice lacking catestatin and their wild type counterparts The gut microbiota is in continuous interaction with the intestinal mucosa via metabolic, neuro- immunological, and neuroendocrine pathways. Disruption in levels of antimicrobial peptides produced by the enteroendocrine cells, such as catestatin, has been associated with changes in the gut microbiota and imbalance in intestinal homeostasis. However, whether the changes in the gut microbiota have a causational role in intestinal dyshomeostasis has remained elusive. To this end, we performed reciprocal fecal microbial transplantation in wild-type mice and mice with a knockout in the catestatin coding region of the chromogranin-A gene (CST-KO mice). Combined microbiota phylogenetic profiling, RNA sequencing, and transmission electron microscopy were employed. Fecal microbiota transplantation from mice deficient in catestatin (CST-KO) to microbiota-depleted wild-type mice induced transcriptional and physiological features characteristic of a distorted colon in the recipient animals, including impairment in tight junctions, as well as an increased collagen area fraction indicating colonic fibrosis. In contrast, fecal microbiota transplantation from wild-type mice to microbiota-depleted CST-KO mice reduced collagen fibrotic area, restored disrupted tight junction morphology, and altered fatty acid metabolism in recipient CST-KO mice. This study provides a comprehensive overview of the murine metabolic- and immune-related cellular pathways and processes that are co-mediated by the fecal microbiota transplantation and supports a prominent role for the gut microbiota in the colonic distortion associated with the lack of catestatin in mice. Overall, the data show that the gut microbiota may play a causal role in the development of features of intestinal inflammation and metabolic disorders, known to be associated with altered levels of catestatin and may, thus, provide a tractable target in the treatment and prevention of these disorders.

sentences: A genome-scale metabolic model of Saccharomyces cerevisiae that integrates expression constraints and reaction thermodynamics Eukaryotic organisms play an important role in industrial biotechnology, from the production of fuels and commodity chemicals to therapeutic proteins. To optimize these industrial systems, a mathematical approach can be used to integrate the description of multiple biological networks into a single model for cell analysis and engineering. One of the current most accurate models of biological systems include metabolism and expression (ME-models), and Expression and Thermodynamics FLux (ETFL) is one such formulation that efficiently integrates RNA and protein synthesis with traditional genome-scale metabolic models. However, ETFL is so far only applicable for E. coli. To therefore adapt this ME-model for Saccharomyces cerevisiae, we herein developed yETFL. To do this, we augmented the original formulation with additional considerations for biomass composition, the compartmentalized cellular expression system, and the energetic costs of biological processes. We demonstrated the predictive ability of yETFL to capture maximum growth rate, essential genes, and the phenotype of overflow metabolism. We envision that the extended ETFL formulation can be applied to ME-model development for a wide range of eukaryotic organisms. The utility of these ME-models can be extended into academic and industrial research.

sentences: A high-throughput yeast display approach to profile pathogen proteomes for MHC-II binding T cells play a critical role in the adaptive immune response, recognizing peptide antigens presented on the cell surface by Major Histocompatibility Complex (MHC) proteins. While assessing peptides for MHC binding is an important component of probing these interactions, traditional assays for testing peptides of interest for MHC binding are limited in throughput. Here we present a yeast display-based platform for assessing the binding of tens of thousands of user-defined peptides in a high throughput manner. We apply this approach to assess a tiled library covering the SARS-CoV-2 proteome and four dengue virus serotypes for binding to human class II MHCs, including HLA-DR401, -DR402, and -DR404. This approach identifies binders missed by computational prediction, highlighting the potential for systemic computational errors given even state-of-the-art training data, and underlines design considerations for epitope identification experiments. This platform serves as a framework for examining relationships between viral conservation and MHC binding, and can be used to identify potentially high-interest peptide binders from viral proteins. These results demonstrate the utility of this approach for determining high-confidence peptide-MHC binding.

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Overactivation of a sleep-active neuron decouples survival from the need to sleep Sleep is a state of behavioral quiescence that is closely associated with survival. Sleep-active neurons promote sleep and survival. It is not known, however, whether sleep-active neurons need to cause sleep to promote survival. Here, we tested how depolarization of the sleep-active RIS neuron in Caenorhabditis elegans controls sleep and survival during larval starvation. Survival always increased with raised RIS depolarization. RIS depolarization promoted sleep over a long range. Unexpectedly, however, high levels of RIS depolarization caused a nearly complete loss of sleep. Similarly, overexpression of sleep-inducing FLP-11 neuropeptides in RIS inhibited sleep, indicating that overactive transmission from RIS inhibits sleep. Despite the loss of sleep, survival was normal following FLP-11 overexpression. Thus, RIS overactivation abolishes sleep yet supports survival. These results indicate that regulation of sleep and survival are separable functions of a sleep-active neuron that are normally coupled but can be uncoupled by sleep-neuron over activation.

Author SummarySleep behavior and survival benefits are independently regulated functions of the sleep neuron RIS in Caenorhabditis elegans.

sentences: Artificial Intelligence-rationalized balanced PPARα/γ dual agonism resets the dysregulated macrophage processes in inflammatory bowel disease A computational platform, the Boolean network explorer (BoNE), has recently been developed to infuse AI-enhanced precision into drug discovery; it enables querying and navigating invariant Boolean Implication Networks of disease maps for prioritizing high-value targets. Here we used BoNE to query an Inflammatory Bowel Disease (IBD)-map and prioritize a therapeutic strategy that involves dual agonism of two nuclear receptors, PPAR/{gamma}. Balanced agonism of PPAR/{gamma} was predicted to modulate macrophage processes, ameliorate colitis in network-prioritized animal models,  reset the gene expression network from disease to health, and achieve a favorable therapeutic index that tracked other FDA-approved targets. Predictions were validated using a balanced and potent PPAR/{gamma}-dual agonist (PAR5359) in two pre-clinical murine models, i.e., Citrobacter rodentium-induced infectious colitis and DSS-induced colitis. Using a combination of selective inhibitors and agonists, we show that balanced dual agonism promotes bacterial clearance more efficiently than individual agonists, both in vivo and in vitro. PPARa is required and its agonism is sufficient to induce the pro-inflammatory cytokines and cellular ROS, which are essential for bacterial clearance and immunity, whereas PPARg-agonism blunts these responses, delays microbial clearance and induces the anti-inflammatory cytokine, IL10; balanced dual agonism achieved controlled inflammation while protecting the gut barrier and  reversal of the transcriptomic network. Furthermore, dual agonism reversed the defective bacterial clearance observed in PBMCs derived from IBD patients. These findings not only deliver a macrophage modulator for use as barrier-protective therapy in IBD, but also highlight the potential of BoNE to rationalize combination therapy.

sentences: Strong regulatory effects of vgll3 genotype on reproductive axis gene expression in immature male Atlantic salmon Age at maturity is a major contributor to the diversity of life history strategies in organisms. The process of maturation is influenced by both genetics and the environment, and includes changes in levels of sex hormones and behavior, but the specific factors leading to variation in maturation timing are not well understood. gnrh1 regulates the transcription of gonadotropin genes at the onset of puberty in many species, but this gene is lacking in certain teleost species including Atlantic salmon (Salmo salar), which raises the possibility of the involvement of other important regulatory factors during this process. Earlier research has reported a strong association of alternative alleles of the vgll3 gene with maturation timing in Atlantic salmon, suggesting it as a potential candidate regulating reproductive axis genes. Here, we investigated the expression of reproductive axis genes in immature Atlantic salmon males with different vgll3 genotypes during the spawning period. We detected strong vgll3 genotype-dependent differential expression of reproductive axis genes (such as fshb, lhb, amh and igf3) tested in the pituitary, and testis of one-year-old immature male Atlantic salmon. In addition, we observed differential expression of jun (ap1) and nr5a1b (sf1), potential upstream regulators of gonadotropins in the pituitary, as well as axin2, id3, insl3, itch, ptgs2a and ptger4b, the downstream targets of amh and igf3 in the testis. Hereby, we provide evidence of strong vgll3 genotype-dependent transcriptional regulation of reproductive axis genes prior to sexual maturation together with models for distinct actions of vgll3 genotypes on the molecular processes controlling spermatogenesis in Atlantic salmon.

sentences: Ecophysiological differentiation between life stages in filmy ferns (Hymenophyllaceae) Desiccation tolerance was a key trait that allowed plants to colonize land. However, little is known about the transition from desiccation tolerant non-vascular plants to desiccation sensitive vascular ones. Filmy ferns (Hymenophyllaceae) represent a useful system to investigate how water-stress strategies differ between non-vascular and vascular stages within a single organism because they have vascularized sporophytes and nonvascular gametophytes that are each capable of varying degrees of desiccation tolerance. To explore this, we surveyed sporophytes and gametophytes of 19 species (22 taxa including varieties) of filmy ferns on Moorea (French Polynesia) and used chlorophyll fluorescence to measure desiccation tolerance and light responses. We conducted phylogenetically informed analyses to identify differences in physiology between life stages and growth habits. Gametophytes had similar or less desiccation tolerance (ability to recover from 2 d desiccation at -86 MPa) and lower photosynthetic optima (maximum electron transport rate of photosystem II and light level at 95% of that rate) than sporophytes. Epiphytes were more tolerant of desiccation than terrestrial species in both life stages. Despite their lack of greater physiological tolerances, gametophytes of several species occurred over a wider elevational range than conspecific sporophytes. Our results demonstrate that filmy fern gametophytes and sporophytes differ in their physiology and niche requirements, and point to the importance of microhabitat in shaping the evolution of water-use strategies in vascular plants.

sentences: Endocytosis-mediated vitellogenin absorption and lipid metabolism in the hindgut-derived pseudoplacenta of the viviparous teleost Xenotoca eiseni Certain viviparous animals possess mechanisms for mother-to-embryo nutrient transport during gestation. Xenotoca eiseni is one such viviparous teleost species in which the mother supplies proteins and other components to the offspring developing in the ovary. The embryo possesses trophotenia, a hindgut-derived pseudoplacenta to receive the maternal supplement. However, the molecular mechanisms underlying viviparous non-mammalian animals remain elusive. We conducted this study to investigate the mechanism for nutrient absorption and degradation in trophoenia of X. eiseni. The tracer assay indicated that a lipid transfer protein, vitellogenin (Vtg), was absorbed into the epithelial layer cells of trophotaenia. Vtg uptake was significantly suppressed by Pitstop-2, an inhibitor of clathrin-mediated endocytosis. Gene expression analysis indicated that the genes involved in endocytosis-mediated lipolysis and lysosomal cholesterol transport were expressed in trophotaenia. In contrast, plasma membrane transporters expressed in the intestinal tract were not functional in trophotaenia. Our results suggested that endocytosis-mediated lysosomal lipolysis is one of the mechanisms underlying maternal component metabolism. Thus, our study demonstrated how viviparous teleost species have acquired a unique developmental system that is based on the hindgut-derived pseudoplacenta.

sentences: YAP1 Activation by Human Papillomavirus E7 Promotes Basal Cell Identity in Squamous Epithelia Persistent human papillomavirus (HPV) infection of stratified squamous epithelial cells causes nearly five percent of cancer cases worldwide. HPV-positive oropharyngeal cancers harbor few mutations in the Hippo signaling pathway compared to HPV-negative cancers at the same anatomical site, prompting the hypothesis that an HPV-encoded protein inactivates the Hippo pathway and activates the Hippo effector YAP1. The HPV E7 oncoprotein is required for HPV infection and for HPV-mediated oncogenic transformation. We investigated the effects of HPV oncoproteins on YAP1 and found that E7 activates YAP1, promoting YAP1 nuclear localization in basal epithelial cells. YAP1 activation by HPV E7 required that E7 bind and degrade the tumor suppressor PTPN14. E7 required YAP1 transcriptional activity to extend the lifespan of primary keratinocytes, indicating that YAP1 activation contributes to E7 carcinogenic activity. Maintaining infection in basal cells is critical for HPV persistence, and here we demonstrate that YAP1 activation causes HPV E7 expressing cells to be retained in the basal compartment of stratified epithelia. We propose that YAP1 activation resulting from PTPN14 inactivation is an essential, targetable activity of the HPV E7 oncoprotein relevant to HPV infection and carcinogenesis.

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Force sensing on cells and tissues by atomic force microscopy Biosensors are aimed to detect tiny physical and chemical stimuli in biological systems. Physical forces are ubiquitous, being implied in all cellular processes, including cell adhesion, migration, and differentiation. Given the strong interplay between cells and their microenvironment, the extracellular matrix (ECM), the structural and mechanical properties of the ECM play an important role in the transmission of external stimuli to single cells within the tissue. Vice versa, also cells themselves use self-generated forces to probe the biophysical properties of the ECM. ECM mechanics influences cell fate, regulates tissue development and show peculiar features in health and disease conditions of living organisms. Force sensing in biological systems is therefore crucial to dissect and understand complex biological processes, such as mechanotransduction.

Atomic Force Microscopy (AFM), which can both sense and apply forces at the nanoscale, with sub-nanoNewton sensitivity, represents an enabling technology and a crucial experimental tool in biophysics and mechanobiology.

In this work, we report on the application of AFM to study of biomechanical fingerprints of different components of biological systems, such as the ECM, the whole cell, and cellular components, like the nucleus and the glycocalyx. We show that physical observables like the (spatially resolved) Youngs modulus of elasticity of ECMs or cells, and the effective thickness and stiffness of the glycocalyx, can be quantitatively characterised by AFM. Their modification can be correlated to changes of the microenvironment, physio-pathological conditions, or gene regulation.

sentences: Dopamine-dependent cAMP dynamics in basal amygdala glutamatergic neurons Dopaminergic inputs to basal amygdala (BA) instruct learning of motivational salience. Here, we investigated the dynamics of dopamine release and downstream signaling during multiple salient events occurring within tens of seconds. We established in vitro and in vivo real-time tracking and manipulation of cAMP - a key intracellular plasticity signal downstream of dopamine receptor activation. Optogenetically-evoked release of dopamine drove proportional increases in cAMP in almost all BA glutamatergic neurons, suggesting widespread actions of dopamine across neurons preferring positive or negative valence. These cAMP responses decayed more slowly than dopamine release, potentially extending the window of plasticity. cAMP levels accumulated following direct photostimulation of cAMP but not repeated stimulation of dopamine axons, due to potent depression of dopamine release. cAMP and protein kinase A (PKA) responses to repeated appetitive or aversive stimuli also exhibited pronounced depression. Thus, history-dependent dynamics of dopamine and cAMP may regulate learning of temporally clustered, salient stimuli.

sentences: Modelling the spread and mitigation of an emerging vector-borne pathogen:citrus greening in the U.S. Predictive models, based upon epidemiological principles and fitted to surveillance data, play an increasingly important role in shaping regulatory and operational policies for emerging outbreaks. Data for parameterising these strategically important models are often scarce when rapid actions are required to change the course of an epidemic invading a new region. We provide a flexible toolkit for landscape-scale disease management, which is applicable to a range of emerging pathogens including vector-borne pathogens for both endemic and invading epidemic vectors. We use the toolkit to analyse and predict the spread of Huanglongbing disease or citrus greening in the U.S. We estimate epidemiological parameters using survey data from one region (Texas) and show how to transfer and test parameters to construct predictive spatio-temporal models for another region (California). The models are used to screen effective coordinated and reactive management strategies for different regions.

sentences: Electrically Controlling and Optically Observing the Membrane Potential of Supported Lipid Bilayers Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipid bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of the equivalent electrical circuit. We also describe the effect of the conducting electrode layer on the fluorescence of the optical probe through metal-induced energy transfer. We conclude with a discussion on possible applications of this platform for the study of voltage-dependent membrane proteins and other processes in membrane biology and surface science.

sentences: Localizing temporal lobe seizure onset zones using deep learning on SEEG cortico-cortical evoked potentials In drug resistant temporal lobe epilepsy, automated tools for seizure onset zone (SOZ) localization using brief interictal recordings would supplement presurgical evaluations and improve care. Thus, we sought to localize SOZs by training a multi-channel convolutional neural network on stereo-EEG (SEEG) cortico-cortical evoked potentials. We performed single pulse electrical stimulation with 10 drug resistant temporal lobe epilepsy patients implanted with SEEG. Using the 500,000 unique post-stimulation SEEG epochs, we trained a multi-channel one-dimensional convolutional neural network to determine whether an SOZ was stimulated. SOZs were classified with a mean leave-one-patient-out testing sensitivity of 78.1% and specificity of 74.6%. To achieve maximum accuracy, the model requires a 0-350 ms post stimulation time period. Post-hoc analysis revealed that the model accurately classified unilateral vs bilateral mesial temporal lobe seizure onset, and neocortical SOZs. This is the first demonstration, to our knowledge, that a deep learning framework can be used to accurately classify SOZs using cortico-cortical evoked potentials. Our findings suggest accurate classification of SOZs relies on a complex temporal evolution of evoked potentials within 350 ms of stimulation. Validation in a larger dataset could provide a practical clinical tool for the presurgical evaluation of drug resistant epilepsy.

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An early decline in ETPs reflects reduced pre-thymic progenitors and altered signals from the thymus microenvironment Age-related thymus involution results in decreased T-cell production, contributing to increased susceptibility to pathogens. Elucidating mechanisms underlying involution will inform strategies to restore thymopoiesis. The thymus is colonized by circulating bone marrow (BM)-derived thymus seeding progenitors (TSPs) that differentiate into early T-cell progenitors (ETPs). We find ETP cellularity declines as early as 3 months (3MO) in mice. This initial ETP reduction could reflect changes in pre-thymic progenitors and/or thymic stromal niches. We demonstrate that the number of functional TSP/ETP niches is not reduced with age. Instead, the number of pre-thymic BM and circulating lymphoid progenitors is substantially reduced by 3MO, although their intrinsic ability to seed and differentiate in the thymus is maintained. Additionally, Notch signaling in BM progenitors and ETPs declines by 3MO, indicating defective niche quality contributes to the reduction in ETPs. Together, these findings indicate that diminished BM lymphopoiesis and thymic stromal support contribute to the initial decline in ETPs, setting the stage for progressive thymus involution.

Summary statementThe number of early T-cell progenitors declines by 3 months of age in mice. This decline reflects a sharp drop in circulating thymus seeding progenitors, fewer bone marrow lymphoid progenitors, and reduced Notch signaling in both bone marrow and thymus.

sentences: A human monoclonal antibody bivalently binding two different epitopes in streptococcal M protein protects against infection Group A streptococci have evolved multiple strategies to evade human antibodies, making it challenging to create effective vaccines or antibody treatments. Here, we have generated antibodies derived from the memory B cells of an individual who had successfully cleared a group A streptococcal infection. The antibodies bind with high affinity in the central region of the surface-bound M protein. Such antibodies are typically non-opsonic. However, one antibody could effectively promote vital immune functions, including phagocytosis and in vivo protection. Remarkably, this antibody primarily interacts through a bivalent dual-Fab cis mode, where the Fabs bind to two distinct epitopes in the M protein. The dual-Fab cis binding phenomenon is conserved across different groups of M types. In contrast, other antibodies binding with normal single-Fab mode to the same region can not bypass the M proteins virulent effects. A broadly binding, protective monoclonal antibody could be a candidate for anti-streptococcal therapy. Our findings highlight the concept of dual-Fab cis binding as a means to access conserved, and normally non-opsonic regions, for protective antibody targeting.

sentences: Crosstalk between AML and stromal cells triggers acetate secretion through the metabolic rewiring of stromal cells Acute myeloid leukaemia (AML) cells interact and modulate components of their surrounding microenvironment into their own benefit. Stromal cells have been shown to support AML survival and progression through various mechanisms. Nonetheless, it is unclear whether AML cells could establish beneficial metabolic interactions with stromal cells. Here, we identify a novel metabolic crosstalk between AML and stromal cells where AML cells prompt stromal cells to secrete acetate for their own consumption to feed the tricarboxylic acid cycle (TCA). By performing transcriptome analysis, tracer-based metabolic NMR analysis and ROS measurements, we observe that stromal cells present a higher rate of glycolysis. We also find that acetate in stromal cells is derived from pyruvate via chemical conversion under the influence of reactive oxygen species (ROS) following ROS transfer from AML to stromal cells via gap junctions. Overall, we present a unique metabolic communication between AML and stromal cells that could potentially be exploited for adjuvant therapy.

sentences: DAXX safeguards pericentromeric heterochromatin formation in embryonic stem cells DNA methylation is essential for heterochromatin formation and repression of DNA repeat transcription, both of which are essential for genome integrity. Loss of DNA methylation is associated with disease, including cancer, but is also required for development. Alternative pathways to maintain heterochromatin are thus needed to limit DNA damage accumulation. Here, we find that DAXX, an H3.3 chaperone, protects pericentromeric heterochromatin and is essential for embryonic stem cells (ESCs) maintenance in the ground-state of pluripotency. Upon DNA demethylation-mediated damage, DAXX relocalizes to pericentromeric regions, and recruits PML and SETDB1, thereby promoting heterochromatin formation. In the absence of DAXX, the 3D-architecture and physical properties of pericentric heterochromatin are disrupted, resulting in derepression of major satellite DNA. Using epigenome editing tools, we demonstrate that H3.3, and specifically H3.3K9 modification, directly contribute to maintaining pericentromeric chromatin conformation. Altogether, our data reveal that DAXX and H3.3 unite DNA damage response and heterochromatin maintenance in ESCs.

sentences: Evolutionary analysis of genomes of SARS-CoV-2-related bat viruses suggests old roots, constant effective population size, and possible increase of fitness It is of vital practical interest to understand the co-evolution of bat {beta}-coronaviruses with their hosts, since a number of these most likely crossed the species boundaries and infected humans. Complete sequences of 47 consensus genomes are available for bat {beta}-coronaviruses related to the SARS-CoV-2 human virus. We carried out several types of evolutionary analyses using these data. First, using the publicly available BEAST 2 software, we generated phylogenetic trees and skyline plots. The roots of the trees, both for the entire sequences and subsequences coding for the E and S proteins as well as the 5 and 3 UTR regions, are estimated to be located from several decades to more than a thousand years ago, while the effective population sizes remained largely constant. Motivated by this, we developed a simple estimator of the effective population size in a Moran model with constant population, which, under the model is equal to the expected age of the MRCA measured in generations. Comparisons of these estimates to those produced by BEAST 2 shows qualitative agreement. We also compared the site frequency spectra (SFS) of the bat genomes to those provided by the Moran Tug-of-War model. Comparison does not exclude the possibility that overall fitness of the bat {beta}-coronaviruses was increasing over time as a result of directional selection. Stability of interactions of bats and their viruses was considered likely on the basis of specific manner in which bat immunity is tuned, and it seems consistent with our analysis.

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Light-weight Electrophysiology Hardware and Software Platform for Cloud-Based Neural Recording Experiments ObjectiveNeural activity represents a functional readout of neurons that is increasingly important to monitor in a wide range of experiments. Extracellular recordings have emerged as a powerful technique for measuring neural activity because these methods do not lead to the destruction or degradation of the cells being measured. Current approaches to electrophysiology have a low throughput of experiments due to manual supervision and expensive equipment. This bottleneck limits broader inferences that can be achieved with numerous long-term recorded samples.

ApproachWe developed Piphys, an inexpensive open source neurophysiological recording platform that consists both hardware and software. It is easily accessed and controlled via a standard web interface through Internet of Things (IoT) protocols.

Main ResultsWe used a Raspberry Pi as the primary processing device and Intan bioamplifier. We designed a hardware expansion circuit board and software to enable voltage sampling and user interaction. This standalone system was validated with primary human neurons, showing reliability in collecting real-time neural activity.

SignificanceThe hardware modules and cloud software allow for remote control of neural recording experiments as well as horizontal scalability, enabling long-term observations of development, organization, and neural activity at scale.

sentences: Bird community structure in a mixed forest-production landscape in the northern Western Ghats, India Production landscapes outside protected areas are important for the conservation of wildlife, especially in countries like India with high biodiversity and human densities. Production landscapes like plantations often occur in close proximity to biodiversity-rich areas. Rubber and cashew are lucrative plantation crops in India, which although grown under similar environmental conditions, vary in their management and productivity. These plantation crops are often found along the edges of forests, thereby creating a buffer between forested and urban landscapes. While these areas have the potential to be a refuge for species otherwise restricted to natural forests, they are poorly-studied. To address this knowledge gap, we studied how habitat type (cashew, rubber or forest) and habitat characteristics affect bird diversity and guild structure in the Tillari landscape of Western Ghats, Maharashtra. Additionally, we examined how these effects are mediated by distance of plantations to nearest forest edge. In each habitat type, in 2018, we sampled birds six times each in 30 locations using fixed radius point counts. We found that bird diversity in cashew plantations (14 species) was comparable to that of adjoining forests (15 species). Rubber plantations, on the other hand, had lower bird diversity (9 species) than that in cashew or forests. When bird diversity was analysed based on dietary guilds, rubber plantations had fewer bird species in all guilds and cashew plantations had higher abundance of nectivores and lower richness of insectivores than in forest. Distance from the forest did not affect bird diversity in rubber plantations, whereas cashew plantations had fewer nectivorous birds and higher insectivorous birds away from the forest edge. Our results show that cashew plantations can serve as an important surrogate habitat for forest birds in the northern Western Ghats. The findings indicate the unsuitability of rubber plantations for sustaining bird diversity. While there are many studies available on bird diversity in rubber plantations, this is among the first studies of bird community structure in cashew plantations. At a time when forests are rapidly being cleared for plantations, our findings provide valuable data to examine the differential impacts of plantation type on biodiversity.

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Single cell trajectory modeling identifies a primitive trophoblast state defined by BCAM enrichment In early placental development, progenitor cytotrophoblasts (CTBs) differentiate along one of two cellular trajectories: the villous or extravillous pathways. CTBs committed to the villous pathway fuse with neighboring CTBs to form the outer multinucleated syncytiotrophoblast (SCT), while CTBs committed to the extravillous pathway differentiate into invasive extravillous trophoblasts (EVT). Unfortunately, little is known about the processes controlling human CTB progenitor maintenance and differentiation. To address this, we established a single cell RNA sequencing (scRNA-seq) dataset from first trimester placentas to identify cell states important in trophoblast progenitor establishment, renewal, and differentiation. Multiple distinct trophoblast states were identified, representing progenitor CTBs, column CTBs, SCT precursors, and EVT. Lineage trajectory analysis identified a progenitor origin that was reproduced in human trophoblast stem cell organoids. Heightened expression of basal cell adhesion molecule (BCAM) defined this primitive state, where BCAM enrichment or gene silencing resulted in enhanced or diminished organoid growth. Together, this work describes at high-resolution trophoblast heterogeneity within the first trimester, resolves gene networks within human CTB progenitors, and identifies BCAM as a primitive progenitor marker and possible regulator.

Summary StatementLineage trajectory modeling identifies multiple human progenitor trophoblast states and defines trophoblast differentiation kinetics, where BCAM-expressing progenitors demonstrate enhanced regenerative ability.

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A method for independent estimation of false localisation rate for phosphoproteomics Phosphoproteomics methods are commonly employed in labs to identify and quantify the sites of phosphorylation on proteins. In recent years, various software tools have been developed, incorporating scores or statistics related to whether a given phosphosite has been correctly identified, or to estimate the global false localisation rate (FLR) within a given data set for all sites reported. These scores have generally been calibrated using synthetic data sets, and their statistical reliability on real datasets is largely unknown. As a result, there is considerable problem in the field of reporting incorrectly localised phosphosites, due to inadequate statistical control.

In this work, we develop the concept of using scoring and ranking modifications on a decoy amino acid, i.e. one that cannot be modified, to allow for independent estimation of global FLR. We test a variety of different amino acids to act as the decoy, on both synthetic and real data sets, demonstrating that the amino acid selection can make a substantial difference to the estimated global FLR. We conclude that while several different amino acids might be appropriate, the most reliable FLR results were achieved using alanine and leucine as decoys, although we have a preference for alanine due to the risk of potential confusion between leucine and isoleucine amino acids. We propose that the phosphoproteomics field should adopt the use of a decoy amino acid, so that there is better control of false reporting in the literature, and in public databases that re-distribute the data. Data are available via ProteomeXchange with identifier PXD028840.

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Minor deviations from randomness have huge repercussions on the functional structuring of sequence space Approaches based on molecular evolution have organized natural proteins into a hierarchy of families, superfamilies, and folds, which are often pictured as islands in a great sea of unrealized and generally non-functional polypeptides. In contrast, approaches based on information theory have substantiated a mostly random scatter of natural proteins in global sequence space. We evaluate these opposing views by analyzing fragments of a given length derived from either a natural dataset or different random models. For this, we compile distances in sequence space between fragments within each dataset and compare the resulting distance distributions between sets. Even for 100-mers, more than 95% of distances can be accounted for by a random sequence model that incorporates the natural amino acid frequency of proteins. When further accounting for the specific residue composition of the respective fragments, which would include biophysical constraints of protein folding, more than 99% of all distances can be modeled. Thus, while the local space surrounding a protein is almost entirely shaped by common descent, the global distribution of proteins in sequence space is close to random, only constrained by divergent evolution through the requirement that all intermediates connecting two forms in evolution must be functional.

Significance StatementWhen generating new proteins by evolution or design, can the entire sequence space be used, or do viable sequences mainly occur only in some areas of this space? As a result of divergent evolution, natural proteins mostly form families that occupy local areas of sequence space, suggesting the latter. Theoretical work however indicates that these local areas are highly diffuse and do not dramatically affect the statistics of sequence distribution, such that natural proteins can be considered to effectively cover global space randomly, though extremely sparsely. By comparing the distance distribution of natural sequences to that of various random models, we find that they are indeed distributed largely randomly, provided that the amino acid composition of natural proteins is respected.

sentences: Weak Coupling Between Spontaneous Local Cortical Activity State Switches Under Anesthesia Leads to Strongly Correlated Global Cortical States Under anesthesia, neural dynamics deviate dramatically from those seen during wakefulness. During recovery from this perturbation, thalamocortical activity abruptly switches among a small set of metastable intermediate states. These metastable states and structured transitions among them form a scaffold that guides the brain back to the waking state. Here, we investigate the mechanisms that constrain cortical activity to discrete states and give rise to abrupt transitions among them. If state transitions were imposed onto the thalamocortical system by changes in the subcortical modulation, different cortical sites should exhibit near-synchronous state transitions. To test this hypothesis, we quantified state synchrony at different cortical sites in anesthetized rats. States were defined by compressing spectra of layer-specific local field potentials (LFPs) in visual and motor cortices. Transition synchrony, mutual information, and canonical correlations all demonstrate that most state transitions in the cortex are local and that coupling between sites is weak. Fluctuations in the LFP in the thalamic input layer 4 were particularly dissimilar from those in supra- and infra-granular layers. Thus, our results suggest that the discrete global cortical states are not imposed by the ascending modulatory pathways but emerge from the multitude of weak pairwise interactions within the cortex.

sentences: Essential regulatory functions of CaMKII T286 phosphorylation in LTP and two distinct forms of LTD The Ca2+/calmodulin-dependent protein kinase II (CaMKII) mediates both long-term potentiation and depression (LTP and LTD) of excitatory synapses, two opposing forms of synaptic plasticity induced by strong versus weak stimulation of NMDA-type glutamate receptors (NMDARs). NMDAR-dependent LTD is prevalent in juvenile hippocampus, but in mature hippocampus, LTD is still readily induced by stimulating metabotropic glutamate receptors (mGluRs). Here we show that mGluR-dependent LTD also requires CaMKII and its T286 autophosphorylation that induces Ca2+-independent autonomous kinase activity. This autophosphorylation (i) accelerated CaMKII movement to excitatory synapses after LTP stimuli and (ii) was required for the movement to inhibitory synapses after NMDAR-LTD stimuli. Similar to NMDAR-LTD, the mGluR-LTD stimuli did not induce any CaMKII movement to excitatory synapses. However, in contrast to NMDAR-LTD, the mGluR-LTD did not involve CaMKII movement to inhibitory synapses and did not require additional T305/306 autophosphorylation. Taken together, even though CaMKII T286 autophosphorylation has a longstanding prominent role in LTP, it is also required for both major forms of LTD in hippocampal neurons, albeit with differential requirements for the heterosynaptic communication of excitatory signals to inhibitory synapses.

sentences: Capturing non-local through-bond effects in molecular mechanics force fields I: Fragmenting molecules for quantum chemical torsion scans Accurate molecular mechanics force fields for small molecules are essential for predicting protein-ligand binding affinities in drug discovery and understanding the biophysics of biomolecular systems. Torsion potentials derived from quantum chemical (QC) calculations are critical for determining the conformational distributions of small molecules, but are computationally expensive and scale poorly with molecular size. To reduce computational cost and avoid the complications of distal through-space intramolecular interactions, molecules are generally fragmented into smaller entities to carry out QC torsion scans. However, torsion potentials, particularly for conjugated bonds, can be strongly affected by through-bond chemistry distal to the torsion it-self. Poor fragmentation schemes have the potential to significantly disrupt electronic properties in the region around the torsion by removing important, distal chemistries, leading to poor representation of the parent molecules chemical environment and the resulting torsion energy profile. Here we show that a rapidly computable quantity, the fractional Wiberg bond order (WBO), is a sensitive reporter on whether the chemical environment around a torsion has been disrupted. We show that the WBO can be used as a surrogate to assess the robustness of fragmentation schemes and identify conjugated bond sets. We use this concept to construct a validation set by exhaustively fragmenting a set of druglike organic molecules and examine their corresponding WBO distributions derived from accessible conformations that can be used to evaluate fragmentation schemes. To illustrate the utility of the WBO in assessing fragmentation schemes that preserve the chemical environment, we propose a new fragmentation scheme that uses rapidly-computable AM1 WBOs, which are available essentially for free as part of standard AM1-BCC partial charge assignment. This approach can simultaneously maximize the chemical equivalency of the fragment and the substructure in the larger molecule while minimizing fragment size to accelerate QC torsion potential computation for small molecules and reducing undesired through-space steric interactions.

sentences: wnt16 exerts pleiotropic effects on bone and lean mass in zebrafish Osteoporosis, a disease of bone fragility, and sarcopenia, a condition of reduced muscle mass and strength, commonly occur in the same individual--a condition termed osteosarcopenia. Genetic variants at the CPED1-WNT16 locus have pleiotropic effects on human bone mineral density (BMD) and lean tissue mass. However, our current understanding of Wnt16 and its influence on bone fails to explain effects on lean mass at this locus. In this study, we sought to better understand the biological mechanism underlying pleiotropy at the CPED1-WNT16 locus on BMD and lean mass. Using single cell analysis, microCT imaging, and genetic approaches in zebrafish, we show that wnt16 exerts dual influence on muscle and bone, and provide evidence that this occurs through a function in somite development, a period in which precursors for both of these tissues are specified. We show that mutations in wnt16 alter vertebral size and lean mass during axial skeletogenesis. We show that wnt16+ cells transcriptionally resemble cells within the amniote dermomyotome, and provide evidence they function as myogenic precursors during embryonic development. We also provide evidence that WNT16 is a gene of major effect on lean mass at the CPED-WNT16 locus. Our studies support the potential for wnt16 to exert pleiotropic effects on bone and lean mass through a function in myogenic precursors.

sentences: Functional insights of two MATE transporters from Vibrio fluvialis Functional characterization of H- and D-MATE (Multidrug and Toxin Extrusion) transporters from clinical isolates of Vibrio fluvialis revealed H-type conferred resistance to fluoroquinolones, ethidium bromide and safranin whereas D-type exhibited marginal resistance towards ethidium bromide only. Both H-/D-type transporters were inhibited by reserpine resulting in increased intracellular norfloxacin concentration. The efflux was facilitated by both Na+/K+ ions, suggesting that these efflux pumps were ion-dependent. In presence of various classes of EPIs, there was decrease in MIC exhibited by H-/D-type efflux pumps towards norfloxacin which didnt translate into transport inhibition. But reserpine presented a conclusive pattern with decrease in MIC towards norfloxacin and increased norfloxacin accumulation inferring maximum inhibition. Substrate binding and electrostatic charge distribution of both the transporters was similar to other known MATE transporters. The H-type exhibited 10 transmembranes and D-type exhibited 11 TMs which was different from other MATE transporters known to have 12 TMs (Transmembranes). Data derived from molecular docking and ion binding studies revealed that Aspartic Acid residue in 1st TM acts as ion binding site with transport mechanism similar to NorM. Electrostatic potential map of both the transporters revealed that there is a cavity formation within the transporters surrounded by charged electronegative amino acid residues. Interestingly, surface models of both transporters revealed that 1st TM forms covalent bond with 7th TM towards extracellular space. Docking studies also revealed that reserpine covalently binds to central pocket of both transporters and serves as excellent EPI against these transporters as evidenced by MIC and drug accumulation assays.

sentences: Adipose triglyceride lipase promotes prostaglandin-dependent actin remodeling by regulating substrate release from lipid droplets A key factor controlling oocyte quality and fertility is lipids. Even though lipid droplets (LDs) are crucial regulators of lipid metabolism, their roles in fertility are poorly understood. During Drosophila oogenesis, LD accumulation in nurse cells coincides with dynamic actin remodeling necessary for late-stage follicle morphogenesis and fertility. Loss of the LD-associated Adipose Triglyceride Lipase (ATGL) disrupts both actin bundle formation and cortical actin integrity, an unusual phenotype also seen when Pxt, the enzyme responsible for prostaglandin (PG) synthesis, is missing. Dominant genetic interactions and PG treatment of follicles in vitro reveal that ATGL and Pxt act in the same pathway to regulate actin remodeling, with ATGL upstream of Pxt. Further, lipidomic analysis detects arachidonic acid (AA) containing triglycerides in ovaries. Because AA is the substrate for Pxt, we propose that ATGL releases AA from LDs to drive PG synthesis necessary for follicle development. We also find that exogenous AA is toxic to follicles in vitro, and LDs modulate this toxicity. This leads to the model that LDs both sequester AA to limit toxicity, and release AA via ATGL to drive PG production. We speculate that the same pathways are conserved across organisms to regulate oocyte development and promote fertility.

sentences: The same ultra-rapid parallel brain dynamics underpin the production and perception of speech. The temporal dynamics by which linguistic information becomes available is one of the key properties to understand how language is organised in the brain. An unresolved debate between different brain language models is whether words, the building blocks of language, are activated in a sequential or parallel manner. In this study we approached this issue from a novel perspective by directly comparing the time course of word component activation in speech production versus perception. In an overt object naming task and a passive listening task we analysed with mixed linear models at the single-trial level the event-related brain potentials elicited by the same lexico-semantic and phonological word knowledge in the two language modalities. Results revealed that both word components manifested simultaneously as early as 75 ms after stimulus onset in production and perception; differences between the language modalities only became apparent after 300 ms of processing. The data provide evidence for ultra-rapid parallel dynamics of language processing and are interpreted within a neural assembly framework where words recruit the same integrated cell assemblies across production and perception. These word assemblies ignite early on in parallel and only later on reverberate in a behaviour-specific manner.

sentences: Protective mitochondrial fission induced by stress responsive protein GJA1-20k The Connexin43 gap junction gene GJA1 has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound, and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 seconds of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin Related Protein 1 (DRP1). We find that GJA1-20k induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.

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ColorBayes: Improved color correction of high-throughput plant phenotyping images to account for local illumination differences BackgroundColor distortion is an inherent problem in image-based phenotyping systems that are illuminated by artificial light. This distortion is problematic when examining plants because it can cause data to be incorrectly interpreted. One of the leading causes of color distortion is the non-uniform spectral and spatial distribution of artificial light. However, color correction algorithms currently used in plant phenotyping assume that a single and uniform illuminant causes color distortion. These algorithms are consequently inadequate to correct the local color distortion caused by multiple illuminants common in plant phenotyping systems, such as fluorescent tubes and LED light arrays. We describe here a color constancy algorithm, ColorBayes, based on Bayesian inference that corrects local color distortions. The algorithm estimates the local illuminants using the Bayes rule, the maximum a posteriori, the observed image data, and prior illuminant information. The prior is obtained from light measurements and Macbeth ColorChecker charts located on the scene.

ResultsThe ColorBayes algorithm improved the accuracy of plant color on images taken by an indoor plant phenotyping system. Compared with existing approaches, it gave the most accurate metric results when correcting images from a dataset of Arabidopsis thaliana images.

The software is available at https://github.com/diloc/Color_correction.git.

sentences: Myoglobin primary structure reveals multiple convergent transitions to semi-aquatic life in the world smallest mammalian divers Identifying the phylogenomic underpinnings of specialized phenotypes that fueled transitions into new adaptive zones is central to evolutionary biology but is often confounded by a fragmentary fossil record, morphological convergence, and unresolved phylogenetic relationships. The speciose mammalian order Eulipotyphla (e.g., moles, shrews, hedgehogs, solenodons) combines an unusual diversity of semi-aquatic, semi-fossorial, and fossorial forms that arose from terrestrial forbearers, yet the ecomorphological pathways leading to these lifestyles have been disputed for a century and more, calling for novel approaches. Here we resolve previously intractable eulipotyphlan intra-family relationships and establish the net surface charge of the oxygen-storing muscle protein myoglobin-readily determined from its primary structure-as a molecular signature to trace ancient lifestyle transitions based on protein sequence alone. Our analyses confidently resolve fossorial habits having evolved twice in talpid moles and reveal five independent origins of a semi-aquatic lifestyle in the order housing the worlds smallest endothermic divers.

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Heterologous expression of Dehalobacter spp. respiratory reductive dehalogenases in Escherichia coli Reductive dehalogenases (RDases) are a family of redox enzymes that are required for anaerobic organohalide respiration, a microbial process that is useful in bioremediation. Structural and mechanistic studies of these enzymes have been greatly impeded due to challenges in RDase heterologous expression, primarily because of their cobamide-dependence. There have been a few successful attempts at RDase production in unconventional heterologous hosts, but a robust method has yet to be developed. In this work we outline a novel respiratory RDase expression system using Escherichia coli as the host. The overexpression of E. colis cobamide transport system, btu, and RDase expression under anaerobic conditions were established to be essential for the expression of active RDases from Dehalobacter - an obligate organohalide respiring bacterium. The expression system was validated on six RDase enzymes with amino acid sequence identities ranging from >30-95%. Dehalogenation activity was verified for each RDase by assaying cell-free extracts of small-scale expression cultures on various chlorinated substrates including chloroalkanes, chloroethenes, and hexachlorocyclohexanes. Two RDases, TmrA from Dehalobacter sp. UNSWDHB and HchA from Dehalobacter sp. HCH1, were purified by nickel affinity chromatography. Incorporation of both the cobamide and iron-sulfur cluster cofactors was verified, and the specific activity of TmrA was found to be consistent with that of the native enzyme. The heterologous expression of respiratory RDases, particularly from obligate organohalide respiring bacteria, has been extremely challenging and unreliable. Here we present a relatively straightforward E. coli expression system that has performed well for a variety of Dehalobacter spp. RDases.

IMPORTANCEUnderstanding microbial reductive dehalogenation is important to refine the global halogen cycle and to improve bioremediation of halogenated contaminants; however, studies of the family of enzymes responsible are limited. Characterization of reductive dehalogenase enzymes has largely eluded researchers due to the lack of a reliable and high-yielding production method. We are presenting an approach to express reductive dehalogenase enzymes from Dehalobacter, a key group of organisms used in bioremediation, in E. coli. This expression system will propel the study of reductive dehalogenases by facilitating their production and isolation, allowing researchers to pursue more in-depth questions about the activity and structure of these enzymes. This platform will also provide a starting point to improve the expression of reductive dehalogenases from many other organisms.

sentences: A combinatorial anticancer drug screen identifies off-target effects of epigenetic chemical probes Anticancer drug response is determined by genetic and epigenetic mechanisms. To identify the epigenetic regulators of anticancer drug response, we conducted a chemical epigenetics screen using chemical probes that target different epigenetic modulators. In this screen, we tested 31 epigenetic probes in combination with 14 mechanistically diverse anticancer agents and identified 8 epigenetic probes that significantly potentiate the cytotoxicity of TAK-243, a first-in-class ubiquitin-activating enzyme (UBA1) inhibitor evaluated in several solid and hematologic malignancies. These probes are TP-472, GSK-864, A-196, UNC1999, SGC-CBP30 and PFI-4 (and its related analogs GSK6853 and GSK5959), and they target BRD9/7, mutant IDH1, SUV420H1/2, EZH2/1, p300/CBP and BRPF1B, respectively. In contrast to epigenetic probes, negative control compounds did not have a significant impact on TAK-243 cytotoxicity. Potentiation of TAK-243 cytotoxicity was associated with reduced ubiquitylation and induction of apoptosis. Mechanistically, epigenetic probes exerted their potentiation by inhibiting the efflux transporter ABCG2 without inducing significant changes in the ubiquitylation pathways or ABCG2 expression levels. The identified probes shared chemical scaffold similarities with TAK-243 and could potentially interact with ABCG2 as assessed by docking analysis. Based on these data, we have developed a cell-based assay that can quantitatively evaluate ABCG2 inhibition by drug candidates. In conclusion, our study identifies epigenetic chemical probes that profoundly potentiate TAK-243 cytotoxicity through off-target ABCG2 inhibition. We also provide experimental evidence that several negative control compounds cannot exclude a subset of off-target effects of chemical probes. Finally, potentiation of TAK-243 cytotoxicity can serve as a quantitative measure of ABCG2-inhibitory activity.

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Staphylococcus aureus overcomes anaerobe-derived short-chain fatty acid stress via FadX and the CodY regulon Chronic rhinosinusitis (CRS) is characterized by immune dysfunction, mucus hypersecretion, and persistent infection of the paranasal sinuses. While Staphylococcus aureus is a primary CRS pathogen, recent sequence-based surveys have found increased relative abundances of anaerobic bacteria, suggesting that S. aureus may experience altered metabolic landscapes in CRS relative to healthy airways. To test this possibility, we characterized the growth kinetics and transcriptome of S. aureus in supernatants of the abundant CRS anaerobe Fusobacterium nucleatum. While growth was initially delayed, S. aureus ultimately grew to similar levels as in the control medium. The transcriptome was significantly affected by F. nucleatum metabolites, with the agr quorum sensing system notably repressed. Conversely, expression of fadX, encoding a putative propionate coA-transferase, was significantly increased, leading to our hypothesis that short chain fatty acids (SCFAs) produced by F. nucleatum could mediate S. aureus growth behavior and gene expression. Supplementation with propionate and butyrate, but not acetate, recapitulated delayed growth phenotypes observed in F. nucleatum supernatants. A fadX mutant was found to be more sensitive than wild type to propionate, suggesting a role for FadX in the S. aureus SCFA stress response. Interestingly, spontaneous resistance to butyrate, but not propionate, was frequently observed. Whole genome sequencing and targeted mutagenesis identified codY mutants as resistant to butyrate inhibition. Together, these data show that S. aureus physiology is dependent on its co-colonizing microbiota and metabolites they exchange, and indicate that propionate and butyrate may act on different targets in S. aureus to suppress its growth.

ImportanceS. aureus is an important CRS pathogen, yet is found in the upper airways of 30-50% of people without complications. The presence of strict and facultative anaerobic bacteria in CRS sinuses has recently spurred research into bacterial interactions and how they influence S. aureus physiology and pathogenesis. We show here that propionate and butyrate produced by one such CRS anaerobe, F. nucleatum, alter the growth and gene expression of S. aureus. We show that fadX is important for S. aureus to resist propionate stress, and that the CodY regulon mediates growth in inhibitory concentrations of butyrate. This work highlights the possible complexity of S. aureus-anaerobe interactions, and implicates membrane stress as a possible mechanism influencing S. aureus behavior in CRS sinuses.

sentences: Hierarchical architecture of dopaminergic circuits enables second-order conditioning in Drosophila Dopaminergic neurons with distinct projection patterns and physiological properties compose memory subsystems in a brain. However, it is poorly understood whether or how they interact during complex learning. Here, we identify a feedforward circuit formed between dopamine subsystems and show that it is essential for second-order conditioning, an ethologically important form of higher-order associative learning. The Drosophila mushroom body comprises a series of dopaminergic compartments, each of which exhibits distinct memory dynamics. We find that a slow and stable memory compartment can serve as an effective "teacher" by instructing other faster and transient memory compartments via a single key interneuron, which we identify by connectome analysis and neurotransmitter prediction. This excitatory interneuron acquires enhanced response to reward-predicting odor after first-order conditioning and, upon activation, evokes dopamine release in the "student" compartments. These hierarchical connections between dopamine subsystems explain distinct properties of first- and second-order memory long known by behavioral psychologists.

sentences: Surface microbiota of Mediterranean loggerhead sea turtles unravelled by 16S and 18S amplicon sequencing Loggerhead sea turtle is considered a keystone species with major ecological role in Mediterranean marine environment. As with other wild reptiles, their outer microbiome is rarely studied. Although there are several studies on sea turtles macro-epibionts and endo-microbiota, there has been little research on epibiotic microbiota associated with sea turtles skin and carapace. Therefore, we provide the first identification of combined epibiotic eukaryotic and prokaryotic microbiota on Mediterranean loggerhead sea turtles. In this study, we sampled skin and carapace of 26 loggerheads from the Mediterranean Sea during 2018 and 2019. To investigate the overall microbial diversity and composition, amplicon sequencing of 16S and 18S rRNA genes was performed. We found that the Mediterranean loggerhead sea turtle epibiotic microbiota is a reservoir of a vast variety of microbial species. Microbial communities between samples mostly varied by different locations and seas, while within prokaryotic communities significant difference was observed between sampled body sites (carapace vs. skin). In terms of relative abundance, Proteobacteria and Bacteroidota were the most represented phyla within prokaryotes, while Alveolata and Stramenopiles thrived among eukaryotes. This study, besides providing a first survey of microbial eukaryotes on loggerheads via metabarcoding, identifies fine differences within both prokaryotic and eukaryotic microbial communities that seem to reflect the host anatomy and habitat. Multi-domain epi-microbiome surveys provide additional layers of information that are complementary with previous morphological studies and enable better understanding of the biology and ecology of these vulnerable marine reptiles.

sentences: Sublayer- and cell-type-specific neurodegenerative transcriptional trajectories in hippocampal sclerosis Hippocampal sclerosis, the major neuropathological hallmark of temporal lobe epilepsy, is characterized by different patterns of neuronal loss. The mechanisms of cell-type specific vulnerability, their progression and histopathological classification remain controversial. Here using single-cell electrophysiology in vivo and immediate early gene expression, we reveal that superficial CA1 pyramidal neurons are overactive in epileptic rats and mice in vivo. Bulk tissue and single-nucleus expression profiling disclosed sublayer-specific transcriptomic signatures and robust microglial pro-inflammatory responses. Transcripts regulating neuronal processes such as voltage-channels, synaptic signalling and cell adhesion molecules were deregulated by epilepsy differently across sublayers, while neurodegenerative signatures primarily involved superficial cells. Pseudotime analysis of gene expression in single-nuclei and in situ validation revealed separated trajectories from health to epilepsy across cell types, and identified a subset of superficial cells undergoing a later stage in neurodegeneration. Our findings indicate sublayer- and cell type-specific changes associated with selective CA1 neuronal damage contributing to progression of hippocampal sclerosis.

sentences: Conserved degronome features governing quality control-associated proteolysis The eukaryotic proteome undergoes constant surveillance by quality control systems that either sequester, refold, or eliminate aberrant proteins by ubiquitin-dependent mechanisms. Ubiquitin-conjugation necessitates the recognition of degradation determinants, termed degrons, by their cognate E3 ubiquitin-protein ligases. To learn about the distinctive properties of quality control degrons, we performed an unbiased peptidome stability screen in yeast. The search identified a large cohort of proteome-derived degrons., some of which exhibited broad E3 ligase specificity. Consequent application of a machine-learning algorithm established constraints governing degron potency, including the amino acids composition and secondary structure propensities. According to the set criteria, degrons with transmembrane domain-like characteristics are the most probable sequences to act as degrons. Similar quality control degrons were identified in viral and human proteins, suggesting for conserved degradation mechanisms. Altogether, the emerging data indicate that transmembrane domain-like degron features have been preserved in evolution as key quality control determinants of proteins half-life.

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Transendothelial migration induces differential migration dynamics of leukocytes in tissue matrix Leukocyte extravasation into inflamed tissue is a complex process that is difficult to capture as a whole in vitro. We employed a blood-vessel-on-a-chip model in which endothelial cells were cultured in a tube-like lumen in a collagen-1 matrix. The vessels are leak-tight, creating a barrier for molecules and leukocytes. Addition of inflammatory cytokine TNF- caused vasoconstriction, actin remodelling and upregulation of ICAM-1. Introducing leukocytes into the vessels allowed real-time visualisation of all different steps of the leukocyte transmigration cascade including migration into the extracellular matrix. Individual cell tracking over time distinguished striking differences in migratory behaviour between T-cells and neutrophils. Neutrophils cross the endothelial layer more efficiently than T-cells, but upon entering the matrix, neutrophils display high speed but low persistence, whereas T-cells migrate with low speed and rather linear migration. In conclusion, 3D imaging in real-time of leukocyte extravasation in a vessel-on-a-chip enables detailed qualitative and quantitative analysis of different stages of the full leukocyte extravasation process in a single assay.

SummaryA functional hydrogel-based blood-vessel-on-a-chip model is used to study the complete leukocyte transendothelial migration process in real time. T-lymphocytes and neutrophils exhibit distinct migration dynamics in the extravascular matrix after transendothelial migration, which can be altered using a chemotactic gradient.

sentences: Identifying Differential Methylation in Cancer Epigenetics via a Bayesian Functional Regression Model DNA methylation plays an essential role in regulating gene activity, modulating disease risk, and determining treatment response. Researchers can obtain insight into methylation patterns at a single nucleotide level utilizing next-generation sequencing technologies. However, complex features inherent in the data obtained via these technologies pose challenges beyond the typical big data problems. Identifying differentially methylated cytosines (dmc) or regions is one of such challenges. Current methodologies for identifying dmcs fall short in handling low read-depth data and missing values, capturing functional data patterns, granting multiple covariates (categorical, continuous, or combination), and multiple group comparisons. We have developed an efficient method to identify dmcs based on a Bayesian functional regression approach, termed DMCFB, that tackles these shortcomings. Through simulation studies, we establish that DMCFB outperforms current methods and results in better smoothing, and efficient imputation. We apply the proposed method to analyze a dataset containing patients with acute promyelocytic leukemia and control samples. With DMCFB, we discovered many new dmcs, and more importantly, exhibited enhanced consistency of differential methylation within islands and at their adjacent shores. Furthermore, we detected differential methylation at more of the binding sites of the fused gene involved in this cancer.

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V367F mutation in SARS-CoV-2 spike RBD emerging during the early transmission phase enhances viral infectivity through increased human ACE2 receptor binding affinity The current global pandemic of COVID-19 is caused by a novel coronavirus SARS-CoV-2. The SARS-CoV-2 spike protein receptor-binding domain (RBD) is the critical determinant of viral tropism and infectivity. To investigate whether naturally occurring mutations in the RBD during the early transmission phase have altered the receptor binding affinity and infectivity, firstly we analyzed in silico the binding dynamics between mutated SARS-CoV-2 RBDs and the human ACE2 receptor. Among 32,123 genomes of SARS-CoV-2 isolates (January through March, 2020), 302 non-synonymous RBD mutants were identified and clustered into 96 mutant types. The six dominant mutations were analyzed applying molecular dynamics simulations. The mutant type V367F continuously circulating worldwide displayed higher binding affinity to human ACE2 due to the enhanced structural stabilization of the RBD beta-sheet scaffold. The increased infectivity of V367 mutants was further validated by performing receptor-ligand binding ELISA, surface plasmon resonance, and pseudotyped virus assays. Genome phylogenetic analysis of V367F mutants showed that during the early transmission phase, most V367F mutants clustered more closely with the SARS-CoV-2 prototype strain than the dual-mutation variants (V367F + D614G) which emerged later and formed a distinct sub-cluster. The analysis of critical RBD mutations provides further insights into the evolutionary trajectory of SARS-CoV-2 under negative selection pressure and supports the continuing surveillance of spike mutations to aid in the development of new COVID-19 drugs and vaccines.

ImportanceA novel coronavirus SARS-CoV-2 has caused the pandemic of COVID-19. The origin of SARS-CoV-2 was associated with zoonotic infections. The spike protein receptor-binding domain (RBD) is identified as the critical determinant of viral tropism and infectivity. Thus, whether the mutations in the RBD of the circulating SARS-CoV-2 isolates have altered the receptor binding affinity and caused them more infectious, should be paid more attentions to. Given that SARS-CoV-2 is a novel coronavirus, the significance of our research is in identifying and validating the RBD mutant types emerging during the early transmission phase that have increased human ACE2 receptor binding affinity and infectivity. The RBD mutation analysis provides insights into SARS-CoV-2 evolution. The continuous surveillance of RBD mutations with increased human ACE2 affinity in human or other animals is important and necessary, particularly when the direct correlation between the virus variations and vaccine effectiveness is underdetermined during the sustained COVID-19 pandemic.

sentences: Integration host factor bends and bridges DNA in a multiplicity of binding modes with varying specificity Nucleoid-associated proteins (NAPs) are crucial in organizing prokaryotic DNA and regulating genes. Vital to these activities are complex nucleoprotein structures, however, how these form remains unclear. Integration host factor (IHF) is an Escherichia coli NAP that creates very sharp bends in DNA at sequences relevant to several functions including transcription and recombination, and is also responsible for general DNA compaction when bound non-specifically. We show that IHF-DNA structural multimodality is more elaborate than previously thought, and provide insights into how this drives mechanical switching towards strongly bent DNA. Using single-molecule atomic force microscopy and atomic molecular dynamics simulations we find three binding modes in roughly equal proportions: "associated" (73{degrees} of DNA bend), "half-wrapped" (107{degrees}) and "fully-wrapped" (147{degrees}), only the latter occurring with sequence specificity. We show IHF bridges two DNA double helices through non-specific recognition that gives IHF a stoichiometry greater than one and enables DNA mesh assembly. We observe that IHF-DNA structural multiplicity is driven through non-specific electrostatic interactions that we anticipate to be a general NAP feature for physical organization of chromosomes.

sentences: A comparison between time-constrained counts and line transects as methods to estimate butterfly diversity and monitor populations in tropical habitats Long-term species monitoring programmes have revealed catastrophic insect population declines and disruption of biological communities that are contributing to biodiversity loss. Such discoveries have been possible because of standardised methods, such as line transects, of counting butterflies and other insects. However, line transects are not feasible in many tropical and mountainous habitats, so alternative methods must be explored. To tackle this issue, we devised time-constrained (30-min) counts and compared butterfly diversity as estimated through this method with that estimated through line transects in three tropical habitats in India (evergreen forest, dry deciduous forest and an urban woodland). We tested the efficacy of the two methods to sample species richness and abundance, as well as numbers of rare, endemic and specialist butterflies. We observed greater species richness, and more species of habitat specialists and endemics per sample in time-constrained counts in evergreen forest, but not in the other two habitats. Thus, time-constrained counts were more efficient at detecting species in the species-rich evergreen habitat. Apart from this difference, the two sampling methods captured similar levels of species richness and other measures of diversity. Our study thus shows that time-constrained counts is a suitable if not a superior alternative to line transects to conduct butterfly diversity surveys and population monitoring in complex tropical landscapes. Due to methodological flexibility and simplicity, this method may be particularly useful to study the impacts of climate change, habitat fragmentation and land use practices on butterfly conservation in populous and tech-ready tropical countries using citizen science frameworks.

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Cholesterol Oxidation Modulates the Formation of Liquid-Ordered Domains in Model Membranes 7-ketocholesterol (KChol) is one of the most cytotoxic oxysterols found in the plasma membrane, and increased levels of KChol are associated with numerous pathologies. It is thought to induce apoptosis via inactivation of the phosphatidylinositol 3-kinase/Akt signaling pathway -- a pathway that depends on lipid-rafts as signaling platforms. By means of coarse-grained molecular dynamics simulations, we demonstrate that KChol disrupts the liquid-liquid phase separation seen in an equimolar mixture of (dipalmitoylphosphatidylcholine) DPPC, (dioleoylphosphatidylcholine) DOPC, and Cholesterol (Chol). This disruption occurs via two mechanisms: i) KChol adopts a wider range of orientations with the membrane, which disrupts the packing of neigh-boring lipids and ii) KChol has no preference for DPPC over DOPC, which is the main driving force for lateral demixing in DPPC/DOPC/Chol membranes. This provides a molecular description of the means by which KChol induces apoptosis, and illustrates that a single chemical substitution to cholesterol can have a profound impact on the lateral organization of lipid membranes.

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Copy number variant heterogeneity among cancer types reflects inconsistent concordance with diagnostic classifications Due to frequent genome instability and accumulation of mutations during the neoplastic process, malignant tumors present with patterns of somatic genome variants on diverse levels of heterogeneity. The delineation of pathophysiological consequences of these patterns remains one of the main challenges in cancer prognosis and treatment.

Although continuous efforts aim for better characterization of cancer entities through inclusion of molecular characteristics, current ontology systems still heavily rely on clinico-pathological features. Traditionally, malignant diseases have been classified using domain-specific or generalized classification systems, based on histopathological features and clinical gestalt. Aside from the general purpose "International Classification for Diseases in Oncology" (ICD-O; WHO), hierarchical terminologies such as NCIt promote data interoperability and ontology-driven computational analysis.

To evaluate two prominent, general cancer classification systems (NCIt and ICD-O) towards their concordance with genomic mutation patterns we have performed a data-driven meta-analysis of 83505 curated cancer samples with genome-wide CNA (copy number aberration) profiles from our Progenetix database. The analysis provides a basis to assess the correspondence level of existing classification systems with respect to homogeneous molecular groups, and how individual codes represent an adequately detailed classification.

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Migrators Within Migrators: Exploring Transposable Element Landscapes, Activity, and Host Gene Associations in the Monarch Butterfly, Danaus plexippus BackgroundLepidoptera (butterflies and moths) are an important model system in ecology and evolution. A high-quality chromosomal genome assembly is available for the monarch butterfly (Danaus plexippus), but it lacks an in-depth transposable element (TE) annotation, presenting an opportunity to explore monarch TE dynamics and the impact of TEs on shaping the monarch genome.

ResultsWe find 6.21% of the monarch genome is comprised of TEs, a reduction of 6.85% compared to the original TE annotation performed on the draft genome assembly. Monarch TE content is low compared to two closely related species with available genomes, Danaus chrysippus (33.97% TE) and Danaus melanippus (11.87% TE). The biggest TE contributions to genome size in the monarch are LINEs and Penelope-like elements, and three newly identified families, r2-hero_dPle (LINE), penelope-1_dPle (Penelope-like), and hase2-1_dPle (SINE), collectively contribute 34.92% of total TE content. We find evidence of recent TE activity, with two novel Tc1 families rapidly expanding over recent timescales (tc1-1_dPle, tc1- 2_dPle). LINE fragments show signatures of genomic deletions indicating a high rate of TE turnover. We investigate associations between TEs and wing colouration and immune genes and identify a three-fold increase in TE content around immune genes compared to other host genes.

ConclusionsWe provide a detailed TE annotation and analysis for the monarch genome, revealing a considerably smaller TE contribution to genome content compared to two closely related Danaus species with available genome assemblies. We identify highly successful novel DNA TE families rapidly expanding over recent timescales, and ongoing signatures of both TE expansion and removal highlight the dynamic nature of repeat content in the monarch genome. Our findings also suggest that insect immune genes are promising candidates for future interrogation of TE-mediated host adaptation.

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Akkermansia Muciniphila induces chronic extramedullary hematopoiesis through cooperative IL-1R and TLR signals Bacterial infections can activate and mobilize hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) to spleen, which is termed as extramedullary hematopoiesis (EMH). Recent studies suggest that commensal bacteria, particularly the microbiota, regulates not only the host immune system but also hematopoietic homeostasis. However, the impact of gut microbial species on hematopoietic pathology remains largely unknown. Here we found that systemic injection of Akkermansia muciniphila (A. m.), a mucin-degrading bacterium abundantly existing in the human gut rapidly activates BM myelopoiesis, and induces a slow but long-lasting hepato-splenomegaly, characterized by the expansion and differentiation of functional HSPCs, which we termed chronic EMH. Genetic deletion of Toll-like receptor-2 and -4 (TLR2/4) partially diminished A. m.-induced chronic EMH, while additional pharmacological inhibition of the interleukin-1 receptor (IL-1R) completely alleviated splenomegaly and EMH. Our results demonstrate that cooperative IL-1R- and TLR-mediated innate immune signals regulate commensal bacteria-driven EMH, which might be relevant for certain autoimmune disorders.

Article SummaryThe aim of our study is to understand how Akkermansia muciniphila (A.m.), one of the major mucin-degrading microbial species in the human gut activate the immune and hematopoietic systems in a mouse model. We found that a single injection of the A.m. membrane fraction can induce long-lasting hepatosplenomegaly with splenic EMH through cooperative IL-1R- and TLR-mediated innate immune signals.

sentences: Protein turnover in the developing Triticum aestivum grain Protein abundance in cereal grains is determined by the relative rates of protein synthesis and protein degradation during grain development. Through combining in vivo stable isotope labelling and in-depth quantitative proteomics, we have measured the turnover of 1400 different types of proteins during wheat grain development. We demonstrate that there is a spatiotemporal pattern to protein turnover rates which explain part of the variation in protein abundances that is not attributable to differences in wheat gene expression. We show that approximately 20% of total grain ATP production is used for grain proteome biogenesis and maintenance, and nearly half of this budget is invested exclusively in storage protein synthesis. We calculate that 25% of newly synthesized storage proteins are turned over during grain development rather than stored. This approach to measure protein turnover rates at proteome scale reveals how different functional categories of grain proteins accumulate, calculates the costs of protein turnover during wheat grain development and identifies the most and the least stable proteins in the developing wheat grain.

sentences: Dynamic model of bovine brucellosis to investigate control strategies in endemic settings Brucellosis imposes substantial impacts on livestock production and public health worldwide. A stochastic, age-structured model incorporating herd demographics was developed describing within- and between-herd transmission of Brucella abortus in dairy cattle herds. The model was fitted to data from a cross-sectional study conducted in Punjab State of India and used to evaluate the effectiveness of control strategies under consideration. Based on model results, stakeholder acceptance and constraints regarding vaccine supply, vaccination of replacement calves in large farms should be prioritised. Test and removal applied at early stages of the control programme where seroprevalence is high would not constitute an effective use of resources. Critically, under current model assumptions, significant numbers of animals  removed (culled or not used for breeding) in this strategy would be removed based on false positive results. To achieve sustained reductions in brucellosis, policymakers must commit to maintaining vaccination in the long term, which may eventually reduce frequency of infection in the livestock reservoir to a low enough level for elimination to be a realistic objective. This exercise provided important insights into the control of brucellosis in India, which has the largest cattle population globally, and a general framework for evaluating control strategies in endemic settings.

sentences: A meta-analysis of epigenome-wide association studies in Alzheimer's disease highlights novel differentially methylated loci across cortex Epigenome-wide association studies of Alzheimers disease have highlighted neuropathology-associated DNA methylation differences, although existing studies have been limited in sample size and utilized different brain regions. Here, we combine data from six DNA methylomic studies of Alzheimers disease (N=1,453 unique individuals) to identify differential methylation associated with Braak stage in different brain regions and across cortex. We identify 236 CpGs in the prefrontal cortex, 95 CpGs in the temporal gyrus and ten CpGs in the entorhinal cortex at Bonferroni significance, with none in the cerebellum. Our cross-cortex meta-analysis (N=1,408 donors) identifies 220 CpGs associated with neuropathology, annotated to 121 genes, of which 84 genes have not been previously reported at this significance threshold. We have replicated our findings using two further DNA methylomic datasets consisting of a further > 600 unique donors. The meta-analysis summary statistics are available in our online data resource (www.epigenomicslab.com/ad-meta-analysis/).

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A Multiscale Model for Quantitative Prediction of Insulin Aggregation Nucleation Kinetics We combined kinetic, thermodynamic, and structural information from single molecule (protein folding) and two molecule (association) explicit-solvent simulations for determination of kinetic parameters in protein aggregation nucleation with insulin as model protein. A structural bioinformatics approach was developed to account for heterogeneity of aggregation-prone species with the transition complex theory found applicable in modeling association kinetics involving non-native species. We show that a key simplification arises from presence of only a few relevant modes for non-native association kinetics. The kinetic parameters thus obtained were used in a population balance model and accurate predictions for aggregation nucleation time varying over two orders of magnitude with changes in concentration of insulin or an aggregation-inhibitor ligand were obtained while an empirical parameter set was not found to be transferable for prediction of ligand effects. This physically determined kinetic parameter set also allowed identification of the rate-limiting step in aggregation nucleation.

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sentences: Functional connectivity of the developing mouse cortex Cross-sectional studies have established a variety of structural, synaptic and cell physiological changes corresponding to key critical periods in cortical development. However, the emergence of functional connectivity (FC) in development has not been fully characterized, and hemodynamic-based measures are vulnerable to any neurovascular coupling changes occurring in parallel. We therefore used optical fluorescence imaging to trace longitudinal calcium FC in the awake, resting-state mouse cortex in the same mice at 5 developmental time points beginning at postnatal day 15 (P15) and ending in early adulthood (P60), resulting in over 500 imaging epochs with both calcium and hemodynamics available as a resource for the field. Proof-of-principle analyses revealed that calcium FC displayed coherent functional maps as early as P15, and FC significantly varied in connections between many regions across development, with the developmental trajectorys shape specific to the functional region. This longitudinal developmental calcium FC dataset provides an essential resource for further algorithm development and studies of healthy development and neurodevelopmental disorders.

sentences: Software Evaluation for de novo Detection of Transposons Transposable elements (TEs) are major genomic components in most eukaryotic genomes and play an important role in genome evolution. However, despite their relevance the identification of TEs is not an easy task and a number of tools were developed to tackle this problem. To better understand how they perform, we tested several widely used tools for de novo TE detection and compared their performance on both simulated data and well curated genomic sequences. As expected, tools that build TE-models performed better than k-mer counting ones, with RepeatModeler beating competitors in most datasets. However, there is a tendency for most tools to identify TE-regions in a fragmented manner and it is also frequent that small TEs or fragmented TEs are not detected. Consequently, the identification of TEs is still a challenging endeavor and it requires a significant manual curation by an experienced expert. The results will be helpful for identifying common issues associated with TE-annotation and for evaluating how comparable are the results obtained with different tools.

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A comparison of hard and soft direct methods for DNA extraction from soil Nucleic acid extraction is the first step in molecular biology studies of soil bacterial communities. The most common used soil DNA extraction method is the direct, hard extraction Mobio method, which uses bead beating to lyse bacteria. In this study we compared the Mobio method with a soft, enzymatic lysis extraction method. Next generation sequencing (Illumina and Pyrosequencing) of amplicons generated from four 16S primer pairs and DNA from 12 soils and 3 composts was used to compare the two extraction methods.

Four bacterial orders, the delta proteobacterial Desulfuromonadales and gamma proteobacterial Pseudomonadales, Enterobacteriales, and Alteromonadales were more common in amplicons from soft extracted DNA, sometimes by two orders of magnitude. These groups can be a significant fraction of the bacterial population. For example the Pseudomonadales made up to 16 % and Enterobacteriales 10% of amplicons from Soft extracted DNA. The JG30-KF-CM45 order was under extracted by the enzymatic lysis extraction method. Results differed more by primer choice than extraction method and the phylogenetic resolution of differences between extraction methods changed with primer choice.

Given how often Mobio extraction is used, these proteobacterial orders are probably under-represented in the studies of soil bacteria that use nucleic acid methods. Further improvements in soil DNA extraction are needed. Amplicons sequencing studies should use a range of different primers to confirm the phylogenetic resolution of their results.

ImportanceSeveral large scale studies of soil bacteria that compare thousands of soil samples across continents have used the Mobio method for DNA extraction. Large scale studies like these are increasing with the recent establishment of the Global Soil Biodiversity Observation Network (Soil BON), which also uses the Mobio method. The results of this work will be used to make policy decisions about how to manage the soil and may be a guide for bioprospectors. As the Mobio method is so widely used, it is important to know its limitations. Studies that use the Mobio method underestimate the fraction of several proteobacterial groups. Most notably the Enterobacteria and Pseudomonas can be under extracted by 10-100 fold. The degree of under extraction varies with different soils.

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SARS-CoV-2 spike protein induces abnormal inflammatory blood clots neutralized by fibrin immunotherapy Blood clots are a central feature of coronavirus disease-2019 (COVID-19) and can culminate in pulmonary embolism, stroke, and sudden death. However, it is not known how abnormal blood clots form in COVID-19 or why they occur even in asymptomatic and convalescent patients. Here we report that the Spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to the blood coagulation factor fibrinogen and induces structurally abnormal blood clots with heightened proinflammatory activity. SARS-CoV-2 Spike virions enhanced fibrin-mediated microglia activation and induced fibrinogen-dependent lung pathology. COVID-19 patients had fibrin autoantibodies that persisted long after acute infection. Monoclonal antibody 5B8, targeting the cryptic inflammatory fibrin epitope, inhibited thromboinflammation. Our results reveal a procoagulant role for the SARS-CoV-2 Spike and propose fibrin-targeting interventions as a treatment for thromboinflammation in COVID-19.

One-Sentence SummarySARS-CoV-2 spike induces structurally abnormal blood clots and thromboinflammation neutralized by a fibrin-targeting antibody.

sentences: Copy-out-paste-in transposition of a Tn6283-like integrative element assists interspecies antimicrobial resistance gene transfer from Vibrio alfacsensis The exchange of antimicrobial resistance (AMR) genes between aquaculture and terrestrial microbial populations has emerged as a serious public health concern. However, the nature of the mobile genetic elements in marine bacteria is poorly documented. To gain insight into the genetic mechanisms underlying AMR gene transfer from marine bacteria, we mated a multidrug-resistant Vibrio alfacsensis strain with an Escherichia coli strain, and then determined the complete genome sequences of the donor and transconjugant strains. Sequence analysis revealed a multidrug resistance conjugative plasmid in the donor strain, which was integrated into the chromosome of the recipient. The plasmid backbone in the transconjugant chromosome was flanked by two copies of a 7.1 kb unclassifiable integrative element harboring a {beta}-lactamase gene. The 7.1 kb element (named SE-6945) and previously reported Tn6283 share four coding sequences, two of which encode the catalytic R-H-R-Y motif of tyrosine recombinases. Polymerase chain reaction and sequencing experiments revealed that SE-6945 generates a circular copy of its one specific strand without generating an empty donor site, in contrast to the movement of integron gene cassette or ICE/IMEs discovered to date. Copy numbers and location of SE-6945 in the chromosome affected antibiotic resistance levels of the transconjugants. SE-6945/Tn6283-related integrative elements are collectively termed SEs (strand-biased circularizing integrative elements). SEs were identified in the genomes of other Vibrio species. Overall, these results suggest that in addition to conjugative plasmids, SEs are involved in the spread of AMR genes among marine bacteria.

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Portable low-cost macroscopic mapping system for all-optical cardiac electrophysiology SignificanceAll-optical cardiac electrophysiology enables the visualization and control of key parameters relevant to the detection of cardiac arrhythmias. Mapping such responses in human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) is of great interest for cardiotoxicity and personalized medicine applications.

AimWe introduce and validate a very low-cost compact mapping system for macroscopic all-optical electrophysiology in layers of hiPSC-CMs.

ApproachThe system uses oblique trans-illumination, low-cost cameras, light-emitting diodes and off-the-shelf components (total < $15,000) to capture voltage, calcium and mechanical waves under electrical or optical stimulation.

ResultsOur results corroborate the equivalency of electrical and optogenetic stimulation of hiPSC-CMs, and Vm - [Ca2+]i similarity in conduction under pacing. Green-excitable optical sensors are combinable with blue optogenetic actuators (Chanelrhodopsin2) only under very low green light (< 0.05mW/mm2). Measurements in warmer culture medium yield larger spread of action potential duration and higher conduction velocities compared to Tyrodes solution at room temperature.

ConclusionsAs multiple optical sensors and actuators are combined, our results can help handle the "spectral congestion" and avoid parameter distortion. We illustrate the utility of the system for uncovering the action of cellular uncoupling agents and show extensibility to an epi-illumination mode for future imaging of thicker native or engineered tissues.

sentences: The effect of vascular health factors on white matter microstructure mediates age-related differences in executive function performance Even within healthy aging, vascular risk factors can detrimentally influence cognition, with executive functions (EF) particularly vulnerable. Fronto-parietal white matter (WM) connectivity in part, supports EF and may be particularly sensitive to vascular risk. Here, we utilized structural equation modeling in 184 healthy adults (aged 20-94 years of age) to test the hypotheses that: 1) fronto-parietal WM microstructure mediates age effects on EF; 2) higher blood pressure (BP) and white matter hyperintensity (WMH) burden influences this association. All participants underwent comprehensive cognitive and neuropsychological testing including tests of processing speed, executive function (with a focus on tasks that require switching and inhibition) and completed an MRI scanning session that included FLAIR imaging for semi-automated quantification of white matter hyperintensity burden and diffusion-weighted imaging for tractography. Structural equation models were specified with age (as a continuous variable) and blood pressure predicting within-tract WMH burden and fractional anisotropy predicting executive function and processing speed. Results indicated that fronto-parietal white matter of the genu of the corpus collosum, superior longitudinal fasciculus, and the inferior frontal occipital fasciculus (but not cortico-spinal tract) mediated the association between age and EF. Additionally, increased systolic blood pressure and white matter hyperintensity burden within these white matter tracts contribute to worsening white matter health and are important factors underlying age-brain-behavior associations. These findings suggest that aging brings about increases in both BP and WMH burden, which may be involved in the degradation of white matter connectivity and in turn, negatively impact executive functions as we age.

sentences: Systematic Single Cell Pathway Analysis (SCPA) reveals novel pathways engaged during early T cell activation Next generation sequencing technologies have revolutionized the study of T cell biology, capturing previously unrecognized diversity in cellular states and functions. Pathway analysis is a key analytical stage in the interpretation of such transcriptomic data, providing a powerful method for detecting alterations in important biological processes. Current pathway analysis tools are built on models developed for bulk-RNA sequencing, limiting their effectiveness when applied to more complex single cell RNA-sequencing (scRNA-seq) datasets. We recently developed a sensitive and distribution-free statistical framework for multisample distribution testing, which we implement here in the open-source R package Single Cell Pathway Analysis (SCPA). After demonstrating the effectiveness of SCPA over commonly used methods, we generate a scRNA-seq T cell dataset and characterize pathway activity over early cellular activation and between T cell populations. This revealed unexpected regulatory pathways in T cells, such as an intrinsic type I interferon system regulating T cell survival and a reliance on arachidonic acid metabolism throughout T cell activation. A systems level characterization of pathway activity in T cells across multiple human tissues also revealed alpha defensin expression as a hallmark of bone marrow derived T cells. Overall, our work here provides a widely applicable tool for single cell pathway analysis, and highlights unexpected regulatory mechanisms of T cells using a novel T cell dataset.

sentences: Comprehensive analyses of RNA-seq and genome-wide data point to enrichment of neuronal cell type subsets in neuropsychiatric disorders Neurological and psychiatric disorders, including substance use disorders share a range of symptoms, which could be the result of shared genetic background. Many genetic loci have been identified for these disorders using genome-wide association studies, but conclusive evidence about cell types wherein these loci are active is lacking. We aimed to uncover implicated brain cell types in neuropsychiatric traits and to assess consistency in results across RNA datasets and methods. We therefore comprehensively employed cell-type enrichment methods by integrating single-cell transcriptomic data from mouse brain regions with an unprecedented dataset of 42 human genome-wide association study results of neuropsychiatric, substance use and behavioral/quantitative brain-related traits (n=12,544,007 individuals). Single-cell transcriptomic datasets from the Karolinska Institute and the 10x Genomics dataset were used. Cell type enrichment was determined using Linkage Disequilibrium Score Regression, Multi-marker Analysis of GenoMic Annotation, and Data-driven Expression Prioritized Integration for Complex Traits. We found the largest degree of consistency across methods for implication of pyramidal cells in schizophrenia and cognitive performance. For other phenotypes, such as bipolar disorder, two methods implicated the same cell types, i.e. medium spiny neurons and pyramidal cells. For autism spectrum disorders and anorexia nervosa, no consistency in implicated cell types was observed across methods. We found no evidence for astrocytes being consistently implicated in neuropsychiatric traits. In conclusion, we provide comprehensive evidence for a subset of neuronal cell types being consistently implicated in several, but not all psychiatric disorders, while non-neuronal cell types seem less implicated.

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Metabolic flux partitioning between the TCA cycle and glyoxylate shunt combined with a reversible methyl citrate cycle provide nutritional flexibility for Mycobacterium tuberculosis The utilisation of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection thereby identifying the Mtb specific metabolic pathways and enzymes required for carbon co-metabolism as potential drug targets. Metabolic flux represents the final integrative outcome of many different levels of cellular regulation that contribute to the flow of metabolites through the metabolic network. It is therefore critical that we have an in-depth understanding of the rewiring of metabolic fluxes in different conditions. Here, we employed 13C-metabolic flux analysis using stable isotope tracers (13C and 2H) and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly on physiologically relevant carbon sources in a steady state chemostat. We demonstrate that Mtb is able to efficiently co-metabolise combinations of either cholesterol or glycerol along with C2 generating carbon substrates. The uniform assimilation of the carbon sources by Mtb throughout the network indicated no compartmentalization of metabolism in these conditions however there were substrate specific differences in metabolic fluxes. This work identified that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle as the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide new insights into the metabolic architecture that affords adaptability of Mtb to divergent carbon substrates.

ImportanceEach year more than 1 million people die of tuberculosis (TB). Many more are infected but successfully diagnosed and treated with antibiotics, however antibiotic-resistant TB isolates are becoming ever more prevalent and so novel therapies are urgently needed that can effectively kill the causative agent. Mtb specific metabolic pathways have been identified as an important drug target in TB. However the apparent metabolic plasticity of this pathogen presents a major obstacle to efficient targeting of Mtb specific vulnerabilities and therefore it is critical to define the metabolic fluxes that Mtb utilises in different conditions. Here, we used 13C-metabolic flux analysis to measure the metabolic fluxes that Mtb uses whilst growing on potential in vivo nutrients. Our analysis identified selective use of the metabolic network that included the TCA cycle, glyoxylate shunt and methyl citrate cycle. The metabolic flux phenotypes determined in this study improves our understanding about the co-metabolism of multiple carbon substrates by Mtb identifying a reversible methyl citrate cycle and the glyoxylate shunt as the critical metabolic nodes which underlie the nutritional flexibility of Mtb.

sentences: Toward a CRISPR-based point-of-care test for tomato brown rugose fruit virus detection Implementing effective monitoring strategies is fundamental to protect crops from pathogens and to ensure the food supply as the world population continues to grow. This is especially important for emergent plant pathogens such as tomato brown rugose fruit virus (ToBRFV), which overcomes the genetic resistance resources used in tomato breeding against tobamoviruses and has become pandemic in less than a decade. Here we report the development of a CRISPR/Cas12a-based test to detect ToBRFV in the laboratory and potentially in a field setting. Using different tobamoviruses to assess specificity, our test showed a clear positive signal for ToBRFV-infected samples, while no cross-reactivity was observed for closely related viruses. Next, we compared the limit of detection of our CRISPR-based test with a reference real-time quantitative PCR test widely used, revealing similar sensitivities for both tests. Finally, to reduce complexity and achieve field-applicability, we used a fast nucleic acid purification step and compared its results side by side with those of a commonly used column-mediated protocol. The new protocol saved time and resources but at the expense of sensitivity. However, it still may be useful to confirm ToBRFV, detection in samples with incipient symptoms of infection. Although there is room for improvement, to our knowledge this is the first field-compatible CRISPR-based test to detect ToBRFV, which combines isothermal amplification with a simplified nucleic acid extraction protocol.

sentences: Regimes and mechanisms of transient amplification in abstract and biological networks We use upper triangular matrices as abstract representations of neuronal networks and directly manipulate their eigenspectra and non-normality to explore different regimes of transient amplification. Counter-intuitively, manipulating the imaginary distribution can lead to highly amplifying regimes. This is noteworthy, because biological networks are constrained by Dales law and the non-existence of neuronal self-loops, limiting the range of manipulations in the real dimension. Within these constraints we can further manipulate transient amplification by controlling global inhibition.

sentences: Cryo-EM reconstructions of inhibitor-bound SMG1 kinase reveal an autoinhibitory state dependent on SMG8 The PI3K-related kinase (PIKK) SMG1 monitors progression of metazoan nonsense-mediated mRNA decay (NMD) by phosphorylating the RNA helicase UPF1. Previous work has shown that the activity of SMG1 is impaired by small molecule inhibitors, is reduced by the SMG1 interactors SMG8 and SMG9, and is downregulated by the so-called SMG1 insertion domain. However, the molecular basis for this complex regulatory network has remained elusive. Here, we present cryo-electron microscopy reconstructions of human SMG1-9 and SMG1-8-9 complexes bound to either a SMG1 inhibitor or a non-hydrolyzable ATP analogue at overall resolutions ranging from 2.8 to 3.6 [A]. These structures reveal the basis with which a small molecule inhibitor preferentially targets SMG1 over other PIKKs. By comparison with our previously reported substrate-bound structure (Langer et al. 2020), we show that the SMG1 insertion domain can exert an autoinhibitory function by directly blocking the substrate binding path as well as overall access to the SMG1 kinase active site. Together with biochemical analysis, our data indicate that SMG1 autoinhibition is stabilized by the presence of SMG8. Our results explain the specific inhibition of SMG1 by an ATP-competitive small molecule, provide insights into regulation of its kinase activity within the NMD pathway, and expand the understanding of PIKK regulatory mechanisms in general.

sentences: METTL18-mediated histidine methylation on RPL3 modulates translation elongation for proteostasis maintenance Protein methylation occurs predominantly on lysine and arginine residues, but histidine also serves as a substrate for the modification. However, a limited number of enzymes responsible for this modification have been reported. Moreover, the biological role of histidine methylation has remained poorly understood. Here, we report that human METTL18 is a histidine methyltransferase for the ribosomal protein RPL3 and that the modification specifically slows ribosome traverse on tyrosine codons, allowing the proper folding of synthesized proteins. By performing an in vitro methylation assay with a methyl donor analog and quantitative mass spectrometry, we found that His245 of RPL3 is methylated at the {tau}-N position by METTL18. Structural comparison of the modified and unmodified ribosomes showed stoichiometric modification and suggested a role in translation tuning. Indeed, genome-wide ribosome profiling revealed suppressed ribosomal translocation at tyrosine codons by RPL3 methylation. Because the slower elongation provides enough time for nascent protein folding, RPL3 methylation protects cells from the cellular aggregation of Tyr-rich proteins. Our results reveal histidine methylation as an example of a "ribosome code" that ensures proteome integrity in cells.

sentences: Ecosystem engineering alters density-dependent feedbacks in an aquatic insect population Ecosystem engineers have large impacts on the communities in which they live, and these impacts may feed back to populations of engineers themselves. In this study, we assessed the effect of ecosystem engineering on density-dependent feedbacks for midges in Lake Myvatn, Iceland. The midge larvae reside in the sediment and build silk tubes that provide a substrate for algal growth, thereby elevating benthic primary production. Benthic algae are in turn the primary food source for the midge larvae, setting the stage for the effects of engineering to feed back to the midges themselves. Using a field mesocosm experiment manipulating larval midge densities, we found a generally positive but nonlinear relationship between density and benthic production. Furthermore, adult emergence increased with the primary production per midge larva. By combining these two relationships in a simple model, we found that the positive effect of midges on benthic production weakened the negative density dependence at low to intermediate larval densities. However, this benefit disappeared at high densities when midge consumption of primary producers exceeded their positive effects on primary production through ecosystem engineering. Our results illustrate how ecosystem engineering can alter density-dependent feedbacks for engineer populations.

sentences: Interpretable machine learning identifies paediatric Systemic Lupus Erythematosus subtypes based on gene expression data Transcriptomic analyses are commonly used to identify differentially expressed genes between patients and controls, or within individuals across disease courses. These methods, whilst effective, cannot encompass the combinatorial effects of genes driving disease. We applied rule-based machine learning (RBML) models and rule networks (RN) to an existing paediatric Systemic Lupus Erythematosus (SLE) blood expression dataset, with the goal of developing gene networks to separate low and high disease activity (DA1 and DA3). The resultant model had an 81% accuracy to distinguish between DA1 and DA3, with unsupervised hierarchical clustering revealing additional subgroups indicative of the immune axis involved or state of disease flare. These subgroups correlated with clinical variables, suggesting that the gene sets identified may further the understanding of gene networks that act in concert to drive disease progression. This included roles for genes i) induced by interferons (IFI35 and OTOF), ii) key to SLE cell types (KLRB1 encoding CD161), or iii) with roles in autophagy and NF-{kappa}B pathway responses (CKAP4). As demonstrated here, RBML approaches have the potential to reveal novel gene patterns from within a heterogeneous disease, facilitating patient clinical and therapeutic stratification.

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Tianeptine, but not fluoxetine, decreases avoidant behavior in a mouse model of early developmental exposure to fluoxetine Depression and anxiety are two of the most common mental health disorders, often sharing symptoms and administrations. Most pharmacological agents available to treat these disorders target monoamine systems. Currently, finding the most effective treatment for an individual is a process of trial and error. Therefore, to better understand how disease etiology may predict treatment response, we studied mice exposed developmentally to the selective serotonin reuptake inhibitor (SSRI) fluoxetine (FLX). These mice show the murine equivalent of anxiety- and depression-like symptoms in adulthood and here we report that these mice are also behaviorally resistant to the antidepressant-like effects of adult SSRI administration. We investigated whether tianeptine (TIA), which exerts its therapeutic effects through the mu-opioid receptor (MOR) instead of directly targeting monoaminergic systems, would be more effective in this model.

We injected C57BL/6J (C57) pups with either FLX (10 mg/kg, i.p) or vehicle from postnatal (PN) day 2 to 11, a period in which mouse brain development parallels that of the third trimester of a human pregnancy. Prior work established that adult 129SvEv (129) mice exposed to FLX in this time period (PN-FLX) showed increased avoidant and decreased hedonic behaviors, which correspond to anxiety- and depressive-like symptoms in humans, respectively. We performed baseline testing in adulthood in C57 PN-FLX animals and confirmed a similar avoidant phenotype to that reported in 129 PN-FLX mice. We then treated these animals with chronic FLX (18 mg/kg in the drinking water) and evaluated effects on two tasks that measure avoidant behavior - the open field and novelty suppressed feeding (NSF) tasks. This administration failed to improve, and even exacerbated, avoidance symptoms in PN-FLX mice. The same animals then underwent chronic administration with TIA (30 mg/kg, 2x/day, i.p.) as an alternative treatment strategy. TIA administration decreased avoidance behavior as measured in the open field and NSF. Overall, this demonstrates that TIA may be a promising alternative treatment to typical antidepressants, especially in patients whose serotonergic system has been altered.

sentences: Local DNA shape is a general principle of transcription factor binding specificity in Arabidopsis thaliana A genome encodes two types of information, the "what can be made" and the "when and where". The "what" are mostly proteins which perform the majority of functions within living organisms and the "when and where" is the regulatory information that encodes when and where DNA is transcribed. Currently, it is possible to efficiently predict the majority of the protein content of a genome but nearly impossible to predict the transcriptional regulation. This regulation is based upon the interaction between transcription factors and genomic sequences at the site of binding motifs1,2,3. Information contained within the motif is necessary to predict transcription factor binding, however, it is not sufficient4, as experimentally verified binding sites are substantially scarcer than the corresponding binding motif. Thus, it remains challenging to derive regulational information from binding motifs. Here we show that a random forest machine learning approach, which incorporates the 3D-shape of DNA, enhances binding prediction for all 216 tested Arabidopsis thaliana transcription factors and improves the resolution of differential binding by transcription factor family members which share the same binding motif. Our results contribute to the understanding of protein-DNA recognition and demonstrate the extraction of binding site features beyond the binding sequence. We observed that those features were individually weighted for each transcription factor, even if they shared the same binding sequence. We show that the gained insights enable a more robust prediction of binding behavior regarding novel, not-in-genome motif sequences. Understanding transcription factor binding as a combination of motif sequence and motif shape brings us closer to predicting gene expression from promoter sequence.

sentences: Intramembrane protease SPP defines a cholesterol-regulated switch of the mevalonate pathway Intramembrane proteolysis regulates important processes such as signaling and transcriptional and posttranslational abundance control of proteins with key functions in metabolic pathways. This includes transcriptional control of mevalonate pathway genes, thereby ensuring balanced biosynthesis of cholesterol and other isoprenoids. Our work shows that, at high cholesterol levels, signal peptide peptidase (SPP) cleaves squalene synthase (SQS), an enzyme that defines the branching point for allocation of isoprenoids to the sterol and non-sterol arms of the mevalonate pathway. This intramembrane cleavage releases SQS from the membrane and targets it for proteasomal degradation. Regulation of this mechanism is achieved by the E3 ubiquitin ligase TRC8 that, in addition to ubiquitinating SQS in response to cholesterol levels, acts as an allosteric activator of SPP-catalyzed intramembrane cleavage of SQS and other substrates. Hence, SPP-TRC8 mediated abundance control of SQS acts as a metabolic switch within the mevalonate pathway.

sentences: Structural analysis of the Spike of the Omicron SARS-COV-2 variant by Cryo-EM and implications for immune evasion The Omicron (B.1.1.529) SARS-COV-2 was reported on November 24, 2021 and declared a variant of concern a couple of days later.1,2 With its constellation of mutations acquired by this variant on its Spike glycoprotein and the speed at which this new variant has replaced the previously dominant variant Delta in South Africa and the United Kingdom, it is crucial to have atomic structural insights to reveal the mechanism of its rapid proliferation. Here we present a high-resolution cryo-EM structure of the Spike protein of the Omicron variant.

sentences: Designing single-cell experiments to harvest fluctuation noise while rejecting measurement noise Measurement error is a complicating factor that reduces or distorts the information contained in any experiment. This problem becomes even more serious in the context of experiments that are specifically designed to quantify single-cell gene expression heterogeneity, where important RNA and protein copy numbers are themselves subject to the inherently random fluctuations of biochemical reactions. It is not clear how measurement noise should be managed, in addition to other experiment design variables such as sampling size and frequency, to ensure that collected data will provide useful insights on gene expression mechanisms of interest. To address these fundamental single-cell experiment design challenges, we propose a computational framework that makes explicit use of measurement error modeling and Fisher Information Matrix-based criteria to decide between experimental methods. This unified approach not only quantifies how different noise characteristics affect uncertainty in parameter estimation, but also enables a systematic approach to design hybrid experiments that combine different measurement modalities.

sentences: Multimodal microscale mechanical mapping of cancer cells in complex microenvironments The mechanical phenotype of the cell is critical for survival following deformations due to confinement and fluid flow. One idea is that cancer cells are plastic and adopt different mechanical phenotypes under different geometries that aid in their survival. Thus, an attractive goal, is to disrupt the cancer cells ability to adopt multiple mechanical states. To begin to address this question, we aimed to quantify the diversity of these mechanical states using in vitro biomimetics to mimic in vivo 2D and 3D extracellular matrix environments. Here, we used two modalities Brillouin microscopy ([~]GHz) and broadband frequency (3-15kHz) optical tweezer microrheology to measure microscale cell mechanics. We measured the response of intracellular mechanics of cancer cells cultured in 2D and 3D environments where we modified substrate stiffness, dimensionality (2D versus 3D), and presence of fibrillar topography. We determined that there was good agreement between two modalities despite the difference in timescale of the two measurements. These findings on cell mechanical phenotype in different environments confirm a correlation between modalities that employ different mechanisms at different temporal scales (Hz-kHz vs. GHz). We also determined that observed heterogeneity in cell shape that is more closely linked to the cells mechanical state. We also determined that individual cells in multicellular spheroids exhibit a lower degree of mechanical heterogeneity when compared to single cells cultured in monodisperse 3D cultures. Moreover, the observed decreased heterogeneity among cells in spheroids suggested that there is mechanical cooperativity between cells that make up a single spheroid.

sentences: CellDrift: Inferring Perturbation Responses in Temporally-Sampled Single Cell Data Cells and tissues respond to perturbations in multiple ways that can be sensitively reflected in alterations of gene expression. Current approaches to finding and quantifying the effects of perturbations on cell-level responses over time disregard the temporal consistency of identifiable gene programs. To leverage the occurrence of these patterns for perturbation analyses, we developed CellDrift (https://github.com/KANG-BIOINFO/CellDrift), a generalized linear model-based functional data analysis method capable of identifying covarying temporal patterns of various cell types in response to perturbations. As compared to several other approaches, CellDrift demonstrated superior performance in the identification of temporally varied perturbation patterns and the ability to impute missing time points. We applied CellDrift to multiple longitudinal datasets, including COVID-19 disease progression and gastrointestinal tract development, and demonstrated its ability to identify specific gene programs associated with sequential biological processes, trajectories, and outcomes.

sentences: Concerted dynamics between conserved histidines in the N-extein region regulates Mycobacterium tuberculosis SufB intein cleavage Inteins are auto-processing domains that implement a multi-step biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe-S] cluster assembly protein SufB from Mycobacterium tuberculosis. Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wild-type precursor protein. Structural analysis and molecular dynamics simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N-S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing.

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Glocal dendritic spikes: variation in branch activity during dendritic spiking indicates multiple functional units in the pyramidal neuron apical tuft There is a lively debate on how many functional units exist within the dendritic tree of pyramidal neurons (PNs). The classical view that the PNs dendritic tree comprises several computational units 1-4 was challenged by recent in-vivo studies that identified mainly widespread dendritic spikes 5-10, suggesting a low degree of dendritic compartmentalization. We reasoned that global spikes do not rule out dendritic compartmentalization if these spikes contain a high variation in branch activity. Therefore, we turned to image Ca2+ activity in many apical dendrites from individual layer (L) 5 PNs of the primary motor cortex (M1). For proper dendritic morphology and Ca2+ activity analysis, we used mice with sparse double labeling of PNs with a structural marker (tdTomato) and a GCaMP6s probe. We first collected data for complete registration of the apical tuft dendritic structure. We then used 3D two-photon microscopy to image Ca2+ activity within these tufts during treadmill running. Using these, we imaged up to 30 apical branches from individual L5 PNs, including branches separated by as many as 11 bifurcations and more than 1000 {micro}m of intracellular distance. We found that local spikes were rare, but global spikes often displayed heterogeneous Ca2+ elevation across branches, giving rise to an activity pattern across the tuft. This spiking pattern was dynamic and according to unsupervised hierarchical cluster analysis, fitted to 2-6 distinct patterns, even over short imaging periods. Taken together, we find a considerable variation in branch activity within and between global dendritic spikes, supporting multiple dendritic functional units.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/484426v1_ufig1.gif" ALT="Figure 1">
View larger version (21K):
org.highwire.dtl.DTLVardef@103ceeaorg.highwire.dtl.DTLVardef@1058a19org.highwire.dtl.DTLVardef@ad7c24org.highwire.dtl.DTLVardef@1b13d94_HPS_FORMAT_FIGEXP  M_FIG C_FIG We detected three main spike types. Local spikes were evident in a minority of the branches and had low amplitude. Contrastingly, global spikes often displayed homogeneous levels of branch activity and were the strongest and most prolonged spikes. An intermediate pattern was the "glocal spike," where a global spike exhibited heterogeneous branch activity. Spikes with heterogeneous Ca2+ elevation across branches constituted at least a quarter of all spikes. In the social sciences, the term "glocalization" refers to global phenomena that are adjusted locally, such as food items sold worldwide but attuned to local tastes 11. Influenced by that, we refer to global spikes with heterogeneous branch activity as "glocal spikes."

sentences: EcoEvoApps: Interactive Apps for Teaching Theoretical Models in Ecology and Evolutionary Biology Theoretical models are fundamental in Ecology and Evolutionary Biology (EEB), but they can be difficult to teach in undergraduate classrooms because students come in with inconsistent mathematical training and varying attitudes towards mathematics. A promising way to make these models more approachable is to use active learning exercises that allow model exploration and simulation. Thus, we present EcoEvoApps, a collection of free, open-source interactive web apps that simulate various theoretical EEB models. By surveying students who used these apps in two remote undergraduate Ecology courses in the U.S.A., we found that using the apps led to considerable gains in students confidence and understanding of the focal models and underlying concepts. We envision EcoEvoApps as a widely available, equitable tool for students around the globe. Consequently, we have developed EcoEvoApps as a fundamentally collaborative platform, with an aim to build an international community of EEB researchers, educators, and students.

sentences: Aqueous extract of Phyllanthus niruri protects against severe malaria by blocking erythrocyte invasion and modulating the host immune response Plasmodium falciparum parasites are the major cause of malaria across Africa. Due to the appearance of multi-drug resistant parasites, new antimalarial drugs are needed. The medicinal plant Phyllanthus niruri is being used to treat fever and other symptoms of malaria in Nigeria; however, little is known about its antimalarial mechanisms. Here, we show that aqueous extract of P. niruri (PE) has multiple antimalarial effects, including anti-parasitic and host immunomodulatory activity. We found that co-culture of P. falciparum with PE drastically reduced parasite number, but PE did not inhibit parasite development or rupture; rather, it blocked erythrocytes invasion. In addition, we identified Astragalin as one of the antimalarial compounds which are contained in PE. Moreover, we found that PE suppresses the inflammatory activity and apoptosis of immune cells (T cells) and astrocytes and neurons in the central nervous system (CNS). Furthermore, we confirmed that oral administration of PE to mice suppressed parasite growth, excessive inflammation, CNS dysfunction, and the development of experimental cerebral malaria in an in vivo murine malaria model. Our findings demonstrate that PE has multiple effects on malaria progression, targeting both parasite and host cells.

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In-Cell Sensitivity-Enhanced NMR of Intact Living Mammalian Cells NMR has the resolution and specificity to determine atomic-level protein structures of isotopically-labeled proteins in complex environments and, with the sensitivity gains conferred by dynamic nuclear polarization (DNP), NMR has the sensitivity to detect proteins at their endogenous concentrations. However, DNP sensitivity enhancements are critically dependent on experimental conditions and sample composition. While some of these conditions are theoretically compatible with cellular viability, the effects of others on cellular sample integrity are unknown. Uncertainty about the integrity of cellular samples limits the utility of experimental outputs. Using several measures, we establish conditions that support DNP enhancements that can enable detection of micromolar concentrations of proteins in experimentally tractable times that are compatible with cellular viability. Taken together, we establish DNP assisted MAS NMR as a technique for structural investigations of biomolecules in intact viable cells that can be phenotyped both before and after NMR experiments.

ClassificationBiophysics and Structural Biology

sentences: Reassembling a cannon in the DNA defense arsenal: genetics of StySA, a BREX phage exclusion system in Salmonella lab strains Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering DNA. Recent investigation has uncovered BREX systems, RM-like activities that employ host protection by DNA modification but replication arrest without evident nuclease action on unmodified phage DNA. We show that the historical stySA RM locus of Salmonella enterica sv Typhimurium is a BREX homolog. The stySA29 allele of the hybrid strain LB5000 carries a mutated version of the ancestral LT2 BREX system. Surprisingly, both a restriction and a methylation defect are observed for this lineage despite lack of mutations in brxX, the modification gene homolog. Instead, flanking genes pglZ and brxC each carry multiple mutations () in C-terminal domains. To avoid plasmid artifacts and potential stoichiometric interference, we chose to investigate this system in situ, replacing the mutated pglZ and brxC genes with wild type (WT). PglZ-WT supports methylation in the presence of either BrxC or BrxC-WT but not in the presence of a deletion/insertion allele, {Delta}brxC::cat. Restriction of phage L requires both BrxC-WT and PglZ-WT, implicating the BrxC C-terminus specifically in restriction activity. Disruption of four other CDS with cat cassettes still permitted modification, suggesting that BrxC, PglZ and BrxX are principal components of the modification activity. BrxL is required for restriction only. A partial disruption of brxL disrupts transcription globally.

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Model selection in occupancy models: inference versus prediction Occupancy models are a vital tool for applied ecologists studying the patterns and drivers of species occurrence, but their use requires a method for selecting between models with different sets of occupancy and detection covariates. The information-theoretic approach, which employs information criteria such as Akaikes Information Criterion (AIC) is arguably the most popular approach for model selection in ecology and is often used for selecting occupancy models. However, the information-theoretic approach risks selecting models which produce inaccurate parameter estimates, due to a phenomenon called collider bias. Using simulations, we investigated the consequences of collider bias (using an illustrative example called M-bias) in the occupancy and detection processes of an occupancy model, and explored the implications for model selection using AIC and a common alternative, the Schwarz Criterion (or Bayesian Information Criterion, BIC). We found that when M-bias was present in the occupancy process, AIC and BIC selected models which inaccurately estimated the effect of the focal occupancy covariate, while simultaneously producing more accurate predictions of the site-level occupancy probability. In contrast, M-bias in the detection process did not impact the focal estimate; all models made accurate inferences, while the site-level predictions of the AIC/BIC-best model were slightly more accurate. Our results demonstrate that information criteria can be used to select occupancy covariates if the sole purpose of the model is prediction, but must be treated with more caution if the purpose is to understand how environmental variables affect occupancy. By contrast, detection covariates can usually be selected using information criteria regardless of the models purpose. These findings illustrate the importance of distinguishing between the tasks of parameter inference and prediction in ecological modelling. Furthermore, our results underline concerns about the use of information criteria to compare different biological hypotheses in observational studies.

Open Research StatementCode to fully reproduce our simulations and analyses is available at: https://zenodo.org/badge/latestdoi/462801230

sentences: The emergence of highly fit SARS-CoV-2 variants accelerated by epistasis and caused by recombination The SARS-CoV-2 pandemic recently entered an alarming new phase with the emergence of the variants of concern (VOC) and understanding their biology is paramount to predicting future ones. Current efforts mainly focus on mutations in the spike glycoprotein (S), but changes in other regions of the viral proteome are likely key. We analyzed more than 900,000 SARS-CoV-2 genomes with a computational systems biology approach including a haplotype network and protein structural analyses to reveal lineage-defining mutations and their critical functional attributes. Our results indicate that increased transmission is promoted by epistasis, i.e., combinations of mutations in S and other viral proteins. Mutations in the non-S proteins involve immune-antagonism and replication performance, suggesting convergent evolution. Furthermore, adaptive mutations appear in geographically disparate locations, suggesting that either independent, repeat mutation events or recombination among different strains are generating VOC. We demonstrate that recombination is a stronger hypothesis, and may be accelerating the emergence of VOC by bringing together cooperative mutations. This emphasizes the importance of a global response to stop the COVID-19 pandemic.

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Disentangling the role of poultry farms and wild birds in the spread of highly pathogenic avian influenza virus H5N8 in Europe Recent outbreaks of highly pathogenic avian influenza H5N8 virus in Europe have caused severe damage to animal health, wildlife conservation and livestock economic sustainability. While epidemiological and phylogenetic studies have generated important clues about the virus spread in Europe, they remained opaque to the specific role of poultry farms and wild birds. Using a phylodynamic framework, we inferred the H5N8 virus transmission dynamics among poultry farms and wild birds in four severely affected countries and investigated drivers of spread between farms across borders during the 2016-17 epidemic. Based on existing genetic data, we showed that the virus was likely introduced into poultry farms during the autumn, in line with the timing of arrival of migratory wild birds. Then, transmission was mainly driven by farm-to-farm transmission in Germany, Hungary and Poland, suggesting that better understanding of how infected farms are connected in those countries would greatly help control efforts. In contrast, the epidemic was dominated by wild bird-to-farm transmission in Czech Republic, meaning that more sustainable prevention strategies should be developed to reduce virus exposure from wild birds. We inferred effective reproduction number Re estimates among poultry farms and wild birds. We expect those estimates being useful to parameterize predictive models of virus spread aiming at optimising control strategies. None of the investigated predictors related to live poultry trade, poultry census and geographic proximity were identified as supportive predictors of the viral spread between farms across borders, suggesting that other drivers should be considered in future studies.

Significance statementIn winter 2016-17, Europe was severely hit by an unprecedented epidemic of highly pathogenic avian influenza (HPAI) H5N8 virus, causing significant impact on animal health, wildlife conservation and livestock economic sustainability. By applying phylodynamic tools to H5N8 sequence data collected from poultry farms and wild birds during the epidemic, we quantified how effectively the first infections were detected, how fast the virus spread, how many infections were missed and how many transmission events occurred at the wildlife-domestic interface. Also, we investigated predictors of the virus spread between farms across borders. These results are crucial to better understand the virus transmission dynamics, with the view to inform policy decision-making and reduce the impact of future epidemics of HPAI viruses.

sentences: Structural connectome quantifies tumor invasion and predicts survival in glioblastoma patients Glioblastoma widely affects brain structure and function, and remodels neural connectivity. Characterizing the neural connectivity in glioblastoma may provide a tool to understand tumor invasion. Here, using a structural connectome approach based on diffusion MRI, we quantify the global and regional connectome disruptions in individual glioblastoma patients and investigate the prognostic value of connectome disruptions and topological properties. We show that the disruptions in the normal-appearing brain beyond the lesion could mediate the topological alteration of the connectome (P <0.001), associated with worse patient performance (P <0.001), cognitive function (P <0.001), and survival (overall survival: HR: 1.46, P = 0.049; progression-free survival: HR: 1.49, P = 0.019). Further, the preserved connectome in the normal-appearing brain demonstrates evidence of remodeling, where increased connectivity is associated with better overall survival (log-rank P = 0.005). Our approach reveals the glioblastoma invasion invisible on conventional MRI, promising to benefit patient stratification and precise treatment.

sentences: Stress-Mediated Attenuation of Translation Undermines T Cell Tumor Control Protein synthesis enables cell growth and survival, but the molecular mechanisms through which T cells suppress or maintain protein translation in the stress of solid tumors are unknown. Using mouse models and human tumors we demonstrate that protein translation in T cells is repressed by the solid tumor microenvironment (TME) due to activation of the unfolded protein response (UPR) via phosphorylation of the  subunit of eukaryotic translation initiation factor 2 (p-eIF2). Given that acute glucose deprivation in T cells exacerbated p-eIF2, we show that metabolic reprogramming toward glycolytic independence allays the UPR and p-eIF2, enabling sustained protein translation in T cells in TME stress. UPR mitigation was associated with enhanced degradation of proteins in antitumor T cells, as proteasome inhibition resulted in eIF2 phosphorylation, attenuation of translation, and loss of antitumor efficacy. In contrast, proteasome stimulation relieved translation inhibition, inducing robust T cell tumor control, offering a new therapeutic avenue to fuel the efficacy of tumor immunotherapy.

sentences: Reversal of the adipostat control of torpor during migration in hummingbirds Many small endotherms use torpor to reduce metabolic rate and manage daily energy balance. However, the physiological "rules" that govern torpor use are unclear. We tracked torpor use and body composition in ruby-throated hummingbirds (Archilochus colubris), a long-distance migrant, throughout the summer using respirometry and quantitative magnetic resonance. During the mid-summer, birds entered torpor at consistently low fat stores ([~]5% of body mass), and torpor duration was negatively related to evening fat load. Remarkably, this energy-emergency strategy was abandoned in the late summer when birds accumulated fat for migration. Migrating birds were more likely to enter torpor on nights when they had higher fat stores, and fat gain was positively correlated with the amount of torpor used. These findings demonstrate the versatility of torpor throughout the annual cycle and suggest a fundamental change in physiological feedback between adiposity and torpor during migration. Moreover, this study highlights the underappreciated importance of facultative heterothermy in migratory ecology.

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Single-cell transcriptomics reveals a conserved metaplasia program in pancreatic injury BACKGROUND & AIMSAcinar to ductal metaplasia (ADM) occurs in the pancreas in response to tissue injury and is a potential precursor for adenocarcinoma. The goal of these studies was to define the populations arising from ADM, the associated transcriptional changes, and markers of disease progression.

METHODSAcinar cells were lineage-traced with enhanced yellow fluorescent protein (EYFP) to follow their fate upon injury. Transcripts of over 13,000 EYFP+ cells were determined using single-cell RNA sequencing (scRNA-seq). Developmental trajectories were generated. Data were compared to gastric metaplasia, KrasG12D-induced neoplasia, and human pancreatitis. Results were confirmed by immunostaining and electron microscopy. KrasG12D was expressed in injury-induced ADM using several inducible Cre drivers. Surgical specimens of chronic pancreatitis from 15 patients were evaluated by immunostaining.

RESULTSscRNA-seq of ADM revealed emergence of a mucin/ductal population resembling gastric pyloric metaplasia. Lineage trajectories suggest that some pyloric metaplasia cells can generate tuft and enteroendocrine cells (EECs). Comparison to KrasG12D-induced ADM identifies populations associated with disease progression. Activation of KrasG12D expression in HNF1B+ or POU2F3+ ADM populations leads to neoplastic transformation and formation of MUC5AC+ gastric-pit-like cells. Human pancreatitis samples also harbor pyloric metaplasia with a similar transcriptional phenotype.

CONCLUSIONSUnder conditions of chronic injury, acinar cells undergo a pyloric-type metaplasia to mucinous progenitor-like populations, which seed disparate tuft cell and EEC lineages. ADM-derived EEC subtypes are diverse. KrasG12D expression is sufficient to drive neoplasia from injury-induced ADM and offers an alternative origin for tumorigenesis. This program is conserved in human pancreatitis, providing insight into early events in pancreas diseases.

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Reinstating olfactory bulb derived limbic gamma oscillations alleviates depression Although the etiology of major depressive disorder remains poorly understood, impairment of gamma oscillations recently emerged as a potential biomarker for major depression. The olfactory bulb (OB) is a major source of brain wide gamma oscillations and bulbectomy is an animal model for depression. Here we demonstrate that chemogenetic suppression of OB neuronal activity or temporally suppressing the OB to pyriform cortex synaptic transmission decreased gamma oscillation power in multiple brain areas associated with depression-like behaviors. To assess the hypothesized link between depression and diffuse depression of gamma oscillations, we employed gamma phase-dependent closed loop neuromodulation of cortical areas, paced by the native OB output. This procedure alleviated depressive-like behaviors in animals and suggests that restoring gamma oscillations may improve depression in humans.

One Sentence SummaryRole of limbic gamma oscillations in depression

sentences: The structural connectome constrains fast brain dynamics Brain activity during rest displays complex, rapidly evolving patterns in space and time. Structural connections comprising the human connectome are hypothesized to impose constraints on the dynamics of this activity. Here, we use magnetoencephalography (MEG) to quantify the extent to which fast neural dynamics in the human brain are constrained by structural connections inferred from diffusion MRI tractography. We characterize the spatio-temporal unfolding of whole-brain activity at the millisecond scale from source-reconstructed MEG data, estimating the probability that any two brain regions will significantly deviate from baseline activity in consecutive time epochs. We find that the structural connectome relates to, and likely affects, the rapid spreading of neuronal avalanches, evidenced by a significant association between these transition probabilities and structural connectivity strengths (r=0.37, p<0.0001). This finding opens new avenues to study the relationship between brain structure and neural dynamics.

sentences: Novel algorithms deriving clinical performance measures from smartphone sensor data collected under a walking test The modern ubiquity of smartphones which incorporate sensors such as inertial measurement units (IMU) and global positioning system (GPS) receivers, raises the possibility that they might be used as inexpensive devices for clinical performance testing. Here, we report on the development and testing of an app and associated statistical signal processing algorithms, to measure the basic gait properties of step rate, walking speed and distance under an outside short walk test experimental protocol, from digital accelerometry and GPS. We provide extensive details on the assumptions underlying the development of the algorithms. A small set of experiments across a range of conditions and individuals, demonstrate the reliability of the combined app, protocol and algorithms. With reasonable adherence to the test protocol, these experiments show 2% error across all metrics. The emphasis in this paper is on illustrating the design principles which all such systems, aiming to re-purpose consumer smartphones as clinical gait measurement devices, must address.

sentences: Short linear motif based interactions and dynamics of the ezrin, radixin, moesin and merlin FERM domains The ERM (ezrin, radixin and moesin) family of proteins and the related protein merlin participate in signaling events at the cell cortex. The proteins share an N-terminal FERM (band Four-point-one (4.1) ERM) domain comprised of three subdomains (F1, F2, and F3) that hold multiple binding sites for short linear peptide motifs. By screening the FERM domains of the ERMs and merlin against a phage library that display peptides representing the intrinsically disordered regions of the human proteome we identified more than 220 FERM binding peptides. The majority of the peptides contained an apparent Yx[FILV] motif, but ligands with alternative motifs were also found. Interactions with thirteen peptides were validated using a fluorescence polarization assay, and interactions with seven full-length proteins were validated through pull-down experiments. We investigated the energy landscapes of interactions between the moesin FERM domain and representative set of ligands using Rosetta FlexPepDock computational peptide docking protocols, which provide a detailed molecular understanding of the binding of peptides with distinct motifs (YxV and E[Y/F]xDFYDF) to different sites on the F3 subdomain. A third motif (FY[D/E]L(4-5x)PLxxx[L/V]) was proposed to bind more diffusely. By combining competition and modeling experiments, we further uncovered interdependencies between different types of ligands. The study expands the motif-based interactomes of the ERMs and merlin, and suggests that the FERM domain acts as a switchable interaction hub where one class of ligands to the F3 subdomain allosterically regulates binding of other F3 ligands.

sentences: Nanoformulated Remdesivir with Extremely Low Content of Poly(2-oxazoline) - Based Stabilizer for Aerosol Treatment of COVID-19 The rise of the novel virus SARS-CoV2 which causes the disease known as COVID-19 has led to a global pandemic claiming millions of lives. With no clinically approved treatment for COVID-19, physicians initially struggled to treat the disease and there is still need for improved anti-viral therapies in this area. We conceived early in the pandemic that an inhalable formulation of the drug Remdesivir which directly targets the virus at the initial site of infection could improve therapeutic outcomes in COVID-19. We developed a set of requirements that would be conducive to rapid drug approval: 1) try to use GRAS or GRAS similar reagents 2) minimize excipient concentration and 3) achieve a working concentration of 5 mg/mL Remdesivir to achieve a deliverable dose which is 5-10% of the IV dose. In this work, we discovered that Poly(2-oxazoline) block copolymers can stabilize drug nanocrystal suspensions and provide suitable formulation characteristics for aerosol delivery while maintaining anti-viral efficacy. We believe POx block copolymers can be used as a semi-ubiquitous stabilizer for the rapid development of nanocrystal formulations for new and existing diseases.

sentences: Sex-Specific Computational Models of Kidney Function in Human Patients with Diabetes The kidney plays an essential role in homeostasis, accomplished through the regulation of pH, electrolytes and fluids, by the building blocks of the kidney, the nephrons. One of the important markers of the proper functioning of a kidney is the glomerular filtration rate. Diabetes is characterised by an enlargement of the glomerular and tubular size of the kidney, affecting the afferent and efferent arteriole resistance and hemodynamics, ultimately leading to chronic kidney disease. We postulate that the diabetes-induced changes in kidney may exhibit significant sex differences as the distribution of renal transporters along the nephron may be markedly different between women and men, as recently shown in rodents. The goals of this study are to (i) analyze how kidney function is altered in male and female patients with diabetes, and (ii) assess the renal effects, in women and men, of an anti-hyperglycemic therapy that inhibits the sodium-glucose cotransporter 2 (SGLT2) in the proximal convoluted tubules. To accomplish these goals, we have developed computational models of kidney function, separate for male and female patients with diabetes. The simulation results indicate that diabetes enhances Na+ transport, especially along the proximal tubules and thick ascending limbs, to similar extents in male and female patients, which can be explained by the diabetes-induced increase in glomerular filtration rate. Additionally, we conducted simulations to study the effects of diabetes and SGLT2 inhibition on solute and water transport along the nephrons. Model simulations also suggest that SGLT2 inhibition, which constricts the afferent arteriole to attenuate glomerular hyperfiltration, can then limit Na+-glucose transport, consequently raising luminal [Cl-] at the macula densa and finally restoring the tubuloglomerular feedback signal. By inducing osmotic diuresis in the proximal tubules, SGLT2 inhibition reduces paracellular transport, eventually leading to diuresis and natriuresis. Those effects on urinary excretion are blunted in women, in part due to their higher distal transport capacity.

sentences: Larval zebrafish exhibit collective motion behaviors in constrained spaces Collective behavior may be elicited or can spontaneously emerge by a combination of interactions with the physical environment and conspecifics moving within that environment. To investigate the relative contributions of these factors in a small millimeter-scale swimming organism, we observed larval zebrafish, interacting at varying densities under circular confinement. Our aim was to understand the biological and physical mechanisms acting on these larvae as they swim together inside circular confinements. If left undisturbed, larval zebrafish swim intermittently in a burst and coast manner and are socially independent at this developmental stage, before shoaling behavioral onset. We report here our analysis of a new observation for this well-studied species: in circular confinement and at sufficiently high densities, the larvae collectively circle rapidly alongside the boundary. This is a new physical example of self-organization of mesoscale living active matter driven by boundaries and environment geometry. We believe this is a step forward toward using a prominent biological model system in a new interdisciplinary context to advance knowledge of the physics of social interactions.

sentences: Identifying protein sites contributing to vaccine escape via statistical comparisons of short-term molecular dynamics simulations The identification of viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. We describe a molecular dynamics (MD) based approach that scales well to a desktop computer with a high-end modern graphics processor and enables the user to identify protein sites that are prone to vaccine escape in a viral antigen. We first implement our MD pipeline to employ site-wise calculation of Kullback-Leibler divergence in atom fluctuation over replicate sets of short-term MD production runs thus enabling a statistical comparison of the rapid motion of influenza hemagglutinin (HA) in both the presence and absence of three well-known neutralizing antibodies. Using this simple comparative method applied to motions of viral proteins, we successfully identified in silico all previously empirically confirmed sites of escape in influenza HA, predetermined via selection experiments and neutralization assays. Upon the validation of our computational approach, we then surveyed potential hot spot residues in the receptor binding domain of the SARS-CoV-2 virus in the presence of COVOX-222 and S2H97 antibodies. We identified many single sites in the antigen-antibody interface that are similarly prone to potential antibody escape and that match many of the known sites of mutations arising in the SARS-CoV-2 variants of concern. In the omicron variant, we find only minimal adaptive evolutionary shifts in the functional binding profiles of both antibodies. In summary, we provide a fast and accurate computational method to monitor hot spots of functional evolution in antibody binding footprints.

sentences: Mitigating biomass composition uncertainties in flux balance analysis using ensemble representations The biomass equation is a critical component in genome-scale metabolic models (GEMs): it is used as the de facto objective function in flux balance analysis (FBA). This equation accounts for the quantities of all known biomass precursors that are required for cell growth based on the macromolecular and monomer compositions measured at certain conditions. However, it is often reported that the macromolecular composition of cells could change across different environmental conditions; the use of the same single biomass equation in FBA, under multiple conditions, is questionable. Thus, we first investigated the qualitative and quantitative variations of macromolecular compositions of three representative host organisms, Escherichia coli, Saccharomyces cerevisiae and Cricetulus griseus, across different environmental/genetic variations. While macromolecular building blocks such as DNA, RNA, protein, and lipid composition vary notably, variations in fundamental biomass monomer units such as nucleotides and amino acids are not appreciable. We further observed that while macromolecular compositions are similar across taxonomically closer species, certain monomers, especially fatty acids, vary substantially. Based on the analysis results, we subsequently propose a new extension to FBA, named "Flux Balance Analysis with Ensemble Biomass (FBAwEB)", to embrace the natural variation in selected components of the biomass equation. The current study clearly highlights that certain components of the biomass equation are very sensitive to different conditions, and the ensemble representation of biomass equation in the FBA framework enables us to account for such natural variations accurately during GEM-guided in silico simulations.

sentences: Niche theory for within-host parasite dynamics: Analogies to food web modules via feedback loops Why do parasites exhibit a wide dynamical range within their hosts? For instance, why can a parasite only sometimes successfully infect its host? Why do some parasites exhibit large fluctuations? Why do two parasites coinfect, exclude each other, or win only sometimes over another (via priority effects)? For insights, we turn to food webs. An omnivory model (IGP) blueprints one parasite competing with immune cells for host energy (PIE), and a competition model (keystone predation, KP) mirrors a new coinfection model (2PIE). We then draw analogies between models using feedback loops. We translate those loops into the intraspecific direct (DE) and indirect effects (IE) that create various dynamics. Three points arise. First, a prey or parasite can flip between stable and oscillatory coexistence with their enemy with weakening IE and strengthening DE. Second, even with comparable loop structure, a parasite cannot exhibit priority effects seen in IGP due to constraints imposed by production of immune cells. Third, despite simpler loop structure, KP predicts parallel outcomes in the two-parasite model due to comparable structure of interactions between competing victims and their resources and enemies. Hence, food web models offer powerful if imperfect analogies to feedbacks underlying the dynamical repertoire of parasites within hosts.

sentences: Synthesis and biological evaluation of new cephalosporin derivatives containing cyclic disulfide moieties Due to a steady increase of microbial resistance, there is a need to increase the effectiveness of antibiotic performance by involving additional mechanisms of their penetration or retention for their better action. Cephalosporins are a successful group of antibiotics to combat pathogenic microorganisms, including drug-resistant strains. In this study, we investigated the effect of newly synthesized cephalosporin derivatives with cyclic disulfide modifications against several Gram-positive and Gram-negative strains as well as against biofilm formation. The incorporation of asparagusic acid was found to be effective in improving the activity of the drug against Gramnegative strains. Furthermore, we could demonstrate the successful inhibition of biofilm formation for S. aureus and P. aeruginosa at similar concentrations as obtained against planktonic cells. We propose that the incorporation of cyclic disulfides is one additional strategy to improve antibiotic activity and to combat bacterial infections.

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On the relation between input and output distributions of scRNA-seq experiments Structured AbstractO_ST_ABSMotivationC_ST_ABSSingle-cell RNA sequencing determines RNA copy numbers per cell for a given gene. However, technical noise poses the question how observed distributions (output) are connected to their cellular distributions (input).

ResultsWe model a single-cell RNA sequencing setup consisting of PCR amplification and sequencing, and derive probability distribution functions for the output distribution given an input distribution. We provide copy number distributions arising from single transcripts during PCR amplification with exact expressions for mean and variance. We prove that the coefficient of variation of the output of sequencing is always larger than that of the input distribution. Experimental data reveals the variance and mean of the input distribution to obey characteristic relations, which we specifically determine for a HeLa data set. We can calculate as many moments of the input distribution as are known of the output distribution (up to all). This, in principle, completely determines the input from the output distribution.

Contactmartin.falcke@mdc-berlin.de

sentences: Harringtonine has the effects of double blocking SARS-CoV-2 membrane fusion Fusion with host cell membrane is the main mechanism of infection of SARS-CoV-2. Here, we propose a new strategy to double block SARS-CoV-2 membrane fusion by using Harringtonine (HT), a small-molecule antagonist. By using cell membrane chromatography (CMC), we found that HT specifically targeted the SARS-CoV-2 S protein and host cell TMPRSS2, and then confirmed that HT can inhibit pseudotyped virus membrane fusion. Furthermore, HT successfully blocked SARS-CoV-2 infection, especially in the delta and Omicron mutant. Since HT is a small-molecule antagonist, it is minimally affected by the continuous variation of SARS-CoV-2. Our findings show that HT is a potential small-molecule antagonist with a new mechanism of action against SARS-CoV-2 infection, and thus HT mainly targets the S protein, and thus, greatly reduces the damage of the S proteins autotoxicity to the organ system, has promising advantages in the clinical treatment of COVID-19.

sentences: Predictive feedback, early sensory representations and fast responses to predicted stimuli depend on NMDA receptors Learned associations between stimuli allow us to model the world and make predictions, crucial for efficient behavior; e.g., hearing a siren, we expect to see an ambulance and quickly make way. While there are theoretical and computational frameworks for prediction, the circuit and receptor-level mechanisms are unclear. Using high-density EEG, Bayesian modeling and machine learning, we show that inferred "causal" relationships between stimuli and frontal alpha activity account for reaction times (a proxy for predictions) on a trial-by-trial basis in an audio-visual delayed match-to-sample task which elicited predictions. Predictive beta feedback activated sensory representations in advance of predicted stimuli. Low-dose ketamine, a NMDA receptor blocker - but not the control drug dexmedetomidine - perturbed behavioral indices of predictions, their representation in higher-order cortex, feedback to posterior cortex and pre-activation of sensory templates in higher-order sensory cortex. This study suggests predictions depend on alpha activity in higher-order cortex, beta feedback and NMDA receptors, and ketamine blocks access to learned predictive information.

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Autophagy is impaired in fetal hypoplastic lungs and rescued by administration of amniotic fluid stem cell extracellular vesicles RationalePulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by reduced branching morphogenesis, which is responsible for poor clinical outcomes. Administration of amniotic fluid stem cell extracellular vesicles (AFSC-EVs) rescues branching morphogenesis in rodent fetal models of pulmonary hypoplasia. Herein, we hypothesized that AFSC-EVs exert their regenerative potential by affecting autophagy, a process required for normal lung development.

ObjectivesTo evaluate autophagy in hypoplastic lungs throughout gestation and establish whether AFSC-EV administration improves branching morphogenesis through autophagy-mediated mechanisms.

MethodsEVs were isolated from c-kit+ AFSC conditioned medium by ultracentrifugation and characterized for size, morphology, and EV markers. Branching morphogenesis was inhibited in rat fetuses by nitrofen administration to dams and in human fetal lung explants by blocking RAC1 activity with NSC23766. Expression of autophagy activators (BECN1 and ATG5) and adaptor (SQSTM1/p62) was analyzed in vitro (rat and human fetal lung explants) and in vivo (rat fetal lungs). Mechanistic studies on rat fetal primary lung epithelial cells were conducted using inhibitors for microRNA-17 and -20a contained in the AFSC-EV cargo and known to regulate autophagy.

Measurements and Main ResultsRat and human models of fetal pulmonary hypoplasia showed reduced autophagy mainly at pseudoglandular and canalicular stages. AFSC-EV administration restored autophagy in both pulmonary hypoplasia models by transferring miR-17[~]92 cluster members contained in the EV cargo.

ConclusionsAFSC-EV treatment rescues branching morphogenesis partly by restoring autophagy through miRNA cargo transfer. This study enhances our understanding of pulmonary hypoplasia pathogenesis and creates new opportunities for fetal therapeutic intervention in CDH babies.

sentences: Initial contact shapes the perception of friction Humans efficiently estimate the grip force necessary to lift a variety of objects, including slippery ones. The regulation of grip force starts with the initial contact, and takes into account the surface properties, such as friction. This estimation of the frictional strength has been shown to depend critically on cutaneous information. However, the physical and perceptual mechanism that provides such early tactile information remains elusive. In this study, we developed a friction-modulation apparatus to elucidate the effects of the frictional properties of objects during initial contact. We found a correlation between participants conscious perception of friction and radial strain patterns of skin deformation. The results provide insights into the tactile cues made available by contact mechanics to the sensorimotor regulation of grip, as well as to the conscious perception of the frictional properties of an object.

sentences: Direct fast estimator for recombination frequency in the F2 cross In this short note, a new unbiased maximum-likelihood estimator is proposed for the recombination frequency in the F2 cross. The estimator is much faster to calculate than its EM algorithm equivalent, yet as efficient. Simulation studies are carried to illustrate the gain over another simple estimate proposed by Benito & Gallego (2004).

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Arabidopsis inositol polyphosphate kinases regulate COP9 signalosome functions in phosphate-homeostasis Targeted protein degradation is essential for physiological development and adaptation to stress. Mammalian INOSITOL PENTAKISPHOSPHATE 2-KINASE (IP5K) and INOSITOL HEXAKISPHOSPHATE KINASE 1 (IP6K1) pair generates inositol polyphosphates (InsPs) to modulate association/dissociation equilibrium of Cullin RING Ubiquitin E3 ligases (CRLs) on the COP9 signalosome (CSN) platform. Deneddylase activity of the CSN5 subunit protects cullins from self-ubiquitination ensuring their functional continuity. In plants, similar regulations by InsP-kinases are not known. Here, we show conserved interactions of Arabidopsis thaliana INOSITOL PENTAKISPHOSPHATE 2-KINASE 1 (IPK1) and INOSITOL 1,3,4-TRISPHOSPHATE 5/6-KINASE 1 (ITPK1), counterparts of the above mammalian InsP-kinase pair, with selective CSN subunits. In ipk1 or itpk1 mutants, deneddylation deficiencies not only cause increased neddylated Cullin1 (CUL1Nedd8) pools more prone to degradation but also impair CSN5 entry/exit shuttles on the CSN holo-complex. Constitutive phosphate-starvation response (PSR), previously known for these mutants are suppressed by pharmacological inhibition of neddylation thus linking CSN-CRL functions to phosphate (Pi)-sensing. Similarly, in wild-type plants exposed to compounds that impair CSN5 deneddylase function affects its dynamics and mimic PSR properties of the mutants. We further show that under Pi-deprivation more CSN5 retentions on the CSN holo-complex and the resulting enhanced CUL1Nedd8 pools is essential for induction of downstream Pi-starvation inducible (PSI) genes. Overall, with our data we present InsP-kinase involvements in maintenance of Pi-homeostasis in plants via CRL-CSN(5) functional synergism.

SignificanceNeddylation modifications on Culling-RING E3 ligases (CRLs) while essential for their role in proteostasis, also threaten their own stability. Selective inositol polyphosphates (InsPs) aid the constitutive photomorphogenesis 9 signalosome (CSN) functions in protecting, deneddylating, and facilitating CRL recycling. Here we demonstrate that plant mutants deficient in these InsPs have disturbed CSN subunit partitioning, are deficient in deneddylase activity, and hyperaccumulate neddylated cullins that lead to constitutive phosphate-starvation response (PSR). Inhibition of CSN functions/consequences mirror the InsP mutant properties indicating pivotal involvement of CSN in phosphate homeostasis. These raises promising possibilities of targeted intervention on CSN functions for nutritional benefit of plants.

sentences: Pharmacokinetics of caffeine: A systematic analysis of reported data for application in metabolic phenotyping and liver function testing Caffeine is by far the most ubiquitous psychostimulant worldwide found in tea, coffee, cocoa, energy drinks, and many other beverages and food. Caffeine is almost exclusively metabolized in the liver by the cytochrome P-450 enzyme system to the main product paraxanthine and the additional products theobromine and theophylline. Besides its stimulating properties, two important applications of caffeine are metabolic phenotyping of cytochrome P450 1A2 (CYP1A2) and liver function testing. An open challenge in this context is to identify underlying causes of the large inter-individual variability in caffeine pharmacokinetics. Data is urgently needed to understand and quantify confounding factors such as lifestyle (e.g. smoking), the effects of drug-caffeine interactions (e.g. medication metabolized via CYP1A2), and the effect of disease. Here we report the first integrative and systematic analysis of data on caffeine pharmacokinetics from 148 publications and provide a comprehensive high-quality data set on the pharmacokinetics of caffeine, caffeine metabolites, and their metabolic ratios in human adults. The data set is enriched by meta-data on the characteristics of studied patient cohorts and subjects (e.g. age, body weight, smoking status, health status), the applied interventions (e.g. dosing, substance, route of application), measured pharmacokinetic time-courses, and pharmacokinetic parameters (e.g. clearance, half-life, area under the curve). We demonstrate via multiple applications how the data set can be used to solidify existing knowledge and gain new insights relevant for metabolic phenotyping and liver function testing based on caffeine. Specifically, we analyzed (i) the alteration of caffeine pharmacokinetics with smoking and use of oral contraceptives; (ii) drug-drug interactions with caffeine as possible confounding factors of caffeine pharmacokinetics or source of adverse effects; (iii) alteration of caffeine pharmacokinetics in disease; and (iv) the applicability of caffeine as a salivary test substance by comparison of plasma and saliva data. In conclusion, our data set and analyses provide important resources which could enable more accurate caffeine-based metabolic phenotyping and liver function testing.

sentences: Label-free imaging of collagen fibers in tissue slices using phase imaging with computational specificity Evaluating the tissue collagen content in addition to the epithelial morphology has been proven to offer complementary information in histopathology, especially in oncology tumor staging and prediction of survival in cancer patients. One imaging modality widely used for this purpose is second harmonic generation microscopy (SHGM), which reports on the nonlinear susceptibility associated with the collagen fibers. Another method is polarization light microscopy (PLM) combined with picrosirius-red (PSR) tissue staining. However, SHGM requires expensive equipment and provides limited throughput, while PLM and PSR staining are not part of the routine surgical pathology workflow. Here, we utilize phase imaging with computational specificity (PICS) to computationally infer the collagen distribution of unlabeled tissue, with high specificity. PICS utilizes deep learning to translate quantitative phase images (QPI) into corresponding PSR images with high accuracy and inference speed of 200 milisecond per forwardpass through the model once trained. We developed a multimodal imaging instrument that yields both Spatial light Inference Microscopy (SLIM) and polarized light microscopy (PLM) images from the same field of view. Our results indicate that the distributions of collagen fiber orientation, length, and straightness reported by PICS closely match the ones from ground truth as defined by KL-divergence.

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CAMAP: Artificial neural networks unveil the role of codon arrangement in modulating MHC-I peptides presentation MHC-I associated peptides (MAPs) play a central role in the elimination of virus-infected and neoplastic cells by CD8 T cells. However, accurately predicting the MAP repertoire remains difficult, because only a fraction of the transcriptome generates MAPs. In this study, we investigated whether codon arrangement (usage and placement) regulates MAP biogenesis. We developed an artificial neural network called Codon Arrangement MAP Predictor (CAMAP), predicting MAP presentation solely from mRNA sequences flanking the MAP-coding codons (MCCs), while excluding the MCC per se. CAMAP predictions were significantly more accurate when using original codon sequences than shuffled codon sequences which reflect amino acid usage. Furthermore, predictions were independent of mRNA expression and MAP binding affinity to MHC-I molecules and applied to several cell types and species. Combining MAP ligand scores, transcript expression level and CAMAP scores was particularly useful to increaser MAP prediction accuracy. Using an in vitro assay, we showed that varying the synonymous codons in the regions flanking the MCCs (without changing the amino acid sequence) resulted in significant modulation of MAP presentation at the cell surface. Taken together, our results demonstrate the role of codon arrangement in the regulation of MAP presentation and support integration of both translational and post-translational events in predictive algorithms to ameliorate modeling of the immunopeptidome.

Author summaryMHC-I associated peptides (MAPs) are small fragments of intracellular proteins presented at the surface of cells and used by the immune system to detect and eliminate cancerous or virus-infected cells. While it is theoretically possible to predict which portions of the intracellular proteins will be naturally processed by the cells to ultimately reach the surface, current methodologies have prohibitively high false discovery rates. Here we introduce an artificial neural network called Codon Arrangement MAP Predictor (CAMAP) which integrates information from mRNA-to-protein translation to other factors regulating MAP biogenesis (e.g. MAP ligand score and transcript expression levels) to improve MAP prediction accuracy. While most MAP predictive approaches focus on MAP sequences per se, CAMAPs novelty is to analyze the MAP-flanking mRNA sequences, thereby providing completely independent information for MAP prediction. We show on several datasets that the integration of CAMAP scores with other known factors involved in MAP presentation (i.e. MAP ligand score and mRNA expression) significantly improves MAP prediction accuracy, and further validate CAMAP learned features using an in-vitro assay. These findings may have major implications for the design of vaccines against cancers and viruses, and in times of pandemics could accelerate the identification of relevant MAPs of viral origins.

sentences: Resistance of Dickeya solani strain IPO 2222 to lytic bacteriophage vB_Dsol_D5 ({Phi}D5) results in fitness tradeoffs for the bacterium during infection Resistance to bacteriophage infections protects bacteria in phage-full environments, allowing them to survive and multiply in the presence of their viral predators. However, such resistance may cause direct costs for strains linked with the ecological fitness expressed as reduced competitiveness for resources or reduced virulence or both. Unfortunately, limited knowledge exists about such costs paid by phage-resistant plant pathogenic bacteria in their natural environments. This study analyzed the costs of phage resistance paid by broad host phytopathogenic pectinolytic bacterium Dickeya solani both in vitro and in potato (Solanum tuberosum L.) plants. Thirteen D. solani IPO 2222 Tn5 mutants were identified that exhibited resistance to infection caused by lytic bacteriophage vB_Dsol_D5 ({Phi}D5). The genes disrupted in these 13 mutants encoded proteins involved in the synthesis of the bacterial envelope components (viz. LPS, EPS and capsule). The ability of {Phi}D5-resistant D. solani mutants to colonize and cause symptoms on potato plants as well as other phenotypes that are known to contribute to the ecological fitness of D. solani in-plant environment, including growth rate, production of effectors, swimming and swarming motility, use of various carbon and nitrogen sources and biofilm formation were assessed. Although phage resistance did not affect most of the phenotypes of {Phi}D5-resistant D. solani evaluated in vitro, all phage resistant mutants were significantly compromised in their ability to survive on and colonize and cause disease symptoms in potato plants. This study is, to our knowledge, one of few to show the direct link between phage resistance and the fitness of plant pathogenic bacteria and the first one to assess phage-host associations for D. solani.

sentences: The development and neuronal complexity of bipinnaria larvae of the sea star Asterias rubens Free-swimming planktonic larvae are a key stage in the development of many marine phyla, and studies of these organisms have contributed to our understanding of major genetic and evolutionary processes. Although transitory, these larvae often attain a remarkable degree of tissue complexity, with well-defined musculature and nervous systems. Amongst the best studied are larvae belonging to the phylum Echinodermata, but with work largely focused on the pleuteus larvae of sea urchins (class Echinoidea). The greatest diversity of larval strategies amongst echinoderms is found in the class Asteroidea (sea-stars), organisms that are rapidly emerging as experimental systems for genetic and developmental studies. However, the bipinnaria larvae of sea stars have only been studied in detail in a small number of species and the full complexity of the nervous system is, in particular, poorly understood. Here we have analysed embryonic development and bipinnaria larval anatomy in the common North Atlantic sea-star Asterias rubens, employing use of a variety of staining methods in combination with confocal microscopy. Importantly, the complexity of the nervous system of bipinnaria larvae was revealed in greater detail than ever before, with identification of at least three centres of neuronal complexity: the anterior apical organ, oral region and ciliary bands. Furthermore, the anatomy of the musculature and sites of cell division in bipinnaria larvae were analysed. Comparisons of developmental progression and molecular anatomy across the Echinodermata provided a basis for hypotheses on the shared evolutionary and developmental processes that have shaped this group of animals. We conclude that bipinnaria larvae appear to be remarkably conserved across ~200 million years of evolutionary time and may represent a strong evolutionary and/or developmental constraint for species utilizing this larval strategy.

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A benchmark of Hi-C scaffolders using reference genomes and de novo assemblies BackgroundStudying a new species using high-throughput sequencing requires a high-quality reference genome. However, assembling chromosome length sequences remains challenging. Recent advances in chromatin conformation capture (Hi-C) have provided a new approach to scaffolding genome assemblies, and the last ten years have seen a proliferation of such methods. However, to our knowledge no comprehensive benchmarking of Hi-C scaffolders has been conducted to date.

ResultsThrough a literature review we identified the most popular Hi-C scaffolders - Lachesis, HiRise, 3d-dna, SALSA, and AllHiC. We tested their ability to scaffold four well studied genomes - S. cerevisiae, L. tarentolae, A. thaliana, and H. sapiens. Scaffolders were tasked with both scaffolding fragmented versions of the reference genome as well as de novo assemblies derived from long read datasets. We found that all scaffolders can exceed 80% accuracy under ideal circumstances but that their performance quickly deteriorates under more challenging conditions. Surprisingly, many scaffolders also showed poor performance on the best assemblies, where contigs are near chromosome length. Overall, we found that HiRise and Lachesis offer the best performance on average across all conditions.

ConclusionsWe compare the performance of five Hi-C scaffolders using multiple reference species under both ideal and real-life conditions, thereby illuminating their strengths and weaknesses.

sentences: Infectious SARS-CoV-2 is emitted in aerosols Respiratory viruses such as SARS-CoV-2 are transmitted in respiratory droplets and aerosols, which are released during talking, breathing, coughing, and sneezing. Non-contact transmission of SARS-CoV-2 has been demonstrated, suggesting transmission in aerosols. Here we demonstrate that golden Syrian hamsters emit infectious SARS-CoV-2 in aerosols, prior to and concurrent with the onset of mild clinical signs of disease. The average emission rate is 25 infectious virions/hour on days 1 and 2 post-inoculation, with average viral RNA levels 200-fold higher than infectious virus in aerosols. Female hamsters have delayed kinetics of viral shedding in aerosols compared to male hamsters, with peak viral emission for females on dpi 2 and for males on dpi 1. The majority of virus is contained within aerosols <8 {micro}m in size. Thus, we provide direct evidence that, in hamsters, SARS-CoV-2 is an airborne virus.

sentences: The Mitochondrial Permeability Transition Pore Activates the Mitochondrial Unfolded Protein Response and Promotes Aging Mitochondrial activity determines aging rate and the onset of chronic diseases. The mitochondrial permeability transition pore (mPTP) is a pathological pore in the inner mitochondrial membrane thought to be composed of the F-ATP synthase (complex V). OSCP, a subunit of F-ATP synthase, helps protect against mPTP formation. How the destabilization of OSCP may contribute to aging, however, is unclear. We have found that loss OSCP in the nematode Caenorhabditis elegans initiates the mPTP and shortens lifespan specifically during adulthood, in part via initiation of the mitochondrial unfolded protein response (UPRmt). Pharmacological or genetic inhibition of the mPTP inhibits the UPRmt and restores normal lifespan. Loss of the putative pore-forming component of F-ATP synthase extends adult lifespan, suggesting that the mPTP normally promotes aging. Our findings reveal how an mPTP/UPRmt nexus may contribute to aging and age-related diseases and how inhibition of the UPRmt may be protective under certain conditions.

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The Central Importance of Hub Proteins in a Disease-Gene Network Model: A New Paradigm of Chronic Myeloid Leukemia Disease Study BackgroundThe network biology of disease-gene association provides a holistic framework to decipher the intrinsic complexity of disease signaling pathways into cellular communication level. Different types of studies including large-scale genome-wide association, multifactor dimensional reduction analysis, whole genome, or exome-based sequencing strategies of diseases are striving to connect genes to diseases. Indeed, these approaches have had some accomplishments, but the cellular communication level needs a more streamlining outcome to understand the mechanistic impact of context. The higher-order combination of disease-gene interaction has a great potential to decipher the intricateness of diseases. The molecular interaction pattern of diseases at the genomic and proteomic level offers a revolutionized platform not only to understand the complexity of particular disease modules and pathways but also leading towards design novel therapeutics.

ResultsThe enrichment and topology analysis was performed by JEPETTO a plugin of Cytoscape software. We identified the chronic myeloid leukemia (CML) disease signaling pathways that appeared first in the ranking order based on XD-score among the bone, breast, and colon genes set and second at kidney and liver. This result validates the highest proximity between CML and five cancerous tissue gene set clusters. The topology analysis also supports the results while (p<0.0001) is considered to be extremely significant between CML and fives cancerous tissues genes set. Enrichment analysis identified that abl-gene acts as an overlapping node which is the major gene for inducing various mutations in CML. Amazingly, we identified 56 common path expansion/added genes among these five cancerous tissues which can be considered the direct cofactors of CML disease. By relative node degree, resolution, possible ligand, stoichiometry, Q-mean, and Z-score analysis we found 11 hubs proteins like SMAD3, GRB2, TP53, SMAD4, RB1, HDAC1, RAF1, ABL1, SHC1, TGFBR1, RELA which can be regarded for further drug target identification.

ConclusionsOur proposed network analysis reflects on the gene set interaction pattern of disease signaling pathways of humans. The integrated multidrug computational and experimental approaches boost up to improve the novel drug target approach. Besides, such a trove can yield unprecedented insights to lead to an enhanced understanding of potential application both in drug target optimization and for drug dislodging.

sentences: Simple morphometrics for predicting lordosis-induced deviations of body-shape in reared Gilthead seabream (Sparus aurata L.) Haemal lordosis, V-shape bending of the haemal vertebrae, is a frequent abnormality of reared fish. Lordosis severity ranges from light deformations of vertebral axis, with insignificant effects on external morphology, to severe axis deformations with significant impact on body-shape. In the present study, we developed a simple morphometric index (PrAn) that links lordosis severity at the juvenile stage with fish body-shape at harvesting, without requiring to radiograph or sacrifice the samples. Examined seabream specimens were part of our previous study (Fragkoulis et al. 2019, Sci. Rep. 9, 9832), which monitored the effects of lordosis on the external morphology of pit-tagged seabream juveniles during their growth, up to harvest size. At both juvenile and adult stages, PrAn was effective in discriminating the normal fish from ca the 70% of lordotic fish. Our results suggest the PrAn as a valuable scale of quality, which quantifies the lordosis effects on fish external morphology, both at the juvenile stage and at harvest. Depending on the lordosis rates, and the hatchery strategy on the maximum allowed abnormality rates, this scale can cull out different rates of lordotic fish, without affecting the fish with normal phenotype or the lordotic fish with high recovery potential.

sentences: Highly Reversible Tunable Thermal-repressible Split-T7 RNA polymerases (Thermal-T7RNAPs) for dynamic gene regulation Temperature is a physical cue that is easy to apply, allowing cellular behaviors to be controlled in a contactless and dynamic manner via heat-inducible/repressible systems. However, existing heat-repressible systems are limited and rely on thermal sensitive mRNA or transcription factors which function at low temperatures, lack tunability, suffer delays or overly-complex. To provide an alternative mode of thermal regulation, we developed a library of compact, reversible and tunable thermal-repressible split-T7 RNA polymerase systems (Thermal-T7RNAPs) which fuses temperature-sensitive domains of Tlpa protein with split-T7RNAP to enable direct thermal control of the T7RNAP activity between 30 - 42 {degrees}C. We generated a large mutant library with varying thermal performances via automated screening framework to extend temperature tunability. Lastly, using the mutants, novel thermal logic circuitry was implemented to regulate cell growth and achieve active thermal control of the cell proportions within co-cultures. Overall, this technology expands avenues for thermal control in biotechnology applications.

sentences: Imaging of the pial arterial vasculature of the human brain in vivo using high-resolution 7T time-of-flight angiography The pial arterial vasculature of the human brain is the only blood supply to the neocortex, but quantitative data on the morphology and topology of these mesoscopic arteries (diameter 50- 300 {micro}m) remains scarce. Because it is commonly assumed that blood flow velocities in these vessels are prohibitively slow, non-invasive time-of-flight MRI angiography (TOF-MRA)--which is well-suited to high 3D imaging resolutions--has not been applied to imaging the pial arteries. Here, we provide a theoretical framework that outlines how TOF-MRA can visualize small pial arteries in vivo, by employing extremely small voxels at the size of individual vessels. We then provide evidence for this theory by imaging the pial arteries at 140-{micro}m isotropic resolution using a 7T MRI scanner and prospective motion correction, and show that pial arteries one voxel-width in diameter can be detected. We conclude that imaging pial arteries is not limited by slow blood flow, but instead by achievable image resolution. This study represents the first targeted, comprehensive account of imaging pial arteries in vivo in the human brain. This ultra-high-resolution angiography will enable the characterization of pial vascular anatomy across the brain to investigate patterns of blood supply and relationships between vascular and functional architecture.

sentences: cGAS recruitment to micronuclei is dictated by pre-existing nuclear chromatin status. Micronuclei (MN) are aberrant cytosolic compartments containing broken genomic fragments or whole lagging chromosomes. MN envelopes irreversibly rupture, allowing the viral receptor cGAS to localize to MN and initiate an inflammatory signalling cascade. Here, we demonstrate that MN envelope rupture is not sufficient for cGAS localization. Unlike MN that arise following ionizing radiation (IR), ruptured MN generated from acute transcription stressors DRB or siSRSF1 are refractory to cGAS localization. Recruitment of cGAS to MN is blocked by inhibiting the histone methyltransferase DOT1L prior to IR exposure, demonstrating that cGAS recruitment to MN is dictated by nuclear chromatin organization at the time of DNA damage. Loss of cGAS+ MN, caused either by acute transcription stressors or by preventing DOT1L-deposited histone methylation, corresponded to significantly decreased cGAS-dependent inflammatory signalling. These results implicate nuclear chromatin organization in micronuclear composition and activity, influencing the ability of damage-induced MN to retain cytosolic proteins upon rupture.

sentences: An ensemble deep learning framework to refine large deletions in linked-reads The detection of structural variants (SVs) remains challenging due to inconsistencies in detected breakpoints and biological complexity of some rearrangements. Linked-reads have demonstrated their superiority in diploid genome assembly and SV detection. Recently developed tools Aquila and Aquila_stLFR use a reference sequence and linked-reads to generate a high quality diploid genome assembly, using which they then detect and phase personal genetic variations. However, they both produce a substantial proportion of false positive deletion SV calls. To take full advantage of linked-reads, an effective downstream filtering and refinement framework is needed pressingly. In this work, we propose AquilaDeepFilter to filter large deletion SVs from Aquila and Aquila_stLFR. AquilaDeepFilter relies on a deep learning ensemble approach by integrating six state-of-the-art CNN backbones. The filtering of deletion SVs is formulated as a binary classification task on image data that are generated through the extraction of multiple alignment signals, including read depth, split reads and discordant read pairs. Three linked-reads libraries sequenced from the well-studied sample NA24385 and the gold standard of GiaB benchmark were used to perform thorough experiments on our proposed method. The results demonstrated that AquilaDeepFilter could increase the precision rate of Aquila while the recall rate of Aquila decreased only slightly, and the overall F1 improved by 20%. Furthermore, AquilaDeepFilter outperformed another deep learning based method for SV filtering, DeepSVFilter. Even though we designed AquilaDeepFilter for linked-reads, the framework could also be used to improve SV detection on short reads.

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SearcHPV: a novel approach to identify and assemble human papillomavirus-host genomic integration events in cancer BackgroundHuman papillomavirus (HPV) is a well-established driver of malignant transformation in a number of sites including head and neck, cervical, vulvar, anorectal and penile squamous cell carcinomas; however, the impact of HPV integration into the host human genome on this process remains largely unresolved. This is due to the technical challenge of identifying HPV integration sites, which includes limitations of existing informatics approaches to discover viral-host breakpoints from low read coverage sequencing data.

MethodsTo overcome this limitation, we developed a new HPV detection pipeline called SearcHPV based on targeted capture technology and applied the algorithm to targeted capture data. We performed an integrated analysis of SearcHPV-defined breakpoints with genome-wide linked read sequencing to identify potential HPV-related structural variations.

ResultsThrough analysis of HPV+ models, we show that SearcHPV detects HPV-host integration sites with a higher sensitivity and specificity than two other commonly used HPV detection callers. SearcHPV uncovered HPV integration sites adjacent to known cancer-related genes including TP63 and MYC, as well as near regions of large structural variation. We further validated the junction contig assembly feature of SearcHPV, which helped to accurately identify viral-host junction breakpoint sequences. We found that viral integration occurred through a variety of DNA repair mechanisms including non-homologous end joining, alternative end joining and microhomology mediated repair.

ConclusionsIn summary, we show that SearcHPV is a new optimized tool for the accurate detection of HPV-human integration sites from targeted capture DNA sequencing data.

sentences: Reactive microglia and cytokine release entails microtubule remodeling from acentrosomal to centrosomal arrays Microglia reactivity entails a large-scale remodeling of cellular geometry, but the role of the microtubule cytoskeleton during these changes remains unexplored. Here we show that reactive proinflammatory microglia provide a heretofore unique example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules. While in the homeostatic state microglia nucleate microtubules at Golgi outposts, proinflammatory signaling induces recruitment of nucleating material nearby the centrosome and inhibition of centrosomal maturation enhances NLRP3 inflammasome activation and secretion of IL-1{beta}. Our results demonstrate that a hallmark of microglia reactivity is a striking remodeling of the microtubule cytoskeleton and suggest that pericentrosomal microtubule nucleation may serve as a distinct marker of microglia activation as well as a novel target to modulate cytokine-mediated inflammatory responses in chronic disease and tissue injury.

sentences: PharmOmics: A Species- and Tissue-specific Drug Signature Database and Online Tool for Drug Repurposing Drug development has been hampered by a high failure rate in clinical trials due to efficacy or safety issues not predicted by preclinical studies in model systems. A key contributor is our incomplete understanding of drug functions across organ systems and species. Therefore, elucidating species- and tissue-specific actions of drugs can provide systems level insights into therapeutic efficacy, potential adverse effects, and interspecies differences that are necessary for more effective translational medicine. Here, we present a comprehensive drug knowledgebase and analytical tool, PharmOmics, comprised of genomic footprints of drugs in individual tissues from human, mouse, and rat transcriptome data from GEO, ArrayExpress, TG-GATEs, and DrugMatrix. Using multi-species and multi-tissue gene expression signatures as molecular indicators of drug functions, we developed gene network-based approaches for drug repositioning. We demonstrate the potential of PharmOmics to predict drugs for new disease indications and validated two predicted drugs for non-alcoholic fatty liver disease in mice. We also examined the potential of PharmOmics to identify drugs related to hepatoxicity and nephrotoxicity. By combining tissue- and species-specific in vivo drug signatures with biological networks, PharmOmics serves as a complementary tool to support drug characterization.

sentences: De novo variants in Chinese ASD trios reveal genetic basis underlying autism without developmental delay and intellectual disabilities Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder that causes a range of social communication and behavioral impairments. ASD typically manifests in young children, often with developmental delay or intellectual disabilities (DD/ID) as comorbidities. Accruing evidence indicates that ASD is highly heritable and genomewide studies on ASD cohorts have defined numerous genetic contributors. Notably, most of these studies have been performed with individuals of European and Hispanic ancestry and thus there is a paucity of genetic analyses of ASD in the East Asian population. Here, we performed whole-exome sequencing on 772 ASD trios from China, combining with a previous study of 369 Chinese ASD trios, to identify de novo variants in a total of 1141 ASD trios. We found that ASD probands without DD/ID carried less disruptive de novo variants, including protein-truncating and missense variants, than ASD with DD/ID. Surprisingly, we showed that expression of genes with de novo variants found in ASD probands without DD/ID were enriched in a specific group of neural progenitor cells, suggesting a potential mechanism underlying high-functioning autism. Importantly, some ASD risk genes from this study are not present in the current ASD gene database, suggesting that there are novel genetic contributors to ASD in East Asian populations. We validated one such novel ASD risk gene - SLC35G1 by showing that mice harboring heterozygous deletion of Slc35g1 exhibited defects in social interaction behaviors. Together, this work nominates novel ASD risk genes and indicates that ASD genetic studies in different geographic populations are essential to reveal the comprehensive genetic architecture of ASD.

sentences: Multi-omics approach reveals dysregulation of protein phosphorylation correlated with lipid metabolism in mouse fatty liver Obesity caused by overnutrition is a major risk factor for non-alcoholic fatty liver disease (NAFLD). Several lipid intermediates such as fatty acids, glycerophospholipids and sphingolipids are implicated in NAFLD, but detailed characterization of lipids and their functional links to proteome and phosphoproteome remain to be elucidated. To characterize this complex molecular relationship, we used multi-omics approach by conducting comparative proteomic, phoshoproteomic and lipidomic analyses of high fat (HFD) and low fat (LFD) diet fed mice livers. We quantified 2447 proteins and 1339 phosphoproteins containing 1650 class I phosphosites (with localization probability > 0.75), of which 669 phosphosites were significantly different between HFD and LFD mice livers. We detected alterations of proteins associated with cellular metabolic processes such as small molecule catabolic process, monocarboxylic acid, long- and medium-chain fatty acid, and ketone body metabolic processes, and peroxisome organization. We observed significant downregulation of protein phosphorylation in HFD fed mice liver in general. Untargeted lipidomics identified upregulation of triacylglycerols, glycerolipids and ether glycerophosphocholines and downregulation of glycerophospholipids such as lysoglycerophospholipids, as well as ceramides and acylcarnitines. Analysis of differentially regulated phosphosites revealed phosphorylation dependent deregulation of insulin signaling as well as lipogenic and lipolytic pathways during HFD induced obesity. Thus, this study reveals a molecular connection between decreased protein phosphorylation and lipolysis, as well as lipid-mediated signaling in diet-induced obesity.

sentences: Generating publication ready visualizations for Single Cell transcriptomics using SCpubr Single Cell transcriptomic analysis has become a widespread technology of choice when it comes to understanding the differences at a transcriptomic level in heterogeneous samples. As a consequence, a plethora of analysis tools have been published to tackle the different analysis steps from count matrix generation to downstream analysis. Many of them provide ways to generate visualizations of the data. While some design choices are made, it is a common practice to provide the user with visualizations as raw as possible so that they can be customized to the user needs. However, in many cases these final customization steps are either time consuming or demand a very specific set of skills. This problem is addressed by SCpubr, which sacrifices some of this initial freedom of choice in aesthetics to provide the user a more streamlined way of generating high quality Single Cell transcriptomic visualizations.

sentences: MYB12 spatiotemporally represses TMO5/LHW-mediated transcription in the Arabidopsis root meristem Transcriptional networks are crucial to integrate various internal and external signals into optimal responses during plant growth and development. Primary root vasculature patterning and proliferation are controlled by a network centred around the basic Helix-Loop-Helix transcription factor complex formed by TARGET OF MONOPTEROS 5 (TMO5) and LONESOME HIGHWAY (LHW), which control cell proliferation and orientation by modulating cytokinin response and other downstream factors. Despite recent progress, many aspects of the TMO5/LHW pathway are not fully understood. In particular, the upstream regulators of TMO5/LHW activity remain unknown. Here, using a forward genetic approach to identify new factors of the TMO5/LHW pathway, we discovered a novel function of the MYB-type transcription factor MYB12. MYB12 physically interacts with TMO5 and dampens the TMO5/LHW-mediated induction of direct target gene expression as well as the periclinal/radial cell divisions. The expression of MYB12 is activated by the cytokinin response, downstream of TMO5/LHW, resulting in a novel MYB12-mediated negative feedback loop that restricts TMO5/LHW activity to ensure optimal cell proliferation rates during root vascular development.

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Minimal phrase composition revealed by intracranial recordings The ability to comprehend phrases is an essential integrative property of the brain. Here we evaluate the neural processes that enable the transition from single word processing to a minimal compositional scheme. Previous research has reported conflicting timing effects of composition, and disagreement persists with respect to inferior frontal and posterior temporal contributions. To address these issues, 19 patients (10 male, 19 female) implanted with penetrating depth or surface subdural intracranial electrodes heard auditory recordings of adjective-noun, pseudoword-noun and adjective-pseudoword phrases and judged whether the phrase matched a picture. Stimulus-dependent alterations in broadband gamma activity, low frequency power and phase-locking values across the language-dominant left hemisphere were derived. This revealed a mosaic located in the posterior superior temporal sulcus (pSTS), in which closely neighboring cortical sites displayed exclusive sensitivity to either lexicality or phrase structure, but not both. Distinct timings were found for effects of phrase composition (210-300 ms) and pseudoword processing (approximately 300-700 ms), and these were localized to neighboring electrodes in pSTS. The pars triangularis and temporal pole encode anticipation of composition in broadband low frequencies, and both regions exhibit greater functional connectivity with pSTS during phrase composition. Our results suggest that the pSTS is a highly specialized region comprised of sparsely interwoven heterogeneous constituents that encodes both lower and higher level linguistic features. This hub in pSTS for minimal phrase processing may form the neural basis for the human-specific computational capacity for forming hierarchically organized linguistic structures.

SignificanceLinguists have claimed that the integration of multiple words into a phrase demands a computational procedure distinct from single word processing. Here, we provide intracranial recordings from a large patient cohort, with high spatiotemporal resolution, to track the cortical dynamics of phrase composition. Epileptic patients volunteered to participate in a task in which they listened to phrases ("red boat"), word-pseudoword or pseudoword-word pairs (e.g., "red fulg"). At the onset of the second word in phrases, greater broadband high gamma activity was found in posterior superior temporal sulcus in electrodes that exclusively indexed phrasal meaning, and not lexical meaning. These results provide direct, high-resolution signatures of minimal phrase composition in humans, a potentially species-specific computational capacity.

sentences: Normal development of the brain: a survey of joint structural-functional brain studies Joint structural-functional (S-F) developmental studies present a novel approach to address the complex neuroscience questions on how the human brain works and how it matures. Joint S-F biomarkers have the inherent potential to model effectively the brains maturation, fill the information gap in temporal brain atlases, and demonstrate how the brains performance matures during the lifespan. This review presents the current state of knowledge on heterochronous and heterogeneous development of S-F links during the maturation period. The S-F relationship has been investigated in early-matured unimodal and prolonged-matured transmodal regions of the brain using a variety of structural and functional biomarkers and data acquisition modalities. Joint S-F unimodal studies have employed auditory and visual stimuli, while the main focus of joint S-F transmodal studies has been resting-state networks and working memory. However, non-significant associations between some structural and functional biomarkers and their maturation show that designing and developing effective S-F biomarkers is still a challenge in the field. Maturational characteristics of brain asymmetries have been poorly investigated by the joint S-F studies, and the results were inconsistent with previous non-joint ones. The inherent complexity of the brain performance can be modeled using multifactorial and nonlinear techniques as promising methods to simulate the impact of age on S-F relations considering their analysis challenges.

sentences: Relating spinal injury-induced neuropathic pain and spontaneous afferent activity to sleep and respiratory dysfunction Spinal cord injury (SCI) can induce dysfunction in a multitude of neural circuits including those that lead to impaired sleep, respiratory dysfunction and neuropathic pain. We used a lower thoracic rodent contusion SCI model - known to develop mechanosensory stimulus hypersensitivity, and spontaneous activity in primary afferents that associates neuropathic pain - and paired this with new approaches that enabled chronic capture of three state sleep and respiration to characterize dysfunction and assess possible interrelations. Noncontact electric field sensors were embedded into home cages for noninvasive capture in naturally behaving mice of the temporal evolution of sleep and respiration changes for 6 weeks after SCI. Hindlimb mechanosensitivity was assessed weekly, and terminal experiments measured primary afferent spontaneous activity in situ from intact lumbar dorsal root ganglia (DRG). We observed that SCI led to increased spontaneous primary afferent activity (both firing rate and the number of spontaneously active DRGs) that correlated with reduced hindpaw mechanical sensitivity, increased respiratory rate variability, and increased sleep fragmentation. This is the first study to measure and link sleep dysfunction and variability in respiratory rate in a SCI model of neuropathic pain, and thereby provide broader insight into the magnitude of overall stress burden initiated by neural circuit dysfunction after SCI.

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Rapid, Reference-Free Human Genotype Imputation with Denoising Autoencoders Genotype imputation is a foundational tool for population genetics. Standard statistical imputation approaches rely on the co-location of large whole-genome sequencing-based reference panels, powerful computing environments, and potentially sensitive genetic study data. This results in computational resource and privacy-risk barriers to access to cutting-edge imputation techniques. Moreover, the accuracy of current statistical approaches is known to degrade in regions of low and complex linkage disequilibrium.

Artificial neural network-based imputation approaches may overcome these limitations by encoding complex genotype relationships in easily portable inference models. Here we demonstrate an autoencoder-based approach for genotype imputation, using a large, commonly-used reference panel, and spanning the entirety of human chromosome 22. Our autoencoder-based genotype imputation strategy achieved superior imputation accuracy across the allele-frequency spectrum and across genomes of diverse ancestry, while delivering at least 4-fold faster inference run time relative to standard imputation tools.

sentences: Memory for stimulus durations is not bound to spatial information Different theories have been proposed to explain how the human brain derives an accurate sense of time. One specific class of theories, intrinsic clock theories, postulate that temporal information of a stimulus is represented much like other features such as color and location, bound together to form a coherent percept. Here we explored to what extent this holds for temporal information after it has been perceived and is held in working memory for subsequent comparison. We recorded EEG of participants who were asked to time stimuli at lateral positions of the screen followed by comparison stimuli presented in the center. Using well-established markers of working memory maintenance, we investigated whether the usage of temporal information evoked neural signatures that were indicative of the location where the stimuli had been presented, both during maintenance and during comparison. Behavior and neural measures including the contralateral delay activity, lateralized alpha suppression and decoding analyses through time all supported the same conclusion: the representation of location was strongly involved during perception of temporal information, but when temporal information was to be used for comparison it no longer showed a relation to spatial information. These results support a model where the initial perception of a stimulus involves intrinsic computations, but that this information is subsequently translated to a stimulus-independent format to be used to further guide behavior.

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The SWI/SNF chromatin remodeling assemblies BAF and PBAF differentially regulate cell cycle exit and cellular invasion in vivo Chromatin remodelers such as the SWI/SNF complex coordinate metazoan development through broad regulation of chromatin accessibility and transcription, ensuring normal cell cycle control and cellular differentiation in a lineage-specific and temporally restricted manner. Mutations in genes encoding the structural subunits of chromatin, such as histone subunits, and chromatin regulating factors (CRFs) are associated with a variety of disease mechanisms including cancer metastasis, in which cancer co-opts cellular invasion programs functioning in healthy cells during development. Here we utilize Caenorhabditis elegans anchor cell (AC) invasion as an in vivo model to identify the suite of chromatin agents and CRFs that promote cellular invasiveness. We demonstrate that the SWI/SNF ATP-dependent chromatin remodeling complex is a critical regulator of AC invasion, with pleiotropic effects on both G0 cell cycle arrest and activation of invasive machinery. Using targeted protein degradation and enhanced RNA interference (RNAi) vectors, we show that SWI/SNF contributes to AC invasion in a dose-dependent fashion, with lower levels of activity in the AC corresponding to aberrant cell cycle entry and increased loss of invasion. Our data specifically implicate the SWI/SNF BAF assembly in the regulation of the G0 cell cycle arrest in the AC, whereas the SWI/SNF PBAF assembly promotes AC invasion via cell cycle-independent mechanisms, including attachment to the basement membrane (BM) and activation of the pro-invasive fos-1/FOS gene. Together these findings demonstrate that the SWI/SNF complex is necessary for two essential components of AC invasion: arresting cell cycle progression and remodeling the BM. The work here provides valuable single-cell mechanistic insight into how the SWI/SNF assemblies differentially contribute to cellular invasion and how SWI/SNF subunit-specific disruptions may contribute to tumorigeneses and cancer metastasis.

SUMMARY STATEMENTCellular invasion through the basement membrane by the C. elegans anchor cell requires both BAF and PBAF SWI/SNF assemblies to arrest the cell cycle and promote the expression of pro-invasive genes.

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Deletion of Rap-Phr systems in Bacillus subtilis influences in vitro biofilm formation and plant root colonization Natural isolates of the soil-dwelling bacterium Bacillus subtilis form robust biofilms under laboratory conditions and colonize plant roots. B. subtilis biofilm gene expression displays phenotypic heterogeneity that is influenced by a family of Rap-Phr regulatory systems. Most Rap-Phr systems in B. subtilis have been studied independently, in different genetic backgrounds and under distinct conditions, hampering true comparison of the Rap-Phr systems impact on bacterial differentiation. Here, we investigated each of the 12 Rap-Phr systems of B. subtilis NCIB 3610 for their effect on biofilm formation. By studying single {Delta}rap-phr mutants, we show that despite redundancy between the cell-cell communication systems, deletion of each of the 12 Rap-Phr systems influences matrix gene expression, which could possibly enable fine-tuning of the timing and level of matrix production in response to specific conditions. Furthermore, some of the {Delta}rap-phr mutants demonstrated altered biofilm formation in vitro and colonization of Arabidopsis thaliana roots, but not necessarily similarly in both processes, indicating that the pathways regulating matrix gene expression and other factors important for biofilm formation may be differently regulated under these distinct conditions.

Significance StatementNatural isolates of Bacillus subtilis form robust biofilms in vitro and on plant roots. The formation of these heterogeneous populations is regulated by diverse Rap-Phr systems. However, most Rap-Phr systems in B. subtilis have been studied independently and in different genetic backgrounds. Here, we report that all 12 Rap-Phr systems affect matrix gene expression, while some of them affect development of in vitro biofilms and plant root colonization. Our study highlights the importance of the Rap-Phr systems in environmental adaptation of B. subtilis, specifically during biofilm formation in the rhizosphere.

sentences: Probabilistic Learning of Treatment Trees in Cancer AO_SCPLOWBSTRACTC_SCPLOWAccurate identification of synergistic treatment combinations and their underlying biological mechnisms is critical across many disease domains, especially cancer. In translational oncology research, preclinical systems such as patient-derived xenografts (PDX) have emerged as a unique study design evaluating multiple treatments administered to samples from the same human tumor implanted into genetically identical mice. In this paper, we propose a novel Bayesian probabilistic tree-based framework for PDX data to investigate the hierarchical relationships between treatments by inferring treatment cluster trees, referred to as treatment trees (Rx-tree). The framework motivates a new metric of mechanistic similarity between two or more treatments accounting for inherent uncertainty in tree estimation; treatments with a high estimated similarity have potentially high mechanistic synergy. Building upon Dirichlet Diffusion Trees, we derive a closed-form marginal likelihood encoding the tree structure, which facilitates computationally efficient posterior inference via a new two-stage algorithm. Simulation studies demonstrate superior performance of the proposed method in recovering the tree structure and treatment similarities. Our analyses of a recently collated PDX dataset produce treatment similarity estimates that show a high degree of concordance with known biological mechanisms across treatments in five different cancers. More importantly, we uncover new and potentially effective combination therapies that confer synergistic regulation of specific downstream biological pathways for future clinical investigations. Our accompanying code, data, and shiny application for visualization of results are available at: https://github.com/bayesrx/RxTree.

sentences: Microstructural development from 9-14 years: evidence from the ABCD Study During late childhood behavioral changes, such as increased risk-taking and emotional reactivity, have been associated with the maturation of cortico-cortico and cortico-subcortical circuits. Understanding microstructural changes in both white matter and subcortical regions may aid our understanding of how individual differences in these behaviors emerge. Restriction spectrum imaging (RSI) is a framework for modelling diffusion-weighted imaging that decomposes the diffusion signal from a voxel into hindered, restricted, and free compartments. This yields greater specificity than conventional methods of characterizing diffusion. Using RSI, we quantified voxelwise restricted diffusion across the brain and measured age associations in a large sample (n=8,086) from the Adolescent Brain and Cognitive Development (ABCD) study aged 9-14 years. Older participants showed a higher restricted signal fraction across the brain, with the largest associations in subcortical regions, particularly the basal ganglia and ventral diencephalon. Importantly, age associations varied with respect to the cytoarchitecture within white matter fiber tracts and subcortical structures, for example age associations differed across thalamic nuclei. This suggests that age-related changes may map onto specific cell populations or circuits and highlights the utility of voxelwise compared to ROI-wise analyses. Future analyses will aim to understand the relevance of this microstructural developmental for behavioral outcomes.

sentences: Immunogenicity of trimetric spike protein associated to Poly(I:C) plus Alum The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid (Poly(I:C)) adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after one or two boosts. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation (spike protein with Alum + Poly(I:C)) in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.

sentences: Coregistration of heading to visual cues in retrosplenial cortex Spatial cognition depends on an accurate representation of orientation within an environment. Head direction cells in distributed brain regions receive a range of sensory inputs, but visual input is particularly important for aligning their responses to environmental landmarks. To investigate how population-level heading responses are aligned to visual input, we recorded from retrosplenial cortex (RSC) of head-fixed mice in a moving environment using two-photon calcium imaging. We show that RSC neurons are tuned to the animals relative orientation in the environment, even in the absence of head movement. Next, we found that RSC receives functionally distinct projections from visual and thalamic areas, and contains several functional classes of neurons. While some functional classes mirror RSC inputs, a newly discovered class coregisters visual and thalamic signals. Finally, decoding analyses reveal unique contributions to heading from each class. Our results suggest an RSC circuit for anchoring heading representations to environmental visual landmarks.

sentences: Altered A-type potassium channel function impairs dendritic spike initiation and temporammonic long-term potentiation in Fragile X syndrome Fragile X syndrome (FXS) is the leading monogenetic cause of cognitive impairment and autism spectrum disorder. Area CA1 of the hippocampus receives current information about the external world from the entorhinal cortex via the temporoammonic (TA) pathway. Given its role in learning and memory, it is surprising that little is known about TA long-term potentiation (TA-LTP) in FXS. We found that TA-LTP was impaired in fmr1 KO mice. Furthermore, dendritic Ca2+ influx was smaller and dendritic spike threshold was depolarized in fmr1 KO mice. Dendritic spike threshold and TA-LTP were restored by block of A-type K+ channels. The impairment of TA-LTP coupled with enhanced Schaffer collateral LTP may contribute to spatial memory alterations in FXS. Furthermore, as both of these LTP phenotypes are attributed to changes in A-type K+ channels in FXS, our findings provide a potential therapeutic target to treat cognitive impairments in FXS.

sentences: Organization and composition of apicomplexan kinetochores reveals plasticity in chromosome segregation across parasite modes of division Kinetochores are multiprotein assemblies directing mitotic spindle attachment and chromosome segregation. In apicomplexan parasites, most known kinetochore components and associated regulators are apparently missing, suggesting a minimal structure with limited control over chromosome segregation. In this study, we use interactomics combined with deep homology searches to identify six divergent eukaryotic components, in addition to a set of eight apicomplexan kinetochore proteins (AKiTs) that bear no detectable sequence similarity to known proteins. The nanoscale organization of the apicomplexan kinetochore includes four subdomains, each displaying different evolutionary rates across the phylum. Functional analyses confirm AKiTs are essential for mitosis and reveal architectures parallel to biorientation at metaphase. Furthermore, we identify a homolog of MAD1 at the apicomplexan kinetochore, suggesting conserved spindle assembly checkpoint signaling. Finally, we show unexpected plasticity in kinetochore composition and segregation throughout the parasite lifecycle, indicating diverse requirements to maintain fidelity of chromosome segregation across apicomplexan modes of division.

sentences: Disentangling the effects of climate change, landscape heterogeneity, and scale on phenological metrics Phenology, the study of the timing of cyclical life history events and seasonal changes, is a fundamental aspect of how individual species, communities, and ecosystems will respond to climate change. Both biotic and abiotic phenological patterns are changing rapidly in response to changing seasonal temperatures and other climate-related drivers, and the consequences of these shifts for individual species and entire ecosystems are largely unknown. Landscape-scale simulations can address some of these needs for better predictions by demonstrating how phenology measures can vary with spatial and temporal grain of observations, and how phenological responses can vary with landscape heterogeneity and climate drivers. To explicitly examine the spatial and temporal scale-dependence of multiple phenology measures, we constructed simulated landscapes populated by virtual plant species with realistic phenologies and environmental sensitivities. This enabled us to examine phenology measures and environmental sensitivities along a continuum of spatial and temporal grains, while also controlling other aspects of sampling design. By relating measures of phenology calculated at a given spatiotemporal grain to average environmental conditions at that same grain size, we are able to determine observed environmental sensitivities for multiple phenological metrics at that spatial and temporal scale. We demonstrate that different phenological events change distinctly and predictably with spatial and temporal measurement scale, opening the way to incorporating scaling laws into predictions. Using plant flowering as our example, we identify that the timing of the beginnings or ends of an event (e.g., First Flower date, Last Flower date), can be especially sensitive to the spatial and temporal grain (or resolution) of observations. Our work provides an initial assessment of the role of observation scale in landscape phenology, and a general approach for incorporating scale-dependence into predictions of a variety of phenological time series.

sentences: Geometrical Study of Virus RNA Sequences In this contribution, some applications of the earlier developed fast algorithm of calculating coordinates of single nucleotides and RNA fragments are considered to create multi-scale geometrical models of RNAs and their mutations. The algorithm allows to plot single nucleotides and RNAs fragments on one figure and to track the RNA mutations of any level visually and numerically using interpolation formulas and point-to-point estimates of coordinates of ATG starting triplets and single nucleotides. The performed study of many samples of SARS CoV-2 viruses shows perturbations of ATG starting triplet coordinates in the vicinity of orf1ab gene end only.

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Enhanced FcγRIII/CD16 activation by discrete ligands as independent correlates of disease severity in COVID-19 patients A dysregulated immune response with high levels of SARS-CoV-2 specific IgG antibodies characterizes patients with severe or critical COVID-19. Although a robust IgG response is traditionally considered to be protective, excessive triggering of activating Fc-gamma-receptors (Fc{gamma}Rs) could be detrimental and cause immunopathology. Here, we document that patients who develop soluble circulating IgG immune complexes (sICs) during infection are subject to enhanced immunopathology driven by Fc{gamma}R activation. Utilizing cell-based reporter systems we provide evidence that sICs are predominantly formed prior to a specific humoral response against SARS-CoV-2. sIC formation, together with increased afucosylation of SARS-CoV-2 specific IgG eventually leads to an enhanced CD16 (Fc{gamma}RIII) activation of immune cells reaching activation levels comparable active systemic lupus erythematosus (SLE) disease. Our data suggest a vicious cycle of escalating immunopathology driven by an early formation of sICs in predisposed patients. These findings reconcile the seemingly paradoxical findings of high antiviral IgG responses and systemic immune dysregulation in severe COVID-19.

Clinical implicationsThe identification of sICs as drivers of an escalating immunopathology in predisposed patients opens new avenues regarding intervention strategies to alleviate critical COVID-19 progression.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/449893v4_ufig1.gif" ALT="Figure 1">
View larger version (17K):
org.highwire.dtl.DTLVardef@b4616corg.highwire.dtl.DTLVardef@682d1aorg.highwire.dtl.DTLVardef@16946cborg.highwire.dtl.DTLVardef@a6ef7d_HPS_FORMAT_FIGEXP  M_FIG C_FIG A vicious cycle of immunopathology in COVID-19 patients is driven by soluble multimeric immune complexes (sICs). SARS-CoV-2 infection triggers sIC formation in prone individuals. Activation of Fc{gamma}RIII/CD16 expressing immune cells by sICs precedes a humoral response to SARS-CoV2 infection. sICs and infection add to IgG afucosylation, further enhancing Fc{gamma}RIII/CD16 activation by opsonized targets. High inflammation induces further sIC mediated immune cell activation ultimately leading to an escalating immunopathology.

sentences: Neural correlates of audio-visual integration of socially meaningful information in macaque monkeys Social interactions rely on the ability to interpret semantic and emotional information, often from multiple sensory modalities. In human and nonhuman primates, both the auditory and visual modalities are used to generate and interpret communicative signals. In individuals with autism, not only are there deficits in social communication, but in the integration of audio-visual information. At present, we know little about the neural mechanisms that subserve the interpretation of complex social events, including the audio-visual integration that is often required with accompanying communicative signals. Based on heart rate estimates and fMRI in two macaque monkeys (Macaca mulatta), we show that individuals systematically associate affiliative facial expressions or social scenes with corresponding affiliative vocalizations, aggressive facial expressions or social scenes with corresponding aggressive vocalizations and escape visual scenes with scream vocalizations. In contrast, vocalizations that are incompatible with the visual information are fully suppressed, suggesting top-down regulation over the processing of sensory input. The process of binding audio-visual semantic and contextual information relies on a core functional network involving the superior temporal sulcus (STS) and lateral sulcus (LS). Peak activations in both sulci co-localize with face or voice patches that have been previously described. While all of these regions of interest (ROIs) respond to both auditory and visual information, LS ROIs have a preference for auditory and audio-visual congruent stimuli while STS ROIs equally respond to auditory, visual and audio-visual congruent stimuli. To further specify the cortical network involved in the control of this semantic association, we performed a whole brain gPPI functional connectivity analysis on the LS and STS cumulated ROIs. This gPPI analysis highlights a functional network connected to the LS and STS, involving the anterior cingulate cortex (ACC), area 46 in the dorsolateral prefrontal cortex (DLPFC), the orbitofrontal cortex (OFC), the intraparietal sulcus (IPS), the insular cortex and subcortically, the amygdala and the hippocampus. Comparing human and macaque results, we propose that the integration of audio-visual information for congruent, meaningful social events involves homologous neural circuitry, specifically, an emotional network composed of the STS, LS, ACC, OFC, and limbic areas, including the amygdala, and an attentional network including the STS, LS, IPS and DLPFC. As such, these networks are critical to the amodal representation of social meaning, thereby providing an explanation for some of deficits observed in autism.

sentences: Genome-wide CRISPR guide RNA design and specificity analysis with GuideScan2 We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly gRNA/library design in custom genomes. GuideScan2 analysis identified widespread confounding effects of low-specificity gRNAs in published CRISPR knockout, interference and activation screens and enabled construction of a ready-to-use gRNA library that reduced off-target effects in a novel gene essentiality screen. GuideScan2 also enabled the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome.

sentences: Gluconobacter oxydans Knockout Collection Finds Improved Rare Earth Element Extraction Rare earth elements (REE) are critical components of our technological society and essential for renewable energy technologies. Traditional thermochemical processes to extract REE from mineral ores or recycled materials are costly and environmentally harmful1, and thus more sustainable extraction methods require exploration. Bioleaching offers a promising alternative to conventional REE extraction2-4, and is already used to extract 5% of the worlds gold, and {approx} 15% of the worlds copper supply5,6. However, the performance of REE bioleaching lags far behind thermochemical processes2,7-9. Despite this, to the best of our knowledge no genetic engineering strategies have yet been used to enhance REE bioleaching, and little is known of the genetics that confer this capability. Here we build a whole genome knockout collection for Gluconobacter oxydans B58, one of the most promising organisms for REE bioleaching10, and use it to comprehensively characterize the genomics of REE bioleaching. In total, we find 304 genes that notably alter production of G. oxydans acidic biolixiviant, including 165 that hold up under statistical comparison with wild-type. The two most impactful groups of genes involved in REE bioleaching have opposing influences on acid production and REE bioleaching. Disruption of genes underlying synthesis of the cofactor pyrroloquinoline quinone (PQQ) and the PQQ-dependent membrane-bound glucose dehydrogenase all but eliminates bioleaching. In contrast, disruption of the phosphate-specific transport system accelerates acid production and enhances bioleaching. We identified 6 disruption mutants, that increase bioleaching by at least 11%. Most significantly, disruption of pstC, encoding part of the phosphate-specific transporter, pstSCAB, enhances bioleaching by 18%. Taken together, these results give a comprehensive roadmap for engineering multiple sites in the genome of G. oxydans to further increase its bioleaching efficiency.

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Network topology and evolution of the gene co-expression of T-cells during immuno-senescence To better understand the potential impact of the gene expression network structure on the dynamics of immune-senescence and defects of cell functions during aging, we investigated network structures in both young and old individuals. We analyzed the gene co-expression networks (GCNs) derived from an aging signature of 130 immune-related genes obtained from CD3+ T-cell splenocytes extracted from FVB/N, C57BL/6N, and BALB/c mice at ages 2 and 22- 24 months. The network structure for the two different mouse age-groups was derived and subsequently analyzed. Analysis of network hubs using clustering coefficients, degree, betweenness, eigenvector, and closeness centralities, as well as local, indirect, and total influence measures, demonstrated changes in gene behavior and network control between the two age groups. Our quantification shows that the young, 2-month old mouse network is more organized than the 22-24-month, old mouse network, while the network structure of the older mouse GCN appears to be far more complicated but far more dispersed. Changes in network structure between the old and young mice suggest deterioration in transcription regulation with age in peripheral T- cells, particularly within the TCR signaling pathway, and potential compensatory mechanisms in older T-cells to overcome loss to regular function resulting from transcriptional irregularity. These results demonstrate the need for more research into gene co-expression in peripheral T-cells in order to better understand both network irregularities and the phenotypic dysfunction observed in older individuals.

Author SummaryIn order to better understand the potential mechanisms of transcriptional irregularities in the immune system with aging, we analyzed the structure of gene co-expression networks of T-cells extracted from the spleens of 2 and 22-24-month old mice. Gene co-expression describes the correlation relationship between two expressed genes; as the expression of one gene goes up, the expression of another gene might also increase (or, conversely, decrease). Strong gene co- expression relationships can signal the existence of a number of important biological phenomena, such as two genes belonging to a transcription pathway or protein structure. Network diagrams visualizing these co-expression relationships in both younger and older mice demonstrated the existence of differences in network structure and properties that may be attributed to the aging of the immune system. Network mathematical methods were used to examine the complexity of each network. We found that the younger mouse network was more organized than the older mouse network. The older mouse group exhibited a 255% increase in co-expression relationships but a decrease of 92% of the connections from the young mouse network. This suggests the older mouse T-cells suffer dysfunction at a transcriptional level. This results in the loss of regular immune and cellular functions. These results demonstrate the importance of future research into gene co-expression to decipher senescence or diseases that perturb gene expression through time.

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High-throughput miRNA-sequencing of the human placenta: expression throughout gestation BackgroundAltered placenta miRNA abundance may impact the maternal-fetal interface and pregnancy outcomes. Understanding miRNA changes across gestation is essential before miRNAs can be used as biomarkers or prognostic indicators during pregnancy.

Materials & MethodsUsing next-generation sequencing, we characterize the normative human placenta miRNA transcriptome in first (N=113) and third trimester (N=47).

ResultsThere are 801 miRNAs expressed in both first and third trimester, including 182 with similar expression across gestation (P[≥]0.05) and 182 significantly different (FDR<0.05). Of placenta-specific miRNA clusters, C14MC is more upregulated in first trimester and C19MC is more highly expressed overall.

ConclusionThis work provides a rich atlas of healthy pregnancies to direct functional studies investigating the epigenetic differences in first and third trimester placentae.

Lay AbstractThe human body produces microRNAs which affect the expression of genes and proteins. This study uses next generation sequencing to identify the microRNA profile of first and third trimester human placentae using a large cohort (N=113 first, N=47 third trimester). All pregnancies resulted in healthy babies. We identify microRNAs with significantly different expression between first and third trimester, as well as stably expressed microRNAs. This work provides a baseline for future studies which may use microRNAs to monitor maternal-fetal health throughout pregnancy.

sentences: A population of neurons that produce hugin and express the diuretic hormone 44 receptor gene projects to the corpora allata in Drosophila melanogaster The corpora allata (CA) are essential endocrine organs that biosynthesize and secrete the sesquiterpenoid hormone, namely juvenile hormone (JH), to regulate a wide variety of developmental and physiological events in insects. Previous studies had demonstrated that the CA are directly innervated with neurons in many insect species, implying the innervations to be important for regulating JH biosynthesis in response to internal physiology and external environments. While this is also true for the model organism, Drosophila melanogaster, which neurotransmitters are produced in the CA-projecting neurons are yet to be clarified. In this study on D. melanogaster, we aimed to demonstrate that a subset of neurons producing the neuropeptide hugin, the invertebrate counterpart of the vertebrate neuromedin U, directly projects to the adult CA. A synaptic vesicle marker in the hugin neurons was observed at their axon termini located on the CA, which were immunolabeled with a newly-generated antibody to the JH biosynthesis enzyme JH acid O-methyltransferase (JHAMT). We also found the CA-projecting hugin neurons to likely express a gene encoding the specific receptor for diuretic hormone 44 (Dh44). Moreover, our data suggested that the CA-projecting hugin neurons have synaptic connections with the upstream neurons producing Dh44. To the best of our knowledge, this is the first study to identify a specific neurotransmitter of the CA-projecting neurons in D. melanogaster, and to anatomically characterize a neuronal pathway of the CA-projecting neurons and their upstream neurons.

sentences: Locus-specific chromatin profiling of evolutionarily young transposable elements Despite a vast expansion in the availability of epigenomic data, our knowledge of the chromatin landscape at interspersed repeats remains highly limited by difficulties in mapping short-read sequencing data to these regions. In particular, little is known about the locus-specific regulation of evolutionarily young transposable elements (TEs), which have been implicated in genome stability, gene regulation and innate immunity in a variety of developmental and disease contexts. Here we propose an approach for generating locus-specific protein-DNA binding profiles at interspersed repeats, which leverages information on the spatial proximity between repetitive and non-repetitive genomic regions. We demonstrate that the combination of HiChIP and a newly developed mapping tool (PAtChER) yields accurate protein enrichment profiles at individual repetitive loci. Using this approach, we reveal previously unappreciated variation in the epigenetic profiles of young TE loci in mouse and human cells. Insights gained using our method will be invaluable for dissecting the molecular determinants of TE regulation and their impact on the genome.

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Transcriptomic response to ISAV infection in the gills, head kidney and spleen of resistant and susceptible Atlantic salmon BackgroundInfectious Salmonid Anaemia virus (ISAV) is an orthomyxovirus responsible of large losses in Atlantic salmon (Salmo salar) aquaculture. Current available treatments and vaccines are not fully effective, and therefore selective breeding to produce ISAV-resistant strains of Atlantic salmon (Salmo salar) is a high priority for the industry. Genomic selection and potentially genome editing can be applied to enhance the disease resistance of aquaculture stocks, and both approaches can benefit from increased knowledge on the genomic mechanisms of resistance to ISAV. To improve our understanding of the mechanisms underlying resistance to ISAV in Atlantic salmon we performed a transcriptomic study in ISAV-infected salmon with contrasting levels of resistance to this virus.

ResultsThree different tissues (gills, head kidney and spleen) were collected on 12 resistant and 12 susceptible fish at three timepoints (pre-challenge, 7 and 14 days post infection) and RNA sequenced. The transcriptomes of Infected and non-infected fish and of resistant and susceptible fish were compared at each timepoint. The results show that the responses to ISAV are organ-specific; an important response to the infection was observed in the head kidney, with up-regulation of immune processes such as interferon and NLR pathways, while in gills and spleen the response was more moderate. In addition to immune related genes our results suggest that other processes such as ubiquitination or ribosomal processing are important during early infection to ISAV. Moreover, the comparison between resistant and susceptible have also highlighted some interesting genes related to ubiquitination, intracellular transport or the inflammasome.

ConclusionsAtlantic salmon infection by ISAV revealed an organ-specific response, implying differential function during the infection. An early immune response was observed in the head kidney, while gills and spleen showed modest responses in comparison. Comparison between resistance and susceptible samples have highlighted genes of interest for further studies, for instance those related to ubiquitination or the inflammasome.

sentences: The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice lightsheet microscopy Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

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Microbial associations with Microscopic Colitis BACKGROUND AND OBJECTIVEMicroscopic colitis is a relatively common cause of chronic diarrhea and may be linked to luminal factors. Given the essential role of the microbiome in human gut health, analysis of microbiome changes associated with microscopic colitis could provide insights into the development of the disease.

METHODSWe enrolled patients who underwent colonoscopy for diarrhea. An experienced pathologist classified patients as having microscopic colitis (n=52) or controls (n=153). Research biopsies were taken from the ascending and descending colon, and the microbiome was characterized with Illumina sequencing. We analyzed the associations between microscopic colitis and microbiome with a series of increasingly complex models adjusted for a range of demographic and health factors.

RESULTSWe found that alpha-diversity was significantly lower in microscopic colitis cases compared to controls in the descending colon microbiome. In the descending colon, a series of models that adjusted for an increasing number of co-variates found taxa significantly associated with microscopic colitis, including Proteobacteria that was enriched in cases and Collinsella enriched in controls. While the alpha-diversity and taxa were not significantly associated with microscopic colitis in the ascending colon microbiome, the inference p-values based on ascending and descending microbiomes were highly correlated.

CONCLUSIONOur study demonstrates an altered microbiome in microscopic colitis cases compared to controls. Because both the cases and controls had diarrhea, we have identified candidate taxa that could be mechanistically responsible for the development of microscopic colitis independent of changes to the microbial community caused by diarrhea.

Significance of this studyO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIMicroscopic colitis is a common cause of chronic diarrhea. The exact etiology of microscopic colitis is unknown, but the gut microbiome is considered to play an important role.
C_LIO_LIPrior studies have reported microbial changes associated with microscopic colitis, but the results are not consistent. Prior studies have generally involved small numbers of patients, fecal samples, and comparison to healthy controls.
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What are the new findings?O_LIThe microbiome was altered in microscopic colitis patients.
C_LIO_LIMicroscopic colitis was associated with lower alpha-diversity, increase of inflammation related taxa and decrease of Collinsella in the descending colon microbiome independent of diarrhea status.
C_LIO_LIThe changes of microbial features were consistent between ascending and descending colon microbiomes, but with smaller changes in the ascending colon microbiome.
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How might it impact on clinical practice in the foreseeable future?O_LIThe altered microbial communities in patients with microscopic colitis suggest that the patients might benefit from prevention or treatment with live biotherapeutic products.
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A constitutively expressed fluorescence ubiquitin cell cycle indicator (FUCCI) in axolotls for studying tissue regeneration Regulation of cell cycle progression is essential for cell proliferation during regeneration following injury. After appendage amputation, the axolotl (Ambystoma mexicanum) regenerates missing structures through an accumulation of proliferating cells known as the blastema. To study cell division during blastema growth, we generated a transgenic line of axolotls that ubiquitously expresses a bicistronic version of the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI). We demonstrate near-ubiquitous expression of FUCCI expression in developing and adult tissues and validate these expression patterns with DNA synthesis and mitosis phase markers. We demonstrate the utility of FUCCI for live and whole-mount imaging, showing the predominantly local contribution of cells during limb and tail regeneration. We also show that spinal cord amputation results in increased proliferation at least 5 mm from the injury. Finally, we use multimodal staining to provide cell type information for cycling cells by combining fluorescence in-situ hybridization, EdU click-chemistry, and immunohistochemistry on a single FUCCI tissue section. This new line of animals will be useful for studying cell cycle dynamics using in-situ endpoint assays and in-vivo imaging in developing and regenerating animals.

Summary statementWe generated a ubiquitous transgenic fluorescence ubiquitin cell cycle indicator (FUCCI) axolotl line for examination of cell cycle dynamics during tissue regeneration.

sentences: Uncovering novel mutational signatures by de novo extraction with SigProfilerExtractor Mutational signature analysis is commonly performed in genomic studies surveying cancer and normal somatic tissues. Here we present SigProfilerExtractor, an automated tool for accurate de novo extraction of mutational signatures for all types of somatic mutations. Benchmarking with a total of 34 distinct scenarios encompassing 2,500 simulated signatures operative in more than 60,000 unique synthetic genomes and 20,000 synthetic exomes demonstrates that SigProfilerExtractor outperforms thirteen other tools across all datasets with and without noise. For genome simulations with 5% noise, reflecting high-quality genomic datasets, SigProfilerExtractor outperforms other approaches by elucidating between 20% and 50% more true positive signatures while yielding more than 5-fold less false positive signatures. Applying SigProfilerExtractor to 4,643 whole-genome sequenced and 19,184 whole-exome sequenced cancers reveals four previously missed mutational signatures. Two of the signatures are confirmed in independent cohorts with one of these signatures associating with tobacco smoking. In summary, this report provides a reference tool for analysis of mutational signatures, a comprehensive benchmarking of bioinformatics tools for extracting mutational signatures, and several novel mutational signatures including a signature putatively attributed to direct tobacco smoking mutagenesis in bladder cancer and in normal bladder epithelium.

sentences: CRISPR/Cas9 gene editing uncovers the role of CTR1 and ROS1 in melon fruit ripening and epigenetic regulation Melon (Cucumis melo L.) has emerged as an alternative model to study fruit ripening due to the coexistence of climacteric and non-climacteric varieties. The previous characterization of a major QTL ETHQV8.1 sufficient to trigger climacteric ripening in a non-climacteric background allowed the identification within the QTL interval of a negative regulator of ripening CmCTR1-like (MELO3C024518), and a putative DNA demethylase CmROS1 (MELO3C024516), the orthologue of DML2, a DNA demethylase regulating fruit ripening in tomato. To understand the role of these genes in climacteric ripening, we generated homozygous CRISPR knockout mutants of CmCTR1-like and CmROS1 in a climacteric genetic background. The climacteric behavior was altered in both loss-of-function mutants in two summer seasons with an advanced ethylene production profile compared to the climacteric wild type, suggesting a role of both genes in climacteric ripening in melon. Single cytosine methylome analyses of the CmROS1 knockout mutant revealed DNA methylation changes in the promoter regions of key ripening genes as ACS1, ETR1 and ACO1, and ripening associated-transcription factors as NAC-NOR, RIN and CNR, suggesting the importance of CmROS1-mediated DNA demethylation for triggering fruit ripening in melon.

sentences: Isolation and characterization of a highly specific monoclonal antibody targeting the botulinum neurotoxin type E exposed SNAP-25 neoepitope Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. Specific detection and quantification of BoNT/E-cleaved SNAP-25 neoepitope is essential for diagnosis of BoNT/E intoxication as well as for characterization of anti-BoNT/E antibody preparations. In order to isolate highly specific monoclonal antibodies suitable for in vitro immuno-detection of the exposed neoepitope, mice and rabbits were immunized with an eight amino acid peptide composed of the C-terminus of the cleaved SNAP-25. Immunized rabbits developed a specific and robust polyclonal antibody response, whereas immunized mice mostly demonstrated a weak antibody response that could not discriminate between the two forms of SNAP-25. An immune scFv phage-display library was constructed from the immunized rabbits and a panel of antibodies was isolated. Sequence alignment of the isolated clones revealed high similarity between both heavy and light chains, with exceptionally short HCDR3 sequences. A chimeric scFv-Fc antibody was further expressed and characterized, exhibiting a selective, ultra-high affinity (pM) towards the SNAP-25 neoepitope. Moreover, this antibody enabled sensitive detection of the cleaved SNAP-25 in BoNT/E treated SiMa cells with no cross reactivity with the intact SNAP-25. This novel antibody can be further used to develop an in vitro cell-based assay to diagnose BoNT/E intoxication and to characterize antitoxin preparations, thus eliminating the use of animals in the standard mouse bioassay.

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Glutamine is essential for overcoming theimmunosuppressive microenvironment in malignant salivary gland tumors RationaleImmunosuppression in the tumor microenvironment (TME) is key to the pathogenesis of solid tumors. Tumor cell-intrinsic autophagy is critical for sustaining both tumor cell metabolism and survival. However, the role of autophagy in the host immune system that allows cancer cells to escape immune destruction remains poorly understood. Here, we determined if attenuated host autophagy is sufficient to induce tumor rejection through reinforced adaptive immunity. Furthermore, we determined whether dietary glutamine supplementation, mimicking attenuated host autophagy, is capable of promoting antitumor immunity.

MethodsA syngeneic orthotopic tumor model in Atg5+/+ and Atg5flox/flox mice was established to determine the impact of host autophagy on the antitumor effects against mouse malignant salivary gland tumors (MSTs). Multiple cohorts of immunocompetent mice were used for oncoimmunology studies, including inflammatory cytokine levels, macrophage, CD4+, and CD8+ cells tumor infiltration at 14 days and 28 days after MST inoculation. In vitro differentiation and in vivo dietary glutamine supplementation were used to assess the effects of glutamine on Treg differentiation and tumor expansion.

ResultsWe showed that mice deficient in the essential autophagy gene, Atg5, rejected orthotopic allografts of isogenic MST cells. An enhanced antitumor immune response evidenced by reduction of both M1 and M2 macrophages, increased infiltration of CD8+ T cells, elevated IFN-{gamma} production, as well as decreased inhibitory Tregs within TME and spleens of tumor-bearing Atg5flox/flox mice. Mechanistically, ATG5 deficiency increased glutamine level in tumors. We further demonstrated that dietary glutamine supplementation partially increased glutamine levels and restored potent antitumor responses in Atg5+/+ mice.

ConclusionsDietary glutamine supplementation exposes a previously undefined difference in plasticity between cancer cells, cytotoxic CD8+ T cells and Tregs.

sentences: Non-alcoholic components of Pelinkovac, a Croatian wormwood-based strong liquor, counteract the inhibitory effect of high ethanol concentration on catalase in vitro Antioxidant enzyme catalase protects the cells against alcohol-induced oxidative stress by scavenging free radicals and metabolizing alcohol. Concentrations of ethanol present in alcoholic beverages can inhibit catalase and foster oxidative stress and alcohol-induced injury. Non-alcoholic components of pelinkovac counteract the inhibitory effects of high ethanol concentration and acidic pH on catalase in vitro.

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Structured decomposition improves systems serology prediction and interpretation Systems serology measurements provide a comprehensive view of humoral immunity by profiling both the antigen-binding and Fc properties of antibodies. Identifying patterns in these measurements will help to guide vaccine and therapeutic antibody development, and improve our understanding of disorders. Furthermore, consistent patterns across diseases may reflect conserved regulatory mechanisms; recognizing these may help to combine modalities such as vaccines, antibody-based interventions, and other immunotherapies to maximize protection. A common feature of systems serology studies is structured biophysical profiling across disease-relevant antigen targets, properties of antibodies interaction with the immune system, and serological samples. These are typically produced alongside additional measurements that are not antigen-specific. Here, we report a new form of tensor factorization, total tensor-matrix factorization (TMTF), which can greatly reduce these data into consistently observed patterns by recognizing the structure of these data. We use a previous study of HIV-infected subjects as an example. TMTF outperforms standard methods like principal components analysis in the extent of reduction possible. Data reduction, in turn, improves the prediction of immune functional responses, classification of subjects based on their HIV control status, and interpretation of these resulting models. Interpretability is improved specifically by applying further data reduction, separation of the Fc from antigen-binding effects, and recognizing consistent patterns across individual measurements. Therefore, we propose that TMTF will be an effective general strategy for exploring and using systems serology.

Summary pointsO_LIStructured decomposition provides substantial data reduction without loss of information.
C_LIO_LIPredictions based on decomposed factors are accurate and robust to missing measurements.
C_LIO_LIDecomposition structure improves the interpretability of modeling results.
C_LIO_LIDecomposed factors represent meaningful patterns in the HIV humoral response.
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sentences: BayesRB: a Markov chain Monte Carlo-based polygenic genetic risk score algorithm for dichotomous traits AO_SCPLOWBSTRACTC_SCPLOWIdentifying high-risk individuals with diseases through reliable prediction models guides screening and preventive treatment. Most complex diseases have a genetic basis influenced by multiple genes and so disease risk can be estimated using polygenic risk score (PRS) algorithms. Many PRS algorithms have been developed so far. Among them, BayesR shows good characteristics of unbiasedness, accuracy, sparseness, and robustness. It detects the associated SNPs, estimates the SNP effects, and makes prediction of disease risks based on all SNPs simultaneously. However, this method assumes that the phenotypes follow a Gaussian distribution, which cannot be met in case-control studies. Here, we made an extension of the BayesR method, called BayesRB, by adding auxiliary variables to the BayesR model. We explored the characteristics, efficacy, and accuracy of BayesRB when estimating SNP effects and predicting disease risks compared with three traditional algorithms under different conditions using both simulated data and real data from the Welcome Trust Case Control Consortium (WTCCC). For SNP effect estimation, BayesRB shows unbiasedness and sparseness for big and small effect SNPs, respectively. For disease risk prediction, BayesRB had the best performance among the methods. This study provides a theoretical basis for complex disease risk prediction and disease prevention

sentences: The Enemy of my Enemy is my Friend: Immune-Mediated Facilitation Contributes to Fitness of Co-Infecting Helminths Our conceptual understanding of immune-mediated interactions between parasites is rooted in the theory of community ecology. One of the limitations of this approach is that most of the theory and empirical evidence has focused on resource or immune-mediated parasite competition and yet, there is ample evidence of positive interactions between species that could be generated by immune-mediated facilitation. Here, we develop an immuno-epidemiological framework and apply it to longitudinal infection data of two gastrointestinal helminths that infect a population of free-living rabbits to investigate, through model testing, the mechanisms of immune-mediated facilitation in dual infections. Simulations show that weakened, species-specific IgA antibody responses and unequal, albeit low, IgA cross-reactions explain higher parasite intensities in dual compared to single infections, for both helminths. Simulations also show that rabbits with dual infections shed more free-living stages that survive fort longer in the environment, implying greater onward transmission than hosts with single infections. These findings support the hypothesis that the two helminths interact through immune-mediated facilitation which contributes to greater fitness and the long-term co-circulation of both species in the host population.

sentences: Profiling Transcription Initiation in Peripheral Leukocytes Reveals Severity-Associated Cis-Regulatory Elements in Critical COVID-19 The contribution of transcription factors (TFs) and gene regulatory programs in the immune response to COVID-19 and their relationship to disease outcome is not fully understood. Analysis of genome-wide changes in transcription at both promoter-proximal and distal cis-regulatory DNA elements, collectively termed the active cistrome, offers an unbiased assessment of TF activity identifying key pathways regulated in homeostasis or disease. Here, we profiled the active cistrome from peripheral leukocytes of critically ill COVID-19 patients to identify major regulatory programs and their dynamics during SARS-CoV-2 associated acute respiratory distress syndrome (ARDS). We identified TF motifs that track the severity of COVID- 19 lung injury, disease resolution, and outcome. We used unbiased clustering to reveal distinct cistrome subsets delineating the regulation of pathways, cell types, and the combinatorial activity of TFs. We found critical roles for regulatory networks driven by stimulus and lineage determining TFs, showing that STAT and E2F/MYB regulatory programs targeting myeloid cells are activated in patients with poor disease outcomes and associated with single nucleotide genetic variants implicated in COVID-19 susceptibility. Integration with single-cell RNA-seq found that STAT and E2F/MYB activation converged in specific neutrophils subset found in patients with severe disease. Collectively we demonstrate that cistrome analysis facilitates insight into disease mechanisms and provides an unbiased approach to evaluate global changes in transcription factor activity and stratify patient disease severity.

sentences: Drug development of an affinity enhanced, broadly neutralizing heavy chain only antibody that restricts SARS-CoV-2 in hamsters We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and -2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and -2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homogeneity and silenced effector functions for enhanced safety, and its stability as well as lack of off-target binding was extensively characterized. Therapeutic systemic administration of a low dose of VHH72_S56A-Fc antibodies strongly restricted replication of both original and D614G mutant variants of SARS-CoV-2 virus in hamsters, and minimized the development of lung damage. This work led to the selection of XVR011 for clinical development, a highly stable anti-COVID-19 biologic with excellent manufacturability. Additionally, we show that XVR011 is unaffected in its neutralizing capacity of currently rapidly spreading SARS-CoV-2 variants, and demonstrate its unique, wide scope of binding across the Sarbecovirus clades.

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Prime-pull immunization of mice with a BcfA-adjuvanted vaccine elicits mucosal immunity and prevents SARS CoV-2 infection and pathology Vaccines against SARS-CoV-2 that induce mucosal immunity capable of preventing infection and disease remain urgently needed. We show that intramuscular priming of mice with an alum and BcfA-adjuvanted Spike subunit vaccine, followed by a BcfA-adjuvanted mucosal booster, generated Th17 polarized tissue resident CD4+ T cells, and mucosal and serum antibodies. The serum antibodies efficiently neutralized SARS-CoV-2 and its Delta variant, suggesting cross-protection against a recent variant of concern (VOC). Immunization with this heterologous vaccine prevented weight loss following challenge with mouse-adapted SARS-CoV-2 and reduced viral replication in the nose and lungs. Histopathology showed a strong leukocyte and polymorphonuclear (PMN) cell infiltrate without epithelial damage in mice immunized with BcfA-containing vaccines. In contrast, viral load was not reduced in the upper respiratory tract of IL-17 knockout mice immunized with the same formulation, suggesting that the Th17 polarized T cell responses are critical for protection. We show that vaccines adjuvanted with alum and BcfA, delivered through a heterologous prime-pull regimen, protect against SARS-CoV-2 infection without causing enhanced respiratory disease.

SIGNIFICANCEThere remains a need for SARS CoV-2 booster vaccines that generate mucosal immunity and prevent transmission. We show that systemic priming followed by a mucosal booster with a BcfA-adjuvanted subunit vaccine generates neutralizing antibodies and Th17 polarized systemic and tissue-resident immune responses that provide sterilizing immunity against wildtype SARS CoV-2, and a variant of concern. Importantly, in contrast to alum alone, the addition of BcfA prevents respiratory pathology. These results suggest that a BcfA-adjuvanted mucosal booster may elicit mucosal immunity in individuals previously immunized systemically with approved vaccines. This foundational study in mice sets the stage for testing our vaccine regimen in larger animal models as a booster vaccine.

sentences: Co-translational assembly counteracts promiscuous interactions During the co-translational assembly of protein complexes, a fully synthesized subunit engages with the nascent chain of a newly synthesized interaction partner. Such events are thought to contribute to productive assembly, but their exact physiological relevance remains underexplored. Here, we examined structural motifs contained in nucleoporins for their potential to facilitate co-translational assembly. We experimentally tested candidate structural motifs and identified several previously unknown co-translational interactions. We demonstrate by selective ribosome profiling that domain invasion motifs of beta-propellers, coiled-coils, and short linear motifs act as co-translational assembly domains. Such motifs are often contained in proteins that are members of multiple complexes (moonlighters) and engage with closely related paralogs. Surprisingly, moonlighters and paralogs assembled co-translationally in only one but not all of the relevant assembly pathways. Our results highlight the regulatory complexity of assembly pathways.

sentences: Triple-helix potential of the mouse genome Certain DNA sequences, including mirror-symmetric polypyrimidine/polypurine runs, are capable of folding into a triple-helix-containing non-B-form DNA structure called H-DNA. Such H-DNA-forming sequences are frequent in many eukaryotic genomes, including in mammals, and multiple lines of evidence indicate that these motifs are mutagenic and can impinge on DNA replication, transcription, and other aspects of genome function. In this study, we show that the triplex-forming potential of H-DNA motifs in the mouse genome can be evaluated using S1-sequencing (S1-seq), which uses the single-stranded DNA (ssDNA)-specific nuclease S1 to generate deep-sequencing libraries that report on the position of ssDNA throughout the genome. When S1-seq was applied to genomic DNA isolated from mouse testis cells and splenic B cells, we observed prominent clusters of S1-seq reads that appeared to be independent of endogenous double-strand breaks, that coincided with H-DNA motifs, and that correlated strongly with the triplex-forming potential of the motifs. Fine-scale patterns of S1-seq reads, including a pronounced strand asymmetry in favor of centrally-positioned reads on the pyrimidine-containing strand, suggested that this S1-seq signal is specific for one of the four possible isomers of H-DNA (H-y5). By leveraging the abundance and complexity of naturally occurring H-DNA motifs across the mouse genome, we further defined how polypyrimidine repeat length and the presence of repeat-interrupting substitutions modify the structure of H-DNA. This study provides a new approach for studying DNA secondary structure genome wide at high spatial resolution.

sentences: A catalogue of omics biological ageing clocks reveals substantial commonality and associations with disease risk Biological age (BA), a measure of functional capacity and prognostic of health outcomes that discriminates between individuals of the same chronological age (chronAge), has been estimated using a variety of biomarkers. Previous comparative studies have mainly used epigenetic models (clocks), we use ~1000 participants to create eleven omics ageing clocks, with correlations of 0.45-0.97 with chronAge, even with substantial sub-setting of biomarkers. These clocks track common aspects of ageing with 94% of the variance in chronAge being shared among clocks. The difference between BA and chronAge - omics clock age acceleration (OCAA) - often associates with health measures. One years OCAA typically has the same effect on risk factors/10-year disease incidence as 0.46/0.45 years of chronAge. Epigenetic and IgG glycomics clocks appeared to track generalised ageing while others capture specific risks. We conclude BA is measurable and prognostic and that future work should prioritise health outcomes over chronAge.

sentences: The CDK8 inhibitor DCA promotes a tolerogenic chemical immunophenotype in CD4+ T cells via a novel CDK8-GATA3-FOXP3 pathway Immune health requires innate and adaptive immune cells to engage precisely balanced pro- and anti-inflammatory forces. We employ the concept of chemical immunophenotypes to classify small molecules functionally or mechanistically according to their patterns of effects on primary innate and adaptive immune cells. The high-specificity, low-toxicity cyclin dependent kinase 8 (CDK8) inhibitor DCA exerts a distinct tolerogenic profile in both innate and adaptive immune cells. DCA promotes Treg and Th2 differentiation, while inhibiting Th1 and Th17 differentiation, in both murine and human cells. This unique chemical immunophenotype led to mechanistic studies showing that DCA promotes Treg differentiation in part by regulating a previously undescribed CDK8-GATA3-FOXP3 pathway that regulates early pathways of Foxp3 expression. These results highlight previously unappreciated links between Treg and Th2 differentiation and extend our understanding of the transcription factors that regulate Treg differentiation and their temporal sequencing. These findings have significant implications for future mechanistic and translational studies of CDK8 and CDK8 inhibitors.

sentences: iGEM comes of age: trends in its research output. The international Genetically Engineered Machine (iGEM) is an educational benchmark in synthetic biology. Eighteen years after its inception, it has also catalysed the infusion of synthetic biology with interdisciplinary fundamental and translational research as well as with inspired young scientists. Here, we communicate a quantitative analysis of compiled published work associated to iGEM projects, highlighting trends in their dissemination and versatility. As iGEM comes of age, we anticipate it will continue to revolutionise, alongside SynBio, several disciplines of science and industries through the development of synthetic biological systems towards a sustainable future.

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PTP1B is an intracellular checkpoint that limits T cell and CAR T cell anti-tumor immunity Immunotherapies aimed at alleviating the inhibitory constraints on T cells have revolutionised cancer management. To date, these have focused on the blockade of cell surface checkpoints such as PD-1. Herein we identify protein-tyrosine-phosphatase 1B (PTP1B) as an intracellular checkpoint that is upregulated in T cells in tumors. We show that the increased PTP1B limits T cell expansion and cytotoxicity to contribute to tumor growth. T cell-specific PTP1B deletion increased STAT-5 signaling and this enhanced the antigen-induced expansion and cytotoxicity of CD8+ T cells to suppress tumor growth. The pharmacological inhibition of PTP1B recapitulated the T cell-mediated repression of tumor growth and enhanced the response to PD-1 blockade. Furthermore, the deletion or inhibition of PTP1B enhanced the efficacy of adoptively-transferred chimeric-antigen-receptor (CAR) T cells against solid tumors. Our findings identify PTP1B as an intracellular checkpoint whose inhibition can alleviate the inhibitory constraints on T cells and CAR T cells to combat cancer.

STATEMENT OF SIGNIFICANCETumors subvert anti-tumor immunity by engaging checkpoints that promote T-cell exhaustion. Here we identify PTP1B as an intracellular checkpoint and therapeutic target. We show that PTP1B is upregulated in intra-tumoral T-cells and that its deletion or inhibition enhances T-cell anti-tumor activity and increases CAR T-cell effectiveness against solid tumors.

sentences: FOXO1 represses Sprouty2 and Sprouty4 expressionin endothelial cells to promote arterial specification and vascular remodeling in the mouse yolk sac The establishment of a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss of function mouse models have shown that FOXO1, a Forkhead family transcription factor, is required for yolk sac vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that loss of FoxO1 in E8.25 endothelial cells results in increased Sprouty2 and Sprouty4 transcripts, reduced expression of arterial genes, and decreased Flk1/Vegfr2 mRNA levels without affecting overall endothelial cell identity, survival, or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, Delta like 4 (Dll4), is significantly reduced in the yolk sac of FoxO1 mutants without being substantially affected in the embryo proper. We show that in the yolk sac, FOXO1 not only binds directly to a subset of previously identified Sprouty2 gene regulatory elements (GREs), as well as newly identified, evolutionarily conserved Sprouty4 GREs, but can also repress their expression. Additionally, over expression of Sprouty4 in transient transgenic embryos largely recapitulates reduced expression of arterial genes seen in endothelial FoxO1 mutant mouse embryos. Together, these data reveal a novel role for FOXO1 as a key early transcriptional repressor controlling both pre-flow arterial specification and subsequent vessel remodeling within the murine yolk sac.

sentences: A motor-based approach to induce chromosome-specific mis-segregations in human cells Various cancer types exhibit highly characteristic and recurrent aneuploidy patterns. The origin of these cancer type-specific karyotypes, and the extent to which they contribute to cancer progression, remains to be elucidated, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to mis-segregate specific chromosomes at will in different human cell types. We employed Tet repressor (TetR) or nuclease dead Cas9 (dCas9) to link a plant-derived microtubule minus-end-directed kinesin (Physcomitrella patens Kinesin14VIb) to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were often counteracted by forces from kinetochore-attached microtubules. This tug of war resulted in chromosome-specific segregation errors during anaphase, and revealed that spindle forces can heavily stretch chromosomal arms. Using chromosome-specific FISH and single-cell whole genome sequencing, we established that motor-induced mis-segregations result in specific arm-level, and to a lesser extent, whole chromosome aneuploidies, after a single cell division. Our kinesin-based strategy to manipulate individual mitotic chromosomes opens up the possibility to investigate the immediate cellular responses to specific (arm level) aneuploidies in different cell types; an important step towards understanding how recurrent aneuploidy patterns arise in different cancer types.

sentences: Toxicity of extracellular alpha-synuclein is independent of intracellular alpha-synuclein Parkinson's disease (PD) pathology progresses throughout the nervous system affecting numerous neuronal structures. It has been postulated that the progression of the pathology is based on a prion-like disease mechanism partly due to the seeding effect of endocytosed alpha-synuclein (ASYN) on the endogenous ASYN. The appearance of the pathology in dopaminergic neurons leads to neuronal cell death and motor symptoms. However, the effect on other neuronal structures is more inconsistent, leading to a higher variability in the prevalence of non-motor symptoms. Thus, the sensitivity to the pathology seems to vary among neuronal subtypes. Here, we analyzed the role of endogenous ASYN in the progression of PD-like pathology and the effect of monomeric and oligomeric ASYN as well as paraquat and rotenone on primary enteric, dopaminergic and cortical neurons from wild-type mice. Our results showed that pathology progression did not occur in the absence of endogenous ASYN and that dopaminergic neurons were more sensitive to ASYN and rotenone when compared to all other neuronal subtypes. Remarkably, the toxic effect of ASYN was independent of the presence of endogenous ASYN and directly related to the disturbance of the mitochondrial membrane potential. Thus, we suggest that the interaction between ASYN and mitochondria plays an important role in the toxicity of trans-synaptically transported ASYN and in the progression of PD pathology. These results question the prion-disease hypothesis and propose that endocytosed ASYN impairs the host's mitochondrial function thereby also contributing to PD-pathology progression.

sentences: Chromosome-level genome assembly for the Aldabra giant tortoise enables insights into the genetic health of a threatened population The Aldabra giant tortoise (Aldabrachelys gigantea) is one of only two giant tortoise species left in the world. The species is endemic to Aldabra Atoll in Seychelles and is considered vulnerable due to its limited distribution and threats posed by climate change. Genomic resources for A. gigantea are lacking, hampering conservation efforts focused on both wild and ex-situ populations. A high-quality genome would also open avenues to investigate the genetic basis of the exceptionally long lifespan. Here, we produced the first chromosome-level de novo genome assembly of A. gigantea using PacBio High-Fidelity sequencing and high-throughput chromosome conformation capture (Hi-C). We produced a 2.37 Gbp assembly with a scaffold N50 of 148.6 Mbp and a resolution into 26 chromosomes. RNAseq-assisted gene model prediction identified 23,953 protein-coding genes and 1.1 Gbp of repetitive sequences. Synteny analyses among turtle genomes revealed high levels of chromosomal collinearity even among distantly related taxa. We also performed a low-coverage re-sequencing of 30 individuals from wild populations and two zoo individuals. Our genome-wide population structure analyses detected genetic population structure in the wild and identified the most likely origin of the zoo-housed individuals. The high-quality chromosome-level reference genome for A. gigantea is one of the most complete turtle genomes available. It is a powerful tool to assess the population structure in the wild population and reveal the geographic origins of ex-situ individuals relevant for genetic diversity management and rewilding efforts.

sentences: Antidepressant Sertraline Hydrochloride Inhibits the Growth of HER2+ AU565 Breast Cancer Cell Line through Induction of Apoptosis, and Arrest of Cell Cycle Breast cancer is one the most aggressive cancer worldwide, especially Pakistan due to limited therapeutic options. This study was conducted to repurpose the use of selective serotonin reuptake inhibitors (SSRIs), in the treatment of breast cancers, and merit to pursue drug re-positioning in oncology. Anti-proliferative activity of SSRIs, such as fluoxetine, paroxetine, and sertraline hydrochloride on the growth of AU-565, MCF-7, MDA-MB-231, and BT-474 breast cancer cell lines, along with human fibroblast BJ cells was determined in vitro. Changes in nuclear morphology (DAPI staining), and induction of apoptosis (flow cytometry, and caspase-3 activation) were also studied. Sertraline hydrochloride most effectively inhibited the growth of breast cancer cells in vitro. Therefore, pharmacological mechanism involved in sertraline mediated cell death was investigated in HER2+ AU565 cell line. Enhanced nuclear fragmentation, increased Annexin (+) cells, and caspase-3/7 activation indicated that sertraline-mediated cell death could be a result of BCl2-independent apoptosis as evidenced by expression of Bax, and BCl2 genes. Taken together, our results identified sertraline hydrochloride, as a potential candidate for the treatment of HER2-positive breast cancer. Even though these are in vitro results, this study opens great opportunity in the field of drug repurposing for the development of chemotherapeutic agents.

sentences: Cryo-EM structures of SARS-CoV-2 Omicron BA.2 spike The BA.2 sub-lineage of the SARS-CoV-2 Omicron variant has gained in proportion relative to BA.1. As differences in spike (S) proteins may underlie differences in their pathobiology, here we determine cryo-EM structures of a BA.2 S ectodomain and compare these to previously determined BA.1 S structures. BA.2 Receptor Binding Domain (RBD) mutations induced remodeling of the internal RBD structure resulting in its improved thermostability and tighter packing within the 3-RBD-down spike. In the S2 subunit, the fusion peptide in BA.2 was less accessible to antibodies than in BA.1. Pseudovirus neutralization and spike binding assays revealed extensive immune evasion while defining epitopes of two RBD-directed antibodies, DH1044 and DH1193, that bound the outer RBD face to neutralize both BA.1 and BA.2. Taken together, our results indicate that stabilization of the 3-RBD-down state through interprotomer RBD-RBD packing is a hallmark of the Omicron variant, and reveal differences in key functional regions in the BA.1 and BA.2 S proteins.

sentences: Neuroimaging signature associated with symptom exacerbation in early-stage psychosis Recent reports have indicated that the occurrence of symptom exacerbation in early-stage psychosis could result in brain changes, which are likely to underlie the poorer disease outcome. Thus, it is important to identify neuroimaging signature associated with symptom exacerbation in early-stage psychosis. We studied 85 patients with psychosis within two years after onset and 94 healthy controls (HC). The patient group was subdivided into two groups: 54 patients who did not experience major symptom exacerbation between the onset and study enrollment (P1), and 31 patients who experienced major symptom exacerbation (P2). We analyzed three brain imaging measures derived from resting-state functional MRI, such as global efficiency, nodal efficiency, and resting-state functional connectivity (rs-FC). After excluding some brain imaging measures that were potentially affected by clinical variables, we conducted a comparison between overall patient group and HC group as well as comparsions between HC, P1, and P2 groups for these three types of brain imaging measures, respectively. By integrating the information, we pinned down the dorsal anterior cingulate cortex and thalamus as key hubs in the context of several large-scale brain networks associated with symptom exacerbations in early-stage psychosis. Our study implies the importance of considering neural mechanism associated with symptom exacerbations in early stages of psychotic disorders.

sentences: Environmental stressors induced strong small-scale phenotypic differentiation in a wide-dispersing marine snail Heterogeneous environments pose a particular challenge for organisms because the same phenotype is unlikely to perform best regardless of the variety of encountered stressors. To understand how species meet this challenge, we investigated the extent to which contrasting environmental pressures induced ecological and phenotypic responses in a natural population of a wide-dispersing marine snail at a small spatial scale. We analyzed several traits of Heleobia australis (Rissooidea: Cochliopidae) collected from heterogeneous, but highly connected, habitats from the intertidal area of the Bahia Blanca estuary, Argentina. We also conducted molecular analyses by amplifying the COI gene in individuals sampled from each habitat. We found that sympatric subpopulations of H. australis exhibited a strong phenotypic divergence in shell characters and body weight in response to thermal, saline, and dehydration stress, crab predation risk, and parasitic castrators. We proved that this differentiation occurred even early in life as most of the characters observed in juveniles mirrored those found in adults. We also found a divergence in penis size in snails collected from each habitat and raised in common garden laboratory conditions. The molecular analyses confirmed that the individuals studied constituted a single species despite the strong phenotypic differences among subpopulations. The small-scale phenotypic differentiation suggests that H. australis experienced a fine-grained environment where conditions imposed by different sources of stress favored the expression of beneficial traits. We discuss the role of plasticity in shaping adaptive phenotypic responses that increase the likelihood of persistence of subpopulations facing environmental stress conditions.

sentences: PRSS2 stimulates tumor growth by remodeling the TME via repression of Tsp1 In the earliest stages of tumor development, epithelial tumors (carcinomas) are physically confined to the area of the tissue in which they form. These nascent lesions (carcinomas in situ) are sequestered from the tissue parenchyma by the basement membrane. Within the tissue parenchyma lie a myriad of cell types comprised of fibroblasts, immune and inflammatory cells and endothelial cells. Upon invasion across the basement membrane and into the tissue parenchyma, tumors must manipulate the expression of pro- and anti-tumorigenic proteins such that pro-tumorigenic factors are produced in excess to anti-tumorigenic proteins. One such anti-tumorigenic protein is Thrombospondin-1 (Tsp-1). We have previously demonstrated that stimulation of Tsp-1 in the tumor microenvironment (TME) potently inhibits tumor growth and progression and in some cases induces tumor regression. Here, we identify a novel tumor-mediated mechanism to repress the expression of Tsp-1 in the TME via secretion of the serine protease PRSS2. We demonstrate that PRSS2 represses Tsp-1, not via its enzymatic activity, but by binding to low-density lipoprotein receptor-related protein 1 (LRP1). These findings describe a novel activity for PRSS2 through binding to LRP1 and represent a potential therapeutic strategy to treat cancer by blocking the PRSS2-mediated repression of Tsp-1.

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The endemic plant species of Bali Ngemba Forest Reserve, Bamenda Highlands Cameroon, with a new Endangered cloud-forest tree species Vepris onanae (Rutaceae). We revise and update the records of strict and near-endemic species of the Bali Ngemba Forest Reserve, the largest known surviving patch (c. 8 km2 in area) of submontane or cloud forest in the Bamenda Highlands, Cameroon which have lost >96 % of their original forest due to human activities. Nine strict endemics, and 11 near endemics are now documented, a drop from the number recorded after the first survey in 2004, since when five of the provisionally named species have been formally published.

We test the hypothesis that a further one of the provisionally named putative Bali Ngemba new species, Vepris sp. A, an 8 - 20 m tall tree from cloud forest in the 1310 - 1600 m altitudinal band, is indeed new to science. We compare it morphologically with other multicarpellate, apocarpous, trifoliolate Cameroon tree species formerly placed in the genus Oricia Pierre until they were subsumed into Vepris by Mziray (1992). These are Vepris trifoliolata (Engl.) Mziray and V. gabonensis (Pierre) Mziray. We conclude that Vepris sp. A is a new undescribed species here named as Vepris onanae. The new species is illustrated, mapped and its conservation status assessed as Endangered using the 2012 IUCN standard due to the threats of habitat clearance from agricultural pressures at its three locations all of which remain formally unprotected.

Vepris onanae appears unique among the Guineo-Congolian African oricioid species of Vepris in occurring in cloud forest, the other species, apart from V. renierii of the Albertine Rift, occurring in lowland forest. It also differs in the very broad, (7.8 -) 11.3 - 18 cm wide leaflets of the flowering stems which have a 6-18(-30) mm long, narrowly triangular acumen (vs leaflets <12 cm wide, acumen absent or short) and in having both subsessile and pedicellate (pedicels 0.25 - 0.3 mm long and 1(- 2) mm long) male flowers (vs male flower pedicels all sessile, or all c. 3 mm long).

We report for the first time on stage-dependent leaf heteromorphy in Vepris and characterise a level of sexual dimorphism more advanced than usual in the genus.

We highlight the importance of protecting Bali Ngemba and other forest patches in the Bamenda Highlands if species such as Vepris onanae are not soon to become extinct.

sentences: T cell stimulation remodels the latently HIV-1 infected cell population by differential activation of proviral chromatin The reservoir of latently HIV-1 infected cells is heterogeneous. To achieve an HIV-1 cure, the reservoir of activatable proviruses should be eliminated while permanently silenced proviruses may be tolerated. We have developed a method to assess the proviral nuclear microenvironment in single cells. In latently HIV-1 infected cells, a zinc finger protein tethered to the HIV-1 promoter produced a fluorescent signal as a protein of interest came in its proximity, such as the viral transactivator Tat when recruited to the nascent RNA. Tat is essential for viral replication. In these cells we assessed the proviral activation and chromatin composition. By linking Tat recruitment to proviral activity, we dissected the mechanisms of HIV-1 latency reversal and the consequences of HIV-1 production. A pulse of promoter-associated Tat was identified that contrasted to the continuous production of viral proteins. As expected, promoter H3K4me3 led to substantial expression of the provirus following T cell stimulation. However, the activation-induced cell cycle arrest and death led to a surviving cell fraction with proviruses encapsulated in repressive chromatin. Further, this cellular model was used to reveal mechanisms of action of small molecules. In a proof-of-concept study we determined the effect of an enhancer specific CBP/P300-inhibitor on HIV-1 latency reversal. Only proviruses resembling active enhancers, associated with H3K4me1 and H3K27ac, efficiently recruited Tat. Tat-independent HIV-1 latency reversal of unknown significance still occurred. We present a method for single cell assessment of the microenvironment of the latent HIV-1 proviruses, used here to reveal how T cell stimulation modulates the proviral activity and how the subsequent fate of the infected cell depends on the chromatin context.

sentences: An egg-derived sulfated N-Acetyllactosamine glycan is an antigenic decoy of influenza virus vaccines Influenza viruses grown in eggs for the purposes of vaccine generation often acquire mutations during egg adaptation or possess differential glycosylation patterns than viruses circulating amongst humans. Here, we report that seasonal influenza virus vaccines possess an egg-derived sulfated N-acetyllactosamine (LacNAc) that is an antigenic decoy. Half of subjects that received an egg-grown vaccine mounted an antibody response against this egg-derived antigen. Egg-binding monoclonal antibodies specifically bind viruses grown in eggs, but not viruses grown in other chicken derived cells, suggesting only egg-grown vaccines can induce anti-LacNAc antibodies. Notably, antibodies against the sulfated LacNAc utilized a restricted antibody repertoire and possessed features of natural antibodies, as most antibodies were IgM and have simple heavy chain complementarity determining region 3. By analyzing a public dataset of influenza virus vaccine induced plasmablasts, we discovered egg-binding public clonotypes that were shared across studies. Together, this study shows that egg-grown vaccines can induce antibodies against an egg-associated glycan, which may divert the host immune response away from protective epitopes.

sentences: Directed differentiation of EA/TEF patient-derived induced pluripotent stem cells into esophageal epithelial organoids reveal SOX2 dysregulation at the anterior foregut stage A series of well-regulated cellular and molecular events result in the compartmentalization of the anterior foregut into the esophagus and trachea. Disruption of the compartmentalization process leads to esophageal atresia/tracheoesophageal fistula (EA/TEF). Therefore, the objective is to differentiate pluripotent stem cells (PSCs), namely, embryonic stem cells and iPSCs from healthy individuals and iPSCs from EA/TEF type C patients, into mature 3-dimensional esophageal organoids expressing Involucrin, Keratin-4, -13, and p63. CXCR4, SOX17, and GATA4 expression was similar in both patient and healthy endodermal cells. Key transcription factor SOX2 was significantly lower in patient-derived anterior foregut. RNA sequencing revealed critical genes GSTM1 and RAB37 to be significantly lower in patient-derived anterior foregut. Furthermore, we observed an abnormal expression of NKX2.1 in the patient-derived mature esophageal organoids. We therefore hypothesize that a transient dysregulation of SOX2 and the abnormal expression of NKX2.1 in patient-derived cells could be responsible for the abnormal foregut compartmentalization.

sentences: Domain-general conflict monitoring predicts neural and behavioral indices of linguistic error processing during reading comprehension The ability to detect and respond to linguistic errors is critical for successful reading comprehension, but these skills can vary considerably across readers. In the current study, healthy adults (age 18-35) read short discourse scenarios for comprehension while monitoring for the presence of semantic anomalies. Using a factor analytic approach, we examined if performance in non-linguistic conflict monitoring tasks (Stroop, AX-CPT) would predict individual differences in neural and behavioral measures of linguistic error processing. Consistent with this hypothesis, domain-general conflict monitoring predicted both readers end-of-trial acceptability judgments and the amplitude of a late neural response (the P600) evoked by linguistic anomalies. Interestingly, the influence on the P600 was non-linear, suggesting that online neural responses to linguistic errors are influenced by both the effectiveness and efficiency of domain-general conflict monitoring. These relationships were also highly specific and remained after controlling for variability in working memory capacity and verbal knowledge. Finally, we found that domain-general conflict monitoring also predicted individual variability in measures of reading comprehension, and that this relationship was partially mediated by behavioral measures of linguistic error detection. These findings inform our understanding of the role of domain-general executive functions in reading comprehension, with potential implications for the diagnosis and treatment of language impairments.

sentences: The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection Summary ParagraphWe identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone1, a compound in clinical trials for anti-fibrotic and anti-inflammatory applications2, as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry3. We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir4. Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginones translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.

sentences: Investigating the structural changes due to adenosine methylation of the Kaposi's sarcoma-associated herpes virus ORF50 transcript Kaposis sarcoma-associated herpes virus (KSHV) is a human oncovirus. KSHV relies on manipulating the host cell N6-methyl adenosine (m6A) RNA modification pathway. Methylation within a RNA stem loop of the open reading frame 50 (ORF50) enhances transcript stability via the recruitment of the m6A reader, SND1. In this contribution we explore the energy landscapes of the unmethylated and methylated RNA stem loops of ORF50 to investigate the effect of methylation on the structure of the stem loop. We observe a significant shift upon methylation between an open and closed configuration of the top of the stem loop. In the unmethylated stem loop the closed configuration is much lower in energy, and, as a result, exhibits higher occupancy.

sentences: Containing Cancer with Personalized Minimum Effective Dose Resistance to treatment is a challenge in many cancer therapies. This is partly due to the heterogeneous nature of tumors, where drug-sensitive and drug-resistant cells compete for the same resources. This competition is largely shaped by cancer treatment. The rapid reduction of drug-sensitive cell population during therapy with a maximum-tolerated dose relaxes competitive stress on the drug-resistant cell population, promoting relapse. Therefore, maintaining a high level of drug-sensitive cell population with a treatment break or lower dose can impose effective competitive stress on drug-resistant cell populations. Adaptive therapy (AT) exploits the competition between cancer cells. However, given the heterogeneous treatment response of individual patients, determining a personalized optimal treatment that can fine-tune competitive stress remains challenging. Using a deterministic model of cancer cell population competition, this study defines an effective dose window (EDW) as a range of doses that conserve sufficient sensitive cells, while maintaining the tumor volume below a threshold (e.g., initial tumor volume), to maintain a sustained competition against resistant cells. As a proof of concept, we sought to determine the EDW for a small cohort of patients with melanoma (n=8). We first fitted the model to longitudinal tumor response data from each patient. We performed structural and practical identifiability analyses to confirm the reproducibility and uniqueness of the estimated parameters. Then, we considered a subset of the cohort with uniquely identifiable parameters and estimated patient-specific EDW. We demonstrated that if the dose belongs to the EDW, the tumor volume for each patient could be indefinitely contained either using continuous or AT strategy. Using the optimal control theory, we concluded that the lower bound of the EDW approximates the minimum effective dose (MED) for containing cancer. Taken together, using tumor biomarker data, this study provides a proof of concept that there may exist a patient-specific EDW that keeps the tumor below a threshold (e.g., initial volume) by maintaining sustained competition on resistant cells.

sentences: 3D Variability Analysis: Resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM Single particle cryo-EM excels in determining static structures of protein molecules, but existing 3D reconstruction methods have been ineffective in modelling flexible proteins. We introduce 3D variability analysis (3DVA), an algorithm that fits a linear subspace model of conformational change to cryo-EM data at high resolution. 3DVA enables the resolution and visualization of detailed molecular motions of both large and small proteins, revealing new biological insight from single particle cryo-EM data. Experimental results demonstrate the ability of 3DVA to resolve multiple flexible motions of -helices in the sub-50 kDa transmembrane domain of a GPCR complex, bending modes of a sodium ion channel, five types of symmetric and symmetry-breaking flexibility in a proteasome, large motions in a spliceosome complex, and discrete conformational states of a ribosome assembly. 3DVA is implemented in the cryoSPARC software package.

sentences: iEC7871 Quercus suber model: the first multi-tissue diel cycle genome-scale metabolic model of a woody tree In the last decade, genome-scale metabolic models have been increasingly used to study plant metabolic behavior at the tissue and multi-tissue level under different environmental conditions. Quercus suber, also known as the cork oak tree, is one of the most important forest communities of the Mediterranean/Iberian region. In this work, we present the genome-scale metabolic model of the Q. suber (iEC7871), the first of a woody plant. The metabolic model comprises 7871 genes, 6231 reactions, and 6481 metabolites across eight compartments. Transcriptomics data was integrated into the model to obtain tissue-specific models for the leaf, inner bark, and phellogen, with specific biomass compositions. The tissue-specific models were merged into a diel multi-tissue metabolic model to predict interactions among the three tissues at the light and dark phases. The metabolic models were also used to analyze the pathways associated with the synthesis of suberin monomers. Nevertheless, the models developed in this work can provide insights into other aspects of the metabolism of Q. suber, such as its secondary metabolism and cork formation.

sentences: Estimating latent positions in social and biological networks using Graph Neural Networks in R with GCN4R Network analysis methods are useful to better understand and contextualize relationships between entities. While statistical and machine learning prediction models generally assume independence between actors, network-based statistical methods for social network data allow for dyadic dependence between actors. While numerous methods have been developed for the R statistical software to analyze such data, deep learning methods have not been implemented in this language. Here, we introduce GCN4R, an R library for fitting graph neural networks on independent networks to aggregate actor covariate information to yield meaningful embeddings for a variety of network-based tasks (e.g. community detection, peer effects models, social influence). We provide an extensive overview of insights and methods utilized by the deep learning community on learning on social and biological networks, followed by a tutorial that demonstrates some of the capabilities of the GCN4R framework to make these methods more accessible to the R research community.

sentences: An integrated model for termination of RNA polymerase III transcription RNA polymerase III (RNAPIII) synthesizes essential and abundant non-coding RNAs such as tRNAs. Controlling RNAPIII span of activity by accurate and efficient termination is a challenging necessity to ensure robust gene expression and to prevent conflicts with other DNA-associated machineries. The mechanism of RNAPIII termination is believed to be simpler than that of other eukaryotic RNA polymerases, solely relying on the recognition of a T-tract in the non-template strand. Here we combine high-resolution genome-wide analyses and in vitro transcription termination assays to revisit the mechanism of RNAPIII transcription termination in budding yeast. We show that T-tracts are necessary but not always sufficient for termination and that secondary structures of the nascent RNAs are important auxiliary cis-acting elements. Moreover, we show that the helicase Sen1 plays a key role in a fail-safe termination pathway. Our results provide a comprehensive model illustrating how multiple mechanisms cooperate to ensure efficient RNAPIII transcription termination.

sentences: Entrainment of Endangered Sturgeon by a Large Water Diversion: Rescue, Enumeration, and Conservation Opportunities The Bonnet Carre Spillway diverts water from the Mississippi River through a floodway into Lake Pontchartrain to reduce river stages at New Orleans and prevent flood damages. Pallid Sturgeon, a federally listed species under the Endangered Species Act, and Shovelnose Sturgeon, listed under the Similarity of Appearance rule, are entrained through the Spillway structure and become trapped in the Spillway canals and other waterbodies. Five openings and corresponding rescue operations occurred between 2008 and 2019 after each Spillway closure. Operational parameters spanned a range of water temperatures and seasons with magnitude and duration of discharge varying across all openings. A total of 70 days with crew number ranging from 6 to 12 were expended to rescue 57 Pallid Sturgeon and 362 Shovelnose Sturgeon after the five openings that spanned 240 total days. More sturgeon were entrained at higher water temperatures, with greater numbers of bays opened, and for longer periods of time. Recovery of sturgeon is initially high but over time declines as sturgeon are depleted from the floodway, stranded in isolated waterbodies in the floodway, and/or displaced further downstream into Lake Pontchartrain during longer openings. Sturgeon that cannot find their way back to the floodway are unlikely to be rescued. Recent population studies indicate that less than 1% of the total population size in the Lower Mississippi River are entrained. However, this does not take into account those individuals entrained but not captured and the potential impacts of more frequent openings of the structure. Conservation recommendations are provided to increase catch efficiency and recovery of the endangered sturgeon.

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A comparison of approaches to scaffolding multiple regions along the 16S rRNA gene for improved resolution MotivationFull length, high resolution 16s rRNA marker gene sequencing has been challenging historically. Short amplicons provide high accuracy reads with widely available equipment, at the cost of taxonomic resolution. One recent proposal has been to reconstruct multiple amplicons along the full-length marker gene, however no barcode-free computationally tractable approach for this is available. To address this gap, we present Sidle (SMURF Implementation Done to acceLerate Efficiency), an implementation of the Short MUltiple Reads Framework algorithm with a novel tree building approach to reconstruct rRNA genes from individually amplified regions.

ResultsUsing simulated and real data, we compared Sidle to two other approaches of leveraging multiple gene region data. We found that Sidle had the least bias in non-phylogenetic alpha diversity, feature-based measures of beta diversity, and the reconstruction of individual clades. With a curated database, Sidle also provided the most precise species-level resolution.

Availability and ImplementationSidle is available under a BSD 3 license from https://github.com/jwdebelius/q2-sidle

sentences: Neurophysiological evidence for cognitive map formation during sequence learning Humans deftly parse statistics from sequences. Some theories posit that humans learn these statistics by forming cognitive maps, or underlying representations of the latent space which links items in the sequence. Here, an item in the sequence is a node, and the probability of transitioning between two items is an edge. Sequences can then be generated from walks through the latent space, with different spaces giving rise to different sequence statistics. Individual or group differences in sequence learning can be modeled by changing the time scale over which estimates of transition probabilities are built, or in other words, by changing the amount of temporal discounting. Latent space models with temporal discounting bear a resemblance to models of navigation through Euclidean spaces. However, few explicit links have been made between predictions from Euclidean spatial navigation and neural activity during human sequence learning. Here, we use a combination of behavioral modeling and intracranial encephalography (iEEG) recordings to investigate how neural activity might support the formation of space-like cognitive maps through temporal discounting during sequence learning. Specifically, we acquire human reaction times from a sequential reaction time task, to which we fit a model that formulates the amount of temporal discounting as a single free parameter. From the parameter, we calculate each individuals estimate of the latent space. We find that neural activity reflects these estimates mostly in the temporal lobe, including areas involved in spatial navigation. Similar to spatial navigation, we find that low dimensional representations of neural activity allow for easy separation of important features, such as modules, in the latent space. Lastly, we take advantage of the high temporal resolution of iEEG data to determine the time scale on which latent spaces are learned. We find that learning typically happens within the first 500 trials, and is modulated by the underlying latent space and the amount of temporal discounting characteristic of each participant. Ultimately, this work provides important links between behavioral models of sequence learning and neural activity during the same behavior, and contextualizes these results within a broader framework of domain general cognitive maps.

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SERS uncovers the link between conformation of cytochrome c heme and mitochondrial membrane potential The balance between the mitochondrial respiratory chain activity and the cells needs in ATP ensures optimal cellular function. Cytochrome c is an essential component of the electron transport chain (ETC), which regulates ETC activity, oxygen consumption, ATP synthesis and can initiate apoptosis. The impact of conformational changes in cytochrome c on its function is not understood for lack of access to these changes in intact mitochondria. We have developed a novel sensor that uses unique properties of label-free surface-enhanced Raman spectroscopy (SERS) to identify conformational changes in heme of cytochrome c and to elucidate their role in functioning mitochondria. We verify that molecule bond vibrations assessed by SERS is a reliable indicator of the heme conformation during changes in the inner mitochondrial membrane potential and ETC activity. We have found that cytochrome c heme reversibly switches between planar and ruffled conformations in response to the inner mitochondrial membrane potential and H+ concentration in the intermembrane space to regulate the efficiency of the mitochondrial respiratory chain, thus, adjusting the mitochondrial respiration to the cells consumption of ATP and the overall activity. The ability of the proposed SERS-based sensor to track mitochondrial function opens wide perspectives on cell bioenergetics.

For Table of Contents Only

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sentences: Dynamic cholesterol redistribution favors membrane fusion pore constriction Previous experiments have shown that cholesterol strongly prefers concave leaflets (which have negative curvature and are typically thin), but cholesterol also orders and thickens bilayers (promoting liquid-ordered phases with positive curvature). Our all-atom molecular dynamics simulations resolve this discrepancy for highly curved fusion pores, similar to those found in the nascent fusion and terminal fission steps of endo-/exocytosis. We find that cholesterol is strongly excluded by bilayer thinning in the fusion pore neck, which is caused by the necks net negative Gaussian (saddle) curvature. Consistent with experiment and our fusion pore simulations, analysis of liquid-disordered planar bilayers indicates that cholesterol prefers overall thicker bilayers, but negative leaflet curvature. The exclusion of cholesterol from the neck because of saddle Gaussian curvature implies that it helps drive fusion pore closure, consistent with literature evidence that membrane reshaping is connected to lateral phase separation.

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Investigating the Role of Chromatin Remodeler FOXA1 in Ferroptotic Cell Death Ferroptosis is a lipid peroxidation-dependent mechanism of regulated cell death known to suppress tumor proliferation and progression. Although several genetic and protein hallmarks have been identified in ferroptotic cell death, it remains challenging to fully characterize ferroptosis signaling pathways and to find suitable biomarkers. Moreover, changes taking place in the epigenome of ferroptotic cells remain poorly studied. In this context, we aimed to investigate the role of chromatin remodeler forkhead box protein A1 (FOXA1) in RSL3-treated multiple myeloma cells because, similar to ferroptosis, this transcription factor has been associated with changes in the lipid metabolism, DNA damage, and epithelial-to-mesenchymal transition (EMT). RNA sequencing and Western blot analysis revealed that FOXA1 expression is consistently upregulated upon ferroptosis induction in different in vitro and in vivo disease models. In silico motif analysis and transcription factor enrichment analysis further suggested that ferroptosis-mediated FOXA1 expression is orchestrated by specificity protein 1 (Sp1), a transcription factor known to be influenced by lipid peroxidation. Remarkably, FOXA1 upregulation in ferroptotic myeloma cells did not alter hormone signaling or EMT, two key downstream signaling pathways of FOXA1. CUT&RUN genome-wide transcriptional binding site profiling showed that GPX4-inhibition by RSL3 triggered loss of binding of FOXA1 to pericentromeric regions in multiple myeloma cells, suggesting that this transcription factor is possibly involved in genomic instability, DNA damage, or cellular senescence under ferroptotic conditions.

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sentences: Speciation dynamics and extent of parallel evolution along a lake-stream environmental contrast in African cichlid fishes Understanding the drivers and dynamics of diversification is a central topic in evolutionary biology. Here, we investigated the dynamics of diversification in the cichlid fish Astatotilapia burtoni that diverged along a lake-stream environmental gradient. Whole-genome and morphometric analyses revealed that divergent selection was essential at the early stages of diversification, but that periods in allopatry were likely involved towards the completion of speciation. While morphological differentiation was continuous, genomic differentiation was not, as shown by two clearly separated categories of genomic differentiation. Reproductive isolation increased along a continuum of genomic divergence, with a "grey zone" of speciation at [~]0.1% net nucleotide divergence. The quantification of the extent of (non-)parallelism in nine lake-stream population pairs from four cichlid species by means of multivariate analyses revealed one parallel axis of genomic and morphological differentiation among seven lake-stream systems. Finally, we found that parallelism was higher when ancestral lake populations were more similar.

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The probability of chromatin to be at the nuclear lamina has no systematic effect on its transcription level. Multiple studies have shown a correlation between gene expression and positioning of genes at nuclear envelope (NE) lined by nuclear lamina. Here, we ask whether there is a causal, systematic connection between the expression level of the groups of genes in topologically associating domains (TADs) of Drosophila nuclei and the probability of TADs to be found at the NE. To investigate the connection, we combine a coarse-grained dynamic model of the entire Drosophila nucleus with the genome-wide gene expression data for both the control and lamins depleted (LD) nuclei. We analyze the TAD averaged transcription levels of genes against the probabilities of individual TADs to be near the NE. Our findings suggest that, within statistical error margin, positioning of Drosophila TADs at the nuclear envelope does not, by itself, systematically affect the average gene expression in these TADs, while the expected negative correlation is confirmed. Verifiable hypotheses of the underlying mechanism for the presence of correlation without causality are discussed.

We introduce a TAD length normalized metric for the average transcription activity of genes in a TAD: number of Reads mapped to all genes in a TAD Per Kilobase of TAD length per Million reads mapped to all TADs (RPKMT).

sentences: Structural and Functional Analyses Explain Pea KAI2 Receptor Diversity and Reveal Stereoselective Catalysis During Signal Perception KAI2 are plant /{beta} hydrolase receptors, which perceive smoke-derived butenolide signals (karrikins) and putative endogenous, yet unidentified phytohormones (KAI2-ligands, KLs). The number of functional KAI2 receptors varies among plant species. It has been suggested that KAI2 gene duplication and sub-functionalization plays an adaptative role for diverse environments or ligand diversification by altering the receptor responsiveness to specific KLs. Legumes represent one of the largest families of flowering plants and contain many essential agronomic crops. Prior to legume diversification, KAI2 underwent duplication, resulting in KAI2A and KAI2B. Integrating plant genetics, ligand perception and enzymatic assays, and protein crystallography, we demonstrate that Pisum sativum KAI2A and KAI2B act as receptors and enzymes with divergent ligand stereoselectivity. KAI2B has a stronger affinity than KAI2A towards the KAI2-ligand (-)-GR24 and remarkably hydrolyses a broader range of substrates including the strigolactone-like isomer (+)-GR24. We determine the crystal structures of PsKAI2B in apo and butenolide-bound states. The biochemical and structural analyses as well as recorded mass spectra of KAI2s reveal a transient intermediate on the catalytic serine and a stable adduct on the catalytic histidine, further illuminating the role of KAI2 not only as receptors but also as bona fide enzymes. Our work uncovers the stereoselectivity of ligand perception and catalysis by evolutionarily diverged KAI2 receptors in KAR/KL signaling pathways and proposes adaptive sensitivity to KAR/KL and strigolactone phytohormones by KAI2B.

sentences: The extracellular matrix promotes breast cancer cell growth under amino acid starvation by promoting tyrosine catabolism Breast cancer tumours are embedded in a collagen I rich extracellular matrix (ECM) network where nutrients are scarce due to limited blood flow and elevated tumour growth. Metabolic adaptation is required for breast cancer cells to endure these conditions. Here, we demonstrated that the presence of ECM supported the growth of invasive breast cancer cells, but not non-transformed mammary epithelial cells, under amino acid starvation, through a mechanism that required ECM uptake. Importantly, we showed that this behaviour was acquired during carcinoma progression. ECM internalisation, followed by lysosomal degradation, contributed to the upregulation of the intracellular levels of several amino acids, including tyrosine and phenylalanine. Finally, we showed that cells on ECM had elevated tyrosine catabolism, leading to elevated fumarate levels, potentially feeding into the tricarboxylic acid cycle. Interestingly, this pathway was required for ECM-dependent cell growth under amino acid starvation, as the knockdown of HPDL, the third enzyme of the pathway, opposed cell growth on ECM without affecting cell proliferation on plastic. Collectively, our results highlight that the ECM surrounding breast cancer tumours represents an alternative source of nutrients to support cancer cell growth, by regulating phenylalanine and tyrosine metabolism.

sentences: Large-scale genetic association and single cell accessible chromatin mapping defines cell type-specific mechanisms of type 1 diabetes risk Translating genome-wide association studies (GWAS) of complex disease into mechanistic insight requires a comprehensive understanding of risk variant effects on disease-relevant cell types. To uncover cell type-specific mechanisms of type 1 diabetes (T1D) risk, we combined genetic association mapping and single cell epigenomics. We performed the largest to-date GWAS of T1D in 489,679 samples imputed into 59.2M variants, which identified 74 novel association signals including several large-effect rare variants. Fine-mapping of 141 total signals substantially improved resolution of causal variant credible sets, which primarily mapped to non-coding sequence. To annotate cell type-specific regulatory mechanisms of T1D risk variants, we mapped 448,142 candidate cis-regulatory elements (cCREs) in pancreas and peripheral blood mononuclear cell types using snATAC-seq of 131,554 nuclei. T1D risk variants were enriched in cCREs active in CD4+ T cells as well as several additional cell types including pancreatic exocrine acinar and ductal cells. High-probability T1D risk variants at multiple signals mapped to exocrine-specific cCREs including novel loci near CEL, GP2 and CFTR. At the CFTR locus, the likely causal variant rs7795896 mapped in a ductal-specific distal cCRE which regulated CFTR and the risk allele reduced transcription factor binding, enhancer activity and CFTR expression in ductal cells. These findings support a role for the exocrine pancreas in T1D pathogenesis and highlight the power of combining large-scale GWAS and single cell epigenomics to provide insight into the cellular origins of complex disease.

sentences: Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals Neurons draw on alternative splicing for their increased transcriptomic complexity throughout animal phylogeny. To delve into the mechanisms controlling the assembly and evolution of this regulatory layer, we characterized the neuronal microexon program in Drosophila and compared it with that of mammals. We found that in Drosophila, this splicing program is restricted to neurons by the post-transcriptional processing of the enhancer of microexons (eMIC) domain in Srrm234 by Elav and Fne. eMIC deficiency or misexpression leads to widespread neurological alterations largely emerging from impaired neuronal activity, as revealed by a combination of neuronal imaging experiments and cell-type-specific rescues. These defects are associated with the genome-wide skipping of short neural exons, which are strongly enriched in ion channels. Remarkably, we found no overlap of eMIC-regulated exons between flies and mice, illustrating how ancient post-transcriptional programs can evolve independently in different phyla to impact distinct cellular modules while maintaining cell-type specificity.

sentences: Fast 3D Clear: A Fast, Aqueous, Reversible Three-Day Tissue Clearing Method for Adult and Embryonic Mouse Brain and Whole Body To date, a variety of optical clearing methods have emerged that serve as powerful tools to study intact organs and neuronal circuits. Here we describe a newly developed, aqueous clearing protocol named "Fast 3D Clear", which relies on tetrahydrofuran (THF) for tissue delipidation, and iohexol (Histodenz) for clearing, such that tissues can be imaged under immersion oil in light sheet imaging systems. Fast 3D Clear requires three days to achieve high transparency of adult and embryonic mouse tissues, while maintaining their anatomical integrity, and preserving a vast array of transgenic and viral/dye fluorophores, such as GCaMP3/6, tdTomato, Fast Blue, and IRF670. A unique advantage of Fast 3D Clear is its complete reversibility and thus compatibility with tissue sectioning and immunohistochemistry. Fast 3D Clear can be easily and quickly applied to a wide range of biomedical studies, thereby greatly facilitating the acquisition of high-resolution, two - and three -dimensional images.

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MS-driven metabolic alterations are recapitulated in iPSC-derived astrocytes ObjectiveAstrocytes play a significant role in the pathology of Multiple Sclerosis (MS). Nevertheless, for ethical reasons, most of the studies in these cells were performed on the Experimental Autoimmune Encephalomyelitis model. As there are significant differences between human and mouse cells, we aimed here to better characterize astrocytes from patients with MS (PwMS), focusing mainly on mitochondrial function and cell metabolism.

MethodsWe obtained and characterized induced pluripotent stem cell (iPSC)-derived astrocytes from three PwMS and three unaffected controls and performed functional assays including electron microscopy, flow cytometry, cytokine measurement, gene expression, in situ respiration, and metabolomics.

ResultsWe detected several differences in MS astrocytes including: (i) enrichment of genes associated with mitophagy and neurodegeneration, (ii) increased mitochondrial fission and decreased mitochondrial to nuclear DNA ratio, indicating disruption of mitochondrial content, (iii) increased production of superoxide and MS-related proinflammatory chemokines, (iv) increased electron transport capacity and proton leak, in line with the increased oxidative stress, and (v) a distinct metabolic profile, with a deficiency in amino acid catabolism and increased sphingolipid metabolism, which have already been linked to MS.

InterpretationTo our knowledge, this is the first study thoroughly describing the metabolic profile of iPSC-derived astrocytes from PwMS, and validating this model as a powerful tool to study disease mechanisms and to perform non-invasive drug targeting assays in vitro. Our findings recapitulate several disease features described in patients and provide new mechanistic insights into the metabolic rewiring of astrocytes in MS, which could be targeted in future therapeutic studies.

sentences: The ventral striatum contributes to energy balance Obesity-related pathologies and anorexia nervosa are increasingly considered as opposite ends of eating disorders (ED) spectrum. Accordingly, accumulating evidence points to opposite dysregulations of common brain systems in these pathologies, the Nucleus Accumbens (NAc) in particular. However, to which extent alterations in NAc activity are sufficient to elicit ED-like behavioral dimensions remains to be established. Using acute, bidirectional, chemogenetic modulation of each NAc medium spiny neuron (MSN) subpopulation, we found that D1-MSNs and D2-MSNs oppositely regulate the balance between food intake and exercise-induced energy expenditure. As opposed to the limited effect of repeated chemogenetic manipulations of each MSN subpopulation, the concomitant manipulation of both MSN subpopulation had sustained effects. Repeated activation of D1-MSNs combined with repeated inhibition of D2-MSNs biased behavior toward activity-related energy expenditure leading to weight/fat loss, whilst the opposite manipulations favored energy intake and hence weight/fat gain. These results suggest that dysregulations of NAc dopaminoceptive MSN network might be at the core of EDs.

sentences: Olanzapine leads to nonalcoholic fatty liver disease through the apolipoprotein A5 pathway The antipsychotic drug olanzapine was reported to induce nonalcoholic fatty liver disease (NAFLD), whereas the underlying mechanism remains incompletely understood. This study investigated whether apolipoprotein A5 (apoA5) and sortilin, two interactive factors involved in NAFLD pathogenesis, are implicated in olanzapine-induced NAFLD. In the present study, at week 8, olanzapine treatment successfully induced hepatic steatosis in male C57 BL/6J mice, even when fed a normal chow diet, which was independent of animal body weight gain. Likewise, olanzapine effectively mediated hepatocyte steatosis in human HepG2 cells in vitro, characterized by substantially elevated intracellular lipid droplets. Increased plasma triglyceride concentration and decreased plasma apoA5 levels were observed in mice treated for 8 weeks with olanzapine. Surprisingly, olanzapine markedly enhanced hepatic apoA5 protein levels in mice, without a significant effect on rodent hepatic ApoA5 mRNA expression. Our in vitro study showed that olanzapine reduced apoA5 protein levels in the medium and enhanced apoA5 protein expression in hepatocytes, whereas this drug exerted no effect on hepatocyte APOA5 mRNA expression. By transfecting APOA5 siRNA into HepG2 cells, it was demonstrated that APOA5 knockdown effectively reversed olanzapine-induced hepatocyte steatosis. In addition, olanzapine drastically increased sortilin mRNA and protein levels in vivo and in vitro. Interestingly, SORT1 knockdown reduced intracellular apoA5 protein expression and increased medium apoA5 protein levels in vitro, without affecting intracellular APOA5 mRNA expression. Furthermore, SORT1 knockdown greatly ameliorated hepatocyte steatosis in vitro. This study provides the first evidence that sortilin inhibits the hepatic apoA5 secretion that is attributable to olanzapine-induced NAFLD, which provides new insight into effective strategies against NAFLD for patients with schizophrenia administered olanzapine.

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Total virome characterizations of game animals in China reveals a spectrum of emerging viral pathogens Game animals are wildlife species often traded and consumed as exotic food, and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1725 game animals, representing 16 species and five mammalian orders, sampled across China. From this we identified 71 mammalian viruses, with 45 described for the first time. Eighteen viruses were considered as potentially high risk to humans and domestic animals. Civets (Paguma larvata) carried the highest number of potentially high risk viruses. We identified the transmission of Bat coronavirus HKU8 from a bat to a civet, as well as cross-species jumps of coronaviruses from bats to hedgehogs and from birds to porcupines. We similarly identified avian Influenza A virus H9N2 in civets and Asian badgers, with the latter displaying respiratory symptoms, as well as cases of likely human-to-wildlife virus transmission. These data highlight the importance of game animals as potential drivers of disease emergence.

HighlightsO_LI1725 game animals from five mammalian orders were surveyed for viruses
C_LIO_LI71 mammalian viruses were discovered, 18 with a potential risk to humans
C_LIO_LICivets harbored the highest number of potential  high risk viruses
C_LIO_LIA species jump of an alphacoronavirus from bats to a civet was identified
C_LIO_LIH9N2 influenza virus was detected in a civet and an Asian badger
C_LIO_LIHumans viruses were also identified in game animals
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Soil microbes mediate the effects of nitrogen supply and co-inoculation on Barley Yellow Dwarf Virus in Avena sativa O_LINutrient supply rates to hosts can mediate host-pathogen interactions. In terrestrial systems, nutrient supply to plants is mediated by soil microbes, suggesting a potential indirect effect of soil microbes on plant-pathogen interactions. Soil microbes also may affect plant pathogens by inducing plant defenses.
C_LIO_LIWe tested the role of soil microbes, nitrogen supply to plant hosts, and co-inoculation on infection by aphid-vectored RNA viruses, Barley Yellow Dwarf Virus (BYDV-PAV) and Cereal Yellow Dwarf Virus (CYDV-RPV), in a grass host grown in soil microbes collected from a long-term nitrogen enrichment experiment.
C_LIO_LIBYDV-PAV incidence declined with high nitrogen supply, co-inoculation, or presence of soil microbes exposed to long-term low nitrogen enrichment. However, when combined, the negative effects of these treatments were sub-additive: nitrogen and co-inoculation did not reduce BYDV-PAV incidence in plants grown with the soil microbes. While soil microbes impacted leaf chlorophyll, they did not alter biomass or CYDV-RPV incidence.
C_LIO_LISoil microbes mediated the effects of nitrogen supply and co-inoculation on infection incidence and the effects of infection on host symptoms. Thus, soil microbial communities can indirectly control disease dynamics, altering the effects of nitrogen enrichment on plant-pathogen and pathogen-pathogen interactions in terrestrial systems.
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sentences: Structural insights on the effects of mutation of a charged binding pocket residue on phosphopeptide binding to 14-3-3 ζ Protein Mutation of an invariant aspartate residue in the binding pocket of 14-3-3{zeta} isoform to alanine dramatically reduced phosphopeptide binding and induced opening of the binding pocket. Here we use extensive molecular dynamics simulations to understand the role of D124 residue in ligand binding. The simulations show that in the absence of phosphopeptide, the D124A mutation leads to binding pocket reorganization including widening up of the binding pocket at the major groove and repositioning of N173, a key residue that interacts with the main chain of phosphopeptide. These structural changes would interfere with the efficient binding of the peptide, corroborating the experimental observations. Both gain and loss of electrostatic interactions in the form of salt bridges strongly indicate a rearrangement of the network of interactions within the binding pocket. Limited proteolysis coupled mass spectrometry (lip-MS) of the apo and holo forms of WT and mutant protein shows a peptide binding helix otherwise buried in the WT protein was particularly accessible to trypsin in the apo form of the mutant protein and the region was mapped to 158-186 amino acid residues of 14-3-3{zeta}). These results further confirm the dynamic nature of D124A mutant. Unlike other basic residues, the invariant D124 facilitates peptide binding by maintaining the geometry of interacting residues and by enforcing the structural integrity of amphipathic pocket.

sentences: A tRNA processing enzyme is a central regulator of the mitochondrial unfolded protein response The mitochondrial unfolded protein response (UPRmt) has emerged as a predominant mechanism that preserves mitochondrial function. Consequently, multiple pathways likely exist to modulate UPRmt. We unexpectedly discovered that the tRNA processing enzyme, homolog of ELAC2 (HOE-1), is central to UPRmt regulation in Caenorhabditis elegans. We find that nuclear HOE-1 is necessary and sufficient to robustly activate UPRmt. We show that HOE-1 acts via transcription factors ATFS-1 and DVE-1 that are crucial for UPRmt. Mechanistically, we show that HOE-1 likely mediates its effects via tRNAs, as blocking tRNA export prevents HOE-1-induced UPRmt. Interestingly, we find that HOE-1 does not act via the integrated stress response, which can be activated by uncharged tRNAs, pointing towards its reliance on a new mechanism. Finally, we show that the subcellular localization of HOE-1 is responsive to mitochondrial stress and is subject to negative regulation via ATFS-1. Together, we have discovered a novel RNA-based cellular pathway that modulates UPRmt.

sentences: Stable 3D Head Direction Signals in the Primary Visual Cortex Visual signals influence the brains computation of spatial position and orientation. Accordingly, the primary visual cortex (V1) is extensively interconnected with areas involved in computing head direction (HD) information. Predictive coding theories posit that higher cortical areas send sensory or motor predictions to lower areas, but whether this includes cognitive variables like the HD signal--and whether HD information is present in V1--is unknown. Here we show that V1 encodes the yaw, roll, and pitch of the head in freely behaving rats, either in the presence or absence of visual cues. HD tuning was modulated by lighting and movement state, but was stable on a population level for over a week. These results demonstrate the presence of a critical spatial orientation signal in a primary cortical sensory area and support predictive coding theories of brain function.

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Potential inhibitors for blocking the interaction of the coronavirus SARS-CoV-2 spike protein and its host cell receptor ACE2 The outbreak of SARS-CoV-2 continues to pose a serious threat to human health and social and economic stability. In this study, we established an anti-coronavirus drug screening platform based on the Homogeneous Time Resolved Fluorescence (HTRF) technology and the interaction between the coronavirus S protein and its host receptor ACE2. This platform is a rapid, sensitive, specific, and high throughput system. With this platform, we screened two compound libraries of 2,864 molecules and identified three potential anti-coronavirus compounds: tannic acid (TA), TS-1276 (anthraquinone), and TS-984 (9-Methoxycanthin-6-one). Our in vitro validation experiments indicated that TS-984 strongly inhibits the interaction of the coronavirus S-protein and the human cell ACE2 receptor. This data suggests that TS-984 is a potent blocker of the interaction between the S-protein and ACE2, which might have the potential to be developed into an effective anti-coronavirus drug.

SIGNIFICANCEThe ongoing pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made a serious threat to public health worldwide. Given the urgency of the situation, researchers are attempting to repurpose existing drugs for treating COVID-19. In this present study, we screened two compound libraries of 2,864 molecules and identified a potent inhibitor (TS-984) for blocking the coronavirus S-protein and the human cell ACE2 receptor. TS-984 might have the potential to be developed into an effective anti-coronavirus drug for treating COVID-19.

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Intracortical microstimulation pulse waveform and frequency recruits distinct spatiotemporal patterns of cortical neuron and neuropil activation. BackgroundNeural prosthetics often use intracortical microstimulation (ICMS) for sensory restoration. To restore natural and functional feedback, we must first understand how stimulation parameters influence the recruitment of neural populations. ICMS waveform asymmetry modulates the spatial activation of neurons around an electrode at 10 Hz; however, it is unclear how asymmetry may differentially modulate population activity at frequencies typically employed in the clinic (e.g. 100 Hz).

ObjectiveWe hypothesized that stimulation waveform asymmetry would differentially modulate preferential activation of certain neural populations, and the differential population activity would be frequency-dependent.

MethodsWe quantified how asymmetric stimulation waveforms delivered at 10 Hz or 100 Hz for 30s modulated spatiotemporal activity of cortical layer II/III pyramidal neurons using in vivo two-photon and mesoscale calcium imaging in anesthetized mice. Asymmetry is defined in terms of the ratio of the leading phase to the return phase of charge-balanced cathodal- and anodal-first waveforms.

ResultsNeurons within 40-60m of the electrode display stable stimulation-induced activity indicative of direct activation, which was independent of waveform asymmetry. The stability of 72% of activated neurons and the preferential activation of 20-90 % of neurons depended on waveform asymmetry. Additionally, this asymmetry-dependent activation of different neural populations was associated with differential progression of population activity. Specifically, neural activity increased over time for some waveforms at 10 Hz, but decreased more at 100 Hz than other waveforms.

ConclusionThese data demonstrate that at frequencies commonly used for sensory restoration, stimulation waveform alters the pattern of activation of different but overlapping populations of excitatory neurons. The impact of these waveform specific responses on the activation of different subtypes of neurons as well as sensory perception merits further investigation.

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Deeplasmid: Deep learning accurately separates plasmids from bacterial chromosomes Plasmids are mobile genetic elements that play a key role in microbial ecology and evolution by mediating horizontal transfer of important genes, such as antimicrobial resistance genes. Many microbial genomes have been sequenced by short read sequencers and have resulted in a mix of contigs that derive from plasmids or chromosomes. New tools that accurately identify plasmids are needed to elucidate new plasmid-borne genes of high biological importance. We have developed Deeplasmid, a deep learning tool for distinguishing plasmids from bacterial chromosomes based on the DNA sequence and its encoded biological data. It requires as input only assembled sequences generated by any sequencing platform and assembly algorithm and its runtime scales linearly with the number of assembled sequences. Deeplasmid achieves an AUC-ROC of over 93%, and it was much more precise than the state-of-the-art methods. Finally, as a proof of concept, we used Deeplasmid to predict new plasmids in the fish pathogen Yersinia ruckeri ATCC 29473 that has no annotated plasmids. Deeplasmid predicted with high reliability that a long assembled contig is part of a plasmid. Using long read sequencing we indeed validated the existence of a 102 Kbp long plasmid, demonstrating Deeplasmids ability to detect novel plasmids.

AvailabilityThe software is available with a BSD license: deeplasmid.sourceforge.io. A Docker container is available on DockerHub under: billandreo/deeplasmid.

Contactwandreopoulos@lbl.gov alevy@mail.huji.ac.il

sentences: A brainstem integrator for self-localization and positional homeostasis To accurately track self-location, animals need to integrate their movements through space. In amniotes, representations of self-location have been found in regions such as the hippocampus. It is unknown whether more ancient brain regions contain such representations and by which pathways they may drive locomotion. Fish displaced by water currents must prevent uncontrolled drift to potentially dangerous areas. We found that larval zebrafish track such movements and can later swim back to their earlier location. Whole-brain functional imaging revealed the circuit enabling this process of positional homeostasis. Position-encoding brainstem neurons integrate optic flow, then bias future swimming to correct for past displacements by modulating inferior olive and cerebellar activity. Manipulation of position-encoding or olivary neurons abolished positional homeostasis or evoked behavior as if animals had experienced positional shifts. These results reveal a multiregional hindbrain circuit in vertebrates for optic flow integration, memory of self-location, and its neural pathway to behavior.

sentences: Massively parallel pooled screening reveals genomic determinants of nanoparticle-cell interactions To accelerate the translation of cancer nanomedicine, we hypothesize that integrated genomic screens will improve understanding of the cellular processes governing nanoparticle trafficking. We developed a massively parallel high-throughput screening method leveraging barcoded, pooled cancer cell lines annotated with multi-omic data to investigate cell association patterns across a nanoparticle library spanning a range of formulations with clinical potential. This approach identified both the materials properties and cell-intrinsic features mediating nanoparticle-cell association. Coupling the data with machine learning algorithms, we constructed genomic nanoparticle trafficking networks and identified nanoparticle-specific biomarkers, including gene expression of SLC46A3. We engineered cell lines to validate SLC46A3 as a biomarker whose expression inversely predicts liposomal nanoparticle uptake both in vitro and in vivo. We further demonstrated the predictive capabilities extend beyond liposomal nanoparticles, regulating both uptake and transfection efficacy of solid lipid nanoparticles. Our work establishes the power of massively parallel pooled cell screens for nanoparticle delivery and enables the identification and utilization of biomarkers to rationally design nanoformulations for specific patient populations.

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Seasonal dynamics are the major driver of microbial diversity and composition in intensive freshwater aquaculture Aquaculture facilities such as fishponds are one of the most anthropogenically impacted freshwater ecosystems. The high fish biomass reared in aquaculture is associated with an intensive input into the water of fish-feed and fish excrements. This nutrients load may affect the microbial community in the water, which in turn can impact the fish health. To determine to what extent aquaculture practices and natural seasonal cycles affect the microbial populations, we characterized the microbiome of an inter-connected aquaculture system at monthly resolution, over three years. The system comprised two fishponds, where fish are grown, and a "control" operational water reservoir in which fish are not actively stocked. Clear natural seasonal cycles of temperature and inorganic nutrients concentration, as well as recurring cyanobacterial blooms during summer, were observed in both the fishponds and the reservoir. The structure of the aquatic bacterial communities in the system, characterized using 16S rRNA sequencing, was explained primarily by the natural seasonality, whereas aquaculture-related parameters had only a minor explanatory power. However, the cyanobacterial blooms were characterized by different cyanobacterial clades dominating at each fishpond, possibly in response to distinct nitrogen and phosphate ratios. In turn, nutrient ratios may have been by the magnitude of fish feed input. Taken together, our results show that, even in strongly anthropogenically impacted aquatic ecosystems, the structure of bacterial communities is mainly driven by the natural seasonality, with more subtle effects if aquaculture-related factors.

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C_LIO_LIThe microbiome changes seasonally, likely driven by temperature and rainwater runoff.
C_LIO_LISummer blooms of toxin-producing cyanobacteria are repeatedly observed.
C_LIO_LIFish food may impact microbiome through changes in nutrient ratios.
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A patient-driven clinicogenomic partnership through the Metastatic Prostate Cancer Project Molecular profiling studies have enabled numerous discoveries for metastatic prostate cancer (MPC), but they have mostly occurred in academic medical institutions focused on select patient populations. We developed the Metastatic Prostate Cancer Project (MPCproject, mpcproject.org), a patient-partnered initiative to empower MPC patients living anywhere in the U.S. and Canada to participate in molecular research and contribute directly to translational discovery. Here we present clinicogenomic results from our partnership with the first 706 MPCproject participants. We found that a patient-centered and remote research strategy enhanced engagement with patients in rural and medically underserved areas. Furthermore, patient-reported data achieved 90% consistency with abstracted health records for therapies and provided a mechanism for patient-partners to share information about their cancer experience not documented in medical records. Among the molecular profiling data from 333 patient-partners (n = 573 samples), whole exome sequencing of 63 tumor samples obtained from hospitals across the U.S. and Canada and 19 plasma cell-free DNA (cfDNA) samples from blood donated remotely recapitulated known findings in MPC and enabled longitudinal study of prostate cancer evolution. Inexpensive ultra-low coverage whole genome sequencing of 318 cfDNA samples from donated blood revealed clinically relevant genomic changes like AR amplification, even in the context of low tumor burden. Collectively, this study illustrates the power of a longitudinal partnership with patients to generate a more representative clinical and molecular understanding of MPC.

NoteTo assist our patient-partners and the wider MPC community interpret the results of this study, we have included a glossary of terms in the Supplementary Materials.

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A unique mineralizing pool of Gli1+ stem cells builds the tendon enthesis and demonstrates therapeutic potential The enthesis, a fibrocartilaginous transition between tendon and bone, is necessary for the transfer of force from muscle to bone to produce joint motion. The enthesis is prone to injury due to mechanical demands, and it cannot regenerate. A better understanding of how the enthesis develops will lead to more effective therapies to prevent pathology and promote regeneration. Here, we used single-cell RNA sequencing to define the development transcriptome of the entheses over postnatal stages. Six resident cell types, including enthesis progenitors and mineralizing chondrocytes, were identified along with their transcription factor regulons and temporal regulation. Following our prior discovery of the necessity of Gli1-lineage cells for enthesis development and healing, we then examined their transcriptomes at single cell resolution and demonstrated their clonogenicity and multipotency. Transplantation of these Gli1- lineage cells to enthesis injuries improved healing, demonstrating their therapeutic potential for enthesis regeneration.

HighlightsO_LIThe transcriptome and differentiation trajectory of enthesis stem cells during postnatal development are defined at single cell resolution.
C_LIO_LITranscription factor regulons drive enthesis stem cell differentiation.
C_LIO_LIGli1-lineage enthesis stem cells demonstrate in vivo and in vitro clonogenicity and multipotency.
C_LIO_LITransplantation of Gli1-lineage enthesis stem cells to enthesis injuries improves healing.
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sentences: An mRNA silencing mechanism reliant on the cooperation between REGE-1/Regnase-1 and RLE-1/Roquin-1 Regnase-1 is an evolutionarily conserved endoribonuclease, degrading diverse mRNAs important, among others, for immune homeostasis, development, and cancer. There are two competing models of Regnase-1 mediated mRNA silencing. One model postulates that Regnase-1 works together with another RNA-binding protein, Roquin-1. The other model proposes that the two proteins function separately. Studying the C. elegans Regnase-1 ortholog, REGE-1, we have uncovered a functional relationship between REGE-1 and the nematode counterpart of Roquin-1, RLE-1. While REGE-1 and RLE-1 associate with mRNA independently of each other, both proteins are essential for mRNA silencing. Intriguingly, the functional interdependence between REGE-1 and RLE-1 is reminiscent of the proposed cooperation between mammalian Regnase-1 and Roquin-1, which may underlie a prototypic silencing mechanism involving both proteins.

sentences: SARS-CoV-2 Infects Syncytiotrophoblast and Activates Inflammatory Responses in the Placenta SARS-CoV-2 infection during pregnancy leads to an increased risk of adverse pregnancy outcomes. Although the placenta itself can be a target of virus infection, most neonates are virus free and are born healthy or recover quickly. Here, we investigated the impact of SARS-CoV-2 infection on the placenta from a cohort of women who were infected late during pregnancy and had tested nasal swab positive for SARS-CoV-2 by qRT-PCR at delivery. SARS-CoV-2 genomic and subgenomic RNA was detected in 23 out of 54 placentas. Two placentas with high virus content were obtained from mothers who presented with severe COVID-19 and whose pregnancies resulted in adverse outcomes for the fetuses, including intrauterine fetal demise and a preterm delivered baby still in newborn intensive care. Examination of the placental samples with high virus content showed efficient SARS-CoV-2 infection, using RNA in situ hybridization to detect genomic and replicating viral RNA, and immunohistochemistry to detect SARS-CoV-2 nucleocapsid protein. Infection was restricted to syncytiotrophoblast cells that envelope the fetal chorionic villi and are in direct contact with maternal blood. The infected placentas displayed massive infiltration of maternal immune cells including macrophages into intervillous spaces, potentially contributing to inflammation of the tissue. Ex vivo infection of placental cultures with SARS-CoV-2 or with SARS-CoV-2 spike (S) protein pseudotyped lentivirus targeted mostly syncytiotrophoblast and in rare events endothelial cells. Infection was reduced by using blocking antibodies against ACE2 and against Neuropilin 1, suggesting that SARS-CoV-2 may utilize alternative receptors for entry into placental cells.

sentences: Balancing selection maintains hyper-divergent haplotypes in C. elegans Across diverse taxa, selfing species have evolved independently from outcrossing species thousands of times. The transition from outcrossing to selfing significantly decreases the effective population size, effective recombination rate, and heterozygosity within a species. These changes lead to a reduction in genetic diversity, and therefore adaptive potential, by intensifying the effects of random genetic drift and linked selection. Within the nematode genus Caenorhabditis, selfing has evolved at least three times and all three species, including in the model organism Caenorhabditis elegans, show substantially reduced genetic diversity relative to outcrossing species. Selfing and outcrossing Caenorhabditis species are often found in the same niches, but we still do not know how selfing species with limited genetic diversity can adapt to these environments. Here, we examine the whole-genome sequences from 609 wild C. elegans strains isolated worldwide and show that genetic variation is concentrated in punctuated hyper-divergent regions that cover 20% of the C. elegans reference genome. These regions are enriched in environmental response genes that mediate sensory perception, pathogen response, and xenobiotic stress response. Population genomic evidence suggests that genetic diversity in these regions has been maintained by long-term balancing selection. Using long-read genome assemblies for 15 wild strains, we show that hyper-divergent haplotypes contain unique sets of genes and show levels of divergence comparable to levels found between Caenorhabditis species that diverged millions of years ago. These results provide an example for how species can avoid the evolutionary "dead end" associated with selfing.

sentences: Diet unmasks genetic variants that regulate lifespan in outbred Drosophila Evolutionary theory suggests that lifespan-associated alleles should be purged from the gene pool, and yet decades of GWAS and model organism studies have shown they persist. Here, we address one potential explanation, the idea that the alleles that regulate lifespan do so only in certain contexts. We exposed thousands of outbred Drosophila to a standard and a high sugar diet. We then sequenced over 10,000 individuals and track genome-wide allele frequency changes over time, as these populations aged. We mapped thousands of lifespan-altering alleles, some associated with early vs late life tradeoffs, late-onset effects, and genotype-by-environment interactions. We find that lifespan-reducing alleles are most likely to be recently derived, have stronger effects on a high-sugar diet, consistent with the hypothesis that historically neutral or beneficial alleles can become detrimental in novel conditions. We also show that the gene midway, a regulator of lipid storage and ortholog of the lifespan-associated gene DGAT1 in mice, also regulates lifespan in Drosophila. Our results provide insight into the highly polygenic and context-dependent genetic architecture of lifespan, as well as the evolutionary processes that shape this key trait.

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Spatial and molecular anatomy of germ layers in the gastrulating primate embryo During mammalian embryogenesis, spatial regulation of gene expression and cell signaling are functionally coupled with lineage specification, patterning of tissue progenitors and germ layer morphogenesis. While the mouse model has been instrumental for our understanding of mammalian development, comparatively little is known about human and non-human primate gastrulation due to the restriction of both technical and ethical issues. Here, we present a morphological and molecular survey of spatiotemporal dynamics of cell types populating the non-human primate embryos during gastrulation. We performed serial sections of Cynomolgus monkeys (Macaca fascicularis) gastrulating embryos at 1-day temporal resolution from E17 to E21, and reconstructed three-dimensional digital models based on high-resolution anatomical atlas that revealed the dynamic changes in the geography of the mesoderm and primitive streaks. Spatial transcriptomics identified unique gene profiles that correspond to distinct germ layers and cross-species spatiotemporal transcriptome analysis revealed a developmental coordinate of germ layer segregation between mouse and primate. Furthermore, we identified species-specific transcription programs during gastrulation. These results offer important insights into evolutionarily conserved and divergent processes during mammalian gastrulation.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/474719v2_ufig1.gif" ALT="Figure 1">
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C_LIO_LICreated a three-dimensional digital template from serial sections of five developmental stages
C_LIO_LIA two-dimensional spatiotemporal transcriptome of the germ layers of gastrulating embryos
C_LIO_LICross-species comparison infers conservation of functional attributes of regulome and signaling activity in germ layer formation
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sentences: West Nile virus detection in horses in three Brazilian states. We report genetic evidence of WNV circulation from southern and northeastern Brazilian states isolated from equine red blood cells. In the northeastern state the tenth human case was also detected, presenting neuroinvasive disease compatible with WNV infection. Our analyses demonstrate that much is still unknown on the virus local epidemiology. We advocate for a shift to active surveillance, to ensure adequate control for future epidemics with spill-over potential to humans.

sentences: Subjectivity of time perception alters choice preference for future rewards through fronto-striatal value signal dynamics Our preference for a reward depends on the time of delay for its delivery. Here we show that changes in the external environment can manipulate the perceived duration of time, which alters the formation of the choice preference through value signals in the cortical and subcortical brain regions. Humans anticipated a real liquid reward delayed by tens of seconds, during which colors of visually presented panels gradually changed. The color-change delay was perceived as shorter than a control color-constant delay. Interestingly, participants with greater perceptual bias of the delay showed stronger preference for rewards with the color-change delay. The ventrolateral prefrontal cortex (vlPFC) and ventral striatum (VS) showed dynamic neural signatures of value components that were modulated by subjectively perceived duration. Crucially, these effects were specifically observed while a future reward was anticipated, and moreover, the vlPFC activity was weaker in participants with greater bias in the duration perception. These results demonstrate that subjective time experience leads to biased choice preference of delayed rewards, which is regulated by dynamic value signals of future rewards and accurate perception of the external world in the vlPFC-VS systems.

sentences: Spatiotemporal expression of regulatory kinases directs the transition from mitosis to cellular morphogenesis Embryogenesis depends on a tightly regulated balance between mitosis, differentiation, and morphogenesis. Understanding how the embryo uses a relatively small number of proteins to transition between growth and morphogenesis is a central question of developmental biology, but the mechanisms controlling mitosis and differentiation are considered to be fundamentally distinct. Here we show the mitotic kinase Polo, which regulates all steps of mitosis [1-3], also directs cellular morphogenesis after cell cycle exit. In mitotic cells, the Aurora kinases activate Polo to control a cytoskeletal regulatory module that directs cytokinesis [4-6]. We show that in the post-mitotic mesoderm, the control of Polo activity transitions from the Aurora kinases to the uncharacterized kinase Back Seat Driver (Bsd), where Bsd and Polo cooperate to regulate muscle morphogenesis. Polo and its effectors therefore direct mitosis and cellular morphogenesis, but the transition from growth to morphogenesis is determined by the spatiotemporal expression of upstream activating kinases.

sentences: Sex-biased response to and brain cell infection by SARS-CoV-2 in a highly susceptible human ACE2 transgenic model The COVID-19 pandemic is caused by SARS-CoV-2 infection. Human angiotensin-converting enzyme II (hACE2) has been identified as the receptor enabling SARS-CoV-2 host entry. To establish a mouse model for COVID-19, we generated transgenic mouse lines using the (HS4)2-pCAG-hACE2-HA-(HS4)2 transgene cassette, which expresses HA-tagged hACE2 under control of the CAG promoter and is flanked by HS4 insulators. Expression levels of the hACE2 transgene are respectively higher in lung, brain and kidney of our CAG-hACE2 transgenic mice and relatively lower in duodenum, heart and liver. The CAG-hACE2 mice are highly susceptibility to SARS-CoV-2 infection, with 100 PFU of SARS-CoV-2 being sufficient to induce 87.5% mortality at 9 days post-infection and resulting in a sole (female) survivor. Mortality was 100% at the higher titer of 1000 PFU. At lower viral titers, we also found that female mice exposed to SARS-CoV-2 infection suffered much less weight loss than male mice, implying sex-biased responses to SARS-CoV-2 infection. We subjected neuronal cultures to SARS-CoV-2 pseudovirus infection to ascertain the susceptibilities of neurons and astrocytes. Moreover, we observed that expression of SARS-CoV-2 Spike protein alters the synaptic responses of cultured neurons. Our transgenic mice may serve as a model for severe COVID-19 and sex-biased responses to SARS-CoV-2 infection, aiding in the development of vaccines and therapeutic treatments for this disease.

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SARS-CoV-2 infection induces inflammatory bone loss in golden Syrian hamsters Extrapulmonary complications of different organ systems have been increasingly recognized in patients with severe or chronic Coronavirus Disease 2019 (COVID-19). However, limited information on the skeletal complications of COVID-19 is known, even though inflammatory diseases of the respiratory tract have been known to perturb bone metabolism and cause pathological bone loss. In this study, we characterized the effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on bone metabolism in an established golden Syrian hamster model for COVID-19. SARS-CoV-2 causes significant multifocal loss of bone trabeculae in the long bones and lumbar vertebrae of all infected hamsters. The bone loss progressively worsens from the acute phase to the post-recovery phase. Mechanistically, the bone loss was associated with SARS-CoV-2-induced cytokine dysregulation which upregulates osteoclastic differentiation of monocyte-macrophage lineage. The pro-inflammatory cytokines further trigger a second wave of cytokine storm in the skeletal tissues to augment their pro-osteoclastogenesis effect. Our findings in this established hamster model suggest that pathological bone loss may be a neglected complication which warrants more extensive investigations during the long-term follow-up of COVID-19 patients. The benefits of potential prophylactic and therapeutic interventions against pathological bone loss should be further evaluated.

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In vitro synthesis and reconstitution using mammalian cell-free lysates enables the systematic study of the regulation of LINC complex assembly Membrane proteins perform numerous important functions in cells and tissues. Approximately 20% of the human genome encodes for membrane proteins, which represent the majority of targets for clinically relevant small molecules. Consequently, understanding their structure and structure-function relationships is a fundamental problem in biomedical research. Given the difficulties inherent to performing mechanistic biochemical and biophysical studies of membrane proteins in vitro, we previously developed a facile HeLa cell-based cell-free expression (CFE) system that enables the efficient reconstitution of full-length (FL) functional membrane proteins in supported lipid bilayers. Despite having shown the directional reconstitution of CFE-synthesized FL inner nuclear membrane SUN proteins (i.e. SUN1 and SUN2), which directly interact with outer nuclear membrane KASH proteins within the nuclear envelope lumen to form linker of nucleoskeleton and cytoskeleton (LINC) complexes that mechanically couple the cytoskeleton and nucleus, the mechanism underlying regulated LINC complex assembly remains unclear. Here, we provide evidence that suggests that the reconstitution of CFE-synthesized FL membrane proteins in supported lipid bilayers occurs primarily through the fusion of endoplasmic reticulum-derived microsomes present within our CFE reactions with our supported lipid bilayers. In addition, we demonstrate the ease with which our synthetic biology platform can be used to investigate the impact of the chemical environment (e.g. calcium ions and redox state) on the ability of CFE-synthesized FL SUN proteins reconstituted in supported lipid bilayers to interact with the luminal domain of the KASH protein nesprin-2. Moreover, we use our platform to study the molecular requirements for the homo- and hetero-typic interactions that can occur between SUN1 and SUN2. Finally, we show that our platform can be used to simultaneously reconstitute three different CFE-synthesized FL membrane proteins in a single supported lipid bilayer. Overall, these results establish our HeLa cell-based CFE and supported lipid bilayer reconstitution platform as a powerful tool for performing mechanistic dissections of the oligomerization and function of FL membrane proteins in vitro. While our platform is not a substitute for cell-based studies of membrane protein biochemistry and function, it does provide important mechanistic insights into the biology of difficult-to-study membrane proteins.

ABSTRACT GRAPHIC O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

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Accurate assignment of disease liability to genetic variants using only population data PurposeThe growing size of public variant repositories prompted us to test the accuracy of predicting pathogenicity of DNA variants using population data alone.

MethodsUnder the a priori assumption that the ratio of the prevalence of variants in healthy and affected populations form two distinct distributions (pathogenic and benign), we used a Bayesian method to assign probability of a variant belonging to either distribution.

ResultsThe approach, termed BayPR, accurately parsed 300 of 313 expertly curated cystic fibrosis transmembrane conductance regulator (CFTR) variants: 284 of 296 pathogenic/likely pathogenic (P/LP) variants in one distribution and 16 of 17 benign/likely benign (B/LB) variants in another. BayPR produced an area under the receiver operating curve (AUC) of 0.99 for 103 functionally-confirmed missense CFTR variants, equal to or exceeding ten commonly used algorithms (AUC range: 0.54 to 0.99). Application of BayPR to expertly curated variants in eight genes associated with seven Mendelian conditions assigned [≥]80% disease-causing probability to 1,350 of 1,374 (98.3%) P/LP variants and [≤]20% to 22 of 23 (95.7%) B/LB variants.

ConclusionAgnostic to variant type or functional effect, BayPR provides probabilities of pathogenicity for DNA variants responsible for Mendelian disorders using only variant counts in affected and unaffected population samples.

sentences: A macroecological description of alternative stable states reproduces intra- and inter-host variability of gut microbiome The most fundamental questions in microbial ecology concern the diversity and variability of communities. Their composition varies widely across space and time, as it is determined by a non-trivial combination of stochastic and deterministic processes. The interplay between non-linear community dynamics and environmental fluctuations determines the rich statistical structure of community variability, with both rapid temporal dynamics fluctuations and non-trivial correlations across habitats. Here we analyze long time-series of gut microbiome and compare intra- and inter-community dissimilarity. Under a macroecological framework we characterize their statistical properties. We show that most taxa have large but stationary fluctuations over time, while a minority is characterized by quick changes of average abundance which cluster in time, suggesting the presence of alternative stable states. We disentangle inter-individual variability in a major stochastic component and a deterministic one, the latter recapitulated by differences in the carrying capacities of taxa. Finally, we develop a model which includes environmental fluctuations and alternative stable states. This model quantitatively predicts the statistical properties of both intra- and inter-individual community variability, therefore summarizing variation in a unique macroecological framework.

sentences: Functional and taxonomic comparison of mouse and human gut microbiotas using extensive culturing and metagenomics Mouse models are essential for biomedical science and drug discovery, yet it is not known how the bacteria in the mouse microbiota - important determinants of phenotypes of health and disease -affect their relevance to human disease. To interrogate the taxonomic and functional differences between the human and mouse gut microbiotas, we developed the Mouse Microbial Genome Collection (MMGC), a compilation of 276 genomes from cultured isolates and 45,218 metagenome-assembled genomes (MAGs) from 1,960 publicly available mouse metagenomes. The MMGC reveals that while only 2.65% of bacterial species are shared between mouse and human, over 80% of annotatable functions are present in both microbiomes. Using drug metabolism and butyrate synthesis as examples, we illustrate that although the species harbouring these key functions can differ between hosts, the MMGC enables identification of functionally equivalent taxa in the mouse and human microbiotas. The MMGC thereby facilitates the informed use of mice in biomedical research by providing access to the conservation and taxonomic locations of bacterial functions of interest.

sentences: Chronic lung diseases are associated with gene expression programs favoring SARS-CoV-2 entry and severity Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyzed the transcriptomes of 605,904 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observed a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD epithelial cells expressed higher levels of genes linked directly to the efficiency of viral replication and innate immune response. Additionally, we identified basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection.

sentences: Slow oscillation-spindle coupling is negatively associated with emotional memoryformation following stress Both stress and sleep enhance emotional memory. They also interact, with the largest effect of sleep on emotional memory being seen when stress occurs shortly before or after encoding. Slow wave sleep (SWS) is critical for long-term episodic memory, facilitated by the temporal coupling of slow oscillations and sleep spindles. Prior work in humans has shown these associations for neutral information in non-stressed participants. Whether coupling interacts with stress to facilitate emotional memory formation is unknown. Here, we addressed this question by reanalyzing an existing dataset of 64 individuals. Participants underwent a psychosocial stressor (32) or comparable control (32) prior to the encoding of 150-line drawings of neutral, positive, and negative images. All participants slept overnight with polysomnography, before being given a surprise memory test the following day. In the stress group, time spent in SWS was positively correlated with memory for images of all valences. Results were driven by those who showed a high cortisol response to the stressor, compared to low responders. The amount of slow oscillation-spindle coupling during SWS was negatively associated with neutral and emotional memory in the stress group only. The association with emotional memory was significantly stronger than for neutral memory within the stress group. These results suggest that stress around the time of initial memory formation impacts the relationship between slow wave sleep and memory.

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TAP: Targeting and analysis pipeline for optimization and verification of coil placement in transcranial magnetic stimulation ObjectiveTranscranial magnetic stimulation (TMS) can modulate brain function via an electric field (E-field) induced in a region of interest (ROI) in the brain. The ROI E-field can be computationally maximized and set to match a specific reference using individualized head models to find the optimal coil placement and stimulus intensity. However, the available software lacks many practical features for prospective planning of TMS interventions and retrospective evaluation of the experimental targeting accuracy.

ApproachThe TMS targeting and analysis pipeline (TAP) software uses an MRI/fMRI-derived brain target to optimize coil placement considering experimental parameters such as the subjects hair thickness and coil placement restrictions. The coil placement optimization is implemented in SimNIBS 3.2, for which an additional graphical user interface (TargetingNavigator) is provided to visualize and adjust procedural parameters. The coil optimization process also computes the E-field at the target, allowing the selection of the TMS device intensity setting to achieve a specific E-field strength. The optimized coil placement information is prepared for neuronavigation software, which supports targeting during the TMS procedure. The neuronavigation system can record the coil placement during the experiment, and these data can be processed in TAP to quantify the accuracy of the experimental TMS coil placement and induced E-field.

Main resultsTAP was demonstrated in a study consisting of three repetitive TMS sessions in five subjects. TMS was delivered by an experienced operator under neuronavigation with the computationally optimized coil placement. Analysis of the experimental accuracy from the recorded neuronavigation data indicated coil location and orientation deviations up to about 2 mm and 2{degrees}, respectively, resulting in an 8% median decrease in the target E-field magnitude compared to the optimal placement.

SignificanceTAP supports navigated TMS with a variety of features for rigorous and reproducible stimulation delivery, including planning and evaluation of coil placement and intensity selection for E-field-based dosing.

sentences: Long-Short Term Memory Networks for Electric Source Imaging with Distributed Dipole Models 1Magneto- and electroencephalography (M/EEG) are widespread techniques to measure neural activity in vivo at a high temporal resolution but relatively low spatial resolution. Locating the sources underlying the M/EEG poses an inverse problem, which is itself ill-posed. In recent years, a new class of source imaging methods was developed based on artificial neural networks. We present a long-short term memory (LSTM) network to solve the M/EEG inverse problem. It integrates several aspects essential for qualitative inverse solutions: Low computational cost, exploitation of both spatial and temporal information, input flexibility and robustness to noise. Using simulation data, the LSTM shows higher accuracy on multiple metrics and for varying numbers of neural sources, compared to classical inverse solutions but also compared to our alternative architectures without integration of temporal information. It successfully integrates the temporal context given by sequential data, which is particularly useful with noisy data. Real data of a median nerve stimulation paradigm was used to show that the LSTM predicts plausible sources that are in concordance with classical inverse solutions. The performance of the LSTM regarding its robustness to noise renders it a promising and easy-to-apply inverse solution to be considered in future source localization studies and for clinical applications.

sentences: Energy status-promoted growth and development of Arabidopsis require copper deficiency response transcriptional regulator SPL7 Copper (Cu) is a cofactor of around 300 Arabidopsis proteins including photosynthetic and mitochondrial electron transfer chain enzymes critical for ATP production and carbon fixation. Plant acclimation to Cu deficiency requires the transcription factor SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE7 (SPL7). We report that in the wild type and in the spl7-1 mutant, respiratory electron flux via Cu-dependent cytochrome c oxidase remained unaffected under both normal and low-Cu cultivation conditions. Contrary to the wild type, supplementing Cu-deficient media with exogenous sugar failed to stimulate growth of spl7-1. The spl7-1 mutant accumulated carbohydrates including the signaling sugar trehalose 6-phosphate, as well as ATP and NADH, also under normal Cu supply and without sugar supplementation. Late flowering of spl7-1 was in agreement with its attenuated sugar responsiveness. Functional TOR and SnRK1 kinase signaling in spl7-1 suggested against fundamental defects in these energy-signaling hubs. Sequencing of chromatin immunoprecipitates combined with transcriptomics identified direct targets of SPL7-mediated positive regulation, including FE SUPEROXIDE DISMUTASE1 (FSD1), COPPER-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR1 (CITF1) and uncharacterized bHLH23 (CITF2), as well as an enriched upstream GTACTRC motif. In summary, transducing energy availability into growth and reproductive development requires the function of SPL7. Our results could help to increase crop yields, especially on Cu-deficient soils.

sentences: Sex- and Subtype-Specific Adaptations in Excitatory Signaling Onto Deep-Layer Prelimbic Cortical Pyramidal Neurons After Chronic Alcohol Exposure Long-term alcohol use results in behavioral deficits including impaired working memory, elevated anxiety, and blunted inhibitory control that are associated with prefrontal cortical (PFC) dysfunction. Preclinical observations demonstrate multiple impairments in GABAergic neurotransmission onto deep-layer principal cells (PCs) in prelimbic cortex that suggest dependence-related cortical dysfunction is the product of elevated excitability in these cells. Despite accumulating evidence showing alcohol-induced changes in interneuron signaling onto PCs differ between sexes, there is limited data explicitly evaluating sex-specific ethanol effects on excitatory signaling onto deep-layer PCs that may further contribute to deficits in PFC-dependent behaviors. To address this, we conducted electrophysiological and behavioral tests in both male and female Sprague-Dawley rats to evaluate the effects of chronic ethanol exposure. Among our observations, we report a marked enhancement in glutamatergic signaling onto deep-layer PCs in male, but not female, rats after alcohol exposure. This phenomenon was furthermore specific to a sub-class of PC, sub-cortically projecting Type-A cells, and coincided with enhanced anxiety-like behavior, but no observable deficit in working memory. In contrast, female rats displayed an alcohol-induced facilitation in working memory performance with no change in expression of anxiety-like behavior. Together, these results suggest fundamental differences in alcohol effects on cell activity, cortical sub-circuits, and PFC-dependent behaviors across male and female rats.

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Unraveling membrane properties at the organelle-level with LipidDyn Cellular membranes are formed from many different lipids in various amounts and proportions depending on the subcellular localization. The lipid composition of membranes is sensitive to changes in the cellular environment, and their alterations are linked to several diseases, including cancer. Lipids not only form lipid-lipid interactions but also interact with other biomolecules, including proteins, profoundly impacting each other.

Molecular dynamics (MD) simulations are a powerful tool to study the properties of cellular membranes and membrane-protein interactions on different timescales and at varying levels of resolution. Over the last few years, software and hardware for biomolecular simulations have been optimized to routinely run long simulations of large and complex biological systems. On the other hand, high-throughput techniques based on lipidomics provide accurate estimates of the composition of cellular membranes at the level of subcellular compartments. The community needs computational tools for lipidomics and simulation data effectively interacting to better understand how changes in lipid compositions impact membrane function and structure. Lipidomic data can be analyzed to design biologically relevant models of membranes for MD simulations. Similar applications easily result in a massive amount of simulation data where the bottleneck becomes the analysis of the data to understand how membrane properties and membrane-protein interactions are changing in the different conditions. In this context, we developed LipidDyn, an in silico pipeline to streamline the analyses of MD simulations of membranes of different compositions. Once the simulations are collected, LipidDyn provides average properties and time series for several membrane properties such as area per lipid, thickness, diffusion motions, the density of lipid bilayers, and lipid enrichment/depletion. The calculations exploit parallelization and the pipelines include graphical outputs in a publication-ready form. We applied LipidDyn to different case studies to illustrate its potential, including membranes from cellular compartments and transmembrane protein domains. LipidDyn is implemented in Python and relies on open-source libraries. LipidDyn is available free of charge under the GNU General Public License from https://github.com/ELELAB/LipidDyn.

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ADAD2 regulates heterochromatin in meiotic and post-meiotic male germ cells via translation of MDC1 Male germ cells establish a unique heterochromatin domain, the XY-body, early in meiosis. How this domain is maintained through the end of meiosis and into post-meiotic germ cell differentiation is poorly understood. ADAD2 is a late meiotic male germ cell specific RNA binding protein, loss of which leads to post-meiotic germ cell defects. Analysis of ribosome association in Adad2 mutants revealed defective translation of Mdc1, a key regulator of XY-body formation, late in meiosis. As a result, Adad2 mutants show normal establishment but failed maintenance of the XY-body. XY-body defects are concurrent with abnormal autosomal heterochromatin and ultimately lead to severely perturbed post-meiotic germ cell heterochromatin and cell death. These findings highlight the requirement of ADAD2 for Mdc1 translation, the role of MDC1 in maintaining meiotic male germ cell heterochromatin, and the importance of late meiotic heterochromatin for normal post-meiotic germ cell differentiation.

SummaryChukrallah et al. demonstrate ADAD2 is required for normal meiotic heterochromatin in male germ cells and loss leads to post-meiotic cell death defining ADAD2 as a heterochromatin maintenance factor.

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Clofoctol inhibits SARS-CoV-2 replication and reduces lung pathology in mice Drug repurposing has the advantage of shortening regulatory preclinical development steps. Here, we screened a library of drug compounds, already registered in one or several geographical areas, to identify those exhibiting antiviral activity against SARS-CoV-2 with relevant potency. Of the 1,942 compounds tested, 21 exhibited a substantial antiviral activity in Vero-81 cells. Among them, clofoctol, an antibacterial drug used for the treatment of bacterial respiratory tract infections, was further investigated due to favorable safety profile and pharmacokinetic properties. Notably, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC50 measured against SARS-CoV-2 in human pulmonary cells. This compound inhibits SARS-CoV-2 at a post-entry step. Lastly, therapeutic treatment of human ACE2 receptor transgenic mice decreased viral load, reduced inflammatory gene expression and lowered pulmonary pathology. Altogether, these data strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients.

SummaryAntivirals targeting SARS-CoV-2 are sorely needed. In this study, we screened a library of drug compounds and identified clofoctol as an antiviral against SARS-CoV-2. We further demonstrated that, in vivo, this compound reduces inflammatory gene expression and lowers pulmonary pathology.

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Synaptic decline precedes dopaminergic neuronal loss in human midbrain organoids harboring a triplication of the SNCA gene 1.Increased levels of the protein alpha-synuclein (-syn) are associated with the development of neurodegenerative diseases like Parkinsons disease (PD). In physiological conditions, -syn modulates synaptic plasticity, neurogenesis and neuronal survival. However, its pathogenic accumulation and aggregation results in toxicity and neurodegeneration.

Here, we used a PD patient specific midbrain organoid model derived from induced pluripotent stem cells harboring a triplication in the SNCA gene to study PD-associated phenotypes. The model recapitulates the two main hallmarks of PD, which are -syn aggregation and loss of dopaminergic neurons. Additionally, impairments in astrocyte differentiation were detected. Transcriptomics data indicate that synaptic function is impaired in PD specific midbrain organoids. This is further confirmed by alterations in synapse number and electrophysiological activity. We found that synaptic decline precedes neurodegeneration. Finally, this study substantiates that patient specific midbrain organoids allow a personalized phenotyping, which make them an interesting tool for precision medicine and drug discovery.

sentences: Silkworm spinning: the programmed self-assembly from natural silk fibroin to superfibre Silkworm silk is one of the best natural protein fibers spun by the silkworm at ambient temperature and pressure using aqueous silk protein solution. It is a great challenge to reproduce high-performance artificial fibers comparable to natural silk by bionics for the incomplete understanding of silkworm spinning mechanism, especially the structure and assembly of natural silk fibroin (NSF) in the silk gland. Here, we studied the structure and assembly of NSF with the assistance of amphipol and digitonin. Our results showed NSFs were present as nanofibrils primarily composed of random coils in the silk gland. Metal ions were vital for the formation of NSF nanofibrils. The successive decrease in pH from posterior silk gland (PSG) to anterior silk gland (ASG) resulted in a gradual increase in NSF hydrophobicity. NSF nanofibrils were randomly arranged from PSG to ASG-1, and then self-assembled into herringbone-like patterns near the spinneret (ASG-2) ready for silkworm spinning. Our study reveals the mechanism by which silkworms cleverly utilize metal ions and pH gradient in the silk gland to drive the programmed self-assembly of NSF from disordered nanofibrils to anisotropic liquid crystalline spinning dope (herringbone-like patterns) for silkworm spinning, thus providing novel insights into silkworm/spider spinning mechanism and bionic creation of high-performance fibers.

sentences: The RIFF: an automated environment for studying the neural basis of auditory-guided complex behavior Behavior consists of the interaction between an organism and its environment, and is controlled by the brain. Brain activity varies at sub-second time scales, but behavioral measures are usually coarse (often consisting of only binary trial outcomes). To overcome this mismatch, we developed the RIFF: a programmable interactive arena for freely-moving rats with multiple feeding areas, multiple sound sources, high-resolution behavioral tracking, and simultaneous electrophysiological recordings. We describe two complex tasks implemented in the RIFF. Rats quickly learned these tasks and developed anticipatory behavior. Neurons in auditory cortex and posterior insula showed sensitivity to non-auditory parameters such as location and pose. Our combination of wireless electrophysiology and detailed behavioral documentation in a controlled environment produces insights into the cognitive capabilities and learning mechanisms of rats and opens the way to a better understanding of how brains control behavior.

sentences: Adolescent social stress decrease of adult morphine-induced behavioral sensitization is dependent upon genetic background 1Deaths related to opioid use have skyrocketed in the United States, leading to a public health epidemic. Research has shown that both biological (genes) and environmental (stress) precursors are linked to opioid use. In particular, stress during adolescence - a critical period of frontal lobe development - influences the likelihood of abusing drugs. However, little is known about the biological mechanisms through which adolescent stress leads to long-term risk of opioid use, or whether genetic background moderates this response. Male and female C57BL/6J and BALB/cJ mice were exposed to chronic variable social stress (CVSS) or control conditions throughout adolescence and then tested for morphine locomotor sensitization or morphine consumption in adulthood. To examine possible mechanisms that underlie stress-induced changes in morphine behaviors, we assessed physiological changes in response to acute stress exposure and prefrontal cortex miRNA gene expression. Adolescent stress did not influence morphine sensitization or consumption in BALB/cJ animals, and there was limited evidence of stress effects in female C57BL/6J mice. In contrast, male C57BL/6J mice exposed to adolescent CVSS had blunted morphine sensitization compared to control animals; no differences were observed in the acute locomotor response to morphine administration or morphine consumption. Physiologically, C57BL/6J mice exposed to CVSS had an attenuated corticosterone recovery following an acute stressor and downregulation of twelve miRNA in the prefrontal cortex compared to control mice. The specificity of the effects for C57BL/6J versus BALB/cJ mice provides evidence of a gene by environmental interaction influencing opioid behaviors. Long-term differences in stress reactivity or miRNA expression suggests two possible biological mechanisms to evaluate in future research.

sentences: Attention-based convolutional neural networks for protein-protein interaction site prediction Proteins usually perform their cellular functions by interacting with other proteins. Accurate identification of protein-protein interaction sites (PPIs) from sequence is import for designing new drugs and developing novel therapeutics. A lot of computational models for PPIs prediction have been developed because experimental methods are slow and expensive. Most models employ a sliding window approach in which local neighbors are concatenated to present a target residue. However, those neighbors are not been distinguished by pairwise information between a neighbor and the target. In this study, we propose a novel PPIs prediction model AttCNNPPISP, which combines attention mechanism and convolutional neural networks (CNNs). The attention mechanism dynamically captures the pairwise correlation of each neighbor-target pair within a sliding window, and therefore makes a better understanding of the local environment of target residue. And then, CNNs take the local representation as input to make prediction. Experiments are employed on several public benchmark datasets. Compared with the state-of-the-art models, AttCNNPPISP significantly improves the prediction performance. Also, the experimental results demonstrate that the attention mechanism is effective in terms of constructing comprehensive context information of target residue.

sentences: Liquid-liquid phase separation in amino acid mixtures is governed by composition Liquid-liquid phase separation (LLPS) has recently come to immense prominence as it is central to the formation of membraneless organelles, leading to a new paradigm of cellular organization. This type of phase transition is mediated by molecular interactions between biomolecules, including nucleic acids and both ordered and disordered proteins. In the latter case, the separation between protein-dense and dilute phases is often interpreted using models adapted from polymer theory. Specifically, the "stickers and spacers" model proposes that LLPS originates from the interplay between two classes of residues and that the main determinants for phase separation are multivalency and sequence patterning. The duality of roles of stickers (aromatics like Phe and Tyr) and spacers (Gly and polar residues) may apply more broadly in protein-like mixtures, and the presence of these two types of components alone may suffice for LLPS to take place. In order to explore this hypothesis, we use atomistic molecular dynamics simulations of capped amino-acid residues as a minimal model system. We study the behaviour of pure amino acids in water for three types of residues that are thought to play critical roles in LLPS, corresponding to the spacer and sticker categories, and their multicomponent mixtures. In agreement with previous observations, we find that the spacer-type amino acids fail to phase-separate on their own, while the sticker is prone to aggregation. However, ternary amino acid mixtures involving both types of amino acids phase-separate into two phases that retain intermediate degrees of compaction and considerable fluidity. Our results suggest that LLPS is an emergent property of amino acid mixtures determined by composition.

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Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells Stem cell research is emerging both as a scientifically and clinically relevant area. One of the current challenges in stem cell research and regenerative medicine is assessment of the pluripotency state of induced pluripotent stem (iPS) cells. Once a stem cell differentiation process is initiated the challenge is how to assess the state of differentiation, and the purity of the differentiated cell population. Stem cell potency and differentiation states are determined by tightly coordinated activity of developmental signaling pathways, such as the Notch, Hedgehog, TGF{beta}, Wnt, PI3K, MAPK-AP1, and NF{kappa}B pathways. Source of the stem cells and culture protocols may influence stem cell phenotype, with potential consequences for pluripotency and in general for experimental reproducibility. Human pluripotent embryonic (hES) and iPS stem cell lines under different culture conditions, organ derived multipotent stem cells, and differentiated cell types, were phenotyped with respect to functional activity of developmental signaling pathways.

MethodsWe previously reported on the development and validation of a novel assay platform for quantitative measurement of activity of multiple signal transduction pathways (STP) simultaneously in a single sample, based on interpreting a preselected set of target mRNA expression levels. Assays were used to calculate Notch, Hedgehog, TGF{beta}, Wnt, PI3K, MAPK-AP1, and NF{kappa}B signal transduction pathway activity scores for individual cell samples, using publicly available Affymetrix expression microarray data.

ResultsCulture conditions (e.g. mouse versus human feeder) influenced pluripotent stem cell pathway activity profiles. hES and iPS stem cell lines cultured in the same lab under similar conditions showed minimal variation in pathway activity profile despite different genetic backgrounds, while across different labs larger variations were measured, even for the same stem cell line. Pathway activity scores for PI3K, MAPK, Hedgehog, Notch, TGF{beta}, and NF{kappa}B pathways rapidly decreased upon pluripotent stem cell differentiation, while increasing for the Wnt pathway. Further differentiation to intestinal progenitor cells resulted in higher PI3K, Wnt and Notch pathway activity. In multipotent intestinal crypt stem cells obtained from intestinal mucosa samples, similar Notch and even higher Wnt pathway activity were measured, which disappeared upon differentiation to mucosal cells.

ConclusionResults support the validity of using these STP assays for quantitative phenotyping of stem cells and differentiated derivatives, and enabled definition of a pluripotency profile with high PI3K, MAPK, Hedgehog, TGF{beta}, Notch, and NF{kappa}B, and low Wnt pathway activity scores.

Measurement of combined signaling pathway activity scores is expected to improve experimental reproducibility and standardization of pluripotent and multipotent stem cell culture and differentiation. It enables controlled manipulation of signaling pathway activity using pathway targeting compounds. An envisioned additional utility may lie in quality control for regenerative medicine purposes.

sentences: Regulation of the Evolutionarily Conserved Muscle Myofibrillar Matrix by Cell Type Dependent and Independent Mechanisms Skeletal muscles play a central role in human movement through forces transmitted by contraction of the sarcomere. We recently showed that mammalian sarcomeres are connected through frequent branches forming a singular, mesh-like myofibrillar matrix. However, the extent to which myofibrillar connectivity is evolutionarily conserved as well as mechanisms which regulate the specific architecture of sarcomere branching remain unclear. Here, we demonstrate the presence of a myofibrillar matrix in the tubular, but not indirect flight (IF) muscles within Drosophila melanogaster. Moreover, we find that loss of transcription factor H15 increases sarcomere branching frequency in the tubular jump muscles, and we show that sarcomere branching can be turned on in IF muscles by salm-mediated conversion to tubular muscles. Finally, we demonstrate that neurochondrin knockdown results in myofibrillar connectivity in IF muscles without conversion to tubular muscles. These data indicate an evolutionarily conserved myofibrillar matrix regulated by both cell-type dependent and independent mechanisms.

sentences: Quantitative Isothermal Amplification on Paper Membranes using Amplification Nucleation Site Analysis Quantitative nucleic acid amplification tests (qNAATs) are critical in treating infectious diseases, such as in HIV viral load monitoring or SARS-CoV-2 testing, in which viral load indicates viral suppression or infectivity. Quantitative PCR is the gold standard tool for qNAATs; however, there is a need to develop point-of-care (POC) qNAATs to manage infectious diseases in outpatient clinics, low- and middle-income countries, and the home. Isothermal amplification methods are an emerging tool for POC NAATs as an alternative to traditional PCR-based workflows. Previous works have focused on relating isothermal amplification bulk fluorescence signals to input copies of target nucleic acids for sample quantification with limited success. In this work, we show that recombinase polymerase amplification (RPA) reactions on paper membranes exhibit discrete fluorescent amplification nucleation sites. We demonstrate that the number of nucleation sites can be used to quantify HIV-1 DNA and RNA in less than 20 minutes. An image-analysis algorithm quantifies nucleation sites and determines the input nucleic acid copies in the range of 67-3,000 copies per reaction. We demonstrate a mobile phone-based system for image capture and onboard processing, illustrating that this method may be used at the point-of-care for qNAATs with minimal instrumentation.

sentences: An efficient and robust HPLC method to determine the sialylation levels of human epithelial cells Sialyltransferase, an enzyme responsible for attaching sialic acid to the cell surface, is reported to play a key role in cancer, making sialyltransferase a potent therapeutic target in drug development for cancer. Hence, this paper aimed to develop a simple method to detect and quantify sialic acids in cancer cells. An efficient method was developed using a reverse-phase ion-pairing HPLC-UV using triisopropanolamine as the ion-pairing agent with a C18 column. Neu5Ac was successfully eluted with the retention time 6.344 min with a flow rate of 0.4 mL/min. The proposed method was validated appropriately according to the AOAC guidelines (2013). This work demonstrates that the proposed method is not only relatively simple but also cost and time effective compared to pre-existing methods to successfully determine both free and protein-bound Neu5Ac in a complex cancer cell matrix. Furthermore, by applying the proposed method, a statistically significant decrease was observed for both HeLa and HuCCT1 cell lines with the application of deoxycholic acid - a known sialyltransferase inhibitor. Hence, the proposed method may be applicable to evaluate the effectiveness of sialyltransferase inhibitors.

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Generation of human chambered cardiac organoids from pluripotent stem cells for improved modelling of cardiovascular diseases Recent progress on murine and human cardiac organoids have provided understanding to the developmental processes of the heart. However, there is still an unfulfilled need for improved modelling of cardiovascular diseases using human cardiac organoids. Herein, we report successful generation of intrinsically formed human chambered cardiac organoids (CCO) and highlight its utility in modelling disease. Single cell transcriptomic profiling of CCOs showed appropriate cardiovascular cell type composition exhibiting improved maturation. Functionally, CCOs recapitulated clinical cardiac hypertrophy by exhibiting thickened chamber walls, reduced ejection fractions, increased myofibrillar disarray and tachycardia. Therefore, CCOs improve current capabilities of disease modelling as an in vitro model bridging the gap to in vivo models, with the ability to assess functional parameters that previously can only be achieved in animal systems.

One sentence summaryModelling cardiac hypertrophy using chambered cardiac organoids derived from human pluripotent stem cells.

sentences: Behavioural synchronization in a multilevel society of feral horses AO_SCPLOWBSTRACTC_SCPLOWBehavioural synchrony among individuals is essential for group-living organisms. It is still largely unknown how synchronization functions in a multilevel society, which is a nested assemblage of multiple social levels between many individuals. Our aim was to build a model that explained the synchronization of activity in a multilevel society of feral horses. We used multi-agent based models based on four hypotheses: A) horses do not synchronize, B) horses synchronize with any individual in any unit, C) horses synchronize only within units and D) horses synchronize across and within units, but internal synchronization is stronger. Our empirical data obtained from drone observations best supported hypothesis D. This result suggests that animals in a multilevel society coordinate with other conspecifics not only within a unit but at an inter-unit level. In this case, inter-individual distances are much longer than those in most previous models which only considered local interaction within a few body lengths.

sentences: An anaphase surveillance mechanism prevents micronuclei formation from mitotic errors Micronuclei are a hallmark of cancer and other human disorders and have recently been implicated in chromothripsis, a series of massive genomic rearrangements that may drive tumor evolution and progression. Here we show that Aurora B kinase mediates a surveillance mechanism that integrates error correction during anaphase with spatial control of nuclear envelope reformation to protect against micronuclei formation during human cell division. Using high-resolution live-cell imaging of human cancer and non-cancer cells we found that anaphase lagging chromosomes are often transient and rarely formed micronuclei. This strong bias against micronuclei formation relied on a midzone-based Aurora B phosphorylation gradient that assisted the mechanical transduction of spindle forces at the kinetochore-microtubule interface required for anaphase error correction, while delaying nuclear envelope reformation on lagging chromosomes, independently of microtubules. Our results uncover a new layer of protection against genomic instability and provide a strategy for the rational design of micronuclei-targeting therapies.

sentences: Structural snapshots of La Crosse virus polymerase reveal the mechanisms underlying Peribunyaviridae replication and transcription Segmented negative-strand RNA bunyaviruses encode a multi-functional polymerase that performs genome replication and transcription. Here, we establish conditions for in vitro activity of La Crosse virus polymerase and visualize by cryo-electron microscopy its conformational dynamics, unveiling the precise molecular mechanics underlying its essential activities. Replication initiation is coupled to distal duplex promoter formation, endonuclease movement, prime-and-realign loop extension and closure of the polymerase core that direct the template towards the active site. Transcription initiation depends on C-terminal region closure and endonuclease movements that firstly prompt primer cleavage and secondly promote primer entry in the active site. Product realignment after priming, observed in replication and transcription, is triggered by the prime-and-realign loop. Switch to elongation results in polymerase reorganization and core region opening to facilitate template-product duplex formation in the active site cavity. The detailed uncovered mechanics will be crucial for future design of antivirals counteracting bunyaviral life-threatening pathogens.

sentences: MARS-Net: Deep learning-based segmentation pipeline for profiling cellular morphodynamics from multiple types of live cell microscopy Quantitative studies of cellular morphodynamics rely on extracting leading-edge velocity time-series based on accurate cell segmentation from live cell imaging. However, live cell imaging has numerous challenging issues about accurate edge localization. Here, we develop a deep learning-based pipeline, termed MARS-Net (Multiple-microscopy- type-based Accurate and Robust Segmentation Network), that utilizes transfer learning and the datasets from multiple types of microscopy to localize cell edges with high accuracy, allowing quantitative profiling of cellular morphodynamics. For effective training with the datasets from multiple types of live cell microscopy, we integrated the pretrained VGG-19 encoder with U-Net decoder and added dropout layers. Using this structure, we were able to train one neural network model that can accurately segment various live cell movies from phase contrast, spinning disk confocal, and total internal reflection fluorescence microscopes. Intriguingly, MARS-Net produced more accurate edge localization than the neural network models trained with single microscopy type datasets, whereas the standard U-Net could not increase the overall accuracy. We expect that MARS-Net can accelerate the studies of cellular morphodynamics by providing accurate segmentation of challenging live cell images.

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Protein detection in blood with single-molecule imaging The ability to identify and characterize individual biomarker protein molecules in patient blood samples could enable diagnosis of diseases at an earlier stage, when treatment is typically more effective. Single-molecule imaging offers a promising approach to accomplish this goal. However, thus far single-molecule imaging methods have only been used to monitor protein molecules in solutions or cell lysates, and have not been translated into the clinical arena. Furthermore, the detection limit of these methods has been confined to the picomolar (10-12 M) range. In many diseases, the circulating concentrations of biomarker proteins fall several orders of magnitude below this range. Here we describe Single-Molecule Augmented Capture (SMAC), a single-molecule imaging technique to visualize, quantify, and characterize individual protein molecules of interest down to the subfemtomolar (<10-15 M) range, even in complex biologic fluids. We demonstrate SMAC in a wide variety of applications with human blood samples, including the analysis of disease-associated secreted proteins, membrane proteins, and rare intracellular proteins. Using ovarian cancer as a model, a lethal malignancy in which high-grade disease is driven almost universally by alterations in the TP53 gene and frequently only diagnosed at a late, incurable stage, we found that mutant pattern p53 proteins are released into the blood in patients at an early stage in disease progression. SMAC opens the door to the application of single-molecule imaging in non-invasive disease profiling and allows for the analysis of circulating mutant proteins as a new class of highly specific disease biomarkers. The SMAC platform can be adapted to multiplex or high-throughput formats to characterize heterogeneous biochemical and structural features of circulating proteins-of-interest.

One Sentence SummaryA single-molecule imaging approach detects individual disease-specific protein molecules, including mutant intracellular proteins, in blood samples.

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gbpA and chiA genes are not uniformly distributed amongst diverse Vibrio cholerae Members of the bacterial genus Vibrio utilise chitin both as a metabolic substrate and a signal to activate natural competence. Vibrio cholerae is a bacterial enteric pathogen, sub-lineages of which can cause pandemic cholera. However, the chitin metabolic pathway in V. cholerae has been dissected using only a limited number of laboratory strains of this species. Here, we survey the complement of key chitin metabolism genes amongst 195 diverse V. cholerae. We show that the gene encoding GbpA, known to be an important colonisation and virulence factor in pandemic isolates, is not ubiquitous amongst V. cholerae. We also identify a putatively novel chitinase, and present experimental evidence in support of its functionality. Our data indicate that the chitin metabolic pathway within the V. cholerae species is more complex than previously thought, and emphasise the importance of considering genes and functions in the context of a species in its entirety, rather than simply relying on traditional reference strains.

Impact statementIt is thought that the ability to metabolise chitin is ubiquitous amongst Vibrio spp., and that this enables these species to survive in aqueous and estuarine environmental contexts. Although chitin metabolism pathways have been detailed in several members of this genus, little is known about how these processes vary within a single Vibrio species. Here, we present the distribution of genes encoding key chitinase and chitin-binding proteins across diverse Vibrio cholerae, and show that our canonical understanding of this pathway in this species is challenged when isolates from non-pandemic V. cholerae lineages are considered alongside those linked to pandemics. Furthermore, we show that genes previously thought to be species core genes are not in fact ubiquitous, and we identify novel components of the chitin metabolic cascade in this species, and present functional validation for these observations.

Data summaryThe authors confirm that all supporting data, code, and protocols have been provided within the article or through supplementary data files.

O_LINo whole-genome sequencing data were generated in this study. Accession numbers for the publicly-available sequences used for these analyses are listed in Supplementary Table 1, Table 2, and the Methods.
C_LIO_LIAll other data which underpin the figures in this manuscript, including pangenome data matrices, modified and unmodified sequence alignments and phylogenetic trees, original images of gels and immunoblots, raw fluorescence data, amplicon sequencing reads, and the R code used to generate Figure 7, are available in Figshare: https://dx.doi.org/10.6084/m9.figshare.13169189
(Note for peer-review: Figshare DOI is inactive but will be activated upon publication, please use temporary URL https://figshare.com/s/7795a2d80c13f694f8fa for review).
C_LI

O_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/430729v1_fig7.gif" ALT="Figure 7">
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org.highwire.dtl.DTLVardef@100c246org.highwire.dtl.DTLVardef@d295aborg.highwire.dtl.DTLVardef@1601f69org.highwire.dtl.DTLVardef@1ae3ecb_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFigure 7.C_FLOATNO ChiA-3-6xHis displays chitobiosidase and endochitinase activities, but not -N-acetylglucosaminidase activity. Lysates and supernatants included in Figures 6b and 6c were assayed for chitinase enzyme activity using a fluorometric chitinase assay kit (see Methods for details). Lysed cells from E. coli cultures harbouring pMJD157 and cultured in the presence of arabinose were the only samples which produced detectable and statistically significant signals on triacetylchitotriose and chitobiose substrates (a, b). No signal was detected in the presence of glucosaminide substrate (c). All plots are scaled equivalently. P = pellet; S = supernatant; EV = empty vector (pBAD33). Parametric t-tests performed where indicated: ns = not significant; *** = p<0.001; **** = p<0.0001.

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sentences: Estimating abundance and phenology from transect count data with GLMs Estimating population abundance is central to population ecology. With increasing concern over declining insect populations, estimating trends in abundance has become even more urgent. At the same time, there is an emerging in interest in quantifying phenological patterns, in part because phenological shifts are one of the most conspicuous signs of climate change. Existing techniques to fit activity curves (and thus both abundance and phenology) to repeated transect counts of insects (a common form of data for these taxa) frequently fail for sparse data, and often require advanced knowledge of statistical computing. These limitations prevent us from understanding both population trends and phenological shifts, especially in the at-risk species for which this understanding is most vital. Here we present a method to fit repeated transect count data with Gaussian curves using linear models, and show how robust abundance and phenological metrics can be obtained using standard regression tools. We then apply this method to eight years of Baltimore checkerspot data using generalized linear models (GLMs). This case study illustrates the ability of our method to fit even years with only a few non-zero survey counts, and identifies a significant negative relationship between population size and annual variation in thermal environment (in growing degree days). We believe our new method provides a key tool to unlock previously-unusable sparse data sets, and may provide a useful middle ground between ad hoc metrics of abundance and phenology and custom-coded mechanistic models.

sentences: Sleep deprivation exacerbates seizures and diminishes GABAergic tonic inhibition Patients with epilepsy report that sleep deprivation is a common trigger for breakthrough seizures. The basic mechanism of this phenomenon is unknown. In the Kv1.1-/- mouse model of epilepsy, daily sleep deprivation indeed exacerbated seizures though these effects were lost after the 3rd day. Sleep deprivation also accelerated mortality in ~52% of Kv1.1-/- mice, not observed in controls. Voltage-clamp experiments on the day after recovery from sleep deprivation showed reductions in GABAergic tonic inhibition in dentate granule cells both in Kv1.1-/- and wild-type mice. Our results suggest that sleep deprivation is detrimental to seizures and survival, possibly due to reductions in GABAergic tonic inhibition.

sentences: Investigating the human host - ssRNA virus interaction landscape using the SMEAGOL toolbox Viruses are intracellular parasites that need their host cell to reproduce. Consequently, they have evolved numerous mechanisms to exploit the molecular machinery of their host cells, including the broad spectrum of host RNA-binding proteins (RBPs). However, the RBP interactome of viral genomes and the consequences of these interactions for infection are still to be mapped for most RNA viruses. To facilitate these efforts we have developed SMEAGOL, a fast and user-friendly toolbox to analyze the enrichment or depletion of RBP binding motifs across RNA sequences (https://github.com/gruber-sciencelab/SMEAGOL). To shed light on the interaction landscape of RNA viruses with human host cell RBPs at a large scale, we applied SMEAGOL to 197 single-stranded RNA (ssRNA) viral genome sequences. We find that the majority of ssRNA virus genomes are significantly enriched or depleted in binding motifs for human RBPs, suggesting selection pressure on these interactions. Our analysis provides an overview of potential virus - RBP interactions, covering the majority of ssRNA viral genomes fully sequenced to date, and represents a rich resource for studying host interactions vital to the virulence of ssRNA viruses. Our resource and the SMEAGOL toolbox will support future studies of virus / host interactions, ultimately feeding into better treatments.

sentences: Photodynamic Inactivation reduces the diversity and changes the composition of bacterial and fungal communities associated with leaf surfaces Plant surfaces are colonized by a myriad of microorganisms including mutualistic strains and pathogens. Particularly in agricultural systems applications are required that protect the plants against pathogens without negative side effects on the environment and humans. Photodynamic Inactivation (PDI) has been demonstrated to be a promising approach to efficiently fight plant pathogens. Based on its mechanism of action, the light-induced and photosensitizer-mediated overproduction of reactive oxygen species in target cells, PDI is likely to generally inactivates microorganisms on plants irrespective of their pathogenicity. In order to prove this hypothesis we used next-generation 16S rRNA gene amplicon sequencing to characterize the bacterial and fungal communities associated with leaf surfaces of Arabidopsis thaliana before and after the photodynamic treatment using the chlorine e6 derivative B17-0024 as photoactive compound and showed that this treatment reduced the microbial richness and altered the microbial community composition. These findings may help to develop effective pathogen-control strategies and may also stimulate research on plant-microbe interactions exploiting the potential of PDI.

sentences: TraB is a novel component of the ER-Mitochondria contact site (EMCS) with dual roles in ER-Mitochondrial tethering and mitophagy ER-mitochondria contact sites (EMCSs) are important for mitochondrial function. Here, we have identified a novel eukaryotic EMCS complex, comprising a family of uncharacterised mitochondrial outer-membrane proteins, TraB1 and the ER protein, VAP27-1. In Arabidopsis, there are two TraB1 isoforms and the trab1a/trab1b double mutant exhibits abnormal mitochondrial morphology, strong starch accumulation and impaired energy metabolism, indicating that these proteins are essential for normal mitochondrial function. Moreover, TraB1 proteins also interact with ATG8 in order to regulate mitochondrial degradation (mitophagy). The turnover of depolarised mitochondria is significantly reduced in both trab1a/b and VAP27 mutants (vap27-1/3/4/6) under mitochondrial stress conditions, with an increased population of dysfunctional mitochondria present in the cytoplasm. Consequently, plant recovery after stress is significantly perturbed. A similar phenotype is found in both autophagy mutants (atg5 and atg7), suggesting that TraB1 regulated mitophagy and ER-mitochondrial tethering are two closely related processes, necessary for normal mitochondrial function. Taken together, we ascribe a dual role to TraB1 which is a novel component of the EMCS complex in eukaryotes, regulating both tethering of the mitochondria to the ER and mitophagy.

sentences: Predicting Individual Task Contrasts From Resting-state Functional Connectivity using a Surface-based Convolutional Network Task-based and resting-state represent the two most common experimental paradigms of functional neuroimaging. While resting-state offers a flexible and scalable approach for characterizing brain function, task-based techniques provide superior localization. In this paper, we build on recent deep learning methods to create a model that predicts task-based contrast maps from resting-state fMRI scans. Specifically, we propose BrainSurfCNN, a surface-based fully-convolutional neural network model that works with a representation of the brains cortical sheet. Our model achieves state of the art predictive accuracy on independent test data from the Human Connectome Project and yields individual-level predicted maps that are on par with the target-repeat reliability of the measured contrast maps. We also demonstrate that BrainSurfCNN can generalize remarkably well to novel domains with limited training data.

sentences: hyperTRIBER: a flexible R package for the analysis of differential RNA editing AO_SCPLOWBSTRACTC_SCPLOWRNA editing by ADAR (adenosine deaminase acting on RNA) is gaining an increased interest in the field of post-transcriptional regulation. Fused to an RNA-binding protein (RBP) of interest, the catalytic activity of ADAR results in A-to-I RNA edits, whose identification will determine RBP-bound RNA transcripts. However, the computational tools available for their identification and differential RNA editing statistical analysis are limited or too specialised for general-purpose usage. Here we present hyperTRIBER, a flexible suite of tools, wrapped into a convenient R package, for the detection of differential RNA editing. hyperTRIBER is applicable to complex scenarios and experimental designs, and provides a robust statistical framework allowing for the control for coverage of reads at a given base, the total expression level and other co-variates. We demonstrate the capabilities of our approach on HyperTRIBE RNA-seq data for the detection of bound RNAs by the N6-methyladenosine (m6A) reader protein ECT2 in Arabidopsis roots. We show that hyperTRIBER finds edits with a high statistical power, even where editing proportions and RNA transcript expression levels are low, together demonstrating its usability and versatility for analysing differential RNA editing.

sentences: A free classified advertising website is a high-risk unregulated source of domestic wild boar, pot-bellied pigs, and their hybrids with domestic swine in Canada: Implications for African Swine Fever Risk Preparedness Invasive wild pigs are a global problem and are established in large areas across Canada. Domestic European wild boar were first introduced to Canadian farms in the 1980s, and their escape and intentional release have since led to free-ranging populations, which have expanded rapidly in numbers and range. Wild pigs are associated with widespread damage to crops and natural environments, as well as risks of disease transmission. African Swine Fever (ASF) is a highly contagious viral disease of global concern, that can spread between domestic and free-ranging swine and cause catastrophic economic impacts. As ASF preparedness requires a detailed understanding of the distribution and movements of all pig types, this paper aimed to characterize an unregulated  grey swine market in Canada to understand risks to domestic swine production and potential contributions to free-ranging wild pig populations. Kijiji.ca is a free Canadian internet classified advertising service, almost exclusively cash-based with no receipts and few, if any, records kept of transactions. We monitored Kijiji for sales of domestic wild boar, pot-bellied pigs, and their hybrids across Canada over two months from April 28 - June 30, 2021. Data was collected from all advertisements, including how the seller labelled pigs breed, age, sex, number for sale, sexual intactness, presence of tattoos and/or ear tags, as well as the date and location of the posting. Locations were mapped and compared to the spatial distribution of existing free-ranging wild pigs in Canada to identify areas they were likely to facilitate new populations or potentially supplement and genetically diversify existing populations. We identified 151 advertisements on Kijiji, the most coming from Ontario (34.4%; n = 52) and Alberta (29.1%; n = 44), followed by Saskatchewan (11.3%; n = 17), British Columbia (10.6%; n = 16), Nova Scotia (7.3%; n = 11), New Brunswick (4.0%; n = 6), Manitoba (2.0%; n = 3), Quebec (0.7%; n = 1), and Prince Edward Island (0.7%; n = 1). No listings were observed in Newfoundland and Labrador, or Yukon, Nunavut and Northwest Territories. Overall, 62 (41%) of advertisements were within watersheds with existing wild pigs that may contribute to supplementing additional swine, and 89 (59%) were within watersheds where wild pigs have not yet been identified. African swine fever preparedness should include policies and action to address this unmonitored and unregulated sale of swine in Canada, including ear tags, tattoos, genetic analysis, and mandatory reporting.

sentences: Positive feedback promotes terrestrial emergence behaviour in an amphibious fish Major ecological transitions such as the invasion of land by aquatic vertebrates have been hypothesised to be facilitated by positive feedback between habitat choice and phenotypic plasticity. We tested whether aquatic hypoxia, emergence behaviour, and plastic changes in gill surface area could create such a positive feedback loop and drive an amphibious fish to spend increasing amounts of time out of water. We found that terrestrially acclimated amphibious mangrove rivulus Kryptolebias marmoratus were more sensitive to, and less tolerant of, aquatic hypoxia relative to water-acclimated fish, which are necessary trade-offs for positive feedback to occur. Next, we acclimated fish to normoxic or hypoxic water with the opportunity to emerge for 7d to test the predictions that fish in hypoxic conditions should regularly leave water, reduce gill surface area, and become less hypoxia tolerant. Consistent with these predictions, fish in severe hypoxia spent almost 50% of the time out of water and coverage of the gill lamellae by an inter-lamellar cell mass almost doubled. Hypoxia acclimated fish were also more sensitive to acute aquatic hypoxia (emergence at higher oxygen levels), and lost equilibrium faster in hypoxic water compared to control fish. Thus, we show that a positive feedback loop develops in amphibious fish where emergence behaviour begets further emergence behaviour, driven by gill remodelling which reduces aquatic respiratory function. Such a scenario may explain how amphibious behaviour has repeatedly evolved in fishes that occupy hypoxic aquatic habitats despite the associated challenges of life on land.

sentences: A murine model of hnRNPH2-related neurodevelopmental disorder recapitulates clinical features of human disease and reveals a mechanism for genetic compensation of HNRNPH2 Mutations in HNRNPH2 cause an X-linked neurodevelopmental disorder with a phenotypic spectrum that includes developmental delay, intellectual disability, language impairment, motor function deficits, and seizures. More than 90% of patients with this disorder have a missense mutation within or adjacent to the nuclear localization signal (NLS) of hnRNPH2, although the specific pathogenic consequences of these mutations have not been examined. Here we found that hnRNPH2 NLS mutations result in reduced interaction with the nuclear transport receptor Kap{beta}2 in vitro and in cultured human cells. These mutations also cause modest accumulation of hnRNPH2 in the cytoplasm, suggesting that mislocalization of the protein might contribute to pathogenesis. We generated two knock-in mouse models with human-equivalent mutations in the endogenous mouse gene Hnrnph2, as well as Hnrnph2 knockout (KO) mice, and subjected them to extensive, deep phenotyping. Mutant knock-in mice displayed a spectrum of phenotypes that recapitulated aspects of the human disorder, including reduced survival in males, craniofacial abnormalities, impaired motor functions, and increased susceptibility to audiogenic seizures. Mutant knock-in male mice developed more severe phenotypes than female mice, likely due to differences in X-chromosome gene dosage. In contrast, two independent lines of Hnrnph2 KO mice showed no detectable phenotypes. Notably, KO mice had upregulated expression of Hnrnph1, a close paralog of Hnrnph2, whereas mutant Hnrnph2 knock-in mice failed to upregulate Hnrnph1. Thus, genetic compensation by Hnrnph1 might be sufficient to counteract the loss of hnRNPH2. These findings suggest that the pathogenesis of HNRNPH2-related disorder in humans may be driven by a toxic gain of function or a complex loss of HNRNPH2 function with impaired compensation by HNRNPH1. The carefully phenotyped mutant knock-in mice described here are an important resource for preclinical studies to assess the potential benefit of either gene replacement or therapeutic knockdown of mutant hnRNPH2.

sentences: Using genomic prediction to detect microevolutionary change of a quantitative trait Detecting microevolutionary responses to natural selection by observing temporal changes in individual breeding values is challenging. The collection of suitable datasets can take many years and disentangling the contributions of the environment and genetics to phenotypic change is not trivial. Furthermore, pedigree-based methods of obtaining individual breeding values have known biases. Here, we apply a genomic prediction approach to estimate breeding values of adult weight in a 35-year dataset of Soay sheep (Ovis aries). During the study period adult body weight decreased, but the underlying genetic component of body weight increased, at a rate that is unlikely to be attributable to genetic drift. Thus cryptic microevolution of greater adult body weight has probably occurred. Using genomic prediction to study microevolution in wild populations can remove the requirement for pedigree data, potentially opening up new study systems for similar research.

sentences: Intestinal organoids expose heterogeneity in SARS-CoV-2 susceptibility Gastrointestinal effects associated with COVID-19 are highly variable for reasons that are not understood. In this study, we used intestinal organoid-derived cultures differentiated from primary human specimens as a model to examine inter-individual variability. Infection of intestinal organoids derived from different donors with SARS-CoV-2 resulted in orders of magnitude differences in virus replication in small intestinal and colonic organoid-derived monolayers. Susceptibility to infection correlated with ACE2 expression level and was independent of donor demographic or clinical features. ACE2 transcript levels in cell culture matched the amount of ACE2 in primary tissue indicating this feature of the intestinal epithelium is retained in the organoids. Longitudinal transcriptomics of organoid-derived monolayers identified a delayed yet robust interferon signature, the magnitude of which corresponded to the degree of SARS-CoV-2 infection. Interestingly, virus with the Omicron variant spike protein infected the organoids with the highest infectivity, suggesting increased tropism of the virus for intestinal tissue. These results suggest that heterogeneity in SARS-CoV-2 replication in intestinal tissues results from differences in ACE2 levels, which may underlie variable patient outcomes.

sentences: JAG1 intracellular domain acts as a transcriptional cofactor that forms an oncogenic transcriptional complex with DDX17/SMAD3/TGIF2 Jagged1 (JAG1) is a Notch ligand that contact-dependently activates Notch receptors and regulates cancer progression. The JAG1 intracellular domain (JICD1) is generated from JAG1, such as the formation of NOTCH1 intracellular domain (NICD1), however, the role of JICD1 in tumorigenicity has not been comprehensively elucidated. Herein, we revealed that JICD1 induced astrocytes to acquire several cancer stem cell properties, including tumor formation, invasiveness, stemness, and resistance to chemotherapy and radiotherapy. The transcriptome, ChIP-sequencing, and proteomic analyses revealed that JICD1 increased SOX2 expression by forming a transcriptional complex with DDX17, SMAD3, and TGIF2. Furthermore, JICD1-driven tumorigenicity was directly regulated by SOX2. Therefore, our results demonstrated that, like NICD1, JICD1 acts as a transcriptional cofactor in the formation of the DDX17/SMAD3/TGIF2 transcriptional complex, leading to oncogenic transformation.

sentences: SARS-CoV-2 bearing a mutation at the S1/S2 cleavage site exhibits attenuated virulence and confers protective immunity Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by host furin protease. Proteolytic cleavage activates the spike protein and influences both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogensis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited a low growth property. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, indicating the attenuated variant nature of S gene mutants. We reproduced the attenuated growth of S gene mutants in primary differenciated human airway epithelial cells. This transient infection was enough to induce protective neutralizing antibodies crossreacting with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of SARS-CoV-2 and highlights the potential benefits of S gene mutants as potential immunogens.

sentences: q-mer analysis: a generalized method for analyzing RNA-Seq data. RNA-Seq data are usually summarized by counting the number of transcript reads aligned to each gene. However, count-based methods do not take alignment information, where and how each read was mapped in the gene, into account. This information is essential to characterize samples accurately. In this study, we developed a method to summarize RNA-Seq data without losing alignment information. To include alignment information, we introduce "q-mer analysis," which summarizes RNA-Seq data with 4q kinds of q-length oligomers. Using publicly available RNA-Seq datasets, we demonstrate that at least q [greater double equals] 9 is required for capturing alignment information in Homo sapiens. Furthermore, principal component analysis showed that q-mer analysis with q = 14 linearly distinguished samples from controls, while a count-based method failed. These results indicate that alignment information is essential to characterize transcriptomics samples. In conclusion, we introduce q-mer analysis to include alignment information in RNA-Seq analysis and demonstrate the superiority of q-mer analysis over count-based methods in that q-mer analysis can distinguish case samples from controls. Combining RNA-Seq research with q-mer analysis could be useful for identifying distinguishing transcriptomic features that could provide hypotheses for disease mechanisms.

sentences: Acetylation regulates the oligomerization state and activity of RNase J, the major ribonuclease of Helicobacter pylori In Helicobacter pylori, post-transcriptional regulation strongly relies on the activity of an RNA degradosome, composed of the essential ribonuclease RNase J and the DEAD-box RNA helicase RhpA. Here, we describe post-translational modifications of this protein complex that affect its activity. Cell-extracted RNase J is acetylated on multiple residues, one of which, K649, strongly impacts RNase J oligomerization, which in turn influences ribonuclease activity. Corroborating the link between oligomerization and activity, mutations targeting K649 and other residues affect the dimerization and in vitro activity of RNase J. Our crystal structure of RNase J reveals three loops that gate access to the active site and rationalizes how oligomerization state influences activity. The acetylated residues of RNase J are important for H. pylori morphology, highlighting that the modifications affect the RNase J cellular function. We propose acetylation as a regulatory level controlling the activity of RNase J and the H. pylori RNA degradosome.

sentences: Comparing two circular distributions: advice for effective implementation of statistical procedures in biology Many biological variables, often involving timings of events or directions, are recorded on a circular rather than linear scale, and need different statistical treatment for that reason. A common question that is asked of such circular data involves comparison between two groups or treatments: Are the populations from which the two samples drawn differently distributed around the circle? For example, we might ask whether the distribution of directions from which a stalking predator approaches its prey differs between sunny and cloudy conditions; or whether the time of day of mating attempts differs between lab mice subject to one of two hormone treatments. An array of statistical approaches to these questions have been developed. We compared 18 of these (by simulation) in terms of both abilities to control type I error rate near the nominal value, and statistical power. We found that only eight tests offered good control of type I error in all our test situations. Of these eight, we are able to identify Watsons U2 test and MANOVA based on trigonometric functions of the data as offering the best power in the overwhelming majority of our test circumstances. There was often little to choose between these tests in terms of power, and no situation where either of the remaining six tests offered substantially better power than either of these. Hence, we recommend the routine use of either Watsons U2 test or MANOVA when comparing two samples of circular data.

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Modelling eDNA transport in river networks reveals highly resolved spatio-temporal patterns of freshwater biodiversity O_LIThe ever-increasing threats to riverine biodiversity call for the development of novel approaches for a complete assessment of biodiversity across highly resolved spatial, temporal and taxonomic scales. Past studies on riverine biodiversity patterns were often restricted to spatially scattered data, focused on specific taxonomic groups, and disregarded the temporal dimension, preventing a universal understanding of relationships between biodiversity and stream size across spatial, temporal and taxonomic scales. Recent advances in the joint use of environmental DNA (eDNA) data and novel mechanistic models for eDNA transport in river networks have the potential to uncover the full structure of riverine biodiversity at an unprecedented spatial resolution, hence providing fundamental insights into ecosystem processes and offering a basis for targeted conservation measures.
C_LIO_LIHere, we applied a mechanistic model (i.e., the eDITH model) to a metabarcoding dataset covering three taxonomic groups (fish, invertebrates and bacteria) and three seasons (spring, summer and autumn) for a 740-km2 Swiss catchment, sampled for eDNA at 73 sites.
C_LIO_LIUsing the mechanistic model, we upscaled eDNA-based biodiversity predictions to more than 1900 individual reaches, allowing an assessment of patterns of - and {beta}-diversity across seasons and taxonomic groups at a space-filling, fine scale over the whole network.
C_LIO_LIWe found that both predicted - and {beta}-diversity varied considerably depending on both season and taxonomic group. Predicted fish -diversity increased in the downstream direction at all seasons, while invertebrate and bacteria -diversity either decreased downstream or was not significantly related to position within network, depending on the season. Spatial {beta}-diversity was mostly found to be decreasing in the downstream direction, and this was the case for all seasons for bacteria. Temporal {beta}-diversity was mostly found to be increasing downstream. In general, genus richness values predicted by the model were found to be higher than those obtained by directly analyzing the eDNA data. Overall, stream size (subsumed by drainage area) was generally a poor predictor of patterns of predicted - and {beta}-diversities. Conversely, riverine biodiversity is shaped by a complex interplay of environmental variables, abiotic and biotic factors, which need be taken into account for a correct assessment of its structure.
C_LI

sentences: Fenbendazole resistance in Heterakis gallinarum, the vector of Histomonas meleagridis, the causative agent of Blackhead Disease in poultry. Due to their ubiquity, management of parasites is a common and important factor for profitable production of poultry. Heterakis gallinarum, the cecal nematode, is the most common nematode parasite of poultry. While typically causing no pathology on its own, H. gallinarum is the vector of Histomonas meleagridis, a protozoan parasite that causes blackhead disease. Histomonas meleagridis is highly pathogenic in turkeys, potentially causing high mortality. In contrast, disease caused by H. meleagridis is much less severe in chickens, where it primarily reduces productivity without manifestations of clinical disease. There are no approved treatments for H. meleagridis, making control reliant on control of the helminth vector through the use of fenbendazole (FBZ) the only drug labeled for treatment of H. gallinarum in the United States We were contacted by an industry veterinarian regarding health-related concerns in a broiler-breeder house due to histomoniasis, despite frequent anthelmintic treatments. Since we had recently diagnosed resistance to FBZ in Ascaridia dissimilis, a closely related nematode of turkeys, we were interested to determine if H. gallinarum had also evolved resistance to FBZ. Heterakis gallinarum eggs were isolated from litter collected from the house and used to infect 108 chickens. Treatment groups included a non-treated control, a label-dose and a 2X-label dose of FBZ, with 36 birds per group divided into two replicate pens of 18 birds each. Birds were placed at 1-day post hatch, and at 3 weeks of age were infected with 150 embryonated eggs via oral gavage. Two weeks post infection, treated birds were administered a minimum of either a label- or 2X label-dose of FBZ in water for 5 days (SafeGuard(R) Aquasol, 1mg/kg BW). To ensure that all birds consumed the full intended dose at a minimum, the dosage was calculated using 1.25 times the average body weight. One-week post treatment, birds were euthanized, ceca removed, and parasites enumerated. Efficacy was calculated by comparing the total numbers of worms recovered from each treatment group to the numbers recovered in the non-treated control group. There were no significant differences in worm numbers recovered from any of the three groups (p-value=0.81). There also was no efficacy benefit to treatment with a 2X dose; H. gallinarum worm counts were reduced by 42.7% and 41.4%, for the label and 2X dosages, respectively. These data provide strong evidence that H. gallinarum has developed resistance to FBZ. Consequently, in houses infected with FBZ-resistant H. gallinarum, H. meleagridis will be able to cycle through the birds in an unrestricted manner. Further investigation is needed to determine the prevalence of resistance in H. gallinarum on chicken farms, but it is clear this has the potential to have a large-scale economic impact on the poultry industry. These data when viewed together with our recent findings of FBZ resistance in A. dissimilis, suggest that drug resistance in ascarid nematodes may be an important emerging problem on poultry operations.

sentences: Engineered chimeras unveil swappable modular features of fatty acid and polyketide synthase acyl carrier proteins The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the E. coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based crosslinking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, -helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step towards the strategic engineering of  unnatural natural products.

sentences: Predicting microbiome compositions through deep learning. Microbes can form complex communities that perform critical functions in maintaining the integrity of their environment or their hosts well-being. Rationally managing these microbial communities requires improving our ability to predict how different species assemblages affect the final species composition of the community. However, making such a prediction remains challenging because of our limited knowledge of the diverse physical, biochemical, and ecological processes governing microbial dynamics. To overcome this challenge, here we present a deep learning framework that automatically learns the map between species assemblages and community compositions from training data only, without knowledge of any of the above processes. First, we systematically validate our framework using synthetic data generated by classical population dynamics models. Then, we apply it to experimental data of both in vitro and in vivo communities, including ocean and soil microbial communities, Drosophila melanogaster gut microbiota, and human gut and oral microbiota. In particular, we show how our framework learns to perform accurate out-of-sample predictions of complex community compositions from a small number of training samples. Our results demonstrate how deep learning can enable us to understand better and potentially manage complex microbial communities.

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Foldseek: fast and accurate protein structure search Highly accurate structure prediction methods are generating an avalanche of publicly available protein structures. Searching through these structures is becoming the main bottleneck in their analysis. Foldseek enables fast and sensitive comparisons of large structure sets. It reaches sensitivities similar to state-of-the-art structural aligners while being four orders of magnitude faster. Foldseek is free open-source software available at foldseek.com and as a webserver at search.foldseek.com.

Contactsoeding@mpinat.mpg.de, martin.steinegger@snu.ac.kr

sentences: Combining metabolomics and experimental evolution reveals key mechanisms underlying longevity differences in laboratory evolved Drosophila melanogaster populations Experimental evolution with Drosophila melanogaster has been used extensively for decades to study aging and longevity. In recent years, the addition of DNA and RNA sequencing to this framework has allowed researchers to leverage the statistical power inherent to experimental evolution study the genetic basis of longevity itself. Here we incorporated metabolomic data into to this framework to generate even deeper insights into the physiological and genetic mechanisms underlying longevity differences in three groups of experimentally evolved D. melanogaster populations with different aging and longevity patterns. Our metabolomic analysis found that aging alters mitochondrial metabolism through increased consumption of NAD+ and increased usage of the TCA cycle. Combining our genomic and metabolomic data produced a list of biologically relevant candidate genes. Among these candidates, we found significant enrichment for genes and pathways associated with neurological development and function, and carbohydrate metabolism. While we do not explicitly find enrichment for aging canonical genes, neurological dysregulation and carbohydrate metabolism are both known to be associated with accelerated aging and reduced longevity. Taken together, our results provide plausible genetic mechanisms for what might be driving longevity differences in this experimental system. More broadly, our findings demonstrate the value of combining multiple types of omic data with experimental evolution when attempting to dissect mechanisms underlying complex and highly polygenic traits like aging.

sentences: Cheetah: a computational toolkit for cybergenetic control Advances in microscopy, microfluidics and optogenetics enable single-cell monitoring and environmental regulation and offer the means to control cellular phenotypes. The development of such systems is challenging and often results in bespoke setups that hinder reproducibility. To address this, we introduce Cheetah - a flexible computational toolkit that simplifies the integration of real-time microscopy analysis with algorithms for cellular control. Central to the platform is an image segmentation system based on the versatile U-Net convolutional neural network. This is supplemented with functionality to robustly count, characterise and control cells over time. We demonstrate Cheetahs core capabilities by analysing long-term bacterial and mammalian cell growth and by dynamically controlling protein expression in mammalian cells. In all cases, Cheetahs segmentation accuracy exceeds that of a commonly used thresholding-based method, allowing for more accurate control signals to be generated. Availability of this easy-to-use platform will make control engineering techniques more accessible and offer new ways to probe and manipulate living cells.

sentences: Loss of transgene expression limits liver gene therapy in primates Introductory ParagraphEfforts to improve liver gene therapy have focused on next-generation adeno-associated virus (AAV) vector capsids, transgene delivery, and immunomodulating drugs, such as corticosteroids, to avoid destructive T-cell responses. We conducted a detailed characterization of AAV transduction in nonhuman primate liver across multiple capsids and transgenes to better define interactions that may limit stable and efficient transgene expression. We show that the initial transduction of hepatocytes is high, but the transduction rapidly diminishes to a lower stable baseline of <1% of cells, even though ~10% of the cells retain vector DNA that is localized within the nucleus. Further characterization showed genomic vector integration at frequencies of 1/100 to 1/1,000 genomes, suggesting that one mechanism for stable expression may occur via genome integration. These studies suggest that strategies to enhance durable transgene expression by activating retained nuclear episomes or by increasing the frequency of vector integrations may improve liver directed AAV gene therapy.

sentences: Chronic obstructive pulmonary disease and cigarette smoke exposure lead to dysregulated MAIT cell activation by bronchial epithelial cells Chronic obstructive pulmonary disease (COPD) is associated with airway inflammation, increased infiltration by CD8+ T lymphocytes, and infection-driven exacerbations. COPD is most commonly caused by cigarette smoke (CS), however the mechanisms driving development of COPD in some smokers but not others are incompletely understood. Lung-resident mucosal-associated invariant T (MAIT) cells play a role in both microbial infections and inflammatory diseases. MAIT cell frequency is reduced in the peripheral blood of individuals with COPD, however the role of MAIT cells in COPD pathology is unknown. Here, we examined MAIT cell activation in response to CS-exposed primary human bronchial epithelial cells (BEC) from healthy, COPD, or smoker donors. We observed significantly higher MAIT cell responses to COPD BEC than healthy BEC. However, COPD BEC stimulated a smaller fold-increase in MAIT cell response despite increased microbial infection. For all donor groups, CS-exposed BEC elicited reduced MAIT cell responses; conversely, CS exposure increased ligand-mediated MR1 surface translocation in healthy and COPD BEC. Our data demonstrate MAIT cell activation is dysregulated in the context of CS and COPD. MAIT cells could contribute to CS- and COPD-associated inflammation through both inappropriate activation and reduced early recognition of bacterial infection, contributing to microbial persistence and COPD exacerbations.

sentences: Dynamic Sex Differences in Reactive and Appetitive Aggression Aggression is an evolutionarily conserved, adaptive component of social behavior. Studies in male mice illustrate that aggression is influenced by numerous factors including the degree to which an individual finds aggression rewarding and will work for access to attack and subordinate mice. While such studies have expanded our understanding of the molecular and circuit mechanisms of male aggression very little is known about female aggression, owed in part to limited availability of valid mouse models in females. Here we use an ethologically relevant model of male vs. female aggression by pair housing adult male and female outbred CFW mice with opposite sex cage mates. We assess reactive (defensive) aggression in the resident intruder (RI) test and appetitive (rewarding) aggression in the aggression conditioned place preference (CPP) and operant self-administration (SA) tests. Our results show dramatic sex differences in both qualitative and quantitative aspects of reactive vs. appetitive aggression. Males exhibit more wrestling and less investigative behavior during RI, find aggression rewarding and will work for access to a subordinate to attack. Females exhibit more bites, alternate between aggressive behaviors and investigative behaviors more readily during RI, however, they do not find aggression to be rewarding or reinforcing. These results establish sex differences in aggression in mice, providing an important resource for the field to better understand the circuit and molecular mechanisms of aggression in both sexes.

sentences: GTP-bound E. coli FtsZ filaments are composed of Tense monomers: A DNP NMR study using interface detection. FtsZ filaments are the major structural component of the bacterial Z-ring and are drivers of bacterial division. While crystal structures for FtsZ from some gram positive bacteria in the presence of GTP-analog like compounds suggest the possibility of a high energy "Tense" conformation, to date it remains an important question to elucidate whether this Tense form is the dominant form in filaments. Using dynamic nuclear polarization (DNP) solid-state NMR and differential isotopic labelling, we directly detect residues located at the inter-monomer interface of GTP-bound WT Escherichia coli FtsZ filaments. We combine chemical shift prediction, homology modelling, and heteronuclear dipolar recoupling techniques to characterize the E. coli FtsZ filament interface and demonstrate that the monomers in active filaments assume a Tense conformation.

sentences: Automated Optimization of Residual Reduction Algorithm Parameters in OpenSim The residual reduction algorithm (RRA) in OpenSim is designed to improve dynamic consistency of kinematics and ground reaction forces in movement simulations of musculoskeletal models. RRA requires the user to select numerous tracking weights for the joint kinematics to reduce residual errors. Selection is often performed manually, which can be time-consuming and is unlikely to yield optimal tracking weights. A multi-heuristic optimization algorithm was used to expedite tracking weight decision making to reduce residual errors. This method produced more rigorous results than manual iterations and although the total computation time was not significantly reduced, this method does not require the user to monitor the algorithms progress to find a solution, thereby reducing manual tuning. Supporting documentation and code to implement this optimization is freely provided to assist the community with developing movement simulations.

sentences: TKGWV2: An ancient DNA relatedness pipeline for ultra-low coverage whole genome shotgun data Estimation of genetically related individuals is playing an increasingly important role in the ancient DNA field. In recent years, the numbers of sequenced individuals from single sites have been increasing, reflecting a growing interest in understanding the familial and social organisation of ancient populations. Although a few different methods have been specifically developed for ancient DNA, namely to tackle issues such as low-coverage homozygous data, they require a 0.1 - 1x minimum average genomic coverage per analysed pair of individuals between. Here we present an updated version of a method that enables estimates of 1st and 2nd-degrees of relatedness with as little as 0.026x average coverage, or around 1.3 million aligned reads per sample - 4 times less data than 0.1x. By using simulated data to estimate false positive error rates, we further show that a threshold even as low as 0.012x, or around 600,000 reads, will always show 1st-degree relationships as related. Lastly, by applying this method to published data, we are able to identify previously undocumented relationships using individuals previously excluded from kinship analysis due to their very low coverage. This methodological improvement has the potential to enable relatedness estimation on ancient whole genome shotgun data during routine low-coverage screening, and therefore improve project management when decisions need to be made on which individuals are to be further sequenced.

sentences: Proximity-based labeling reveals DNA damage-induced N-terminal phosphorylation of fused in sarcoma (FUS) leads to distinct changes in the FUS protein interactome. Cytoplasmic accumulation of the RNA/DNA binding protein, fused in sarcoma (FUS), into inclusions is a common hallmark of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) pathology. We have previously shown that DNA damage can trigger the cytoplasmic accumulation of an N-terminally phosphorylated FUS. However, the functional consequences of N-terminal FUS phosphorylation are unknown. To gain insight into this question, we utilized proximity-dependent biotin labeling via ascorbate peroxidase 2 (APEX2) paired with mass-spectrometry (MS) to investigate whether N-terminal phosphorylation shifts the FUS protein-protein interaction network (interactome), and subsequently, its function. We report the first comparative analysis of the interactomes for three FUS variants: homeostatic wild-type FUS (FUS WT), a phosphomimetic variant of FUS (a proxy for N-terminally phosphorylated FUS, FUS PM), and a toxic FUS P525L mutant (a mutation that causes juvenile ALS, FUS P525L). Data are available via ProteomeXchange with identifier PXD026578. We demonstrate that compared to FUS WT and FUS P525L, the FUS PM interactome uniquely enriches for a set of cytoplasmic proteins that mediate mRNA metabolism and translation and nuclear proteins involved in spliceosome and DNA repair functions, respectively. We further identify and validate three proteins, VPS35, MOV10, and CLTA, as novel interacting partners of all three FUS variants. Lastly, we provide functional evidence that N-terminally phosphorylated FUS may disrupt homeostatic translation and steady state levels of specific mRNA transcripts. Taken together, these results highlight phosphorylation as a unique modulator of the FUS interactome and function.

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RapidoPGS: A rapid polygenic score calculator for summary GWAS data without a test dataset MotivationPolygenic scores (PGS) aim to genetically predict complex traits at an individual level. PGS are typically trained on genome-wide association summary statistics and require an independent test dataset to tune parameters. More recent methods allow parameters to be tuned on the training data, removing the need for independent test data, but approaches are computationally intensive. Based on fine-mapping principles, we present RapidoPGS, a flexible and fast method to compute PGS requiring summary-level GWAS datasets only, with little computational requirements and no test data required for parameter tuning.

ResultsWe show that RapidoPGS performs slightly less well than two out of three other widely-used PGS methods (LDpred2, PRScs, and SBayesR) for case-control datasets, with median r2 difference: -0.0092, -0.0042, and 0.0064, respectively, but up to 17,000-fold faster with reduced computational requirements. RapidoPGS is implemented in R and can work with user-supplied summary statistics or download them from the GWAS catalog.

Availability and implementationOur method is available with a GPL license as an R package from GitHub.

sentences: Neural systems underlying the learning of cognitive effort costs People balance the benefits of cognitive work against the costs of cognitive effort. Models that incorporate prospective estimates of the costs of cognitive effort into decision making require a mechanism by which these costs are learned. However, it remains open what brain systems are important for this learning, particularly when learning is not tied explicitly to a decision about what task to perform. In this fMRI experiment, we parametrically manipulated the level of effort a task requires by increasing task switching frequency across six task contexts. In a scanned learning phase, participants implicitly learned about the task switching frequency in each context. In a subsequent test phase, participants made selections between pairs of these task contexts. We modeled learning within a reinforcement learning framework, and found that effort expectations that derived from task-switching probability and response time (RT) during learning were the best predictors of later choice behavior. Prediction errors (PE) from these two models were associated with FPN during distinct learning epochs. Specifically, PE derived from expected RT was most correlated with the fronto-parietal network early in learning, whereas PE derived from expected task switching frequency was correlated with the fronto-parietal network late in learning. These results suggest that multiple task-related factors are tracked by the brain while performing a task that can drive subsequent estimates of effort costs.

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Pigment pattern morphospace of Danio fishes: evolutionary diversification and mutational effects Molecular and cellular mechanisms underlying differences in adult form remain largely unknown. Adult pigment patterns of fishes in the genus Danio, which includes zebrafish, D. rerio, include horizontal stripes, vertical bars, spots and uniform patterns, and provide an outstanding opportunity to identify causes of species level variation in a neural crest derived trait. Yet understanding such variation requires quantitative approaches to assess phenotypes, and such methods have been mostly lacking for pigment patterns. We introduce metrics derived from information theory that describe patterns and pattern variation in Danio fishes. We find that such metrics used singly and in multivariate combinations are suitable for distinguishing general pattern types, and can reveal even subtle phenotypic differences attributable to mutations. Our study provides new tools for analyzing pigment pattern in Danio and potentially other groups, and sets the stage for future analyses of pattern morphospace and its mechanistic underpinnings.

Summary statementWe provide quantitative metrics for studying pigment patterns of zebrafish and other species. These metrics are applicable to changes between species as well as impacts of laboratory induced mutations

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ModPhred: an integrative toolkit for the analysis and storage of nanopore sequencing DNA and RNA modification data MotivationDNA and RNA modifications can now be identified using Nanopore sequencing. However, we currently lack a flexible software to efficiently encode, store, analyze and visualize DNA and RNA modification data.

ResultsHere we present ModPhred, a versatile toolkit that facilitates DNA and RNA modification analysis from nanopore sequencing reads in a user-friendly manner. ModPhred integrates probabilistic DNA and RNA modification information within the FASTQ and BAM file formats, can be used to encode multiple types of modifications simultaneously, and its output can be easily coupled to genomic track viewers, facilitating the visualization and analysis of DNA and RNA modification information in individual reads in a simple and computationally efficient manner.

Availability and ImplementationModPhred is available at https://github.com/novoalab/modPhred, is implemented in Python3, and is released under an MIT license.

Supplementary DataSupplementary Data are available at Bioinformatics online.

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Effects of Spike Mutations in SARS-CoV-2 Variants of Concern on Human or Animal ACE2-Mediated Virus Entry and Neutralization SARS-CoV-2 is a zoonotic agent capable of infecting humans and a wide range of animal species. Over the duration of the pandemic, mutations in the SARS-CoV-2 Spike protein (S) have arisen in circulating viral populations, culminating in the spread of several variants of concern (VOC) with varying degrees of altered virulence, transmissibility, and neutralizing antibody escape. In this study, we employed lentivirus-based pseudotyped viruses that express specific SARS-CoV-2 S protein substitutions and cell lines that stably express ACE2 from nine different animal species to gain insights into the effects of VOC mutations on viral entry and antibody neutralization capability. All animal ACE2 receptors tested, except mink, support viral cell entry for pseudoviruses expressing the parental (prototype Wuhan-1) S at levels comparable to human ACE2. Most single S substitutions (e.g., 452R, 478K, 501Y) did not significantly change virus entry, although 614G and 484K resulted in a decreased efficiency in viral entry. Conversely, combinatorial VOC substitutions in the S protein were associated with significantly increased entry capacity of pseudotyped viruses compared to that of the parental Wuhan-1 pseudotyped virus. Similarly, infection studies using live ancestral (USA-WA1/2020), Alpha, and Beta SARS-CoV-2 viruses in hamsters revealed a higher replication potential for the Beta variant compared to the ancestral prototype virus. Moreover, neutralizing titers in sera from various animal species, including humans, were significantly reduced by single substitutions of 484K or 452R, double substitutions of 501Y-484K, 452R-484K and 452R-478K and the triple substitution of 501Y-484K-417N, suggesting that 484K and 452R are particularly important for evading neutralizing antibodies in human, cat, and rabbit sera. Cumulatively, this study reveals important insights into the host range of SARS-CoV-2 and the effect of recently emergent S protein substitutions on viral entry, virus replication and antibody-mediated viral neutralization.

Author summaryCells stably expressing ACE2 from various animals and a lentivirus-based SARS-CoV-2 pseudotyped virus assay were established to study SARS-CoV-2 cell entry. The results demonstrated that ACE2 from a wide range of animal species facilitate S-mediated virus entry into cells, which is supported by in silico data as well as natural and experimental infection studies. Pseudotyped viruses containing mutations in the RBD of S representative of the Alpha, Gamma, and especially Beta, variants of concern demonstrated that certain mutations are associated with increased viral entry compared to the parental S. The Beta variant was also observed to have a replicative advantage in vitro and in vivo compared to the prototype virus. Pseudotyped viruses containing combinatorial substitutions of 501Y-484K-417K, 614G-501Y-484K and 614G-501Y-484K-417N increased viral entry via ACE2 across multiple species. The 501Y or 478K single substitution did not significantly affect neutralizing capacity of immune sera compared to the prototype strain, but the addition of 484K or 452R substitutions significantly reduced the neutralizing titers.

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Repeated translocation of a supergene underlying rapid sex chromosome turnover in Takifugu fish Recent studies have revealed a surprising diversity of sex chromosomes in vertebrates. However, the detailed mechanism of their turnover is still elusive. To understand this process, it is necessary to compare closely related species in terms of sex-determining genes and the chromosomes harboring them. Here, we explored the genus Takifugu, in which one strong candidate sex-determining gene, Amhr2, has been identified. To trace the processes involved in transitions in the sex determination system in this genus, we studied 12 species and found that while the Amhr2 locus likely determines sex in the majority of Takifugu species, three species have acquired sex-determining loci at different chromosomal locations. Nevertheless, the generation of genome assemblies for the three species revealed that they share a portion of the male-specific supergene that contains a candidate sex-determining gene, GsdfY, along with genes that potentially play a role in male fitness. The shared supergene span approximately 100 kb and are flanked by two duplicated regions characterized by CACTA transposable elements. These results suggest that the shared supergene has taken over the role of sex-determining locus from Amhr2 in lineages leading to the three species, and repeated translocations of the supergene underlie the turnover of sex chromosomes in these lineages. These findings highlight the underestimated role of a mobile supergene in the turnover of sex chromosomes in vertebrates.

SignificanceAlthough turnover of sex chromosomes is very common in many vertebrate lineages, the transition process is still elusive. We studied the sex-determining region (SDR) of 12 congeneric fish species. We found that while nine species retained their ancestral SDR, three species had acquired derived SDRs. Although the derived SDRs resided in three different chromosomes, they harbored a shared supergene flanked by two putative transposable elements. The results highlight the underestimated role of a mobile supergene in turnover of sex chromosomes in vertebrates.

sentences: Copper dependent ERK1/2 phosphorylation is essential for the viability of neurons and not glia Intracellular Copper [Cu(I)] has been hypothesized to play role in the differentiation of the neurons. This necessitates understanding the role of Cu(I) not only in the neurons but also in the glia considering their anatomical proximity, contribution towards ion homeostasis, neuronal physiology, and neurodegeneration. In this study we did a systematic investigation of the changes in the cellular copper homeostasis during neuronal and glial differentiation and the pathways triggered by them. Our study demonstrates increased mRNA for the plasma membrane copper transporter CTR1 leading to an increased Cu(I) during neuronal (PC-12) differentiation. ATP7A is retained in the Trans Golgi Network (TGN) despite high Cu(I) demonstrating its utilization in triggering the pathways towards the neuronal differentiation. One of these pathways is ERK1/2 phosphorylation accompanying the differentiation of both PC-12 and human fetal brain derived neuronal progenitor cells. The study demonstrates that the ERK1/2 phosphorylation is essential for the viability of the neurons. In contrast, differentiated C-6 (glia) cells contain low intracellular copper and significant downregulation of the ERK1/2 phosphorylation. Interestingly ATP7A shows vesicular localization despite the low copper in the glia. In addition to the TGN in the perinuclear region, ATP7A localizes into RAB11 positive recycling endosomes in the glial neurites, not observed in the neurons. Our study demonstrates role of the copper dependent ERK1/2 phosphorylation in the neuronal differentiation. Whereas glial differentiation largely involves sequestration of Cu(I) into the endosomes potentially (i) for ready release to the neurons (ii) rendering cytosolic copper unavailable for pathways like the ERK1/2 activation.

sentences: Tempo of degeneration across independently evolved non-recombining regions Recombination is beneficial over the long term, allowing more effective selection. Despite long-term advantages of recombination, local recombination suppression can evolve and lead to genomic degeneration, in particular on sex chromosomes. Here, we investigated the tempo of degeneration in non-recombining regions, i.e., the function curve for the accumulation of deleterious mutations over time, leveraging on 22 independent events of recombination suppression identified on mating-type chromosomes of anther-smut fungi, including newly identified ones. Using previously available and newly generated high-quality genome assemblies of alternative mating types of 13 Microbotryum species, we estimated degeneration levels in terms of accumulation of non-optimal codons and non-synonymous substitutions in non-recombining regions. We found a reduced frequency of optimal codons in the non-recombining regions compared to autosomes, that was not due to less frequent GC-biased gene conversion or lower ancestral expression levels compared to recombining regions. The frequency of optimal codons rapidly decreased following recombination suppression and reached an asymptote after ca 3 Mya. The strength of purifying selection remained virtually constant at dN/dS = 0.55, i.e. at an intermediate level between purifying selection and neutral evolution. Accordingly, non-synonymous differences between mating-type chromosomes increased linearly with stratum age, at a rate of 0.015 per MY. We thus develop a method for disentangling effects of reduced selection efficacy from GC-biased gene conversion in the evolution of codon usage and we quantify the tempo of degeneration in non-recombining regions, which is important for our knowledge on genomic evolution and on the maintenance of regions without recombination.

sentences: Cyanolichen microbiome contains novel viruses that encode genes to promote microbial metabolism Lichen thalli are formed through the symbiotic association of a filamentous fungus and photosynthetic green alga and/or cyanobacterium. Recent studies have revealed lichens also host highly diverse communities of secondary fungal and bacterial symbionts, yet few studies have examined the viral component within these complex symbioses. Here, we describe viral biodiversity and functions in cyanolichens collected from across North America and Europe. As current machine learning viral-detection tools are not trained on complex eukaryotic metagenomes, we first developed efficient methods to remove eukaryotic reads prior to viral detection and a custom pipeline to validate viral contigs predicted with three machine-learning methods. Our resulting high-quality viral data illustrate that every cyanolichen thallus contains diverse viruses that are distinct from viruses in other terrestrial ecosystems. In addition to cyanobacteria, predicted viral hosts include other lichen-associated bacterial lineages and algae, although a large fraction of viral contigs had no host prediction. Functional annotation of cyanolichen viral sequences reveals numerous viral-encoded auxiliary metabolic genes (AMGs) involved in amino acid, nucleotide, and carbohydrate metabolism, including AMGs for secondary metabolism (antibiotics and antimicrobials) and fatty acid biosynthesis. Overall, the diversity of cyanolichen AMGs suggests that viruses may alter microbial interactions within these complex symbiotic assemblages.

sentences: Mathematical models of tumor volume dynamics in response to radiotherapy From the beginning of the usage of radiotherapy (RT) for cancer treatment, mathematical modeling has been integral to understanding radiobiology and for designing treatment approaches and schedules. There has been extensive modeling of response to RT with the inclusion of various degrees of biological complexity. Here we focus on models of tumor volume dynamics. There has been much discussion on the implications of different models of tumor growth, and it is just important to consider the implications of selecting different models for response to RT. In this study, we compare three models of tumor volume dynamics: (1) exponential growth with RT directly reducing tumor volume, (2) logistic growth with direct tumor volume reduction, and (3) logistic growth with RT reducing the tumor carrying capacity. For all three models, we: performed parameter sensitivity and identifiability analyses; investigated the impact of the parameter sensitivity on the tumor volume trajectories; and examined the rates of change in tumor volume ({Delta}V/{Delta}t) during and RT treatment course. The parameter identifiability and sensitivity analyses revealed the interdependence of the different model parameters and may inform parameter calibration in any further usage of these models. In examining the {Delta}V/{Delta}t trends, we coined a new metric - the point of maximum reduction of tumor volume (MRV) - to quantify the magnitude and timing of the expected largest impact of RT during a treatment course. Ultimately, the results of these analyses help us to better understand the implications of model selection while simultaneously generating many hypotheses about the underlying radiobiology that need to be tested on time-resolved measurements of tumor volume from appropriate pre-clinical or clinical data. The answers to these questions and more detailed study of these and similar models of tumor volume dynamics may enable more appropriate model selection on a disease-site or patient-by-patient basis.

sentences: Predicting modular functions and neural coding of behavior from a synaptic wiring diagram How much can connectomes with synaptic resolution help us understand brain function? An optimistic view is that a connectome is a major determinant of brain function and a key substrate for simulating a brain. Here we investigate the explanatory power of connectomics using a wiring diagram reconstructed from a larval zebrafish brainstem. We identify modules of strongly connected neurons that turn out to be specialized for different behavioral functions, the control of eye and body movements. We then build a neural network model using a synaptic weight matrix based on the reconstructed wiring diagram. This leads to predictions that statistically match the neural coding of eye position as observed by calcium imaging. Our work shows the promise of connectome-based brain modeling to yield experimentally testable predictions of neural activity and behavior, as well as mechanistic explanations of low-dimensional neural dynamics, a widely observed phenomenon in nervous systems.

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The lncRNA APOLO interacts with the transcription factor WRKY42 to trigger root hair cell expansion in response to cold Plant long noncoding RNAs (lncRNAs) have emerged as important regulators of chromatin dynamics, impacting on transcriptional programs leading to different developmental outputs. The lncRNA AUXIN REGULATED PROMOTER LOOP (APOLO) directly recognizes multiple independent loci across the Arabidopsis genome and modulates their three-dimensional chromatin conformation, leading to transcriptional shifts. Here, we show that APOLO recognizes the locus encoding the root hair (RH) master regulator ROOT HAIR DEFECTIVE 6 (RHD6) and controls RHD6 transcriptional activity leading to cold-enhanced RH elongation through the consequent activation of the transcription factor gene RHD6-like RSL4. Furthermore, we demonstrate that APOLO interacts with the transcription factor WRKY42 and modulates its binding to the RHD6 promoter. WRKY42 is required for the activation of RHD6 by low temperatures and WRKY42 deregulation impairs cold-induced RH expansion. Collectively, our results indicate that a novel ribonucleoprotein complex involving APOLO and WRKY42 forms a regulatory hub which activates RHD6 by shaping its epigenetic environment and integrates signals governing RH growth and development.

SUMMARYThe lncRNA APOLO directly regulates the transcription of the root hair-master gene RHD6. In response to cold, APOLO is induced and it decoys the H3K27me3-binding protein LHP1 away from RHD6. In addition, APOLO modulates the binding of the transcription factor WRKY42 to the RHD6 promoter at low temperatures.

sentences: The presence of corticomuscular coherence during unipedal stance Standing in unipedal stance requires higher effort to maintain posture balance within narrow base-of-support. Although changes in cortical activity are known to occur as standing task difficulty increased, it is unclear whether it indicates a change in cortical control of muscle activity. To elucidate this point, this study examined corticomuscular coherence (CMC) between the sensorimotor cortex and ankle joint muscles during three tasks such as bipedal stance, unipedal stance, and isometric contraction tasks. For twenty-one healthy participants, we measured the maximal peak of CMC (CMCmax) between electroencephalograms overlying the foot representation area and surface electromyograms from the tibialis anterior (TA), medial gastrocnemius (MG), lateral gastrocnemius (LG), and soleus (SOL), respectively, for each task. We also measured the center of pressure (COP) during both stance tasks. Although there was no significant CMC during bipedal stance in all participants, most participants (n = 14) showed significant CMC for all muscles during unipedal stance with larger COP fluctuation in most participants. Indeed, there was significant difference in CMCmax between unipedal and bipedal stance tasks. The greater CMC would indicate increased cortical control of muscle activity to compensate for postural instability during unipedal stance.

sentences: RNA sequencing on muscle biopsy from a 5-week bedrest study reveals the effect of exercise and potential interactions with dorsal root ganglion neurons Lack of physical activity is a predictor of poor health outcomes that can be prevented or reversed by exercise. Sedentary lifestyle, chronic disease or microgravity can cause muscle deconditioning that then has an impact on other physiological systems. An example is the nervous system, which is adversely affected by decreased physical activity resulting in increased incidence of neurological problems such as chronic pain. We sought to better understand how this might occur by conducting RNA sequencing experiments on muscle biopsies from human volunteers in a 5-week bed-rest study with an exercise intervention arm. We also used a computational method for examining ligand-receptor interactions between muscle and human dorsal root ganglion (DRG) neurons, the latter of which play a key role in nociception and are generators of signals responsible for chronic pain. We identified 1352 differentially expressed genes (DEGs) in bed rest subjects without an exercise intervention but only 132 DEGs in subjects with the intervention. Thirty-six genes were shared between the exercise and no intervention groups. Among 591 upregulated muscle genes in the no intervention arm, 26 of these were ligands that have receptors that are expressed by human DRG neurons. We detected a specific splice variant of one of these ligands, placental growth factor (PGF), in deconditioned muscle that binds to neuropilin 1, a receptor that is highly expressed in DRG neurons and known to promote neuropathic pain. We conclude that exercise intervention protects muscle from deconditioning transcriptomic changes, and prevents changes in expression of ligands that might sensitize DRG neurons that promote pain. Our work creates a set of actionable hypotheses to better understand how deconditioned muscle may influence the function of sensory neurons that innervate the entire body.

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Physiological relevance, localization and substrate specificity of the alternative (type II) mitochondrial NADH dehydrogenases of Ogataea parapolymorpha Mitochondria from Ogataea parapolymorpha harbor a branched electron-transport chain containing a proton-pumping Complex I NADH dehydrogenase and three alternative (type II) NADH dehydrogenases (NDH2s). To investigate the physiological role, localization and substrate specificity of these enzymes, growth of various NADH dehydrogenase mutants was quantitatively characterized in shake-flask and chemostat cultures, followed by oxygen-uptake experiments with isolated mitochondria. Furthermore, NAD(P)H:quinone oxidoreduction of the three NDH2s were individually assessed. Our findings show that the O. parapolymorpha respiratory chain contains an internal NADH-accepting NDH2 (Ndh2-1/OpNdi1), at least one external NAD(P)H-accepting enzyme and likely additional mechanisms for respiration-linked oxidation of cytosolic NADH. Metabolic regulation appears to prevent competition between OpNdi1 and Complex I for mitochondrial NADH. With the exception of OpNdi1, the respiratory chain of O. parapolymorpha exhibits metabolic redundancy and tolerates deletion of multiple NADH-dehydrogenase genes without compromising fully respiratory metabolism.

ImportanceTo achieve high productivity and yields in microbial bioprocesses, efficient use of the energy substrate is essential. Organisms with branched respiratory chains can respire via the energy-efficient proton-pumping Complex I, or make use of alternative NADH dehydrogenases (NDH2s). The yeast Ogataea parapolymorpha contains three uncharacterized, putative NDH2s which were investigated in this work. We show that O. parapolymorpha contains at least one  internal NDH2, which provides an alternative to Complex I for mitochondrial NADH oxidation, albeit at a lower efficiency. The use of this NDH2 appeared to be limited to carbon excess conditions and the O. parapolymorpha respiratory chain tolerated multiple deletions without compromising respiratory metabolism, highlighting opportunities for metabolic (redox) engineering. By providing a more comprehensive understanding of the physiological role of NDH2s, including insights into their metabolic capacity, orientation and substrate specificity this study also extends our fundamental understanding of respiration in organisms with branched respiratory chains.

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RNA is required for the maintenance of multiple cytoplasmic and nuclear membrane-less organelles. Numerous membrane-less organelles composed of a combination of RNA and proteins, referred to as RNP granules, are observed in the nucleus and cytoplasm of eukaryotic cells, including stress granules, processing bodies, Cajal bodies, and nuclear speckles. An unresolved issue is how frequently RNA molecules are required for the maintenance of RNP granules in either the nucleus or cytosol. To address this issue, we degraded intracellular RNA in either the cytosol or the nucleus by the activation of RNase L and examined the impact of RNA loss on several RNP granules. Strikingly, we find the majority of RNP granules, including stress granules, processing bodies, Cajal bodies, nuclear speckles and the nucleolus are altered by the degradation of their RNA components. In contrast, super-enhancer complexes and TIS granules were largely unaffected by widespread intracellular RNA degradation. This highlights a critical and widespread role of RNA in the organization of many, but not all, RNP granules.

SUMMARYDecker et al. examine the role of RNA in maintaining membrane-less organelles composed of RNA and protein by activating RNase L in the cytoplasm or nucleus. Degradation of RNA alters the majority of membrane-less organelles indicating that RNA plays a widespread role in their organization.

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PsiNorm: a scalable normalization for single-cell RNA-seq data Single-cell RNA sequencing (scRNA-seq) enables transcriptome-wide gene expression measurements at single-cell resolution providing a comprehensive view of the compositions and dynamics of tissue and organism development. The evolution of scRNA-seq protocols has led to a dramatic increase of cells throughput, exacerbating many of the computational and statistical issues that previously arose for bulk sequencing. In particular, with scRNA-seq data all the analyses steps, including normalization, have become computationally intensive, both in terms of memory usage and computational time. In this perspective, new accurate methods able to scale efficiently are desirable.

Here we propose PsiNorm, a between-sample normalization method based on the power-law Pareto distribution parameter estimate. Here we show that the Pareto distribution well resembles scRNA-seq data, independently of sequencing depths and technology. Motivated by this result, we implement PsiNorm, a simple and highly scalable normalization method. We benchmark PsiNorm with other seven methods in terms of cluster identification, concordance and computational resources required. We demonstrate that PsiNorm is among the top performing methods showing a good trade-off between accuracy and scalability. Moreover PsiNorm does not need a reference, a characteristic that makes it useful in supervised classification settings, in which new out-of-sample data need to be normalized.

PsiNorm is available as an R package available at https://github.com/MatteoBlla/PsiNorm

sentences: Scars of hope and forewarn of a bleak future: Post-mortem findings of dugongs (Dugong dugon) belonging to a relict population in the Gulf of Kachchh, India A relict dugong population resides in the Gulf of Kachchh in Gujarat state of India. Very little is known on this population stemming from lack of focused studies and inadequate examinations of previous strandings. In this study, crucial ecological information is gathered through a systematic necropsy on stranded dugongs in the gulf. As indicated through dorsal tusk-rake scars on both the carcasses, this study presents first records of derivative physical evidence to the presence of a socially interacting population. Progressive healing and differences in width of the scars indicated more than two individuals had participated in a sexual agnostic or courtship event. Conversely, our findings report that both the animals suffered relative effects of asphyxiation after fishing net entanglement. An implication of a prior pathological condition(s) in the form of dermal cysts, swollen mesenteric lymph nodes, and endoparasites are also reported. Stomach content was examined for a qualitative dietary characterization. Other potential threats along with fishing net microfilaments found in the stomach contents of both dugongs are discussed in brief.

sentences: Unraveling the intracellular cross-talk governing the balance between TNFα mediated survival and apoptosis signaling Tumor necrosis factor alpha (TNF), a pleiotropic cytokine, helps maintain a balance between proliferation and apoptosis in normal cells. This balance is often sacrificed in a diseased cell, such as that of a cancer, by preferring survival phenotype over apoptosis. Restoring this balance requires a detailed understanding of the causal intracellular mechanisms that govern TNF stimulated apoptotic response. In this study, we use a systems biology approach to unravel the interplay between the intracellular signaling markers that orchestrate apoptosis levels. Our approach deciphered the synergism between the early intracellular markers phosphorylated JNK (pJNK) and phosphorylated AKT (pAKT) that modulate the activation of Caspase3, an important apoptotic regulator. We demonstrate that this synergism depends critically on the survival pathway signaling mediated by NF{kappa}B which plays a dominant role in controlling the extent of the overall apoptotic response. By systematic inhibition of the signaling markers, we establish that the dynamic cross-talk between the pJNK and pAKT transients directs the apoptosis phenotype via accumulated Caspase3 response. Interestingly, superposition of the semi-quantitative correlation between apoptosis and Caspase3 transient levels on the proposed TNF network model permits quantification of the dynamic apoptotic response under different stimulation conditions. Thus, the predictive model can be leveraged towards arriving at useful insights that can identify potential targeted therapeutic strategies for altering apoptotic response.

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Curcumin and NCLX Inhibitors Share Anti-Tumoral Mechanisms in Microsatellite-Instability-Driven Colorectal Cancer Colorectal cancer (CRC) is associated with high mortality worldwide and new targets are needed to overcome treatment resistance. Recent evidences highlight a role of the mitochondria calcium homeostasis in the development of CRC. In this context, we aimed to evaluate the role of the mitochondrial sodium-calcium-lithium exchanger (NCLX) and its targeting in CRC. We also identified curcumin as a new potential inhibitor of NCLX.

In vitro, curcumin exerted strong anti-tumoral activity through its action on NCLX with mtCa2+ and reactive oxygen species overload associated with a mitochondrial membrane depolarization, leading to reduced ATP production and apoptosis through mitochondrial permeability transition pore opening concomitant with G2/M cell cycle arrest. NCLX inhibition with either CGP37157 (a benzodiazepine derivative), small interfering RNA-mediated knock-down or knockout approaches reproduced the effects of curcumin. Altered mitochondrial respiration, cellular aerobic glycolysis and endoplasmic reticulum-mitochondria membrane perturbations participated in these mechanisms. In a xenograft mouse model, NCLX inhibitors decreased CRC tumor growth. Both transcriptomic analysis of The Cancer Genome Atlas dataset and immunohistochemical analysis of tissue microarrays from 381 patients with microsatellite instability (MSI)-driven CRC demonstrated that higher NCLX expression was associated with MSI status and for the first time NCLX expression was significantly associated with recurrence-free survival in MSI CRC patients.

Our findings provide strong evidence that blocking NCLX inhibits CRC in vitro and in vivo. We highlight a novel anti-tumoral mechanism of curcumin through its action on NCLX and mitochondria calcium overload that could benefit for therapeutic treatment of patients with MSI CRC.

sentences: Decoding regulatory specificity of human ribosomal proteins on global gene expression Human ribosomes, made of around 80 ribosomal proteins (RPs) and four ribosomal RNAs, have long been thought as uniform passive protein-making factories with little regulatory function. Recently, accumulating evidence showed heterogeneity of RP composition in ribosomes responsible for regulating gene expression in development and tumorigenesis. However, a comprehensive understanding of regulatory spectrum of RPs is still lacking. In this study, we conducted a systematic survey of regulatory specificity of human RPs on global gene expression. We quantified translational and transcriptional changes of gene expression upon deficiency of 75 RPs, including 44 from the large subunit (60S) and 31 from the small subunit (40S), by ribosomal profiling and RNA sequencing analysis. We showed that RP deficiency induced diverse expression changes, particularly at the translational level. RPs were subjected to co-translational regulation under ribosomal stress where deficiency of the 60S and the 40S RPs had opposite effects on the two subunits. RP deficiency perturbed expression of genes related to cell cycle, cellular metabolism, signal transduction and development. Deficiency of RPs from the 60S led to a greater repression effect on cell growth than that from the 40S by perturbing P53 signaling and cell cycle pathways. To demonstrate functional specificity of RPs, we showed that RPS8 deficiency stimulated cellular apoptosis and RPL13 or RPL18 deficiency promoted cellular senescence. We also showed that RPL11 and RPL15 played important roles in retina development and angiogenesis, respectively. Overall, our study demonstrated a widespread regulatory role of RPs in controlling cellular activity, providing an important resource which can offer novel insights into ribosome regulation in human diseases and cancer.

sentences: Evolutionary assembly of cooperating cell types in an animal chemical defense system A long-standing challenge in biology is explaining how the functions of multicellular organs emerge from the underlying evolution of cell types. We deconstructed evolution of an organ novelty: a rove beetle gland that secretes a defensive cocktail. We show that gland function was pieced together via assembly of two cell types that manufacture distinct compounds. One cell type forms a chemical reservoir in the beetles abdomen and produces alkane and ester compounds. We demonstrate that this cell type is a hybrid of cuticle cells and ancient pheromone and adipocyte-like cells, and executes its function via a mosaic of enzymes sourced from each parental cell type. The second cell type synthesizes noxious benzoquinones using a chimeric pathway derived from conserved cellular energy and cuticle formation pathways. We present evidence that evolution of each cell type was shaped by coevolution between the two cell types: the benzoquinones produced by the second cell type dissolve in solvents produced by the first, yielding a potent secretion that confers adaptive value onto the gland as a whole. Our findings illustrate how cooperation between cell types can arise, generating new, organ-level behaviors.

sentences: Counting of Enzymatically Amplified Affinity Reactions in Hydrogel Particle-Templated Drops Counting of numerous compartmentalized enzymatic reactions underlies quantitative and high sensitivity immunodiagnostic assays. However, digital enzyme-linked immunosorbent assays (ELISA) require specialized instruments which have slowed adoption in research and clinical labs. We present a lab-on-a-particle solution to digital counting of thousands of single enzymatic reactions. Hydrogel particles are used to bind enzymes and template the formation of droplets that compartmentalize reactions with simple pipetting steps. These hydrogel particles can be made at a high throughput, stored, and used during the assay to create ~500,000 compartments within 2 minutes. These particles can also be dried and rehydrated with sample, amplifying the sensitivity of the assay by driving affinity interactions on the hydrogel surface. We demonstrate digital counting of {beta}-galactosidase enzyme at a femtomolar detection limit with a dynamic range of 3 orders of magnitude using standard benchtop equipment and experiment techniques. This approach can faciliate the development of digital ELISAs with reduced need for specialized microfluidic devices, instruments, or imaging systems.

sentences: Electrochemical measurements reveal reactive oxygen species in stress granules. Stress granules (SGs) are membraneless organelles that assemble in the cytoplasm to organize cellular contents and promote rapid adaptation during stress. To understand how SGs contribute to physiological functions, we used electrochemical measurements to detect electroactive species in SGs. With amperometry, we discovered that reactive oxygen species (ROS) are encapsulated inside arseniteinduced SGs, and H2O2 is the main species. The release kinetics of H2O2 from single SGs and the number of H2O2 molecules were quantified. The discovery that SGs contain ROS implicates them as communicators of the cellular stresses rather than a simple end-point. This may explain how SGs regulate cellular metabolism and stress responses. This may also help better understand their cytoprotective functions in pathological conditions associated with SGs such as neurodegenerative diseases, cancers and viral infections.

sentences: Detection and Spatiotemporal analysis of in-vitro 3D migratory Triple-Negative Breast cancer cells The invasion of cancer cells into the surrounding tissues is one of the hallmarks of cancer. However, a precise quantitative understanding of the spatiotemporal patterns of cancer cell migration and invasion still remains elusive. A promising approach to investigate these patterns are 3D cell cultures, which provide more realistic models of cancer growth compared to conventional 2D monolayers. Quantifying the spatial distribution of cells in these 3D cultures yields great promise for understanding the spatiotemporal progression of cancer. In the present study, we present an image processing and segmentation pipeline for the detection of 3D GFP-fluorescent Triple-Negative Breast Cancer cell nuclei, and we perform quantitative analysis of the formed spatial patterns and their temporal evolution. The performance of the proposed pipeline was evaluated using experimental 3D cell culture data, and was found to be comparable to manual segmentation, outperforming four alternative automated methods. The spatiotemporal statistical analysis of the detected distributions of nuclei revealed transient, non-random spatial distributions that consisted of clustered patterns across a wide range of neighbourhood distances, as well as dispersion for larger distances. Overall, the implementation of the proposed framework revealed the spatial organization of cellular nuclei with improved accuracy, providing insights into the 3 dimensional inter-cellular organization and its progression through time.

sentences: Learning inverse folding from millions of predicted structures We consider the problem of predicting a protein sequence from its backbone atom coordinates. Machine learning approaches to this problem to date have been limited by the number of available experimentally determined protein structures. We augment training data by nearly three orders of magnitude by predicting structures for 12M protein sequences using AlphaFold2. Trained with this additional data, a sequence-to-sequence transformer with invariant geometric input processing layers achieves 51% native sequence recovery on structurally held-out backbones with 72% recovery for buried residues, an overall improvement of almost 10 percentage points over existing methods. The model generalizes to a variety of more complex tasks including design of protein complexes, partially masked structures, binding interfaces, and multiple states.

sentences: An Aerobic Anoxygenic Phototrophic Bacterium Fixes CO2 via the Calvin-Benson-Bassham Cycle Aerobic anoxygenic phototrophic bacteria (AAnPB) are photoheterotrophs, which use light as auxiliary energy and require organic carbon (OC) for growth. Herein, we report the unusual strain B3, which is a true AAnPB because it requires oxygen for growth, harbours genes for cbb3- and bd-type cytochromes and acsF, and produces bacteriochlorophyll. The B3 genome encodes the complete metabolic pathways for AAnPB light utilization, CO2 fixation via Calvin-Benson-Bassham (CBB) cycle and oxidation of sulfite and H2, and the transcriptome indicated that all components of these pathways were fully transcribed. Expression of the marker genes related to photosynthesis, including pufM for light harnessing and rbcL for CO2 fixation, and the activity of RubisCO, the key enzyme in the Calvin-Benson-Bassham (CBB) cycle, increased in response to decreased OC supply. Large amounts of cell biomass were obtained in liquid BG11 medium under illumination. The strain thus likely photoautotrophically grows using sulfite or H2 as an electron donor. Similar GC contents between photosynthesis, the CBB cycle and 16S rRNA genes and the consistency of their phylogenetic topologies implied that light harnessing and carbon fixation genes evolved vertically from an anaerobic phototrophic ancestor of Rhodospirillaceae in Alphaproteobacteria. In conclusion, strain B3 represents a novel AAnPB characterized by photoautotrophy using the CBB cycle. This kind of AAnPB may be ecologically significant in the global carbon cycle.

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A method to estimate the effective point spread function of static single molecule localization microscopy images Single molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM dataset is an essential aspect of the image processing and analysis pipeline. Here we present a simple method that uses the pair autocorrelation function evaluated both in space and time to measure the time-interval dependent effective point spread function of SMLM images of static samples. Using this approach, we demonstrate that experimentally obtained images typically have effective point spread functions that are broader than expected from the localization precision alone, due to additional uncertainty arising from factors such as drift and drift correction algorithms. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labelled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion proteins.

STATEMENT OF SIGNIFICANCESingle molecule localization microscopy (SMLM) is a class of imaging methods that resolve fluorescently labeled structures beyond the optical resolution limit of visible light. SMLM detects stochastically blinking labels over time, and localizes each blink with precision of order 10 nm. The effective resolution depends on factors such as signal-to-noise ratio, localization algorithm, and several post-processing steps such as stage drift correction. We present a method to evaluate this effective resolution by taking advantage of temporal correlations of fluorophore blinking to separate the distribution of pairs of localizations from the same molecule from those from different molecules. The method is robust on useful timescales for a range of SMLM probes.

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Rare copy number variants (CNVs) and breast cancer risk BackgroundCopy number variants (CNVs) are pervasive in the human genome but potential disease associations with rare CNVs have not been comprehensively assessed in large datasets. We analysed rare CNVs in genes and non-coding regions for 86,788 breast cancer cases and 76,122 controls of European ancestry with genome-wide array data.

ResultsGene burden tests detected the strongest association for deletions in BRCA1 (P= 3.7E-18). Nine other genes were associated with a p-value < 0.01 including known susceptibility genes CHEK2 (P= 0.0008), ATM (P= 0.002) and BRCA2 (P= 0.008). Outside the known genes we detected associations with p-values < 0.001 for either overall or subtype-specific breast cancer at nine deletion regions and four duplication regions. Three of the deletion regions were in established common susceptibility loci.

ConclusionsThis is the first genome-wide analysis of rare CNVs in a large breast cancer case-control dataset. We detected associations with exonic deletions in established breast cancer susceptibility genes. We also detected suggestive associations with non-coding CNVs in known and novel loci with large effects sizes. Larger sample sizes will be required to reach robust levels of statistical significance.

sentences: Severe drought events trigger long-term salt marsh dieback in the Southeastern and Gulf of Mexico USA The frequency of salt marsh dieback events has been increasing in the last 25 years with unknown consequences to the resilience of the ecosystem to accelerated sea level rise (SLR). Sudden dieback events triggered by extreme climatic events were previously thought to recover naturally within a few months to years. We provide evidence that about 15% of total marsh area has not revegetated 10- and 20-years after dieback events in two case studies in the Southeast and Gulf of Mexico USA, respectively. In both case studies, dieback onset coincided with severe drought events. In West Ashley, SC, dieback areas were associated with lower elevation zones in the interior of the marsh. Restoration by grass planting was shown to be effective in the South Carolina urban marsh, indicating that the stressors that caused dieback may be overcome despite that most of dieback areas have not recovered naturally. A mechanistic modelling approach was used to predict the resilience of the South Carolina salt marsh to accelerated SLR under different scenarios of primary productivity, ecological management, and rates of SLR. Predictions indicate that a highly productive restored marsh (2000 g m-2 y-1) would persist at a moderate SLR rate of 60 cm 100 y-1, whereas a non-restored mudflat would rapidly lose all of its elevation capital. Further, at an elevated SLR rate of 122 cm 100 y-1, most likely an extreme climate change scenario, even highly productive ecosystems augmented by sediment placement would not keep pace with SLR. Thus, both climate change adaptation and mitigation actions are urgently needed to preserve highly valuable present-day marsh ecosystems close to urban environments.

sentences: P5A-ATPases control the ER translocation of Wnt for neuronal migration Wnt family are conserved secretory proteins required for developmental patterning and tissue homeostasis. The mechanisms underlying intracellular maturation and intercellular signal transduction of Wnt proteins have been extensively studied. However, how Wnt is targeted to the endoplasmic reticulum (ER) for processing and secretion remains elusive. Here we report that CATP-8/P5A-ATPase directs neuronal migration non-cell autonomously in C. elegans by regulating EGL-20/Wnt biogenesis. CATP-8 functions as a translocase to translocate EGL-20/Wnt nascent polypeptide into the ER by interacting with the hydrophobic core region of EGL-20 signal sequence. Such regulation of Wnt biogenesis by P5A-ATPase is conserved in human cells. These findings reveal physiological roles of P5A-ATPase in neural development and identify Wnt proteins as direct substrates of P5A-ATPase for ER translocation.

sentences: Order of arrival promotes coexistence via spatial niche preemption by the weak competitor Historical contingency, such as the order of species arrival, can modify competitive outcomes via niche modification or preemption. However, how these mechanisms ultimately modify stabilising niche and average fitness differences remains largely unknown. By experimentally assembling two congeneric spider mite species feeding on tomato plants during two generations, we show that order of arrival affects species competitive ability and changes the outcome of competition. Contrary to expectations, order of arrival did not cause positive frequency dependent priority effects. Instead, coexistence was predicted when the inferior competitor (Tetranychus urticae) arrived first. In that case, T. urticae colonised the preferred feeding stratum (leaves) of T. evansi leading to spatial niche preemption, which equalised fitness and reduced niche differences, driving community assembly to a close-to-neutrality scenario. Our study demonstrates how the order of species arrival and the spatial context of competitive interactions can jointly determine whether species can coexist.

sentences: Prediction of coronavirus 3C-like protease cleavage sites using machine-learning algorithms The coronavirus 3C-like (3CL) protease is a Cysteine protease. It plays an important role in viral infection and immune escape by not only cleaving the viral polyprotein ORF1ab at 11 sites, but also cleaving the host proteins. However, there is still a lack of effective tools for determining the cleavage sites of the 3CL protease. This study systematically investigated the diversity of the cleavage sites of the coronavirus 3CL protease on the viral polyprotein, and found that the cleavage motif were highly conserved for viruses in the genera of Alphacoronavirus, Betacoronavirus and Gammacoronavirus. Strong residue preferences were observed at the neighboring positions of the cleavage sites. A random forest (RF) model was built to predict the cleavage sites of the coronavirus 3CL protease based on the representation of residues at cleavage site and neighboring positions by amino acid indexes, and the model achieved an AUC of 0.96 in cross-validations. The RF model was further tested on an independent test dataset composed of cleavage sites on host proteins, and achieved an AUC of 0.88 and a prediction precision of 0.80 when considering the accessibility of the cleavage site. Then, 1,079 human proteins were predicted to be cleaved by the 3CL protease by the RF model. These proteins were enriched in pathways related to neurodegenerative diseases and pathogen infection. Finally, a user-friendly online server named 3CLP was built to predict the cleavage sites of the coronavirus 3CL protease based on the RF model. Overall, the study not only provides an effective tool for identifying the cleavage sites of the 3CL protease, but also provides insights into the molecular mechanism underlying the pathogenicity of coronaviruses.

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ISOTOPE: ISOform-guided prediction of epiTOPEs in cancer Immunotherapies provide effective treatments for previously untreatable tumors and identifying tumor-specific epitopes can help elucidate the molecular determinants of therapy response. Here, we describe a pipeline, ISOTOPE (ISOform-guided prediction of epiTOPEs In Cancer), for the comprehensive identification of tumor-specific splicing-derived epitopes. Using RNA sequencing and mass spectrometry for MHC-I associated proteins, ISOTOPE identified neoepitopes from tumor-specific splicing events that are potentially presented by MHC-I complexes. Analysis of multiple samples indicates that splicing alterations may affect the production of self-epitopes and generate more candidate neoepitopes than somatic mutations. Although there was no difference in the number of splicing-derived neoepitopes between responders and non-responders to immune therapy, higher MHC-I binding affinity was associated with a positive response. Our analyses highlight the diversity of the immunogenic impacts of tumor-specific splicing alterations and the importance of studying splicing alterations to fully characterize tumors in the context of immunotherapies. ISOTOPE is available at https://github.com/comprna/ISOTOPE

Author summaryImmune cells have the ability to attack tumor cells upon the identification of tumor-specific peptides, i.e., epitopes, that are presented by the major histocompatibility complex (MHC). New cancer immunotherapies that help trigger this process provide a promising therapeutic strategy. One crucial aspect for their success is the ability to determine the molecular properties of a tumor that are informative about the effectiveness of the therapy. Alterations in the way genes are processed to express RNA molecules could lead to the production of new peptides, with some of them potentially being presented as tumor epitopes and facilitate the attack of immune cells. It is therefore essential to facilitate the identification of these splicing-derived epitopes. In this work, we describe a computational pipeline that performs a comprehensive identification of splicing alterations in a tumor and the potential epitopes that they would produce. Analysis of tumor samples with our pipeline show that responders and non-responders to immune therapy do not show differences in the number of splicing-derived epitopes, but splicing neoepitopes have higher affinity to the MHC complex in responders. Our new pipeline facilitates the genome-scale analysis of the role of splicing alterations in shaping the molecular properties that influence response to immunotherapy.

sentences: Lysosomal Exocytosis Releases Pathogenic α-Synuclein Species from Neurons Considerable evidence supports the release of pathogenic aggregates of the neuronal protein -Synuclein (Syn) into the extracellular space. While this release is proposed to instigate the neuron-to-neuron transmission and spread of Syn pathology in synucleinopathies including Parkinsons disease, the molecular-cellular mechanism(s) remain unclear. Here we show that pathogenic species of Syn accumulate within neuronal lysosomes in mouse brains and primary neurons. We then find that neurons release these pathogenic Syn species via SNARE-dependent lysosomal exocytosis; proposing a central mechanism for exocytosis of aggregated and degradation-resistant proteins from neurons.

sentences: No support for a meiosis suppressor in Daphnia pulex: Comparison of linkage maps reveals normal recombination in males of obligate parthenogenetic lineages. It is often assumed that obligate parthenogenesis (OP) evolves by a disruption of meiosis and recombination. One emblematic example that appears to support this view is the crustacean Daphnia pulex. Here, by constructing high-density linkage maps, we estimate genome-wide recombination rates in males that are occasionally produced by OP lineages, as well as in males and females of cyclical parthenogenetic (CP) lineages. The results show no significant differences in recombination rates and patterns between CP and OP males nor between CP male and CP females. The observation that recombination is not suppressed in OP males invalidates the hypothesis of a general meiosis suppressor responsible for OP. Rather, our findings suggest that in D. pulex, as in other species where OP evolves from CP ancestors, the CP to OP transition evolves through a re-use of the parthenogenesis pathways already present in CP and through their extension to the entire life cycle, at least in females. In addition to the implications for the evolution of OP, the genetic maps produced by this study constitute an important genomic resource for the model species Daphnia.

sentences: Sequence-based correction of barcode bias in massively parallel reporter assays Massively parallel reporter assays (MPRA) are a high-throughput method for evaluating in vitro activities of thousands of candidate cis-regulatory elements (CREs). In these assays, candidate sequences are cloned upstream or downstream of a reporter gene tagged by unique DNA sequences. However, tag sequences may themselves affect reporter gene expression and lead to major potential biases in the measured cis-regulatory activity. Here, we present a sequence-based method for correcting tag sequence-specific effects and demonstrate that our method can significantly reduce this source of variation, and improve the identification of functional regulatory variants by MPRAs. We also show that our model captures sequence features associated with post-transcriptional regulation of mRNA. Thus, this new method helps to not only improve detection of regulatory signals in MPRA experiments but also to design better MPRA protocols.

sentences: Prolonged and extended impacts of SARS-CoV-2 on the olfactory neurocircuit The impact of SARS-CoV-2 on the olfactory pathway was studied over several time points using Syrian golden hamsters. We found an incomplete recovery of the olfactory sensory neurons, prolonged activation of glial cells in the olfactory bulb, and a decrease in the density of dendritic spines within the hippocampus. These data may be useful for elucidating the mechanism underlying long-lasting olfactory dysfunction and cognitive impairment as a post-acute COVID-19 syndrome.

sentences: SUCCESSFUL IN VITRO EMBRYO PRODUCTION WITH OOCYTES ASPIRATED FROM LIVE WHITE-TAILED DEER (Odocoileus virginianus texanus) DONNORS UNDER CAPTIVITY IN NORTHEAST MEXICO Assisted reproductive technologies (ARTs), such as artificial insemination, semen sorting and freezing, embryo production in vivo and in vitro, are all methods in which animal reproduction management of several species has become considerably more efficient. These ARTs have been applied both in domestic and wild ruminants. In this study, in vitro embryo production was attempted with oocytes collected surgically during the mating season, from live white-tailed deer (WTD) hinds, maintained under captivity, in Northeast Mexico. The study was conducted in two WTD farms nearby Cd. Victoria, Tamps., Mexico, and another WTD farm located in Guerrero Coahuila, Mexico. The laboratory work was carried at the Centro de Desarrollo de la Capacidad Productiva y Mejoramiento Genetico de la Ganaderia, property of the Union Ganadera Regional de Tamaulipas (Centro-UGRT). The deer hinds are kept in captivity year-around, with feeding and health management provided accordingly to their requirements (50-60 kg females). Fresh green forage and clean fresh water is supplied daily. Oocytes were collected on each farm, while the hinds were maintained under general anesthesia and by means of a mid-ventral laparotomy, ovaries were exposed and follicles greater than 1-2 mm were aspirated with a 20 G needle, connected by a two-way plastic and latex hose to a vacum machine (WTA, Brazil); the oocytes were collected into a 50 ml. centrifuge tube containing wash media with heparine (Vitrogen, Brazil). Each oocyte collection lasted approximately 20 minutes, after which and while still on the farm, the oocytes were filtered with 50 {micro} mesh filters and rinsed several times with wash media, then placed on search Petri plates. Oocytes were then counted and classified (total, viable and non-viable oocytes) and placed into cryovials with maturation media (3 ml. MIV, Vitrogen, Brazil) and placed into a portable incubator with 5 % CO2 gas mix (LabiMixWTA, Brazil), after all hinds were done, oocytes were transported to the in vitro fertilization laboratory (IVF Lab) at the Centro-UGRT. Once at the IVF Lab, the oocytes were placed on a larger incubator (Eve, WTA, Brazil) and kept there for 18-20 hours, after which, the MIV media was changed for fertilization media (FIV, Vitrogen, Brazil) and fertilization was initiated by adding 10,000-12,000 live sperm per cryovial, and incubated for an additional period of 24 hours; after which, FIV media was replaced by the same media and at this point, cleavage rate was estimated by counting the oocytes that initiated cell division. At 72 hours after cell division started, fertilization rate was estimated; and 7 days after, the blastocysts were counted and classified. The whole process of oocyte maturation and embryo production in vitro, was conducted based on a beef cattle embryo production system and adapted to a deer embryo production system using media for small ruminants (Vitrogen, Brazil). Data collected per hind included total number of oocytes, viable and non-viable oocytes, cleavage rate (ratio of viable oocytes that initiated cell division over viable oocytes), fertilization rate (ratio of embryos that initiated cleavage over those that continued development to the blastocyst stage) and blastocyst rate (embryos reaching the blastocyst stage over cleaved embryos); averages were calculted for each parameter. The main results from this study on a per hind basis for total viable and non-viable oocytes were 9.8, 6.3 and 4.5, respectively; cleavage and blastocyst rates were 39.5 and 36.8, respectively and 2.3 blastocysts. In conclusion, oocyte collection from live WTD hinds and in vitro embryo production was succesfully done under farming conditions in Northeast Mexico.

sentences: Systemic Tissue and Cellular Disruption from SARS-CoV-2 Infection revealed in COVID-19 Autopsies and Spatial Omics Tissue Maps The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has infected over 115 million people and caused over 2.5 million deaths worldwide. Yet, the molecular mechanisms underlying the clinical manifestations of COVID-19, as well as what distinguishes them from common seasonal influenza virus and other lung injury states such as Acute Respiratory Distress Syndrome (ARDS), remains poorly understood. To address these challenges, we combined transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues, matched with spatial protein and expression profiling (GeoMx) across 357 tissue sections. These results define both body-wide and tissue-specific (heart, liver, lung, kidney, and lymph nodes) damage wrought by the SARS-CoV-2 infection, evident as a function of varying viral load (high vs. low) during the course of infection and specific, transcriptional dysregulation in splicing isoforms, T cell receptor expression, and cellular expression states. In particular, cardiac and lung tissues revealed the largest degree of splicing isoform switching and cell expression state loss. Overall, these findings reveal a systemic disruption of cellular and transcriptional pathways from COVID-19 across all tissues, which can inform subsequent studies to combat the mortality of COVID-19, as well to better understand the molecular dynamics of lethal SARS-CoV-2 infection and other viruses.

sentences: Plagl1 is part of the mammalian retinal injury response and a critical regulator of Muller glial cell quiescence Retinal damage triggers reactive gliosis in Muller glia across vertebrate species, but only in regenerative animals, such as teleost fish, do Muller glia initiate repair; proliferating and undergoing neurogenesis to replace lost cells. By mining scRNA-seq and bulk RNA-seq datasets, we found that Plagl1, a maternally imprinted gene, is dynamically regulated in reactive Muller glia post-insult, with transcript levels transiently increasing before stably declining. To study Plagl1 retinal function, we examined Plagl1+/-pat null mutants postnatally, revealing defects in retinal architecture, visual signal processing and a reactive gliotic phenotype. Plagl1+/-pat Muller glia proliferate ectopically and give rise to inner retinal neurons and photoreceptors. Transcriptomic and ATAC-seq profiles revealed similarities between Plagl1+/-pat retinas and neurodegenerative and injury models, including an upregulation of pro-gliogenic and pro-proliferative pathways, such as Notch, not observed in wild-type retinas Plagl1 is thus an essential component of the transcriptional regulatory networks that retain mammalian Muller glia in quiescence.

sentences: Microsecond resolved infrared spectroscopy on non-repetitive protein reactions by applying caged-compounds and quantum cascade laser frequency combs Infrared spectroscopy is ideally suited for the investigation of protein reactions at the atomic level. Many systems were investigated successfully by applying Fourier transform infrared (FTIR) spectroscopy. While rapid-scan FTIR spectroscopy is limited by time resolution (about10 ms with 16 cm-1 resolution), step-scan FTIR spectroscopy reaches a time-resolution of about 10 ns but is limited to cyclic reactions that can be repeated hundreds of times under identical conditions. Consequently, FTIR with high time resolution was only possible with photoactivable proteins that undergo a photocycle. The huge number of non-repetitive reactions, e.g. induced by caged compounds, were limited to the ms time domain. The advent of dual comb quantum cascade laser allows now for a rapid reaction monitoring in the s time domain. Here we investigate the potential to apply such an instrument to the huge class of G-proteins. We compare caged-compound induced reactions monitored by FTIR and dual comb spectroscopy, respectively, by applying the new technique to the  subunit of the inhibiting Gi protein and to the larger protein-protein complex of Gi with its cognate regulator of G-protein signaling (RGS). We observe good data quality with 4 s time resolution with a wavelength resolution comparable to FTIR. This is more than three orders of magnitude faster than any FTIR measurement on G-proteins in the literature. This study paves the way for infrared spectroscopic studies in the so far unresolvable s time regime for non-repetitive biological systems including all GTPases and ATPases.

sentences: The physical properties of the stick insect pad secretion are independent of body size Many insects use adhesive organs to climb. The ability to cling to surfaces is advantageous but is increasingly challenged as animals grow, due to the associated reduction in surface-to-volume ratio. Previous work has demonstrated that some climbing animals overcome this scaling problem by systematically altering the maximum force per area their adhesive pads can sustain; their adhesive organs become more efficient as they grow, an observation which is also of substantial relevance for the design of bio-inspired adhesives. What is the origin of this change in efficiency? In insects, adhesive contact is mediated by a thin film of a liquid, thought to increase adhesive performance via capillary and viscous forces. Here, we use interference reflection microscopy and dewetting experiments to measure the contact angle and dewetting speed of the pad secretion of Indian stick insects varying in mass by over two orders of magnitude. Neither contact angle nor dewetting speed change significantly with body mass, suggesting that the key physical properties of the pad secretion - its surface tension and viscosity - are size-invariant. Thus, the observed change in pad efficiency is unlikely to arise from systematic changes of the physical properties of the pad secretion; the functional role of the secretion remains unclear.

sentences: NMDAR-independent acetylcholine, serotonin, and norepinephrine mediated modulation of synaptic eligibility traces into LTD in mice visual cortex In reward-based learning, synaptic eligibility traces are a well-defined theoretical solution for the conversion of initial co-activation of pre and postsynaptic neurons into long-term changes in synaptic strength by reward-linked neuromodulators. However, the types of neuromodulators involved in such a phenomenon in mouse visual cortex remain unknown. To characterize the Ex vivo condition, we used optogenetic stimulation of channelrhodopsin-(ChR2) expressing Cre/Ai32(ChR2-eYFP); Tph2-Cre/Ai32(ChR2-eYFP); Thi-Cre/Ai32(ChR2-eYFP) homozygous mice, which release acetylcholine, serotonin, and norepinephrine, respectively. With these mice it is possible to measure the transformation of eligibility traces into long-term changes by endogenous neuromodulators. Here we delineated that layer 2/3 neurons in the visual cortex showed no LTD after conditioning with paired-pulse low-frequency stimulation (ppLFS; 2Hz, 15 min). However, if conditioning was paired with acetylcholine, serotonin, or norepinephrine release upon 473 nm optical stimulation in brain slices, LTD occurs in every case. Thus, our data suggests a new pathway to connect the gap between stimulus and reward. Moreover, we found that stimulation by theta-glass or metal stimulators evoked IPSC traces with the same amplitudes but differences in decay kinetics, further questioning the appropriate use of stimulators in brain slices for evoking an event.

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Host tissue proteomics reveal insights into the molecular basis of Schistosoma haematobium-induced bladder pathology BackgroundUrogenital schistosomiasis remains a major public health concern worldwide. In response to egg deposition, the host bladder undergoes gross and molecular morphological changes relevant for disease manifestation. However, limited mechanistic studies to date imply that the molecular mechanisms underlying pathology have not been well-defined. We leveraged a mouse model of urogenital schistosomiasis to perform for the first time, proteome profiling of the early molecular events that occur in the bladder after exposure to S. haematobium eggs, and to elucidate the protein pathways involved in urogenital schistosomiasis-induced pathology.

MethodsPurified S. haematobium eggs or control vehicle were microinjected into the bladder walls of mice. Mice were sacrificed seven days post-injection and bladder proteins isolated and processed for proteome profiling using mass spectrometry.

ResultsWe demonstrate that biological processes including carcinogenesis, immune and inflammatory responses, increased protein translation or turnover, oxidative stress responses, reduced cell adhesion and epithelial barrier integrity, and increased glucose metabolism were significantly enriched in S. haematobium infection.

ConclusionS. haematobium egg deposition in the bladder results in significant changes in proteins and pathways that play a role in pathology. Our findings highlight the potential bladder protein indicators for host-parasite interplay and provide new insights into the complex dynamics of pathology and characteristic bladder tissue changes in urogenital schistosomiasis. The findings will be relevant for development of improved interventions for disease control.

Author summaryThe molecular mechanisms underlying the urinary and genital pathology from urogenital schistosomiasis have not been well-defined. This has mainly been due to limited mechanistic studies and the lack of a suitable animal model for S. haematobium infection. We leveraged a mouse model of urogenital schistosomiasis, along with proteomics analysis, to determine the early molecular events that occur in the bladder after exposure to S. haematobium eggs, and to define protein pathways that may be involved in morbidity from urogenital schistosomiasis infection. Our results show that S. haematobium egg deposition in the bladder results in significant changes in proteins and pathways that play a role in pathology. Biological processes including carcinogenesis, immune and inflammatory responses, increased protein translation or turnover, oxidative stress responses, reduced cell adhesion and epithelial barrier integrity, and increased glucose metabolism are significantly enriched in S. haematobium infection. Our findings highlight important protein indicators of host-parasite interactions in the bladder and their association with tissue changes in urogenital schistosomiasis. The findings presented here will be relevant for development of improved interventions for disease control, and contribute to realizing the 2021-2030 NTD goals by demonstrating evidence on the early events that occur during schistosome infection.

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The spatiotemporal link of temporal expectations: contextual temporal expectation is independent of spatial attention Temporal expectation is the ability to construct predictions regarding the timing of events, based on previously-experienced temporal regularities of different types. For example, cue-based expectations are constructed when a cue validly indicates when a target is expected to occur. However, in the absence of such cues, expectations can be constructed based on contextual temporal information, including the events hazard-rate function - its moment-by-moment conditional probability that changes over time; and prior experiences, which provide probabilistic information regarding the events predicted timing (sequential effects).

It was previously suggested that cue-based temporal expectation is exerted via synchronization of spatially-specific neural activity at a target's predictable time, within receptive fields corresponding to the targets expected location. Here, we tested if the same theoretical model holds for contextual temporal effects. Participants (n = 40) performed a speeded spatial-cueing detection task, with two-thirds valid spatial cues. The targets hazard-rate function was modulated by varying the foreperiod - the interval between the spatial cue and the target - among trials, and was manipulated between groups by changing the interval distribution. Reaction times were analyzed using both frequentist and Bayesian generalized linear mixed models, accounting for hazard and sequential effects. Results showed that the effects of contextual temporal structures on reaction times were independent of spatial attention. This suggests that the spatiotemporal mechanisms, thought to account for cue-based expectation, cannot explain other sources of temporal expectations. We conclude that expectations based on contextual structures have different characteristics than cue-based temporal expectation, suggesting reliance on distinct neural mechanisms.

Significance statementTemporal expectation is the ability to predict an event onset based on temporal regularities. A neurophysiological model suggested that temporal expectation relies on the synchronization of spatially-specific neurons whose receptive fields represent the attended location. This model predicts that temporal expectation would be evident solely within the locus of spatial attention. Existing evidence supported this model for expectation based on associations between a temporal cue and a target, but here we show that it cannot account for another source of temporal expectation - expectation that is based on contextual information, i.e. hazard-rate and recent priors. These findings reveal the existence of different predictive mechanisms for cued and contextual temporal predictions, with the former depending on spatial attention and the latter non-spatially-specific.

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SPIRO - the automated Petri plate imaging platform designed by biologists, for biologists The imaging of plant seedlings, fungal mycelia and bacterial colonies grown on Petri plates is common in phenotyping assays, and is typically done manually despite the procedures being time-consuming and laborious. The main reason for this is the still limited availability of existing automated phenotyping tools and facilities. Additionally, constructing a custom-made automated solution is a daunting task for most research groups specializing in biology.

Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photos of up to four vertically oriented Petri plates in a single experiment. SPIRO was designed for biologists by biologists; thus, its assembly does not require experience in engineering or programming and its operation is sufficiently intuitive to be carried out without training. SPIRO has a small footprint optimal for fitting into standard incubators for plants and microbes, and is equipped with an LED light source for imaging in the dark, thus allowing acquisition of photos under optimal growth conditions. SPIROs web-based user interface allows setting up experiments and downloading data remotely, without interfering with samples growth. SPIROs excellent image quality is optimal for automated image processing, which we demonstrate with two semi-automated assays for analysis of commonly used phenotypic traits: seed germination and root growth.

Moreover, the robot can be easily customized for a specific use, as all information about SPIRO, including the models for 3D-printed structural components, control software, and scripts for image analysis, are released under permissive open-source licenses.

sentences: Glutamatergic dysfunction leads to a hyper-dopaminergic phenotype through deficits in short-term habituation: a mechanism for aberrant salience. Psychosis in disorders like schizophrenia is commonly associated with aberrant salience and elevated striatal dopamine. However, the underlying cause(s) of this hyper-dopaminergic state remain elusive. Various lines of evidence point to glutamatergic dysfunction and impairments in synaptic plasticity in the aetiology of schizophrenia, including deficits associated with the GluA1 AMPAR subunit. GluA1 knockout (Gria1-/-) mice provide a model of impaired synaptic plasticity in schizophrenia and exhibit a selective deficit in a form of short-term memory which underlies short-term habituation. As such, these mice are unable to reduce attention to recently presented stimuli. In this study we used fast-scan cyclic voltammetry to measure phasic dopamine responses in the nucleus accumbens of Gria1-/- mice to determine whether this behavioral phenotype might be a key driver of a hyper-dopaminergic state. There was no effect of GluA1 deletion on electrically-evoked dopamine responses in anaesthetized mice, demonstrating normal endogenous release properties of dopamine neurons in Gria1-/- mice. Furthermore, dopamine signals were initially similar in Gria1-/- mice compared to controls in response to both sucrose rewards and neutral light stimuli. They were also equally sensitive to changes in the magnitude of delivered rewards. In contrast, however, these stimulus-evoked dopamine signals failed to habituate with repeated presentations in Gria1-/- mice, resulting in a task-relevant, hyper-dopaminergic phenotype. Thus, here we show that GluA1 dysfunction, resulting in impaired short-term habituation, is a key driver of enhanced striatal dopamine responses, which may be an important contributor to aberrant salience and psychosis in psychiatric disorders like schizophrenia.

sentences: Comparative analysis of 16S rRNA gene and metagenome sequencing in pediatric gut microbiomes The colonization of the human gut microbiome begins at birth, and, over time, these microbial communities become increasingly complex. Most of what we currently know about the human microbiome, especially in early stages of development, was described using culture-independent sequencing methods that allow us to identify the taxonomic composition of microbial communities using genomic techniques, such as amplicon or shotgun metagenomic sequencing. Each method has distinct tradeoffs, but there has not been a direct comparison of the utility of these methods in stool samples from very young children, which have different features than those of adults. We compared the effects of profiling the human infant gut microbiome with 16S rRNA amplicon versus shotgun metagenomic sequencing techniques in 130 fecal samples; younger than 15, 15-30, and older than 30 months of age. We demonstrate that observed changes in alpha-diversity and beta-diversity with age occur to similar extents using both profiling methods. We also show that 16S rRNA profiling identified a larger number of genera and we find several genera that are missed or underrepresented by each profiling method. We present the link between alpha diversity and shotgun metagenomic sequencing depth for children of different ages. These findings provide a guide for selecting an appropriate method and sequencing depth for the three studied age groups.

sentences: CRISPR perturbations at many coronary artery disease loci impair vascular endothelial cell functions Genome-wide association studies have identified 161 genetic variants associated with coronary artery disease (CAD), but the causal genes and biological pathways remain unknown at most loci. Here, we used CRISPR knockout, inhibition and activation to target 1998 variants at 83 CAD loci to assess their effect on six vascular endothelial cell phenotypes (E-selectin, ICAM1, VCAM1, nitric oxide, reactive oxygen species, calcium signalling). We identified 42 significant variants located within 26 CAD loci. Detailed characterization of the RNA helicase DHX38 and CRISPR activation at the FURIN/FES, CCDC92/ZNF664 and CNNM2 loci revealed a strong effect on vascular endothelial cell senescence.

sentences: DPSleep: Open-Source Longitudinal Sleep Analysis From Accelerometer Data Wearable devices are now widely available to collect continuous objective behavioral data from individuals and to measure sleep. Here we introduce a pipeline to infer sleep onset, duration, and quality from raw accelerometer data and then quantify relationships between derived sleep metrics and other variables of interest. The pipeline released here for the deep phenotyping of sleep, as the "DPSleep" software package, uses (a) a stepwise algorithm to detect missing data; (b) within-individual, minute-based, spectral power percentiles of activity; and (c) iterative, forward- and backward-sliding windows to estimate the major Sleep Episode onset and offset. Software modules allow for manual quality control adjustment of derived sleep features and correction for time zone changes. In this report, we illustrate the pipeline with data from participants studied for more than 200 days each. Actigraphy-based measures of sleep duration are associated with self-report rating of sleep quality. Simultaneous measures of smartphone use and GPS location data support the validity of the sleep timing inferences and reveal how phone measures of sleep timing can differ from actigraphy data. We discuss the uses of DPSleep in relation to other available sleep estimation approaches and provide example use cases that include multi-dimensional, deep longitudinal phenotyping, extended measurement of dynamics associated with mental illness, and the possibility of combining wearable actigraphy and personal electronic device data (e.g., smartphone, tablet) to measure individual differences across a wide range of behavioral variation in health and disease.

sentences: Cylcop: An R Package for Circular-Linear Copulae with Angular Symmetry The R package cylcop extends the copula package to allow modeling of correlated circular-linear random variables using copulae that are symmetric in the circular dimension. We present and derive several new circular-linear copulae with this property and demonstrate how they can be implemented in the cylcop package to model animal movements in discrete time. The package contains methods for estimating copulae parameters, plotting probability density and cumulative distribution functions, and simulating data.

sentences: How much do model organism phenotypes contribute to the computational identification of human disease genes? Computing phenotypic similarity has been shown to be useful in identification of new disease genes and for rare disease diagnostic support. Genotype-phenotype data from orthologous genes in model organisms can compensate for lack of human data to greatly increase genome coverage. Work over the past decade has demonstrated the power of cross-species phenotype comparisons, and several cross-species phenotype ontologies have been developed for this purpose. The relative contribution of different model organisms to identifying diseaseassociated genes using computational approaches is not yet fully explored. We use methods based on phenotype ontologies to semantically relate phenotypes resulting from loss-of-function mutations in different model organisms to disease-associated phenotypes in humans. Semantic machine learning methods are used to measure how much different model organisms contribute to the identification of known human gene-disease associations. We find that only mouse phenotypes can accurately predict human gene-disease associations. Our work has implications for the future development of integrated phenotype ontologies, as well as for the use of model organism phenotypes in human genetic variant interpretation.

sentences: Neural pathways linking hypoxia with pectoral fin movements in Danio rerio Zebrafish larvae respond to hypoxia by increasing a number of ventilatory behaviors. During development, these animals switch from skin-resident to gill-resident neuroendocrine cells around 7 days post fertilization (d.p.f.) to detect hypoxia and drive adaptive behaviors. Here, we probe the neural pathways that receive inputs from skin-resident neuroendocrine cells and alter pectoral fin movements. We first show that a 5 d.p.f. larva increases its pectoral fin movements and heart activity upon hypoxia exposure. Next, we map the downstream neural circuitry and show that individual vagal sensory neurons receive inputs from multiple oxygen-sensing neuroendocrine cells. We then use calcium imaging to show that neurons in the second, but not third, vagal sensory ganglia show increases in the magnitude of their hypoxia-evoked responses. Finally, we link purinergic signaling between neuroendocrine cells and second vagal sensory neurons to increases in pectoral fin movements. Collectively, we suggest that vagal sensory neurons transform hypoxic stimuli into respiratory behaviors.

sentences: SARS-CoV-2 comprehensive receptor profiling: mechanistic insight to drive new therapeutic strategies Here we describe a hypothesis free approach to screen for interactions of SARS-CoV-2 spike (S) protein with human cell surface receptors. We used a library screening approach to detect binding interactions across one of the largest known panels of membrane-bound and soluble receptors, comprising 5845 targets, expressed recombinantly in human cells. We were able confirm and replicate SARS-CoV-2 binding to ACE2 and other putative coreceptors such as CD209 and CLEC4M. More significantly, we identified interactions with a number of novel SARS-CoV-2 S binding proteins. Three of these novel receptors, NID1, CNTN1 and APOA4 were specific to SARS-CoV-2, and not SARS-COV, with APOA4 binding the S-protein with equal affinity as ACE2. With this knowledge we may further understand the disease pathogenesis of COVID-19 patients and how infection by SARS-CoV-2 may lead to differences in pathology in specific organs or indeed the virulence observed in different ethnicities. Importantly we illustrate a methodology which can be used for rapid, unbiassed identification of cell surface receptors, to support drug screening and drug repurposing approaches for this and future pandemics.

sentences: Histone H3K4me1 and H3K27ac play roles in nucleosome depletion and eRNA transcription, respectively, at enhancers Histone H3K4me1 and H3K27ac are enhancer specific modifications and are required for enhancers to activate transcription of target genes. However the reciprocal effects of these histone modifications on each other and their roles in enhancers are not clear. Here to comparatively analyze the role of these modifications, we inhibited H3K4me1 and H3K27ac by deleting SET domain of histone methyltransferases MLL3 and MLL4 and HAT domain of histone acetyltransferase p300, respectively, in erythroid K562 cells. The loss of H3K4me1 reduced H3K27ac at the {beta}-globin enhancer LCR HSs, but H3K27ac reduction did not affect H3K4me1. This unequal relationship between two modifications was revealed in putative enhancers by genome-wide analysis using ChIP-seq. Histone H3 depletion at putative enhancers was weakened by the loss of H3K4me1 but not by the loss of H3K27ac. Chromatin remodeling complexes were recruited into the {beta}-globin LCR HSs in a H3K4me1-dependent manner. In contrast, H3K27ac was required for enhancer RNA (eRNA) transcription, and H3K4me1 was not enough for it. Forced H3K27ac induced eRNA transcription without affecting H3K4me1 at the {beta}-globin LCR HSs. These results indicate that H3K4me1 and H3K27ac affect each other in different ways and play more direct roles in nucleosome depletion and eRNA transcription, respectively, at enhancers.

sentences: Multiple Causal Variants Underlie Genetic Associations in Humans The majority of associations between genetic variation and human traits and diseases are non-coding and in strong linkage disequilibrium (LD) with surrounding genetic variation. In these cases, a single causal variant is often assumed to underlie the association, however no systematic assessment of the number of causal variants has been performed. In this study, we applied a massively parallel reporter assay (MPRA) in lymphoblastoid cells to functionally evaluate 49,256 allelic pairs, representing 30,893 genetic variants in high, local linkage disequilibrium for 744 independent cis-expression quantitative trait loci (eQTL) and assessed each for colocalization across 114 traits. We identified 8,502 allele-independent regulatory regions containing 1,264 allele-specific regulatory variants, and found that 17.7% of eQTL contained more than one significant allelic effect. We show that detected regulatory variants are highly and specifically enriched for activating chromatin structures and allelic transcription factor binding, for which ETS-domain family members are a large driver. Integration of MPRA profiles with eQTL/complex trait colocalizations identified causal variant sets for associations with blood cell measurements, Asthma, Multiple Sclerosis, Inflammatory Bowel Disease, and Crohns Disease. These results demonstrate that a sizable number of association signals are manifest through multiple, tightly-linked causal variants requiring high-throughput functional assays for fine-mapping.

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Surfactin stimulated by pectin molecular patterns and root exudates acts as a key driver of Bacillus-plant mutualistic interaction Bacillus velezensis is considered as model species belonging to the so-called B. subtilis complex that typically evolved to dwell in the soil rhizosphere niche and establish intimate association with plant roots. This bacterium provides protection to its natural host against diseases and represents one of the most promising biocontrol agents. However, the molecular basis of the cross-talk that this bacterium establishes with its natural host has been poorly investigated. We show here that these plant-associated bacteria have evolved some polymer-sensing system to perceive their host and that in response, they increase the production of the surfactin-type lipopeptide. Furthermore, we demonstrate that surfactin synthesis is favoured upon growth on root exudates and that this lipopeptide is a key component used by the bacterium to optimize biofilm formation, motility and early root colonization. In this specific nutritional context, the bacterium also modulates qualitatively the pattern of surfactin homologues co-produced in planta and mainly forms variants that are the most active at triggering plant immunity. Surfactin represents a shared good as it reinforces the defensive capacity of the host.

ImportanceWithin the plant-associated microbiome, some bacterial species are of particular interest due to the disease protective effect they provide via direct pathogen suppression and/or stimulation of host immunity. While these biocontrol mechanisms are quite well characterized, we still poorly understand the molecular basis of the cross talk these beneficial bacteria initiate with their host. Here we show that the model species Bacillus velezensis stimulates production of the surfactin lipopeptide upon sensing pectin as cell surface molecular pattern and upon feeding on root exudates. Surfactin favors bacterial rhizosphere fitness on one hand and primes the plant immune system on the other hand. Our data therefore illustrate how both partners use this multifunctional compound as unique shared good to sustain mutualistic interaction.

sentences: Targeted isolation of panels of diverse human protective broadly neutralizing antibodies against SARS-like viruses The emergence of current SARS-CoV-2 variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy 1-7. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed 8, 9. Notably, several recent studies have revealed that vaccination of recovered COVID-19 donors results in enhanced nAb responses compared to SARS-CoV-2 infection or vaccination alone 10-13. Here, we utilized a targeted donor selection strategy to isolate a large panel of broadly neutralizing antibodies (bnAbs) to sarbecoviruses from two such donors. Many of the bnAbs are remarkably effective in neutralization against sarbecoviruses that use ACE2 for viral entry and a substantial fraction also show notable binding to non-ACE2-using sarbecoviruses. The bnAbs are equally effective against most SARS-CoV-2 VOCs and many neutralize the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor binding domain (RBD) as opposed to strain-specific nAbs to the receptor binding site that are commonly elicited in SARS-CoV-2 infection and vaccination 14-18. Consistent with targeting of conserved sites, select RBD bnAbs exhibited in vivo protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model. The generation of a large panel of potent bnAbs provides new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and, importantly, provides a molecular basis for effective design of pan-sarbecovirus vaccines.

sentences: Approximated Gene Expression Trajectories (AGETs) for Gene Regulatory Network Inference on Cell Tracks The study of pattern formation has benefited from reverse-engineering gene regulatory network (GRN) structure from spatio-temporal quantitative gene expression data. Traditional approaches omit tissue morphogenesis, hence focusing on systems where the timescales of pattern formation and morphogenesis can be separated. In such systems, pattern forms as an emergent property of the underlying GRN. This is not the case in many animal patterning systems, where patterning and morphogenesis are simultaneous. To address pattern formation in these systems we need to adapt our methodologies to explicitly accommodate cell movements and tissue shape changes. In this work we present a novel framework to reverse-engineer GRNs underlying pattern formation in tissues experiencing morphogenetic changes and cell rearrangements. By combination of quantitative data from live and fixed embryos we approximate gene expression trajectories (AGETs) in single cells and use a subset to reverse-engineer candidate GRNs using a Markov Chain Monte Carlo approach. GRN fit is assessed by simulating on cell tracks (live-modelling) and comparing the output to quantitative data-sets. This framework outputs candidate GRNs that recapitulate pattern formation at the level of the tissue and the single cell. To our knowledge, this inference methodology is the first to integrate cell movements and gene expression data, making it possible to reverse-engineer GRNs patterning tissues undergoing morphogenetic changes.

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Deep and superficial layers of the primary somatosensory cortex are critical for whisker-based texture discrimination in mice The neocortex, comprised of multiple distinct layers, processes sensory input from the periphery, makes decisions, and executes actions. Despite extensive investigation of cortical anatomy and physiology, the contributions of different cortical layers to sensory guided behaviors remain unknown. Here, we developed a two-alternative forced choice (2AFC) paradigm in which head-fixed mice use a single whisker to either discriminate textures of parametrically varied roughness or detect the same textured surfaces. Lesioning the barrel cortex revealed that 2AFC texture discrimination, but not detection, was cortex-dependent. Paralyzing the whisker pad had little effect on performance, demonstrating that passive can rival active perception and cortical dependence is not movement-related. Transgenic Cre lines were used to target inhibitory opsins to excitatory cortical neurons of specific layers for selective perturbations. Both deep and superficial layers were critical for texture discrimination. We conclude that even basic cortical computations require coordinated transformation of sensory information across layers.

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sentences: Light-dependent modulation of protein localization and function in living bacteria cells Most bacteria lack membrane-enclosed organelles to compartmentalize cellular processes. In lieu of physical compartments, bacterial proteins are often recruited to macromolecular scaffolds at specific subcellular locations to carry out their functions. Consequently, the ability to modulate a proteins subcellular location with high precision and speed bears the potential to manipulate its corresponding cellular functions. Here we demonstrate that the CRY2/CIB1 system from Arabidopsis thaliana can be used to rapidly direct proteins to different subcellular locations inside live E. coli cells including the nucleoid, the cell pole, membrane, and the midcell division plane. We further show that such light-induced re-localization can be used to rapidly inhibit cytokinesis in actively dividing E. coli cells. Finally, we demonstrate that the CRY2/CIBN binding kinetics can be modulated by green light, adding a new dimension of control to the system.

sentences: GLOBAL LAND-USE AND LAND-COVER DATA FOR ECOLOGISTS: HISTORICAL, CURRENT AND FUTURE SCENARIOS Land-use land-cover (LULC) data are important predictors of species occurrence and biodiversity threat. Although there are LULC datasets available under current conditions, there is a lack of such data under historical and future climatic conditions. This hinders, for example, projecting niche and distribution models under global change scenarios at different time scenarios. The Land Use Harmonization Project (LUH2) is a global terrestrial dataset at 0.25{degrees} spatial resolution that provides LULC data from 850 to 2300 for 12 LULC state classes. The dataset, however, is compressed in a file format (NetCDF) that is incompatible for many analyses and requires layer extractions and transformations that are intractable for most researchers. Here we selected and transformed the LUH2 in a standard GIS format data to make it more user-friendly. We provide LULC for every year from 850 to 2100, and from 2015 on, the LULC dataset is provided under two Shared Socioeconomic Pathways (SSP2-4.5 and SSP5-8.5). We provide two types of files for each year: separate files with continuous values for each of the 12 LULC state classes, and a single categorical file with all state classes combined. To create the categorical layer, we assigned the state with the highest value in a given pixel among the 12 continuous data. LUH2 predicts a pronounced decrease in primary forest, particularly noticeable in the Amazon, the Brazilian Atlantic Forest, the Congo Basin and the boreal forests, an equally pronounced increase in secondary forest and non-forest lands, and in croplands in the Brazilian Atlantic Forest and sub-Saharan Africa. The final dataset provides LULC data for 1251 years that will be of interest for macroecology, ecological niche modeling, global change analysis, and other applications in ecology and conservation.

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The Arabidopsis HOP2 Gene Has a Role in Preventing illegitimate Exchanges between Nonhomologous Chromosomes Meiotic homologous chromosomes pair up and undergo crossing over. In many eukaryotes both intimate pairing and crossing over require the induction of double stranded breaks (DSBs) and subsequent repair via Homologous Recombination (HR). In these organisms, two key proteins are the recombinases RAD51 and DMC1. Recombinase-modulators HOP2 and MND1 have been identified as proteins that assist RAD51 and DMC1 and are needed to promote stabilized pairing. We have probed the nature of the genetic lesions seen in hop2 mutants and looked at the role of HOP2 in the fidelity of genetic exchanges. Using {gamma}H2Ax as a marker for unrepaired DSBs we found that hop2-1 and mnd1 mutants have different appearance/disappearance for DSBs than wild type, but all DSBs are repaired by mid-late pachytene. Therefore, the bridges and fragments seen from metaphase I onward are due to mis-repaired DSBs, not unrepaired ones. Studying Arabidopsis haploid meiocytes we found that wild type haploids produced the expected five univalents, but hop2-1 haploids suffered many illegitimate exchanges that were stable enough to produce bridged chromosomes during segregation. Our results suggest that HOP2 has a significant active role in preventing nonhomologous associations. We also found evidence that HOP2 plays a role in preventing illegitimate exchanges during repair of radiation-induced DSBs in rapidly dividing petal cells. Thus, HOP2 plays both a positive role in promoting homologous chromosome synapsis and a separable role in preventing nonhomologous chromosome exchanges. Possible mechanisms for this second important role are discussed.

SIGNIFICANCE STATEMENTThe fidelity of Homologous Recombination (HR) during meiosis is essential to the production of viable gametes and for maintaining genome integrity in vegetative cells. HOP2 is an important protein for accurate meiotic HR in plants. We find high levels of illegitimate repairs between nonhomologous chromosomes during meiosis and in irradiated petal cells in HOP2 mutants. We consider mechanisms of how this second role might be accomplished.

sentences: Chemical and genetic screens identify new regulators of tetracycline-inducible gene expression system in mammalian cells The tetracycline repressor (tetR)-regulated system is a widely used tool to specifically control gene expression in mammalian cells. Based on this system, we generated a human osteosarcoma cell line which allows for inducible expression of an EGFP-fusion of the TAR DNA-binding protein 43 (TDP-43), which has been linked to neurodegenerative diseases. Consistent with previous findings, TDP-43 overexpression led to the accumulation of aggregates and limited the viability of U2OS. Using this inducible system, we conducted a chemical screen with a library that included FDA-approved drugs. While the primary screen identified several compounds that prevented TDP-43 toxicity, further experiments revealed that these chemicals abrogated doxycyclinedependent TDP-43 expression. This antagonistic effect was observed with both doxycycline and tetracycline, and in several Tet-On cell lines expressing different genes, confirming the general effect of these compounds as inhibitors of the tetR system. Using the same cell line, a genome-wide CRISPR/Cas9 screen identified epigenetic regulators such as the G9a methyltransferase or TRIM28 as potential modifiers of TDP-43 toxicity. Yet again, further experiments revealed that G9a inhibition or TRIM28 loss prevented doxycycline-dependent expression of TDP-43. Together, these results suggest that none of the medically approved drugs significantly mitigates TDP-43 toxicity, identify new chemical and genetic regulators of the tetR system, and raise awareness on the limitations of this approach to conduct chemical or genetic screenings in mammalian cells.

sentences: Hippocampal-medial prefrontal event segmentation and integration contribute to episodic memory formation How do we encode our continuous life experiences for later retrieval? Theories of event segmentation and integration suggest that the hippocampus binds separately represented events into an ordered narrative. Using a functional Magnetic Resonance Imaging (fMRI) movie watching-recall dataset, we quantified two types of neural similarities (i.e., activation pattern similarity and within-region voxel-based connectivity pattern similarity) between separate events during movie watching and related them to subsequent retrieval of events as well as retrieval of sequential order. We demonstrated that compared to forgotten events, successfully remembered events were associated with distinct activation patterns in the hippocampus and medial prefrontal cortex. By contrast, similar connectivity patterns between events were associated with memory formation and were also relevant for retaining events in the correct order. We applied the same approaches to an independent movie watching fMRI dataset as validation and highlighted again the role of hippocampal activation pattern and connectivity pattern in memory formation. We propose that distinct activation patterns represent neural segmentation of events while similar connectivity patterns encode context information, and therefore integrate events into a narrative. Our results provide novel evidence for the role of hippocampal-medial prefrontal event segmentation and integration in episodic memory formation of real-life experience.

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Constitutive signal bias mediated by the human GHRHR splice variant 1 Alternative splicing of G protein-coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to {beta}-arrestins. Based on the cryo-electron microscopy structures of SV1 in the apo state or in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs vs. {beta}-arrestins. Suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias towards {beta}-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.

Significance StatementThe mechanism of functional changes induced by alternative splicing of GHRHR is largely unknown. Here, we demonstrate that GHRH-elicited signal bias towards {beta}-arrestin recruitment is constitutively mediated by SV1. The cryo-electron microscopy structures of SV1 and molecular dynamics simulations reveal the different functionalities between GHRHR and SV1 at the near-atomic level, i.e., the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs vs. {beta}-arrestins. Our findings provide valuable insights into functional diversity of class B1 GPCRs which may aid in the design of better therapeutic agents against certain cancers.

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Comparison of linear combination modeling strategies for GABA-edited MRS at 3T PurposeJ-difference-edited spectroscopy is a valuable approach for the in vivo detection of {gamma}-aminobutyric-acid (GABA) with MRS. A recent expert consensus article recommends linear combination modeling (LCM) of edited MRS but does not give specific details of implementation. This study explores different modeling strategies to adapt LCM for GABA-edited MRS.

Methods61 medial parietal lobe GABA-edited MEGA-PRESS spectra from a recent 3T multi-site study were modeled using 102 different strategies combining six different approaches to account for co-edited macromolecules, three modeling ranges, three baseline knot spacings, and the use of basis sets with or without homocarnosine. The resulting GABA and GABA+ estimates (quantified relative to total creatine), the residuals at different ranges, SDs and CVs, and Akaike information criteria, were used to evaluate the models performance.

ResultsSignificantly different GABA+ and GABA estimates were found when a well-parameterized MM3co basis function was included in the model. The mean GABA estimates were significantly lower when modeling MM, while the CVs were similar. A sparser spline knot spacing led to lower variation in the GABA and GABA+ estimates, and a narrower modeling range - only including the signals of interest - did not substantially improve or degrade modeling performance. Additionally, results suggest that LCM can separate GABA and the underlying co-edited MM3co. Incorporating homocarnosine into the modeling did not significantly improve variance in GABA+ estimates.

ConclusionGABA-edited MRS is most appropriately quantified by LCM with a well-parameterized co-edited MM3co basis function with a constraint to the non-overlapped MM0.93, in combination with a sparse spline knot spacing (0.55 ppm) and a modeling range between 0.5 and 4 ppm.

Graphical Abstract

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org.highwire.dtl.DTLVardef@129ce60org.highwire.dtl.DTLVardef@1ac3797org.highwire.dtl.DTLVardef@1759d69org.highwire.dtl.DTLVardef@b190a3_HPS_FORMAT_FIGEXP  M_FIG C_FIG 102 strategies to model GABA-edited MRS with linear combination modeling were evaluated to quantify GABA and GABA+ in Osprey. Significantly different GABA and GABA+ estimates were found when a well-parameterized macro-molecule at 3 ppm was included. The findings suggest that linear combination modeling needs to be adapted for quantification of GABA-edited MRS.

sentences: Elys deficiency constrains Kras-driven tumour burden by amplifying oncogenic stress The nucleoporin ELYS, encoded by AHCTF1, is a large multifunctional protein with essential roles in nuclear pore assembly and mitosis. Using a zebrafish model of hepatocellular carcinoma, in which the expression of an inducible mutant kras transgene (krasG12V) drives hepatocyte-specific hyperplasia and liver enlargement, we show that reducing ahctf1 gene dosage by 50% markedly shrinks tumour burden, while non-hyperplastic tissues are unaffected. We demonstrate that ahctf1 heterozygosity impairs nuclear pore formation, mitotic spindle assembly and chromosome segregation, leading to DNA damage and activation of TP53-dependent and independent mechanisms of cell death and cell cycle arrest. This selective vulnerability of cancer cells to mild disruption of Elys function uncovers a novel synthetic lethal interaction between ahctf1 and kras mutations that could be exploited therapeutically. Heterozygous expression of both ahctf1 and ranbp2, or treatment of heterozygous ahctf1 larvae with the nucleocytoplasmic transport inhibitor, Selinexor, completely blocked krasG12V-driven hepatocyte hyperplasia, revealing promising avenues for combinatorial treatments.

sentences: The  prion and HSP104 modulate cytochrome c oxidase deficiency caused by deletion of COX12 Cytochrome c oxidase is a pivotal enzyme of the mitochondrial respiratory chain, which sustains bioenergetics of eukaryotic cells. Cox12, a peripheral subunit of cytochrome c oxidase, is required for full activity of the enzyme, but its exact function is unknown. Here, experimental evolution of a Saccharomyces cerevisiae {Delta}cox12 strain for ~300 generations allowed to restore the activity of cytochrome c oxidase. In one population, the enhanced bioenergetics was caused by a A375V mutation in the AAA+ disaggregase Hsp104. Deletion or overexpression of Hsp104 also increased respiration of the {Delta}cox12 ancestor strain. This beneficial effect of Hsp104 was related to the loss of the [PSI+] prion, which forms cytosolic amyloid aggregates of the Sup35 protein. Overall, our data demonstrate that cytosolic aggregation of a prion impairs the mitochondrial metabolism of cells defective for Cox12. These findings identify a new functional connection between cytosolic proteostasis and biogenesis of the mitochondrial respiratory chain.

sentences: The Skeletal Muscle Circadian Clock Regulates Titin Splicing Through RBM20 Circadian rhythms are maintained by a cell autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression through both RNAseq and LC-MS. This alteration in titins spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMSBmal1-/- muscle. Finally, we identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20, a potent splicing regulator of titin. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies.

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Urban tree deaths from invasive alien forest insects in the United States, 2020-2050 O_LIUrban trees are important nature-based solutions for future wellbeing and livability but are at high risk of mortality from insect pests. In the United States (US), 82% of the population live in urban settings and this number is growing, making urban tree mortality a matter of concern for most of its population. Until now, the magnitudes and spatial distributions of risks were unknown.
C_LIO_LIHere, we combine new models of street tree populations in [~]30,000 US communities, species-specific spread predictions for 57 invasive insect species, and estimates of tree death due to insect exposure for 48 host tree genera.
C_LIO_LIWe estimate that 1.4 million street trees will be killed by invasive insects from 2020 through 2050, costing an annualized average of US$ 30M. However, these estimates hide substantial variation: 23% of urban centers will experience 95% of all insect-induced mortality. Further, 90% of all mortality will be due to emerald ash borer (Agrilus planipennis, EAB), which is expected to kill virtually all ash trees (Fraxinus spp.) in >6000 communities.
C_LIO_LIWe define an EAB high-impact zone spanning 902,500km2, largely within the southern and central US, within which we predict the death of 98.8% of all ash trees. "Mortality hotspot cities" include Milwaukee, WI; Chicago, IL; and New York, NY.
C_LIO_LIWe identify Asian wood borers of maple and oak trees as the highest risk future invaders, where a new establishment could cost US$ 4.9B over 30 years.
C_LIO_LIPolicy implications: To plan effective mitigation, managers must know which tree species in which communities will be at the greatest risk, as well as the highest-risk insects. We provide the first country-wide, spatial forecast of urban tree mortality due to invasive insect pests. This framework identifies dominant pest insects and spatial impact hotspots, which can provide the basis for spatial prioritization of spread control efforts such as quarantines and biological control release sites. Our results highlight the need for EAB early-detection efforts as far from current infestations as Seattle, WA. Further, these findings produce a list of biotic and spatiotemporal risk factors for future high-impact US urban forest insect pests.
C_LI

sentences: ATP6AP2-to-MMP14, a key pathway for osteoblast to osteocyte transition Osteocytes, derived from osteoblast (OB)-lineage, occupy lacunae within bone matrix and exhibit unique morphology with dendrite-like projections to form an inter-connected network. Such a network is essential for osteocytes to monitor and orchestrate bone homeostasis. Thus, it is of considerable interest to investigate how osteocytes are formed and how they built the network. Here we provide evidence for ATP6AP2, an accessory subunit of V-ATPase, in OB-lineage cells to be critical for OB-to-osteocyte transition, and osteocyte distribution, maturation, and morphogenesis. Mice (ATP6AP2Ocn-Cre) that selectively deplete ATP6AP2 in OB-lineage cells results in altered osteocyte distribution and morphology, impaired osteocyte maturation and dendrite-like processes, increased osteocyte cell death and cortical woven bone formation. Further mechanistic studies identify MMP14 (matrix metalloproteinase-14) as a critical downstream of ATP6AP2 for osteocyte differentiation. ATP6AP2 interacts with MMP14 and promotes MMP14 surface distribution largely in immature- or osteoid-osteocytes, where this pathway regulates bone matrix remodeling and osteocyte differentiation. Expression of MMP14 into ATP6AP2 knock out OB-lineage cells in the mouse cortical bone could diminish the deficits in osteocyte maturation, dendrite-like process formation, and survival. These results thus demonstrate an un-recognized function of ATP6AP2 in promoting OB-to-osteocyte transition and uncover a pathway from ATP6AP2-to-MMP14 in osteocyte differentiation.

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ColabFold - Making protein folding accessible to all ColabFold offers accelerated protein structure and complex predictions by combining the fast homology search of MMseqs2 with AlphaFold2 or RoseTTAFold. ColabFolds 40 - 60x faster search and optimized model use allows predicting close to a thousand structures per day on a server with one GPU. Coupled with Google Colaboratory, ColabFold becomes a free and accessible platform for protein folding. ColabFold is open-source software available at github.com/sokrypton/ColabFold. Its novel environmental databases are available at colabfold.mmseqs.com

Contactmilot.mirdita@mpibpc.mpg.de, so@fas.harvard.edu, martin.steinegger@snu.ac.kr

sentences: Experimental characterisation of de novoproteins and their unevolved random-sequencecounterparts De novo gene emergence provides a route for new proteins to be formed from previously non-coding DNA. Proteins born in this way are considered random sequences, and typically assumed to lack defined structure. While it remains unclear how likely a de novo protein is to assume a soluble and stable tertiary structure, intersecting evidence from random-sequence and de novo-designed proteins suggests that native-like biophysical properties are abundant in sequence space. Taking putative de novo proteins identified in human and fly, we experimentally characterise a library of these sequences to assess their solubility and structure propensity. We compare this library to a set of synthetic random proteins with no evolutionary history. Bioin-formatic prediction suggests that de novo proteins may have remarkably similar distributions of biophysical properties to unevolved random sequences of a given length and amino acid composition. However, upon expression in vitro, de novo proteins exhibit higher solubility which is further induced by the DnaK chaperone system. We suggest that while synthetic ran-dom sequences are a useful proxy for de novo proteins in terms of structure propensity, de novo proteins may be better integrated in the cellular system given their higher solubility.

sentences: Learning the rules of cell competition without prior scientific knowledge AO_SCPLOWBSTRACTC_SCPLOWDeep learning is now a powerful tool in microscopy data analysis, and is routinely used for image processing applications such as segmentation and denoising. However, it has rarely been used to directly learn mechanistic models of a biological system, owing to the complexity of the internal representations. Here, we develop an end-to-end machine learning model capable of learning the rules of a complex biological phenomenon, cell competition, directly from a large corpus of time-lapse microscopy data. Cell competition is a quality control mechanism that eliminates unfit cells from a tissue and during which cell fate is thought to be determined by the local cellular neighborhood over time. To investigate this, we developed a new approach ({tau}-VAE) by coupling a probabilistic encoder to a temporal convolution network to predict the fate of each cell in an epithelium. Using the{tau} -VAEs latent representation of the local tissue organization and the flow of information in the network, we decode the physical parameters responsible for correct prediction of fate in cell competition. Remarkably, the model autonomously learns that cell density is the single most important factor in predicting cell fate - a conclusion that is in agreement with our current understanding from over a decade of scientific research. Finally, to test the learned internal representation, we challenge the network with experiments performed in the presence of drugs that block signalling pathways involved in competition. We present a novel discriminator network that, using the predictions of the{tau} -VAE, can identify conditions which deviate from the normal behaviour, paving the way for automated, mechanism-aware drug screening.

sentences: Balancing conservation priorities for grassland and forest specialist bird communities in agriculturally dominated landscapes Effective conservation planning often requires difficult decisions when at-risk species inhabit economically valuable landscapes or if the needs of multiple threatened species do not align. In the agriculture-dominated landscape of eastern Ontario and southwestern Quebec, Canada, conflicting habitat requirements exist between threatened grassland birds benefiting from certain agriculture practices and those of a diverse woodland bird community dependent on forest recovery. Using multi-scale species distribution models with Breeding Bird Survey (BBS) data, we assessed habitat suitability for 8 threatened grassland and forest specialists within this region. We also identified landscapes that jointly maximize occurrence of the 8 focal species and diversity of the overall grassland and forest communities. Influential habitat associations differed among species at the territory (200m radius) and landscape level (1km), highlighting the importance of considering multiple spatial scales. Species diversity was maximized when forest or grassland/pasture cover approached 40-50%, indicating a positive response to land cover heterogeneity. We identified species diversity hotspots near Lake Huron, as well as along the shore and southeast of the St. Lawrence River. These areas represent mosaic landscapes, balancing forest patches, wetland, grassland/pasture, and row crops such as corn, soybean, and cereals. Despite drastic landscape changes associated with agroecosystems, we demonstrate that targeted habitat protection and enhancement that prioritizes land cover diversity can maximize protection of bird communities with directly contrasting needs. We highlight multiple pathways to achieve this balance, including forest retention or separating row crops with hedgerows and wooded fence-lines, improving flexibility in conservation approaches.

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Heterogeneity of monocyte subsets and susceptibility to influenza virus contribute to inter-population variability of protective immunity There is considerable inter-individual and inter-population variability in response to viruses. The potential of monocytes to elicit type-I interferon responses has attracted attention to their role in viral infections. Here, we use an ex vivo model to characterize the role of cellular heterogeneity in human variation of monocyte responses to influenza A virus (IAV) exposure. Using single-cell RNA-sequencing, we show widespread inter-individual variability in the percentage of IAV-infected monocytes. We show that cells escaping viral infection display increased mRNA expression of type-I interferon stimulated genes and decreased expression of ribosomal genes, relative to both infected cells and those never exposed to IAV. While this host defense strategy is shared between CD16+/CD16- monocytes, we also uncover CD16+-specific mRNA expression of IL6 and TNF in response to IAV, and a stronger resistance of CD16+ monocytes to IAV infection. Notably, individuals with high cellular susceptibility to IAV are characterized by a lower activation at basal state of an IRF/STAT-induced transcriptional network, which includes antiviral genes such as IFITM3, MX1, and OAS3. Finally, using flow cytometry and bulk RNA-sequencing across 200 individuals of African and European ancestry, we observe a higher number of CD16+ monocytes and lower susceptibility to IAV infection among monocytes from individuals of African-descent. Collectively, our results reveal the effects of IAV infection on the transcriptional landscape of human monocytes and highlight previously unappreciated differences in cellular susceptibility to IAV infection between individuals of African and European ancestry, which may account for the greater susceptibility of Africans to severe influenza.

Significance StatementMonocytes may play a critical role during severe viral infections. Our study tackles how heterogeneity in monocyte subsets and activation contributes to shape individual differences in the transcriptional response to viral infections. Using single-cell RNA-sequencing, we reveal heterogeneity in monocyte susceptibility to IAV infection, both between CD16+/CD16- monocytes and across individuals, driven by differences in basal activation of an IRF/STAT-induced antiviral program. Furthermore, we show a decreased ability of IAV to infect and replicate in monocytes from African-ancestry individuals, with possible implications for antigen presentation and lymphocyte activation. These results highlight the importance of early cellular activation in determining an individuals innate immune response to viral infection.

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Adaptive learning through temporal dynamics of state representation People adjust their learning rate rationally according to local environmental statistics and calibrate such adjustments based on the broader statistical context. To date, no theory has captured the observed range of adaptive learning behaviors or the complexity of its neural correlates. Here, we attempt to do so using a neural network model that learns to map an internal context representation onto a behavioral response via supervised learning. The network shifts its internal context upon receiving supervised signals that are mismatched to its output, thereby changing the "state" to which feedback is associated. A key feature of the model is that such state transitions can either increase learning or decrease learning depending on the duration over which the new state is maintained. Sustained state transitions that occur after changepoints facilitate faster learning and mimic network reset phenomena observed in the brain during rapid learning. In contrast, state transitions after one-off outlier events are short-lived, thereby limiting the impact of outlying observations on future behavior. State transitions in our model provide the first mechanistic interpretation for bidirectional learning signals, such the p300, that relate to learning differentially according to the source of surprising events and may also shed light on discrepant observations regarding the relationship between transient pupil dilations and learning. Taken together, our results demonstrate that dynamic latent state representations can afford normative inference and provide a coherent framework for understanding neural signatures of adaptive learning across different statistical environments.

Significance StatementHow humans adjust their sensitivity to new information in a changing world has remained largely an open question. Bridging insights from normative accounts of adaptive learning and theories of latent state representation, here we propose a feed-forward neural network model that adjusts its learning rate online by controlling the speed of transitioning its internal state representations. Our model proposes a mechanistic framework for explaining learning under different statistical contexts, explains previously observed behavior and brain signals, and makes testable predictions for future experimental studies.

sentences: The Trypanosoma brucei RNA-binding protein DRBD18 ensures correct mRNA trans splicing and polyadenylation patterns The parasite Trypanosoma brucei grows as bloodstream forms in mammals, and as procyclic forms in tsetse flies. Transcription is polycistronic, all mRNAs are trans spliced, and polyadenylation sites are defined by downstream splicing signals. Expression regulation therefore depends heavily on post-transcriptional mechanisms. The RNA-binding protein DRBD18 was previously implicated in the export of some mRNAs from the nucleus in procyclic forms. It copurifies the outer ring of the nuclear pore, mRNA export factors and exon-junction-complex proteins. We show that for >200 mRNAs, DRBD18 depletion caused preferential accumulation of versions with shortened 3-untranslated regions, arising from use of polyadenylation sites that were either undetectable or rarely seen in non-depleted cells. The shortened mRNAs were often, but not always, more abundant in depleted cells than the corresponding longer versions in normal cells. Their appearance was linked to the appearance of trans spliced, polyadenylated RNAs containing only downstream 3-untranslated-region-derived sequences. Experiments with one mRNA suggested that nuclear retention alone, through depletion of MEX67, did not affect mRNA length, suggesting a specific effect of DRBD18 on processing. Since DRBD18-bound mRNAs were enriched in polypyrimidine tract motifs, and it is found in both the nucleus and the cytoplasm, we suggest that DRBD18 acts in the nucleus by binding to polypyrimidine tracts in 3-UTRs. DRBD18 binding might both prevent polypyrimidine tract recognition by splicing factors, and promote export of the bound RNAs to the cytosol.

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Predicting potentially permissive substitutions that improve the fitness of A(H1N1)pdm09 viruses bearing the H275Y NA substitution Oseltamivir-resistant influenza viruses arise due to amino-acid mutations in key residues, but these changes often reduce their replicative and transmission fitness. Widespread oseltamivir-resistance has not yet been observed in A(H1N1)pdm09 viruses. However, it is known that permissive mutations in the neuraminidase (NA) of former seasonal A(H1N1) viruses from 2007-2009 buffered the detrimental effect of the NA H275Y mutation, resulting in fit oseltamivir-resistant viruses that circulated widely. This study explored two approaches to predict permissive mutations that may enable a fit H275Y A(H1N1)pdm09 variant to arise.

A computational approach used phylogenetic and in silico protein stability analyses to predict potentially permissive mutations, which were then evaluated by in vitro NA enzyme activity and expression analysis, followed by in vitro replication. The second approach involved the generation of a virus library which encompassed all possible individual 2.9 x 104 codon mutations in the NA whilst keeping H275Y fixed. To select for variant viruses with the greatest fitness, the virus library was serially passaged in ferrets (via contact and aerosol transmission) and resultant viruses were deep sequenced.

The computational approach predicted three NA permissive mutations, and even though they only offset the in vitro impact of H275Y on NA enzyme expression by 10%, they could restore replication fitness of the H275Y variant in A549 cells. In our experimental approach, a diverse virus library (97% of 8911 possible single amino-acid substitutions were sampled) was successfully transmitted through ferrets, and sequence analysis of resulting virus pools in nasal washes identified three mutations that improved virus transmissibility. Of these, one NA mutation, I188T, has been increasing in frequency since 2017 and is now present in 90% of all circulating A(H1N1)pdm09 viruses.

Overall, this study provides valuable insights into the evolution of the influenza NA protein and identified several mutations that may potentially facilitate the emergence of a fit H275Y A(H1N1)pdm09 variant.

sentences: LncRNA Miat and Metadhedrin maintain a treatment resistant stem-like niche of medulloblastoma cells Medulloblastoma is a pediatric brain tumor arising from the cerebellum and brainstem that is curable with surgery and aggressive cytotoxic therapy including craniospinal irradiation and chemotherapy. Preclinical models indicate that a small pool of de-differentiated, stem cell-like medulloblastoma cells are resistant to cytotoxic treatment and contribute to medulloblastoma relapse after aggressive therapy. Here, we identify Miat as a Shh and Myc regulated long noncoding RNA (lncRNA) that is required for maintenance of a treatment-resistant medulloblastoma stem-like phenotype. Loss of Miat delays medulloblastoma formation in genetically defined mouse models of Shh medulloblastoma and enforces differentiation of tumorigenic stem-like medulloblastoma cells into a non-tumorigenic cell state. Miat facilitates treatment resistance in part by downregulating p53 signaling and impairing radiation induced cell death in stem-like MB cells, which can be reversed by therapeutic inhibition of Miat with antisense oligonucleotides. The RNA binding protein Metadhedrin (Mtdh), which is associated with resistance to cytotoxic therapy in numerous types of cancer, co-localizes with Miat in stem-like medulloblastoma cells. Further, loss of Mtdh activates p53 signaling and reduces tumorigenicity in stem-like medulloblastoma cells. Taken together, these data reveal a critical role for the lncRNA Miat in sustaining a treatment resistant pool of tumorigenic stem-like medulloblastoma cells.

sentences: Discovery of pan-ErbB inhibitors protecting from SARS-CoV-2 replication, inflammation, and lung injury by a drug repurposing screen Effective therapies are needed to combat emerging viruses. Seventeen candidates that rescue cells from SARS-CoV-2-induced lethality and target diverse functions emerged in a screen of 4,413 compounds. Among the hits was lapatinib, an approved inhibitor of the ErbB family of receptor tyrosine kinases. Lapatinib and other pan-ErbB inhibitors suppress replication of SARS-CoV-2 and unrelated viruses with a high barrier to resistance. ErbB4, but not lapatinibs cancer targets ErbB1 and ErbB2, is required for SARS-CoV-2 entry and encephalitis alphavirus infection and is a molecular target mediating lapatinibs antiviral effect. In human lung organoids, lapatinib protects from SARS-CoV-2-induced activation of pathways implicated in non-infectious acute lung injury and fibrosis downstream of ErbBs (p38-MAPK, MEK/ERK, and AKT/mTOR), pro-inflammatory cytokine production, and epithelial barrier injury. These findings reveal regulation of viral infection, inflammation, and lung injury via ErbBs and propose approved candidates to counteract these effects with implications for pandemic coronaviruses and unrelated viruses.

sentences: Chromatic bacteria version 2 - A Himar1 transposon based delivery vector to extend the host range of a toolbox to fluorescently tag bacteria A recent publication described the construction and utility of a comprehensive "Chromatic Bacteria" toolbox containing a set of genetic tools that allows for fluorescently tagging a variety of Proteobacteria. In an effort to expand the range of bacteria taggable with the Chromatic Bacteria toolbox, a Himar1 transposon vector was constructed to mediate insertion of fluorescent protein and abiotic resistant genes. The Himar1 transposon was chosen as it is known to function in a wide range of bacteria. To test the use of the Himar1 derivative of the Chromatic Bacteria plasmid series for fluorescently tagging bacteria, recently isolated non-model organisms were conjugated with the Himar1 transposon plasmids. We were unsuccessful in delivering the plasmids into Gram positive bacterial isolates but were able to successfully integrate the transposon in isolates that we were previously unable to modify such as Sphingomonas sp. Leaf357 and Acidovorax sp. Leaf84. This manuscript reports on the currently available plasmids and transposition success in different bacteria. The manuscript will be updated regularly until a full plasmid series is available and a large range of recipient strains was tested.

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RENANO: a REference-based compressor for NANOpore FASTQ files Nanopore sequencing technologies are rapidly gaining popularity, in part, due to the massive amounts of genomic data they produce in short periods of time (up to 8.5 TB of data in less than 72 hours). In order to reduce the costs of transmission and storage, efficient compression methods for this type of data are needed. Unlike short-read technologies, nanopore sequencing generates long noisy reads of variable length. In this note we introduce RENANO, a reference-based lossless FASTQ data compressor, specifically tailored to compress FASTQ files generated with nanopore sequencing technologies. RENANO builds on the recent compressor ENANO, which is the current state of the art. RENANO focuses on improving the compression of the base call sequence portion of the FASTQ file, leaving the other parts of ENANO intact. Two novel reference-based compression algorithms are introduced, contemplating different scenarios: in the first scenario, a reference genome is available without cost to both the compressor and the decompressor; in the second, the reference genome is available only on the compressor side, and a compacted version of the reference is transmitted to the decompressor as part of the compressed file. To evaluate the proposed algorithms, we compare RENANO against ENANO on several publicly available nanopore datasets. In the first scenario considered, RENANO improves the base call sequences compression of ENANO by 39.8%, on average, over all the datasets. As for total compression (including the other parts of the FASTQ file), the average improvement is 12.7%. In the second scenario considered, the base call compression improvements of RENANO over ENANO range from 15.2% to 49.0%, depending on the coverage of the compressed dataset, while in terms of total size, the improvements range from 5.1% to 16.5%.

Implementations of the proposed algorithms are freely available for download at https://github.com/guilledufort/RENANO.

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Localization, induction, and cellular effects of tau-phospho-threonine 217 IntroductionTau phosphorylation at T217 is a promising AD biomarker, but its functional consequences were unknown.

MethodsHuman brain and cultured mouse neurons were analyzed by immunoblotting and immunofluorescence for total tau, taupT217, taupT181, taupT231 and taupS396/pS404. dSTORM super resolution microscopy was used to localize taupT217 in cultured neurons. EGFP-tau was expressed in fibroblasts as wild type and T217E pseudo-phosphorylated tau, and fluorescence recovery after photobleaching (FRAP) reported tau turnover rates on microtubules.

ResultsIn brain, taupT217 appears in neurons at Braak stages I-II, becomes more prevalent later and co-localizes partially with other phospho-tau epitopes. In cultured neurons taupT217, is increased by extracellular tau oligomers (xcTauOs), and is associated with developing post-synaptic sites. FRAP recovery was fastest for EGFP-tauT217E.

ConclusionTaupT217 increases in brain as AD progresses and is induced by xcTauOs. Post-synaptic taupT217 suggests a role for T217 phosphorylation in synapse impairment. T217 phosphorylation reduces taus affinity for microtubules.

sentences: The morning burst: Shifting daily patterns of ATP production in Drosophila and temporal windows for their improvement in ageing Mitochondria produce energy for cell function via adenosine triphosphate (ATP) and are regulated by a molecular 24h clock. Here we use Drosophila melanogaster to reveal shifts in whole animal ATP production over 24h, showing a marked peak in the morning that declines around midday and remains low from then through to the following morning. Mitochondrial membrane potential and ATP production has been shown previously to improve after long wavelength exposure, but apparently not at all times. Hence, to explore this further we exposed flies to 670nm at different times. Exposures between 08.00 and 11.00 resulted in a significant increase in ATP, while exposures at other times had no effect. Within the morning window, not all times were equally effective, however, 670nm exposure mid-morning when ATP production was maximal did not increase ATP, possibly because mitochondria lacked spare capacity at this time. Hence, in the morning there is a complex dynamic relationship between long wavelength light and mitochondria. Mitochondrial function and the influence of long wavelengths are conserved across species from fly to human, and determining the time points for light administration to improve function in ageing and disease is of key importance. Our data progress this search and reveal the outline of these times.

sentences: scFlow: A Scalable and Reproducible Analysis Pipeline for Single-Cell RNA Sequencing Data Advances in single-cell RNA-sequencing technology over the last decade have enabled exponential increases in throughput: datasets with over a million cells are becoming commonplace. The burgeoning scale of data generation, combined with the proliferation of alternative analysis methods, led us to develop the scFlow toolkit and the nf-core/scflow pipeline for reproducible, efficient, and scalable analyses of single-cell and single-nuclei RNA-sequencing data. The scFlow toolkit provides a higher level of abstraction on top of popular single-cell packages within an R ecosystem, while the nf-core/scflow Nextflow pipeline is built within the nf-core framework to enable compute infrastructure-independent deployment across all institutions and research facilities. Here we present our flexible pipeline, which leverages the advantages of containerization and the potential of Cloud computing for easy orchestration and scaling of the analysis of large case/control datasets by even non-expert users. We demonstrate the functionality of the analysis pipeline from sparse-matrix quality control through to insight discovery with examples of analysis of four recently published public datasets and describe the extensibility of scFlow as a modular, open-source tool for single-cell and single nuclei bioinformatic analyses.

sentences: Complex Activity and Short-term Memories in Reciprocally Connected Cerebral Organoids Macroscopic axonal connections in the human brain distribute information and neuronal activity across the brain. Although brain organoid technologies have recently provided novel avenues to investigate human brain function by replicating small neuronal networks of the brain in vitro, functionality of the macroscopic connections between distant regions has not been investigated with organoids. Here, we describe the neural activity of human cerebral organoids reciprocally connected by a bundle of axons. The connected organoids produced significantly more intense and complex oscillatory activity than conventional cerebral organoids. Optogenetic manipulations revealed that the connected organoids could respond to external stimuli and maintain the elevated frequency of neuronal activity for a short term, indicating that the connected organoids exhibit plasticity as a neuronal network. Our findings highlight functional importance of inter-regional connections and suggest that connected organoids can be used as a powerful tool for investigating the development and functions of macroscopic circuits in the human brain. can allow researchers to develop and test compounds to modulate activity of in vitro neuronal network models in unprecedented ways.

sentences: Undersampling and the inference of coevolution in proteins Protein structure, function, and evolution depend on local and collective epistatic interactions between amino acids. A powerful approach to defining these interactions is to construct models of couplings between amino acids that reproduce the empirical statistics (frequencies and correlations) observed in sequences comprising a protein family. The top couplings are then interpreted. Here, we show that as currently implemented, this inference is always biased, a problem that fundamentally arises from the distinct scales at which epistasis occurs in proteins in the context of limited sampling. We show that these issues explain the ability of current approaches to predict tertiary contacts between amino acids and the inability to obviously expose larger networks of functionally-relevant, collectively evolving residues called sectors. This work provides a necessary foundation for more deeply understanding and improving evolution-based models of proteins.

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The Development of Sleep/Wake Disruption and Cataplexy as Hypocretin/Orexin Neurons Degenerate in Male vs. Female Orexin/tTA; TetO-DTA Mice Narcolepsy Type 1 (NT1), a sleep disorder with similar prevalence in both sexes, is thought to be due to loss of the hypocretin/orexin (Hcrt) neurons. Several transgenic strains have been created to model this disorder and are increasingly being used for preclinical drug development and basic science studies, yet most studies have solely used male mice. We compared the development of narcoleptic symptomatology in male vs. female orexin-tTA; TetO-DTA mice, a model in which Hcrt neuron degeneration can be initiated by removal of doxycycline (DOX) from the diet. EEG, EMG, body temperature, gross motor activity and video recordings were conducted for 24-h at baseline and 1, 2, 4 and 6 weeks after DOX removal. Female DTA mice exhibited cataplexy, the pathognomonic symptom of NT1, by Week 1 in the DOX(-) condition but cataplexy was not consistently present in males until Week 2. By Week 2, both sexes showed an impaired ability to sustain long wake bouts during the active period, the murine equivalent of excessive daytime sleepiness in NT1. Body temperature appeared to be regulated at lower levels in both sexes as the Hcrt neurons degenerated. During degeneration, both sexes also exhibited the "Delta State", characterized by sudden cessation of activity, high delta activity in the EEG, maintenance of muscle tone and posture, and the absence of phasic EMG activity. Since the phenotypes of the two sexes were indistinguishable by Week 6, we conclude that both sexes can be safely combined in future studies to reduce cost and animal use.

Statement of SignificanceAlthough narcolepsy is a disorder that affects both men and women with similar frequency, most basic research and preclinical development studies of sleep have utilized male experimental subjects. The identification of the hypocretin/orexin (Hcrt) neuron loss as the likely cause of human narcolepsy has led to the development of transgenic mouse strains that model this disorder. Here, we compare the emergence of narcoleptic symptoms in male vs. female bigenic orexin-tTA; TetO DTA mice, a state-of-the-art narcolepsy model in which degeneration of the Hcrt neurons can be triggered by dietary manipulation. We find that female mice develop the narcoleptic phenotype more rapidly than males but that both sexes are equally symptomatic by the end of the degeneration period.

sentences: Primary brain cell infection by Toxoplasma gondii reveals the extent and dynamics of parasite differentiation and its impact on neuron biology. Toxoplasma gondii is a eukaryotic parasite that forms latent cysts in the brain of immunocompetent individuals. The latent parasite infection of the immune-privileged central nervous system is linked to most complications. With no drug currently available to eliminate the latent cysts in the brain of infected hosts, the consequences of neurons long-term infection are unknown. It has long been known that T. gondii specifically differentiates into a latent form (bradyzoite) in neurons, but how the infected neuron responds to the infection remains to be elucidated. We have established a new in vitro model resulting in the production of mature bradyzoite cysts in brain cells. Using dual, host and parasite RNA-seq, we characterized the dynamics of differentiation of the parasite, revealing the involvement of key pathways in this process. Moreover, we identified how the infected brain cells responded to the parasite infection revealing the drastic changes that take place. We showed that neuronal-specific pathways are strongly affected, with synapse signaling being particularly affected, especially glutamatergic synapse signaling. The establishment of this new in vitro model allows investigating both the dynamics of parasite differentiation and the specific response of neurons to the long-term infection by this parasite.

sentences: Machine learning guided signal enrichment for ultrasensitive plasma tumor burden monitoring In solid tumor oncology, circulating tumor DNA (ctDNA) is poised to transform care through accurate assessment of minimal residual disease (MRD) and therapeutic response monitoring. To overcome the sparsity of ctDNA fragments in low tumor fraction (TF) settings and increase MRD sensitivity, we previously leveraged genome-wide mutational integration through plasma whole genome sequencing (WGS). We now introduce MRD-EDGE, a composite machine learning-guided WGS ctDNA single nucleotide variant (SNV) and copy number variant (CNV) detection platform designed to increase signal enrichment. MRD-EDGE uses deep learning and a ctDNA-specific feature space to increase SNV signal to noise enrichment in WGS by 300X compared to our previous noise suppression platform MRDetect. MRD-EDGE also reduces the degree of aneuploidy needed for ultrasensitive CNV detection through WGS from 1Gb to 200Mb, thereby expanding its applicability to a wider range of solid tumors. We harness the improved performance to track changes in tumor burden in response to neoadjuvant immunotherapy in non-small cell lung cancer and demonstrate ctDNA shedding in precancerous colorectal adenomas. Finally, the radical signal to noise enrichment in MRD-EDGE enables de novo mutation calling in melanoma without matched tumor, yielding clinically informative TF monitoring for patients on immune checkpoint inhibition.

sentences: PhosPiR: An automated phospho-proteomic pipeline in R Large-scale phospho-proteome profiling using mass spectrometry (MS) provides functional insight that is crucial for disease biology and drug discovery. However, extracting biological understanding from this data is an arduous task requiring multiple analysis platforms that are not adapted for automated high-dimensional data analysis. Here, we introduce an integrated pipeline that combines several R packages to extract high-level biological understanding from largescale phosphoproteomic data by seamless integration with existing databases and knowledge resources. In a single run, PhosPiR provides data clean-up, fast data overview, multiple statistical testing, differential expression analysis, phospho-site annotation and translation across species, multi-level enrichment analyses, proteome-wide kinase activity and substrate mapping and network hub analysis. Data output includes graphical formats such as heatmap, box-, volcano- and circos-plots. This resource is designed to assist proteome-wide data mining of pathophysiological mechanism without a need for programming knowledge.

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Decoding the olfactory map: targeted transcriptomics link olfactory sensory neurons to glomeruli Sensory processing in vertebrate olfactory systems is organized across olfactory bulb glomeruli, wherein axons of peripheral sensory neurons expressing the same olfactory receptor co-terminate to transmit receptor-specific activity to central neurons. Understanding how receptors map to glomeruli is therefore critical to understanding olfaction. High-throughput spatial transcriptomics is a rapidly advancing field, but low-abundance olfactory receptor expression within glomeruli has previously precluded high-throughput mapping of receptors to glomeruli. Here we combined spatial sectioning along the anteroposterior, dorsoventral, and mediolateral axes with target capture enrichment sequencing to overcome low-abundance target expression. This strategy allowed us to spatially map 86% of olfactory receptors across the olfactory bulb and uncover a relationship between OR sequence and glomerular position.

ONE SENTENCE SUMMARYTargeted enrichment of olfactory receptor mRNA in olfactory bulb sections determines spatial positions for murine glomeruli.

sentences: Time-reversal symmetric component of the flagellar beat enhances the translational and rotational velocities of isolated Chlamydomonas flagella The beating of cilia and flagella is essential to perform many important biological functions, including generating fluid flows on the cell surface or propulsion of micro-organisms. In this work, we analyze the motion of isolated and demembranated flagella from green algae Chlamydomonas reinhardtii, which act as ATP-driven micro-swimmers. The waveform of the Chlamydomonas beating flagella has an asymmetric waveform that is known to involve the superposition of a static component, corresponding to a fixed, intrinsic curvature, and a dynamic wave component traveling in the base-to-tip direction at the fundamental beat frequency, plus higher harmonics. Here, we demonstrate that these modes are not sufficient to reproduce the observed flagella waveforms. We find that two extra modes play an essential role to describe the motion: first, a time-symmetric mode, which corresponds to a global oscillation of the axonemal curvature, and second, a secondary tip-to-base wave component at the fundamental frequency that propagates opposite to the dominant base-to-tip wave, albeit with a smaller amplitude. Although the time-symmetric mode cannot, by itself, contribute to propulsion (scallop theorem), it does enhance the translational and rotational velocities of the flagellum by approximately a factor of 2. This mode highlights a long-range coupled on/off activity of force-generating dynein motors and can provide further insight into the underling biology of the ciliary beat.

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Effects of long-term high-temperature stress on reproductive growth and seed development in development in Brassica napus Brassica napus is the second most important oilseed crop worldwide. Increasing average temperatures and extreme weather have a severe impact on rapeseed yield. We determined the response of three cultivars to different temperature regimes (21/18 {degrees}C, 28/18 {degrees}C and 34/18 {degrees}C), focusing on the plant appearance, seed yield, seed quality, seed viability, and embryo development. Our microscopic observations identified that embryo development is affected by high temperatures. We noticed an acceleration of its development, in addition to pattern defects. Reduced fertilization rate, increased abortion rate, and preharvest sprouting would be responsible for the low seed yield at the high-temperature regime. Hormone profiling indicates that reduced auxin levels in young seeds may cause the observed embryo pattern defects. Moreover, reduced seed dormancy may result from low ABA and IAA levels in mature seeds. Glucosinolates and oil composition measurements suggest reduced seed quality. These identified cues help understand seed thermomorphogenesis and pave the way to the development of thermoresilient rapeseed plants.

HighlightBrassica napus flowering plants growth at high temperatures accelerates embryo development, causing a high seed abortion rate and reduced seed quality. Temperature-reduced ABA levels cause premature seed sprouting.

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Screening and Identification of Azo Dye Decolourizers from Mangrove Rhizospheric soil Removal of synthetic textile dye poses a challenge to the textile industry and a threat to environment flora and fauna. These dyes are marginally degradable and recalcitrant, hence alternatives to physical and chemical techniques like various bioremediation studies involving plants, plant roots, single or consortium of microbes have been used as environment friendly methods for the removal of textile dye. In the present study potent bacteria for dye decolourization were isolated from rhizospheric soil collected from Kamothe, Navi Mumbai, India. Of the 20 isolates obtained after enrichment, seven isolates were used for further screening of efficient decolourization ability in MBM media containing 10% glucose, 2.5 % trace metal solution and 0.1% of MO dye concentration. Physiological parameters to optimize the decolourization of dye at optimum pH, temperature and incubation time was studied for all the seven isolates. UV-vis and Fourier Transform Infrared spectroscopy were used to investigate dye decolorization. The seven isolates were characterized morphologically, biochemically, and molecular identification of these strains were performed by 16S rRNA sequence analysis. The isolates were identified as Bacillus paramycoides, Pseudomonas taiwanensis, Citrobacter murliniae, Acinetobacter pitti, Exiguobacterium acetylicum, Psychrobacter celer, and Aeromonas taiwanensis. Out of these Aeromonas taiwanensis has shown exceptional capacity by 100% decolorization of azo dye in minimum time.

1. Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/481723v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@e54071org.highwire.dtl.DTLVardef@e9d6dforg.highwire.dtl.DTLVardef@19ccfeorg.highwire.dtl.DTLVardef@1e14201_HPS_FORMAT_FIGEXP  M_FIG C_FIG Highlights of the studyO_LICollection of soil samples from rhizospheric and non-rhizospheric region of the mangroves plant.
C_LIO_LIEnrichment of soil sample in media containing azo dye for dye decolourization.
C_LIO_LIScreening and isolation of potential dye decolourizers.
C_LIO_LIOptimize the condition for dye decolourization.
C_LIO_LIFTIR analysis and application based studies.
C_LIO_LIIdentification of microbes using 16SrRNA.
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sentences: Seasonal resistome diversity and dissemination of WHO priority antibiotic-resistant pathogens in Lebanese estuaries Anthropogenic activities are demonstrated to be the key drivers of antimicrobial resistance (AMR) dissemination in the environment. Lebanese rivers that lead to the Mediterranean Sea were sampled at estuaries sites, under high anthropogenic pressure, in spring 2017 and winter 2018 to study seasonal variation of antimicrobial-resistant bacteria (ARBs) and antimicrobial resistance genes (ARGs). Methods: A combined approach using culture techniques and high throughput qPCR identified hotspots for antimicrobial resistance and anthropogenic pressure in particular locations along the Lebanese coast. Results: Multi-resistant Gram-negative (Enterobacterales and Pseudomonas spp) and Gram-positive bacterial pathogens were isolated. A high abundance of certain ARGs (vanB, blaBIC-1, blaGES, tetM, and mcr-1) was detected in 5 Lebanese estuaries. The relative abundance of ARGs was highest in winter and areas with high anthropogenic activities and population growth with an influx of refugees. Conclusion: Qualitative analysis of ARB and the analysis of the Lebanese estuaries resistome revealed critical levels of contamination with pathogenic bacteria and provided significant information about the spread of ARGs in anthropogenically impacted estuaries.

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Pooling in a predictive model of V1 explains functional and structural diversity across species Neurons in the primary visual cortex are selective to orientation with various degrees of selectivity to the spatial phase, from high selectivity in simple cells to low selectivity in complex cells. Various computational models have suggested a possible link between the presence of phase invariant cells and the existence of cortical orientation maps in higher mammals V1. These models, however, do not explain the emergence of complex cells in animals that do not show orientation maps. In this study, we build a model of V1 based on a convolutional network called Sparse Deep Predictive Coding (SDPC) and show that a single computational mechanism, pooling, allows the SDPC model to account for the emergence of complex cells as well as cortical orientation maps in V1, as observed in distinct species of mammals. By using different pooling functions, our model developed complex cells in networks that exhibit orientation maps (e.g., like in carnivores and primates) or not (e.g., rodents and lagomorphs). The SDPC can therefore be viewed as a unifying framework that explains the diversity of structural and functional phenomena observed in V1. In particular, we show that orientation maps emerge naturally as the most cost-efficient structure to generate complex cells under the predictive coding principle.

SignificanceCortical orientation maps are among the most fascinating structures observed in higher mammals brains: In such maps, similar orientations in the input image activate neighboring cells in the cortical surface. However, the computational advantage brought by these structures remains unclear, as some species (rodents and lagomorphs) completely lack orientation maps. In this study, we introduce a computational model that links the presence of orientation maps to a class of nonlinear neurons called complex cells. In particular, we propose that the presence or absence orientation maps correspond to different strategies employed by different species to generate invariance to complex stimuli.

sentences: Significant organic carbon acquisition by Prochlorococcus in the oceans Marine phytoplankton are responsible for about half of the photosynthesis on Earth. Many are mixotrophs, combining photosynthesis with heterotrophic assimilation of organic carbon but the relative contribution of these two carbon sources is not well quantified. Here, single-cell measurements reveal that Prochlorococcus at the base of the photic zone in the Eastern Mediterranean Sea are obtaining only ~20% of carbon required for growth by photosynthesis. Consistently, laboratory-calibrated evaluations of Prochlorococcus photosynthesis indicate that carbon fixation is systematically too low to support published in situ growth rates in the deep photic layer of the Pacific Ocean. Furthermore, agent-based model simulations show that mixotrophic cells maintain realistic growth rates and populations 10s of meters deeper than obligate photo-autotrophs, deepening the nutricline and Deep Chlorophyll Maximum by ~20 m. Time-series of Prochlorococcus ecotype-abundance from the subtropical North Atlantic and North Pacific suggest that up to 30% of the Prochlorococcus cells live where light intensity is not enough to sustain obligate photo-autotrophic populations during warm, stratified periods. Together, these data and models suggest that mixotrophy underpins the ecological success of a large fraction of the global Prochlorococcus population and its collective genetic diversity.

sentences: Integrated quality control of allele-specific copy numbers, mutations and tumour purity from cancer whole genome sequencing assays Cancer genomes contain thousands of somatic point mutations, chromosome copy alterations and more complex structural variants, which contribute to tumour growth and therapy response. Whole genome sequencing is a well established approach for somatic variant identification, but its broad application comes with complications, particularly in how proposed calls are quality assessed. To address this issue, we present CNAqc, a quantitative framework to quality control somatic mutations and allele-specific copy numbers, both in clonal and subclonal settings while accounting for variations in tumour purity, as commonly seen in bulk sampling. We test the model via extensive simulations, validate it using low-pass single-cell data, and apply it to 2778 single-sample PCAWG whole-genomes, 10 in-house multi-region whole-genomes and 48 TCGA whole-exomes. CNAqc is compatible with common bioinformatic pipelines and designed to support automated parameterization processes that are crucial in the era of large-scale whole genome sequencing.

sentences: An Investigation of the Mechanism for a Novel Influenza Inhibitor Designed to Target the Import of PB1 A novel inhibitor of PB1 import, referred to here as GM30, previously displayed nuclear retention of viral nucleoprotein (NP), which spurred the question of whether the compound directly or indirectly blocked viral ribonucleoprotein (vRNP) complex export. vRNPs are exported from the nucleus via exportin 1 (XPO1). Using verdinexor (VNXR), a direct inhibitor of vRNP export that binds to XPO1 and also blocks nuclear factor kappa B (NF-{kappa}B) export, we found that GM30 does not block nuclear export of NF-{kappa}B. GM30 likewise demonstrated high nuclear retention of NP but not as much as the direct inhibition of nuclear export by VNXR. When the compound was added hours after infection, the compound lost its ability to block nuclear export but VNXR retained its ability to block nuclear export. GM30 is therefore likely an indirect inhibitor of nuclear export because it disrupts the vRNP complex formation and impedes export from the nucleus by reducing nuclear import of PB1.

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SARS-CoV-2 expresses a microRNA-like small RNA able to selectively repress host genes Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease (COVID-19), continues to be a pressing health concern. In this study, we investigated the impact of SARS-CoV-2 infection on host microRNA (miRNA) populations in three human lung-derived cell lines, as well as in nasopharyngeal swabs from SARS-CoV-2 infected individuals. We did not detect any major and consistent differences in host miRNA levels after SARS-CoV-2 infection. However, we unexpectedly discovered a viral miRNA-like small RNA, named vmiR-5p (for viral miRNA), derived from the SARS-CoV-2 ORF7a transcript. Its abundance ranges from low to moderate as compared to host miRNAs. vmiR-5p functionally associates with Argonaute proteins -- core components of the RNA interference pathway -- leading to downregulation of host transcripts. One such host messenger RNA encodes Basic Leucine Zipper ATF-Like Transcription Factor 2 (BATF2), which is linked to interferon signaling. We demonstrate that vmiR-5p production relies on cellular machinery, yet is independent of Drosha protein, and is enhanced by the presence of a strong and evolutionarily conserved hairpin formed within the ORF7a sequence.

Significance statementWe discovered that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses a small viral non-coding RNA, named vmiR-5p (for viral miRNA), derived from the ORF7a transcript. vmiR-5p associates with the cellular RNA interference machinery to regulate host transcripts likely via target silencing. The production of vmiR-5p relies on cellular machinery and the formation of a strong hairpin within ORF7a sequences. This newly-described vmiR-5p may contribute to SARS-CoV-2 pathogenesis and could become a target for therapeutic intervention.

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Analyses of cephalic and non-cephalic sensory cell types provide insight into joint photo- and mechanoreceptor evolution Rhabdomeric Opsins (r-Opsins) are light-sensors in cephalic eye photoreceptors, but also function in additional sensory organs. This has prompted questions on the evolutionary relationship of these cell types, and if ancient r-Opsins cells were non-photosensory. Our profiling of cephalic and non-cephalic r-opsin1-expressing cells of the marine bristleworm Platynereis dumerilii reveals shared and distinct features. Non-cephalic cells possess a full set of phototransduction components, but also a mechanosensory signature. We determine that Pdu-r-Opsin1 is a Gq-coupled blue-light receptor. Profiling of cells from r-opsin1 mutants versus wild-types, and a comparison under different light conditions reveals that in the non-cephalic cells, light - mediated by r-Opsin1 - adjusts the expression level of a calcium transporter relevant for auditory mechanosensation in vertebrates. We establish a deep learning-based quantitative behavioral analysis for animal trunk movements, and identify a light-and r-Opsin-1-dependent fine-tuning of the worms undulatory movements in headless trunks, which are known to require mechanosensory feedback.

Our results suggest an evolutionary concept in which r-Opsins act as ancient, light-dependent modulators of mechanosensation, and suggest that light-independent mechanosensory roles of r-Opsins likely evolved secondarily.

sentences: Coupling Comprehensive Transcriptome-Metabolome Association and Phylogenetic Analysis Speed Dissecion of Polyphyllins Biosynthetic Pathway The genes in polyphyllins pathway mixed with other steroid biosynthetic genes form an extremely complex biosynthetic network in Paris polyphylla with a giant genome. The lack of genomic data and tissue specificity causes the study of the biosynthetic pathway notably difficult. Here, we report an effective method for the prediction of key genes of polyphyllin biosynthesis. Full-length transcriptome from eight different organs via hybrid sequencing of next generation sequencingand third generation sequencing platforms annotated two 2,3-oxidosqualene cyclases (OSCs), 216 cytochrome P450s (CYPs), and 199 UDP glycosyltransferases (UGTs). Combining metabolic differences, gene-weighted co-expression network analysis, and phylogenetic trees, the candidate ranges of OSC, CYP, and UGT genes were further narrowed down to 2, 15, and 24, respectively. Beside the three previously characterized CYPs, we identified the OSC involved in the synthesis of cycloartenol and the UGT (PpUGT73CR1) at the C-3 position of diosgenin and pennogenin in P. polyphylla. This study provides a idea for the investigation of gene cluster deficiency biosynthesis pathways in medicinal plants.

sentences: De novo deletions and duplications at recombination hotspots in mouse germlines Numerous DNA double-strand breaks (DSBs) arise during meiosis to initiate homologous recombination. These DSBs are usually repaired faithfully, but here we uncover a new type of mutational event in which deletions form via joining of ends from two closely spaced DSBs (double cuts) within a single hotspot or at adjacent hotspots on the same or different chromatids. Deletions occur in normal meiosis but are much more frequent when DSB formation is dysregulated in the absence of the ATM kinase. Events between chromosome homologs point to multi-chromatid damage and aborted gap repair. Some deletions contain DNA from other hotspots, indicating that double cutting at distant sites creates substrates for insertional mutagenesis. End joining at double cuts can also yield tandem duplications or extrachromosomal circles. Our findings highlight the importance of DSB regulation and reveal a previously hidden potential for meiotic mutagenesis that is likely to affect human health and genome evolution.

sentences: Mammary lineage dictates homologous recombination repair and PARP inhibitor vulnerability It has long been assumed that all normal cells have the same capacity to engage homologous recombination (HR) and non-homologous end joining (NHEJ) to repair DNA double-strand breaks (DSBs), a concept exploited for DNA-damaging chemotherapeutics. We show that mammary epithelial lineage dictates the DSB repair pathway choice. Primary mammary proteomes and DSB repair enumeration by {gamma}-H2AX, Rad51 and DNA-PKc foci reveal that NHEJ operates in all epithelial cells, but high-fidelity HR is restricted to the luminal lineage. This translates to divergent poly (ADP-ribose) polymerase inhibitor (PARPi) vulnerability of mammary epithelial progenitor activity in both mouse and human, irrespective of the BRCA1/2 status. Proteome-defined lineage-specific signatures correlate to breast cancer subtypes and predict PARPi response of triple-negative human breast cancer xenografts. These intrinsically divergent HR characteristics of mammary cell types underpin a new strategy for identifying PARPi responders.

sentences: STARsolo: accurate, fast and versatile mapping/quantification of single-cell and single-nucleus RNA-seq data We present STARsolo, a comprehensive turnkey solution for quantifying gene expression in single-cell/nucleus RNA-seq data, built into RNA-seq aligner STAR. Using simulated data that closely resembles realistic scRNA-seq, we demonstrate that STARsolo is highly accurate and significantly outperforms pseudoalignment-to-transcriptome tools. STARsolo can replicate the results of, but is considerably faster than CellRanger, currently the most widely used tool for pre-processing scRNA-seq data. In addition to uniquely mapped reads, STARsolo takes account of multi-gene reads, necessary to detect certain classes of biologically important genes. It has a flexible cell barcode processing scheme, compatible with many established scRNA-seq protocols, and extendable to emerging technologies. STARsolo can quantify transcriptomic features beyond gene expression, which we illustrate by analyzing cell-type-specific alternative splicing in the Tabula Muris project.

sentences: Evidence for a vocal signature in the rat and its reinforcing effects While the term "language" is used for human and non-human primates, "vocal communication" is rather used for rodents or other species. The main difference is that there is, to date, no evidence for a vocal signature in the well-known 50- and 22-kHz ultrasonic vocalizations (USV) emitted by rats. Here, we show that rats can recognize the identity of the USV emitter since they self-administer preferentially playback of 50-kHz USV emitted by a stranger rat over those emitted by their cage-mate. In a second experiment, we show that the familiarity with the USV emitter also modulate the effect of USV playback during cocaine self-administration, since only stranger, but not familiar, 50-kHz USV decrease drug intake. Finally, to study the neurobiological substrate of those processes, we have tested the effects of the subthalamic nucleus (STN) lesion on these various conditions. STN-lesioned rats did not lever press much for any USV playback, whatever their emotional valence, nor did they seem able to differentiate familiar from stranger peer. Advocating for the existence of a vocal signature in rats, these results highlight the importance of ultrasonic communication in socio-affective influence of behavior, such as the influence of proximal social factors on drug consumption and confirm the role of the subthalamic nucleus on this influence.

sentences: Contribution of Uremia to Ureaplasma-Induced Hyperammonemia Lung transplant recipients (LTRs) are vulnerable to unexplained hyperammonemia syndrome (HS) in the early post-operative period, a condition typically unresponsive to non-antibiotic interventions. Recently, we showed that HS in LTRs is strongly correlated with Ureaplasma infection of the respiratory tract. It is not well-understood what makes LTRs preferentially susceptible to this phenomenon, compared to other immunocompromised hosts. Ureaplasma species harbor highly active ureases that convert urea to ammonia and CO2, utilizing the generated transmembrane potential to synthesize ATP. Post-operative LTRs commonly experience renal failure, resulting in uremia. We hypothesized that uremia could be a potentiating comorbidity to the development of HS secondary to Ureaplasma infection in LTRs by providing increased substrate for ureaplasmal ureases. We designed a novel dialyzed flow system to test the ammonia producing capacity of four isolates of Ureaplasma parvum and six isolates of Ureaplasma urealyticum in media formulations relating to normal and uremic host conditions. For all isolates, growth under uremic conditions resulted in significantly increased ammonia production over 24 hours, despite similar end-point bacterial quantities. Specifically, the isolates produced, on average, 1776.52 [standard deviation=263.98] mol/L more ammonia when grown under uremic compared to normal conditions. This suggests that uremia, common in early post-operative LTRs, is a plausible contributing factor to the phenomenon of Ureaplasma-induced HS in this patient population.

sentences: Succinate dehydrogenase/complex II is critical for metabolic and epigenetic regulation of T cell proliferation and inflammation Robust and effective T cell-mediated immune responses require the proper allocation of metabolic resources to sustain energetically costly processes like growth, proliferation, and cytokine production. Epigenetic control of the genome also governs T cell transcriptome and T cell lineage commitment and maintenance. Cellular metabolic programs interact with epigenetic regulation by providing substrates for covalent modifications of chromatin. By employing complementary genetic, epigenetic, and metabolic approaches, we revealed that tricarboxylic acid (TCA) cycle flux fuels biosynthetic processes while controlling the ratio of -ketoglutarate/succinate to modulate the activities of dioxygenases that are critical for driving T cell inflammation. In contrast to cancer cells, where succinate dehydrogenase (SDH)/complex II inactivation drives cell transformation and growth, SDH/complex II deficiency in T cells causes proliferation and survival defects when the TCA cycle is truncated, blocking carbon flux to support nucleosides biosynthesis. Accordingly, replenishing the intracellular nucleoside pool partially relieved the dependence of T cells on SDH/complex II for proliferation and survival. Conversely, SDH deficiency induces a pro-inflammatory gene signature in T cells and promotes T helper 1 (TH1) and T helper 17 (TH17) lineage differentiation. Mechanistically, the hypoxia-inducible factor 1 (HIF-1) is not required for succinate-induced inflammation in T cells. A reduced -ketoglutarate/succinate ratio in SDH deficient T cells promotes inflammation through changing the pattern of the transcriptional and chromatin-accessibility signatures and consequentially increasing the expression of the transcription factor, B lymphocyte-induced maturation protein-1 (Blimp-1). Collectively, our studies revealed a critical role of SDH/complex II in allocating carbon resources for anabolic processes and epigenetic regulation in T cell proliferation and inflammation.

sentences: Sapogenin based self-assembly structures activating a non-apoptotic cell death via multiple pathways Induction of distinct cell death pathways is critical to deal with tumor heterogeneity and therapeutic resistance. In our previous study, we reported a promising saponin analog (AG-08) for cancer therapy inducing non-canonical necrotic cell death. Here, we describe that AG-08 forms unique supramolecular structures responsible for its biological activity. After internalization via non-canonical endocytosis pathway, these structures affect several cell signaling pathways including unfolded protein response, immune response, oxidative stress and heat stress. Moreover, we prepared 18 analogs to reveal the role of residues on the formation of supramolecular structures and biological activities. The results have demonstrated that unique structural features are required for particulate structures and unprecedented cell death mechanism. Although small molecule based supramolecular assemblies have widely been accepted as nuisance for drug discovery studies, our results indicate that they may provide a new research field for anti-cancer drug development studies.

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Senescence-Induced Immune Remodeling Facilitates Metastatic Adrenal Cancer in a Sex-Dimorphic Manner Aging is a carcinogen that markedly increases cancer risk, yet we have limited mechanistic understanding of cancer initiation in aged cells. Here, we demonstrate induction of the hallmark aging process cellular senescence, triggered by loss of Wnt inhibitor ZNRF3, remodels the tissue microenvironment and ultimately permits metastatic adrenal cancer. Detailed characterization reveals a striking sexual dimorphism. Males exhibit earlier senescence activation and a greater innate immune response. This results in high myeloid cell accumulation and lower incidence of malignancy. Conversely, females present a dampened immune response and are more prone to metastatic cancer. Senescence-recruited myeloid cells become increasingly depleted with advanced tumor progression, which is recapitulated in patients where a low myeloid signature is associated with worse outcome. Collectively, our study reveals a novel role for myeloid cells in restraining adrenal cancer progression with significant prognostic value, and provides a model for interrogating pleiotropic effects of cellular senescence in cancer.

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sentences: Cell-cell communication through FGF4 generates and maintains robust proportions of differentiated cell types in embryonic stem cells During embryonic development and tissue homeostasis, reproducible proportions of differentiated cell types are specified from populations of multipotent precursor cells. Molecular mechanisms that enable both robust cell type proportioning despite variable initial conditions in the precursor cells, as well as the re-establishment of these proportions upon perturbations in a developing tissue remain to be characterized. Here we report that the differentiation of robust proportions of epiblast-like and primitive endoderm-like cells in mouse embryonic stem cell cultures emerges at the population level through cell-cell communication via a short-range FGF4 signal. We characterize the molecular and dynamical properties of the communication mechanism, and show how it controls both robust cell type proportioning from a wide range of experimentally controlled initial conditions, as well as the autonomous re-establishment of these proportions following the isolation of one cell type. The generation and maintenance of reproducible proportions of discrete cell types is a new function for FGF signaling that may operate in a range of developing tissues.

sentences: Platr4 is an ESC-specific lncRNA that exhibits its function downstream on meso/endoderm lineage commitment The mammalian genome encodes thousands of long non-coding RNAs (lncRNAs) that are developmentally regulated and differentially expressed across tissues, suggesting possible roles in cellular differentiation. Despite this expression pattern, little is known about how lncRNAs influence lineage commitment at the molecular level. Here, we reveal that perturbation of an embryonic stem cell (ESC)-specific lncRNA, Pluripotency associated transcript 4 (Platr4), in ESCs directly influences the downstream meso/endoderm differentiation program without affecting pluripotency. We further show that Platr4 interacts with the TEA domain transcription factor 4 (Tead4) to regulate the expression of a downstream target gene crucial in the cardiac lineage program known as connective tissue growth factor (Ctgf). Importantly, Platr4 knockout mice exhibit myocardial atrophy, valve mucinous degenration associated with reduced cardiac output and sudden heart failure. Together, our findings provide evidence that Platr4 expression in undifferentiated ESCs is critical for downstream lineage differentiation, highlighting its importance in disease modeling and regenerative medicine.

sentences: DNA metabarcoding reveals adaptive seasonal variation of individual trophic traits in a critically endangered fish Dietary studies are critical for understanding foraging strategies and have important applications in conservation and habitat management. We applied a robust metabarcoding protocol to characterize the diet of the critically endangered freshwater fish Zingel asper and conducted modelling and simulation analyses to characterize and identify some of the drivers of individual trophic trait variation in this species. We found that intra-specific competition and ontogeny had minor effects on the trophic niche of Z. asper. Instead, our results suggest that the majority of trophic niche variation was driven by seasonal variation in ecological opportunity (in our case, the seasonal variation in the availability of preferred prey types). Overall, our results are in line with the optimal foraging theory and suggest that Z. asper is specialized on a few ephemeropteran prey species (Baetis fuscatus and Ecdyonurus) but adapts its foraging by becoming more opportunistic as its favoured prey seasonally decline. Despite the now widespread usage of metabarcoding, very few studies have attempted to study inter- and intra-populational individual trophic traits variation with metabarcoding data. This study illustrates how metabarcoding data obtained from feces can be combined with modelling and simulation approaches to test hypotheses in the conventional analytic framework of trophic analysis.

sentences: Light-inducible T cell engagers trigger, tune and shape the activation of primary T cells Cells perceive overtime complex sequences of receptor stimulation that they integrate to mount an appropriate response. Yet, the influence of signal dynamics on cell responses has been poorly characterized due to technical limitations. Here, we present a generalizable approach to control receptor stimulation on unmodified primary cells. Indeed, for applications on primary murine T cells, we have engineered the LiTe system, a new recombinant optogenetics-based Light-inducible T cell engager which allows tunable and reversible spatiotemporal control of the T Cell Receptor (TCR) stimulation. We also provided in vitro evidence that this system enables efficient T cell activation with light, leading to cytokine secretion or tumor cell killing. Using specific time-gated stimulations, we have been able to orient the outcome of the activation of T cells. Overall, the LiTe system constitutes a versatile ON/OFF molecular switch allowing to decipher the cellular response to stimulation dynamics. Its original control over T cell activation opens new avenues for future precision cancer immunotherapy.

sentences: A novel tubulin binding molecule drives differentiation of acute myeloid leukaemia cells Acute Myeloid Leukaemia (AML) continues to have a poor prognosis, especially in the elderly. One reason for this is that many treatment regimens are not well tolerated by elderly patients. Much current focus is on the development of therapies that can target specific vulnerabilities of AML while having fewer toxic side effects. However, despite much recent progress in developing better drugs, many patients with AML still die within a year of diagnosis, partly due to the fact that it is difficult to identify therapeutic targets that are effective across multiple AML subtypes. One common factor across AML subtypes is the presence of a block in differentiation. Thus screening for compounds that can overcome this block in genetically diverse AML models should allow for the identification of agents that are not dependent on a specific mutation for their efficacy. Here, we used a phenotypic screen to identify novel compounds that stimulate differentiation in several AML cell lines. Lead compounds were shown to decrease tumour burden and to increase survival in vivo. Using multiple complementary target deconvolution approaches, these compounds were revealed to be anti-mitotic tubulin disruptors that cause differentiation by inducing a G2-M mitotic arrest. Together, these results reveal a novel function for tubulin disruptors in causing differentiation of AML cells.

sentences: Prediction of hERG potassium channel PAS-domain variants trafficking via machine learning 1Long QT syndrome (LQTS) is a cardiac condition characterized by a prolonged QT-interval. This prolongation can precipitate fatal arrhythmias despite otherwise normal metrics of cardiac function. Hence, genetic screening of individuals remains a standard for the initial identification of individuals susceptible to LQTS. Several genes, including KCNH2 that encodes for the Kv11.1 channel, are known to cause LQTS2. However, only a small percentage of variants found in the human population are established as pathogenic. As an example, the majority of KCNH2 missense mutations are either classified as benign or are a variant of unknown significance (VUS). Here, we evaluate whether structure-based molecular dynamics (MD) simulations and machine learning (ML) can improve the accuracy of classifying Kv11.1 channel variants with normal or loss-of-function phenotypes. We focus on defective Kv11.1 trafficking, as it is the most common cause of KCHN2 loss-of-function phenotypes. Specifically, we use these computational techniques to correlate structural and dynamic changes in a Kv11.1 subdomain, the PAS-domain (PASD), with the channels ability to traffic normally to the plasma membrane. These simulations unveiled several molecular features, including the number of hydrating waters and H-Bonds, as well as FoldX scores, that are predictive of trafficking. We then used statistical and machine learning (ML) (Decision tree (DT), Random forest (RF), and Support vector machine (SVM)) techniques to classify KNCH2 variants using these simulation-derived features. Together with bioinformatics data, such as sequence conservation and folding energies, we are able to predict with reasonable accuracy ({approx}75%) the KCNH2 variants that do not traffic. Therefore, structure-based simulations of KCNH2 variants localized to the Kv11.1 PAS domain lead to a significant improvement ({approx}10%) in classification accuracy and may ultimately complement standard genetic testing.

sentences: Pou5f3 and Sox19b determine gene expression repertoire at Zygotic Genome Activation Awakening of zygotic transcription in animal embryos relies on maternal pioneer transcription factors. The interplay of global and specific functions of these proteins remains poorly understood. Here, we analyze chromatin accessibility and time-resolved transcription in single and double mutant zebrafish embryos lacking pluripotency factors Pou5f3 and Sox19b. We show that two factors modify chromatin in a largely independent manner. We distinguish four types of direct enhancers by differential requirements for Pou5f3 or Sox19b. We demonstrate that changes in chromatin accessibility of enhancers underlie the changes in zygotic expression repertoire in the double mutants. Pou5f3 or Sox19b promote chromatin accessibility of enhancers linked to the genes involved in gastrulation and ventral fate specification. The genes regulating mesendodermal and dorsal fates are primed for activation independently of Pou5f3 and Sox19b. Strikingly, simultaneous loss of Pou5f3 and Sox19b leads to premature expression of genes, involved in regulation of organogenesis and differentiation.

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TSC2-extracellular matrix crosstalk controls pulmonary vascular proliferation and pulmonary hypertension Increased proliferation and survival of resident cells in small pulmonary arteries (PA) are important drivers of pulmonary hypertension (PH). Tuberous sclerosis complex 2 (TSC2) is a negative regulator of mTOR complex 1 and cell growth. Here we show that TSC2 is deficient in small remodeled PA/PA vascular smooth muscle cells (PAVSMC) from human PAH and experimental PH lungs. TSC2 deficiency was reproduced in vitro by maintaining PAVSMC on pathologically stiff substrates and was required for stiffness-induced proliferation, accumulation of transcriptional co-activators YAP/TAZ and up-regulation of mTOR. Depletion of TSC2 reproduced PH features in vitro in human PAVSMC and in vivo in SM22-Tsc2+/- mice. TSC2 loss in PAVSMC was supported by YAP and led to the up-regulation of YAP/TAZ and mTOR via modulating the extracellular matrix (ECM) composition. ECM, produced by TSC2-deficient PAVSMC, promoted growth of non-diseased PA adventitial fibroblasts and PAVSMC, which, in turn, was prevented by 5{beta}1 integrin receptor antagonist ATN161. In vitro, molecular and pharmacological (SRT2104) restoration of TSC2 down-regulated YAP/TAZ, mTOR, and ECM production, inhibited proliferation and induced apoptosis in human PAH PAVSMC. In vivo, orally administrated SRT2104 restored TSC2, resolved pulmonary vascular remodeling, PH, and improved right heart in two rodent models of PH. Thus, PAVSMC TSC2 is a critical integrator of ECM composition and stiffness with pro-proliferative signaling and PH, and the restoration of functional TSC2 could be an attractive therapeutic option to treat PH.

One Sentence SummaryTSC2 acts as mechanosensor and mechanotransducer, integrating ECM composition and stiffness with pro-proliferative signaling in pulmonary vasculature; its deficiency in PA vascular smooth muscle cells results in ECM remodeling, hyper-proliferation and pulmonary hypertension, which could be reversed by pharmacological restoration of functional TSC2.

sentences: The endoplasmic reticulum-resident protein TMEM-120/TMEM120A promotes fat storage in C. elegans and mammalian cells The synthesis of triacylglycerol (TAG) is essential for the storage of excess fatty acids, which can subsequently be used for energy or cell growth. A series of enzymes act in the endoplasmic reticulum (ER) to synthesize TAG, prior to its transfer to lipid droplets (LDs), which are conserved organelles for fat storage. Here, we report that the deficiency of TMEM-120/TMEM120A, a protein with 6-transmembrane helices, retards TAG synthesis and LD expansion in C. elegans. A missense mutation near the predicted coenzyme A binding site of TMEM-120 confers strong loss of function phenotypes. GFP fusion proteins of TMEM-120, expressed at the endogenous level in live worms, were observed throughout the ER network. Using Stimulated Raman Scattering, we discovered a specific requirement of TMEM-120 in the storage of exogenous fatty acids in LDs. Knockdown of TMEM120A impedes adipogenesis of pre-adipocytes in vitro, while its over-expression is sufficient to promote LD expansion. Pharmacological studies indicate that TMEM120A most likely acts upstream of diacylglycerol O-acyltransferase 1 (DGAT1). Our results suggest that TMEM-120/TMEM120A plays a conserved role in increasing the efficiency of TAG synthesis.

sentences: m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5 splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the ACAGA box is modified by N6 methylation, but the role of this m6A modification is poorly understood. Here we show that m6A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIO1, is required for Arabidopsis U6 snRNA m6A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5 splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m6A influences 3 splice site usage and reinforces splicing fidelity at elevated temperature. We generalise these findings to reveal two major classes of 5 splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA ACAGA box. We conclude that U6 snRNA m6A modification contributes to the selection of degenerate 5 splice sites crucial to alternative splicing.

sentences: Improved correspondence of fMRI visual field localizer data after macroanatomical alignment Studying the visual system with fMRI often requires using localizer paradigms to define regions of interest (ROIs). However, the considerable interindividual variability of the cerebral cortex represents a crucial confound for group-level analyses. Cortex-based alignment (CBA) techniques reliably reduce interindividual macroanatomical variability. Yet, their utility has not been assessed for visual field localizer paradigms, which map specific parts of the visual field within retinotopically organized visual areas. We evaluated CBA for an attention-enhanced visual field localizer mapping homologous parts of each visual quadrant in 50 participants. We compared CBA with volume-based alignment and a surface-based analysis, which did not include macroanatomical alignment. CBA led to the strongest increase in the probability of activation overlap (up to 40 percent). On the group level, CBA led to the most consistent increase in ROI size while preserving vertical ROI symmetry. Overall, our results indicate, that in addition to the increased signal-to-noise ratio of a surface-based analysis macroanatomical alignment considerably improves statistical power. These findings confirm and extend the utility of CBA for the study of the visual system in the context of group analyses. CBA should be particularly relevant when studying neuropsychiatric disorders with abnormally increased interindividual macroanatomical variability.

sentences: Cell tip growth underlies injury response of marine macroalgae Regeneration is a widely observed phenomenon by which the integrity of an organism is recovered after damage. So far, studies on the molecular and cellular mechanisms of regeneration have been limited to a handful of model multicellular organisms. Here, we systematically surveyed the regeneration ability of marine macroalgae (Rhodophyta, Phaeophyceae, Chlorophyta) after thallus severing and applied live cell microscopy on them to uncover the cellular response to the damage. We observed three types of responses - budding, rhizoid formation and/or sporulation - in 25 species among 66 examined, demonstrating the high potential of regeneration of macroalgae. In contrast, callus formation, which often accompanies plant regeneration, was never observed. We monitored the cellular and nuclear dynamics during cell repair or rhizoid formation of four phylogenetically diverged Rhodophyta and Chlorophyta species (Colaconema sp., Dasya sessilis, Cladophora albida, Codium fragile). We observed tip growth of the cells near the damaged site as a common response, despite the difference in the number of nuclei and cells across species. Nuclear translocation follows tip growth, enabling overall uniform distribution of multinuclei (Dasya sessilis, Cladophora albida, Codium fragile) or central positioning of the mononucleus (Colaconema sp.). In contrast, the control of cell cycle events, such as nuclear division and septation, varied in these species. In Dasya sessilis, the division of multinuclei was synchronised, whereas it was not the case in Cladophora albida. Septation was tightly coupled with nuclear division in Colaconema and Dasya but not in others. These observations show that marine macroalgae utilise a variety of regeneration pathways, with some common features. This study also provides a novel methodology of live cell biology in macroalgae, offering a foundation for the future of this under-studied taxon.

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Landscape change and alien invasions drive shifts in native lady beetle communities over a century AimUnderstanding drivers of insect population declines is essential for the development of successful conservation plans, but data limitations restrict assessment across spatial and temporal scales. Museum records represent a unique source of historical data that can be leveraged to investigate temporal trends in insect communities. Native lady beetle decline has been attributed to competition with established alien species and landscape change, but the relative importance of these drivers is difficult to measure with short-term field-based studies. Here we assessed distribution patterns for native lady beetle species over 12 decades using museum records and evaluated the relative importance of alien species and landscape change as long-term drivers contributing to changes in lady beetle communities.

LocationOhio, USA.

MethodsWe compiled occurrence records for 28 lady beetle species collected in Ohio, USA from 1900-2018. Incidence-based measures of taxonomic beta-diversity were used to evaluate changes in lady beetle community composition over time. To evaluate the relative influence of temporal, spatial, landscape, and community drivers on the captures of native lady beetles, we constructed negative binomial generalized additive models.

ResultsWe report evidence of declines in captures for several native species, including Adalia bipunctata, Coccinella novemnotata, Hippodamia convergens, and Coleomegilla maculata. Importantly, the timing, severity, and drivers of these documented declines were species-specific. Changes in lady beetle species composition began in the 1980s, when processes of species loss/gain and turnover shifted communities towards dominance by a few alien lady beetle species. Land cover change also was associated with declines in captures, particularly for C. novemnotata which declined prior to the arrival of alien species in the state.

Main conclusionsOur study documented shifts in Ohios lady beetle communities beginning in the 1980s as alien species supplanted natives. Drivers of declines in captures of native lady beetle species were highly species-specific, emphasizing that mechanisms driving population losses cannot be generalized even among closely related species. These findings also indicate the importance of museum holdings and the analysis of species-level data when studying temporal trends in insect populations.

sentences: Evaluation of flow cytometry for cell count and detection of bacteria in biological fluids. The analysis of biological fluids is crucial for the diagnosis and monitoring of diseases causing effusions and helps in the diagnosis of infectious diseases. The gold standard method for cell count in biological fluids is the manual method using counting chambers. The microbiological routine procedures consist of Direct Gram staining and culture on solid or liquid media. We evaluate the analytical performance of SYSMEX UF4000 (Sysmex, Kobe, Japan) and Sysmex XN10 (Sysmex, Kobe, Japan) in comparison with cytological and microbiological routine procedures. A total of 526 biological fluid samples were included in this study (42 ascitic, 31 pleural, 31 peritoneal, 125 cerebrospinal, 281 synovial, and 16 peritoneal dialysis fluids). All samples were analysed by flow cytometry and subsequently processed following cytological and/or microbiological routine procedures. With regards to cell counts, UF4000 (Sysmex, Kobe, Japan) showed a performance which was at least equivalent to those of the reference methods and superior to those of XN10 (Sysmex, Kobe, Japan). Moreover, the bacterial count obtained with UF4000 (Sysmex, Kobe, Japan) was significantly higher among culture or Direct Gram stain positive samples. We established 3 optimal cut-off points to predict Direct Gram stain positive samples for peritoneal (465.0 bacteria/L), synovial (1200.0 bacteria/L), and cerebrospinal fluids (17.2 bacteria/L) with maximum sensitivity and negative predictive values. Cell count and detection of bacteria by flow cytometry could be used upstream cytological and microbiological routine procedures to improve and accelerate the diagnosis of infection of biological fluid samples.

sentences: Mechanistic Insights of High Temperature-Interfered Meiosis in Autotetraploid Arabidopsis thaliana Environmental temperature has a huge impact on multiple meiosis processes in flowering plants. Polyploid plants derived from whole genome duplication are believed to have an enhanced abiotic stress tolerance. In this study, the impact of high temperatures on male meiosis in autotetraploid Arabidopsis thaliana was investigated. We found that autotetraploid Columbia (Col-0) plants generate a subpopulation of aberrant meiotic products under normal temperature, which is significantly increased under heat stress. Cytological studies revealed that, as the case in diploid Arabidopsis thaliana, assembly of microtubular cytoskeleton network, pairing and segregation of homologous chromosomes, and meiotic recombination in autotetraploid Arabidopsis are compromised under the high temperatures. Immunostaining of {ramshorn}H2A.X and recombinase DMC1 suggested that heat stress inhibits formation of DNA double-strand breaks; additionally, it specifically destabilizes ASY1 and ASY4, but not SYN1 on chromosomes. The loading defects of ASY1 and ASY4 overlap in the syn1 mutant, which supports that the building of lateral element of synaptonemal complex occurs downstream of a SYN1-ASY4-ASY3 stepwise assembly of axis. Remarkably, heat-induced abnormalities of ASY1 and ASY4 co-localize on chromosomes of both diploid and autotetraploid Arabidopsis, suggesting that high temperatures interfere with ASY1-associated SC via an impacted stability of chromosome axis. Furthermore, ZYP1-dependent transverse filament of SC is disrupted by heat stress. Taken together, these findings suggest that polyploidization negatively contributes to instability of chromosome axis and meiotic recombination in Arabidopsis thaliana under heat stress.

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Reconstructing the history of variation in effective population size along phylogenies. AO_SCPLOWBSTRACTC_SCPLOWThe nearly-neutral theory predicts specific relations between effective population size (Ne) and patterns of divergence and polymorphism, which depend on the shape of the distribution of fitness effects (DFE) of new mutations. However, testing these relations is not straightforward, owing to the difficulty in estimating Ne. Here, we introduce an integrative framework allowing for an explicit reconstruction of the phylogenetic history of Ne, thus leading to a quantitative test of the nearly-neutral theory and an estimation of the allometric scaling of the ratios of non-synonymous over synonymous polymorphism ({pi}N /{pi}S) and divergence (dN/dS) with respect to Ne. As an illustration, we applied our method to primates, for which the nearly-neutral predictions were mostly verified. Under a purely nearly-neutral model with a constant DFE across species, we find that the variation in{pi} N /{pi}S and dN/dS as a function of Ne is too large to be compatible with current estimates of the DFE based on site frequency spectra. The reconstructed history of Ne shows a ten-fold variation across primates. The mutation rate per generation u, also reconstructed over the tree by the method, varies over a three-fold range and is negatively correlated with Ne. As a result of these opposing trends for Ne and u, variation in{pi} S is intermediate, primarily driven by Ne but substantially influenced by u. Altogether, our integrative framework provides a quantitative assessment of the role of Ne and u in modulating patterns of genetic variation, while giving a synthetic picture of their history over the clade.

SO_SCPLOWIGNIFICANCEC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWSTATEMENTC_SCPLOWNatural selection tends to increase the frequency of mutants of higher fitness and to eliminate less fit genetic variants. However, chance events over the life of the individuals in the population are susceptible to introduce deviations from these trends, which are expected to have a stronger impact in smaller populations. In the long-term, these fluctuations, called random drift, can lead to the accumulation of mildly deleterious mutations in the genomes of living species, and for that reason, the effective population size (usually denoted Ne, and which captures the relative strength of drift, relative to selection) has been proposed as a major determinant of the evolution of genome architecture and content. A proper quantitative test of this hypothesis, however, is hampered by the fact that Ne is difficult to estimate in practice. Here, we propose a Bayesian integrative approach for reconstructing the broad-scale variation in Ne across an entire phylogeny, which in turns allows for quantifying how Ne correlates with life history traits and with various measures of genetic diversity and selection strength, between and within species. We apply this approach to the phylogeny of primates, and observe that selection is indeed less efficient in primates characterized by smaller effective population sizes.

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Environmental Conditions and Vigilance Behaviour in the Smooth-coated Otter, Lutrogale perspicillata Vigilance is an ecologically important behaviour that acts as a defence mechanism against threats such as predators, competitors, and environmental pressures. Being vigilant can place energetic constraints on an individual, as conducting other behaviours such as grooming can be limited. The smooth-coated otter is a vulnerable species, and understanding baseline behaviour, such as vigilance, and stressor effects can be a crucial step in implementing any conservation strategies. However, literature on this charismatic species is relatively sparse, with the majority of research covering prey items, habitat use and extent of occurrence.

Using camera trap footage along with historical climate datasets, the current study assessed various aspects of vigilance behaviour of the smooth-coated otter. These aspects include how long individuals dedicated to being vigilant, what they prioritised being vigilant over, and how intense their vigilance efforts were

Variables that are expected to change with global climate change such as temperature, and to an extent, air pressure, were associated with increases in vigilance duration. Wind speeds, which are also expected to be affected by climate change, along with increasing numbers of conspecifics, were linked to increased intensity possibly highlighting that increasing ambient movement results in more frequent head rotations. When vigilance is ineffectual however, such during periods of higher rainfall, cloud cover or at night, intensity decreases, and gazing towards conspecifics increases - possibly to reaffirm where safety lies when threats cannot be adequately monitored.

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Genome sequencing of 196 Treponema pallidum strains from six continents reveals additional variability in vaccine candidate genes and dominance of Nichols clade strains in Madagascar In spite of its immutable susceptibility to penicillin, Treponema pallidum (T. pallidum) subsp. pallidum continues to cause millions of cases of syphilis each year worldwide, resulting in significant morbidity and mortality and underscoring the urgency of developing an effective vaccine to curtail the spread of the infection. Several technical challenges, including absence of an in vitro culture system until very recently, have hampered efforts to catalog the diversity of strains collected worldwide. Here, we provide near-complete genomes from 196 T. pallidum strains - including 191 T. pallidum subsp. pallidum - sequenced directly from patient samples collected from 8 countries and 6 continents. Maximum likelihood phylogeny revealed that samples from most sites were predominantly SS14 clade. However, 99% (84/85) of the samples from Madagascar formed two of the five distinct Nichols subclades. Although recombination was uncommon in the evolution of modern circulating strains, we found multiple putative recombination events between T. pallidum subsp. pallidum and subsp. endemicum, shaping the genomes of several subclades. Temporal analysis dated the most recent common ancestor of Nichols and SS14 clades to 1717 (95% HPD: 1543-1869), in agreement with other recent studies. Rates of SNP accumulation varied significantly among subclades, particularly among different Nichols subclades, and was associated in the Nichols A subclade with a C394F substitution in TP0380, a ERCC3-like DNA repair helicase. Our data highlight the role played by variation in genes encoding putative surface-exposed outer membrane proteins in defining separate lineages, and provide a critical resource for the design of broadly protective syphilis vaccines targeting surface antigens.

Author SummaryEach year, millions of new cases of venereal and congenital syphilis, caused by the bacterium Treponema pallidum (T. pallidum) subsp. pallidum, are diagnosed worldwide, resulting in significant morbidity and mortality. Alongside endemic circulation of syphilis in low-income countries, disease resurgence in high-income nations has underscored the need for a vaccine. Due to prior technological limitations in culturing and sequencing the organism, the extent of the genetic diversity within modern strains of T. pallidum subsp. pallidum remains poorly understood, hampering development of a broadly protective vaccine. In this study, we obtained 196 near-complete T. pallidum genomes directly from clinical swabs from eight countries across six continents. Of these, 191 were identified as T. pallidum subsp. pallidum, including 90 Nichols clade genomes. Bayesian analysis revealed a high degree of variance in mutation rate among subclades. Interestingly, a Nichols subclade with a particularly high mutation rate harbors a non-synonymous mutation in a putative DNA repair helicase. Coupling sequencing data with protein structure prediction, we identified multiple novel amino acid variants in several proteins previously identified as potential vaccine candidates. Our data help inform current efforts to develop a broadly protective syphilis vaccine.

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A single-cell atlas of human fetal lung development between 14 and 19 weeks of gestation RationaleHuman lung development has been mainly described in morphologic studies and the potential underlying molecular mechanisms were extrapolated from animal models. Therefore, there is a need to gather knowledge from native human lung tissue. In this study we describe changes at a single-cell level in human fetal lungs during the pseudoglandular stage.

MethodsWe report the cellular composition, cell trajectories and cell-to-cell communication in developing human lungs with single-nuclei RNA sequencing (snRNA-seq) on 23,251 nuclei isolated from nine human fetuses with gestational ages between 14 to 19 weeks of gestation.

ResultsWe identified nine different cell types, including a rare pulmonary neuroendocrine cells population. For each cell type, marker genes are reported, and selected marker genes are used for spatial validation with fluorescent RNA in situ hybridization. Enrichment and developmental trajectory analysis provide insight into molecular mechanisms and signaling pathways within individual cell clusters according to gestational age. Lastly, ligand-receptor analysis highlights determinants of cell-to-cell communication among the different cell types through the pseudoglandular stage, including general developmental pathways (NOTCH and TGFB), as well as more specific pathways involved in vasculogenesis, neurogenesis, and immune system regulation.

ConclusionThese findings provide a clinically relevant background for research hypotheses generation in projects studying normal or impaired lung development and help to develop and validate surrogate models to study human lung development, such as human lung organoids.

TAKE HOME MESSAGEUsing a single-cell transcriptomic approach (single-nuclei RNA sequencing), we describe here, for the first time, the cellular landscape, cell developmental trajectories, and cell-to-cell communication in the developing human lung during the pseudoglandular stage.

sentences: Deep Learning-based Pseudo-Mass Spectrometry Imaging Analysis for Precision Medicine Liquid chromatography-mass spectrometry (LC-MS) based untargeted metabolomics provides systematic profiling of metabolic. Yet its applications in precision medicine (disease diagnosis) have been limited by several challenges, including metabolite identification, information loss, and low reproducibility. Here, we present the deepPseudoMSI project (https://www.deeppseudomsi.org/), which converts LC-MS raw data to pseudo-MS images and then processes them by deep learning for precision medicine, such as disease diagnosis. Extensive tests based on real data demonstrated the superiority of deepPseudoMSI over traditional approaches and the capacity of our method to achieve an accurate individualized diagnosis. Our framework lays the foundation for future metabolic-based precision medicine.

sentences: Long-read transcriptome sequencing analysis with IsoTools Long-read transcriptome sequencing (LRTS) holds the promise to boost our understanding of alternative splicing. Recent advances in accuracy and throughput have diminished the major limitations and enabled the direct quantification of isoforms. Considering the complexity of the data and the broad range of potential applications, it is clear that highly flexible, accurate analysis tools are crucial. Here, we present IsoTools, a comprehensive Python-based analysis package, for the improvement of alternative and differential splicing analysis. Iso-Tools provides a comprehensive data structure that integrates genomic information from LRTS transcripts together with the reference annotation, and enables broad functionality to quality control, visualize and analyze the data. Additionally, we implemented a graph-based method for the identification of alternative splicing events and a statistical approach based on the beta binomial distribution for the detection of differential events. To demonstrate our methods, we generated PacBio Iso-Seq data of human hepatocytes treated with the HDAC inhibitor valproic acid, a compound known to induce widespread transcriptional changes. Contrasted with short read RNA-Seq of the same samples, this analysis shows that LRTS provides valuable additional insights for a better understanding of alternative splicing, in particular with respect to complex novel and differential splicing events. IsoTools is made available for the community along with extensive documentation at https://github.com/MatthiasLienhard/isotools.

sentences: MLF2 modulates phase separated nuclear envelope condensates that provoke dual proteotoxicity DYT1 dystonia is a highly debilitating neurological movement disorder arising from mutation in the AAA+ ATPase TorsinA. The hallmark of Torsin dysfunction is nuclear envelope blebbing resulting from defects in nuclear pore complex biogenesis. Whether blebs actively contribute to disease manifestation is presently unknown. We report that FG-nucleoporins in the bleb lumen undergo phase separation and contribute to DYT1 dystonia by provoking two proteotoxic insults. Short-lived ubiquitinated proteins that are normally rapidly degraded in healthy cells partition into the bleb lumen and become stabilized. Additionally, blebs selectively sequester a chaperone network composed of HSP70s and HSP40s. The composition of this chaperone network is altered by the bleb component MLF2. We further demonstrate that MLF2 is a catalyst of phase separation that suppresses the ectopic accumulation of FG-nucleoporins and modulates the selective properties and size of condensates in vitro. Our studies identify unprecedented, dual mechanisms of proteotoxicity in the context of liquid-liquid phase separation with direct implications for our understanding of disease etiology and treatment.

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Design and execution of a Verification, Validation, and Uncertainty Quantification plan for a numerical model of left ventricular flow after LVAD implantation Backgroundleft ventricular assist devices (LVADs) are implantable pumps that act as a life support therapy for patients with severe heart failure. Despite improving the survival rate, LVAD therapy can carry major complications. Particularly, the flow distortion introduced by the LVAD in the left ventricle (LV) may induce thrombus formation. While previous works have used numerical models to study the impact of multiple variables in the intra-LV stagnation regions, a comprehensive validation analysis has never been executed. The main goal of this work is to present a model of the LV-LVAD system and to design and follow a verification, validation and uncertainty quantification (VVUQ) plan based on the ASME V&V40 and V&V20 standards to ensure credible predictions.

MethodsThe experiment used to validate the simulation is the SDSU cardiac simulator, a bench mock-up of the cardiovascular system that allows mimicking multiple operation conditions for the heart-LVAD system. The numerical model is based on Alya, the BSCs in-house platform for numerical modelling. Alya solves the Navier-Stokes equation with an Arbitrarian Lagrangian-Eulerian (ALE) formulation in a deformable ventricle and includes pressure-driven valves, a 0D Windkessel model for the arterial output and a LVAD boundary condition modeled through a dynamic pressure-flow performance curve. The designed VVUQ plan involves: (a) a risk analysis and the associated credibility goals; (b) a verification stage to ensure correctness in the numerical solution procedure; (c) a sensitivity analysis to quantify the impact of the inputs on the four quantities of interest (QoIs) (average aortic root flow [Formula], maximum aortic root flow [Formula], average LVAD flow [Formula], and maximum LVAD flow [Formula]); (d) an uncertainty quantification using six validation experiments that include extreme operating conditions.

ResultsNumerical code verification tests ensured correctness of the solution procedure and numerical calculation verification showed small numerical errors. The total Sobol indices obtained during the sensitivity analysis demonstrated that the ejection fraction, the heart rate, and the pump performance curve coefficients are the most impactful inputs for the analysed QoIs.

The Minkowski norm is used as validation metric for the uncertainty quantification. It shows that the midpoint cases have more accurate results when compared to the extreme cases. The total computational cost of the simulations was above 100 [core-years] executed in around three weeks time span in Marenostrum IV supercomputer.

ConclusionsThis work details a novel numerical model for the LV-LVAD system, that is supported by the design and execution of a VVUQ plan created following recognised international standards. We present a methodology demonstrating that stringent VVUQ according to ASME standards is feasible but computationally expensive.

sentences: Self-ubiquitination of a pathogen type-III effector traps and blocks the autophagy machinery to promote disease Beyond its role in cellular homeostasis, autophagy plays anti- and pro-microbial roles in host-microbe interactions, both in animals and plants. One prominent role of anti-microbial autophagy is to degrade intracellular pathogens or microbial molecules, in a process termed xenophagy. Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination. Although well-described in animals, the extent to which xenophagy contributes to plant-bacteria interactions remains unknown. Here, we provide evidence that Xanthomonas campestris pv. vesicatoria (Xcv) suppresses host autophagy by utilizing type-III effector XopL. XopL interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection. Intriguingly, XopL is targeted for degradation by defense-related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery. Our results implicate plant antimicrobial autophagy in depletion of a bacterial virulence factor and unravels an unprecedented pathogen strategy to counteract defense-related autophagy.

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Synergistic neural integration is greater downstream of recurrent information flow in organotypic cortical cultures The directionality of network information flow dictates how networks process information. A central component of information processing in both biological and artificial neural networks is their ability to perform synergistic integration-a type of computation. We established previously that synergistic integration varies directly with the strength of feedforward information flow. However, the relationships between both recurrent and feedback information flow and synergistic integration remain unknown. To address this, we analyzed the spiking activity of hundreds of neurons in organotypic cultures of mouse cortex. We asked how empirically observed synergistic integration varied with local functional network structure that was categorized into motifs with varying recurrent and feedback information flow. We found that synergistic integration was elevated in motifs with greater recurrent information flow beyond that expected from the local feedforward information flow. Feedback information flow was interrelated with feedforward information flow and was associated with decreased synergistic integration. Our results indicate that synergistic integration is distinctly influenced by the directionality of local information flow.

Author SummaryNetworks compute information. That is, they modify inputs to generate distinct outputs. These computations are an important component of network information processing. Knowing how the routing of information in a network influences computation is therefore crucial. Here we asked how a key form of computation--synergistic integration--is related to the direction of local information flow in networks of spiking cortical neurons. Specifically, we asked how information flow between input neurons (i.e., recurrent information flow) and information flow from output neurons to input neurons (i.e., feedback information flow) was related to the amount of synergistic integration performed by output neurons. We found that greater synergistic integration occurred where there was more recurrent information flow. And, lesser synergistic integration occurred where there was more feedback information flow relative to feedforward information flow. These results show that computation, in the form of synergistic integration, is distinctly influenced by the directionality of local information flow. Such work is valuable for predicting where and how network computation occurs and for designing networks with desired computational abilities.

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Akkermansia muciniphila secretome promotes α-synuclein aggregation in enteroendocrine cells. The notion that the gut microbiota play a role in neurodevelopment, behavior and outcome of neurodegenerative disorders is recently taking place. A number of studies have consistently reported a greater abundance of Akkermansia muciniphila in Parkinsons disease (PD) fecal samples. Nevertheless, a functional link between A.muciniphila and sporadic PD remained unexplored. Here, we investigated whether A.muciniphila secretome could initiate the misfolding process of -synuclein (Syn) in enteroendocrine cells (EECs), which are part of the gut epithelium and possess many neuron-like properties. We found that A.muciniphila secretome is directly modulated by mucin, induces intracellular calcium (Ca2+) release, and causes increased mitochondrial Ca2+ uptake in EECs, which in turn leads to production of reactive oxygen species (ROS) and Syn aggregation. However, these events were efficiently inhibited once we buffered mitochondrial Ca2+. Thereby, these molecular insights provided here offer evidence that bacterial secretome is capable of inducing Syn aggregation in enteroendocrine cells.

SYNOPSIS FIGURE DESCRIPTIONThe secretome isolated from the commensal gut bacterium Akkermansia muciniphila triggers intracellular Ca2+ signaling in enteroendocrine cells, leading to increased mitochondrial Ca2+ uptake. Mitochondrial Ca2+ overload leads to ROS generation culminating with Syn phosphorylation and aggregation (left panel). All these events were inhibited once mitochondrial Ca2+ is buffered (right panel).

O_LIGram-negative gut bacterium Akkermansia muciniphila is consistently found more abundant in Parkinsons disease patients.
C_LIO_LIAkkermansia muciniphila protein secretome composition is directly modulated by mucin and induces an IP3-independent endoplasmic reticulum (ER)-calcium release in enteroendocrine cells.
C_LIO_LIThis Ca2+ release is triggered by direct activation of Ryanodine Receptors leading to increased mitochondrial Ca2+ uptake.
C_LIO_LIMitochondrial Ca2+ overload leads to ROS generation culminating with Syn aggregation.
C_LIO_LIBuffering mitochondrial Ca2+ efficiently inhibits A.muciniphila-induced Syn aggregation in enteroendocrine cells.
C_LI

sentences: The p53 transactivation domain 1-dependent response to acute DNA damage in endothelial cells protects against radiation-induced cardiac injury Thoracic radiation therapy can cause endothelial injury in the heart, leading to cardiac dysfunction and heart failure. Although it has been demonstrated that the tumor suppressor p53 functions in endothelial cells to prevent the development of radiation-induced myocardial injury, the key mechanism(s) by which p53 regulates the radiosensitivity of cardiac endothelial cells is not completely understood. Here, we utilized genetically engineered mice that express mutations in p53 transactivation domain 1 (TAD1) (p5325,26) or mutations in p53 TAD1 and TAD2 (p5325,26,53,54) specifically in endothelial cells to study the p53 transcriptional program that protects cardiac endothelial cells from ionizing radiation in vivo. p5325,26,53,54 loses the ability to drive transactivation of p53 target genes after irradiation while p5325,26 can induce transcription of a group of non-canonical p53 target genes, but not the majority of classic radiation-induced p53 targets critical for p53-mediated cell cycle arrest and apoptosis. After 12 Gy whole-heart irradiation, we found that both p5325,26 and p5325,26,53,54 sensitized mice to radiation-induced cardiac injury, in contrast to wild-type p53. Histopathological examination suggested that mutation of TAD1 contributes to myocardial necrosis after whole-heart irradiation, while mutation of both TAD1 and TAD2 abolishes the ability of p53 to prevent radiation-induced heart disease. Taken together, our results show that the transcriptional program downstream of p53 TAD1, which activates the acute DNA damage response after irradiation, is necessary to protect cardiac endothelial cells from radiation injury in vivo.

sentences: Atp{Theta} is an inhibitor of F0F1 ATP synthase to arrest ATP hydrolysis during low-energy conditions in cyanobacteria Biological processes in all living cells are powered by ATP, a nearly universal molecule of energy transfer. ATP synthases produce ATP utilizing proton gradients that are usually generated by either respiration or photosynthesis. However, cyanobacteria are unique in combining photosynthetic and respiratory electron transport chains in the same membrane system, the thylakoids. How cyanobacteria prevent the futile reverse operation of ATP synthase under unfavorable conditions pumping protons while hydrolyzing ATP is mostly unclear. Here, we provide evidence that the small protein Atp{Theta}, which is widely conserved in cyanobacteria, is mainly fulfilling this task. The expression of Atp{Theta} becomes induced under conditions such as darkness or heat shock, which can lead to a weakening of the proton gradient. Translational fusions of Atp{Theta} to the green fluorescent protein revealed targeting to the thylakoid membrane. Immunoprecipitation assays followed by mass spectrometry and far Western blots identified subunits of ATP synthase as interacting partners of Atp{Theta}. ATP hydrolysis assays with isolated membrane fractions as well as purified ATP synthase complexes demonstrated that Atp{Theta} inhibits ATPase activity in a dose-dependent manner similar to the F0F1-ATP synthase inhibitor N,N-dicyclohexylcarbodimide. The results show that, even in a well-investigated process, crucial new players can be discovered if small proteins are taken into consideration and indicate that ATP synthase activity can be controlled in surprisingly different ways.

sentences: Cellular remodeling and JAK inhibition promote zygotic gene expression in the Ciona germline During development, remodeling of the cellular transcriptome and proteome underlies cell fate decisions and, in somatic lineages, transcription control is a major determinant of fateful biomolecular transitions. By contrast, early germline fate specification in numerous vertebrate and invertebrate species relies extensively on RNA-level regulation, exerted on asymmetrically inherited maternal supplies, with little-to-no zygotic transcription. However delayed, a maternal-to-zygotic transition is nevertheless poised to complete the deployment of pre-gametic programs in the germline. Here, we focused on early germline specification in the tunicate Ciona to study zygotic genome activation. We first demonstrate that a peculiar cellular remodeling event excludes localized postplasmic mRNAs, including Pem-1, which encodes the general inhibitor of transcription. Subsequently, zygotic transcription begins in Pem-1-negative primordial germ cells (PGCs), as revealed by histochemical detection of elongating RNA Polymerase II (RNAPII), and nascent transcripts from the Mef2 locus. Using PGC-specific Mef2 transcription as a read-out, we uncovered a provisional antagonism between JAK and MEK/BMPRI/GSK3 signaling, which controls the onset of zygotic gene expression, following cellular remodeling of PGC. We propose a 2-step model for the onset of zygotic transcription in the Ciona germline, which relies on successive cellular remodeling and JAK inhibition, and discuss the significance of germ plasm dislocation and remodeling in the context of developmental fate specification.

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Mechanism underlying the DNA-binding preferences of the Vibrio cholerae and vibriophage VP882 VqmA quorum-sensing receptors Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. In the global pathogen Vibrio cholerae, one quorum-sensing receptor and transcription factor, called VqmA (VqmAVc), activates expression of the vqmR gene encoding the small regulatory RNA VqmR, which represses genes involved in virulence and biofilm formation. Vibriophage VP882 encodes a VqmA homolog called VqmAPhage that activates transcription of the phage gene qtip, and Qtip launches the phage lytic program. Curiously, VqmAPhage can activate vqmR expression but VqmAVc cannot activate expression of qtip. Here, we investigate the mechanism underlying this asymmetry. We find that promoter selectivity is driven exclusively by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity. A protein sequence-guided mutagenesis approach revealed that the residue E194 of VqmAPhage and A192, the equivalent residue in VqmAVc, in the helix-turn-helix motifs contribute to promoter-binding specificity. A genetic screen to identify VqmAPhage mutants that are incapable of binding the qtip promoter but maintain binding to the vqmR promoter delivered additional VqmAPhage residues located immediately C-terminal to the helix-turn-helix motif as required for binding the qtip promoter. Surprisingly, these residues are conserved between VqmAPhage and VqmAVc. A second, targeted genetic screen revealed a region located in the VqmAVc DNA-binding domain as necessary to prevent VqmAVc from binding the qtip promoter, thus restricting DNA-binding to the vqmR promoter. We propose that the VqmAVc helix-turn-helix motif and the C-terminal flanking residues function together to prohibit VqmAVc from binding the qtip promoter.

AUTHOR SUMMARYBacteria use a chemical communication process called quorum sensing (QS) to orchestrate collective behaviors. Recent studies demonstrate that bacteria-infecting viruses, called phages, also employ chemical communication to regulate collective activities. Phages can encode virus-specific QS-like systems, or they can harbor genes encoding QS components resembling those of bacteria. The latter arrangement suggests the potential for chemical communication across domains, i.e., between bacteria and phages. Ramifications stemming from such cross-domain communication are not understood. Phage VP882 infects the global pathogen Vibrio cholerae, and "eavesdrops" on V. cholerae QS to optimize the timing of its transition from living as a parasite to killing the host, and moreover, to manipulate V. cholerae biology. To accomplish these feats, phage VP882 relies on VqmAPhage, the phage-encoded homolog of the V. cholerae VqmAVc QS receptor and transcription factor. VqmAVc, by contrast, is constrained to the control of only V. cholerae genes and is incapable of regulating phage biology. Here, we discover the molecular mechanism underpinning the asymmetric transcriptional preferences of the phage-encoded and bacteria-encoded VqmA proteins. We demonstrate how VqmA transcriptional regulation is crucial to the survival and persistence of both the pathogen V. cholerae, and the phage that preys on it.

sentences: Integration of chemosensing and carbon catabolite repression impacts fungal enzyme regulation and plant associations Fungal metabolism and enzyme production are regulated by nutrient availability and by interactions with the living environment. We investigated the mechanisms underpinning adaptation of the biotechnological fungus Trichoderma reesei to decaying plant biomass versus living plants. We found that concentration-gated response to glucose, the main molecule sensed from dead plant biomass, is mediated by a conserved signaling pathway downstream of G protein-coupled receptors (GPCRs), while the carbon catabolite repressor CRE1 is critical for glucose concentration gating. The GPCRs CSG1 and CSG2 are further required for root colonization and formation of appressorium like structures on plant surfaces. Acceleration of sexual development in the presence of plant roots and their interactions with fruiting bodies indicates preferential association with plants. Our results reveal a complex sensing network governing resource distribution, enzyme production and fungal development that explains previously observed phenomena in fermentations and opens new perspectives for industrial strain improvement and agriculture.

sentences: Insertions and deletions as phylogenetic signal in alignment-free sequence comparison Most methods for phylogenetic tree reconstruction are based on sequence alignments; they infer phylogenies from substitutions that may have occurred at the aligned sequence positions. Gaps in alignments are usually not employed as phylogenetic signal. In this paper, we explore an alignment-free approach that uses insertions and deletions (indels) as an additional source of information for phylogeny inference. For a set of four or more input sequences, we generate so-called quartet blocks of four putative homologous segments each. For pairs of such quartet blocks involving the same four sequences, we compare the distances between the two blocks in these sequences, to obtain hints about indels that may have happened between the blocks since the respective four sequences have evolved from their last common ancestor. A prototype implementation is presented to infer phylogenetic trees from these data, using a quartet-tree approach or, alternatively, under the maximum-parsimony paradigm. This approach should not be regarded as an alternative to established methods, but rather as a complementary source of phylogenetic information. Interestingly, however, our software is able to produce phylogenetic trees from putative indels alone that are comparable to trees obtained with existing alignment-free methods.

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Complete genome sequence of Xylella taiwanensis and comparative analysis of virulence gene content with Xylella fastidiosa The bacterial genus Xylella contains plant pathogens that are major threats to agriculture in America and Europe. Although extensive research was conducted to characterize different subspecies of Xylella fastidiosa (Xf), comparative analysis at above-species levels were lacking due to the unavailability of appropriate data sets. Recently, a bacterium that causes pear leaf scorch (PLS) in Taiwan was described as the second Xylella species (i.e., Xylella taiwanensis; Xt). In this work, we report the complete genome sequence of Xt type strain PLS229T. The genome-scale phylogeny provided strong support that Xf subspecies pauca (Xfp) is the basal lineage of this species and Xylella was derived from the paraphyletic genus Xanthomonas. Quantification of genomic divergence indicated that different Xf subspecies share [~]87-95% of their chromosomal segments, while the two Xylella species share only [~]66-70%. Analysis of overall gene content suggested that Xt is most similar to Xf subspecies sandyi (Xfs). Based on the existing knowledge of Xf virulence genes, the homolog distribution among 28 Xylella representatives was examined. Among the 11 functional categories, those involved in secretion and metabolism are the most conserved ones with no copy number variation. In contrast, several genes related to adhesins, hydrolytic enzymes, and toxin-antitoxin systems are highly variable in their copy numbers. Those virulence genes with high levels of conservation or variation may be promising candidates for future studies. In summary, the new genome sequence and analysis reported in this work contributed to the study of several important pathogens in the family Xanthomonadaceae.

Contribution to the FieldXylella fastidiosa is a plant-pathogenic bacterium with multiple subspecies that are major threats to agriculture in America and Europe. Although extensive research has been conducted, comparative analysis of this species with other bacteria is lacking due to the unavailability of known close relatives. In this work, we report the complete genome sequence of Xylella taiwanensis, a newly described species within the same genus. This new data set and our focused analysis helped to better understand the evolutionary relationships among different Xylella lineages and their genomic diversity. Moreover, detailed examination of their virulence genes identified those that are either highly conserved or variable, providing promising candidates for future studies to further investigate the molecular mechanisms of Xylella virulence.

sentences: Expression signatures based on pyroptosis-related genes can robustly diagnose skin cutaneous melanoma and predict the prognosis. Skin cutaneous melanoma (SKCM) is a chronically malignant tumor with a high mortality rate. Pyroptosis, a kind of pro-inflammatory programmed cell death, has been linked to cancer in recent studies. However, the value of pyroptosis in the diagnosis and prognosis of SKCM is not clear. In this study, it was discovered that 20 pyroptosis-related genes (PRGs) differed in expression between SKCM and normal tissues, which were related to diagnosis and prognosis. Firstly, based on these genes, nine machine-learning algorithms were shown to perform well in constructing diagnostic classifiers, including K-Nearest Neighbor (KNN), logistic regression, Support Vector Machine (SVM), Artificial Neural Network (ANN), decision tree, random forest, XGBoost, LightGBM, and CatBoost. Secondly, the least absolute shrinkage and selection operator (LASSO) Cox regression analysis was applied and the prognostic model was constructed based on 9 PRGs. Subgroups in low and high risks determined by the prognostic model were shown to have different survival. Thirdly, functional enrichment analyses were performed by applying the gene set enrichment analysis (GSEA), and results suggested that the risk was related to immune response. Finally, immune infiltration analysis was carried out, which showed that fractions of activated CD4+ memory T cells, {gamma}{delta} T cells, Ml macrophages, and M2 macrophages were significantly different between subgroups. In conclusion, the expression signatures of pyroptosis-related genes are effective and robust in the diagnosis and prognosis of SKCM, which is related to immunity.

sentences: A network model of glymphatic flow under different experimentally-motivated parametric scenarios Rapidly growing evidence demonstrates that flow of cerebrospinal fluid (CSF) through perivascular spaces (PVSs) - annular channels surrounding vasculature in the brain - is a critically-important component of neurophysiology. CSF inflow contributes during physiological conditions to clearance of metabolic waste and in pathological situations to edema formation. However, brain-wide imaging methods cannot resolve PVSs, and high-resolution methods cannot access deep tissue or be applied to human subjects, so theoretical models provide essential insight. We model this CSF pathway as a network of hydraulic resistances, built from published parameters. A few parameters have very wide uncertainties, so we focus on the estimated limits of their feasible ranges by analyzing different parametric scenarios. We identify low-resistance PVSs and high-resistance parenchyma (brain tissue) as the scenario that best explains experimental observations. Our results point to the most important parameters that should be measured in future experiments. Extensions of our modeling may help predict stroke severity or lead to neurological disease treatments and drug delivery methods.

sentences: Applications of the indole-alkaloid gramine modulate the assembly of individual members of the barley rhizosphere microbiota Microbial communities proliferating at the root-soil interface, collectively referred to as the rhizosphere microbiota, represent an untapped beneficial resource for plant growth, development and health. Integral to a rational manipulation of the microbiota for sustainable agriculture is the identification of the molecular determinants of these communities. In plants, biosynthesis of allelochemicals is centre stage in defining inter-organismal relationships in the environment. Intriguingly, this process has been moulded by domestication and breeding selection. The indole-alkaloid gramine, whose occurrence in barley (Hordeum vulgare L.) is widespread among wild genotypes but has been counter selected in several modern varieties, is a paradigmatic example of this phenomenon. This prompted us to investigate how exogenous applications of gramine impacted on the rhizosphere microbiota of two, gramine-free, elite barley varieties grown in a reference agricultural soil. High throughput 16S rRNA gene amplicon sequencing revealed that applications of gramine interfere with the proliferation of a subset of soil microbes with a relatively broad phylogenetic assignment. Strikingly, growth of these bacteria appeared to be rescued by barley plants in a genotype- and dosage-independent manner. In parallel, we discovered that host recruitment cues can interfere with the impact of gramine application in a host genotype-dependent manner. Interestingly, this latter effect displayed a bias for members of the phyla Proteobacteria. These initial observations indicate that gramine can act as a determinant of the prokaryotic communities inhabiting the root-soil interface.

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Towards end-to-end disease prediction from raw metagenomic data Analysis of the human microbiome using metagenomic sequencing data has demonstrated high ability in discriminating various human diseases. Raw metagenomic sequencing data require multiple complex and computationally heavy bioinformatics steps prior to data analysis. Such data contain millions of short sequences read from the fragmented DNA sequences and are stored as fastq files. Conventional processing pipelines consist in multiple steps including quality control, filtering, alignment of sequences against genomic catalogs (genes, species, taxonomic levels, functional pathways, etc.). These pipelines are complex to use, time consuming and rely on a large number of parameters that often provide variability and impact the estimation of the microbiome elements.

Training Deep Neural Networks directly from raw sequencing data is a promising approach to bypass some of the challenges associated with mainstream bioinformatics pipelines. Most of these methods use the concept of word and sentence embeddings that create a meaningful and numerical representation of DNA sequences, while extracting features and reducing the dimensionality of the data. In this paper we present an end-to-end approach that classifies patients into disease groups directly from raw metagenomic reads: metagenome2vec. This approach is composed of four steps (i) generating a vocabulary of k-mers and learning their numerical embeddings; (ii) learning DNA sequence (read) embeddings; (iii) identifying the genome from which the sequence is most likely to come and (iv) training a multiple instance learning classifier which predicts the phenotype based on the vector representation of the raw data. An attention mechanism is applied in the network so that the model can be interpreted, assigning a weight to the influence of the prediction for each genome. Using two public real-life data-sets as well a simulated one, we demonstrated that this original approach reaches high performance, comparable with the state-of-the-art methods applied directly on processed data though mainstream bioinformatics workflows. These results are encouraging for this proof of concept work. We believe that with further dedication, the DNN models have the potential to surpass mainstream bioinformatics workflows in disease classification tasks.

sentences: Local conjugation of auxin by the GH3 amido synthetases is required for normaldevelopment of roots and flowers in Arabidopsis Gretchen Hagen 3 (GH3) amido synthetases conjugate amino acids to a carboxyl group of small molecules including hormones auxin, jasmonate, and salicylic acid. The Arabidopsis genome harbors 19 GH3 genes, whose exact roles in plant development have been difficult to define because of genetic redundancy among the GH3 genes. Here we use CRISPR/Cas9 gene editing technology to delete the Arabidopsis group II GH3 genes, which are able to conjugate indole-3-acetic acid (IAA) to amino acids. We show that plants lacking the eight group II GH3 genes (gh3 octuple mutants) accumulate free IAA and fail to produce IAA-Asp and IAA-Glu conjugates. Consequently, gh3 octuple mutants have extremely short roots, long and dense root hairs, and long hypocotyls and petioles. Our characterization of gh3 septuple mutants, which provide sensitized backgrounds, reveals that GH3.17 and GH3.9 play prominent roles in root elongation and seed production, respectively. We show that GH3 functions correlate with their expression patterns, suggesting that local deactivation of auxin also contributes to maintaining auxin homeostasis and is important for plant development. Moreover, this work provides a method for elucidating functions of individual members of a gene family, whose members have overlapping functions.

sentences: Gene set enrichment analysis for genome-wide DNA methylation data DNA methylation is one of the most commonly studied epigenetic marks, due to its role in disease and development. Illumina methylation arrays have been extensively used to measure methylation across the human genome. Methylation array analysis has primarily focused on preprocessing, normalisation and identification of differentially methylated CpGs and regions. GOmeth and GOregion are new methods for performing unbiased gene set testing following differential methylation analysis. Benchmarking analyses demonstrate GOmeth outperforms other approaches and GOregion is the first method for gene set testing of differentially methylated regions. Both methods are publicly available in the missMethyl Bioconductor R package.

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The Dendritic Ergic: Microtubule And Actin Cytoskeletons Mediate Stop-And-Go Movement Of Mobile Carriers Between Stable Structures The ER-to-Golgi intermediate compartment (ERGIC) is a membranous organelle that mediates protein transport between the endoplasmic reticulum (ER) and Golgi apparatus. In neurons, clusters of these vesiculotubular structures are situated in throughout the cell in proximity to the ER, passing cargo to the cis-Golgi cisternae located mainly in the perinuclear region. Although ERGIC markers have been identified in neurons, the distribution and dynamics of neuronal ERGIC structures have not been characterized.

Here, we argue that long-distance ERGIC transport occurs via an intermittent mechanism in neurons, with mobile elements moving between stationary structures. Using immunofluorescence microscopy, we detected discrete, irregular ERGIC structures in neural soma and dendrites. Slow live-cell imaging (2 frames/minute; 15 minutes) indicated that 8% of dendritic ERGIC structures were stable, remaining in place over long periods. On the other hand, fast live-cell imaging (2 frames/second; 180 seconds) captured mobile ERGIC structures advancing very short distances along dendrites. Importantly, these distances were consistent with the lengths between the stationary ERGIC structures. Kymography revealed ERGIC elements that moved intermittently, emerging from and fusing with stationary ERGIC structures. Surprisingly, this movement was apparently dependent not only on the integrity of the microtubule cytoskeleton, as has been previously reported, but on the actin cytoskeleton as well.

Our results indicate that the dendritic ERGIC has a dual nature, with both stationary and mobile structures. The neural ERGIC network transports proteins via a stop-and-go movement that is mediated by the microtubule and actin cytoskeletons.

sentences: Sex differences in the immediate extinction deficit and renewal of extinguished fear in rats Extinction learning is central to exposure-based behavioral therapies for reducing fear and anxiety in humans. However, patients with fear and anxiety disorders are often resistant to extinction. Moreover, trauma and stress-related disorders are highly prone to relapse and are twice as likely to occur in females compared to males, suggesting that females may be more susceptible to extinction deficits and fear relapse phenomena. In this report, we tested this hypothesis by examining sex differences in a stress-induced extinction learning impairment, the immediate extinction deficit (IED), and renewal, a common form of fear relapse. In contrast to our hypothesis, there were no sex differences in the magnitude of the immediate extinction deficit in two different rat strains (Long-Evans and Wistar). However, we did observe a sex difference in the renewal of fear when the extinguished conditioned stimulus was presented outside the extinction context. Male Wistar rats exhibited significantly greater renewal than female rats, a sex difference that has previously been reported after appetitive extinction. Collectively, these data reveal that stress-induced extinction impairments are similar in male and female rats, though the context-dependence of extinction is more pronounced in males.

sentences: Spatially coherent diffusion of human RNA Pol II depends on transcriptional state rather than chromatin motion Gene transcription by RNA polymerase II (RNAP II) is a tightly regulated process in the genomic, temporal, and spatial context. Transcriptionally active genes often spatially cluster at RNA Pol II foci, called transcription factories, causing long-range interactions between distal sites of the genome. Recently, we have shown that chromatin exhibits spatially long-range coherently moving regions over the entire nucleus, and transcription enhances this long-range correlated DNA movement. Yet, it remains unclear how the mobility of RNA Pol II molecules is affected by transcription regulation and whether this response depends on the coordinated chromatin movement. We applied our Dense Flow reConstruction and Correlation method to analyze nucleus-wide coherent movements of RNA Pol II in living human cancer cells. We quantify the spatial correlation length of RNA Pol II in the context of DNA motion. We observe a spatially coherent movement of RNA Pol II molecules over ~1 m, considerably less than for DNA, suggesting that spatially coherent RNA Pol II motion does not solely result from the DNA motion. In contrast to DNA, inducing transcription in quiescent cells decreased the coherent motion of RNA Pol II, while the inhibition of transcription elongation by using DRB slightly increased coherent RNA Pol II motion. The spatially coherent movement of RNA Pol II domains is affected by the transcriptional state and largely independent of the underlying chromatin domains. Our study reveals the nucleus-wide interplay between chromatin and RNA Pol II in the dynamic regulation of chromatin organization.

sentences: ChAlPred: A Web Server for Prediction of Allergenicity of Chemical Compounds Allergy is the abrupt reaction of the immune system that may occur after the exposure with allergens like protein/peptide or chemical allergens. In past number of methods of have been developed for classifying the protein/peptide based allergen. To the best of our knowledge, there is no method to classify the allergenicity of chemical compound. Here, we have proposed a method named "ChAlPred", which can be used to fill the gap for predicting the chemical compound that might cause allergy. In this study, we have obtained the dataset of 403 allergen and 1074 non-allergen chemical compounds and used 2D, 3D and FP descriptors to train, test and validate our prediction models. The fingerprint analysis of the dataset indicates that PubChemFP129 and GraphFP1014 are more frequent in the allergenic chemical compounds, whereas KRFP890 is highly present in non-allergenic chemical compounds. Our XGB based model achieved the AUC of 0.89 on validation dataset using 2D descriptors. RF based model has outperformed other classifiers using 3D descriptors (AUC = 0.85), FP descriptors (AUC = 0.92), combined descriptors (AUC = 0.93), and hybrid model (AUC = 0.92) on validation dataset. In addition, we have also reported some FDA-approved drugs like Cefuroxime, Spironolactone, and Tioconazole which can cause the allergic symptoms. A user user-friendly web server named "ChAlPred" has been developed to predict the chemical allergens. It can be easily accessed at https://webs.iiitd.edu.in/raghava/chalpred/.

sentences: Somatostatin interneurons activated by 5-HT2A receptor suppress slow oscillations in medial entorhinal cortex Serotonin (5-HT) is one of the major neuromodulators present in the mammalian brain and has been shown to play a role in multiple physiological processes. The mechanisms by which 5-HT modulates cortical network activity, however, are not yet fully understood. We investigated the effects of 5-HT on slow oscillations (SOs), a synchronized cortical network activity universally present across species. SOs are observed during anesthesia and are considered to be the default cortical activity pattern. We discovered that ({+/-})3,4-methylenedioxymethamphetamine (MDMA) and fenfluramine, two potent 5-HT releasers, inhibit SOs within the entorhinal cortex (EC) in anesthetized mice. Combining opto- and pharmacogenetic manipulations with in vitro electrophysiological recordings, we uncovered that somatostatin-expressing (Sst) interneurons activated by the 5-HT2A receptor (5-HT2AR) play an important role in the suppression of SOs. Since 5-HT2AR signaling is involved in the etiology of different psychiatric disorders and mediates the psychological effects of many psychoactive serotonergic drugs, we propose that the newly discovered link between Sst interneurons and 5-HT will contribute to our understanding of these complex topics.

sentences: In-solution buffer-free digestion for the analysis of SARS-CoV-2 RBD proteins allows a full sequence coverage and detection of post-translational modifications in a single ESI-MS spectrum Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, are among the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage ([≥] 99 %) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, glutathionylation, cyanilation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90 %) and did not detect peptides carrying some of the above-mentioned post-translational modifications. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD and the low-abundance scrambling variants, free cysteine residues, O-glycoforms and incomplete processing of the N-terminal end, if present. Artifacts that might be generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented and we foresee that it can be also helpful to the characterization of mutated RBD.

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Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1)-mediated plasticity in the paraventricular thalamic nucleus promotes a fundamental adaptation to stress BACKGROUNDHabituation is defined as a progressive decline in response to repeated exposure to a familiar and predictable stimulus and is highly conserved across species. Disrupted habituation is a signature of post-traumatic stress disorder (PTSD). In rodents, habituation is observed in neural, neuroendocrine and behavioral responses to repeated exposure to the predictable and moderately intense stress or restraint. We previously demonstrated that lesions to the posterior division of the paraventricular thalamic nucleus (pPVT) impairs habituation. However, the underlying molecular mechanisms and specific neural connections among the pPVT and other brain regions that underlie habituation are unknown.

METHODSBehavioral and neuroendocrine habituation was assessed in adult male Sprague-Dawley restraints using the repeated restraint paradigm. Pan neuronal and Cre-dependent Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) were used to chemogenetically inhibit the pPVT and the subpopulation of pPVT neurons that project to the medial prefrontal cortex (mPFC), respectively. Activity-regulated cytoskeleton-associated protein (Arc) expression was knocked down in the pPVT using siRNA directed towards Arc. Golgi staining was used to assess structural plasticity of pPVT neurons. Local field potential recordings were used to assess coherent neural activity between the pPVT and mPFC. The attentional set-shifting task was used to assess mPFC-dependent behavior.

RESULTSHere, we show that Arc promotes habituation by increasing stress-induced spinogenesis in the pPVT, increasing coherent neural activity with the mPFC, and improving mPFC-mediated cognitive flexibility.

CONCLUSIONOur results demonstrate that Arc induction in the pPVT regulates habituation to repeated restraint and mPFC function.

One Sentence SummaryWe demonstrate that Arc in the posterior division of the paraventricular thalamic nucleus promotes habituation to repeated stress by increasing dendritic spines.

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A Guide to Pre-Processing High-Throughput Animal Tracking Data O_LIModern, high-throughput animal tracking studies collect increasingly large volumes of data at very fine temporal scales. At these scales, location error can exceed the animals step size, leading to mis-estimation of key movement metrics such as speed.  Cleaning the data to reduce location errors prior to analyses is one of the main ways movement ecologists deal with noisy data, and has the advantage of being more scalable to massive datasets than more complex methods. Though data cleaning is widely recommended, and ecologists routinely consider cleaned data to be the ground-truth, inclusive uniform guidance on this crucial step, and on how to organise the cleaning of massive datasets, is still rather scarce.
C_LIO_LIA pipeline for cleaning massive high-throughput datasets must balance ease of use and computationally efficient signal vs. noise screening, in which location errors are rejected without discarding valid animal movements. Another useful feature of a pre-processing pipeline is efficiently segmenting and clustering location data for statistical methods, while also being scalable to large datasets and robust to imperfect sampling. Manual methods being prohibitively time consuming, and to boost reproducibility, a robust pre-processing pipeline must be automated.
C_LIO_LIIn this article we provide guidance on building pipelines for pre-processing high-throughput animal tracking data in order to prepare it for subsequent analysis. Our recommended pipeline, consisting of removing outliers, smoothing the filtered result, and thinning it to a uniform sampling interval, is applicable to many massive tracking datasets. We apply this pipeline to simulated movement data with location errors, and also show a case study of how large volumes of cleaned data can be transformed into biologically meaningful  residence patches, for quick biological inference on animal space use. We use calibration data to illustrate how pre-processing improves its quality, and to verify that the residence patch synthesis accurately captures animal space use. Finally, turning to tracking data from Egyptian fruit bats (Rousettus aegyptiacus), we demonstrate the pre-processing pipeline and residence patch method in a fully worked out example.
C_LIO_LITo help with fast implementation of standardised methods, we developed the R package atlastools, which we also introduce here. Our pre-processing pipeline and atlastools can be used with any high-throughput animal movement data in which the high data-volume combined with knowledge of the tracked individuals movement capacity can be used to reduce location errors. The atlastools function is easy to use for beginners, while providing a template for further development. The use of common pre-processing steps that are simple yet robust promotes standardised methods in the field of movement ecology and leads to better inferences from data.
C_LI

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Proteasome dysfunction disrupts adipogenesis and induces inflammation via ATF3 ObjectiveRegulation of proteasomal activity is an essential component of cellular proteostasis and function. This is evident in patients with mutations in proteasome subunits and regulators, who suffer from proteasome-associated autoinflammatory syndromes (PRAAS). These patients display lipodystrophy and fevers, which may be partly related to adipocyte malfunction and abnormal thermogenesis in adipose tissue. However, the cell-intrinsic pathways that could underlie these symptoms are unclear. Here, we investigate the impact of two proteasome subunits implicated in PRAAS, Psmb4 and Psmb8, on differentiation, function and proteostasis of brown adipocytes.

MethodsIn immortalized mouse brown pre-adipocytes, levels of Psmb4, Psmb8, and downstream effectors genes were downregulated through reverse transfection with siRNA. Adipocytes were differentiated and analyzed with various assays of adipogenesis, lipogenesis, lipolysis, inflammation, and respiration.

ResultsLoss of Psmb4, but not Psmb8, disrupted proteostasis and adipogenesis. Proteasome function was reduced upon Psmb4 loss, but partly recovered by the activation of Nuclear factor, erythroid-2, like-1 (Nfe2l1). In addition, cells displayed higher levels of surrogate inflammation and stress markers, including Activating transcription factor-3 (Atf3). Simultaneous silencing of Psmb4 and Atf3 lowered inflammation and restored adipogenesis.

ConclusionsOur study shows that Psmb4 is required for adipocyte development and function in cultured adipocytes. These results imply that in humans with PSMB4 mutations, PRAAS-associated lipodystrophy is partly caused by disturbed adipogenesis. While we uncover a role for Nfe2l1 in the maintenance of proteostasis under these conditions, Atf3 is a key effector of inflammation and blocking adipogenesis. In conclusion, our work highlights how proteasome dysfunction is sensed and mitigated by the integrated stress response in adipocytes with potential relevance for PRAAS patients and beyond.

Highlights- PRAAS-associated PSMB4 is required for brown adipocyte differentiation
- Loss of PSMB4 activates NFE2L1 to counteract proteasome dysfunction
- The ATF3 pathway regulates adipocyte dysfunction and inflammation
- Loss of ATF3 restores adipogenesis in cells with loss of PSMB4

Graphical abstract

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Therapeutic potential of macrophage colony-stimulating factor (CSF1) in chronic liver disease Resident and recruited macrophages control the development and proliferation of the liver. We showed previously in multiple species that treatment with a macrophage colony stimulating factor (CSF1)-Fc fusion protein initiated hepatocyte proliferation and promoted repair in models of acute hepatic injury in mice. Here we investigated the impact of CSF1-Fc on resolution of advanced fibrosis and liver regeneration, utilizing a non-resolving toxin-induced model of chronic liver injury and fibrosis in C57BL/6J mice. Co-administration of CSF1-Fc with exposure to thioacetamide (TAA) exacerbated inflammation consistent with monocyte contributions to initiation of pathology. After removal of TAA, either acute or chronic CSF1-Fc treatment promoted liver growth, prevented progression and promoted resolution of fibrosis. Acute CSF1-Fc treatment was also anti-fibrotic and pro-regenerative in a model of partial hepatectomy in mice with established fibrosis. The beneficial impacts of CSF1-Fc treatment were associated with monocyte-macrophage recruitment and increased expression of remodeling enzymes and growth factors. These studies indicate that CSF1-dependent macrophages contribute to both initiation and resolution of fibrotic injury and that CSF1-Fc has therapeutic potential in human liver disease.

Summary statementMacrophages contribute to both progression and resolution of chronic tissue injury and fibrogenesis. Administration of a macrophage growth factor promoted liver regeneration and resolution of advanced liver fibrosis in mice.

sentences: Phenotypic analysis of catastrophic childhood epilepsy genes: The Epilepsy Zebrafish Project Genetic engineering techniques have contributed to the now widespread use of zebrafish to investigate gene function, but zebrafish-based human disease studies, and particularly for neurological disorders, are limited. Here we used CRISPR-Cas9 to generate 40 single-gene mutant zebrafish lines representing catastrophic childhood epilepsies. We evaluated larval phenotypes using electrophysiological, behavioral, neuro-anatomical, survival and pharmacological assays. Phenotypes with unprovoked electrographic seizure activity (i.e., epilepsy) were identified in zebrafish lines for 8 genes; ARX, EEF1A, GABRB3, GRIN1, PNPO, SCN1A, STRADA and STXBP1. A unifying epilepsy classification scheme was developed based on local field potential recordings and blinded scoring from ~3300 larvae. We also created an open-source database containing sequencing information, survival curves, behavioral profiles and representative electrophysiology data. We offer all zebrafish lines as a resource to the neuroscience community and envision them as a starting point for further functional analysis and/or identification of new therapies.

sentences: Tubulinopathy mutations in TUBA1A that disrupt neuronal morphogenesis and migration override XMAP215/Stu2 regulation of microtubule dynamics Heterozygous, missense mutations in - or {beta}-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutations impact at the molecular and cellular levels scale with the severity of brain malformation. Here we focus on two mutations at the valine 409 residue of TUBA1A, V409I and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A-V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and delayed retraction in primary neuronal cultures, accompanied by increased microtubule acetylation. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends and ablate tubulin binding to TOG domains. In each assay tested, the TUBA1A-V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A-V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations may limit tubulin conformational states and thereby disrupt microtubule regulation during neuronal morphogenesis and migration.

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Epicardial HDAC3 promotes myocardial growth through a novel microRNA pathway RationalEstablishment of the myocardial wall requires proper growth cues from nonmyocardial tissues. During heart development, the epicardium and epicardium-derived cells (EPDCs) instruct myocardial growth by secreting essential factors including fibroblast growth factor 9 (FGF9) and insulin-like growth factor 2 (IGF2). However, it is poorly understood how the epicardial secreted factors are regulated, in particular by chromatin modifications for myocardial formation.

ObjectiveTo understand whether and how histone deacetylase 3 (HDAC3) in the developing epicardium regulates myocardial growth.

Methods and ResultsWe deleted Hdac3 in the developing murine epicardium and mutant hearts showed ventricular myocardial wall hypoplasia with reduction of EPDCs. The cultured embryonic cardiomyocytes with supernatants from Hdac3 knockout (KO) mouse epicardial cells (MECs) also showed decreased proliferation. Genome-wide transcriptomic analysis revealed that Fgf9 and Igf2 were significantly down-regulated in Hdac3 KO MECs. We further found that Fgf9 and Igf2 expression is dependent on HDAC3 deacetylase activity. The supplementation of FGF9 or IGF2 can rescue the myocardial proliferation defects treated by Hdac3 KO supernatant. Mechanistically, we identified that microRNA (miR)-322 and miR-503 were upregulated in Hdac3 KO MECs and Hdac3 epicardial KO hearts. Overexpression of miR-322 or miR-503 repressed FGF9 and IGF2 expression, while knockdown of miR-322 or miR-503 restored FGF9 and IGF2 expression in Hdac3 KO MECs.

ConclusionsOur findings reveal a critical signaling pathway in which epicardial HDAC3 promotes compact myocardial growth by stimulating FGF9 and IGF2 through repressing miR-322/miR-503, providing novel insights in elucidating etiology of congenital heart defects, and conceptual strategies to promote myocardial regeneration.

sentences: Transcriptional condensates formed by phase-separated ALOG family proteins control shoot meristem maturation for flowering Plants have evolved remarkable diversity in inflorescence architecture. At the center of this diversity lies a meristem maturation program featured by transition of stem cell populations from a vegetative state into a reproductive growth, determining when, where, and how many flowers are produced on inflorescences. Here we identified a new meristem maturation regulator TMF FAMILY MEMBER3 (TFAM3) that encodes an ALOG family transcription factor. Loss of TFAM3 results in early flowering and simplified inflorescences with fewer flowers. Genetic analysis by creating high-order mutants of TFAM3 with three key regulators of tomato shoot meristem maturation, TERMINATING FLOWER (TMF), TMF FAMILY MEMBER1 (TFAM1) and TMF FAMILY MEMBER2 (TFAM2), suggested that they synergistically control flowering transition and inflorescence architecture. The four paralogous ALOG proteins share the prion-like properties and undergo liquid-liquid phase separation in vitro. Strikingly, TMF can recognize cognate TFAM proteins and selectively recruit them into phase separated condensates. Supporting this, they interact with themselves and each other to form biomolecular condensates in the nucleus. Their interaction induces formation of transcriptional condensates that directly repress expression of floral identity gene ANANTHA. Our study revealed a selective-recruitment phase separation mechanism for transcriptional condensation by which plants achieve optimal coordination of functional overlapped paralogs within a protein family to enable precise control of shoot meristem maturation for flowering and production of compound inflorescences.

sentences: Homeostasis Equation: An Approach to Theoretical Medicine Homeostasis is kind of force that makes living organism to live. In this study, we suggest an integral equation that models homeostasis in living organism. We also showed that various situations can be modeled by homeostasis, and give mathematical interpretation of mechanism of living organism. With our proposed integral equation, one can handle homeostasis quantitatively, and this approach is expected to unveil various hidden properties of living organism.

sentences: Genetic, bioacoustic and morphological analyses reveal cryptic speciation in the warbling vireo complex (Vireo gilvus: Vireonidae: Passeriformes) Cryptic species are closely related taxa that are difficult to separate morphologically, but are reproductively isolated. Here we examine the warbling vireo complex (Vireo gilvus), a widespread songbird speculated to be comprised of more than one cryptic species. We included three taxa within the complex: two of the western (Vireo gilvus swainsonii and V. g. brewsteri) subspecies and the single eastern (V. g. gilvus) subspecies. We used mtDNA and microsatellite loci to assess the congruence of genetic data to the current subspecies boundaries. We then incorporated bioacoustic, morphometric, and ecological niche modeling analyses to further examine differences. We found two genetic groups with mtDNA analysis. Microsatellite analyses revealed four genetic groups: an eastern group, a Black Hills group and two western groups that do not agree with current western subspecies boundaries based on phenotypic data. Our results suggest that eastern and western warbling vireos have been reproductively isolated for a long period of time and therefore, may be best treated as separate species; however, more research into areas of contact to examine the presence of hybridization is advised before making a taxonomic revision. Differences between the two western genetic groups appear less clear, requiring additional research.

sentences: Monitoring promoter activity by RNA editing based reporter Traditional methods monitoring the promoter activity require the insertion of reporter protein (e.g. fluorescent protein) downstream of a targeted promoter. These approaches suffer from low sensitivity and potential interference to endogenous transcripts especially when the targeted transcript is a noncoding RNA. Here, we develop a mechanistically different reporter system to monitor promoter activity based on ribozyme processed ADAR engaging RNA directed editing (REDDIT). We show that REDDIT can be used to monitor the promoter activity of protein coding, long noncoding RNA, and microRNA (miRNA) genes. Furthermore, REDDIT reporter can also be adapted to use bioluminescence imaging to monitor the promoter activity, which is more suitable for in vivo live imaging. Finally, the reporter sensitivity may be further increased by the circularization of ADAR recruiting RNA. REDDIT provides a powerful platform that is promising in a variety of applications such as monitoring the promoter activity, cell lineage tracing, and processing and manipulating genetic information for synthetic biology applications.

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Plasmodium falciparum genetic diversity in coincident human and mosquito hosts Population genetic diversity of P. falciparum antigenic loci is high despite large bottlenecks in population size during the parasite life cycle. The extent of this diversity in human blood-stage infections, following expansion from a small number of liver-stage schizonts, has been well described. However, little is known about parasite genetic diversity in the vector, where a similar bottleneck and expansion occurs following parasite mating and where parasite genotypes from several different human infections may accumulate. We assessed parasite genetic diversity within human and mosquito P. falciparum infections collected from the same households during a 14-month longitudinal cohort study using amplicon deep sequencing of two antigenic gene fragments (ama1 and csp). To a prior set of infected humans (n=1175/2813; 86.2% sequencing success) and mosquito abdomens (n=199/1448; 95.5% sequencing success), we added sequences from infected mosquito heads (n=134/1448; 98.5% sequencing success). Across all sample types we observed 456 ama1 and 289 csp unique haplotypes. While both hosts contained many rare haplotypes, population genetic metrics indicated that the overall and sample-level parasite populations were more diverse in mosquitoes than in humans, and infections were more likely to harbor a dominant haplotype in humans than in mosquitoes (based on relative read abundance). Finally, within a given mosquito there was little overlap in genetic composition of abdomen and head infections, suggesting that infections may be cleared from the abdomen during a mosquitos lifespan. Taken together, our observations provide evidence for the role of the mosquito vector in maintaining sequence diversity of malaria parasite populations.

Significance statementConcurrent infections with multiple strains of Plasmodium falciparum, the leading causative agent of death due to malaria, are common in highly endemic regions. During transitions within and between the parasites mosquito and human hosts, population bottlenecks occur, and distinct parasite strains may have differential fitness in the various environments encountered. These bottlenecks and fitness differences may lead to differences in strain prevalence and diversity between hosts. We investigated differences in genetic diversity between P. falciparum parasites in human and mosquito hosts and found that, compared to human parasite populations and infections, mosquito populations and infections were more diverse. This suggests that the mosquito vector may play a role in in maintaining sequence diversity in malaria parasite populations.

sentences: Single-cell transcriptomic analysis unveils spinal motor neuron subtype diversity underpinning the water-to-land transition in vertebrates Spinal motor neurons (MNs) integrate sensory stimuli and brain commands to generate motor movements in vertebrates. Distinct MN populations and their diversity has long been hypothesized to co-evolve with motor circuit to provide the neural basis from undulatory to ambulatory locomotion during aquatic-to-terrestrial transition of vertebrates. However, how these subtypes are evolved remains largely enigmatic. Using single-cell transcriptomics, we investigate heterogeneity in mouse MNs and discover novel segment-specific subtypes. Among limb-innervating MNs, we reveal a diverse neuropeptide code for delineating putative motor pool identities. We further uncovered that axial MNs are subdivided by three conserved and molecularly distinct subpopulations, defined by Satb2, Nr2f2 or Bcl11b expression. Although axial MNs are conserved from cephalochordates to humans, subtype diversity becomes prominent in land animals and appears to continue evolving in humans. Overall, our study provides a unified classification system for spinal MNs and paves the way towards deciphering how neuronal subtypes are evolved.

sentences: mRNA- and Factor-Driven Dynamic Variability Controls eIF4F-Cap Recognition for Translation Initiation. mRNA 5' cap recognition by eIF4F is a key step in eukaryotic translational control. While different mRNAs respond differently to eIF4F-directed regulation, the molecular basis for this variability remains unclear. We developed single-molecule fluorescence assays to directly observe eIF4F- mRNA interactions. We uncovered a complex interplay of mRNA features with factor activities that differentiates cap recognition between mRNAs. eIF4E-cap association rates are anticorrelated with mRNA length. eIF4A leverages ATP binding to differentially accelerate eIF4E- mRNA association; the extent of this acceleration correlates with translation efficiency in vivo. eIF4G lengthens eIF4E-cap binding to persist on the initiation timescale. The full eIF4F complex discriminates between mRNAs in an ATP-dependent manner. After eIF4F-mRNA binding, eIF4E is ejected from the cap by eIF4A ATP hydrolysis. Our results suggest features throughout mRNA coordinate in controlling cap recognition at the 5' end, and suggest a model for how eIF4F-mRNA dynamics establish mRNA sensitivity to translational control processes.

sentences: Endothelial-derived sphingolipids are required for vascular development and systemic lipid homeostasis Serine palmitoyl transferase (SPT), the rate-limiting enzyme in the de novo synthesis of sphingolipids (SL), is needed for embryonic development, physiological homeostasis, and response to stress. The functions of de novo SL synthesis in vascular endothelial cells (EC), which line the entire circulatory system, are not well understood. Here we show that the EC de novo synthesis not only impacts vascular development but also maintains normal SL metabolic homeostasis in the circulatory system and peripheral organs. Mice with an endothelial-specific gene knockout of SPTLC1 (Sptlc1 ECKO), an essential subunit of the SPT complex, exhibited EC-intrinsic effects including reduced EC proliferation and tip/stalk cell differentiation, resulting in delayed retinal vascular development. In addition, Sptlc1 ECKO mice had reduced pathological retinal neovascularization in the oxygen-induced retinopathy model, suggesting that EC SL produced from the de novo pathway are needed for efficient VEGF signaling within the vascular system. Post-natal deletion of the EC Sptlc1also showed cell-extrinsic effects, including rapid reduction of several SL metabolites in plasma, red blood cells and peripheral organs (lung and liver) but not in the retina, part of the central nervous system (CNS). In the liver, EC de novo SL synthesis was required for acetaminophen-induced ceramide elevation and hepatotoxicity. These results suggest that EC-derived SL metabolites are in constant flux between the vasculature, circulatory elements, and parenchymal cells of non-CNS organs. Taken together, our data point to the central role of the endothelial SL biosynthesis in maintaining vascular development and neovascular proliferation, non-CNS tissue metabolic homeostasis and hepatocyte response to stress.

sentences: Velodrome: Out-of-Distribution Generalization from Labeled and Unlabeled Gene Expression Data for Drug Response Prediction Data discrepancy between preclinical and clinical datasets poses a major challenge for accurate drug response prediction based on gene expression data. Different methods of transfer learning have been proposed to address this data discrepancy. These methods generally use cell lines as source domains and patients, patient-derived xenografts, or other cell lines as target domains. However, they assume that they have access to the target domain during training or fine-tuning and they can only take labeled source domains as input. The former is a strong assumption that is not satisfied during deployment of these models in the clinic. The latter means these methods rely on labeled source domains which are of limited size. To avoid these assumptions, we formulate drug response prediction as an out-of-distribution generalization problem which does not assume that the target domain is accessible during training. Moreover, to exploit unlabeled source domain data, which tends to be much more plentiful than labeled data, we adopt a semi-supervised approach. We propose Velodrome, a semi-supervised method of out-of-distribution generalization that takes labeled and unlabeled data from different resources as input and makes generalizable predictions. Velodrome achieves this goal by introducing an objective function that combines a supervised loss for accurate prediction, an alignment loss for generalization, and a consistency loss to incorporate unlabeled samples. Our experimental results demonstrate that Velodrome outperforms state-of-the-art pharmacogenomics and transfer learning baselines on cell lines, patient-derived xenografts, and patients. Finally, we showed that Velodrome models generalize to different tissue types that were well-represented, under-represented, or completely absent in the training data. Overall, our results suggest that Velodrome may guide precision oncology more accurately.

sentences: Reconstructing co-dependent cellular crosstalk in lung adenocarcinoma using REMI Cellular crosstalk in tissue microenvironments is fundamental to normal and pathological biological processes. Global assessment of cell-cell interactions (CCI) is not yet technically feasible, but computational efforts to reconstruct these interactions have been proposed. Current computational approaches that identify CCI often make the simplifying assumption that pairwise interactions are independent of one another, which can lead to reduced accuracy. We present REMI (REgularized Microenvironment Interactome), a graph-based algorithm that predicts ligand-receptor (LR) interactions by accounting for LR dependencies on high-dimensional, small sample size datasets. We apply REMI to reconstruct the human lung adenocarcinoma (LUAD) interactome from a bulk flow-sorted RNA-seq dataset, then leverage single-cell transcriptomics data to increase its resolution and identify LR prognostic signatures. We experimentally confirmed colocalization of CTGF:LRP6 as an interaction predicted to be associated with LUAD progression. Our work presents a novel way to reconstruct interactomes and a new approach to identify clinically-relevant cell-cell interactions.

sentences: Binding affinity landscapes constrain the evolution of broadly neutralizing anti-influenza antibodies Over the past two decades, several broadly neutralizing antibodies (bnAbs) that confer protection against diverse influenza strains have been isolated1,2. Structural and biochemical characterization of these bnAbs has provided molecular insight into how they bind distinct antigens1. However, our understanding of the evolutionary pathways leading to bnAbs, and thus how best to elicit them, remains limited. Here, we measure equilibrium dissociation constants of combinatorially complete mutational libraries for two naturally isolated influenza bnAbs3-5 (CR-9114, 16 mutations; CR-6261, 11 mutations), reconstructing all possible intermediates back to the unmutated germline sequences. We find that these two libraries exhibit strikingly different patterns of breadth: while many variants of CR-6261 display moderate affinity to diverse antigens, those of CR-9114 display appreciable affinity only in specific, nested combinations. By examining the extensive pairwise and higher-order epistasis between mutations, we find key sites with strong synergistic interactions that are highly similar across antigens for CR-6261 and different for CR-9114. Together, these features of the binding affinity landscapes strongly favor sequential acquisition of affinity to diverse antigens for CR-9114, while the acquisition of breadth to more similar antigens for CR-6261 is less constrained. These results, if generalizable to other bnAbs, may explain the molecular basis for the widespread observation that sequential exposure favors greater breadth6-8, and such mechanistic insight will be essential for predicting and eliciting broadly protective immune responses.

sentences: gen3sis: the general engine for eco-evolutionary simulations on the origins of biodiversity Understanding the origins of biodiversity has been an aspiration since the days of early naturalists. The immense complexity of ecological, evolutionary and spatial processes, however, has made this goal elusive to this day. Computer models serve progress in many scientific fields, but in the fields of macroecology and macroevolution, eco-evolutionary models are comparatively less developed. We present a general, spatially-explicit, eco-evolutionary engine with a modular implementation that enables the modelling of multiple macroecological and macroevolutionary processes and feedbacks across representative spatio-temporally dynamic landscapes. Modelled processes can include environmental filtering, biotic interactions, dispersal, speciation and evolution of ecological traits. Commonly observed biodiversity patterns, such as , {beta} and {gamma} diversity, species ranges, ecological traits and phylogenies, emerge as simulations proceed. As a case study, we examined alternative hypotheses expected to have shaped the latitudinal diversity gradient (LDG) during the Earths Cenozoic era. We found that a carrying capacity linked with energy was the only model variant that could simultaneously produce a realistic LDG, species range size frequencies, and phylogenetic tree balance. The model engine is open source and available as an R-package, enabling future exploration of various landscapes and biological processes, while outputs can be linked with a variety of empirical biodiversity patterns. This work represents a step towards a numeric and mechanistic understanding of the physical and biological processes that shape Earths biodiversity.

sentences: STORM-Net: Simple and Timely Optode Registration Method for Functional Near-Infrared Spectroscopy (fNIRS) We introduce a robust video-based method for estimating the positions of fNIRS optodes on the scalp. The method is fast, requires no special hardware, and is intuitive to use with developmental populations. Co-registration is a crucial step for reliable analysis of FNIRS data, yet it still remains an open problem when considering these populations. Existing methods pose motion constraints, require expert annotation, or are only applicable in laboratory conditions. Using novel computer-vision technologies, we implement a fully-automatic appearance-based method that estimates the registration parameters of a mounted cap to the scalp from a raw video of the subject. We validate our method on 10 adult subjects and demonstrate its usability with infants. We compare our method to the standard 3D digitizer, and to other photogrammetry based approaches. We show our method achieves comparable accuracy to current appearance-based methods, while being orders of magnitude faster. Our fast registration facilitates more spatially precise fNIRS analysis with developmental populations even in unconventional environments. The method is implemented as a open-source toolbox at https://github.com/yoterel/STORM-Net.

sentences: A compact spatial map in V2 visual cortex Vision plays a critical role in guiding spatial navigation. A traditional view of the visual cortex is to compute a world-centered map of visual space, and visual neurons exhibit diverse tunings to simple or complex visual features. The neural representation of spatio-visual map in the visual cortex is thought to be transformed from spatial modulation signals at the hippocampal-entorhinal system. Although visual thalamic and cortical neurons have been shown to be modulated by spatial signals during navigation, the exact source of spatially modulated neurons within the visual circuit has never been identified, and the neural correlate underpinning a visuospatial or spatio-visual map remains elusive. To search for direct visuospatial and visuodirectional signals, here we record in vivo extracellular spiking activity in the secondary visual cortex (V2) from freely foraging rats in a naturalistic environment. We identify that V2 neurons forms a complete spatio-visual map with a wide range of spatial tunings, which resembles the classical spatial map that includes the place, head-direction, border, grid and conjunctive cells reported in the hippocampal-entorhinal network. These spatially tuned V2 neurons display stable responses to external visual cues, and are robust with respect to non- spatial environmental changes. Spatially and directionally tuned V2 neuronal firing persists in darkness, suggesting that this spatio-visual map is not completely dependent on visual inputs. Identification of functionally distinct spatial cell types in visual cortex expands its classical role of information coding beyond a retinotopic map of the eye-centered world.

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Every individual makes a difference: A trinity derived from linking individual brain morphometry, connectivity and mentalising ability Mentalising ability, indexed as the ability to understand others beliefs, feelings, intentions, thoughts, and traits, is a pivotal and fundamental component of human social cognition. How-ever, considering the multifaceted nature of mentalising ability, little research has focused on characterising individual differences in different mentalising components. And even less research has been devoted to investigating how the variance in the structural and functional patterns of the amygdala and hippocampus, two vital subcortical regions of the  social brain, are related to inter-individual variability in mentalising ability. Here, as a first step toward filling these gaps, we exploited inter-subject representational similarity analysis (IS-RSA) to assess relationships between amygdala and hippocampal morphometry (surface-based multivariate morphometry statistics, MMS), connectivity (resting-state functional connectivity, rs-FC) and mentalising ability (interactive mentalisation questionnaire (IMQ) scores) across the participants (N = 24). In IS-RSA, we proposed a novel pipeline, i.e., computing patching and pooling operations-based surface distance (CPP-SD), to obtain a decent representation for high-dimensional MMS data. On this basis, we found significant correlations (i.e., secondorder isomorphisms) between these three distinct modalities, indicating that a trinity existed in idiosyncratic patterns of brain morphometry, connectivity and mentalising ability. Notably, a region-related mentalising specificity emerged from these associations: self-self and self-other mentalisation are more related to the hippocampus, while other-self mentalisation shows a closer link with the amygdala. Furthermore, by utilising the dyadic regression analysis, we observed significant interactions such that subject pairs with similar morphometry had even greater mentalising similarity if they were also similar in rs-FC. Altogether, we demonstrated the feasibility and illustrated the promise in using IS-RSA to study individual differences, deepening our understanding of how individual brains give rise to their mentalising abilities.

O_QD But this general character is contained in every individual character; without individual character there can be no general character. If all individual character were removed, what general character would remain?

C_QD

(MO_SCPLOWAOC_SCPLOW TO_SCPLOWSEC_SCPLOWO_SCPCAP-C_SCPCAPO_SCPLOWTUNGC_SCPLOW - OO_SCPLOWNC_SCPLOW CO_SCPLOWONTRADICTIONC_SCPLOW1)

sentences: Implementation and acceleration of optimal control for systems biology Optimal control theory provides insight into complex resource allocation decisions. The forward-backward sweep method (FBSM) is an iterative technique commonly implemented to solve two-point boundary value problems (TPBVPs) arising from the application of Pontryagins Maximum Principle (PMP) in optimal control. In this review we discuss the PMP approach to optimal control and the implementation of the FBSM. By conceptualising the FBSM as a fixed point iteration process, we leverage and adapt existing acceleration techniques to improve its rate of convergence. We show that convergence improvement is attainable without prohibitively costly tuning of the acceleration techniques. Further, we demonstrate that these methods can induce convergence where the underlying FBSM fails to converge. All code used in this work to implement the FBSM and acceleration techniques is available on GitHub at https://github.com/Jesse-Sharp/Sharp2021.

sentences: Single nucleotide polymorphism induces divergent dynamic patterns in CYP3A5: a microsecond scale biomolecular simulation of variants identified in Sub-Saharan African populations Pharmacogenomics aims to reveal variants associated with drug response phenotypes. Genes whose roles involve the absorption, distribution, metabolism, and excretion of drugs, are highly polymorphic between populations. High coverage whole genome sequencing showed that a large proportion of the variants for these genes are rare in African populations. This study investigates the impact of such variants on protein structure to assess their functional importance. We use genetic data of CYP3A5 from 458 individuals from sub-Saharan Africa to conduct a structural bioinformatics analysis. Five missense variants were modeled and microsecond scale molecular dynamics simulations were conducted for each, as well as for the CYP3A5 wildtype, and the Y53C variant, which has a known deleterious impact on enzyme activity. The binding of ritonavir and artemether to CYP3A5 variant structures was also evaluated. Our results showed different conformational characteristics between all the variants. No significant structural changes were noticed. However, the genetic variability acts on the plasticity of the protein. The impact on drug binding may be drug dependant. We conclude that rare variants hold relevance in determining the pharmacogenomics properties of populations. This could have a significant impact on precision medicine applications in sub-Saharan Africa.

sentences: Transcriptomic Analysis of Human Skin Wound Healing and Rejuvenation Following Ablative Fractional Laser Treatment Ablative fractional laser treatment is considered the gold standard for skin rejuvenation. In order to understand how fractional laser works to rejuvenate skin, we performed microarray profiling on skin biopsies to identify temporal and dose-response changes in gene expression following fractional laser treatment. The backs of 14 women were treated with ablative fractional laser (Fraxel(R)) and 4 mm punch biopsies were collected from an untreated site and at the treated sites 1, 3, 7, 14, 21 and 28 days after the single treatment. In addition, in order to understand the effect that multiple fractional laser treatments have on skin rejuvenation, several sites were treated sequentially with either 1, 2, 3, or 4 treatments (with 28 days between treatments) followed by the collection of 4 mm punch biopsies. RNA was extracted from the biopsies, analyzed using Affymetrix U219 chips and gene expression was compared between untreated and treated sites. We observed dramatic changes in gene expression as early as 1 day after fractional laser treatment with changes remaining elevated even after 1 month. Analysis of individual genes demonstrated significant and time related changes in inflammatory, epidermal, and dermal genes, with dermal genes linked to extracellular matrix formation changing at later time points following fractional laser treatment. When comparing the age-related changes in skin gene expression to those induced by fractional laser, it was observed that fractional laser treatment reverses many of the changes in the aging gene expression. Finally, multiple fractional laser treatments resulted in continued changes in gene expression, with many genes either differentially regulated or continuously upregulated with increasing number of treatments, indicating that maximal skin rejuvenation requires multiple fractional laser treatments. In conclusion, fractional laser treatment of skin activates several biological processes involved in wound healing and tissue regeneration, all of which significantly contribute to the rejuvenating effect of fractional laser treatment on aged skin.

sentences: The high-resolution structure of cell membranes revealed by in situ cryo-electron tomography As the structural unit of life, cell is defined by the membrane system. The cell membrane separates the internal and external environment of the cell, and the endomembrane system defines the organelles to perform different functions1-3. However, lack of tools to in situ observe membrane proteins at a molecular resolution has limited our understanding of membrane organization and membrane protein interactions. Here we characterize the high-resolution 3D structure of human red blood cell (hRBC) membranes and the membrane proteins for the first time in situ by cryo-electron tomography (CryoET)4-7. By analyzing tomograms, we have obtained the first fine three-dimensional (3D) structure of hRBC membranes and found the asymmetrical distribution of membrane proteins on both sides of the membranes. We found that the membrane proteins are mainly located on the cytoplasmic side of hRBC membranes, with protein sizes ranging from 6nm to 8nm, in contrast to the ectoplasmic side with basically no proteins. Quantitative analysis of the density of hRBC membrane proteins shows that the membranes with higher protein occupancy have less phospholipid, making the membranes more rigid. Meanwhile, we obtained the channel protein-like structures by preliminary analysis of the membrane protein. Our results represent the first in situ structure characterization of the cell membranes and membrane proteins through cryoET and opens the door for understanding the biological functions of cell membranes in their physiological environments.

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Phenotypic architecture of sociality and its associated genetic polymorphisms in zebrafish Sociality is often seen as a single phenotypic trait, but it relies on motivational and cognitive components implemented by specific causal mechanisms. Hence, these components may have evolved independently, or may have been linked by phenotypic correlations driven by a shared selective pressure for increased social competence. Furthermore, these components may be domain-specific or of general domain across social and non-social contexts. Here we have characterized the phenotypic architecture of sociality in zebrafish, which has been increasingly used as a model organism in social neuroscience. For this purpose, we have behaviorally phenotyped zebrafish from different wild type lines in four tests: social tendency, social and non-social recognition, and open-field test. Our results indicate that: (1) sociality has two main components that are independent from each other (social tendency and social recognition), hence not supporting the occurrence of a sociality syndrome; (2) both social traits are phenotypically linked to non-social traits (non-social exploration and non-social memory, respectively), forming two general behavioral modules, general inspection and general recognition, and suggesting that sociality traits have been co-opted from general-domain motivational and cognitive traits. Moreover, the study of the association between genetic polymorphisms (i.e. single nucleotide polymorphisms, SNPs) and each behavioral module further supports this view, since several SNPs from a list of candidate "social" genes, are statistically associated with the general inspection (motivational), but not with a general recognition (cognitive), behavioral module. The SNPs associated with general inspection are widespread across different chromosomes and include neurotransmitters, neuromodulators, and synaptic plasticity genes, suggesting that this behavioral module is regulated by multiple genes, each of them with small effects. Together, these results support the occurrence of general domain motivational and cognitive behavioral modules in zebrafish, which have been co-opted for the social domain.

Author summarySocial living has been considered one of the major transitions in evolution and it has been considered to act as a major selective force shaping the evolution of brain and behavior in animals. Sociality relies on two basic behavioral mechanisms: (1) the willingness to approach and be near others (aka social tendency); and (2) the ability to distinguish between others (aka social recognition) in order to adjust the behavior expressed during social interactions according to the identity of the interactant. There is an ongoing debate on to what extent these social abilities have specifically evolved in response to social living and are domain specific, or if they were selected as a broad response to cognitive demands and are of general domain. Here, we used zebrafish to test the domain-specific vs. general domain hypotheses and to assess the association of social tendency and social recognition with a set of candidate "social" genes (i.e. genes that have been linked to social behavior in other studies with different vertebrate species). We found that both social traits are not correlated to each other and are of general domain, and that only social tendency is associated with candidate "social" genes, suggesting that social tendency and social recognition are independent behavioral modules that rely on separate genetic architectures and that can evolve separately.

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Oral Glucosamine Ameliorates Aggravated Neurological Phenotype in Mucopolysaccharidosis III Type C Mouse Model Expressing Misfolded HGSNAT Variant ObjectiveOver 55% of mucopolysaccharidosis IIIC (MPS IIIC) patients have at least one allelic missense variant responsible for misfolding of heparan sulfate acetyl-CoA:-glucosaminide N- acetyltransferase (HGSNAT). These variants are potentially treatable with pharmacological chaperones, such as a competitive HGSNAT inhibitor, glucosamine. Since the constitutive HGSNAT knockout mice, we generated previously cannot be used to test such strategy in vivo, we generated a novel model, the HgsnatP304L strain, expressing misfolded mutant HGSNAT with human missense mutation Pro311Leu (Pro304Leu in the mouse enzyme).

ResultsHgsnatP304L mice present deficits in short-term (novel object recognition test) and working/spatial (Y-maze test) memory at 4 months of age, 2-4 months earlier than previously described gene-targeted Hgsnat-Geo mice, which lack HGSNAT protein. HgsnatP304L mice also show increased severity of synaptic deficits in CA1 neurons, and accelerated course of CNS pathology including neuronal storage of heparan sulfate, accumulation of misfolded proteins, increase of simple gangliosides, and neuroinflammation as compared with Hgsnat-Geo mice. Expression of misfolded human Pro311Leu HGSNAT protein in cultured hippocampal Hgsnat- Geo neurons aggravated reduction of synaptic proteins. Memory deficits and majority of pathological changes in the brain were rescued in mice receiving daily doses of oral glucosamine.

InterpretationAltogether, our data for the first time demonstrate dominant-negative effects of the misfolded HGSNAT Pro304Leu variant and show that these effects are treatable by oral administration of glucosamine, suggesting that patients, affected with missense mutations preventing normal folding of the enzyme, could benefit from chaperone therapy.

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Synthetic Cooling Agents in US-marketed E-cigarette Refill Liquids and Popular Disposable E-cigarettes: Chemical Analysis and Risk Assessment. BackgroundMenthol, through its cooling and pleasant sensory effects, facilitates smoking and tobacco product initiation, resulting in the high popularity of mint/menthol-flavored E-cigarettes. More recently, E-cigarette vendors started marketing synthetic cooling agents as additives that impart a cooling effect but lack a characteristic minty odor. Knowledge about content of synthetic coolants in US-marketed E-cigarette products and associated health risks is limited.

MethodsE-liquid vendor sites were searched with the terms "koolada", "kool/cool", "ice", or WS-3/WS-23, denoting individual cooling agents, and relevant refill E-liquids were purchased. "Ice" flavor varieties of Puffbar, the most popular disposable E-cigarette brand, were compared with non-"Ice" varieties. E-liquids were characterized, and synthetic coolants quantified using GC/MS. Margin of exposure (MOE), a risk assessment parameter, was calculated to assess the risk associated with synthetic coolant exposure from E-cigarette use.

ResultsWS-3 was detected in 24/25 refill E-liquids analyzed. All Puffbar flavor varieties contained either WS-23 (13/14) or WS-3 (5/14), in both "Ice"- and non-"Ice" flavors. Modeling consumption of WS-3 from vaped E-liquids, resulted in MOEs below the safe margin of 100 for most daily use scenarios. MOEs for WS-23 were <100 for 10/13 Puffbar flavors in all use scenarios. Puffbar power specifications are identical to Juul devices.

ConclusionsSynthetic cooling agents (WS-3/WS-23) were present in US-marketed E-cigarettes, at levels that may result in consumer exposures exceeding safety thresholds set by regulatory agencies. Synthetic coolants are not only found in mint-or menthol-flavored products, but also in fruit- and candy-flavored products, including popular disposable E-cigarette products such as Puffbar.

ImplicationsSynthetic cooling agents are widely used in "kool/cool"- and "ice"-flavored E-liquids and in E-liquids without these labels, both as a potential replacement for menthol or to add cooling  notes to non-menthol flavors. These agents may be used to bypass current and future regulatory limits on menthol content in tobacco products, and not just E-cigarettes. Since synthetic cooling agents are odorless, they may not fall under the category of "characterizing flavor", potentially circumventing regulatory measures based on this concept. Regulators need to consider the additional health risks associated with exposure to synthetic cooling agents.

sentences: Perturb-map enables CRISPR genomics with spatial resolution and identifies regulators of tumor immune composition The cellular architecture of a tumor, particularly immune composition, has a major impact on cancer outcome, and thus there is an interest in identifying genes that control the tumor microenvironment (TME). While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying gene functions operating extracellularly or within a tissue context. To address this, we developed an approach for spatial functional genomics called Perturb-map, which utilizes protein barcodes (Pro-Code) to enable spatial detection of barcoded cells within tissue. We show >120 Pro-Codes can be imaged within a tumor, facilitating spatial mapping of 100s of cancer clones. We applied Perturb-map to knockout dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Additionally, we paired Perturb-map and spatial transcriptomics for unbiased molecular analysis of Pro-Code/CRISPR lesions. Our studies found in Tgfbr2 knockout lesions, the TME was converted to a mucinous state and T-cells excluded, which was concomitant with increased TGF{beta} expression and pathway activation, suggesting Tgfbr2 loss on lung cancer cells enhanced suppressive effects of TGF{beta} on the TME. These studies establish Perturb-map for functional genomics within a tissue at single cell-resolution with spatial architecture preserved.

sentences: Frequent PIK3CA mutations in eutopic endometrium of patients with ovarian clear cell carcinoma Recent studies have reported cancer-associated mutations in normal endometrium. Mutations in eutopic endometrium may lead to endometriosis and endometriosis-associated ovarian cancer. We investigated PIK3CA mutations (PIK3CAm) for three hotspots (E542K, E545K, H1047R) in eutopic endometrium in patients with ovarian cancer and endometriosis from formalin-fixed paraffin-embedded specimens by laser-capture microdissection and droplet digital PCR. The presence of PIK3CAm in eutopic endometrial glands with mutant allele frequency [≥]15% were as follows: ovarian clear cell carcinoma (OCCC) with PIK3CAm in tumors, 20/300 hotspots in 11/14 cases; OCCC without PIK3CAm, 42/78 hotspots in 11/12 cases; high-grade serous ovarian carcinoma, 8/45 hotspots in 3/5 cases; and endometriotic cysts, 5/63 hotspots in 5/6 cases. These rates were more frequent than in non-cancer non-endometriosis controls (7/309 hotspots in 5/17 cases). In OCCC without PIK3CAm, 7/12 (58%) cases showed multiple hotspot mutations in the same eutopic endometrial glands. In 3/54 (5.6%) cases, PIK3CAm was found in eutopic endometrial stroma. Multi-sampling of the OCCC tumors with PIK3CAm showed intratumor heterogeneity in three of eight cases. In two cases, PIK3CAm was detected in the stromal component of the tumor. Homogenous PIK3CAm in the epithelial component of the tumor matched the mutation in eutopic endometrial glands in only one case. Eutopic endometrial glands in ovarian cancer and endometriosis show high frequency of PIK3CAm that is not consistent with tumors, and multiple hotspot mutations are often found in the same glands. Our results suggest that most PIK3CAm in eutopic endometrial glands are passenger rather than driver mutations.

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Intracellular phase separation of globular proteins facilitated by short cationic peptides Phase separation provides intracellular organization and underlies a variety of cellular processes. These biomolecular condensates exhibit distinct physical and material properties. Current strategies for engineering condensate formation include using intrinsically disordered domains and altering protein surface charge by chemical supercharging or site-specific mutagenesis. We add to this toolbox by designing short, highly charged peptide tags that provide several key advantages for engineering protein phase separation. Herein, we report the use of short cationic peptide tags for sequestration of proteins of interest into bacterial condensates. Using a panel of GFP variants, we demonstrate how cationic tag and globular domain charge contribute to intracellular phase separation in E. coli and observe that the tag can affect condensate disassembly at a given net charge near the phase separation boundary. We showcase the broad applicability of these tags by appending them onto enzymes and demonstrating that the sequestered enzymes remain catalytically active.

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sentences: Discovery and visualization of uncharacterized drug-protein adducts using mass spectrometry. Drugs are often metabolized to reactive intermediates that form protein adducts. Adducts can inhibit protein activity, elicit immune responses, and cause life threatening adverse drug reactions. The masses of reactive metabolites are frequently unknown, rendering traditional mass spectrometry-based proteomics incapable of adduct identification. Here, we present Magnum, an open-mass search algorithm optimized for adduct identification, and Limelight, a web-based data processing package for analysis and visualization of data from all existing algorithms. Limelight incorporates tools for sample comparisons and xenobiotic-adduct discovery. We validate our tools with two drug/protein combinations and apply our workflow to identify novel xenobiotic-protein adducts in CYP3A4. Our new methods and software enable accurate identification of xenobiotic-protein adducts with no prior knowledge of adduct masses or protein targets. Magnum outperforms existing tools in xenobiotic-protein adduct discovery, while Limelight fulfills a major need in the rapidly developing field of open-mass searching, which until now lacked comprehensive data visualization tools.

sentences: Adenylate kinase 1 overexpression increases locomotor activity in medaka fish Maintenance of the energy balance is indispensable for cell survival and function. Adenylate kinase (Ak) is a ubiquitous enzyme highly conserved among many organisms. Ak plays an essential role in energy regulation by maintaining adenine nucleotide homeostasis in cells. However, its role at the whole organism level, especially in animal behavior, remains unclear. Here, we established a model using medaka fish (Oryzias latipes) to examine the function of Ak in environmental adaptation. Medaka overexpressing the major Ak isoform Ak1 exhibited increased locomotor activity compared to that of the wild type. Interestingly, this increase was temperature dependent. Our findings suggest that cellular energy balance can modulate locomotor activity.

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Electrical Stimulated Glut4 Signaling Attenuates Critical Illness-Associated Muscle Wasting BackgroundCritical illness myopathy (CIM) is a debilitating condition characterized by the preferential loss of the motor protein myosin. CIM is a byproduct of critical care, attributed to impaired recovery, long-term complications, and mortality. CIM pathophysiology is complex, heterogeneous and remains incompletely understood, however loss of mechanical stimuli contributes to critical illness associated muscle atrophy and weakness. Passive mechanical loading (ML) and electrical stimulation (ES) therapies augment muscle mass and function. While having beneficial outcomes, the mechanistic underpinning of these therapies is less known. Therefore, here we aimed to assess the mechanism by which chronic supramaximal ES ameliorates CIM in a unique experimental rat model of critical care.

MethodsRats were subjected to 8 days critical care conditions entailing deep sedation, controlled mechanical ventilation, and immobilization with and without direct soleus ES. Muscle size and function were assessed at the single cell level. RNAseq and Western blotting were employed to understand the mechanisms driving ES muscle outcomes in CIM.

ResultsFollowing 8 days of controlled mechanical ventilation and immobilization, soleus muscle mass, Myosin:Actin ratio and single muscle fiber maximum force normalized to cross-sectional area (specific force) were reduced by 40-50% (p< 0.0001). ES significantly reduced the loss of soleus muscle fiber cross-sectional area (CSA) and Myosin:Actin ratio by approximately 30% (p< 0.05) yet failed to effect specific force. RNAseq pathway analysis revealed downregulation of insulin signaling in the soleus muscle following critical care and GLUT4 trafficking was reduced by 55% leading to an 85% reduction of muscle glycogen content (p< 0.01). ES promoted phosphofructokinase and insulin signaling pathways to control levels (p< 0.05), consistent with the maintenance of GLUT4 translocation and glycogen levels. AMPK, but not AKT, signaling pathway was stimulated following ES, where the downstream target TBC1D4 increased 3 logFC (p= 0.029) and AMPK-specific P-TBC1D4 levels were increased approximately 2-fold (p= 0.06). Reduction of muscle protein degradation rather than protein synthesis promoted soleus CSA, as ES reduced E3 ubiquitin proteins, Atrogin-1 (p= 0.006) and MuRF1 (p= 0.08) by approximately 50%, downstream of AMPK-FoxO3.

ConclusionsES maintained GLUT4 translocation through increased AMPK-TBC1D4 signaling leading to improved muscle glucose homeostasis. Soleus CSA and myosin content was promoted through reduced protein degradation via AMPK-FoxO3 E3 ligases, Atrogin-1 and MuRF1. These results demonstrate chronic supramaximal ES reduces critical care associated muscle wasting, preserved glucose signaling and reduced muscle protein degradation in CIM.

sentences: Sensory target detection at local and global time scales dissociates modality-specific and supramodal dynamics in the cortical hierarchy How do cortical networks support the detection of irregular sensory events for different senses? Here we reconcile two views of cortical function according to which supramodal processing is either deferred to associative cortices in higher levels of a unimodal-to-supramodal cortical hierarchy or features across all cortical processing stages. In a series of hierarchical multisensory target detection studies using EEG, we applied a temporal cross-decoding approach to reveal the cortical dynamics elicited by rule-based global and feature-based local sensory deviations within and between the visual, somatosensory and auditory modality. We uncovered simple feature-based sensory deviations presented in temporal vicinity to a monotonous input stream triggered an MMN-like local negativity which decayed quickly and early in different sensory modalities. Conversely, complex rule-based targets tracked across time evoked a P3b-like global effect which generalised across a late time window. Ultimately, a series of temporal cross-modality decoding analyses demonstrated that global effects are maintained in supramodal networks whereas local effects propagate along modality-specific cortical networks and evolve into short-lived supramodal activity. Taken together, each sense implements a cortical hierarchy which orchestrates target detection responses operating on local and global time scales at successive processing stages. Converging evidence from different analyses and studies, we show that cortical organisation largely follows a gradient in which modality-specific processes dominate local responses and higher-order processes encode abstract supramodal information fed forward from modality-specific cortices. In conclusion, the cortex implements a hierarchy which hosts increasingly supramodal processes in each level rather than a strict hierarchy which delays supramodal function to associative cortices.

sentences: Single-fiber nucleosome density shapes the regulatory output of a mammalian chromatin remodeling enzyme ATP-dependent chromatin remodelers regulate the DNA accessibility required of virtually all nuclear processes. Biochemical studies have provided insight into remodeler action at the nucleosome level, but how these findings translate to activity on chromatin fibers in vitro and in vivo remains poorly understood. Here, we present a massively multiplex single-molecule platform allowing high-resolution mapping of nucleosomes on fibers assembled on mammalian genomic sequences. We apply this method to distinguish between competing models for chromatin remodeling by the essential ISWI ATPase SNF2h: linker-length-dependent dynamic positioning versus fixed-linker-length static clamping. Our single-fiber data demonstrate that SNF2h operates as a density-dependent, length-sensing chromatin remodeler whose ability to decrease or increase DNA accessibility depends on single-fiber nucleosome density. In vivo, this activity manifests as different regulatory modes across epigenomic domains: at canonically-defined heterochromatin, SNF2h generates evenly-spaced nucleosome arrays of multiple nucleosome repeat lengths; at SNF2h-dependent accessible sites, SNF2h slides nucleosomes to increase accessibility of motifs for the essential transcription factor CTCF. Overall, our generalizable approach provides molecularly-precise views of the processes that shape nuclear physiology. Concurrently, our data illustrate how a mammalian chromatin remodeling enzyme can effectively sense nucleosome density to induce diametrically-opposed regulatory effects within the nucleus.

sentences: Mature parvalbumin interneuron function in prefrontal cortex requires activity during a postnatal sensitive period Sensitive periods in which experience-driven changes in activity persistently shape circuit function are well-described in sensory cortex. Whether comparable periods govern the development of associative cortical areas, like the prefrontal cortex, remains unclear. Here, we focus on the role of activity in the maturation and circuit integration of prefrontal parvalbumin-expressing interneurons, as these cells play an essential role in sensory cortical maturation and develop in lockstep with overall prefrontal circuit function. We found that transiently decreasing prefrontal parvalbumin activity during peripubertal and adolescent development results in persistent impairments in adult functional connectivity, in vivo network function and set-shifting behavior that can be rescued by targeted activation of these interneurons in the adult animal. In contrast, comparable adult inhibition had no lasting effects. These findings identify an activity-dependent sensitive period for prefrontal parvalbumin maturation and highlight how abnormal parvalbumin activity early in life can persistently alter adult circuit function and behavior.

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Spatial phylogenetics of Japanese ferns: Patterns, processes, and implications for conservation PremiseBiodiversity is often only measured with species richness. However, this ignores evolutionary history and is not sufficient for making conservation decisions. Here, we characterize multiple facets and drivers of biodiversity to understand how these relate to bioregions and conservation status in the ferns of Japan.

MethodsWe compiled a community dataset of 1,239 20 km x 20 km grid-cells including 672 taxa based on > 300,000 specimen records. We combined this with a phylogeny and functional traits to analyze taxonomic, phylogenetic, and functional diversity, and modeled biodiversity metrics in response to environmental factors and reproductive mode. Hierarchical clustering was used to delimit bioregions. Conservation status and threats were assessed by comparing the overlap of significantly diverse grid-cells with conservation zones and range maps of native Japanese deer.

ResultsTaxonomic richness was highest at mid-latitudes. Phylogenetic and functional diversity and phylogenetic endemism were highest in small southern islands. Relative phylogenetic and functional diversity were high at high and low latitudes, and low at mid-latitudes. Grid-cells were grouped into three (phylogenetic) or four (taxonomic) major bioregions. Temperature and apomixis were identified as drivers of biodiversity patterns. Conservation status was generally high for grid-cells with significantly high biodiversity, but the threat due to herbivory by deer was greater for taxonomic richness than other metrics.

ConclusionsOur integrative approach reveals previously undetected patterns and drivers of biodiversity in the ferns of Japan. Future conservation efforts should recognize that threats can vary by biodiversity metric and consider multiple multiple metrics when establishing conservation priorities.

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In-situ energy budget of needle-leaves reveals shift from evaporative to 'air cooling' under drought O_LIThe modulation of the leaf energy budget and the balance between its sensible heat (H) and latent heat (LE) fluxes is vital for vegetation functioning and survival, as it is linked to maintaining leaf temperature below the thermal threshold, an increasingly important mechanism under a drying and warming climate, when evaporative cooling is suppressed.
C_LIO_LICombining measurements and theoretical estimates using a new methodology, we obtained rare and comprehensive energy budgets of leaves on twigs under field conditions in droughted and non-droughted plots of a semi-arid pine forest with low and high evapotranspiration rates, respectively.
C_LIO_LIAn examination of all components of the needle-leaf energy budget indicated that under the same radiative load, leaf cooling shifts from nearly equal contributions to H and LE in non-droughted trees to almost exclusively H in droughted ones while maintaining a similar leaf temperature.
C_LIO_LIThis LE-to-H shift in leaves of droughted trees highlights the efficiency of the  air cooling mechanism in maintaining temperature, which can enhance the resilience of trees to drying conditions. Additionally, leaf energy budgets are a fundamental tool to help understand leaf cooling and aerodynamic resistance under field conditions, and to improve modelling of ecosystem activity and its effect on the climate system.
C_LI

sentences: Gene network simulations provide testable predictions for the molecular domestication syndrome The domestication of plant species lead to repeatable morphological evolution, often referred to as the phenotypic domestication syndrome. Domestication is also associated with important genomic changes, such as the loss of genetic diversity compared to adequately large wild populations, and modifications of gene expression patterns. Here, we explored theoretically the effect of a domestication-like scenario on the evolution of gene regulatory networks. We ran population genetics simulations in which individuals were featured by their genotype (an interaction matrix encoding a gene regulatory network) and their gene expressions, representing the phenotypic level. Our domestication scenario included a population bottleneck and a selection switch mimicking human-mediated directional and canalizing selection, i.e., change in the optimal gene expression level and selection towards more stable expression across environments. We showed that domestication profoundly alters genetic architectures. Based on four examples of plant domestication scenarios, our simulations predict (i) a drop in neutral allelic diversity, (ii) a change in gene expression variance that depends upon the domestication scenario, (iii) transient maladaptive plasticity, (iv) a deep rewiring of the gene regulatory networks, with a trend towards gain of regulatory interactions, and (v) a global increase in the genetic correlations among gene expressions, with a loss of modularity in the resulting coexpression patterns and in the underlying networks. We provide empirically testable predictions on the differences of genetic architectures between wild and domesticated forms. The characterization of such systematic evolutionary changes in the genetic architecture of traits contributes to define a molecular domestication syndrome.

sentences: Reconstructing essential active zone functions within a synapse Active zones are molecular machines that control neurotransmitter release through synaptic vesicle docking and priming, and through coupling of these vesicles to Ca2+ entry. The complexity of active zone machinery has made it challenging to determine which mechanisms drive these roles in release. Here, we induce RIM+ELKS knockout to eliminate active zone scaffolding networks, and then reconstruct each active zone function. Re-expression of RIM1-Zn fingers positioned Munc13 on undocked vesicles and rendered them release-competent. Reconstitution of release-triggering required docking of these vesicles to Ca2+ channels. Fusing RIM1-Zn to CaV{beta}4-subunits sufficed to restore docking, priming and release-triggering without reinstating active zone scaffolds. Hence, exocytotic activities of the 80 kDa CaV{beta}4-Zn fusion protein bypassed the need for megadalton-sized secretory machines. These data define key mechanisms of active zone function, establish that fusion competence and docking are mechanistically separable, and reveal that active zone scaffolding networks are not required for release.

sentences: IDENTIFICATION OF HIGH SEED OIL YIELD AND HIGH OLEIC ACID CONTENT IN BRAZILIAN GERMPLASM OF WINTER SQUASH (Cucurbita moschata D.) Cucurbita moschata D. seed oil contains approximately 75% unsaturated fatty acids, with high levels of monounsaturated fatty acids and antioxidant compounds such as vitamin E and carotenoid, constituting a promising food in nutritional terms. Associated to this, the Brazilian germplasm of C. moschata exhibits remarkable variability, representing an important source for the genetic breeding of this vegetable and other cucurbits. In this context, the present study evaluated the productivity and profile of the seed oil of 91 C. moschata accessions from different regions of Brazil and maintained in the Vegetable Germplasm Bank of the Federal University of Vicosa (BGH-UFV). A field experiment was conducted between January and July 2016. The tested C. moschata accessions showed high genetic variability in terms of characteristics related to seed oil productivity (SOP), such as the mass of seeds per fruit and productivity of seeds, providing predicted selection gains of 29.39 g and 0.26 t ha-1, respectively. Based on the phenotypic and genotypic correlations, greater SOP can be achieved while maintaining high oleic acid content and low linoleic acid content, providing oil of better nutritional and chemical quality. In variability analysis, the accessions were clustered into five groups, which presented different averages for SOP and fatty acid content of seed oil; approach that will guide the use of appropriate germplasm in programs aimed at genetic breeding for SOP and seed oil profile. Per se analysis identified BGH-4610, BGH-5485A, BGH-6590, BGH-5556A, BGH-5472A, and BGH-5544A as the most promising accessions in terms of SOP, with average (+g) of approximately 0.20 t ha-1. The most promising accessions for higher oleic acid content of seed oil were BGH-5456A, BGH-3333A, BGH-5361A, BGH-5472A, BGH-5544A, BGH-5453A, and BGH-1749, with average (+g) of approximately 30%, and almost all of these accessions were also the most promising in terms of lower linoleic acid content of seed oil, with average (+g) of approximately 45%. Overall, part of the C. moschata accessions evaluated in the present study can serve as a promising resource in genetic breeding programs for SOP and fatty acid profile, aiming at the production of oil with better nutritional and physicochemical quality.

sentences: Evidence that faecal carriage of resistant Escherichia coli by 16-week-old dogs in the United Kingdom is associated with raw feeding and that E. coli from these dogs are shared with humans and cause opportunistic infections. We report a survey (August 2017 to March 2018) and risk factor analysis of faecal carriage of antibacterial-resistant (ABR) Escherichia coli in 223 sixteen-week-old dogs in the United Kingdom. Raw feeding was associated with the presence of E. coli resistant to fluoroquinolones, tetracycline, amoxicillin, and streptomycin, but not to cefalexin or cefotaxime. Whole genome sequencing of 30 fluoroquinolone-resistant (FQ-R), 22 cefotaxime-resistant (CTX-R) and seven dual FQ-R/CTX-R E. coli isolates showed a wide range of sequence types (STs), an approximately 50:50 split of CTX-M:AmpC-mediated CTX-R, and almost exclusively mutational FQ-R dominated by ST744 and ST162. Comparisons between E. coli isolates from puppies known to be located within a 50 x 50 km region with those isolated from human urinary tract and bloodstream infections (isolated in parallel in the same region) identified a clone of ST963 E. coli carrying chromosomal blaCMY.2 in two puppies and causing two urinary tract infections and one bloodstream infection. Furthermore, an ST744 FQ-R clone was carried by one puppy and caused one urinary tract infection. Accordingly, we conclude that raw feeding is associated with carriage of ABR E. coli in dogs even at sixteen weeks of age and that bacteria carried by dogs are shared with humans.

sentences: Time cell encoding in deep reinforcement learning agents depends on mnemonic demands The representation of "what happened when" is central to encoding episodic and working memories. Recently discovered hippocampal time cells are theorized to provide the neural substrate for such representations by forming distinct sequences that both encode time elapsed and sensory content. However, little work has directly addressed to what extent cognitive demands and temporal structure of experimental tasks affect the emergence and informativeness of these temporal representations. Here, we trained deep reinforcement learning (DRL) agents on a simulated trial-unique nonmatch-to-location (TUNL) task, and analyzed the activities of artificial recurrent units using neuroscience-based methods. We show that, after training, representations resembling both time cells and ramping cells (whose activity increases or decreases monotonically over time) simultaneously emerged in the same population of recurrent units. Furthermore, with simulated variations of the TUNL task that controlled for (1) memory demands during the delay period and (2) the temporal structure of the episodes, we show that memory demands are necessary for the time cells to encode information about the sensory stimuli, while the temporal structure of the task only affected the encoding of "what" and "when" by time cells minimally. Our findings help to reconcile current discrepancies regarding the involvement of time cells in memory-encoding by providing a normative framework. Our modelling results also provide concrete experimental predictions for future studies.

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Matrix stiffness controls ciliogenesis and centriole position Mechanical stress and the stiffness of the extracellular matrix are key drivers of tissue development and homeostasis. Aberrant mechanosensation is associated with a wide range of pathologies, including diseases such as osteoarthritis. Substrate stiffness is one of the well-known mechanical properties of the matrix that enabled establishing the central dogma of an integrin-mediated mechanotransduction using stem cells. However, how specific cells  feel or sense substrate stiffness requires further study. The primary cilium is an essential cellular organelle that senses and integrates mechanical and chemical signals from the extracellular environment. We hypothesised that the primary cilium dynamically alters its length and position to fine-tune cell mechanosignalling based on substrate stiffness alone. We used a hydrogel system of varying substrate stiffness to examine the role of substrate stiffness on cilia frequency, length and centriole position as well as cell and nuclei area over time. Contrary to other cell types, we show that chondrocyte primary cilia shorten on softer substrates demonstrating tissue-specific mechanosensing which is aligned with the tissue stiffness the cells originate from. We further show that stiffness alone determines centriole positioning to either the basal or apical membrane during attachment and spreading, with centriole positioned towards the basal membrane on stiffer substrates. These phenomena are mediated by force generation actin-myosin stress fibres in a time-dependent manner. Based on these findings, we propose that substrate stiffness plays a central role in cilia positioning, regulating cellular response to external forces, and may be a key driver of mechanosignalling-associated diseases.

Significance StatementThe primary cilium has been thrust into the limelight owing to its role as a cellular sensor in embryonic development and adult tissue maintenance. How the primary cilium interacts with the mechanical environment still remains unclear. We show that substrate stiffness dynamically regulates primary cilium length and position through integrin-mediated traction forces, the cilia are a key determinant of cell shape on certain stiffnesses. Our data support the promising potential of primary cilia as a novel target in mechanotherapy for improved clinical outcomes in cartilage pathobiology.

sentences: Comparative evaluation of plasma protein purification and 2-D gel electrophoresis protocols for analysis of HIV-1 infected human plasma proteins Purification of proteins from human plasma is a herculean task to perform 2-D gel electrophoresis. Human plasma contains nearly 70% albumin and globulin. The removal of such high abundance high molecular weight proteins is very difficult before performing 2-D gel electrophoresis. It becomes more difficult when we intent to investigate in infectious diseases like HIV/AIDS. We tried to the best of our efforts adopting various organic and non-organic based protocols based on various published papers. After failure of these protocols in results of 2-D gel-electrophoresis Aurum serum mini kit (Bio-Rad, USA) was adopted for plasma protein purification for performing 2-D gel electrophoresis. The low-abundance proteins were better resolved by 10% SDS-PAGE in 2-D gel-electrophoresis. Then,we extended the MALDI-TOF/TOF analysis of low-abundance proteins in human plasma by adopting the Aurum serum mini kit (Bio-Rad, USA) for 2-D gel electrophoresis. Thus, we concluded that, depletion of high abundant proteins like albumin and globulin, the use of the Aurum serum mini kit (Bio-Rad, USA) is the protocol of choice to perform the 2-D gel electrophoresis of HIV-1 infected human plasma.

sentences: Genomic variations and epigenomic landscape of the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel The teleost medaka (Oryzias latipes) is a well-established vertebrate model system, with a long history of genetic research, and multiple high-quality reference genomes available for several inbred strains (HdrR, HNI and HSOK). Medaka has a high tolerance to inbreeding from the wild, thus allowing one to establish inbred lines from wild founder individuals. We have exploited this feature to create an inbred panel resource: the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel. This panel of 80 near-isogenic inbred lines contains a large amount of genetic variation inherited from the original wild population. We used Oxford Nanopore Technologies (ONT) long read data to further investigate the genomic and epigenomic landscapes of a subset of the MIKK panel. Nanopore sequencing allowed us to identify a much greater variety of high-quality structural variants compared with Illumina sequencing. We also present results and methods using a pan-genome graph representation of 12 individual medaka lines from the MIKK panel. This graph-based reference MIKK panel genome revealed novel differences between the MIKK panel lines compared to standard linear reference genomes. We found additional MIKK panel-specific genomic content that would be missing from linear reference alignment approaches. We were also able to identify and quantify the presence of repeat elements in each of the lines. Finally, we investigated line-specific CpG methylation and performed differential DNA methylation analysis across the 12 lines. We thus present a detailed analysis of the MIKK panel genomes using long and short read sequence technologies, creating a MIKK panel specific pan genome reference dataset allowing for the investigation of novel variation types that would be elusive using standard approaches.

sentences: The polyol pathway is an evolutionarily conserved system for sensing glucose uptake Cells must adjust the expression levels of metabolic enzymes in response to fluctuating nutrient supply. For glucose, such metabolic remodeling is highly dependent on a master transcription factor ChREBP/MondoA. However, it remains elusive how glucose fluctuations are sensed by ChREBP/MondoA despite the stability of major glycolytic pathways. Here we show that in both flies and mice, ChREBP/MondoA activation in response to glucose ingestion depends on an evolutionarily conserved glucose-metabolizing pathway: the polyol pathway. The polyol pathway converts glucose to fructose via sorbitol. It has been believed that this pathway is almost silent, and its activation in hyperglycemic conditions has deleterious effects on human health. We show that the polyol pathway is required for the glucose-induced nuclear translocation of Mondo, a Drosophila homologue of ChREBP/MondoA, which directs gene expression for organismal growth and metabolism. Likewise, inhibition of the polyol pathway in mice impairs ChREBPs nuclear localization and reduces glucose tolerance. We propose that the polyol pathway is an evolutionarily conserved sensing system for the glucose uptake that allows metabolic remodeling.

sentences: Meta-analysis reveals the global picture of antibiotic resistance gene prevalence across environments The environment is an important component in the emergence and transmission of antimicrobial resistance (AMR). Despite that, current AMR monitoring initiatives lack comprehensive reference data for the vast majority of environments. To enable monitoring to detect deviations from the normal background resistance levels in the environment, it is necessary to establish the typical baseline of AMR in a variety of settings. In an attempt to establish this baseline level, we here performed a comprehensive literature survey across 2516 scientific papers, 150 of which contained relevant qPCR data on antibiotic resistance genes (ARGs) in environments associated with potential routes of AMR dissemination. The collected data include 1487 samples distributed across 30 different countries and 17 environmental types, in a time span from 2005 to 2018. More than 330 different genes were identified from the collected studies. Most surveyed environments contained a diverse set of ARGs, but generally at low abundances. We used linear mixed models and overrepresentation analysis to identify time trends and associations between ARGs and specific environments. Altogether these data represent a comprehensive overview of the occurrence and levels of ARGs in different environments, providing background data for risk assessment models within current and future AMR monitoring frameworks.

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Assessment of AlphaFold2 residue conformations for human proteins MotivationAs only 35% of human proteins feature (often partial) PDB structures, the protein structure prediction tool AlphaFold2 (AF2) could have massive impact on human biology and medicine fields, making independent benchmarks of interest. We studied AF2s ability to describe the backbone solvent exposure as an easily interpretable "natural coordinate" of protein conformation, using human proteins as test case.

ResultsAfter screening for appropriate comparative sets, we matched 1818 human proteins predicted by AF2 against 7585 unique experimental PDBs, and after curation for sequence overlap, we assessed 1264 comparative pairs comprising 115 unique AF2-structures and 652 unique experimental structures. AF2 performed markedly worse for multimers, whereas ligands, cofactors and experimental resolution were interestingly not very important for performance. AF2 performed excellently for monomer proteins. Challenges relating to specific groups of residues and multimers were analyzed. We identify larger errors for lower-confidence scores (pLDDT) and exposed residues, and polar residues (Asp, Glu, Asn e.g.) being less accurately described than hydrophobic residues. Proline conformations were the hardest to predict, probably due to common location in dynamic solvent-accessible parts. In summary, using solvent exposure as a natural metric of local conformation, we quantify the performance of AF2 for human proteins and provide estimates of the expected error as a function of ligand presence, multimer/monomer status, resolution, local residue solvent exposure, pLDDT, and amino acid type. Overall performance was found to be excellent.

Availability and ImplementationScripts used to perform benchmarking are available at https://github.com/ktbaek/AlphaFold.

sentences: RNA viral communities are structured by host plant phylogeny in oak and conifer leaves Wild plants can suffer devastating diseases, experience asymptomatic, persistent infections, and serve as reservoirs for viruses of agricultural crops, yet we have a limited understanding of the natural plant virosphere. To access representatives of locally and globally distinct wild plants and investigate their viral diversity, we extracted and sequenced dsRNA from leaves from 16 healthy oak and conifer trees in the UC Davis Arboretum (Davis, California). From de novo assemblies, we recovered 389 RNA-dependent RNA polymerase (RdRp) gene sequences from 384 putative viral species, and a further 580 putative viral contigs were identified with virus prediction software followed by manual confirmation of virus annotation. Based on similarity to known viruses, most recovered viruses were predicted to infect plants or fungi, with the highest diversity and abundance observed in the Totiviridae and Mitoviridae families. Phyllosphere viral community composition differed significantly by host plant phylogeny, suggesting the potential for host-specific viromes. The phyllosphere viral community of one oak tree differed substantially from other oak viral communities and contained a greater proportion of putative mycoviral sequences, potentially due to the trees more advanced senescence at the time of sampling. These results suggest that oaks and conifers harbor a vast diversity of viruses with as-yet unknown roles in plant health and phyllosphere microbial ecology.

sentences: Advanced Complexity and Plasticity of Neural Activity in Reciprocally Connected Human Cerebral Organoids Macroscopic axonal connections in the human brain distribute information and neuronal activity across the brain. Although brain organoid technologies have recently provided novel avenues to investigate human brain function by replicating small neuronal networks of the brain in vitro, functionality of the macroscopic connections between distant regions has not been investigated with organoids. Here, we describe the neural activity of human cerebral organoids reciprocally connected by a bundle of axons. The connected organoids produced significantly more intense and complex oscillatory activity than conventional cerebral organoids. Optogenetic manipulations revealed that the connected organoids could respond to external stimuli and maintain the elevated frequency of neuronal activity for a short term, indicating that the connected organoids exhibit plasticity as a neuronal network. Our findings highlight functional importance of inter-regional connections and suggest that connected organoids can be used as a powerful tool for investigating the development and functions of macroscopic circuits in the human brain. can allow researchers to develop and test compounds to modulate activity of in vitro neuronal network models in unprecedented ways.

sentences: Modelling of filamentous phage-induced antibiotic tolerance of P.aeruginosa Filamentous molecules tend to spontaneously assemble into liquid crystalline droplets with a tactoid morphology in the environments with the high concentration on non-adsorbing molecules. Tactoids of filamentous Pf bacteriophage, such as those produced by Pseudomonas aeruginosa, have been linked with increased antibiotic tolerance. We modelled this system and show that tactoids, composed of filamentous Pf virions, can lead to antibiotic tolerance by acting as an adsorptive diffusion barrier. The continuum model, reminiscent of descriptions of reactive diffusion in porous media, has been solved numerically and good agreement was found with the analytical results, obtained using a homogenisation approach. We find that the formation of tactoids significantly increases antibiotic diffusion times leading to stronger antibiotic resistance.

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RASSF1A independence and early Galectin-1 upregulation in PIK3CA induced hepatocarcinogenesis: new therapeutic venues Aberrant activation of the PI3K/AKT/mTOR and Ras/Mitogen-Activated Protein Kinase pathways is a hepatocarcinogenesis hallmark. In a subset of hepatocellular carcinomas (HCC), PI3K/AKT/mTOR signaling dysregulation depends on PIK3CA mutations, while RAS/MAPK activation is partly attributed to promoter methylation of the tumor suppressor RASSF1A. To evaluate a possible co-carcinogenic effect of PIK3CA activation and RASSF1A knockout, plasmids expressing oncogenic forms of PIK3CA (E545K or H1047R mutants) were delivered to the liver of RASSF1A knockout and wildtype mice by hydrodynamic tail vein injection combined with Sleeping Beauty-mediated somatic integration. Transfection of either PIK3CA E545K or H1047R mutants sufficed to induce hepatocellular carcinomas in mice irrespective of RASSF1A mutational background. The related tumors displayed a lipogenic phenotype with upregulation of Fatty acid synthase and Stearoyl-CoA desaturase-1 (SCD1). Galectin-1, which was commonly upregulated in preneoplastic lesions and tumors, emerged as a regulator of SCD1. Co-inhibitory treatment with PIK3CA inhibitors and the Galectin-1 inhibitor OTX-008 resulted in synergistic cytotoxicity in human HCC cell lines, suggesting novel therapeutic venues.

Graphical AbstractHydrodynamic tail vein injection of Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutant forms E545K and H1047R induces stepwise hepatocarcinogenesis in mice, independent of Ras association domain-containing protein 1 (RASSF1A) status. Gene expression analyses revealed an early increase in Galectin-1, which regulates the lipogenic enzyme Stearoyl-CoA desaturase-1 (SCD1). PIK3CA- and Galectin1 inhibitors act synergistically, pointing at novel therapeutic strategies.

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Cerebral biodistribution and real-time particle tracking in vivo of ultrabright fluorescent PLGA nano-carriers Poly(lactic-co-glycolic acid) (PLGA)-based drug formulations are approved for the use in humans, however, the potential of PLGA to design nanoparticles (NPs) and target the central nervous system remains to be exploited.

The aim of the current study was design PLGA NPs, loading them with bulky fluorophores thereby increasing single particle fluorescence to a level visible by in vivo microscopy, and investigate their brain biodistribution. We developed, highly fluorescent 70 nm PLGA NPs significantly brighter then quantum dots enabling their visualization by intravital real-time 2-photon microscopy. We found that PLGA NPs coated with pluronic F-68 (PF-68) had a substantially longer plasma half-life than uncoated NPs and were taken up by cerebro-vascular endothelial cells. High resolution confocal microscopy revealed that coated PLGA NPs were present in late endothelial endosomes of cerebral vessels within 1 hour after systemic injection and were more readily taken up by endothelial cells in peripheral organs.

The current data suggest that PF-68 coated PLGA NPs are taken up by mouse cerebral and peripheral endothelial cells in vivo. The combination of ultra-bright NPs and in vivo imaging may thus represent a promising approach to reduce the gap between development and clinical application of nanoparticle-based drug carriers.

sentences: Population genetics of polymorphism and divergence in rapidly evolving populations In rapidly evolving populations, numerous beneficial and deleterious mutations can arise and segregate within a population at the same time. In this regime, evolutionary dynamics cannot be analyzed using traditional population genetic approaches that assume that sites evolve independently. Instead, the dynamics of many loci must be analyzed simultaneously. Recent work has made progress by first analyzing the fitness variation within a population, and then studying how individual lineages interact with this traveling fitness wave. However, these "traveling wave" models have previously been restricted to extreme cases where selection on individual mutations is either much faster or much slower than the typical coalescent timescale Tc. In this work, we show how the traveling wave framework can be extended to intermediate regimes in which the scaled fitness effects of mutations (Tcs) are neither large nor small compared to one. This enables us to describe the dynamics of populations subject to a wide range of fitness effects, and in particular, in cases where it is not immediately clear which mutations are most important in shaping the dynamics and statistics of genetic diversity. We use this approach to derive new expressions for the fixation probabilities and site frequency spectra of mutations as a function of their scaled fitness effects, along with related results for the coalescent timescale Tc and the rate of adaptation or Mullers ratchet. We find that competition between linked mutations can have a dramatic impact on the proportions of neutral and selected polymorphisms, which is not simply summarized by the scaled selection coefficient Tcs. We conclude by discussing the implications of these results for population genetic inferences.

sentences: Genetic and chemotherapeutic causes of germline hypermutation Mutation in the germline is the source of all evolutionary genetic variation and a cause of genetic disease. Previous studies have shown parental age to be the primary determinant of the number of new germline mutations seen in an individuals genome. Here we analysed the genome-wide sequences of 21,879 families with rare genetic diseases and identified 12 hypermutated individuals with between two and seven times more de novo single nucleotide variants (dnSNVs) than expected. In most of these families (8/12) the excess mutations could be attributed to the father. We determined that two of these families had genetic drivers of germline hypermutation, with the fathers carrying damaging genetic variation in known DNA repair genes, causing distinctive mutational signatures. For five families, by analysing clinical records and mutational signatures, we determined that paternal exposure to chemotherapeutic agents prior to conception was a key driver of hypermutation. Our results suggest that the germline is well protected from mutagenic effects, hypermutation is rare and relatively modest in degree and that most hypermutated individuals will not have a genetic disease.

sentences: Variation in the response to malaria in Diversity Outbred mice We infected Diversity Outbred mice with Plasmodium chabaudi to better understand how the host response to infection can vary and to try to identify genetic loci responsible for this variation. We identified two loci correlating with binary traits: one on chromosome two was linked to undetectable parasite loads and another on chromosome ten which was linked to death. Though we tested many variable traits, none of those reached statistical significance using the 489 mice we tested.

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Rhythmic Cilium in SCN Neuron is a Gatekeeper for the Intrinsic Circadian Clock The internal circadian rhythm is controlled by the central pacemaker in the hypothalamic suprachiasmatic nucleus (SCN). SCN drives coherent and synchronized circadian oscillations via intercellular coupling, which are resistant to environmental perturbations. Here we report that primary cilium is a critical device for intercellular coupling among SCN neurons and acts as a gatekeeper to maintain the internal clock in mice. A subset of SCN neurons, namely neuromedin S-producing (NMS) neurons, exhibit cilia dynamics with a pronounced circadian rhythmicity. Genetic ablation of ciliogenesis in NMS neurons enables a rapid phase shift of the internal clock under experimental jet lag conditions. The circadian rhythms of individual neurons in cilia-deficient SCN slices lose their coherence following external perturbations. Rhythmic cilia dynamics drive oscillations of Sonic Hedgehog (Shh) signaling and oscillated expressions of multiple circadian genes in SCN neurons. Genetic and chemical inactivation of Shh signaling in NMS neurons phenocopies the effect of cilia ablation. Our findings establish ciliary signaling as a novel interneuronal coupling mechanism in the SCN and may lead to novel therapy of circadian disruption-linked diseases.

One-Sentence SummaryRhythmic cilium is a critical device for intercellular coupling among SCN neurons and acts as gatekeeper for the internal clock.

sentences: A thermal fuse in methionine biosynthesis arrests growth and protects Escherichia coli at elevated temperatures Adaptive stress resistance in microbes is mostly attributed to the expression of stress response genes, such as heat shock proteins, which prevent deterioration of cellular material. Here, we report a novel response of E. coli to heat stress: induction of a growth-arrested state, caused by degradation of an enzyme in the methionine biosynthesis pathway (MetA). While MetA degradation is detrimental for proliferation, we show that the resulting growth arrest has a direct benefit for survival at high temperatures; it protects cells when temperatures rise beyond 50{degrees}C, increasing the survival chances by over 1000-fold. Using a combination of experiments and mathematical modelling, we show that degradation of MetA leads to the coexistence of growing and non-growing cells, allowing microbes to bet-hedge between continued growth if conditions remain bearable and survival if conditions worsen. We test our model experimentally and verify quantitatively how protein expression, degradation rates and environmental stresses affect the partitioning between growing and non-growing cells. Because growth arrest can be abolished with simple mutations, such as point mutations of MetA and knock-outs of proteases, we interpret the breakdown of methionine synthesis as a system that has evolved to disintegrate at high temperature and shut off growth, analogous to  thermal fuses used in engineering to shut off electricity when the device could be damaged by overheating.

sentences: vRhyme enables binning of viral genomes from metagenomes Genome binning has been essential for characterization of bacteria, archaea, and even eukaryotes from metagenomes. Yet, no approach exists for viruses. We developed vRhyme, a fast and precise software for construction of viral metagenome-assembled genomes (vMAGs). vRhyme utilizes single- or multi-sample coverage effect size comparisons between scaffolds and employs supervised machine learning to identity nucleotide feature similarities, which are compiled into iterations of weighted networks and refined bins. Using simulated viromes, we displayed superior performance of vRhyme compared to available binning tools in constructing more complete and uncontaminated vMAGs. When applied to 10,601 viral scaffolds from human skin, vRhyme advanced our understanding of resident viruses, highlighted by identification of a Herelleviridae vMAG comprised of 22 scaffolds, and another vMAG encoding a nitrate reductase metabolic gene, representing near-complete genomes post-binning. vRhyme will enable a convention of binning uncultivated viral genomes and has the potential to transform metagenome-based viral ecology.

sentences: Binned Relative Environmental Change Indicator (BRECI): A tool to communicate the nature of differences between environmental niche model outputs Niche models are now widely used in many branches of the biological sciences and are often used to contrast the distribution of favourable environments between regions or under changes in environmental conditions such as climate change. Evaluating model performance and selecting optimal models is now accepted as best-practice, and a number of methods are available assist this process. One aspect of ENM application which has not received as much attention is developing methods to communicate the degree and nature of changes between model outputs (typically as raster maps). The method described in this paper, Binned Relative Environmental Change Index (BRECI), seeks to address this shortfall in communicating model results.

sentences: In silico discovery of small molecules for efficient stem cell differentiation into definitive endoderm Improving methods for human embryonic stem cell differentiation represents a challenge in modern regenerative medicine research. Using drug repurposing approaches, we discover small molecules that regulate the formation of definitive endoderm. Among them are inhibitors of known processes involved in endoderm differentiation (mTOR, PI3K, and JNK pathways) and a new compound, with an unknown mechanism of action, capable of inducing endoderm formation in the absence of growth factors in the media. Optimization of the classical protocol by including this compound achieves the same differentiation efficiency with a 90% cost reduction. The gene expression profile induced by the compound suggests that it is an inhibitor of the MYC pathway. The proposed in silico procedure for candidate molecule selection has broad potential for improving stem cell differentiation protocols.

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Amplicon sequencing of single-copy protein-coding genes reveals accurate diversity for sequence-discrete microbiome populations An in-depth understanding of microbial function and the division of ecological niches requires accurate delineation and identification of microbes at a fine taxonomic resolution. Microbial phylotypes are typically defined using a 97% small subunit (16S) rRNA threshold. However, increasing evidence has demonstrated the ubiquitous presence of taxonomic units of distinct functions within phylotypes. These so-called sequence-discrete populations (SDPs) have used to be mainly delineated by disjunct sequence similarity at the whole-genome level. However, gene markers that could accurately identify and quantify SDPs are lacking in microbial community studies. Here we developed a pipeline to screen single-copy protein-coding genes that could accurately characterize SDP diversity via amplicon sequencing of microbial communities. Fifteen candidate marker genes were evaluated using three criteria (extent of sequence divergence, phylogenetic accuracy, and conservation of primer regions) and the selected genes were subject to test the efficiency in differentiating SDPs within Gilliamella, a core honeybee gut microbial phylotype, as a proof-of-concept. The results showed that the 16S V4 region failed to report accurate SDP diversities due to low taxonomic resolution and changing copy numbers. In contrast, the single-copy genes recommended by our pipeline were able to successfully quantify Gilliamella SDPs for both mock samples and honeybee guts, with results highly consistent with those of metagenomics. The pipeline developed in this study is expected to identify single-copy protein coding genes capable of accurately quantifying diverse bacterial communities at the SDP level.

IMPORTANCEMicrobial communities can be distinguished by discrete genetic and ecological characteristics. These sequence-discrete populations are foundational for investigating the composition and functional structures of microbial communities at high resolution. In this study, we screened for reliable single-copy protein-coding marker genes to identify sequence-discrete populations through our pipeline. Using marker gene amplicon sequencing, we could accurately and efficiently delineate the population diversity in microbial communities. These results suggest that single copy protein-coding genes can be an accurate, quantitative and economical alternative for characterizing population diversity. Moreover, the feasibility of a gene as marker for any bacterial population identification can be quickly evaluated by the pipeline proposed here.

sentences: Data-driven large-scale genomic analysis reveals an intricate phylogenetic and functional landscape in J-domain proteins The 70 kDalton Heat shock protein (Hsp70) chaperone system is emerging as a central node of the proteostasis network that supervises protein homeostasis in all organisms. Hsp70 recruitment to perform a vast array of different cellular functions is regulated by a family of co-chaperones known as J-domain proteins (JDP) that bear a small namesake J-domain, which is required to interact and drive the ATPase cycle of Hsp70s. Both prokaryotic and in eukaryotic JDPs display a staggering diversity in domain architecture (besides the ubiquitous J-domain), function and cellular localization. This is mirrored by a comparatively small number of Hsp70 paralogs, suggesting a degree of specificity, but also of promiscuity, in their mutual partnering. Very little is known about this family of cochaperones, despite their essential role in cellular proteostasis, development and the link to a broad range of human diseases. The numbers of JDP sequences identified as a consequence of efficient sequencing techniques has exponentially increased where it is now beyond the ability of careful manual curation. In this work, we first provide a broad view of the JDP repertoire accessible from public databases, and then we then use an automated classification scheme, based on Artificial Neural Networks (ANNs), to demonstrate that the sequences of the J-domains carry sufficient information to recover with high reliability the phylogeny, localization and domain composition of the corresponding full length JDP. Exploiting the interpretability of the ANNs employed, we find that several of the sequence positions that are most useful for the classification match to residues that form the interaction interface between the J-domain and Hsp70, revealing that a set of residues within the J-domains have co-evolved with their obligatory Hsp70 partners to build chaperone circuits for specific functions.

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Microscopic and ultramicroscopic anatomical characteristics of root nodules in Podocarpus macrophyllus during development To understand the morphological and structural characteristics of root nodules in Podocarpus macrophyllus and their development, this study prepared P. macrophyllus root nodule samples at the young, mature, and senescent stages. Optical microscopy and transmission electron microscopy (SEM) revealed that new nodules can be formed on roots and senescent nodules; new nodules formed on the roots are nearly spherical and have an internal structure similar to finite nodules; new nodules on senescent nodules are formed by extension and differentiation of the vascular cylinder of the original nodules; and these new nodules are nested at the base of the original nodules, which create growth space for new nodules by dissociating the cortical tissue; clusters of nodules are formed after extensive accumulation, and the growth pattern is similar to that of infinite nodules; the symbiotic bacteria of P. macrophyllus root nodules mainly invade from the epidermal intercellular space of the roots and migrate along the intercellular space of the nodule cortex; infected nodule cortex cells have a well-developed inner membrane system and enlarged and loose nuclei; and unique Frankia vesicles, and rhizobia cysts, and bacteriophages can all develop. Compared with common leguminous and nonleguminous plant nodules, P. macrophyllus root nodules are more complex in morphology, structure and composition. From the perspective of plant system evolution, the rhizobium nodules in leguminous angiosperms and Frankia nodules in nonleguminous angiosperms are most likely two branches derived from the nodules in gymnosperms, such as P. macrophyllus. The conclusions of this study can provide a theoretical basis for the developmental biology of P. macrophyllus root nodules and the evolutionary pattern of plant symbionts.

HighlightsWe discuss from the perspective of cell developmental biology, the rhizobium nodules in leguminous angiosperms and Frankia nodules in nonleguminous angiosperms are most likely two branches derived from the nodules in gymnosperms, such as Podocarpus macrophyllus.

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Systems analysis of gut microbiome influence on metabolic disease in HIV and high-risk populations Poor metabolic health, characterized by insulin resistance and dyslipidemia, is higher in people living with HIV and has been linked with inflammation, anti-retroviral therapy (ART) drugs, and ART-associated lipodystrophy (LD). Metabolic disease is associated with gut microbiome composition outside the context of HIV but has not been deeply explored in HIV infection nor in high-risk men who have sex with men (HR-MSM), who have a highly altered gut microbiome composition. Furthermore, the contribution of increased bacterial translocation and associated systemic inflammation that has been described in HIV-positive and HR-MSM individuals has not been explored. We used a multi-omic approach to explore relationships between impaired metabolic health, defined using fasting blood markers, gut microbes, immune phenotypes and diet. Our cohort included ART-treated HIV positive MSM with and without LD, untreated HIV positive MSM, and HR-MSM. For HIV positive MSM on ART, we further explored associations with the plasma metabolome. We found that elevated plasma lipopolysaccharide binding protein (LBP) was the most important predictor of impaired metabolic health and network analysis showed that LBP formed a hub joining correlated microbial and immune predictors of metabolic disease. Taken together, our results suggest the role of inflammatory processes linked with bacterial translocation and interaction with the gut microbiome in metabolic disease among HIV positive and negative MSM.

Importance StatementThe gut microbiome in people living with HIV (PLWH) is of interest as chronic infection often results in long term comorbidities. Metabolic disease is prevalent in PLWH even in well-controlled infection and has been linked with the gut microbiome in previous studies, but little attention has been given to PLWH. Furthermore, integrated analyses that consider gut microbiome together with diet, systemic immune activation, metabolites, and demographics have been lacking. In a systems-level analysis of predictors of metabolic disease in PLWH and men who are at high risk of acquiring HIV, we found that increased LBP, an inflammatory marker indicative of compromised intestinal barrier function, was associated with worse metabolic health. We also found impaired metabolic health associated with specific dietary components, gut microbes, and host and microbial metabolites. This work lays the framework for mechanistic studies aimed at targeting the microbiome to prevent or treat metabolic endotoxemia in HIV-infected individuals.

sentences: Cell cycle-independent integration of stress signals promotes Non-G1/G0 cellular quiescence Cellular quiescence is a non-proliferative state required for cell survival under stress and during development. In most quiescent cells, proliferation is stopped in a reversible state of low Cdk1 kinase activity; in many organisms, however, quiescent states with high Cdk1 activity can also be established through still uncharacterized stress or developmental mechanisms. Here, we used a microfluidics approach coupled to phenotypic classification by machine learning to identify stress pathways associated with starvation-triggered high-Cdk1 quiescent states in Saccharomyces cerevisiae. We found that low- and high-Cdk1 quiescent states shared a core of stress-associated processes, such as autophagy, protein aggregation, and mitochondrial upregulation, but differed in the nuclear accumulation of the stress transcription factors Xbp1, Gln3, and Sfp1. The decision between low- or high-Cdk1 quiescence was controlled by cell cycle-independent accumulation of Xbp1, which acted as a time-delayed integrator of the duration of stress stimuli. Our results show how cell cycle-independent stress-activated factors promote cellular quiescence outside of G1/G0.

sentences: DYNAMITE: a phylogenetic tool for identification of dynamic transmission epicenters Molecular data analysis is invaluable in understanding the overall behavior of a rapidly spreading virus population when epidemiological surveillance is problematic. It is also particularly beneficial in describing subgroups within the population, often identified as clades within a phylogenetic tree, that represent individuals connected via direct transmission or transmission via differing risk factors in viral spread. However, transmission patterns or viral dynamics within these smaller groups should not be expected to exhibit homogeneous behavior over time. As such, standard phylogenetic approaches that identify clusters based on summary statistics (e.g., median genetic distance over the clade) would not be expected to capture dynamic clusters of transmission. For this purpose, we have developed DYNAMITE (DYNAMic Identification of Transmission Epicenters), a cluster identification algorithm based on a branch-wise (rather than traditional clade-wise) search for cluster criteria, allowing partial clades to be recognized as clusters. Using simulated viral outbreaks with varying cluster types and dynamics, we show that DYNAMITE is consistently more sensitive than existing tools in detecting both static and dynamic transmission clusters. DYNAMITE has been implemented in R and released as open source at: github.com/ProsperiLab/DYNAMITE.

sentences: Impact of ploidy and pathogen life cycle on resistance durability The breeding of resistant hosts based on the gene-for-gene interaction is crucial to address epidemics of plant pathogens in agroecosystems. Resistant host deployment strategies are developed and studied worldwide to decrease the probability of resistance breakdown and increase the resistance durability in various pathosystems. A major component of deployment strategies is the proportion of resistant hosts in the landscape. However, the impact of this proportion on resistance durability remains unclear for diploid pathogens with complex life cycles. In this study, we modelled pathogen population dynamics and genetic evolution at the virulence locus to assess the impact of the ploidy (haploid or diploid) and the pathogens life cycle (with or without host alternation) on resistance durability. Ploidy has a strong impact on evolutionary trajectories, with much greater stochasticity and delayed times of resistance breakdown for diploids. This result emphasises the importance of genetic drift in this system: as the virulent allele is recessive, positive selection on resistant hosts only applies to homozygous (virulent) individuals, which may lead to population collapse at low frequencies of the virulent allele. We also observed differences in the effect of host deployment depending on the pathogens life cycle. With host alternation, the probability that the pathogen population collapses strongly increases with the proportion of resistant hosts in the landscape. Therefore, resistance breakdown events occurring at high proportions of resistant hosts frequently amount to evolutionary rescue. Last, life cycles correspond to two selection regimes: without host alternation (soft selection) the resistance breakdown is mainly driven by the migration rate. Conversely, host alternation (hard selection) resembles an all-or-nothing game, with stochastic trajectories caused by the recurrent allele redistributions on the alternate host.

sentences: Spatial transcriptome sequencing revealed spatial trajectory in the Non-Small Cell Lung Carcinoma Deepening understanding in the heterogeneity of tumors is critical for clinical treatment. Here we investigate tissue-wide gene expression heterogeneity throughout a multifocal lung cancer using the spatial transcriptomics (ST) technology. We identified gene expression gradients in stroma adjacent to tumor regions that allow for re-understanding of the tumor micro-environment. The establishment of these profiles was the first step towards an unbiased view of lung cancer and can serve as a dictionary for future studies. Tumor subclones were detected by ST technology in our research, while we contrast the EMT ability among in subclones which inferred the possible trajectory of tumor metastasis and invasion, which was confirmed by constructing the pseudo-time model of spatial transition within subclones. Together, these results uncovered lung cancer spatial heterogeneity, highlight potential tumor micro-environment differences and spatial evolution trajectory, and served as a resource for further investigation of tumor microenvironment.

sentences: Cross-feeding shapes both competition and cooperation in microbial ecosystems Recent work suggests that cross-feeding - the secretion and consumption of metabolic biproducts by microbes - is essential for understanding microbial ecology. Yet how cross-feeding and competition combine to give rise to ecosystem-level properties remains poorly understood. To address this question, we analytically analyze the Microbial Consumer Resource Model (MiCRM), a prominent ecological model commonly used to study microbial communities. Our mean-field solution exploits the fact that unlike replicas, the cavity method does not require the existence of a Lyapunov function. We use our solution to derive new species-packing bounds for diverse ecosystems in the presence of cross-feeding, as well as simple expressions for species richness and the abundance of secreted resources as a function of cross-feeding (metabolic leakage) and competition. Our results show how a complex interplay between competition for resources and cooperation resulting from metabolic exchange combine to shape the properties of microbial ecosystems.

sentences: UNIGEMS: plasmids and parts to facilitate teaching on assembly, gene expression control and logic in E. coli Synthetic biology is as an excellent vehicle for education, as it enables creative combination of engineering and molecular biology approaches for quantitative characterisations of the assembled constructs. However, there is a limited number of resources available for such applications in the educational context, where straightforward setup, easily measurable phenotypes and extensibility are of particular importance. To expand the availability of education-friendly resources to teach synthetic biology and genetic engineering, we developed Unigems, a set of 10 plasmids that enable out-of-the-box investigations of principles of gene expression control, as well as more complex designs a biological logic gate. The system uses a common high-copy plasmid backbone and a common set of primers to enable Gibson-assembly of PCR-generated or synthesised parts into a target vector. It currently has two reporter genes with either two constitutive (high- or low-level) or two inducible (lactose- or arabinose-) promoters, as well as a single-plasmid implementation of an AND logic gate. The Unigems system has already been employed in undergraduate teaching settings, during outreach events and for training of iGEM teams. All plasmids have been deposited in Addgene.

sentences: Mechanism of LolCDE as a molecular extruder of bacterial triacylated lipoproteins Present in all bacteria, lipoproteins are central in bacterial growth and antibiotic resistance. These proteins use lipid acyl chains attached to the N-terminal cysteine residue to anchor on the outer surface of cytoplasmic membrane. In Gram-negative bacteria, many lipoproteins are transported to the outer membrane (OM), a process dependent on the ATP-binding cassette (ABC) transporter LolCDE which extracts the OM-targeted lipoproteins from the cytoplasmic membrane for subsequent trafficking across the periplasm. Lipid-anchored proteins pose a unique challenge for transport machinery as they have both hydrophobic lipid moieties and soluble protein component, and the underlying mechanism is poorly understood. Here we determined the cryo-EM structures of nanodisc-embedded LolCDE in the nucleotide-free and nucleotide-bound states at 3.8-[A] and 3.5-[A] resolution, respectively. The structural analyses, together with biochemical and mutagenesis studies, uncover how LolCDE specifically recognizes its substrate by establishing multiple interactions with the lipid and N-terminal peptide moieties of the lipoprotein, and identify the amide-linked acyl chain as the key element for LolCDE interaction. Upon nucleotide binding, the transmembrane helices and the periplasmic domains of LolCDE undergo large-scale, asymmetric movements, resulting in extrusion of the captured lipoprotein. Comparison of LolCDE and MacB reveals the conserved mechanism of type VII ABC transporters and emphasizes the unique properties of LolCDE as a molecule extruder of triacylated lipoproteins.

sentences: Metabolic resilience is encoded in genome plasticity Metabolism plays a central role in evolution, as resource conservation is a selective pressure for fitness and survival. Resource-driven adaptations offer a good model to study evolutionary innovation more broadly. It remains unknown how resource-driven optimization of genome function integrates chromatin architecture with transcriptional phase transitions. Here we show that tuning of genome architecture and heterotypic transcriptional condensates mediate resilience to nutrient limitation. Network genomic integration of phenotypic, structural, and functional relationships reveals that fat tissue promotes organismal adaptations through metabolic acceleration chromatin domains and heterotypic PGC1A condensates. We find evolutionary adaptations in several dimensions; low conservation of amino acid residues within protein disorder regions, nonrandom chromatin location of metabolic acceleration domains, condensate-chromatin stability through cis-regulatory anchoring and encoding of genome plasticity in radial chromatin organization. We show that environmental tuning of these adaptations leads to fasting endurance, through efficient nuclear compartmentalization of lipid metabolic regions, and, locally, human-specific burst kinetics of lipid cycling genes. This process reduces oxidative stress, and fatty-acid mediated cellular acidification, enabling endurance of condensate chromatin conformations. Comparative genomics of genetic and diet perturbations reveal mammalian convergence of phenotype and structural relationships, along with loss of transcriptional control by diet-induced obesity. Further, we find that radial transcriptional organization is encoded in functional divergence of metabolic disease variant-hubs, heterotypic condensate composition, and protein residues sensing metabolic variation. During fuel restriction, these features license the formation of large heterotypic condensates that buffer proton excess, and shift viscoelasticity for condensate endurance. This mechanism maintains physiological pH, reduces pH-resilient inflammatory gene programs, and enables genome plasticity through transcriptionally driven cell-specific chromatin contacts. In vivo manipulation of this circuit promotes fasting-like adaptations with heterotypic nuclear compartments, metabolic and cell-specific homeostasis. In sum, we uncover here a general principle by which transcription uses environmental fluctuations for genome function, and demonstrate how resource conservation optimizes transcriptional self-organization through robust feedback integrators, highlighting obesity as an inhibitor of genome plasticity relevant for many diseases.

sentences: Cellular genome wide association study identifies common genetic variation influencing lithium induced neural progenitor proliferation Lithium is used in the treatment of bipolar disorder (BD) and is known to increase neural progenitor cell (NPC) proliferation. Though the mechanism of lithiums therapeutic effect is not understood, evidence suggests that genetic variation influences response to treatment. Here, we used a library of genetically diverse human NPCs to identify common genetic variants that modulate lithium induced proliferation. We identified a locus on chr3p21.1 associated with lithium induced proliferation that colocalizes with BD risk. One lithium responsive gene, GNL3, was detected within the locus. The allele associated with increased baseline and lithium-induced GNL3 expression was also associated with increased lithium-induced NPC proliferation. Experimental manipulation of GNL3 expression using CRISPRa/i in NPCs showed that GNL3 was necessary for lithiums full proliferative effects, and sufficient to induce proliferation without lithium treatment. In all, our data suggest that GNL3 expression sensitizes NPCs for a stronger proliferative response to lithium.

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Accurate and sensitive detection of microbial eukaryotes from whole metagenome shotgun sequencing BackgroundMicrobial eukaryotes are found alongside bacteria and archaea in natural microbial systems, including host-associated microbiomes. While microbial eukaryotes are critical to these communities, they are challenging to study with shotgun sequencing techniques and are therefore often excluded.

ResultsHere we present EukDetect, a bioinformatics method to identify eukaryotes in shotgun metagenomic sequencing data. Our approach uses a database of 521,824 universal marker genes from 241 conserved gene families, which we curated from 3,713 fungal, protist, non-vertebrate metazoan, and non-streptophyte archaeplastid genomes and transcriptomes. EukDetect has a broad taxonomic coverage of microbial eukaryotes, performs well on low-abundance and closely related species, and is resilient against bacterial contamination in eukaryotic genomes. Using EukDetect, we describe the spatial distribution of eukaryotes along the human gastrointestinal tract, showing that fungi and protists are present in the lumen and mucosa throughout the large intestine. We discover that there is a succession of eukaryotes that colonize the human gut during the first years of life, mirroring patterns of developmental succession observed in gut bacteria. By comparing DNA and RNA sequencing of paired samples from human stool, we find that many eukaryotes continue active transcription after passage through the gut, though some do not, suggesting they are dormant or nonviable. We analyze metagenomic data from the Baltic Sea and find that eukaryotes differ across locations and salinity gradients. Finally, we observe eukaryotes in Arabidopsis leaf samples, many of which are not identifiable from public protein databases.

ConclusionsEukDetect provides an automated and reliable way to characterize eukaryotes in shotgun sequencing datasets from diverse microbiomes. We demonstrate that it enables discoveries that would be missed or clouded by false positives with standard shotgun sequence analysis. EukDetect will greatly advance our understanding of how microbial eukaryotes contribute to microbiomes.

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Catalase protects against non-enzymatic decarboxylations during photorespiration Photorespiration recovers carbon that would be otherwise lost following the oxygenation reaction of rubisco and production of glycolate. Photorespiration is essential in plants and recycles glycolate into usable metabolic products through reactions spanning the chloroplast, mitochondrion, and peroxisome. Catalase in peroxisomes plays an important role in this process by disproportionating H2O2 resulting from glycolate oxidation into O2 and water. We hypothesize that catalase in the peroxisome also protects against non-enzymatic decarboxylations between hydrogen peroxide and photorespiratory intermediates (glyoxylate and/or hydroxypyruvate). We test this hypothesis by detailed gas exchange and biochemical analysis of Arabidopsis thaliana mutants lacking peroxisomal catalase. Our results strongly support this hypothesis, with catalase mutants showing gas exchange evidence for an increased stoichiometry of CO2 release from photorespiration, specifically an increase in the CO2 compensation point, a photorespiratory-dependent decrease in the quantum efficiency of CO2 assimilation, increase in the 12CO2 released in a 13CO2 background and an increase in the post-illumination CO2 burst. Further metabolic evidence suggests this excess CO2 release occurred via the non-enzymatic decarboxylation of hydroxypyruvate. Specifically, the catalase mutant showed an accumulation of photorespiratory intermediates during a transient increase in rubisco oxygenation consistent with this hypothesis. Additionally, end products of alternative hypotheses explaining this excess release were similar between wild type and catalase mutants. Furthermore, the calculated rate of hydroxypyruvate decarboxylation in catalase mutant is much higher than that of glyoxylate decarboxylation. This work provides evidence that these non-enzymatic decarboxylation reactions, predominately hydroxypyruvate decarboxylation, can occur in vivo when photorespiratory metabolism is genetically disrupted.

One Sentence SummaryCatalase guards against additional carbon loss from photorespiration arising from non-enzymatic decarboxylations of photorespiratory intermediates.

sentences: Protective Effects of Matrine on Acute Myocardial Hypertrophy Induced by Isoproterenol via Akt/mTOR/p70S6K/eIF4E Signaling Pathway in Rats This study aimed to investigate whether matrine (Ma) attenuates isoproterenol (ISO)-induced acute myocardial hypertrophy via activating Akt/mTOR/p70S6K/eIF4E signaling pathway in rats. We collected 42 male Sprague-Dawley rats weighing 300{+/-}20g, randomly divided into seven groups (n=6). The myocardial hypertrophy (MH) model was well established by 85 mg/kg ISO. Changes in hemodynamic parameters were recorded using electrocardiogram after 24h with ISO injection. Western blot and real-time polymerase chain reaction were used to evaluate the expression of Akt, mechanistic target of rapamycin (mTOR), p70S6K, and eIF4E. Tissue arrangement of the 200 and 100 mg/kg Ma+ISO groups was regularly smaller than that of the ISO group. For the protein expression, Akt values in the 200 and 100 mg/kg Ma+ISO groups were 1.57- and 1.81-fold higher than in the ISO group, respectively. Moreover, compared with the ISO group, the expression trends of mTOR in the 200, 100, and 50 mg/kg Ma+ISO groups significantly downregulated. The levels of p70S6K and eIF4E reduced in the 200, 100, and 50 mg/kg Ma+ISO groups according to the ISO group (P<0.05). MRNA expression of p70S6K and eIF4E in the ISO group were 1.90- and 6.38-fold higher compared with that in the 100 mg/kg Ma+ISO group. Ma exerted neuroprotective effects against pachyntic injury. Akt activity was accelerated, but activities of mTOR, p70S6K, and eIF4E were inhibited by Ma. Activation of the Akt/mTOR/p70S6K/eIF4E signaling pathway might be the targets for the protective effects of Ma on acute myocardial hypertrophy in rats.

sentences: Embryonic development of fully biocompatible organic light-emitting diodes Prototype fully biocompatible organic light-emitting diodes are investigated, with a view to creating a suitable and high-performance light source as a medical implant device. A selection of organic LED materials that have potential suitability for the biological environment are examined. First, the biocompatibility of selected OLED materials was evaluated by the study of cell adhesion and cytotoxicity of HeLa cells cultured on the candidate materials. Thus it was possible to design a device structure composed entirely of biocompatible materials. Second, the characterization of the electroluminescence properties of the prototype OLED is shown and its limitation evaluated. Third, the aqueous stability of the fully biocompatible light source is examined. There is strong evidence that fully biocompatible and stable light-emitting implant devices can be easily constructed. This is the first time a fully biocompatible organic light-emitting diode, albeit embryonic, is reported, with the hope that it may lead to further research to optimize the device performance. Some suggestions on suitable device properties towards in vivo transition are provided.

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A vitamin D-RelB/NF-κB pathway limits Chandipura virus multiplication by rewiring the homeostatic state of autoregulatory type 1 interferon-IRF7 signaling Besides its functions in the skeletomuscular system, vitamin D also promotes protective immunity against viral pathogens. Viral sensing by mammalian cells triggers nuclear activation of RelA/NF-{kappa}B and IRF3 factors, which collaborate in mediating the early induction of antiviral type 1 interferons (T1-IFNs). Autocrine T1-IFN signaling further accumulates otherwise negligibly expressed IRF7 in virus-infected cells that then sustains T1-IFN production in a positive feedback. Surprisingly, prior cell-culture studies revealed that vitamin D actually suppresses signal-induced RelA activation. Indeed, it remains unclear how vitamin D limits viral multiplication in a cell-autonomous manner. Here, we examined the role of vitamin D in controlling cellular infections by the Chandipura virus (CHPV), a cytoplasmic RNA virus implicated in human epidemics. We found that vitamin D conditioning produced an altered cell state less permissive for CHPV multiplication because of the heightened expression of T1-IFNs. It is thought that viruses also induce a distinct RelB/NF-{kappa}B activity, which counteracts RelA-driven T1-IFN expressions in infected cells. Our analyses instead characterized a basal nuclear RelB activity, which was downregulated upon vitamin D-mediated suppression of RelB synthesis. Interestingly, this vitamin D-RelB pathway provoked IRF7-mediated positive autoregulation augmenting constitutive T1-IFN expressions even in the absence of viral infections. Accordingly, RelB deficiency rendered redundant, while IRF7 depletion abrogated antiviral vitamin D actions. In sum, our study suggests that the homeostatic state of the signaling circuitry comprising of the NF-{kappa}B and T1-IFN pathways connects micronutrients to antiviral immunity at the cellular level.

Significance statementVitamin D limits viral infections, but the underlying mechanism remains unclear. Linking micronutrients to antiviral immunity, Ratra et al. characterize an immune signaling circuitry engaged by vitamin D that generates a cellular state less permissive to infections by Chandipura virus, a pathogen of public health importance.

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Large-scale exploration of whole-brain structural connectivity in anorexia nervosa: alterations in the connectivity of frontal and subcortical networks BackgroundAnorexia nervosa (AN) is characterized by disturbances in cognition and behavior surrounding eating and weight. The severity of AN combined with the absence of localized brain abnormalities suggests distributed, systemic underpinnings that may be identified using diffusion-weighted MRI (dMRI) and tractography to reconstruct white matter pathways.

MethodsdMRI data acquired from female patients with AN (n = 147) and female healthy controls (HC; n = 119), aged 12-40 years, were combined across five studies. Probabilistic tractography was completed, and full cortex connectomes describing streamline counts between 84 brain regions generated and harmonized. Graph theory methods were used to describe alterations in network organization in AN. The network-based statistic tested between-group differences in brain subnetwork connectivity. The metrics strength and efficiency indexed the connectivity of brain regions (network nodes), and were compared between groups using multiple linear regression.

ResultsIndividuals with AN, relative to HC, had reduced connectivity in a network comprising subcortical regions and greater connectivity between frontal cortical regions (p < 0.05, FWE corrected). Node-based analyses indicated reduced connectivity of the left hippocampus in patients relative to HC (p < 0.05, permutation corrected). Severity of illness, assessed by BMI, was associated with subcortical connectivity (p < 0.05, uncorrected).

ConclusionsAnalyses identified reduced structural connectivity of subcortical networks and regions, and stronger cortical network connectivity, amongst individuals with AN relative to HC. These findings are consistent with alterations in feeding, emotion and executive control circuits in AN, and may direct hypothesis-driven research into mechanisms of persistent restrictive eating behavior.

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Structural basis for potent antibody neutralization of SARS-CoV-2 variants including B.1.1.529 With B.1.1.529 SARS-CoV-2 variants rapid spread and substantially increased resistance to neutralization by vaccinee and convalescent sera, monoclonal antibodies with potent neutralization are eagerly sought. To provide insight into effective neutralization, we determined cryo-EM structures and evaluated potent receptor-binding domain (RBD) antibodies for their ability to bind and neutralize this new variant. B.1.1.529 RBD mutations altered 16% of the RBD surface, clustering on a ridge of this domain proximal to the ACE2-binding surface and reducing binding of most antibodies. Significant inhibitory activity was retained, however, by select monoclonal antibodies including A19-58.1, B1-182.1, COV2-2196, S2E12, A19-46.1, S309 and LY-CoV1404, which accommodated these changes and neutralized B.1.1.529 with IC50s between 5.1-281 ng/ml, and we identified combinations of antibodies with potent synergistic neutralization. Structure-function analyses delineated the impact of resistance mutations and revealed structural mechanisms for maintenance of potent neutralization against emerging variants.

Summary SentenceWe show potent B.1.1.529 neutralization by select antibodies and use EM structures to reveal how potency can be retained.

sentences: Engineered extracellular matrices reveal stiffness-mediated chemoresistance in patient-derived pancreatic cancer organoids Pancreatic ductal adenocarcinoma (PDAC) is characterized by its fibrotic and stiff extracellular matrix (ECM); however, the role that altered cell-ECM signaling may play in driving PDAC phenotype has historically been difficult to dissect. Here, we design an engineered matrix that recapitulates key hallmarks of the tumor ECM and show that patient-derived PDAC organoids develop gemcitabine chemoresistance when cultured within high stiffness matrices mechanically matched to in vivo tumors. Using genetic barcoding, we find that while matrix-specific clonal selection occurs, cellular heterogeneity is not the main driver of chemoresistance. Instead, stiffness-induced chemoresistance occurs due to the development of a plastic cancer stem cell phenotype - mediated by hyaluronan mechanosignaling - with increased expression of drug efflux transporters. Moreover, PDAC chemoresistance is reversible following transfer from high to low stiffness matrices, suggesting that mechanotherapeutics targeting the fibrotic ECM may sensitize chemoresistant tumors. Overall, we demonstrate the power of engineered matrices and patient-derived organoids to elucidate how ECM properties influence human disease pathophysiology.

sentences: Phylodynamic Pattern of Genetic Clusters, Paradigm Shift on Spatio-temporal Distribution of Clades, and Impact of Spike Glycoprotein Mutations of SARS-CoV-2 Isolates from India Background: The COVID-19 pandemic is associated with high morbidity and mortality, with the emergence of numerous variants. The dynamics of SARS-CoV-2 with respect to clade distribution is uneven, unpredictable and fast changing. Aims: Retrieving the complete genomes of SARS-CoV-2 from India and subjecting them to analysis on phylogenetic clade diversity, Spike (S) protein mutations and their functional consequences such as immune escape features and impact on infectivity. Methods: Whole genome of SARS-CoV-2 isolates (n=4,326) deposited from India during the period from January 2020 to December 2020 is retrieved from GISAID and various analyses performed using in silico tools. Results: Notable clade dynamicity is observed indicating the emergence of diverse SARS-CoV-2 variants across the country. GR clade is predominant over the other clades and the distribution pattern of clades is uneven. D614G is the commonest and predominant mutation found among the S-protein followed by L54F. Mutation score prediction analyses reveal that there are several mutations in S-protein including the RBD and NTD regions that can influence the virulence of virus. Besides, mutations having immune escape features as well as impacting the immunogenicity and virulence through changes in the glycosylation patterns are identified. Conclusions: The study has revealed emergence of variants with shifting of clade dynamics within a year in India. It is shown uneven distribution of clades across the nation requiring timely deposition of SARS-CoV-2 sequences. Functional evaluation of mutations in S-protein reveals their significance in virulence, immune escape features and disease severity besides impacting therapeutics and prophylaxis.

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Resolving complex structures at oncovirus integration loci with conjugate graph Oncovirus integrations cause copy number variations (CNVs) and complex structural variations (SVs) on host genomes. However, the understanding of how inserted viral DNA impacts the local genome remains limited. The linear structure of the oncovirus integrated local genomic map (LGM) will lay the foundations to understand how oncovirus integrations emerge and compromise the host genome s functioning. We propose a conjugate graph model to reconstruct the rearranged local genomic map at integrated loci. Simulation tests prove the reliability and credibility of the algorithm. Applications of the algorithm to whole-genome sequencing data of Human papillomavirus (HPV) and hepatitis B virus (HBV)-infected cancer samples gained biological insights on oncovirus integrations. We observed five affection patterns of oncovirus integrations from the HPV and HBV-integrated cancer samples, including the exon loss, promoter gain, hyper-amplification of tumor gene, the viral cis-regulation inserted at the single intron and at the intergenic region. We found that the focal duplicates and host SVs are frequent in the HPV-integrated LGMs, while the focal deletions and complex virus SVs are prevalent in HBV-integrated LGMs. Furthermore, with the results yields from our method, we found the enhanced microhomology-mediated end joining (MMEJ) might lead to both HPV and HBV integrations, and conjectured that the HPV integrations might mainly occur during the DNA replication process. The conjugate graph algorithm code and LGM construction pipeline, available at https://github.com/deepomicslab/FuseSV.

Key pointsO_LIConjugate graph model resolves complex local genomic strcuture at oncovirus integration loci.
C_LIO_LILocal genome maps reveal five affection patterns of oncovirus integrations.
C_LIO_LIMicrohomology bases and small insertions are enriched at the junctions of structural variations and virus integrations.
C_LIO_LIHPV and HBV integrations may be induced by the enhanced microhomology-meditated mechanism.
C_LI

sentences: Chromosome-scale assembly of the lablab genome - A model for inclusive orphan crop genomics Orphan crops (also described as underutilised and neglected crops) hold the key to diversified and climate-resilient food systems. After decades of neglect, the genome sequencing of orphan crops is gathering pace, providing the foundations for their accelerated domestication and improvement. Recent attention has however turned to the gross under-representation of researchers in Africa in the genome sequencing efforts of their indigenous orphan crops. Here we report a radically inclusive approach to orphan crop genomics using the case of Lablab purpureus (L.) Sweet (syn. Dolichos lablab, or hyacinth bean) - a legume native to Africa and cultivated throughout the tropics for food and forage. Our Africa-led South-North plant genome collaboration produced a high-quality chromosome-scale assembly of the lablab genome - the first chromosome-scale plant genome assembly locally produced in Africa. We also re-sequenced cultivated and wild accessions of lablab from Africa confirming two domestication events and examined the genetic diversity in lablab germplasm conserved in Africa. Our approach provides a valuable resource for lablab improvement and also presents a model that could be explored by other researchers carrying out sequencing projects of indigenous crops particularly from Low and middle income countries (LMIC).

sentences: T-CoV: a comprehensive portal of HLA-peptide interactions affected by SARS-CoV-2 mutations Rapidly appearing SARS-CoV-2 mutations can affect T cell epitopes, which can help the virus to evade either CD8 or CD4 T-cell responses. We developed T-cell COVID-19 Atlas (T-CoV, https://t-cov.hse.ru) - the comprehensive web portal, which allows one to analyze how SARS-CoV-2 mutations alter the presentation of viral peptides by HLA molecules. The data are presented for common virus variants and the most frequent HLA class I and class II alleles. Binding affinities of HLA molecules and viral peptides were assessed with accurate in silico methods. The obtained results highlight the importance of taking HLA alleles diversity into account: mutation-mediated alterations in HLA-peptide interactions were highly dependent on HLA alleles. For example, we found that the essential number of peptides tightly bound to HLA-B*07:02 in the reference Wuhan variant ceased to be tight binders for the Indian (Delta) and the UK (Alpha) variants. In summary, we believe that T-CoV will help researchers and clinicians to predict the susceptibility of individuals with different HLA genotypes to infection with variants of SARS-CoV-2 and/or forecast its severity.

sentences: Chimeric a-subunit isoforms generate functional yeast V-ATPases with altered regulatory properties in vitro and in vivo V-ATPases are highly regulated, multi-subunit proton pumps that acidify organelles. The V-ATPase a-subunit is a two-domain protein containing a C-terminal transmembrane domain that participates in proton transport and a N-terminal cytosolic domain (aNT) that acts as a regulatory hub, integrating environmental inputs to regulate assembly, localization, and V-ATPase activity. Tissue- and organelle-specific a-subunit isoforms exist in most organisms, but how regulatory inputs are decoded by aNT isoforms is unknown. The yeast S. cerevisiae encodes only two organelle-specific a-isoforms, Stv1 in the Golgi and Vph1 in the vacuole. Based on recent structures, we designed chimeric yeast aNTs in which the globular proximal and distal ends are exchanged. The Vph1 proximal-Stv1 distal (VPSD) aNT chimera binds to the glucose-responsive RAVE assembly factor in vitro but exhibits little binding to phosphoinositide lipids that activate V-ATPases. The Stv1 proximal-Vph1 distal (SPVD) aNT lacks RAVE binding but binds more tightly to phosphoinositides than Vph1 or Stv1. When attached to the Vph1 C-terminal domain in vivo, both chimeras complement growth defects of a vph1{Delta} mutant, but only the SPVD chimera exhibits wild-type V-ATPase activity. Cells containing the SPVD chimera adapt more slowly to a poor carbon source than wild-type cells but grow more rapidly than wild-type after a shift to alkaline pH. This is the first example of a "redesigned" V-ATPase with altered regulatory properties and adaptation to specific stresses.

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Association of EBV (Type 1 and 2) with Histopathological Outcomes in Breast Cancer in Pakistani Women IntroductionBreast cancer is one of the major and frequent tumors in the public health sector globally. The rising global prevalence of breast cancer has aroused attention in a viral etiology. Other than genetic and hormonal roles, viruses like Epstein - Barr virus (EBV) also participate in the development and advancement of breast cancer.

AimThis study was conducted to detect the frequency of EBV genotypes in breast cancer patients and compare it with histopathological breast cancer changes.

MethodsFormalin-fixed paraffin-embedded samples of breast cancer (N=60) ages ranged from 22-70 years were collected. EBV DNA was isolated, amplified, typed through PCR, and correlated with histopathological outcomes of breast cancer using SPSS software version 26.

ResultsOur findings suggest that among breast cancer factors, Invasive ductal carcinoma (IDC) was the most common pathological pattern found among patients (90%), observed statistically significant (p= 0.01275). In regards to clinical staging, 8 (13.3 %) patients diagnosed with stage I, 39 (65 %) with stage II, and 13 (21.6 %) with stage III reported statistically significant association (p=0.0003). EBV DNA was detected in 68.3% (41/60) breast cancer patients, reported a statistically significant difference between the prevalence of EBV in breast cancer patients and normal samples (p = 0.001). Of 41 EBV-positive samples, 40 were EBV-1, while only 1 had EBV-2 infection (p < 0.001). No influence on cancer histology was observed. Regarding the association of breast cancer with EBV, histological type (P =0.209), tumor stage (P = 0.48), tumor grade (0.356), tumor sizes (p= 0.976), age (p= 0.1055), tumor laterality (p= 0.533) and ER/PR status (p=0.773) showed no significant association.

ConclusionEBV-1 is prevalent in breast cancer patients and associated with IDC in the study area. For conclusive evidence, more studies are required based on a large sample size and by using more sensitive techniques.

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Characteristics of genetically diverse Antarctic sea ice bacteriophages allow adaptation to changing environmental conditions Despite generally appreciated significant roles of microbes in sea ice and polar waters, detailed studies of virus-host systems from such environments have been so far limited by only a few isolates. Here, we investigated infectivity under various conditions, infection cycles, and genetic diversity of Antarctic sea ice bacteriophages: PANV1, PANV2, OANV1, and OANV2. The phages infect common sea ice bacteria belonging to the genera Paraglaciecola or Octadecabacter. Although the phages are marine and cold-active, replicating at 0-5 {degrees}C, they all survived temporal incubations at [≥]30 {degrees}C and remained infectious without any salts or supplemented only with magnesium, suggesting a robust virion assembly maintaining integrity under a wide range of conditions. Host recognition in the cold proved to be effective, and the release of progeny viruses occurred as a result of cell lysis. The analysis of viral genome sequences showed that nearly half of the gene products of each virus are unique, highlighting that sea ice harbors unexplored virus diversity. Based on predicted genes typical for tailed double-stranded DNA phages, we suggest placing the four studied viruses in the class Caudoviricetes. Searching against viral sequences from metagenomic assemblies revealed that related viruses are not restricted to Antarctica, but also found in distant marine environments.

ImportanceVery little is known about sea ice microbes despite the significant role of sea ice in the global oceans as well as microbial input into biogeochemical cycling. Studies on the sea ice viruses have been typically limited to -omics-based approaches and microscopic examinations of sea ice samples. Up to date, only four cultivable viruses have been isolated from Antarctic sea ice. Our study of these unique isolates advances the understanding of the genetic diversity of viruses in sea ice environments, their interactions with host microbes, and possible links to other biomes. Such information contributes to more accurate future sea ice biogeochemical models.

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Zooplankton trophic dynamics drive carbon export efficiency The magnitude and efficiency of particulate carbon export from the ocean surface depends not only on net primary production (NPP) but also on how carbon is consumed, respired, and repackaged by organisms. We contend that several of these processes can be captured by the size spectrum of the plankton community. However, most global models have relatively simple food-web structures that are unable to generate plankton sizespectra. Moreover, the life-cycles of multicellular zooplankton are typically not resolved, restricting the ability of models to represent time-lags that are known to impact carbon export and its efficiency (pe-ratio). Here, we use a global mechanistic size-spectrum model of the marine plankton community to investigate how particulate export and pe-ratio relate to the community size spectrum, community composition, and time-lags between predators and prey. The model generates emergent food-webs with associated size distributions for organisms and detrital particles. To resolve time-lags between phytoplankton and zooplankton, we implement the life-cycle of multicellular zooplankton (here represented by copepods). The simulation successfully captures observed patterns in biomass and energy fluxes across regions. We find that carbon export correlates best with copepod biomass and trophic level, whereas the pe-ratio correlates best with the exponent of the size spectrum and sea surface temperature (SST). Community metrics performed better than NPP or SST for both deep export and pe-ratio. Time-lags between phytoplankton and copepods did not strongly affect export or pe-ratio. We conclude by discussing how can we reconcile size-spectrum theory with field sampling.

Plain Language SummaryPlankton are tiny but extremely abundant aquatic organisms. Plankton lock CO2 away from the atmosphere as they sink to the deep ocean, where carbon can be stored for hundreds of years. However, how much carbon is locked away and for how long depends on how organisms eat, defecate, and respire. We argue that these processes are reflected in the size composition of the plankton community. The size composition shows a clear relationship between the number of organisms and their body-size. The steepness of this "size-abundance relationship" describes the balance between small vs. large organisms, and has been argued to reflect how energy is transferred from small to large organisms. Since large organisms create fast-sinking particles, the size-abundance relationship could be used to estimate how much carbon is being stored in the deep ocean. Here we use a computer simulation of the global plankton community to investigate how the removal of carbon relates to the plankton community and the steepness of the sizeabundance relationship. The model successfully captures patterns observed in nature. We found that the size-abundance relationship, together with the quantity of large zooplankton better explained carbon export than other measures typically used, such as photosynthesis and temperature.

Key PointsO_LIWe use a global mechanistic size-spectrum model to investigate the relation between particulate export and plankton community metrics.
C_LIO_LIWe find a good correlation between export efficiency and the exponent of the size spectrum.
C_LIO_LITotal carbon export correlated well with copepod biomass and trophic level of active copepods in the model.
C_LI

sentences: Endometrial Gap Junction Expression - Early Indicators of Endometriosis and Integral to Invasiveness Endometriosis is an invasive disease, and a leading cause of pain, infertility and disability among women, with an incidence 10 fold that of cancer. A more complete understanding of disease pathogenesis is essential for the development of non-surgical diagnostic assays and non-hormonal therapeutics. Avoidance of immune clearance and implantation of endometrial tissue on peritoneal surfaces are features of endometriosis lesion formation that overlap with cancer metastasis. Connexins, and the gap junctions they form, have been implicated in cancer progression, and may be associated endometriosis pathophysiology. Single cell transcriptomic profiling of endometrial epithelial and stromal cells from women with endometriosis reveals a striking and progressive shift in expression of connexins and related regulatory and junctional genes. We demonstrate that gap junction coupling between endometrial cells and the peritoneal mesothelium is dramatically induced, specifically in endometriosis patients, and is required for invasion by inducing breakdown of the mesothelial barrier function.

sentences: Cdk5 drives formation of heterogeneous pancreatic neuroendocrine tumors Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous population of neoplasms that arise from hormone-secreting islet cells of the pancreas and have increased markedly in incidence over the past four decades. Non-functional PanNETs, which occur more frequently than hormone-secreting tumors, are often not diagnosed until later stages of tumor development and have poorer prognoses. Development of successful therapeutics for PanNETs has been slow, partially due to a lack of diverse animal models for pre-clinical testing. Here, we report development of an inducible, conditional mouse model of PanNETs by using a bitransgenic system for regulated expression of the aberrant activator of Cdk5, p25, specifically in {beta}-islet cells. This model produces a heterogeneous population of PanNETs that includes a subgroup of well-differentiated, non-functional tumors. The utility of this model is enhanced by ability to form tumor-derived allografts. Production of these tumors demonstrates the causative potential of aberrantly active Cdk5 for generation of PanNETs. Further, we show that human PanNETs express Cdk5 pathway components, are dependent on Cdk5 for growth, and share genetic and transcriptional overlap with the INS-p25OE model. This new model of PanNETs will facilitate molecular delineation of Cdk5-dependent PanNETs and the development of new targeted therapeutics.

sentences: Classification and genetic targeting of cell types in the primary taste and premotor center of the Drosophila brain Neural circuits carry out complex computations that allow animals to evaluate food, select mates, move toward attractive stimuli, and move away from threats. In insects, the subesophageal zone (SEZ) is a brain region that receives gustatory, pheromonal, and mechanosensory inputs and contributes to the control of diverse behaviors, including feeding, grooming, and locomotion. Despite its importance in sensorimotor transformations, the study of SEZ circuits has been hindered by limited knowledge of the underlying diversity of SEZ neurons. Here, we generate a collection of split-GAL4 lines that provides precise genetic targeting of 138 different SEZ cell types in D. melanogaster, comprising approximately one third of all SEZ neurons. We characterize the single cell anatomy of these neurons and find that they cluster by morphology into six supergroups that organize the SEZ into discrete anatomical domains. We find that the majority of local SEZ interneurons are not classically polarized, suggesting rich local processing, whereas SEZ projection neurons tend to be classically polarized, conveying information to a limited number of higher brain regions. This study provides insight into the anatomical organization of the SEZ and generates resources that will facilitate further study of SEZ neurons and their contributions to sensory processing and behavior.

sentences: Host PDZ-containing proteins targeted by SARS-Cov-2 Small linear motif targeting protein interacting domains called PDZ have been identified at the C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins E, 3a, and N. Using a high-throughput approach of affinity-profiling against the full human PDZome, we identified sixteen human PDZ binders of SARS-CoV-2 proteins E, 3A and N showing significant interactions with dissociation constants values ranging from 3 M to 82 M. Six of them (TJP1, PTPN13, HTRA1, PARD3, MLLT4, LNX2) are also recognized by SARS-CoV while three (NHERF1, MAST2, RADIL) are specific to SARS-CoV-2 E protein. Most of these SARS-CoV-2 protein partners are involved in cellular junctions/polarity and could be also linked to evasion mechanisms of the immune responses during viral infection. Seven of the PDZ-containing proteins among binders of the SARS-CoV-2 proteins E, 3a or N affect significantly viral replication under knock-down gene expression in infected cells. This PDZ profiling identifying human proteins potentially targeted by SARS-CoV-2 can help to understand the multifactorial severity of COVID19 and to conceive effective anti-coronaviral agents for therapeutic purposes.

sentences: Ultra-rapid somatic variant detection via real-time threshold sequencing Molecular markers are becoming increasingly important for cancer diagnosis, proper clinical trial enrollment, and even surgical decision making, motivating ultra-rapid, intraoperative variant detection. Sequencing-based detection is considered the gold standard approach, but typically takes hours to perform. In this work, we present Threshold Sequencing, a methodology for designing protocols for targeted variant detection on real-time sequencers with a minimal time to result. Threshold Sequencing analytically identifies a time-optimal threshold to stop target amplification and begin sequencing. To further reduce diagnostic time, we explore targeted Loop-mediated Isothermal Amplification (LAMP) and design a LAMP-specific bioinformatics tool--LAMPrey--to process sequenced LAMP product. LAMPreys concatemer aware alignment algorithm is designed to maximize recovery of diagnostically relevant information leading to a more rapid detection versus standard read alignment approaches. Coupled with time-optimized DNA extraction and library preparation, we demonstrate confirmation of a hot-spot mutation (250x support) from tumor tissue in less than 30 minutes.

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MagnaExtract, a novel magnetic bead-based extraction method for the molecular detection of antimicrobial resistance genes in fresh water. BackgroundThe environmental is increasingly recognised as an important reservoir of antimicrobial resistance (AMR) genes. Polymerase chain reaction (PCR) and whole genome sequencing (WGS) have great potential in the surveillance of AMR genes. However, molecular methods are dependent upon the isolation of high-quality DNA yields. Currently, there is no consensus for the optimum DNA extraction strategies from complex environmental matrices for downstream molecular applications.

MethodsWe present a novel magnetic bead-based method for the isolation of antimicrobial resistance genes (ARGs) from river water in Malawi, named MagnaExtract. We present this with analytic limit of detection (LOD) as well as a case study in Southern Malawi. Here we compare the DNA yield and subsequent PCR output from MagnaExtract with commercially available QIAGEN kits and the crude boil and spin method, utilising a high-resolution melt analysis (HRM) PCR panel designed for the detection of third generation cephalosporin and carbapenem resistant genes.

ResultsOf the 98 water samples evaluated we found the MagnaExtract method to be comparable, and in some instances superior to commercially available kits for the isolation of ARGs from river water samples. In addition, we found overnight incubation to promote the recovery of extended spectrum beta-lactamase (ESBL) genes and simultaneous reduction in the detection of carbapenemase genes.

ConclusionThe MagnaExtract approach offers a simple, affordable, high yielding extraction method that could be used for the detection of ARGs isolated from river water samples in environmental surveillance campaigns in East Africa.

sentences: FLASHIda: Intelligent data acquisition for top-down proteomics that doubles proteoform level identification count Top-down proteomics (TDP) has gained a lot of interest in biomedical application for detailed analysis and structural characterization of proteoforms. Data-dependent acquisition (DDA) of intact proteins is non-trivial due to the diversity and complex signal of proteoforms. Dedicated acquisition methods thus have the potential to greatly improve TDP. We present FLASHIda, an intelligent online data acquisition algorithm for TDP that ensures the real-time selection of high-quality precursors of diverse proteoforms. FLASHIda combines fast charge deconvolution algorithms and machine learning-based quality assessment for optimal precursor selection. In analysis in E. coli lysates, FLASHIda increased the number of unique proteoform level identifications from 800 to 1,500, or generated a near-identical number of identifications in [1/3] of instrument time when compared to standard DDA mode. Furthermore, FLASHIda enabled sensitive mapping of post translational modifications and detection of chemical adducts. As an extension module to the instrument, FLASHIda can be readily adopted for TDP studies of complex samples to enhance proteoform identification rates.

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Presynaptic Gq-coupled receptors drive biphasic dopamine transporter trafficking that modulates dopamine clearance and motor function Extracellular dopamine (DA) is temporally constrained by the presynaptic DA transporter (DAT). Decades of work stemming from primarily in vitro systems support that DAT is dynamically regulated by endocytic trafficking. However, despite its pivotal role in DA neurotransmission, DAT regulation in situ is poorly understood, and it is unknown whether regulated DAT trafficking impacts dopaminergic signaling and/or DA-dependent behaviors. Here, we leveraged chemogenetics and conditional gene silencing in mice to probe the endogenous signaling mechanisms that regulate DAT trafficking, and their impact on DA signaling and DA-dependent behaviors. Activating presynaptic Gq-coupled receptors, either hM3Dq or mGluR5, drove rapid DAT membrane insertion followed by endocytic retrieval, with region-specific differences in ventral and dorsal striata. Gq-stimulated DAT membrane insertion required DRD2 autoreceptors and intact Vps35+ retromer complex, whereas subsequent DAT retrieval required PKC activation and the neuronal GTPase, Rit2. Ex vivo voltammetry in striatal slices revealed that DAT trafficking impacts DA clearance. Conditional mGluR5 silencing in DA neurons demonstrated that presynaptic mGluR5 is specifically required for biphasic DAT trafficking, and its loss increased baseline DAT membrane presentation. Locomotor studies revealed that DAergic mGluR5 silencing abolished motor learning, and selective DAT inhibition rescued this motor deficit, consistent with a causal role for DAT trafficking dysfunction in motor performance. These studies report that striatal presynaptic DAT trafficking is complex, multimodal, and region-specific, and identify cell autonomous mechanisms required for DAT trafficking. Importantly, the findings establish a link between regulated DAT trafficking, DA clearance, and motor function.

Significance StatementDA transmission is central to motivation, learning, and motor function, and mechanisms that impact DA signaling exert profound control over these behaviors. The DAT is pivotal in constraining the DA signal, but little is known about how DAT is regulated in DA terminals, or whether DAT regulation may impact DA-dependent behaviors. Using a variety of complementary genetic approaches in mice, this study provides the first evidence that regulated DAT trafficking plays a strong contributory role in DA signaling and DA-dependent behaviors.

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CaPTure: Calcium Peak Toolbox for analysis of in vitro calcium imaging data BackgroundCalcium imaging is a powerful technique for recording cellular activity across large populations of neurons. However, analysis methods capable of single-cell resolution in cultured neurons, especially for cultures derived from human induced pluripotent stem cells (hiPSCs), are lacking. Existing methods lack scalability to accommodate high-throughput comparisons between multiple lines, across developmental timepoints, or across pharmacological manipulations.

ResultsWe developed a scalable, automated Ca2+ imaging analysis pipeline called CaPTure (https://github.com/LieberInstitute/CaPTure). This method detects neurons, classifies and quantifies spontaneous activity, quantifies synchrony metrics, and generates cell- and network-specific metrics that facilitate phenotypic discovery. The method is compatible with parallel processing on computing clusters without requiring significant user input or parameter modification.

ConclusionCaPTure allows for rapid assessment of neuronal activity in cultured cells at cellular resolution, rendering it amenable to high-throughput screening and phenotypic discovery. The platform can be applied to both human- and rodent-derived neurons and is compatible with many imaging systems.

sentences: The influence of the respiratory cycle on reaction times in sensory-cognitive paradigms Behavioural and electrophysiological studies point to widespread influence of the state of respiration on brain activity and cognitive performance. Still, the prevalence and relevance of such respiratory-behavioural relations in typical sensory-cognitive tasks remain unclear. We here used a battery of six tasks probing sensory detection, discrimination and short-term memory to address the questions of whether and by how much behaviour covaries with the respiratory cycle. Our results show that participants tended to align their respiratory cycle to the experimental paradigm. Furthermore, their reaction times, but not so much their response accuracy, consistently and significantly covaried with the respiratory cycle, and this effect was strongest when analyzed contingent on the respiratory state at participants responses. The respective effect sizes where comparable to those seen in many typical neurocognitive experimental manipulations. These results support a prominent relation between respiration and sensory-cognitive function and suggest that sensation is intricately linked to rhythmic bodily or interoceptive functions.

sentences: Assessing bivalve phylogeny using Deep Learning and Computer Vision approaches Phylogenetic analyses using morphological data currently require hand-crafted character matrices, limiting the number of taxa that can be included. Here I explore how Deep Learning and Computer Vision approaches typically applied to image classification tasks, may be used to infer phylogenetic relationships among bivalves. A convolutional neural network (CNN) was trained on thousands of images showing species of 75 bivalve families. The predictions of the CNN on a large number of bivalve images are then interpreted as an indication of how similar these bivalves are to each other, are averaged by the families to which the species belonged, and visualized in a cluster diagram. In this cluster diagram, significantly more families clustered with members of their subclasses than expected by chance, confirming the feasibility of the approach. To address the issue of convergent evolution, two further CNNs were trained, on the same images but grouped by the orders and subclasses to which the species belonged. Combining predictions for the same images but on different taxonomic levels improved the inferred phylogenetic relationships also of families that the CNNs had not been trained on. Finally, this combined tree is merged with five published phylogenetic trees into a supertree, representing the largest single phylogeny of the Bivalvia to date, encompassing 128 families, including six exclusively fossil families and nine extant families for which presently no molecular data are available. Issues inherent to the approach and suggestions for future directions are discussed.

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Complete and high-quality genomes of novel microbial species from a meromictic lake using a workflow combining long- and short-read sequencing platforms BackgroundMost of Earths bacteria have yet to be cultivated. The metabolic and functional potentials of these uncultivated microorganisms thus remain mysterious, and the metagenome-assembled genome (MAG) approach is the most robust method for uncovering these potentials. However, MAGs discovered by conventional metagenomic assembly and binning methods are usually highly fragmented genomes with heterogeneous sequence contamination, and this affects the accuracy and sensitivity of genomic analyses. Though the maturation of long-read sequencing technologies provides a good opportunity to fix the problem of highly fragmented MAGs as mentioned above, the methods error-prone nature causes severe problems of long-read-alone metagenomics. Hence, methods are urgently needed to retrieve MAGs by a combination of both long- and short-read technologies to advance genome-centric metagenomics.

ResultsIn this study, we combined Illumina and Nanopore data to develop a new workflow to reconstruct 233 MAGs--six novel bacterial orders, 20 families, 66 genera, and 154 species--from Lake Shunet, a secluded meromictic lake in Siberia. Those new MAGs were underrepresented or undetectable in other MAGs studies using metagenomes from human or other common organisms or habitats. Using this newly developed workflow and strategy, the average N50 of reconstructed MAGs greatly increased 10-40-fold compared to when the conventional Illumina assembly and binning method were used. More importantly, six complete MAGs were recovered from our datasets, five of which belong to novel species. We used these as examples to demonstrate many novel and intriguing genomic characteristics discovered in these newly complete genomes and proved the importance of high-quality complete MAGs in microbial genomics and metagenomics studies.

ConclusionsThe results show that it is feasible to apply our workflow with a few additional long reads to recover numerous complete and high-quality MAGs from short-read metagenomes of high microbial diversity environment samples. The unique features we identified from five complete genomes highlight the robustness of this method in genome-centric metagenomic research. The recovery of 154 novel species MAGs from a rarely explored lake greatly expands the current bacterial genome encyclopedia and broadens our knowledge by adding new genomic characteristics of bacteria. It demonstrates a strong need to recover MAGs from diverse unexplored habitats in the search for microbial dark matter.

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The sex-specific VC neurons are mechanically activated motor neurons that facilitate serotonin-induced egg laying in C. elegans Successful execution of behavior requires coordinated activity and communication between multiple cell types. Studies using the relatively simple neural circuits of invertebrates have helped to uncover how conserved molecular and cellular signaling events shape animal behavior. To understand the mechanisms underlying neural circuit activity and behavior, we have been studying a simple circuit that drives egg-laying behavior in the nematode worm C. elegans. Here we show that the sex-specific, Ventral C (VC) motor neurons are important for vulval muscle contractility and egg laying in response to serotonin. Ca2+ imaging experiments show the VCs are active during times of vulval muscle contraction and vulval opening, and optogenetic stimulation of the VCs promotes vulval muscle Ca2+ activity. Blocking VC neurotransmission inhibits egg laying in response to serotonin and increases the failure rate of egg-laying attempts, indicating that VC signaling facilitates full vulval muscle contraction and opening of the vulva for efficient egg laying. We also find the VCs are mechanically activated in response to vulval opening. Optogenetic stimulation of the vulval muscles is sufficient to drive VC Ca2+ activity and requires muscle contractility, showing the presynaptic VCs and the postsynaptic vulval muscles can mutually excite each other. Together, our results demonstrate that the VC neurons facilitate efficient execution of egg-laying behavior by coordinating postsynaptic muscle contractility in response to serotonin and mechanosensory feedback.

Significance StatementMany animal motor behaviors are modulated by the neurotransmitters serotonin and acetylcholine. Such motor circuits also respond to mechanosensory feedback, but how neurotransmitters and mechanoreceptors work together to coordinate behavior is not well understood. We address these questions using the egg-laying circuit in C. elegans where we can manipulate presynaptic neuron and postsynaptic muscle activity in behaving animals while recording circuit responses through Ca2+ imaging. We find that the cholinergic VC motoneurons are important for proper vulval muscle contractility and egg laying in response to serotonin. Muscle contraction also activates the VCs, forming a positive feedback loop that promotes full contraction for egg release. In all, mechanosensory feedback provides a parallel form of modulation that shapes circuit responses to neurotransmitters.

sentences: The link between static and dynamic brain functional network connectivity and genetic risk of Alzheimer's disease Apolipoprotein E (APOE) polymorphic alleles are genetic factors associated with Alzheimers disease (AD) risk. Although previous studies have explored the link between AD genetic risk and static functional network connectivity (sFNC), to the best of our knowledge, no previous studies have evaluated the association between dynamic FNC (dFNC) and AD genetic risk. Here, we examined the link between sFNC, dFNC, and AD genetic risk with a reproducible, data-driven approach. We used rs-fMRI, demographic, and APOE data from cognitively normal individuals (N=894) between 42 to 95 years of age (mean = 70 years). We divided individuals into low, moderate, and high-risk groups. Using Pearson correlation, we calculated sFNC across seven brain networks. We also calculated dFNC with a sliding window and Pearson correlation. The dFNC windows were partitioned into three distinct states with k-means clustering. Next, we calculated the amount of time each subject spent in each state, called occupancy rate or OCR. We compared both sFNC and OCR, estimated from dFNC, across individuals with different genetic risk and found that both sFNC and dFNC are related to AD genetic risk. We found that higher AD risk reduces within-visual sensory network (VSN) sFNC and that individuals with higher AD risk spend more time in a state with lower within-VSN dFNC. Additionally, we found that AD genetic risk affects whole-brain sFNC and dFNC in women but not in men. In conclusion, we presented novel insights into the links between sFNC, dFNC, and AD genetic risk.

sentences: The Shape of Trees - Limits of Current Diversification Models To investigate how biodiversity arose, the field of macroevolution largely relies on model-based approaches to estimate rates of diversification and what factors influence them. The number of available models is rising steadily, facilitating the modeling of an increasing number of possible diversification dynamics, and multiple hypotheses relating to what fueled or stifled lineage accumulation within groups of organisms. However, growing concerns about unchecked biases and limitations in the employed models suggest the need for rigorous validation of methods used to infer. Here, we address two points: the practical use of model adequacy testing, and what model adequacy can tell us about the overall state of diversification models. Using a large set of empirical phylogenies, and a new approach to test models using aspects of tree shape, we test how a set of staple models performs with regards to adequacy. Patterns of adequacy are described across trees and models and causes for inadequacy - particularly if all models are inadequate - are explored. The findings make clear that overall, only few empirical phylogenies cannot be described by at least one model. However, finding that the best fitting of a set of models might not necessarily be adequate makes clear that adequacy testing should become a step in the standard procedures for diversification studies.

sentences: Functional ultrasound imaging of the spreading activity following optogenetic stimulation of the rat visual cortex Optogenetic stimulation of the primary visual cortex (V1) is a promising therapy for sight restoration, but it remains unclear what total cerebral volume is activated after surface stimulation. In this study, we expressed the red-shifted opsin ChrimsonR in excitatory neurons within V1 in rats, and used the fine spatial resolution provided by functional ultrasound imaging (fUS) over the whole depth of the brain to investigate the brain response to focal surface stimulation. We observed optogenetic activation of a high proportion of the volume of V1. Extracellular recordings confirmed the neuronal origin of this activation. Moreover, neuronal responses were even located in deep layers under conditions of low irradiance, spreading to the LGN and V2, consistent with a normal visual information process. This study paves the way for the use of optogenetics for cortical therapies, and highlights the value of coupling fUS with optogenetics.

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Emotional context sculpts action goal representations in the lateral frontal pole Emotional states provide an ever-present source of contextual information that should inform behavioral goals. Despite the ubiquity of emotional signals in our environment, the neural mechanisms underlying their influence on goal-directed action remains unclear. Prior work suggests that the lateral frontal pole (FPl) is uniquely positioned to integrate affective information into cognitive control representations. We used pattern similarity analysis to examine the content of representations in FPl and interconnected mid-lateral prefrontal and amygdala circuitry. Healthy participants (n=37; n=21 females) were scanned while undergoing an event-related Affective Go/No-Go task, which requires goal-oriented action selection during emotional processing. We found that FPl contained conjunctive emotion-action goal representations that were related to successful cognitive control during emotional processing. These representations differed from conjunctive emotion-action goal representations found in the basolateral amygdala. While robust action goal representations were present in mid-lateral prefrontal cortex, they were not modulated by emotional valence. Finally, converging results from functional connectivity and multivoxel pattern analyses indicated that FPls emotional valence signals likely originated from interconnected subgenual ACC (BA25), which was in turn functionally coupled with the amygdala. Thus, our results identify a key pathway by which internal emotional states influence goal-directed behavior.

Significance statementOptimal functioning in everyday life requires behavioral regulation that flexibly adapts to dynamically changing emotional states. However, precisely how emotional states influence goal-directed action remains unclear. Unveiling the neural architecture that supports emotion-goal integration is critical for our understanding of disorders such as psychopathy, which is characterized by deficits in incorporating emotional cues into goals, as well as mood and anxiety disorders, which are characterized by impaired goal-based emotion regulation. Our study identifies a key circuit through which emotional states influence goal-directed behavior. This circuitry comprised the lateral frontal pole (FPl), which represented integrated emotion-goal information, as well as interconnected amygdala and subgenual ACC, which conveyed emotional signals to FPl.

sentences: Analysis of SARS-CoV-2 mutations reveals three types of temporal dynamics and one is correlated with international travels Coronavirus disease 2019 (COVID-19) is a contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This disease has spread globally, causing more than 161.5 million cases and 3.3 million deaths to date. Surveillance and monitoring of new mutations in the virus genome are crucial to our understanding of the adaptation of SARS-CoV-2. Moreover, how the temporal dynamics of these mutations is influenced by control measures and non-pharmaceutical interventions (NPIs) is poorly understood. Using 1 058 020 SARS-CoV-2 from sequenced COVID-19 cases from 98 countries (totaling 714 country-month combinations), we perform a normalization by COVID-19 cases to calculate the relative frequency of SARS-CoV-2 mutations and explore their dynamics over time. We found 115 mutations estimated to be present in more than 3 % of global COVID-19 cases and determined three types of mutation dynamics: High-Frequency, Medium-Frequency, and Low-Frequency. Classification of mutations based on temporal dynamics enable us to examine viral adaptation and evaluate the effects of implemented control measures in virus evolution during the pandemic. We showed that Medium-Frequency mutations are characterized by high prevalence in specific regions and/or in constant competition with other mutations in several regions. Finally, taking N501Y mutation as representative of High-Frequency mutations, we showed that level of control measure stringency negatively correlates with the effective reproduction number of SARS-CoV-2 with High-Frequency or not-High-Frequency and both follows similar trends in different levels of stringency.

sentences: TAFKAP: An improved method for probabilistic decoding of cortical activity Cortical activity can be difficult to interpret. Neural responses to the same stimulus vary between presentations, due to random noise and other sources of variability. This unreliable relationship to external stimuli renders any pattern of activity open to a multitude of plausible interpretations. We have previously shown that this uncertainty in cortical stimulus representations can be characterized using a probabilistic decoding algorithm, which inverts a generative model of stimulus-evoked cortical responses. Here, we improve upon this method in two important ways, which both target the precision with which the generative model can be estimated from limited, noisy training data. We show that these improvements lead to considerably better estimation of the presented stimulus and its associated uncertainty. Estimates of the presented stimulus are recovered with an accuracy that exceeds that of standard decoding methods (SVMs), and in some cases even approaches the behavioral accuracy of human observers. Moreover, the uncertainty in the decoded probability distributions better characterizes the precision of cortical stimulus information from trial to trial.

sentences: Time-adaptive Unsupervised Auditory Attention Decoding Using EEG-based Stimulus Reconstruction The goal of auditory attention decoding (AAD) is to determine to which speaker out of multiple competing speakers a listener is attending based on the brain signals recorded via, e.g., electroencephalography (EEG). AAD algorithms are a fundamental building block of so-called neuro-steered hearing devices that would allow identifying the speaker that should be amplified based on the brain activity. A common approach is to train a subject-specific stimulus decoder that reconstructs the amplitude envelope of the attended speech signal. However, training this decoder requires a dedicated  ground-truth EEG recording of the subject under test, during which the attended speaker is known. Furthermore, this decoder remains fixed during operation and can thus not adapt to changing conditions and situations. Therefore, we propose an online time-adaptive unsupervised stimulus reconstruction method that continuously and automatically adapts over time when new EEG and audio data are streaming in. The adaptive decoder does not require ground-truth attention labels obtained from a training session with the end-user and instead can be initialized with a generic subject-independent decoder or even completely random values. We propose two different implementations: a sliding window and recursive implementation, which we extensively validate on three independent datasets based on multiple performance metrics. We show that the proposed time-adaptive unsupervised decoder outperforms a time-invariant supervised decoder, representing an important step toward practically applicable AAD algorithms for neuro-steered hearing devices.

sentences: Developmental and Intergenerational Landscape of Human Circulatory Lipidome and its Association with Obesity Risk Lipids play a vital role in human health and development, but changes to their circulatory levels during gestation and in early life are poorly understood. Here we present the first developmental and intergenerational landscape of the human circulatory lipidome, derived by profiling of 480 lipid species representing 25 lipid classes, in mothers and their offspring (n=2491). Levels of 66% of the profiled lipids increased in maternal circulation during gestation, while cord blood had higher concentrations of acylcarnitines and lysophospholipids. The offspring lipidome at age six years revealed striking similarities with postnatal maternal lipidome (adult) in its lipid composition and concentrations. Comparison of lipids associated with child and maternal adiposity identified a 92% overlap, implying intergenerational similarities in the lipid signatures of obesity risk. We also catalogued lipid signatures linked with maternal adiposity during gestation and offspring birthweight, and validated (>70% overlap) the findings in an independent birth-cohort (n=1935).

sentences: A Francisella tularensis L,D-carboxypeptidase plays important roles in cell morphology, envelope integrity, and virulence. Francisella tularensis is a Gram-negative, intracellular bacterium that causes the zoonotic disease tularemia. Intracellular pathogens, including F. tularensis, have evolved mechanisms to survive in the harsh environment of macrophages and neutrophils, where they are exposed to cell envelope-damaging molecules. The bacterial cell wall, primarily composed of peptidoglycan (PG), maintains cell morphology, structure, and membrane integrity. Intracellular Gram-negative bacteria protect themselves from macrophage and neutrophil killing by recycling and repairing damaged PG - a process that involves over 50 different PG synthesis and recycling enzymes. Here, we identified a PG recycling enzyme, L,D-carboxypeptidase A (LdcA), of F. tularensis that is responsible for converting PG tetrapeptide stems to tripeptide stems. Unlike E. coli LdcA and most other orthologs, F. tularensis LdcA does not localize to the cytoplasm and also exhibits L,D-endopeptidase activity, converting PG pentapeptide stems to tripeptide stems. Loss of F. tularensis LdcA led to altered cell morphology and membrane integrity, as well as attenuation in a mouse pulmonary infection model and in primary and immortalized macrophages. Finally, an F. tularensis ldcA mutant protected mice against virulent Type A F. tularensis SchuS4 pulmonary challenge.

sentences: Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains, rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C-terminus through the central pore of the hexameric double-ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.

sentences: Meta-analysis of scRNA seq data to define the landscape of human liver immune homeostasis. The liver is unique in both its ability to maintain immune homeostasis and in its potential for immune tolerance following solid organ transplantation. Single-cell RNA sequencing (scRNA seq) is a powerful approach to generate highly dimensional transcriptome data to understand cellular phenotypes. However, when scRNA data is produced by different groups, with different data models, different standards, and samples processed in different ways, it can be challenging to draw meaningful conclusions from the aggregated data. The goal of this study was to establish a method to combine  human liver scRNA seq datasets by 1) characterizing the heterogeneity between studies and 2) using the meta-atlas to define the dominant phenotypes across immune cell subpopulations in healthy human liver. Publicly available scRNA seq data generated from liver samples obtained from a combined total of 17 patients and [~]32,000 cells were analyzed. Liver-specific immune cells (CD45+) were extracted from each dataset, and immune cell subpopulations (myeloid cells, NK and T cells, plasma cells, and B cells) were examined using dimensionality reduction (UMAP), differential gene expression, and ingenuity pathway analysis. All datasets co-clustered, but cell proportions differed between studies. Gene expression correlation demonstrated similarity across all studies, and canonical pathways that differed between datasets were related to cell stress and oxidative phosphorylation rather than immune-related function. Next, a meta-atlas was generated via data integration and compared against PBMC data to define gene signatures for each hepatic immune subpopulation. This analysis defined key features of hepatic immune homeostasis, with decreased expression across immunologic pathways and enhancement of pathways involved with cell death. This method for meta-analysis of scRNA seq data provides a novel approach to broadly define the features of human liver immune homeostasis. Specific pathways and cellular phenotypes described in this human liver immune meta-atlas provide a critical reference point for further study of immune mediated disease processes within the liver.

sentences: Staying in touch: how highly specialised moth pollinators track host plant phenology in unpredictable climates For pollinating insects that visit just a single flowering species, the co-occurrence of flowers and insects in time is likely to have critical implications for both plant and pollinator. Insects often utilise diapause to persist through periods in which resources are unavailable, timing their re-emergence by responding to the same environmental cues as their host plants. The obligate pollination mutualisms (OPMs) between Epicephala moths (Gracillariidae) and their leaf flower host plants are some of the most specialised interactions between plants and insects. However, to date there have been very few studies of Epicephala moth lifecycles and none of how they synchronise their activity with the flowering of their host plants. Breynia oblongifolia (Phyllanthaceae) is known to be exclusively pollinated by two highly specific species of Epicephala moth (Gracillariidae). We surveyed populations of both the host plant and its pollinators over multiple years to determine their annual phenology and then modelled the climatic factors that drive their activity. Using our newly gained knowledge of moth and host plant phenology, we then looked for evidence of diapause at both the egg and pre-pupal stages. Our phenology surveys showed that although female flowers were present throughout the entire year, the abundance of flowers and fruits was highly variable between sites and strongly associated with local rainfall and photoperiod. Fruit abundance, but not flower abundance, was a significant predictor of adult Epicephala activity, suggesting that eggs or early instar larvae diapause within dormant flowers and emerge as fruits mature. Searches of overwintering flowers confirmed this, with many containing evidence of pollen and diapausing pollinators. We also observed the behaviour of adult Epicephala prior to pupation and found that ~10% of the Autumn emerging Epicephala enter diapause, eclosing to adulthood after 38-56 weeks. The remaining 90% of autumn emerging adults pupate directly with no diapause, suggesting a bet hedging strategy for adult emergence. As such, Epicephala moths appear to utilise diapause at multiple stages in their lifecycle, and possibly bet hedging, in order to deal with variable flowering phenology and climatic unpredictability.

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Effects of theta transcranial alternating current stimulation (tACS) on exploration and exploitation during uncertain decision-making Exploring ones surroundings may yield unexpected rewards, but is associated with uncertainty and risk. Alternatively, exploitation of certain outcomes is related to low risk, yet potentially better outcomes remain unexamined. As such, risk-taking behavior depends on perceived uncertainty and a trade-off between exploration-exploitation. Previously, it has been suggested that risk-taking may relate to theta activity in the prefrontal cortex. Furthermore, previous studies hinted at a relationship between a right-hemispheric bias in frontal theta asymmetry and risky behavior. In the present double-blind sham-controlled within-subject study, we applied bifrontal transcranial alternating current stimulation (tACS) at the theta frequency (5 Hz) on eighteen healthy volunteers during a gambling task. Two tACS montages with either left-right or posterior-anterior current flow were employed at an intensity of 1 mA. Results showed that, compared to sham, theta tACS increased perceived uncertainty irrespective of current flow direction. Despite this observation, no direct effect of tACS on exploration behavior and general risk-taking was observed. Furthermore, frontal theta asymmetry was more right-hemispherically biased after posterior-anterior tACS, compared to sham. Finally, we used electric field simulation to identify which regions were targeted by the tACS montages as an overlap in regions may explain why the two montages resulted in comparable outcomes. Our findings provide a first step towards understanding the relationship between frontal theta oscillations and different features of risk-taking.

Highlights- Risk taking is related to uncertainty, exploration and exploitation
- Frontal theta tACS was applied to modulate aspects of risk taking
- tACS did increase perceived uncertainty, but not exploration behavior
- tACS induced a right hemispheric shift in frontal theta asymmetry

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Peroxide antimalarial drugs target redox homeostasis in Plasmodium falciparum infected red blood cells Plasmodium falciparum causes the most lethal form of malaria. Peroxide antimalarials based on artemisinin underpin the frontline treatments for malaria, but artemisinin resistance is rapidly spreading. Synthetic peroxide antimalarials, known as ozonides, are in clinical development and offer a potential alternative. Here, we used chemoproteomics to investigate the protein alkylation targets of artemisinin and ozonide probes, including an analogue of the ozonide clinical candidate, artefenomel. We greatly expanded the list of protein targets for peroxide antimalarials and identified significant enrichment of redox-related proteins for both artemisinins and ozonides. Disrupted redox homeostasis was confirmed by dynamic live imaging of the glutathione redox potential using a genetically encoded redox-sensitive fluorescence-based biosensor. Targeted LC-MS-based thiol metabolomics also confirmed changes in cellular thiol levels. This work shows that peroxide antimalarials disproportionately alkylate proteins involved in redox homeostasis and that disrupted redox processes are involved in the mechanism of action of these important antimalarials.

ImportanceThe frontline treatments for malaria are combination therapies based on the peroxide antimalarial, artemisinin. Concerningly, artemisinin resistance has emerged in malaria-endemic regions, and now poses a major threat to malaria treatment and eradication efforts. New medicines are urgently required to replace the artemisinins, and some of the most advanced candidates are the fully synthetic peroxide antimalarials, OZ277 (arterolane) and OZ439 (artefenomel). The mechanism of action of peroxide antimalarials involves the reductive activation of the peroxide bond by intra-parasitic haem, but there is no consensus regarding the specific protein targets of the resulting radical species for artemisinins and/or the ozonides. This study provides a comprehensive and unbiased chemoproteomic profile of over 400 target proteins, and confirms the specific impact of peroxide antimalarials on redox metabolism. The key role of redox targets is particularly relevant considering that the mechanism of artemisinin resistance appears to involve modulation of peroxide activation and redox homeostasis.

sentences: Screening and selection of 21 novel microhaplotype markers for ancestry inference in ten Chinese Subpopulations Genetic findings suggested that ethnolinguistically diverse populations in China harbor differentiated genetic structure and complex evolutionary admixture history, which provide the genetic basis and theoretical foundation for forensic biogeographical ancestry inference (BGAI). Forensic assays for BGAI among intracontinental eastern Eurasians were previously conducted mainly based on the SNPs or InDels. Microhaplotypes, as a set of closely linked SNPs within 200 base pairs, possess the advantages of both STR and SNP and have great potential in forensic ancestry inference. However, the developed forensic assay based on the ancestry informative microhaplotypes in the BGAI remained to be comprehensively explored, especially in China with enriching genetic diversity. Here, we described a new BGAI panel based on 21 novel identified ancestry informative microhaplotypes that focused on dissected finer-scale ancestry composition of Chinese populations. We initially screened all possible microhaplotypes with high Fst values among five East Asian populations and finally employed 21 candidate microhaplotypes in two multiplex SNaPshot assays. Forensic amplification efficiency and statistically/physically phased haplotypes of the 21 microhaplotypes were validated using both SNaPshot and massively parallel sequencing (MPS) platforms. Followingly, we validated the efficiency of these microhaplotypes for BGAI in 764 individuals from ten Chinese populations. Fine-scale ancestry source and ancestry proportion estimated by the principal component analysis (PCA), multidimensional scaling (MDS), phylogenetic tree and model-based STRUCTURE among worldwide populations and East Asians showed that our customized panel could provide a higher discrimination resolution in both continental population stratification and East Asian regional substructure. East Asian populations could be further classified into linguistically/geographically different intracontinental subpopulations (Tibeto-Burman, Tai-Kadai and others). Finally, we obtained a higher estimated accuracy using training and tested datasets in the microhaplotype-based panel than traditional SNP-based panels. Generally, the above results demonstrated that this microhaplotype panel was robust and suitable for forensic BGAI in Chinese populations, which not only provided a high discriminatory power for continental populations but also discriminated East Asians into linguistically restricted subpopulations.

sentences: Morphine exposure bidirectionally alters c-Fos expression in a sex-, age-, and brain region-specific manner during adolescence Drug and alcohol use during adolescence is common, and data in both humans and preclinical animal models clearly indicate drug exposure during adolescence increases the risk of substance use and other mental health disorders later in life. Adolescence is a period of social, emotional, and cognitive development, and is characterized by increased exploratory behavior, risk-taking, and peer-centered social interactions. These are thought to be behavioral manifestations of developmental plasticity in  reward regions of the brain. Human data indicate that adolescence is not a unitary developmental period, but rather different neural and behavioral sequelae can be observed in early vs. late adolescence. However, most studies with rodent models examine a single adolescent age compared to a mature adult age, and often only in males. Herein, we sought to determine whether the acute response to the opioid morphine would also differ across adolescence, and by sex. By quantifying c-Fos positive cells, a proxy for neural activity, at different stages during adolescence (pre-, early, mid-, and late adolescence) and in multiple reward regions (prefrontal cortex, nucleus accumbens, caudate/putamen), we determined that acute morphine can either reduce or increase c-Fos expression dependent on adolescent age, sex, and brain region. These data suggest that heterogeneity in the consequences of adolescent opioid exposure may be due to the interaction between age- and sex-specific developmental profiles of reward processing in individual brain regions. In future studies, it will be important to add age within adolescence as an independent variable to fully capture the consequences of healthy or abnormal reward-related neural development.

sentences: The voltage and spiking responses of subthreshold resonant neurons to structured and fluctuating inputs: persistence and loss of resonance and variability We systematically investigate the response of neurons to oscillatory currents and synaptic-like inputs and we extend our investigation to non-structured synaptic-like spiking inputs with more realistic distributions of presynaptic spike times. We use two types of chirp-like inputs consisting of (i) a sequence of cycles with discretely increasing frequencies over time, and (ii) a sequence having the same cycles arranged in an arbitrary order. We develop and use a number of frequency-dependent voltage response metrics to capture the different aspects of the voltage response, including the standard impedance (Z) and the peak-to-trough amplitude envelope (VENV) profiles. We show that Z-resonant cells (cells that exhibit subthreshold resonance in response to sinusoidal inputs) also show VENV-resonance in response to sinusoidal inputs, but generally do not (or do it very mildly) in response to square-wave and synaptic-like inputs. In the latter cases the resonant response using Z is not predictive of the preferred frequencies at which the neurons spike when the input amplitude is increased above subthreshold levels. We also show that responses to conductance-based synaptic-like inputs are attenuated as compared to the response to current-based synaptic-like inputs, thus providing an explanation to previous experimental results. These response patterns were strongly dependent on the intrinsic properties of the participating neurons, in particular whether the unperturbed Z-resonant cells had a stable node or a focus. In addition, we show that variability emerges in response to chirp-like inputs with arbitrarily ordered patterns where all signals (trials) in a given protocol have the same frequency content and the only source of uncertainty is the subset of all possible permutations of cycles chosen for a given protocol. This variability is the result of the multiple different ways in which the autonomous transient dynamics is activated across cycles in each signal (different cycle orderings) and across trials. We extend our results to include high-rate Poisson distributed current- and conductance-based synaptic inputs and compare them with similar results using additive Gaussian white noise. We show that the responses to both Poisson-distributed synaptic inputs are attenuated with respect to the responses to Gaussian white noise. For cells that exhibit oscillatory responses to Gaussian white noise (band-pass filters), the response to conductance-based synaptic inputs are low-pass filters, while the response to current-based synaptic inputs may remain band-pass filters, consistent with experimental findings. Our results shed light on the mechanisms of communication of oscillatory activity among neurons in a network via subthreshold oscillations and resonance and the generation of network resonance.

sentences: Improved neutralization of the SARS-CoV-2 Omicron variant after Pfizer-BioNTech BNT162b2 COVID-19 vaccine boosting In late November 2021, the World Health Organization declared the SARS-CoV-2 lineage B.1.1.529 the fifth variant of concern, Omicron. This variant has acquired 15 mutations in the receptor binding domain of the spike protein, raising concerns that Omicron could evade naturally acquired and vaccine-derived immunity. We utilized an authentic virus, multicycle neutralisation assay to demonstrate that sera collected 1, 3, and 6 months post-two doses of Pfizer-BioNTech BNT162b2 has a limited ability to neutralise SARS-CoV-2. However, four weeks after a third dose, neutralizing antibody titres are boosted. Despite this increase, neutralising antibody titres are reduced 4-fold for Omicron compared to lineage A.2.2 SARS-CoV-2.

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β-importins Tnpo-SR and Cadmus and the small GTPase Ran promote ovarian cyst formation in Drosophila Germ cells undergo mitotic expansion via incomplete cytokinesis, forming cysts of undifferentiated cells that remain interconnected prior to meiotic initiation, through mechanisms that are not well-defined. In somatic cells, Ras-related nuclear protein (Ran) spatiotemporally regulates mitotic spindle assembly, cleavage furrow formation and abscission. Here, we identify Ran and {beta}-importins as critical regulators of cyst development in the Drosophila ovary. Depletion of Ran or the {beta}-importins Tnpo-SR and cadmus disrupts oocyte selection and results in egg chambers with variable numbers of germ cells, suggesting abnormal cyst development and cyst fragmentation. We demonstrate that Ran, Tnpo-SR, and Cadmus regulate key cellular processes during cyst formation, including cell cycle dynamics, fusome biogenesis, and ring canal stability, yet do so independently of mitotic spindle assembly. Further, Tnpo-SR and Cadmus control cyclin accumulation and suppress cytokinesis independent of Ran-GTP, suggesting that {beta}-importins sequester protein cargos that normally promote the mitotic-to-meiotic transition. Our data demonstrates that Ran and {beta}-importins are critical for germ cell cyst formation, a role that is likely conserved in other organisms.

SUMMARY STATEMENTRan and two {beta}-importins function coordinately to promote oocyte selection and cyst development in the Drosophila ovary.

sentences: You will know by its tail: a method for quantification of heterogeneity of bacterial populations using single cell MIC profiling Severe non-healing infections are often caused by multiple pathogens or by genetic variants of the same pathogen exhibiting different levels of antibiotic resistance. For example, polymicrobial diabetic foot infections double the risk of amputation compared to monomicrobial infections. Although these infections lead to increased morbidity and mortality, standard antimicrobial susceptibility methods are designed for homogenous samples and are impaired in quantifying heteroresistance. Here, we propose a droplet-based label-free method for quantifying the antibiotic response of the entire population at the single-cell level. We used Pseudomonas aeruginosa and Staphylococcus aureus samples to confirm that the shape of the profile informs about the coexistence of diverse bacterial subpopulations, their sizes, and antibiotic heteroresistance. These profiles could therefore indicate the outcome of antibiotic treatment in terms of the size of remaining subpopulations. Moreover, we studied phenotypic variants of a S. aureus strain to confirm that the profile can be used to identify tolerant subpopulations, such as small colony variants, associated with increased risks for the development of persisting infections. Therefore, the profile is a versatile instrument for quantifying the size of each bacterial subpopulation within a specimen as well as their individual and joined heteroresistance.

sentences: Markerless mouse tracking for social experiments Automated behavior quantification requires accurate tracking of animals. Simultaneous tracking of multiple animals, particularly those lacking visual identifiers, is particularly challenging. Problems of mistaken identities and lost information on key anatomical features are common in existing methods. Here we propose a markerless video-based tool to simultaneously track two socially interacting mice of the same appearance. It incorporates conventional handcrafted tracking and deep learning based techniques, which are trained on a small number of labeled images from a very basic, uncluttered experimental setup. The output consists of body masks and coordinates of the snout and tail-base for each mouse. The method was tested on a series of cross-setup videos recorded under commonly used experimental conditions including bedding in the cage and fiberoptic or headstage implants on the mice. Results obtained without any human intervention showed the effectiveness of the proposed approach, evidenced by a near elimination of identities switches and a 10% improvement in tracking accuracy over a pure deep-learning-based keypoint tracking approach trained on the same data. Finally, we demonstrated an application of this approach in studies of social behaviour of mice, by using it to quantify and compare interactions between pairs of mice in which some are anosmic, i.e. unable to smell. Our results indicated loss of olfaction impaired typical snout-directed social recognition behaviors of mice, while non-snout-directed social behaviours were enhanced. Together, these results suggest that the hybrid approach could be valuable for studying group behaviors in rodents, such as social interactions.

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BWA-MEME: BWA-MEM emulated with a machine learning approach MotivationThe growing use of next-generation sequencing and enlarged sequencing throughput require efficient short-read alignment, where seeding is one of the major performance bottlenecks. The key challenge in the seeding phase is searching for exact matches of substrings of short reads in the reference DNA sequence. Existing algorithms, however, present limitations in performance due to their frequent memory accesses.

ResultsThis paper presents BWA-MEME, the first full-fledged short read alignment software that leverages learned indices for solving the exact match search problem for efficient seeding. BWA-MEME is a practical and efficient seeding algorithm based on a suffix array search algorithm that solves the challenges in utilizing learned indices for SMEM search which is extensively used in the seeding phase. Our evaluation shows that BWA-MEME achieves up to 3.45x speedup in seeding throughput over BWA-MEM2 by reducing the number of instructions by 4.60x, memory accesses by 8.77x, and LLC misses by 2.21x, while ensuring the identical SAM output to BWA-MEM2.

AvailabilityThe source code and test scripts are available for academic use at https://github.com/kaist-ina/BWA-MEME/.

Contacttom418@kaist.ac.kr, dhan.ee@kaist.ac.kr

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Allicin From Garlic Disrupts the Cytoskeleton in Plants Allicin is a defence substance produced by garlic cells upon injury. It is a thiosulfinate showing redox-activity and a broad range of antimicrobial and biocidal activity. It is known that allicin efficiently oxidizes thiol-groups and it has been described as a redox toxin. In order to learn more about the effect of allicin on plants we used pure synthetized allicin, and investigated cytoplasmic streaming in sterile filaments of Tradescantia fluminensis, organelle movement using transgenic Arabidopsis with organelle-specifics GFP-tags, and effects on actin and tubulin in the cytoskeleton using GFP-tagged lines. Auxin distribution in roots was investigated using PIN1:GFP, PIN3:GFP, DR5:GFP and DII-VENUS Arabidopsis reporter lines.

Allicin inhibited cytoplasmic streaming in T. fluminensis and organelle movement of peroxisomes and the Golgi apparatus in a concentration-dependent manner, inhibited root growth and destroyed the correct root tip distribution of auxin.

We speculate that the cytoskeleton can be a primary "receptor" for allicins oxidizing properties and as a consequence cytoskeleton-dependent cellular processes are disrupted.

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A Novel Sequence Labeling Framework for Extracting Drug-Protein Relations from Biomedical Literature Automatic extracting interactions between chemical compound/drug and gene/protein is significantly beneficial to drug discovery, drug repurposing, drug design, and biomedical knowledge graph construction. To promote the development and evaluation of systems that are able to automatically detect in relations between drug and protein, the DrugProt track was organized during the BioCreative VII challenge. This paper describes our approach to this challenging task. In addition to the conventional text classification framework that has been widely used in relation extraction tasks, we propose a novel sequence labeling framework to drug-protein relation extraction. We first comprehensively compared the cutting-edge biomedical pre-trained language models for both frameworks. Then, we explored several ensemble methods to further improve the final performance. In the challenge, our best submission (i.e., the ensemble of models in two frameworks via major voting) achieved the F1-score of 0.795 on the official test set. Further, we realized the sequence labeling framework is more efficient and achieves better performance than does the text classification framework. Finally, our ensemble of the sequence labeling models with majority voting achieves the best F1-score of 0.800 on the test set.

Database URLhttps://github.com/lingluodlut/BioCreativeVII_DrugProt

sentences: Pupil correlates of decision variables in mice playing a competitive mixed-strategy game In a competitive game involving an animal and an opponent, the outcome is contingent on the choices of both players. To succeed, the animal must continually adapt to competitive pressure, or else risk being exploited and lose out on rewards. In this study, we demonstrate that head-fixed mice can be trained to play the iterative competitive game  matching pennies against a virtual computer opponent. We find that the animals performance is well described by a hybrid computational model that includes Q-learning and choice kernels. Comparing between matching pennies and a non-competitive two-armed bandit task, we show that the tasks encourage animals to operate at different regimes of reinforcement learning. To understand the involvement of neuromodulatory mechanisms, we measure fluctuations in pupil size and use multiple linear regression to relate the trial-by-trial transient pupil responses to decision-related variables. The analysis reveals that pupil responses are modulated by observable variables, including choice and outcome, as well as latent variables for value updating, but not action selection. Collectively, these results establish a paradigm for studying competitive decision-making in head-fixed mice and provide insights into the role of arousal-linked neuromodulation in the decision process.

sentences: Antibiotic inhibition of the Plasmodium apicoplast decreases haemoglobin degradation and antagonises dihydroartemisinin action The World Health Organisation (WHO) recommends artemisinin (ART) combinations for treatment of uncomplicated Plasmodium falciparum malaria. Understanding the interaction between co-administered drugs within combination therapies is clinically important to prevent unintended consequences. The WHO guidelines recommend second line treatments that combine artesunate with tetracycline, doxycycline, or clindamycin--antibiotics that target the Plasmodium relict plastid, the apicoplast. In addition, antibiotics can be used simultaneously against other infectious diseases, leading to their inadvertent combination with ARTs. One consequence of apicoplast inhibition is a perturbation to haemoglobin uptake and trafficking--a pathway required for activation of ART derivatives. Here, we show that apicoplast-targeting antibiotics reduce the abundance of the catalyst of ART activation (free haem) in P. falciparum, likely through diminished haemoglobin digestion. We demonstrate antagonism between ART and these antibiotics, suggesting that apicoplast inhibitors reduce ART activation. These data have potential clinical implications due to the reliance on--and widespread use of--both ARTs and these antibiotics in malaria endemic regions.

sentences: Single caudate neurons encode temporally discounted value for formulating motivation for action The term  temporal discounting describes both choice preferences and motivation for delayed rewards. Here we show that neuronal activity in the dorsal part of the primate caudate head (dCDh) signals the temporally discounted value needed to compute the motivation for delayed rewards. Macaque monkeys performed an instrumental task, in which visual cues indicated the forthcoming size and delay duration before reward. Single dCDh neurons represented the temporally discounted value without reflecting changes in the animals physiological state. Bilateral pharmacological or chemogenetic inactivation of dCDh markedly distorted the normal task performance based on the integration of reward size and delay, but did not affect the task performance for different reward sizes without delay. These results suggest that dCDh is involved in encoding the integrated multidimensional information critical for motivation.

sentences: COVID-19 dominant D614G mutation in the SARS-CoV-2 spike protein desensitizes its temperature-dependent denaturation The D614G mutation in the spike protein of SARS-CoV-2 alters the fitness of the virus, making it the dominant form in the COVID-19 pandemic. Here we demonstrated by cryo-electron microscopy that the D614G mutation does not significantly perturb the structure of the spike protein, but multiple receptor binding domains are in an upward conformation poised for host receptor binding. The impact of the mutation lies in its ability to eliminate the unusual cold-induced unfolding characteristics, and to significantly increase the thermal stability under physiological pH. Our findings shed light on how the D614G mutation enhances the infectivity of SARS-CoV-2 through a stabilizing mutation, and suggest an approach for better design of spike-protein based conjugates for vaccine development.

sentences: Bone mechanoregulation allows subject-specific load estimation based on time-lapsed micro-CT and HR-pQCT in vivo Patients at high risk of fracture due to metabolic diseases frequently undergo long-term antiresorptive therapy. However, in some patients treatment is unsuccessful in preventing fractures or causes severe adverse health outcomes. Understanding load driven bone remodelling, i.e. mechanoregulation, is critical to understand which patients are at risk for progressive bone degeneration and may enable better patient selection or adaptive therapeutic intervention strategies. Bone microarchitecture assessment using high-resolution peripheral quantitative computed tomography (HR-pQCT) combined with computed mechanical loads has successfully been used to investigate bone mechanoregulation at the trabecular level. To obtain the required mechanical loads that induce local variances in mechanical strain and cause bone remodelling, estimation of physiological loading is essential. Yet, current models homogenise strain patterns throughout the bone to estimate load distribution in vivo, which may be a flawed assumption for investigating alterations in bone mechanoregulation. By further utilising available spatiotemporal information of time-lapsed bone imaging studies, we developed a mechanoregulation-based load estimation algorithm (MR). MR calculates organ scale loads by scaling and superimposing a set of predefined independent unit loads to optimise measured bone formation in high, quiescence in medium and resorption in low strain regions. We benchmarked our algorithm against a previously published load history algorithm (LH) using synthetic data, micro-CT images of murine vertebrae under defined experimental in vivo loadings and HR-pQCT images from seven patients. Our algorithm consistently outperformed LH in all three datasets. In silico generated time evolutions of distal radius geometries (n = 5) indicated significantly higher sensitivity, specificity, and accuracy for MR than LH (p < 0.01). This increased performance led to substantially better discrimination between physiological and extra-physiological loading in mice (n = 8). Moreover, a significantly (p < 0.01) higher association between remodelling events and computed local mechanical signals were found using MR (CCR = 0.42) than LH (CCR = 0.38) to estimate human distal radius loading. Future applications of MR may enable clinicians to link subtle changes in bone strength to changes in day-to-day loading, identifying weak spots in the bone micro-structure for local intervention and personalised treatment approaches.

sentences: Clumpy coexistence in phytoplankton: The role of functional similarity in community assembly AO_SCPLOWBSTRACTC_SCPLOWEmergent neutrality (EN) suggests that species must be sufficiently similar or sufficiently different in their niches to avoid interspecific competition. Such a scenario results in a transient pattern with clumps and gaps of species abundance along the niche axis (e.g., represented by body size). From this perspective, clumps are groups of coexisting species with negligible fitness differences and stochastic abundance fluctuations. Plankton is an excellent model system for developing and testing ecological theories, especially those related to size structure and species coexistence. We tested EN predictions using the phytoplankton community along the course of a tropical river considering (i) body size structure, (ii) functional clustering of species in terms of morphology-based functional groups (MBFG), and (iii) the functional similarity among species concerning their functional traits. Two main clumps in the body size axis (clump I and II) were conspicuous through time and were detected in different stretches of the river. Clump I comprised medium-sized species from the MBFGs IV, V, and VI while clump II included large-bodied species from the MBFGs V and VI. Pairwise differences in species biovolume correlated with species functional similarity when the whole species pool was considered, but not among species within the same clump. Although clumps comprised multiple MBFGs, the dominant species within the clump belonged always to the same MBFG. Also, within-clump species biovolume increased with functional distinctiveness considering both seasons and stretches, except the lower course. These results suggest that species within clumps behave in a quasi-neutral state, but even minor shifts in trait composition may affect species biovolume. Our findings point that EN belongs to the plausible mechanisms explaining community assembly in river ecosystems.

sentences: A combined experimental-computational approach uncovers a role for the Golgi matrix protein Giantin in breast cancer progression Few studies so far have investigated the impact of different cell migration traits on tumor progression. To address this, we developed a mathematical model wherein cells migrate in two-dimensional space, divide, die or intravasate into the vasculature. Exploring a wide range of speed and persistence combinations, we find that tumor growth positively correlates with increasing speed and higher persistence. As a biologically relevant example, we focused on Golgi fragmentation induced by depletion of Giantin, a Golgi matrix protein, the downregulation of which correlates with poor patient survival. Applying the migration and invasion traits of Giantin depleted cells to our mathematical model, we predict that loss of Giantin increases the number of intravasating cells. This prediction was validated, by showing that circulating tumor cells express significantly less Giantin than primary tumor cells. Altogether, our computational model identifies cell migration traits that regulate tumor progression and uncovers a role of Giantin in breast cancer progression.

sentences: AIMP1-derived peptide secreted from hair follicle stem cells activates dermal papilla cells to promote hair growth Hair follicle stem cells (HFSCs) and dermal papilla cells (DPCs) are crucial in the biogenesis and maintenance of hair follicles (HFs). In this study, a fragment derived from aminoacyl-tRNA synthetase-interacting multifunctional protein1 (AIMP1) was secreted from HFSCs to activate DPCs to maintain hair follicle homeostasis. A histological analysis revealed that AIMP1 levels in hair follicles decreased with hair loss. Hair regrowth in AIMP1-induced mice was faster than that in non-induced mice. Deletion mapping revealed 41 amino acids (TN41, aa 6-46) as the active region of AIMP1. The N-terminal peptide fragment of AIMP1 generated by MMP1 was secreted from Wnt-treated HFSCs to activate DPCs via FGFR2. TN41 activated Akt and ERK, increased {beta}-catenin, and enhanced DPCs activation. TN41 also promoted hair shaft elongation in cultured human hair follicles and improved the hair-inducing activity of cultured DPC spheroids. In summation, the AIMP1 fragment secreted from HFSCs appears to stimulate active hair regrowth through activating DPCs.

sentences: A novel class of long small RNAs associates with Argonaute1 and is up-regulated by nutrient deprivation in the alga Chlamydomonas Small RNAs (sRNAs) associate with Argonaute (AGO) proteins forming effector complexes with key roles in gene regulation and defense responses against molecular parasites. In multicellular eukaryotes, extensive duplication and diversification of RNA interference (RNAi) components have resulted in intricate pathways for epigenetic control of gene expression. The unicellular alga Chlamydomonas reinhardtii also has a complex RNAi machinery, including three AGOs and three Dicer-like (DCL) proteins. However, little is known about the biogenesis and function of most endogenous sRNAs. We demonstrate here that Chlamydomonas contains uncommonly long sRNAs (>26 nt), which associate preferentially with AGO1. Somewhat reminiscent of animal PIWI-interacting RNAs, these long sRNAs are derived from moderately repetitive genomic clusters and their biogenesis appears to be Dicer-independent. Interestingly, long sRNA encoding sequences have been conserved and amplified in phylogenetically related Chlamydomonas species. Additionally, expression of several long sRNAs increases substantially under nutrient deprivation, correlating with the downregulation of predicted target transcripts. We hypothesize that the transposon-like sequences encoding long sRNAs might have been ancestrally targeted for silencing by the RNAi machinery but, during evolution, some long sRNAs might have fortuitously acquired endogenous target genes and become integrated into gene regulatory networks.

sentences: A New Old Tale: Dopamine Transporter implications in an animal model of Attention-Deficit Hyperactivity Disorder Attention deficit/Hyperactivity disorder (ADHD) is one of the most diagnosed psychiatric disorders nowadays. The core symptoms of the condition include hyperactivity, impulsiveness and inattention. The main pharmacological treatment consists of psychostimulant drugs affecting Dopamine Transporter (DAT) function. We have previously shown that genetically modified mice lacking p35 protein (p35KO), which have reduced Cdk5 activity, present key hallmarks resembling those described in animal models useful for studying ADHD. The p35KO mouse displays spontaneous hyperactivity and shows a calming effect of methylphenidate or amphetamine treatment. Interestingly, dopaminergic neurotransmission is altered in these mice as they have an increased Dopamine (DA) content together with a low DA turnover. This led us to hypothesize that the lack of Cdk5 activity affects DAT expression and/or function in this animal model. In this study, we performed biochemical assays, cell-based approaches, quantitative fluorescence analysis and functional studies that allowed us to demonstrate that p35KO mice exhibit decreased DA uptake and reduced cell surface DAT expression levels in the striatum (STR). These findings are supported by in vitro observations in which the inhibition of Cdk5 activity in N2a cells induced a significant increase in constitutive DAT endocytosis with a concomitant increase in DAT localization to recycling endosomes. Taken together, these data provide evidences regarding the role of Cdk5/p35 in DAT expression and function, thus contributing to the knowledge of DA neurotransmission physiology and also providing therapeutic options for the treatment of DA pathologies such as ADHD.

sentences: Local Shifts in Inflammatory and Resolving Lipid Mediators in Response to Tendon Overuse Tendon inflammation has been implicated in both adaptive connective tissue remodeling and overuse-induced tendinopathy. Lipid mediators control the initiation and resolution of inflammation, but their roles within tendon are largely unknown. Here we profiled local shifts in intratendinous lipid mediators via liquid chromatography-tandem mass spectrometry in response to synergist ablation-induced plantaris tendon overuse. Sixty-four individual lipid mediators were detected in homogenates of habitually loaded plantaris tendons from healthy ambulatory rats. This included many bioactive metabolites of the cyclooxygenase (COX), lipoxygenase (LOX), and epoxygenase (CYP) pathways. Synergist ablation induced a robust inflammatory response at day 3 post-surgery characterized by epitenon infiltration of polymorphonuclear leukocytes (PMNs) and macrophages (M{Phi}), heightened expression of inflammation-related genes, and increased intratendinous concentrations of the pro-inflammatory eicosanoids thromboxane B2 (TXB2) and prostaglandin E2 (PGE2). By day 7, M{Phi} became the predominant myeloid cell type in tendon and there were further delayed increases in other COX metabolites including PGD2, PGF2 and PGI2. Specialized pro-resolving mediators (SPMs) including protectin D1 (PD1) and resolvin D6 (RvD6), as well as related pathway markers of D-resolvins (17-HDoHE), E-resolvins (18-HEPE) and lipoxins (15-HETE) were also increased locally in response to tendon overuse, as were many anti-inflammatory fatty acid epoxides of the CYP pathway (e.g. EpETrEs). Nevertheless, intratendinous prostaglandins remained markedly increased even following 28 days of tendon overuse together with a lingering M{Phi} presence. These data reveal a delayed and prolonged local inflammatory response to tendon overuse characterized by an overwhelming predominance of pro-inflammatory eicosanoids and a relative lack of pro-resolving lipid mediators.

sentences: Brain network motifs are markers of loss and recovery of consciousness Motifs are patterns of inter-connections between nodes of a network, and have been investigated as building blocks of directed networks. This study explored the re-organization of 3-node motifs during loss and recovery of consciousness. Nine healthy subjects underwent a 3-hour anesthetic protocol while 128-channel electroencephalography (EEG) was recorded. In the alpha (8-13 Hz) band, five-minute epochs of EEG were extracted for: baseline; induction; unconscious; 30-, 10- and 5-minutes pre-recovery of responsiveness; 30- and 180-minutes post-recovery of responsiveness. We constructed a functional brain network using the weighted and directed phase lag index, on which we calculated the frequency and topology of 3-node motifs. Three motifs (motifs 1, 2 and 5) were significantly present across participants and epochs, when compared to random networks (p<0.05). The topology of motifs 1 and 5 changed significantly between responsive and unresponsive epochs (p<0.01). Motif 1 was constituted by long-range chain-like connections, while motif 5 was constituted by short-range, loop-like connections. Our results suggest that anesthetic-induced unconsciousness is associated with a topological re-organization of network motifs. As motif topological re-organization may precede (motif 5) or accompany (motif 1) the return of responsiveness, motifs could contribute to the understanding of the neural correlates of consciousness.

sentences: Helical Ordering of Envelope Associated Proteins and Glycoproteins in Respiratory Syncytial Virus Filamentous Virions Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Treatments for RSV disease are however limited and efforts to produce an effective vaccine have so far been unsuccessful. Understanding RSV virion structure is an important prerequisite for developing interventions to treat or prevent infection but has been challenging because of the fragility of virions propagated in cell culture. Here we show, using cryogenic electron microscopy (cryoEM) and cryogenic electron tomography (cryoET) of RSV particles cultivated directly on transmission electron microscopy (TEM) grids, that there is extensive helical symmetry in RSV filamentous virions. We have calculated a 16 [A] resolution three-dimensional reconstruction of the viral envelope, targeting the matrix protein (M) that forms an endoskeleton below the viral membrane. These data define a helical lattice of M proteins, showing how M is oriented relative to the viral envelope and that helical ordering of viral glycoproteins that stud the viral envelope is coordinated by the M layer. Moreover, the helically ordered viral glycoproteins in RSV filamentous virions cluster in pairs, which may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV filamentous virions. Overall, the structural data obtained provides molecular insight into the organization of the virion and the mechanism of its assembly.

sentences: Biogenic action of Lactobacillus plantarum SBT2227 promotes sleep in Drosophila melanogaster Lactic acid bacteria (LAB) influence multiple aspects of host brain function via the production of active metabolites in the gut, which is known as the pre/pro-biotic action. However, little is known about the biogenic effects of LAB on host brain function. Here, we reported that the Lactobacillus plantarum SBT2227 promoted sleep in Drosophila melanogaster. Administration of SBT2227 primarily increased the amount of sleep and decreased sleep latency at the beginning of night-time. The sleep-promoting effects of SBT2227 were independent of the existing gut flora. Furthermore, heat treatment or mechanical crushing of SBT2227 did not suppress the sleep-promoting effects, indicative of biogenic action. Transcriptome analysis, and RNAi mini-screening for gut-derived peptide hormones revealed the requirement of neuropeptide F, a homologue of the mammalian neuropeptide Y, for the action of SBT2227. These biogenic effects of SBT2227 on the host sleep provide new insights into the interaction between the brain and gut bacteria.

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Cholesteryl Hemiazelate Induces Lysosome Dysfunction and Exocytosis in Macrophages OBJECTIVEA key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins in lysosomes. This event culminates with the loss of cell homeostasis, inflammation and cell death. In this study we propose to identify the chemical etiological factors and understanding the molecular and cellular mechanisms responsible for the impairment of lysosome function in macrophages.

APPROACH AND RESULTSUsing shotgun lipidomics we have discovered that a family of oxidized lipids (cholesteryl hemiesters, ChE), end products of oxidation of polyunsaturated cholesteryl esters, occurs at higher concentrations in the plasma of two cohorts of cardiovascular disease patients than in the plasma of a control cohort. Macrophages exposed to the most prevalent ChE, cholesteryl hemiazelate (ChA) exhibit lysosome enlargement, peripheral lysosomal positioning, lysosome dysfunction and lipidosis which are irreversible. The transcriptomic profile of macrophages exposed to ChA indicates that the lysosome pathway is deeply affected and is well correlated with lysosome phenotypic and functional changes. Interestingly, the dysfunctional peripheral lysosomes are more prone to fuse with the plasma membrane, secreting their undigested luminal content into the extracellular milieu with potential consequences to the pathology.

CONCLUSIONWe identify ChA not only as one of the molecules involved in the etiology of irreversible lysosome dysfunction culminating with lipidosis but also as a promoter of exocytosis of the dysfunctional lysosomes. The latter event is a new mechanism that may be important in the pathogenesis of atherosclerosis.

sentences: Controlling taxa abundance improves metatranscriptomics differential analysis A common task in analyzing metatranscriptomics data is to identify microbial metabolic pathways with differential RNA abundances across multiple sample groups. With information from paired metagenomics data, current differential methods control for either DNA or taxa abundances to address their strong correlation with RNA abundance. We discovered that when either DNA or taxa abundance is controlled for, RNA abundance still has a strong partial correlation with the other factor. Thus, both factors need to be controlled for in the differential model. In both simulation studies and a real data analysis, we demonstrated that controlling for both DNA and taxa abundances leads to superior performance compared to only controlling for one factor.

sentences: Stimulus dependence of theta rhythmic activity in primate V1 and its potential relevance for visual perception A growing body of psychophysical research reports theta (3-8 Hz) rhythmic fluctuations in visual perception that are often attributed to an attentional sampling mechanism arising from theta rhythmic neural activity in mid- to high-level cortical association areas. However, it remains unclear to what extent such neuronal theta oscillations might already emerge at early sensory cortex like the primary visual cortex (V1), e.g. from the stimulus filter properties of neurons. To address this question, we recorded multi-unit neural activity from V1 of two macaque monkeys viewing a static visual stimulus with variable sizes, orientations and contrasts. We found that among the visually responsive electrode sites, more than 50 % showed a spectral peak at theta frequencies. Theta power varied with varying basic stimulus properties. Within each of these stimulus property domains (e.g. size), there was usually a single stimulus value that induced the strongest theta activity. In addition to these variations in theta power, the peak frequency of theta oscillations increased with increasing stimulus size and also changed depending on the stimulus position in the visual field. Further analysis confirmed that this neural theta rhythm was indeed stimulus-induced and did not arise from small fixational eye movements (microsaccades). When the monkeys performed a detection task of a target embedded in a theta-generating visual stimulus, reaction times also tended to fluctuate at the same theta frequency as the one observed in the neural activity. The present study shows that a highly stimulus-dependent neuronal theta oscillation can be elicited in V1 that appears to influence the temporal dynamics of visual perception.

sentences: Impact of bacterial microcompartment-dependent ethanolamine and propanediol metabolism on Listeria monocytogenes interactions with Caco-2 cells Bacterial microcompartment (BMC) dependent ethanolamine (eut) and propanediol utilization (pdu) has recently been shown to stimulate anaerobic growth of Listeria monocytogenes. This metabolic repertoire conceivably contributes to the competitive fitness of L. monocytogenes in the human gastrointestinal (GI) tract, where these compounds become available following phospholipid degradation and mucus-derived rhamnose metabolism by commensal microbiota. Previous transcriptomics and mutant studies of eut and pdu L. monocytogenes suggested a possible role of eut and pdu BMC metabolism in transmission in foods and pathogenicity, but data on a potential role of L. monocytogenes interaction with human cells is currently absent. First, we ask which cellular systems are expressed in the activation of eut and pdu BMC metabolism and the extent to which these systems are conserved between the states. We find common and unique systems related to metabolic shifts, stress and virulence factors. Next, we hypothesize that these common and unique activated cellular systems contribute to a role in the interaction of L. monocytogenes interaction with human cells. We present evidence that metabolically primed L. monocytogenes with active eut and pdu BMCs, as confirmed by metabolic analysis, transmission electron microscopy and proteomics, show significantly enhanced translocation efficacy compared to non-induced cells in a trans-well assay using Caco-2 cells, while adhesion and invasion capacity was similar. Taken together, our results provide insights into the possible key cellular players that drive translocation efficacy upon eut and pdu BMC activation.

sentences: Selection towards lighter seeds in Danish grasslands over an eight-year period Time-series analysis of cover data from 236 Danish grassland sites demonstrated community selection on seed mass. Across four grassland habitat types and during an eight-year period there was significant community selection towards lighter seeds. In the analysis, the continuous seed size variable was used to group plant species into functional types. This method was chosen in order to account for the sampling process of the abundance data, and is in contrast to most other analyses of trait selection, where the community weighted mean of the traits is used as the dependent variable. Generally, little is known on the underlying causes of possible plant trait community selection in semi-natural grasslands that are not subjected to successional processes.

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Extensive duplication and convergent evolution of an antimicrobial peptide gene Antimicrobial peptides (AMPs) are host-encoded antibiotics that combat invading pathogens. These genes commonly encode multiple products as post-translationally cleaved polypeptides. Recent studies have highlighted roles for AMPs in neurological contexts suggesting functions for these defence molecules beyond infection. During our immune study characterizing the antimicrobial peptide gene Baramicin, we recovered multiple Baramicin paralogs in Drosophila melanogaster and other species, united by their N-terminal IM24 domain. Not all paralogs were immune-induced. Here, through careful dissection of the Baramicin familys evolutionary history, we find that these non-immune paralogs result from repeated events of duplication and subsequent truncation of the coding sequence from an immune-inducible ancestor. These truncations leave only the IM24 domain as the prominent gene product. Surprisingly, using mutation and targeted gene silencing we demonstrate that two such genes are adapted for function in neural contexts in D. melanogaster. We also show enrichment in the head for independent Baramicin genes in other species. The Baramicin evolutionary history reveals that the IM24 Baramicin domain is not strictly useful in an immune context. We thus provide a case study for how an AMP-encoding gene might play dual roles in both immune and non-immune processes via its multiple peptide products. We reflect on these findings to highlight a blind spot in the way researchers approach AMP research in in vivo contexts.

Significance statementAntimicrobial peptides are immune proteins recently implicated in neurological roles. To date little attention has been paid to the contributions of different gene products in this function. Here we show that an antimicrobial peptide gene encodes multiple products with either immune-specific or neurological roles.

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Necroptosis contributes to chronic inflammation and fibrosis in aging liver Inflammaging, characterized by an increase in low-grade chronic inflammation with age, is a hallmark of aging and is strongly associated with various age-related diseases, including chronic liver disease (CLD) and hepatocellular carcinoma (HCC). Because necroptosis is a cell death pathway that induces inflammation through the release of DAMPs, we tested the hypothesis that age-associated increase in necroptosis contributes to chronic inflammation in aging liver. Phosphorylation of MLKL and MLKL-oligomers, markers of necroptosis, as well as phosphorylation of RIPK3 and RIPK1 were significantly upregulated in the livers of old mice relative to young mice and this increase occurred in the later half of life (i.e., after 18 months of age). Markers of M1 macrophages, expression of proinflammatory cytokines (TNF, IL6 and IL-1{beta}), and markers of fibrosis were significantly upregulated in the liver with age and the change in necroptosis paralleled the changes in inflammation and fibrosis. Hepatocytes and liver macrophages isolated from old mice showed elevated levels of necroptosis markers as well as increased expression of proinflammatory cytokines relative to young mice. Short term treatment with the necroptosis inhibitor, necrostatin-1s (Nec-1s), reduced necroptosis, markers of M1 macrophages, expression of proinflammatory cytokines, and markers of fibrosis in the livers of old mice. Thus, our data show for the first time that liver aging is associated with increased necroptosis and necroptosis contributes to chronic inflammation in the liver, which in turn appears to contribute to liver fibrosis and possibly CLD.

Graphical abstract

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sentences: Arf6 anchors Cdr2 nodes at the cell cortex to control cell size at division Fission yeast cells prevent mitotic entry until a threshold cell surface area is reached. The protein kinase Cdr2 contributes to this size control system by forming multiprotein nodes that inhibit Wee1 at the medial cell cortex. Cdr2 node anchoring at the cell cortex is not fully understood. Through a genomic screen, we identified the conserved GTPase Arf6 as a component of Cdr2 signaling. Cells lacking Arf6 failed to divide at a threshold surface area and instead shifted to volume-based divisions at increased overall size. Arf6 stably localized to Cdr2 nodes in its GTP-bound but not GDP-bound state, and its GEF (guanine nucleotide exchange factor) Syt22 was required for both Arf6 node localization and proper size at division. In arf6{Delta} mutants, Cdr2 nodes detached from the membrane and exhibited increased dynamics. These defects were enhanced when arf6{Delta} was combined with other node mutants. Our work identifies a regulated anchor for Cdr2 nodes that is required for cells to sense surface area.

sentences: KnowMore: An Automated Knowledge Discovery Tool for the FAIR SPARC Datasets This manuscript provides the methods and outcomes of KnowMore, the Grand Prize winning automated knowledge discovery tool developed by our team during the 2021 NIH SPARC FAIR Data Codeathon. The National Institutes of Health Stimulating Peripheral Activity to Relieve Conditions (NIH SPARC) program generates rich datasets from neuromodulation researches, curated according to the Findable, Accessible, Interoperable, and Reusable (FAIR) SPARC data standards. These datasets are publicly available through the SPARC Data Portal at sparc.science. Currently, the process of simultaneously comparing and analyzing multiple SPARC datasets is tedious because it requires investigating each dataset of interest individually and downloading all of them to conduct cross-analyses. It is crucial to enhance this process to enable rapid discoveries across SPARC datasets. To fill this need, we created KnowMore, a tool integrated into the SPARC Portal that only requires the user to select their datasets of interest to launch an automated discovery process. KnowMore uses several SPARC resources (Pennsieve, o2S2PARC, SciCrunch, protocols.io, Biolucida), data science methods, and machine learning algorithms in the back end to generate various visualizations in the front end intended to help the user identify potential similarities, differences, and relations across the datasets. These visualizations can lead to a new discovery, new hypothesis, or simply guide the user to the next logical step in their discovery process. The outcome of this project is a SPARC portal-ready code architecture that helps researchers to use SPARC datasets more efficiently and fully leverages their FAIR characteristics. The tool has been built and documented such that more data analysis methods and visualization items could be easily added. The potential for automated discoveries from SPARC datasets is huge given the unique SPARC data ecosystem promoting FAIR data practices, and KnowMore has only demonstrated a small highlight of what could be achieved to speed up discoveries from SPARC datasets.

sentences: Selective Chemical Labeling and Sequencing of 5-Hydroxymethylcytosine in DNA at Single-Base Resolution 5-Hydroxymethylcytosine (5hmC), the oxidative product of 5-methylcytosine (5mC) catalyzed by ten-eleven translocation (TET) enzymes, plays an important role in many biological processes as an epigenetic mediator. Prior studies have shown that 5hmC can be selectively labeled with chemically-modified glucose moieties and enriched using click chemistry with biotin affinity approaches. Besides, DNA deaminases of the AID/APOBEC family can discriminate modified 5hmC bases from cytosine (C)or 5-methylcytosine (5mC). Herein, we developed a method based on ESC whole-genome analysis which could enrich 5hmC-containing DNA by selective chemical labeling and locate 5hmC sites at single-base resolution with enzyme-based deamination. The combination experimental design is an extension of previous methods, and we hope that this cost-effective single base resolution 5hmC sequencing method could be used to promote the mechanism and diagnosis research of 5hmC.

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Developmental dynamics of the neural crest-mesenchymal axis in creating the thymic microenvironment The thymic stroma is composed of epithelial and non-epithelial cells that collectively provide separate microenvironments controlling the homing of blood-born precursors to the tissue, and their subsequent differentiation to functionally mature and correctly selected T cells. While thymic epithelial cells are well characterized for their role in thymopoiesis, a comparably comprehensive analysis of the non-epithelial thymic stroma is lacking. Here we explore at single cell resolution the complex composition and dynamic changes that occur over time in the non-epithelial stromal compartment. We detail across different developmental stages in human and mouse thymus, and in an experimental model of Di George syndrome, the most common form of human thymic hypoplasia, the separate transcriptomes of mouse mesothelium, fibroblasts, neural crest cells, endothelial and vascular mural cells. The detected gene expression signatures identify novel stromal subtypes and relate their individual molecular profiles to separate differentiation trajectories and functions. Specifically, we demonstrate an abundance and unprecedented heterogeneity of diverse fibroblast subtypes that emerge at discrete developmental stages and vary in their expression of key regulatory signalling circuits and components of the extracellular matrix. Taken together, these findings highlight the dynamic complexity of the non-epithelial thymus stroma and link the cells specific gene expression profiles to separate instructive roles essential for normal thymus organogenesis and tissue maintenance.

TeaserSingle cell profiling of thymic stroma identifies a dynamic contribution from neural crest cells to the thymic mesenchyme.

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Phylovar: Towards scalable phylogeny-aware inference of single-nucleotide variations from single-cell DNA sequencing data Single-nucleotide variants (SNVs) are the most common variations in the human genome. Recently developed methods for SNV detection from single-cell DNA sequencing (scDNAseq) data, such as SCI{Phi} and scVILP, leverage the evolutionary history of the cells to overcome the technical errors associated with single-cell sequencing protocols. Despite being accurate, these methods are not scalable to the extensive genomic breadth of single-cell whole-genome (scWGS) and whole-exome sequencing (scWES) data.

Here we report on a new scalable method, Phylovar, which extends the phylogeny-guided variant calling approach to sequencing datasets containing millions of loci. Through benchmarking on simulated datasets under different settings, we show that, Phylovar outperforms SCI{Phi} in terms of running time while being more accurate than Monovar (which is not phylogeny-aware) in terms of SNV detection. Furthermore, we applied Phylovar to two real biological datasets: an scWES triple-negative breast cancer data consisting of 32 cells and 3375 loci as well as an scWGS data of neuron cells from a normal human brain containing 16 cells and approximately 2.5 million loci. For the cancer data, Phylovar detected somatic SNVs with high or moderate functional impact that were also supported by bulk sequencing dataset and for the neuron dataset, Phylovar identified 5745 SNVs with non-synonymous effects some of which were associated with neurodegenerative diseases. We implemented Phylovar and made it publicly available at https://github.com/mae6/Phylovar.git.

sentences: A linear programming-based strategy to save pipette tips in automated DNA assembly Laboratory automation and mathematical optimisation are key to improving the efficiency of synthetic biology research. While there are algorithms optimising the construct designs and synthesis strategies for DNA assembly, the optimisation of how DNA assembly reaction mixes are prepared remains largely unexplored. Here, we focus on reducing the pipette tip consumption of a liquid-handling robot as it delivers DNA parts across a multi-well plate where several constructs are being assembled in parallel. We propose a linear programming formulation of this problem based on the capacitated vehicle routing problem, along with an algorithm which applies a linear programming solver to our formulation, hence providing a strategy to prepare a given set of DNA assembly mixes using fewer pipette tips. The algorithm performed well in randomly generated and real-life scenarios concerning several modular DNA assembly standards, proving capable of reducing the pipette tip consumption by up to 61% in large-scale cases. Combining automatic process optimisation and robotic liquid-handling, our strategy promises to greatly improve the efficiency of DNA assembly, either used alone or in combination with other algorithmic methods.

sentences: LNCRNA expression landscape and specificity between brain regions Long noncoding RNAs (lncRNAs) are transcribed into low potential protein coding RNA molecules, which account for over 70% of mammalian transcriptional products. The role of lncRNAs and their expression is still largely unknown, and the subject of recent investigations. Here, we used bulk RNA sequencing data from the Genotype-Tissue Expression (GTEx) project to reveal the occurrence and identify the specificity of lncRNAs in 13 brain regions (1000 samples). We observed that these highly specific lncRNA were co-expressed with previously known mRNA markers for the 13 study regions of the brain. Further investigation revealed that splicing could influence the divergent biogenesis and enrichment of specific lncRNA alleles in different brain regions. Overall, we demonstrate the use of lncRNA as an independent tool for deconvolving brain regions and further highlights its use for cell-type identification from bulk transcriptome data.

sentences: DNA as a Recyclable Natural Polymer Nature has the ability of circularly re-using its components to produce molecules and materials it needs. An example is the ability of most living organisms of digesting proteins they feed off into amino acids and then using such amino acids in the ribosomal synthesis of new proteins. Recently, we have shown that such recycling of proteins can be reproduced outside living organisms. The key proteins feature that allows for this type of recycling is their being sequence-defined polymers. Arguably, Natures most famous sequence-defined polymer is DNA. Here we show that it is possible starting from sheared calf-DNA to obtain all the four nucleotides as monophosphate-nucleotides (dNMPs). These dNMPs were phosphorylated in a one-pot, multi-enzymes, phosphorylation reaction to generate triphosphate-nucleotides (dNTPs). Finally, we used the dNTPs so achieved (with a global yield of [~]60%) as reagents for PCR (polymerase chain reaction) to produce target DNA strands, and for the diagnose of targeted DNA by quantitative PCR (qPCR). This approach is an efficient, convenient, and environmentally friendly way to produce dNTPs and DNA through recycling according to the paradigm of circular economy.

sentences: Massive export of diazotrophs across the tropical south Pacific Ocean Diazotrophs are widespread microorganisms that alleviate nitrogen limitation in 60% of our oceans, regulating marine productivity. Yet, their contribution to organic matter export has not been quantified, making an assessment of their impact on the biological carbon pump impossible. Here, we demonstrate that cyanobacterial and non-cyanobacterial diazotrophs are massively exported down to 1000 m-depth in the western subtropical South Pacific Ocean (WTSP), accounting for up to 52-100% of the total particulate nitrogen export fluxes. We further demonstrate that small size unicellular diazotrophs (UCYN, 1-8 {micro}m) are exported more efficiently than filamentous diazotrophs (>100-1000 {micro}m) under the form of large (>50 {micro}m) aggregates linked by an extracellular organic matrix. Beyond the WTSP, our data are supported by analysis of the Tara Oceans metagenomes collected in other ocean basins, showing that diazotrophs are systematically detected in mesopelagic waters when present at the surface. We thus conclude that diazotrophs are key players in carbon sequestration in the ocean and need to be considered in future studies to improve the accuracy of current regional and global estimates of export.

sentences: SARS-CoV-2 infects and replicates in photoreceptor and retinal ganglion cells of human retinal organoids Several studies have pointed to retinal involvement in COVID-19 disease, yet many questions remain regarding the ability of SARS-CoV-2 to infect and replicate in retinal cells and its effects on the retina. Here we have used human stem cell-derived retinal organoids to study retinal infection by the SARS-CoV-2 virus. Indeed, SARS-CoV-2 can infect and replicate in retinal organoids, as it is shown to infect different retinal lineages, such as retinal ganglion cells and photoreceptors. SARS-CoV-2 infection of retinal organoids also induces the expression of several inflammatory genes, such as interleukin 33, a gene associated with acute COVID-19 disease and retinal degeneration. Finally, we show that the use of antibodies to block the ACE2 receptor significantly reduces SARS-CoV-2 infection of retinal organoids, indicating that SARS-CoV-2 infects retinal cells in an ACE2-dependent manner. These results suggest a retinal involvement in COVID-19 and emphasize the need to monitor retinal pathologies as potential sequelae of "long COVID".

sentences: Emotional states as distinct configurations of functional brain networks The conceptualization of emotional states as patterns of interactions between large-scale brain networks has recently gained support. Yet, few studies have directly examined the brains network structure during emotional experiences. Here, we investigated the brains functional network organization during experiences of sadness, amusement, and neutral states elicited by movies, in addition to a resting-state. We tested the effects of the experienced emotion on individual variability in the brains functional connectome. Next, for each state, we defined a community structure of the brain and quantified its segregation and integration. Our results show that emotional states increase the similarity between and within individuals in the brains functional connectome. Second, in the brains modular organization, sadness, relative to amusement, was associated with higher modular integration and increased connectivity of cognitive control networks: the salience and fronto-parietal networks. Last, in both the functional connectome and the emotional report, we found higher similarity among women. Our results suggest that the experience of emotion is linked to a reconfiguration of whole-brain distributed, not emotion-specific, functional networks and that the brains topological structure carries information about the subjective emotional experience.

sentences: Continuous sensing of nutrients and growth factors by the mTORC1-TFEB axis mTORC1 senses nutrient and growth factor status and phosphorylates downstream targets, including the transcription factor TFEB, to coordinate metabolic supply and demand. The molecular mechanisms of mTORC1 activation are thought to enforce a strict requirement for simultaneous amino acid and growth factor stimuli, but this model has not been evaluated with quantitative or single-cell methods. Here, we develop a series of fluorescent protein-TFEB fusions and investigate how combinations of stimuli jointly regulate signaling from mTORC1 to TFEB at the single-cell level. Live-cell imaging of individual cells revealed that mTORC1-TFEB signaling responds with graded changes to individual amino acid and growth factor inputs, rather than behaving as a logical "AND" gate. We find that mTORC1 inputs can be sequentially sensed, with responses that vary between mTORC1 substrates and are amplified by input from other kinases, including GSK3{beta}. In physiologically relevant concentrations of amino acids, we observe fluctuations in mTORC1-TFEB signaling that indicate continuous responsiveness to nutrient availability. Our results clarify how the molecular regulation of mTORC1 enables homeostatic processes at the cellular level and provide a more precise understanding of its behavior as an integrator of multiple inputs.

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Differential proteomic analysis of laser-microdissected penetration glands of avian schistosome cercariae with a focus on proteins involved in host invasion Schistosome invasive stages, cercariae, leave intermediate snail hosts, penetrate the skin of definitive hosts, and transform to schistosomula migrating to final localization. During invasion, cercariae employ histolytic and other bioactive products of specialized holocrine secretory cells - postacetabular (PA) and circumacetabular (CA) penetration glands. Although several studies attempted to characterize protein composition of the in vitro induced gland secretions in Schistosoma mansoni and Schistosoma japonicum, the results were inconsistent and dependent on the method of sample collection and processing. Products of both gland types mixed during their secretion did not allow localization of identified proteins to a particular gland. Here we compared proteomes of separately isolated cercarial gland cells of the avian schistosome Trichobilharzia szidati employing laser-assisted microdissection and shotgun LC-MS/MS, thus obtaining the largest dataset so far concerning the representation and localization of cercarial penetration gland proteins. We optimized the methods of sample processing with cercarial bodies (heads) first. Alizarin-pre-stained, chemically non-fixed samples provided optimal results of MS analyses, and enabled distinguishing PA and CA glands for microdissection. Using 7.5 x 106 m3 sample volume per gland replicate, we identified 3347 peptides assigned to 792 proteins, from which 461 occurred in at least 2 of 3 replicates in either gland type (PA = 455, 40 exclusives; CA = 421, 6 exclusives; 60 proteins differed significantly in their abundance between the glands). Peptidases of 5 catalytic types accounted for ca. 8 % and 6 % of reliably identified proteins in PA and CA glands, respectively. Invadolysin, nardilysin, cathepsins B2 and L3, and elastase 2b orthologs were the major gland endopeptidases. Two cystatins and a serpin were highly abundant peptidase inhibitors in the glands. CA glands were rich in venom allergen-like proteins. The assembled total cercarial body proteome included 1631 identified proteins and revealed additional interesting factors possibly related to tissue invasion.

HighlightsO_LIProteomes of two penetration gland types in schistosome cercariae greatly differ
C_LIO_LIPostacetabular glands possess 40 unique proteins and are abundant in hydrolases
C_LIO_LICircumacetabular glands posses 6 unique proteins and are rich in VAL proteins
C_LIO_LIPeptidases make up 8 % of postacetabular and 6 % of circumacetabular gland proteins
C_LIO_LICercarial elastase is unique to circumacetabular glands of Trichobilharzia szidati
C_LI

Note: Supplementary data associated with this article All supplementary data files can be accessed from the following link: http://www.helminthology.cz/supplementary_files.html

sentences: Spatial regulation of AMPK signaling revealed by a sensitive kinase activity reporter AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics which coordinates metabolism by phosphorylating a plethora of substrates throughout the cell. But whether AMPK activity is regulated at different subcellular locations to provide precise spatial and temporal control over metabolism is unclear. Genetically encoded AMPK activity reporters (AMPKAR) have provided a window into spatial AMPK activity, but the limited dynamic range of current AMPKARs hinders detailed study. To monitor the dynamic activity of AMPK with high sensitivity, we developed a single-fluorophore AMPK activity reporter (ExRai AMPKAR) that exhibits an excitation ratiometric fluorescence change upon phosphorylation by AMPK, with over 3-fold greater response compared to previous AMPKARs. Using subcellularly localized ExRai AMPKAR, we found that the activity of AMPK at the lysosome and mitochondria are differentially regulated. While different activating conditions, irrespective of their effects on ATP, robustly yet gradually increase mitochondrial AMPK activity, lysosomal AMPK activity accumulates with much faster kinetics. Genetic deletion of the canonical upstream kinase liver kinase B1 (LKB1) resulted in slower AMPK activity at lysosomes but did not affect the response amplitude at either location, in sharp contrast to the necessity of LKB1 for maximal cytoplasmic AMPK activity. We further discovered AMPK activity in the nucleus, which resulted from LKB1-mediated cytoplasmic activation of AMPK followed by nuclear shuttling. Thus, a new, sensitive reporter for AMPK activity, ExRai AMPKAR, in complement with mathematical and biophysical methods, captured subcellular AMPK activity dynamics in living cells and unveiled complex regulation of AMPK signaling within subcellular compartments.

sentences: Odor motion sensing enables complex plume navigation Studies dating back a century (Flugge, 1934) have stressed the critical role of the wind as the primary directional cue in odor plume navigation. Here, we show that Drosophila shape their navigational decisions using a second directional cue - the direction of motion of odors - which they detect from the temporal correlations of the odor signal between their two antennae. Using a high-resolution virtual reality paradigm to deliver spatiotemporally complex fictive odors to freely-walking flies, we demonstrate that such odor direction sensing is computationally equivalent to motion detection algorithms underlying motion detection in vision. Simulations and theoretical analysis of turbulent plumes reveal that odor motion contains valuable directional information absent from the airflow; indeed, this information is used by both Drosophila and virtual agents to navigate naturalistic odor environments. The generality of our findings suggests that odor direction sensing is likely used throughout the animal kingdom, and could significantly improve olfactory robot navigation in harsh chemical environments.

sentences: Accumulation of the GSK3 target protein β-catenin is lethal for B cell precursors and malignant B cells Glycogen synthase kinase 3 (GSK3) is a ubiquitously expressed kinase involved in a myriad of biological processes. Although GSK3 mediated phosphorylation has been shown to induce the degradation of many pro-survival and pro-proliferation factors, cancer cells of different origin show reduced proliferation or survival after GSK3 inhibition. Our current understanding of the role GSK3 plays in normal mature B cells, B cell precursors and transformed B cells is incomplete and does not allow to assess whether GSK3 inhibitors can be used to treat B cell derived malignancies. Here we identify {beta}-catenin as the major factor driving GSK3-inhibition induced changes in B cells. We show that {beta}-catenin accumulation has opposing effects on cell metabolism and survival in mature B cells and B cell precursors. Moreover, we demonstrate that {beta}-catenin destabilizes the commitment to the B cell lineage. In summary, our study identifies {beta}-catenin induced signaling as a factor that can be exploited to limit the survival of malignant B cells.

sentences: Lack of blocking activity in anti-CTLA-4 antibodies reduces toxicity, but not anti-tumor efficacy Anti-CTLA-4 antibodies such as ipilimumab were among the first immune-oncology agents to show significantly improved outcomes for patients. However, existing anti-CTLA-4 therapies fail to induce a response in a majority of patients and can induce severe, immune-related adverse events. It has been assumed that checkpoint inhibition, i.e., blocking the interaction between CTLA-4 and its ligands, is the primary mechanism of action for ipilimumab. In this study we present evidence that checkpoint inhibition is not a primary mechanism of action for efficacy of anti-CTLA-4 antibodies. Instead, the primary mechanism for efficacy is FcR-mediated Treg depletion in the tumor microenvironment. First, we identified a monoclonal antibody (mAb) that binds to CTLA-4 at an epitope that differs from ipilimumabs by only a few amino acids, yet has limited checkpoint inhibitor activity. Surprisingly, the weak checkpoint inhibitor has superior anti-tumor activity compared to ipilimumab in a murine model. The weak checkpoint inhibitor also induces less Treg proliferation and has increased ability to induce in vitro FcR signaling and in vivo depletion of intratumoral Tregs. Further experiments showed that the enhanced FcR activity of the weak checkpoint inhibitor likely contributes to its enhanced anti-tumor activity. Importantly, we also showed that weak checkpoint inhibition was associated with lower toxicity in murine models. Our work suggests that new anti-CTLA-4 drugs should be optimized for Treg depletion rather than checkpoint inhibition.

sentences: Lateralized deficits in arousal processing after insula lesions: behavioral and autonomic evidence A large body of evidence ascribes a pivotal role in emotion processing to the insular cortex. However, the complex structure and lateralization of emotional deficits following insular damage are not understood. Here, we investigated emotional ratings of valence and arousal and skin conductance responses (SCR) to a graded series of emotionally arousing scenes in patients with left (n = 10) or right (n = 9) insular damage and in healthy controls (n = 18). We found a significant reduction in overall SCRs, arousal ratings and valence extremity scores in right-lesioned patients, as compared to left-lesioned patients and healthy controls. Additional analyses of correlations between subjective arousal ratings resp. SCR and normative arousal ratings revealed that both lesion groups had evaluative and physiological difficulties to discover changes in stimulus arousal. Although no group differences emerged on overall ratings of valence, analysis of correlations between subjective and normative valence ratings displayed markedly reduced accuracy in right-lesioned patients, as compared to left-lesioned patients and healthy controls. Our findings support the hypothesis that the left and right insulae subserve different functions in emotion processing, potentially due to asymmetrical representations of autonomic information in the left and right human forebrain. The right insula may serve as integral node for sympathetic arousal and cognitive affective processing.

sentences: Rapid profiling of protein complex re-organization in perturbed systems Protein complexes constitute the primary functional modules of cellular activity. To respond to perturbations, complexes undergo changes in their abundance, subunit composition or state of modification. Understanding the function of biological systems requires global strategies to capture this contextual state information on protein complexes and interaction networks. Methods based on co-fractionation paired with mass spectrometry have demonstrated the capability for deep biological insight but the scope of studies using this approach has been limited by the large measurement time per biological sample and challenges with data analysis. As such, there has been little uptake of this strategy beyond a few expert labs into the broader life science community despite rich biological information content. We present a rapid integrated experimental and computational workflow to assess the re-organization of protein complexes across multiple cellular states. It enables complex experimental designs requiring increased sample/condition numbers. The workflow combines short gradient chromatography and DIA/SWATH mass spectrometry with a data analysis toolset to quantify changes in complex organization. We applied the workflow to study the global protein complex rearrangements of THP-1 cells undergoing monocyte to macrophage differentiation and a subsequent stimulation of macrophage cells with lipopolysaccharide. We observed massive proteome organization in functions related to signaling, cell adhesion, and extracellular matrix during differentiation, and less pronounced changes in processes related to innate immune response induced by the macrophage stimulation. We therefore establish our integrated differential pipeline for rapid and state-specific profiling of protein complex organization with broad utility in complex experimental designs.

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Comethyl: A network-based methylome approach to investigate the multivariate nature of health and disease Health outcomes are frequently shaped by difficult to dissect inter-relationships between biological, behavioral, social, and environmental factors. DNA methylation patterns reflect such multi-variate intersections, providing a rich source of novel biomarkers and insight into disease etiologies. Recent advances in whole-genome bisulfite sequencing (WGBS) enable investigation of DNA methylation over all genomic CpGs, but existing bioinformatic approaches lack accessible system-level tools. Here, we develop the R package Comethyl, for weighted gene correlation network analysis (WGCNA) of user-defined genomic regions that generates modules of comethylated regions, which are then tested for correlations with sample traits. First, regions are defined by CpG genomic location or regulatory annotation and filtered based on CpG count, sequencing depth, and variability. Next, correlation networks are used to find modules of interconnected nodes using methylation values within the selected regions. Each module containing multiple comethylated regions is reduced in complexity to a single eigennode value, which is then tested for correlations with experimental metadata. Comethyl has the ability to cover the noncoding regulatory regions of the genome with high relevance to interpretation of genome-wide association studies and integration with other types of epigenomic data. We demonstrate the utility of Comethyl on a dataset of male cord blood samples from newborns later diagnosed with autism spectrum disorder (ASD) versus typical development. Comethyl successfully identified an ASD-associated module containing gene regions with brain glial functions. Comethyl is expected to be useful in uncovering the multi-variate nature of health disparities for a variety of common disorders. Comethyl is available at github.com/cemordaunt/comethyl.

Description of the AuthorsCharles E. Mordaunt, Ph.D. developed Comethyl while a postdoctoral fellow in the department of Medical Microbiology and Immunology at UC Davis. He is currently a Computational Biologist at GSK.

Julia S. Mouat is a doctoral student in the Integrative Genetics and Genomics graduate group at UC Davis with interests in health disparities and intergenerational epigenetic risk factors for autism spectrum disorders.

Rebecca J. Schmidt, Ph.D. is an Associate Professor of Public Health Sciences at UC Davis, with expertise in the use of epigenetics in epidemiology and neurodevelopmental disorders.

Janine M. LaSalle, Ph.D. is a Professor of Medical Microbiology and Immunology, Co-Director of the Perinatal Origins of Disparities Center, and Deputy Director of the Environmental Health Sciences Center at UC Davis, with expertise in epigenomics and neurodevelopmental disorders.

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Exploring the structural basis to develop efficient multi-epitope vaccines displaying interaction with HLA and TAP and TLR3 molecules to prevent NIPAH infection, a global threat to human health BackgroundNipah virus (NiV) is an emerging zoonotic virus that caused several serious outbreaks in the South Asian region with high mortality rates ranging from 40 to 90% since 2001. NiV infection causes lethal encephalitis and respiratory disease with the symptom of endothelial cell-cell fusion. No specific vaccine has yet been reported against NiV.

Methodology and Principal FindingsRecently, the design of some Multi-Epitope Vaccines (MEV) has been proposed but that involves vary limited number of epitopes which limits the potential of vaccine. To address the urgent need for a specific and effective vaccine against NiV infection, in the present study, we have designed two MEVs composed of 33 Cytotoxic T lymphocyte (CTL) epitopes and 38 Helper T lymphocyte (HTL) epitopes. Both the MEVs carry potential B cell linear epitope overlapping regions, B cell discontinuous epitopes as well as IFN-{gamma} inducing epitopes. Hence the designed MEVs carry potential to elicit cell-mediated as well as humoral immune response. Selected CTL and HTL epitopes were validated for their stable molecular interactions with HLA class I and II alleles as well as in case of CTL epitopes, with human transporter associated with antigen processing (TAP). Human {beta}-defensin 2 and {beta}-defensin 3 were used as adjuvants to enhance the immune response of both the MEVs. Molecular dynamics simulation studies of MEVs-TLR3 ectodomain (Toll-Like Receptor 3) complex indicate the stable molecular interaction. Further, the codon optimized cDNA of both the MEVs has shown high expression potential in the mammalian host cell line (Human). Hence for further studies, the designed MEV constructs could be expressed and tried in-vivo as potential vaccine candidates against NiV.

ConclusionWe conclude that the MEVs designed and in silico validated here could be highly potential vaccine candidate to combat NiV, with greater effectiveness, high specificity and large human population coverage worldwide.

AUTHOR SUMMARYNipah Virus (NiV) has caused several outbreaks in past two decades calming large number of human lives. Our present work aims to design and in silico validate Multi-Epitope Vaccine against NiV. The current approach to design vaccine involves whole virus or full length proteins as vaccine candidates against NiV. These approaches carry chances of raising the unwanted non-neutralizing antibodies which have been found to cause clinical complexities. Recently few Multi-Epitope vaccines have also been proposed, but they have involved limited number of epitopes for vaccine design in result limiting the effectiveness and human population coverage. Here in our MEVs we have involved all the proteins of NiV to design the vaccine. Moreover since we have used in silico validated epitopes we may conclude that the here proposed MEVs would be highly specific, effective and potential vaccine candidate to combat NiV with large human population coverage worldwide.

sentences: Isolation and characterization of a Cyprinid herpesvirus strain YZ01 from apparently healthy goldfish after rising water temperature A Cyprinid herpesvirus 2 (CyHV-2) strain YZ01 was isolated from an apparently healthy goldfish with history of exposure to CyHV-2 when the water temperature was heated from 3-6{square} to 25{square}. Brain, kidney, spleen (referred to as BKS) and intestine of sick and dead fish as well as crucian carp (CrCB) or goldfish brain cell (GFB) suspensions inoculated with above tissue homogenate were collected for PCR assay.Infected cell layers were prepared into ultrathin sections for transmission electron microscopy. Virus DNA were purified from cell suspensions for whole genome sequencing and analysis. Intestine and mixed brain, kidney and spleen fluid (BKS) of dead goldfish and BKS-infected CrCB suspensions as well as their subsequent passages were tested positive by PCR. Nucleus deformation, lattice-like arrangement of inclusion bodies and various forms of immature and mature virions in the nucleus, cytoplasm and outside of cell membranes were observed. Whole genome analysis of 7 CyHV-2 strains available in GeneBank showed that all strains genomes share very high homology while YZ01 was more close to CyHV-2 CNDF-TB2015 and SY-C1 which are classified as C genotype.

sentences: From the Modern Synthesis to the Molecular Synthesis: updating how we teach and assess evolution by natural selection Evolution by natural selection is recognized as both the most important concept in undergraduate biology and the most difficult to teach. Unfortunately, teaching and assessment of evolution have been impaired by legacy approaches that focus on Darwins original insights and the Modern Synthesis integration of Mendelian genetics, but ignore or downplay advances from what we term the Molecular Synthesis. To create better alignment between instructional approaches and contemporary research in the biosciences, we propose that the primary learning goal in teaching evolution should be for students to connect genotypes, phenotypes, and fitness. To support this approach, we developed and tested assessment questions and scoring rubrics called the Extended Assessing Conceptual Reasoning of Natural Selection (E-ACORNS) instrument. Initial E-ACORNS data suggest that after traditional instruction, few students recognize the molecular synthesis--prompting us to propose that introductory course sequences be re-organized with the molecular synthesis as their central theme.

sentences: Development of 3D breast cancer models with human T cells expressing engineered MAIT cell receptors Immunotherapy has revolutionized cancer treatment with the advent of advanced cell engineering techniques aimed at targeted therapy with reduced systemic toxicity. However, understanding the underlying immune-cancer interactions require development of advanced three-dimensional (3D) models of human tissues. In this study, we developed proof-of-concept 3D tumor models with increasing complexity to study the cytotoxic responses of CD8+ T cells, genetically engineered to express mucosal-associated invariant T (MAIT) cell receptors, towards MDA-MB-231 breast cancer cells. Homotypic MDA-MB-231 and heterotypic MDA-MB-231/human dermal fibroblast (HDF) tumor spheroids were primed with precursor MAIT cell ligand 5-amino-6-D-ribitylaminouracil (5-ARU). Engineered T cells effectively eliminated tumors after a 3-day culture period, demonstrating that the engineered TCR recognized MR1 expressing tumor cells in the presence of 5-ARU. Tumor cell killing efficiency of engineered T cells were also assessed by encapsulating these cells in fibrin, mimicking a tumor extracellular matrix microenvironment. Expression of proinflammatory cytokines such as IFN{gamma}, IL-13, CCL-3 indicated immune cell activation in all tumor models, post immunotherapy. Further, in corroborating the cytotoxic activity, we found that granzymes A and B were also upregulated, in homotypic as well as heterotypic tumors. Finally, a 3D bioprinted tumor model was employed to study the effect of localization of T cells with respect to tumors. T cells bioprinted proximal to the tumor had reduced invasion index and increased cytokine secretion, which indicated a paracrine mode of immune-cancer interaction. Development of 3D tumor-T cell platforms may enable studying the complex immune-cancer interactions and engineering MAIT cells for cell-based cancer immunotherapies.

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Helminth infection is associated with dampened cytokine responses to viral and bacterial stimulations in Tsimane hunter-horticulturalists. BackgroundSoil-transmitted helminth (STH) infections can catalyze immunological changes that affect the response to subsequent infections, particularly those that elicit strong inflammatory responses. As globalization heightens the risk that remote communities with high STH prevalence will encounter novel pathogens, understanding how STHs shape immune responses to these downstream infections becomes increasingly crucial.

MethodologyWe worked with Tsimane forager-horticulturalists in the Bolivian Amazon, where STHs are prevalent. We tested whether STHs and eosinophil levels--most likely indicative of infection in this population--are associated with dampened immune responses to in vitro stimulation with H1N1 and lipopolysaccharide (LPS) antigens. Whole blood samples (n = 179) were treated with H1N1 vaccine and LPS and assayed for 13 cytokines (interferon gamma [INF-{gamma}], interleukin [IL]-1{beta}, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, Granulocyte-macrophage colony-stimulating factor [GM-CSF], and Tumor necrosis factor-alpha [TNF-]). We evaluated how STHs and eosinophil levels affected cytokine responses and T helper (Th) 1 and Th2-cytokine suite responses to stimulation.

ResultsInfection with Ascaris lumbricoides was significantly (p [≤] 0.05) associated with lower response of some cytokines to H1N1 and LPS in women. Eosinophils were significantly negatively associated with some cytokine responses to H1N1 and LPS, with the strongest effects in women, and associated with a reduced Th1- and Th2-cytokine response to H1N1 and LPS in women and men.

Conclusions and implicationsWe find that STHs were associated with dampened cytokine responses to certain viral and bacterial antigens, and suggest that this mitigation of host-induced damage may reduce the incidence of cytokine storms in populations with high STH prevalence.

sentences: Succession comprises a sequence of threshold-induced community assembly processes towards multidiversity Research on successions and community assembly both address the same processes such as dispersal, species sorting, and biotic interactions but lack unifying concepts. Recent theoretical advances integrated both research lines proposing a sequence of stochastic and niche-based processes along successional gradients. Shifts in assembly processes are predicted to occur abruptly once abiotic and biotic factors dominate over dispersal as main driver. Considering the multidiversity composed of five organismal groups including plants, animals, and microbes, we empirically show that stochastic dispersal-dominated community assembly is replaced by environmental filters and biotic interactions after around 60 years of succession in a glacier forefield. The niche-based character of later successional processes is further supported by a decline in multi-beta-diversity. Our results may update concepts of community assembly by considering multiple taxa, help to bridge the gap between research on successions and community assembly, and provide new insights into the emergence of multidiverse and complex ecosystems.

sentences: A curved manifold orients rotational dynamics in motor cortex Controlling arm movements requires complex, time-varying patterns of muscle activity 1,2. Accordingly, the responses of neurons in motor cortex are complex, time-varying, and heterogeneous during reaching 2-4. When examined at the population level, patterns of neural activity evolve over time according to dynamical rules 5,6. During reaching, these rules have been argued to be "rotational" 7 or variants thereof 8,9, containing coordinated oscillations in the spike rates of individual neurons. While these models capture key aspects of the neural responses, they fail to capture others - accounting for only 20-50% of the neural response variance. Here, we consider a broader class of dynamical models. We find that motor cortex dynamics take an unexpected form: there were 3-4 rotations at fixed frequencies in M1 and PMd explaining more than 90% of neural responses, but these rotations occurred in different portions of state space when movements differ. These rotations appear to reflect a curved manifold of fixed points in state space, around which dynamics are locally rotational. These fixed-frequency rotations obeyed a simple relationship with movement: the orientation of rotations in motor cortex activity were related almost linearly to the movement the animal made, allowing linear decoding of reach kinematic time-courses on single trials. This model constitutes a fundamentally novel way to consider pattern generation: like placing a record player in a large bowl, the frequency of activity is fixed, but the location of motor cortex activity on a curved manifold sets the orientation of locally-rotational dynamics. This system simplifies motor control, helps reconcile conflicting frameworks for interpreting motor cortex, and enables greatly improved neural decoding.

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Individual-based quantitative temporal beta-diversity: Novel indices for assessing community compositional shifts accounting for changes in the properties of individuals AimRevealing patterns of temporal biodiversity change is an urgent issue in light of the effects of global climate change. In addition, climate change is known to modify not only biodiversity but also individual life-history. Nevertheless, although the connection between individual physiology and large-scale ecological patterns is a major challenge for the mechanistic understanding of macroecology, large gaps exist between individual life-history and biodiversity. In this context, novel methods can be used to evaluate changes in individual properties and their effects on biodiversity.

InnovationNovel indices that can evaluate compositional shifts across time by explicitly considering the contributions of the life-histories (i.e., growth, mortality, and recruitment) of individuals in a community were successfully developed. By applying subset data of the Forest Inventory and Analysis database for the state of Rhode Island in the USA, changes in the contribution of individual life-histories to biodiversity change were successfully identified.

Main conclusionsThe development of the proposed novel indices not only represents methodological progress in community ecology and macroecology but also a conceptual advance, bridging studies of biodiversity and individual life-history and physiology. The individual-based diversity index opens the door to the possibility of individual-based biodiversity science, which could facilitate understanding the comprehensive effects of climate change across different hierarchies of biological organisation.

sentences: Partial genome assembly of the medicinal plant Ephedra sinica. Ephedra sinica is a high-value medicinal plant that produces important phenylpropylamino alkaloids pseudoephedrine and ephedrine. Few genomics resources exist for E. sinica, which has been characterized as a tetraploid with a monoploid genome size of 8.56 Gb. Here we reported a partial genome assembly of E. sinica (12.8 Gb) based on Illumina short-read sequencing at low coverage.

sentences: A comparative baseline of coral disease across the central Red Sea The Red Sea is a unique environment for corals with a strong environmental gradient characterized by temperature extremes and high salinities, but minimal terrestrial runoff or riverine input and their associated pollution. Disease surveys were conducted along 22 reefs in the central Red Sea along the Saudi Arabian coast in October 2015, which coincided with a bleaching event. Our objectives were to 1) document types, prevalence, and distribution of coral diseases in a region with minimal terrestrial input, 2) compare regional differences in diseases and bleaching along a latitudinal gradient of environmental conditions, and 3) use histopathology to characterize disease lesions at the cellular level. Coral reefs of the central Red Sea had a widespread but a surprisingly low prevalence of disease (<0.5%), based on the examination of >75,750 colonies. Twenty diseases were recorded affecting 16 coral taxa and included black band disease, white syndromes, endolithic hypermycosis, skeletal eroding band, growth anomalies and focal bleached patches. The three most common diseases were Acropora white syndrome (59.1% of the survey sites), Porites growth anomalies (40.9%), and Porites white syndrome (31.8%). Over half of the coral genera within transects had lesions and corals from the genera Acropora, Millepora and Lobophyllia were the most commonly affected. Cell-associated microbial aggregates were found in four coral genera resembling patterns found in the Indo-Pacific. Differences in disease prevalence, coral cover, amount of heat stress as measured by degree heating weeks (DHW) and extent of bleaching was evident among sites. Disease prevalence was not explained by coral cover or DHW, and a negative relationship between coral bleaching and disease prevalence was found. The northern-most sites off the coast of Yanbu had the highest average DHW values but absence of bleaching and the highest average disease prevalence was recorded. Our study provides a foundation and baseline data for coral disease prevalence in the Red Sea, which is projected to increase as a consequence of increased frequency and severity of ocean warming.

sentences: A comprehensive score reflecting memory-related fMRI activations and deactivations as potential biomarker for neurocognitive aging Older adults and particularly those at risk for developing dementia typically show a decline in episodic memory performance, which has been associated with altered memory network activity detectable via functional magnetic resonance imaging (fMRI). To quantify the degree of these alterations, a score has been developed as a putative imaging biomarker for successful aging in memory for older adults (Functional Activity Deviations during Encoding, FADE; Duzel et al., 2011). Here, we introduce and validate a more comprehensive version of the FADE score, termed FADE-SAME (Similarity of Activations during Memory Encoding), which differs from the original FADE score by considering not only activations but also deactivations in fMRI contrasts of stimulus novelty and successful encoding, and by taking into account the variance of young adults activations. We computed both scores for novelty and subsequent memory contrasts in a cohort of 217 healthy adults, including 106 young and 111 older participants, as well as a replication cohort of 117 young subjects. We further tested the stability and generalizability of both scores by controlling for different MR scanners and gender, as well as by using different data sets of young adults as reference samples. Both scores showed robust age-group-related differences for the subsequent memory contrast, and the FADE-SAME score additionally exhibited age-group-related differences for the novelty contrast. Furthermore, both scores correlate with behavioral measures of cognitive aging, namely memory performance. Taken together, our results suggest that single-value scores of memory-related fMRI responses may constitute promising biomarkers for quantifying neurocognitive aging.

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Whole-Colony Modeling of Escherichia coli Bacterial behavior is the outcome of both molecular mechanisms within each cell and interactions between cells in the context of their environment. Whereas whole-cell models simulate a single cells behavior using molecular mechanisms, agent-based models simulate many agents independently acting and interacting to generate complex collective phenomena. To synthesize agent-based and whole-cell modeling, we used a novel model integration software, called Vivarium, to construct an agent-based model of E. coli colonies where each agent is represented by a current source code snapshot from the E. coli Whole-Cell Modeling Project and interacts with other cells in a shared spatial environment. The result is the first "whole-colony" computational model that mechanistically links expression of individual proteins to a population-level phenotype. Simulated colonies exhibit heterogeneous effects on their environments, heterogeneous gene expression, and media-dependent growth. Extending the cellular model with mechanisms of antibiotic susceptibility and resistance, our model also suggested that variation in the expression level of the betalactamase AmpC, and not of the multi-drug efflux pump AcrAB-TolC, was the key mechanistic driver of survival in the presence of nitrocefin. We see this as a significant step forward in the creation of more comprehensive multi-scale models, and it broadens the range of phenomena that can be modeled in mechanistic terms.

Author summaryThis work combines several models of molecular and physical processes that impact the physiology and behavior of the common microbe Escherichia coli into a multiscale model. Colonies comprised of multiple individual cells are simulated as they grow and divide--each with complex internal mechanisms, and with physical interactions and molecular diffusion in their environments. The integrative modeling methodology supports the addition of new submodels. The flexibility of this methodology is demonstrated by adding models of antibiotic resistance and simulating the colonys response to antibiotic treatment.

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Drivers of Transcriptional Variance in Human Intestinal Epithelial Organoids Background & AimsHuman intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity.

MethodsRNA-sequencing datasets from 251 experiments performed on 35 human enteroid and colonoid lines from 28 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and Gene Set Enrichment Analysis (GSEA) was used to identify differentially expressed genes and enriched of pathways.

ResultsPERMANOVA, Pearson correlations, and dendrogram analysis of all data indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3D, transwell, and monolayer) had the largest effect (7,271-1,305 differentially expressed genes-DEGs); segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect (5,977-420 DEGs), and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell vs. monolayer cultures, and cholesterol biosynthesis genes enriched in Matrigel vs. collagen cultures.

ConclusionsSurprisingly large differences in organoid transcriptome were driven by variations in culture methods such as format and substrate, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal organoids.

sentences: Identification of Lagopus muta japonica plant food resources in the Northern Japan Alps using DNA metabarcoding DNA metabarcoding was employed to identify plant-derived food resources of the Japanese rock ptarmigan (Lagopus muta japonica), registered as a natural living monument in Japan, in the Northern Japanese Alps in Toyama Prefecture, Japan, in July to October, 2015-2018. By combined use of rbcL and ITS2 local databases of 74 alpine plant species found in the study area, a total of 43 plant taxa were identified and could be assigned to 40 species (93.0%), two genera (4.7%), and one family (2.3%). Rarefaction analysis of each sample collection period showed that this study covered more than 90% of the plant food resources found in the study area. Of the 21 plant families identified using the combined rbcL and ITS2 local databases, the most dominant families were Ericaceae (98.1% of 105 fecal samples), followed by Rosaceae (42.9%), Apiaceae (35.2%), and Poaceae (19.0%). In all fecal samples examined, the most frequently encountered plant species were Vaccinium ovalifolium var. ovalifolium (69.5%), followed by Empetrum nigrum var. japonicum (68.6%), Vaccinium sp. (54.3%), Kalmia procumbens (42.9%), and Tilingia ajanensis (34.3%). Rarefaction analysis of each collection period in the study revealed that this study covered more than 90% (from 91.0% in July to 97.5% in September) of the plant food resources found in the study area, and 98.1% of the plant food taxa were covered throughout the entire study period. Thus, DNA metabarcoding using the rbcL and ITS2 local databases of alpine plants in combination and rarefaction analysis are considered to be well suited for estimating the dominant food plants in the diet of Japanese rock ptarmigans. Further, the local database constructed in this study can be used to survey other areas with similar flora.

sentences: GxEsum: a novel approach to estimate the phenotypic variance explained by genome-wide GxE interaction based on GWAS summary statistics for biobank-scale data Genetic variation in response to the environment is fundamental in the biology of complex traits and diseases, i.e. genotype-by-environment interaction (GxE). However, existing methods are computationally demanding and infeasible to handle biobank-scale data. Here we introduce GxEsum, a method for estimating the phenotypic variance explained by genome-wide GxE based on GWAS summary statistics. Through comprehensive simulations and analysis of UK Biobank with 288,837 individuals, we show that GxEsum can handle a large-scale biobank dataset with controlled type I error rates and unbiased GxE estimates, and its computational efficiency can be hundreds of times higher than existing GxE methods.

sentences: Functional crosstalk between the carrier translocase machinery and YME1 complex maintains mitochondrial proteostasis and integrity The TIM22 pathway cargos are essential for sustaining mitochondrial proteostasis as an excess of these proteins leads to proteostatic stress and cell death. Yme1 is an inner membrane metalloprotease that regulates proteostasis with its chaperone-like and proteolytic activities. Although the mitochondrial translocase and protease machinery are critical for organelle health, the functional link between these complexes remains unexplored. The present study unravels a novel genetic connection between the TIM22 complex and YME1 machinery in maintaining mitochondrial proteostasis and quality control. Our genetic analyses indicate that impairment in the TIM22 complex rescues the respiratory growth defects of cells without Yme1. We further demonstrate that Yme1 is essential for the stability of the TIM22 complex and regulating the proteostasis of the TIM22 pathway substrates. Moreover, impairment in the TIM22 complex suppressed the mitochondrial structural and functional defects of Yme1 devoid cells. Notably, the functional dependence between the TIM22 and YME1 complexes remains functionally conserved from yeast to humans. Our findings suggest that excessive levels of the TIM22 pathway substrates could be one of the reasons for the respiratory growth defects of cells lacking Yme1 and compromising the TIM22 complex compensate for the imbalance in mitochondrial proteostasis caused by loss of Yme1.

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HDX-MS performed on BtuB in E. coli outer membranes delineates the luminal domain's allostery and unfolding upon B12 and TonB binding To import large metabolites across the outer membrane of Gram-negative bacteria, TonB dependent transporters (TBDTs) undergo significant conformational change. After substrate binding in BtuB, the E. coli vitamin B12 TBDT, TonB binds and couples BtuB to the inner membrane proton motive force that powers transport (1). But, the role of TonB in rearranging the plug domain to form a putative pore remains enigmatic. Some studies focus on force-mediated unfolding (2) while others propose force-independent pore formation (3) by TonB binding leading to breakage of a salt bridge termed the "Ionic Lock". Our hydrogen exchange/mass spectrometry measurements in E. coli outer membranes find that the region surrounding the Ionic Lock, far from the B12 site, is fully destabilized upon substrate binding. A comparison of the exchange between the B12 bound and the B12&TonB bound complexes indicates that B12 binding is sufficient to unfold the Ionic Lock region with the subsequent binding of a TonB fragment having much weaker effects. TonB binding accelerates exchange in the third substrate binding loop, but pore formation does not obviously occur in this or any region. This study provides a detailed structural and energetic description of the early stages of B12 passage that provides support both for and against current models of the transport process.

Significance StatementTonB dependent transporters such as BtuB are found in the outer membranes of Gram-negative bacteria. They import scarce nutrients essential for growth, such as B12, the substrate of BtuB. Many transport steps remain enigmatic. Recent studies have emphasized force-mediated unfolding or the breakage of the "Ionic Lock", a moiety far from the B12 binding site. A strong dependence on the membrane environment has been noted. Accordingly, we measured hydrogen exchange on BtuB still embedded in native outer membranes and found that B12 binding is sufficient to break the Ionic Lock. The amino terminus then extends into the periplasm to bind TonB. But we find no evidence of pore formation, which likely requires energy transduction from the inner membrane by TonB.

sentences: Host immune responses to enzootic and invasive pathogen lineages vary in magnitude, timing, and efficacy Infectious diseases of wildlife continue to pose a threat to biodiversity worldwide, yet pathogens are far from monolithic in virulence. Within the same pathogen species, virulence can vary considerably depending on strain or lineage, in turn eliciting variable host responses. One pathogen that has caused extensive biodiversity loss is the amphibian-killing fungus, Batrachochytrium dendrobatidis (Bd), which is comprised of a globally widespread hypervirulent lineage (Bd-GPL), and multiple geographically restricted lineages. Whereas host immunogenomic responses to Bd-GPL have been characterized in a number of amphibian species, immunogenomic responses to geographically-restricted, enzootic Bd lineages are unknown. To examine lineage- specific host immune responses to Bd, we exposed a species of pumpkin toadlet, Brachycephalus pitanga, which is endemic to Brazils Southern Atlantic Forest, to either the Bd-GPL or the enzootic Bd-Asia-2/Brazil (hereafter Bd-Brazil) lineage. We quantified functional immunogenomic responses over the course of infection using differential gene expression tests and coexpression network analyses. Host immune responses varied significantly with Bd lineage. Toadlet responses to Bd-Brazil were weak at early infection (26 genes differentially expressed), peaked by mid-stage infection (435 genes) and were nearly fully resolved by late-stage disease (9 genes). In contrast, responses to Bd-GPL were magnified and delayed; toadlets differentially expressed 97 genes early, 86 genes at mid-stage infection, and 728 genes by late-stage infection. Given that infection intensity did not vary between mid- and late-stage disease, this suggests that pumpkin toadlets may be at least partially tolerant to the geographically-restricted Bd- Brazil lineage. In contrast, mortality was higher in Bd-GPL-infected toadlets, suggesting that late-stage immune activation against Bd-GPL was not protective and was consistent with immune dysregulation previously observed in other species. Our results demonstrate that both the timing of immune response and the particular immune pathways activated are specific to Bd lineage. Within regions where multiple Bd lineages co-occur, and given continued global Bd movement, these differential host responses may influence not only individual disease outcome, but transmission dynamics at the population and community levels.

sentences: Human IL-10-producing B cells have diverse states induced from multiple B cell subsets Regulatory B cells (Bregs) can suppress immune responses through the secretion of IL-10 and other anti-inflammatory cytokines. This immunomodulatory capacity holds therapeutic potential, yet a definitional immunophenotype for enumeration and prospective isolation of B cells capable of IL-10 production remains elusive. We therefore applied mass cytometry to simultaneously quantify cytokine production and immunophenotype in human peripheral B cells across a range of stimulatory conditions and timepoints. While multiple B cell subsets produced IL-10, no phenotype uniquely identified IL-10+ B cells and a significant portion of IL-10+ B cells co-expressed the proinflammatory cytokines IL-6 and TNF. Despite this heterogeneity, we found operationally tolerant liver transplant recipients had a unique enrichment of IL-10+, but not TNF+ or IL-6+, B cells as compared to transplant recipients receiving immunosuppression. Thus, human IL-10-producing B cells constitute an induced, transient state arising from a diversity of B cell subsets that may contribute to maintenance of immune homeostasis.

sentences: Whole brain network analysis of neural synchrony and information flow during transition from wakefulness to light non-rapid eye movement sleep Sleep is a highly stereotyped phenomenon, requiring robust spatial and temporal coordination of neural activity. How the brain coordinates neural activity with sleep onset can provide insight into the physiological functions subserved by sleep and pathologic phenomena associated with sleep onset. Here, we quantified whole-brain network changes during the transition from wake to non-rapid eye movement (NREM) sleep using magnetoencephalography imaging in healthy subjects. The sleep transition was identified to be encoded in spatially and temporally specific patterns of local and long-range neural synchrony. Patterns of information flow revealed that mesial frontal regions receive hierarchically organized inputs from broad cortical regions upon sleep onset. Finally, biophysical neural mass modeling demonstrated spatially heterogeneous properties of cortical excitation-to-inhibition from wake to NREM. Together, these findings provide evidence for the structured corticocortical modulation in sleep-wake behavior and demonstrate the orchestration of local and long-range, frequency-specific cortical interactions that are fundamental to sleep onset.

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In Vivo Multi-Day Calcium Imaging of CA1 Hippocampus in Freely Moving Rats Reveals a High Preponderance of Place Cells with Consistent Place Fields Calcium imaging using GCaMP indicators and miniature microscopes has been used to image cellular populations during long timescales and in different task phases, as well as to determine neuronal circuit topology and organization. Because the hippocampus (HPC) is essential for tasks of memory, spatial navigation, and learning, calcium imaging of large populations of HPC neurons can provide new insight on cell changes over time during these tasks. All reported HPC in vivo calcium imaging experiments have been done in mouse. However, rats have many behavioral and physiological experimental advantages over mice. In this paper, we present the first (to our knowledge) in vivo calcium imaging from CA1 hippocampus in freely moving male rats. Using the UCLA Miniscope, we demonstrate that, in rat, hundreds of cells can be visualized and held across weeks. We show that calcium events in these cells are highly correlated with periods of movement, with few calcium events occurring during periods without movement. We additionally show that an extremely large percent of cells recorded during a navigational task are place cells (77.3{+/-}5.0%, surpassing the percent seen during mouse calcium imaging), and that these cells enable accurate decoding of animal position and can be held over days with consistent place fields in a consistent spatial map. A detailed protocol is included, and implications of these advancements on in vivo imaging and place field literature are discussed.

Significance statementIn vivo calcium imaging in freely moving animals allows the visualization of cellular activity across days. In this paper, we present the first in vivo Ca2+ recording from CA1 hippocampus in freely moving rats. We demonstrate that hundreds of cells can be visualized and held across weeks, and that calcium activity corresponds to periods of movement. We show that a high percentage (77.3{+/-}5.0%) of imaged cells are place cells, and that these place cells enable accurate decoding and can be held stably over days with little change in field location. Because the hippocampus is essential for many tasks involving memory, navigation, and learning, imaging of large populations of HPC neurons can shed new insight on cellular activity changes and organization.

sentences: A histidine cluster determines YY1-compartmentalized coactivators and chromatin elements in phase-separated super-enhancers As an oncogenic transcription factor, Yin Yang 1 (YY1) regulates enhancer and promoter connection. However, gaps still exist in understanding how YY1 coordinates coactivators and chromatin elements to assemble super-enhancers. Here, we demonstrate that YY1 activates FOXM1 gene expression through forming liquid-liquid phase separation to compartmentalize both coactivators and enhancer elements. In the transactivation domain of YY1, a histidine cluster is essential for its activities of forming phase separation, which can be extended to additional proteins. Coactivators EP300, BRD4, MED1 and active RNA polymerase II are components of YY1-rich nuclear puncta. Consistently, histone markers for gene activation, but not repression, colocalize with YY1. Importantly, multiple enhancer elements and the FOXM1 promoter are bridged by YY1 to form super-enhancers. These studies propose that YY1 is a general transcriptional activator, and promotes phase separation with incorporation of major coactivators and stabilization by distal enhancers to activate target gene expression.

sentences: Modeling vital rates and age-sex structure of Pacific arctic phocids: influence on aerial survey correction factors To estimate abundance, surveys of marine mammals often rely on samples of satellite-tagged individuals to correct counts for the proportion of animals that are unavailable to be detected. However, naive application of this correction relies on the key assumption that availability of the tagged sample resembles that of the population. Here, we show how matrix population models can be used to estimate the stable stage structure of bearded seals (Erignathus barbatus), ribbon seals (Histriophoca fasciata), ringed seals (Pusa hispida), and spotted seals (Phoca largha) in the Bering and Chukchi Seas, and how these proportions can be used to adjust aerial survey correction factors so that they represent population-level availability. We find that correction factors ignoring age-sex composition can positively bias spotted seal abundance by an average of 13% and negatively bias ribbon seal abundance by an average of 5%. Note that we did not examine potential bias for bearded or ringed seals due to low sample sizes; as such, we urge caution in interpretation of abundance estimates for these species.

sentences: Neuromodulators enable overlapping synaptic memory regimes and nonlinear transition dynamics in recurrent neural networks Mood, arousal, and other internal states can drastically alter behavior, even in identical external circumstances -- a cold glass of water when you are thirsty is much more desirable than when you are sated. Neuromodulators are critical controllers of such neural states, with dysfunctions linked to various neuropsychiatric disorders. Although biological aspects of neuromodulation have been well studied, the computational principles underlying how large-scale neuromodulation of distributed neural populations shifts brain states remain unclear. We use recurrent neural networks to model how synaptic weight modulation -- an important function of neuromodulators -- can achieve nuanced alterations in neural computation, even in a highly simplified form. We find that under structural constraints like those in brains, this provides a fundamental mechanism that can increase the computational capability and flexibility of a neural network by enabling overlapping storage of synaptic memories able to generate diverse, even diametrically opposed, behaviors. Our findings help explain how neuromodulators "unlock" specific behaviors by creating task-specific hypertubes in the space of neural activities and motivate more flexible, compact and capable machine learning architectures.

sentences: Transcriptomic responses of Galapagos finches to avian pox virus infection Emerging pathogens can have devastating effects on naive hosts, but disease outcomes often vary among hosts. Comparing the cellular response of different host species to infection can provide insight into mechanisms of host defense and the basis of host susceptibility to disease. Here, we used RNA-seq to characterize the transcriptomic response of Darwins finches to avian poxvirus, which is introduced to the Galapagos Islands. We tested whether gene expression differs between infected and uninfected birds, and whether transcriptomic differences were related either to known antiviral mechanisms and/or the co-option of the host cellular environment by the virus. We compared two species, the medium ground finch (Geospiza fortis) and the vegetarian finch (Platyspiza crassirostris), to determine whether related species have similar responses to the same novel pathogen. We found that medium ground finches had a strong transcriptomic response to infection, upregulating genes involved in the innate immune response including interferon production, inflammation, and other immune signaling pathways. In contrast, vegetarian finches had a more limited response to infection. Our results also revealed evidence of viral manipulation of the hosts cellular function and metabolism, providing insight into the ways in which poxviruses affect their hosts. Many of the transcriptomic responses to infection mirrored known processes seen in model and in-vitro studies of poxviruses indicating that many pathways of host defense against poxviruses are conserved among vertebrates and present even in hosts without a long evolutionary history with the virus. At the same time, the variation we observed between closely related species indicates that some endemic species of Galapagos finch may be more susceptible to avian pox than others.

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Synergistic protection against secondary pneumococcal infection by human monoclonal antibodies targeting distinct epitopes Streptococcus pneumoniae persists as a leading cause of bacterial pneumonia despite the widespread use of polysaccharide-based vaccines. The limited serotype coverage of current vaccines has led to increased incidence of non-vaccine serotypes, as well as an increase in antibiotic resistance among these serotypes. Pneumococcal infection often follows a primary viral infection such as influenza virus, which hinders host defense and results in bacterial spread to the lungs. We previously isolated human monoclonal antibodies (mAbs) against the conserved surface antigen pneumococcal histidine triad protein D (PhtD), and we demonstrated that mAbs to this antigen are protective against lethal pneumococcal challenge prophylactically and therapeutically. In this study, we elucidated the mechanism of protection of a protective anti- pneumococcal human mAb, PhtD3, which is mediated by the presence of complement and macrophages in a mouse model of pneumococcal infection. Treatment with mAb PhtD3 reduced blood and lung bacterial burden in mice, and mAb PhtD3 is able to bind to bacteria in the presence of the capsular polysaccharide, indicating exposure of surface PhtD on encapsulated bacteria. In a mouse model of secondary pneumococcal infection, protection mediated by mAb PhtD3 and another mAb targeting a different epitope, PhtD7, was reduced, however, robust protection was restored by combining mAb PhtD3 with mAb PhtD7, indicating a synergistic effect. Overall, these studies provide new insights into anti-pneumococcal mAb protection and demonstrate for the first time that mAbs to pneumococcal surface proteins can protect against secondary pneumococcal infection in the mouse model.

Author SummaryThe persistence of Streptococcus pneumoniae as a leading cause of bacterial pneumonia despite numerous approved pneumococcal vaccines is a serious threat to public health globally. Currently, prophylactic and therapeutic options for Streptococcus pneumoniae are constrained by the limited serotype coverage of vaccines and the emergence of antibiotic resistant strains. An additional hurdle to overcome is the incidence of secondary pneumococcal infection following a viral infection, which leads to increased mortality. Here, we determined the mechanism of action of a monoclonal antibody (mAb) that targets Streptococcus pneumoniae. We found that mAb PhtD3 operates through macrophage and complement mediated functions. mAb PhtD3 was also discovered to reduce bacterial titers in the lungs and blood and bind to a related antigen PhtE. We also tested additional mAbs and discovered that two unique mAbs to the antigen PhtD conferred protection in a pneumococcal-influenza virus co-infection model. Our study provides new insights into the mechanisms and therapeutic potential of mAbs targeting conserved proteins of Streptococcus pneumoniae.

sentences: Meta-analysis reveals consistent immune response patterns in COVID-19 infected patients at single-cell resolution A number of single-cell RNA studies looking at the human immune response to the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recently published. However, the number of samples used in each individual study typically is small, moreover the technologies and protocols used in different studies vary, thus somewhat restricting the range of conclusions that can be made with high confidence. To better capture the cellular gene expression changes upon SARS-CoV-2 infection at different levels and stages of disease severity and to minimise the effect of technical artefacts, we performed meta-analysis of data from 9 previously published studies, together comprising 143 human samples, and a set of 16 healthy control samples (10X). In particular, we used generally accepted immune cell markers to discern specific cell subtypes and to look at the changes of the cell proportion over different disease stages and their consistency across the studies. While half of the observations reported in the individual studies can be confirmed across multiple studies, half of the results seem to be less conclusive. In particular, we show that the differentially expressed genes consistently point to upregulation of type I Interferon signal pathway and downregulation of the mitochondrial genes, alongside several other reproducibly consistent changes. We also confirm the presence of expanded B-cell clones in COVID-19 patients, however, no consistent trend in T-cell clonal expansion was observed.

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ACSS2 Regulates HIF-2α Degradation through the E3-Ubiquitin Ligase MUL1 in Clear Cell Renal Cell Carcinoma Clear cell renal cell carcinoma (ccRCC) is an aggressive kidney cancer driven by VHL loss and aberrant HIF-2 signaling. Acetate metabolism may contribute to this axis by ACSS2-dependent acetylation of HIF-2 and may provide opportunities to intervention. Here we tested the effects of pharmacological and genetic manipulation of ACSS2 on HIF-2, ccRCC cells, and tumors. ACSS2 inhibition led to HIF-2 degradation and suppressed ccRCC growth in vitro, in vivo, and in primary cell cultures of ccRCC patient tumors. This treatment resulted in reduced glucose and cholesterol metabolism, mitochondrial biogenesis and altered cristae deformation, that are consistent with loss of HIF-2. Mechanistically, HIF-2 protein levels are regulated through proteolytic degradation and we found, in parallel to VHL, HIF-2 stability was dependent on ACSS2 activity to prevent direct interaction with the E3 ligase MUL1. These findings highlight ACSS2 as a critical upstream regulator of HIF-2 that may be exploited to overcome resistance to HIF-2 inhibitor therapies.

STATEMENT OF SIGNIFICANCEWe have unveiled ACSS2 as a critical upstream regulator of HIF-2 in ccRCC. Targeting ACSS2 potently promotes HIF-2 degradation via MUL1 to effectively deplete mitochondrial activity and block ccRCC primary tumor models and growth models resistant to HIF-2 inhibitor therapy.

sentences: Dynamic collateral sensitivity profiles highlight challenges and opportunities for optimizing antibiotic sequences As traditional antimicrobial therapies fail at escalating rates, recent focus has shifted to evolution-based therapies to slow resistance. Collateral sensitivity-the increased susceptibility to one drug associated with evolved resistance to a different drug-offers a potentially exploitable evolutionary constraint, but the manner in which collateral effects emerge over time is not well understood. Here, we use laboratory evolution in the opportunistic pathogen E. faecalis to phenotypically characterize collateral profiles through evolutionary time. Specifically, we measure collateral profiles for 400 strain-antibiotic combinations over the course of 4 evolutionary time points as strains are selected in increasing concentrations of antibiotic. We find that collateral resistance dominates during early phases of adaptation, whereas a diverse set of collateral profiles are accessible with further selection. Using simple numerical simulations, we illustrate how these temporally dynamic profiles potentially impact sequential drug therapies. Finally, we show experimentally how dynamic collateral sensitivity relationships can create optimal dosing windows that depend on finely timed switching between drugs.

sentences: A new replication medium enables a rapid identification of {varphi}x-174 virus by synchronous fluorescence of Tryptophan. Development of rapid methods for detection of virus particles based on their intrinsic fluorescence is challenging. Pure viruses may be detected in filtered solutions, based on the strong fluorescence of the amino acid Tryptophan (Trp) in their proteins. Nevertheless, Trp also exists in high quantities in the hosts and host cultivation media. In this work, we show that a separation of the bacteriophage{varphi} x-174 from its E. coli host (grown on the standard cultivation medium "Nutrient Agar") by simple extraction and filtration is not sufficient for its detection based on the intrinsic fluorescence since ~70 % of the Trp fluorescence is derived from impurities. We formulate a new cultivation medium with very low Trp concentration. We apply synchronous fluorescence measurements to show that no Trp fluorescence is detected in the extract solution upon incubation of this medium substrate with ammonium acetate extraction buffer. Finally, we detect{varphi} x-174 based on the spectral fingerprint of its intrinsic Trp content by synchronous fluorescence measurements. The concept of coupling intrinsic fluorescence-based methods to impurities reduction in the source, may pave the way towards future development of simple, cheap, and rapid methods for detection of viral pathogens.

sentences: Stable modulation of neuronal oscillations produced through brain-machine equilibrium Brain stimulation is predominantly delivered independent of ongoing activity, whereas closed-loop systems can modify activity through interaction. They have the unrealised potential to continuously bind external stimulation to specific dynamics of a neural circuit. Such manipulations are particularly suited to rhythmic activities, where neuronal activity is organised in oscillatory cycles. Here, we developed a fast algorithm that responds on a cycle-by-cycle basis to stimulate basal ganglia nuclei at predetermined phases of successive cortical beta cycles in parkinsonian rats. Using this approach, we demonstrate a stable brain-machine interaction. An equilibrium emerged between the modified brain signal and feedback-dependent stimulation pattern, which led to sustained amplification or suppression of the oscillation. Sustained beta amplification slowed movement speed by altering the mode of locomotion. Integrating an external stimulus with network activity in this way could be used to correct maladaptive activities and to define the role of oscillations in fundamental brain functions.

sentences: Vertebrate whole genome duplications shaped the current 3D genome architecture Topologically associated domains (TADs) are interaction sub-networks of 3D genomes. TAD boundaries frequently coincide with genome breaks while their deletion is under negative selection, suggesting that TADs act as modules facilitating genome rearrangements and metazoan evolution. However, the role of TADs in the evolution of gene regulation and essentiality is not well understood. Here, we show that TADs play a role organising ancestral functions and evolutionary novelty. We discovered that genes co-localise by evolutionary age in the human and mouse genomes, resulting in TADs that have different proportions of younger and older genes. A major transition in the TAD age co-localisation patterns is observed between the genes born as a result of the vertebrate whole genome duplications (WGDs) or before, and those born afterwards. We also found that primate- and rodentspecific genes are more frequently essential when they are located in  aged TADs and connected to genes that have not duplicated since the WGD. Our data suggests that evolutionary success of recent genes may increase when located in functionally relevant TADs with established regulatory networks.

sentences: Integrated single-cell analysis of enteric glial cells reveals a molecular basis for postnatal neurogenesis and its therapeutic application The enteric nervous system (ENS) consists of glial cells (EGCs) and neurons derived from neural crest precursors. EGCs retain capacity for large-scale neurogenesis in culture, and in vivo lineage tracing has identified neurons derived from glial cells in response to inflammation. We thus hypothesize that EGCs possess a chromatin structure poised for neurogenesis. We use single-cell multiome sequencing to assess EGCs undergoing spontaneous neurogenesis in culture, as well as freshly isolated small intestine myenteric plexus EGCs. Cultured EGCs maintain open chromatin at genomic loci accessible in neurons, and neurogenesis from EGCs involves dynamic chromatin rearrangements with a net decrease in accessible chromatin. Multiome analysis of freshly isolated EGCs reveals transcriptional diversity, with open chromatin at neuron-associated genomic elements. A subset of EGCs, highly enriched within the myenteric ganglia, has a gene expression program and chromatin state consistent with neurogenic potential.

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Eliminating Animal Agriculture Would Negate 56 Percent of Anthropogenic Greenhouse Gas Emissions Through 2100 Animal agriculture contributes significantly to global warming through ongoing emissions of the potent greenhouse gases methane and nitrous oxide, and displacement of biomass carbon on the land used to support livestock. However, because estimates of the magnitude of the effect of ending animal agriculture often focus on only one factor, the full potential benefit of a more radical change remains underappreciated. Here we quantify the full "climate opportunity cost" of current global livestock production, by modeling the combined, long-term effects of emission reductions and biomass recovery that would be unlocked by a phaseout of animal agriculture. We show that, even in the absence of any other emission reductions, persistent drops in atmospheric methane and nitrous oxide levels, and slower carbon dioxide accumulation, following a phaseout of livestock production would, through the end of the century, have the same cumulative effect on the warming potential of the atmosphere as a 25 gigaton per year reduction in anthropogenic CO2 emissions, providing half of the net emission reductions necessary to limit warming to 2{degrees}C. The magnitude and rapidity of these potential effects should place the reduction or elimination of animal agriculture at the forefront of strategies for averting disastrous climate change.

Significance StatementThe use of animals to produce food has a negative impact on the climate, but the benefits of a global switch to a plant based diet are underappreciated. We show that the global warming impact, through the rest of this century, of eliminating greenhouse gas emissions from livestock and allowing native ecosystems to regrow on the land currently used to house and feed livestock, would be equivalent to a 68% reduction in carbon dioxide emissions. We hope putting clearer numbers on the "climate opportunity cost" of our continued use of animals as food technology will help policymakers and the public properly prioritize dietary change as a climate defense strategy.

Declaration of Conflict of InterestPatrick Brown is the founder and CEO of Impossible Foods, a company developing alternatives to animals in food-production. Michael Eisen is an advisor to Impossible Foods. Both are shareholders in the company and thus stand to benefit financially from reduction of animal agriculture.

sentences: Charting functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders Targeted protein degradation is a new pharmacologic paradigm established by drugs that recruit target proteins to E3 ubiquitin ligases via a ternary ligase-degrader-target complex. Based on the structure of the degrader and the neosubstrate, different E3 ligase interfaces are critically involved in this process, thus forming defined "functional hotspots". Understanding disruptive mutations in functional hotspots informs on the architecture of the underlying assembly, and highlights residues prone to cause drug resistance. Until now, their identification was driven by structural methods with limited scalability. Here, we employ haploid genetics to show that hotspot mutations cluster in the substrate receptors of the hijacked ligases and find that type and frequency of mutations are shaped by the essentiality of the harnessed ligase. Intersection with deep mutational scanning data revealed hotspots that are either conserved, or specific for chemically distinct degraders or recruited neosubstrates. Biophysical and structural validation suggest that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we identified and validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and experimentally widely accessible methodology that empowers the characterization of small-molecule degraders and informs on associated resistance mechanisms.

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Metagenomes from the Loxahatchee wildlife refuge in the Florida Everglades The Florida Everglades ecosystem represents a significant wetlands area and serves as a terrestrial carbon reservoir mediated in large part by microorganisms. Shotgun metagenome sequencing provides a snapshot of microbial diversity and the frequency of metabolic and functional gene content. Here, we present an analysis of 20 sediment samples collected from the Arthur R. Marshall Loxahatchee National Wildlife Refuge to characterize the taxonomic and functional potential of the microbial and viral communities, and reconstructed metagenome-assembled genomes. A total of 122 medium-quality and 6 high-quality MAGs are reported, three of which likely represent a novel species within the class Dehalococcoidia.

The most abundant phyla of bacteria and archaea were Proteobacteria and Euryarchaeota, respectively. Caudovirales was the most abundant viral order. Significant differences in taxonomic composition and diversity were observed among collection sites. Additionally, water samples were analyzed for pH, total nitrogen, total organic carbon, elements (P, K, Mg, Fe, Mn, Pb, Ca, S), chloride, electric conductivity, orthophosphate, nitrate, and ammonia, while the sediment samples were analyzed for carbon, nitrogen, and pH. Differences in measured aquatic and sediment analytes revealed significant correlations with numerous phyla. Significant correlations were observed between estimated gene frequencies of both aquatic and sediment analytes, most notably between kup/kdpB and dsrA/cysC with potassium and sulfur, respectively, as well as phoD/phnX and cysC with pH. Together, these data provide an important view into the functional and metabolic potential encoded within the sediment microbial communities in the Florida Everglades.

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Transcriptomic and metabolomic responses to carbon and nitrogen sources in Methylomicrobium album BG8 Methanotrophs use methane as their sole carbon and energy source and represent an attractive platform for converting single-carbon feedstocks into value-added compounds. Optimizing these species for biotechnological applications involves choosing an optimal growth substrate based on an understanding of cellular responses to different nutrients. Although many studies of methanotrophs have examined growth rate, yield, and central carbon flux in cultures grown with different carbon and nitrogen sources, few studies have examined more global cellular responses to different media. Here, we evaluated global transcriptomic and metabolomic profiles of Methylomicrobium album BG8 when grown with methane or methanol as the carbon source and nitrate or ammonium as the nitrogen source. We identified five key physiological changes during growth on methanol: M. album BG8 cultures upregulated transcripts for the Entner-Doudoroff and pentose phosphate pathways for sugar catabolism, produced more ribosomes, remodeled its phospholipid membrane, activated various stress response systems, and upregulated glutathione-dependent formaldehyde detoxification. When using ammonium, M. album BG8 upregulated haoAB hydroxylamine dehydrogenase and the overall central metabolic activity; whereas when using nitrate, cultures upregulated genes for nitrate assimilation and conversion. Overall, we identified several nutrient source-specific responses that could provide a valuable basis for future research on the biotechnological optimization of these species.

IMPORTANCEMethanotrophs are gaining increasing interest for their biotechnological potential to convert single-carbon compounds into value-added products such as industrial chemicals, fuels, and bioplastics. Optimizing these species for biotechnological applications requires a detailed understanding of how cellular activity and metabolism varies across different growth substrates. Although each of the two most commonly used carbon sources (methane or methanol) and nitrogen sources (ammonium or nitrate) in methanotroph growth media have well-described advantages and disadvantages in an industrial context, their effects on global cellular activity remain poorly characterized. Here, we comprehensively describe the transcriptomic and metabolomic changes that characterize the growth of an industrially promising methanotroph strain on multiple combinations of carbon and nitrogen sources. Our results represent a more holistic evaluation of cellular activity than previous studies of core metabolic pathways and provide a valuable basis for the future biotechnological optimization of these species.

sentences: Detection and quantification of the vacuolar H ATPase using the Legionella effector protein SidK Acidification of secretory and endocytic organelles is required for proper receptor recycling, membrane traffic, protein degradation, and solute transport. Proton-pumping vacuolar ATPases (V-ATPases) are responsible for this luminal acidification, which increases progressively as secretory and endocytic vesicles mature. An increasing density of V-ATPase complexes is thought to account for the gradual decrease in pH, but available reagents have not been sufficiently sensitive nor specific to test this hypothesis. We introduce a new probe to localize and quantify V-ATPases in eukaryotic cells. The probe is derived from SidK, a Legionella pneumophila effector protein that binds to the V-ATPase A subunit. We generated plasmids encoding fluorescent chimeras of SidK1-278, and labeled recombinant SidK1-278 with AlexaFluor-568 to visualize and quantify V-ATPases with high specificity in live and fixed cells, respectively. We show that V-ATPases are acquired progressively during phagosome maturation, that they distribute in discrete membrane subdomains, and that their density in lysosomes depends on the subcellular localization of the lysosome.

sentences: Enabling high throughput target-based drug discovery in 3D cell cultures through novel bioprinting workflow Advanced three-dimensional cell culture techniques have been adopted in many laboratories to better model in vivo tissue by recapitulating multi-cellular architecture and the presence of extracellular matrix features. We describe here a 3D cell culture platform in a small molecule screening workflow that uses traditional biomarker and intracellular kinase end point assay readouts. By combining the high throughput bioprinter Rastrum with the high throughput screening assay AlphaLISA, we demonstrate the utility of the workflow in 3D synthetic hydrogel cultures with breast cancer (MDA-MB-231 and MCF-7) and fibroblast cells. To establish and validate the workflow, we treated the breast cancer cultures with doxorubicin, while fibroblast cultures were stimulated with the pro-inflammatory lipopolysaccharide. 3D and 2D MDA-MB-231 cultures were equally susceptible to doxorubicin treatment, while showing opposite ERK phosphorylation changes. Doxorubicin readily entered embedded MCF-7 spheroids and markedly reduced intracellular GSK3{beta} phosphorylation. Furthermore, quantifying extracellular interleukin 6 levels showed a very similar activation profile for fibroblasts in 2D and 3D cultures, with 3D fibroblast networks being more resistant against the immune challenge. Through these validation experiments we demonstrate the full compatibility of the bioprinted 3D cell cultures with several widely-used 2D culture assays. The efficiency of the workflow, minimal culture handling, and applicability of traditional screening assays, demonstrates that advanced encapsulated 3D cell cultures can be used in 2D cell culture screening workflows, while providing a more holistic view on cell biology to increase the predictability to in vivo drug response.

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Prior expectations in visual speed perception predict encoding characteristics of neurons in area MT Bayesian inference provides an elegant theoretical framework for understanding the characteristic biases and discrimination thresholds in visual speed perception. However, the framework is difficult to validate due to its flexibility and the fact that suitable constraints on the structure of the sensory uncertainty have been missing. Here, we demonstrate that a Bayesian observer model constrained by efficient coding not only well explains human visual speed perception but also provides an accurate quantitative account of the tuning characteristics of neurons known for rep-resenting visual speed. Specifically, we found that the population coding accuracy for visual speed in area MT ("neural prior") is precisely predicted by the power-law, slow-speed prior extracted from fitting the Bayesian model to psychophysical data ("behavioral prior") to the point that the two priors are indistinguishable in a cross-validation model comparison. Our results demonstrate a quantitative validation of the Bayesian observer model constrained by efficient coding at both the behavioral and neural levels.

Significance StatementStatistical regularities of the environment play an important role in shaping both neural representations and perceptual behavior. Most previous works addressed these two aspects independently. Here we present a quantitative validation of a theoretical framework that makes joint predictions for neural coding and behavior, based on the assumption that neural representations of sensory information are efficient but also optimally used in generating a percept. Specifically, we demonstrate that the neural tuning characteristics for visual speed in brain area MT are precisely predicted by the statistical prior expectations extracted from psychophysical data. As such, our results provide a normative link between perceptual behavior and the neural representation of sensory information in the brain.

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Can proteomics of snake venoms help drug discovery? A study on the venom of spectacled cobra (Naja naja) from the Western Ghats Venom proteome profiling of Naja naja from the Western Ghats region in Kerala was achieved through SDS-PAGE and RP-HPLC followed by Q-TOF LC-MS/MS analysis, incorporating PEAKS and Novor assisted de novo sequencing methodologies. A total of 115 proteins distributed across 17 different enzymatic and non-enzymatic venom protein families were identified through conventional and 39 peptides through homology-driven proteomics approaches. Fourteen peptides derived through de novo complements the Mascot data indicating the importance of homology-driven approaches in improving protein sequence information. Among the protein families identified, glutathione peroxidase and endonuclease were reported for the first time in the Indian cobra venom. Immunological cross-reactivity assessed using Indian polyvalent antivenoms suggested that VINS showed better EC50 (2.48 g/mL) value than that of PSAV (6.04 g/mL) and Virchow (6.03 g/mL) antivenoms. Western blotting experiments indicated that all the antivenoms elicited poor binding specificities, especially towards low molecular mass proteins. Second-generation antivenomics studies revealed that VINS antivenom was less efficient to detect many low molecular mass proteins such as three-finger toxins and Kunitz-type serine protease Inhibitors. Taken together, the present study enabled a large-scale characterization of the venom proteome of Naja naja from the Western Ghats and emphasized the need for developing more efficient antivenoms.

HighlightsO_LIProteomics of cobra venom resulted in the identification of 115 proteins representing 17 snake venom protein families.
C_LIO_LIDe novo approaches exclusively yielded 39 peptides harbouring multiple amino acid mutations.
C_LIO_LIGlutathione peroxidase and endonuclease were identified for the first time in Indian cobra venom.
C_LIO_LIIndian polyvalent antivenoms showed varying cross-reactivity towards cobra venom.
C_LIO_LIVINS antivenom was less efficient to detect many low molecular mass proteins (< 20 kDa).
C_LI

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The damage-independent evolution of ageing by selective destruction Ageing is currently believed to reflect the accumulation of molecular damage due to energetic costs of maintenance, as proposed in disposable soma theory (DST). Here we use agent-based modelling to describe an alternative theory by which ageing could undergo positive selection independent of energetic costs. We suggest that the selective advantage of aberrant cells with fast growth might necessitate a mechanism of counterselection we name selective destruction that specifically removes the faster cells from tissues, preventing the morbidity and mortality risks they pose. The resulting survival advantage of slower mutants could switch the direction of selection, allowing them to outcompete both fast mutants and wildtype cells, causing them to spread and induce ageing in the form of a metabolic slowdown.

Selective destruction could therefore provide a proximal cause of ageing that is both consistent with the gene expression hallmarks of ageing, and independent of accumulating damage. Furthermore, negligible senescence would acquire a new meaning of increased basal mortality.

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Glucose dynamics during ozone exposure measured using radiotelemetry: Stress drivers and human concordance BackgroundStress-related neurobehavioral and metabolic disorders are associated with altered circulating adrenal-derived hormones and hyperglycemia. Temporal assessment of glucose and these hormones is critical for insights on an individuals health.

ObjectivesHere we used implantable-telemetry in rats to assess real-time changes in circulating glucose during and after exposure to the air pollutant ozone, and link responses to circulating neuroendocrine stress and metabolic hormones. We also compared rodent glucose and corticosterone (cortisol in humans) responses to humans exposed to ozone.

MethodsFirst, using a cross-over design, we monitored glucose levels during single or repeated ozone exposures (0.0, 0.2, 0.4 and 0.8-ppm) and non-exposure periods in male Wistar-Kyoto-rats implanted with glucose-telemeters. A second cohort of un-implanted rats was exposed to ozone (0.0, 0.4 or 0.8-ppm) for 30-min, 1-hour, 2-hour, or 4-hour with hormones measured immediately after exposure. Then we assessed glucose metabolism in sham and adrenalectomized rats with or without pharmacological interventions of adrenergic and glucocorticoid receptors. Finally, we assessed glucose and cortisol in serum samples from a clinical study involving exposure of human volunteers to air or 0.3 ppm ozone.

ResultsOzone (0.8-ppm) caused hyperglycemia and hypothermia beginning 90-min into exposure, with reversal of effects 4-6 hours post-exposure. Glucose monitoring during four daily 4-hour ozone exposures revealed duration of hyperglycemia, adaptation, and diurnal variations. Ozone-induced hyperglycemia was preceded by increased adrenocorticotropic hormone, corticosterone, and epinephrine, but depletion of thyroid-stimulating, prolactin, and luteinizing hormones. Hyperglycemia was inhibited in rats that were adrenalectomized and/or treated with glucocorticoid inhibitor. The depletion of cortisol was dampened in humans exposed to ozone during intermittent exercise.

DiscussionWe demonstrate for the first time the temporality of neuroendocrine-stress-mediated biological sequalae responsible for ozone-induced metabolic dysfunction as exposure occurs. Real-time glucose monitoring with stress hormones assessment may be useful in identifying interactions among pollutants and stress-related illnesses.

sentences: White Matter Signals Reflect Information Transmission Between Brain Regions During Seizures White matter supports critical brain functions such as learning and memory, modulates the distribution of action potentials, and acts as a relay of neural communication between different brain regions. Interestingly, neuronal cell bodies exist in deeper white matter tissue, neurotransmitter vesicles are released directly in white matter, and white matter blood-oxygenation level dependent (BOLD) signals are detectable across a range of different tasks--all appearing to reflect intrinsic electrical signals in white matter. Yet, such signals within white matter have largely been ignored. Here, we elucidate the properties of white matter signals using intracranial EEG. We show that such signals capture the communication between brain regions and differentiate pathophysiologies of epilepsy. In direct contradiction to past assumptions about white matter in-activity, we show that white matter recordings can elucidate brain function and pathophysiology not apparent in gray matter. Broadly, white matter functional recordings acquired through implantable devices may provide a wealth of currently untapped knowledge about the neurobiology of disease.

sentences: Growth allometry and dental topography in Upper Triassic conodonts supports trophic differentiation and molar-like element function Conodont dental elements are distinguished by their high disparity and rapid morphological evolution. P1 elements located in the pharynx are the most rapidly evolving, but their function in the animal has been only investigated in a handful of taxa and proposed to be analogous to mammal molars. This hypothesis predicts that their surface area should show positive allometry with respect to element length, as has been previously identified in 2D projections in two Carboniferous taxa. Here we apply the same method to test this hypothesis in 3D models of platform-bearing P1 elements of two common Late Triassic taxa, Metapolygnathus communisti and Epigondolella rigoi. We further hypothesise that these commonly co-occurring taxa differed in their growth allometry, reflecting their different trophic niches. Platform length grew isometrically with respect to element length, whereas log-transformed platform area showed positive allometry with respect to element length, with slopes equal 3.86 in M. communisti and 4.16 in E. rigoi, supporting a function of the platform analogous to molars and trophic differentiation. We cross-tested the latter interpretation by dental topographic analysis using Dirichlet Normal Energy (DNE). Specimens of the adult growth stage of E. rigoi showed higher DNE values than specimens of the same growth stage in M. communisti, consistent with stronger positive allometry of platform surface and with a higher demand for energy in this species. DNE values of platform surface increased linearly in function of element length and log-transformed platform area, indicating no ontogenetic changes. Based on DNE values available for primates, those of the adult growth stages were similar to those reported for insectivores or folivores in the case of E. rigoi and for folivores or omnivores in the case of M. communisti. Previous studies applying morphological and ultrastructural proxies for the dietary position of conodonts addressed mostly stratigraphically older conodont taxa, but our results indicate that Late Triassic species occupied the predator/scavenger niche in spite of the highly developed diversity of gnathostomes in this niche. We also show that within this broad niche, co-occurring taxa differed in their diets, which supports trophic diversification as an important driver of the remarkable disparity of their elements.

sentences: Homologous or Heterologous Booster of Inactivated Vaccine Reduces SARS-CoV-2 Omicron Variant Escape from Neutralizing Antibodies The massive and rapid transmission of SARS-CoV-2 has led to the emergence of several viral variants of concern (VOCs), with the most recent one, B.1.1.529 (Omicron), which accumulated a large number of spike mutations, raising the specter that this newly identified variant may escape from the currently available vaccines and therapeutic antibodies. Using VSV-based pseudovirus, we found that Omicron variant is markedly resistant to neutralization of sera form convalescents or individuals vaccinated by two doses of inactivated whole-virion vaccines (BBIBP-CorV). However, a homologous inactivated vaccine booster or a heterologous booster with protein subunit vaccine (ZF2001) significantly increased neutralization titers to both WT and Omicron variant. Moreover, at day 14 post the third dose, neutralizing antibody titer reduction for Omicron was less than that for convalescents or individuals who had only two doses of the vaccine, indicating that a homologous or heterologous booster can reduce the Omicron escape from neutralizing. In addition, we tested a panel of 17 SARS-CoV-2 monoclonal antibodies (mAbs). Omicron resists 7 of 8 authorized/approved mAbs, as well as most of the other mAbs targeting distinct epitopes on RBD and NTD. Taken together, our results suggest the urgency to push forward the booster vaccination to combat the emerging SARS-CoV-2 variants.

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mRNA Vaccination Induces Durable Immune Memory to SARS-CoV-2 with Continued Evolution to Variants of Concern SARS-CoV-2 mRNA vaccines have shown remarkable efficacy, especially in preventing severe illness and hospitalization. However, the emergence of several variants of concern and reports of declining antibody levels have raised uncertainty about the durability of immune memory following vaccination. In this study, we longitudinally profiled both antibody and cellular immune responses in SARS-CoV-2 naive and recovered individuals from pre-vaccine baseline to 6 months post-mRNA vaccination. Antibody and neutralizing titers decayed from peak levels but remained detectable in all subjects at 6 months post-vaccination. Functional memory B cell responses, including those specific for the receptor binding domain (RBD) of the Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) variants, were also efficiently generated by mRNA vaccination and continued to increase in frequency between 3 and 6 months post-vaccination. Notably, most memory B cells induced by mRNA vaccines were capable of cross-binding variants of concern, and B cell receptor sequencing revealed significantly more hypermutation in these RBD variant-binding clones compared to clones that exclusively bound wild-type RBD. Moreover, the percent of variant cross-binding memory B cells was higher in vaccinees than individuals who recovered from mild COVID-19. mRNA vaccination also generated antigen-specific CD8+ T cells and durable memory CD4+ T cells in most individuals, with early CD4+ T cell responses correlating with humoral immunity at later timepoints. These findings demonstrate robust, multi-component humoral and cellular immune memory to SARS-CoV-2 and current variants of concern for at least 6 months after mRNA vaccination. Finally, we observed that boosting of pre-existing immunity with mRNA vaccination in SARS-CoV-2 recovered individuals primarily increased antibody responses in the short-term without significantly altering antibody decay rates or long-term B and T cell memory. Together, this study provides insights into the generation and evolution of vaccine-induced immunity to SARS-CoV-2, including variants of concern, and has implications for future booster strategies.

GRAPHICAL ABSTRACT

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sentences: Cortico-striatal activity driving compulsive reward seeking Addicted individuals compulsively seek drugs. Cortico-striatal projections have been implicated in persevering to seek rewards even when punished. The temporo-spatial determinants of the activity underlying the compulsive reward seeking however remains elusive. Here we trained mice in a seek-take chain, rewarded by optogenetic dopamine neuron self-stimulation (oDASS). Mice that persevered when seeking was punished, exhibited an increased AMPA/NMDA ratio selectively at orbitofrontal cortex (OFC) to dorsal striatum (DS) synapses. In addition, an activity peak of spiny projection neurons (SPNs) in the DS at the moment of signalled reward availability was detected. Chemogenetic inhibition of OFC neurons curbed the activity peak and reduced punished reward seeking, as did optogenetic hyperpolarization of SPNs time locked to the cue predicting reward availability, establishing a causal link. Taken together, we conclude that the strengthening of OFC-DS synapses drives SPNs activity when a reward predictive cue is delivered, thus encouraging reward seeking in subsequent trials.

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Single-cell multi-omics analyses reveal EZH2 as a main driver of retinoic acid resistance in PLZF-RARA leukemia. Cancer relapse is caused by a subset of malignant cells that are resistant to treatment. To characterize resistant cells and their vulnerabilities, we studied the retinoic acid (RA)-resistant PLZF-RARA acute promyelocytic leukemia (APL) using single-cell multi-omics. We uncovered transcriptional and chromatin heterogeneity in leukemia cells and identified a subset of cells resistant to RA that depend on a fine-tuned transcriptional network targeting the epigenetic regulator Enhancer of Zeste Homolog 2 (EZH2). Epigenomic and functional analyses validated EZH2 selective dependency of PLZF-RARA leukemia and its driver role in RA resistance. Targeting pan-EZH2 activities (canonical/non-canonical) was necessary to eliminate leukemia relapse initiating cells, which underlies a dependency of resistant cells on an EZH2 non-canonical activity and the necessity to degrade EZH2 to overcome resistance.

Our study provides critical insights into the mechanisms of RA resistance that allow us to eliminate treatment-resistant leukemia cells by targeting EZH2, thus highlighting a potential targeted therapy approach.

HIGHLIGHTS- sc-RNAseq identifies PLZF-RARA leukemia heterogeneity and retinoic acid resistant cells
- sc-ATACseq refines leukemic cell identity and resolves retinoic acid resistant networks
- EZH2 is a selective dependency of PLZF-RARA leukemia and drives retinoic acid resistance
- Targeting pan-EZH2 activities (canonical/non-canonical) is necessary to overcome leukemia onset

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Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9. Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9 thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled the first-time isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin/microtubule cytoskeleton.

SummaryHuman septins in cells form hetero-octameric complexes containing the ubiquitously expressed SEPT9. Iv et al. describe the first-time isolation of recombinant human septin octamers with distinct SEPT9 isoforms. Reconstitution studies show that octamers with either a long or a short SEPT9 isoform form higher-order filament assemblies and directly bind and cross-link actin filaments.

sentences: A DEEP LEARNING APPROACH TO ESTIMATING INITIAL CONDITIONS OF BRAIN NETWORK MODELS IN REFERENCE TO MEASURED FMRI DATA 1.Brain Network Models (BNMs) are a family of dynamical systems that simulate whole brain activity using neural mass models to represent local activity in different brain regions that influence each other via a global structural network. Research has been interested in using these network models to explain measured whole brain activity measured via resting state functional magnetic resonance imaging (rs-fMRI). Properties computed over longer periods of simulated and measured data such as average functional connectivity (FC), have shown to be comparable with similar properties estimated from measured rs-fMRI data. While this shows that these network models have similar properties over the dynamical landscape, it is unclear how well simulated trajectories compare with empirical trajectories on a timepoint-by-timepoint basis. Previous studies have shown that BNMs are able to produce relevant features at shorter timescales, but analysis of short-term trajectories or transient dynamics as defined by synchronized predictions from BNM made at the same timescale as the collected data has not yet been conducted. Relevant neural processes exist in the time frame of measurements and are often used in task fMRI studies to understand neural responses to behavioral cues. Therefore, it is important to investigate how much of these dynamics are captured by our current brain simulations. To test the nature of BNMs short term trajectories against observed data, we utilize a deep learning technique known as Neural ODE that based on an observed sequence of fMRI measurements, estimates the initial conditions such that the BNMs simulation is synchronized to produce the closest trajectory relative to the observed data. We test to see if the parameterization of a specific well studied BNM, the Firing Rate Model, calculated by maximizing its accuracy in reproducing observed short term trajectories matches with the parameterized model that produces the best average long-term metrics. Our results show that such an agreement between parameterization using long and short simulation analysis exists if also considering other factors such as the sensitivity in accuracy with relative to changes in structural connectivity. Therefore, we conclude that there is evidence that by solving for initial conditions, BNMs can be simulated in a meaningful way when comparing against measured data trajectories, although future studies are necessary to establish how BNM activity relate to behavioral variables or to faster neural processes during this time period.

sentences: Mechanosensitive Osteogenesis on Native Cellulose Scaffolds for Bone Tissue Engineering. In recent years, plant-derived cellulosic biomaterials have become a popular way to create scaffolds for a variety of tissue engineering applications. Moreover, such scaffolds possess similar physical properties (porosity, stiffness) that resemble bone tissues and have been explored as potential biomaterials for tissue engineering applications. Here, plant-derived cellulose scaffolds were seeded with MC3T3-E1 pre-osteoblast cells. Moreover, to assess the potential of these biomaterials, we also applied cyclic hydrostatic pressure (HP) to the cells and scaffolds over time to mimic a bone-like environment more closely. After one week of proliferation, cell-seeded scaffolds were exposed to HP up to 270 KPa at a frequency of 1Hz, once per day, for up to two weeks. Scaffolds were incubated in osteogenic inducing media (OM) or regular culture media (CM). The effect of cyclic HP combined with OM on cell-seeded scaffolds resulted in an increase of differentiated cells. This corresponded to an upregulation of alkaline phosphatase activity and scaffold mineralization. Importantly, the results reveal that well known mechanosensitive pathways cells which regulate osteogenesis appear to remain functional even on novel plant-derived cellulosic biomaterials.

sentences: Light activation of Orange Carotenoid Protein reveals initial C8'-C7' double bond trans/cis isomerization Orange Carotenoid protein (OCP) is the only known photoreceptor which uses carotenoid for its activation1,2. It is found exclusively in cyanobacteria, where it functions to control light-harvesting of the photosynthetic machinery. However, the photochemical reactions and structural dynamics of this unique photosensing process are not yet resolved. We present time-resolved crystal structures at second-to-minute delays under bright illumination, capturing the early photoproduct and structures of the subsequent reaction intermediates. The first stable photoproduct shows carotenoid trans/cis isomerization at the C7-C8double bond and structural changes in the N-terminal domain with minute timescale kinetics. These are followed by a thermally-driven cis/trans isomerization that recovers to the dark state carotenoid configuration. Structural changes propagate to the C-terminal domain, resulting, at later time, in the H-bond rupture of the carotenoid keto group with protein residues. The isomerization and its transient nature are confirmed in OCP crystals and solution by FTIR and UV/Vis spectroscopy. This study reveals the isomerization of the carotenoid and subsequent thermal structural reactions as the basis of OCP photoreception. Understanding and potentially controlling the OCP dynamics offers the prospect of novel applications in biomass engineering3 as well as in optogenetics and bioimaging.

sentences: Perilipin 5 interacts with Fatp4 at membrane contact sites to promote lipid droplet-to-mitochondria fatty acid transport Cells adjust their metabolism by remodeling membrane contact sites that channel metabolites to different fates. Lipid droplet (LD)-mitochondria contacts change in response to fasting, cold exposure, and exercise. However, their function and mechanism of formation have remained controversial. We focused on perilipin 5 (Plin5), an LD protein that tethers mitochondria, to probe the function and regulation of LD-mitochondria contacts. We demonstrate that efficient LD-to-mitochondria fatty acid (FA) trafficking and {beta}-oxidation during starvation of myoblasts requires both phosphorylation of Plin5 and an intact Plin5 mitochondrial tethering domain. We further identified the acyl-CoA synthetase, Fatp4 (ACSVL4) as a novel mitochondrial interactor of Plin5. The C-terminal domains of Plin5 and Fatp4 constitute a minimal protein interaction capable of inducing organelle contacts. Our work suggests that starvation leads to phosphorylation of Plin5, lipolysis, and subsequent channeling of FAs from LDs to Fatp4 on mitochondria for conversion to fatty-acyl-CoAs and subsequent oxidation.

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Selenium in wheat from farming to food Selenium (Se) plays an important role in human health. Approximately 80% of the worlds population does not consume enough Se which recommended by WHO (World Health Organization). Wheat is an important staple food and Se source for most people in the world. This article summarizes literatures about Se from 1936 to 2020 to investigate Se in wheat farming soil, wheat, and its derived foods. Se fortification and the recommended Se level in wheat were also discussed. Results showed that Se contents in wheat farming soil, grain, and its derived foods around the world were 3.8-552 (mean, 220.99), 0-8,270 (mean, 347.30), and 15-2,372 (mean, 211.86) g{middle dot}kg-1, respectively. Adopting suitable agronomic measures could effectively realize Se fortification in wheat. The contents in grain, flour, and its derived foods could be improved from 93.94 to 1,181.92, 73.06 to 1,007.75, and 86.90 to 587.61 g{middle dot}kg-1 in average after leaf Se fertilizer application in the field. There was a significant positive correlation between Se content in farming soil and grain, and it was extremely the same between foliar Se fertilizer concentration rate and grain Se increased rate. The recommended Se fortification level in cultivation of wheat in China, India, and Spain was 18.53-23.96, 2.65-3.37, and 3.93-9.88 g{middle dot}hm-2 respectively. Milling processing and food type could greatly affect Se content of wheat derived food and should be considered seriously to meet peoples Se requirement by wheat.

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sentences: Brace root phenotypes predict root lodging susceptibility and the contribution to anchorage in maize Plant mechanical failure (lodging) causes global yield losses of 7-66% in cereal crops. We have previously shown that the above-ground nodal roots (brace roots) in maize are critical for anchorage. However, it is unknown how brace root phenotypes vary across genotypes and the functional consequence of this variation. This study quantifies the contribution of brace roots to anchorage, brace root traits, plant height, and root lodging susceptibility in 52 maize inbred lines. We show that the contribution of brace roots to anchorage and root lodging susceptibility varies among genotypes and this contribution can be explained by plant architectural variation. Additionally, supervised machine learning models were developed and show that multiple plant architectural phenotypes can predict the contribution of brace roots to anchorage and root lodging susceptibility. Together these data define the plant architectures that are important in lodging resistance and show that the contribution of brace roots to anchorage is a good proxy for root lodging susceptibility.

sentences: A unified view of LCRs across species Low-complexity regions (LCRs) in proteins are important for higher-order assemblies of organisms, yet we lack a unified view of their sequences, features, relationships, and functions. Here, we use dotplots and dimensionality reduction to systematically define LCR type/copy relationships and create a map of LCR sequence space capable of integrating LCR features and functions. By defining LCR relationships across the proteome, we provide insight into how LCR type and copy number contribute to higher-order assemblies, such as the importance of K-rich LCR copy number for assembly of the nucleolar protein RPA43 in vivo and in vitro. With LCR maps, we reveal the underlying structure of LCR sequence space, and relate differential occupancy in this space to the conservation and emergence of higher-order assemblies, including the metazoan extracellular matrix and plant cell wall. Together, LCR relationships and maps uncovered the distribution and prevalence of E-rich LCRs in the nucleolus, and revealed previously undescribed regions of LCR sequence space with properties of higher order assemblies, including a teleost-specific T/H-rich sequence space. Thus, this unified view of LCRs enables discovery of how LCRs encode higher-order assemblies of organisms.

sentences: Four novel, immortal cell lines derived from embryos of Xenopus tropicalis The diploid anuran, Xenopus tropicalis, has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10, and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10, and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30{degrees}C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus.

sentences: Macromolecular crowding limits growth under pressure Cells that grow in confined spaces eventually build up mechanical compressive stress. This growth-induced pressure (GIP) decreases cell growth. GIP is important in a multitude of contexts from cancer[1-3], to microbial infections[4], to biofouling, yet our understanding of its origin and molecular consequences remains limited. Here, we combine microfluidic confinement of the yeast Saccha-romyces cerevisiae[5],with rheological measurements using genetically encoded multimeric nanoparticles (GEMs)[6] to reveal that growth-induced pressure is accompanied with an increase in a key cellular physical property: macromolecular crowding. We develop a fully calibrated model that predicts how increased macromolecular crowding hinders protein expression and thus diminishes cell growth. This model is sufficient to explain the coupling of growth rate to pressure without the need for specific molecular sensors or signaling cascades. As molecular crowding is similar across all domains of life, this could be a deeply conserved mechanism of biomechanical feedback that allows environmental sensing originating from the fundamental physical properties of cells.

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Analytical Performance of a Standardized Kit for Mass Spectrometry-based Measurements of Human Glycosaminoglycans Glycosaminoglycans (GAGs) are long linear sulfated polysaccharides implicated in processes linked to disease development such as mucopolysaccharidosis, respiratory failure, cancer, and viral infections, thereby serving as potential biomarkers. A successful clinical translation of GAGs as biomarkers depends on the availability of standardized GAG measurements. However, owing to the analytical complexity associated with the quantification of GAG concentration and structural composition, a standardized method to simultaneously measure multiple GAGs is missing. In this study, we sought to characterize the analytical performance of a ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS)-based kit for the quantification of 17 GAG disaccharides. The kit showed acceptable linearity, selectivity and specificity, accuracy and precision, and analyte stability in the absolute quantification of 15 GAG disaccharides. In native human samples, here using urine as a reference matrix, the analytical performance of the kit was acceptable for the quantification of CS disaccharides. Intra- and inter-laboratory tests performed in an external laboratory demonstrated robust reproducibility of GAG measurements showing that the kit was acceptably standardized. In conclusion, these results indicated that the UHPLC-MS/MS kit was standardized for the simultaneous measurement of GAG disaccharides allowing for comparability of measurements and enabling translational research.

SummaryAnalytical performance of a kit for standardized GAG measurements, based on an established UHPLC-MS/MS method

sentences: MitoVisualize: A resource for analysis of variants in human mitochondrial RNAs and DNA We present MitoVisualize, a new tool for analysis of the human mitochondrial DNA (mtDNA). MitoVisualize enables visualization of: (1) the position and effect of variants in mitochondrial transfer RNA (tRNA) and ribosomal RNA (rRNA) secondary structures alongside curated variant annotations, (2) data across RNA structures, such as to show all positions with disease-associated variants or with post-transcriptional modifications, and (3) the position of a base, gene or region in the circular mtDNA map, such as to show the location of a large deletion. All visualizations can be easily downloaded as figures for reuse. MitoVisualize can be useful for anyone interested in exploring mtDNA variation, though is designed to facilitate mtDNA variant interpretation in particular. MitoVisualize can be accessed via https://www.mitovisualize.org/. The source code is available at https://github.com/leklab/mito_visualize/.

sentences: Biophysical and proteomic analyses suggest functions of Pseudomonas syringae pv tomato DC3000 extracellular vesicles in bacterial growth during plant infection Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. Bacterial EVs contain molecular cargo from the donor bacterium and play important roles in bacterial survival and growth. Here, we describe EV production in plant-pathogenic Pseudomonas syringae pv. tomato DC3000 (Pto DC3000), the causal agent of bacterial speck disease. Cultured Pto DC3000 exhibited EV structures both on the cell surface and in the vicinity of bacterial cells, observed as outer membrane vesicle (OMV) release. We used in-solution trypsin digestion coupled to mass spectrometry to identify 369 proteins enriched in EVs recovered from cultured Pto DC3000. The predicted localization profile of EV proteins supports the production of EVs also in the form of outer-inner-membrane vesicles (OIMVs). EV production varied slightly between bacterial lifestyles and also occurred in planta. The potential contribution of EVs to Pto DC3000 plant infection was assessed using plant treatments and bioinformatic analysis of the EV-enriched proteins. While these results identify immunogenic activities of the EVs, they also point at roles for EVs in bacterial defences and nutrient acquisition by Pto DC3000.

sentences: Cellular Fitness Phenotypes of Cancer Target Genes from Oncobiology to Cancer Therapeutics To define the growing significance of cellular targets of cancer drugs, we examined the fitness dependency of cellular targets or effectors of cancer drug targets across human cancer cells from 19 cancer types. We observed that the deletion of 35 out of 47 cellular mediators or targets of oncology drugs did not result in the expected loss of cell fitness in appropriate cancer types for which drugs targeting or utilizing these molecules were approved. Additionally, our analysis recognized 43 cellular targets as fitness genes in several cancer types in which these drugs were not approved, and thus, providing clues repurposing approved oncology drugs in cancer types. For example, we found the widespread upregulation and fitness dependency of the components of the mevalonate and purine biosynthesis pathways (currently targeted by bisphosphonates, statins, and pemetrexed in certain cancers) and an association between the overexpression of these targets and reduction in the overall survival duration of patients with breast and other hard-to-treat cancers, for which such drugs are not approved. In brief, the present analysis raised cautions about off-target and undesirable effects of certain oncology drugs in a subset of cancers where the in-tended cellular effectors of drug might not be fitness genes and offers a potential rationale for repurposing certain approved oncology drugs for targeted therapeutics in additional cancer types.

sentences: Adar-mediated A-to-I editing is required for establishment of embryonic body axes in zebrafish Adenosine deaminases (ADARs) catalyze the deamination of adenosine to inosine, also known as A-to-I editing, in RNA. Although A-to-I editing occurs widely across animals, and is well studied, new biological roles are still being discovered. Here, we study the role of A-to-I editing in early zebrafish development. We demonstrate that Adar, the zebrafish orthologue of mammalian ADAR1, is essential for establishing the antero-posterior and dorso-ventral axes and patterning. Genome-wide editing discovery revealed pervasive editing in maternal and the earliest zygotic transcripts, the majority of which occurred in the 3-UTR. Interestingly, transcripts implicated in gastrulation as well as dorso-ventral and antero-posterior patterning were found to contain multiple editing sites. Adar knockdown or overexpression affected gene expression and global editing patterns at 12 hpf, but not earlier. Our study established that RNA editing by Adar is necessary for the earliest steps of embryonic patterning along the zebrafish antero-posterior and dorso-ventral axes.

sentences: Self-organization of Tissue Growth by Interfacial Mechanical Interactions in Multi-layered Systems Morphogenesis is a spatially and temporally regulated process involved in various physiological and pathological transformations. In addition to the associated biochemical factors, the physical regulation of morphogenesis has attracted increasing attention. However, the driving force of morphogenesis initiation remains elusive. Here, we show that during the growth of multi-layered tissues, morphogenetic process can be self-organized by the progression of compression gradient stemmed from the interfacial mechanical interactions between layers. In tissues with low fluidity, the compression gradient is progressively strengthened during growth and induces stratification by triggering symmetric-to-asymmetric cell division reorientation at the critical tissue size. In tissues with high fluidity, compression gradient is dynamic and induces cell junction remodelling regulated cell rearrangement leading to 2D in-plane morphogenesis instead of 3D deformation. Morphogenesis can be tuned by manipulating tissue fluidity, cell adhesion forces and mechanical properties to influence the progression of compression gradient during the development of cultured cell sheets and chicken embryos. Together, the dynamics of compression gradient arised from interfacial mechanical interaction provides a conserved mechanism underlying morphogenesis initiation and size control during tissue growth.

sentences: Use of Raman and Raman optical activity to extract atomistic details of saccharides in aqueous solution Sugars are crucial components in biosystems and industrial applications. In aqueous environments, the natural state of short saccharides or charged glycosaminoglycans is floating and wiggling in solution. Therefore, tools to characterize their structure in a native aqueous environment are crucial but not always available. Here, we show that a combination of Raman/ROA and, on occasions, NMR experiments with Molecular Dynamics (MD) and Quantum Mechanics (QM) is a viable method to gain insights into structural features of sugars in solutions. Combining these methods provides information about accessible ring puckering conformers and their proportions. It also provides information about the conformation of the linkage between the sugar monomers, i.e., glycosidic bonds, allowing for identifying significantly accessible conformers and their relative abundance. For mixtures of sugar moieties, this method enables the deconvolution of the Raman/ROA spectra to find the actual amounts of its molecular constituents, serving as an effective analytical technique. For example, it allows calculating anomeric ratios for reducing sugars and analyzing more complex sugar mixtures to elucidate their real content. Altogether, we show that combining Raman/ROA spectroscopies with simulations is a versatile method applicable to saccharides. It allows for accessing many features with precision comparable to other methods routinely used for this task, making it a viable alternative. Furthermore, we prove that the proposed technique can scale up by studying the complicated Raffinose trisaccharide, and therefore, we expect its wide adoption to characterize sugar structural features in solution.

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Disrupting inferior frontal cortex activity alters affect decoding efficiency from clear but not from ambiguous affective speech The evaluation of socio-affective sound information is accomplished by the primate neural auditory cortex in collaboration with limbic and inferior frontal brain nodes. For the latter, activity in inferior frontal cortex (IFC) is often observed during classification of voice sounds, especially if they carry affective information. Partly opposing views have been proposed, with IFC either coding cognitive processing challenges in case of sensory ambiguity or representing categorical object and affect information for clear vocalizations. Here, we presented clear and ambiguous affective speech to two groups of human participants during neuroimaging, while in one group we inhibited right IFC activity with transcranial magnetic stimulation (TMS) prior to brain scanning. Inhibition of IFC activity led to partly faster affective decisions, more accurate choice probabilities and reduced auditory cortical activity for clear affective speech, while fronto-limbic connectivity increased for clear vocalizations. This indicates that IFC inhibition might lead to a more intuitive and efficient processing of affect information in voices. Contrarily, normal IFC activity might represent a more deliberate form of affective sound processing (i.e., enforcing cognitive analysis) that flags categorial sound decisions with precaution (i.e., representation of categorial uncertainty). This would point to an intermediate functional property of the IFC between previously assumed mechanisms.

TeaserInferior frontal cortex enforces cognitive analyses during affect decisions with different levels of sensory ambiguity.

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Y recombination arrest and degeneration in the absence of sexual dimorphism Current theory proposes degenerated sex chromosomes evolve via three successive steps: recombination arrest, which links male-beneficial alleles to the Y chromosome; degeneration of these regions due to the inefficacy of natural selection in the absence of recombination; and lastly, the evolution of dosage compensation to correct the resulting low expression of X-linked genes in males. Here we investigate new models of sex chromosome evolution incorporating the coevolution of cis- and trans-regulators of gene expression. We show that the early emergence of dosage compensation favors the maintenance of Y-linked inversions by creating sex-antagonistic regulatory effects. This is followed by inversion degeneration caused by regulatory divergence between the X and Y chromosomes. In stark contrast to the current theory, the whole process occurs without any selective pressure related to sexual dimorphism.

One Sentence SummaryTurning sex chromosome theory on its head: early evolution of dosage compensation can maintain successively forming Y chromosome strata that undergo genetic degeneration.

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Age-related deficits in rapid visuomotor decision-making Many goal-directed actions that require rapid visuomotor planning and perceptual decision-making are affected in older adults, causing difficulties in execution of many functional activities of daily living. Visuomotor planning and perceptual decision-making are mediated by the dorsal and ventral visual streams, respectively, but it is unclear how age-induced changes in sensory processing in these streams contribute to declines in goal-directed actions. Previously, we have shown that in healthy adults task demands affect the integration of sensory information between the two streams and more motorically demanding tasks induce earlier decisions and more decision errors. Here, we asked the question if older adults would exhibit larger declines in interactions between the two streams during demanding motor tasks. Older adults (n=15) and young controls (n=26) performed a simple reaching task and a more demanding interception task towards virtual objects. In some blocks of trials, participants also had to select an appropriate movement based on the shape of the object. Our results showed that older adults made a similar number of initial decision errors during both the reaching and interception tasks but corrected fewer of those errors during movement. During the more demanding interception decision task, older adults made more decision- and execution-related errors than young adults, which were related to early initiation of their movements. Together, these results suggest that older adults have a reduced ability to integrate new perceptual information to guide online action, which may reflect impaired ventral-dorsal stream interactions.

HighlightsO_LIOlder adults showed reduced performance in a visuomotor decision-making task
C_LIO_LIInitial decision errors were similar between young and older adults
C_LIO_LIOlder adults were less likely to correct initial decision errors
C_LIO_LIMore demanding movements were associated with earlier and less accurate decisions
C_LI

sentences: Hydroxyzine inhibits SARS-CoV-2 Spike protein binding to ACE2 in a qualitative in vitro assay COVID-19 currently represents a major public health problem. Multiple efforts are being performed to control this disease. Vaccinations are already in progress. However, no effective treatments have been found so far. The disease is caused by the SARS-CoV-2 coronavirus that through the Spike protein interacts with its cell surface receptor ACE2 to enter into the host cells. Therefore, compounds able to block this interaction may help to stop disease progression. In this study, we have analyzed the effect of compounds reported to interact and modify the activity of ACE2 on the binding of the Spike protein. Among the compounds tested, we found that hydroxyzine could inhibit the binding of the receptor-binding domain of Spike protein to ACE2 in a qualitative in vitro assay. This finding supports the reported clinical data showing the benefits of hydroxyzine on COVID-19 patients, raising the need for further investigation into its effectiveness in the treatment of COVID-19 given its well-characterized medical properties and affordable cost.

sentences: A memristor-based neuromodulation device for real-time monitoring and adaptive control of in vitro neuronal populations Neurons are specialized cells in information transmission and information processing. Following this, many neurologic disorders are directly linked not to cellular viability/homeostasis issues but rather to specific anomalies in electrical activity dynamics. Acknowledging this fact, therapeutic strategies based on direct modulation of neuronal electrical activity have been producing remarkable results, with successful examples ranging from cochlear implants to deep brain stimulation. Development on these implantable devices are hindered, however, by important challenges: power requirements, size factor, signal transduction, and adaptability/computational capabilities. Memristors, nanoscale electronic components able to emulate natural synapses, provide unique properties to address these constraints and their use in neuroprosthetic devices is being actively explored. Here we demonstrate for the first time the use of memristive devices in a clinically relevant setting where communication between two neuronal populations is conditioned to specific activity patterns in the source population. In our approach, the memristor device performs a simple pattern detection computation and acts as a synapstor capable of reversible short-term plasticity. Using in vitro hippocampal neuronal cultures, we show real-time adaptive control with a high degree of reproducibility using our monitor-compute-actuate paradigm. We envision very similar systems being used for automatic detection and suppression of seizures in epileptic patients.

sentences: Iron Deficiency Induces Heart Failure with Ectopic Cardiac Calcification in Mice with Metabolic Syndrome Iron deficiency is linked to worse clinical status and outcomes in heart failure. Although metabolic syndrome contributes to the development of heart failure, the impact of iron deficiency in heart failure complicated with metabolic syndrome remains obscure. KKAy mice were used as a model of metabolic syndrome. Four-week-old male C57BL/6J and KKAy mice were fed either a normal diet or iron-restricted (IR) diet for 12 weeks. During the experiment, 40% of mice died due to pulmonary congestion in KKAy mice with IR diet (KKAy-IR), while no mice died in other groups. Necropsy showed the presence of multiple white lesions on the cardiac surface in those KKAy-IR mice. Echocardiography and histological analyses revealed that KKAy-IR mice exhibited cardiac hypertrophy and cardiac dysfunction with cardiac calcification. Cardiac mRNA of ectonucleotide pyrophosphatase/phosphodiesterase-1 (Enpp1), a key enzyme for bone mineralization, was highly abundant in KKAy-IR mice. Of note, iron restriction-induced cardiac calcification and dysfunction were attenuated by etidronate, an inhibitor of bone mineralization, with decreased cardiac Enpp1 mRNA abundance in KKAy-IR mice. In conclusion, iron deficiency leads to ectopic cardiac calcification and dysfunction with the increase of Enpp1 in metabolic syndrome model mice.

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Heritable differences in synaptic zinc-transporter levels drive variation in learned birdsong Complex learned behaviors exhibit striking variation within populations, yet how heritable factors contribute to such inter-individual differences remains largely unknown. Here, we used behavioral-genetic analysis within a Bengalese finch population (Lonchura striata domestica) to investigate molecular and circuit mechanisms underlying heritable differences in the tempo of learned birdsong. We identified a genomic locus encoding the zinc transporter ZIP11 and found that zip11(SLC39A11) transcript was expressed at higher levels in song control circuitry of faster singing birds. Reducing soluble zinc increased synaptic currents in motor circuitry and accelerated song, whereas reducing ZIP11 slowed song. Our results reveal a novel zinc-dependent mechanism that modulates neural activity to drive differences in behavior and suggest that natural variation in learning may preferentially target modulatory processes rather than core neural machinery.

One sentence summaryHeritable levels of a synaptic zinc transporter drive inter-individual differences in circuit excitability and learned song

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Selective cleavage of ncRNA and antiviral activity by human RNase2/EDN in a macrophage infection model RNase2, also named the Eosinophil derived Neurotoxin (EDN), is one of the main proteins secreted by the eosinophil secondary granules. RNase2 is also expressed in other leukocyte cells and is the member of the human ribonuclease A family most abundant in macrophages. The protein is endowed with a high ribonucleolytic activity and participates in the host antiviral activity. Although RNase2 displays a broad antiviral activity, it is mostly associated to the targeting of single stranded RNA viruses. To explore RNase2 mechanism of action in antiviral host defence we knocked out RNase2 expression in the THP1 monocyte cell line and characterized the cell response to human Respiratory Syncytial Virus (RSV). We observed that RSV infection induced the RNase2 expression and protein secretion in THP1 macrophage-derived cells, whereas the knockout (KO) of RNase2 resulted in higher RSV burden and reduced cell viability. Next, by means of the cP-RNAseq methodology, which uniquely amplifies the RNA 23cyclic-phosphate-end products released by an endonuclease cleavage, we compared the ncRNA population in native and RNase2-KO cell lines. Among the ncRNAs accumulated in WT versus KO cells, we found mostly tRNA-derived fragments and secondly miRNAs. Analysis of the differential sequence coverage of tRNAs molecules in native and KO cells identified fragments derived from only few parental tRNAs, revealing a predominant cleavage at anticodon loops and secondarily at D-loops. Inspection of cleavage region identified U/C and A, at 5 and 3 sides of cleavage sites respectively (namely RNase B1 and B2 base binding subsites). Likewise, only few selected miRNAs were significantly more abundant in WT versus RNase2-KO cells, with cleavage sites located at the end of stem regions with predominance for pyrimidines at B1 but following an overall less defined nucleotide specificity.

Complementarily, by screening of a tRF&tiRNA PCR array we identified an enriched population of tRNA-derived fragments associated to RNase2 expression and RSV infection. The present results confirm the contribution of the protein in macrophage response against virus infection and provide the first evidence of its cleavage selectivity against ncRNA population. A better understanding of the mechanism of action of RNase2 recognition of cellular RNA during the antiviral host defence should pave the basis for the design of novel antiviral drugs.

sentences: Cell division drives DNA methylation loss in late-replicating domains in primary human cells DNA methylation undergoes dramatic age-related changes, first described more than four decades ago1-4. Loss of DNA methylation at late-replicating regions of the genome attached to the nuclear lamina advances with age in normal tissues, and is further exacerbated in cancer5-7. We present here the first experimental evidence that this DNA hypomethylation is directly driven by proliferation-associated DNA replication. Loss of DNA methylation at low-density CpGs in A:T-rich, partially methylated domains (PMD solo-WCGWs), tracks cumulative population doublings in primary cell culture. Cell cycle deceleration resulted in a proportional decrease in the rate of DNA hypomethylation. Blocking DNA replication via Mitomycin C treatment halted methylation loss. Loss of methylation continued unabated after TERT immortalization until finally reaching a severely hypomethylated equilibrium. Ambient oxygen culture conditions increased the rate of methylation loss compared to low-oxygen conditions, suggesting that some methylation loss may occur during unscheduled, oxidative damage repair-associated DNA synthesis. Finally, we present and validate a model to estimate the relative cumulative replicative histories of human cells, which we call "RepliTali" (Replication Times Accumulated in Lifetime).

sentences: BinChecker: a new algorithm for quality assessment of microbial draft genomes In the reconstruction of microbial genomes from metagenomic sequence data, the estimation of the final completeness and possible contamination is crucial for quality control. In metagenomics candidate genomes are usually obtained from a metagenome assembly and a subsequent binning of the assembled contigs. BinChecker provides a novel approach to quality assessment that is based on a fast protein domain search and a clustering approach for identification of marker domain ("feature") sets. The feature sets that are used for estimation are not pre-computed for a given database of reference genomes, but are individually found for each bin by adaptive clustering and feature selection. In particular, the adaptivity facilitates the creation and extension of the underlying database, which just requires to add protein feature profiles of reference genomes. Tests with simulated bins indicate that the prediction accuracy of BinChecker meets the current state of the art while providing significant advantages in terms of speed and flexibility.

sentences: High coverage of single cell genomes by T7-assisted enzymatic methyl-sequencing Conventional approaches to studying 5mC marks in single cells or samples with picogram input DNA amounts usually suffer from low genome coverage due to DNA degradation. Many methods have been developed to optimize the library construction efficiency for bisulfite-treated DNA. However, most of these approaches ignored the amplification bias of bisulfite-treated DNA, which leads to shallow genome coverage. In this study, we developed the T7-assisted enzymatic methyl-sequencing method (TEAM-seq), which adopts enzymatic conversion to minimize DNA degradation and T7 polymerase-assisted unbiased amplification. We demonstrate that TEAM-seq delivered, to the best of our knowledge, the highest reported coverage(70% for 100pg, 35% for 20pg) of single cell genomes in whole-genome 5mC sequencing.

sentences: Spatial cumulant models enable spatially informed treatment strategies and analysis of local interactions in cancer systems Theoretical and applied cancer studies that use individual-based models (IBMs) have been limited by the lack of a mathematical formulation that enables rigorous analysis of these models. However, spatial cumulant models (SCMs), which have arisen from recent advances in theoretical ecology, can be used to describe population dynamics generated by a specific family of IBMs, namely spatio-temporal point processes (STPPs). SCMs are spatially resolved population models formulated by a system of ordinary differential equations that approximate the dynamics of two STPP-generated summary statistics: first order spatial cumulants (densities), and second order spatial cumulants (spatial covariances). Here, we exemplify how SCMs can be used in mathematical oncology by modelling a theoretical cancer cell population comprising interacting growth factor producing and non-producing cells. To formulate our models, we use the Unified Framework Software which was recently developed by Cornell et al. (2019) and enables the generation of STPPs, SCMs and mean-field population models (MFPMs) from user-defined model descriptions. Our results demonstrate that SCMs can capture STPP-generated population density dynamics, even when MFPMs fail to do so. From both MFPM and SCM equations, we derive treatment-induced cell death rates required to achieve non-growing cell populations. When testing these treatment strategies in STPP-generated cell populations, our results demonstrate that SCM-informed strategies outperform MFPM-informed strategies in terms of inhibiting population growths. We argue that SCMs provide a new framework in which to study cell-cell interactions, and can be used to deepen the mathematical analysis of IBMs and thereby increase IBMs' applicability in cancer research.

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Experiencing surprise: the temporal dynamics of its impact on memory To efficiently process information, the brain shifts between encoding and retrieval states, prioritising bottom-up or top-down processing accordingly. Expectation violation before or during learning has been shown to trigger an adaptive encoding mechanism, resulting in better memory for unexpected events. Using fMRI we explored (1) whether this encoding mechanism is also triggered during retrieval, and if so, (2) what the temporal dynamics of its mnemonic consequences are. Male and female participants studied object images, then, with new objects, they learned a contingency between a cue and a semantic category. Rule-abiding (expected) and violating (unexpected) targets and similar foils were used at test. We found interactions between previous and current similar events expectation, such that when an expected event followed a similar but unexpected event, its performance was boosted, underpinned by activation in the hippocampus, midbrain, and occipital cortex. In contrast, a sequence of two unexpected similar events also triggered occipital engagement, however, this did not enhance memory performance. Taken together, our findings suggest that when the goal is to retrieve, encountering surprising events engages an encoding mechanism, supported by bottom-up processing, that may enhance memory for future related events.

Significance statementOptimising the balance between new learning and the retrieval of existing knowledge is an ongoing process, at the core of human cognition. Previous research into memory encoding suggests experiencing surprise leads to the prioritisation of the leaning of new memories, forming an adaptive encoding mechanism. We examined whether this mechanism is also engaged when the current goal is to retrieve information. Our results demonstrate that an expectation-driven shift towards an encoding state, supported by enhanced perceptual processing, is beneficial for the correct identification of subsequent expected similar events. These findings have important implications for our understanding of the temporal dynamics of the adaptive encoding of information into memory.

sentences: SARS-CoV-2 RdRp uses NDPs as a substrate and is able to incorporate NHC into RNA from diphosphate form molnupiravir The coronavirus disease 2019 (COVID-19) has been ravaging throughout the world for more than two years and has severely impaired both human health and the economy. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) employs the viral RNA-dependent RNA polymerase (RdRp) complex for genome replication and transcription, making RdRp an appealing target for antiviral drug development. Here, we reveal that RdRp can recognize and utilize nucleoside diphosphates (NDPs) as a substrate to synthesize RNAs with an efficiency of about two thirds of using nucleoside triphosphates (NTPs) as a substrate. NDPs incorporation is also template-specific and has high fidelity. Moreover, RdRp can incorporate {beta}-d-N4-hydroxycytidine (NHC) into RNA while using diphosphate form molnupiravir (MDP) as a substrate. We also observed that MDP is a better substrate for RdRp than the triphosphate form molnupiravir (MTP).

sentences: A neural progenitor mitotic wave is required for asynchronous axon outgrowth and morphology Spatiotemporal mechanisms generating neural diversity are fundamental for understanding neural processes. Here, we investigated how neural diversity arises from neurons coming from identical progenitors. In the dorsal thorax of Drosophila, rows of mechanosensory organs originate from the division of sensory organ progenitor (SOPs). We show that in each row of the notum, a central SOP divides first, then neighboring SOPs divide, and so on. This centrifugal wave of mitoses depends on cell-cell inhibitory interactions mediated by SOP cytoplasmic protrusions and Scabrous, a secreted protein interacting with the Delta/Notch complex. Furthermore, when the mitotic wave was abolished, axonal growth was more synchronous, axonal terminals had a complex branching pattern and fly behavior was impaired. We show that the temporal order of progenitor divisions influences the birth order of sensory neurons, axon branching and impact on grooming behavior. These data support the idea that developmental timing controls axon wiring neural diversity.

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Links between memory, synapses, amyloid-beta, tau and microglial changes after tau pathology induction by Alzheimer's brain inoculation in amyloid-beta-plaque bearing mice BackgroundAlzheimers disease (AD) is characterized by the presence of both extracellular amyloid-{beta} (A{beta}) plaques and intracellular tau accumulations. The causative interactions between these lesions, and the subsequent neuroinflammation, synaptic alterations and memory impairment are still debated. Intracerebral infusion of human AD brain extracts in A{beta} bearing mice that do not overexpress pathological tau proteins induces widespread A{beta} and tau pathologies following heterotopic seeding of mouse tau. These models provide a unique opportunity to assess relationships between AD lesions and downstream events.

MethodsHuman AD and control-brain extracts (ADbe and Ctrlbe) were infused in the hippocampus of A{beta} plaque-bearing APPswe/PS1dE9 mice. We evaluated the links between A{beta} plaques, tau aggregates, microgliosis, astrogliosis at the inoculation site and in connected regions (perirhinal/entorhinal cortex), and memory impairment and synaptic density 4 and 8 months post-inoculation.

ResultsADbe-inoculated animals displayed memory deficit and synaptic loss in addition to A{beta} and tau pathological aggregates. A{beta} plaques were detected in both ADbe- and Ctrlbe-inoculated APPswe/PS1dE9 mice, but their load was increased close to the inoculation site in ADbe- inoculated animals. Tau-positive neuropil threads and neurofibrillary tangles occurred next to the inoculation site only in ADbe-inoculated animals and were not detected in Ctrlbe mice. These lesions spread to connected regions notably the perirhinal/entorhinal cortex. Tau-positive neuritic plaques were detected in both ADbe- and Ctrlbe-inoculated animals. Unexpectedly, ADbe inoculation did not increase the number of neuritic plaques close to the inoculation site as compared to Ctrlbe inoculation. ADbe inoculation however increased the area of tau-positive elements within these neuritic plaques. Increased tau pathology as well as lower Iba1-positive microglial load in the hippocampus and perirhinal/entorhinal cortex were correlated with memory impairment and with synaptic loss.

ConclusionsThe integrative analysis of pathological events associated with ADbe inoculation in A{beta}-bearing mice highlighted memory and synaptic loss as well as the induction of tau lesions that spread in the brain. Increased tau lesions and lower microglial load are the two main events correlated with memory impairment and synaptic loss. The study suggests that microglial activity may be protective.

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sentences: Dual RNA modulation of protein mobility and stability within phase-separated condensates One of the key mechanisms employed by cells to control their spatiotemporal organization is the formation and dissolution of phase-separated condensates. The balance between condensate assembly and disassembly can be critically regulated by the presence of RNA. In this work, we use a novel sequence-dependent coarse-grained model for proteins and RNA to unravel the impact of RNA in modulating the transport properties and stability of biomolecular condensates. We explore the phase behavior of several RNA-binding proteins such as FUS, hnRNPA1 and TDP-43 proteins along with that of their corresponding prion-like domains and RNA-recognition motifs, from absence to moderately high RNA concentration. By characterising the phase diagram, key molecular interactions, surface tension and transport properties of the condensates, we report a dual RNA-induced behavior: On the one hand, RNA enhances phase separation at low concentration as long as the RNA radius of gyration is comparable to that of the proteins, whilst at high concentration it inhibits the ability of proteins to self-assemble independently of its length. On the other hand, along with the stability modulation, the viscosity of the condensates can be considerably reduced at high RNA concentration as long as the length of the RNA chains is shorter than that of the proteins. Conversely, long RNA strands increase viscosity, even at high concentration, but barely modify protein self-diffusion, which mainly depends on RNA concentration and on its own effect on droplet density. On the whole, our work rationalizes the different routes by which RNA can regulate phase separation and condensate dynamics, as well as the subsequent aberrant rigidification implicated in the emergence of various neuropathologies and age-related diseases.

sentences: Harmoni: a Method for Eliminating Spurious Interactions due to the Harmonic Components in Neuronal Data Cross-frequency synchronization (CFS) has been proposed as a mechanism for integrating spatially and spectrally distributed information in the brain. However, investigating CFS in Magneto- and Electroencephalography (MEG/EEG) is hampered by the presence of spurious neuronal interactions due to the non-sinusoidal waveshape of brain oscillations. Such waveshape gives rise to the presence of oscillatory harmonics mimicking genuine neuronal oscillations. Until recently, however, there has been no methodology for removing these harmonics from neuronal data. In order to address this long-standing challenge, we introduce a novel method (called HARMOnic miNImization - Harmoni) that removes the signal components which can be harmonics of a non-sinusoidal signal. Harmonis working principle is based on the presence of CFS between harmonic components and the fundamental component of a non-sinusoidal signal. We extensively tested Harmoni in realistic EEG simulations. The simulated couplings between the source signals represented genuine and spurious CFS and within-frequency phase synchronization. Using diverse evaluation criteria, including ROC analyses, we showed that the within- and cross-frequency spurious interactions are suppressed significantly, while the genuine activities are not affected. Additionally, we applied Harmoni to real resting-state EEG data revealing intricate remote connectivity patterns which are usually masked by the spurious connections. Given the ubiquity of non-sinusoidal neuronal oscillations in electrophysiological recordings, Harmoni is expected to facilitate novel insights into genuine neuronal interactions in various research fields, and can also serve as a steppingstone towards the development of further signal processing methods aiming at refining within- and cross-frequency synchronization in electrophysiological recordings.

sentences: CRY-BARs: Versatile light-gated molecular tools for the remodeling of membrane architectures BAR (Bin, Amphiphysin and Rvs) protein domains are responsible for the generation of membrane curvature and represent a critical mechanical component of cellular functions. Thus, BAR domains have great potential as components of membrane-remodeling tools for cell biologists. In this work, we describe the design and implementation of a family of versatile light-gated I-BAR domain containing tools ( CRY-BARs) with applications in the remodeling of membrane architectures and the control of cellular dynamics. By taking advantage of the intrinsic membrane binding propensity of the I-BAR domain, CRY-BARs can be used for spatial and temporal control of cellular processes that require induction of membrane protrusions. Using cell lines and primary neuron cultures, we demonstrate that the CRY-BAR optogenetic tool reports membrane dynamic changes associated with cellular activity. Moreover, we provide evidence that Ezrin acts as a relay between the plasma membrane and the actin cytoskeleton and therefore is an important mediator of switch function. Overall, CRY-BARs hold promise as a useful addition to the optogenetic toolkit to study membrane remodeling in live cells.

sentences: Deficiency of p38α-MAPK in myeloid cells ameliorates symptoms and pathology of APP-transgenic Alzheimer's disease mice Microglial activation is a hall marker of Alzheimers disease (AD); its pathogenic role and regulating mechanisms are unclear. p38-MAPK, a stress-responding kinase, is activated in AD brain in early disease stages. In APP-transgenic mice, we deleted p38-MAPK in whole myeloid cells from birth or specifically in microglia from 9 months, and analysed AD pathology at the age of 4, 9 and 12 months. In both experimental settings, p38-MAPK deficiency decreased cerebral A{beta} and improved cognitive function of AD mice; however, p38-MAPK-deficient myeloid cells were more effective than p38-MAPK-deficient microglia in preventing AD pathogenesis. Deficiency of p38-MAPK in myeloid cells inhibited the inflammatory activation of individual microglia by 4 months, but enhanced it by 9 months. Inflammatory activation was essential for p38-MAPK deficiency to promote microglial internalization of A{beta}. Interestingly, p38-MAPK deficiency in peripheral myeloid cells reduced il-17a transcription in CD4-positive spleen cells. By cross-breeding APP-transgenic mice and IL-17a knockout mice, we further observed that IL-17a deficiency activated microglia and decreased A{beta} deposits in AD mouse brain. Thus, p38-MAPK deficiency in myeloid cells prevents AD pathogenesis, perhaps through reducing IL-17a-expressing T lymphocytes, and promoting A{beta} clearance in the brain. Our study supports p38-MAPK as a novel target for AD therapy.

sentences: Nicotinamide riboside alleviates Parkinson's disease symptoms but downregulates dopamine metabolism upon lactacystin-induced proteostasis failure Activation of mitochondrial metabolism and proteostasis with the NAD+ precursor nicotinamide riboside (NR) has emerged as a potential therapeutic approach for neurodegenerative disorders including Parkinsons disease (PD). However, despite recently started clinical trials, studies on NR in animal models of PD are scarce. In this study, we investigated the effect of NR in multiple models of PD. In transgenic C. elegans overexpressing -synuclein, a protein of which aggregation is believed to promote PD, NR rescued PD-like phenotypes including mitochondrial dysfunction and motility defects, decreased oxidative stress, and age-related dopamine (DA) neuron loss. We found that NR eased symptoms of disease by activating the mitochondrial unfolded protein response (UPRmt) via the transcription factor atfs-1. Similarly, in a proteasome inhibitor, lactacystin, -induced mouse model of PD, NR rescued mitochondrial dysfunction and behavioural deficits caused by lactacystin lesion. However, long-term NR supplementation, in conjunction with proteasome inhibition, resulted in decreased DA levels in both the lesioned and unlesioned sides of the substantia nigra with concomitant downregulation of key genes in DA metabolism. Our results suggest specific endpoints that should be monitored in ongoing NR clinical trials.

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Nanoviricides platform technology based NV-CoV-2 polymer increases the half-life of Remdesivir in vivo So far, there are seven coronaviruses identified that infect humans and only 4 of them belong to the beta family of coronavirus (HCoV-HKU1, SARS-CoV-2, MERS-CoV and SARS-CoV). SARS family are known to cause severe respiratory disease in humans. In fact, SARS-CoV-2 infection caused a pandemic COVID-19 disease with high morbidity and mortality. Remdesivir (RDV) is the only antiviral drug so far approved for COVID-19 therapy by the FDA. However, the efficacy of RDV in vivo is limited due to its low stability in presence of plasma. This is the report of analysis of the non-clinical pharmacology study of NV-CoV-2 (Polymer) and NV-CoV-2-R (Polymer encapsulated Remdesivir) in both infected and uninfected rats with SARS-CoV-2.

Detection and quantification of NV-CoV-2-R in plasma samples was done by MS-HPLC chromatography analyses of precipitated plasma samples from rat subjects.

O_LINV-CoV-2-R show RDV peak in MS-HPLC chromatography, whereas only NV-CoV-2 does not show any RDV-Peak, as expected.
C_LIO_LINV-CoV-2 polymer encapsulation protects RDV in vivo from plasma-mediated catabolism.
C_LIO_LIBody weight measurements of the normal (uninfected) rats after administration of the test materials (NV-CoV-2, and NV-CoV-2-R) show no toxic effects on them.
C_LI

Our platform technology based NV-387-encapsulated-RDV (NV-CoV-2-R) drug has a dual effect on coronaviruses. First, NV-CoV-2 itself as an antiviral regimen. Secondly, RDV is protected from plasma-mediated degradation in transit, rendering altogether the safest and an efficient regimen against COVID-19.

sentences: Somatodendritic release of cholecystokinin potentiates GABAergic synapses onto ventral tegmental area dopamine cells Neuropeptides are contained in nearly every neuron in the central nervous system and can be released from somatodendritic sites as well as from nerve terminals. Cholecystokinin (CCK), among the most abundant neuropeptides in the brain, is expressed in the majority of midbrain dopamine neurons. Here we report that ventral tegmental area (VTA) dopamine neurons release CCK from somatodendritic regions, where it triggers long-term potentiation of GABAergic synapses. The somatodendritic release occurs with trains of action potentials or prolonged but modest depolarization and is dependent on synaptotagmin 7 and T-type Ca2+ channels. Depolarization-induced LTP is blocked by the CCK2R antagonist, LY225910, and mimicked by exogenously added CCK. To test the behavioral role of CCK, we infused it into the mouse VTA. Ca2+ imaging in vivo demonstrated that infused CCK reduced dopamine cell signals during fasted food consumption. Moreover, local infusion of CCK also inhibited food consumption and decreased distance traveled in an open field test. Together our experiments introduce somatodendritic neuropeptide release as a previously unknown feedback regulator of VTA dopamine cell excitability and dopamine-related behaviors.

sentences: Post-weaning social isolation modulates reward-seeking behavior in a sex-specific manner in mice Social isolation is a growing concern in public health. Although isolation at any age is harmful, previous studies have shown that isolation during adolescence, correlating with critical periods of brain development, can impair cognitive function and increase the risk for psychiatric illness later in life. In this study, we utilized a mouse model of adolescent social isolation (SI) and compared performance of isolated and group-housed mice on touchscreen-based continuous performance test (CPT) and fixed ratio/progressive ratio (FR/PR) tasks in adulthood. SI increased sensitivity in the CPT in male mice and had no effect in female mice. The increase in sensitivity was consistent across time bins within the 45-minute testing session and there were no SI effects on reaction times or reward retrieval latencies. A possible confound for performance in the CPT would be SI-induced changes in reward-seeking or motivation for the strawberry milk reward. We next compared the SI mice to their group-housed littermate controls on both FR and PR schedules of reinforcement and found that male SI mice earned significantly more reinforcers on FR schedules of reinforcement and had higher breakpoints on PR schedules compared to their group-housed littermates. SI had no effect on FR or PR performance in female mice. These data indicate that SI during adolescence has striking, sex-specific effects on reward-seeking behavior in adult mice and may provide a useful behavioral model for studying the link between SI and risk for neuropsychiatric disorders.

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Atlas of tissue-specific and tissue-preferential gene expression in ecologically and economically significant conifer Pinus sylvestris Despite their ecological and economical importance, conifers genomic resources are limited, mainly due to the large size and complexity of their genomes. Additionally, the available genomic resources lack complete structural and functional annotation. Transcriptomic resources have been commonly used to compensate for these deficiencies, though for most conifer species they are limited to a small number of tissues, or capture only a fraction of the genes present in the genome.

Here we provide an atlas of gene expression patterns for conifer Pinus sylvestris across five tissues: embryo, megagametophyte, needle, phloem, and vegetative bud. We used a wide range of tissues and focused our analyses on the expression profiles of genes at tissue level. We provide comprehensive information of the per-tissue normalized expression level, indication of tissue preferential upregulation and tissue-specificity of expression. We identified a total of 48,001 tissue preferentially upregulated and tissue specifically expressed genes, of which 28% have annotation in the Swiss-Prot database. Even though most of the putative genes identified do not have functional information in current biological databases, the tissue-specific patterns discovered provide valuable information about their potential functions for further studies, as for example in the areas of plant physiology, population genetics, and genomics in general. As we provide information on tissue specificity at both diploid and haploid life stages, our data will also contribute to the understanding of evolutionary rates of different tissue types and ploidy levels.

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Processive translocation of cohesive and non-cohesive cohesin in vivo Cohesin is a central architectural element of chromosomes that regulates numerous DNA-based events. The complex holds sister chromatids together until anaphase onset and organizes individual chromosomal DNAs into loops. Purified cohesin translocates along DNA in a diffusive fashion that can be propelled by transcribing RNA polymerase. The complex also extrudes DNA loops in a process that consumes ATP. In this study we examine processive genomic translocation of cohesin in vivo. To this end, obstacles of increasing size were tethered to DNA to act as roadblocks to complexes mobilized by transcription in yeast. The obstacles were built from a GFP-lacI core fused to one or more mCherries. A chimera with four mCherries blocked cohesin passage in late G1. During M phase, the threshold barrier depended on the state of cohesion: non-cohesive complexes were also blocked by four mCherries whereas cohesive complexes were blocked by as few as three mCherries. Furthermore, cohesive complexes that were stalled at obstacles, in turn, blocked the passage of non-cohesive complexes. That synthetic barriers alter cohesin redistribution demonstrates that the complex translocates processively on chromatin in vivo. The approach provides a relative measure of the maximum size of the DNA binding chamber(s) of cohesin. Together, this study reveals unexplored limitations to cohesin movement on chromosomes.

Significance StatementCohesin is an architectural protein that brings distant chromosomal DNA sites together. The complex links sister chromatids cohesion but it also binds to single pieces of DNA in ways that do not generate cohesion. One class of non-cohesive complexes organizes chromosomal DNA into loops. All cohesin complexes move on DNA but the constraints on such movement are not fully explored. Here, we use size-calibrated obstacles in yeast to interrogate cohesin and the properties of its movement on DNA. We show that both cohesive and non-cohesive complexes translocate processively on chromosomes. In addition, we show that cohesive and non-cohesive complexes are blocked by obstacles of different size. Lastly, we show that stalled cohesive complexes block passage of non-cohesive complexes.

sentences: Glucocorticoids Unmask Silent Non-Coding Genetic Risk Variants for Common Diseases Understanding the function of non-coding genetic variants represents a formidable challenge for biomedicine. We previously identified genetic variants that influence gene expression only after exposure to a hormone or drug. Using glucocorticoid signaling as a model system, we have now demonstrated, in a genome-wide manner, that exposure to glucocorticoids triggered disease risk variants with previously unclear function to influence the expression of genes involved in autoimmunity, metabolic and mood disorders, osteoporosis and cancer. Integrating a series of pharmacogenomic and pharmacoepigenomic datasets, we identified the cis-regulatory elements and 3-dimensional interactions underlying the ligand-dependent associations between those genetic variants and distant risk genes. These observations increase our understanding of mechanisms of non-coding genetic variant-chemical environment interactions and advance the fine-mapping of disease risk and pharmacogenomic loci.

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NtrC expression at lag-phase improves competitive fitness under low and fluctuating nutrient condition The enteric pathogens cycle between nutrient rich host and nutrient poor external environment. These pathogens compete for nutrients in the host as well as in external environment and also often experience starvation. In this context, we have studied the role of a global nitrogen regulator (NtrC) of Salmonella Typhimurium. The ntrC mutation caused extended lag phase and slow growth in minimal media. In lag phase the wild-type cells showed [~]60 fold more expression of ntrC as compared to log phase cells. The role of ntrC gene is often studied with respect to nitrogen scavenging in a low nitrogen growing condition. However, our observation indicates that, even in the adequate supply of nitrogen, the ntrC null mutants failed to adapt to new nutrient conditions and were slow to exit from the lag phase. Gene expression studies at lag phase showed down-regulation of the genes involved in carbon/nitrogen transportation and metabolism in {Delta}ntrC mutant. In the co-culture competition studies, we observed that ntrC knockout was unable to survive with the wild-type Salmonella and E. coli. We also observed that the {Delta}ntrC mutant did not survive long term nitrogen starvation (150 days). Critical analysis of starvation survival revealed that, ntrC gene is essential for recycling of nutrients from the dead cells. The nutrient recycling efficiency of {Delta}ntrC mutant was [~] 12 times less efficient than wild-type. Hence, the current work establishes that ntrC expression at the lag phase is essential for competitive fitness of Salmonella to survive in an environment having low and fluctuating nutrient conditions.

ImportanceSalmonella Typhimurium, both inside or outside of the host organism face enormous competition from other microorganisms. The competition may take place either in steady or in fluctuating climatic conditions. Thus, how Salmonella Typhimurium survive under such overlapping stress conditions, remained unclear. Therefore, here we report that, a global regulator NtrC, which is also a part of two-component system, activates the set of genes and operons involved in rapid adaptation and efficient nutrient recycling/scavenging. These properties, make cell able to compete with other microbes, under characteristic feast- or-famine life style of Salmonella Typhimurium. Therefore, this piece of work helps us to understand the starvation physiology of enteric bacterial pathogen Salmonella Typhimurium.

sentences: Environmental nucleic acids: a field-based comparison for monitoring freshwater habitats using eDNA and eRNA Nucleic acids released by organisms and isolated from environmental substrates are increasingly being used for molecular biomonitoring. While environmental DNA (eDNA) has received attention recently, the potential of environmental RNA as a biomonitoring tool remains less explored. Several recent studies using paired DNA and RNA metabarcoding of bulk samples suggest that RNA might better reflect "metabolically active" parts of the community. However, such studies mainly capture organismal eDNA and eRNA. For larger eukaryotes, isolation of extra-organismal RNA will be important, but viability needs to be examined in a field-based setting. In this study we evaluate (a) whether extra-organismal eRNA release from macroeukaryotes can be detected given its supposedly rapid degradation, and (b) if the same field collection methods for eDNA can be applied to eRNA. We collected eDNA and eRNA from water in lakes where fish community composition is well documented, enabling a comparison between the two nucleic acids in two different seasons with monitoring using conventional methods. We found that eRNA is released from macroeukaryotes and can be filtered from water and metabarcoded in a similar manner as eDNA to reliably provide species composition information. eRNA had a small but significantly greater true positive rate than eDNA, indicating that it correctly detects more species known to exist in the lakes. Given relatively small differences between the two molecules in describing fish community composition, we conclude that if eRNA provides significant advantages in terms of lability, it is a strong candidate to add to the suite of molecular monitoring tools.

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ksrates: positioning whole-genome duplications relative to speciation events using rate-adjusted mixed paralog-ortholog KS distributions SummaryWe present ksrates, a user-friendly command-line tool to position ancient whole-genome duplication (WGD) events with respect to speciation events in a phylogeny by comparing paralog and ortholog KS distributions derived from genomic or transcriptomic sequences, while adjusting for substitution rate differences among the lineages involved.

Availability and implementationksrates is implemented in Python 3 and as a Nextflow pipeline. The source code, Singularity and Docker containers, documentation and tutorial are available via https://github.com/VIB-PSB/ksrates.

Contactsteven.maere@ugent.vib.be, rolf.lohaus@ugent.vib.be

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Identification of Antimalarial Compounds that Inhibit Apicomplexan AP2 Proteins in the Human Malaria Parasite Plasmodium falciparum Plasmodium parasites are reliant on the Apicomplexan AP2 (ApiAP2) transcription factor family to regulate gene expression programs. AP2 DNA binding domains have no homologs in the human or mosquito host genomes, making them potential antimalarial drug targets. Using an in-silico screen to dock thousands of small molecules into the crystal structure of the AP2-EXP (Pf3D7_1466400) AP2 domain (PDB:3IGM), we identified compounds that interact with this domain. Four compounds were found to compete for DNA binding with AP2-EXP and at least one additional ApiAP2 protein. Our top ApiAP2 competitor compound perturbs the transcriptome of P. falciparum trophozoites and results in a decrease in abundance of log2 fold change > 2 for 50% (46/93) of AP2-EXP target genes. Additionally, two ApiAP2 competitor compounds have multi-stage anti-Plasmodium activity against blood and mosquito stage parasites. In summary, we describe a novel set of antimalarial compounds that are targeted against the ApiAP2 family of proteins. These compounds may be used for future chemical genetic interrogation of ApiAP2 proteins or serve as starting points for a new class of antimalarial therapeutics.

Author SummaryPlasmodium parasites are the causative agent of malaria, which resulted in over 600,000 deaths in 2021. Due to resistance arising for every antimalarial therapeutic deployed to date, new drug targets and druggable pathways must be explored. To address this concern, we used a molecular docking screen to predict competitors of DNA binding by the parasite specific family of Apicomplexan AP2 (ApiAP2) transcription factor proteins for testing in vitro and in vivo. We find that ApiAP2 competing compounds have antimalarial activity consistent with the disruption of gene regulation. This work will further our understanding of both the biological role and targetability of parasite transcriptional regulation.

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Understanding C4 photosynthesis in Setaria by a proteomic and kinetic approach Plants performing C4 photosynthesis have a higher productivity per crop area related to an optimized use of water and nutrients. This is achieved through a series of anatomical and biochemical features that allow the concentration of CO2 around RuBisCO. In C4 plants the photosynthetic reactions are distributed between two cell types, they initially fix the carbon to C4 acids within the mesophyll cells (M) and then transport these compounds to the bundle sheath cells (BS), where they are decarboxylated so that the resulting CO2 is incorporated into the Calvin cycle (CC).

This work is focused on the comparative analysis of the proteins present in M and BS of Setaria viridis, a C4 model close relative of several major feed, fuel, and bioenergy grasses. The integration of kinetic and proteomic approaches agrees that the C4 compound malate is mainly decarboxylated in the chloroplasts of BS cells by NADP-malic enzyme (NADP-ME). Besides, NAD-malic enzyme (NAD-ME) located in the mitochondria could also contribute to the C4 carbon shuttle. We presented evidence of metabolic strategies that involve chloroplastic, mitochondrial and peroxisomal proteins to avoid the leakage of C4 intermediates in order to sustain an efficient photosynthetic performance.

HighlightProteomic and kinetic analyses show metabolic strategies involving chloroplastic, mitochondrial and peroxisomal proteins to maintain the C4 cycle performance in parallel to other metabolic pathways.

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Multiplexed Imaging Mass Cytometry of Chemokine Milieus in Metastatic Melanoma Characterizes Features of Response to Immunotherapy Intratumoral immune cells are crucial for tumor control and anti-tumor responses during immunotherapy. Immune cell trafficking into tumors is mediated by chemokines, which are expressed and secreted upon various stimuli and interact with specific receptors. To broadly characterize chemokine expression and function in tumors, we have used multiplex mass cytometry-based imaging of protein markers and RNA transcripts to analyze the chemokine landscape and immune infiltration in metastatic melanoma samples. Tumors that lacked immune infiltration were devoid of most chemokines and exhibited particularly low levels of antigen presentation and inflammation. Infiltrated tumors were characterized by expression of multiple chemokines. CXCL9 and CXCL10 were often localized in patches associated with dysfunctional T cells expressing CXCL13 which was strongly associated with B cell patches and follicles. TCF7+ naive-like T cells, which predict response to immunotherapy, were enriched in the vicinity of B cell patches and follicles. Our data highlight the strength of RNA and protein co-detection which was critical to deconvolve specialized immune microenvironments in inflamed tumors based on chemokine expression. Our findings further suggest that the formation of tertiary lymphoid structures is accompanied by naive and naive- like T cell recruitment, which ultimately boosts anti-tumor activity.

One sentence summaryInflammatory chemokine milieus in metastatic melanoma are hotspots of T cell dysfunction and CXCL13 expression, which likely guide the recruitment of B cells and the formation of B cell follicles responsible for anti-tumor immunity.

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scatterHatch: an R/Bioconductor package for colorblind accessible visualization of single-cell data SummaryColor is often used as a primary differentiating factor in visualization of single-cell and multi-omics analyses. However, color-based visualizations are extremely limiting and require additional considerations to account for the wide range of color perceptions in the population. The scatterHatch package provides software for accessible single-cell visualizations that use patterns in conjunction with colors to amplify the distinction between different cell types, states, and groups.

AvailabilityscatterHatch is available on Github at https://github.com/FertigLab/scatterHatch.

Contactadeshpande@jhu.edu.

Supplementary informationSupplementary figures are available in the attached document.

sentences: Computational search of hybrid human/ SARS-CoV-2 dsRNA reveals unique viral sequences that diverge from those of other coronavirus strains The role of the RNAi/Dicer/Ago system to degrade RNA viruses has been elusive in mammals, which prompted authors to think that interferon (IFN) synthesis is essential in this clade relegating the RNAi defense strategy against viral infection as accessory function. We explore the theoretical possibilities that RNAi triggered by SARS-CoV-2 might degrade some host transcripts in the opposite direction although this hypothesis seems counter intuitive. SARS-CoV-2 genome was therefore computational searched for exact intra pairing within the viral RNA and also hybrid exact pairing with human transcriptome over a minimum 20 bases length. Minimal segments of 20 bases length of SARS-CoV-2 RNA were found based on the theoretical matching with existing complementary strands in the human host transcriptome. Few human genes potentially annealing with SARS-CoV-2 RNA, among them mitochondrial deubiquitinase USP30, a subunit of ubiquitin protein ligase complex FBXO21 along with two long coding RNAs were retrieved. The hypothesis that viral originated RNAi might mediate degradation of messengers of the host transcriptome was corroborated by clinical observation and phylogenetic comparative analysis indicating a strong specificity of these hybrid pairing sequences for both SARS-CoV-2 and human genomes.

sentences: A cortico-hippocampal scaffold for representing and recalling lifelike events Real-world events are complex, featuring elements that may be unique to, or shared across, multiple situations. In the present study, we used fMRI to identify how different event components are represented in real-time and during memory retrieval. Twenty participants viewed and recalled eight videos depicting real-world events, combining people, contexts, and context types. Multi-voxel pattern similarity analyses revealed specific  person representations, persistent across contexts, in regions of an Anterior-Temporal Network. Conversely, we found specific  context representations, persistent across people, in regions of a Posterior-Medial Network. We also found schema-like generalization across contexts in medial prefrontal cortex, and episodic specificity in the hippocampus. Event patterns were reinstated during recall, and hippocampal reinstatement predicted the number of details retrieved. Finally, we observed distinct representational timescales across the hippocampus and cortical regions. These findings reveal mechanisms for scaffolding different aspects of lifelike event representations in cortico-hippocampal networks as experiences are observed and recalled.

sentences: High-quality complete genome resource of tomato rhizosphere strain Pseudomonas donghuensis P482, a representative of a species with biocontrol activity against plant pathogens Strain P482 was isolated from a tomato rhizosphere and classified as Pseudomonas donghuensis. The P. donghuensis species was first established in 2015 and currently comprises only four strains: P482, HYST, SVBP6, and 22G5. P. donghuensis strains antagonize plant pathogens, including bacteria, fungi and oomycetes, and therefore are of high interest regarding their biological control potential to combat plant diseases. The antimicrobial activity of P. donghuensis P482 is based on the production of iron scavenging compound 7-hydroxytropolone, antifungal volatile organic compounds, and yet unidentified secondary metabolite(s). Here, we report a complete genome resource for P. donghuensis strain P482. The genome consists of a single chromosome (5 656 185 bp) with 5258 ORFs (5158 protein-coding genes, 74 tRNAs, 22 rRNAs, 3 ncRNAs and 1 tmRNA) and no plasmid. We believe that the information of the first high-quality, complete genome of P. donghuensis will provide resources for analyses targeting the biological control potential of this species and understanding the traits essential for plant-microbe interaction.

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Comprehensive analysis of behavioral dynamics in the protochordate Ciona intestinalis Vertebrate nervous systems can generate a remarkable diversity of behaviors. However, our understanding of how behaviors may have evolved in the chordate lineage is limited by the lack of neuroethological studies leveraging our closest invertebrate relatives.

Here we combine high-throughput video acquisition with pharmacological perturbations of bioamine signaling to systematically reveal the global structure of the motor behavioral repertoire in the Ciona intestinalis larvae. Most of Cionas postural variance can be captured by six basic shapes, which we term "Eigencionas". Motif analysis of postural time series further demonstrated that bioamines influence numerous stereotyped behavioral maneuvers including "startle-like" and "beat-and-glide". Employing computational modeling of swimming dynamics and spatio-temporal embedding of postural features revealed that subtle behavioural differences are generated at the levels of motor modules and the transitions between, both modulated by bioamines. Finally, we show that flexible motor module usage gives rise to diverse behaviors in response to different light stimuli.

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Plant trait heterosis is quantitatively associated with expression heterosis of plastid ribosomal proteins The use of hybrids is widespread in agriculture, yet the molecular basis for hybrid vigor (heterosis) remains obscure. To identify molecular components that may contribute to the known higher photosynthetic capacity of maize hybrids, we analyzed proteomic and transcriptomic data from seedling leaf tissues of the hybrid, B73xMo17, and its inbred parents. Subunits of complexes required for protein synthesis in the chloroplast and for the light reactions of photosynthesis were expressed above mid-parent and high-parent levels, respectively. Nuclear and plastid-encoded subunits were expressed similarly and in a dominant pattern with B73 as the high-parent for most proteins. The reciprocal hybrid displayed the same pattern with B73 still the dominant parent. Protein expression patterns were mostly the same in the adult leaf blade. To rank the relevance of expression differences to trait heterosis, we compared seedling leaf protein levels to adult plant heights of 15 hybrids. Expression heterosis (hybrid/mid-parent expression levels) was most positively correlated with plant height heterosis (hybrid/mid-parent plant height) for the plastid-encoded and nuclear-encoded chloroplast ribosomal proteins; the cytosolic ribosomal proteins were slightly less correlated. Ethylene biosynthetic enzymes were expressed below mid-parent levels in the hybrids, and the ethylene biosynthesis mutant, acs2/acs6, largely phenocopied the hybrid proteome, indicating that a reduction in ethylene biosynthesis may mediate the differences between inbreds and their hybrids. Levels of jasmonic acid biosynthetic enzymes were reduced in both acs2/acs6 and the hybrid, and expression heterosis levels of these proteins were the most negatively correlated with plant height heterosis.

Significance StatementHeterosis (hybrid vigor) boosts the productivity and resilience of crops and livestock above the levels of both parents, yet its underlying mechanisms remain unknown. We analyzed expression patterns of proteins in maize hybrids and their inbred parents. Differences in several molecular machines and biochemical pathways were found and quantitatively assessed using a panel of 15 hybrids. Seedling leaf chloroplast ribosomal proteins were able to quantitatively infer levels of adult plant heterosis. Expression levels of biosynthetic enzymes for the stress hormone, ethylene, were reduced in hybrids as was previously reported for the dicot Arabidopsis. Mutation of these genes in a maize inbred caused the proteome to resemble a hybrid. Repression of ethylene biosynthesis may be a conserved component of heterosis physiology.

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MRG proteins are shared by multiple protein complexes with distinct functions MRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain recognizing H3K36me3 in chromatin. Intriguingly, it has been reported in the literature to interact with several different factors involved in chromatin modifications, gene regulation, alternative mRNA splicing and DNA repair by homologous recombination. In order to get a complete and reliable picture of associations in physiological conditions, we used genome editing and tandem affinity purification to analyze the stable native interactome of human MRG15, its paralog MRGX/MORF4L2 that lacks the chromodomain, and MRGBP (MRG-binding protein) in isogenic K562 cells. We found stable interchangeable association of MRG15 and MRGX with the NuA4/TIP60 histone acetyltransferase/chromatin remodeler, Sin3B histone deacetylase/demethylase, ASH1L histone methyltransferase and PALB2/BRCA2 DNA repair protein complexes. These associations were further confirmed and analyzed by CRISPR-tagging of endogenous proteins and comparison of expressed isoforms. Importantly, based on structural information, point mutations could be introduced that can specifically disrupt MRG15 association with some complexes but not others. Most interestingly, we also identified a new abundant native complex formed by MRG15/X-MRGBP-BRD8-EP400NL that is functionally similar to the yeast TINTIN (Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes) complex. Our results show that EP400NL, being homologous to the N-terminal region of NuA4/TIP60 subunit EP400, creates TINTIN by competing for BRD8 association. Functional genomics indicate that human TINTIN plays a role in transcription of specific genes. This is most likely linked to the H4ac-binding bromodomain of BRD8 along the H3K36me3-binding chromodomain of MRG15 on the coding region of transcribed genes. Taken together, our data provide a complete detailed picture of human MRG proteins-associated protein complexes which is essential to understand and correlate their diverse biological functions in chromatin-based nuclear processes.

HighlightsO_LIMRG15 and MRGX are stably associated with several different protein complexes important for genome expression and stability.
C_LIO_LISeveral MRG-containing complexes are chromatin modifiers.
C_LIO_LISpecific point mutations in the MRG domain differentially affect associated complexes.
C_LIO_LIA major human complex homologous to the yeast TINTIN complex is identified.
C_LIO_LIThe protein EP400NL competes with EP400 to functionally separate TINTIN from the NuA4/TIP60 complex.
C_LIO_LITINTIN contains a bromodomain and a chromodomain to regulate transcription.
C_LI

sentences: Tetrazoles as PPARγ ligands: A Structural and Computational Investigation. Diabetes is an important chronic disease affecting about 10% of the adult population in the US and over 420 million people worldwide, resulting in 1.6 million deaths every year, according to the World Health Organization. The most common type of the disease, type 2 diabetes, can be pharmacologically managed using oral hypoglycemic agents or thiazolidinediones (TZDs), such as pioglitazone, which act by activating the Peroxisome Proliferated-Activated Receptor {gamma}. Despite their beneficial effects in diabetes treatment, TZDs like rosiglitazone and troglitazone were withdrawn due to safety reasons, creating a void in the pharmacological options for the treatment of this important disease. Here, we explored a structure-based approach in the screening for new chemical probes for a deeper investigation of the effects of PPAR{gamma} activation. A class of tetrazole compounds was identified and the compounds named T1, T2 and T3 were purchased and evaluated for their ability to interact with the PPAR{gamma} ligand binding domain (LBD). The compounds were binders with micromolar range affinity, as determined by their IC50 values. A Monte Carlo simulation of the compound T2 revealed that the tetrazole ring makes favorable interaction with the polar arm of the receptor binding pocket. Finally, the crystal structure of the PPAR{gamma}-LBD-T2 complex was solved at 2.3 [A], confirming the binding mode for this compound. The structure also revealed that, when the helix H12 is mispositioned, an alternative binding conformation is observed for the ligand suggesting an H12-dependent binding conformation for the tetrazole compound.

sentences: An allosteric ligand stabilizes distinct conformations in the M2 muscarinic acetylcholine receptor Allosteric modulators provide therapeutic advantages over orthosteric drugs. A plethora of allosteric modulators have been identified for several GPCRs, particularly for muscarinic receptors (mAChRs)1,2. To study the molecular mechanisms governing allosteric modulation, we utilized a recently developed NMR system to investigate the conformational changes in the M2 muscarinic receptor (M2R) in response to the positive allosteric modulator (PAM) LY2119620. Our studies provide the first biophysical data showing that LY2119620 can substantially change the structure and dynamics of M2R in both the extracellular and G-protein coupling domains during the activation process. These NMR data suggest that LY2119620 may function by stabilizing distinct sets of conformations not observed in the presence of orthosteric agonists alone, which may account for the different signaling behaviors of the M2R when bound to LY2119620. Our studies provide new structural information for understanding the mechanism of GPCR allostery, and may facilitate the rational design of allosteric therapeutics targeting muscarinic receptors.

sentences: The ER membrane chaperone Shr3 acts in a progressive manner to assist the folding of related plasma membrane transport proteins Proteins with multiple membrane-spanning segments (MS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. In Saccharomyces cerevisiae, Shr3 is an ER membrane-localized chaperone that is specifically required for the functional expression of amino acid permeases (AAP), a family of eighteen transporters comprised of 12 MS. Here, comprehensive scanning mutagenesis and deletion analysis of Shr3, combined with a modified split-ubiquitin approach, were used to probe chaperone-substrate interactions with seven different AAP in vivo. Our findings indicate that Shr3 specifically recognizes AAP substrates, largely independent of sequence-specific interactions involving membrane and luminally oriented domains. Shr3 selectively and robustly interacts with nested C-terminal AAP truncations in marked contrast to similar truncations of non-Shr3 substrate polytopic sugar transporters. Strikingly, Shr3-AAP interactions initiate with the first 4 MS of AAP and successively strengthen, but abruptly weaken when all 12 MS are present. The data are consistent with Shr3 acting in a temporal manner as a scaffold preventing AAP translation intermediates from engaging in non-productive interactions.

sentences: Identification of neural progenitor cells and their progeny reveals long distance migration in the developing octopus brain Cephalopods have evolved nervous systems that parallel the complexity of mammalian brains in terms of neuronal numbers and richness in behavioral output. How the cephalopod brain develops has only been described at the morphological level, and it remains unclear where the progenitor cells are located and what molecular factors drive neurogenesis. Using histological techniques, we located dividing cells, neural progenitors and postmitotic neurons in Octopus vulgaris embryos. Our results indicate that progenitors are located outside the central brain cords in the lateral lips adjacent to the eyes, suggesting that newly formed neurons migrate into the cords. Lineage tracing experiments then showed that progenitors, depending on their location in the lateral lips, generate neurons for the different lobes. The finding that octopus newborn neurons migrate over long distances is reminiscent of vertebrate neurogenesis and suggests it might be a fundamental strategy for large brain development.

sentences: Actin-microtubule dynamic composite forms responsive active matter with memory Active cytoskeletal materials in vitro demonstrate self-organising properties similar to those observed in their counterparts in cells. However, the search to emulate phenomena observed in the living matter has fallen short of producing a cytoskeletal network that would be structurally stable yet possessing adaptive plasticity. Here, we address this challenge by combining cytoskeletal polymers in a composite, where self-assembling microtubules and actin filaments collectively self-organise due to the activity of microtubules-percolating molecular motors. We demonstrate that microtubules spatially organise actin filaments that in turn guide microtubules. The two networks align in an ordered fashion using this feedback loop. In this composite, actin filaments can act as structural memory and, depending on the concentration of the components, microtubules either write this memory or get guided by it. The system is sensitive to external stimuli suggesting possible autoregulatory behaviour in changing mechanochemical environment. We thus establish artificial active actin-microtubule composite as a system demonstrating architectural stability and plasticity.

sentences: Sex-specific interactions between procedural and deliberative decision-making systems in a mouse model of Alzheimer's disease A central question in aging and Alzheimers disease (AD) is when and how neural substrates underlying decision-making are altered. Here we show that while APP mice, a commonly used mouse model of AD, were able to learn Restaurant Row, a complex neuroeconomic decision-making task, they were significantly impaired in procedural, habit-forming, aspects of cognition and relied heavily on deliberation when making decisions. Surprisingly, these behavioral changes are associated with amyloid-beta (A{beta}) pathology and network remodeling in the striatum, a key brain region involved in procedural cognition. Furthermore, APP mice and control mice relied on distinct sex-specific strategies in this neuroeconomic task. These findings provide foundational pillars to examine how aging and age-related neurodegenerative diseases impact decision-making across sexes. They also highlight the need for complex behavioral tasks that allow for the dissociation of competing neurally-distinct decision-making circuits to get an accurate picture of changes in neurodegenerative models of human disease.

sentences: Insula to mPFC reciprocal connectivity differentially underlies novel taste neophobic response and learning To survive in an ever-changing environment, animals must detect and learn salient information. The anterior insular cortex (aIC) and medial prefrontal cortex (mPFC) are heavily implicated in salience and novelty processing, and specifically, the processing of taste sensory information. Here, we examined the role of aIC-mPFC reciprocal connectivity in novel taste neophobia and memory formation, in mice. Using pERK and neuronal intrinsic properties as markers for neuronal activation, and retrograde AAV (rAAV) constructs for connectivity, we demonstrate a correlation between aIC-mPFC activity and novel taste experience. Furthermore, by expressing inhibitory chemogenetic receptors in these projections, we show that aIC-to-mPFC activity is necessary for both taste neophobia and its attenuation. However, activity within mPFC-to-aIC projections is essential only for the neophobic reaction but not for the learning process. These results provide an insight into the cortical circuitry needed to detect, react to- and learn salient stimuli, a process critically involved in psychiatric disorders.

sentences: Protein kinase J regulates Rv0642c expression and sensitivity to rifampicin of mycobacteria Mycobacterium tuberculosis (M.tb) persists for long-duration inside the human host in both active and latent form by modulating the immune response. The mechanisms employed by M.tb to survive inside the host and evade the host immune system need to be explored in greater depth for the rational design of novel treatment strategies. The phosphorylation and methylation of biomolecules need to be addressed in mycobacteria because it has an important role in infection establishment and persistence. In the present study, we elaxborate on the role of PknJ in the slow growth of BCG and its association with mmaA4 protein during extracellular and intracellular growth. The pknJ knock-out (KO) BCG has been used to decode the functional significance in mycobacteria. The mmaA4 expression and methyltransferase activity is decreased in knocked-out BCG strain (pknJ-/-) during extracellular growth, while mmaA4 expression and methyltransferase activity is increased during intracellular growth of mycobacteria. The knocked-out BCG strain is highly sensitive to the rifampicin antibiotics during extracellular growth in compared to control. A significant association of pknJ and mmaA4 was found in our studies during the growth and intracellular persistence of mycobacteria.

sentences: metaMIC: reference-free Misassembly Identification and Correction of de novo metagenomic assemblies Evaluating the quality of metagenomic assemblies is important for constructing reliable metagenome-assembled genomes and downstream analyses. Here, we present metaMIC (https://github.com/ZhaoXM-Lab/metaMIC), a machine-learning based tool for identifying and correcting misassemblies in metagenomic assemblies. Benchmarking results on both simulated and real datasets demonstrate that metaMIC outperforms existing tools when identifying misassembled contigs. Furthermore, metaMIC is able to localize the misassembly breakpoints, and the correction of misassemblies by splitting at misassembly breakpoints can improve downstream scaffolding and binning results.

sentences: Transcriptomic landscapes of SARS-CoV-2-infected and bystander lung cells reveal a selective upregulation of NF-κB-dependent coding and non-coding proviral transcripts Detailed knowledge of cellular networks that are modulated by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to understand viral replication and host response. So far, transcriptomic analyses of interactions between SARS-CoV-2 and cells were performed on mixed populations of infected and uninfected cells or using single-cell RNA sequencing, both leading to inaccurate or low-resolution gene expression interpretations. Moreover, they generally focused on annotated messenger RNAs (mRNAs), ignoring other transcripts, such as long non-coding RNAs (lncRNAs) and unannotated RNAs. Here, we performed deep polyA+ transcriptome analyses of lung epithelial A549 cells infected with SARS-CoV-2, which were sorted based on the expression of the viral protein spike (S). To increase the sequencing depth and improve the robustness of the analysis, the samples were depleted of viral transcripts. Infection caused a massive reduction in mRNAs and lncRNAs, including transcripts coding for antiviral innate immune proteins, such as interferons (IFNs). This absence of IFN response probably explains the poor transcriptomic response of bystander cells co-cultured with spike positive (S+) ones. NF-{kappa}B and inflammatory response were among the pathways that escaped the global shutoff in S+ cells. In agreement with the RNA-seq analysis, inflammatory cytokines, but not IFNs, were produced and secreted by infected cells. Functional investigations revealed the proviral function of the NF-kB subunit p105/p50 and some of its known target genes, including IL32 and IL8, as well as the lncRNA ADIRF-AS1, which we identified as a novel NF-kB target gene. Thus, analyzing the polyA+ transcriptome of sorted populations of infected lung cells allowed unprecedented identification of cellular functions that are directly affected by infection and the recovery of coding and non-coding genes that contribute to SARS-CoV-2 replication.

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Single cell multi-omics analysis of chromothriptic medulloblastoma highlights genomic and transcriptomic consequences of genome instability Chromothripsis is a form of genome instability, whereby a presumably single catastrophic event generates extensive genomic rearrangements of one or few chromosome(s). However, little is known about the heterogeneity of chromothripsis across different clones from the same tumor, as well as changes in response to treatment. We analyzed single-cell genomic and transcriptomic alterations linked with chromothripsis in human p53-deficient medulloblastoma (n=7). We reconstructed the order of somatic events, identified early alterations likely linked to chromothripsis and depicted the contribution of chromothripsis to malignancy. We characterized subclonal variation of chromothripsis and its effects on double-minute chromosomes, cancer drivers and putatively druggable targets. Furthermore, we highlighted the causative role and the fitness consequences of specific rearrangements in neural progenitors.

Graphical Abstract

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sentences: Cannabis Vapour Exposure Alters Neural Circuit Oscillatory Activity In A Neurodevelopmental Model Of Schizophrenia: Exploring The Differential Impact Of Cannabis Constituents Cannabis use is highly prevalent in patients with schizophrenia and worsens the course of the disorder. To understand the causal impacts of cannabis on schizophrenia-related oscillatory disruptions, we herein investigated the impact of exposure to cannabis vapour (containing delta-9-tetrahydrocannabinol [THC] or balanced THC and cannabidiol [CBD]) on oscillatory activity in the neonatal ventral hippocampal lesion (NVHL) rat model of schizophrenia. Male Sprague Dawley rats underwent NVHL or sham surgeries on postnatal day 7. In adulthood, electrodes were implanted targeting the cingulate cortex (Cg), the prefrontal cortex (PFC), the dorsal hippocampus (HIP), and the nucleus accumbens (NAc). Local field potential recordings were obtained following exposure to two strains of vapourized cannabis flower (with ~10% THC or ~10% balanced THC:CBD) in a cross-over design with a two-week wash-out period between exposures. Compared to controls, NVHL rats had reduced baseline gamma power in the Cg, dHIP, and NAc, and reduced high-gamma coherence between the dHIP-Cg. THC-only vapour broadly suppressed oscillatory power and coherence, even beyond the baseline suppressions observed in NHVL rats. Balanced THC:CBD vapour appeared to ameliorate the THC-induced impacts on power and coherence in both sham and NVHL rats. For NVHL rats, THC-only vapour also normalized the baseline dHIP-Cg high-gamma coherence deficits. NHVL rats also demonstrated a 20ms delay in dHIP theta to high-gamma phase coupling, which was ameliorated by both exposures in the PFC and NAc. In conclusion, THC-only cannabis vapour suppressed oscillatory activity in NVHL and sham rats, while balanced THC:CBD vapour may ameliorate some of these effects.

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The Magnaporthe oryzae MAP kinase Pmk1 regulates polycomb repressive complex 2 to reprogram genes expression for biotrophic growth Biotrophic and hemibiotrophic fungi have evolved the ability to colonize living plant cells, but how they establish biotrophic growth by remodeling gene expression is poorly understood. By using in planta invasive hyphae (IH) of Magnaporthe oryzae to perform an integrated Chromatin immunoprecipitation sequencing (ChIPseq) and RNA-seq analysis, combining with biological and cellular analyses, we found Polycomb repressive complex 2 (PRC2)-mediated epigenetic repression plays a key role in regulating biotrophic growth. ChIPseq for biotrophic IH samples identified 1701 PRC2 target genes. RNA-seq analysis showed that expression of 462 PRC2 target genes were up-regulated in the {Delta}suz12 mutant, while 82 were down-regulated, indicating a major role of PRC2 in gene repression of IH. During biotrophic growth, PRC2 repressed fungal cell wall synthesis genes and extracellular enzyme genes required for penetration, and secondary metabolites biosynthesis genes required for necrotrophic growth. A great number of effector-encoding genes were repressed by PRC2, which were highly expressed during penetration stage, suggesting PRC2 coordinates biotrophic growth by regulating effector suppression for immune evasion. This regulation was finely coordinated by Pmk1, through regulating phosphorylation, nuclear localization and protein abundance of Suz12. Our results indicate that the Pmk1-PRC2 regulatory module is required for gene remodeling to facilitate biotrophic growth in M. oryzae.

IMPORTANCEBiotrophic and hemibiotrophic fungi establish a biotrophic stage for infection in host cells. For example, M. oryzae forms appressoria to penetrate host cell and establish a biotrophic growth stage for infection. How gene expression patterns are elaborately controlled for fungal biotrophic growth is largely unknown. In this study, we found that, the PRC2-mediated H3K27me3 repressed fungal penetration-required cell wall synthesis genes and extracellular enzyme genes, and necrotrophic growth-required secondary metabolites biosynthesis genes for biotrophic growth. Interestingly, a great number of effector-encoding genes were also repressed by PRC2 at biotrophic stage, which were highly expressed at penetration stage, suggesting PRC2 coordinates biotrophic growth by regulating effector suppression for immune evasion. The PRC2-mediated epigenetic repression is therefore required for the gene expression remodeling during fungal infection. This regulation was finely coordinated by Pmk1, through regulating nuclear localization and protein abundance of the PRC2 component Suz12.

sentences: pre-mRNA spatial distributions suggest that splicing can occur post-transcriptionally Splicing is the molecular process by which introns are removed from pre-mRNA and exons are joined together to form the sequence of the mature mRNA. While the biochemical steps of splicing have been largely worked out, the ordering and spatial distribution of these steps, especially in relation to the transcriptional process itself, remain controversial. Here, we use single molecule RNA FISH together with expansion microscopy to measure the spatial distribution of nascent and partially spliced transcripts in mammalian cells, allowing us to infer the order in which an intron is transcribed and spliced out of the pre-mRNA. We show that 4 out of 4 genes we interrogated exhibit clear post-transcriptional splicing of at least one intron, and that introns can be spliced in any order. Expansion microscopy further revealed the presence of a transcription site proximal zone in which the motion of RNA is slower than in the nucleoplasm. Full length pre-mRNA undergo continuous splicing as they move through this zone after transcription. Upon leaving this zone, some genes transcripts localize to speckles during the process of splicing while others appear to traffic freely through the nucleus without localizing to any other nuclear compartment. Our results suggest a model in which transcription and splicing are largely independent, and the existence of a slow moving zone around the transcription site allows for the unification of co-transcriptional and post-transcriptional models of splicing.

sentences: Heat shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition While the RNA binding protein TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) with TDP-43-containing liquid outer shells and liquid centers of HSP70 family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including ALS. Here we show that transient oxidative stress, proteasome inhibition, or inhibition of HSP70s ATP-dependent chaperone activity provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independent of RNA binding or stress granules. Isotope labeling mass spectrometry is used to identify that phase separated cytoplasmic TDP-43 is primarily bound by the small heat shock protein HSPB1. Binding is direct, mediated through TDP-43s RNA binding and low complexity domains. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and is essential for disassembly of stress-induced, TDP-43 droplets. Decrease of HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion is identified within ALS-patient spinal motor neurons containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation.

sentences: Alternative developmental and transcriptomic responses to host plant water limitation in a butterfly metapopulation Predicting how climate change affects biotic interactions and their evolution poses a challenge. Plant-insect herbivore interactions are particularly sensitive to climate change, as climate-induced changes in plant quality cascade into the performance of insect herbivores. Whereas the immediate survival of herbivore individuals depends on plastic responses to climate change induced nutritional stress, long-term population persistence via evolutionary adaptation requires genetic variation for these responses. In order to assess the prospects for population persistence under climate change, it is therefore crucial to characterise response mechanisms to climate change induced stressors, and quantify their variability in natural populations. Here, we test developmental and transcriptomic responses to water limitation induced host plant quality change in a Glanville fritillary butterfly (Melitaea cinxia) metapopulation. We combine nuclear magnetic resonance spectroscopy on the plant metabolome, larval developmental assays and an RNA seq analysis of the larval transcriptome. We observed that responses to feeding on water limited plants, in which amino acids and aromatic compounds are enriched, showed marked intrapopulation variation, with individuals of some families performing better on control and others on water limited plants. The transcriptomic responses were concordant with the developmental responses: Families exhibiting opposite developmental responses also produced opposite transcriptomic responses, e.g. in growth associated intracellular signalling. The opposite developmental and transcriptomic responses are associated with between families differences in organic compound catabolism and storage protein production. The results reveal heritable intrapopulation variability in plasticity, suggesting potential for evolutionary responses to drought-induced changes in host plant quality in the Finnish M. cinxia metapopulation.

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Fusobacterium nucleatum metabolically integrates commensals and pathogens in oral biofilms Fusobacterium nucleatum is a common constituent of the oral microbiota in both periodontal health and disease. Previously, we discovered ornithine cross-feeding between F. nucleatum and Streptococcus gordonii, where S. gordonii secretes ornithine via an arginine-ornithine antiporter (ArcD), which in turn supports the growth and biofilm development of F. nucleatum; however, broader metabolic aspects of F. nucleatum within polymicrobial communities and their impact on periodontal pathogenesis have not been addressed. Here, we show that when co-cultured with S. gordonii, F. nucleatum increased amino acid availability to enhance the production of butyrate and putrescine, a polyamine produced by ornithine decarboxylation. Co-culture with Veillonella parvula, another common inhabitant of the oral microbiota, also increased lysine availability, promoting cadaverine production by F. nucleatum. We confirmed that ArcD-dependent ornithine excretion by S. gordonii results in synergistic putrescine production, and mass spectrometry imaging revealed this metabolic capability creates a putrescine-rich microenvironment inside F. nucleatum biofilms. We further demonstrated that polyamines caused significant changes in the biofilm phenotype of a periodontal pathogen, Porphyromonas gingivalis, with putrescine being a potent stimulator of biofilm development and dispersal, and confirmed that F. nucleatum-mediated conversion of ornithine to putrescine enhances biofilm formation by P. gingivalis. Lastly, analysis of plaque samples revealed cooccurrence of P. gingivalis with genetic modules for putrescine production by S. gordonii and F. nucleatum. Overall, our results highlight the ability of F. nucleatum to induce synergistic polyamine production within multi-species consortia, and provide insight into how the trophic web in oral biofilm ecosystems can eventually shape disease-associated communities.

Significance StatementPeriodontitis is caused by the pathogenic transition of subgingival microbiota ecosystems, which is accompanied by alterations to microbiome functions including metabolic systems and the establishment of metabolic cross-feeding. While Fusobacterium nucleatum is a major constituent of the periodontal microbiota, its metabolic integration within polymicrobial communities and the impact on periodontal pathogenesis are poorly understood. Here, we report that amino acids supplied by other commensal bacteria induce polyamine production by F. nucleatum, creating polyamine-rich microenvironments. We further show that this trophic web results in enhancement of biofilm formation and dispersal of a periodontal pathogen, Porphyromonas gingivalis. This work provides mechanistic insight into how cooperative metabolism within oral biofilms can tip the balance toward periodontitis.

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Investigating the conformational dynamics of SARS-CoV-2 NSP6 protein with emphasis on non-transmembrane 91-112 & 231-290 regions The NSP6 protein of SARS-CoV-2 is a transmembrane protein, with some regions lying outside the membrane. Besides, a brief role of NSP6 in autophagosome formation, this is not studied significantly. Also, there is no structural information available till date. Based on the prediction by TMHMM server for transmembrane prediction, it is found that the N-terminal residues (1-11), middle region residues (91-112) and C-terminal residues (231-290) lies outside the membrane. Molecular Dynamics (MD) simulations showed that NSP6 consisting of helical structures, whereas membrane outside lying region (91-112) showed partial helicity, which further used as model and obtain disordered type conformation after 1.5 microsecond. Whereas, the residues 231-290 has both helical and beta sheet conformations in its structure model. A 200ns simulations resulted in the loss of beta sheet structures, while helical regions remained intact. Further, we have characterized the residue 91-112 by using reductionist approaches. The NSP6 (91-112) was found disordered like in isolation, which gain helical conformation in different biological mimic environmental conditions. These studies can be helpful to study NSP6 (91-112) interactions with host proteins, where different protein conformation might play significant role. The present study adds up more information about NSP6 protein aspect, which could be exploited for its host protein interaction and pathogenesis.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/451329v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1b6e8aborg.highwire.dtl.DTLVardef@165564eorg.highwire.dtl.DTLVardef@551f7org.highwire.dtl.DTLVardef@1485518_HPS_FORMAT_FIGEXP  M_FIG C_FIG The schematic representation of NSP6 membrane topology and conformational dynamics of residue 91-112. The N-terminal and C-terminal are shown in cytoplasmic side based on the experimental evidence on coronaviruses reported by Oostra et al., 2008. The membrane anchoring domain are shown based on the TMHMM server prediction.

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How Communication Pathways Bridge Local and Global Conformations in an IgG4 Antibody: a Molecular Dynamics Study The affinity of an antibody for its antigen is primarily determined by the specific sequence and structural arrangement of the complementarity-determining regions (CDRs). Recently, however, evidence has accumulated that points toward a nontrivial relation between the CDR and distal sites on the antibody structure: variations in the binding strengths have been observed upon mutating amino acids separated from the paratope by several nanometers, thus suggesting the existence of a communication network within antibodies whose extension and relevance might be deeper than insofar expected. In this work, we test this hypothesis by means of molecular dynamics (MD) simulations of the IgG4 monoclonal antibody pembrolizumab, an approved drug that targets the programmed cell death protein 1 (PD-1). The molecule is simulated in both the apo and holo states, totalling 4s of MD trajectory. The analysis of these simulations shows that the bound antibody explores a restricted range of conformations with respect to the apo one, and that the global conformation of the molecule correlates with that of the CDR; a pivotal role in this relationship is played by the relatively short hinge, which mechanically couples Fab and Fc domains. These results support the hypothesis that pembrolizumab behaves as a complex machinery, with a multi-scale hierarchy of global and local conformational changes that communicate with one another. The analysis pipeline developed in this work is general, and it can help shed further light on the mechanistic aspects of antibody function.

SynopsisAntigen binding restricts the conformational variability of the therapeutic antibody pembrolizumab in an interplay between the paratope and hinge region, mediated by a full-scale interaction network.

Graphical TOC Entry

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sentences: Structured illumination microscopy with noise-controlled image reconstructions Super-resolution structured illumination microscopy (SIM) has become a widely used method for biological imaging. Standard reconstruction algorithms, however, are prone to generate noise-specific artefacts that limit their applicability for lower signal-to-noise data. Here, we present a physically realistic noise model that explains the structured noise artefact and that is used to motivate new complementary reconstruction approaches. True Wiener-filtered SIM optimizes contrast given the available signal-to-noise ratio, flat-noise SIM fully overcomes the structured noise artefact while maintaining resolving power. Both methods eliminate ad-hoc user adjustable reconstruction parameters in favour of physical parameters, enhancing objectivity. The new reconstructions point to a trade-off between contrast and a natural noise appearance. This trade-off can be partly overcome by additional notch filtering, but at the expense of a decrease in signal-to-noise ratio. The benefits of the proposed approaches are demonstrated on focal adhesion and tubulin samples in 2D and 3D, and on nano-fabricated fluorescent test patterns.

sentences: Structural and functional networks involved in empathy for positive and negative emotions in late adulthood Empathy, among other social-cognitive processes, changes across adulthood. More specifically, cognitive components of empathy (understanding anothers perspective) appear to decline with age, while findings for affective empathy (sharing anothers emotional state) are rather mixed. Structural and functional correlates underlying cognitive and affective empathy in aging and the extent to which valence affects empathic response in brain and behavior are not well understood yet. To fill these research gaps, younger and older adults completed a modified version of the Multifaceted Empathy Test, which measures both cognitive and affective empathy as well as empathic responding to both positive and negative stimuli (i.e., positive vs. negative empathy). Adopting a multimodal imaging approach and applying multivariate analysis, the study found that for cognitive empathy to negative emotions, regions of the salience network including the anterior insula and anterior cingulate were more involved in older than younger adults. For affective empathy to positive emotions, in contrast, younger and older adults recruited a similar brain network including main nodes of the default mode network. Additionally, increased structural integrity (fractional anisotropy values) of the posterior, but not the anterior, cingulum bundle was related to activation of default mode regions during affective empathy for positive emotions in both age groups. These findings provide novel insights into the functional networks subserving cognitive and affective empathy in younger and older adults and highlight the importance of considering valence in empathic response in aging research. Further this study, for the first time, underscores the role of the posterior cingulum bundle in higher-order social-cognitive processes such as empathy, specifically for positive emotions, in aging.

sentences: Differential response of Candida species morphologies and isolates to fluconazole and boric acid Candida albicans is the most prevalent cause of vulvovaginal candidiasis ( yeast infection) and recurrent vulvovaginal candidiasis, though the incidence of non-albicans yeast species is increasing. The azole fluconazole is the primary antifungal drug used to treat R/VVC yet isolates from some species have intrinsic resistance to fluconazole, and recurrent infection can occur even with fluconazole-susceptible populations. The second-line broad-spectrum antimicrobial drug, boric acid, is an alternative treatment that has been found to successfully treat complicated VVC infections. Far less is known about how boric acid inhibits growth of yeast isolates in different morphologies compared to fluconazole. We found significant differences in drug resistance and drug tolerance (the ability of a subpopulation to grow slowly in high levels of drug) between C. albicans, C. glabrata, and C. parapsilosis isolates, with the specific relationships dependent on both drug and phenotype. Population-level variation for both susceptibility and tolerance was broader for fluconazole than boric acid in all species. Unlike fluconazole, which neither prevented hyphal formation nor disrupted mature biofilms, boric acid inhibited C. albicans hyphal formation and reduced mature biofilm biomass and metabolic activity in all isolates in a dose-dependent manner. Variation in planktonic response did not generally predict biofilm phenotypes. Overall, our findings illustrate that boric acid is broadly effective at inhibiting growth across many isolates and morphologies, which could explain why it is an effective treatment for R/VVC.

sentences: Interplay between PML NBs and HIRA for H3.3 deposition on transcriptionally active interferon-stimulated genes Promyelocytic Leukemia Nuclear Bodies (PML NBs) are nuclear membrane-less organelles physically associated with chromatin underscoring their crucial role in genome function. The H3.3 histone chaperone complex HIRA accumulates in PML NBs upon senescence, viral infection or IFN-I treatment in primary cells. Yet, the molecular mechanisms of this partitioning and its function in regulating histone dynamics have remained elusive. Here, by using specific siRNAs and protein Affimers, we identify intermolecular SUMO-SIM interactions as an essential mechanism for HIRA recruitment in PML NBs. In addition, we demonstrate that HIRA localization in the nuclear bodies is intimately linked to the presence of a soluble pool of H3.3-H4 dimers inside PML NBs, that is not found in cancer cells. Transcription inhibition prevents HIRA accumulation in PML NBs underscoring the importance of transcriptional activity to drive HIRA through PML NBs. Finally, in the context of inflammatory responses, HIRA and PML are necessary for the prolonged H3.3 deposition at the transcriptional end sites of interferon-stimulated genes (ISGs), well beyond the peak of transcription. We thus propose that HIRA partitioning in PML NBs is essential to regulate H3.3 deposition on transcriptionally active regions.

sentences: Proton-gated coincidence detection is a common feature of GPCR signaling The evolutionary expansion of G protein-coupled receptors (GPCRs) has produced a rich diversity of transmembrane sensors for many physical and chemical signals. In humans alone, over 800 GPCRs detect stimuli such as light, hormones, and metabolites to guide cellular decision making primarily using intracellular G protein signaling networks. This diversity is further enriched by GPCRs that function as molecular logic gates capable of discerning multiple inputs to transduce cues encoded in complex, context-dependent signals. Here, we show that many GPCRs are switch-like Boolean-gated coincidence detectors that link proton (H+) binding to GPCR signaling. Using a panel of 28 receptors, covering 280 individual GPCR-G coupling combinations, we show that H+ gating both positively and negatively modulates and controls GPCR signaling. Notably, these observations extend to all modes of GPCR pharmacology including ligand efficacy, potency, and cooperativity. Additionally, we show that GPCR antagonism and constitutive activity are regulated by H+ gating and report the discovery of a new acid sensor, the adenosine A2a receptor (ADORA2A), which can be activated solely by acidic pH. Together, these findings establish a new paradigm for GPCR biology and pharmacology in acidified microenvironments such as endosomes, synapses, tumors, and ischemic vasculature.

sentences: Activation of the membrane-bound Nrf1 transcription factor by USP19, a ubiquitinspecific protease C-terminally tail-anchored in the endoplasmic reticulum The membrane-bound transcription factor Nrf1 (i.e., encoded by Nfe2l1) is activated by sensing glucose deprivation, cholesterol excess, proteasomal inhibition and oxidative stress, and then mediates distinct signaling responses in order to maintain cellular homeostasis. Herein, we found that Nrf1 stability and transactivity are both enhanced by USP19, a ubiquitin-specific protease tail-anchored in the endoplasmic reticulum (ER) through its C-terminal transmembrane domain. Further experiments revealed that USP19 directly interacts with Nrf1 in proximity to the ER and topologically acts as a deubiquitinating enzyme to remove ubiquitin moieties from this protein, and hence allows it to circumvent the potential proteasomal degradation. Such USP19-mediated effect takes place only after Nrf1 is retrotranslocated by p97 out of ER membranes to dislocate the cytoplasmic side. Conversely, knockout of USP19 causes significant decreases in the Nrf1 abundance and its active isoform entering the nucleus, resulting in down-regulation of its target proteasomal subunits. This led to a modest reduction of USP19-/--derived tumor growth in xenograft mice, when compared with wild-type controls. Altogether, these demonstrate that USP19 serves as a novel mechanistic modulator of Nrf1, but not Nrf2, enabling Nrf1 to be rescued from putative ubiquitin-directed ER-associated degradation pathway. In turn, our additional experimental evidence has unraveled that transcriptional expression of endogenous USP19 and its promoter-driven reporter genes is differentially regulated by Nrf2, as well by Nrf1, at distinct layers within a complex hierarchical regulatory network.

sentences: The limits of evolutionary convergence in sympatry: reproductive interference and developmental constraints leading to local diversity in aposematic signals Mutualistic interactions between defended species represent a striking case of evolutionary convergence in sympatry, driven by the increased protection against predators brought by mimicry in warning traits. However, such convergence is often limited: sympatric defended species frequently display different or imperfectly similar warning traits. The phylogenetic distance between sympatric species may indeed prevent evolution towards the exact same signal. Moreover, warning traits are also involved in mate recognition, so that trait convergence might result in heterospecific courtship and mating. Here, we develop a mathematical model to investigate the strength and direction of evolution of warning trait in defended species with different ancestral traits. Specifically, we determine the effect of phenotypic distances between ancestral trait states of sympatric defended species and of costs of heterospecific sexual interactions on imperfect mimicry and trait divergence. Our analytical results confirm that reproductive interference and historical constraints limit the convergence of warning traits, leading to either complete divergence, or imperfect mimicry. Our model reveals that imperfect mimicry evolves only when ancestral trait values differ between species, due to historical constraints and highlights the importance of female and predator discriminations in the evolution of such imperfect mimicry. Our study thus provides new predictions on how reproductive interference interacts with historical constraints and may promote the emergence of novel warning traits, enhancing mimetic diversity.

sentences: Mathematical expressions describing enzyme velocity and inhibition at high enzyme concentration Enzyme behaviour is typically characterised in the laboratory using very diluted solutions of enzyme. However, in vivo processes usually occur at [ST] {approx} [ET] {approx} Km. Furthermore, the study of enzyme action usually involves analysis and characterisation of inhibitors and their mechanisms. However, to date, there have been no reports proposing mathematical expressions that can be used to describe enzyme activity at high enzyme concentration apart from the simplest single substrate, irreversible case. Using a continued fraction approach, equations can be easily derived to apply to the most common cases in monosubstrate reactions, such as irreversible or reversible reactions and small molecule (inhibitor or activator) kinetic interactions. These expressions are simple and can be understood as an extension of the classical Michaelis-Menten equations. A first analysis of these expressions permits to deduce some differences at high vs low enzyme concentration, such as the greater effectiveness of allosteric inhibitors compared to catalytic ones. Also, they can be used to understand catalyst saturation in a reaction. Although they can be linearised following classical approaches, these equations also show some differences that need to be taken into account. The most important one may be the different meaning of line intersection points in Dixon plots. All in all, these expressions may be useful tools for the translation in vivo of in vitro experimental data or for modelling in vivo and biotechnological processes.

sentences: Antibody Evasion Properties of SARS-CoV-2 Omicron Sublineages The identification of the Omicron variant (B.1.1.529.1 or BA.1) of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in Botswana in November 20211 immediately raised alarms due to the sheer number of mutations in the spike glycoprotein that could lead to striking antibody evasion. We2 and others3-6 recently reported results in this Journal confirming such a concern. Continuing surveillance of Omicron evolution has since revealed the rise in prevalence of two sublineages, BA.1 with an R346K mutation (BA.1+R346K) and B.1.1.529.2 (BA.2), with the latter containing 8 unique spike mutations while lacking 13 spike mutations found in BA.1. We therefore extended our studies to include antigenic characterization of these new sublineages. Polyclonal sera from patients infected by wild-type SARS-CoV-2 or recipients of current mRNA vaccines showed a substantial loss in neutralizing activity against both BA.1+R346K and BA.2, with drops comparable to that already reported for BA.12,3,5,6. These findings indicate that these three sublineages of Omicron are antigenically equidistant from the wild-type SARS-CoV-2 and thus similarly threaten the efficacies of current vaccines. BA.2 also exhibited marked resistance to 17 of 19 neutralizing monoclonal antibodies tested, including S309 (sotrovimab)7, which had retained appreciable activity against BA.1 and BA.1+R346K2-4,6. This new finding shows that no presently approved or authorized monoclonal antibody therapy could adequately cover all sublineages of the Omicron variant.

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Differential Requirement of DICER1 Activity during Development of Mitral and Tricuspid Valves Mitral and tricuspid valves are essential for unidirectional blood flow in the heart. They are derived from similar cell sources, and yet congenital dysplasia affecting both valves is clinically rare, suggesting the presence of differential regulatory mechanisms underlying their development. We specifically inactivated Dicer1 in the endocardium during cardiogenesis, and unexpectedly found that Dicer1-deletion caused congenital mitral valve stenosis and regurgitation, while it had no impact on other valves. We showed that hyperplastic mitral valves were caused by abnormal condensation and extracellular matrix (ECM) remodeling. Our single-cell RNA Sequencing analysis revealed impaired maturation of mesenchymal cells and abnormal expression of ECM genes in mutant mitral valves. Furthermore, expression of a set of miRNAs that target ECM genes was significantly lower in tricuspid valves compared to mitral valves, consistent with the idea that the miRNAs are differentially required for mitral and tricuspid valve development. Our study thus reveals miRNA-mediated gene regulation as a novel molecular mechanism that differentially regulates mitral and tricuspid valve development, thereby enhancing our understanding of the non-association of inborn mitral and tricuspid dysplasia observed clinically.

Summary statementAlthough mitral and tricuspid heart valves are both derived from same cell sources in the atrioventricular region through similar initiation and remodeling processes, congenital dysplasia simultaneously affecting both valves is clinically uncommon. We show that DICER1 activity is only essential for mitral valve development whereas it is dispensable for other valves. Our study thus reveals miRNA-mediated gene regulation as a novel molecular mechanism that is differentially required to regulate mitral and tricuspid valve development, thereby enhancing our understanding of the non-association of inborn mitral and tricuspid dysplasia observed clinically.

sentences: circFL-seq reveals full-length circular RNAs with rolling circular reverse transcription and nanopore sequencing Circular RNAs (circRNAs) act through multiple mechanisms with their sequence features to fine-tune gene expression networks. Due to overlapping sequences with linear cognates, identifying internal sequences of circRNAs remains a great challenge, which hinders comprehensive understanding of circRNA functions and mechanisms. Here, based on rolling circular reverse transcription (RCRT) and nanopore sequencing, we developed circFL-seq, a full-length circRNA sequencing method, to profile circRNA at the isoform level. With a customized computational pipeline circfull to directly identify full-length sequences from rolling circular reads, we reconstructed 77,606 high-quality circRNAs from seven human cell lines and two human tissues. Benefiting from rolling circles and long-read sequencing, circFL-seq showed more than tenfold enrichment of circRNA reads and advantages for both detection and quantification at the isoform level compared to short-read RNA sequencing. The concordance of RT-qPCR and circFL-seq results for the identification of differential alternative splicing suggested wide application prospects for functional studies of internal variants in circRNAs. Moreover, the detection of cancer-related fusion circRNAs at the omics scale may further expand the application of circFL-seq. Together, the accurate identification and quantification of full-length circRNAs make circFL-seq a potential tool for large-scale screening of functional circRNAs.

sentences: Oxytocin modulates neurocomputational mechanisms underlying prosocial reinforcement learning Humans often act in the best interests of others. However, how we learn which actions result in good outcomes for other people and the neurochemical systems that support this  prosocial learning remain poorly understood. Using computational models of reinforcement learning, functional magnetic resonance imaging and dynamic causal modelling, we examined how different doses of intranasal oxytocin, a neuropeptide linked to social cognition, impact how people learn to benefit others (prosocial learning) and whether this influence could be dissociated from how we learn to benefit ourselves (self-oriented learning). We show that a low dose of oxytocin prevented decreases in prosocial performance over time, despite no impact on self-oriented learning. Critically, oxytocin produced dose-dependent changes in the encoding of prediction errors (PE) in the midbrain-subgenual anterior cingulate cortex (sgACC) pathway specifically during prosocial learning. Our findings reveal a new role of oxytocin in prosocial learning by modulating computations of PEs in the midbrain-sgACC pathway.

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Theory of local k-mer selection with applications to long-read alignment MotivationSelecting a subset of k-mers in a string in a local manner is a common task in bioinformatics tools for speeding up computation. Arguably the most well-known and common method is the minimizer technique, which selects the  lowest-ordered k-mer in a sliding window. Recently, it has been shown that minimizers are a sub-optimal method for selecting subsets of k-mers when mutations are present. There is however a lack of understanding behind the theory of why certain methods perform well.

ResultsWe first theoretically investigate the conservation metric for k-mer selection methods. We derive an exact expression for calculating the conservation of a k-mer selection method. This turns out to be tractable enough for us to prove closed-form expressions for a variety of methods, including (open and closed) syncmers, (, b, n)-words, and an upper bound for minimizers. As a demonstration of our results, we modified the minimap2 read aligner to use a more optimal k-mer selection method and demonstrate that there is up to an 8.2% relative increase in number of mapped reads.

Availability and supplementary informationSimulations and supplementary methods available at https://github.com/bluenote-1577/local-kmer-selection-results. os-minimap2 is a modified version of minimap2 and available at https://github.com/bluenote-1577/os-minimap2.

Contactjshaw@math.toronto.edu

sentences: Characterization of morpho-functional traits in mesophotic corals reveals optimized light capture and photosynthesis The morphology and skeleton architecture of photosynthetic corals modulates the light capture and functioning of the coral-algal symbiosis on shallow-water corals. Since corals can thrive on mesophotic reefs under extreme light-limited conditions, we hypothesized that microskeletal coral features optimize light capture under low-light environments. Using micro-computed tomography scanning, we conducted a comprehensive three-dimensional (3D) assessment of small-scale skeleton morphology of the depth-generalist coral Stylophora pistillata collected from shallow (5 m) and mesophotic (45 m) depths. We detected a high phenotypic diversity between depths, resulting in two distinct morphotypes, with calyx diameter, theca height, and corallite marginal spacing contributing to most of the variation between depths. To determine whether such depth-specific morphotypes affect coral light capture and photosynthesis on the corallite-scale, we developed 3D simulations of light propagation based on photosynthesis-irradiance parameters. We found that corals associated with shallow morphotypes dissipated excess light through self-shading microskeletal features; while mesophotic morphotypes facilitated enhanced light absorption and photosynthesis under low-light conditions. We conclude that the mesophotic coral architecture provides a greater ability to trap solar energy and efficiently exploit the limited light conditions, and suggest that morphological modifications play a key role in the photoadaptation response to low-light.

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Proteome-wide cellular thermal shift assay reveals novel crosstalk between brassinosteroid and auxin signaling Despite the growing interest in using chemical genetics in plant research, small-molecule target identification remains a major challenge. The cellular thermal shift assay coupled with high-resolution mass-spectrometry (CETSA MS) that monitors changes in the thermal stability of proteins caused by their interactions with small molecules, other proteins, or post-translational modifications allows the identification of drug targets, or the study of protein-metabolite and protein-protein interactions mainly in mammalian cells. To showcase the applicability of this method in plants, we applied CETSA MS to intact Arabidopsis thaliana cells and identified the thermal proteome of the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, bikinin. A comparison between the thermal- and the phospho-proteomes of bikinin revealed the auxin efflux carrier PIN-FORMED1 (PIN1) as a novel substrate of the Arabidopsis GSK3s that negatively regulate the brassinosteroid signaling. We established that PIN1 phosphorylation by the GSK3s is essential for maintaining its intracellular polarity that is required for auxin-mediated regulation of vascular patterning in the leaf thus, revealing a novel crosstalk between brassinosteroid and auxin signaling.

Significance StatementChemical genetics, which investigates the biological processes using small molecules, is gaining interest in plant research. However, a major challenge is to uncover the mode of action of the small molecule. Here, we applied the cellular thermal shift assay coupled with mass spectrometry (CETSA MS) to intact Arabidopsis cells and showed that bikinin, the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, changed the thermal stability of some of its direct targets and putative GSK3 interacting proteins. In combination with phosphoproteomics, we also revealed that GSK3s phosphorylate the auxin carrier PIN-FORMED1 (PIN1) and regulated its polarity that is required for the vascular patterning in the leaf.

sentences: A recurrent circuit links antagonistic cerebellar modules during associative motor learning The neural architecture of the cerebellum is thought to be specialized for performing supervised learning: specific error-related climbing fiber inputs are used to teach sensorimotor associations to small ensembles of Purkinje cells located in functionally distinct modules that operate independently of each other in a purely feedforward manner. Here, we test whether the basic operation of the cerebellum complies with this basic architecture in mice that learned a simple sensorimotor association during eyeblink conditioning. By recording Purkinje cells in different modules and testing whether their responses rely on recurrent circuits, our results reveal three operational principles about the functional organization of the cerebellum that stand in stark contrast to the conventional view: (1) Antagonistic organization, (2) Recurrent network dynamics, and (3) Intermodular communication. We propose that the neural architecture of the cerebellum implements these three operational principles to achieve optimal performance and solve a number of problems in motor control.

sentences: Fitting quantum machine learning potentials to experimental free energy data: Predicting tautomer ratios in solution The computation of tautomer ratios of druglike molecules is enormously important in computer-aided drug discovery, as over a quarter of all approved drugs can populate multiple tautomeric species in solution. Unfortunately, accurate calculations of aqueous tautomer ratios--the degree to which these species must be penalized in order to correctly account for tautomers in modeling binding for computer-aided drug discovery--is surprisingly diffcult. While quantum chemical approaches to computing aqueous tautomer ratios using continuum solvent models and rigid-rotor harmonic-oscillator thermochemistry are currently state of the art, these methods are still surprisingly inaccurate despite their enormous computational expense. Here, we show that a major source of this inaccuracy lies in the breakdown of the standard approach to accounting for quantum chemical thermochemistry using rigid rotor harmonic oscillator (RRHO) approximations, which are frustrated by the complex conformational landscape introduced by the migration of double bonds, creation of stereocenters, and introduction of multiple conformations separated by low energetic barriers induced by migration of a single proton. Using quantum machine learning (QML) methods that allow us to compute potential energies with quantum chemical accuracy at a fraction of the cost, we show how rigorous relative alchemical free energy calculations can be used to compute tautomer ratios in vacuum free from the limitations introduced by RRHO approximations. Furthermore, since the parameters of QML methods are tunable, we show how we can train these models to correct limitations in the underlying learned quantum chemical potential energy surface using free energies, enabling these methods to learn to generalize tautomer free energies across a broader range of predictions.

sentences: Two Competing Guilds as a Core Microbiome Signature for Chronic Diseases Summary ParagraphGut microbiota may work as an essential organ and its members interact closely with each other and form a higher-level organization called guilds. How such guild-level structure supports the gut microbiota to stably provide essential health-relevant functions to the host remains elusive. With high quality metagenome-assembled genomes as network nodes, here we identified a core microbiome signature made up of two robust competing guilds that together correlate with a wide range of host health conditions. Genomes in these two guilds kept their ecological relationship unchanged despite experiencing profound abundance changes during a 3-month high fiber intervention and 1-year follow-up in patients with type 2 diabetes. The genomes of one guild harbored more genes for plant polysaccharide degradation and butyrate production, while the other guild had more genes for virulence or antibiotic resistance. A Random Forest regression model showed that the abundance distributions of these genomes were associated with 41 out of 43 bio-clinical parameters in the study cohort. With these genomes as reference, Random Forest modeling successfully classified case and control of 8 chronic diseases in 12 independent metagenomic datasets from 1,816 participants across ethnicity and geography. This core microbiome signature may facilitate ecological management of chronic diseases.

sentences: Gustatory cortex is involved in evidence accumulation during food choice Food choice is one of the most fundamental and most frequent value-based decisions for all animals including humans. However, the neural circuitry involved in food-based decisions is only recently being addressed. Given the relatively fast dynamics of decision formation, EEG-informed fMRI analysis is highly beneficial for localizing this circuitry in humans. Here by using the EEG correlates of evidence accumulation in a simultaneously recorded EEG-fMRI dataset, we found a significant role for the right temporal-parietal operculum (PO) and medial insula including gustatory cortex (GC) in binary choice between food items. These activations were uncovered by using the "EEG energy" (power 2) as the BOLD regressor and were missed if conventional analysis with the EEG signal itself were to be used, in agreement with theoretical predictions for EEG and BOLD relations. No significant positive correlations were found with higher powers of EEG (powers 3 or 4) pointing to specificity and sufficiency of EEG energy as the main correlate of the BOLD response. This finding extends the role of cortical areas traditionally involved in palatability processing to value-based decision making and offers the "EEG energy" as a key regressor of BOLD response in simultaneous EEG-fMRI designs.

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Structural covariance and heritability of the optic tract and primary visual cortex in living human brains Individual differences among human brains exist at many scales, spanning gene expression, white matter tissue properties, and the size and shape of cortical areas. One notable example is an approximately 3-fold range in the size of human primary visual cortex (V1), a much larger range than is found in overall brain size. A previous study (Andrews et al., 1997) reported a correlation between optic tract cross-section area and V1 size in post-mortem human brains, suggesting that there may be a common developmental mechanism for multiple components of the visual pathways. We evaluated the relationship between properties of the optic tract and V1 in a much larger sample of living human brains by analyzing the Human Connectome Project 7 Tesla Retinotopy Dataset. This dataset includes retinotopic maps measured with functional MRI (fMRI) and fiber tract data measured with diffusion MRI (dMRI). We found a negative correlation between optic tract fractional anisotropy and V1 surface area (r = -0.2). This correlation, though small, was consistent across multiple dMRI datasets differing in acquisition parameters. Further, we found that both V1 size and optic tract properties were correlated among twins, with higher correlations for monozygotic than dizygotic twins, indicating a high degree of heritability for both properties. Together, these results demonstrate covariation across individuals in properties of the retina (optic tract) and cortex (V1) and show that each is influenced by genetic factors.

Significance statementThe size of human primary visual cortex (V1) has large inter-individual differences. These differences cannot be explained by differences in overall brain size. A previous post-mortem study reported a correlation between the size of the human optic tract and V1. In this study, we evaluated the relationship between the optic tract and V1 in living humans by analyzing a neuroimaging dataset that included functional and diffusion MRI data. We found a small, but robust correlation between optic tract tissue properties and V1 size, supporting the existence of structural covariance between the optic tract and V1 in living humans. The results suggest that characteristics of retinal ganglion cells, reflected in optic tract measurements, are related to individual differences in human V1.

sentences: Single cell transcriptomic profiling of tauopathy in a novel 3D neuron-astrocyte coculture model The use of iPSC derived brain organoid models to study neurodegenerative disease has been hampered by a lack of systems that accurately and expeditiously recapitulate pathogenesis in the context of neuron-glial interactions. Here we report development of a system, termed AstTau, which propagates toxic human tau oligomers in iPSC derived neuron-astrocyte spheroids. The AstTau system develops much of the neuronal and astrocytic pathology observed in tauopathies including misfolded, phosphorylated, oligomeric, and fibrillar tau, strong neurodegeneration, and reactive astrogliosis. Single cell transcriptomic profiling combined with immunochemistry characterizes a model system that can more closely recapitulate late-stage changes in adult neurodegeneration. The transcriptomic studies demonstrate striking changes in neuroinflammatory and heat shock protein (HSP) chaperone systems in the disease process. Treatment with the HSP90 inhibitor PU-H71 was used to address the putative dysfunctional HSP epichaperome and produced a strong reduction of pathology and neurodegeneration, highlighting the potential of AstTau as a rapid and reproducible tool for drug discovery.

sentences: The Origin of Conductive-Pulse Sensing Inside a Nanopore and the Role of Electro-Hydrodynamics Nanopore sensing is nearly synonymous with resistive pulse sensing due to the characteristic reduction of ionic flux during molecular occupancy of a pore, particularly at high salt concentrations. However, conductive pulses are widely reported at low salt conditions wherein electroosmotic flow can be quite significant. Aside from transporting molecules like DNA, we investigated whether electroosmotic flow has other potential impacts on sensing attributes such current enhancements due to the analyte molecule. The overwhelming majority of literature reports counterions as the dominant mechanism of conductive events (a moleculecentric theory for conductive events). Conductive events are not well understood due to the complex interplay between (charged) nanopore walls, DNA grooves, ion mobility, and counterion clouds. Yet, the prevailing consensus of counterions being introduced into the pore by the molecule does not fit well with a growing number of experiments including the fact that proteins can generate conductive events despite having a heterogeneous surface charge. Herein, we demonstrate theory and experiments underpinning the translocation mechanism (i.e., electroosmosis or electrophoresis), pulse direction (i.e., conductive or resistive) and shape (e.g., monophasic or biphasic) through fine control of chemical, physical, and electronic parameters. Results from these studies predict strong electroosmosis plays a role in driving DNA events and generating conductive events due to polarization effects (i.e. a pore-centric theory). We believe these findings will stimulate a useful discussion on the nature of conductive events and their impact on molecular sensing in nanoscale pores.

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Soil protists can actively redistribute beneficial bacteria along Medicago truncatula roots The rhizosphere is the region of soil directly influenced by plant roots. The microbial community in the rhizosphere includes fungi, protists, and bacteria, all of which play a significant role in plant health. The beneficial bacterium Sinorhizobium meliloti infects growing root hairs on nitrogen starved leguminous plants. Infection leads to the formation of a root nodule, where S. meliloti converts atmospheric nitrogen to ammonia, the usable form of nitrogen for plants. However, S. meliloti, often found in biofilms, travels slowly; whereas infectible root hairs are found at the growing root tip, potentially causing many root hairs to remain uninfected by S. meliloti when it is delivered as a seed inoculant. Soil protists are an important component of the rhizosphere system who prey on soil bacteria and have been known to egest undigested phagosomes. We show that the soil protist, Colpoda sp., plays an important role in transporting S. meliloti down Medicago truncatula roots. By using pseudo-3D soil microcosms we directly observed the presence of fluorescently labelled S. meliloti along M. truncatula roots and track the displacement of bacteria over time. In the presence of Colpoda sp., S. meliloti was detected 44 mm, on average, farther down the roots, compared with the Bacteria Only Treatment. Facilitating bacterial transport may be an important mechanism whereby soil protists promote plant health. Protist facilitated transport as a sustainable agriculture biotechnology has the potential to boost efficacy of bacterial inoculants, avoid overuse of nitrogen fertilizers, and enhance performance of no-till farming practices.

ImportanceSoil protists are an important part of the microbial community in the rhizosphere. Plants grown with protists fare better than plants grown without protists. Mechanisms through which protists support plant health include nutrient cycling, alteration of the bacterial community through selective feeding and consumption of plant pathogens. Here we provide data in support of an additional mechanism: protists act as a transport vectors for bacteria. We show that protist-facilitated transport can deliver plant-beneficial bacteria to the growing tips of roots that may otherwise be sparsely inhabited with bacteria from a seed-associated inoculum. By co-inoculating Medicago truncatula roots with both Sinorhizobium meliloti, a nitrogen fixing legume symbiont, and the ciliated protist, Colpoda sp., we show bacteria are distributed an average of 44 mm farther down plant roots than when the plants are inoculated by bacteria alone. Co-inoculation with shelf-stable encysted soil protists may be employed as a sustainable agriculture biotechnology to better distribute beneficial bacteria and enhance the performance of inoculants.

sentences: Production of a Highly Immunogenic Antigen from SARS-CoV-2 by Covalent Coupling of the Receptor Binding Domain of Spike Protein to a Multimeric Carrier Since the discovery of SARS-CoV-2, several antigens have been proposed to be part of COVID-19 vaccines. The receptor binding domain (RBD) of Spike protein is one of the promising candidates to develop effective vaccines since it can induce potent neutralizing antibodies. We previously reported the production of RBD in Pichia pastoris and showed it is structurally identical to the protein produced in mammalian HEK-293T cells. In this work we designed an RBD multimer construct with the purpose of increasing RBD immunogenicity. We produced multimeric particles by a transpeptidation reaction between the RBD expressed in P. pastoris and Lumazine Synthase from Brucella abortus (BLS), which is a highly immunogenic and very stable decameric protein of 170 kDa. We vaccinated mice with two doses 30 days apart, and then we measured humoral immune response. When the number of RBD copies coupled to BLS was high (6-7 RBD molecules per BLS decamer, in average), the immune response was significantly better than that elicited by RBD alone or even by RBD-BLS comprising low number of RBD copies (1-2 RBD molecules per BLS decamer). Remarkably, the construct with high number of RBD copies induced high IgG titers with high neutralizing capacity. Furthermore, a superior immune response was observed when Al(OH)3 adjuvant was added to this formulation, exhibiting a higher titer of neutralizing antibodies. Altogether our results suggest that RBD covalent coupled to BLS forming a multimer-particle shows an advantageous architecture to the antigen-presentation to the immune system which enhances immune responses. This new antigen should be considered a potent candidate for a protein-based vaccine.

sentences: An Ensemble Learning and Slice Fusion Strategy for Three-Dimensional Nuclei Instance Segmentation Automated microscopy image analysis is a fundamental step for digital pathology and computer aided diagnosis. Most existing deep learning methods typically require post-processing to achieve instance segmentation and are computationally expensive when directly used with 3D microscopy volumes. Supervised learning methods generally need large amounts of ground truth annotations for training whereas manually annotating ground truth masks is laborious especially for a 3D volume. To address these issues, we propose an ensemble learning and slice fusion strategy for 3D nuclei instance segmentation that we call Ensemble Mask R-CNN (EMR-CNN) which uses different object detectors to generate nuclei segmentation masks for each 2D slice of a volume and propose a 2D ensemble fusion and a 2D to 3D slice fusion to merge these 2D segmentation masks into a 3D segmentation mask. Our method does not need any ground truth annotations for training and can inference on any large size volumes. Our proposed method was tested on a variety of microscopy volumes collected from multiple regions of organ tissues. The execution time and robustness analyses show that our method is practical and effective.

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Empirical estimates of the mutation rate for an alphabaculovirus Mutation rates are of key importance for understanding evolutionary processes and predicting their outcomes. Empirical estimates of mutation rate are available for a number of RNA viruses, but few are available for DNA viruses, which tend to have larger genomes. Whilst some viruses have very high mutation rates, lower mutation rates are expected for viruses with large genomes to ensure genome integrity. Alphabaculoviruses are insect viruses with large genomes and often have high levels of polymorphism, suggesting high mutation rates despite evidence of proofreading activity by the replication machinery. Here, we report an empirical estimate of the mutation rate per base per strand copying (s/n/r) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). To avoid biases due to selection, we analyzed mutations that occurred in a stable, non-functional genomic insert after five serial passages in Spodoptera exigua larvae. Population bottlenecks, viral mode of replication and thresholds for mutation detection likely affect mutation rate estimates, and we therefore used population genetic models that account for these processes to infer the mutation rate. We estimated a mutation rate of 1x10-7 s/n/r. This estimate was not sensitive to different model assumptions or including whole genome data. The rates at which different classes of mutations accumulate provide good evidence for neutrality of mutations occurring within the inserted region. We therefore present a robust approach for mutation rate estimation for viruses with stable genomes, and strong evidence of a much lower alphabaculovirus mutation rate than supposed based on the high levels of polymorphism observed.

Author SummaryVirus populations can evolve rapidly, driven by the large number of mutations that occur during virus replication. It is challenging to measure mutation rates because selection will affect which mutations are observed: beneficial mutations are overrepresented in virus populations, while deleterious mutations are selected against and therefore underrepresented. Few mutation rates have been estimated for viruses with large DNA genomes, and there are no estimates for any insect virus. Here, we estimate the mutation rate for an alphabaculovirus, a virus that infects caterpillars and has a large, 134 kilobase pair DNA genome. To ensure that selection did not bias our estimate of mutation rate, we studied which mutations occurred in a large artificial region inserted into the virus genome, where mutations did not affect viral fitness. We deep sequenced evolved virus populations, and compared the distribution of observed mutants to predictions from a simulation model to estimate mutation rate. We found evidence for a relatively low mutation rate, of one mutation in every 10 million bases replicated. This estimate is in line with expectations for a virus with self-correcting replication machinery and a large genome.

sentences: Cephalopod Retinal Development Shows Vertebrate-like Mechanisms of Neurogenesis Neurogenesis, the regulation of cellular proliferation and differentiation in the developing nervous system, is the process that underlies the diversity of size and cell type found in animal nervous systems. Our understanding of how this process has evolved is limited because of the lack of high resolution data and live-imaging methods across species. The retina is a classic model for the study of neurogenesis in vertebrates and live-imaging of the retina has shown that during development, progenitor cells are organized in a pseudostratified neuroepithelium and nuclei migrate in coordination with the cell cycle along the apicobasal axis of the cell, a process called interkinetic nuclear migration. Eventually cells delaminate and differentiate within the boundaries of the epithelium. This process has been considered unique to vertebrates and thought to be important in maintaining organization during the development of a complex nervous system. Coleoid cephalopods, including squid, cuttlefish and octopus, have the largest nervous system of any invertebrate and convergently-evolved camera-type eyes, making them a compelling comparative system to vertebrates. Here we have pioneered live-imaging techniques to show that the squid, Doryteuthis pealeii, displays cellular mechanisms during cephalopod retinal neurogenesis that are hallmarks of vertebrate processes. We find that retinal progenitor cells in the squid undergo interkinetic nuclear migration until they exit the cell cycle, we identify retinal organization corresponding to progenitor, post-mitotic and differentiated cells, and we find that Notch signaling regulates this process. With cephalopods and vertebrates having diverged 550 million years ago, these results suggest that mechanisms thought to be unique to vertebrates may be common to highly proliferative neurogenic primordia contributing to a large nervous system.

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X upregulation is not global and extent of upregulation differs between ancestral and acquired X-linked genes Evolution of sex chromosome dosage compensation in mammals remains poorly understood. Ohnos hypothesis state that evolution of dosage compensation occurred through two steps: first, to compensate the dosage imbalance created due to the degradation of Y chromosome in male, upregulation of X-chromosome happened. Subsequently, transmission of X-chromosome upregulation (XCU) in female led to the evolution of X-chromosome inactivation (XCI) to counteract extra dosage of X-linked genes in female cells. Here, we have profiled gene-wise dynamics of XCU in pre-gastrulation mouse embryos at single cell level and found that XCU is dynamically linked with XCI, however, XCU is not global or chromosome-wide like XCI. Therefore, our result raises question whether XCU driven the evolution of XCI. If so, then why XCI is chromosome wide while XCU is not. We propose that XCI might have evolved independent of XCU and therefore refining the current model is necessary. Separately, we show that higher occupancy of different activating factors at upregulated X-linked genes leads to enhanced transcriptional burst frequency and thereby leads to upregulation. On the other hand, our analysis indicates that extent of upregulation, enrichment of different activating marks differs between ancestral and newly acquired X-linked genes. Altogether, our study provides significant insight into the dynamics and mechanistic basis of evolution of sex chromosome dosage compensation.

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: Collective action or individual choice: Spontaneity and individuality contribute to decision-making in Drosophila Our own unique character traits make our behavior consistent and define our individuality. Yet, this consistency does not entail that we behave repetitively like machines. Like humans, animals also combine personality traits with spontaneity to produce adaptive behavior: consistent, but not fully predictable. Here, we study an iconically rigid behavioral trait - insect phototaxis - that that nevertheless also contains both components of individuality and spontaneity. In a light/dark T-maze, approximately 70% of a group of Drosophila fruit flies choose the bright arm of the T-Maze, while the remaining 30% walk into the dark. Taking the photopositive and the photonegative subgroups and re-testing them reveals the spontaneous component: a similar 70-30 distribution emerges in each of the two subgroups. Increasing the number of choices to ten choices, reveals the individuality component: flies with extremely negative first choices were more likely to show photonegative behavior in subsequent choices and vice versa. General behavioral traits, independent of light/dark preference, contributed to the development of this individuality. The interaction of individuality and spontaneity together explains why group averages, even for such seemingly stereotypical behaviors, are poor predictors of individual choices.

sentences: Resveratrol And Pterostilbene Potently Inhibit SARS-CoV-2 Replication In Vitro The current COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has an enormous impact on human health and economy. In search for therapeutic options, researchers have proposed resveratrol, a food supplement with known antiviral, anti-inflammatory and anti-oxidant properties as an advantageous antiviral therapy for SARS-CoV-2 infection. Here, we provide evidence that both resveratrol and its metabolically more stable structural analog, pterostilbene, exhibit potent antiviral properties against SARS-CoV-2 in vitro. Resveratrol and pterostilbene showed antiviral activity in African green monkey kidney cells and in human primary bronchial epithelial cells cultured in an air-liquid interface system. Both compounds actively inhibit virus replication within infected cells as reduced virus progeny production was observed when the compound was added at post-inoculation conditions. Without replenishment of the compound, antiviral activity was observed up to roughly 5 rounds of replication, demonstrating the long-lasting effect of these compounds. Collectively, our data indicate that resveratrol and pterostilbene are promising antiviral compounds to treat SARS-CoV-2 infection. Because these results represent laboratory findings in cells, we advocate evaluation of these compounds in clinical trials before statements are made whether or not these drugs are advantageous for COVID-19 treatment.

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Development of opioid-induced hyperalgesia depends on reactive astrocytes controlled by Wnt5a signaling Opioid analgesics are the frontline pain medicine for managing various types of pain. Paradoxically, repeated use of opioid analgesics may cause an exacerbated pain state known as opioid-induced hyperalgesia (OIH). OIH significantly contributes to dose escalation and consequently opioid overdose. In addition to neuronal malplasticity, emerging evidence suggests a critical role of reactive glia in OIH development. A potential astrocytic underpinning of OIH pathogenesis is indicated by their prominently activation in OIH animal models. However, this hypothesis has not been conclusively tested and the mechanism underlying the astrocyte activation remains unclear. Here, we show that reactive astrocytes (a.k.a. astrogliosis) are critical for OIH development in mice. Genetic ablation of astrogliosis inhibited the expression of OIH and morphine-induced neural circuit polarization (NCP) in the spinal dorsal horn (SDH). We also found that Wnt5a is a neuron-to-astrocyte signal that is required for morphine-induced astrogliosis. Conditional knock-out of Wnt5a in neurons or its co-receptor ROR2 in astrocytes blocked not only morphine-induced astrogliosis but also OIH and NCP. Furthermore, we showed that the Wnt5a-ROR2 signaling-dependent astrogliosis contributes to OIH via inflammasome-regulated IL-1{beta}. Our results reveal an important role of morphine-induced astrogliosis in OIH pathogenesis and elucidate a neuron-to-astrocyte intercellular Wnt signaling pathway that controls the astrogliosis.

One sentence summaryNeuron-to-astrocyte Wnt5a signaling controls the pathogenesis of opioid-induced hyperalgesia via astrogliosis

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The consequences of reciprocally exchanging the genomic sites of Integration Host Factor (IHF) subunit production for subunit stoichiometry and bacterial physiology in Salmonella enterica serovar Typhimurium Integration host factor (IHF) is a heterodimeric nucleoid-associated protein that plays roles in bacterial nucleoid architecture and genome-wide gene regulation. The ihfA and ihfB genes encode the subunits and are located 350 kilobase pairs apart, in the Right replichore of the Salmonella chromosome. IHF is composed of one IhfA and one IhfB subunit. Despite this 1:1 stoichiometry, mass spectrometry revealed that IhfB is produced in 2-fold excess over IhfA. We re-engineered Salmonella to exchange reciprocally the protein-coding regions of ihfA and ihfB, such that each relocated protein-encoding region was driven by the expression signals of the others gene. Mass spectrometry showed that in this  rewired strain, IhfA is produced in excess over IhfB, correlating with enhanced stability of the hybrid ihfB-ihfA mRNA that was expressed from the ihfB promoter. Nevertheless, the rewired strain grew at a similar rate to the wild type, had identical cell morphology, and was similar in competitive fitness. However, compared to the wild type, it was less motile, had growth-phase-specific reductions in SPI-1 and SPI-2 gene expression and was engulfed at a higher rate by RAW macrophage. Our data show that while exchanging the physical locations of its ihf genes and the rewiring of their regulatory circuitry are well tolerated in Salmonella, genes involved in the production of type 3 secretion systems exhibit dysregulation accompanied by altered phenotypes.

IMPACT STATEMENTIntegration Host Factor (IHF) is an abundant nucleoid-associated protein that organises DNA architecturally, influencing gene expression globally in Salmonella and other bacteria. IHF is composed of two related, non-identical, subunits, produced by genes that are 350 kilobase pairs apart. Each ihf gene has unique expression controls and is embedded in a complex genetic network that supports mRNA translation. Given that the subunits are thought to be required in a 1:1 ratio to form functional IHF, we were surprised by this physical and regulatory separation. We rewired the Salmonella genome so that each subunit was produced using the others regulatory signals and gene location. This revealed a high degree of tolerance to the effects of this rewiring. However, we discovered that bacterial motility was disrupted, as was the expression of virulence genes that have been acquired by horizontal gene transfer. Proteomic analysis using mass spectroscopy (MS) showed the extent of the alterations to cell composition. Our MS data also demonstrated that the subunits of IHF are not produced in a 1:1 ratio in either the wild type or the rewired strain. We discuss this finding in terms of the ability of each subunit to stabilise its partner.

DATA SUMMARYO_LIWhole genome sequence data for strain OrfSwapihfA-ihfB are available from the European Nucleotide Archive with accession number ERS4653309.
C_LIO_LIData from mass spectrometry analyses are available via ProteomeXchange with identifier PXD027465 (login: reviewer_pxd027465@ebi.ac.uk and password: GVeNIUB2).
C_LIO_LIAll supporting data have been provided in the article or through supplementary data files.
C_LI

RepositoriesWhole genome sequence data for strain OrfSwapihfA-ihfB are available from the European Nucleotide Archive with accession number ERS4653309. Data from mass spectrometry analyses are available via ProteomeXchange with identifier PXD027465 (login: reviewer_pxd027465@ebi.ac.uk and password: GVeNIUB2).

sentences: Rats emit unique distress calls in social inequality conditions Humans show aversion toward inequality of social reward, and this aversion plays important roles for the establishment of social cooperation. However, it has remained unknown whether commonly used experimental animals show negative responses to social reward inequality. In this study, we found that rats showed bonding-like behavior to an affiliative human who repeatedly stroked the rats. In addition, these rats emitted distress calls, an index of negative emotion, when an affiliative human stroked another rat in front of them. These distress calls had acoustic characteristics different from those emitted in response to physical stress stimuli such as air-puff. Rats emitted calls with higher frequency (28 kHz) and shorter durations (0.05 sec) in an inequality condition than the frequency and durations of calls emitted when receiving air-puff. Our results suggested that rats exhibited negative emotion with unique distress calls in response to a social inequality condition.

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Spatial transcriptomics reveals unique molecular fingerprints of human nociceptors Nociceptors are specialized sensory neurons that detect damaging or potentially damaging stimuli and are found in the dorsal root (DRG) and trigeminal ganglia. These neurons are critical for the generation of neuronal signals that ultimately create the perception of pain. These neurons are also primary targets for acute and chronic pain therapeutics. Single-cell transcriptomics on mouse nociceptors has transformed our understanding of pain mechanisms. We sought to generate equivalent information for human nociceptors with the goal of identifying transcriptomic signatures of nociceptors, identifying species differences and elucidating new drug targets. We used spatial transcriptomics to molecularly characterize transcriptomes of single dorsal root ganglion (DRG) neurons from 8 organ donors. We identified 12 clusters of human sensory neurons, 5 of which are C nociceptors; as well as 1 A{beta} nociceptor, 2 A{delta}, 2 A{beta} and 1 proprioceptor subtypes. By focusing on expression profiles for ion channels, G-protein coupled receptors (GPCRs) and other pharmacological targets, we provide a rich map of drug targets in the human DRG with direct comparison to mouse sensory neuron transcriptomes. We also compare human DRG neuronal subtypes to non-human primates showing conserved patterns of gene expression among many cell types, but divergence among specific nociceptor subsets. Finally, we identify sex differences in human DRG subpopulation transcriptomes, including a marked increase in CALCA expression in female pruritogen receptor enriched nociceptors. Our data open the door to development of drug discovery programs for new pain targets and unparalleled molecular characterization of clinical sensory disorders.

One Sentence SummaryWe used spatial transcriptomics to molecularly characterize human sensory neurons, comparing them to mouse and non-human primate finding similarities but also divergence, in particular among drug targets.

sentences: Adult medial habenula neurons require GDNF receptor GFRalpha1 for synaptic stability and function The medial habenula (mHb) is an understudied small brain nucleus linking forebrain and midbrain structures controlling anxiety and fear behaviors. The mechanisms that maintain the structural and functional integrity of mHb neurons and their synapses remain unknown. Using spatio-temporally controlled Cre-mediated recombination in adult mice, we found that the GDNF receptor alpha 1 (GFR1) is required in adult mHb neurons for synaptic stability and function. mHb neurons express some of the highest levels of GFR1 in the mouse brain, and acute ablation of GFR1 results in loss of septo-habenular and habenulo-interpeduncular glutamatergic synapses, with the remaining synapses displaying reduced numbers of presynaptic vesicles. Chemo- and opto-genetic studies in mice lacking GFR1 revealed impaired circuit connectivity, reduced AMPA receptor postsynaptic currents, and abnormally low rectification index of AMPARs, suggesting reduced Ca2+-permeability. Further biochemical and proximity ligation assay studies defined the presence of GluA1/GluA2 (Ca2+-impermeable) as well as GluA1/GluA4 (Ca2+-permeable) AMPAR complexes in mHb neurons, as well as clear differences in the levels and association of AMPAR subunits in mHb neurons lacking GFR1. Finally, acute loss of GFR1 in adult mHb neurons reduced anxiety-like behavior and potentiated context-based fear responses, phenocopying the effects of lesions to septal projections to the mHb. These results uncover an unexpected function for GFR1 in the maintenance and function of adult glutamatergic synapses, and reveal a potential new mechanism for regulating synaptic plasticity in the septo-habenulo-interpeduncular pathway and attuning of anxiety and fear behaviors.

sentences: Relative water content consistently predicts drought mortality risk in seedling populations with different morphology, physiology, and times to death Predicted increases in forest drought mortality highlight the need for predictors of incipient drought-induced mortality (DIM) risk that enable proactive large-scale management. Such predictors should be consistent across plants with varying morphology and physiology. Because of their integrative nature, indicators of water status are promising candidates for real time monitoring of DIM, particularly if they standardize morphological differences among plants. We assessed the extent to which differences in morphology and physiology between Pinus ponderosa populations influence time to mortality and the predictive power of key indicators of DIM risk. Time to incipient mortality differed between populations but occurred at the same relative water content (RWC) and water potential (WP). RWC and WP were accurate predictors of drought mortality risk. These results highlight that variables related to water status capture critical thresholds during DIM and the associated dehydration processes. Both WP and RWC are promising candidates for large-scale assessments of DIM risk. RWC is of special interest because it allows comparisons across different morphologies and can be remotely sensed. Our results offer promise for real-time landscape-level monitoring of DIM and its global impacts in the near term.

sentences: A Variable Clock Underlies Internally Generated Hippocampal Sequences Humans have the ability to store and retrieve memories with various degrees of specificity, and recent advances in reinforcement learning have identified benefits to learning when past experience is represented at different levels of temporal abstraction. How this flexibility might be implemented in the brain remains unclear. We analyzed the temporal organization of rat hippocampal population spiking to identify potential substrates for temporally flexible representations. We examined activity both during locomotion and during memory-associated population events known as sharp wave-ripples (SWRs). We found that spiking during SWRs is rhythmically organized with higher event-to-event variability than spiking during locomotion-associated population events. Decoding analyses using clusterless methods further indidate that similar spatial experience can be replayed in multiple SWRs, each time with a different rhythmic structure whose periodicity is sampled from a lognormal distribution. This variability increases with experience despite the decline in SWR rates that occurs as environments become more familiar. We hypothesize that the variability in temporal organization of hippocampal spiking provides a mechanism for storing experiences with various degrees of specificity.

sentences: From telomere to telomere: the transcriptional and epigenetic state of human repeat elements Mobile elements and highly repetitive genomic regions are potent sources of lineage-specific genomic innovation and fingerprint individual genomes. Comprehensive analyses of large, composite or arrayed repeat elements and those found in more complex regions of the genome require a complete, linear genome assembly. Here we present the first de novo repeat discovery and annotation of a complete human reference genome, T2T-CHM13v1.0. We identified novel satellite arrays, expanded the catalog of variants and families for known repeats and mobile elements, characterized new classes of complex, composite repeats, and provided comprehensive annotations of retroelement transduction events. Utilizing PRO-seq to detect nascent transcription and nanopore sequencing to delineate CpG methylation profiles, we defined the structure of transcriptionally active retroelements in humans, including for the first time those found in centromeres. Together, these data provide expanded insight into the diversity, distribution and evolution of repetitive regions that have shaped the human genome.

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DNA methylation dynamics during stress-response in woodland strawberry (Fragaria vesca) O_LIEnvironmental stresses can result in a wide range of physiological and molecular responses in plants. These responses can also impact epigenetic information in genomes especially at the level of DNA methylation. DNA methylation is the hallmark heritable epigenetic modification and plays a key role in silencing transposable elements (TEs). Although DNA methylation is an essential epigenetic mechanism, fundamental aspects of its contribution to stress responses and adaptation remain obscure.
C_LIO_LIWe investigated epigenome dynamics of wild strawberry (Fragaria vesca) in response to variable environmental conditions at DNA methylation level. F. vesca methylome responded with great plasticity to ecologically relevant abiotic and hormonal stresses. Thermal stress resulted in substantial genome-wide loss of DNA methylation. Notably, all tested stress conditions resulted in marked hot spots of differential DNA methylation near centromeric or pericentromeric regions, particularly in non-symmetrical DNA methylation context. Additionally, we identified differentially methylated regions (DMRs) within promoter regions of transcription factor (TF) superfamilies involved in plant stress-response and assessed the effects of these changes on gene expression.
C_LIO_LIThese findings improve our understanding on stress-response at the epigenome level by highlighting the correlation between DNA methylation, TEs and gene expression regulation in plants subjected to a broad range of environmental stresses.
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sentences: Comprehensive assessment of functional effects of commonly used sweeteners on ex vivo human gut microbiome The gut microbiome composition and function are associated with health and diseases. Sweeteners are widely used food additives, although many studies using animal models have linked sweetener consumption to gut microbial changes and health issues. Whether sweeteners directly change the human gut microbiome functionality remains largely unknown. In this study, we systematically investigated the responses of five human gut microbiomes to 21 common sweeteners, using an approach combining high-throughput ex vivo microbiome culturing and metaproteomics to quantify functional changes in different taxa. Hierarchical clustering based on metaproteomic responses of individual microbiomes resulted in two clusters. The first cluster was composed of non-caloric artificial sweeteners (NAS) and two sugar alcohols with shorter carbon backbones (4-5 carbon atoms), and the second cluster was composed of sugar alcohols with longer carbon backbones. The metaproteomic functional responses of the second cluster were similar to the prebiotic fructooligosaccharides and kestose, indicating that these sugar alcohol-type sweeteners have potential prebiotic functions. This study provides a comprehensive evaluation of the direct effects of commonly used sweeteners on the functions of the human gut microbiome using a functional metaproteomics approach, improving our understanding of the roles of sweeteners on microbiome-associated human health and disease issues.

sentences: A recombinant murine rotavirus with Nano-Luciferase expression reveals tissue tropism, replication dynamics, and virus transmission Rotaviruses (RVs) are one of the main causes of severe gastroenteritis, diarrhea, and death in children and young animals. Although suckling mice prove to be highly useful small animal models of RV infection and pathogenesis, direct visualization tools are lacking to track the temporal dynamics of RV replication and transmissibility in vivo. Here, we report the generation of the first recombinant murine RV that encodes a Nano-Luciferase reporter (NLuc) using a newly optimized RV reverse genetics system. The NLuc-expressing RV was replication-competent in cell culture and both infectious and virulent in neonatal mice in vivo. Strong luciferase signals were detected in the proximal and distal small intestines, colon, and mesenteric lymph nodes. We showed, via a noninvasive in vivo imaging system, that RV intestinal replication peaked at day 2 and day 5 post infection. Moreover, we successfully tracked RV transmission to uninoculated littermates as early as 3 days post infection, 1 day prior to clinically apparent diarrhea and 3 days prior to detectable fecal RV shedding in the uninoculated littermates. We also observed significantly increased viral replication in Stat1 knockout mice that lack the host interferon signaling. Our results suggest that the NLuc RV represents a non-lethal powerful tool for the studies of tissue tropism and host and viral factors that regulate RV replication and spread, as providing a new mechanism to facilitate the testing of prophylactic and therapeutic interventions in the future.

sentences: Assembling Long Accurate Reads Using Multiplex de Bruijn Graphs Although most existing genome assemblers are based on the de Bruijn graphs, it remains unclear how to construct these graphs for large genomes and large k-mer sizes. This algorithmic challenge has become particularly important with the emergence of long high-fidelity (HiFi) reads that were recently utilized to generate a semi-manual telomere-to-telomere assembly of the human genome and to get a glimpse into biomedically important regions that evaded all previous attempts to sequence them. To enable automated assemblies of long and accurate reads, we developed a fast LJA algorithm that reduces the error rate in these reads by three orders of magnitude (making them nearly error-free) and constructs the de Bruijn graph for large genomes and large k-mer sizes. Since the de Bruijn graph constructed for a fixed k-mer size is typically either too tangled or too fragmented, LJA uses a new concept of a multiplex de Bruijn graph with varying k-mer sizes. We demonstrate that LJA improves on the state-of-the-art assemblers with respect to both accuracy and contiguity and enables automated telomere-to-telomere assemblies of entire human chromosomes.

sentences: Constitutive, calcium-independent endoplasmic reticulum-plasma membrane contact site oscillations and its implications in store-operated calcium entry The endoplasmic reticulum and plasma membrane (ER-PM) contact site dynamics plays a central role for store-operated calcium entry (SOCE). ER localized calcium sensor STIM1 translocates to the contact sites, interacts with Orai and mediates calcium influx from the extracellular environment. Different species of phosphoinositides (PIPs) have been reported to be involved in contact site dynamics as well as STIM1 translocation. However, most of the studies were based on loss-of-function experiments or conditions that generate massive calcium store depletion. The kinetics of ER-PM contact site dynamics during physiological stimuli -induced calcium oscillations are not well understood. Using total internal reflection fluorescence microscopy (TIRFM), we investigated the relationship between dynamics of STIM1 as well as cortical ER (cER) proteins and calcium oscillations in rat basophilic leukemia (RBL) mast cells. Surprisingly, a significant percentage of cells displayed cyclic STIM1 and cER dynamics that were calcium-independent. Using specific lipid sensors, we showed that cyclic ER-PM contact site assembly was in phase with PI(4)P oscillation, but preceded phases of PI(4,5)P2 or PI(3,4,5)P3 oscillation. Optogenetic recruitment of the phosphoinositide 5-phosphatase from INPP5E, which decreased PI(4,5)P2 and increased PI(4)P levels on the plasma membrane, stimulated the translocation of STIM1 and inhibited calcium oscillations. Interestingly, prolonged stable translocation of STIM1 to the plasma membrane had an inhibitory effect on calcium oscillations. Collectively, our findings suggest that ER-PM contact sites formation is PI(4)P-dependent. In addition, reversibility of ER-PM contact sites dynamics and intermediate strength of ER-PM contact are needed for calcium oscillation.

sentences: Nanoparticle Internalization Promotes the Survival of Primary Macrophages Macrophages, a class of tissue resident innate immune cells, are responsible for sequestering foreign objects through the process of phagocytosis, making them a promising target for immune-modulation via particulate engineering. Here, we report that nanoparticle (NP) dosing and cellular internalization via phagocytosis significantly enhances survival of ex vivo cultures of primary bone marrow-derived, alveolar, and peritoneal macrophages over particle-free controls. The enhanced survival is attributed to suppression of caspase-dependent apoptosis and is linked to phagocytosis and lysosomal signaling, which was also found to occur in vivo. Uniquely, poly(ethylene glycol)-based NP treatment does not alter macrophage polarization or lead to inflammatory effects. The enhanced survival phenomenon is also applicable to NPs of alternative chemistries, indicating the potential universality of this phenomenon with relevant drug delivery particles. These findings provide a framework for extending the lifespan of primary macrophages ex vivo for drug screening, vaccine studies, and cell therapies and has implications for any in vivo particulate immune-engineering applications.

sentences: Boundary domain genes were recruited to suppress bract growth and promote branching in maize Grass inflorescence development is diverse and complex and involves sophisticated but poorly understood interactions of genes regulating branch determinacy and leaf growth. Here, we use a combination of transcript profiling, genetic and phylogenetic analyses to investigate tasselsheath1 (tsh1) and tsh4, two maize genes that simultaneously suppress inflorescence leaf growth and inhibit branching. We identify a regulatory network of inflorescence leaf suppression that involves the phase change gene tsh4 upstream of tsh1 and the ligule identity gene liguleless2 (lg2). We also find that a series of duplications in the tsh1 gene lineage facilitated its shift from boundary domain in non-grasses to suppressed inflorescence leaves of grasses. Collectively, these results suggest that the boundary domain genes tsh1 and lg2 were recruited to inflorescence leaves where they suppress growth and regulate a non-autonomous signaling center that promotes inflorescence branching, an important component of yield in cereal grasses.

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DeepPerVar: a multimodal deep learning framework for functional interpretation of genetic variants in personal genome MotivationUnderstanding the functional consequence of genetic variants, especially the noncoding ones, is important but particularly challenging. Genome-wide association studies or quantitative trait locus analyses may be subject to limited statistical power and linkage disequilibrium, and thus are less optimal to pinpoint the causal variants. Moreover, most existing machine learning approaches, which exploit the functional annotations to interpret and prioritize putative causal variants, cannot accommodate the heterogeneity of personal genetic variations and traits in a population study, targeting a specific disease.

ResultsBy leveraging paired whole genome sequencing data and epigenetic functional assays in a population study, we propose a multi-modal deep learning framework to predict genome-wide quantitative epigenetic signals by considering both personal genetic variations and traits. The proposed approach can further evaluate the functional consequence of noncoding variants on an individual level by quantifying the allelic difference of predicted epigenetic signals. By applying the approach to the ROSMAP cohort studying Alzheimers disease (AD), we demonstrate that the proposed approach can accurately predict quantitative genome-wide epigenetic signals and in key genomic regions of AD causal genes, learn canonical motifs reported to regulate gene expression of AD causal genes, improve the partitioning heritability analysis, and prioritize putative causal variants in a GWAS risk locus. Finally, we release the proposed deep learning model as a stand-alone Python toolkit and a web server.

Availabilityhttps://github.com/lichen-lab/DeepPerVar

sentences: Agrochemical control of gene expression using evolved split RNA polymerase Chemical inducible gene expression systems are valuable tools for rational control of gene expression both for basic research and biotechnology. However, most chemical inducers are confined to certain groups of organisms. Therefore, dissecting interaction between different organisms could be challenging using existing chemical inducible systems. We engineered a mandipropamid induced gene expression system (Mandi-T7) based on evolved split T7 RNAP system. As a proof-of-principle, we induced GFP expression in E. coli cells grown inside plant tissue.

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Feline leukemia virus (FeLV) endogenous and exogenous recombination events result in multiple FeLV-B subtypes during natural infection Feline leukemia virus (FeLV) is associated with a range of clinical signs in felid species. The primary hosts of FeLV are domestic cats of the Felis genus that also harbor endogenous FeLV (enFeLV) elements stably integrated in their genomes. EnFeLV elements display 86% nucleotide identity to exogenous, horizontally transmitted FeLV (FeLV-A). Variation between enFeLV and FeLV-A is primarily in the long terminal repeat (LTR) and env regions, which potentiates generation of FeLV-B recombinant subtypes during natural infection with enhanced virulence. The aim of this study was to examine exogenous FeLV (exFeLV) and enFeLV recombination events in a natural FeLV epizootic. We previously described that of 32 individuals in a closed colony with productive FeLV-A infection, 22 had detectable circulating FeLV-B. We cloned and sequenced the env gene of FeLV-B, FeLV-A, and enFeLV spanning known recombination breakpoints, examining between 1-13 clones per individual to assess sequence diversity and recombination sites. We documented multiple recombination breakpoints resulting in the production of unique FeLV-B genotypes. At least half of the cats harbored more than one FeLV-B variant, and almost all animals had variants similar to those recovered from at least one other individual in the colony. This analysis reveals that FeLV-B is predominantly generated de novo within each host, though horizontal transmission may be inferred based upon FeLV-B sequence identities between individuals. This work represents a comprehensive analysis of endogenous-exogenous retroviral interactions with important insights into host-viral interactions that underlie disease pathogenesis in a natural setting.

ImportanceFeline leukemia virus (FeLV) is a felid retrovirus associated with a variety of disease outcomes. Exogenous FeLV-A is the most common horizontally transmitted virus subgroup. Domestic cats (Felis catus) harbor endogenous copies of FeLV (enFeLV) in their genomes. Recombination between FeLV-A and enFeLV may result in emergence of largely replication-defective, but highly virulent recombinant strains. FeLV-B, the most common recombinant form, results when enFeLV env recombines with FeLV-A during FeLV replication. This study evaluated endogenous-exogenous recombination outcomes in a naturally-infected closed colony of domestic cats to determine recombination sites and FeLV-B genotypic heterogeneity associated with enhanced disease virulence. While FeLV-A and enFeLV genotypes were highly conserved, a large number of unique FeLV-B variants were identified in association with predicted recombination hotspots. The findings provide insight into recombination events between viral and host genomes that result in new, and potentially more pathogenic, viral strains.

sentences: SIRV: Spatial inference of RNA velocity at the single-cell resolution Studying cellular differentiation using single-cell RNA sequencing (scRNA-seq) rapidly expands our understanding of cellular development processes. Recently, RNA velocity has created new possibilities in studying these cellular differentiation processes, as differentiation dynamics can be obtained from measured spliced and unspliced mRNA expression. However, to map these differentiation processes to developments within a tissue, the spatial context of the tissue should be taken into account, which is not possible with current approaches as they start from dissociated cells. We present SIRV (Spatially Inferred RNA Velocity), a method to infer spatial differentiation trajectories within the spatial context of a tissue at the single-cell resolution. SIRV integrates spatial transcriptomics data with reference scRNA-seq data, to enrich the spatially measured genes with spliced and unspliced expressions from the scRNA-seq data. Next, SIRV calculates RNA velocity vectors for every spatially measured cell and maps these vectors to the spatial coordinates within the tissue. We tested SIRV on the Developing Mouse Brain Atlas data and obtained biologically relevant spatial differentiation trajectories. Additionally, SIRV annotates spatial cells with cellular identities and the region of origin which are transferred from the annotated reference scRNA-seq data. Altogether, with SIRV, we introduce a new tool to enrich spatial transcriptomics data that can assist in understanding how tissues develop.

sentences: Collateral damage: Identification and characterisation of spontaneous mutations causing deafness from a targeted knockout programme Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but the process of culturing embryonic stem cells during the making of a targeted allele offers opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We have identified 8 different mutations causing deafness in 16 of these 25 lines and characterised the resulting phenotypes. Our data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes.

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Predicted Impact of the Viral Mutational Landscape on the Cytotoxic Response against SARS-CoV-2 The massive assessment of immune evasion due to viral mutations that potentially increase COVID-19 susceptibility can be computationally facilitated. The adaptive cytotoxic T response is critical during primary infection and the generation of long-term protection. Potential epitopes in the SARS-CoV-2 proteome were predicted for 2,915 human alleles of 71 HLA class I families. Allele families showed extreme differences in number of recognized epitopes, underscoring genetic variability of protective capacity between humans. Up to 1,222 epitopes were associated with any of the twelve supertypes, that is, allele clusters covering 90% population. Among them, the B27 supertype showed the lowest number of epitopes. Epitope escape mutations identified in ~118,000 NCBI isolates mainly involved non-conservative substitutions at the second and C-terminal position of the ligand core, or total ligand removal by large recurrent deletions. Escape mutations affected 47% of supertype epitopes, which in 21% of cases concerned isolates from two or more sub-continental areas. Some of these changes were coupled, but never surpassed 15% evaded epitopes for the same supertype in the same isolate, except for B27, which reached up to 33%. In contrast to most supertypes, eight particular allele families mostly contained alleles with few SARS-CoV-2 ligands. Isolates harboring cytotoxic escape mutations for these families co-existed geographically within sub-Saharan and Asian populations enriched in these alleles. Collectively, these data indicate that independent escape mutation events have already occurred for half of HLA class I supertype epitopes. However, it is presently unlikely that, overall, it poses a threat to the global population. In contrast, single and double mutations for susceptible alleles may be associated with viral selective pressure and alarming local outbreaks. This study highlights the automated integration of genomic, geographical and immunoinformatic information for surveillance of SARS-CoV-2 variants potentially affecting the population as a whole, as well as minority subpopulations.

AUTHOR SUMMARYThe cytotoxic T response, a type of immune response dependent upon an individual's genetics that does not require antibodies, is critical for neutralizing SARS-CoV-2 infection. The potential bypass of the cytotoxic T response by mutations acquired by the virus after one year of the pandemic is therefore of maximal concern. We have approached the complexity of human variability and more than 100.000 viral genomes in this respect using a computational strategy. We have detected numerous mutations in these genomes that mask some viral regions involved in the cytotoxic response. However, the accumulation of these changes in independent isolates is still too low to threaten the global human population. In contrast, our protocol has identified mutations that may be relevant for specific populations and minorities with cytotoxic genetic backgrounds susceptible to SARS-CoV-2 infection. Some viral variants co-existed in the same country with these human communities which warrants deeper surveillance in these cases to prevent local outbreaks. Our study support the integration of massive data of different natures in the surveillance of viral pandemics.

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BiSulfite Bolt: A BiSulfite Sequencing Analysis Platform BackgroundBisulfite sequencing is commonly employed to measure DNA methylation. Processing bisulfite sequencing data is often challenging due to the computational demands of mapping a low complexity, asymmetrical library and the lack of a unified processing toolset to produce an analysis ready methylation matrix from read alignments. To address these shortcomings, we have developed BiSulfite Bolt (BSBolt); a fast and scalable bisulfite sequencing analysis platform. BSBolt performs a pre-alignment sequencing read assessment step to improve efficiency when handling asymmetrical bisulfite sequencing libraries.

FindingsWe evaluated BSBolt against simulated and real bisulfite sequencing libraries. We found that BSBolt provides accurate and fast bisulfite sequencing alignments and methylation calls. We also compared BSBolt to several existing bisulfite alignment tools and found BSBolt outperforms Bismark, BSSeeker2, BISCUIT, and BWA-Meth based on alignment accuracy and methylation calling accuracy.

ConclusionBSBolt offers streamlined processing of bisulfite sequencing data through an integrated toolset that offers support for simulation, alignment, methylation calling, and data aggregation. BSBolt is implemented as a python package and command line utility for flexibility when building informatics pipelines. BSBolt is available at https://github.com/NuttyLogic/BSBolt under an MIT license.

sentences: Sensory conflict disrupts circadian rhythms in the sea anemone Nematostella vectensis. Circadian clocks allow organisms to optimize their phenotypes in relation to rhythmic environmental conditions. Clocks must synchronize, or entrain, to environmental signals called zeitgebers (such as light and temperature) while maintaining internal rhythms that are robust to environmental noise. We know surprisingly little about how clocks function in the presence of multiple zeitgebers, although these are the conditions that exist in nature. Misalignment between zeitgeber cycles (here, "sensory conflict") can disrupt circadian rhythms in behavior and gene expression, but it is not clear whether this is generally true across animals. In order to understand general rules about animal clocks, and how clocks have evolved throughout Metazoa, it is necessary to study non-bilaterian animals such as cnidarians, which include corals, jellies, and sea anemones. The sea anemone Nematostella vectensis has emerged as the principal model system for cnidarian circadian biology. In this study, we show that temperature cycles entrain circadian locomotor rhythms in Nematostella, and then conduct extensive behavioral experiments across a range of light and temperature cycles to show that Nematostellas circadian behavior is disrupted by sensory conflict. Chronic exposure to conflicting light and temperature cycles also causes substantial changes to the rhythmic transcriptome, including the breakdown of rhythmic expression of various metabolic processes. These results have important implications for our understanding of cnidarian circadian clocks at both the molecular and organismal levels, and for how sensory information is processed by clocks in the absence of a central nervous system.

sentences: Mixed culture metagenomics of the microbes making sour beer Mixed microbial cultures create sour beers, but many brewers do not know which microbes comprise their cultures. The objective of this work was to use deep sequencing to identify microorganisms in sour beers brewed by spontaneous and non-spontaneous methods. Twenty samples were received from brewers, which were processed for microbiome analysis by next generation sequencing. For bacteria, primers were used to amplify the V3-V4 region of the 16S rRNA gene; fungal DNA detection was performed using primers to amplify the entire internal transcribed spacer region. The sequencing results were then used for taxonomy assignment, sample composition, and diversity analyses, as well as nucleotide BLAST searching. We identified 60 genera and 140 species of bacteria, of which the most prevalent were Lactobacillus acetotolerans, Pediococcus damnosus, and Ralstonia picketti/mannitolilytica. In fungal identification, 19 genera and 26 species were found, among which the most common yeasts were Brettanomyces bruxellensis and Saccharomyces cerevisiae. In some cases, genetic material from more than 60 microorganisms was found in a single sample. In conclusion, we were able to determine the microbiomes of various mixed cultures used to produce beer, providing useful information to better understand the sour beer fermentation process and brewing techniques.

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Effects of learning and escitalopram administration on serum BDNF levels, a randomised placebo-controlled trial BackgroundThe brain-derived neurotrophic factor (BDNF) has been implicated in processes essential for neuroplasticity. Learning and serotonin reuptake inhibitors (SSRI) foster neuronal reorganization, a mechanism potentially related to BDNF. This study aims to assess the effects of associative learning and escitalopram on serum BDNF (sBDNF) levels, to gain further knowledge on their dynamic interplay.

MethodsFor three weeks, 37 participants performed one of two associative learning paradigms with either emotional or semantic content daily. During a subsequent three-week period of reversal learning, subjects either received escitalopram (10mg per day) or placebo. Before and after each learning period sBDNF values were assessed. Citalopram plasma levels were measured at the last time point. Linear mixed effects models (LME) and partial Spearmans rank and Pearson correlations were used for statistical analyses.

ResultsOne-way LME resulted in a significant effect of time during the first learning period over both groups (p<0.01). Two-way LME revealed a significant interaction effect of the emotional content learning group and time (p=0.02). Three-way LME (time x reversal learning group x substance) showed no significant effects (all p> 0.05). Furthermore, correlation between citalopram and sBDNF level after three weeks of escitalopram administration exhibit a negative trend (partial Pearson correlation: r=-0.30, p=0.05; partial Spearmans rank: r=-0.22, p=0.15).

ConclusionThe results suggest that three weeks of associative emotional content learning affect sBDNF levels, while subsequently assessed citalopram plasma and sBDNF levels tend to correlate negatively.

Key PointsO_LIEmotional learning may affect serum BDNF levels in healthy human subjects
C_LIO_LIBlood levels of citalopram and serum BDNF exhibit a negative correlation
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Augmented Transcutaneous Stimulation Using an Injectable Electrode Minimally invasive neuromodulation technologies seek to marry the neural selectivity of implantable devices with the low-cost and non-invasive nature of transcutaneous electrical stimulation (TES). The Injectrode(R) is a needle-delivered electrode that is injected onto neural structures under image guidance. Power is then transcutaneously delivered to the Injectrode using surface electrodes. The Injectrode serves as a low-impedance conduit to guide current to the deep on-target nerve, reducing activation thresholds by an order of magnitude compared to using only surface stimulation electrodes. To minimize off-target recruitment of cutaneous fibers, the energy transfer efficiency from the surface electrodes to the Injectrode must be optimized.

TES energy is transferred to the Injectrode through both capacitive and resistive mechanisms. Electrostatic finite element models generally used in TES research consider only the resistive means of energy transfer by defining tissue conductivities. Here, we present an electroquasistatic model, taking into consideration both the conductivity and permittivity of tissue, to understand transcutaneous power delivery to the Injectrode. The model was validated with measurements taken from (n=4) swine cadavers. We used the validated model to investigate system and anatomic parameters that influence the coupling efficiency of the Injectrode energy delivery system. Our work suggests the relevance of electroquasistatic models to account for capacitive charge transfer mechanisms when studying TES, particularly when high-frequency voltage components are present, such as those used for voltage-controlled pulses and sinusoidal nerve blocks.

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Time course of alterations in adult spinal motoneuron properties in the SOD1(G93A) mouse model of ALS Although ALS is an adult-onset neurodegenerative disease, motoneuron electrical properties are already altered during embryonic development. Motoneurons must therefore exhibit a remarkable capacity for homeostatic regulation to maintain a normal motor output for most of the life of the patient. In the present paper, we demonstrate how maintaining homeostasis could come at a very high cost. We studied the excitability of spinal motoneurons from young adult SOD1(G93A) mice to end-stage. Initially homeostasis is highly successful in maintaining their overall excitability. This initial success, however, is achieved by pushing some cells far above the normal range of passive and active conductances. As the disease progresses, both passive and active conductances shrink below normal values in the surviving cells. This shrinkage may thus promote survival, implying the previously large values contribute to degeneration. These results support the hypothesis that motoneuronal homeostasis may be "hyper-vigilant" in ALS and a source of accumulating stress.

Significance StatementDuring ALS, motoneurons exhibit a remarkable ability to maintain a normal motor output despite continuous alterations of their electrophysiological properties, up to the point when overt symptoms become apparent. We show that this homeostatic process can sometimes push motoneurons beyond the normal range, which may be causing long-lasting harm.

sentences: Identification of bacteria strains using the Recombinase Polymerase Amplification assay on a miniaturized solid-state pH sensor Rapid identification of bacteria based on nucleic acid amplification allows dealing with the detection of pathogens in clinical, food, and environmental samples. Amplification product must be detected and analyzed by external devices or integrated complicated optical systems. Here, we developed a solid-state pH electrode based on iridium oxide (IrO2) films to measure released hydrogen ions (H+) from isothermal nucleic acid (NA) amplification of bacterial samples. By recombinase polymerase amplification (RPA), we achieved rapid (< 15 min) and sensitive (<30 copies) detection with an accuracy of about 0.03 pH. The RPA-based hydrogen ion sensing assay shows higher specificity, sensitivity, and efficiency as the same polymerase chain reaction (PCR) methods. We initially used the RPA-based sensor to detect E. coli species in laboratory samples. Among, 27 random laboratory samples of E. coli samples, 6 were found to be DH5alpha, 9 BL21, 3 HB101, 6 TOP10, and 3 JM109. The electrical detection of amplification provides generally applicable techniques for the detection of nucleic acid amplification, enabling molecular diagnostic tests in the field and integrating data transmission to the mobile device. These results can be future developed into an efficient tool for rapid on-site detection of bacterial pathogens in clinical samples.

sentences: The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutL{beta} heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol {delta}) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutL{beta} recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutL{beta} ensemble inhibits Pif1-stimulated D-loop extension by Pol {delta} and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutL{beta} may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1s activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutL{beta} ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.

sentences: Socioeconomics drive population change in the world's largest carnivores Land-use1,2 and climate change3-5 have been linked to wildlife population declines, but the role of socioeconomic factors in driving declines, and promoting population recoveries, remains relatively unexplored despite its likely importance. Here, we evaluate a comprehensive array of potential drivers of population changes observed in some of the worlds most charismatic species - large mammalian carnivores. Our results reveal a strong role of human socioeconomic development, which we find has a greater impact on population change than habitat loss and climate change. Increases in socioeconomic development are linked to sharp population declines but, importantly, once development is high, carnivore populations have the potential to recover. These links between human development and wildlife population health highlight the challenges ahead to achieve the different UN Sustainable development goals.

sentences: LIFELONG CYTOMEGALOVIRUS AND EARLY-LIFE IRRADIATION SYNERGISTICALLY POTENTIATE AGE-RELATED DEFECTS IN RESPONSE TO VACCINATION AND INFECTION A popular "DNA-damage theory" of aging posits that unrepaired DNA damage leads to cellular (and organismal) senescence. Indeed, some hallmarks of immune aging are more prevalent in individuals exposed to Whole-Body Irradiation (WBI). To test this hypothesis in a model relevant to human immune aging, we examined separate and joint effects of lifelong latent Murine Cytomegalovirus (MCMV) and early-life WBI (i) over the course of the lifespan; (ii) in response to a West Nile virus (WNV) live attenuated vaccine; and (iii) following lethal WNV challenge subsequent to vaccination. We recently published that a single dose of non-lethal WBI in youth, on its own, was not sufficient to accelerate aging of the murine immune system despite causing widespread DNA damage and repopulation stress in hematopoietic cells. However, 4Gy sub-lethal WBI caused manifest reactivation of MCMV. Following vaccination and challenge with WNV in the old age, MCMV-infected animals experiencing 4Gy, but not lower, dose of sub-lethal WBI in youth had reduced survival. By contrast, old irradiated mice lacking MCMV and MCMV-infected, but not irradiated, mice were both protected to the same high level as the old non-irradiated, uninfected controls. Analysis of the quality and quantity of anti-WNV immunity showed that higher mortality in MCMV-positive WBI mice correlated with increased levels of MCMV-specific immune activation during WNV challenge. Moreover, we demonstrate that infection, including that by WNV, led to MCMV reactivation. Our data suggest that MCMV reactivation may be an important determinant of increased late-life mortality following early-life irradiation and late-life acute infection.

sentences: TACAN is a novel regulator of PKD2 Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in membrane receptor PKD1 or cation channel PKD2. TACAN (also named TMEM120A), recently reported as an ion channel in neuron cells for mechano and pain sensing, is also distributed in diverse non-neuronal tissues such as kidney, heart and intestine, suggesting its involvement in other functions. In this study, we found that TACAN is in complex with PKD2 in native renal cell lines. Using the two-electrode voltage clamp in Xenopus oocytes we found that TACAN inhibited the channel activity of PKD2 gain-of-function mutant F604P. The first and last transmembrane domains of TACAN were found to interact with the PKD2 C-and N-terminal portions, respectively. We showed that the TACAN N-terminus acted as a blocking peptide and that TACAN inhibits the PKD2 function through the PKD2/TACAN binding. By patch clamping in mammalian cells, we found that TACAN inhibits both the single channel conductance and open probability of PKD2 and mutant F604P. PKD2 co-expressed with TACAN, but not PKD2 alone, exhibited pressure sensitivity. Furthermore, we also found that TACAN aggravates PKD2-dependent tail curvature and pronephric cysts in larval zebrafish, in support of the in vitro inhibitory effects of TACAN. In summary, this study revealed that TACAN acts as a PKD2 inhibitor and mediates mechano sensitivity of the PKD2/TACAN channel complex.

sentences: More comprehensive proprioceptive stimulation of the hand amplifies its cortical processing Corticokinematic coherence (CKC) quantifies the phase coupling between limb kinematics and cortical neurophysiological signals reflecting proprioceptive feedback to the primary sensorimotor (SM1) cortex. We studied CKC to proprioceptive stimulation (i.e. movement-actuator-evoked movements) of right-hand digits (index, middle, ring and little) performed simultaneously or separately. CKC was computed between magnetoencephalography (MEG) and finger acceleration signals. The strongest CKC was obtained by stimulating the fingers simultaneously at fixed 3-Hz frequency, and can, therefore, be recommended as design for fast functional localization of the hand area in the primary sensorimotor (SM1) cortex using MEG. The peaks of CKC sources were concentrated in the hand region of the SM1 cortex, but did not follow consistent somatotopic order. This result suggests that spatial specificity of MEG is not sufficient to separate proprioceptive finger representations of the same hand adequately or that their representations are overlapping.

sentences: Functional evolution of a bark beetle odorant receptor clade detecting monoterpenoids of different ecological origins Insects detect odors using an array of odorant receptors (ORs), which may expand through gene duplication. How specificities evolve and new functions arise in related ORs within a species remain poorly investigated. We addressed this question by functionally characterizing ORs from the Eurasian spruce bark beetle Ips typographus, in which antennal detection and behavioral responses to pheromones, volatiles from host and non-host trees, and fungal symbionts are well described. In contrast, knowledge of OR function is restricted to two receptors detecting the pheromone compounds (S)-(-)-ipsenol (ItypOR46) and (R)-(-)-ipsdienol (ItypOR49). These receptors belong to a species-specific OR-lineage comprising seven ItypORs. To gain insight into the functional evolution of related ORs, we characterized the five remaining ORs in this clade, using Xenopus oocytes. Two receptors responded primarily to the host tree monoterpenes (+)-3-carene (ItypOR25) and p-cymene (ItypOR27). Two receptors responded to oxygenated monoterpenoids produced in relatively large amounts by the beetle-associated fungi, with ItypOR23 specific for (+)-trans-(1R,4S)-4-thujanol, and ItypOR29 responding to (+)-isopinocamphone and similar ketones. ItypOR28 responded to the pheromone E-myrcenol from the competitor Ips duplicatus. Overall, the OR responses match well with those of previously characterized olfactory sensory neuron classes except that neurons detecting E-myrcenol have not been identified. The characterized ORs are under strong purifying selection and demonstrate a shared functional property in that they all primarily respond to monoterpenoids. The variation in functional groups among OR ligands and their diverse ecological origins suggest that neofunctionalization has occurred early in the evolution of this OR-lineage following gene duplication.

sentences: RTT109 AND Fun30 proteins mediate epigenetic regulation of the DNA damage response pathway in C. albicans Fun30, an ATP-dependent chromatin remodeller, from S. cerevisiae mediates both regulation of gene expression as well as DNA damage response/repair. In this paper, we have characterized the biochemical and physiological function of Fun30 from the opportunistic fungi, C. albicans. Biochemically, the protein shows DNA-stimulated ATPase activity. Physiologically, the protein co-regulates transcription of RTT109, TEL1, MEC1, and SNF2-genes that encode for proteins involved in DNA damage response and repair pathway. The expression of FUN30, in turn, is regulated by histone H3 acetylation catalysed by Rtt109 encoded by RTT109. The RTT109Hz/FUN30Hz mutant strain shows sensitivity to oxidative stress and resistance to MMS as compared to the wild type strain. Quantitative PCR showed that the sensitivity to oxidative stress results from downregulation of MEC1, RAD9, MRC1 and RAD5 expression; ChIP experiments showed Fun30 but not H3ac regulates the expression of these genes in response to oxidative stress. In contrast, on treatment with MMS, the expression of RAD9 is upregulated and this upregulation is co-regulated by both Fun30 and H3 acetylation catalysed by Rtt109. Thus, Fun30 and H3 acetylation mediate the response of the fungal cell to genotoxic agents in C. albicans by regulating the expression of DNA damage response and repair pathway genes.

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Guidance for RNA-seq co-expression estimates: the importance of data normalization, batch effects, and correlation measures MotivationGene co-expression analysis is an attractive tool for leveraging enormous amounts of public RNA-seq datasets for the prediction of gene functions and regulatory mechanisms. However, the optimal data processing steps for the accurate prediction of gene co-expression from such large datasets remain unclear. Especially the importance of batch effect correction is understudied.

ResultsWe processed RNA-seq data of 68 human and 76 mouse cell types and tissues using 50 different workflows into 7,200 genome-wide gene co-expression networks. We then conducted a systematic analysis of the factors that result in high-quality co-expression predictions, focusing on normalization, batch effect correction, and measure of correlation. We confirmed the key importance of high sample counts for high-quality predictions. However, choosing a suitable normalization approach and applying batch effect correction can further improve the quality of co-expression estimates, equivalent to a >80% and >40% increase in samples. In larger datasets, batch effect removal was equivalent to a more than doubling of the sample size. Finally, Pearson correlation appears more suitable than Spearman correlation, except for smaller datasets.

ConclusionA key point for accurate prediction of gene co-expression is the collection of many samples. However, paying attention to data normalization, batch effects, and the measure of correlation can significantly improve the quality of co-expression estimates.

sentences: To denoise or to cluster? That is not the question. Optimizing pipelines for COI metabarcoding and metaphylogeography The recent blooming of metabarcoding applications to biodiversity studies comes with some relevant methodological debates. One such issue concerns the treatment of reads by denoising or by clustering methods, which have been wrongly presented as alternatives. It has also been suggested that denoised sequence variants should replace clusters as the basic unit of metabarcoding analyses, missing the fact that sequence clusters are a proxy for species-level entities, the basic unit in biodiversity studies. We argue here that methods developed and tested for ribosomal markers have been uncritically applied to highly variable markers such as cytochrome oxidase I (COI) without conceptual or operational (e.g., parameter setting) adjustment. COI has a naturally high intraspecies variability that should be assessed and reported, as it is a source of highly valuable information. We contend that denoising and clustering are not alternatives. Rather, they are complementary and both should be used together in COI metabarcoding pipelines. Using a typical dataset from benthic marine communities, we compared two denoising procedures (based on the UNOISE3 and the DADA2 algorithms), set suitable parameters for denoising and clustering COI datasets, and compared the outcome of applying these processes in different orders. Our results indicate that denoising based on the UNOISE3 algorithm preserves a higher intra-cluster variability. We suggest and test ways to improve this algorithm taking into account the natural variability of each codon position in coding genes. The order of the steps (denoising and clustering) has little influence on the final outcome. We recommend researchers to consider reporting their results in terms of both denoised sequences (a proxy for haplotypes) and clusters formed (a proxy for species), and to avoid collapsing the sequences of the latter into a single representative. This will allow studies at the cluster (ideally equating species-level diversity) and at the intra-cluster level, and will ease additivity and comparability between studies.

sentences: Classification of Pacemaker Dynamics in the Mouse Intestine by Field Potential Microimaging The flexible and sophisticated movement of the gastrointestinal (GI) tract implies the involvement of mechanisms other than enteric neural circuits, to coordinate excitation in microregions. We thus performed microimaging of pacemaker dynamics in the small intestine of mice since it contains typical network-forming pacemaker cells. A dialysis membrane-reinforced low-impedance microelectrode array (MEA) enabled field potentials over a wide frequency range to be stably measured in microregions. The pacemaker dynamics were classified into basic patterns despite large variations. In the developmental process, pacemaker activity was categorized as either an  expanding or a  migrating pattern that was initiated in or propagated to the MEA sensing area, respectively. The intercellular current of the volume conductor complicated the waveform of both activities. The existence of  expanding and  migrating patterns was attributable to duplicated pacemaker systems such as intracellular Ca2+ oscillation-activated and voltage-gated mechanisms. Additionally, from the spatio-temporal feature during the period of pacemaker events, the  bumpy/aberrant pattern was defined by aberrant, incoherent propagation, and associated with local impairment of excitability, while the  colliding/converging pattern involved the interaction of multiple activities in the MEA area. Interconversion between the four micro-coordination patterns occurred in the same microregion. 5-Hydroxytryptamine (5-HT) promoted  migrating activity, implying an improvement or restoration of spatial conductivity. These results agree well with the action of 5-HT to change GI movement toward propulsion. In conclusion, our MEA method of microimaging classification enables the quantitative assessment of spatio-temporal electric coordination underlying GI motility, suggesting its application to small model animals.

sentences: Differential Conditioning Produces Merged Long-term Memory in Drosophila Multiple spaced trials of aversive differential conditioning can produce two independent long-term memories (LTMs) of opposite valence. One is an aversive memory for avoiding the conditioned stimulus (CS+), and the other is a safety memory for approaching the non-conditioned stimulus (CS-). Here, we show that a single trial of aversive differential conditioning yields one merged LTM (mLTM) for avoiding both CS+ and CS-. Such mLTM can be detected after sequential exposures to the shock-paired CS+ and unpaired CS-, and be retrieved by either CS+ or CS-. The formation of mLTM relies on triggering aversive-reinforcing dopaminergic neurons and subsequent new protein synthesis. Expressing mLTM involves {beta} Kenyon cells and corresponding approach-directing mushroom body output neurons (MBONs), in which similar-amplitude long-term depression of responses to CS+ and CS- seems to signal the mLTM. Our results suggest that animals can develop distinct strategies for occasional and repeated threatening experiences.

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Hair phenotype diversity across Indriidae lemurs ObjectivesHair (i.e., pelage/fur) is a salient feature of primate (including human) diversity and evolution-- serving functions tied to thermoregulation, protection, camouflage, and signaling--but wild primate pelage evolution remains relatively understudied. Specifically, assessing multiple hypotheses across distinct phylogenetic scales is essential but is rarely conducted. We examine whole body hair color and density variation across Indriidae (Avahi, Indri, Propithecus)--a lineage that, like humans, exhibits vertical posture (i.e., their whole bodies are vertical to the sun).

Materials and MethodsOur analyses consider multiple phylogenetic scales (family-level, genus-level) and hypotheses (e.g., Glogers rule, the body cooling hypotheses). We obtain hair color and density from museum and/or wild animals, opsin genotypes from wild animals, and climate data from WorldClim. To analyze our data, we use phylogenetic generalized linear mixed models (PGLMM) using Markov chain Monte Carlo algorithms.

ResultsOur results show that across the Indriidae family, darker hair is typical in wetter regions. However, within Propithecus, dark black hair is common in colder forest regions. Results also show pelage redness increases in populations exhibiting enhanced color vision. Lastly, we find follicle density on the crown and limbs increases in dry and open environments.

DiscussionThis study highlights how different selective pressures across distinct phylogenetic scales have likely acted on primate hair evolution. Specifically, our data across Propithecus may implicate thermoregulation and is the first empirical evidence of Bogerts rule in mammals. Our study also provides rare empirical evidence supporting an early hypothesis on hominin hair evolution.

sentences: Individualized event structure drives individual differences in whole-brain functional connectivity Resting-state functional connectivity is typically modeled as the correlation structure of whole-brain regional activity. It is studied widely, both to gain insight into the brains intrinsic organization but also to develop markers sensitive to changes in an individuals cognitive, clinical, and developmental state. Despite this, the origins and drivers of functional connectivity, especially at the level of densely sampled individuals, remain elusive. Here, we leverage novel methodology to decompose functional connectivity into its precise framewise contributions. Using two dense sampling datasets, we investigate the origins of individualized functional connectivity, focusing specifically on the role of brain network "events" - short-lived and peaked patterns of high-amplitude cofluctuations. Here, we develop a statistical test to identify events in empirical recordings. We show that the patterns of cofluctuation expressed during events are repeated across multiple scans of the same individual and represent idiosyncratic variants of template patterns that are expressed at the group level. Lastly, we propose a simple model of functional connectivity based on event cofluctuations, demonstrating that group-averaged cofluctuations are suboptimal for explaining participant-specific connectivity. Our work complements recent studies implicating brief instants of high-amplitude cofluctuations as the primary drivers of static, whole-brain functional connectivity. Our work also extends those studies, demonstrating that cofluctuations during events are individualized, positing a dynamic basis for functional connectivity.

sentences: An alignment free approach confirms semantic properties of species proteomes Alignment-based methods dominate molecular biology. However, by primarily allowing one-to-one comparisons, these methods are focused on a gene-centered viewpoint and lack the broad context essential to analyze how complex biological systems function and evolve. In actuality, a gene is part of genome where more than one sequence contributes to the functional network and evolutionary trajectory of the cell. The need for conservation of established interactions, is arguably more important to the evolutionary success of species than conservation of individual function. To test whether such contextual information exists, a distributional semantics method - Latent Semantic Analysis (LSA), was applied to thousands of species proteomes. Using natural language processing, Latent Taxonomic Signatures (LTSs) were identified that outperformed existing alignment-based BLAST methods when random protein sequences were being mapped to annotated taxonomy according to GenBank. LTSs are a novel proteome distributed feature, suggesting the existence of evolutionary constraints imposed on individual proteins by their proteome context. Even orphan proteins are exhibiting LTSs, which makes their uniqueness linked to a specific taxonomic level questionable. Unlike more simple bias, LTSs represent a self-similarity pattern, where random sets of species proteins show the same statistical properties of a complete proteome at many scales. Natural language processing and machine learning provide insights not easily discernable using alignment based methods suggestive there is more to species related differences than just translational optimization.

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Optimizing Sequencing Resources in Genotyped Livestock Populations Using Linear Programming BackgroundLow-cost genome-wide single-nucleotide polymorphisms (SNPs) are routinely used in animal breeding programs. Compared to SNP arrays, the use of whole-genome sequence data generated by the next-generation sequencing technologies (NGS) has great potential in livestock populations. However, a large number of animals are required to be sequenced to exploit the full potential of whole-genome sequence data. Thus, novel strategies are desired to allocate sequencing resources in genotyped livestock populations such that the entire population can be sequenced or imputed efficiently.

MethodsWe present two applications of linear programming models called LPChoose for sequencing resources allocation. The first application is to identify the minimum number of animals for sequencing while meeting the criteria that each haplotype in the population is contained in at least one of the animals selected for sequencing. The second is to sequence a fixed number of animals whose haplotypes include as large a proportion as possible of the haplotypes present in the population given a limited sequencing budget.

ResultsIn both applications LPChoose has similar or better performance than some other methods. The linear programming models we proposed are based on rigorous and well defined optimization techniques and easy and straightforward to implement. LPChoose is available as an open-source package.

sentences: Drs2 regulates TRAPPIII in Atg9 transport: exposing the interplay of P4-ATPases and Multisubunit Tethering Complexes Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether transported vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are just partially understood. Here we systematically investigate the interactome that regulates the function of MTCs. We found that P4-ATPases, a family of lipid transporters involved in the biogenesis of vesicles, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We used the lipid flippase Drs2 as a model to investigate the mechanism and biological relevance of such interplay during the transport of Atg9 vesicles. Binding to the N-terminal tail of Drs2 stabilizes TRAPPIII on membrane compartments loaded with Atg9 and it is required for the delivery of Atg9 during selective autophagy, a role that is independent of previously reported functions of the P4-ATPase. This mechanism relies on the I(S/R)TTK motif nested in the N-terminal tail cavity of Drs2, a motif that is required for the interaction with MTCs.

sentences: Robust Inference of Kinase Activity using Functional Networks Mass spectrometry enables high-throughput screening of phospho-proteins across a broad range of biological contexts. When complemented by computational algorithms, phospho-proteomic data allows the inference of kinase activity, facilitating the identification of dysregulated kinases in various diseases including cancer, Alzheimers disease and Parkinsons disease. To enhance the reliability of kinase activity inference, we present a network-based framework, RoKAI, that integrates various sources of functional information to capture coordinated changes in signaling. Through computational experiments, we show that phosphorylation of sites in the functional neighborhood of a kinase are significantly predictive of its activity. The incorporation of this knowledge in RoKAI consistently enhances the accuracy of kinase activity inference methods while making them more robust to missing annotations and quantifications. This enables the identification of understudied kinases and will likely lead to the development of novel kinase inhibitors for targeted therapy of many diseases. RoKAI is available as web-based tool at http://rokai.io.

sentences: Exogenous pigments shield microorganisms from spaceflight-induced changes Research has indicated that pigments commonly produced by microorganisms may be protective against the environmental stresses inherent to spaceflight. However, few studies have directly tested the protective capabilities of microbial pigments applied externally as shielding materials. In this study, liquid cultures of Bacillus subtilis were shielded by various pigment solutions, and solid media cultures of Bacillus subtilis were co-inoculated with the highly pigmented microorganisms Aspergillus niger and Neurospora crassa. These experiments were conducted in a compact, automated payload aboard the International Space Station (ISS) interior for 30 days. Post-flight phenotypic analyses of liquid cultures showed that solutions of carotenoid pigments were effective at minimizing detrimental effects of spaceflight. Elevated growth rate was observed for solid cultures, and distinct morphology changes were identified in both liquid and solid samples and quantified as markers of spaceflight-induced stress. These findings collectively progress our understanding of microbial pigments for the development of space-related applications.

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Competent immune responses to SARS-CoV-2 variants in older adults following mRNA vaccination Aging is associated with a reduced magnitude of primary immune responses to vaccination and constriction of immune receptor repertoire diversity. Clinical trials demonstrated high efficacy of mRNA based SARS-CoV-2 vaccines in older adults but concerns about virus variant escape have not been well addressed. We have conducted an in-depth analysis of humoral and cellular immunity against an early-pandemic viral isolate and compared that to the P.1. (Gamma) and B.1.617.2 (Delta) variants in <50 and >55 age cohorts of mRNA vaccine recipients. We have further measured neutralizing antibody titers for B.1.617.1 (Kappa) and B.1.595; a SARS-CoV-2 isolate bearing Spike mutation E484Q. As reported, robust immunity required the second dose of vaccine. Older vaccinees manifested robust cellular immunity against early-pandemic SARS-CoV-2 and more recent variants, which remained statistically comparable to the adult group. The older cohort had lower neutralizing capacity at the first time point following the second dose, but at later time points immunity was indistinguishable between them. While the duration of these immune responses remains to be determined over longer periods of time, these results provide reasons for optimism regarding vaccine protection of older adults against SARS-CoV-2 variants and inform thinking about boost vaccination with variant vaccines.

eTOC summaryVaccine responses are often diminished with aging, but we found strong responses to SARS-CoV-2 in older adults following mRNA vaccination. T cell responses were not diminished when confronted by SARS-CoV-2 variants. Neutralizing Ab were reduced but not more than those in adults.

Graphical Abstract

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Allosteric Cross-Talk Among SARS-CoV-2 Spike's Receptor-Binding Domain Mutations Triggers an Effective Hijacking of Human Cell Receptor The rapid and relentless emergence of novel highly transmissible SARS-CoV-2 variants, possibly decreasing vaccine efficacy, currently represents a formidable medical and societal challenge. These variants frequently hold mutations on the Spike proteins Receptor-Binding Domain (RBD), which, binding to the Angiotensin-Converting Enzyme 2 (ACE2) receptor, mediates viral entry into the host cells.

Here, all-atom Molecular Dynamics simulations and Dynamical Network Theory of the wild-type and mutant RBD/ACE2 adducts disclose that while the N501Y mutation (UK variant) enhances the Spikes binding affinity towards ACE2, the N501Y, E484K and K417N mutations (South African variant) aptly adapt to increase SARS-CoV-2 propagation via a two-pronged strategy: (i) effectively grasping ACE2 through an allosteric signaling between pivotal RBD structural elements; and (ii) impairing the binding of antibodies elicited by infected/vaccinated patients. This information, unlocking the molecular terms and evolutionary strategies underlying the increased virulence of emerging SARS-CoV-2 variants, set the basis for developing the next-generation anti-COVID-19 therapeutics.

TOC GRAPHICS

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Distinct cellular immune signatures in acute Zika virus infection are associated withhigh or low persisting neutralizing antibody titers Although the formation of a durable neutralizing antibody response after an acute viral infection is a key component of protective immunity, little is known about why some individuals generate high versus low neutralizing antibody titers to infection or vaccination. Infection with Zika virus (ZIKV) during pregnancy can cause devastating fetal outcomes, and efforts to understand natural immunity to this infection are essential for optimizing vaccine design. In this study, we leveraged the high-dimensional single-cell profiling capacity of mass cytometry (CyTOF) to deeply characterize the cellular immune response to acute and convalescent ZIKV infection in a cohort of blood donors in Puerto Rico incidentally found to be viremic during the 2015-2016 epidemic in the Americas. During acute ZIKV infection, we identified widely coordinated responses across innate and adaptive immune cell lineages. High frequencies of multiple activated innate immune subsets, as well as activated follicular helper CD4+ T cells and proliferating CD27-IgD-B cells, during acute infection were associated with high titers of ZIKV neutralizing antibodies at 6 months post-infection. On the other hand, low titers of ZIKV neutralizing antibodies were associated with immune features that suggested a cytotoxic-skewed immune "set-point." Our study offers insight into the cellular coordination of immune responses and identifies candidate cellular biomarkers that may offer predictive value in vaccine efficacy trials for ZIKV and other acute viral infections aimed at inducing high titers of neutralizing antibodies.

One Sentence SummaryMass cytometry reveals acute ZIKV infection cellular immune signatures that predict high or low neutralizing antibody titers 6 months post-infection.

sentences: Sensory plasticity in a socially plastic bee The social Hymenoptera have contributed much to our understanding of the evolution of sensory systems. Attention has focussed chiefly on how sociality and sensory systems have evolved together. In the Hymenoptera, the antennal sensilla are important for optimising the perception of olfactory social information. Social species have denser antennal sensilla than solitary species, which is thought to enhance social cohesion through nest-mate recognition. In the current study, we test whether sensilla numbers vary between regional populations of the socially plastic sweat bee Halictus rubicundus. We found region level differences in both olfactory and hygro/thermoreceptive sensilla numbers. We also found evidence that olfactory sensilla density is developmentally plastic: when we transplanted bees from Scotland to the south-east of England, their offspring (which developed in the south) had more olfactory hairs than the transplanted individuals themselves (which developed in Scotland). The transplanted bees displayed a mix of social (a queen plus workers) and solitary nesting, but neither individual nor nest phenotype was related to sensilla density. We suggest that this general, rather than caste-specific sensory plasticity provides a flexible means to optimise sensory perception according to the most pressing demands of the environment, and may support social plasticity in H. rubicundus.

sentences: The miR-424/503 cluster modulates Wnt/β-catenin signaling in the mammary epithelium by regulating the expression of the LRP6 co-receptor During the female lifetime, the enlargement of the epithelial compartment dictated by the ovarian cycles is supported by a transient increase in the MaSC population. Notably, activation of Wnt/{beta}-catenin signaling is an important trigger for MaSC expansion. Here, we report that the miR-424/503 cluster is a novel modulator of canonical Wnt-signaling in the mammary epithelium that exerts its function by targeting the LRP6 co-receptor. Additionally, we show that the loss of this microRNA cluster is associated with breast cancers possessing high levels of Wnt/{beta}-catenin signaling.

sentences: Performance Scaling for Structural MRI Surface Parcellations: A Machine Learning Analysis in the ABCD Study The use of pre-defined parcellations on surface-based representations of the brain as a method for data reduction is common across neuroimaging studies. In particular, prediction-based studies typically employ parcellation-driven summaries of brain measures as input to predictive algorithms, but the choice of parcellation and its influence on performance is often ignored. Here we employed pre-processed structural magnetic resonance imaging data (sMRI) from the ABCD Study(R) to examine the relationship between 220 parcellations and out-of-sample predictive performance across 45 phenotypic measures in a large sample of 9-10-year-old children (N=9,432). Choice of Machine Learning (ML) pipeline and use of alternative multiple parcellation-based strategies were also assessed. Relative parcellation performance was dependent on the spatial resolution of the parcellation, with larger numbers of parcels (up to [~]4000) outperforming coarser parcellations, according to a power-law scaling of between 1/4 and 1/3. Performance was further influenced by the type of parcellation, ML pipeline, and general strategy, with existing literature-based parcellations, a support vector based pipeline, and ensembling across multiple parcellations, respectively, as the highest performing. These findings highlight the choice of parcellation as an important influence on downstream predictive performance, showing in some cases that switching to a higher resolution parcellation can yield a relatively large boost to performance.

sentences: Quantitative modeling of pentose phosphate pathway response to oxidative stress reveals a cooperative regulatory strategy Living cells use signaling and regulatory mechanisms to adapt to environmental stresses. In the case of oxidative stress due for instance to hydrogen peroxide exposure, the adaptation response relies on co-regulation of enzymes in both glycolysis and pentose phosphate pathways (PPP), so as to support PPP-dependent NADPH and redox homeostasis. To understand the regulatory logic underlying early oxidative stress response, available metabolomics and 13C fluxomics dataset are used to infer a probabilistic ensemble of kinetic models. Model ensemble properties of parameter distributions, transient dynamics, dose-response curves and loss-of-function phenotypes all highlights significant and cooperative effects of allosteric regulations of G6PD, PGI and GAPD in early oxidative response. Indeed, efficient flux rerouting into PPP is shown to require dose-dependent coordination between upregulated G6PD enzyme and increased G6P metabolite, the latter requiring fine-tuned inhibition of upper and lower glycolytic enzymes. This set of allosteric regulation also combines negative and positive feedback loops in a subtle manner prone to generate paradoxical perturbation phenotypes for instance related to 6PGD modulation.

sentences: General and cell-type-specific aspects of the motor neuron maturation transcriptional program Building a nervous system is a protracted process that starts with the specification of individual neuron types and ends with the formation of mature neural circuits. The molecular mechanisms that regulate the temporal progression of maturation in individual cell types remain poorly understood. In this work, we have mapped the gene expression and chromatin accessibility changes in mouse spinal motor neurons throughout their lifetimes. We found that both motor neuron gene expression and putative regulatory elements are dynamic during the first three weeks of postnatal life, when motor circuits are maturing. Genes that are up-regulated during this time contribute to adult motor neuron diversity and function. Almost all of the chromatin regions that gain accessibility during maturation are motor neuron specific, yet a majority of the transcription factor binding motifs enriched in these regions are shared with other mature neurons. Collectively, these findings suggest that a core transcriptional program operates in a context-dependent manner to access cell-type-specific cis-regulatory systems associated with maturation genes. Discovery of general principles governing neuronal maturation might inform methods for transcriptional reprogramming of neuronal age and for improved modelling of age-related neurodegenerative diseases.

sentences: cLD: Rare-variant disequilibrium between genomic regions identifies novel genomic interactions Introductory ParagraphLinkage disequilibrium (LD) is a fundamental concept in genetics; critical for studying genetic associations and molecular evolution. However, LD measurements are only reliable for common genetic variants, leaving low-frequency variants unanalyzed. In this work, we introduce cumulative LD (cLD), a stable statistic that captures the rare-variant LD between genetic regions and opens the door for furthering biological knowledge using rare genetic variants. In application, we find cLD reveals an increased genetic association between genes in 3D chromatin interactions, a phenomenon recently reported negatively by calculating standard LD between common variants. Additionally, we show that cLD is higher between gene pairs reported in interaction databases, identifies unreported protein-protein interactions, and reveals interacting genes distinguishing case/control samples in association studies.

sentences: ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. The binding of the TCR-specific repair factor CSB triggers DNA damage-induced ubiquitylation of RNA polymerase II (RNAPII) at a single lysine (K1268) by the CRL4CSA ubiquitin ligase. However, how the CRL4CSA ligase is specifically directed toward the K1268 site is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to the K1268 site, revealing ELOF1 as a specificity factor that positions CRL4CSA for optimal RNAPII ubiquitylation. Furthermore, drug-genetic interaction screening reveals an unanticipated compensatory TCR pathway in which ELOF1 together with known factors DOT1L and HIRA protect CSB-deficient cells from collisions between transcription and replication machineries. Our study provides a genetic framework of the transcription stress response and reveals key insights into the molecular mechanism of TCR.

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Direct regulation of cell cycle regulatory gene expression by NtrX to promote Sinorhizobium meliloti cell division Cell division of the alfalfa symbiont, Sinorhizobium meliloti, is regulated by the CtrA signaling network. The gene expression of regulatory proteins in the network is affected by nutrient signaling. In this study, we found that NtrX, one of the regulators of nitrogen metabolic response, can directly regulate the expression of several regulatory genes from the CtrA signaling network. Three sets of S. meliloti ntrX mutants, including the plasmid insertion strain, the depletion strain and the substitution of the 53rd aspartate (ntrXD53E) from a plasmid in the wild-type strain (Sm1021), showed similar cell division defects, such as slow growth, abnormal morphology of partial cells and delayed DNA synthesis. Transcript quantitative evaluation indicated that the transcription of genes such as ctrA and gcrA was up-regulated, while the transcription of genes such as dnaA and ftsZl was down-regulated in the insertion mutant and the strain of Sm1021 expressing ntrXD53E. Correspondingly, inducible transcription of ntrX activates the expression of dnaA and ftsZ1, but represses ctrA and gcrA in the depletion strain. The expression levels of CtrA and GcrA were confirmed by western blotting, which were consistent with the transcription data. The transcriptional regulation of these genes requires phosphorylation of the conserved 53rd aspartate in the NtrX protein. The NtrX protein binds directly to the promoter regions of ctrA, gcrA, dnaA and ftsZ1 by recognizing the characteristic sequence CAAN2-5TTG Our findings reveal that NtrX is a novel transcriptional regulator of the CtrA signaling pathway genes, and positively affects bacterial cell division, associated with nitrogen metabolism.

IMPORTANCESinorhizobium meliloti infects the host alfalfa and induces formation of nitrogen-fixing nodules. Proliferation of rhizobia in plant tissues and cells is strictly controlled in the early stage of symbiotic interactions. However, the control mechanism is not very clear. Cell division of S. meliloti in the free-living state is regulated by the CtrA signaling network, but molecular mechanisms by which the CtrA system is associated with environmental nutrient signals (e.g., ammonia nitrogen) need to be further explored. This study demonstrates that NtrX, a regulator of nitrogen metabolism, required for symbiotic nodulation and nitrogen fixation by S. meliloti 1021, can act as a transcriptional regulator of the CtrA signaling system. It may link nitrogen signaling to cell cycle regulation in Rhizobium species.

sentences: UPF3A and UPF3B are redundant and modular activators of nonsense-mediated mRNA decay in human cells The paralogous human proteins UPF3A and UPF3B are involved in recognizing mRNAs targeted by nonsense-mediated mRNA decay (NMD). While UPF3B has been demonstrated to support NMD, contradicting reports describe UPF3A either as an NMD activator or inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using combinatory experimental approaches. Overexpression or knockout of UPF3A as well as knockout of UPF3B did not detectably change global NMD activity. In contrast, the co-depletion of UPF3A and UPF3B resulted in a marked NMD inhibition and a transcriptome-wide upregulation of NMD substrates, demonstrating a functional redundancy between both NMD factors. Although current models assume that UPF3 bridges NMD-activating exon-junction complexes (EJC) to the NMD factor UPF2, UPF3B exhibited only slightly impaired NMD activity in rescue experiments when UPF2 or EJC binding was impaired. Further rescue experiments revealed partially redundant functions of UPF3B domains in supporting NMD, involving both UPF2 and EJC interaction sites and the central region of UPF3. Collectively, UPF3A and UPF3B serve as fault-tolerant NMD activators in human cells.

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Bacterial, phytoplankton, and viral dynamics of meromictic Lake Cadagno offer insights into the Proterozoic ocean microbial loop Lake Cadagno, a permanently stratified high-alpine lake with a persistent microbial bloom in its anoxic chemocline, has long been considered a model for the low-oxygen, high-sulfide Proterozoic ocean where early microbial life gave rise to Earths oxygenated atmosphere. Although the lake has been studied for over 25 years, the absence of concerted study of the bacteria, phytoplankton, and viruses, together with primary and secondary production, has hindered a comprehensive understanding of its microbial food web. Here, the identities, abundances, and productivity of microbes were evaluated in the context of Lake Cadagno biogeochemistry. Photo-synthetic pigments and chloroplast 16S rRNA gene phylogenies suggested high abundances of eukaryotic phytoplankton, primarily Chlorophyta, through the water column. Of these, a close relative of Ankyra judayi, a high-alpine adapted chlorophyte, peaked with oxygen in the mixolimnion, while Closteriopsis-related chlorophytes peaked in the chemocline and monimolimnion. Anoxygenic phototrophic sulfur bacteria, Chromatium, dominated the chemocline along with Lentimicrobium, a newly observed genus of known fermenters. Secondary production peaked in the chemocline, suggesting anoxygenic primary producers depended on heterotrophic nutrient remineralization. Virus-to-microbe ratios spanned an order of magnitude, peaking with high phytoplankton abundances and at a minimum at the peak of Chromatium, dynamic trends that suggest viruses may play a role in the modulation of oxygenic and anoxygenic photo- and chemosynthesis in Lake Cadagno. Through the combined analysis of bacterial, eukaryotic, viral, and biogeochemical dynamics of Lake Cadagno, this study provides a new perspective on the biological and geochemical connections that comprised the food webs of the Proterozoic ocean.

IMPORTANCEAs a window to the past, the study offers insights into the role of microbial guilds of Proterozoic ocean chemoclines in the production and recycling of organic matter of sulfur- and ammonia-containing ancient oceans. The new observations described here suggest that eukaryotic algae were persistent in the low oxygen upper-chemocline in association with purple and green sulfur bacteria in the lower half of the chemocline. Further, this study provides the first insights into Lake Cadagno viral ecology. High viral abundances suggested viruses may be essential components of the chemocline where their activity may result in the release and recycling of organic matter. The framework developed in this study through the integration of diverse geochemical and biological data types lays the foundation for future studies to quantitatively resolve the processes performed by discrete populations comprising the microbial loop in this early anoxic ocean analogue.

sentences: Adipose gene expression profiles reveal novel insights into the adaptation of northern Eurasian semi-domestic reindeer (Rangifer tarandus) Reindeer (Rangifer tarandus) are semi-domesticated animals adapted to the challenging arctic conditions of northern Eurasia. Adipose tissues play a crucial role in animals living in northern environments by altering gene expression in their tissues to regulate energy homeostasis and thermogenic activity. Here, we performed transcriptome profiling by RNA sequencing of adipose tissues from three different anatomical depots: metacarpal (bone marrow), perirenal, and prescapular fat in Finnish and Even reindeer (in Sakha) during two seasonal time points (spring and winter). On average 36.5 million pair-ended clean reads were obtained for each sample, and a total of 16,362 genes were expressed in our data. Gene expression profiles in metacarpal tissue were distinct and clustered separately from perirenal and prescapular adipose tissues. Notably, metacarpal adipose tissue appeared to have a significant role in the regulation of the energy metabolism of reindeer in spring when their nutritional condition is poor after winter. During spring, when the animals are in less optimal condition, genes associated with the immune system (e.g., CCL2, CCL11, CXCL14, IGSF3, IGHM, IGLC7, IGKC, JCHAIN, and IGSF10) were upregulated in the perirenal and prescapular adipose tissue, while genes involved in energy metabolism (e.g., ACOT2, APOA1, ANGPTL1, ANGPTL8, ELOVL7, MSMO1, PFKFB1, and ST3GAL6) were upregulated in metacarpal tissue. Even reindeer harboured relatively fewer significantly differentially expressed genes than Finnish reindeer, irrespective of the season, possibly owing to climatic and management differences. Moreover, blood and tissue parameters reflecting general physiological and metabolic status showed less seasonal variation in Even reindeer than in Finnish reindeer. This study identified adipose candidate genes potentially involved in immune response, fat deposition, energy metabolism, development, cell growth, and organogenesis. Taken together, this study provides new information on the mechanisms by which reindeer adapt to less optimal arctic conditions.

sentences: The global exposure of species ranges and protected areas to forest management The majority of vertebrate species globally are dependent on forests, most of which require active protection to safeguard global biodiversity. Forests, however, are increasingly either being disturbed, planted or managed in the form of timber or food plantations. Because of a lack of spatial data, forest management has commonly been ignored in previous conservation assessments. Here we show - using a new global map of forest management - that disturbed and human managed forests cover the distributional ranges of most forest-associated species. Even more worrying, protected areas are increasingly being established in areas dominated by disturbed forests. Our results imply that species extinction risk and habitat assessments might have been overly optimistic with forest management practices being ignored. With forest restoration being in the centre of climate and conservation policies in this decade, we caution that policy makers should explicitly consider forest management.

sentences: Biotechnological exploitation of Saccharomyces jurei and its hybrids in craft beer fermentation uncovers new aroma combinations. Hybridization is an important evolutionary mechanism to bring about novel phenotypes and may produce new hybrids with advantageous combinations of traits of industrial importance. Within the Saccharomyces group, Saccharomyces jurei is a newly discovered species and its biotechnological potential has not yet been fully explored. This yeast was found to be able to grow well in synthetic wort and at low temperatures, qualities necessary in good candidates for fermentated bevarages. Here, we analysed its fermentation and aroma profile and created novel non-GMO hybrids between S. jurei and S. cerevisiae ale yeasts to develop new starter strains with interesting flavours for the craft beer industry. Pilot beer fermentations with specific hybrids showed a good fermentation performance, similar to the ale parent strain, while presenting a better sugar attenuation and a more complex flavour profile. This study exploits the genetic diversity of yeasts and show how inter-specific hybridisation and clone selection can be effectively used in brewing to create new products and to eliminate or increase specific traits.

sentences: Antisense non-coding transcription represses the PHO5 model gene via remodelling of promoter chromatin structure Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. By enhancing or impairing the level of overlapping antisense transcription through specific mutant backgrounds and the use of the CRISPRi system, we demonstrated a negative role for antisense transcription at the PHO5 gene locus. Furthermore, enhanced elongation of PHO5 antisense leads to a more repressive chromatin configuration at the PHO5 gene promoter, which is remodeled more slowly upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated by inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, we extend the model of PHO5 gene regulation by demonstrating a gene silencing function of antisense transcription through a chromatin-based mechanism.

sentences: Multiplexed triage of candidate biomarkers in plasma using internal standard triggered-parallel reaction monitoring mass spectrometry Despite advances in proteomic technologies, clinical translation of plasma biomarkers remains low, partly due to a major bottleneck between the discovery of candidate biomarkers and downstream costly clinical validation studies. Due to a dearth of multiplexable assays, generally only a few candidate biomarkers are tested, and the validation success rate is accordingly low. Here, we demonstrate the capability of internal standard triggered-parallel reaction monitoring (IS-PRM) to prioritize candidate biomarkers for validation studies. A 5,176-plex assay coupling immunodepletion and fractionation with IS-PRM was developed and implemented in human plasma to quantify peptides representing 1,314 breast cancer biomarker candidates. Compared to prior approaches using data-dependent analysis, IS-PRM showed improved sensitivity (912 vs 295 proteins quantified) and precision (CV 0.1 vs 0.27) enabling rank-ordering of candidate biomarkers for validation studies. The assay greatly expands capabilities for quantification of large numbers of proteins and is well suited for prioritization of viable candidate biomarkers.

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A Syx-RhoA-Dia1 signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in glioblastoma Glioblastomas (GBM) are aggressive tumors that lack effective treatments. Here, we show that the Rho family guanine nucleotide exchange factor Syx promotes GBM cell growth both in vitro and in orthotopic GBM patient-derived xenografts. Growth defects upon Syx depletion are attributed to prolonged mitosis, increased DNA damage, G2/M cell cycle arrest, and cell apoptosis, mediated by altered mRNA and protein expression of various cell cycle regulators. These effects are phenocopied by depletion of the Rho downstream effector Dia1 and are due at least in part to increased cytoplasmic retention and reduced activity of the YAP/TAZ transcriptional coactivators. Further, targeting Syx signaling cooperates with radiation treatment and temozolomide (TMZ) to decrease viability in GBM cells irrespective of their inherent response to TMZ. Taken together, the data indicate that a Syx-RhoA-Dia1-YAP/TAZ signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in GBM and argue for its targeting for cancer treatment.

One Sentence SummarySyx promotes growth and therapy resistance in glioblastoma.

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An NMR fingerprint matching approach for the identification and structural re-evaluation of Pseudomonas lipopeptides. Cyclic lipopeptides (CLiPs) are secondary metabolites secreted by a range of bacterial phyla. CLiPs from Pseudomonas in particular display diverse structural variations in terms of the number of amino acid residues, macrocycle size, amino acid identity and stereochemistry (e.g. D- vs. L-amino acids). Reports detailing the discovery of novel or already characterized CLiPs from new sources appear regularly in literature. Increasingly however, the lack of detailed characterization threatens to cause considerable confusion, especially if configurational heterogeneity is present for one or more amino acids. Using Pseudomonas CLiPs from the Bananamide, Orfamide and Xantholysin groups as test cases, we demonstrate and validate that 1H and 13C NMR chemical shifts of CLiPs are sufficiently sensitive to differentiate between possible diastereomers of a particular sequence even when they only differ in a single D/L configuration. Rapid screening, involving simple comparison of the NMR fingerprint of a newly isolated CLiP with that of a reference CLiP of known stereochemistry, can then be applied to resolve dead-ends in configurational characterization and avoid the much more cumbersome chemical characterization protocols. Even when the stereochemistry of a particular reference CLiP remains to be established, NMR fingerprinting still allows verifying whether a CLiP from a novel source is already present in the reference collection, thus contributing to dereplication. To benefit research involving CLiPs, we have made a publicly available  knowledge base at https://www.rhizoclip.be, where we present an overview of published NMR fingerprint data of characterized CLiPs, together with literature data on the originally determined structures.

Significance StatementPseudomonas CLiPs, are ubiquitous specialized metabolites, impacting the producers lifestyle and interactions with the (a)biotic environment. Consequently, they generate interest for agricultural and clinical applications. Establishing structure-activity relationships as premise to their development is hindered because full structural characterization including stereochemistry requires labor-intensive analyses, without guarantee for success. Moreover, increasing use of superficial comparison with previously characterized CLiPs introduces or propagates erroneous attributions, clouding further scientific progress. We provide a generally applicable characterization methodology for structural comparison of newly isolated CLiPs to reference compounds with (un)known stereochemistry based on NMR fingerprints. The reference compound database available for the wide scientific community promises to facilitate structural assessment and dereplication of newly isolated CLiPs, and to support genome mining for novel CLiPs.

sentences: Measuring competitive exclusion in non-small cell lung cancer Therapeutic strategies for tumor control have traditionally assumed that maximizing reduction in tumor volume correlates with clinical efficacy. Unfortunately, this rapid decrease in tumor burden is almost invariably followed by the emergence of therapeutic resistance. Evolutionary based treatment strategies attempt to delay resistance via judicious treatments that maintain a significant treatable subpopulation. While these strategies have shown promise in recent clinical trials, they often rely on biological conjecture and intuition to derive parameters. In this study we experimentally measure the frequency-dependent interactions between a gefitinib resistant non-small cell lung cancer (NSCLC) population and its sensitive ancestor via the evolutionary game assay. We show that cost of resistance is insufficient to accurately predict competitive exclusion and that frequency-dependent growth rate measurements are required. In addition, we show that frequency-dependent growth rate changes may ultimately result in a safe harbor for resistant populations to safely accumulate, even those with significant cost of resistance. Using frequency-dependent growth rate data we then show that gefitinib treatment results in competitive exclusion of the ancestor, while absence of treatment results in a likely, but not guaranteed exclusion of the resistant strain. Finally, using our empirically derived growth rates to constrain simulations, we demonstrate that incorporating ecological growth effects can dramatically change the predicted time to sensitive strain extinction. In addition, we show that higher drug concentrations may not lead to the optimal reduction in tumor burden. Taken together, these results highlight the potential importance of frequency-dependent growth rate data for understanding competing populations, both in the laboratory and the clinic.

sentences: Antibacterial T6SS effectors with a VRR-Nuc domain induce target cell death via DNA Double-Strand Breaks The T6SS (Type VI secretion System) secretes antibacterial effectors into target competitors. Salmonella spp. encode five phylogenetically distinct T6SSs. Here we characterize the function of the SPI-22 T6SS of S. bongori, showing that it has antibacterial activity. We identify a group of antibacterial T6SS effectors (TseV1-4) containing an N-terminal PAAR-like domain and a C-terminal VRR-Nuc domain encoded next to cognate immunity proteins that contain the DUF3396 domain (TsiV1-4). TseV2 and TseV3 are toxic when expressed in Escherichia coli and bacterial competition assays confirm that TseV2 and TseV3 are secreted by the SPI-22 T6SS. Phylogenetic analysis reveals that TseV1-4 are evolutionarily related to enzymes involved in DNA repair. TseV2 and TseV3 maintained the ability to bind DNA, but instead cause specific DNA double-strand breaks and induce the SOS response in target cells. The crystal structure of the TseV3:TsiV3 complex reveals that the immunity protein likely blocks the effector interaction with the DNA substrate. These results expand our knowledge on the function of Salmonella pathogenicity islands, the evolution of toxins used in biological conflicts, and the endogenous mechanism regulating the activity of these toxins.

sentences: Isoform-specific roles of the Drosophila filamin-type protein Jitterbug (Jbug) during development Filamins are highly conserved actin-crosslinking proteins that regulate organization of the actin cytoskeleton. As key components of versatile signaling scaffolds, filamins are implicated in developmental anomalies and cancer. Multiple isoforms of filamins exist, raising the possibility of distinct functions for each isoform during development and in disease. Here, we provide an initial characterization of jitterbug (jbug), which encodes one of the two filamin-type proteins in Drosophila. We generate Jbug antiserum that recognizes all of the spliced forms and reveals differential expression of different Jbug isoforms during development, and a significant maternal contribution of Jbug protein. To reveal the function of Jbug isoforms, we create new genetic tools, including a null allele that deletes all isoforms, hypomorphic alleles that affect only a subset, and UAS lines for Gal4-driven expression of the major isoforms. Using these tools, we demonstrate that Jbug is required for viability and that specific isoforms are required in the formation of actin-rich protrusions including thoracic bristles in adults and ventral denticles in the embryo. We also show that specific isoforms of Jbug show differential localization within epithelia and that maternal and zygotic loss of jbug disrupts Crumbs (Crb) localization in several epithelial cell types.

sentences: Acentrosomal spindles assemble from branching microtubule nucleation near chromosomes Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstituted microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and found that chromosomes alone can form spindles. We visualized microtubule nucleation at chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. The initial branches nucleate near and towards kinetochores, helping explain how kinetochores might be efficiently captured. By depleting molecular motors, we find that the organization of the resultant polar branched networks is consistent with a theoretical model where the effectors for branching nucleation are released by chromosomes, forming a concentration gradient around them that spatially biases branching nucleation. In the presence of motors, these branched networks are organized into multipolar spindles.

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SALTS: SURFR (small noncoding RNA) And LAGOOn (long noncoding RNA) Transcriptomics Suite The widespread utilization of high-throughput sequencing technologies has unequivocally demonstrated that eukaryotic transcriptomes consist primarily (>98%) of non-coding RNA (ncRNA) transcripts significantly more diverse than their protein-coding counterparts.

ncRNAs are typically divided into two categories based on their length. (1) ncRNAs less than 200 nucleotides (nt) long are referred as small non-coding RNAs (sncRNAs) and include microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), transfer ribonucleic RNAs (tRNAs), etc., and the majority of these are thought to function primarily in controlling gene expression. That said, the full repertoire of sncRNAs remains fairly poorly defined as evidenced by two entirely new classes of sncRNAs only recently being reported, i.e., snoRNA-derived RNAs (sdRNAs) and tRNA-derived fragments (tRFs). (2) ncRNAs longer than 200 nt long are known as long ncRNAs (lncRNAs). lncRNAs represent the 2nd largest transcriptional output of the cell (behind only ribosomal RNAs), and although functional roles for several lncRNAs have been reported, most lncRNAs remain largely uncharacterized due to a lack of predictive tools aimed at guiding functional characterizations.

Importantly, whereas the cost of high-throughput transcriptome sequencing is now feasible for most active research programs, tools necessary for the interpretation of these sequencings typically require significant computational expertise and resources markedly hindering widespread utilization of these datasets. In light of this, we have developed a powerful new ncRNA transcriptomics suite, SALTS, which is highly accurate, markedly efficient, and extremely user-friendly. SALTS stands for SURFR (sncRNA) And LAGOOn (lncRNA) Transcriptomics Suite and offers platforms for comprehensive sncRNA and lncRNA profiling and discovery, ncRNA functional prediction, and the identification of significant differential expressions among datasets. Notably, SALTS is accessed through an intuitive Web-based interface, can be used to analyze either user-generated, standard next-generation sequencing (NGS) output file uploads (e.g., FASTQ) or existing NCBI Sequence Read Archive (SRA) data, and requires absolutely no dataset pre-processing or knowledge of library adapters/oligonucleotides.

SALTS constitutes the first publically available, Web-based, comprehensive ncRNA transcriptomic NGS analysis platform designed specifically for users with no computational background, providing a much needed, powerful new resource capable of enabling more widespread ncRNA transcriptomic analyses. The SALTS WebServer is freely available online at http://salts.soc.southalabama.edu.

sentences: Using the glial mitochondrial machinery to prevent multilevel dysfunctions observed in a mouse model of Alzheimer s disease Alzheimers disease (AD) is becoming increasingly prevalent worldwide. It represents one of the greatest medical challenge as no pharmacologic treatments are available to prevent disease progression. Astrocytes play crucial functions within neuronal circuits by providing metabolic and functional support, regulating interstitial solute composition, and modulating synaptic transmission. In addition to these physiological functions, growing evidence points to an essential role of astrocytes in neurodegenerative diseases like AD. Early stage AD is associated with hypometabolism and oxidative stress. Contrary to neurons that are vulnerable to oxydative stress, astrocytes are particularly resistant to mitochondrial dysfunction and are therefore more resilient cells. In our study, we leveraged astrocytic mitochondrial uncoupling and examined neuronal function in the 3xTg AD mouse model. We overexpressed the mitochondrial uncoupling protein 4 (UCP4), which has been shown to improve neuronal survival in vitro. We found that this treatment efficiently prevented alterations of hippocampal metabolite levels observed in AD mice, along with hippocampal atrophy and reduction of basal dendrite arborization of subicular neurons. This approach also averted aberrant neuronal excitability observed in AD subicular neurons and preserved episodic-like memory in AD mice assessed in a spatial recognition task. These findings show that targeting astrocytes and their mitochondria is an effective strategy to prevent the decline of neurons facing AD-related stress at the early stages of the disease.

sentences: Teosinte introgression modulates phosphatidylcholine levels and induces early maize flowering time Native Americans domesticated maize (Zea mays ssp. mays) from lowland teosinte parviglumis (Zea mays ssp.parviglumis) in the warm Mexican southwest and brought it to the highlands of Mexico and South America where it was exposed to lower temperatures that imposed strong selection on flowering time. Phospholipids are important metabolites in plant responses to low-temperature and phosphorus availability, and have been suggested to influence flowering time. Here, we combined linkage mapping with genome scans to identify High PhosphatidylCholine 1 (HPC1), a gene that encodes a phospholipase A1 enzyme, as a major driver of phospholipid variation in highland maize. Common garden experiments demonstrated strong genotype-by-environment interactions associated with variation at HPC1, with the highland HPC1 allele leading to higher fitness in highlands, possibly by hastening flowering. The highland maize HPC1 variant resulted in impaired function of the encoded protein due to a polymorphism in a highly conserved sequence. A meta-analysis across HPC1 orthologs indicated a strong association between the identity of the amino acid at this position and optimal growth in prokaryotes. Mutagenesis of HPC1 via genome editing validated its role in regulating phospholipid metabolism. Finally, we showed that the highland HPC1 allele entered cultivated maize by introgression from the wild highland teosinte Zea mays ssp. mexicana and has been maintained in maize breeding lines from the Northern US, Canada and Europe. Thus, HPC1 introgressed from teosinte mexicana underlies a large metabolic QTL that modulates phosphatidylcholine levels and has an adaptive effect at least in part via induction of early flowering time.

sentences: River phosphorus cycling constrains lake cyanobacteria blooms Bioavailable phosphorus exports from rivers during high flow often fuel downstream harmful cyanobacterial blooms; yet whether river phosphorus cycles affect these exports is unclear. Here, we examined river phosphorus cycling during high flow events in a large agricultural watershed that drives cyanobacterial blooms in Lake Erie. We show that between 2003 and 2019 river phosphorus cycles, through phosphorus sorption, reduced bioavailable phosphorus exports by 24%, potentially constraining Lake Erie cyanobacterial blooms by 61%. Over the last 45-years, phosphorus sorption has declined with suspended sediment exports due to increases in soil-erosion-minimizing agricultural practices, likely contributing to recent cyanobacterial blooms. In this, and likely other agricultural watersheds, rivers perform an unrecognized ecosystem service during high flow creating field-river-lake linkages that need to be incorporated into phosphorus management.

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Attention Networks and the Intrinsic Network Structure of the Human Brain Attention network theory states that attention is not a unified construct but consists of three independent systems that are supported by separable distributed networks: an alerting network to deploy attentional resources in anticipation of upcoming events, an orienting network to direct attention to a cued location, and a control network to select relevant information at the expense of concurrently available information. Ample behavioral and neuroimaging evidence supports the dissociation of the three attention domains. The strong assumption that each attentional system is realized through a separable network, however, raises the question how these networks relate to the intrinsic network structure of the brain.

Our understanding of brain networks has advanced majorly in the past years due to the increasing focus on brain connectivity. It is well established that the brain is intrinsically organized into several large-scale networks whose modular structure persists across task states. Existing proposals on how the presumed attention networks relate to intrinsic networks rely mostly on anecdotal and partly contradictory arguments. We addressed this issue by mapping different attention networks with highest spatial precision at the level of cifti-grayordinates. Resulting group maps were compared to the group-level topology of 23 intrinsic networks which we reconstructed from the same participants resting state fMRI data. We found that all attention domains recruited multiple and partly overlapping intrinsic networks and converged in the dorsal fronto-parietal and midcingulo-insular network. While we observed a preference of each attentional domain for its own set of intrinsic networks, implicated networks did not match well to those proposed in the literature. Our results indicate a necessary refinement of the attention network theory.

sentences: Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination The Nsp1 protein of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates translation termination in the stop codon recognition stage at all three stop codons. This stimulation targets the release factor 1 (eRF1) and does not affect the release factor 3 (eRF3). The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and removal them from the pool of the active ribosomes.

sentences: The individuality of shape asymmetries of the human cerebral cortex Asymmetries of the cerebral cortex are found across diverse phyla and are particularly pronounced in humans, with important implications for brain function and disease. Here, we introduce a novel approach to characterize asymmetries of the whole cortical shape across different spatial frequencies using magnetic resonance imaging data in three independent datasets. We find that cortical shape asymmetry is highly individualized and robust, akin to a cortical fingerprint, and identifies individuals more accurately than size-based descriptors, such as cortical thickness and surface area, or measures of inter-regional functional coupling of brain activity. Individual identifiability is optimal at coarse spatial scales ([~]37 mm wavelength), and shape asymmetries show scale-specific associations with sex and cognition, but not handedness. While unihemispheric cortical shape shows significant heritability at coarse scales ([~]65 mm wavelength), shape asymmetries are determined primarily by subject-specific environmental effects. Thus, coarse-scale shape asymmetries are highly personalized, sexually dimorphic, linked to individual differences in cognition, and are primarily driven by stochastic environmental influences.

sentences: Characterizing Hydrogen Bonds in Intact RNA from MS2 Bacteriophage Using Solid State Magic Angle Spinning NMR Ribonucleic acid (RNA) is a polymer with pivotal functions in many biological processes. RNA structure determination is thus a vital step towards understanding its function. The secondary structure of RNA is stabilized by hydrogen bonds formed between nucleotide base pairs and it defines the positions and shapes of functional stem-loops, internal loops, bulges, and other functional and structural elements. In this work we present a methodology for studying large intact RNA biomolecules using homonuclear 15N solid state nuclear magnetic resonance (NMR) spectroscopy. We show that Proton Driven Spin Diffusion (PDSD) experiments with long mixing times, up to 16s, improved by the incorporation of multiple rotor-synchronous 1H inversion pulses (termed Radiofrequency Dipolar Recoupling, RFDR, pulses), reveal key hydrogen-bond contacts. In the full-length RNA isolated from MS2 phage, we observed strong and dominant contributions of G-C Watson-Crick base pairs, and beyond these common interactions, we observe a significant contribution of the G-U wobble base pairs. Moreover, we can differentiate base-paired and non-base-paired nitrogen atoms. Using the improved technique facilitates characterization of hydrogen-bond types in intact large-scale RNA using solid-state NMR. It can be highly useful to guide secondary structure prediction techniques, and possibly structure determination methods.

sentences: A versatile CRISPR toolbox to study neurological CaV3.2 channelopathies by promoter-mediated transcription control Precise genome editing in combination with viral delivery systems provides a valuable tool for neuroscience research. Traditionally, the role of genes in neuronal circuits has been addressed by overexpression or knock-out/knock-down systems. However, those techniques do not manipulate the endogenous loci and therefore have limitations. Those constraints include that many genes exhibit extensive alternative splicing, which can be regulated by neuronal activity. This complexity cannot be easily reproduced by overexpression of one protein variant. The CRISPR activation and interference/inhibition systems (CRISPRa/i) directed to promoter sequences can modulate the expression of selected target genes in a highly specific manner. This strategy could be particularly useful for the overexpression of large proteins and for alternatively spliced genes, e.g. for studying large ion channels known to be affected in ion channelopathies in a variety of neurological diseases. Here, we demonstrate the feasibility of a newly developed CRISPRa/i toolbox to manipulate the promoter activity of the Cacna1h gene. Impaired, function of the low-voltage-activated T-Type calcium channel CaV3.2 is involved in genetic/mutational as well as acquired/transcriptional channelopathies that emerge with epileptic seizures. We show CRISPR-induced activation and inhibition of the Cacna1h locus in NS20Y cells and primary cortical neurons, as well as activation in mouse organotypic slice cultures. In future applications, the system offers the intriguing perspective to study functional effects of gain-of-function or loss-of-function variations in the Cacna1h gene in more detail. A better understanding of CaV3.2 channelopathies might result in a major advancement in the pharmacotherapy of CaV3.2 channelopathy diseases.

sentences: Hippocampal replay of experience at real-world speeds Representations related to past experiences play a critical role in memory and decision-making processes. The rat hippocampus expresses these types of representations during sharp-wave ripple (SWR) events, and previous work identified a minority of SWRs that contain "replay" of spatial trajectories at ~20x the movement speed of the animal. Efforts to understand replay typically make multiple assumptions about which events to examine and what sorts of representations constitute replay. We therefore lack a clear understanding of both the prevalence and the range of representational dynamics associated with replay. Here we develop a state space model that uses a combination of movement dynamics of different speeds to capture the spatial content and time evolution of replay during SWRs. Using this model, we find that the large majority of replay events contain spatially coherent, interpretable content. Furthermore, many events progress at real-world, rather than accelerated, movement speeds, consistent with actual experiences.

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Stem cell-free therapy for glaucoma to preserve vision Glaucoma is the leading cause of irreversible blindness with trabecular meshwork (TM) dysfunction resulting in elevated intraocular pressure and retinal ganglion cell (RGC) damage leading to vision loss. In this study, we discovered that secretome, derived from human TM stem cells, via minimal invasive periocular injection, can reduce intraocular pressure, restore TM homeostasis, protect RGC, and restore RGC function in both steroid-induced and genetic myocilin mutant mouse models of glaucoma. The secretome upregulated the COX2-PGE2 axis via mitochondrial TMEM177 and led to activation of endogenous stem cells and TM regeneration. Inhibition of COX2 abolished the protective effect of secretome on TM cells. Secretome treatment also enhanced RGC survival and function. Proteomic analysis revealed that the secretome is enriched with proteins involved in extracellular matrix modulation leading to the remodeling of TM to restore homeostasis. This study highlights the feasibility of stem cell-free therapy for glaucoma with minimal invasive administration and the involvement of multiple novel pathways for a cumulative regenerative effect on the TM to protect RGC.

Brief summaryThis study describes a cell-free treatment using stem cell secretome in two animal models of glaucoma and explores the potential mechanisms

sentences: Compartment-Specific Neurexin Nanodomains Orchestrate Tripartite Synapse Assembly At tripartite synapses, astrocytes enmesh synaptic contacts, but how astrocytes contribute to the formation, maturation and plasticity of synapses remains elusive. Here we show that both astrocytes and neurons abundantly express neurexin-1, a presynaptic adhesion molecule that controls synaptic properties. Using super-resolution imaging, we demonstrate that presynaptic neuronal and astrocytic neurexin-1 form discrete nanoclusters at excitatory synapses. We find that distinct patterns of heparan sulfate modification and alternative splicing confer onto astrocytic and neuronal neurexin-1 different ligand specificities, thereby enabling compartment-specific signaling by neurexin-1. At hippocampal Schaffer-collateral synapses, deletion of neurexin-1 from either astrocytes or neurons did not alter synapse numbers, but differentially impaired synapse function. Neuronal neurexin-1 was essential for NMDA-receptor-mediated synaptic responses, whereas astrocytic neurexin-1 was required for maturation of silent synapses, AMPA-receptor recruitment, and long-term potentiation. Thus, astrocytes and neurons surprisingly use the same synaptic adhesion molecule to control distinct synapse properties.

sentences: In-scanner head motion and structural covariance networks In-scanner head motion systematically reduces estimated regional gray matter volumes obtained from structural brain MRI. Here, we investigate how head motion affects structural covariance networks that are derived from regional gray matter volumetric estimates. We acquired motion-affected and motion-free whole brain T1-weighted MRI in 29 healthy adult subjects and estimated relative regional gray matter volumes using a voxel-based morphometry approach. Structural covariance network analyses were undertaken while systematically increasing the number of motion-affected scans included in the analysis. We demonstrate that the standard deviation in regional gray matter estimates increases as the number of motion-affected scans increases. This subsequently increases pair-wise correlations between regions, a key determinant for construction of structural covariance networks. We further demonstrate that head motion systematically alters graph theoretic metrics derived from these networks. Our findings suggest that in-scanner head motion is a source of error that violates the assumption that structural covariance networks reflect neuroanatomical connectivity between brain regions. Results of structural covariance studies should be interpreted with caution, particularly when subject groups are likely to move their heads in the scanner.

sentences: Preserving protein homeostasis prevents motor impairment in DNA Damage Response-compromised C. elegans To maintain genome integrity, cells rely on a complex system of DNA repair pathways and cell cycle checkpoints, together referred to as the DNA damage response (DDR). Impairments in DDR pathways are linked to cancer, but also to a wide range of degenerative processes, frequently including progressive neuropathy and accelerated aging. How defects in mechanistically distinct DDR pathways can drive similar degenerative phenotypes is not understood. Here we show that defects in various DDR components are linked to a loss of protein homeostasis in Caenorhabditis elegans. Prolonged silencing of atm-1, brc-1 or ung-1, central components in respectively checkpoint signaling, double strand break repair and base excision repair enhances the global aggregation of proteins occurring in adult animals, and accelerates polyglutamine protein aggregation in a model for neurodegenerative diseases. Overexpression of the molecular chaperone HSP-16.2 prevents enhanced protein aggregation in atm-1, brc-1 or ung-1-compromised animals. Strikingly, rebalancing protein homeostasis with HSP-16.2 almost completely rescues age-associated impaired motor function in these animals as well. This reveals that the consequences of a loss of atm-1, brc-1 or ung-1 converge on an impaired protein homeostasis to cause degeneration. These findings indicate that a loss of protein homeostasis is a crucial downstream consequence of DNA repair defects, and thereby provide an attractive novel framework for understanding the broad link between DDR defects and degenerative processes.

sentences: Low-disturbance Farming Regenerates Healthy Deep Soil towards Sustainable Agriculture Intensive conventional farming has degraded farmland topsoil and seriously threaten food and environment security globally. Although low-disturbance practices have been widely adapted to restore soil health, whether this measure in a long run can potentially recover the critical deep soil to meet sustainable intensification of crop production are still unclear. Here we compared soil microbiome, physicochemical parameters along 3-m deep soil profiles, and crop yield in Northeast China subjected to ten years of farming practices at 3 levels of disturbance, including conventional tillage (CT), no-tillage without stover mulching (NTNS), and no-tillage with stover mulching (NTSM). We found that low-disturbance practices (NTNS and NTSM) promoted the ability of the deep soil to retain water, nitrogen and salt-extractable organic, regenerated whole-soil microbial diversity and metabolic function, improved topsoil organic carbon stock and corn yield in the drought year, showed the potential to reduce energy consumption and greenhouse gas emissions, thus regenerating highly efficient, sustainable agriculture.

sentences: The evolution of information transmission in mammalian brain networks Brain communication, defined as information transmission through white-matter connections, is at the foundation of the brain's computational capacities that virtually subtend all aspects of behavior: from sensory perception shared across mammalian species, to complex cognitive functions in humans. How did communication strategies in macroscale brain networks adapted across evolution to accomplish increasingly complex functions? By applying a novel approach to measure information transmission in mouse, macaque and human brains, we found an evolutionary gradient from selective information processing, where brain regions share information through single polysynaptic pathways, to parallel information processing, where regions communicate through multiple parallel pathways. In humans, parallel processing acts as a major connector between unimodal and transmodal systems. Communication strategies are unique to individuals across different mammalian species, pointing at the individual-level specificity of information routing architecture. Our work provides compelling evidence that different communication strategies are tied to the evolutionary complexity of mammalian brain networks.

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Somatic mutation detection in plants: a critical evaluation through simulations and reanalyses O_LIMutation, the source of genetic diversity, is the raw material of evolution; however, the mutation process remains understudied, especially in plants. Using both a simulation and reanalysis framework, we set out to test the performance of two types of variant callers, generic ones and those developed for cancer research, to detect de novo somatic mutations.
C_LIO_LIIn an in silico experiment, we generated Illumina-like sequence reads spiked with simulated mutations at different allele frequencies to compare the performance of seven commonly-used variant callers to recall them. More empirically, we then reanalyzed two of the largest datasets available for plants, both developed for identifying within-individual variation in long-lived pedunculate oaks.
C_LIO_LIEven in plants, variant callers developed for cancer research outperform generic callers regarding mutation recall and precision, especially at low allele frequency. Such variants at low allele frequency are typically expected for within-individual de novo plant mutations. Reanalysis of published oak data with the best-performing caller based on our simulations identified up to 7x more somatic mutations than initially reported.
C_LIO_LIOur results advocate the use of cancer research callers to boost de novo mutation research in plants, and to reconcile empirical reports with theoretical expectations.
C_LI

sentences: Implicit anticipation of probabilistic regularities: Larger CNV emerges for unpredictable events Anticipation of upcoming events plays a crucial role in automatic behaviors. It is, however, still unclear whether the event-related brain potential (ERP) markers of anticipation could track the implicit acquisition of probabilistic regularities that can be considered as building blocks of automatic behaviors. Therefore, in a four-choice reaction time (RT) task performed by young adults (N = 36), the contingent negative variation (CNV) as an ERP marker of anticipation was measured from the onset of a cue stimulus until the presentation of a target stimulus. Due to the probability structure of the task, target stimuli were either predictable or unpredictable, but this was unknown to participants. The cue did not contain predictive information on the upcoming target. Results showed that the CNV amplitude during response preparation was larger before the unpredictable than before the predictable target stimuli. In addition, although RTs increased, the P3 amplitude decreased for the unpredictable as compared with the predictable target stimuli, possibly due to the stronger response preparation that preceded stimulus presentation. These results suggest that enhanced attentional resources are allocated to the implicit anticipation and processing of unpredictable events. This might originate from the formation of internal models on the probabilistic regularities of the stimulus stream, which primarily facilitates the processing of predictable events. Overall, we provide ERP evidence that supports the role of implicit anticipation and predictive processes in the acquisition of probabilistic regularities.

sentences: Defining dysfunction due to loss of MECP2 in Rett Patient Brain Rett Syndrome is characterized by a postnatal loss of neurophysiological function and regression of childhood development. Because the syndrome is X-linked and males with MECP2 mutations generally do not survive birth, the study of this syndrome has been complicated by the fact that in female brain, a portion of neurons express wild type MECP2, and another portion express a non-functional allele of MECP2. Therefore, bulk-RNA-sequencing of Rett brain is confounded by the presence of chimerism of neurons for functional MECP2 in neurons. We developed an approach that allows for single-nuclei transcriptional profiling of individual neurons and a direct comparison between neurons that express functional MECP2 with those that express the disease-causing allele. We found that mutant neurons from Rett brain show patterns of aberrant expression of synaptic and metabolic genes, both of which can be detected in in vitro models of Rett Syndrome. We used these resources to identify a role for POU2F1/OCT1 transcription factor in mediating the response to stress due to loss of MECP2, highlighting a potential key molecular regulator of stress in Rett neurons. Together, our new sorting approach enables us to highlight defective molecular and metabolic pathways in Rett brain neurons and suggests that in vitro models could serve as valuable tools to further study this syndrome and potentially for development of novel therapeutics.

sentences: Modeling minimum viable population size with multiple genetic problems of small populations An important goal for conservation is to define minimum viable population (MVP) sizes for long-term persistence. Although many MVP size estimates focus on ecological processes, with increasing evidence for the role of genetic problems in population extinction, conservation practitioners have also increasingly started to incorporate inbreeding depression (ID). However, small populations also face other genetic problems such as mutation accumulation (MA) and loss of genetic diversity through genetic drift that are usually factored into population viability assessments only via verbal arguments. Comprehensive quantitative theory on interacting genetic problems is missing. Here we develop eco-evolutionary quantitative models that track both population size and levels of genetic diversity. Our models assume a biallelic multilocus genome whose loci can be under either a single or interacting genetic forces. In addition to mutation-selection-drift balance (for loci facing ID and MA), we include three forms of balancing selection (for loci where variation is lost through genetic drift). We define MVP size as the lowest population size that avoids an eco-evolutionary extinction vortex after a time sufficient for an equilibrium allele frequency distribution to establish. Our results show that MVP size decreases rapidly with increasing mutation rates for populations whose genomes are only under balancing selection, while for genomes under mutation-selection-drift balance, the MVP size increases rapidly. MVP sizes also increase rapidly with increasing number of loci under the same or different selection mechanisms until a point is reached at which even arbitrarily large populations cannot survive anymore. In the case of fixed number of loci under selection, interaction of genetic problems did not necessarily increase MVP sizes. To further enhance our understanding about interaction of genetic problems, there is need for more empirical studies to reveal how different genetic processes interact in the genome.

sentences: Plasticity facilitates rapid evolution Developmental plasticity enables organisms to cope with new environmental challenges. If deploying such plasticity is costly in terms of time or energy, the same adaptive behavior could subsequently evolve through piecemeal genomic reorganisation that replaces the requirement to acquire that adaptation by individual plasticity. Here we report a new dimension to the way in which plasticity can drive evolutionary change leading to ever greater complexity in biological organization. Our model deploys the concept of partially overlapping functional systems. We found that plasticity accelerated dramatically the evolutionary accumulation of adaptive systems in model organisms with relatively low rates of mutation. The effect of plasticity on the evolutionary growth of complexity was even greater when the number of elements needed to construct a functional system was increased. These results suggest that as the difficulty of challenges from the environment become greater, so plasticity exerts an ever more powerful role in meeting those challenges and in opening up new avenues for the subsequent evolution of complex adaptations.

sentences: Unique functions for Notch4 in murine embryonic lymphangiogenesis In mice, embryonic dermal lymphatic development is well-understood and used to study gene functions in lymphangiogenesis. Notch signaling is an evolutionarily conserved pathway that modulates cell fate decisions, which has been shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. Actively growing embryonic dermal lymphatics expressed NOTCH1, NOTCH4 and DLL4 which correlated with Notch activity. In lymphatic endothelial cells (LECs), DLL4 activation of Notch induced a subset of Notch effectors and lymphatic genes, which were distinctly regulated by Notch1 and Notch4 activation. Treatment of LECs with VEGF-A or VEGF-C upregulated Dll4 transcripts, and differentially and temporally regulated the expression of Notch1 and Hes/Hey genes. Mice nullizygous for Notch4 had an increase in the closure of the lymphangiogenic fronts which correlated with reduced vessel caliber in the maturing lymphatic plexus at E14.5, and reduced branching at E16.5. Activation of Notch4 suppressed LEC migration in a wounding assay significantly more than Notch1, suggesting a dominant role for Notch4 in regulating LEC migration. Unlike Notch4 nulls, inhibition of canonical Notch signaling by expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ LECs led to increased lymphatic density consistent with an increased in LEC proliferation, described for the loss of LEC Notch1. Moreover, loss of Notch4 did not affect LEC canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis.

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Investigating age- and sex-specific effects of socialization on voluntary ethanol administration using a novel vapor paradigm IntroductionAdolescence is characterized as a transitional developmental period between childhood and adulthood that is associated with increased freedom and novel experiences that are frequently peer-influenced. Due to newfound independence, there is a higher prevalence of alcohol consumption, which is heightened by the rewarding effects of alcohol. However, the contributions of social interaction and sexual dimorphism to alcohol intake are not fully understood. Here we explore the use a novel self-administration ethanol (EtOH) vapor system to investigate the sexual dimorphic nature of socially facilitated ethanol exposure.

MethodsAdolescent and adult male and female Sprague-Dawley rats underwent a novel voluntary intermittent EtOH vapor paradigm. Nosepoke initiated self-administration vapor chambers administered 20mg/L of vaporized EtOH or air into the chamber following each nosepoke. Beginning on postnatal day 30 (PND30), during the onset of adolescence, or 70 (PND70), at the onset of adulthood, animals were placed in vapor chambers for 4hr every other day for 40 sessions. All animals underwent 10 sessions with their cagemate (social access) followed by 10 sessions in isolation (isolated access), a 10-day forced abstinence period, 10 sessions isolated access, and 10 sessions social access.

ResultsThese data reveal that despite low EtOH consumption across all groups, adolescent (PND30) and adult (PND70) female rats voluntarily self-administered more EtOH vapor per body weight than age-matched males, while male rats increased EtOH preference over sessions regardless of age. In addition, all rats regardless of sex or age voluntarily self-administered more EtOH vapor per body weight during the social access session than during the subsequent isolated access sessions.

ConclusionThese data demonstrate that under these experimental parameters, male and female rats regardless of age do not self-administer high quantities of EtOH vapor using this paradigm. Further work is required to determine whether the nose-poke EtOH vapor self-administration apparatus can be modified to promote high voluntary EtOH consumption that can be socially facilitated. These data demonstrate that with further investigation, the self-administration EtOH vapor system could be an effective alternative to other methods of voluntary EtOH administration to further our understanding of socially facilitated drinking.

sentences: Magnetic Bead-Based Separation (MBS) of Pneumococcal Serotypes The separation of pneumococcal serotypes from a complex polymicrobial mixture may be required for different applications. For instance, a minority strain could be present at a low frequency in a clinical sample, making it difficult to identify and isolate by traditional culture-based methods. We therefore developed an assay to separate mixed pneumococcal samples using serotype-specific antiserum and a magnetic bead-based separation method. Using qPCR and colony counting methods, we first show that serotypes (12F, 23F, 3, 14, 19A and 15A) present at [~]0.1% of a dual serotype mixture can be enriched to between 10% and 90% of the final sample. We demonstrate two applications for this method: extraction of a known pneumococcal serotype from saliva samples and efficient purification of capsule switch variants from experimental transformation experiments. Moreover, this method may have further laboratory or clinical applications when the selection of specific serotypes is required.

sentences: Eicosanoid signaling as a therapeutic target in middle-aged mice with severe COVID-19 Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Resolution of the COVID-19 pandemic has been advanced by the recent development of SARS-CoV-2 vaccines, but vaccine efficacy is partly compromised by the recent emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially in aged populations. Here, we describe the isolation of a new set of highly virulent mouse-adapted viruses and use them to test a novel therapeutic drug useful in infections of aged animals. Initially, we show that many of the mutations observed in SARS-CoV-2 during mouse adaptation (at positions 417, 484, 501 of the spike protein) also arise in humans in variants of concern (VOC)2. Their appearance during mouse adaptation indicates that immune pressure is not required for their selection. Similar to the human infection, aged mice infected with mouse-adapted SARS-CoV-2 develop more severe disease than young mice. In murine SARS, in which severity is also age-dependent, we showed that elevated levels of an eicosanoid, prostaglandin D2 (PGD2) and of a phospholipase, PLA2G2D, contributed to poor outcomes in aged mice3,4. Using our virulent mouse-adapted SARS-CoV-2, we show that infection of middle-aged mice lacking expression of DP1, a PGD2 receptor, or PLA2G2D are protected from severe disease. Further, treatment with a DP1 antagonist, asapiprant, protected aged mice from a lethal infection. DP1 antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, and demonstrates that the PLA2G2D-PGD2/DP1 pathway is a useful target for therapeutic interventions. (Words: 254)

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Circadian rhythm shows potential for mRNA efficiency and self-organized division of labor in multinucleate cells Multinucleate cells occur in every biosphere and across the kingdoms of life, including in the human body as muscle cells and bone-forming cells. Data from filamentous fungi suggest that, even when bathed in a common cytoplasm, nuclei are capable of autonomous behaviors, including division. How does this potential for autonomy affect the organization of cellular processes between nuclei? Here we analyze a simplified model of circadian rhythm, a form of cellular oscillator, in a mathematical model of the filamentous fungus Neurospora crassa. Our results highlight the role played by mRNA-protein phase separation to keep mRNAs close to the nuclei from which they originate, while allowing proteins to diffuse freely between nuclei. Our modeling shows that syncytism allows for extreme mRNA efficiency -- we demonstrate assembly of a robust oscillator with transcription levels 104-fold less than in comparable uninucleate cells. We also show self-organized division of the labor of mRNA production, with one nucleus in a two-nucleus syncytium producing at least twice as many mRNAs as the other in 30% of cycles. This division can occur spontaneously, but division of labor can also be controlled by regulating the amount of cytoplasmic volume available to each nucleus. Taken together, our results show the intriguing richness and potential for emergent organization among nuclei in multinucleate cells. They also highlight the role of previously studied mechanisms of cellular organization, including nuclear space control and localization of mRNAs through RNA-protein phase separation, in regulating nuclear coordination.

Author summaryCircadian rhythms are among the most researched cellular processes, but limited work has been done on how these rhythms are coordinated between nuclei in multinucleate cells. In this work, we analyze a mathematical model for circadian oscillations in a multinucleate cell, motivated by frequency mRNA and protein data from the filamentous fungus Neurospora crassa. Our results illuminate the importance of mRNA-protein phase separation, in which mRNAs are kept close to the nucleus in which they were transcribed, while proteins can diffuse freely across the cell. We demonstrate that this phase separation allows for a robust oscillator to be assembled with very low mRNA counts. We also investigate how the labor of transcribing mRNAs is divided between nuclei, both when nuclei are evenly spaced across the cell and when they are not. Division of this labor can be regulated by controlling the amount of cytoplasmic volume available to each nucleus. Our results show that there is potential for emergent organization and extreme mRNA efficiency in multinucleate cells.

sentences: Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis Cleavage of membrane proteins in the lipid bilayer by intramembrane proteases is crucial for health and disease. Although different lipid environments can potently modulate their activity, how this is linked to their structural dynamics is unclear. Here we show that the carboxy-peptidase-like activity of the archaeal intramembrane protease PSH, a homolog of the Alzheime[r]s disease-associated presenilin/{gamma}-secretase is impaired in micelles and promoted in a lipid bilayer. Comparative molecular dynamics simulations revealed that important elements for substrate binding such as transmembrane domain 6a of PSH are more labile in micelles and stabilized in the lipid bilayer. Moreover, consistent with an enhanced interaction of PSH with a transition- state analog inhibitor, the bilayer promoted the formation of the enzymes catalytic active site geometry. Our data indicate that the lipid environment of an intramembrane protease plays a critical role in structural stabilization and active site arrangement of the enzyme-substrate complex thereby promoting intramembrane proteolysis.

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From contigs to chromosomes: automatic Improvement of Long Read Assemblies (ILRA) Recent advances in long read technologies not only enable large consortia to aim to sequence all eukaryotes on Earth, but they also allow many laboratories to sequence their species of interest. Although there is a promise to obtain "perfect genomes" with long read technologies, the number of contigs often exceeds the number of chromosomes significantly, containing many insertion and deletion errors around homopolymer tracks. To overcome these issues, we implemented ILRA to correct long reads-based assemblies, a pipeline that orders, names, merges, and circularizes contigs, filters erroneous small contigs and contamination, and corrects homopolymer errors with Illumina reads. We successfully tested our approach to assemble the genomes of four novel Plasmodium falciparum samples, and on existing assemblies of Trypanosoma brucei and Leptosphaeria spp. We found that correcting homopolymer tracks reduced the number of genes incorrectly annotated as pseudogenes, but an iterative correction seems to be needed to reduce high numbers of homopolymer errors. In summary, we described and compared the performance of a new tool, which improves the quality of long read assemblies. It can be used to correct genomes of a size of up to 300 Mb.

AvailabilityThe tool is available at GitHub: https://github.com/ThomasDOtto/ILRA.

sentences: Isolation and Purification of Lipoarabinomannan from Urine of Adults with Active Tuberculosis Lipoarabinomannan (LAM) is a cell wall component of Mycobacterium tuberculosis that is excreted in the urine of persons with active tuberculosis (TB). Limited diagnostic sensitivity of LAM immunoassays has been due to selecting antibodies against LAM derived from in vitro cultured M. tuberculosis, rather than LAM purified from in vivo clinical urine specimens. Urinary LAM (uLAM) is critical to enable the development of and/or screening of novel uLAM-specific antibodies but is typically dilute and in heterogeneous mixtures with other urine components. We used physical, enzymatic, and chemical processes for the scaled isolation and purification of uLAM. The purified material may then be used to develop more sensitive uLAM diagnostic tests for active TB disease.

sentences: The molecular evolution of spermatogenesis across mammals The testis is a key male reproductive organ that produces gametes through the process of spermatogenesis. Testis morphologies and spermatogenesis evolve rapidly in mammals, presumably due to the evolutionary pressure on males to be reproductively successful1,2. The rapid evolution of the testis was shown to be reflected at the molecular level based on bulk-tissue work3-8, but the molecular evolution of individual spermatogenic cell types across mammalian lineages remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from eleven species that cover the three major mammalian lineages (eutherians, marsupials, egg-laying monotremes) and birds (the evolutionary outgroup), and include seven key primates. Our analyses reveal that the rapid evolution of the testis is driven by accelerated fixation rates of gene expression changes, amino acid altering substitutions, and newly emerged genes in late spermatogenic stages - likely facilitated by reduced pleiotropic constraints, haploid selection, and a transcriptionally permissive chromatin environment. We identify temporal expression changes of individual genes across species, which may have contributed to the emergence of species-specific phenotypes, but also conserved expression programs underlying ancestral spermatogenic processes. Sex chromosome analyses show that genes predominantly expressed in spermatogonia (i.e., germ cells fueling spermatogenesis) and Sertoli cells (i.e., somatic supporting cells) independently accumulated on X chromosomes across mammals during evolution, presumably due to male-beneficial selective forces. Further work uncovered that the process of meiotic sex chromosome inactivation (MSCI) also occurs in monotremes and hence is common to the different mammalian sex chromosome systems, contrary to previous inferences9. Thus, the general mechanism of meiotic silencing of unsynapsed chromatin (MSUC), which underlies MSCI, represents an ancestral mammalian feature. Together, our study illuminates the cellular and molecular evolution of mammalian spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals.

sentences: Simultaneous dual recordings from hair cells and their calyx afferents reveal the functional significance of these specialized endings in the vestibular periphery. In the vestibular periphery, transmission via conventional synaptic boutons is supplemented by postsynaptic calyceal endings surrounding Type I hair cells. This review focusses on the multiple modes of communication between these receptors and their enveloping calyces as revealed by simultaneous dual-electrode recordings. Classic orthodromic transmission is accompanied by two forms of bidirectional communication enabled by the extensive cleft between the Type I hair cell and its calyx. The slowest cellular communication low-pass filters the transduction current with a time constant 10 -- 100 milliseconds: potassium ions accumulate in the synaptic cleft, depolarizing both the hair cell and afferent to potentials greater than necessary for rapid vesicle fusion in the receptor and potentially triggering action potentials in the afferent. On the millisecond timescale, conventional glutamatergic quantal transmission occurs when hair cells are depolarized to potentials sufficient for calcium influx and vesicle fusion. Depolarization also permits a third form of transmission which occurs over tens of microseconds, resulting from the large voltage- and ion-sensitive cleft-facing conductances in both the hair cell and the calyx that are open at their resting potentials. Current flowing out of either the hair cell or the afferent divides into the fraction flowing across the cleft into its cellular partner, and the remainder flowing out of the cleft and into the surrounding fluid compartment. These findings suggest multiple biophysical bases for the extensive repertoire of response dynamics seen in the population of primary vestibular afferent fibers. The results further suggest that evolutionary pressures drive selection for the calyx afferent.

sentences: Antigen-derived peptides directly engage the unfolded-protein sensor IRE1α to curb cross-presentation by dendritic cells Dendritic cells (DCs) promote adaptive immunity by cross-presenting antigen-based epitopes to CD8+ T cells. DCs process internalized protein antigens into peptides that enter the endoplasmic reticulum (ER) and upload onto major histocompatibility type I (MHC-I) protein complexes for cell-surface transport and cross-presentation. Perplexingly, DCs often exhibit activation of the ER-stress sensor IRE1 in the absence of classical ER stress--leaving the underlying mechanism unexplained. Here we show that antigen-derived hydrophobic peptides directly engage ER-resident IRE1 by masquerading as unfolded proteins. Furthermore, IRE1 activation depletes MHC-I heavy-chain mRNAs through regulated IRE1-dependent decay (RIDD), thereby curtailing antigen cross-presentation. In tumor-bearing mice, IRE1 disruption increased MHC-I expression on tumor-infiltrating DCs, and enhanced recruitment and activation of CD8+ T cells. Moreover, IRE1 inhibition synergized with anti-PD-L1 antibody treatment to cause tumor regression. Our findings elucidate the mechanism and consequence of antigen-driven IRE1 activation in DCs, yielding a promising combination strategy for cancer immunotherapy.

sentences: Saturation mutagenesis of a predicted ancestral Syk-family kinase Many tyrosine kinases cannot be expressed readily in E. coli, limiting facile production of these proteins for biochemical experiments. We used ancestral sequence reconstruction to generate a spleen tyrosine kinase (Syk) variant that can be expressed in bacteria and purified in soluble form, unlike the human members of this family (Syk and ZAP-70). The catalytic activity, substrate specificity, and regulation by phosphorylation of this Syk variant are similar to the corresponding properties of human Syk and ZAP-70. Taking advantage of the ability to express this novel Syk-family kinase in bacteria, we developed a two-hybrid assay that couples the growth of E.coli in the presence of an antibiotic to successful phosphorylation of a bait peptide by the kinase. Using this assay, we screened a site-saturation mutagenesis library of the kinase domain of this reconstructed Syk-family kinase. Sites of loss-of-function mutations identified in the screen correlate well with residues established previously as critical to function and/or structure in protein kinases. We also identified activating mutations in the regulatory hydrophobic spine and activation loop, which are within key motifs involved in kinase regulation. Strikingly, one mutation in an ancestral Syk-family variant increases the soluble expression of the protein by 75-fold. Thus, through ancestral sequence reconstruction followed by deep mutational scanning, we have generated Syk-family kinase variants that can be expressed in bacteria with very high yield.

sentences: Angiomotin isoform 2 promotes binding of PALS1 to KIF13B at the base of primary cilia and suppresses ciliary elongation The kinesin-3 motor KIF13B functions in endocytosis, vesicle transport, and regulation of ciliary length and signaling. Direct binding of the membrane-associated guanylate kinase (MAGUK) DLG1 to KIF13Bs MAGUK-binding stalk (MBS) domain relieves motor autoinhibition and promotes microtubule plus end-directed cargo transport. Here we characterize Angiomotin isoform 2 (Ap80) as a novel KIF13B interactor that promotes binding of another MAGUK, the polarity protein and Crumbs complex component PALS1, to KIF13B. Live cell imaging analysis indicated that Ap80 is concentrated at the base of primary cilia and recruits PALS1 to this site, but is not itself a cargo of KIF13B. Consistent with a ciliary function for Ap80, its depletion led to elongated primary cilia while its overexpression caused ciliary shortening. Our results suggest that Ap80 may specifically activate KIF13B cargo binding at the base of primary cilia to regulate ciliary length.

sentences: GABABR agonist baclofen promotes central nervous system remyelination Promoting remyelination - the endogenous response by which lost myelin sheaths are regenerated - is considered as a potential neuroprotective strategy to prevent/limit the development of permanent neurological disability in patients with multiple sclerosis (MS). To this end, a number of clinical trials are investigating the potential of existing drugs to enhance oligodendrocyte progenitor cell (OPC) differentiation, the process that fails in chronic MS lesions. As we previously reported that oligodendroglia lineage cells express GABAB receptors (GABABRs) both in vitro and in vivo, and that GABABR-mediated signaling enhances OPC differentiation and myelination in vitro, here we focused on the remyelinating potential of the best-known GABABR agonist baclofen (Bac), already approved to treat spasticity in MS. We demonstrated that Bac increases myelin protein production following lysolecithin (LPC)-induced demyelination in cerebellar ex vivo slices. In addition, Bac administration enhanced OPC differentiation and remyelination in LPC-induced spinal cord lesions in adult mice. Thus, our results suggest that Bac should be considered as a potential therapeutic agent, not only to treat spasticity, but also to improve remyelination in patients with MS.

sentences: RNF168-mediated localization of BARD1 recruits the BRCA1-PALB2 complex to DNA damage DNA damage prompts a diverse range of alterations to the chromatin landscape. The RNF168 E3 ubiquitin ligase catalyzes the mono-ubiquitination of histone H2A at lysine (K)13/15 (mUb-H2A), forming a binding module for DNA repair proteins. BRCA1 promotes homologous recombination (HR), in part, through its interaction with PALB2, and the formation of a larger BRCA1-PALB2-BRCA2-RAD51 (BRCA1-P) complex. The mechanism by which BRCA1-P is recruited to chromatin surrounding DNA breaks is unclear. In this study, we reveal that an RNF168-governed signaling pathway is responsible for localizing the BRCA1-P complex to DNA damage. Using mice harboring a Brca1CC (coiled coil) mutation that blocks the Brca1-Palb2 interaction, we uncovered an epistatic relationship between Rnf168- and Brca1CC alleles, which disrupted development, and reduced the efficiency of Palb2-Rad51 localization. Mechanistically, we show that RNF168-generated mUb-H2A recruits BARD1 through a BRCT domain ubiquitin-dependent recruitment motif (BUDR). Subsequently, BARD1-BRCA1 accumulate PALB2-RAD51 at DNA breaks via the CC domain-mediated BRCA1-PALB2 interaction. Together, these findings establish a series of molecular interactions that connect the DNA damage signaling and HR repair machinery.

sentences: Vascular dysregulation following SARS-CoV-2 infection involves integrin signaling through a VE-Cadherin mediated pathway The vascular barrier is heavily injured following SARS-CoV-2 infection and contributes enormously to life-threatening complications in COVID-19. This endothelial dysfunction is associated with the phlogistic phenomenon of cytokine storms, thrombotic complications, abnormal coagulation, hypoxemia, and multiple organ failure. The mechanisms surrounding COVID-19 associated endotheliitis have been widely attributed to ACE2-mediated pathways. However, integrins have emerged as possible receptor candidates for SARS-CoV-2, and their complex intracellular signalling events are essential for maintaining endothelial homeostasis. Here, we showed that the spike protein of SARS-CoV-2 depends on its RGD motif to drive barrier dysregulation through hijacking integrin V{beta}3. This triggers the redistribution and internalization of major junction protein VE-Cadherin which leads to the barrier disruption phenotype. Both extracellular and intracellular inhibitors of integrin V{beta}3 prevented these effects, similarly to the RGD-cyclic peptide compound Cilengitide, which suggests that the spike protein - through its RGD motif - binds to V{beta}3 and elicits vascular leakage events. These findings support integrins as an additional receptor for SARS-CoV-2, particularly as integrin engagement can elucidate many of the adverse endothelial dysfunction events that stem from COVID-19.

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Single-cell analysis of microglial transcriptomic diversity in subarachnoid hemorrhage BackgroundSubarachnoid hemorrhage (SAH) is a severe stroke and the advanced treatment for SAH is still limited. Recent studies have shown that microglia-mediated neuroinflammation plays a critical role in the pathogenesis of SAH. Microglia can transform their states in response to central nervous system injury. However, the transcriptomic features of microglia remained unknown in SAH. Recent developed single-cell RNA sequencing (scRNA-seq) provides a possible way to solve this problem.

MethodsEndovascular perforation (EVP) murine SAH model was established to reproduce experimental SAH. Microglia states are examined with immune staining and quantitate analysis. Post-SAH microglial single-cell suspension were harvest and sequenced using 10X scRNA-seq platform. Then, the detailed single-cell transcriptomic characterization of post-SAH microglia were analyzed with bioinformatics.

ResultsTranscriptional analysis revealed at least ten diverse microglial subgroups, including SAH-associated microglia (SAM), inflammatory-associated microglia (IAM) and proliferation-associated microglia (PAM), which all exhibit distinct marker gene expression patterns. Microglia subsets interaction reveals the functional relationship between elevated signaling pathways and microglial sub-populations in SAH. Receptor-ligand pair analysis revealed that complex inter-cellular interactions exist between the microglia subsets and other cell types, and indicated that microglia are important mediators of neuroinflammation after SAH. Integrated analysis with normal microglia further proved the existence of these microglia subpopulations and different gene markers associated with SAH were clarified.

ConclusionsCollectively, we first report the single-cell transcriptome of post-SAH microglia and found specific biomarkers related to the neuroinflammation in SAH. These results enhanced our understanding of the pathological mechanisms of microglial response to SAH, and may guide future development of SAH monitoring methods and therapeutics.

sentences: Both Prey and Predator Features Determine Predation Risk and Survival of Schooling Prey Predation is one of the main evolutionary drivers of social grouping. While it is well appreciated that predation risk is likely not shared equally among individuals within groups, its detailed quantification has remained difficult due to the speed of attacks and the highly-dynamic nature of collective prey response. Here, using high-resolution tracking of solitary predators (Northern pike) hunting schooling fish (golden shiners), we not only provide detailed insights into predator decision-making but show which key spatial and kinematic features of predator and prey influence individuals risk to be targeted and survive attacks. Pike tended to stealthily approach the largest groups, and were often already inside the school when launching their attack, making prey in this frontal "strike zone" the most vulnerable to be targeted. From the preys perspective, those fish in central locations, but relatively far from, and less aligned with, neighbours, were most likely to be targeted. While the majority of attacks (70%) were successful, targeted individuals that did manage to avoid capture exhibited a higher maximum acceleration response just before the attack and were further away from the pike s head. Our results highlight the crucial interplay between predators attack strategy and response of prey in determining predation risk in mobile animal groups.

sentences: Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling pathway-regulated directed movement of cell clusters. OR is instructed by several signaling events, including the EGFR and Notch pathways, and planar cell polarity (PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification and differentiation. Here, we show that Abl kinase negatively regulates ommatidial rotation through its activity in the R3/R4 pair. Interestingly in wild-type, Abl is localized to apical junctional regions in R4 but not in R3 during OR, and this apical enrichment requires Notch signaling. We further demonstrate that Abl and Notch genetically interact during OR, and Abl co-immunoprecipitates in complexes with Notch in the developing eye disc. Perturbations of Abl interfere with adherens junction dynamics of the ommatidial preclusters, which are critical for the OR process. Taken together, our data suggest a model in which Abl kinase acts directly downstream of the Notch receptor in R4 to fine-tune OR via its input into adherens junction complexes.

sentences: A cooperative network at the nuclear envelope counteracts LINC-mediated forces during oogenesis in C. elegans Oogenesis involves meiosis and oocyte maturation. Both processes rely on mechanical forces (Lee et al., 2015; Nagamatsu et al., 2019; Rog and Dernburg, 2015; Sato et al., 2009; Tsatskis et al., 2020; Wynne et al., 2012), which can be transduced from the cytoskeleton to the nuclear envelope (NE) through linker of nucleoskeleton and cytoskeleton (LINC) complexes (Burke, 2018; Chang et al., 2015; Fan et al., 2020; Link et al., 2014). Gametes must protect their genomes from damage in this mechanically stressful environment. In C. elegans, oocyte nuclei lacking the single lamin protein LMN-1 are vulnerable to nuclear collapse. Here we deploy the auxin-inducible degradation system to investigate the balance of forces that drive this collapse and protect oocyte nuclei. We find that nuclear collapse is not a consequence of apoptosis. It is promoted by dynein and a LINC complex comprised of SUN-1 and ZYG-12, which assumes polarized distribution at the NE in response to dynein-mediated forces. We also show that the lamin meshwork works in parallel with other inner nuclear membrane (INM) proteins to counteract mechanical stress at the NE during oogenesis. We speculate that a similar network may protect oocyte integrity during the long arrest period in mammals.

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BSG/CD147 and ACE2 receptors facilitate SARS-CoV-2 infection of human iPS cell-derived kidney podocytes BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the Coronavirus disease 2019 (COVID-19), which was declared a pandemic by the World Health Organization (WHO) in March 2020. The disease has caused more than 5.1 million deaths worldwide. While cells in the respiratory system are frequently the initial target for SARS-CoV-2, clinical studies suggest that COVID-19 can become a multi-organ disease in the most severe cases. Still, the direct affinity of SARS-CoV-2 for cells in other organs such as the kidneys, which are often affected in severe COVID-19, remains poorly understood.

MethodIn this study, we employed a human induced pluripotent stem (iPS) cell-derived model to investigate the affinity of SARS-CoV-2 for kidney glomerular podocytes. We studied uptake of the live SARS-CoV-2 virus as well as pseudotyped viral particles by human iPS cell derived podocytes using qPCR, western blot, and immunofluorescence. Global gene expression and qPCR analyses revealed that human iPS cell-derived podocytes express many host factor genes (including ACE2, BSG/CD147, PLS3, ACTR3, DOCK7, TMPRSS2, CTSL CD209, and CD33) associated with SARS-CoV-2 binding and viral processing.

ResultInfection of podocytes with live SARS-CoV-2 or spike-pseudotyped lentiviral particles revealed viral uptake by the cells at low Multiplicity of Infection (MOI of 0.01) as confirmed by RNA quantification and immunofluorescence studies. Our results also indicate that direct infection of human iPS cell-derived podocytes by SARS-CoV-2 virus can cause cell death and podocyte foot process retraction, a hallmark of podocytopathies and progressive glomerular diseases including collapsing glomerulopathy observed in patients with severe COVID-19 disease. Additionally, antibody blocking experiments identified BSG/CD147 and ACE2 receptors as key mediators of spike binding activity in human iPS cell-derived podocytes.

ConclusionThese results show that SARS-CoV-2 can infect kidney glomerular podocytes in vitro. These results also show that the uptake of SARS-CoV-2 by kidney podocytes occurs via multiple binding interactions and partners, which may underlie the high affinity of SARS-CoV-2 for kidney tissues. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism.

Significant statementMany patients with COVID19 disease exhibit multiorgan complications, suggesting that SARS-CoV-2 infection can extend beyond the respiratory system. Acute kidney injury is a common COVID-19 complication contributing to increased morbidity and mortality. Still, SARS-Cov-2 affinity for specialized kidney cells remain less clear. By leveraging our protocol for stem cell differentiation, we show that SARS-CoV-2 can directly infect kidney glomerular podocytes by using multiple Spike-binding proteins including ACE2 and BSG/CD147. Our results also indicate that infection by SARS-CoV-2 virus can cause podocyte cell death and foot process effacement, a hallmark of podocytopathies including collapsing glomerulopathy observed in patients with severe COVID-19 disease. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism.

sentences: Diversity and metagenome analysis of a hydrocarbon-degrading bacterial consortium from asphalt lakes located in Wietze, Germany The pollution of terrestrial and aquatic environments by petroleum contaminants, especially diesel fuel, is a persistent environmental threat requiring cost-effective and environmentally sensitive remediation approaches. Bioremediation is one such approach, but is dependent on the availability of microorganisms with the necessary metabolic abilities and environmental adaptability. The aim of this study was to examine the microbial community in a petroleum contaminated site, and isolate organisms potentially able to degrade hydrocarbons. Through successive enrichment of soil microorganisms from samples of an historic petroleum contaminated site in Wietze, Germany, we isolated a bacterial consortium using diesel fuel hydrocarbons as sole carbon and energy source. The 16S rRNA gene analysis revealed the dominance of Alphaproteobacteria. We further reconstructed a total of 18 genomes from both the original soil sample and the isolated consortium. The analysis of both the metagenome of the consortium and the reconstructed metagenome-assembled genomes show that the most abundant bacterial genus in the consortium, Acidocella, possess many of the genes required for the degradation of diesel fuel aromatic hydrocarbons, which are often the most toxic component. This can explain why this genus proliferated in all the enrichment cultures. Therefore, this study reveals that the microbial consortium isolated in this study and its dominant genus, Acidocella, could potentially serve as an effective inoculum for the bioremediation of sites polluted with diesel fuel or other organic contaminants.

sentences: Sex chromosomes, sex ratios and sex gaps in longevity in plants In animals, males and females can display markedly different longevity (also called sex gap in longevity, SGLs). Recent work has revealed that sex chromosomes contribute to establishing these SGLs. X-hemizygosity and toxicity of the Y chromosomes are two mechanisms that have been suggested to reduce male longevity (Z-hemizygosity and W toxicity in females in ZW systems). In plants, SGLs are known to exist but the role of sex chromosomes remains to be established. Here, by using adult sex ratio as a proxy for measuring SGLs, we explored the relationship between sex chromosome and SGLs across 43 plant species. Based on the knowledge recently accumulated in animals, we specifically asked whether: (i) species with XY systems tend to have female-biased sex ratios (reduced male longevity) and species with ZW ones tend to have male-biased sex ratios (reduced female longevity), and (ii) this patterns was stronger in heteromorphic systems compared to homomorphic ones. Our results tend to support these predictions although we lack statistical power because of a small number of ZW systems and the absence of any heteromorphic ZW system in the dataset. We discuss the implications of these findings, which we hope will stimulate further research on sex-differences in lifespan and ageing across plants.

sentences: In vivo KRAS G12D/V Degradation Mediated by CANDDY Using a Modified Proteasome Inhibitor "Undruggable" proteins, such as RAS proteins, remain problematic despite efforts to discover inhibitors against them. KRAS mutants are prevalent in human cancers. Recently, the KRAS G12C inhibitor have been clinically approved, but inhibitors for KRAS G12D/V are still under development. Here, we described the development of a novel chemical knockdown strategy, termed CANDDY (Chemical knockdown with Affinity aNd Degradation DYnamics). This strategy involves a CANDDY tag modified from a proteasome inhibitor inducing direct proteasomal degradation. We constructed TUS-007 as a multispecific small molecule tethered from a KRAS interactor and CANDDY tag to target KRAS G12D/V. We confirmed that the degradation by TUS-007 was independent of target ubiquitination. This allows to solve a laborious design of matchmaker in the current ubiquitination-dependent proteolysis technology. And TUS-007 successfully suppressed tumors due to in vivo degradation of KRAS G12D/V. The CANDDY technology could represent a simple and rational strategy to degrade currently "undruggable" proteins.

sentences: Mismatch repair deficiency is not sufficient to increase tumor immunogenicity DNA mismatch repair deficiency (MMRd) in human cancer is associated with high tumor mutational burden (TMB), frameshift mutation-derived neoantigens, increased T cell infiltration, and remarkable responsiveness to immune checkpoint blockade (ICB) therapy. Nevertheless, about half of MMRd tumors do not respond to ICB for unclear reasons. While tumor cell line transplant models of MMRd have reinforced the importance of TMB in immune response, critical questions remain regarding the role of immunosurveillance in the evolution of MMRd tumors induced in vivo. Here, we developed autochthonous mouse models of lung and colon cancer with highly efficient ablation of MMR genes via in vivo CRISPR/Cas9 targeting. Surprisingly, MMRd in these models did not result in increased immunogenicity or response to ICB. Mechanistically, we showed this lack of immunogenicity to be driven by profound intratumoral heterogeneity (ITH). Studies in animals depleted of T cells further demonstrated that immunosurveillance in MMRd tumors has no impact on TMB but shapes the clonal architecture of neoantigens by exacerbating ITH. These results provide important context for understanding immune evasion in cancers with high TMB and have major implications for therapies aimed at increasing TMB.

sentences: Horizontal transfer and subsequent explosive expansion of a DNA transposon in sea kraits (Laticauda) Transposable elements (TEs) are self replicating genetic sequences and are often described as important "drivers of evolution". This driving force is because TEs promote genomic novelty by enabling rearrangement, and through exaptation as coding and regulatory elements. However, most TE insertions will be neutral or harmful, therefore host genomes have evolved machinery to supress TE expansion. Through horizontal transposon transfer (HTT) TEs can colonise new genomes, and since new hosts may not be able to shut them down, these TEs may proliferate rapidly. Here we describe HTT of the Harbinger-Snek DNA transposon into sea kraits (Laticauda), and its subsequent explosive expansion within Laticauda genomes. This HTT occurred following the divergence of Laticauda from terrestrial Australian elapids ~15-25 Mya. This has resulted in numerous insertions into introns and regulatory regions, with some insertions into exons which appear to have altered UTRs or added sequence to coding exons. Harbinger-Snek has rapidly expanded to make up 8-12% of Laticauda spp. genomes; this is the fastest known expansion of TEs in amniotes following HTT. Genomic changes caused by this rapid expansion may have contributed to adaptation to the amphibious-marine habitat.

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Natural loss of function of ephrin-B3 shapes spinal flight circuitry in birds Flight in birds evolved through patterning of the wings from forelimbs and transition from alternating gait to synchronous flapping. In mammals, the spinal midline guidance molecule ephrin-B3 instructs the wiring that enables limb alternation, and its deletion leads to synchronous hopping gait. Here we show that the ephrin-B3 protein in birds lacks several motifs present in other vertebrates, diminishing its affinity for the EphA4 receptor. The avian ephrin-B3 gene lacks an enhancer that drives midline expression, and is missing in Galliformes. The morphology and wiring at brachial levels of the chick spinal cord resemble those of ephrin-B3 null mice. Importantly, dorsal midline decussation, evident in the mutant mouse, is apparent at the chick brachial level, and is prevented by expression of exogenous ephrin-B3 at the roof plate. Our findings support a role for loss of ephrin-B3 function in shaping the avian brachial spinal cord circuitry and facilitating synchronous wing flapping.

TeaserWalking vs flying: Deciphering the organization and evolution of the neuronal network that controls wing flapping in birds.

sentences: Bacterial but not fungal rhizosphere communities differ among perennial grass ecotypes under abiotic environmental stress Environmental change, especially frequent droughts, is predicted to detrimentally impact the North American perennial grasslands. Consistent dry spells will affect plant communities as well as their associated rhizobiomes, possibly altering the plant host performance under environmental stress. Therefore, there is a need to understand the impact of drought on the rhizobiome, and how the rhizobiome may modulate host performance and ameliorate its response to drought stress. In this study, we analyzed bacterial and fungal communities in the rhizospheres of three ecotypes (dry, mesic, and wet) of a dominant prairie grass, Andropogon gerardii. The ecotypes were established in 2010 in a common garden design and grown for a decade under persistent dry conditions at the arid margin of the species range in Colby Kansas. The experiment aimed to answer whether and to what extent do the different ecotypes maintain or recruit distinct rhizobiomes after ten years in an arid climate. In order to answer this question, we screened the bacterial and fungal rhizobiome profiles of the ecotypes under the arid conditions of western KS as a surrogate for future climate environmental stress using 16S rRNA and ITS2 metabarcoding sequencing. Under these conditions, bacterial communities differed compositionally among the A. gerardii ecotypes, whereas the fungal communities did not. The ecotypes were instrumental in driving the differences among bacterial rhizobiomes, as the ecotypes maintained distinct bacterial rhizobiomes even after ten years at the edge of the host species range. This study will aid us to optimize plant productivity through the use of different ecotypes under future abiotic environmental stress, especially drought.

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Exosomal TAR DNA binding protein 43 profile in canine model of amyotrophic lateral sclerosis: A preliminary study in developing blood-based biomarker for neurodegenerative diseases ObjectiveBlood-based biomarkers provide a crucial information in progress of neurodegenerative diseases with minimally invasive sampling method. Validated blood-based biomarker application in people with amyotrophic lateral sclerosis would derive numerous benefits. Canine degenerative myelopathy is a naturally occurring animal disease model to study the biology of human amyotrophic lateral sclerosis. Serum derived exosomes are potential carriers for cell-specific cargoes making them ideal venue to study biomarkers for a variety of diseases and biological processes. This study assessed the exosomal proteins that may be assigned as surrogate biomarker that may reflect biochemical changes in central nervous system.

MethodsExosomes were isolated from canine serum using commercial exosome isolation reagents. Exosomes target proteins contents were analysed by Western blotting method.

ResultsThe profiles of potential biomarker candidates in spinal cord homogenate and that of serum-derived exosomes were found elevated in dogs with degenerative myelopathy as compare to control subjects.

ConclusionsSerum-derived exosomal biomolecules can serve as surrogate biomarkers in neuro degenerative diseases.

Key MessagesO_LIA canine with degenerative myelopathy can serve as a model animal to study human amyotrophic lateral sclerosis.
C_LIO_LISerum-derived exosomes contains Transactive Response DNA Binding Protein 43 (TDP-43), potential biomarker candidate.
C_LIO_LIThe levels of spinal cord TDP-43 proteins and that of serum-derived exosomes exhibited a similar profiling. Therefore, serum derived exosomes may be used as a venue for establishing blood-based biomarkers for neurodegenerative diseases.
C_LI

sentences: Directed evolution of colE1 plasmid replication compatibility: a fast tractable tunable model for investigating biological orthogonality. Plasmids of the ColE1 family are among the most frequently used plasmids in molecular biology. They were adopted early in the field for many biotechnology applications, and as model systems to study plasmid biology. The mechanism of replication of ColE1 plasmids is well understood, involving the interaction between a plasmid-encoded sense-antisense gene pair (RNAI and RNAII). Because of its mechanism of replication, bacterial cells cannot maintain two different plasmids with the same origin, with one being rapidly lost from the population - a process known as plasmid incompatibility. While mutations in the regulatory genes RNAI and RNAII have been reported to make colE1 plasmids more compatible, there has been no attempt to engineer compatible colE1 origins, which can be used for multi-plasmid applications and that can bypass design constrains created by the current limited plasmid origin repertoire available. Here, we show that by targeting sequence diversity to the loop regions of RNAI (and RNAII), it is possible to select new viable colE1 origins that are compatible with the wild-type one. We demonstrate origin compatibility is not simply determined by sequence divergence in the loops, and that pairwise compatibility is not an accurate guide for higher order interactions. We identify potential principles to engineer plasmid copy number independently from other regulatory strategies and we propose plasmid compatibility as a tractable model to study biological orthogonality. New characterised plasmid origins increase flexibility and accessible complexity of design for challenging synthetic biology applications where biological circuits can be dispersed between multiple independent genetic elements.

sentences: Evolutionary conservation of centriole rotational asymmetry in the human centrosome Centrioles are formed by microtubule triplets in a nine-fold symmetric arrangement. In flagellated protists and in animal multiciliated cells, accessory structures tethered to specific triplets render the centrioles rotationally asymmetric, a property that is key to cytoskeletal and cellular organization in these contexts. In contrast, centrioles within the centrosome of animal cells display no conspicuous rotational asymmetry. Here, we uncover rotationally asymmetric molecular features in human centrioles. Using ultrastructure expansion microscopy, we show that LRRCC1, the ortholog of a protein originally characterized in flagellate green algae, associates preferentially to two consecutive triplets in the distal lumen of human centrioles. LRRCC1 partially co-localizes and affects the recruitment of another distal component, C2CD3, which also has an asymmetric localization pattern in the centriole lumen. Together, LRRCC1 and C2CD3 delineate a structure reminiscent of a filamentous density observed by electron microscopy in flagellates, termed the  acorn. Functionally, the depletion of LRRCC1 in human cells induced defects in centriole structure, ciliary assembly and ciliary signaling, supporting that LRRCC1 cooperates with C2CD3 to organizing the distal region of centrioles. Since a mutation in the LRRCC1 gene has been identified in Joubert syndrome patients, this finding is relevant in the context of human ciliopathies. Taken together, our results demonstrate that rotational asymmetry is an ancient property of centrioles that is broadly conserved in human cells. Our work also reveals that asymmetrically localized proteins are key for primary ciliogenesis and ciliary signaling in human cells.

sentences: A protein-based biosensor for detecting calcium by magnetic resonance imaging Calcium-responsive contrast agents for magnetic resonance imaging (MRI) offer an attractive approach to noninvasively image neural activity with wide coverage in deep brain regions. However, current MRI sensors for calcium are based on synthetic architectures fundamentally incompatible with genetic technologies for in vivo delivery and targeting. Here, we present a protein-based MRI sensor for calcium, derived from a calcium-binding protein known as calprotectin. Calcium-binding causes calprotectin to sequester manganese. We demonstrate that this mechanism allows calprotectin to alter T1 and T2 weighted contrast in response to biologically relevant calcium concentrations. Corresponding changes in relaxation times are comparable to synthetic calcium sensors and exceed those of previous protein-based MRI sensors for other neurochemical targets. The biological applicability of calprotectin was established by detecting calcium in lysates prepared from a neuronal cell line. Calprotectin thus represents a promising path towards imaging neural activity by combining the benefits of MRI and protein sensors.

sentences: Spatial turnover of soil viral populations and genotypes overlain by cohesive responses to moisture in grasslands Although soil viral abundance, diversity, and potential roles in microbial community dynamics and biogeochemical cycling are beginning to be appreciated1-5, little is known about the patterns and drivers of soil viral community composition that underlie their contributions to terrestrial ecology. Here, we analyzed 43 soil viromes from a precipitation manipulation experiment in a Mediterranean grassland in California, USA. We recovered 5,315 viral population sequences (vOTUs), and viral community composition exhibited a highly significant distance-decay relationship within the 18 m long field. This pattern was recapitulated in the microheterogeneity of 130 prevalent vOTUs (detected in >=90% of the viromes), which tended to exhibit significant negative correlations between genomic similarity of their predominant allelic variants and distance. Although spatial turnover was also observed in the bacterial and archaeal communities from the same soils, the signal was dampened relative to the viromes, suggesting differences in assembly drivers at local scales for viruses and their microbial hosts and/or differences in the temporal scales captured by viromes and total DNA. Despite the overwhelming spatial signal, vOTUs responsive to a decrease in soil moisture were significantly enriched in a predicted protein-sharing subnetwork of 326 vOTUs linked to 191 known actinobacteriophages, suggesting a genomically cohesive viral response to soil moisture evocative of environmental filtering, potentially by way of actinobacterial hosts. Overall, soil viral ecological processes appear to be highly constrained in space and tightly coupled to the heterogeneous, dynamic soil environment and thus fundamentally different from those of their well-mixed and more thoroughly studied marine counterparts.

sentences: PBRM1 BD2 and BD4 associate with RNA to facilitate chromatin association PBRM1 is a subunit of the PBAF chromatin remodeling complex, which is mutated in 40-50% of clear cell renal cell carcinoma patients. It is thought to largely function as a chromatin binding subunit of the PBAF complex, but the molecular mechanism underlying this activity is not fully known. PBRM1 contains six tandem bromodomains which are known to cooperate in binding of nucleosomes acetylated at histone H3 lysine 14 (H3K14ac). Here we demonstrate that the second and fourth bromodomains from PBRM1 also bind nucleic acids, selectively associating with double stranded RNA elements. Disruption of the RNA binding pocket is found to compromise PBRM1 chromatin binding and inhibit PBRM1-mediated cellular growth effects.

sentences: Tissue-wide metabolomics reveals wide impact of gut microbiota on mice metabolite composition The essential role of gut microbiota in health and disease is well-recognized, but the biochemical details underling beneficial impact remain largely undefined. Dysbiosis of gut bacteria results in the alteration of certain microbial and host metabolites, and identifying these markers could enhance the early detection of certain diseases. We report LC-MS based non-targeted metabolic profiling to demonstrate a large effect of gut microbiota on mammalian tissue metabolites. It was hypothesized that gut microbiota influences the overall biochemistry of the host metabolome and this effect is tissue-specific. Thirteen different tissues from germ-free and conventional mice were selected and their metabolic differences were analyzed. Our study demonstrated a large effect of the microbiome on mammalian biochemistry at different tissue levels and resulted in significant modulation of metabolites from multiple metabolic pathway (p [≤] 0.05). A vast metabolic response of host to metabolites generated by the microbiota was observed, Hundreds of molecular features were detected exclusively in one mouse group, with the majority of these being unique to specific tissue, suggesting direct impact gut microbiota on host metabolism.

sentences: Flexible control of Pavlovian-instrumental transfer based on expected reward value The Pavlovian-instrumental transfer (PIT) paradigm is widely used to assay the motivational influence of reward-predictive cues, reflected by their ability to invigorate instrumental behavior. Leading theories of incentive learning assume that cues are assigned motivational properties based on the value of the rewards they predict. We outline an alternative account of PIT which recognizes that reward-predictive cues may suppress rather than motivate instrumental behavior under certain conditions. We posit that cues capable of triggering a strong reward expectancy will tend to inhibit instrumental behavior, which is exploratory by nature, in order to facilitate retrieval of the expected reward before it is lost or stolen. According to this view, the motivation to engage in instrumental behavior during a cue should be inversely related to the value of the predicted reward, since there is more to lose by failing to secure a high-value reward than a low-value reward. We tested this hypothesis in rats using a high-conflict PIT protocol which elicits competing instrumental and food-port entry responses. In Experiment 1, cues signaling different reward magnitudes (1, 3, or 9 pellets) differentially influenced these behaviors. Whereas the 1-pellet cue increased instrumental behavior, cues signaling 3 or 9 pellets suppressed instrumental behavior and elicited high levels of food-port activity. Experiment 2 found that reward-predictive cues suppressed instrumental behavior and increased food-port activity in a flexible manner that was disrupted by post-training reward devaluation. We discuss how the PIT task provides a useful tool for studying cognitive control over cue-motivated behavior in rodents.

sentences: In situ imaging of bacterial membrane projections and associated protein complexes using electron cryo-tomography The ability to produce membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of [~] 90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified MEs and MVs in 13 species and classified several major ultrastructures: 1) tubes with a uniform diameter (with or without an internal scaffold), 2) tubes with irregular diameter, 3) tubes with a vesicular dilation at their tip, 4) pearling tubes, 5) connected chains of vesicles (with or without neck-like connectors), 6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.

sentences: A functionally conserved STORR gene fusion in Papaver species that diverged 16.8 million years ago The STORR gene fusion event is considered a key step in the evolution of benzylisoquinoline alkaloid (BIA) metabolism in opium poppy as the resulting bi-modular protein performs the isomerization of (S)- to (R)-reticuline which is required for morphinan biosynthesis. Our previous analysis of the opium poppy genome suggested the STORR gene fusion event occurred before a whole genome duplication event 7.2 million years ago. Here we use a combination of phylogenetic, transcriptomic, metabolomic, biochemical and genomic analysis to investigate the origin of the STORR gene fusion across the Papaveraceae family. The pro-morphinan/morphinan subclass of BIAs was present in a subset of 10 Papaver species including P. somniferum (opium poppy) and this correlated with the presence of the STORR gene fusion with one important exception. P. californicum does not produce morphinans but it does contain a STORR gene fusion that epimerizes (S)- to (R)-reticuline when heterologously expressed in yeast. The high similarity of the amino acid sequence linking the two modules of STORR along with phylogenetic gene tree analysis strongly suggests the gene fusion occurred only once and between 17-25 million years ago before the separation of P. californicum from the other Papaver species. We discovered that the most abundant BIA in P. californicum is (R)-glaucine, a member of the aporphine subclass of BIAs. Only the (S) isomer of this compound has previously been reported from nature. These results lead us to conclude that the function of the STORR gene fusion is not exclusive to morphinan production in the Papaveraceae.

sentences: Brain-to-brain concordance in child-parent dyads underlies children's psychological wellbeing Negative family environments can be harmful to childrens psychological wellbeing via altered child-parent interactions. Single-brain paradigms have uncovered changes in brain systems related to negative family environments, but how neurobiological reciprocity between child and parent brains contribute to childrens psychological wellbeing remains unknown. Across two studies examining 395 child-parent dyads, we show that children from negative family environments experienced more psychopathological symptoms. Dyad-based analysis of movie-watching functional magnetic resonance imaging data revealed higher inter-subject correlations in dorsal- and ventral-MPFC, and greater concordance of activity with regions critical for socioemotional skills in child-parent than child-stranger dyads. Negative family environment reduced inter-subject correlation of ventral-MPFC with the hippocampus in child-parent dyads, which further mediated childrens internalizing symptoms. The ventral-MPFC and hippocampus exhibited reciprocal activity, which helped child-parent dyads form shared meaning during movie watching. Our findings suggest child-parent brain-to-brain concordance may be a neurobiological mechanism for how family environments affect psychological wellbeing.

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Quantitative Single-Cell Proteomics as a Tool to Characterize Cellular Hierarchies Large-scale single-cell analyses are of fundamental importance in order to capture biological heterogeneity within complex cell systems, but have largely been limited to RNA-based technologies. Here we present a comprehensive benchmarked experimental and computational workflow, which establishes global single-cell mass spectrometry-based proteomics as a tool for large-scale single-cell analyses.

By exploiting a primary leukemia model system, we demonstrate both through pre-enrichment of cell populations and through a non-enriched unbiased approach that our workflow enables the exploration of cellular heterogeneity within this aberrant developmental hierarchy. Our approach is capable of consistently quantifying approximately 1000 proteins per cell across thousands of individual cells using limited instrument time. Furthermore, we developed a computational workflow (SCeptre) that effectively normalizes the data, integrates available FACS data and facilitates downstream analysis. The approach presented here lays a solid foundation for implementing global single-cell proteomics studies across the world.

sentences: Inference of tissue relative proportions of the breast epithelial cell types luminal progenitor, basal, and luminal mature Single-Cell Analysis has revolutionised genomic science in recent years. However, due to cost and other practical considerations, single-cell analyses are impossible for studies based on medium or large patient cohorts. For example, a single-cell analysis usually costs thousands of euros for one tissue sample from one volunteer, meaning that typical studies using single-cell analyses are based on very few individuals. While single-cell genomic data can be used to examine the phenotype of individual cells, cell-type deconvolution methods are required to track the quantities of these cells in bulk-tissue genomic data. Hormone receptor negative breast cancers are highly aggressive, and are thought to originate from a subtype of epithelial cells called the luminal progenitor. In this paper, we show how to quantify the number of luminal progenitor cells as well as other epithelial subtypes in breast tissue samples using DNA and RNA based measurements. We find elevated levels of cells which resemble these hormone receptor negative luminal progenitor cells in breast tumour biopsies of hormone receptor negative cancers, as well as in healthy breast tissue samples from BRCA1 (FANCS) mutation carriers. We also find that breast tumours from carriers of heterozygous mutations in non-BRCA Fanconi Anaemia pathway genes are much more likely to be hormone receptor negative. These findings have implications for understanding hormone receptor negative breast cancers, and for breast cancer screening in carriers of heterozygous mutations of Fanconi Anaemia pathway genes.

sentences: Localized delivery of β-NGF via injectable microrods accelerates endochondral fracture repair Currently, there are no biological approaches to accelerate bone fracture repair. Osteobiologics that promote endochondral ossification are an exciting alternative to surgically implanted bone grafts, however, the translation of osteobiologics remains elusive because of the need for localized and sustained delivery that is both safe and effective. In this regard, an injectable system composed of hydrogel-based microparticles designed to release osteobiologics in a controlled and localized manner is ideal in the context of bone fracture repair. Here, we describe poly (ethylene glycol) dimethacrylate (PEGDMA)-based microparticles, in the form of microrods, engineered to be loaded with beta nerve growth factor ({beta}-NGF) for use in a murine tibial fracture model. In-vitro studies demonstrated that protein-loading efficiency is readily altered by varying PEGDMA macromer concentration and that {beta}-NGF loaded onto PEGDMA microrods exhibited sustained release over a period of 7 days. In-vitro bioactivity of {beta}-NGF was confirmed using a tyrosine receptor kinase A (Trk-A) expressing cell line, TF-1. Moreover, TF-1 cell proliferation significantly increased when incubated with {beta}-NGF loaded PEGDMA microrods versus {beta}-NGF in media. In-vivo studies show that PEGDMA microrods injected into the fracture calluses of mice remained in the callus for over 7 days. Importantly, a single injection of {beta}-NGF-loaded PEGDMA microrods resulted in significantly improved fracture healing as indicated by significant increases in bone volume, trabecular connective density, and bone mineral density and a significant decrease in cartilage despite a remarkably lower dose ([~]111 fold) than the {beta}-NGF in media. In conclusion, we demonstrate a novel and translational method of delivering {beta}-NGF via injectable PEGDMA microrods to improve bone fracture repair.

sentences: Beyond BOLD: in search of genuine diffusion fMRI contrast in human brain Functional Magnetic Resonance Imaging (fMRI) is an essential method to measure brain activity non-invasively. While fMRI almost systematically relies on the blood oxygenation level-dependent (BOLD) contrast, there is an increasing interest in alternative methods that would not rely on neurovascular coupling. A promising but controversial such alternative is diffusion fMRI (dfMRI), which relies instead on dynamic fluctuations in apparent diffusion coefficient (ADC) due to microstructural changes underlying neuronal activity, i.e. neuromorphological coupling. However, it is unclear whether genuine dfMRI contrast, distinct from BOLD contamination, can be detected in the human brain in physiological conditions. Here, we present the first dfMRI study in humans attempting to minimize BOLD contamination sources and comparing functional responses at two field strengths (3T and 7T). Our study benefits from unprecedented high spatio-temporal resolution, harnesses novel denoising strategies and examines characteristics of not only task but also resting-state dfMRI. We report task-induced decrease in ADC with temporal and spatial features distinct from the BOLD response and yielding more specific activation maps. Furthermore, we report dfMRI resting-state functional connectivity which, compared to its BOLD counterpart, is essentially free from physiological artifacts and preserves positive correlations but preferentially suppresses anti-correlations, which are likely of vascular origin. A careful acquisition and processing design thus enable the detection of genuine dfMRI contrast on clinical MRI systems. As opposed to BOLD, diffusion functional contrast could be particularly well suited for low-field MRI.

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Niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes diarrheal disease in humans and animals. During salmonellosis, S. Typhimurium colonizes epithelial cells lining the gastrointestinal tract. S. Typhimurium has an unusual lifestyle in epithelial cells that begins within an endocytic-derived Salmonella-containing vacuole (SCV), followed by escape into the cytosol, epithelial cell lysis and bacterial release. The cytosol is a more permissive environment than the SCV and supports rapid bacterial growth. The physicochemical conditions encountered by S. Typhimurium within the cytosol, and the bacterial genes required for cytosolic colonization, remain unknown. Here we have exploited the parallel colonization strategies of S. Typhimurium in epithelial cells to decipher the two niche-specific bacterial virulence programs. By combining a population-based RNA-seq approach with single-cell microscopic analysis, we identified bacterial genes/sRNAs with cytosol-specific or vacuole-specific expression signatures. Using these genes/sRNAs as environmental biosensors, we defined that Salmonella is exposed to iron and manganese deprivation and oxidative stress in the cytosol and zinc and magnesium deprivation in the SCV. Furthermore, iron availability was critical for optimal S. Typhimurium replication in the cytosol, as well as entC, fepB, soxS and sitA-mntH. Virulence genes that are typically associated with extracellular bacteria, namely Salmonella pathogenicity island 1 (SPI1) and SPI4, had a cytosolic-specific expression profile. Our study reveals that the cytosolic and vacuolar S. Typhimurium virulence gene programs are unique to, and tailored for, residence within distinct intracellular compartments. Therefore, this archetypical vacuole-adapted pathogen requires extensive transcriptional reprogramming to successfully colonize the mammalian cytosol.

Author SummaryIntracellular pathogens reside either within a membrane-bound vacuole or are free-living in the cytosol and their virulence programs are tailored towards survival within a particular intracellular compartment. Some bacterial pathogens (such as Salmonella enterica) can successfully colonize both intracellular niches, but how they do so is unclear. Here we have exploited the parallel intracellular lifestyles of S. enterica in epithelial cells to identify the niche-specific bacterial expression profiles and environmental cues encountered by S. enterica. We have also discovered bacterial genes that are required for colonization of the cytosol, but not the vacuole. Our results advance our understanding of pathogen-adaptation to alternative replication niches and highlight an emerging concept in the field of bacteria-host cell interactions.

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Predicting butyrate- and propionate-forming bacteria of gut microbiota from sequencing data BackgroundThe bacteria-derived short chain fatty acids (SCFAs) butyrate and propionate play important (distinct) roles in health and disease and understanding the ecology of respective bacteria on a community-wide level is a top priority in microbiome research. The aim of this study was to reveal members harboring main pathways for the production of those metabolites and assess the applicability of sequence data (metagenomics and 16S rRNA gene) to predict SCFAs production in vitro and in vivo.

ResultsA clear split between butyrate- and propionate-forming bacteria was detected with only very few taxa exhibiting pathways for the production of both SCFAs. After in vitro growth of fecal communities from distinct donors (n=8) on different substrates (n=7) abundances of bacteria exhibiting pathways correlated with respective SCFA concentrations, in particular in the case of butyrate. While final growth differed markedly between cultures, communities showed high functional redundancies with comparable yields, i.e., concentration of metabolite per grown bacterium exhibiting pathway(s), irrespective of the donor and substrate used. For propionate, correlations were weaker indicating that its production is less imprinted into the core metabolism compared with butyrate-forming bacteria. Longitudinal measurements in vivo (five samples derived from 20 subjects) also revealed a correlation between abundances of pathway-carrying bacteria and concentrations of the two SCFAs. Additionally, lower bacterial cell concentrations, together with higher stool moisture, promoted overall bacterial activity (measured by flow cytometry and coverage patterns of metagenome-assembled genomes) that led to elevated SCFAs concentrations with over-proportional levels of butyrate. Butyrate concentrations displayed lower temporal stability than propionate, however, abundances of bacteria exhibiting the butyrate-forming pathway were more stable than those carrying pathways for propionate production. Predictions on pathway abundances based on 16S rRNA gene data using our in-house database worked well yielding similar results as metagenomic-based analyses.

ConclusionsWe demonstrated that pathway abundances enable predictions on concentrations of SCFAs indicating that stimulating bacterial growth directly leads to more production of those compounds. The strong separation of gut microbiota into two functional communities facilitates the development of precision intervention strategies targeting either metabolite.

sentences: Genetic architecture of spatially complex color patterning in hybrid Mimulus Interspecies hybridization generates inter-genomic interactions, which may result in unique traits not seen in either parent species. Here we explore the genetic basis of two types of floral pigment phenotypes, in hybrids between monkeyflower species Mimulus cupreus and M. luteus var. variegatus. Mimulus cupreus has abundant yellow carotenoid pigmentation in its petal lobes, while M. l. variegatus has a derived reduction in carotenoid intensity. Thus, as expected, carotenoid intensity segregates in an F2 hybrid population. More surprisingly, both species appear to have petal lobes solidly and identically covered in magenta anthocyanin pigment (leading to an orange color in the high-carotenoid species M. cupreus), yet F1 and F2 hybrids exhibit novel and complex spatial patterns of anthocyanin spotting. A rare yellow morph of M. cupreus, which lacks petal anthocyanins, also generates spatially patterned offspring when hybridized with M. l. variegatus. We use this cross, together with newly developed genomic and image analysis tools, to investigate the genetic architecture of color and pattern variation in an F2 hybrid population. We report that the non-patterned carotenoid reduction in M. l. variegatus is genetically simple, and is explained by a single QTL which contains the Beta-carotene hydroxylase-1 (BCH1) gene. HPLC results show that beta-carotene content differs between dark yellow and light yellow petals, which supports a causal role for BCH1. The hybrid-specific anthocyanin patterning phenotypes are more complex, with one QTL of large effect and four detectable QTLs of small effect. These results illustrate how different types of traits may have predictably distinct genetic architectures, and provide candidate genomic regions for investigating the molecular mechanisms of both simple and complex floral color patterning.

sentences: DeepVASP-E: A Flexible Analysis of Electrostatic Isopotentials for Finding and Explaining Mechanisms that Control Binding Specificity Amino acids that play a role in binding specificity can be identified with many methods, but few techniques identify the biochemical mechanisms by which they act. To address a part of this problem, we present DeepVASP-E, an algorithm that can suggest electrostatic mechanisms that influence specificity. DeepVASP-E uses convolutional neural networks to classify an electrostatic representation of ligand binding sites into specificity categories. It also uses class activation mapping to identify regions of electrostatic potential that are salient for classification. We hypothesize that electrostatic regions that are salient for classification are also likely to play a biochemical role in achieving specificity. Our findings, on two families of proteins with electrostatic influences on specificity, demonstrate that large salient regions can identify amino acids that have an electrostatic role in binding, and that DeepVASP-E is an effective classifier of ligand binding sites.

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FiNN: A toolbox for neurophysiological network analysis In recent years, neuroscience has seen a shift from localist approaches to network-wide investigations of brain function. Neurophysiological signals across different spatial and temporal scales provide an informative insight into neural communication. However, additional methodological considerations arise when investigating network-wide brain dynamics rather than local effects. Specifically, larger amounts of data, investigated across a higher dimensional space, are necessary.

Here, we present FiNN (Find Neurophysiological Networks), a novel toolbox for the analysis of neurophysiological data with a focus on functional and effective connectivity. FiNN provides a wide range of data processing methods, introduces new methodological developments to acquire efficient and reliable connectivity estimates that build on already existing concepts, and statistical and visualization tools to facilitate inspection of connectivity estimates and the resulting metrics of brain dynamics. The toolbox is freely available in Python (https://github.com/neurophysiological-analysis/FiNN), and complemented by online documentation (https://neurophysiological-analysis.github.io/FiNN/).

To highlight the properties of our toolbox, we evaluated FiNN against a number of established frameworks on both a conceptual and an implementation level. We found FiNN to require much less processing time and memory than other toolboxes. In addition, FiNN adheres to a design philosophy of easy access and modifiability, while providing efficient data processing implementations. Since the investigation of network-level neural dynamics is experiencing increasing interest, we place FiNN at the disposal of the neuroscientific community as open-source software.

Highlights- Investigation of network-wide brain dynamics requires specialized software.
- We provide a toolbox to ease the pre-processing and analysis steps of the investigation.
- FiNN requires less processing time and memory in comparison to other toolboxes.
- Extensive documentation facilitates usage by users from various technical backgrounds.

sentences: Genetic mapping and identification of a QTL determining tolerance to freezing stress in Fragaria vesca L. Extreme cold and frost cause significant stress to plants which can potentially be lethal. Low temperature freezing stress can cause significant and irreversible damage to plant cells and can induce physiological and metabolic changes that impact on growth and development. Low temperatures cause physiological responses including winter dormancy and autumn cold hardening in strawberry (Fragaria) species, and some diploid F. vesca accessions have been shown to have adapted to low-temperature stresses. To study the genetics of freezing tolerance, a F. vesca mapping population of 142 seedlings segregating for differential responses to freezing stress was raised. The progeny was mapped using  Genotyping-by-Sequencing and a linkage map of 2,918 markers at 851 loci was resolved. The mapping population was phenotyped for freezing tolerance response under controlled and replicated laboratory conditions and subsequent quantitative trait loci analysis using interval mapping revealed a single significant quantitative trait locus on Fvb2 in the physical interval 10.6 Mb and 15.73 Mb on the F. vesca v4.0 genome sequence. This physical interval contained 896 predicted genes, several of which had putative roles associated with tolerance to abiotic stresses including freezing. Differential expression analysis of the 896 QTL-associated gene predictions in the leaves and crowns from  Alta and  NCGR1363 parental genotypes revealed genotype-specific changes in transcript accumulation in response to low temperature treatment as well as expression differences between genotypes prior to treatment for many of the genes. The putative roles, and significant interparental differential expression levels of several of the genes reported here identified them as good candidates for the control of the effects of freezing tolerance at the QTL identified in this investigation and the possible role of these candidate genes in response to freezing stress is discussed.

sentences: Insights into the regulatory mechanisms of Clostridioides difficile biofilm formation Mucosal biofilms play an important role in intestinal health; however, the mucosal bacterial community has been implicated in persistent infections. Clostridioides difficile is an important nosocomial pathogen, with an unacceptable high rate of recurrence following antibiotic treatment. As C. difficile is a known biofilm producer, a property which may contribute to this suboptimal therapeutic response, we have investigated the transcriptional changes and regulatory pathways during the transition from planktonic to biofilm mode of growth. Widespread metabolic reprogramming during biofilm formation was detected, characterised by an increased usage of glycine metabolic pathways to yield key metabolites, which are used for energy production and synthesis of short chain fatty acids. We detected the expression of 107 small non-coding RNAs that appear to, in some part, regulate these pathways; however, 25 of these small RNAs were specifically expressed during biofilm formation, indicating they may play a role in regulating biofilm-specific genes. Similar to Bacillus subtilis, biofilm formation is a multi-regulatory process and SinR negatively regulates biofilm formation independently of other known mechanisms. This comprehensive analysis furthers our understanding of biofilm formation in C. difficile, identifies potential targets for anti-virulence factors, and provides evidence of the link between metabolism and virulence traits.

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Review and performance evaluation of trait-based between-community dissimilarity measures O_LIIn the recent years a variety of indices have been proposed with the aim of quantifying functional dissimilarity between communities. These indices follow different approaches to account for between-species similarities in the calculation of community dissimilarity, yet they all have been proposed as straightforward tools.
C_LIO_LIIn this paper we reviewed the trait-based dissimilarity indices available in the literature, contrasted the approaches they follow, and evaluated their performance in terms of correlation with an underlying environmental gradient using individual-based community simulations with different gradient lengths. We tested how strongly dissimilarities calculated by different indices correlate with environmental distances. Using random forest models we tested the importance of gradient length, the choice of data type (abundance vs. presence/absence), the transformation of between-species similarities (linear vs. exponential), and the dissimilarity index in the predicting correlation value.
C_LIO_LIWe found that many indices behave very similarly and reach high correlation with environmental distances. There were only a few indices (e.g. Raos DQ, and representatives of the nearest neighbour approach) which performed regularly poorer than the others. By far the strongest determinant of correlation with environmental distance was the gradient length, followed by the data type. The dissimilarity index and the transformation method seemed not crucial decisions when correlation with an underlying gradient is to be maximized.
C_LIO_LISynthesis: We provide a framework of functional dissimilarity indices and discuss the approaches they follow. Although, these indices are formulated in different ways and follow different approaches, most of them perform similarly well. At the same time, sample properties (e.g. gradient length) determine the correlation between trait-based dissimilarity and environmental distance more fundamentally.
C_LI

sentences: Large-scale cellular-resolution imaging of neural activity in freely behaving mice Miniaturized microscopes for head-mounted fluorescence imaging are powerful tools for visualizing neural activity during naturalistic behaviors, but the restricted field of view of first-generation  miniscopes limits the size of neural populations accessible for imaging. Here we describe a novel miniaturized mesoscope offering cellular-resolution imaging over areas spanning several millimeters in freely moving mice. This system enables comprehensive visualization of activity across entire brain regions or interactions across areas.

sentences: Mindfulness Training Alters Resting-State EEG Dynamics in Novice Practitioners via Mindful Breathing and Body-scan Mindfulness-based stress reduction (MBSR) has been proven to improve mental health and quality of life. This study examined how mindfulness training and various types of mindfulness practices altered brain activity. Specifically, the spectral powers of scalp electroencephalography (EEG) of the MBSR group who underwent an 8-week mindfulness training--including mindful breathing and body-scan--were evaluated and compared with those of the waitlist controls. Empirical results indicated that the long-term mindfulness intervention effect significantly elevated the resting-state beta powers and reduced resting-state delta powers in both practices; such changes were not observed in the waitlist control. Compared with mindful breathing, body-scanning resulted in an overall decline in EEG spectral powers at both delta and gamma bands among trained participants. Together with our preliminary data of expert mediators, the aforementioned spectral changes were salient after intervention, but mitigated along with expertise. Additionally, after receiving training, the MBSR groups mindfulness and emotion regulation levels improved significantly, which were correlated with the EEG spectral changes in the theta, alpha, and low-beta bands. This study elaborated the neurophysiological correlates of mindfulness practices, suggesting that MBSR might function as a unique internal processing that involves increased vigilant capability and induces alterations similar to other cognitive training.

sentences: A minimal yet flexible likelihood framework to assess correlated evolution An evolutionary process is reflected in the sequence of changes of any trait (e.g. morphological, molecular) through time. Yet, a better understanding of evolution would be procured by characterizing correlated evolution, or when two or more evolutionary processes interact. Many previously developed parametric methods often require significant computing time as they rely on the estimation of many parameters. Here we propose a minimal likelihood framework modelling the joint evolution of two traits on a known phylogenetic tree. The type and strength of correlated evolution is characterized by few parameters tuning mutation rates of each trait and interdependencies between these rates. The framework can be applied to study any discrete trait or character ranging from nucleotide substitution to gain or loss of a biological function. More specifically, it can be used to 1) test for independence between two evolutionary processes, 2) identify the type of interaction between them and 3) estimate parameter values of the most likely model of interaction. In its current implementation, the method takes as input a phylogenetic tree together with mapped discrete evolutionary events on it and then maximizes the likelihood for one or several chosen scenarios. The strengths and limits of the method, as well as its relative power when compared to a few other methods, are assessed using both simulations and data from 16S rRNA sequences in a sample of 54 {gamma}-enterobacteria. We show that even with datasets of fewer than 100 species, the method performs well in parameter estimation and in the selection of evolutionary scenario.

sentences: Structure-Functional-Selectivity Relationship Studies on A-86929 Analogs and Small biaryl Fragments toward Discovery of biased D1 agonists Dopamine regulates normal functions such as movement, reinforcement learning, and cognition, and its dysfunction has been implicated in multiple psychiatric and neurological disorders. Dopamine acts through the D1- (D1R and D5R) and D2-class (D2R, D3R and D4R) of seven transmembrane receptors, and activates both G-protein- and {beta}-arrestin-dependent signaling pathways, to mediate its physiological effects. Current dopamine receptor-based therapies are used to ameliorate motor deficits in Parkinsons disease, or as antipsychotic medications for schizophrenia. These drugs show efficacy for ameliorating only some symptoms caused by dopamine dysfunction and are plagued by debilitating side-effects. Studies in primates and rodents have shown that shifting the balance of dopamine receptor signaling towards the arrestin pathway can be beneficial for inducing normal movement, while reducing motor side-effects such as dyskinesias, and can be efficacious at enhancing cognitive function compared to balanced agonists. Several structure-activity-relationship (SAR) studies have embarked on discovering {beta}-arrestin-biased dopamine agonists, focused on D2 partial agonists, non-catechol D1 agonists, and mixed D1/D2R dopamine receptor agonists. Here, we describe an SAR study to identify novel D1R {beta}-arrestin biased ligands using A-86929, a high-affinity D1R catechol agonist, as a core scaffold. Previously described and novel analogs of A-86929 were synthesized and screened in vitro for structure-functional-selectivity relationships (SFSR) studies to identify chemical motifs responsible for {beta}-arrestin biased activity at both D1 and D2Rs. Most of the A-86929 analogs screened were G protein biased but none of them were exclusively arrestin-biased. Additionally, various catechol aryl fragments were designed and synthesized. Other compounds surveyed included hydroxyl and chloro analogs of dopamine to test for hydrogen bonding and ionic interactions. Some of these small molecular probes displayed weak bias towards the {beta}-arrestin pathway. Continued in-depth SFSR studies informed by structure determination, molecular modeling, and mutagenesis studies will facilitate discovery of potent and efficacious arrestin-biased dopamine receptor ligands.

sentences: Reciprocalspaceship: A Python Library for Crystallographic Data Analysis AO_SCPLOWBSTRACTC_SCPLOWX-ray crystallography is an invaluable technique for studying the atomic structure of macromolecules. Much of crystallographys success is due to the software packages developed to enable the automated processing of diffraction data. However, the analysis of unconventional diffraction experiments can still pose significant challenges--many existing programs are closed-source, sparsely documented, or are challenging to integrate with modern libraries for scientific computing and machine learning. Here we describe reciprocalspaceship, a Python library for exploring reciprocal space. It provides a tabular representation for reflection data from diffraction experiments that extends the widely-used pandas library with built-in methods for handling space group, unit cell, and symmetry-based operations. As we illustrate, this library facilitates new modes of exploratory data analysis while supporting the prototyping, development, and release of new methods.

sentences: "Playing mysterious": occurrence of Bio-duck calls in Brazil reveals a complex acoustic behaviour for the Antarctic minke whale (Balaenoptera bonaerensis) Acoustic methods can provide important data on the occurrence and distribution of migratory species. Information about Antarctic Minke whale (Balaenoptera bonaerensis) occurrence in the winter breeding grounds is scarce, mostly limited to old records from whaling stations before 1960s international moratory, such as Costinha Station in Northeastern Brazil (6{degrees} S / 34{degrees} W). This work describes the occurrence of the Antarctic minke whale (AMW) through Bio-duck acoustic detections in the Santos Basin, South-Southeastern Brazil (22{degrees} and 28{degrees} S / 42{degrees} and 48{degrees} W), registered between November 12 and December 19, 2015. AMW calls were detected for 12 days. We detected and classified 9 different Bio-duck calls in Brazilian coast waters, evidencing a high diverse acoustic behaviour for the minke whale breeding ground. This is the first study to describe the acoustic diversity of AMW vocalizations in lower latitudes, constituting important information to the conservation and management of cetaceans and their habitat. Therefore, our study presents the foremost acoustic evidence of the Antarctic minke whale in Brazil, utilizing high technological passive acoustic methods, such as autonomous underwater vehicle (SeaGlider) sampling.

sentences: Adjustment for SNPs instead of demographic and clinical covariates in GWAS With capabilities of sequencing ancient DNA to high coverage often limited by sample quality or cost, imputation of missing genotypes presents a possibility to increase power of inference as well as cost-effectiveness for the analysis of ancient data. However, the high degree of uncertainty often associated with ancient DNA poses several methodological challenges, and performance of imputation methods in this context has not been fully explored. To gain further insights, we performed a systematic evaluation of imputation of ancient data using Beagle 4.0 and reference data from phase 3 of the 1000 Genomes project, investigating the effects of coverage, phased reference and study sample size. Making use of five ancient samples with high-coverage data available, we evaluated imputed data with respect to accuracy, reference bias and genetic affinities as captured by PCA. We obtained genotype concordance levels of over 99% for data with 1x coverage, and similar levels of accuracy and reference bias at levels as low as 0.75x. Our findings suggest that using imputed data can be a realistic option for various population genetic analyses even for data in coverage ranges below 1x. We also show that a large and varied phased reference set as well as the inclusion of low-to moderate-coverage ancient samples can increase imputation performance, particularly for rare alleles. In-depth analysis of imputed data with respect to genetic variants and allele frequencies gave further insight into the nature of errors arising during imputation, and can provide practical guidelines for post-processing and validation prior to downstream analysis.

sentences: Physiological roles of short-chain and long-chain menaquinones (vitamin K2) in Lactococcus cremoris Lactococcus cremoris and L. lactis are well-known for their occurrence and applications in dairy fermentations, but their niche extends to a range of natural and food production environments. L. cremoris and L. lactis produce MKs (vitamin K2), mainly as the long-chain forms represented by MK-9 and MK-8, and a detectable amount of short-chain forms represented by MK-3. The physiological significance of the different MK forms in the lifestyle of these bacterial species has not been investigated extensively. In this study, we used L. cremoris MG1363 to construct mutants producing different MK profiles by deletion of genes encoding (i) a menaquinone-specific isochorismate synthase, (ii) a geranyltranstransferase and (iii) a prenyl diphosphate synthase. These gene deletions resulted in (i) a non-MK producer ({Delta}menF), (ii) a presumed MK-1 producer ({Delta}ispA) and (iii) a MK-3 producer ({Delta}llmg_0196), respectively. By examining the phenotypes of the MG1363 wildtype strain and respective mutants, including biomass accumulation, stationary phase survival, oxygen consumption, primary metabolites, azo dye/copper reduction, and proteomes, under aerobic, anaerobic and respiration-permissive conditions, we could infer that short-chain MKs like MK-1 and MK-3 are preferred to mediate extracellular electron transfer and reaction with extracellular oxygen, while the long-chain MKs like MK-9 and MK-8 are more efficient in aerobic respiratory electron transport chain. The different electron transfer routes mediated by short-chain and long-chain MKs likely support growth and survival of L. cremoris in a range of (transiently) anaerobic and aerobic niches including food fermentations, highlighting the physiological significance of diverse MKs in L. cremoris.

sentences: Comparison of Neutralizing Antibody Titers Elicited by mRNA and Adenoviral Vector Vaccine against SARS-CoV-2 Variants The increasing prevalence of SARS-CoV-2 variants has raised concerns regarding possible decreases in vaccine efficacy. Here, neutralizing antibody titers elicited by mRNA-based and an adenoviral vector-based vaccine against variant pseudotyped viruses were compared. BNT162b2 and mRNA-1273-elicited antibodies showed modest neutralization resistance against Beta, Delta, Delta plus and Lambda variants whereas Ad26.COV2.S-elicited antibodies from a significant fraction of vaccinated individuals were of low neutralizing titer (IC50 <50). The data underscore the importance of surveillance for breakthrough infections that result in severe COVID-19 and suggest the benefit of a second immunization following Ad26.COV2.S to increase protection against the variants.

sentences: Molluscan dorsal-ventral patterning relying on bmp2/4 and chordin provides insights into spiralian development and bilaterian evolution Although a conserved mechanism relying on bmp2/4 and chordin is suggested in animal dorsal-ventral (DV) patterning, this mechanism has not been reported in spiralians, one of the three major clades of bilaterians. Studies on limited spiralian representatives have suggested markedly diverse DV patterning mechanisms, a considerable amount of which no longer deploy BMP signaling. Here, we showed that bmp2/4 and chordin regulated DV patterning in the mollusk Lottia goshimai, which was predicted in spiralians but not reported before. In the context of the diverse reports in spiralians, it conversely represents a relatively unusual case. We then showed that bmp2/4 and chordin coordinated to mediate signaling from the D-quadrant organizer to induce the DV axis, among which chordin transferred breakdown-of-symmetry information. Further investigations on the L. goshimai embryos with influenced DV patterning suggested roles of BMP signaling in regulating the localization of the blastopore and the organization of the nervous system, indicating a cooption of DV patterning and the transition of these key characteristics at the origin of bilaterians. These findings provide insights into the evolution of animal DV patterning, the unique development mode of spiralians driven by the D-quadrant organizer, and the evolution of bilaterian body plans.

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Seasonal dynamics of phytoplankton and bacterioplankton at the ultra-oligotrophic southeastern Mediterranean Sea The Eastern Mediterranean Sea (EMS) is a poorly studied ultra-oligotrophic marine environment, dominated by small-size phyto- and bacterioplankton. Here, we describe the dynamics of a single annual cycle (2018-19) of phyto- and bacterioplankton (abundances, pigments and productivity) in relation to the physical and chemical conditions in the photic water column at an offshore EMS site (Station THEMO-2, [~]1,500m depth, 50km offshore). We show that phytoplankton biomass (as chlorophyll a), primary and bacterial productivity differed between the mixed winter (January-April) and the thermally stratified (May-December) periods. Prochlorococcus and Synechococcus numerically dominated the picophytoplankton populations, with each clade revealing different temporal and depth changes indicative to them, while pico-eukaryotes (primarily haptophytes) were less abundant, yet likely contributed significant biomass. Estimated primary productivity ([~]32 gC m-2 y-1) was lower compared with other well-studied oligotrophic locations, including the north Atlantic and Pacific (BATS and HOT observatories), the western Mediterranean (DYFAMED observatory) and the Red Sea, and was on-par with the ultra-oligotrophic South Pacific Gyre. In contrast, integrated bacterial production ([~]11 gC m-2 y-1) was similar to other oligotrophic locations. Phytoplankton seasonal dynamics were similar to those at BATS and the Red Sea, suggesting an observable effect of winter mixing in this ultra-oligotrophic location. These results highlight the ultra-oligotrophic conditions in the EMS and provide, for the first time in this region, a full-year baseline and context to ocean observatories in the region.

HighlightsO_LIBacterioplankton dynamics were assessed monthly in the Eastern Mediterranean Sea
C_LIO_LISmall-sized picophytoplankton numerically dominated the phytoplankton community
C_LIO_LISeasonal phytoplankton dynamics are similar to BATS and Red Sea, but not to HOT
C_LIO_LIAnnual primary productivity is among the lowest in the worlds oceans
C_LIO_LIBacterial to primary production ratio is higher than most oligotrophic seas
C_LI

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Intratumoral Heterogeneity Promotes Collective Cancer Invasion Through NOTCH1 Variation Cellular and molecular heterogeneity within tumors has long been associated with the progression of cancer to an aggressive phenotype and a poor prognosis. However, how such intratumoral heterogeneity contributes to the invasiveness of cancer is largely unknown. Here, using a multidisciplinary approach, we investigate the interaction between molecular subtypes within bladder microtumors and the corresponding effects on their invasiveness. Our results reveal heterogeneous microtumors formed by multiple molecular subtypes possess enhanced invasiveness compared to individual cells, even when both cells are not invasive individually. To examine the molecular mechanism of intratumoral heterogeneity mediated invasiveness, live single cell biosensing, RNA interference, and CRISPR-Cas9 gene editing approaches were applied to investigate and control the composition of the microtumors. An agent-based computational model was also developed to evaluate the influence of NOTCH1 variation on DLL4 expression within a microtumor. The data indicate that variation in NOTCH1 expression can lead to upregulation of DLL4 expression within the microtumor and enhancement of microtumor invasiveness. Overall, our results reveal a novel mechanism of heterogeneity mediated invasiveness through intratumoral variation of gene expression.

Summary statementThis study reveals a mechanism that Notch1 variation, instead of the average value, promotes the invasiveness of microtumor, providing a link between intratumoral heterogeneity and collective cancer invasion.

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Genome size evolution in the diverse insect order Trichoptera BackgroundGenome size is implicated in form, function, and ecological success of a species. Two principally different mechanisms are proposed as major drivers of eukaryotic genome evolution and diversity: Polyploidy (i.e., whole genome duplication: WGD) or smaller duplication events and bursts in the activity of repetitive elements (RE). Here, we generated de novo genome assemblies of 17 caddisflies covering all major lineages of Trichoptera. Using these and previously sequenced genomes, we use caddisflies as a model for understanding genome size evolution in diverse insect lineages.

ResultsWe detect a ~14-fold variation in genome size across the order Trichoptera. We find strong evidence that repetitive element (RE) expansions, particularly those of transposable elements (TEs), are important drivers of large caddisfly genome sizes. Using an innovative method to examine TEs associated with universal single copy orthologs (i.e., BUSCO genes), we find that TE expansions have a major impact on protein-coding gene regions, with TE-gene associations showing a linear relationship with increasing genome size. Intriguingly, we find that expanded genomes preferentially evolved in caddisfly clades with a higher ecological diversity (i.e., various feeding modes, diversification in variable, less stable environments).

ConclusionOur findings provide a platform to test hypotheses about the potential evolutionary roles of TE activity and TE-gene associations, particularly in groups with high species, ecological, and functional diversities.

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Neural dynamics of illusory tactile pulling sensations The sensation of directional forces and their associated sensorimotor commands are inextricably intertwined, complicating the identification of brain circuits responsible for tactile pulling sensations. One hypothesis is that, like tactile frequency discrimination, pulling sensations are generated by early sensory-frontal activity. Alternatively, they may be generated later in the somatosensory association cortex. To dissociate these accounts and uncouple the pulling sensation from unrelated but correlated sensory and motor processing, we combined high-density EEG with an oddball paradigm and asymmetric vibration, which creates an illusory sensation of the hand being directionally pulled. Oddballs that created a pulling sensation in the opposite direction to common stimuli were compared to the same oddballs in the context of neutral common stimuli (symmetric vibration) and to neutral oddballs. Brain responses to having directional pulling expectations violated by directional stimuli were therefore isolated. Contrary to the sensory-frontal account, frontal N140 brain activity was actually larger for neutral than pulling oddballs. Instead, pulling sensations were associated with amplitude and latency modulations of midline P200 and P3b potentials, and specifically, to contralateral parietal lobe activity 280ms post-stimulus. The timing of this activity suggested pulling sensations involve spatial processing, such as tactile remapping between coordinate frames. Source localization showed this activity to be centered on the postcentral sulcus, superior parietal lobule and intraparietal sulcus, suggesting that pulling sensations arise via the processing of body position, tactile orientation and peripersonal space. Our results demonstrate how tactile illusions can uniquely disambiguate parietal contributions to somatosensation by removing unrelated sensory processing.

Significance statementThe neural mechanisms of tactile pulling sensations are poorly understood. Competing early sensory-frontal and later somatosensory association cortex accounts are hard to dissociate due to confounding sensory and motor signals present when forces are applied to the skin. Here, we used EEG and a novel asymmetric vibration approach to induce an illusory pulling sensation, which circumvents these issues. We found that pulling sensations were associated with parietal lobe activity 280ms post-stimulus and modulations of the P200. The timing and location of this activity suggested that pulling sensations necessitate spatial processing and supported a somatosensory association cortex account of the pulling sensation.

sentences: A nesprin-4/kinesin-1 cargo model for nuclear positioning in cochlear outer hair cells Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, nesprin-4 (encoded by Syne4) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in-vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

sentences: Optimized liquid and gas phase fractionation increase HLA-peptidome coverage for primary cell and tissue samples Mass spectrometry is the most effective method to directly identify peptides presented on HLA molecules. However, current standard approaches often require many millions of cells for input material to achieve high coverage of the immunopeptidome and are therefore not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples off-line followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared to samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8,107 distinct peptides starting with as few as 100 million cells or 150 milligrams of wet weight tumor tissue. This increased sensitivity can improve HLA binding prediction algorithms and enable detection of clinically relevant epitopes such as neoantigens.

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Knockout of Sorbs2 in Cardiomyocytes Leads to Dilated Cardiomyopathy in Mice 2.RationaleSorbs2 is a cardiomyocyte-enriched, cytoskeletal adaptor protein, and given some evidence for its dysregulated expression in failing hearts, there is growing interest in understanding its roles in cardiac biology and disease. While Sorbs2 global knockout mice display lethal cardiomyopathy with severe arrhythmias, the underlying mechanisms remain unclear, and whether this results from intrinsic loss of Sorbs2 in cardiomyocytes is unknown, as Sorbs2 is also well-expressed in the nervous system and vasculature. In addition, the potential relevance of Sorbs2 in human cardiomyopathy remains underexplored.

ObjectiveTo characterize the effects and potential underlying mechanisms of cardiomyocyte- specific deletion of Sorbs2 on cardiac structure and function in mice, and to further examine Sorbs2 dysregulation in failing hearts and explore potential links between Sorbs2 genetic variations and human cardiovascular disease phenotypes.

Methods and ResultsWe report that myocardial Sorbs2 expression is consistently upregulated in humans with ischemic and idiopathic cardiomyopathies, and in experimental animal models of heart failure (HF). We generated mice with cardiomyocyte-specific loss of Sorbs2 (Sorbs2-cKO) and found early atrial and ventricular conduction abnormalities, despite unaltered expression of primary action potential ion channels and gap junction proteins. At mid-life, Sorbs2-cKO mice exhibit impaired cardiac contractility with cardiomyofibers failing to maintain adequate mechanical tension. As a result, these mice develop progressive diastolic and systolic dysfunction, enlarged cardiac chambers, and die with congestive HF at approximately one year of age. Comprehensive survey of potential underlying mechanisms revealed that Sorbs2-cKO hearts exhibit defective microtubule polymerization and compensatory upregulation of structural proteins desmin, vinculin, and tubulins. Finally, consistent with our observations in mice, we identified suggestive links between Sorbs2 genetic variants and related human cardiac phenotypes, including conduction abnormalities, atrial enlargement, and dilated cardiomyopathy.

ConclusionsOur studies show that Sorbs2 is essential for maintaining cytoskeletal structural integrity in cardiomyocytes likely through strengthening the interactions between microtubules and other structural proteins at crosslink sites. Overall, this study provides key insights into the critical role for Sorbs2 in cardiomyocytes and likely other cell types in maintaining normal cardiac structure and function and highlights its potential clinical relevance.

sentences: The Effect of A1/A2 Reactive Astrocyte Expression on Hydrocephalus Shunt Failure Understanding the composition characteristics of the glial scar contributing to the high failure rate of neuroprosthetic devices implanted in the brain has been limited, to date, with the evaluation of cells, tissue, and biomarkers obstructing the implant. However, there remains a critical knowledge gap in gene expression profiles of the obstructing cells. This first-time study investigates the phenotypic expression specific to astrocyte scarring from those cells on hydrocephalus shunt surfaces at the time of failure, aimed at the development of therapeutic approaches to target reactive astrocytes for improved functional outcome. Recent evidence has indicated that the tissue obstructing shunts is over 80% inflammatory, with a more exaggerated astrocytic response. To understand how to mitigate the astrocyte immune response to shunts, we performed gene expression profiling of the C3 and EMP1 genes to quantify if astrocytes were classically activated and pro-inflammatory (A1) or alternatively activated and anti-inflammatory (A2), respectively. Shunt catheters were removed from patients at the time of failure and categorized by obstructed vs non-obstructed shunts. RNAscope fluorescent in situ hybridization and quantitative PCR analysis of the C3 and EMP1 expressed genes revealed that a heterogeneous mixed population of both the A1 and A2 reactive phenotype exist on the shunt surface. However, the number of A2 reactive astrocytes are significantly higher on obstructed shunts compared to A1 reactive astrocytes. ELISA data also confirmed higher levels of IL-6 for obstructed shunts involved in A2 reactive astrocyte proliferation and glial scar formation on the shunt surface. Since TNF- and IL-1{beta} propel resting astrocytes into an A2 reactive state, by simply blocking the secretion or action of these cytokines, astrocyte activation and attachment on obstructing shunts could be inhibited.

sentences: Acoustic frequency-dependent physical mechanism of sub-MHz ultrasound neurostimulation Ultrasound is an innovative physical modality allowing non-invasive and reversible modulation of neural circuit activity in the brain with high spatial resolution. Despite growing interest in clinical applications, the safe and effective use of ultrasound neuromodulation has been limited by a lack of understanding of the physical mechanisms underlying its effects. Here, we demonstrate acoustic frequency-dependent physical effects that underlie ultrasound neuromodulation, where cavitation and radiation forces are the dominant sources of low- and high-frequency stimulation, respectively. We used 39.5 kHz and 500 kHz acoustic frequencies to stimulate cultured neural and glial cells, excised from rat cortex, to study acoustic frequency-dependent neural responses. We demonstrate increased evoked responses due to increased cavitation activity at the 39.5 kHz acoustic frequency. In contrast, notable cavitation activity was not detected at 500 kHz despite detection of evoked responses. Our work highlights the dependence of ultrasound neuromodulation on acoustic frequencies, with different physical effects underlying cell responses to low and high sub-MHz acoustic frequency ranges.

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Tracking extracellular vesicle (EV) cargo as a platform for studying EVomics, signaling, and targeting in vivo Extracellular vesicle (EV)-based signaling is a challenge to study, due to EV small size, heterogeneity, and limited information on cargo content in vivo. We present Caenorhabditis elegans as a discovery platform that allows single EV tracking from source to target tissue in living animals. We enriched ciliary EVs using GFP-tagged PKD-2 cargo followed by mass spectrometry analysis to identify 2,888 cargo candidates. By integrating our dataset with single-cell transcriptomic data, we identified EV cargo produced by individual neurons and other cell and tissue types. A single cilium produces multiple EVs with distinct protein content. Ciliary EVs carry nucleic acid binding proteins. We observed transfer of EV cargo from the male reproductive tract to the hermaphrodite uterus during mating, a direct demonstration of animal-to-animal EV targeting.

One-Sentence SummaryHere we present a discovery platform for studying animal extracellular vesicle composition and biogenesis.

sentences: Motivated semantic control: Exploring the effects of extrinsic reward and self-reference on semantic retrieval in semantic aphasia Recent insights show increased motivation can benefit executive control, but this effect has not been explored in relation to semantic cognition. Patients with deficits of controlled semantic retrieval in the context of semantic aphasia (SA) after stroke may benefit from this approach since  semantic control is considered an executive process. Deficits in this domain are partially distinct from domain-general deficits of cognitive control. We assessed the effect of both extrinsic and intrinsic motivation in healthy controls and semantic aphasia patients. Experiment 1 manipulated extrinsic reward using high or low levels of points for correct responses during a semantic association task. Experiment 2 manipulated the intrinsic value of items using self-reference; allocating pictures of items to the participant ( self) or researcher ( other) in a shopping game before people retrieved their semantic associations. These experiments revealed that patients, but not controls, showed better performance when given an extrinsic reward, consistent with the view that increased external motivation may help to ameliorate patients semantic control deficits. However, while self-reference was associated with better episodic memory, there was no effect on semantic retrieval. We conclude that semantic control deficits can be reduced when extrinsic rewards are anticipated; this enhanced motivational state is expected to support proactive control, for example, through the maintenance of task representations. It may be possible to harness this modulatory impact of reward to combat the control demands of semantic tasks in SA patients.

sentences: Distinct complexes containing the cytosolic type III secretion system ATPase resolved by 3D single-molecule tracking in live Yersinia enterocolitica The membrane-embedded injectisome, the structural component of the virulence-associated type III secretion system (T3SS), is used by gram-negative bacterial pathogens to inject species-specific effector proteins into eukaryotic host cells. The cytosolic injectisome proteins are required for export of effectors and display both stationary, injectisome-bound populations as well as freely-diffusing cytosolic populations. How the cytosolic injectisome proteins interact with each other in the cytosol and associate with membrane-embedded injectisomes remains unclear. Here, we utilize 3D single-molecule tracking to resolve distinct cytosolic complexes of injectisome proteins in living Yersinia enterocolitica cells. Tracking of the eYFP-labeled ATPase, YeSctN, and its regulator, YeSctL, reveals that these proteins form a cytosolic complex with each other and then further with YeSctQ. YeSctNL and YeSctNLQ complexes can be observed in both in wild type cells and in{Delta} sctD mutants, which cannot assemble injectisomes. In{Delta} sctQ mutants, the relative abundance of the YeSctNL complex is considerably increased. These data indicate that distinct cytosolic complexes of injectisome proteins can form prior to injectisome binding - a feature that could contribute to injectisome functional regulation.

sentences: Object manifold geometry across the mouse cortical visual hierarchy Despite variations in appearance we robustly recognize objects. Neuronal populations responding to objects presented under varying conditions form object manifolds and hierarchically organized visual areas untangle pixel intensities into linearly decodable object representations. However, the associated changes in the geometry of object manifolds along the cortex remain unknown. Using home cage training we showed that mice are capable of invariant object recognition. We simultaneously recorded the responses of thousands of neurons to measure the information about object identity across the visual cortex and found that lateral areas LM, LI and AL carry more linearly decodable object information compared to other visual areas. We applied the theory of linear separability of manifolds, and found that the increase in classification capacity is associated with a decrease in the dimension and radius of the object manifold, identifying the key features in the geometry of the population neural code that enable invariant object coding.

sentences: Artisanal and farmers bread making practices differently shape fungal species diversity in French sourdoughs Preserving microbial diversity in food systems is one of the many challenges to be met to achieve food security and quality. Although industrialization led to the selection and spread of specific fermenting microbial strains, there are still ongoing artisanal processes that may allow the conservation of a wider diversity of microbial species. We examined whether the diversity of artisanal practices could lead to an increased level in fungal species diversity for bread making. We used an interdisciplinary participatory research approach including bakers, psycho-sociologists and microbiologists to analyse French bread making practices and describe fungal communities in naturally fermented sourdough of 27 bakers and 12 farmer bakers. Their bread making practices were classified in two groups: the farmer- like practices group and the artisanal-like practices group. Surprisingly, the well-known bakery yeast, Saccharomyces cerevisiae, was dominant (i.e. with a relative abundance over 50%) in only 24% of sourdoughs while other yeast species of the closely related Kazachstania genus were dominant in 54% of sourdoughs. Bread making practices were found to drive the distribution of yeast species. The most remarkable difference was the occurrence of Kazachstania humilis in sourdoughs made with artisanal- like practices and the occurrence of Kazachstania bulderi in sourdoughs made with farmer-like practices. Phenotyping of these two species in laboratory sourdough mimicking media revealed signatures of domestication for K. humilis but not for K. bulderi. Overall, our results showed that preserving bread making practices diversity allows the preservation of a higher taxonomic and phenotypic diversity in microbial communities.

sentences: Antagonistic inhibitory subnetworks control cooperation and competition across cortical space The cortical microcircuit can dynamically adjust to dramatic changes in the strength, scale, and complexity of its input. In the primary visual cortex (V1), pyramidal cells (PCs) integrate widely across space when signals are weak, but narrowly when signals are strong, a phenomenon known as contrast-dependent surround suppression. Theoretical work has proposed that local interneurons could mediate a shift from cooperation to competition of PCs across cortical space, underlying this computation. We combined calcium imaging and electrophysiology to constrain a stabilized supralinear network model that explains how the four principal cell types in layer 2/3 (L2/3) of mouse V1- somatostatin (SST), parvalbumin (PV), and vasoactive intestinal peptide (VIP) interneurons, and PCs- transform inputs from layer 4 (L4) PCs to encode drifting gratings of varying size and contrast. Using bidirectional optogenetic perturbations, we confirmed key predictions of the model. Our data and modeling showed that recurrent amplification drives a transition from a positive PC[->]VIP{dashv}SST{dashv}PC feedback loop at small size and low contrast to a negative PC[->]SST{dashv}PC feedback loop at large size and high contrast to contribute to this flexible computation. This may represent a widespread mechanism for gating competition across cortical space to optimally meet task demands.

sentences: Transferability of Geometric Patterns from Protein Self-Interactions to Protein-Ligand Interactions There is significant interest in developing machine learning methods to model protein-ligand interactions but a scarcity of experimentally resolved protein-ligand structures to learn from. Protein self-contacts are a much larger source of structural data that could be leveraged, but currently it is not well understood how this data source differs from the target domain. Here, we characterize the 3D geometric patterns of protein self-contacts as probability distributions. We then present a flexible statistical framework to assess the transferability of these patterns to protein-ligand contacts. We observe that the level of transferability from protein self-contacts to protein-ligand contacts depends on contact type, with many contact types exhibiting high transferability. We then demonstrate the potential of leveraging information from these geometric patterns to aid in ligand pose-selection problems in protein-ligand docking. We publicly release our extracted data on geometric interaction patterns to enable further exploration of this problem.

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SWAP1-SFPS-RRC1 splicing factor complex modulates pre-mRNA splicing to promote photomorphogenesis in Arabidopsis Light signals perceived by a group of photoreceptors have profound effects on the physiology, growth, and development of plants. The red/far-red light absorbing phytochromes modulate these aspects by intricately regulating gene expression at multiple levels. Previously, we reported that two splicing factors SFPS (SPLICING FACTOR FOR PHYTOCHROME SIGNALING) and RRC1 (REDUCED RED LIGHT RESPONSES IN CRY1CRY2 BACKGROUND 1), interact with photoactivated phyB to regulate light-mediated pre-mRNA alternative splicing (AS). Here, we report the identification and functional characterization of an RNA binding splicing factor, SWAP1 (SUPPRESSOR-OF-WHITE-APRICOT/SURP RNA-BINDING DOMAIN-CONTAINING PROTEIN1). Loss-of-function swap1-1 mutant is hyposensitive to red light and exhibits a day light-independent early flowering phenotype. SWAP1 physically interacts with both SFPS and RRC1 in a light-independent manner and forms a ternary complex. In addition, SWAP1 also physically interacts with photoactivated phyB and colocalizes with nuclear phyB photobodies. Deep RNA-seq analyses show that SWAP1 regulates the gene expression and pre-mRNA alternative splicing of a large number of genes including those involved in plant responses to light signaling. A comparison with SFPS- and RRC1-regulated events shows that all three splicing factors coordinately regulate the alternative splicing of a subset of genes. Collectively, our study uncovered the function of a new splicing factor, which interacts with photoactivated phyB, in modulating light-regulated development in plants.

SIGNIFICANCERegulation of transcription and pre-mRNA alternative splicing is essential for the transcript diversity and modulation of light signaling in plants. Although several transcription factors involved in light signaling have been discovered and characterized in-depth, only a few splicing factors have been shown to be involved in the regulation of light signaling pathways. In this study, we describe the identification and characterization of a new splicing factor SWAP1, which interact with two previously characterized splicing factors, SFPS and RRC1, forming a ternary complex. We show that, like SFPS and RRC1, SWAP1 also interacts with photoactivated phyB, and consistently, swap1 seedlings are hyposensitive to red light. SWAP1 modulates alternative splicing of a large number of genes and a subset of these genes are coordinately regulated by SFPS, RRC1 and SWAP1. These results highlight the importance of not only the transcription factors but also the phyB-interacting splicing factors in light-regulated plant development.

sentences: Interaction of thyroid hormones and gonadotropin inhibitory hormone in the multifactorial control of zebrafish (Danio rerio) spermatogenesis Reproduction is under multifactorial control of neurohormones, pituitary gonadotropins, as well as a number of gonadal hormones including sex steroids and growth factors. Gonadotropin-inhibitory hormone (Gnih), a novel RFamide neuropeptide, was shown to be involved in the control of pituitary gonadotropin production, as well as being involved as a paracrine factor in the regulation of gonadal function. In this context, recent studies have demonstrated that Gnih inhibited gonadotropin-induced spermatogenesis in the zebrafish testicular explants. Thyroid hormones are known to interact with the reproductive axis, and are, in particular, involved in the regulation of testicular function. Based on this background, we investigated the interaction between Gnih and thyroid hormones in the control of zebrafish spermatogenesis. To this end, zebrafish adult males were treated with the goitrogen methimazole (1mM for 21 days) in order to generate a hypothyroid model organism. Subsequently, a factorial design using an ex vivo testis culture system in combination with histomorphometrical and FACScan cell cycle analyses were adopted. Our results showed that methimazole treatment affected both basal and gonadotropin-induced spermatogenesis, in particular, meiosis and spermiogenesis. Moreover, the goitrogen treatment nullified the inhibitory actions of Gnih on the gonadotropin-induced spermatogenesis, specifically in the haploid cell population. We have demonstrated that thyroid hormones interaction with gonadotropin and Gnih are important components for the regulation of zebrafish spermatogenesis. The results provide a support for the hypothesis that thyroid hormones are important contributors in multifactorial control of spermatogenesis in zebrafish.

sentences: Modelling 3D tumour microenvironment in vivo: a tool to predict cancer fate Recently, many studies demonstrated the fundamental role of tumour microenvironment (TME) in cancer progression. Here, we describe a state-of-the-art method to visualize in 3D the behaviour of tumours in zebrafish embryos. We highlight two major actors of TME, macrophages and vessels. This valuable tool is transposable to Patients Derived Xenograft imaging in order to predict the fate of malignant tumours according to the dynamics of their TME.

sentences: Nulliparity affects the expression of a limited number of genes and pathways in Day 8 equine embryos Nulliparous mares produce lighter and smaller foals compared to mares having previously foaled, with effects observed at least until 4 months of age. The need for a first gestation priming for the uterus to reach its full capacity has been proposed to explain this observation. Embryo developmental defects could be hypothesized but effects of maternal parity on the embryo have only been described once, in old mares, thus combining effects of parity and old age. The aim of this study was to determine effects of mare parity on embryo gene expression. Day-8 post ovulation blastocysts were collected from young (5/6 years old) nulliparous (YN, N=6) or multiparous (YM, N=4) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure (TE_part) or inner-cell-mass-enriched (ICMandTE) trophoblast were obtained by embryo bisection for RNA sequencing (paired end, non-oriented, Illumina, NextSeq500). Deconvolution was performed on the ICMandTE dataset. Differential expression, with embryo sex and diameter as cofactors and gene set enrichment analysis (GO BP, KEGG, REACTOME databases) were performed using a false discovery rate <0.05 cutoff. Only a few genes were altered (ICM: n=18; TE: n=6) but several gene sets were perturbed (ICM: n=62; TE: n=50) by maternal parity. In YM, only pathways related to transcription, RNA processing and vesicle transport functions were enriched in the ICM whereas only pathways related to RNA localization were enriched in TE. In YN, while only gene sets related to ribosomes and extracellular matrix were enriched in the ICM, functions related to energy and lipid metabolism, lipid transport and interleukin-1 signaling were enriched in the TE. In conclusion, several genes and pathways are affected in embryos collected from nulliparous mares, with different effects on TE and ICM. Embryo development is altered in nulliparous mares, which could partially explain the term phenotype. Whether differences in gene expression result/induce poor embryo-maternal communication remains to be determined.

sentences: DNA Breaks and Gaps Target Retroviral Integration Integration into a host genome is essential for retrovirus infection and is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and reverse transcribed (RT) copy DNA (cDNA). Previous studies demonstrated DNA site recognition limited intasome integration. Using single molecule Forster resonance energy transfer (smFRET), we show Prototype Foamy Virus (PFV) intasomes pause at DNA strand breaks and gaps. The break/gap discontinuities are similar to base excision repair (BER) lesion-processing intermediates, which affect retrovirus integration in vivo. Pausing targeted site-directed integration at the break/gap without inducing intasome conformational alterations. An 8-oxo-guanine lesion normally processes by BER and a G/T mismatch or a +T nucleotide insertion that induce flexibility or a bend in the DNA backbone did not promote intasome pausing or targeted integration. These results suggest that repair intermediates can modulate dynamic intasome-DNA interactions which target retroviral integration.

sentences: The return of the rings: evolutionary role of aromatic residues in liquid-liquid phase separation Aromatic residues appeared relatively late in the evolution of protein sequences. They stabilize the hydrophobic core of globular proteins and are typically absent from intrinsically disordered regions (IDRs). However, recent advances in protein liquid-liquid phase separation (LLPS) studies have shown that aromatic residues in IDRs often act as important "stickers", promoting multivalent interactions and the formation of higher-order oligomers. To reconcile this apparent contradiction, we compared levels of sequence disorder in RNA binding proteins and the human proteome and found that aromatic residues appear more frequently than expected in the IDRs of RNA binding proteins, which are often found to undergo LLPS. Phylogenetic analysis shows that aromatic residues are highly conserved among chordates, highlighting their importance in LLPS-driven functional assembly. These results suggest therefore that aromatic residues have contributed twice to evolution: in stabilizing structured proteins and in the assembly of biomolecular condensates.

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Rule-based and stimulus-based cues bias auditory decisions via different computational and physiological mechanisms Expectations, such as those arising from either learned rules or recent stimulus regularities, can bias subsequent auditory perception in diverse ways. However, it is not well understood if and how those diverse effects depend on the source of the expectations. Further, it is unknown whether these different sources of bias use the same or different computational and physiological mechanisms. We examined how rule-based and stimulus-based expectations influenced behavior and pupil-linked arousal, a marker of certain forms of expectation-based processing, of human subjects performing an auditory frequency-discrimination task. Rule-based cues biased choices and response times (RTs) toward the more-probable stimulus. In contrast, stimulus-based cues had a complex combination of effects, including choice and RT biases toward and away from the frequency of recently presented stimuli. These different behavioral patterns also had: 1) distinct computational signatures, including different modulations of key components of a novel form of a drift-diffusion decision model and 2) distinct physiological signatures, including substantial bias-dependent modulations of pupil size in response to rule-based but not stimulus-based cues. These results imply that different sources of expectations can modulate auditory processing via distinct mechanisms: one that uses arousal-linked, rule-based information and another that uses arousal-independent, stimulus-based information to bias the speed and accuracy of auditory perceptual decisions.

Author summaryPrior information about upcoming stimuli can bias our perception of those stimuli. Whether different sources of prior information bias perception in similar or distinct ways is not well understood. We compared the influence of two kinds of prior information on tone-frequency discrimination: rule-based cues, in the form of explicit information about the most-likely identity of the upcoming tone; and stimulus-based cues, in the form of sequences of tones presented before the to-be-discriminated tone. While both types of prior information biased auditory decision-making, they demonstrated distinct behavioral, computational, and physiological signatures. Our results suggest that the brain processes prior information in a form-specific manner rather than utilizing a general-purpose prior. Such form-specific processing has implications for understanding decision biases real-world contexts, in which prior information comes from many different sources and modalities.

sentences: Regulation of adult neurogenesis and neuronal differentiation by Neural Cell Adhesion Molecule 2 (NCAM2) Adult neurogenesis persists in mammals in the neurogenic zones where newborn neurons are incorporated into existing neuronal circuits. Relevant molecular elements of the neurogenic niches include the family of Cell Adhesion Molecules (CAM), which participate in signal transduction and regulate radial glial progenitors (RGPs) survival, division and differentiation. The Neural Cell Adhesion Molecule 2 (NCAM2) is expressed in brain development and in adult stages, and controls dendrite arborisation and synaptic formation and maintenance during development. Nevertheless, the role of NCAM2 in neurogenesis and lineage progression is not well understood. Here we analyse the functions of NCAM2 in the regulation of RGPs in adult neurogenesis in the dentate gyrus and during corticogenesis, by using different lentiviral-mediated genetic approaches to modulate its expression, both in vivo and in vitro. First, we characterized the expression of NCAM2 among the main actors of the neurogenic process revealing different levels of NCAM2 amid the progression of RGPs and the formation of juvenile neurons. Further, we show that overexpression of NCAM2 arrest infected cells in a RGP-like state, with characteristic morphological, immunocytochemical and electron microscopy features. In contrast, NCAM2 overexpression in embryonic cortical progenitors does not seems to alter cell fate, but causes transient migration deficits. These results reveal a differential role of NCAM2 in the regulation of adult and embryonic RGPs, and specifically, a significant implication of NCAM2 in the regulation and progression of RGPs during adult neurogenesis in the hippocampus.

sentences: Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors. In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits.

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An automated method for precise axon reconstruction from recordings of high-density micro-electrode arrays Neurons communicate with each other by sending action potentials through their axons. The velocity of axonal signal propagation describes how fast electrical action potentials can travel, and can be affected in a human brain by several pathologies, including multiple sclerosis, traumatic brain injury and channelopathies. High-density microelectrode arrays (HD-MEAs) provide unprecedented spatio-temporal resolution to extracellularly record neural electrical activity. The high density of the recording electrodes enables to image the activity of individual neurons down to subcellular resolution, which includes the propagation of axonal signals. However, axon recon-struction, to date, mainly relies on a manual approach to select the electrodes and channels that seemingly record the signals along a specific axon, while an automated approach to track multiple axonal branches in extracellular action-potential recordings is still missing.

In this article, we propose a fully automated approach to reconstruct axons from extracellular electrical-potential landscapes, so-called "electrical footprints" of neurons. After an initial electrode and channel selection, the proposed method first constructs a graph, based on the voltage signal amplitudes and latencies. Then, the graph is interrogated to extract possible axonal branches. Finally, the axonal branches are pruned and axonal action-potential propagation velocities are computed.

We first validate our method using simulated data from detailed reconstructions of neurons, showing that our approach is capable of accurately reconstructing axonal branches. We then apply the reconstruction algorithm to experimental recordings of HD-MEAs and show that it can be used to determine axonal morphologies and signal-propagation velocities at high throughput.

We introduce a fully automated method to reconstruct axonal branches and estimate axonal action-potential propagation velocities using HD-MEA recordings. Our method yields highly reliable and reproducible velocity estimations, which constitute an important electrophysiological feature of neuronal preparations.

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The Molecular Subtyping Resource (MouSR): a user-friendly tool for rapid biological discovery from human or mouse transcriptional data Generation of transcriptional data has dramatically increased in the last decade, driving the development of analytical algorithms that enable interrogation of the biology underpinning the profiled samples. However, these resources require users to have expertise in data wrangling and analytics, reducing opportunities for biological discovery by "wet-lab" users with a limited programming skillset. Although commercial solutions exist, costs for software access can be prohibitive for academic research groups.

To address these challenges, we have developed an open source and user-friendly data analysis platform for on-the-fly bioinformatic interrogation of transcriptional data derived from human or mouse tissue, called "MouSR". This internet-accessible analytical tool, https://mousr.qub.ac.uk/, enables users to easily interrogate their data using an intuitive "point and click" interface, which includes a suite of molecular characterisation options including QC, differential gene expression, gene set enrichment and microenvironmental cell population analyses from RNA-Seq. Users are provided with adjustable options for analysis parameters to generate results that can be saved as publication-quality images. To highlight its ability to perform high quality data analysis, we utilise the MouSR tool to interrogate our recently published tumour dataset, derived from genetically engineered mouse models and matched organoids, where we rapidly reproduced the key transcriptional findings.

The MouSR online tool provides a unique freely-available option for users to perform rapid transcriptomic analyses and comprehensive interrogation of the signalling underpinning transcriptional datasets, which alleviates a major bottleneck for biological discovery.

sentences: Regulation of CHK1 inhibitor resistance by a c-Rel and USP1 dependent pathway We previously discovered that deletion of c-Rel in the E-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-{kappa}B subunit in B-cell malignancies. Here we report that c-rel -/- E{micro}-Myc cells have an unexpected and major defect in the CHK1 pathway, with almost undetectable levels of CHK1 and CLSPN protein leading to therapeutic resistance to the highly specific CHK1 inhibitor (CHK1i) CCT244747. Similar downregulation of CHK1 levels was also seen in CCT244747 resistant U20S osteosarcoma cells. Further investigation revealed that downregulation of the deubiquitinase USP1 is responsible, at least in part, for these effects. Importantly, we demonstrate that c-rel -/- E{micro}-Myc lymphoma cells survive though upregulation of compensatory PI3K/AKT pathway activity. Moreover, targeting this pathway with Pictilisib (GDC-0941) effectively killed c-rel -/- E{micro}-Myc in vivo, while having no effect on wild type E-Myc cells. This data reveals an NF-{kappa}B regulated pathway controlling CHK1 activity in cancer cells and identifies a potential mechanism for both acquiring and overcoming CHK1i resistance in cancer patients.

sentences: Benchmarking small-variant genotyping in polyploids Genotyping from sequencing is the basis of emerging strategies in the molecular breeding of polyploid plants. However, compared with the situation for diploids, where genotyping accuracies are confidently determined with comprehensive benchmarks, polyploids have been neglected; there are no benchmarks measuring genotyping error rates for small variants using real sequencing reads. We previously introduced a variant calling method - Octopus - that accurately calls germline variants in diploids and somatic mutations in tumors. Here, we evaluate Octopus and other popular tools on whole-genome tetraploid and hexaploid datasets created using in silico mixtures of diploid Genome In a Bottle samples. We find that genotyping errors are abundant for typical sequencing depths, but that Octopus makes 25% fewer errors than other methods on average. We supplement our benchmarks with concordance analysis in real autotriploid banana datasets.

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Deciphering conformational selectivity in the A2A adenosine G protein-coupled receptor by Free Energy simulations Transmembranal G Protein-Coupled Receptors (GPCRs) transduce extracellular chemical signals to the cell, via conformational change from a resting (inactive) to an active (canonically bound to a G-protein) conformation. Receptor activation is normally modulated by extracellular ligand binding, but mutations in the receptor can also shift this equilibrium by stabilizing different conformational states. In this work, we built structure-energetic relationships of receptor activation based on original thermodynamic cycles that represent the conformational equilibrium of the prototypical A2A adenosine receptor (AR). These cycles were solved with efficient free energy perturbation (FEP) protocols, allowing to distinguish the pharmacological profile of different series of A2AAR agonists with different efficacies. The modulatory effects of point mutations on the basal activity of the receptor or on ligand efficacies could also be detected. This methodology can guide GPCR ligand design with tailored pharmacological properties, or allow the identification of mutations that modulate receptor activation with potential clinical implications.

Author SummaryThe design of new ligands as chemical modulators of G protein-coupled receptors (GPCRs) has benefited considerably during the last years of advances in both the structural and computational biology disciplines. Within the last, area, the use of free energy calculation methods has arisen as a computational tool to predict ligand affinities to explain structure-affinity relationships and guide lead optimization campaigns. However, our comprehension of the structural determinants of ligands with different pharmacological profile is scarce, and knowledge of the chemical modifications associated with an agonistic or antagonistic profile would be extremely valuable. We herein report an original implementation of the thermodynamic cycles associated with free energy perturbation (FEP) simulations, to mimic the conformational equilibrium between active and inactive GPCRs, and establish a framework to describe pharmacological profiles as a function of the ligands selectivity for a given receptor conformation. The advantage of this method resides into its simplicity of use, and the only consideration of active and inactive conformations of the receptor, with no simulation of the transitions between them. This model can accurately predict the pharmacological profile of series of full and partial agonists as opposed to antagonists of the A2A adenosine receptor, and moreover, how certain mutations associated with modulation of basal activity can influence this pharmacological profiles, which enables our understanding of such clinically relevant mutations.

sentences: PathoClock and PhysioClock in mice recapitulate human multimorbidity and heterogeneous aging Multimorbidity is a public health concern and an essential component of aging and healthspan but understudied because investigative tools are lacking that can be translatable to capture similarities and differences of the aging process across species and variability between individuals and individual organs. To help address this need, body organ disease number (BODN) borrowed from human studies was applied to C57BL/6 (B6) and CB6F1 mouse strains at 8, 16, 24 and 32 months of age, as a measure of systems morbidity based on pathology lesions to develop a mouse PathoClock resembling clinically based Body Clock in humans, using Bayesian inference. A mouse PhysioClock was also developed based on measures of physiological domains including cardiovascular, neuromuscular, and cognitive function in the same two mouse strains so that alignment with BODN was predictable. The results revealed between- and within-age variabilities in PathoClock and PhysioClock, as well as between-strain variabilities. Both PathoClock and PhysioClock correlated with chronological age more strongly in CB6F1 than C57BL/6. Prediction models were then developed, designated as PathoAge and PhysioAge, using regression models of pathology and physiology measures on chronological age. PathoAge better predicted chronological age than PhysioAge as the predicted chronological and observed chronological age for PhysioAge were complex rather than linear. In conclusion, PathoClock and PhathoAge can be used to capture biological changes that predict BODN, a metric developed in human, and compare multimorbidity across species. These mouse clocks are potential translational tools that could be used in aging intervention studies.

sentences: Within-species trait diversity reorganizes consumer competitive hierarchy and prey community structure in an experimental microbial ecosystem A popular idea in ecology is that trait variation among individuals from the same species may promote the coexistence of competing species. However, theoretical and empirical tests of this idea have yielded inconsistent findings. We manipulated intraspecific trait diversity in a ciliate competing with a nematode for bacterial prey in experimental microcosms. We found that intraspecific trait variation inverted the original competitive hierarchy to favor the consumer with variable traits, ultimately resulting in competitive exclusion. This competitive outcome was driven by foraging traits (size, speed, and directionality) that increased the ciliates fitness ratio and niche overlap with the nematode. The interplay between consumer trait variation and competition resulted in non-additive cascading effects - mediated through prey defense traits - on prey community assembly. Our results suggest that predicting consumer competitive population dynamics and the assembly of prey communities will require understanding the complexities of trait variation within consumer species.

sentences: Mechanical stress in pancreatic cancer: Signaling pathway adaptation activates cytoskeletal remodeling and enhances cell migration New treatments for patients with advanced or metastatic pancreatic cancers are urgently needed due to their resistance to all current therapies. Current studies focus on alternative treatment approaches that target or normalize the abnormal microenvironment of pancreatic tumors, which among others, is responsible for elevated mechanical stress in the tumor interior. Nevertheless, the underlying mechanisms by which mechanical stress regulates pancreatic cancer metastatic potential remain elusive. Herein, we used a large-scale proteomic assay to profile mechanical stress-induced signaling cascades that drive the motility of pancreatic cancer cells. Proteomic analysis, together with selective protein inhibition and siRNA treatments, revealed that mechanical stress enhances cell migration through activation of the p38 MAPK/HSP27 and JNK/c-Jun signaling axes, and activation of the actin cytoskeleton remodelers: Rac1, cdc42, and Myosin II. Our results highlight targeting aberrant signaling in cancer cells that are adapted to the mechanical tumor microenvironment as a novel approach to effectively limit pancreatic cancer cell migration.

sentences: Golden pompano genome resource enables discovery of valuable gene determining growth traits One important goal of fish genetic breeding is to identify valuable loci and genes that can facilitate growth and thereby productivity. Few such loci or genes have been identified in golden pompano (Trachinotus ovatus), a species of significant economic value. In this study, we produced a high-quality chromosome-level genome assembly of the golden pompano by de novo sequencing and assemblies for 2 parents and 200 F1 offspring by genome re-sequencing. We exploited these assemblies to identify loci and genes by QTL mapping, Kompetitive Allele Specific PCR (KASP) genotyping, and haplotype-based regional association analysis based on growth records of a 64 biparental and 147 individuals from a naturally occurring population. At a locus 291kb from BSNP21031, we identified a somatostatin receptor type 1-like (designated as gpsstr1) gene in which the BSNP1369 of the promoter region was highly associated with growth. Loss of sstr1a, the homolog of gpsstr1 in zebrafish, caused growth retardation. Sstr1a mediated growth via sstr2 and Wnt-gsk-3{beta} signaling pathways. Our findings provide new insights into the underlying mechanisms controlling growth. Our strategy can serve as an effective way to uncover novel genomic information and facilitate improvement of fish growth.

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Neural speech tracking shifts from the syllabic to the modulation rate of speech as intelligibility decreases The most prominent acoustic features in speech are intensity modulations, represented by the amplitude envelope of speech. Synchronization of neural activity with these modulations is vital for speech comprehension. As the acoustic modulation of speech is related to the production of syllables, investigations of neural speech tracking rarely distinguish between lower-level acoustic (envelope modulation) and higher-level linguistic (syllable rate) information. Here we manipulated speech intelligibility using noise-vocoded speech and investigated the spectral dynamics of neural speech processing, across two studies at cortical and subcortical levels of the auditory hierarchy, using magnetoencephalography. Overall, cortical regions mostly track the syllable rate, whereas subcortical regions track the acoustic envelope. Furthermore, with less intelligible speech, tracking of the modulation rate becomes more dominant. Our study highlights the importance of distinguishing between envelope modulation and syllable rate and provides novel possibilities to better understand differences between auditory processing and speech/language processing disorders.

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CSDE1 is a Post-Transcriptional Regulator of the LDL Receptor The low-density lipoprotein receptor (LDLR) controls cellular delivery of cholesterol and clears LDL from the bloodstream, protecting against atherosclerotic heart disease, the leading cause of death in the United States. We therefore sought to identify regulators of the LDLR beyond the targets of current clinical therapies and known causes of familial hypercholesterolemia. We show that Cold Shock Domain-Containing Protein E1 (CSDE1) enhances hepatic LDLR mRNA decay via its 3 untranslated region to regulate atherogenic lipoproteins in vivo. Using parallel phenotypic genome-wide CRISPR interference screens, we found 40 specific regulators of the LDLR left unidentified by observational human genetics. Among these, we show that CSDE1 regulates the LDLR at least as strongly as the mechanistically distinct pathways exploited by the best available clinical therapies: statins and PCSK9 inhibitors. Additionally, we show that hepatic gene silencing of Csde1 treats diet-induced dyslipidemia in mice better than that of Pcsk9. Our results reveal the therapeutic potential of manipulating a newly identified key factor in the post-transcriptional regulation of the LDLR mRNA for the prevention of cardiovascular disease. We anticipate that our approach of modelling a clinically relevant phenotype in a forward genetic screen, followed by mechanistic pharmacologic dissection and in vivo validation, will serve as a generalizable template for the identification of therapeutic targets in other human disease states.

One Sentence SummaryA genome-wide CRISPR screen identifies CSDE1 as a key regulator of hepatic LDLR mRNA decay in vivo, making it a promising target for heart disease.

Graphical Abstract

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sentences: Population structure and genetic connectivity of the scalloped hammerhead shark (Sphyrna lewini) across nursery grounds from the Eastern Tropical Pacific: implications for management and conservation Defining demographically independent units and understanding gene flow between them is essential for managing and conserving exploited populations. The scalloped hammerhead shark, Sphyrna lewini, is a coastal semi-oceanic species found worldwide in tropical and subtropical waters. Pregnant females give birth in shallow coastal estuarine habitats that serve as nursery grounds for neonates and small juveniles, and adults move offshore and become highly migratory. We evaluated the population structure and connectivity of S. lewini in coastal areas across the Eastern Tropical Pacific (ETP) using both sequences of the mitochondrial DNA control region (mtCR) and nuclear-encoded microsatellite loci. The mtCR defined two genetically discrete geographic groups: the Mexican Pacific and the central-southern Eastern Tropical Pacific (Guatemala, Costa Rica, Panama, and Colombia). Overall, the mtCR data showed low levels of haplotype diversity ranged from 0.000 to 0.608, while nucleotide diversity ranged from 0.000 to 0.0015. A more fine-grade population structure analysis was detected using microsatellite loci where Guatemala, Costa Rica, and Panama differed significantly. Genetic diversity analysis with nuclear markers revealed an observed heterozygosity ranging from 0.68 to 0.71 and an allelic richness from 5.89 to 7.00. Relatedness analysis revealed that individuals within nursery areas were more closely related than expected by chance, suggesting that S. lewini may exhibit reproductive philopatric behaviour within the ETP. Findings of at least two different management units, and evidence of philopatric behaviour call for intensive conservation actions for this critically endangered species in the ETP.

sentences: Rewiring glucose metabolism improves 5-FU efficacy in glycolytic p53-deficient colorectal tumors 5-fluorouracil (5-FU) is the backbone for chemotherapy in colorectal cancer (CRC). Response rates in patients are, however, limited to 50%. Despite the importance of 5-FU, the molecular mechanisms by which it induces toxicity remain unclear, limiting the development of strategies to improve efficacy. How fundamental aspects of cancer, such as driver mutations and phenotypic intra-tumor heterogeneity, relate to the 5-FU response is also ill-defined. This is largely due to the shortage of mechanistic studies executed in pre-clinical models that can faithfully recapitulate key CRC features. Here, we analyzed the 5-FU response in human organoids genetically engineered to reproduce the different stages of CRC progression. We find that 5-FU induces pyrimidine imbalance, which leads to DNA damage and cell death. Actively proliferating cancer (stem) cells are accordingly efficiently targeted by 5-FU. Importantly, p53 behaves as a discriminating factor for 5-FU sensitivity, whereas p53-deficiency leads to DNA damage-induced cell death, active p53 protects from these effects through inducing cell cycle arrest. Moreover, we find that targeting the Warburg effect, by rewiring glucose metabolism, enhances 5-FU toxicity by further altering the nucleotide pool and without increasing toxicity in healthy-non-transformed cells. Thus, targeting cancer metabolism in combination with replication stress-inducing chemotherapies emerges as a promising strategy for CRC treatment.

sentences: The Genetics and Evolution of Eye Color in Domestic Pigeons (Columba livia) The avian eye color, generally referred to the color of the iris, results from both pigments and structural coloration. Avian iris colors exhibit striking interspecific and, in some domestic species, intraspecific variations, suggesting unique evolutionary and ecological histories. Here we tackled the genetic basis of the pearl (white) iris color in domestic pigeons (Columba livia), to elucidate the largely unknown genetic mechanism underlying the evolution of avian iris coloration. Using a genome-wide association study (GWAS) in 92 pigeons, we mapped the pearl iris trait to a 9 kb region and a facilitative glucose transporter gene SLC2A11B. A nonsense mutation W49X leading to a premature stop codon in SLC2A11B was identified as the causal variant. Transcriptome analysis suggested that SLC2A11B loss-of-function may downregulate the xanthophore-differentiation gene CSF1R, and a key gene GCH1 involved in biosynthesis of pteridine, whose absence results in pearl iris. Coalescence and phylogenetic analyses indicated the mutation originated about 5,400 years ago coinciding with the onset of pigeon domestication, while positive selection was detected likely associated with artificial breeding. Within Aves, potentially impaired SLC2A11B was found in 10 species from six distinct lineages correlated to their signature brown or blue eyes. Analysis of vertebrate SLC2A11B orthologs revealed relaxed selection in the avian clade, consistent with the scenario that, during and after avian divergence from reptile ancestor, the SLC2A11B-involved development of dermal chromatophores likely degenerated due to feather coverage. Our findings provide new insight into the mechanism of avian iris color variations and the evolution of pigmentation in vertebrates.

sentences: CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly contagious presenting a significant public health issue. Current therapies used to treat coronavirus disease 2019 (COVID-19) include monoclonal antibody cocktail, convalescent plasma, antivirals, immunomodulators, and anticoagulants, though the current therapeutic options remain limited and expensive. The vaccines from Pfizer and Moderna have recently been authorized for emergency use, which are invaluable for the prevention of SARS-CoV-2 infection. However, their long-term side effects are not yet to be documented, and populations with immunocompromised conditions (e.g., organ-transplantation and immunodeficient patients) may not be able to mount an effective immune response. In addition, there are concerns that wide-scale immunity to SARS-CoV-2 may introduce immune pressure that could select for escape mutants to the existing vaccines and monoclonal antibody therapies. Emerging evidence has shown that chimeric antigen receptor (CAR)- natural killer (NK) immunotherapy has potent antitumor response in hematologic cancers with minimal adverse effects in recent studies, however, the potentials of CAR-NK cells in preventing and treating severe cases of COVID-19 has not yet been fully exploited. Here, we improve upon a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2 and its D614G mutant. CAR-NK cells were generated using the scFv domain of S309 (henceforward, S309-CAR-NK), a SARS-CoV and SARS-CoV-2 neutralizing antibody that targets the highly conserved region of SARS-CoV-2 spike (S) glycoprotein, therefore would be more likely to recognize different variants of SARS-CoV-2 isolates. S309-CAR-NK cells can specifically bind to pseudotyped SARS-CoV-2 virus and its D614G mutant. Furthermore, S309-CAR-NK cells can specifically kill target cells expressing SARS-CoV-2 S protein in vitro and show superior killing activity and cytokine production, compared to that of the recently published CR3022-CAR-NK cells. Thus, these results pave the way for generating  off-the-shelf S309-CAR-NK cells for treatment in high-risk individuals as well as provide an alternative strategy for patients unresponsive to current vaccines.

sentences: Activation of IP10/CXCR3 signaling with highly coincidental with PrPSc deposit in the brains of scrapie infected mice Activation of chemokine IP10, also named as CXCL10, and its receptor CXCR3 in CNS is described in some neurodegenerative diseases. Our previous study has also demonstrated an increased brain IP10 levels in several scrapie infected rodent models. However, the detailed alteration of IP10/CXCR3 signaling in CNS during prion infection remains unsettled. Here, we found the increased IP10 signals in the brains of scrapie infected mice mainly localized in the neurons and the activated microglia using various methodologies. The levels of CXCR3 were markedly increased in brains of the scrapie infected mice and in the prion infected cell line SMB-S15. The increased CXCR3 mainly distributed in neurons. Obviously morphological colocalizations of PrP/PrPSc with IP10 and CXCR3 in the brains of scrapie infected mice were observed in the assays of immunohistochemistry (IHC) and immunofluorescence. Additionally, IHC analysis with whole brain sections demonstrated that the increased IP10 and CXCR3 accumulated in the brain regions with more PrPSc deposits. Co-immunoprecipitation and biomolecular interaction assays identified the evidence for the molecular interactions of PrP with IP10 and CXCR3. Compared to the normal partner cell line SMB-PS, the more portion of IP10 accumulated insides of prion infected SMB-S15 cells. Removal of prion replication in SMB-S15 cells by resveratrol converted the pattern of the accumulation and secretion of cellular IP10. Our data here demonstrate an activation of IP10/CXCR3 signaling in the brain tissues of prion infection, highly coincidental with PrPSc deposit. Modulation of brain IP10/CXCR3 signaling is potential therapeutic target for reducing the progression of prion diseases.

sentences: Epstein-Barr Virus Induced Cytidine Metabolism Roles in Transformed B-cell Growth and Survival Epstein-Barr virus (EBV) is associated with 200,000 cancers annually, including B-cell lymphomas in immunosuppressed hosts. Hypomorphic mutations of the de novo pyrimidine synthesis pathway enzyme cytidine 5 triphosphate synthase 1 (CTPS1) suppress cell mediated immunity, resulting in fulminant EBV infection and EBV+ central nervous system (CNS) lymphomas. Since CTP is a critical precursor for DNA, RNA and phospholipid synthesis, this observation raises the question of whether the isozyme CTPS2 or cytidine salvage pathways help meet CTP demand in EBV-infected B-cells. Here, we found that EBV upregulated CTPS1 and CTPS2 with distinct kinetics in newly infected B-cells. While CRISPR CTPS1 knockout caused DNA damage and proliferation defects in lymphoblastoid cell lines (LCL), which express the EBV latency III program observed in CNS lymphomas, double CTPS1/2 knockout caused stronger phenotypes. EBNA2, MYC and non-canonical NF-{square}B positively regulated CTPS1 expression. CTPS1 depletion impaired EBV lytic DNA synthesis, suggesting that latent EBV may drive pathogenesis with CTPS1 deficiency. Cytidine rescued CTPS1/2 deficiency phenotypes in EBV-transformed LCL and Burkitt B-cells, highlighting CTPS1/2 as a potential therapeutic target for EBV-driven lymphoproliferative disorders. Collectively, our results suggest that CTPS1 and CTPS2 have partially redundant roles in EBV-transformed B-cells and provide insights into EBV pathogenesis with CTPS1 deficiency.

sentences: Novel CHD8 genomic targets identified in fetal mouse brain by in vivo Targeted DamID Genetic studies of autism spectrum disorder (ASD) have revealed a causal role for mutations in chromatin remodeling genes. Chromodomain helicase DNA binding protein 8 (CHD8) encodes a chromatin remodeler with one of the highest de novo mutation rates in sporadic ASD. However, the relationship between CHD8 genomic function and autism-relevant biology remains poorly elucidated. CHD8 binding studies have relied on Chromatin Immunoprecipitation followed by sequencing (ChIP-seq), however, these datasets exhibit significant variability. ChIP-seq has technical limitations in the context of weak or indirect protein-DNA interactions or when high-performance antibodies are unavailable. Thus, complementary approaches are needed overall, and, specifically, to establish CHD8 genomic targets and regulatory function. Here we used Targeted DamID in utero to characterize CHD8 binding in developing embryonic mouse cortex. CHD8 Targeted DamID followed by sequencing (CHD8 TaDa-seq) revealed binding at previously identified targets as well as loci sensitive to Chd8 haploinsufficiency. CHD8 TaDa-seq highlighted CHD8 binding distal to a subset of genes specific to neurodevelopment and neuronal function. These studies establish TaDa-seq as a useful alternative for mapping protein-DNA interactions in vivo and provide insights into the relationship between chromatin remodeling by CHD8 and autism-relevant pathophysiology associated with CHD8 mutations.

sentences: MYC overexpression leads to increased chromatin interactions at superenhancers and c-Myc binding sites The MYC oncogene encodes for the c-Myc protein and is frequently dysregulated across multiple cancer cell types, making it an attractive target for cancer therapy. There have been many difficulties in targeting c-Myc, due to its complex network of regulators and the unstructured nature of its protein. Thus, we are interested in looking at the downstream cancer-specific functions of c-Myc. Overexpression of MYC leads to c-Myc binding at active enhancers, resulting in a global transcriptional amplification of active genes. However, the mechanism underlying this c-Myc enhancer invasion has not been well studied. To that end, we performed ChIP-seq, RNA-seq, 4C-seq and SIQHiC (Spike-in Quantitative Hi-C) on the U2OS osteosarcoma cell line with tetracycline-inducible MYC. MYC overexpression in U2OS cells modulated histone acetylation and increased c-Myc binding at superenhancers. SIQHiC analysis revealed increased global chromatin contact frequency, particularly at chromatin interactions connecting c-Myc binding sites. Our results suggest that c-Myc molecules are recruited to and accumulates within zones of high transcription activity, binding first at stable promoter binding sites at low expression levels, then at superenhancer binding sites when overexpressed. At the same time, the recruitment of c-Myc and other transcription factors may stabilize chromatin interactions to increase chromatin contact frequency. The accumulation of c-Myc at cancer-type specific superenhancers may then drive the expression of interacting oncogenes that each cancer is highly reliant on. By elucidating the chromatin landscape of c-Myc driven cancers, we can potentially target these chromatin interactions for cancer therapy, without affecting physiological c-Myc signaling.

sentences: Heat activation and inactivation of bacterial spores. Is there an overlap? Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore germination. This treatment also has potential to be employed in food processing to eliminate undesired bacterial spores by enhancing their germination, and then inactivating the less heat resistant germinated spores at a milder temperature. However, incorrect heat treatment could also generate heat damage in spores, and lead to more heterogeneous spore germination. Here, the heat activation and heat damage profile of Bacillus subtilis spores was determined by testing spore germination and outgrowth at both population and single spore levels. The heat treatments used were 40-80{degrees}C, and for 0-300 min. The results were as follows. 1) Heat activation at 40-70{degrees}C promoted L-valine and L-asparagine-glucose-fructose-potassium (AGFK) induced germination in a time dependent manner. 2) The optimal heat activation temperatures for AGFK and L-valine germination via the GerB plus GerK or GerA germinant receptors were 65 and 50-65{degrees}C, respectively. 3) Heat inactivation of dormant spores appeared at 70{degrees}C, and the heat damage of molecules essential for germination and growth began at 70 and 65{degrees}C, respectively. 4) Heat treatment at 75{degrees}C resulted in both activation of germination and damage to the germination apparatus, and 80{degrees}C treatment caused more pronounced heat damage. 5) For the spores that should withstand adverse environmental temperatures in nature, heat activation seems functional for a subsequent optimal germination process, while heat damage affected both germination and outgrowth.

sentences: Protoporphyrin-IX nanostructures modulate their protein aggregation ability via differential oxidation and protein binding Porphyrias are caused by genetic defects in the heme biosynthetic pathway and are associated with accumulation of high levels of porphyrins that become cytotoxic. Porphyrins, due to their amphipathic nature, spontaneously associate into different nanostructures but very little is known about the effect of porphyrin speciation on the cytotoxic effects of porphyrins. Previously we demonstrated the unique ability of fluorescent biological porphyrins, including protoporphyrin IX (PP-IX), to cause organelle selective protein aggregation, which we posit to be a major mechanism by which porphyrins exerts their cytotoxic effect. Herein, we tested the hypothesis that PP-IX-mediated protein aggregation is modulated by different PP-IX nanostructures via a mechanism that depends on their oxidizing potential and protein binding ability. We demonstrate that PP-IX nanostructure formation is reversible in nature, and that nanostructure size modulates consequent protein oxidation and aggregation potential. We also show that albumin, the most abundant serum protein, preferentially binds PP-IX dimers and enhances their oxidizing ability. Additionally, extracellular albumin protects from intracellular porphyrinogenic stress and protein aggregation by acting as a PP-IX sponge. This work highlights the importance of PP-IX speciation in the context of the porphyrias, and offers insights into potential novel therapeutic approaches.

sentences: Legionella pneumophila regulates host cell motility by targeting Phldb2 with a 14-3-3ζ-dependent protease effector The cytoskeleton network of eukaryotic cells is essential for diverse cellular processes, including vesicle trafficking, cell motility and immunity, thus is a common target for bacterial virulence factors. A number of effectors from the bacterial pathogen Legionella pneumophila have been shown to modulate the function of host actin cytoskeleton to construct the Legionella-containing vacuole (LCV) permissive for its intracellular replication. In this study, we identified the Dot/Icm effector Lem8 (Lpg1290) as a protease that interferes with host motility. We show that the protease activity of Lem8 is catalyzed by a Cys-His-Asp motif known to be associated with diverse biochemical activities. Intriguingly, we found that Lem8 interacts with the host regulatory protein 14-3-3{zeta}, which activates its protease activity. Furthermore, Lem8 undergoes self-cleavage in a process that requires 14-3-3{zeta}. We identified the PH domain-containing protein Phldb2 involved in cell migration as a target of Lem8 and demonstrate that Lem8 plays a role in the inhibition of host cell migration. Our results reveal a novel mechanism of inhibiting host cell motility by L. pneumophila for its virulence.

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Countering reproducibility issues in mathematical models with software engineering techniques: A case study using a one-dimensional mathematical model of the atrioventricular node One should assume that in silico experiments in systems biology are less susceptible to reproducibility issues than their wet-lab counterparts, because they are free from natural biological variations and their environment can be fully controlled. However, recent studies show that only half of the published mathematical models of biological systems can be reproduced without substantial effort. In this article we examine the potential causes for failed or cumbersome reproductions in a case study of a one-dimensional mathematical model of the atrioventricular node, which took us four months to reproduce. The model demonstrates that even otherwise rigorous studies can be hard to reproduce due to missing information, errors in equations and parameters, a lack in available data files, non-executable code, missing or incomplete experiment protocols, and missing rationales behind equations. Many of these issues seem similar to problems that have been solved in software engineering using techniques such as unit testing, regression tests, continuous integration, version control, archival services, and a thorough modular design with extensive documentation. Applying these techniques, we reimplement the examined model using the modeling language Modelica. The resulting workflow is independent of the model and can be translated to SBML, CellML, and other languages. It guarantees methods reproducibility by executing automated tests in a virtual machine on a server that is physically separated from the development environment. Additionally, it facilitates results reproducibility, because the model is more understandable and because the complete model code, experiment protocols, and simulation data are published and can be accessed in the exact version that was used in this article. We found the additional design and documentation effort well justified, even just considering the immediate benefits during development such as easier and faster debugging, increased understandability of equations, and a reduced requirement for looking up details from the literature.

Author summaryReproducibility is one of the cornerstones of the scientific method. In order to draw reliable conclusions, an experiment must yield the same results when it is repeated using the same methods. However, biological systems are complex, which makes experiments cumbersome. It is therefore desirable to build a mathematical representation of the biological system, which captures its essential behavior in a set of variables and equations and allows for easier and faster experimentation. Unfortunately, recent studies have shown that half of the published mathematical models are not immediately reproducible due to missing information, mathematical errors, and incomplete documentation. These issues are similar to those faced in software engineering: A single missing file or a buggy line of code can render any kind of software useless. Software engineering has turned to rigorous software testing, automated development pipelines, and version control systems to overcome these challenges, but these techniques are not yet widely applied to mathematical modeling. In this paper we demonstrate their benefit for the reproducibility of a large mathematical model of the atrioventricular node. The software engineering solutions that we employ can be applied to any mathematical model and could therefore facilitate scientific progress by encouraging and simplifying model reuse.

sentences: Metabarcoding metacommunities: time, space, and land use interact to structure aquatic macroinvertebrate communities in streams There is an increasing need to move beyond evaluating the effect of land use on stream communities by only studying local variables, and instead incorporate a metacommunity perspective which integrates environmental and spatial factors across larger spatial scales. The use of molecular tools (DNA metabarcoding) to identify bioindicator groups, such as aquatic macroinvertebrates, can provide greater taxonomic resolution to explore patterns in stream metacommunities. In this study, we collected aquatic macroinvertebrates from streams in southern Ontario which spanned a gradient of agricultural disturbance and used DNA metabarcoding to identify the species composition from these samples. We address a significant knowledge gap in previous stream aquatic macroinvertebrate metacommunity studies by incorporating molecular identification as well as a temporal component. We observed that a combination of local habitat conditions, regional agricultural land use, and spatial position influenced aquatic macroinvertebrate community composition, suggesting there is an interaction between environmental filtering and dispersal processes that structures these communities. However, aquatic macroinvertebrate communities were also highly dissimilar between streams and composed of many rare species, and a large percentage of unexplained variation suggests that there is a strong stochastic component to community assembly. We also observed that there is a seasonal component to metacommunity dynamics, with different water quality variables being significant to community composition in each sampling month. While we expected that an increased percentage of surrounding agricultural land use would result in more homogenous macroinvertebrate communities, we only detected this relationship in May and found evidence that a larger riparian buffer width can mitigate the effects of agricultural land use. We demonstrate the utility of DNA metabarcoding for revealing patterns in metacommunity dynamics that may not be detectable using coarse taxonomic identifications, and reveal the importance of incorporating a seasonal component when evaluating the influence of land use on community composition.

sentences: Metabolism of glucose activates TORC1 through multiple mechanisms in Saccharomyces cerevisiae Target of Rapamycin Complex 1 (TORC1) is a conserved eukaryotic protein complex that links the presence of nutrients with cell growth. In Saccharomyces cerevisiae, TORC1 activity is positively regulated by the presence of amino acids and glucose in the medium. However, mechanisms underlying nutrient-induced TORC1 activation remain poorly understood. By utilizing a TORC1 activation assay, we demonstrate that differential metabolism of glucose activates TORC1 through three distinct pathways in yeast. The first  canonical Rag GTPase-dependent pathway requires conversion of glucose to fructose 1,6-bisphosphate which activates TORC1 via the Rag GTPase heterodimer Gtr1GTP/Gtr2GDP. The second  non-canonical Rag GTPase-dependent pathway requires conversion of glucose to glucose 6-phosphate which activates TORC1 via Gtr1GTP/Gtr2GTP. The third  Rag GTPase-independent pathway requires complete glycolysis and vacuolar ATPase reassembly for TORC1 activation. Glucose-induced TORC1 activation can be uncoupled from glucose-induced AMPK inactivation. We have established a roadmap to deconstruct the link between glucose metabolism and TORC1 activation.

sentences: Structure of the lysosomal membrane fusion machinery Lysosomes are of central importance in cellular recycling, nutrient signaling 1,2 and endocytosis, and are tightly connected to autophagy 3 and the invasion of pathogenic bacteria and viruses 1,4. Lysosomal fusion events are fundamental to cell survival and require HOPS, a conserved heterohexameric tethering complex 5,6. HOPS recognizes and binds small membrane-associated GTPases on lysosomes and organelles, and assembles membrane bound SNAREs for fusion 7,8. Through tethering, HOPS brings membranes in close proximity to each other and significantly increases fusion efficacy by catalysing SNARE assembly. Consequently, different HOPS mutations are causative for severe diseases 6. Despite its fundamental cellular duties, it remained speculative how HOPS fulfils its function as high-resolution structural data were unavailable. Here, we used cryo-electron microscopy to reveal the structure of HOPS. In the complex, two central subunits form the backbone and an assembly hub for the functional domains. Two GTPase binding units extend to opposing ends, while the SNARE binding module points to the side, resulting in a triangular shape of the complex. Unlike previously reported, HOPS is surprisingly rigid and extensive flexibility is confined to its extremities. We show that HOPS complex variants with mutations proximal to the backbone can still tether membranes but fail to efficiently promote fusion indicating, that the observed integrity of HOPS is essential to its function. In our model, the core of HOPS acts as a counter bearing between the flexible GTPase binding domains. This positions the SNARE binding module exactly between the GTPase anchored membranes to promote fusion. Our structural and functional analysis reveals the link between the spectacular architecture of HOPS and its mechanism that couples membrane tethering and SNARE assembly, to catalyse lysosomal fusion.

sentences: The energy requirements of ion homeostasis determine the lifespan of starving bacteria The majority of microbes on earth, whether they live in the ocean, the soil or in animals, are not growing, but instead struggling to survive starvation1-6. Some genes and environmental conditions affecting starvation survival have been identified7-13, but despite almost a century of study14-16, we do not know which processes lead to irreversible loss of viability, which maintenance processes counteract them and how lifespan is determined from the balance of these opposing processes. Here, we used time-lapse microscopy to capture and characterize the cell death process of E. coli during carbon starvation for the first time. We found that a lack of nutrients results in the collapse of ion homeostasis, triggering a positive-feedback cascade of osmotic swelling and membrane permeabilization that ultimately results in lysis. Based on these findings, we hypothesized that ion transport is the major energetic requirement for starving cells and the primary determinant of the timing of lysis. We therefore developed a mathematical model that integrates ion homeostasis and  cannibalistic nutrient recycling from perished cells16,17 to predict lifespan changes under diverse conditions, such as changes of cell size, medium composition, and prior growth conditions. Guided by model predictions, we found that cell death during starvation could be dramatically slowed by replacing inorganic ions from the medium with a non-permeating osmoprotectant, removing the cost of ion homeostasis and preventing lysis. Our quantitative and predictive model explains how survival kinetics are determined in starvation and elucidates the mechanistic underpinnings of starvation survival.

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Characterization of the utility of three nebulizers in investigating infectivity of airborne viruses Laboratory-generated bioaerosols are widely used in aerobiology studies of viruses, however few comparisons of alternative nebulizers exist. We compared aerosol production and virus survival for a Collison nebulizer, vibrating mesh nebulizer (VMN), and hydraulic spray atomizer (HAS). We also measured the dry size distribution of the aerosols produced, calculated the droplet sizes before evaporation and the dry size distribution from normal saline solution. Dry count median diameters of 0.25, 0.63 and 0.76 {micro}m were found for normal saline from the Collison nebulizer, VMN and HSA, respectively. The volume median diameters were 2.91, 3.2 and 2.43 {micro}m, respectively. The effect of nebulization on the viability of two influenza A viruses (IAVs) (H1N1, H3N2) and human rhinovirus (HRV)-16, was assessed by direct nebulization into an SKC Biosampler. The HSA had least impact on surviving fractions (SFs) of H1N1 and H3N2 (89{+/-}5%, 94{+/-}3%), followed by the Collison nebulizer (82{+/-}2%, 82{+/-}3%). The VMN yielded SFs of 78{+/-}2% and 76{+/-}2%, respectively. Conversely, for HRV-16, the VMN produced higher SFs (86{+/-}15%). Our findings indicate that although the VMN had the greatest impact on IAV survival, it produced higher aerosol concentrations within the airborne-size range making it more suitable where high aerosol mass production is required.

ImportanceViral respiratory tract infections cause millions of lost days of work and physician visits globally, accounting for significant morbidity and mortality. Respiratory droplet and droplet nuclei from infected hosts are the substantial potential carriers of such viruses within indoor environments. Laboratory-generated bioaerosols are applied in understanding the transmission and infection of viruses, simulating the physiological aspects of bioaerosol generation in a controlled environment. However, little comparative characterization exists for nebulizers used in infectious disease aerobiology, including Collison nebulizer, Vibrating mesh nebulizer, and hydraulic spray atomizer. This study characterized the physical features of aerosols generated by laboratory nebulizers, and their performance in producing aerosols at a size relevant to airborne transmission used in infectious disease aerobiology. We also determined the impact of nebulization mechanisms of these nebulizers on the viability of human respiratory viruses, including IAV H1N1, IAV H3N2 and HRV-16.

sentences: Potentiation of drug toxicity through virus latency reversal promotes preferential elimination of HIV infected cells Eliminating latently infected cells is a highly challenging, indispensable step towards the overall cure for HIV/AIDS. We recognized that the unique HIV protease cut site (Phe-Pro) can be reconstructed using a potent toxin, monomethyl auristatin F (MMAF), which features Phe at its C-terminus. We hypothesized that this presents opportunities to design prodrugs that are specifically activated by the HIV protease. To investigate this, a series of MMAF derivatives was synthesized and evaluated in cell culture using latently HIV-infected cells. Cytotoxicity of compounds was enhanced upon latency reversal by up to 11-fold. In a mixed cell population, nanomolar concentrations of the lead compound depleted predominantly the HIV-infected cells and in doing so markedly enriched the pool with the uninfected cells. Despite expectation, mechanism of action of the synthesized toxins was not as HIV protease-specific prodrugs, but likely through the synergy of toxicities between the toxin and the reactivated virus.

sentences: Predicting protein domain temperature adaptation across the prokaryote-eukaryote divide Protein thermostability is important for fitness but difficult to measure across the proteome. Fortunately, protein thermostability is correlated with prokaryote optimal growth temperatures (OGTs), which can be predicted from genome features. Models that can predict temperature sensitivity across the prokaryote-eukaryote divide would help inform how eukaryotes adapt to elevated temperatures, such as those predicted by climate change models. In this study we test whether prediction models can cross the prokaryote-eukaryote divide to predict protein stability in both prokaryotes and eukaryotes. We compare models built using a) the whole proteome, b) Pfam domains, and c) individual amino acid residues. Proteome-wide models accurately predict prokaryote optimal growth temperatures (r2 up to 0.93), while site-specific models demonstrate that nearly half of the proteome is associated with optimal growth temperature in both Archaea and Bacteria. Comparisons with the small number of eukaryotes with temperature sensitivity data suggest that site-specific models are the most transferable across the prokaryote-eukaryote divide. Using the site-specific models, we evaluated temperature sensitivity for 323,850 amino acid residues in 2,088 Pfam domain clusters in Archaea and Bacteria species separately. 59.0% of tested residues are significantly associated with OGT in Archaea and 75.2% of tested residues are significantly associated with OGT in Bacteria species at a 5% false discovery rate. These models make it possible to identify which Pfam domains and amino acid residues are involved in temperature adaptation and facilitate future research questions about how species will fare in the face of increasing environmental temperatures.

sentences: Integration and comparison of multi-omics profiles of NGLY1 deficiency plasma and cellular models to identify clinically relevant molecular phenotypes NGLY1 (N-glycanase 1) deficiency is a rare congenital recessive disorder of protein deglycosylation unaddressed by the current standard of care. Using combined metabolomics and proteomics profiling, we show that NGLY1 deficiency activates the immune response and disturbs lipid metabolism, biogenic amine synthesis, and glutathione metabolism. These alterations were also observed in NGLY1 deficient patient-derived induced pluripotent stem cells (iPSCs) and differentiated neural progenitor cells (NPCs), which serve as personalized cellular models of the disease. These findings provide molecular insight into the pathophysiology of NGLY1 deficiency and suggest potential therapeutic strategies.

sentences: Pericentriolar matrix integrity relies on cenexin and Polo-Like Kinase (PLK)1 Polo-Like-Kinase (PLK) 1 activity is associated with maintaining the functional and physical properties of the centrosomes pericentriolar matrix (PCM). In this study, we use a multimodal approach of human cells (HeLa) and zebrafish embryos in parallel to phylogenic analysis to test the role of a PLK1 binding protein, cenexin, in regulating the PCM. Our studies identify that cenexin is required for tempering microtubule nucleation and that a conserved C-terminal PLK1 binding site between humans and zebrafish is needed for PCM maintenance through mediating PLK1-dependent substrate phosphorylation events. PCM architecture in cenexin-depleted zebrafish embryos was rescued with wild-type human cenexin, but not with a C-terminal cenexin mutant (S796A) deficient in PLK1 binding. We propose a model where cenexins C-terminus acts in a conserved manner in eukaryotes, excluding nematodes and arthropods, to anchor PLK1 moderating its potential to phosphorylate PCM substrates required for PCM maintenance and function.

sentences: Termites have developed wider thermal limits to cope with environmental conditions in savannas The most diverse and abundant family of termites, the Termitidae, evolved in warm, wet African tropical forests. Since then, they have colonised grassy biomes such as savannas. These environments have more extreme temperatures than tropical forests, and greater temporal fluctuations (both annually and diurnally) that are challenging for soft-bodied ectotherms. We propose that that a likely mechanism that facilitated the expansion from forest to savanna was the widening of physiological limits of savanna termite species in order to cope with more extreme environmental conditions. We sampled termites directly from mound structures across an environmental gradient in Ghana, and recorded the thermal tolerance of individual termites, both critical thermal maximum (CTmax) and critical thermal minimum (CTmin). We estimated colony thermal tolerance by taking an average of each tested individual, and modelled these data against several environmental factors (canopy cover above the mound, rainfall and temperature). We found that savanna termite species had significantly higher CTmax values, and significantly lower CTmin values, than forest species. In addition, areas with high canopy cover were significantly associated with low CTmax values, and areas with higher average daily rainfall were significantly associated with higher CTmin values. Our results suggest that the widening of thermal tolerances has occurred in savanna termite species, probably in response to the more extreme temperatures found in those environments.

sentences: Receptor-like kinases FER and SRK mediate Lotus japonicus freezing tolerance and climate adaptation Many plant species have succeeded in colonizing a wide range of diverse climates through local adaptation, but the underlying molecular genetics remain obscure. We previously found that winter survival was a direct target of selection during colonization of Japan by the perennial legume Lotus japonicus and identified associated candidate genes. Here, we show that two of these, the FERONIA-receptor like kinase (LjFER) and a novel S-receptor-like kinase (LjSRK) are required for non-acclimated freezing tolerance and show haplotype-dependent cold-responsive expression. Our work demonstrates that recruiting a conserved growth regulator, FER, and a novel receptor-like kinase, SRK, into the set of cold-responsive genes has contributed to freezing tolerance and local climate adaptation in L. japonicus, offering new functional genetic insight into perennial herb evolution.

sentences: An Evolutionary Process Without Variation and Selection Natural selection successfully explains how organisms accumulate adaptive change despite that traits acquired over a lifetime are eliminated at the end of each generation. However, in some domains that exhibit cumulative, adaptive change--e.g., cultural evolution, and earliest life--acquired traits are retained; these domains do not face the problem that Darwins theory was designed to solve. Lack of transmission of acquired traits occurs when germ cells are protected from environmental change, due to a self-assembly code used in two distinct ways: (i) actively interpreted during development to generate a soma, and (ii) passively copied without interpretation during reproduction to generate germ cells. Early life and cultural evolution appear not to involve a self-assembly code used in these two ways. We suggest that cumulative, adaptive change in these domains is due to a lower-fidelity evolutionary process, and model it using Reflexively Autocatalytic and Foodset-generated networks. We refer to this more primitive evolutionary process as Self-Other Reorganisation (SOR) because it involves internal self-organising and self-maintaining processes within entities, as well as interaction between entities. SOR encompasses learning but in general operates across groups. We discuss the relationship between SOR and Lamarckism, and illustrate a special case of SOR without variation.

sentences: Genome-wide analysis of PTR/POT transporters in Candida species and their functional characterization in the newly emerged pathogen Candida auris The PTR or proton-dependent oligopeptide transporter (POT) family exploits the inwardly directed proton motive force to facilitate the cellular uptake of di- and tripeptides. Interestingly, representatives from the family have shown efficacy in delivering peptide-based antifungal derivatives in certain Candida species. Given the increased incidences of fungal infections by Candida species and the associated escalating orders of resistance against frontline antifungals, peptide derivatives are attractive therapeutic options. In that direction, the identification and characterization of PTR transporters serve as an essential first step in the translation of peptide-based antifungals as next-gen therapeutics. Herein, we present a genome-wide inventory of the PTR transporters in five prominent Candida species. Our study identifies 2 PTR transporters each in C. albicans and C. dubliniensis, 1 in C. glabrata, 4 in C. parapsilosis, and 3 in C. auris. Notably, despite all representatives retaining the conserved features seen in the PTR family, there exist two distinct classes of PTR transporters that differ in terms of their sequence identities and membrane topology. Further, we also evaluated the contribution of each PTR protein of the newly emerged multidrug-resistant C. auris in di-/tripeptide uptake. Notably, deletion of the PTR transporters encoded by BNJ08_003830 and BNJ08_005124 led to a marked reduction in the transport capabilities of several tested di-/tripeptides. Besides, BNJ08_005124 deletion also resulted in increased resistance towards the peptide-nucleoside drug Nikkomycin Z, pointing towards its predominant role in the uptake mechanism. Altogether, the study provides an important template for future structure-function investigations of PTR transporters in Candida species.

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Schema Playground: A tool for authoring, extending, and using metadata schemas to improve FAIRness of biomedical data BackgroundBiomedical researchers are strongly encouraged to make their research outputs more Findable, Accessible, Interoperable, and Reusable (FAIR). While many biomedical research outputs are more readily accessible through open data efforts, finding relevant outputs remains a significant challenge. Schema.org is a metadata vocabulary standardization project that enables web content creators to make their content more FAIR. Leveraging Schema.org could benefit biomedical research resource providers, but it can be challenging to apply Schema.org standards to biomedical research outputs. We created an online browser-based tool that empowers researchers and repository developers to utilize schema.org or other biomedical schema projects.

ResultsOur browser-based tool includes features which can help address many of the barriers towards schema.org-compliance such as: The ability to easily browse for relevant Schema.org classes, the ability to extend and customize a class to be more suitable for biomedical research outputs, the ability to create data validation to ensure adherence of a research output to a customized class, and the ability to register a custom class to our schema registry enabling others to search and re-use it. We demonstrate the use of our tool with the creation of the Outbreak.info schema--a large multi-class schema for harmonizing various COVID-19 related resources.

ConclusionsWe have created a browser-based tool to empower biomedical research resource providers to leverage Schema.org classes to make their research outputs more FAIR.

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MafF is an antiviral host factor that suppresses transcription from Hepatitis B Virus core promoter Hepatitis B Virus (HBV) is a stealth virus that exhibits only minimal induction of the interferon system that is required for both innate and adaptive immune responses. However, 90% of acutely infected adults can clear the virus, suggesting the presence of additional mechanisms that facilitate viral clearance. Herein, we report that Maf bZIP transcription factor F (MafF) promotes host defense against infection with HBV. Using siRNA library and an HBV/NL reporter virus, we screened to identify anti-HBV host factors. Our data showed that silencing of MafF led to a 6-fold increase in luciferase activity after HBV/NL infection. Overexpression of MafF reduced HBV core promoter transcriptional activity, which was relieved upon mutating the putative MafF binding region. Loss of MafF expression by CRISPR/CAS9 (in HepG2-hNTCP-C4 cells) or siRNA silencing (in primary hepatocytes [PXB]), induced HBV core and HBV pregenomic RNA (pgRNA) levels, respectively, after HBV infection. MafF physically binds to HBV core promoter and competitively inhibits HNF-4 binding to an overlapping sequence in HBV enhancer II sequence (EnhII) as seen by ChIP analysis. MafF expression was induced by IL-1{beta}/TNF- treatment in both HepG2 and PXB cells, in an NF-{kappa}B-dependent manner. Consistently, MafF expression levels were significantly enhanced and positively correlated with the levels of these cytokines in patients with chronic HBV infection, especially in the immune clearance phase.

ImportanceHBV is a leading cause of chronic liver diseases, infecting about 250 million people worldwide. HBV has developed strategies to escape interferon-dependent innate immune responses. Hence, the identification of other anti-HBV mechanisms is important for understanding HBV pathogenesis, and developing anti-HBV strategies. MafF was shown to suppress transcription from HBV core promoter, leading to a significant suppression of HBV life cycle. Furthermore, MafF expression was induced in chronic HBV patients and in primary human hepatocytes (PXB). This induction correlated with the levels of inflammatory cytokines (IL-1{beta} and TNF-). These data suggest that the induction of MafF contributes to the hosts antiviral defense by suppressing transcription from selected viral promoters. Our data shed light on a novel role for MafF as anti-HBV host restriction factor.

sentences: Discovery of Flaviviridae-derived endogenous viral elements in shrew genomes provide novel insights into Pestivirus ancient history As viral genomic imprints in host genomes, endogenous viral elements (EVEs) shed light on the deep evolutionary history of viruses, ancestral host ranges, and ancient viral-host interactions. In addition, they may provide crucial information for calibrating viral evolutionary timescales. In this study, we conducted a comprehensive in silico screening of a large dataset of available mammalian genomes for EVEs deriving from members of the viral family Flaviviridae, an important group of viruses including well-known human pathogens. We identified two novel pestivirus-like EVEs in the reference genome of the Indochinese shrew (Crocidura indochinensis). Homologs of these novel EVEs were subsequently detected in vivo by molecular detection and sequencing in 27 shrew species, including 26 species representing a wide distribution within the Crocidurinae subfamily and one in the Soricinae subfamily. Based on this wide distribution, we estimate that the integration event occurred before the last common ancestor of the subfamily, about 10.8 million years ago, attesting to an ancient origin of pestiviruses and Flaviviridae in general. Moreover, we provide the first description of Flaviviridae-derived EVEs in mammals even though the family encompasses numerous mammal-infecting members, including major human pathogens such as Zika, dengue, or hepatitis C viruses. This also suggests that shrews were past and perhaps also current natural reservoirs of pestiviruses. Taken together, our results expand the current known Pestivirus host range and provide novel insight into the ancient evolutionary history of pestiviruses and the Flaviviridae family in general.

sentences: Identification of Catechins Binding Pockets in Monomeric Aβ42 Through Ensemble Docking and MD Simulations The assembly of the Amyloid-{beta} peptide (A{beta}) into toxic oligomers and fibrils is associated with Alzheimers disease and dementia. Therefore, disrupting amyloid assembly by direct targeting of the A{beta} monomeric form with small molecules or antibodies is a promising therapeutic strategy. However, given the dynamic nature of A{beta}, standard computational tools cannot be easily applied for high-throughput structure-based virtual screening in drug discovery projects. In the current study, we propose a computational pipeline - in the framework of the ensemble docking strategy - to identify catechins binding pockets in monomeric A{beta}42. It is shown that both hydrophobic aromatic interactions and hydrogen bonding are crucial for the binding of catechins to A{beta}42. Also, it has been found that all the studied ligands, especially the EGCG, can act as potent inhibitors against amyloid aggregation by blocking the central hydrophobic region of the A{beta}. Our findings are evaluated and confirmed with multi-microsecond MD simulations. Finally, it is suggested that our proposed pipeline, with low computational cost in comparison with MD simulations, is a suitable approach for the virtual screening of ligand libraries against A{beta}.

sentences: Structural insights into the peptide selectivity and activation of human neuromedin U receptors Neuromedin U receptors (NMURs), including NMUR1 and NMUR2, are a group of Gq/11-coupled G protein-coupled receptors (GPCRs) related to pleiotropic physiological functions. Upon stimulation by two endogenous neuropeptides, neuromedin U and S (NMU and NMS) with similar binding affinities, NMUR1 and NMUR2 primarily display distinct peripheral tissue and central nervous system (CNS) functions, respectively, due to their distinct tissue distributions. These NMU receptors have triggered extensive attention as drug targets for obesity and immune inflammation. Specifically, selective agonists for NMUR1 in peripheral tissue show promising long-term anti-obesity effects with fewer CNS-related side effects. However, the mechanisms of peptide binding specificity and receptor activation remain elusive due to the lack of NMU receptor structures, which hamper drug design targeting NMU receptors. Here, we report four cryo-electron microscopy structures of Gq chimera-coupled NMUR1 and NMUR2 bound with NMU and NMS. These structures present the conserved overall peptide-binding mode and reveal the mechanism of peptide selectivity for specific NMURs, as well as the common activation mechanism of the NMUR subfamily. Together, these findings provide insights into the molecular basis of the peptide recognition selectivity and offer a new opportunity for designing selective drugs targeting NMURs.

sentences: A new hypothesis for type 1 diabetes risk: The at-risk allele at rs3842753 associates with increased beta cell INS mRNA in a meta-analysis of single cell RNA sequencing data Type 1 diabetes is characterized by the autoimmune destruction of insulin secreting {beta} cells. Genetic variations upstream at the insulin (INS) locus contribute to ~10% of type 1 diabetes heritable risk. Multiple studies showed an association between rs3842753 C/C genotype and type 1 diabetes susceptibility, but the molecular mechanisms remain unclear. To date, no large-scale studies have looked at the effect of genetic variation at rs3842753 on INS mRNA at the single cell level. We aligned all human islet single cell RNA sequencing datasets available to us in 2020 to the reference genome GRCh38.98 and genotyped rs3842753, integrating 2315 {beta} cells and 1223 {beta}-like cells from 13 A/A protected donors, 23 A/C heterozygous donors, and 35 C/C at-risk donors, including adults without diabetes and with type 2 diabetes. INS expression mean and variance were significantly higher in single {beta} cells from females compared with males. Comparing across {beta} cells and {beta}-like cells, we found that rs3842753 C containing cells (either homozygous or heterozygous) had the highest INS expression. We also found that {beta} cells with the rs3842753 C allele had significantly higher ER stress marker gene expression compared to the A/A homozygous genotype. These findings support the emerging concept that inherited risk of type 1 diabetes may be associated with inborn, persistent elevated insulin production which may lead to {beta} cell ER stress and fragility.

sentences: Effects of fine-scale population structure on inbreeding in a long-term study of snapdragons (Antirrhinum majus) Many studies have quantified the distribution of heterozygosity and relatedness in natural populations, but surprisingly few have examined the demographic processes driving these patterns. In this study we take a novel approach by studying how population structure affects both pairwise identity and the distribution of heterozygosity in a natural population of a self-incompatible plant Antirrhinum majus. We look at a measure of the variance in heterozygosity within a population, identity disequilibrium (g2), together with FST using a panel of 91 SNPs in 22,353 individuals collected over 11 years. We find that pairwise relatedness (FST) declines rapidly over short spatial scales, and the excess variance in heterozygosity between individuals (g2) reflects significant variation in inbreeding. Additionally, we detect an excess of individuals with around half the average heterozygosity, indicating that some are due to selfing or matings between close relatives. We use two types of simulation to ask whether variation in heterozygosity is consistent with fine-scale spatial population structure. First, by simulating offspring using parents drawn from a range of spatial scales, we show that the known pollen dispersal kernel explains g2. Second, we simulate a 1000-generation pedigree using the known dispersal and spatial distribution and find that the resulting g2 is consistent with that observed from the field data. In contrast, a simulated population with uniform density underestimates g2, indicating that heterogeneous density promotes identity disequilibrium. Our study shows that heterogeneous density and leptokurtic dispersal can together explain the distribution of heterozygosity. Furthermore, our study highlights the limitations of making theoretical predictions from simulations that only assume simple density and dispersal distributions.

sentences: Exo-enzymatic addition of diazirine-modified sialic acid to cell surfaces enables photocrosslinking of glycoproteins Glycan binding often mediates extracellular macromolecular recognition events. Accurate characterization of these binding interactions can be difficult because of dissociation and scrambling that occur during purification and analysis steps. Use of photocrosslinking methods has been pursued to covalently capture glycan-dependent interactions in situ however use of metabolic glycan engineering methods to incorporate photocrosslinking sugar analogs is limited to certain cell types. Here we report an exo-enzymatic labeling method to add a diazirine-modified sialic acid (SiaDAz) to cell surface glycoconjugates. The method involves chemoenzymatic synthesis of diazirine-modified CMP-sialic acid (CMP-SiaDAz), followed by sialyltransferase-catalyzed addition of SiaDAz to desialylated cell surfaces. Cell surface SiaDAz-ylation is compatible with multiple cell types and is facilitated by endogenous extracellular sialyltransferase activity present in Daudi B cells. This method for extracellular addition of 2-6-linked SiaDAz enables UV-induced crosslinking of CD22, demonstrating the utility for covalent capture of glycan-mediated binding interactions.

sentences: A trade-off between stress resistance and tolerance underlies the adaptive response to hydrogen peroxide In response to environmental stress, cellular defense strategies may be divided into two categories: those, as in homeostatic systems, that seek to maintain cell proliferation by degrading the stressor (i.e., resistance); and those that ensure cell survival (i.e. tolerance), even if this is often at the expense of cell proliferation. In this study, we have explored the genetic bases of the antagonism between resistance and tolerance during the response to hydrogen peroxide (H2O2) in budding yeast. We show that inactivation of protein kinase A (PKA) by H2O2 signaling induces an abrupt transition from normal homeostatic function to a stress-tolerant state by protecting the growth machinery, hence maximizing cellular fitness in a changing environment. This model system paves the way for developing antiproliferative strategies that target both resistance and tolerance mechanisms to prevent relapse.

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NLoed: A Python package for nonlinear optimal experimental design in systems biology MotivationModelling in systems and synthetic biology relies on accurate parameter estimates and predictions. Accurate model calibration relies, in turn, on data, and on how well-suited the available data is to a particular modelling task. Optimal experimental design (OED) techniques can be used to identify experiments and data collection procedures that will most efficiently contribute to a given modelling objective. However, implementation of OED is limited by currently available software tools that are not well-suited for the diversity of nonlinear models and non-normal data commonly encountered in biological research. Moreover, existing OED tools do not make use of the state-of-the-art numerical tools, resulting in inefficient computation.

ResultsHere we present the NLoed software package. NLoed is an open-source Python library providing convenient access to OED methods, with particular emphasis on experimental design for systems biology research. NLoed supports a wide variety of nonlinear, multi-input/output, and dynamic models, and facilitates modelling and design of experiments over a wide variety of data types. To support OED investigations, the NLoed package implements maximum likelihood fitting and diagnostic tools, providing a comprehensive modelling workflow. NLoed offers an accessible, modular, and flexible OED tool-set suited to the wide variety of experimental scenarios encountered in systems biology research. We demonstrate NLOEDs capabilities by applying it to experimental design for characterization of a bacterial optogenetic system.

AvailabilityNLoed is available via pip from the PyPi repository; https://pypi.org/project/nloed/. Source code, documentation and examples can be found on Github at https://github.com/ingallslab/NLoed.

Contactbingalls@uwaterloo.ca

Supplementary informationSupplementary materials are available online.

sentences: Genomic consequences of colonisation, migration and genetic drift in barn owl insular populations of the eastern Mediterranean The study of insular populations was key in the development of evolutionary theory. The successful colonisation of an island depends on the geographic context, and specific characteristics of the organism and the island, but also on stochastic processes. As a result, apparently identical islands may harbour populations with contrasting histories. Here, we use whole genome sequences of 65 barn owls to investigate the patterns of inbreeding and genetic diversity of insular populations in the eastern Mediterranean Sea. We focus on Crete and Cyprus, islands with similar size, climate and distance to mainland, that provide natural replicates for a comparative analysis of the impacts of microevolutionary processes on isolated populations. We show that barn owl populations from each island have a separate origin, Crete being genetically more similar to other Greek islands and mainland Greece, and Cyprus more similar to the Levant. Further, our data show that their respective demographic histories following colonisation were also distinct. On the one hand, Crete harbours a small population and maintains very low levels of gene flow with neighbouring populations. This has resulted in low genetic diversity, strong genetic drift, increased relatedness in the population and remote inbreeding. Cyprus, on the other hand, appears to maintain enough gene flow with the mainland to avoid such an outcome. Our work provides a comparative population genomic analysis of the effects of neutral processes on a classical island-mainland model system. It provides empirical evidence for the role of stochastic processes in determining the fate of diverging isolated populations.

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Genomic and physiological characterization of Novosphingobium terrae sp. nov., an alphaproteobacterium isolated from Cerrado soil containing a megasized chromid A novel bacterial strain, designated GeG2T, was isolated from soils of native Cerrado, a highly biodiverse savanna-like Brazilian biome. 16S rRNA gene sequence analysis of strain GeG2T revealed high sequence identity (100%) to the alphaproteobacterium Novosphingobium rosa, however, comparisons with N. rosa DSM7285T showed several distinctive features, prompting a full characterization of the new strain in terms of growth, morphology, biochemistry and, ultimately, its genome. GeG2T cells were Gram-stain negative bacilli, facultatively anaerobic, motile, positive for catalase and oxidase activities and for starch hydrolysis. Strain GeG2T presented planktonic-sessile dimorphism and cell aggregates surrounded by extracellular matrix and nanometric spherical structures were observed in liquid cultures, suggesting the production of exopolysaccharides (EPS) and outer membrane vesicles (OMVs). Whole genome assembly revealed four circular replicons: a 4.1 Mb chromosome, a 2.7 Mb extrachromosomal megareplicon and two plasmids (212.7 and 68.6 kb). The megareplicon contains few core genes and plasmid-type replication/maintenance systems, consistent with its classification as a chromid. Genome annotation shows a vast repertoire of carbohydrate active enzymes and genes involved in the degradation of aromatic compounds, highlighting the biotechnological potential of the new isolate obtained from Cerrado soils, especially regarding EPS production and biodegradation of recalcitrant compounds. Chemotaxonomic features, including polar lipid and fatty acid profiles, as well as physiological, molecular and whole genome comparisons showed significant differences between strain GeG2T and a N. rosa, clearly indicating that it represents a novel species, for which the name Novosphingobium terrae is proposed. The type strain is GeG2T (=CBMAI 2313T =CBAS 753T).

IMPORTANCENovosphingobium is an alphaproteobacterial genus presenting diverse physiological profiles and broad biotechnological applications. However, many aspects regarding the biology of this important bacterial group remain elusive. A novel Novosphingobium strain was isolated from soils of Cerrado, an important Brazilian biome. Despite 100% 16S rRNA gene identity with Novosphingobium rosa, polyphasic characterizations, including physiological, chemotaxonomic, and whole genome- based analyses revealed significant differences between GeG2T and N. rosa DSM7285T, reinforcing resolution limitations in phylogenetic analysis based solely on 16S RNA and highlighting the importance of employing different approaches for the description of bacterial species. Using short and long read sequencing approaches, a high-quality fully resolved genome assembly was generated and one of the largest chromids reported to date was identified. A comprehensive characterization of environmental isolates allows us to better elucidate the diversity and biology of members of this bacterial group with potential biotechnological importance, guiding future bioprospecting efforts and genomic studies.

sentences: A unique Toxoplasma gondii haplotype under strong selection has accompanied domestic cats in their global expansion Toxoplasma gondii is a cyst-forming apicomplexan parasite of virtually all warm-blooded species, with all true cats (Felidae) as definitive hosts. It is the etiologic agent of toxoplasmosis, a disease causing substantial public health burden worldwide. Its wide range of host species and its global occurrence probably complicate the study of its evolutionary history, and conflicting scenarios have been proposed to explain its current global distribution. In this study, we analyse a global set of 156 genomes (including 105 new genomes) and we provide the first direct estimate of T. gondii mutation rate and the first estimate of its generation time. We elucidate how the evolution of T. gondii populations is intimately linked to the major events that have punctuated the recent history of cats. We show that a unique haplotype --whose length represents only 0.16% of the whole T. gondii genome-- is common to all domestic T. gondii strains worldwide and has accompanied wild cats (Felis silvestris) during their emergence from the wild to domestic settlements, their dispersal in the Old World and their recent expansion to the Americas in the last six centuries. By combining environmental and functional data to selection inference tools, we show that selection of this domestic haplotype is most parsimoniously explained by its role in initiation of sexual reproduction of T. gondii in domestic cats.

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Improving replicability using interaction with laboratories: a multi-lab experimental assessment Experimentation with mouse and rat models has become a central strategy for discovering mammalian gene function, and for preclinical testing of pharmacological treatments, yet the utility of any findings critically depends on their replicability in other laboratories. In previous publications we proposed a statistical approach for estimating the inter-laboratory replicability of novel discoveries made in a single laboratory. We demonstrated that previous phenotyping results from multi-lab databases can be used to derive a Genotype-by-Lab (GxL) adjustment factor to greatly enhance the replicability of the single-lab findings, for similarly measured phenotypes, even before making the effort of replicating these finding in additional laboratories.

This demonstration, however, still raised several important questions that could only be answered by an additional large-scale prospective experiment: 1) Does GxL-adjustment work in single-lab experiments that were not intended to be standardized across laboratories, and with genotypes that were not included in the previous experiments? And 2) Can it be used to adjust the results of pharmacological experiments? We investigated these questions by attempting to replicate, across three laboratories, results from five single-lab studies in the Mouse Phenome Database (MPD), offering 212 comparisons, including 60 involving a pharmacological treatment: 18 mg/kg/day fluoxetine. In addition, we define and use a dimensionless GxL factor, by dividing the GxL variance by the standard deviation between animals within groups, as a more robust vehicle to transfer the adjustment from the multi-lab analysis to very different labs and genotypes.

For genotype comparisons, GxL-adjustment reduced the rate of non-replicable discoveries from 60% to 12%, for the price of reducing the power to make replicable discoveries from 87% to 66%. In absolute numbers, the adjustment prevented 23 non-replicable discoveries for the price of missing only three replicated ones. Tools and data needed for deployment of this method across other mouse experiments are publicly available in MPD. Our results further point at some phenotypes as more prone to produce non-replicable results, while others, known to be more difficult to measure, are as likely to produce replicable results (once adjusted) such as the physiological measure, body weight.

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Bacteriophages antagonize cGAS-like immunity in bacteria The recently discovered cyclic-oligonucleotide-based anti-phage signaling system (CBASS) is related to eukaryotic cGAS-STING anti-viral immunity and is present in diverse prokaryotes. However, our understanding of how CBASS detects, inhibits, and co-evolves with phages is limited because CBASS function has only been studied in reconstituted heterologous systems. Here, we identify a phage-encoded CBASS antagonist (acbIIA1, anti-cbass type II-A gene 1) necessary for phage replication in the presence of endogenous CBASS immunity in Pseudomonas aeruginosa. acbIIA1 homologs are encoded by numerous lytic and temperate phages infecting Gram-negative bacteria. Deletion of acbIIA1 renders multiple phages susceptible to CBASS, but phages can then escape immune function via mutations in the major capsid gene. These mutants suggest that CBASS is activated by, or targets, the late-expressed phage capsid. Together, we establish a native model system to study CBASS and identify a common phage-encoded CBASS antagonist, demonstrating that CBASS is a bona fide anti-phage immune system in nature.

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Mapping drug-microenvironment-genetic interplay in CLL reveals trisomy 12 as a modulator of microenvironmental signals The tumour microenvironment and genetic alterations collectively influence drug efficacy in cancer, but current evidence is limited to small scale studies and systematic analyses are lacking. We chose Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia in adults, as a model disease to study this complex interplay systematically. We performed a combinatorial assay using 12 drugs individually co-applied with each of 17 microenvironmental stimuli in 192 primary CLL samples, generating a comprehensive map of drug-microenvironment interactions in CLL. This data was combined with whole-exome sequencing, DNA-methylation, RNA-sequencing and copy number variant annotation. Our assay identified four distinct CLL subgroups that differed in their responses to the panel of microenvironmental stimuli. These subgroups were characterized by distinct clinical outcomes independently of known prognostic markers. We investigated the effect of CLL- specific recurrent genetic alterations on microenvironmental responses and identified trisomy 12 as an amplifier of multiple microenvironmental stimuli. We further quantified the impact of microenvironmental stimuli on drug response, confirmed known interactions such as Interleukin (IL) 4 mediated resistance to B cell receptor (BCR) inhibitors, and identified new interactions such as Interferon-{gamma} induced resistance to BCR inhibitors. Finally, we identified interactions which were limited to genetic subgroups. Resistance to chemotherapeutics, such as Fludarabine, induced by Toll-Like Receptor (TLR) agonists could be observed in IGHV unmutated patient samples and IGHV mutated samples with trisomy 12. In-vivo relevance was investigated in CLL-infiltrated lymph nodes, which showed increased IL4 and TLR signalling activity compared to healthy samples (p<0.001). High IL4 activity in lymph nodes correlated with faster disease progression (p=0.038).

We provide a publicly available resource (www.dietrichlab.de/CLL_Microenvironment/) which uncovers tumour cell extrinsic influences on drug response and disease progression in CLL, and how these interactions are modulated by cell intrinsic molecular features.

sentences: Discovery of small molecule antagonists of human Retinoblastoma Binding Protein 4 (RBBP4) RBBP4 is a nuclear WD40 motif-containing protein widely implicated in various cancers and a putative drug target. It interacts with multiple proteins within diverse complexes such as nucleosome remodeling and deacetylase (NuRD) complex and polycomb repressive complex 2 (PRC2), as well as histone H3 and H4 through two distinct binding sites. B-cell lymphoma/leukemia 11A (BCL11A), friend of GATA-1 (FOG-1), plant homeodomain finger protein 6 (PHF6) and histone H3 bind to the top of the donut-shaped seven-bladed {beta}-propeller fold of RBBP4, while suppressor of zeste 12 (SUZ12), metastasis associated protein 1 (MTA1) and histone H4 bind to a pocket on the side of the WD40 repeats of this protein. Here, we report the discovery of the first small molecule antagonists of the RBBP4 top pocket, competing with interacting peptides from proteins such as BCL11A and histone H3. We also determined the first crystal structure of RBBP4 in complex with a small molecule (OICR17251), paving the path for structure-guided design and optimization towards more potent antagonists.

sentences: Cancer-specific loss of TERT activation sensitizes glioblastoma to DNA damage Most glioblastomas (GBMs) achieve cellular immortality by acquiring a mutation in the telomerase reverse transcriptase (TERT) promoter. TERT promoter mutations create a binding site for a GA binding protein (GABP) transcription factor complex, whose assembly at the promoter is associated with TERT reactivation and telomere maintenance. Here, we demonstrate increased binding of a specific GABPB1L isoform-containing complex to the mutant TERT promoter. Furthermore, we find that TERT promoter mutant GBM cells, unlike wild-type cells, exhibit a critical near-term dependence on GABPB1L for proliferation, both in cell culture and post-tumor establishment in vivo. Upregulation of the protein paralogue GABPB2, which is normally expressed at very low levels, can rescue this dependence. More importantly, when combined with frontline temozolomide (TMZ) chemotherapy, inducible GABPB1L knockdown and the associated TERT reduction led to an impaired DNA damage response that resulted in profoundly reduced growth of intracranial GBM tumors. Together, these findings provide new insights into the mechanism of cancer-specific TERT regulation, uncover rapid effects of GABPB1L-mediated TERT suppression in GBM maintenance, and establish GABPB1L inhibition in combination with chemotherapy as a novel therapeutic strategy for TERT promoter mutant GBM.

sentences: Larval zebrafish display dynamic learning of aversive stimuli in a constant visual surrounding Balancing exploration and anti-predation are fundamental to the fitness and survival of all animal species from early life stages. How these basic survival instincts drive learning remains poorly understood. Here, employing a light/dark preference paradigm with well-controlled luminance history and constant visual surrounding in larval zebrafish, we analyzed intra- and inter-trial dynamics for two behavioral components, dark avoidance and center avoidance. We uncover that larval zebrafish display short-term learning of dark avoidance with initial sensitization followed by habituation; they also exhibit long-term learning that is sensitive to trial interval length. We further show that such stereotyped learning patterns is stimulus specific, as they are not observed for center avoidance. Finally, we demonstrate at individual levels that long-term learning is under homeostatic control. Together, our work has established a novel paradigm to understand learning, uncovered sequential sensitization and habituation, and demonstrated stimulus specificity, individuality, as well as dynamicity in learning.

sentences: Ribosome stalling caused by the Argonaute-microRNA-SGS3 complex regulates the production of secondary siRNAs in plants The path of ribosomes on mRNAs can be impeded by various obstacles. One such example is halting of ribosome movement by microRNAs, though the exact mechanism and physiological role remain unclear. Here, we find that ribosome stalling caused by the Argonaute-microRNA-SGS3 complex regulates the production of secondary small interfering RNAs (siRNAs) in plants. We show that the double-stranded RNA-binding protein SGS3 directly interacts with the 3' end of the microRNA in an Argonaute protein, resulting in ribosome stalling. Importantly, microRNA-mediated ribosome stalling positively correlates with efficient production of secondary siRNAs from target mRNAs. Our results illustrate a role for paused ribosomes in regulation of small RNA function that may have broad biological implications across the plant kingdom.

sentences: Tie-lines reveal interactions driving heteromolecular condensate formation Phase separation of biomolecules give rise to membraneless organelles that contribute to the spatiotemporal organisation of the cell. In most cases, such biomolecular condensates contain multiple components, but the manner in which interactions between components control the stability of condensates remained challenging to elucidate. Here, we develop an approach to determine tie-line gradients in ternary liquid-liquid phase separation (LLPS) systems, based on measurements of the dilute phase concentration of only one component. We show that the sign of the tie-line gradient is related to the cross-interaction energy between the polymers in the system and discriminates between competitive and cooperative phase separation. Using this approach, we studied the interaction between protein Fused in Sarcoma (FUS) and polyethylene glycol (PEG) polymer chains, and measured positive tie-line gradients. Our results show that PEG drives LLPS through an associative interaction with FUS and is not an inert crowder. We further studied the interaction between PolyA RNA (3.0{+/-}0.5kDa) and the protein G3BP1, and using the tie-line gradient as a reporter for the stoichiometry of polymers in the condensate we determined a G3BP1-to-PolyA RNA molar ratio of 1:4 in the dense phase. Our framework for measuring tie-line gradients opens up a route for the characterisation of interaction types and compositions in ternary LLPS systems.

sentences: A non-adaptive demographic mechanism for genome expansion in Streptomyces The evolution of microbial genome size is driven by gene acquisition and loss events that occur at scales from individual genomes to entire pangenomes. The equilibrium between gene gain and loss is shaped by evolutionary forces, including selection and drift, which are in turn influenced by population demographics. There is a well-known bias towards deletion in microbial genomes, which promotes genome streamlining. Less well described are mechanisms that promote genome expansion, giving rise to the many microbes, such as Streptomyces, that have unusually large genomes. We find evidence of genome expansion in Streptomyces sister-taxa, and we hypothesize that a recent demographic range expansion drove increases in genome size through a non-adaptive mechanism. These Streptomyces sister-taxa, NDR (northern-derived) and SDR (southern-derived), represent recently diverged lineages that occupy distinct geographic ranges. Relative to SDR genomes, NDR genomes are larger, have more genes, and their genomes are enriched in intermediate frequency genes. We also find evidence of relaxed selection in NDR genomes relative to SDR genomes. We hypothesize that geographic range expansion, coupled with relaxed selection, facilitated the introgression of non-adaptive horizontally acquired genes, which accumulated at intermediate frequencies through a mechanism known as genome surfing. We show that similar patterns of pangenome structure and genome expansion occur in a simulation that models the effects of population expansion on genome dynamics. We show that non-adaptive evolutionary phenomena can explain expansion of microbial genome size, and suggest that this mechanism might explain why some bacteria with large genomes can be found in soil.

sentences: The ultrasound guide: katydid ear pinnae code for bat call detection Early predator detection is a key component of the predator-prey arms race, and has driven the evolution of multiple animal hearing systems. Katydids (Insecta) have sophisticated ears, each consisting of paired tympana on each foreleg that receive sound directly externally, and internally via a narrowing ear canal through the acoustic spiracle. These ears are pressure-time difference receivers capable of sensitive and accurate directional hearing across a wide frequency range, despite the small size of katydids. Many katydid species have cuticular pinnae which form cavities around the outer tympanal surfaces, but their function is unknown. We investigated pinnal function in the katydid Copiphora gorgonensis by combining experimental biophysics and numerical modelling using 3D ear geometries. Results show that the pinnae in C. gorgonensis do not assist in directional hearing for specific call frequencies, but instead pinnae act as ultrasound detector devices. Pinnae induced large sound pressure gains that enhanced sound detection at high ultrasonic frequencies (> 60 kHz), matching the echolocation range of co-occurring insectivorous bats. Comparing pinnal mechanics of sympatric katydid species supports these findings, and suggests that pinnae evolved in katydids primarily for enhanced predator detection. Audiograms (both behavioural and neural) and tympanal cavity resonances obtained from living specimens corroborate our findings.

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Expression analysis of Huntington disease mouse models reveals robust striatum disease signatures Huntingtons disease is caused by expanded trinucleotide repeats in the huntingtin gene (HTT), and a higher number of repeats is associated with an earlier age of disease onset. Although the causative gene has been identified, its connections to the observed disease phenotypes is still unclear. Mouse models engineered to contain increasing numbers of trinucleotide repeats were sacrificed at different ages to detect RNA-level and protein-level changes specific to each repeat length and age in order to examine the transcriptional and translational characteristics of the disease. RNA-seq and quantitative proteomics data were collected on 14 types of tissues at up to 8 repeat lengths and in up to 3 different ages, and differential gene and protein expression were examined. A modified method of imputing missing proteomics data was employed and is described here. The most dysregulated tissue at both the RNA and protein levels was the striatum, and a strong gender effect was observed in all of the liver experiments. The full differential expression results are available to the research community on the HDinHD.org website. The results of multiple expression tests in the striatum were combined to generate an RNA disease signature and a protein disease signature, and the signatures were validated in external data sets. These signatures represent molecular readouts of disease progression in HD mice, which further characterizes their HD-related phenotype and can be useful in the preclinical evaluation of candidate therapeutic interventions.

Author SummaryMouse models of Huntingtons disease were engineered to allow a detailed examination of how the disease causes changes in gene activity in a variety of tissues. Among the 14 tissues studied, the one most affected by the disease in our experiments was the striatum, a brain region involved in voluntary movement. The liver results showed large differences in gene activity between the male and female mice. In our analysis, we propose a minor change in how proteomics data is typically analyzed in order to improve the ranking of significant results. Using the striatum data in this study and in others, we identified robust genetic signatures of disease at both the RNA and protein levels.

sentences: mRNA-decapping associated DcpS enzyme controls critical steps of neuronal development Homozygous mutations in the gene encoding the scavenger mRNA-decapping enzyme, DcpS, have been shown to underlie developmental delay and intellectual disability. Intellectual disability is associated with both abnormal neocortical development and mRNA metabolism. However, the role of DcpS and its scavenger decapping activity in neuronal development is unknown. Here, we show that human neurons derived from patients with a DcpS mutation have compromised differentiation and neurite outgrowth. Moreover, in the developing mouse neocortex, DcpS is required for the radial migration, polarity, neurite outgrowth and identity of developing glutamatergic neurons. Collectively, these findings demonstrate that the scavenger mRNA decapping activity contributes to multiple pivotal roles in neural development, and further corroborate that mRNA metabolism and neocortical pathologies are associated with intellectual disability.

sentences: Pro-apoptotic caspase deficiency reveals a cell-extrinsic mechanism of NK cell regulation AO_SCPLOWBSTRACTC_SCPLOWRegulated cell death is essential for the maintenance of cellular and tissue homeostasis. In the hematopoietic system, genetic defects in apoptotic cell death generally produce the accumulation of immune cells, inflammation and autoimmunity. In contrast, we found that genetic deletion of caspases of the mitochondrial apoptosis pathway reduces natural killer (NK) cell numbers and makes NK cells functionally defective in vivo and in vitro. Caspase deficiency results in constitutive activation of a type I interferon (IFN) response, due to leakage of mitochondrial DNA and activation of the cGAS/STING pathway. The NK cell defect in caspase-deficient mice is independent of the type I IFN response, but the phenotype is partially rescued by cGAS or STING deficiency. Finally, caspase deficiency alters NK cells in a cell-extrinsic manner. Type I IFNs and NK cells are two essential effectors of antiviral immunity, and our results demonstrate that they are both regulated in a caspase-dependent manner. Beyond caspase-deficient animals, our observations may have implications in infections that trigger mitochondrial stress and caspase-dependent cell death.

sentences: Nutrient dominance governs the assembly of microbial communities in mixed nutrient environments A major open question in microbial community ecology is whether we can predict how the components of a diet collectively determine the taxonomic composition of microbial communities. Motivated by this challenge, we investigate whether communities assembled in pairs of nutrients can be predicted from those assembled in every single nutrient alone. We first find that although the null, naturally additive model generally predicts well the family-level community composition, there exist systematic deviations from the additive predictions that reflect generic patterns of nutrient dominance at the family-level. Pairs of more similar nutrients (e.g. two sugars) are on average more additive than pairs of more dissimilar nutrients (one sugar-one organic acid). Second, a simple dominance rule emerges: sugars generally dominate organic acids. These findings may be explained by family-level asymmetries in nutrient benefits. Overall, our results suggest that regularities in how nutrients interact may help predict communities responses to dietary changes.

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Long-term cross-scale comparison of grazing and mowing on plant diversity and community composition in a salt-marsh system O_LILand abandonment is increasing in recent decades in Europe, usually accompanied by a decline in biodiversity. Whether livestock grazing and mowing can safeguard biodiversity across spatial scales in the long term is unclear.
C_LIO_LIUsing a 48-year experiment in a salt marsh, we compared land abandonment (without grazing and mowing) and seven management regimes including cattle grazing, early season mowing, late season mowing, both early and late season mowing, and grazing plus each of the mowing regimes on plant diversity at the local (i.e. plot) and landscape scales (i.e. across plots). Also, we compared their effects on community composition (both in identities and abundances) in time and space.
C_LIO_LIUnder land abandonment, plant diversity declined in the local communities and this decline became more apparent at the landscape scale, particularly for graminoids and halophytes. All management regimes, except the late season mowing, maintained plant diversity at these scales.
C_LIO_LILocal plant communities under all treatments underwent different successional trajectories, in the end, diverged from their initial state except for that under grazing (a cyclic succession). Interannual composition change remained stable over time under land abandonment and grazing plus early season mowing. It increased over time under grazing and late season mowing, it increased in the second half of the experiment under other treatments. Vegetation homogenized in the landscape over time under land abandonment while vegetation was heterogeneous under all management regimes.
C_LIO_LISynthesis. Our experiment suggests that late season mowing may not be sustainable to conserve plant diversity in salt marshes. Other management regimes can maintain plant diversity across scales and vegetation heterogeneity in the landscape in the long term, but local community composition may change over time.
C_LI

sentences: Paucimyces polynucleatus gen. nov, sp. nov., a novel polycentric genus of anaerobic gut fungi from the feces of a wild blackbuck antelope The anaerobic gut fungi (AGF, phylum Neocallimastigomycota) reside in the alimentary tracts of herbivores. Multiple novel, yet-uncultured AGF taxa have recently been identified in culture-independent diversity surveys. Here, we report on the isolation and characterization of the first representative of the RH5 lineage from fecal samples of a wild blackbuck (Indian Antelope) from Sutton County, Texas, USA. The isolates displayed medium sized (2-4 mm) compact circular colonies on agar roll tubes and thin loose biofilm-like growth in liquid medium. Microscopic examination revealed monoflagellated zoospores and polycentric thalli with highly branched nucleated filamentous rhizomycelium, a growth pattern encountered in a minority of described AGF genera so far. The obtained isolates are characterized by formation of spherical vesicles at the hyphal tips from which multiple sporangia formed either directly on the spherical vesicles or at the end of sporangiophores. Phylogenetic analysis using the D1/D2 regions of the large ribosomal subunit (D/D2 LSU) and the ribosomal internal transcribed spacer 1 (ITS1) revealed sequence similarities of 93.5%, and 81.3%, respectively, to the closest cultured relatives (Orpinomyces joyonii strain D3A (D1/D2 LSU), and Joblinomyces apicalis strain GFH681 (ITS1). Substrate utilization experiments using the type strain (BB-3) demonstrated growth capabilities on a wide range of mono-, oligo-, and polysaccharides, including glucose, xylose, mannose, fructose, cellobiose, sucrose, maltose, trehalose, lactose, cellulose, xylan, starch, and raffinose. We propose accommodating these novel isolates in a new genus and species, for which the name Paucimyces polynucleatus is proposed. The type species is strain BB-3.

sentences: Dimensionality and modularity of adaptive variation: Divergence in threespine stickleback from diverse environments In nature, populations are subjected to a wide variety of environmental conditions that affect fitness and induce adaptive or plastic responses in traits, resulting in phenotypic divergence between populations. The dimensionality of that divergence, however, remains contentious. At the extremes, some contend that populations diverge along a single axis of trait covariance with greatest availability of heritable variation, even if this does not lead a population directly to its fitness optimum. Those at the other extreme argue that selection can push populations towards their fitness optima along multiple phenotype axes simultaneously, resulting in divergence in numerous dimensions. Here, we address this debate using populations of threespine stickleback (Gasterosteus aculeatus) in the Cook Inlet region of southern Alaska from lakes with contrasting ecological conditions. We calculated effective dimensionality of divergence in several trait suites (defensive, swimming, and trophic) thought to be under correlated selection pressures, as well as across all traits. We also tested for integration among the trait suites and between each trait suite and the environment. We found that populations in the Cook Inlet radiation exhibit dimensionality of phenotype high enough to preclude a single axis of divergence.

sentences: How accurately can we assess zoonotic risk? The authors have withdrawn their manuscript as a website associated with a zoonotic risk prediction tool mentioned here was not public. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author

sentences: Tuneable wide-field illumination and single-molecule photoswitching with a single MEMS mirror Homogeneous illumination in single-molecule localization microscopy (SMLM) is key for the quantitative analysis of super-resolution images. Therefore, different approaches for flat-field illumination have been introduced as alternative to the conventional Gaussian illumination. Here, we introduce a single microelectromechanical systems (MEMS) mirror as a tuneable and cost-effective device for adapting wide-field illumination in SMLM. In flat-field mode the MEMS allowed for consistent SMLM metrics across the entire field of view. Employing single-molecule photoswitching, we developed a simple yet powerful routine to benchmark different illumination schemes on the basis of local emitter brightness and ON-state lifetime. Moreover, we propose that tuning the MEMS beyond optimal flat-field conditions enables to study the kinetics of photoswitchable fluorophores within a single acquisition.

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A Self-Replicating Radiation-Shield for Human Deep-Space Exploration: Radiotrophic Fungi can Attenuate Ionizing Radiation aboard the International Space Station The greatest hazard for humans on deep-space exploration missions is radiation. To protect astronauts venturing out beyond Earths protective magnetosphere, advanced passive radiation protection is highly sought after. In search of innovative radiation-shields, biotechnology appeals with suitability for in-situ resource utilization (ISRU), self-regeneration, and adaptability.

Certain fungi thrive in high-radiation environments on Earth, such as the contamination radius of the Chernobyl Nuclear Power Plant. Analogous to photosynthesis, these organisms appear to perform radiosynthesis, utilizing ionizing radiation to generate chemical energy. It has been postulated that the absorption of radiation is attributable to the pigment melanin. It is further hypothesized that this phenomenon translates to radiation-shielding properties.

Here, growth of Cladosporium sphaerospermum and its capability to attenuate ionizing radiation, was studied aboard the International Space Station (ISS) over a period of 26 days, as an analog to habitation on the surface of Mars. At full maturity, radiation beneath a {approx} 1.7 mm thick lawn of the dematiaceous radiotrophic fungus was approx. 0.84% lower as compared to the negative-control. In addition, a growth advantage in Space of [~] 21% was observed, substantiating the thesis that the fungus radiotropism is extendable to Space radiation.

sentences: A Miniaturized 3D-Printed Pressure Regulator (μPR) for Microfluidic Cell Culture Applications Controlled fluid flows are the hallmark feature of microfluidic culture systems and provide precise definition over the biophysical and biochemical microenvironment. Flow control is commonly achieved using displacement-based (e.g., syringe or peristaltic pumps) or pressure-based techniques. These methods offer complex flow capabilities but can be challenging to integrate into incubators or other confined environments due to their large form factors and accompanying peripheral equipment. Since many microfluidic cell culture studies use a single controlled flow rate to maintain or stimulate cells, a portable flow control platform that fits easily into an incubator will benefit the microfluidic community. Here, we demonstrate that a tunable, 3D printed micro pressure regulator (PR), combined with a battery-powered miniature air pump, can operate as a stand-alone pneumatic flow control platform for microfluidic applications. We detail the design and fabrication of the PR and demonstrate: i) a tunable outlet pressure range relevant for microfluidic applications (1-10 kPa), ii) highlight dynamic control in a microfluidic network, and iii) maintain human umbilical vein endothelial cells (HUVECs) in a multi-compartment membrane-based culture device under continuous flow conditions. We anticipate that our 3D-printed fabrication approach and open access designs will allow other laboratories to rapidly customize PRs to support a broad range of applications.

sentences: Overexpression of the WAPO-A1 gene increases the number of spikelets per spike in bread wheat Two homoeologous QTLs for number of spikelets per spike (SPS) were mapped on chromosomes 7AL and 7BL using two wheat MAGIC populations. Sets of lines contrasting for the QTL on 7AL were developed which allowed for the validation and fine mapping of the 7AL QTL and for the identification of a previously described candidate gene, WHEAT ORTHOLOG OF APO1 (WAPO1). Using transgenic overexpression in both a low and a high SPS line, we provide a functional validation for the role of this gene in determining SPS also in hexaploid wheat. We show that the expression levels of this gene positively correlate with SPS in multiple MAGIC founder lines under field conditions as well as in transgenic lines grown in the greenhouse. This work highlights the potential use of WAPO1 in hexaploid wheat for further yield increases. The impact of WAPO1 and SPS on yield depends on other genetic and environmental factors, hence, will require a finely balanced expression level to avoid the development of detrimental pleiotropic phenotypes.

sentences: Population bottleneck has only marginal effect on fitness evolution and its repeatability in dioecious C. elegans The predictability of evolution is expected to depend on the relative contribution of deterministic and stochastic processes. This ratio is modulated by effective population size. Smaller effective populations harbor less genetic diversity and stochastic processes are generally expected to play a larger role, leading to less repeatable evolutionary trajectories. Empirical insight into the relationship between effective population size and repeatability is limited and focused mostly on asexual organisms. Here, we tested whether fitness evolution was less repeatable after a population bottleneck in obligately outcrossing populations of Caenorhabditis elegans. Replicated populations founded by 500, 50, or 5 individuals (no/moderate/strong bottleneck) were exposed to a novel environment with a different bacterial prey. As a proxy for fitness, population size was measured after one week of growth before and after 15 weeks of evolution. Surprisingly, we found no significant differences among treatments in their fitness evolution. Even though the strong bottleneck reduced the relative contribution of selection to fitness variation, this did not translate to a significant reduction in the repeatability of fitness evolution. Thus, although a bottleneck reduced the contribution of deterministic processes, we conclude that the predictability of evolution may not universally depend on effective population size, especially in sexual organisms.

sentences: Environmental enrichment mitigates stroke-induced change in sharp-wave associated ripple characteristics Cognitive and memory impairments are common sequelae after stroke, yet how middle cerebral artery (MCA) stroke chronically affects the neural activity of the hippocampus, a brain region critical for memory but remote from the stroke epicenter, is poorly understood. Environmental enrichment (EE) improves cognition following stroke; however, the electrophysiology that underlies this behavioral intervention is still elusive. We recorded local field potentials simultaneously from sensorimotor cortex and hippocampus in rats following MCA occlusion and subsequent EE treatment. We found that MCA stroke significantly impacted the electrophysiology in the hippocampus, in particular it disrupted characteristics of sharp-wave associated ripples (SPW-Rs) which are known correlates of memory and cognition. Importantly, we show that EE mitigates stroke-induced changes to SPW-R characteristics. These results begin to uncover the complex interaction between cognitive deficit following stroke and EE treatment, providing a testbed to assess different strategies for therapeutics following stroke.

sentences: Gene conversion facilitates the adaptive evolution of self-resistance in highly toxic newts Reconstructing the histories of complex adaptations and identifying the evolutionary mechanisms underlying their origins are two of the primary goals of evolutionary biology. Taricha newts, which contain high concentrations of the deadly toxin tetrodotoxin (TTX) as an antipredator defense, have evolved resistance to self-intoxication, which is a complex adaptation requiring changes in six paralogs of the voltage-gated sodium channel (Nav) gene family, the physiological target of TTX. Here, we reconstruct the origins of TTX self-resistance by sequencing the entire Nav gene family in newts and related salamanders. We show that moderate TTX resistance evolved early in the salamander lineage in three of the six Nav paralogs, preceding the proposed appearance of tetrodotoxic newts by [~]100 million years. TTX-bearing newts possess additional unique substitutions across the entire Nav gene family that provide physiological TTX resistance. These substitutions coincide with signatures of positive selection and relaxed purifying selection, as well as gene conversion events, that together likely facilitated their evolution. We also identify a novel exon duplication within Nav1.4 encoding an expressed TTX-binding site. Two resistance-conferring changes within newts appear to have spread via nonallelic gene conversion: in one case, one codon was copied between paralogs, and in the second, multiple substitutions were homogenized between the duplicate exons of Nav1.4. Our results demonstrate that gene conversion can accelerate the coordinated evolution of gene families in response to a common selection pressure.

sentences: Genome-wide association study of open fieldbehavior in outbred heterogeneous stock ratsidentifies multiple loci implicated inpsychiatric disorders Many personality traits are influenced by genetic factors. Rodents models provide an efficient system for analyzing genetic contribution to these traits. Using 1,246 adolescent heterogeneous stock (HS) male and female rats, we conducted a genome-wide association study (GWAS) of behaviors measured in an open field, including locomotion, novel object interaction, and social interaction. We identified 30 genome-wide significant quantitative trait loci (QTL). Using multiple criteria, including the presence of high impact genomic variants and co-localization of cis-eQTL, we identified 13 candidate genes (Adarb2, Ankrd26, Cacna1c, Clock, Crhr1, Ctu2, Cyp26b1, Eva1a, Fam114a1, Kcnj9, Mlf2, Rab27b, Sec11a) for these traits. Most of these genes have been implicated by human GWAS of various psychiatric traits. For example, Cacna1c, a gene known to be critical for social behavior in rodents and implicated in human schizophrenia and bipolar disorder, is a candidate gene for distance to the social zone. In addition, the QTL region for total distance to the novel object zone, on Chr1 at 144 Mb, is syntenic to a hotspot on human Chr15 (82.5-90.8 Mb) that contains 14 genes associated with psychiatric or substance abuse traits. Although some of the genes identified by this study appear to replicate findings from prior human GWAS, others likely represent novel findings that can be the catalyst for future molecular and genetic insights into human psychiatric diseases. Together, these findings provide strong support for the use of the HS population to study psychiatric disorders.

sentences: Visualizing translation dynamics at atomic detail inside a bacterial cell Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells. Here, we use cryo-electron tomography and sub-tomogram analysis to visualize the dynamics of translation inside the prokaryote Mycoplasma pneumoniae. We first obtain an in-cell atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves thirteen ribosome states that differ in conformation and composition and reflect intermediates during translation. Based on these states, we animate translation elongation and demonstrate how antibiotics reshape the translation landscape inside cells. During translation elongation, ribosomes often arrange in a defined manner to form polysomes. By mapping the intracellular three-dimensional organization of translating ribosomes, we show that their association into polysomes exerts a local coordination mechanism that is mediated by the ribosomal protein L9. Our work demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.

sentences: Causal evidence for a cerebellar role in prefrontal-hippocampal interaction in spatial working memory decision-making Spatial working memory (SWM) is a cognitive skill supporting survival-relevant behaviors, such as optimizing foraging behavior by remembering recent routes and visited sites. It is known that SWM decision-making in rodents requires the medial prefrontal cortex (mPFC) and dorsal hippocampus. The decision process in SWM tasks carries a specific electrophysiological signature of a brief, decision-related increase in neuronal communication in the form of an increase in the coherence of neuronal theta oscillations (4-12 Hz) between the mPFC and dorsal hippocampus, a finding we replicated here during spontaneous exploration of a plus maze in freely moving mice. We further evaluated SWM decision-related coherence changes within frequency bands above theta. Decision-related coherence increases occurred in seven frequency bands between 4 and 200 Hz and decision-outcome related differences in coherence modulation occurred within the beta and gamma frequency bands and in higher frequency oscillations up to 130 Hz. With recent evidence that Purkinje cells in the cerebellar lobulus simplex (LS) represent information about the phase and phase differences of gamma oscillations in the mPFC and dorsal hippocampus, we hypothesized that LS might be involved in the modulation of mPFC-hippocampal gamma coherence. We show that optical stimulation of LS significantly impairs SWM performance and decision-related mPFC-dCA1 coherence modulation, providing causal evidence for an involvement of cerebellar LS in SWM decision making at the behavioral and neuronal level. Our findings suggest that the cerebellum might contribute to SWM decision-making by optimizing the decision-related modulation of mPFC-dCA1 coherence.

sentences: Lichen speciation is sparked by a substrate requirement shift and reproduction mode differentiation We show that obligate lignicoles in lichenized Micarea are predominately asexual whereas most facultative lignicoles reproduce sexually. Our 3 loci phylogenetic analyses (ITS, mtSSU, Mcm7) together with ancestral state reconstruction show that the shift in reproduction mode has evolved independently several times within the group and that facultative and obligate lignicoles are sister species. The analyses support the assumption that the ancestor of these species was a facultative lignicole. We hypotezise that a shift in substrate requirement from bark to wood leads to differentiation in reproduction mode and becomes a driver of speciation. This is the first example of lichenized fungi where reproduction mode is linked to substrate requirement. This is also the first example where such a linkage is demonstrated to spark lichen speciation. Our main hypothesis is that obligate species on dead wood need to colonize new suitable substrata relatively fast and asexual reproduction is more effective a strategy for successful colonization. Our main hypothesis for explaining the discovered phenomenon involves the species life cycle: species on decaying wood face a significant challenge because the ecological properties of their substratum change relatively fast. When this happens, species need to colonize new suitable substrata. This may set a time limit, where asexual reproduction is a faster and more effective strategy for successful colonization.

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Accurate Genome-Wide Phasing from IBD Data As genotype databases increase in size, so too do the number of detectable segments of identity by descent (IBD): segments of the genome where two individuals share an identical copy of one of their two parental haplotypes, due to shared ancestry. We show that given a large enough genotype database, these segments of IBD collectively overlap entire chromosomes and can be used to phase them accurately. Furthermore, primarily using instances of IBD that span multiple chromosomes, we can accurately phase an entire genome.

We are able to separate the DNA inherited from each parent completely, across the entire genome, with 98% median accuracy in a test set of 30,000 individuals. We estimate the IBD data requirements for accurate phasing, and we propose a method for estimating confidence in the resulting phase. We show that our methods do not require the genotypes of close family, and that they are robust to genotype errors and missing data. In fact, our method can impute missing data accurately and correct genotype errors.

Author summaryWe present a method for phasing, separating the DNA inherited from each parent, of an entire genome using short segments of DNA that are shared between the genome of the person we wish to phase and those of distant cousins. Essentially, if we have enough of these distant cousins genotypes available, we can piece together overlapping segments until we have phased the entire genome.

We have developed a method that can phase large numbers of individuals, and that makes special considerations for potential close family and potential genotype errors in data.

We report results on experiments phasing 30,000 individuals. We analyze how many such segments are required to phase accurately, and propose a model-based method to estimate accuracy. We also show that our method can accurately impute missing data and correct genotype errors.

sentences: Striatal BOLD and midfrontal theta power express motivation for action Action selection is biased by the valence of anticipated outcomes. To assess mechanisms by which these motivational biases are expressed and controlled, we measured simultaneous EEG-fMRI during a motivational Go/NoGo learning task (N=36), leveraging the temporal resolution of EEG and subcortical access of fMRI. VmPFC BOLD encoded cue valence, importantly predicting trial-by-trial valence-driven response speed differences and EEG theta power around cue onset. In contrast, striatal BOLD encoded selection of active Go responses and correlated with theta power around response time. Within trials, theta power ramped in the fashion of an evidence accumulation signal for the value of making a  Go response, capturing the faster responding to reward cues. Our findings reveal a dual nature of midfrontal theta power, with early components reflecting the vmPFC contribution to motivational biases, and late components reflecting their striatal translation into behavior, in line with influential recent "value of work" theories of striatal processing.

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A MALDI-TOF assay identifies nilotinib as an inhibitor of inflammation in acute myeloid leukaemia Inflammatory responses are important in cancer, particularly in the context of monocyte-rich aggressive myeloid neoplasm. We developed a label-free cellular phenotypic drug discovery assay to identify anti-inflammatory drugs in human monocytes derived from acute myeloid leukaemia (AML), by tracking several biological features ionizing from only 2,500 cells using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. A proof-of-concept screen showed that the BCR-ABL inhibitor nilotinib, but not the structurally similar imatinib, blocks inflammatory responses. In order to identify the cellular (off-)targets of nilotinib, we performed thermal proteome profiling (TPP). Unlike imatinib, nilotinib and other later generation BCR-ABL inhibitors inhibit the p38-MK2/3 signalling axis which suppressed the expression of inflammatory cytokines, cell adhesion and innate immunity markers in activated human monocytes derived from AML. Thus, our study provides a tool for the discovery of new anti-inflammatory drugs, which could contribute to the treatment of inflammation in myeloid neoplasms and other diseases.

Key PointsLabel-free cell-based assay identifies new anti-inflammatory drugs using MALDI-TOF MS. Nilotinib reduces inflammation by inhibition of MAPK14-MK2/3 signalling axis in AML.

sentences: Post-infection treatment with a protease inhibitor increases survival of mice with a fatal SARS-CoV-2 infection Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and was proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 hr post infection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2-treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2.

sentences: Integrative analysis of scRNAs-seq and scATAC-seq revealed transit-amplifying thymic epithelial cells expressing autoimmune regulator Medullary thymic epithelial cells (mTECs) are critical for self-tolerance induction in T cells via promiscuous expression of tissue-specific antigens (TSAs), which are controlled by transcriptional regulator AIRE. Whereas AIRE-expressing (Aire+) mTECs undergo constant turnover in the adult thymus, mechanisms underlying differentiation of postnatal mTECs remain to be discovered. Integrative analysis of single-cell assays for transposase accessible chromatin (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) suggested the presence of proliferating mTECs with a specific chromatin structure, which express high levels of Aire and co-stimulatory molecules CD80 (Aire+CD80hi). Proliferating Aire+CD80hi mTECs detected by using Fucci technology express a minimal level of Aire-dependent TSAs and are converted into quiescent Aire+CD80hi mTECs expressing high levels of TSAs after a transit amplification. These data provide evidence for the existence of transit amplifying Aire+mTEC precursors during Aire+mTEC differentiation process of the postnatal thymus.

sentences: Identification of CDRH3 Loops in the Naive B Cell Receptor Repertoire that Can Be Engaged by Candidate Immunogens B cell lineages that are the current focus of vaccine development efforts against HIV-1, influenza or coronaviruses, often contain rare features, such as long heavy chain complementarity determining regions (CDRH3) loops. These unusual characteristics may limit the number of available B cells in the natural immunoglobulin repertoire that can respond to pathogen vaccinations. To measure the ability of a given immunogen to engage naturally occurring B cell receptors of interest, here we describe a mixed experimental and bioinformatic approach for determining the frequency and sequence of CDRH3 loops in the immune repertoire that can be recognized by a vaccine candidate. By combining deep mutational scanning and B cell receptor database analysis, CDRH3 loops were found that can be engaged by two HIV-1 germline-targeting immunogens, thus illustrating how the methods described here can be used to evaluate candidate immunogens based on their ability to engage diverse B cell lineage precursors.

sentences: Non-destructive chemical sensing within bulk soil using 1000 biosensors per gram of matrix Gene expression can be monitored in hard-to-image environmental materials using gas-reporting biosensors, but these outputs have only been applied in autoclaved matrices that are hydrated with rich medium. To better understand the compatibility of indicator gas reporting with environmental samples, we evaluated how matrix hydration affects the gas signal of an engineered microbe added to a sieved soil. A gas-reporting microbe presented a gas signal in a forest soil (Alfisol) when hydrated to an environmentally-relevant osmotic pressure. When the gas signal was concentrated prior to analysis, a biosensor titer of 103 cells/gram soil produced a significant signal when soil was supplemented with halides. A signal was also observed without halide amendment, but a higher cell titer (106 cells/gram soil) was required. A sugar-regulated gas biosensor was able to report with a similar level of sensitivity when added to an unsterilized soil matrix, illustrating how gas concentration enables biosensing within a soil containing environmental microbes. These results establish conditions where engineered microbes can report on gene expression in living environmental matrices with minimal perturbation, using biosensor titers that are orders of magnitude lower than the number of cells typically observed in a gram of soil.

sentences: Proteomic analysis of the actin cortex in interphase and mitosis In animal cells, many shape changes are driven by gradients in the contractile tension of the actomyosin cortex, a thin cytoskeletal network that supports the plasma membrane. Elucidating how cortical tension is controlled is thus essential for understanding cell and tissue morphogenesis. Increasing evidence shows that alongside myosin II activity, actin network organisation and composition are key to cortex tension regulation. However, how cortex composition changes when cortical tension changes remains poorly understood. Here, we compared cortices from cells in interphase and in mitosis, as mitosis entry is associated with a strong increase in cortical tension. We purified cortex-enriched cellular fractions and analysed their composition using mass spectrometry, identifying 922 proteins consistently represented in both interphase and mitotic cortices. We curated this dataset by focusing on actin-related proteins, narrowing down to 238 candidate regulators of the cortex during the mitotic cortical tension increase. Among these candidates, our analysis pointed to a role for septins, and in particular septin 9, in the control of mitotic cell rounding. Overall, our study brings insight into the regulation of mitotic rounding, and paves the way for systematic investigations of the regulation of cell surface mechanics.

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Voltage imaging in Drosophila using a hybrid chemical-genetic rhodamine voltage reporter We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in Drosophila melanogaster. Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach in vivo to achieve selective neuron labeling in intact fly brains. We generate a Drosophila UAS-HaloTag reporter line in which the HaloTag enzyme is expressed on the surface of cells. We validate the voltage sensitivity of this new construct in cell culture before driving expression of HaloTag in specific brain neurons in flies. We show that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in either larval neuromuscular junction (NMJ) or in whole adult brains. Finally, we validate the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, show the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and perform voltage imaging of carbachol-evoked depolarization and osmolarity-evoked hyperpolarization in projection neurons and in interoceptive subesophageal zone neurons in fly brain explants following in vivo labeling. We envision the turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes, will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila.

Significance StatementVoltage imaging is a powerful method for interrogating neurobiology. Chemical indicators possess fast response kinetics, turn-on responses to membrane depolarization, and can be compatible with two-photon excitation. However, selective cell labeling in intact tissues and in vivo remains a challenge for completely synthetic fluorophores. Here, we show that a chemical - genetic hybrid approach in Drosophila enables cell-specific staining in vivo and voltage imaging in whole-brain explants and at neuromuscular junction synapses.

sentences: Joint copy number and mutation phylogeny reconstruction fromsingle-cell amplicon sequencing data Reconstructing the history of somatic DNA alterations that occurred in a tumour can help understand its evolution and predict its resistance to treatment. Single-cell DNA sequencing (scDNAseq) can be used to investigate clonal heterogeneity and to inform phylogeny reconstruction. However, existing phylogenetic methods for scDNAseq data are designed either for point mutations or for large copy number variations, but not for both types of events simultaneously. Here, we develop COMPASS, a computational method for inferring the joint phylogeny of mutations and copy number alterations from targeted scDNAseq data. We evaluate COMPASS on simulated data and show that it outperforms existing methods. We apply COMPASS to a large cohort of 123 patients with acute myeloid leukemia (AML) and detect copy number alterations, including subclonal ones, which are in agreement with and extend current knowledge of AML development. We further used bulk sequencing and SNP array data to orthogonally validate our findings.

sentences: L-WNK1 is required for BK channel activation in intercalated cells BK channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high K+ diet. In cell culture, the long isoform of the kinase WNK1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high K+ diet. Mice with an IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high K+ (5% K+ as KCl) diet for at least 10 days. IC-L-WNK1-KO mice exhibited higher blood K+ concentrations ([K+]) than controls. BK channel-dependent whole-cell currents in ICs from cortical collecting ducts of high K+ fed IC-L-WNK1-KO mice were reduced compared to controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice have higher blood [K+] and reduced IC BK channel currents are consistent with impaired urinary K+ secretion, and suggest that IC L-WNK1 has a role in the renal adaptation to a high K+ diet.

sentences: A unique form of collective epithelial migration is crucial for tissue fusion in the secondary palate and can overcome loss of epithelial apoptosis Tissue fusion is an oft-employed process in morphogenesis which often requires the removal of the epithelia intervening multiple distinct primordia to form one continuous structure. In the mammalian secondary palate, a midline epithelial seam (MES) forms between two palatal shelves and must be removed to allow mesenchymal confluence. Abundant apoptosis and cell extrusion in this epithelial seam support their importance in its removal. However, by genetically disrupting the intrinsic apoptotic regulators BAX and BAK within the MES, we find a complete loss of cell death and cell extrusion, but successful removal of the MES, indicating that developmental compensation enables fusion. Novel static and live imaging approaches reveal that the MES is removed through a unique form of collective epithelial cell migration in which epithelial trails and islands stream through the mesenchyme to reach the oral and nasal epithelial surfaces. These epithelial trails and islands begin to express periderm markers while retaining expression of the basal epithelial marker {Delta}Np63, suggesting their migration to the oral and nasal surface is concomitant with their differentiation to an epithelial intermediate. Live imaging reveals anisotropic actomyosin contractility within epithelial trails that drives their peristaltic movement, and genetic loss of non-muscle myosin IIA-mediated actomyosin contractility results in dispersion of epithelial collectives and dramatic failure of normal MES migration. These findings demonstrate redundancy between cellular mechanisms of morphogenesis and reveal a crucial role for a unique form of collective epithelial migration during tissue fusion.

sentences: Hypusinated eIF5A is required for the translation of collagen The evolutionary conserved elongation factor eIF5A is required for the translation of mRNAs that encode protein sequences with consecutive prolines or combined with glycine and charged amino acids. Mammalian collagens are enriched in putative eIF5A-dependent Pro-Gly-containing tripeptides. Here, we show that eIF5A is needed for heterologous expression of collagen in yeast, and using a dual luciferase reporter system we confirmed that eIF5A depletion interrupts translation at Pro-Gly-collagenic motifs. Using mouse fibroblasts, we showed that depletion of active eIF5A reduced collagen 1 (Col1a1) content, which became concentrated around the nuclei, in contrast to a stronger and all over the cell collagen signal in untreated cells. Active eIF5A-depleted mouse fibroblast showed upregulation of endoplasmic reticulum (ER) stress markers, suggesting retention of partially synthesized Col1a1 in the ER. A dramatically lower level of Col11 protein was also observed in functional eIF5A-depleted human hepatic stellate cells treated with the profibrotic cytokine TGF-{beta}1. Our results show that collagen expression requires eIF5A and imply its potential as a target for regulating collagen production in fibrotic diseases.

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Localized and Distributed Representations of Person Knowledge for Faces Modular neural models of face processing posit that face-associated person-knowledge is localized in, or accessed through, the ventral anterior temporal lobe (vATL). However, some studies have suggested that person-knowledge is more widely distributed within a larger face-processing network that includes the mid-fusiform gyrus ( fusiform face area). Here, we conducted an fMRI study to distinguish whether person-knowledge is localized or distributed by comparing brain responses evoked by synthetic faces, for which participants had learned person-knowledge (biographical facts) and faces for which the subjects had learned only physical facts. After extensive training, participants were cued to recall a particular biographical or physical fact about the upcoming face. In an alternate passive-viewing task, participants were shown the same faces but were not cued to recall biographical or physical facts. Classification analyses (MVPA) were performed on several a priori chosen face-selective regions (ROIs) in the ventral temporal cortex. Within each ROI tested in isolation, MVPA discriminated faces associated with person-knowledge from faces only associated with physical facts. This result is consistent with a distributed model for person-knowledge. However, when tested in a single model to separate shared and unique information, unique information was limited to the left mid-fusiform and vATL posterior (vATL-p) ROIs. Moreover, the feature weights from these two areas showed that only left vATL-p was specialized for processing biographical facts. This latter result was obtained only when these biographical facts were explicitly retrieved in the cueing task. Thus, our results indicate that the left vATL-p represents explicit recall of face-associated person-knowledge.

New & NoteworthyWhether person knowledge for faces is localized in a domain-specific region, vATL, or distributed in many domain-general brain regions, including the mid-fusiform gyrus, is hotly contested. We resolve this debate by using multivariate analyses to partial fMRI signal from different brain regions into unique and shared variance. Our findings show that unique information for person knowledge is found in both the vATL and mid-fusiform but only the vATL represents explicit recall of face-associated person knowledge.

sentences: An EMG-assisted Muscle-Force Driven Finite Element Analysis Pipeline to Investigate Joint- and Tissue-Level Mechanical Responses in Functional Activities: Towards a Rapid Assessment Toolbox Joint tissue mechanics (e.g., stress and strain) are believed to have a major involvement in the onset and progression of musculoskeletal disorders, e.g., knee osteoarthritis (KOA). Accordingly, considerable efforts have been made to develop musculoskeletal finite element (MS-FE) models to estimate highly-detailed tissue mechanics that predict cartilage degeneration. However, creating such models is time-consuming and requires advanced expertise. This limits these complex, yet promising MS-FE models to research applications with few participants and making the models impractical for clinical assessments. Also, these previously developed MS-FE models are not assessed for any activities other than the gait. This study introduces and validates a semi-automated rapid state-of-the-art MS-FE modeling and simulation toolbox incorporating an electromyography (EMG) assisted MS model and a muscle-force driven FE model of the knee with fibril-reinforced poro(visco)elastic cartilages and menisci. To showcase the usability of the pipeline, we estimated joint- and tissue-level knee mechanics in 15 KOA individuals performing different daily activities. The pipeline was validated by comparing the estimated muscle activations and joint mechanics to existing experimental data. Also, to examine the importance of EMG-assisted MS analyses, results were compared against outputs from the same FE models but driven by static-optimization-based MS models. The EMG-assisted MS-FE pipeline bore a closer resemblance to experiments, compared to the static-optimization-based MS-FE pipeline. More importantly, the developed pipeline showed great potentials as a rapid MS-FE analysis toolbox to investigate multiscale knee mechanics during different activities of individuals with KOA.

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Metabolism is the major driver of hydrogen isotope fractionation recorded in tree-ring glucose of Pinus nigra - Stable isotope abundances convey valuable information about plant physiological processes and underlying environmental controls. Central gaps in our mechanistic understanding of hydrogen isotope abundances impede their widespread application within the plant and biogeosciences.
- To address these gaps, we analysed intramolecular deuterium abundances in glucose of Pinus nigra extracted from an annually resolved tree-ring series (1961 to 1995).
- We found fractionation signals (i.e., temporal variability in deuterium abundance) at glucose H1 and H2 introduced by closely related metabolic processes. Regression analysis indicates that these signals (and thus metabolism) respond to drought and atmospheric CO2 concentration beyond a response change point. They explain {approx}60% of the whole-molecule deuterium variability. Altered metabolism is associated with below-average yet not exceptionally low growth.
- We propose the signals are introduced at the leaf-level by changes in sucrose-to-starch carbon partitioning and anaplerotic carbon flux into the Calvin-Benson cycle. In conclusion, metabolism can be the main driver of hydrogen isotope variation in plant glucose.

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Nanoceutical Fabric Prevents COVID-19 Spread through Expelled Respiratory Droplets: A Combined Computational, Spectroscopic and Anti-microbial Study Centers for Disease Control and Prevention (CDC) warns the use of one-way valves or vents in free masks for potential threat of spreading COVID-19 through expelled respiratory droplets. Here, we have developed a nanoceutical cotton fabric duly sensitized with non-toxic zinc oxide nanomaterial for potential use as membrane filter in the one way valve for the ease of breathing without the threat of COVID-19 spreading. A detailed computational study revealed that zinc oxide nanoflowers (ZnO NF) with almost two-dimensional petals trap SARS-CoV-2 spike proteins, responsible to attach to ACE-2 receptors in human lung epithelial cells. The study also confirm significant denaturation of the spike proteins on the ZnO surface, revealing removal of virus upon efficient trapping. Following the computational study, we have synthesized ZnO NF on cotton matrix using hydrothermal assisted strategy. Electron microscopic, steady-state and picosecond resolved spectroscopic studies confirm attachment of ZnO NF to the cotton (i.e., cellulose) matrix at atomic level to develop the nanoceutical fabric. A detailed antimicrobial assay using Pseudomonas aeruginosa bacteria (model SARS-CoV-2 mimic) reveals excellent anti-microbial efficiency of the developed nanoceutical fabric. To our understanding the novel nanoceutical fabric used in one-way valve of a face mask would be the choice to assure breathing comfort along with source control of COVID-19 infection. The developed nanosensitized cloth can also be used as antibacterial/anti CoV-2 washable dress material in general.

GRAPHICAL ABSTRACT O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

A novel nanoceutical cotton fabric duly sensitized with non-toxic zinc oxide nanoflower can potentially be used as membrane filter in the one way valve of face mask to assure breathing comfort along with source control of COVID-19 infection. The nanoceutical fabric denatures the SARS-CoV-2 spike protein and makes the microorganism ineffective.

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Chromosome-scale assembly of the Sparassis latifolia genome obtained using long-read and Hi-C sequencing Sparassis latifolia is a valuable edible mushroom cultivated in China. In 2018, our research group reported an incomplete and low quality genome of S. latifolia was obtained by Illumina HiSeq 2500 sequencing. These limitations in the available genome have constrained genetic and genomic studies in this mushroom resource. Herein, an updated draft genome sequence of S. latifolia was generated by Oxford Nanopore sequencing and the Hi-C technique. A total of 8.24 Gb of Oxford Nanopore long reads representing ~198.08X coverage of the S. latifolia genome were generated. Subsequently, a high-quality genome of 41.41 Mb, with scaffold and contig N50 sizes of 3.31 Mb and 1.51 Mb, respectively, was assembled. Hi-C scaffolding of the genome resulted in 12 pseudochromosomes containing 93.56% of the bases in the assembled genome. Genome annotation further revealed that 17.47% of the genome was composed of repetitive sequences. In addition, 13,103 protein-coding genes were predicted, among which 98.72% were functionally annotated. BUSCO assay results further revealed that there were 92.07% complete BUSCOs. The improved chromosome-scale assembly and genome features described here will aid further molecular elucidation of various traits, breeding of S. latifolia, and evolutionary studies with related taxa.

ImportanceSparassis latifolia Y.C. Dai et Z. Wang (Sparassidaceae, Polyporales, Agaricomycetes), collections from Asian Sparassis (Dai et al., 2006) exhibits diverse biological and pharmacologic activities. To date, total fresh fruit production in Chinese factories is over 20 tons/d. Despite the significant economic and medical value of S. latifolia, the assembly level of genome was under chromosome-scale. This study assembles a high-quality chromosome-scale reference genome of S. latifolia using Oxford Nanopore sequencing combined with Hi-C (High-through chromosome conformation capture) scaffolding. The improved reference genome will facilitate molecular breeding of S. latifolia and advance our understanding of its genetics and evolution.

sentences: Infanticide by females is a leading source of juvenile mortality in a large social carnivore Social animals benefit from their groupmates, so why do they sometimes kill each others offspring? Using 30 years of data from multiple groups of wild spotted hyenas, we address three critical aims for understanding infanticide in any species: (1) quantify the contribution of infanticide to overall mortality (2) describe the circumstances under which infanticide occurs and (3) evaluate hypotheses about the evolution of infanticide. We find that, although observed only rarely, infanticide is in fact a leading source of juvenile mortality. Infanticide accounted for 24% of juvenile mortality, and 1 in 10 hyenas born in our population perished due to infanticide. In all observed cases of infanticide, killers were adult females, but victims could be of both sexes. Of four hypotheses regarding the evolution of infanticide, we found the most support for the hypothesis that infanticide in spotted hyenas reflects competition over social status among matrilines.

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Multi-Factor Authentication of Potential 5' Splice Sites by the Saccharomyces cerevisiae U1 snRNP In eukaryotes, splice sites define the introns of pre-mRNAs and must be recognized and excised with nucleotide precision by the spliceosome to make the correct mRNA product. In one of the earliest steps of spliceosome assembly, the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5 splice site (5 SS) through a combination of base pairing, protein-RNA contacts, and interactions with other splicing factors. Previous studies investigating the mechanisms of 5 SS recognition have largely been done in vivo or in cellular extracts where the U1/5 SS interaction is difficult to deconvolute from the effects of trans-acting factors or RNA structure. In this work we used co-localization single-molecule spectroscopy (CoSMoS) to elucidate the pathway of 5 SS selection by purified yeast U1 snRNP. We determined that U1 reversibly selects 5 SS in a sequence-dependent, two-step mechanism. A kinetic selection scheme enforces pairing at particular positions rather than overall duplex stability to achieve long-lived U1 binding. Our results provide a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5 SS and preferentially accumulate at these sequences rather than on close cognates.

IMPACT STATEMENTThe yeast U1 snRNP recognizes multiple features of target RNAs to reversibly identify splicing-competent 5 splice sites.

sentences: Relation between the number of peaks and the number of reciprocal sign epistatic interactions Empirical essays of fitness landscapes suggest that they may be rugged, that is having multiple fitness peaks. Such fitness landscapes, those that have multiple peaks, necessarily have special local structures, called reciprocal sign epistasis ([14]). Here, we investigate the quantitative relationship between the number of fitness peaks and the number of reciprocal sign epistatic interactions. Previously it has been shown ([14]) that pairwise reciprocal sign epistasis is a necessary but not sufficient condition for the existence of multiple peaks. Applying discrete Morse theory, which to our knowledge has never been used in this context, we extend this result by giving the minimal number of reciprocal sign epistatic interactions required to create a given number of peaks.

sentences: The annotation and analysis of complex 3D plant organs using 3DCoordX A fundamental question in biology concerns how molecular and cellular processes become integrated during morphogenesis. In plants, characterization of 3D digital representations of organs at single-cell resolution represents a promising approach to addressing this problem. A major challenge is to provide organ-centric spatial context to cells of an organ. We developed several general rules for the annotation of cell position and embodied them in 3DCoordX, a user-interactive computer toolbox implemented in the open-source software MorphoGraphX. It enables rapid spatial annotation of cells even in highly curved biological shapes. With the help of 3DCoordX we obtained new insight by analyzing cellular growth patterns in organs of several species. For example, the data indicated the presence of a basal cell proliferation zone in the ovule primordium of Arabidopsis thaliana. Proof-of-concept analyses suggested a preferential increase in cell length associated with neck elongation in the archegonium of Marchantia polymorpha and variations in cell volume linked to central morphogenetic features of a trap of the carnivorous plant Utricularia gibba. Our work demonstrates the broad applicability of the developed strategies as they provide organ-centric spatial context to cellular features in plant organs of diverse shape complexity.

sentences: Selective Inhibitory Circuit Dysfunction after Chronic Frontal Lobe Contusion Traumatic brain injury (TBI) is a leading cause of neurologic disability; the most common deficits affect prefrontal cortex-dependent functions such as attention, working memory, social behavior, and mental flexibility. Despite this prevalence, little is known about the pathophysiology that develops in frontal cortical microcircuits after TBI. We investigated if alterations in subtype-specific inhibitory circuits are associated with cognitive inflexibility in a mouse model of frontal lobe contusion that recapitulates aberrant mental flexibility as measured by deficits in rule reversal learning. Using patch clamp recordings and optogenetic stimulation, we identified selective vulnerability in the non-fast spiking, somatostatin-expressing (SOM+) subtype of inhibitory neurons in layer V of the orbitofrontal cortex (OFC) two months after injury. These neurons exhibited reduced intrinsic excitability and a decrease in their synaptic output onto pyramidal neurons. By contrast, fast spiking, parvalbumin-expressing (PV+) interneurons did not show changes in intrinsic excitability or synaptic output. Impairments in SOM+ inhibitory circuit function were also associated with network hyperexcitability. These findings provide evidence for selective disruptions within specific inhibitory microcircuits that may guide the development of novel therapeutics for TBI.

sentences: Mutational footprint of platinum chemotherapy in a secondary thyroid cancer Although papillary thyroid carcinoma (PTC) is the most frequent endocrine tumor with a generally excellent prognosis, a patient developed a clinically aggressive PTC eleven years after receiving platinum chemotherapy for ovarian endometrioid adenocarcinoma. Germline and somatic analyses of multi-temporal and multi-regional molecular profiles indicated that ovarian and thyroid tumors did not share common genetic alterations. PTC tumors had driver events associated with aggressive PTC behavior, an RBPMS-NTRK3 fusion and a TERT promoter mutation. Spatial and temporal genomic heterogeneity analysis indicated a close link between anatomical locations and molecular patterns of PTC. Mutational signature analyses demonstrated a molecular footprint of platinum exposure, and that aggressive molecular drivers of PTC were linked to prior platinum-associated mutagenesis. This case provides a direct association between platinum chemotherapy exposure and secondary solid tumor evolution, in specific aggressive thyroid carcinoma, and suggests that uniform clinical assessments for secondary PTC after platinum chemotherapy may warrant further evaluation.

sentences: Inactivation of SARS-CoV-2 by β-propiolactone Causes Aggregation of Viral Particles and Loss of Antigenic Potential Inactivated viral preparations are important resources in vaccine and antisera industry. Of the many vaccines that are being developed against COVID-19, inactivated whole-virus vaccines are also considered effective. {beta}-propiolactone (BPL) is a widely used chemical inactivator of several viruses. Here, we analyze various concentrations of BPL to effectively inactivate SARS-CoV-2 and their effects on the biochemical properties of the virion particles. BPL at 1:2000 (v/v) concentrations effectively inactivated SARS-CoV-2. However, higher BPL concentrations resulted in the loss of both protein content as well as the antigenic integrity of the structural proteins. Higher concentrations also caused substantial aggregation of the virion particles possibly causing undesirable outcomes including a potential immune escape by infectious virions, and a loss in antigenic potential. We also identify that the viral RNA content in the culture supernatants can be a direct indicator of their antigenic content. Our findings may have important implications in the vaccine and antisera industry during COVID-19 pandemic.

sentences: Shift-inducible  increase in recombination rate as an evolving strategy in a periodic environment: a numerical model Numerous empirical studies have witnessed a plastic increase in meiotic recombination rate in organisms experiencing physiological stress due to unfavourable environmental conditions. Yet, it is not clear enough which characteristics of an ecological factor (intensity, duration, variability, etc.) make it stressogenic and therefore recombinogenic for an organism. Several previous theoretical models proceeded from the assumption that organisms increase their recombination rate when the environment becomes more severe, and demonstrated the evolutionary advantage of such recombination strategy. Here we explore another stress-associated recombination strategy, implying a reversible increase in recombination rate each time when the environment alternates. We allow such plastic changes in the organisms, grown in an environment different from that of their parents, and, optionally, also in their offspring. We show that such shift-inducible recombination is always favoured over intermediate constant optimal recombination. Besides, it sometimes outcompetes also zero and free optimal constant recombination, therefore making selection on recombination less polarized. Shift-inducible strategies with a longer, transgenerational plastic effect, are favoured under slightly stronger selection and longer period. These results hold for both panmixia and partial selfing, although selfing makes the dynamics of recombination modifier alleles faster. Our results suggest that epigenetic factors, presumably underlying the environmental plasticity of recombination, may play an important evolutionary role.

sentences: Combining GWAS and TWAS to identify candidate causal genes for tocochromanol levels in maize grain Tocochromanols (tocopherols and tocotrienols, collectively vitamin E) are lipid-soluble antioxidants important for both plant fitness and human health. The main dietary sources of vitamin E are seed oils that often accumulate high levels of tocopherol isoforms with lower vitamin E activity. The tocochromanol biosynthetic pathway is conserved across plant species but an integrated view of the genes and mechanisms underlying natural variation of tocochromanol levels in seed of most cereal crops remains limited. To address this issue, we utilized the high mapping resolution of the maize Ames panel of [~]1,500 inbred lines scored with 12.2 million single-nucleotide polymorphisms to generate metabolomic (mature grain tocochromanols) and transcriptomic (developing grain) data sets for genetic mapping. By combining results from genome- and transcriptome-wide association studies, we identified a total of 13 candidate causal gene loci, including five that had not been previously associated with maize grain tocochromanols: four biosynthetic genes (arodeH2 paralog, dxs1, vte5, and vte7) and a plastid S-adenosyl methionine transporter (samt1). Expression quantitative trait locus (eQTL) mapping of these 13 gene loci revealed that they are predominantly regulated by cis-eQTL. Through a joint statistical analysis, we implicated cis-acting variants as responsible for co-localized eQTL and GWAS association signals. Our multi-omics approach provided increased statistical power and mapping resolution to enable a detailed characterization of the genetic and regulatory architecture underlying tocochromanol accumulation in maize grain and provided insights for ongoing biofortification efforts to breed and/or engineer vitamin E and antioxidant levels in maize and other cereals.

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BDSF is a degradation-prone quorum-sensing signal detected by the histidine kinase RpfC of Xanthomonas campestris pv. campestris Diffusible signal factors (DSFs) are medium-chain fatty acids that induce bacterial quorum sensing. Among these compounds, BDSF is a structural analog of DSF that is commonly detected in bacterial species (e.g., Xanthomonas, Pseudomonas, and Burkholderia). Additionally, BDSF contributes to the interkingdom communication regulating fungal life stage transitions. How BDSF is sensed in Xanthomonas spp. and the functional diversity between BDSF and DSF remain unclear. In this study, we generated genetic and biochemical evidence that BDSF is a low-active regulator of X. campestris pv. campestris quorum sensing, whereas trans-BDSF seems not a signaling compound. BDSF is detected by the sensor histidine kinase RpfC. Although BDSF has relatively low physiological activities, it binds to the RpfC sensor with a high affinity and activates RpfC autophosphorylation to a level that is similar to that induced by DSF in vitro. The inconsistency in the physiological and biochemical activities of BDSF is not due to RpfC- RpfG phosphorylation or RpfG hydrolase. Neither BDSF nor DSF controls the phosphotransferase and phosphatase activities of RpfC or the ability of RpfG hydrolase to degrade the bacterial second messenger cyclic di-GMP. We demonstrated that BDSF is prone to degradation by RpfB, a critical fatty acyl-CoA ligase involved in the turnover of DSF-family signals. rpfB mutations lead to substantial increases in BDSF-induced quorum sensing. Although DSF and BDSF are similarly detected by RpfC, our data suggest that their differential degradation in cells is the major factor responsible for the diversity in their physiological effects.

IMPORTANCEDiffusible signal factor (DSF) family are quorum-sensing signals employed by gram-negative bacteria. These signals are a group of cis-2-unsaturated fatty acids, such as DSF, BDSF, IDSF, CDSF, and SDSF. However, the functional divergence of various DSF signals remains unclear. The present study demonstrates that though BDSF is a low active quorum-sensing signal than that of DSF, it binds histidine kinase RpfC with a higher affinity and activates RpfC autophosphorylation to the similar level as DSF. Rather than regulation of enzymatic activities of RpfC and its cognate response regulator RpfG encoding a c-di-GMP hydrolase, BDSF is prone to degradation in bacterial cells by RpfB, which effectively avoided the inhibition of bacterial growth by accumulating high concentration of BDSF. Therefore, our study shed new light on the functional difference of quorum-sensing signals and revealed that bacteria balance quorum-sensing and growth by fine-turning concentration of signaling chemical.

sentences: Driving potent neutralization of a SARS-CoV-2 Variant of Concern with a heterotypic boost The emergence of SARS-CoV-2 Variants of Concern (VOCs) with mutations in key neutralizing antibody epitopes threatens to undermine vaccines developed against the pandemic founder variant (Wu-Hu-1). Widespread vaccine rollout and continued transmission are creating a population that has antibody responses of varying potency to Wu-Hu-1. Against this background, it is critical to assess the outcomes of subsequent immunization with variant antigens. It is not yet known whether heterotypic vaccine boosts would be compromised by original antigenic sin, where pre-existing responses to a prior variant dampen responses to a new one, or whether the primed memory B cell repertoire would bridge the gap between Wu-Hu-1 and VOCs. Here, we show that a single adjuvanted dose of receptor binding domain (RBD) protein from VOC 501Y.V2 (B.1.351) drives an extremely potent neutralizing antibody response capable of cross-neutralizing both Wu-Hu-1 and 501Y.V2 in rhesus macaques previously immunized with Wu-Hu-1 spike protein. Passive immunization with plasma sampled following this boost protected K18-hACE2 mice from lethal challenge with a 501Y.V2 clinical isolate, whereas only partial protection was afforded by plasma sampled after two Wu-Hu-1 spike immunizations.

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Early neuroadaptations to an obesogenic diet identify the schizophrenia-related ErbB4 receptor in obesity-induced hippocampal abnormalities Childhood obesity leads to hippocampal atrophy and altered cognition. However, the molecular mechanisms underlying these impairments are poorly understood. The neurotrophic factor neuregulin-1 (NRG1) and its cognate ErbB4 receptor play critical roles in hippocampal maturation and function. This study aimed to determine whether altered NRG1-ErbB4 activities may partly explain hippocampal abnormalities in rats exposed to an obesogenic Western-like diet (WD). Lewis rats were randomly divided into four groups (12 rats/group): 1) control diet+vehicle (CDV); 2) CD+NRG1 (CDN) (daily intraperitoneal injections: 5 g/kg/day; between postnatal day, PND 21-PND 41); 3) WD+VEH (WDV); 4) WD+NRG1 (WDN). Neurobehavioral assessments were performed at PND 43-49. Brains were harvested for MRI and molecular analyses at PND 49. We found that NRG1 administration reduced hippocampal volume (7%) and attenuated hippocampal-dependent cued fear conditioning in CD rats (56%). NRG1 administration reduced PSD-95 protein expression (30%) and selectively reduced hippocampal cytokine levels (IL-33, GM-CSF, CCL-2, IFN-{gamma}) while significantly impacting microglia morphology (increased span ratio and reduced circularity). WD rats exhibited reduced right hippocampal volume (7%), altered microglia morphology (reduced density and increased lacunarity), and increased levels of cytokines implicated in neuroinflammation (IL-1, TNF-, IL-6). Notably, NRG1 synergized with the WD to increase hippocampal ErbB4 phosphorylation and the tumor necrosis alpha converting enzyme (TACE/ADAM17) protein levels. Together, these data suggest a novel interaction between obesogenic diet exposure and TACE/ADAM17-NRG1-ErbB4 signaling during hippocampal maturation. Our results indicate that supraoptimal ErbB4 activities may contribute to the abnormal hippocampal structure and cognitive vulnerabilities observed in obese individuals.

HighlightsO_LIObesogenic diet consumption during adolescence induces anxiety-like behaviors before the onset of obesity-related changes in metabolism.
C_LIO_LIObesogenic diet-driven abnormal behaviors co-occurred with alterations in hippocampal pro-inflammatory cytokine profiles.
C_LIO_LIObesogenic diet consumption attenuates exogenous NRG1 effects on hippocampal-related behaviors and structure.
C_LIO_LIExogenous NRG1 administration during adolescence resulted in reduced hippocampal volumes and domain-specific cognitive impairments.
C_LIO_LIExogenous NRG1 administration has potent immunomodulatory actions and alters hippocampal microglia morphology.
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sentences: Effect of luminal surface structure of decellularized aorta on thrombus formation and cell behavior As the number of cardiovascular diseases increases, artificial heart valves and blood vessels have been developed. Although cardiovascular application using decellularized tissue have been studied, the mechanism of their functionality is still unknown. To find the important factor for preparing decellularized cardiovascular prothesis which shows good in vivo performance, the effect of luminal surface structure of decellularized aorta on thrombus formation and cell behavior was investigated. Various luminal surface structures of decellularized aorta were prepared by heating, drying and peeling. The luminal surface structure and collagen denaturation was evaluated by immunohistological staining, collagen hybridizing peptide (CHP) staining and scanning electron microscopy (SEM) analysis. To evaluate the effect of luminal surface structure of decellularized aorta on thrombus formation and cell behavior, blood clotting test and recellularization of endothelial cells and smooth muscular cells were performed. The results of blood clotting test showed that the closer the luminal surface structure is to native aorta, the higher the anti-coagulant property. From the result of cell seeding-test, it was suggested that vascular cells recognize the luminal surface structure and regulate adhesion, proliferation and functional expression. These results will provide important factor for preparing decellularized cardiovascular prothesis and lead to future development on decellularized cardiovascular applications.

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Progranulin promotes immune evasion of pancreatic adenocarcinoma through regulation of MHCI expression Immune evasion is indispensable for cancer initiation and progression, although its underlying mechanisms in pancreatic ductal adenocarcinoma (PDAC) remain elusive. Here, we unveiled a cancer cell-autonomous function of PGRN in driving immune evasion in primary PDAC. Tumor- but not macrophage-derived PGRN was associated with poor overall survival in PDAC. Multiplex immunohistochemistry revealed low MHC class I (MHCI) expression and lack of CD8+ T cells infiltration in PGRN-high tumors. Inhibition of PGRN abrogated autophagy-dependent MHCI degradation and restored MHCI expression on PDAC cells. Antibody-based blockade of PGRN in a genetic PDAC mouse model remarkably decelerated tumor initiation and progression. Notably, tumors expressing LCMV-gp33 as model antigen were sensitized towards cytotoxic gp33-TCR transgenic T cells upon anti-PGRN antibody treatment. Overall, our study uncovered an unprecedented role of tumor-derived PGRN in regulating immunogenicity of primary PDAC.

STATEMENT OF SIGNIFICANCEImmune evasion is a key property of PDAC, rendering it refractory to immunotherapy. Here we demonstrate that tumor-derived PGRN promotes autophagy-dependent MHCI degradation, while anti-PGRN increases intratumoral CD8 infiltration and blocks tumor progression. With recent advances in T cell-mediated approaches, PGRN represents a pivotal target to enhance tumor antigen-specific cytotoxicity.

sentences: Human endogenous oxytocin and its neural correlates show adaptive responses to social touch based on recent social context Both oxytocin (OT) and touch are key mediators of social attachment. In rodents, tactile stimulation elicits endogenous release of OT, potentially facilitating attachment and other forms of prosocial behavior, yet the relationship between endogenous OT and neural modulation remains unexplored in humans. Using serial sampling of plasma hormone levels during functional neuroimaging, we show that contextual circumstances of social touch facilitate or inhibit not only current hormonal and brain responses, but also calibrate later responses. Namely, touch from a romantic partner enhanced subsequent OT release for touch from an unfamiliar stranger, yet OT responses to partner touch were dampened following stranger touch. Hypothalamus and dorsal raphe activation reflected plasma OT changes during the initial interaction. In the subsequent social interaction, OT modulation depended on the previous interaction, mediated by precuneus and parietal-temporal cortex pathways, including a region of medial prefrontal cortex that also covaried with plasma cortisol. These findings demonstrate that hormonal neuromodulation during successive human social interactions is adaptive to social context, and they point to mechanisms that flexibly calibrate receptivity in social encounters.

sentences: Inferring genome-wide correlations of mutation fitness effects between populations The effect of a mutation on fitness may differ between populations depending on environmental and genetic context, but little is known about the factors that underlie such differences. To quantify genome-wide correlations in mutation fitness effects, we developed a novel concept called a joint distribution of fitness effects (DFE) between populations. We then proposed a new statistic w to measure the DFE correlation between populations. Using simulation, we showed that inferring the DFE correlation from the joint allele frequency spectrum is statistically precise and robust. Using population genomic data, we inferred DFE correlations of populations in humans, Drosophila melanogaster, and wild tomatoes. In these specices, we found that the overall correlation of the joint DFE was inversely related to genetic differentiation. In humans and D. melanogaster, deleterious mutations had a lower DFE correlation than tolerated mutations, indicating a complex joint DFE. Altogether, the DFE correlation can be reliably inferred, and it offers extensive insight into the genetics of population divergence.

sentences: Non-phylogenetic identification of co-evolving genes for reconstructing the archaeal Tree of Life Assessing the phylogenetic compatibility between individual gene families is a crucial and often computationally demanding step in many phylogenomics analyses. Here we describe the Evolutionary Similarity Index (IES) to assess shared evolution between gene families using a weighted Orthogonal Distance Regression applied to sequence distances. This approach allows for straightforward pairing of paralogs between co-evolving gene families without resorting to multiple tests, or a priori assumptions of molecular interactions between protein products from assessed genes. The utilization of pairwise distance matrices, while less informative than phylogenetic trees, circumvents error-prone comparisons between trees whose topologies are inherently uncertain. Analyses of simulated gene family evolution datasets showed that IES was more accurate and less susceptible to noise than popular tree-based methods (Robinson-Foulds and geodesic distance) for assessing evolutionary signal compatibility, since it bypasses phylogenetic reconstruction and its inherent uncertainty. Applying IES to a real dataset of 1,322 genes from 42 archaeal genomes identified eight major clusters of gene families with compatible evolutionary trends. Four of these clusters included genes with a taxonomic distribution across all archaeal phyla, while other clusters included a subset of taxa that do not map to generally accepted archaeal clades, indicating possible shared horizontal transfers by clustered gene families. We identify one strongly connected set of 62 genes from the same cluster, occurring as both single-copy and multiple homologs per genome, with compatible phylogenetic reconstructions closely matching previously published species trees for Archaea. An IES implementation is available at https://github.com/lthiberiol/evolSimIndex.

sentences: A diagnostic primer pair to distinguish between wMel and wAlbB Wolbachia infection - A simplified real-time PCR approach for use in field collected Aedes aegypti in dengue reduction trials Detection of the Wolbachia endosymbiont in Aedes aegypti mosquitoes through real-time PCR assays is widely used during and after Wolbachia releases in dengue reduction trials involving the wMel and wAlbB strains. However, primers applied in current successful Wolbachia releases cannot be used in a single assay to distinguish between these strains. Here, we developed a new diagnostic primer pair wMA which can detect wMel or wAlbB infection in the same assay. We also tested current Wolbachia primers and show that there is variation in their performance when they are used to assess the relative density of Wolbachia. The new wMA primers provide an accurate and efficient estimate of the presence and density of both Wolbachia infections.

sentences: A comprehensive library of canonical and non-canonical MHC class I antigens for cancer vaccine development. A longstanding disconnect between the growing number of MHC Class I immunopeptidomic studies and genomic medicine hinders cancer vaccine design. We develop COD-dipp to genomically map the full spectrum of detected canonical and non-canonical (non-exonic) MHC Class I antigens from 26 cancer studies. We demonstrate that patient mutations in regions overlapping physically identified antigens better predict immunotherapy response when compared to neoantigen predictions. We suggest a vaccine design approach using 140,966 highly immune-visible regions of the genome annotated by their expression and haplotype frequency in the human population. These regions tend to be highly conserved, mutated in cancer and harbor 7.8 times more immunogenicity. Intersecting pan-cancer mutations with these immune surveilled regions revealed a potential to create off-the-shelf multi-epitope vaccines against public neoantigens. Here we release COD-dipp, a cancer vaccine toolkit as a web-application (https://www.proteogenomics.ca/COD-dipp) and open-source high-throughput resource.

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The mating type transcription factor MAT1-1-1 from the fungal human pathogen Aspergillus fumigatus: synthesis, purification, and crystallization of the DNA binding domain. Mating type (MAT) loci are the most important and significant regulators of sexual reproduction and development in ascomycetous fungi. Usually, they encode two transcription factors (TFs), named MAT1-1-1 or MAT1-2-1. Mating-type strains carry only one of the two TF genes, which control expression of pheromone and pheromone receptor genes, involved in the cell-cell recognition process. The present work presents the crystallization for the alpha1 (1) domain of MAT1-1-1 from the human pathogenic fungus Aspergillus fumigatus (AfMAT1-1-1). Crystals were obtained for the complex between a polypeptide containing the 1 domain and DNA carrying the AfMAT1-1-1 recognition sequence. A streak seeding technique was applied to improve native crystal quality, resulting in diffraction data to 3.2 [A] resolution. Further, highly redundant data sets were collected from the crystals of selenomethionine-substituted AfMAT1-1-1 with a maximum resolution of 3.2 [A]. This is the first report of structural studies on the 1 domain MAT regulator involved in the mating of ascomycetes.

SynopsisAn optimized purification and crystallization protocol together with initial X-ray datasets are described for this mating type transcription factor from human pathogenic fungus Aspergillus fumigatus.

sentences: Cohort Profile: Genetic data in the German Socio-Economic Panel Innovation Sample (Gene-SOEP) The German Socio-Economic Panel (SOEP) serves a global research community by providing representative annual longitudinal data of private households in Germany. The sample provides a detailed life course perspective based on a rich collection of information about living conditions, socio-economic status, family relationships, personality, values, preferences, and health. We collected genetic data from 2,598 individuals in the SOEP Innovation Sample, yielding the first genotyped sample that is representative of the entire German population (Gene-SOEP). The Gene-SOEP sample is a longitudinal study that includes 107 full-sibling pairs, 501 parent-offspring pairs, and 152 parent-offspring trios that are overlapping with the parent-offspring pairs. We constructed a repository of 66 polygenic indices in the Gene-SOEP sample based on results from well-powered genome-wide association studies. The Gene-SOEP data provides a valuable resource to study individual differences, inequalities, life-course development, health, and interactions between genetic predispositions and environment.

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The potyviral protein 6K1 reduces plant protease activity during Turnip mosaic virus infection O_LIPotyviral genomes encode just 11 major proteins and multifunctionality is associated to most of these proteins at different stages of virus life cycle. The potyviral protein 6K1 is required for potyvirus replication at the early stages of viral infection and may mediate cell-to-cell movement at later stages.
C_LIO_LIOur study demonstrates that the 6K1 protein from Turnip mosaic virus (TuMV) reduces the abundance of transcripts related to jasmonic acid biosynthesis and transcripts that encode cysteine protease inhibitors when expressed in trans in Nicotiana benthamiana relative to controls. Furthermore, 6K1 stability increases when lipoxygenase and cysteine protease activity is inhibited chemically, linking a mechanism to the rapid turnover of 6K1 when expressed in trans.
C_LIO_LIUsing transient expression, we show 6K1 is degraded rapidly at early time points in the infection process, whereas at later stages of infection protease activity is reduced and 6K1 becomes more stable, resulting in higher TuMV accumulation in systemic leaves. There was no impact of 6K1 transient expression on TuMV accumulation in local leaves.
C_LIO_LITogether, these results suggest a novel function for the TuMV 6K1 protein which has not been reported previously and enhances our understanding of the complex interactions occurring between plants and potyviruses.
C_LI

sentences: The BASP1 transcriptional repressor modifies chromatin through lipid-dependent and lipid-independent mechanisms The transcriptional corepressor BASP1 requires N-terminal myristoylation for its activity and functions through interactions with nuclear lipids. Here we determine the role of BASP1 lipidation in histone modification and the modulation of chromatin accessibility. We find that the removal of the active histone modifications H3K9ac and H3K4me3 by BASP1 requires the N-terminal myristoylation of BASP1. In contrast, the placement of the repressive histone modification, H3K27me3, by BASP1 does require BASP1 lipidation. RNA-seq and ATAC-seq analysis finds that BASP1 regulates the activity of multiple transcription factors and induces extensive changes in chromatin accessibility. We find that [~]50% of BASP1 target genes show lipidation-dependent chromatin compaction and transcriptional repression. Our results suggest that BASP1 elicits both lipid-dependent and lipid-independent functions in histone modification and transcriptional repression. In accordance with this, we find that the tumor suppressor activity of BASP1 is also partially dependent on its myristoylation.

sentences: Pseudomonas arenae sp. nov., Pseudomonas glycinis sp. nov. and Pseudomonas harudinis sp. nov., three novel bacterial species and plant endophytes Three novel Pseudomonas species associated with healthy plants are described from the United States. They are Pseudomonas arenae sp. nov. from soybean in Missouri and Phragmites sp. in New Jersey; Pseudomonas glycinis sp. nov. from Vaccinium macrocarpon fruit in Massachusetts, groundwater in Tennessee and soybean in Indiana; and Pseudomonas harudinis sp. nov. from Phragmites sp. in New Jersey. No pathogenic strains are known for any of the novel species based on genome comparisons to assemblies in GenBank.

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Critical Assessment of Metaproteome Investigation (CAMPI): a Multi-Lab Comparison of Established Workflows Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear.

Here, we carried out the first community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluated the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample.

We observed that variability at the peptide level was predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappeared at the protein group level. While differences were observed for predicted community composition, similar functional profiles were obtained across workflows.

CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

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Cryptic individual-level variation in propensity for selfing in a hermaphroditic snail For mating-system evolution, individual-level variation is essential. In self-compatible hermaphrodites, individuals may vary in their lifetime propensity for selfing, which consists of an individual and environmental component. According to the reproductive assurance hypothesis explaining partial selfing, a key environmental factor is mate availability, which fluctuates with population density.

We quantified individual variation in selfing propensity in a hermaphroditic snail by manipulating mate availability, recording mating behaviour, estimating selfing rates from progeny arrays, and measuring female lifetime fitness. We found four classes of individuals: pure outcrossers, pure selfers, and two types of plastic individuals. These classes only became apparent in the laboratory; the field population is outcrossing. All classes were present both under low and increased mate availability. This large variation in selfing propensities rendered effects of differential mate availability on selfing rates non-significant despite large effect sizes and sufficient statistical power.

We suspect that selfing propensities have a genetic component and when selected on cause mean selfing rates to evolve. We propose that heritable variation in selfing propensities offers a reconciliation between the reproductive assurance hypothesis and its weak empirical support: distributions of selfing propensities vary temporally and spatially, thus obscuring the relationship between population density and realised selfing rates.

sentences: Proteomic Characterization of Isolated Arabidopsis Clathrin-Coated Vesicles Reveals Evolutionarily Conserved and Plant Specific Components In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor- protein AP-1 complex operates as part of the secretory pathway at the trans-Golgi network, while the AP-2 complex and the TPLATE complex (TPC) jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant- specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched trans-Golgi network/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

sentences: A multiplexed in vivo approach to identify driver genes in small cell lung cancer Small cell lung cancer (SCLC) is a highly lethal form of lung cancer. The high mutation burden in SCLC cells makes it challenging to predict key drivers of SCLC from genome sequencing data, thereby hindering the identification of possible therapeutic targets. Here we develop a quantitative multiplexed approach based on lentiviral barcoding with somatic CRISPR/Cas9-mediated genome editing to functionally investigate candidate regulators of tumor initiation and growth in genetically engineered mouse models of SCLC. Lentiviral vector-mediated SCLC initiation was greatly enhanced by naphthalene pre-treatment, enabling high multiplicity of tumor clones for analysis through high-throughput sequencing methods. Based on a meta-analysis across multiple human SCLC genomic datasets, we quantified the impact of inactivating 39 genes across many candidate pathways and captured both positive and detrimental effects on SCLC initiation and progression upon gene inactivation. This analysis and subsequent validation in human SCLC cells identified TSC1 in the PI3K-AKT-mTOR pathway as a robust tumor suppressor in SCLC. This new approach should illuminate novel drivers of SCLC, facilitate the development of precision therapies for defined SCLC genotypes, and identify new therapeutic targets.

sentences: Dysfunction of exocytosis causes catecholamine hypersecretion in patient with pheochromocytoma Pheochromocytoma (Pheo) is a neuroendocrine tumor that develops from chromaffin cells of the adrenal medulla, and is responsible of an excess of catecholamines secretion leading to severe clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Surprisingly, hypersecretory activity of Pheo has never been explored at the cellular and molecular levels from individual tumor cells. In the present study, we have combined catecholamine secretion measurement by carbon fiber amperometry on human tumor cells directly cultured from freshly resected Pheo, with the analysis by mass spectrometry of the exocytotic proteins differentially expressed between the tumor and the matched adjacent non-tumor tissue. Catecholamine secretion recordings from individual Pheo cells obtained from most patients reveal a higher number of exocytic events per cell associated with faster kinetic parameters. Accordingly, we unravel significant tumor-associated modifications in the expression of key proteins involved in different steps of the calcium-regulated exocytic pathway. Altogether, our findings indicate that dysfunction of the calcium-regulated exocytosis at the level of individual Pheo cell is a cause of the tumor-associated hypersecretion of catecholamines.

sentences: Mechanical counterbalance of kinesin and dynein motors in microtubular network regulates cell mechanics, 3D architecture, and mechanosensing Microtubules (MTs) and MT motor proteins form active 3D networks made of unstretchable cables with rod-like bending mechanics that provide cells with a dynamically changing structural scaffold. In this study, we report an antagonistic mechanical balance within the dynein-kinesin microtubular motor system. Dynein activity drives microtubular network inward compaction, while isolated activity of kinesins bundles and expands MTs into giant circular bands that deform the cell cortex into discoids. Furthermore, we show that dyneins recruit MTs to sites of cell adhesion increasing topographic contact guidance of cells, while kinesins antagonize it via retraction of MTs from sites of cell adhesion. Actin-to-microtubules translocation of septin-9 enhances kinesins-MTs interactions, outbalances activity of kinesins over dyneins and induces discoid architecture of cells. These orthogonal mechanisms of MT network reorganization highlight the existence of an intricate mechanical balance between motor activities of kinesins and dyneins that controls cell 3D architecture, mechanics, and cell-microenvironment interactions.

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Cerebellar Activation Bidirectionally Regulates Nucleus Accumbens Shell and Core Traditionally viewed as a motor control center, the cerebellum (CB) is now recognized as an integral part of a broad, long-range brain network that serves limbic functions and motivates behavior. This diverse CB functionality has been at least partly attributed to the multiplicity of its outputs. However, most studies have focused on cerebellar-cerebral cortical connections, at the expense of subcortical limbic structures. Nothing is known about how the CB connects to the nucleus accumbens (NAc), a subcortical region with which the CB shares functionality in motivated behaviors. Here, we report findings from in vivo electrophysiological experiments that investigated the functional connectivity between CB and NAc. We found that electrical microstimulation of deep cerebellar nuclei (DCN) modulates NAc spiking activity. This modulation differs in terms of directionality (excitatory vs. inhibitory) and temporal characteristics, in a manner that depends on NAc subregion: in the medial shell of NAc (NAcMed), slow inhibitory responses prevail over excitatory ones, whereas the proportion of fast excitatory responses is greater in the NAc core (NAcCore) compared to NAcMed.

Differences also exist in response onset latencies and dependence on CB stimulation intensity, which further argues for differential connectivity. If different pathways provide signal to each subregion, the divergence likely occurs downstream of the CB because we did not find any response-type clustering within DCN. We propose that the fast excitatory responses would be well poised to support rapid communication of information critical to the control of motivated behavior, such as prediction or prediction-error signals. The slower, less synchronous and longer-lasting modulation may be suggestive of a regulatory function, such as gain control of the communication between NAc and other brain regions. Finally, because there are no direct monosynaptic connections between CB and NAc, we performed viral tracing experiments to chart disynaptic pathways that could potentially mediate the newly discovered CB-NAc communication. We identified two anatomical pathways, which recruit the ventral tegmental area and intralaminar thalamus as nodes. These pathways and the functional connectivity they support could underlie the role of the CB in motivated behaviors.

sentences: Downregulation of Extraembryonic Tension Controls Body Axis Formation in Avian Embryos Embryonic tissues undergoing shape change draw mechanical input from extraembryonic substrates. In avian eggs, the early blastoderm disk is under the tension of the vitelline membrane (VM). Here we report that chicken VM characteristically downregulates tension and stiffness to facilitate stage-specific embryo morphogenesis. While early relaxation of the VM impairs blastoderm expansion, maintaining VM tension in later stages resists the convergence of the posterior body causing stalled elongation, open neural tube, and axis rupture. Biochemical and structural analysis shows that VM weakening follows the reduction of its outer-layer glycoprotein fibers, which is caused by an increasing albumen pH due to CO2 release from the egg. Our results identify a previously unrecognized mechanism of body axis defects through mis-regulation of extraembryonic tissue tension.

sentences: Rethinking habitat occupancy modeling and the role of diel activity in an anthropogenic world Current methods to model species habitat use through space and diel time are limited. Development of such models is critical when considering rapidly changing habitats where species are forced to adapt to anthropogenic change, often by shifting their diel activity across space. We use an occupancy modeling framework to develop a new model, the multi-state diel occupancy model (MSDOM), which can evaluate species diel activity against continuous response variables which may impact diel activity within and across seasons or years. We used two case studies, fosa in Madagascar and coyote in Chicago, USA, to conceptualize the application of this model and to quantify the impacts of human activity on species spatial use in diel time. We found support that both species varied their habitat use by diel states--in and across years, and by human disturbance. Our results exemplify the importance of understanding animal diel activity patterns and how human disturbance can lead to temporal habitat loss. The MSDOM will allow more focused attention in ecology and evolution studies on the importance of the short temporal scale of diel time in animal-habitat relationships and lead to improved habitat conservation and management.

sentences: The β-lactam Ticarcillin is a Staphylococcus aureus UDP-N-acetylglucosamine 2-epimerase binder. Infectious diseases account for 25% of the causes of death worldwide and this rate is expected to increase due to antibiotic resistance. Among the bacteria associated with healthcare infections, Staphylococcus aureus is a prevalent pathogen and about 50% of the isolates are found to be methicillin-resistant. Here we describe the identification of ticarcillin as a weak binder of the S. aureus UDP-N-acetylglucosamine 2-epimerase. After a docking screening, ticarcillin was identified as a ligand in using the recently proposed isothermal analysis of differential scanning fluorimetry data. Finally, an equilibrium MD simulation confirmed the docking binding mode as a stable pose, with large contributions to the binding energy coming from interactions between Arg206 and Arg207 and the carboxylate groups in ticarcillin.

sentences: Dystrophin regulates peripheral circadian SRF signalling Dystrophin is a sarcolemmal protein essential for muscle contraction and maintenance, absence of which leads to the devastating muscle wasting disease Duchenne muscular dystrophy (DMD)[1, 2]. Dystrophin has an actin-binding domain [3-5], which specifically binds and stabilises filamentous (F)-actin[6], an integral component of the RhoA-actin-serum response factor (SRF)-pathway[7]. The RhoA-actin-SRF-pathway plays an essential role in circadian signalling whereby the hypothalamic suprachiasmatic nucleus, transmits systemic cues to peripheral tissues, activating SRF and transcription of clock target genes[8, 9]. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised that dystrophin loss causes circadian deficits. Here we show for the first time alterations in the RhoA-actin-SRF-signalling-pathway, in both dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios and nuclear MRTF, dysregulation of core clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from DMD patients harbouring an array of mutations. Further, disrupted circadian locomotor behaviour was observed in dystrophic mice indicative of disrupted SCN signalling, and indeed dystrophin protein was absent in the SCN of dystrophic animals. Dystrophin is thus a critically important component of the RhoA-actin-SRF-pathway and a novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation.

sentences: Energy content of anchovy and sardine using surrogate calorimetry methods European anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus) are crucial species for the marine ecosystem of the Northwestern Mediterranean Sea. They account for a high percentage of fish landings and they represent an important economic income. Concerns over their stock status are rising in recent years as biomass, growth, reproductive capacity and body condition of both species are declining. Therefore, there is an urgent need for a continuous and fast body condition monitoring scheme. Energy storage variability has important implications for both fish recruitment and population structure. Direct condition indices, such as bomb calorimetry, are highly reliable for measuring the energy content, but extremely time-consuming. Alternatively, fatmeter analysis and relative condition index (Kn) have been proposed as effective indirect methods. The aim of this study is to test the application of fatmeter as a surrogate of bomb calorimetry to infer the energy content of both small pelagic fishes. For the validation, fatmeter values were compared with both energy density (ED; via bomb calorimetry) and Kn values. Individuals of both species were sampled monthly in Barcelona harbor for a year in order to assess seasonal variations in energy content. Our results highlight that fatmeter measurements are strongly correlated with calorimetry for sardine, while a weaker but significant correlation was found for anchovy. The observed differences between the two species are related to their breeding strategies. Based on this study, fatmeter analysis appears to be a faster and suitable method to evaluate the energy content of both species routinely., In addition, we provide a linear model to infer ED from fatmeter values of both small pelagic fish. Eventually, these findings could allow for the avoidance of bomb calorimetry and could be used to implement body condition monitoring protocols, and to boost continuous large-scale monitoring.

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Glass Half Full: Preserved Anatomical Bypasses Predict Variance in Language Functions After Stroke The severity of post-stroke aphasia is related to damage to white matter connections. However, neural signaling can route not only through direct connections, but also along multi-step network paths. When brain networks are damaged by stroke, paths can bypass around the damage to restore communication. The shortest network paths between regions could be the most efficient routes for mediating bypasses. We examined how shortest-path bypasses after left hemisphere strokes were related to language performance. Regions within and outside of the canonical language network could be important in aphasia recovery. Therefore, we innovated methods to measure the influence of bypasses in the whole brain.

Distinguishing bypasses from all residual shortest paths is difficult without pre-stroke imaging. We identified bypasses by finding shortest paths in subjects with stroke that were longer than those observed in the average network of the most reliably observed connections in age-matched controls. We tested whether features of those bypasses predicted scores in four orthogonal dimensions of language performance derived from a factor analysis of a battery of language tasks. The features were the length of each bypass in steps, and how many bypasses overlapped on each individual direct connection. We related these bypass features to language factors using grid-search cross-validated Support Vector Regression, a technique that extracts robust relationships in high-dimensional data analysis.

We discovered that the length of bypasses reliably predicted variance in lexical production (R2 = .576) and auditory comprehension scores (R2 = .164). Bypass overlaps reliably predicted variance in Lexical Production scores (R2 = .247). The predictive elongation features revealed that bypass efficiency along the dorsal stream and ventral stream were most related to Lexical Production and Auditory Comprehension, respectively. Among the predictive bypass overlaps, increased bypass routing through the right hemisphere putamen was negatively related to lexical production ability.

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Is Vaccination a Viable Method to Control Johne's Disease Caused by Mycobacterium avium subsp. paratuberculosis? Data from 12 Million Ovine Vaccinations and 7.6 Million Carcass Examinations in New South Wales, Australia from 1999-2009 BackgroundMycobacterium avium subsp. paratuberculosis (MAP) causes Johnes disease (or paratuberculosis), a chronic wasting disease of ruminants and other animals resulting from granulomatous enteritis. There are increasing concerns that MAP is zoonotic. The prevalence of Johnes disease is increasing worldwide. In an attempt to control an epidemic of ovine Johnes disease (OJD) in New South Wales (NSW), a government/industry sponsored voluntary vaccination/on-farm management program commenced in 2000. We report herein an observational study of changes in disease prevalence as vaccination progressed, based on abattoir surveillance data for OJD from 1999 to 2009. We also discuss the epidemiological, policy, regulatory, research, economic and sociological elements that contributed to the development of a mature control program, whose aim was to halt the epidemic spread of OJD in a naive sheep population.

MethodsNSW was divided into areas of "High" (HPA), "Medium" (MPA) and "Low" (LPA) OJD prevalence. A killed whole cell vaccine (Gudair(R)) was administered to sheep from 2000 to 2009. Trained examiners evaluated the viscera of adult sheep carcasses at slaughter for gross evidence of OJD. MAP infection was confirmed by histopathology.

Principal findingsFrom 2000-2009, 12 million vaccine doses were administered in NSW (91%; 10.9 million in the HPA). Many of the vaccinated flocks were suffering > 5% annual mortality in adult sheep, with some individual flocks with 10-15% losses attributable to OJD. A total of 7.6 million carcasses were examined (38%; 2.9 million from the HPA). Overall, 16% of slaughter consignments (sheep consigned to the abattoir from a single vendor) were positive for OJD, of which 94% were from the HPA. In the HPA, the percentage of animals with lesions attributable to OJD at slaughter fell progressively from 2.4% (10,406/432,860) at commencement of vaccination in 2000 to 0.8% (1,573/189,564) by 2009. Herd immunity from vaccination in the HPA was estimated at 70% by 2009. This coincided with a progressive decrease in reports of clinical disease and mortalities in vaccinated flocks.

SignificanceWe show a decrease in the prevalence of lesions attributable to OJD in NSW concomitant with initiation of voluntary vaccination, on-farm management plans, abattoir monitoring and feedback of animal prevalence data to sheep producers. We conclude that a target of [≤] 1% regional prevalence of OJD affected sheep at slaughter is achievable using these interventions.

sentences: Improved fluorescent Listeria spp. biosensors for analysis of antimicrobials by flow cytometry The global increase in antibiotic resistances of pathogenic microorganisms require identification and characterization of novel antimicrobials. Bacterial biosensors expressing fluorescent proteins such as pHluorin variants are suitable for high-throughput screenings. Here, we present Listeria spp. pH-sensitive biosensors with improved fluorescence for single cell analysis of antimicrobials by flow cytometry.

sentences: Eicosanoids in the pancreatic tumor microenvironment: a multicellular, multifaceted progression Eicosanoids, oxidized fatty acids that serve as cell-signaling molecules, have been broadly implicated in tumorigenesis. To identify eicosanoids relevant to pancreatic tumorigenesis, we profiled normal pancreas and pancreatic ductal adenocarcinoma (PDAC) in mouse models and patient samples using mass spectrometry. We interrogated RNA sequencing datasets for eicosanoid synthase or receptor expression. Findings were confirmed by immunostaining. In murine models, we identified elevated levels of PGD2, prostacyclin, and thromboxanes in neoplasia while PGE2, 12-HHTre, HETEs, and HDoHEs are elevated specifically in tumors. Analysis of scRNA-seq datasets suggests that PGE2 and prostacyclins are derived from fibroblasts, PGD2 and thromboxanes from myeloid cells, and PGD2 and 5-HETE from tuft cells. In patient samples, we identified a transition from PGD2 to PGE2-producing enzymes in the epithelium during the transition to PDAC, fibroblast/tumor expression of PTGIS, and myeloid/tumor cell expression of TBXAS1. Altogether, our analyses identify key changes in eicosanoid species during pancreatic tumorigenesis and the cell types responsible for their synthesis.

sentences: Sequence determinants of the aggregation of proteins within condensates generated by liquid-liquid phase separation The transition between the native and amyloid states of proteins can proceed via a deposition pathway via oligomeric intermediates or via a condensation pathway via liquid droplet intermediates generated through liquid-liquid phase separation. Here we investigate the sequence determinants of aggregation from within the droplet state based on generic interactions. We describe a model in which these sequence determinants can be captured by three features, the droplet-promoting propensity, the aggregation-promoting propensity and the binding mode entropy. By using this approach, we propose a formula to identify aggregation-promoting mutations in droplet-forming proteins. This analysis provides insights into the amino acid code for the conversion of proteins between liquid-like and solid-like condensates.

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Interannual climate variability data improves niche estimates in species distribution models AimClimate is an essential element of species niche estimates in many current ecological applications such as species distribution models (SDMs). Climate predictors are often used in the form of long-term mean values. Yet, climate can also be described as spatial or temporal variability for variables like temperature or precipitation. Such variability, spatial or temporal, offers additional insights into niche properties. Here, we test to what degree spatial variability and long-term temporal variability in temperature and precipitation improve SDM predictions globally.

LocationGlobal.

Time period1979-2013

Major taxa studiesMammal, Amphibians, Reptiles

MethodsWe use three different SDM algorithms, and a set of 833 amphibian, 779 reptile, and 2211 mammal species to quantify the effect of spatial and temporal climate variability in SDMs. All SDMs were cross-validated and accessed for their performance using the Area under the Curve (AUC) and the True Skill Statistic (TSS).

ResultsMean performance of SDMs with climatic means as predictors was TSS=0.71 and AUC=0.90. The inclusion of spatial variability offers a significant gain in SDM performance (mean TSS=0.74, mean AUC=0.92), as does the inclusion of temporal variability (mean TSS=0.80, mean AUC=0.94). Including both spatial and temporal variability in SDMs shows similarly high TSS and AUC scores.

Main conclusionsAccounting for temporal rather than spatial variability in climate improved the SDM prediction especially in exotherm groups such as amphibians and reptiles, while for endotermic mammals no such improvement was observed. These results indicate that more detailed information about temporal climate variability offers a highly promising avenue for improving niche estimates and calls for a new set of standard bioclimatic predictors in SDM research.

sentences: Delineation of insula dynorphin and kappa opioid receptor circuit elements in alcohol consumption Alcohol use disorder is complex and multi-faceted, involving the engagement of multiple signaling systems across numerous brain regions to drive pathological behavior. Previous work has indicated that both the insular cortex and dynorphin (DYN)/Kappa opioid receptor (KOR) systems contribute to excessive alcohol use. More recently, we identified a microcircuit in the medial aspect of the insular cortex that signals through DYN/KOR. Here, we explored the role of insula DYN/KOR circuit elements on alcohol intake in a long-term intermittent access (IA) procedure. Using a combination of conditional knockout strategies and site-directed pharmacology, we discovered distinct and sex-specific roles for insula DYN and KOR systems in alcohol drinking and related behavior. Our findings show that insula DYN deletion blocked escalated consumption and decreased overall intake of and preference for alcohol in male and female mice. This effect was specific to alcohol in male mice, as DYN deletion did not impact their sucrose intake. In male mice, insula KOR antagonism reduced alcohol intake and preference during the early phase of IA only. Alcohol consumption as not affected by insula KOR knockout. In addition, we found that long-term IA decreased the intrinsic excitability of DYN and deep layer pyramidal neurons (DLPN) in the insula of male mice. Excitatory synaptic transmission was also impacted by IA, as it drove an increase in excitatory synaptic drive in both DYN neurons and DLPN. Combined, our findings suggest that there is a dynamic interplay between excessive alcohol consumption and insula DYN/KOR circuit elements.

sentences: Exit from quiescence couples epithelial stress amplification to fluidization Cellular quiescence is a state of reversible cell cycle arrest that is associated with tissue dormancy. Timely regulated entry into and exit from quiescence is important for processes such as tissue homeostasis, tissue repair, stem cell maintenance, developmental processes and immunity. Here we show that quiescent human keratinocyte monolayers contain an actinomyosin-based system that facilitates global viscoelastic flow upon serum-stimulated exit from quiescence. Mechanistically, serum exposure causes rapid amplification of pre-existing contractile sites leading to a burst in monolayer stress that subsequently drives monolayer fluidization. The stress magnitude after quiescence exit correlates with quiescence depth, and a critical stress level must be reached to overcome the cell sheet displacement barrier. The study shows that static quiescent cell monolayers are mechanically poised for motility and identifies global stress amplification as a mechanism for tissue fluidization.

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Chemogenetic enhancement of cAMP signaling renders hippocampal synaptic plasticity resilient to the impact of acute sleep deprivation Sleep facilitates memory storage and even brief periods of sleep loss lead to impairments in memory, particularly memories that are hippocampus dependent. In previous studies, we have shown that the deficit in memory seen after sleep loss is accompanied by deficits in synaptic plasticity. Our previous work has also found that sleep deprivation is associated with reduced levels of cyclic adenosine monophosphate (cAMP) in the hippocampus, and that the reduction of cAMP mediates the diminished memory performance. Based on these findings, we hypothesized that cAMP acts as a mediator for not only the cognitive deficits caused by sleep deprivation, but also the observed deficits in synaptic plasticity. In this study, we expressed the heterologous Drosophila melanogaster Gs-protein coupled octopamine receptor (DmOct{beta}1R) in mouse hippocampal neurons. This receptor is selectively activated by the systemically injected ligand (octopamine), thus allowing us to increase cAMP levels in hippocampal neurons during a five-hour sleep deprivation period. Our results show that chemogenetic enhancement of cAMP during the period of sleep deprivation prevents deficits in a persistent form of long-term potentiation (LTP) that is induced at the Schaffer collateral synapses in the hippocampal CA1 region. We also found that elevating cAMP levels only in the early or later half of sleep deprivation successfully prevented LTP deficits. These findings reveal that cAMP-dependent signaling pathways are key mediators of sleep deprivation at the synaptic level. Targeting these pathways could be useful in designing strategies to prevent the impact of sleep loss.

Significance statementInsufficient sleep is an issue with significant health and socioeconomic implications. This includes a negative impact on memory consolidation. Previous studies in mice found that acute sleep deprivation leads to deficits in hippocampal synaptic plasticity and memory, which are associated with reduced levels of the signaling molecule cAMP. In this study, we used a chemogenetic strategy to enhance cAMP levels in specific hippocampal neurons during sleep deprivation. We found that this made synaptic plasticity resilient to the negative effects of sleep deprivation. These findings reveal that cAMP-dependent signaling pathways are key mediators of sleep deprivation and that targeting these pathways could be useful in designing strategies to prevent the impact of sleep loss.

sentences: Humans reconfigure target and distractor processing to address distinct task demands When faced with distraction, we can focus more on goal-relevant information (targets) or focus less goal-conflicting information (distractors). How people decide to distribute cognitive control across targets and distractors remains unclear. To help address this question, we developed a parametric attentional control task that can index both target discriminability and distractor interference. We find that participants exert independent control over target and distractor processing. We measured control adjustments through the influence of incentives and previous conflict on target and distractor sensitivity, finding that these have dissociable influences on control. Whereas incentives preferentially led to target enhancement, conflict on the previous trial preferentially led to distractor suppression. These distinct drivers of control altered sensitivity to targets and distractors early in the trial, promptly followed by reactive reconfiguration towards task-appropriate feature sensitivity. Finally, we provide a process-level account of these findings by show that these control adjustments are well-captured by an evidence accumulation model with attractor dynamics over feature weights. These results help establish a process-level account of control configuration that provides new insights into how multivariate attentional signals are optimized to achieve task goals.

sentences: Unperturbed dormancy recording reveals stochastic awakening strategies in endocrine treated breast cancer cells Hormone dependent breast cancer (HDBC) is the most commonly diagnosed tumor type in women. Adjuvant endocrine therapies (ET) have been the cornerstone in the clinical management of HDBC patients for over forty years. A vast proportion of HDBC patients incur long periods of clinical dormancy following ET, with tumour awakening appearing at a steady pace for up to 25 years (Pan et al., 2017). Extensive genomic studies have demonstrated that 15-30% of clinical relapses develop recurrent genomic changes which contribute to drug resistance (i.e. ESR1 activating mutations) (Bertucci et al., 2019; Magnani et al., 2017; Razavi et al., 2018). However, even in these cases, there is no conclusive evidence around the pre-existence vs. de novo nature of these events. We previously showed that ETs can trigger and select for dormancy in subpopulations of breast cancer(Hong et al., 2019). In this work we took two novel approaches to investigate the dormancy and awakening roadmap of HDBC cells in unprecedented detail. Firstly, we leveraged a rare cohort of n=5 patients which were treated with primary adjuvant ETs in the absence of surgery (TRACING-HT) to dissect the contribution of genomic aberrations to tumor awakening. Next, we developed a first of its kind evolutionary study in vitro to systematically annotate cancer cells adaptive strategies at single cell level in unperturbed systems during a period of several months (TRADITIOM). Collectively our data suggest that ETs steer HDBC cells into an inherently unstable dormant state. Over time, routes to awakening emerge sporadically and spontaneously in single lineages. Each dormant cell retains an intrinsic awakening probability which we propose is a function of epigenetic decay. Awakening occurs without an external trigger and involves multiple apparent endpoint phenotypes that cannot be fully explained by conventional Darwinian genetic selection processes. Finally, our data show that common genetic hits associated with resistance happen downstream of awakening. Overall, our data have uncovered previously unsuspected roles for stochastic nongenetic events during dormancy with profound clinical implications.

sentences: Genomic taxonomic assignment of individual root-knot nematodes Root-knot nematodes (RKN; genus Meloidogyne) are polyphagous plant pathogens of great economic importance to agriculturalists globally. These species are small, diverse, and can be challenging for accurate taxonomic identification. Many of the most important crop pests confound analysis with simple genetic marker loci as they are polyploids of likely hybrid origin. Here we take a low-coverage, long-read genome sequencing approach to characterisation of individual root-knot nematodes. We demonstrate library preparation for Oxford Nanopore Technologies Flongle sequencing of low input DNA from individual juveniles and immature females, multiplexing up to twelve samples per flow cell. Taxonomic identification with Kraken 2 (a k-mer-based taxonomic assignment tool) is shown to reliably identify individual nematodes to species level, even within the very closely related Meloidogyne incognita group. Our approach forms a robust, low-cost, and scalable method for accurate RKN species diagnostics.

sentences: Sex-specific lipid profiles in muscle of Atlantic salmon juveniles Energy allocation in juvenile fish can have important implications for future life-history progression. Inherited and environmental factors determine when and where individuals allocate energy, and timely and sufficient energy reserves are crucial for reaching key life stages involved in the timing of maturation and sea migration. In Atlantic salmon, lipid reserves are predominantly found in the viscera and myosepta in the muscle and have been shown to play a key role in determining the timing of maturity. This life-history trait is tightly linked to fitness in many species and can be different between males and females, however, the details of relative energy allocation in juveniles of different sexes is not well understood. Therefore, the aim of this study was to investigate the effects of sex, genetics and environment during juvenile development of salmon on the amount and composition of their lipid reserves. To do so, juvenile salmon were fed one of two different lipid food contents during their first summer and autumn under common-garden conditions. Muscle lipid composition and concentrations were determined by thin layer chromatography. The muscle lipid class concentrations covaried negatively with body length and males showed higher concentrations than females for phosphatidylcholine, cholesterol, sphingomyelin, and triacylglycerol. This sex-specific difference in major lipid classes presents a new scope for understanding the regulation of lipids during juvenile development and gives direction for understanding how lipids may interact and influence major life-history traits in Atlantic salmon.

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Chromatin accessibility changes induced by the microbial metabolite butyrate reveal possible mechanisms of anti-cancer effects Butyrate is a four-carbon fatty acid produced in large quantities by bacteria found in the human gut. It is the major source of colonic epithelial cell energy, can bind to and agonize short-chain fatty acid G-protein coupled receptors and functions as a histone deacetylase (HDAC) inhibitor. Anti-cancer effects of butyrate are attributed to a global increase in histone acetylation in colon cancer cells; however, the role that corresponding chromatin remodeling plays in this effect is not fully understood. We used longitudinal paired ATAC-seq and RNA-seq on HCT-116 colon cancer cells to determine how butyrate-related chromatin changes functionally associate with cancer. We detected distinct temporal changes in chromatin accessibility in response to butyrate with less accessible regions enriched in transcription factor binding motifs and distal enhancers. These regions significantly overlapped with regions maintained by the SWI/SNF chromatin remodeler, and were further enriched amongst chromatin regions that are associated with ARID1A/B synthetic lethality. Finally, we found that butyrate-induced chromatin regions were enriched for both colorectal cancer GWAS loci and somatic mutations in cancer. These results demonstrate the convergence of both somatic mutations and GWAS risk variants for colon cancer within butyrate-responsive chromatin regions, providing a molecular map of the mechanisms by which this microbial metabolite might confer anti-cancer properties.

HighlightsO_LIChromatin accessibility changes longitudinally upon butyrate exposure in colon cancer cells.
C_LIO_LIChromatin regions that close in response to butyrate are enriched among distal enhancers.
C_LIO_LIThere is strong overlap between butyrate-induced peaks and peaks associated with SWI/SNF synthetic lethality.
C_LIO_LIButyrate-induced peaks are enriched for colorectal cancer GWAS loci and somatic variation in colorectal cancer.
C_LI

sentences: Integrative small and long RNA-omics analysis of human healing and non-healing wounds discovers cooperating microRNAs as therapeutic targets MicroRNAs (miR) are important posttranscriptional regulators and exhibit a high potential to be utilized in diagnosis and therapy. However, our insufficient knowledge of the miR-mediated gene regulation in human skin wound healing severely hinders the identification of clinically relevant miRs. Here, we performed paired small RNA and long RNA sequencing in human tissue samples, including matched skin and acute wounds collected at each healing stage and chronic non-healing venous ulcers (VU). With integrative small and long RNA-omics analysis, we developed a compendium (https://www.xulandenlab.com/humanwounds-mirna-mrna), which will be an open, comprehensive resource to broadly aid wound healing research. With this first clinical, wound-centric resource of miRs and mRNAs, we identified 17 pathologically relevant miRs that exhibited abnormal VU expression and displayed their targets enriched explicitly in the VU gene signature. Intermeshing regulatory networks controlled by these miRs revealed their high cooperativity in contributing to chronic wound pathology characterized by persistent inflammation and proliferative phase initiation failure. Furthermore, we demonstrated that miR-34a, miR-424, and miR-516, upregulated in VU, cooperatively suppressed keratinocyte growth while promoting inflammatory response. Collectively, our study opens the possibility of developing innovative wound treatment that targets pathologically relevant cooperating miRs to attain higher therapeutic efficacy and specificity.

sentences: Cell Types of Origin in the Cell Free Transcriptome in Human Health and Disease Cell-free RNA (cfRNA) can be used to noninvasively measure dynamic and longitudinal physiological changes throughout the body. While there is considerable effort in the liquid biopsy field to determine disease tissue-of-origin, pathophysiology occurs at the cellular level. Here, we describe two approaches to identify cell type contributions to cfRNA. First we used Tabula Sapiens, a transcriptomic cell atlas of the human body to computationally deconvolve the cell-free transcriptome into a sum of cell type specific transcriptomes, thus revealing the spectrum of cell types readily detectable in the blood. Second, we used individual tissue transcriptomic cell atlases in combination with the Human Protein Atlas RNA consensus dataset to create cell type signature scores which can be used to infer the implicated cell types from cfRNA for a variety of diseases. Taken together, these results demonstrate that cfRNA reflects cellular contributions in health and disease from diverse cell types, potentially enabling determination of pathophysiological changes of many cell types from a single blood test.

sentences: Appetitive learning relies on octopamine and dopamine in ants Associative learning relies on the detection of coincidence between a stimulus and a reward or punishment. In the insect brain, this process is thought to be carried out in the mushroom bodies under control of octopaminergic and dopaminergic neurons. It was assumed that appetitive learning is governed by octopaminergic neurons, while dopamine is required for aversive learning. This view has been recently challenged: Both neurotransmitters seem to be involved in both types of memory in bees and flies. Here, we test which neurotransmitters are required for appetitive learning in ants. We trained Lasius niger ant workers to discriminate two mixtures of linear hydrocarbons and associate one of them with a sucrose reward. We analysed the behaviour of the trained ants using machine learning and found that they preferred the rewarded odour over the other, a preference that was stable for at least 24 hours. We then treated the ants before learning with either epinastine, an octopamine receptor blocker, or with flupentixol, a dopamine receptor blocker. Ants with blocked octopamine receptors did not remember the rewarded odour. Octopamine signalling is thus necessary for the formation of appetitive memory. In contrast, ants with blocked dopamine receptors initially learned the rewarded odour but failed to retrieve this memory 24 hours later. Dopamine is thus required for long-term memory consolidation during appetitive conditioning, independent of short-term memory formation. Our results show that appetitive learning depends on both octopamine and dopamine signalling in ants.

sentences: The incentive circuit: memory dynamics in the mushroom body of Drosophila melanogaster Insects adapt their response to stimuli, such as odours, according to their pairing with positive or negative reinforcements, such as sugar or shock. Recent electrophysiological and imaging findings in Drosophila melanogaster allow detailed examination of the neural mechanisms supporting the acquisition, forgetting and assimilation of memories. We propose that this data can be explained by the combination of a dopaminergic plasticity rule that supports a variety of synaptic strength change phenomena, and a circuit structure (derived from neuroanatomy) between dopaminergic and output neurons that creates different roles for specific neurons. Computational modelling shows that this circuit allows for rapid memory acquisition, transfer from short-term to long-term, and exploration/exploitation trade-off. The model can reproduce the observed changes in the activity of each of the identified neurons in conditioning paradigms and can be used for flexible behavioural control.

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Modelling the visual world of a velvet worm In many animal phyla, eyes are small and provide only low-resolution vision for general orientation in the environment. Because these primitive eyes rarely have a defined image plane, traditional visual-optics principles cannot be applied. To assess the functional capacity of such eyes we have developed modelling principles based on ray tracing in 3D reconstructions of eye morphology, were refraction on the way to the photoreceptors and absorption in the photopigment are calculated incrementally for ray bundles from all angles withing the visual field. From the ray tracing, we calculate the complete angular acceptance function of each photoreceptor in the eye, revealing the visual acuity for all parts of the visual field. We then use this information to generate visual filters that can be applied to high resolution images or videos to convert them to accurate representations of the spatial information seen by the animal. The method is here applied to the 0.1 mm eyes of the velvet worm Euperipatoides rowelli (Onychophora). These eyes of these terrestrial invertebrates consist of a curved cornea covering an irregular but optically homogeneous lens directly joining a retina packed with photoreceptive rhabdomeres. 3D reconstruction from histological sections revealed an asymmetric eye, where the retina is deeper in the forward-pointing direction. The calculated visual acuity also reveals performance differences across the visual field, with a maximum acuity of about 0.11 cycles/deg in the forward direction despite laterally pointing eyes. The results agree with previous behavioural measurements of visual acuity, and suggest that velvet worm vision is adequate for orientation and positioning within the habitat.

Author summaryIt is difficult to understand the roles that vision may have in animals with visual performance very different to our own. Many invertebrates such as flatworms, polychaetes, onychophorans, gastropod molluscs and numerous arthropods have tiny eyes with unknown visual abilities. At best, behavioural experiments can reveal visual performance limits for specific behaviours but they will not give general information about what is visible to the animal, which is crucial for understanding the roles vision may have. Here we use ray tracing applied to accurate anatomical/optical models of the eyes of a velvet worm to reconstruct the visual acuity in all parts of the visual field. We also use the calculated visual performance to closely simulate what animals would see in their natural habitat. The method can be applied to any (preferably small) eye and offers an alternative strategy that may yield information about the visual capacity that is otherwise hard to obtain.

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Structural requirements for dihydrobenzoxazepinone anthelmintics: actions against medically important and model parasites - Trichuris muris, Brugia malayi, Heligmosomoides polygyrus and Schistosoma mansoni Nine hundred million people are infected with the soil-transmitted helminths Ascaris lumbricoides (roundworm), hookworm, and Trichuris trichiura (whipworm). However, low single-dose cure rates of the benzimidazole drugs, the mainstay of preventative chemotherapy for whipworm, together with parasite drug resistance, mean that current approaches may not be able to eliminate morbidity from Trichuriasis. We are seeking to develop new anthelmintic drugs specifically with activity against whipworm as a priority, and previously identified a hit series of dihydrobenzoxazepinone (DHB) compounds that block motility of ex vivo Trichuris muris. Here we report a systematic investigation of the structure-activity relationship of the anthelmintic activity of DHB compounds. We synthesised 47 analogues, which allowed us to define features of the molecules essential for anthelmintic action, as well as broadening the chemotype by identification of dihydrobenzoquinolinones (DBQ) with anthelmintic activity. We investigated the activity of these compounds against other parasitic nematodes, identifying DHB compounds with activity against Brugia malayi and Heligmosomoides polygyrus. We also demonstrated activity of DHB compounds against the trematode Schistosoma mansoni, a parasite that causes schistosomiasis. These results demonstrate the potential of DHB and DBQ compounds for further development as broad-spectrum anthelmintics.

Author summaryAround a billion people are infected by the soil transmitted helminths Ascaris, hookworm and whipworm. In the case of whipworm, the benzimidazole drugs, which are distributed to school children in affected areas, have low cure rates. This means that finding an improved treatment for whipworm is a priority. We previously identified five DHB compounds in a screen for new compounds active against whipworm. Here we systematically dissect these molecules, making 47 modified versions of the compounds. This allowed us to define the features of these compounds that are important for activity against whipworm. We also demonstrate activity of DHB compounds against other parasitic nematodes, and against Schistosoma mansoni, a trematode parasite. These results show the potential for further development of DHB compounds as broad-spectrum anthelmintics.

sentences: Laminar dynamics of beta bursts in human motor cortex Modulation of motor cortical activity in the beta frequency range is one of the strongest and most studied movement-related neural signals. At the single trial level, beta band activity is often characterized by transient bursting events rather than slowly modulating oscillations, suggesting a more rapid, information-encoding functional role than previously believed. Insight into how beta bursts are generated in sensorimotor circuits can provide important constraints to theories about their functional role for movement control. To this end, we leverage and extend recent developments in high precision MEG for temporally resolved laminar analysis of burst activity, combined with a neocortical circuit model that simulates the biophysical generators of the electrical currents which drive beta bursts. This approach pinpoints the generation of beta bursts in human motor cortex to distinct excitatory synaptic inputs to deep and superficial cortical layers, which drive current flow in opposite directions. These laminar dynamics of beta bursts in motor cortex align with prior invasive animal recordings within the somatosensory cortex, and suggest a conserved mechanism for somatosensory and motor cortical beta bursts. More generally, we demonstrate the ability for uncovering the laminar dynamics of event-related neural signals in human non-invasive recordings.

sentences: Manipulating the microbiome alters regenerative outcomes in Xenopus laevis tadpoles via lipopolysaccharide signalling Xenopus laevis tadpoles can regenerate functional tails, containing spinal cord, notochord, muscle, fin, blood vessels and nerves, except for a brief refractory period at around one week of age. At this stage, amputation of the tadpoles tail may either result in scarless wound healing, or the activation of a regeneration programme, which replaces the lost tissues. We recently demonstrated a link between bacterial lipopolysaccharides and successful tail regeneration in refractory stage tadpoles, and proposed that this could result from lipopolysaccharides binding to Toll-like receptor 4 (TLR4). Here, we have used 16S rRNA sequencing to show that the tadpole skin microbiome is highly variable between sibships and that the community can be altered by raising embryos in the antibiotic gentamicin. Six gram-negative genera, including Delftia and Chryseobacterium, were over-represented in tadpoles that underwent tail regeneration. Lipopolysaccharides purified from a commensal Chryseobacterium spp. XDS4, an exogenous Delftia spp. or Escherichia coli could significantly increase the number of antibiotic-raised tadpoles that attempted regeneration. Conversely, the quality of regeneration was impaired in native-raised tadpoles exposed to the antagonistic lipopolysaccharide of Rhodobacter sphaeroides. Editing TLR4 using CRISPR/Cas9 also reduced regeneration quality, but not quantity, at the level of the cohort. However, we found that the editing level of individual tadpoles was a poor predictor of regenerative outcome. In conclusion, our results suggest that variable regeneration in refractory stage tadpoles depends at least in part on the skin microbiome and lipopolysaccharide signalling, but that signalling via TLR4 cannot account for all of this effect.

sentences: Multimodal-Neural Predictive Models of Children's General Intelligence That Are Stable Across Two Years of Development Childhood intelligence is strikingly predictive of major life outcomes. Understanding the brain underpinnings of early-life intelligence is of prime clinical and public health importance, but has so far remained elusive. Here, we demonstrate that it is possible to arrive at models of the brain that are both predictive and explanatory of childhood intelligence. For this we leverage the unique power of the large-scale, longitudinal, multimodal MRI data from the Adolescent Brain Cognitive Development (ABCD) study (n [~]11k) and create a novel predictive modeling framework that integrates machine-learning and structural-equation-based methodologies. Our predictive models combine six MRI modalities (task-fMRI from three tasks, resting-state fMRI, structural MRI, DTI) using machine-learning. In terms of prediction, our models achieve an unprecedented longitudinal association (r=.41) with childhood intelligence across two years in unseen data. We found fronto-parietal networks during a working-memory task to drive childhood-intelligence prediction. In terms of explanation, our models significantly explain variance in childhood intelligence due to (1) key socio-demographic and psychological factors (proportion mediated=18.65% [17.29%-20.12%]) and (2) genetic liability, as reflected by the polygenic score of cognitive ability (proportion mediated=15.6% [11%-20.7%]). In summary, our work shows that novel models of human multimodal neuroimaging data are powerful in helping us predict and explain variation in childhood intelligence.

sentences: Global FAW population genomic signature supports complex introduction events across the Old World Native to the Americas, the invasive Spodoptera frugiperda (fall armyworm; FAW) was reported in West Africa in 2016, followed by chronological order of detections across the Old World and the hypothesis of an eastward Asia expansion. We explored population genomic signatures of American and Old World FAW and identified 12 maternal mitochondrial DNA genome lineages across the invasive range, while from 870 high-quality nuclear single nucleotide polymorphic DNA markers five distinct New World population clusters were identified, broadly reflected their native geographical ranges and absence of host-plant preferences. We identified unique admixed Old World populations, and admixed and non-admixed Asian FAW individuals, all of which suggested multiple introductions underpinned the pests global spread. Directional gene flow from the East into eastern Africa was also detected, in contrast to the west-to-east spread hypothesis. Our study demonstrated population genomic approaches via international partnership to address global emerging pest threats and biosecurity challenges.

sentences: Transposable elements mediate genetic effects altering the expression of nearby genes in colorectal cancer Transposable elements (TEs) are interspersed repeats that contribute to more than half of the human genome, and TE-embedded regulatory sequences are increasingly recognized as major components of the human regulome. Perturbations of this system can contribute to tumorigenesis, but the impact of TEs on gene expression in cancer cells remains to be fully assessed. Here, we analyzed 275 normal colon and 276 colorectal cancer (CRC) samples from the SYSCOL colorectal cancer cohort and discovered 10,111 and 5,152 TE expression quantitative trait loci (eQTLs) in normal and tumor tissues, respectively. Amongst the latter, 376 were exclusive to CRC, likely driven by changes in methylation patterns. We identified that transcription factors are more enriched in tumor-specific TE-eQTLs than shared TE-eQTLs, indicating that TEs are more specifically regulated in tumor than normal. Using Bayesian Networks to assess the causal relationship between eQTL variants, TEs and genes, we identified that 1,758 TEs are mediators of genetic effect, altering the expression of 1,626 nearby genes significantly more in tumor compared to normal, of which 51 are cancer driver genes. We show that tumor-specific TE-eQTLs trigger the driver capability of TEs subsequently impacting expression of nearby genes. Collectively, our results highlight a global profile of a new class of cancer drivers, thereby enhancing our understanding of tumorigenesis and providing potential new candidate mechanisms for therapeutic target development.

sentences: Integrative genomics identifies lncRNA regulatory networks across 1,044 pediatric leukemias and extra-cranial solid tumors Long non-coding RNAs (lncRNAs) play an important role in gene regulation and contribute to tumorigenesis. While pan-cancer studies of lncRNA expression have been performed for adult malignancies, the lncRNA landscape across pediatric cancers remains largely uncharted. Here, we curate RNA sequencing data for 1,044 pediatric leukemia and solid tumors and integrate paired tumor whole genome sequencing and epigenetic data in relevant cell line models to explore lncRNA expression, regulation, and association with cancer. We report a total of 2,657 robustly expressed lncRNAs across six pediatric cancers, including 1,142 exhibiting histotype-specific expression. DNA copy number alterations contributed to lncRNA dysregulation at a proportion comparable to protein coding genes. Application of a multi-dimensional framework to identify and prioritize lncRNAs impacting gene networks revealed that lncRNAs dysregulated in pediatric cancer are associated with proliferation, metabolism, and DNA damage hallmarks. Analysis of upstream regulation via cell-type specific transcription factors further implicated distinct histotype-specific and developmental lncRNAs. We integrated our analyses to prioritize lncRNAs for experimental validation and showed that silencing of TBX2-AS1, our top-prioritized neuroblastoma-specific lncRNA, resulted in significant growth inhibition of neuroblastoma cells, confirming our computational predictions. Taken together, these data provide a comprehensive characterization of lncRNA regulation and function in pediatric cancers and pave the way for future mechanistic studies.

sentences: The eukaryotic translation initiation factor eIF4E reprogrammes the splicing machinery anddrives alternative splicing Aberrant RNA splicing contributes to the pathogenesis of many malignancies including Acute Myeloid Leukemia (AML). While mutation is the best described mechanism underpinning aberrant splicing, recent studies show that predictions based on mutations alone likely underestimate the extent of this dysregulation. Here, we show that elevation of the eukaryotic translation initiation factor eIF4E reprogrammes splicing of nearly a thousand RNAs in model cell lines. In AML patient specimens which did not harbour known splice factor mutations, ~4000 transcripts were differentially spliced based on eIF4E levels and this was associated with poor prognosis. Inhibition of eIF4E in cell lines reverted the eIF4E-dependent splice events examined. Splicing targets of eIF4E act in biological processes consistent with its role in malignancy. This altered splicing program likely arose from eIF4E-dependnet increases in the production of many components of the spliceosome including SF3B1 and U2AF1 which are frequently mutated in AML. Notably, eIF4E did not drive mutation of these factors, only their production. eIF4E also physically associated with many splice factors including SF3B1, U2AF1, and UsnRNAs. Importantly, many eIF4E-dependent splice events differed from those arising from SF3B1 mutation, and were more extensive highlighting that these splicing profiles arise from distinct mechanisms. In all, our studies provide a paradigm for how dysregulation of a single factor, eIF4E, can alter splicing.

sentences: Better together - Relative retention time plus spectral matching improves automated glycan characterization using PGC-nLC-IT-ESI-MS/MS Porous Graphitized Carbon nano-liquid chromatography tandem mass spectrometry (PGC-nLC-MS/MS) is a glycomics technique with the unique capacity to differentiate isobaric glycans. The lack of suitable software tools integrating chromatography and MS-information delivered by PGC-nLC-MS/MS has been limiting fast and robust glycan identification and quantitation. We report a LC-system-independent strategy called GlycoRRT that combines relative retention time (RRT) and negative ion fragment spectra analyses for isobaric structure-specific glycomics of PGC-nLC-MS/MS data. The GlycoRRT toolset is fully customizable and easily adaptable enabling semi-automated high-throughput structural assignments. The current library contains over 200 entries and their individual meta-data (MS instrumentation, experimental conditions, retention times, fragmentation profiles and glycan structural diagnostic ion features) relevant for reliable data analyses. The GlycoRRT workflow was employed to map the N- and O-glycome in blood group matched human plasma and urine as well as decipher Immunoglobulin (IgG) glycosylation features from 13 different animal species. We have also developed visualization tools to enable a consistent, reliable, and reproducible analysis of large sets of multidimensional PGC-nLC-MS/MS glycomics data. This comprehensive glycan resource provides the glycan map of human and animal species, will serve as a reference in dissecting the role of glycans in host pathogen interaction and zoonotic disease transmission.

sentences: Evolutionary dynamics of sex-biased genes expressed in cricket brains and gonads Sex-biased gene expression, particularly sex-biased expression in the gonad, has been linked to rates of protein sequence evolution (nonsynonymous to synonymous substitutions, dN/dS) in animals. However, in insects, sex-biased expression studies remain centered on a few holometabolous species, and moreover, other major tissue types such as the brain remain underexplored. Here, we studied sex-biased gene expression and protein evolution in a hemimetabolous insect, the cricket Gryllus bimaculatus. We generated novel male and female RNA-seq data for two sexual tissue types, the gonad and somatic reproductive system, and for two core components of the nervous system, the brain and ventral nerve cord. From a genome-wide analysis, we report several core findings. Firstly, testis-biased genes had accelerated evolution, as compared to ovary-biased and unbiased genes, which was associated with positive selection events. Secondly, while sex-biased brain genes were much less common than for the gonad, they exhibited a striking tendency for rapid protein evolution, an effect that was stronger for the female than male brain. Further, some sex-biased brain genes were linked to sexual functions and mating behaviors, which we suggest may have accelerated their evolution via sexual selection. Thirdly, a tendency for narrow cross-tissue expression breadth, suggesting low pleiotropy, was observed for sex-biased brain genes, suggesting relaxed purifying selection, which we speculate may allow enhanced freedom to evolve adaptive protein functional changes. The findings of rapid evolution of testis-biased genes and male and female-biased brain genes are discussed with respect to pleiotropy, positive selection, and the mating biology of this cricket.

sentences: Exploitation of the genetic variability in the genotype of Egyptian Wheat by studding Molecular Markers, Molecular Docking and Nano Composites. Regulation of leaf and stem rust This manuscript has been withdrawn by the authors as it was submitted and made public without the full consent of all the authors. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

sentences: Increased connectivity among sensory and motor regions during visual and audiovisual speech perception In everyday conversation, we usually process the talkers face as well as the sound of their voice. Access to visual speech information is particularly useful when the auditory signal is degraded. Here we used fMRI to monitor brain activity while adult humans (n = 60) were presented with visual-only, auditory-only, and audiovisual words. The audiovisual words were presented in quiet and several signal-to-noise ratios. As expected, audiovisual speech perception recruited both auditory and visual cortex, with some evidence for increased recruitment of premotor cortex in some conditions (including in substantial background noise). We then investigated neural connectivity using psychophysiological interaction (PPI) analysis with seed regions in both primary auditory cortex and primary visual cortex. Connectivity between auditory and visual cortices was stronger in audiovisual conditions than in unimodal conditions, including a wide network of regions in posterior temporal cortex and prefrontal cortex. In addition to whole-brain analyses, we also conducted a region-of-interest analysis on the left posterior superior temporal sulcus (pSTS), implicated in many previous studies of audiovisual speech perception. We found evidence for both activity and effective connectivity in pSTS for visual-only and audiovisual speech, although these were not significant in whole-brain analyses. Taken together, our results suggest a prominent role for cross-region synchronization in understanding both visual-only and audiovisual speech that complements activity in "integrative" brain regions like pSTS.

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Multi-echo Quantitative Susceptibility Mapping: How to Combine Echoes for Accuracy and Precision PurposeTo compare different multi-echo combination methods for MRI quantitative susceptibility mapping (QSM). Given the current lack of consensus, we aimed to elucidate how to optimally combine multi-echo gradient-recalled echo (GRE) signal phase information, either before or after applying Laplacian-base methods (LBMs) for phase unwrapping or background field removal.

MethodsMulti-echo GRE data were simulated in a numerical head phantom, and multiecho GRE images were acquired at 3 T in ten healthy volunteers. To enable image-based estimation of GRE signal noise, five volunteers were scanned twice in the same session without repositioning. Five QSM processing pipelines were designed: one applied nonlinear phase fitting over echo times (TEs) before LBMs; two applied LBMs to the TE-dependent phase and then combined multiple TEs via either TE-weighted or signal-to-noise ratio (SNR)-weighted averaging; two calculated TE-dependent susceptibility maps via either multi-step or single-step QSM and then combined multiple TEs via magnitude-weighted averaging. Results from different pipelines were compared using visual inspection; summary statistics of susceptibility in deep gray matter, white matter, and venous regions; phase noise maps (error propagation theory); and, in the healthy volunteers, regional fixed bias analysis (Bland-Altman) and regional differences between the means (nonparametric tests).

ResultsNonlinearly fitting the multi-echo phase over TEs before applying LBMs provided the highest regional accuracy of{chi} and the lowest phase noise propagation compared to averaging the LBM-processed TE-dependent phase. This result was especially pertinent in high-susceptibility venous regions.

ConclusionFor multi-echo QSM, we recommend combining the signal phase by nonlinear fitting before applying LBMs.

sentences: A new mathematical model of phyllotaxis to solve the genuine puzzle spiromonostichy The view is widely accepted that the inhibitory effect of existing leaf primordia on new primordium formation determines phyllotactic patterning. Previous studies have shown that mathematical models based on such inhibitory effect can generate most of phyllotactic patterns. However, a few types of phyllotaxis still remain unaddressed. A notable example is costoid phyllotaxis showing spiromonostichy, which is characterized by a steep spiral with a small divergence angle and is unique to Costaceae plants. Costoid phyllotaxis has been called a "genuine puzzle" because it seems to disagree with the inhibitory effect-based mechanism. In an attempt to produce a steep spiral pattern, we developed a new mathematical model assuming that each leaf primordium emits not only the inhibitory effect but also some inductive effect. Computer simulations with the new model successfully generated a steep spiral pattern when these two effects met a certain relationship. The obtained steep spiral matched the real costoid phyllotaxis observed with Costus megalobractea. We also found by the mathematical model analysis that the early phyllotactic transition in the seedlings of this plant can be explained by the SAM enlargement.

sentences: ARID1A-mutant and deficient bladder cancer is sensitive to EZH2 pharmacologic inhibition Metastatic urothelial carcinoma of the bladder is generally incurable by current systemic therapy. Molecular characterization of bladder cancer (BLCa) has revealed multiple candidate driver genes for BLCa tumorigenesis. Epigenetic/chromatin modifiers have been shown to be frequently mutated in BLCa, with ARID1A mutations highly prevalent in nearly 20% of early and late stage tumors. EZH2 is a histone methyltransferase that acts as an oncogene. The data herein show that ARID1A deficient tumors, but not ARID1A wild-type tumors are sensitive to EZH2 inhibition. Specifically, EZH2 inhibitor-treated ARID1A deficient bladder cancer cells show significantly reduced cell viability, colony formation, and in vivo tumor growth relative to ARID1A-wild type bladder cancer cells. Thus, our study suggests that a specific subset of bladder cancer patients with ARID1A mutations can be therapeutically treated with pharmacologic inhibitors targeting EZH2.

sentences: ABA homeostasis and long-distance translocation is redundantly regulated by ABCG ABA importers The effects of abscisic acid (ABA) on plant growth, development and response to the environment depend on local ABA concentrations. Here, we exploited a genome-scale amiRNA screen, targeting the Arabidopsis transportome, to show that ABA homeostasis is regulated by two previously unknown ABA transporters. ABCG17 and ABCG18 are localized to the plasma membranes of leaf mesophyll and stem cortex cells to redundantly promote ABA import, leading to conjugated inactive ABA sinks, thus restricting stomatal closure. ABCG17 and ABCG18 double knockdown revealed that the transporters encoded by these genes not only limit stomatal aperture size, conductance and transpiration while increasing water-use efficiency but also control ABA translocation from the shoot to the root to regulate lateral root emergence. The proposed ABCG17- and ABCG18-dependent ABA glucosyl ester shoot sink mechanism is restrained under abiotic stress conditions to further activate the ABA responses.

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Computational modelling of cell motility modes emerging from cell-matrix adhesion dynamics Lymphocytes have been described to perform different motility patterns such as Brownian random walks, persistent random walks, and Levy walks. Depending on the conditions, such as confinement or the distribution of target cells, either Brownian or Levy walks lead to more efficient interaction with the targets. The diversity of these motility patterns may be explained by an adaptive response to the surrounding extracellular matrix (ECM). Indeed, depending on the ECM composition, lymphocytes either display a floating motion without attaching to the ECM, or sliding and stepping motion with respectively continuous or discontinuous attachment to the ECM, or pivoting behaviour with sustained attachment to the ECM. Moreover, on the long term, lymphocytes either perform a persistent random walk or a Brownian-like movement depending on the ECM composition. How the ECM affects cell motility is still incompletely understood. Here, we integrate essential mechanistic details of the lymphocyte-matrix adhesions and lymphocyte intrinsic cytoskeletal induced cell propulsion into a Cellular Potts model (CPM). We show that the combination of de novo cell-matrix adhesion formation, adhesion growth and shrinkage, adhesion rupture, and feedback of adhesions onto cell propulsion recapitulates multiple lymphocyte behaviours, for different lymphocyte subsets and various substrates. With an increasing attachment area and increased adhesion strength, the cells speed and persistence decreases. Additionally, the model can predict short-term persistent with long-term subdiffusive motility, showing a pivoting motion. For small adhesion areas, we observe that the spatial distribution of adhesions influences cell motility. Small adhesions at the front allow for more persistent motion than larger clusters at the back, despite a similar total adhesion area. In conclusion, we present an integrated framework to simulate the effects of ECM proteins on cell-matrix adhesion dynamics. The model reveals a sufficient set of principles explaining the plasticity of lymphocyte motility.

Author summaryDuring immunosurveillance, lymphocytes patrol through tissues to interact with cancer cells, other immune cells, and pathogens. The efficiency of this process depends on the kinds of trajectories taken, ranging from simple Brownian walks to Levy walks. The composition of the extracellular matrix (ECM), a network of macromolecules, affects the formation of cell-matrix adhesions, thus strongly influencing the way lymphocytes move. Here, we present a model of lymphocyte motility driven by adhesions that grow, shrink and rupture in response to the ECM and cellular forces. Compared to other models, our model is computationally light making it suitable for generating long term cell track data, while still capturing actin dynamics and adhesion turnover. Our model suggests that cell motility is affected by the force required to break adhesions and the rate at which new adhesions form. Adhesions can promote cell protrusion by inhibiting retrograde actin flow. After introducing this effect into the model, we found that it reduces the cellular diffusivity and that it promotes stick-slip behaviour. Furthermore, location and size of adhesion clusters determined cell persistence. Overall, our model explains the plasticity of lymphocyte behaviour in response to the ECM.

sentences: Genome-wide development of lncRNA-derived-SSR markers for Dongxiang wild rice (Oryza rufipogon Griff.) Dongxiang wild rice (Oryza rufipogon Griff., DXWR) is the northernmost distributed common wild rice found in the world. It contains a large number of agronomically valuable genes, which makes it a natural gene pool for rice breeding. Molecular markers, especially simple repeat sequence (SSR) markers, play important roles in crop breeding. Although a large number of SSR markers have been developed, most of them are derived from the genome coding sequences, rarely from non-coding sequences. Meanwhile, long non-coding RNAs (lncRNAs), which are derived from the transcription of non-coding sequences, play vital roles in plant growth, development and stress responses. In this study, 1878 SSR loci were detected from the lncRNA sequences of DXWR, and 1258 lncRNA-derived-SSR markers were developed on the genome-wide scale. To verify the validity and applicability of these markers, 72 pairs of primers were randomly selected to test 44 rice materials. The results showed that 42 (58.33%) pairs of primers have abundant polymorphism among these rice materials; the polymorphism information content (PIC) values ranged from 0.04 to 0.87 with an average of 0.50; the genetic diversity index of SSR loci varied from 0.04 to 0.88 with an average of 0.56; and the number of alleles per marker ranged from 2 to 11 with an average of 4.36. Thus, we concluded that these lncRNA-derived-SSR markers are a very useful source for future basic and applied research, including genetic diversity analysis, QTL mapping, and molecular breeding programs, to make good use of the elite lncRNA genes from DXWR.

sentences: Transcranial direct current stimulation does not improve performance in a whole-body movement task Research has investigated the use of non-invasive brain interventions, such as transcranial direct current stimulation (tDCS), to enhance motor learning and rehabilitation. Much research has shown that tDCS improves motor learning and that bilateral tDCS is more beneficial than unilateral tDCS in improving motor learning. However, past research has primarily utilised simple motor tasks in measuring motor skill learning. These are not ecologically reliable as whole-body movement is required for everyday activities. This study involved two experiments. Each experiment involved participants learning 12 Ballroom and Latin dance moves whilst undergoing tDCS. All participants underwent three sessions of tDCS, (unilateral, bilateral and sham), over three consecutive days. Participants in the first experiment (n=30) had stimulation to the primary motor cortex (PMC) and those in the second experiment (n=31) had stimulation to the dorsolateral prefrontal cortex (DLPFC). In each experiment, a baseline was taken before the training sessions and two outcome measures were taken; a day after the last training session and two weeks later. In each testing session participants dance ability was measured. Our results showed that bilateral tDCS impaired performance in both experiments. Unilateral stimulation impaired performance in the first experiment, and did not significantly improve performance any better than the sham stimulation in the second experiment. These results suggest that task complexity plays a crucial role when tDCS procedures are used to modulate motor performance and highlights possible limitations of tDCS in practice.

sentences: Using expert-elicitation to deliver biodiversity monitoring priorities on a Mediterranean island Biodiversity monitoring plays an essential role in tracking changes in ecosystems, species distributions and abundances across the globe. Data collected through both structured and unstructured biodiversity recording can inform conservation measures designed to reduce, prevent, and reverse declines in valued biodiversity of many types. However, resources for biodiversity monitoring are limited, it is therefore important that funding bodies prioritise actions relative to the requirements in any given region. We addressed this prioritisation requirement through a three-stage process of expert-elicitation, resulting in a prioritised list of twenty biodiversity monitoring needs for Cyprus. Equal priority was assigned to the twenty monitoring needs within three categories: a top nine, a middle five, and a bottom six. The most highly prioritised biodiversity monitoring needs were those related to the development of robust methodologies, and those ensuring a geographic spread of sufficiently skilled and informed contributors. We suggest ways that the results of our expert-elicitation process could be used to support current and future biodiversity monitoring in Cyprus.

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TDFragMapper: a visualization tool for evaluating experimental parameters in top-down proteomics MotivationWe present a new software-tool allowing an easy visualization of fragment ions and thus a rapid evaluation of key experimental parameters on the sequence coverage obtained for the MS/MS analysis of intact proteins. Our tool can deal with multiple fragmentation methods.

ResultsWe demonstrate that TDFragMapper can rapidly highlight the experimental fragmentation parameters that are critical to the characterization of intact proteins of various size using top-down proteomics.

AvailabilityTDFragMapper, a demonstration video and user tutorial are freely available at https://msbio.pas-teur.fr/tdfragmapper, for academic use; all data are thus available from the ProteomeXchange consortium (identifier PXD024643).

Contactdiogobor@gmail.com or julia.chamot-rooke@pasteur.fr

sentences: A rapid prototyping approach for intensive field courses to build scientific and professional skills This paper reports on the design and evaluation of Field Studies in Functional Ecology (FSFE), a two-week intensive residential field course that engages students to master core content in functional ecology alongside skills that facilitate their transition from  student to  scientist. This paper provides an overview of the course structure, showing how the constituent elements have been designed and refined over successive iterations of the course. We detail how FSFE students are guided first to develop their own research questions (Field Problems), and then through a structured reflective approach of rapid prototyping and iterative refinement of each stage of the research process. Using extensive evaluation data, we show how, coached by experts within a cognitive apprenticeship framework, FSFE students develop a coherent and nuanced understanding of how to approach and execute ecological studies. The sophisticated knowledge and skills as ecology researchers that they develop during the course is highlighted through high quality presentations and peer-reviewed publications in a student-led journal. We illustrate how field course elements can be used to provide cognitive, affective and reflective tools that allow students to gain maximum value from their educational journey, and the confidence to consider research in their future careers.

sentences: Hunchback activates Bicoid in post-mitotic Pair1 neurons to regulate synapse number The proper formation and function of neural circuits is crucial for cognition, sensation, and behavior. Neural circuits are highly-specific, and this specificity is dependent on neurons developing key features of their individual identities: morphology, anatomical location, molecular expression and biophysiological properties. Previous research has demonstrated that a neurons identity is, in part, generated by the temporal transcription window the neuron is born in, and the homeodomain transcription factors expressed in the mature neuron. However, whether temporal transcription factors and homeodomain transcription factors regulate neural circuit formation, maintenance and function remains unknown. Here, we utilize a well-characterized neural circuit in the Drosophila larvae, the Pair1 neuron. We determined that in the Pair1 neuron, the temporal transcription factor Hunchback activates the homeodomain transcription factor Bicoid (Bcd). Both Hunchback and Bcd are expressed in Pair1 throughout larval development. Interestingly, Hunchback and Bcd were not required in Pair1 for neurotransmitter identity or axonal morphology, but were required for synapse density. We found that these transcription factors were functioning post-mitotically in Pair1 to regulate synapse density. Additionally, knocking down Hunchback and Bcd in Pair1 neurons disrupted the behavioral output of the circuit. We utilized the genetic tool TransTango to determine that Hunchback function in Pair1 is to repress forming synapses with erroneous neurons. To our knowledge, these data are the first to show Hunchback activating Bcd expression, as well as the first to demonstrate a role for Hunchback and Bcd post-mitotically.

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Intrinsic and tumor-induced JAK/STAT signaling regulate developmental timing by the Drosophila prothoracic gland Development involves tightly paced, reproducible sequences of events, yet it must adjust to conditions external to it, such as resource availability and organismal damage. A major mediator of damage-induced immune responses in vertebrates and insects is JAK/STAT signaling. At the same time, JAK/STAT activation by the Drosophila Upd cytokines is pleiotropically involved in normal development of multiple organs. Whether inflammatory and developmental roles of JAK/STAT intersect is unknown. Here, we show that JAK/STAT is active during development of the prothoracic gland (PG), the organ that controls metamorphosis onset through ecdysone production. Reducing JAK/STAT signaling decreased PG size and slightly advanced metamorphosis. Conversely, JAK/STAT hyperactivation, achieved through overexpression of pathway components or SUMOylation loss, caused PG hypertrophy and metamorphosis delay. Interestingly, tissue damage and tumors, known to secrete Upd cytokines, also activated JAK/STAT in the PG and delayed metamorphosis. Finally, we show that expression of transcription factor Apontic, a JAK/STAT target in the PG, recapitulates PG hypertrophy and metamorphosis delay. JAK/STAT damage signaling, therefore, regulates metamorphosis onset at least in part by coopting its developmental role in the PG.

Summary statementDamage signaling from tumors mediated by JAK/STAT-activating Upd cytokines delays the Drosophila larva-pupa transition through cooption of a JAK/STAT developmental role in the prothoracic gland.

sentences: On the inference of positive and negative species associations and their relation to abundance The prevalence of rare species in ecosystems begs the question of how they persist. In a recent paper, Calatayuda et al. (CEA) provided a new hypothesis that rare species, in contrast to common species, share unique microhabitats and/or preferentially engage in mutualistic interactions. CEA support this hypotheses by reconstructing association networks from spatially replicated abundance data finding that rare species are over-representing in positive association networks while common species are over-representing in negative association networks. However, the use of abundance and co-occurrence data to infer true species associations is difficult and often inaccurate. Here, I show that the finding of rare species being more represented in positive association networks can be explained by statistical artifacts in the inference of species associations from abundance data. I caution against the inference of ecological association networks from abundance data alone.

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How do King Cobras move across a major highway? Unintentional wildlife crossing structures may facilitate movement. O_LIGlobal road networks continue to expand, and the wildlife responses to these landscape-level changes need to be understood to advise long-term management decisions. Roads have high mortality risk to snakes because snakes typically move slowly and can be intentionally targeted by drivers.
C_LIO_LIWe investigated how radio-tracked King Cobras (Ophiophagus hannah) traverse a major highway in northeast Thailand, and if reproductive cycles were associated with road hazards.
C_LIO_LIWe surveyed a 15.3km stretch of Highway 304 to determine if there were any locations where snakes, and other wildlife, could safely move across the road (e.g., culverts, bridges). We used recurse analysis to detect possible road-crossing events, and used subsets of King Cobra movement data to create dynamic Brownian Bridge Movement Models (dBBMM) in an attempt to show movement pathways association with possible unintentional crossing structures. We further used Integrated Step Selection Functions (ISSF) to assess seasonal differences in avoidance of major roads for adult King Cobras in relation to reproductive state.
C_LIO_LIWe discovered 32 unintentional wildlife crossing locations capable of facilitating King Cobra movement across the highway. Our dBBMMs failed to show if underpasses were being used by telemetered individuals; however, the tracking locations pre- and post-crossing provided strong evidence of underpass use. Our ISSF suggested a lower avoidance of roads during the breeding season, though the results were inconclusive. With the high volume of traffic, large size of King Cobras and a 98.8% success rate of crossing the road in our study, we strongly suspect that individuals are using the unintentional crossing structures to safely traverse the road.
C_LIO_LIFurther research is needed to determine the extent of wildlife underpass use at our study site and globally, alongside using previously proven fencing to facilitate their use. We propose that more consistent integration of drainage culverts and bridges could help mitigate the impacts of roads on some terrestrial wildlife, particularly in areas where roads fragment forests and wildlife corridors.
C_LI

sentences: Enhancing Sampling of Water Rehydration on Ligand Binding: A Comparison of Techniques 1Water often plays a key role in protein structure, molecular recognition, and mediating protein-ligand interactions. Thus, free energy calculations must adequately sample water motions, which often proves challenging in typical MD simulation timescales. Thus, the accuracy of methods relying on MD simulations ends up limited by slow water sampling. Particularly, as a ligand is removed or modified, bulk water may not have time to fill or rearrange in the binding site. In this work, we focus on several molecular dynamics (MD) simulation-based methods attempting to help rehydrate buried water sites: BLUES, using nonequilibrium candidate Monte Carlo (NCMC); grand, using grand canonical Monte Carlo (GCMC); and normal MD. We assess the accuracy and efficiency of these methods in rehydrating target water sites. We selected a range of systems with varying numbers of waters in the binding site, as well as those where water occupancy is coupled to the identity or binding mode of the ligand. We analyzed rehydration of buried water sites in binding pockets using both clustering of trajectories and direct analysis of electron density maps. Our results suggest both BLUES and grand enhance water sampling relative to normal MD and grand is more robust than BLUES, but also that water sampling remains a major challenge for all of the methods tested. The lessons we learned for these methods and systems are discussed.

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Complete loss of CASK causes severe ataxia through cerebellar degeneration in human and mouse Heterozygous loss of X-linked genes like CASK and MeCP2 (Rett syndrome) causes neurodevelopmental disorders (NDD) in girls, while in boys such loss leads to profound encephalopathy. The cellular basis for these disorders remains unknown. CASK is presumed to work through the Tbr1-reelin pathway in neuronal migration during brain development. Here we report our clinical and histopathological analysis of a deceased 2-month-old boy with a CASK-null mutation. We demonstrate that although smaller in size, the CASK-null human brain exhibits normal lamination without defective neuronal differentiation, migration, or axonal guidance, excluding the role of reelin in CASK-linked pathology. The disproportionately hypoplastic cerebellum in humans without CASK expression is associated with cerebellar astrogliosis, a marker for neuronal loss. Cerebellum-specific deletion in mouse confirms a post-developmental degeneration of cerebellar granular neurons that results in a small cerebellum. Mechanistically, cerebellar hypoplasia in CASK mutation thus results from neurodegeneration rather that developmental defects. Zygosity-pathology correlation suggests that NDDs like CASK mutation and Rett syndrome are pathologically neurodegenerative; however, random X-chromosome inactivation in the typical heterozygous mutant girls results in 50% of cells expressing the functional gene, resulting in a non-progressive pathology, whereas complete loss of the only allele in boys leads to unconstrained degeneration and encephalopathy.

One sentence summary of studyCASK loss causes cerebellar degeneration. The authors have declared that no conflict of interest exists.

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Murine Norovirus infection results in anti-inflammatory response downstream of amino acids depletion in macrophages Murine norovirus (MNV) infection results in a late translation shut-off, that is proposed to contribute to the attenuated and delayed innate immune response observed both in vitro and in vivo. Recently, we further demonstrated the activation of the eIF2 kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signalling during metabolic stress. While viral infection is usually associated with activation of dsRNA binding pattern recognition receptor PKR, we hypothesised that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterise cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signalling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation.

ImportanceDuring viral infection, host defences are typically characterised by the secretion of pro-inflammatory autocrine and paracrine cytokines, potentiation of the IFN response and induction of the anti-viral response via activation of JAK and Stat signalling. To avoid these and propagate viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signalling. This highlights novel tools in the viral countermeasures tool-kit, and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.

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Network States Classification based on Local Field Potential Recordings in the Awake Mouse Neocortex Recent studies using intracellular recordings in awake behaving mice revealed that cortical network states, defined based on membrane potential features, modulate sensory responses and perceptual outcomes. Single cell intracellular recordings are difficult to achieve and have low yield compared to extracellular recordings of population signals, such as local field potentials (LFPs). However, it is currently unclear how to identify these behaviorally-relevant network states from the LFP. We used simultaneous LFP and intracellular recordings in the somatosensory cortex of awake mice to design and calibrate a model-based analysis method, the Network State Index (NSI), that enables network state classification from the LFP. We used the NSI to analyze the relationship between single-cell (intracellular) and population (LFP) signals over different network states of wakefulness. We found that graded levels of population signal faithfully predicted the levels of single cell depolarization in non-rhythmic regimes whereas, in delta ([2-4 Hz]) oscillatory regimes, the graded levels of rhythmicity in the LFP mapped into a stereotypical oscillatory pattern of membrane potential. Finally, we showed that the variability of network states, beyond the occurrence of slow oscillatory activity, critically shaped the average correlations between single cell and population signals. NSI-based characterization provides a ready-to-use tool to understand from LFP recordings how the modulation of local network dynamics shapes the flexibility of sensory processing during behavior.

Significance statementSensation during behaviour is strongly modulated by the animals internal state. Such context-dependent modulation of sensory processing is believed to largely stem from top-down control of network states in sensory cortices, with different network states being associated with distinct computational properties of the circuit. So far, a detailed characterization of network states in the awake cortex has mostly been achieved through single-cell intracellular recordings, which however cannot be easily recorded. Here, we developed a new method to classify network states from the easily accessible extracellular LFP recordings of population activity. Given the widespread use of LFPs, our work provides a critical methodology to greatly expand our understanding of the mechanisms underlying state-dependent computations in neocortex.

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Different DGAT1s show different TAG synthesis abilities and a specific amino acid substitution enhances oil accumulation Triacylglycerols (TAGs) are the major component of plant storage lipids. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the Kennedy pathway, and responsible for plant oil accumulation. We previously found DGAT activity of Vernonia galamensis DGAT1 was distinctively higher than that of Arabidopsis thaliana DGAT1 and soybean DGAT1 in a yeast microsome assay. In this study, the DGAT1 cDNAs of Arabidopsis, Vernonia, soybean, and castor were introduced into Arabidopsis (ecotype Col-0). All Vernonia DGAT1 expressing lines showed a significantly higher oil content (average 49% relative increase compared to the wild type) followed by soybean, and castor. Most Arabidopsis DGAT1 over-expressing lines did not show a significant increase. In addition to these four DGAT1s, sunflower, Jatropha and sesame DGAT1 genes were introduced into the TAG biosynthesis defective yeast mutant (H1246). In the yeast expression culture, DGAT1s from Arabidopsis, castor, and soybean only slightly increased TAG content, however, DGAT1s from Vernonia, sunflower, Jatropha, and sesame remarkably increased TAG content more than 10 times higher than the former three DGAT1s. Three amino acid residues were characteristically common in the latter four DGAT1s. Using soybean DGAT1, these amino acid substitutions by site-directed mutagenesis was performed and analyzed. These substitutions substantially increased the TAG content.

HighlightDGAT1s from several plant species were tested their TAG accumulation promotion in Arabidopsis and yeast. They were divided into high and low function and single amino acid substitution enhanced function

sentences: Multi-GBS: A massively multiplexed GBS-based protocol optimized for large, repetitive conifer genomes Reduced representation library approaches are still a valuable tool for breeding and population and ecological genomics, even with impressive increases in sequencing capacity in recent years. Unfortunately, current approaches only allow for multiplexing up to 384 samples. To take advantage of increased sequencing capacity, we present Multi-GBS, a massively multiplexable extension to Genotyping-by-Sequencing that is also optimized for large conifer genomes. In Norway Spruce, a highly repetitive 20Gbp diploid genome with high population genetic variation, we call over a million variants in 32 genotypes from three populations, two natural forest in the Alps and Bohemian Alps, and a managed population from southeastern Austria using the existing TASSEL GBSv2 pipeline. Metric MDS analysis of replicated genotypes shows that technical bias in resulting genotype calling is minimal and that populations cluster in biologically meaningful ways.

sentences: Benchmarking brain organoid recapitulation of fetal corticogenesis Brain organoids are becoming increasingly relevant to dissect the molecular mechanisms underlying psychiatric and neurological conditions. The in vitro recapitulation of key features of human brain development affords the unique opportunity of investigating the developmental antecedents of neuropsychiatric conditions in the context of the actual patients genetic backgrounds. Specifically, multiple strategies of brain organoid (BO) differentiation have enabled the investigation of human cerebral corticogenesis in vitro with increasing accuracy. However, the field lacks a systematic investigation of how closely the gene co-expression patterns seen in cultured BO from different protocols match those observed in fetal cortex, a paramount information for ensuring the sensitivity and accuracy of modeling disease trajectories. Here we benchmark BO against fetal corticogenesis by integrating transcriptomes from in-house differentiated cortical BO (CBO), other BO systems, human fetal brain samples processed in-house, and prenatal cortices from the BrainSpan Atlas. We identified co-expression patterns and prioritized hubs of human corticogenesis and CBO differentiation, highlighting both well-preserved and discordant trends across BO protocols, including different degrees of heterochronicity in differentiation across BO models compared to fetal cortex. Our approach provides a framework to directly compare the extent of in vivo/in vitro alignment of neurodevelopmentally relevant processes and their attending temporalities, structured as an accessible resource to query for modelling human corticogenesis and the neuropsychiatric outcomes of its alterations.

sentences: Isolation, Molecular identification, Molecular docking  In Vitro and In Silico Evaluation study against Fusarium oxysporum sp. Lactucae bioRxiv has withdrawn this preprint as it was submitted and made public without the full consent of all the contributing authors.

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Control of N2 fixation and NH3 excretion in Azorhizobium caulinodans ORS571 Due to the costly energy demands of N2 fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the N2-fixing catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity NH3 assimilation by glutamine synthetase (GS), preventing excess release of excess NH3 for plants. Diazotrophic bacteria can be engineered to excrete NH3 by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH3 to non-target plants. Here, we attempted two strategies to control GS regulation and NH3 excretion in our model cereal symbiont Azorhizobium caulinodans AcLP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both PII homologues glnB and glnK stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyltransferases (uATs) in a {Delta}glnE mutant of AcLP which permitted strong GS shutdown and excretion of NH3 derived from N2 fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a uAT allele under control of the NifA-dependent promoter PnifH, permitting GS shutdown and NH3 excretion specifically under microaerobic conditions, the same cue that initiates N2 fixation, then deleted nifA and transferred a rhizopine-inducible nifAL94Q/D95Q-rpoN controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N2 fixation with NH3 excretion. In future, this highly sophisticated and multi-layered control circuitry could be used to activate N2 fixation and NH3 excretion specifically by AcLP colonising transgenic rhizopine producing cereals, targeting delivery of fixed N to the crop, and preventing interaction with non-target plants.

Author SummaryInoculation of cereal crops with associative "diazotrophic" bacteria that convert atmospheric N2 to NH3 could be used to sustainably improve delivery of nitrogen in agriculture. However, due to the costly energy demands of N2 fixation, natural diazotrophic bacteria have evolved to conserve energy by preventing excess production of NH3 and release to the plants. Diazotrophs can be engineered for excess NH3 production and release, however genetic control is required to minimise growth penalties and prevent unintended provision of NH3 to non-target weed species. Here, we engineer control of N2 fixation and NH3 excretion in response to the signalling molecule rhizopine which is produced by transgenic barley. This control could be used to establish plant host-specific activation of N2 fixation and NH3 release following root colonisation in the field, minimising bacterial energy requirements in the bulk soil and preventing provision of NH3 to non-target plants.

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Dependency of Lower Limb Joint Reaction Forces on Femoral Anteversion BackgroundMusculoskeletal (MSK) models based on literature data are meant to represent a generic anatomy and are a popular tool employed by biomechanists to estimate the internal loads occurring in the lower limb joints, such as joint reaction forces (JRFs). However, since these models are normally just linearly scaled to an individuals anthropometry, it is unclear how their estimations would be affected by the personalization of key features of the MSK anatomy, one of which is the femoral anteversion angle.

Research QuestionHow are the lower limb JRF magnitudes computed through a generic MSK model affected by changes in the femoral anteversion?

MethodsWe developed a bone-deformation tool in MATLAB (https://simtk.org/projects/bone_deformity) and used it to create a set of seven OpenSim models spanning from 2{degrees} femoral retroversion to 40{degrees} anteversion. We used these models to simulate the gait of an elderly individual with an instrumented prosthesis implanted at their knee joint (5th Grand Challenge dataset) and quantified both the changes in JRFs magnitude due to varying the skeletal anatomy and their accuracy against the correspondent in vivo measurements at the knee joint.

ResultsHip and knee JRF magnitudes were affected by the femoral anteversion with variations from the unmodified generic model up to 11.7{+/-}5.5% at the hip and 42.6{+/-}31.0% at the knee joint. The ankle joint was unaffected by the femoral geometry. The MSK models providing the most accurate knee JRFs (root mean squared error: 0.370{+/-}0.069 body weight, coefficient of determination: 0.764{+/-}0.104, largest peak error: 0.36{+/-}0.16 body weight) were those with the femoral anteversion angle closer to that measured on the segmented bone of the individual.

SignificanceFemoral anteversion substantially affects hip and knee JRFs estimated with generic MSK models, suggesting that personalizing key MSK anatomical features might be necessary for accurate estimation of JRFs with these models.

sentences: Glutamatergic projections from ventral hippocampus to nucleus accumbens cholinergic neurons are altered in a mouse model of depression The cholinergic interneurons (ChATs) of the nucleus accumbens (NAc) have a critical role in the activity of this region, specifically in the context of major depressive disorder. To understand the circuitry regulating this behavior we sought to determine the areas that directly project to these cells/interneurons by utilizing the monosynaptic cell-specific tracing technique. Mapping showed monosynaptic projections that are exclusive to NAc ChATs. To determine if some of these projections are altered in a depression mouse model, we used mice that do not express the calcium binding protein p11 specifically in ChATs (ChAT-p11 cKO) and display a depressive-like phenotype. Our data demonstrated that while the overall projection areas remain similar between wild type and in ChAT-p11 cKO mice, the number of projections coming from the ventral hippocampus (vHIP) is significantly reduced in the ChAT-p11 cKO mice. Furthermore, using optogenetics and electrophysiology we showed that glutamatergic projections from vHIP to NAc ChATs are severely altered in mutant mice. These results show that specific alterations in the circuitry of the accumbal ChAT interneurons could play an important role in the regulation of depressive-like behavior, reward seeking behavior in addictions, or psychiatric symptoms in neurodegenerative diseases.

sentences: Trait variation between and within Andes and coastal mountain ranges in the iconic South American tree Araucaria araucana in Chile As global commitments to restoration are underway, science is needed to support capacity to achieve meaningful gains for ecosystems and human communities. In Chile, identification and generation of appropriate plant material is a barrier to achieving major restoration goals under the Paris Climate Agreement. Understanding genetic differentiation among plant populations is needed to maximize restoration success. For Araucaria araucana, a highly threatened iconic South American tree, this information is greatly needed to guide restoration and conservation efforts because this species occurs across a strong climate gradient. We grew seedlings from 12 populations of A. araucana across its range in Chile in a common garden to assess regional (coastal versus Andes mountain ranges) and population variation in key plant traits and relate this variation to environmental variables. We demonstrate that A. araucana is differentiated within regions and populations across its range in Chile by a suite of traits, particularly branch number and length (showing plant architectural differences) and needle width (showing leaf investment differences). We show that this variation is at least partly explained by climate and soil variables, with the most variation explained by differences between regions in temperature annual range. Thus, we recommend that restoration efforts focus on conserving genetic variation among and within regions and their populations and preventing the translocations of genotypes between coastal and Andes populations.

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Galbut virus infection minimally influences Drosophila melanogaster fitness traits in a strain and sex-dependent manner Galbut virus (family Partitiviridae) infects Drosophila melanogaster and can be transmitted vertically from infected mothers or infected fathers with near perfect efficiency. This form of super-Mendelian inheritance should drive infection to 100% prevalence, and indeed galbut virus is ubiquitous in wild D. melanogaster populations. But on average only about 60% of individual flies are infected. One possible explanation for this apparent paradox is that a subset of flies are resistant to infection. Although galbut virus infected flies seem healthy, infection may be sufficiently costly to drive selection for resistant hosts, thereby decreasing overall prevalence. To test this hypothesis, we quantified a variety of fitness-related traits in galbut virus infected flies from two lines from the Drosophila Genetic Reference Panel (DGRP). Galbut virus infected flies had slightly decreased average lifespan and total offspring production, but these decreases were mostly not statistically significant. Galbut virus DGRP-517 flies pupated and eclosed slightly faster than their uninfected counterparts. Some galbut virus infected flies exhibited altered sensitivity to viral, bacterial, and fungal pathogens. Galbut virus infection produced minimal changes to host mRNA levels as measured by RNA sequencing, consistent with minimal phenotypic changes. The microbiome composition of flies was not measurably perturbed by galbut virus infection. Differences in phenotype attributable to galbut virus infection varied as a function of fly sex and DGRP strain and were generally dwarfed by larger differences attributable to strain and sex. Thus, galbut virus infection does produce measurable phenotypic changes, with changes being minor, offsetting, and possibly net negative.

ImportanceVirology has largely focused on viruses that cause unmistakable phenotypic changes. But metagenomic surveys are revealing that persistent virus infections are extremely common, even in apparently healthy organisms. The extent to which these persistent viruses impact host fitness and evolution remains largely unclear. Here we study fitness impacts of a partitivirus named galbut virus that is ubiquitous in wild D. melanogaster populations. Despite efficient biparental vertical transmission, galbut virus is present in only just over half of wild flies. We quantified various fitness-related traits in galbut virus infected and uninfected fly lines and found that infection produced small but measurable changes in host phenotype that in aggregate may reduce fly fitness. Further studies that take advantage of this virus that naturally infects a premier model organism that is easy to study in the wild will shed further light on the persistent virus-host dynamics that may actually represent most viral infections.

sentences: The mutation effect reaction norm (MERN) highlights environmentally dependent mutation effects and genetic interactions Since the modern synthesis, the fitness effects of mutations and epistasis have been central yet provocative concepts in evolutionary and population genetics. Studies of how the interactions between parcels of genetic information can change as a function of environmental context have added a layer of complexity to these discussions. Here I introduce the "mutation effect reaction norm" (Mu-RN), a new instrument through which one can analyze the phenotypic consequences of mutations and interactions across environmental contexts. It embodies the fusion of measurements of genetic interactions with the reaction norm, a classic depiction of the performance of genotypes across environments. I demonstrate the utility of the Mu-RN through the signature of a "compensatory ratchet" mutation that undermines reverse evolution of antimicrobial resistance. More broadly, I argue that the mutation effect reaction norm may help us resolve the dynamism and unpredictability of evolution, with implications for theoretical biology, genetic modification technology, and public health.

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In lupus nephritis, specific in situ inflammatory states are associated with refractory disease and progression to renal failure In human lupus nephritis (LN), tubulointerstitial inflammation (TII) on biopsy predicts progression to end stage renal disease (ESRD). However, while approximately half of patients with moderate or severe TII develop ESRD, half do not. Therefore, we hypothesized that TII is heterogeneous, with distinct inflammatory states associated with different renal outcomes. We interrogated renal biopsies from LN longitudinal and cross-sectional cohorts using both conventional and highly multiplexed confocal microscopy. To accurately segment cells across whole biopsies, and to understand their spatial relationships, we developed unique computational pipelines by training and implementing several deep learning models and other computer vision techniques. Surprisingly, across biopsies, high B cell densities were strongly associated with protection from ESRD. In contrast, elevated CD4-T cell population densities, which included CD8, {gamma}{delta} and double negative (CD4-CD8-{delta}-, DN) T cells, were associated with both acute refractory renal failure and gradual progression to ESRD. Interestingly, lymphocytes and dendritic cells were organized into discrete clusters or neighborhoods that could be characterized by the enrichment for specific cell populations. B cells were often organized into large neighborhoods with CD4+ T cells including T follicular helper-like cells. In contrast, the CD4-T cell populations formed small cellular neighborhoods whose frequency predicted subsequent progression to ESRD. These data reveal that in LN, specific in situ inflammatory states are associated with refractory disease and progression to ESRD.

One sentence summaryUsing deep machine learning to analyze confocal microscopy data, we demonstrate that in lupus nephritis, CD4-T cell populations, including CD8+ and {gamma}{delta} T cells, organize into specific spatial neighborhoods that predict progression to renal failure.

sentences: A hierarchy of linguistic predictions during natural language comprehension Understanding spoken language requires transforming ambiguous acoustic streams into a hierarchy of representations, from phonemes to meaning. It has been suggested that the brain uses prediction to guide the interpretation of incoming input. However, the role of prediction in language processing remains disputed, with disagreement about both the ubiquity and representational nature of predictions. Here, we address both issues by analysing brain recordings of participants listening to audiobooks, and using a deep neural network (GPT-2) to precisely quantify contextual predictions. First, we establish that brain responses to words are modulated by ubiquitous, probabilistic predictions. Next, we disentangle model-based predictions into distinct dimensions, revealing dissociable signatures of syntactic, phonemic and semantic predictions. Finally, we show that high-level (word) predictions inform low-level (phoneme) predictions, supporting hierarchical predictive processing. Together, these results underscore the ubiquity of prediction in language processing, showing that the brain spontaneously predicts upcoming language at multiple levels of abstraction.

sentences: Guided Axon Outgrowth of Neurons by Real-Time Femtosecond Laser Penetration on a 4 μm Thick Thin-Glass Sheet Transplantation of scaffold-embedded guided neurons has been reported to increase neuronal regeneration following brain injury. However, precise axonal integration between host and transplant neurons to form functional synapses remains a major problem. This study aims to develop a real-time femtosecond (fs) laser penetration on a 4 m thick thin-glass sheet to promote guided axon outgrowth influenced by molecular gradients in a microfluidic device. The device enables the introduction of the guidance molecule (i.e., netrin-1), neuronal culture, and manipulation by fs laser. After fabricating multiple micro-holes on the thin-glass sheet using fs laser, netrin-1 gradients with radial concentrations are generated in the chamber, affecting axon outgrowth and guidance. A majority of axons (~92%) experiences guided outgrowth with positive angular changes towards netrin-1 gradients. These results demonstrate the capability of the precise and real-time manipulation system based on a fs laser and a microfluidic device to control the growth of neurons.

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Spontaneous Facet Joint Osteoarthritis in NFAT1-Mutant Mice ObjectivesAlthough rodent models of traumatically or chemically induced intervertebral facet joint osteoarthritis (FJOA) were previously described, the characteristics of spontaneous FJOA animal models have not been documented. This study aimed to identify the characteristics of a murine model of spontaneous FJOA and its underlying mechanisms.

MethodsThe lumbar facet joints of mutant mice carrying a disrupted NFAT1 (nuclear factor of activated T cells 1) allele and of wild-type control mice were examined by histochemistry, quantitative gene expression analysis, immunohistochemistry, and histomorphometry using a novel FJOA scoring system at 2, 6, 12, and 18 months of age. The reproducibility of the FJOA scoring system was analyzed by inter-observer and intra-observer variability tests. Tissue-specific histomorphometric and gene expression changes were statistically analyzed.

ResultsNFAT1-mutant facet joints displayed dysfunction of articular chondrocytes and synovial cells with aberrant gene and protein expression in cartilage and synovium as early as 2 months, followed by osteoarthritic structural changes such as articular surface fissuring and chondro-osteophyte formation at 6 months. Deeper cartilage lesions, synovitis, separation of cartilage from thickened subchondral bone, and tissue-specific molecular and cellular alterations in NFAT1-mutant facet joints became evident at 12 and 18 months. Osteoarthritic structural changes were not detected in wild-type facet joints at any ages, though age-related cartilage degeneration was observed at 18 months.

ConclusionsUsing NFAT1-mutant mice, this study has identified for the first time an animal model of spontaneous FJOA with age-dependent osteoarthritic characteristics, developed the first FJOA scoring system, and elucidated the molecular mechanisms of NFAT1 mutation-mediated FJOA.

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Deep Learning for MYC binding site recognition MotivationThe definition of the genome distribution of the Myc transcription factor is extremely important since it may help predict its transcriptional activity particularly in the context of cancer. Myc is among the most powerful oncogenes involved in the occurrence and development of more than 80% of different types of pediatric and adult cancers. Myc regulates thousands of genes which can be in part different, depending on the type of tissues and tumours. Myc distribution along the genome has been determined experimentally through chromatin immunoprecipitation This approach, although powerful, is very time consuming and cannot be routinely applied to tumours of individual patients. Thus, it becomes of paramount importance to develop in-silico tools that can effectively and rapidly predict its distribution on a given cell genome. New advanced computational tools (DeeperBind) can then be successfully employed to determine the function of Myc in a specific tumour, and may help to devise new directions and approaches to experiments first and personalized and more effective therapeutic treatments for a single patient later on.

ResultsThe use of DeeperBind with DeepRAM on Colab platform can effectively predict the binding sites for the MYC factor with an accuracy above 0.96 AUC, when trained with multiple cell lines. The analysis of the filters in DeeperBind trained models shows, besides the consensus sequence CACGTG classically associated to the MYC factor, also the other consensus sequences G/C box or TGGGA, respectively bound by the SP1 and MIZ-1 transcription factors, which are known to mediate the MYC repressive response. Overall, our findings suggest a stronger sinergy between the machine learning tools as DeeperBind and biological experiments, which may reduce the time consuming experiments by providing a direction to guide them.

sentences: Prior induction of cellular antiviral pathways limits frog virus 3 replication in two permissive Xenopus laevis skin epithelial-like cell lines Frog virus 3 (FV3) causes mortality in a range of amphibian species. Despite the importance of the skin epithelium as a first line of defence against FV3, the interaction between amphibian skin epithelial cells and FV3 remains largely uncharacterized. Here, we used newly established Xenopus laevis skin epithelial-like cell lines, Xela DS2 and Xela VS2, to study the susceptibility and permissiveness of frog skin epithelial cells to FV3, and the innate immune antiviral and proinflammatory gene regulatory responses of these cells to FV3. Both cell lines are susceptible and permissive to FV3, yet do not exhibit appreciable transcript levels of scavenger receptors recently demonstrated to be used by FV3 for cellular entry. Xela DS2 and Xela VS2 upregulate antiviral and proinflammatory cytokine transcripts in response to poly(I:C) but not to FV3 or UV-inactivated FV3. Poly(I:C) pretreatment limited FV3 replication and FV3-induced cytopathic effects in both cell lines. Thus, Xela DS2 and Xela VS2 can support FV3 propagation, represent in vitro systems to investigate antiviral responses of frog skin epithelial cells, and are novel tools for screening compounds that initiate effective antiviral programs to limit FV3 replication.

sentences: Gain control by sparse, ultra-slow glycinergic synapses SO_SCPLOWUMMARYC_SCPLOWRetinal ON starburst amacrine cells (SACs) play a critical role in computing stimulus direction, partly in service of image stabilization by optokinetic nystagmus. ON SAC responses are sculpted by rich GABAergic innervation, mostly from neighbouring SACs. Surprisingly, however, we find that glycinergic narrow field amacrine cells (NACs) serve as their dominant source of inhibition during sustained activity. Although NAC inputs constitute only [~]5% of inhibitory synapses to ON SACs, their distinct input patterns enable them to drive glycine inhibition during the both light increments and decrements. NAC-to-ON-SAC inhibition appears to be mediated by ultra-slow non-canonical glycine receptors containing the 4 subunit, which effectively summate during repetitive stimulation. Glycinergic inhibition strongly decreases the output gain of the SACs, ensuring that their direction-selective output is maintained over their operating range. These results reveal an unexpected role for glycinergic pathways and receptor kinetics in modulating direction selectivity in the retina.

sentences: Ptbp1 deletion does not induce glia-to-neuron conversion in adult mouse retina and brain. Direct reprogramming of glia into neurons is a potentially promising approach for the replacement of neurons lost to injury or neurodegenerative disorders. Knockdown of the polypyrimidine tract-binding protein Ptbp1 has been recently reported to induce efficient conversion of retinal Muller glia and brain astrocytes into functional neurons. However, genetic analysis of Ptbp1 function in adult glia has not been conducted. Here, we use a combination of genetic lineage tracing, scRNA-Seq, and electrophysiological analysis to show that specific deletion of Ptbp1 in adult retinal Muller glia and brain astrocytes does not lead to any detectable level of glia-to-neuron conversion. Only a few changes in gene expression are observed in glia following Ptbp1 deletion, and glial identity is maintained. These findings highlight the importance of using genetic manipulation and lineage tracing methods in studying cell type conversion.

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Truncation of IFT80 causes early embryonic loss in cattle Recessive alleles represent a risk in populations that have undergone bottleneck events. We present a comprehensive framework for identification and validation of these genetic defects, including haplotype-based detection, variant selection from sequence data, and validation using knockout embryos. Holstein haplotype 2 (HH2), which causes embryonic death, was used to demonstrate the approach. HH2 was identified using a deficiency-of-homozygotes approach and confirmed to negatively affect conception rate and stillbirths. Five carriers were present in a group of 183 sequenced Holstein bulls selected to maximize the coverage of unique haplotypes. Three variants concordant with haplotype calls were found in HH2: a high-priority frameshift mutation resulting in a deletion, and two low-priority variants (1 synonymous variant, 1 premature stop codon). The frameshift in intraflagellar protein 80 (IFT80) was confirmed in a separate group of Holsteins from the 1000 Bull Genomes Project that shared no animals with the discovery set. IFT80-null embryos were generated by truncating the IFT80 transcript at exon 2 or 11 using a CRISPR-Cas9 system. Abattoir-derived oocytes were fertilized in vitro and embryos were injected at the one-cell stage either with CRISPR-Cas9 complex (n=100) or Cas9 mRNA (control, n=100) before return to culture, and replicated 3 times. IFT80 is activated at the 8-cell stage, and IFT80-null embryos arrested at this stage of development, which is consistent with data from mouse hypomorphs and HH2 carrier-to-carrier matings. This frameshift in IFT80 on chromosome 1 at 107,172,615 bp (p.Leu381fs) disrupts WNT and hedgehog signaling, and is responsible for the death of homozygous embryos.

Significance StatementHolstein haplotype 2 is an embryonic lethal present in 1.21% of the US Holstein cattle population, and unrecognized carrier-to-carrier matings are responsible for >$2 million/year in additional breeding expenses. A high-impact frameshift mutation in exon 11 of intraflagellar protein 80 (IFT80) was identified as the putative causal variant. Biallelic IFT80 knockout embryos were produced in vitro and compared to wild-type embryos. IFT80-null embryos consistently arrested at the 8-cell stage of development. The IFT80 protein expressed in knockout embryos had substantially altered protein structure, resulting in a loss of functional domains. These results validate the putative causal mutation observed in Holsteins. This system is a good model for investigating possible causal variants that affect livestock fertility early in development.

sentences: Kronos scRT: a uniform framework for single-cell replication timing analysis Mammalian genomes are replicated in a cell-type specific order and in coordination with transcription and chromatin organization. Although the field of replication is also entering the single-cell era, current studies require cell sorting, individual cell processing and have yielded a limited number (<100) of cells. Here, we have developed Kronos scRT (https://github.com/CL-CHEN-Lab/Kronos_scRT), a software for single-cell Replication Timing (scRT) analysis. Kronos scRT does not require a specific platform nor cell sorting, allowing the investigation of large datasets obtained from asynchronous cells. Analysis of published available data and droplet-based scWGS data generated in the current study, allows exploitation of scRT data from thousands of cells for different mouse and human cell lines. Our results demonstrate that, although most cells replicate within a close timing range for a given genomic region, replication can also occur stochastically throughout S phase. Altogether, Kronos scRT allows investigating the RT program at a single-cell resolution for both homogeneous and heterogeneous cell populations in a fast and comprehensive manner.

sentences: What canonical online and offline measures of statistical learning can and cannot tell us Statistical learning (SL) allows individuals to rapidly detect regularities in the sensory environment. We replicated previous findings showing that adult participants become sensitive to the implicit structure in a continuous speech stream of repeating tri-syllabic pseudowords within minutes, as measured by standard tests in the SL literature: a target detection task and a 2AFC word recognition task. Consistent with previous findings, we found only a weak correlation between these two measures of learning, leading us to question whether there is overlap between the information captured by these two tasks. Representational similarity analysis on reaction times measured during the target detection task revealed that reaction time data reflect sensitivity to transitional probability, triplet position, word grouping, and duplet pairings of syllables. However, individual performance on the word recognition task was not predicted by similarity measures derived for any of these four features. We conclude that online detection tasks provide richer and multi-faceted information about the SL process, as compared with 2AFC recognition tasks, and may be preferable for gaining insight into the dynamic aspects of SL.

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Semi-automated analysis of an optical ATP indicator in neurons SignificanceThe firefly enzyme luciferase has been used in a wide range of biological assays, including bioluminescence imaging of ATP. The biosensor Syn-ATP utilizes subcellular targeting of luciferase to nerve terminals for optical measurement of ATP in this compartment. Manual analysis of Syn-ATP signals is challenging due to signal heterogeneity and cellular motion in long imaging sessions. Here, we have leveraged machine learning tools to develop a method for analysis of bioluminescence images.

AimOur goal was to create a semi-automated pipeline for analysis of bioluminescence imaging to improve measurements of ATP content in nerve terminals.

ApproachWe developed an image analysis pipeline that applies machine learning toolkits to distinguish neurons from background signals, and excludes neural cell bodies, while also incorporating user input.

ResultsSide-by-side comparison of manual and semi-automated image analysis demonstrated that the latter improves precision and accuracy of ATP measurements.

ConclusionsOur method streamlines data analysis and reduces user-introduced bias, thus enhancing the reproducibility and reliability of quantitative ATP imaging in nerve terminals.

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Microbiota analysis of rural and urban surface waters and sediments in Bangladesh identifies human waste as driver of antibiotic resistance In many low- and middle-income countries antibiotic resistant bacteria spread in the environment due to inadequate treatment of wastewater and the poorly regulated use of antibiotics in agri- and aquaculture. Here we characterised the abundance and diversity of antibiotic-resistant bacteria and antibiotic resistance genes in surface waters and sediments in Bangladesh through quantitative culture of Extended-Spectrum Beta-Lactamase (ESBL)-producing coliforms and shotgun metagenomics. Samples were collected from highly urbanised settings (n = 7), from rural ponds with a history of aquaculture-related antibiotic use (n = 11) and from rural ponds with no history of antibiotic use (n = 6). ESBL-producing coliforms were found to be more prevalent in urban samples than in rural samples. Shotgun sequencing showed that sediment samples were dominated by the phylum Proteobacteria (on average 73.8% of assigned reads), while in the water samples Cyanobacteria (on average 60.9% of assigned reads) were the predominant phylum. Antibiotic resistance genes were detected in all samples, but their abundance varied 1,525-fold between sites, with the highest levels of antibiotic resistance genes being present in urban surface water samples. We identified an IncQ1 sulphonamide resistance plasmid ancestral to the widely studied RSF1010 in one of the urban water samples. The abundance of antibiotic resistance genes was significantly correlated (R2 = 0.73; P = 8.9 x 10-15) with the abundance of bacteria originating from the human gut, which suggests that the release of untreated sewage is a driver for the spread of environmental antibiotic resistance genes in Bangladesh, particularly in highly urbanised settings.

ImportanceLow- and middle-income countries (LMICs) have higher burdens of multidrug-resistant infections than high-income countries and there is thus an urgent need to elucidate the drivers of the spread of antibiotic-resistant bacteria in LMICs. Here we study the diversity and abundance of antibiotic resistance genes in surface water and sediments from rural and urban settings in Bangladesh. We found that urban surface waters are particularly rich in antibiotic resistance genes, with a higher number of them associated with plasmids indicating that they are more likely to spread horizontally. The abundance of antibiotic resistance genes was strongly correlated with the abundance of bacteria that originate from the human gut, suggesting that uncontrolled release of human waste is a major driver for the spread of antibiotic resistance in the urban environment. Improvements in sanitation in LMICs may thus be a key intervention to reduce the dissemination of antibiotic resistant bacteria.

sentences: Significance of linkage disequilibrium and epistasis on the genetic variances and covariance between relatives in non-inbred and inbred populations Because no feasible theoretical model can depict the complexity of phenotype development from a genotype, the joint significance of linkage disequilibrium (LD), epistasis, and inbreeding on the genetic variances remains unclear. The objective of this investigation was to assess the impact of LD and epistasis on the genetic variances and covariances between relatives in non-inbred and inbred populations using simulated data. We provided the theoretical background and simulated grain yield assuming 400 genes in 10 chromosomes of 200 and 50 cM. We generated five populations with low to high LD levels, assuming 10 generations of random cross and selfing. The analysis of the parametric LD in the populations shows that the LD level depends mainly on the gene density. The significance of the LD level is impressive on the magnitude of the genotypic and additive variances, which is the most important component of the genotypic variance, regardless of the LD level and the degree of inbreeding. Regardless of the type of epistasis, the ratio epistatic variance/genotypic variance is proportional to the percentage of the epistatic genes. For the epistatic variances, except for duplicate epistasis and dominant and recessive epistasis, with 100% of epistatic genes, their magnitudes are much lower than the magnitude of the additive variance. The additive x additive variance is the most important epistatic variance. Our results explain why LD for genes and relationship information are key factors affecting the genomic prediction accuracy of complex traits and the efficacy of association studies.

sentences: Quantifying full-length circular RNAs in cancer Circular RNAs (circRNAs) are abundantly expressed in cancer. Their resistance to exonucleases enables them to have potentially stable interactions with different types of biomolecules. Alternative splicing can create different circRNA isoforms that have different sequences and unequal interaction potentials. The study of circRNA function thus requires knowledge of complete circRNA sequences. Here we describe psirc, a method that can identify full-length circRNA isoforms and quantify their expression levels from RNA sequencing data. We confirm the effectiveness and computational efficiency of psirc using both simulated and actual experimental data. Applying psirc on transcriptome profiles from nasopharyngeal carcinoma and normal nasopharynx samples, we discover and validate circRNA isoforms differentially expressed between the two groups. Compared to the assumed circular isoforms derived from linear transcript annotations, some of the alternatively spliced circular isoforms have 100 times higher expression and contain substantially fewer microRNA response elements, demonstrating the importance of quantifying full-length circRNA isoforms.

sentences: Effects of low intensity pulsed ultrasound stimulation on metabolic lipolysis of adipocytes Obesity is closely related to several metabolic diseases along with abnormal increase in fat cells. Reducing size and number of fat cells, a procedure known as lipolysis, may be used to prevent obesity as a potential therapy, which also requires fundamental understanding of the mechanisms of lipolysis at molecular level upon different types of stimulations. Here, we used low intensity pulsed ultrasound (LIPUS) stimulation to investigate underlying mechanisms of the activation of lipolysis and autophagy related genes and signaling pathways of adipocytes differentiated from 3T3-L1 cells. LIPUS with the center frequency of 2 MHz was applied for 10 minutes per day for three days. After LIPUS stimulation, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot were used to determine the regulation of lipolytic factors such as adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase (MGL). At RNA level, all three factors were upregulated while only MGL was upregulated at protein level, which presents slightly different activation pattern of lipolysis compared to widely used chemical stimulation. These results demonstrate that LIPUS stimulation can promote the lipolytic capacity of adipocytes in the differentiated state. The differences between transcriptional genes and metabolites were analyzed by transcript analysis and metabolomic profiling experiments. Cellular RNA-sequencing (RNA-Seq) showed an increase in lipolysis and immune-related genes and autophagy related genes after LIPUS stimulation. This study may provide an important experimental basis for the clinical applications and a fundamental understanding of the mechanisms of lipolysis using LIPUS stimulation.

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The effects of an 8-week mindful eating intervention on anticipatory reward responses in striatum and midbrain Obesity is a highly prevalent disease, usually resulting from chronic overeating. Accumulating evidence suggests that increased neural responses during the anticipation of high-calorie food play an important role in overeating. A promising method for counteracting enhanced food anticipation in overeating might be mindfulness-based interventions (MBIs). However, the neural mechanisms by which MBIs can affect food reward anticipation are unclear. In this randomized, actively controlled study, the primary objective was to investigate the effect of an 8-week mindful eating intervention on reward anticipation. On the neural level, we hypothesized that mindful eating would decrease striatal reward anticipation responses. Additionally, responses in the midbrain - from which the reward pathways originate - were explored. Using functional magnetic resonance imaging (fMRI), we tested 58 healthy participants with a wide body mass index range (BMI: 19-35 kg/m2), motivated to change their eating behavior. During scanning they performed an incentive delay task, measuring neural reward anticipation responses to caloric and monetary cues before and after 8 weeks of mindful eating or educational cooking (active control). Compared with the educational cooking intervention, mindful eating affected neural reward anticipation responses, with relatively reduced caloric versus monetary reward responses. This effect was, however, not seen in the striatum, but only in the midbrain. The secondary objective was to assess temporary and long-lasting (one year follow-up) intervention effects on self-reported eating behavior and anthropometric measures (BMI, waist circumference, waist-to-hip-ratio (WHR)). We did not observe effects of the mindful eating intervention on eating behavior. Instead, the control intervention showed temporary beneficial effects on BMI, waist circumference, and diet quality, but not on WHR or self-reported eating behavior, as well as long-lasting increases in knowledge about healthy eating. These results suggest that an 8-week mindful eating intervention may have decreased the relative salience of food cues by affecting midbrain but not striatal reward responses. However, these exploratory results should be verified in confirmatory research.

The primary and secondary objectives of the study were registered in the Dutch Trial Register (NTR): NL4923 (NTR5025).

sentences: Cellular reprogramming with ATOH1, GFI1, and POU4F3 implicate epigenetic changes and cell-cell signaling as obstacles to hair cell regeneration in mature mammals Reprogramming of the cochlea with hair cell-specific transcription factors such as ATOH1 has been proposed as a potential therapeutic strategy for hearing loss. ATOH1 expression in the developing cochlea can efficiently induce hair cell regeneration but the efficiency of hair cell reprogramming declines rapidly as the cochlea matures. We developed Cre-inducible mice to compare hair cell reprogramming with ATOH1 alone or in combination with two other hair cell transcription factors, GFI1 and POU4F3. In newborn mice, all transcription factor combinations tested produced large numbers of cells with the morphology of hair cells and rudimentary mechanotransduction properties. However, one week later, only a combination of ATOH1, GFI1 and POU4F3 could reprogram non-sensory cells of the cochlea to a hair cell fate, and these new cells were less mature than cells generated by reprogramming one week earlier. We used scRNA-seq and combined scRNA-seq and ATAC-seq to suggest at least two impediments to hair cell reprogramming in older animals. First, hair cell gene loci become less epigenetically accessible in non-sensory cells of the cochlea with increasing age. Second, signaling from hair cells to supporting cells, including Notch signaling, can prevent reprogramming of many supporting cells to hair cells, even with three hair cell transcription factors. Our results shed light on the molecular barriers that must be overcome to promote hair cell regeneration in the adult cochlea.

sentences: Population modelling insights of extinct environments: the case of the Kem Kem palaeocommunity The Kem Kem beds are well-known palaeontological deposits. Among the species that lived there, there are some large theropods, such as Deltadromeus agilis, Carcharodontosaurus saharicus, and Spinosaurus aegyptiacus. It is possible that these large predators were facultative scavengers, and they could compete for carrion. In the present paper, I simulate a small community module of this environment, consisting of Carrion, Fishes, Spinosaurus, and a functional group composed of large terrestrial Theropods. I assume that these top predators feed on carrion, but they also have exclusive food sources. I show that these exclusive food sources could have assured the possibility of coexistence, and in their absence, one top predator could be locally extinct.

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Modeling and Characterization of Inter-Individual Variability in CD8 T Cell Responses in Mice To develop vaccines it is mandatory yet challenging to account for inter-individual variability during immune responses. Even in laboratory mice, T cell responses of single individuals exhibit a high heterogeneity that may come from genetic backgrounds, intra-specific processes (e.g. antigen-processing and presentation) and immunization protocols.

To account for inter-individual variability in CD8 T cell responses in mice, we propose a dynamical model coupled to a statistical, nonlinear mixed effects model. Average and individual dynamics during a CD8 T cell response are characterized in different immunization contexts (vaccinia virus and tumor). On one hand, we identify biological processes that generate inter-individual variability (activation rate of naive cells, the mortality rate of effector cells, and dynamics of the immunogen). On the other hand, introducing categorical covariates to analyze two different immunization regimens, we highlight the steps of the response impacted by immunogens (priming, differentiation of naive cells, expansion of effector cells and generation of memory cells). The robustness of the model is assessed by confrontation to new experimental data.

Our approach allows to investigate immune responses in various immunization contexts, when measurements are scarce or missing, and contributes to a better understanding of inter-individual variability in CD8 T cell immune responses.

sentences: Quantifying the Morphology and Mechanisms of Cancer Progression in 3D in-vitro environments: Integrating Experiments, Multiscale Models, and Spatial Validation Mathematical models of cancer growth have become increasingly more accurate both in the space and time domains. However, the limited amount of data typically available has resulted in a larger number of qualitative rather than quantitative studies. In the present study, we provide an integrated experimental-computational framework for the quantification of the morphological characteristics and the mechanistic modelling of cancer progression in 3D environments. The proposed framework allows for the calibration of multiscale, spatiotemporal models of cancer growth using state-of-the-art 3D cell culture data, and their validation based on the resulting experimental morphological patterns. Its implementation enabled us to pursue two goals; first, the quantitative description of the morphology of cancer progression in 3D cultures, and second, the relation of tumour morphology with underlying biophysical mechanisms that govern cancer growth and migration. We applied this framework to the study of the spatiotemporal progression of Triple Negative Breast Cancer cells cultured in Matrigel scaffolds, and validated the hypothesis of chemotactic migration using a multiscale Keller-Segel model. The results revealed transient, non-random spatial distributions of cancer cells that consist of clustered, and dispersion patterns. The proposed model was able to describe the general characteristics of the experimental observations and suggests that cancer cells exhibited chemotactic migration and accumulation, as well as random motion during the examined time period of development. To our knowledge, this is the first time that a multiscale model is used to quantify the relationship between the spatial patterns and the underlying mechanisms of cancer growth in 3D environments.

sentences: Affinity maturation for an optimal balance between long-term immune coverage and short-term resource constraints In order to target threatening pathogens, the adaptive immune system performs a continuous reorganization of its lymphocyte repertoire. Following an immune challenge, the B cell repertoire can evolve cells of increased specificity for the encountered strain. This process of affinity maturation generates a memory pool whose diversity and size remain difficult to predict. We assume that the immune system follows a strategy that maximizes the long-term immune coverage and minimizes the short-term metabolic costs associated with affinity maturation. This strategy is defined as an optimal decision process on a finite dimensional phenotypic space, where a pre-existing population of naive cells is sequentially challenged with a neutrally evolving strain. We unveil a trade-off between immune protection against future strains and the necessary reorganization of the repertoire. This plasticity of the repertoire drives the emergence of distinct regimes for the size and diversity of the memory pool, depending on the density of naive cells and on the mutation rate of the strain. The model predicts power-law distributions of clonotype sizes observed in data, and rationalizes antigenic imprinting as a strategy to minimize metabolic costs while keeping good immune protection against future strains.

sentences: An immunogenic model of KRAS-mutant lung cancer for study of targeted therapy and immunotherapy combinations Mutations in oncogenes such as KRAS and EGFR cause a high proportion of lung cancers. Drugs targeting these proteins cause tumour regression but ultimately fail to cure these cancers, leading to intense interest in how best to combine them with other treatments, such as immunotherapies. However, preclinical systems for studying the interaction of lung tumours with the host immune system are inadequate, in part due to the low tumour mutational burden in genetically engineered mouse models. Here we set out to develop mouse models of mutant KRAS-driven lung cancer with an elevated tumour mutational burden by expressing the human DNA cytosine deaminase, APOBEC3B, to mimic the mutational signature seen in human lung cancer. This failed to substantially increase clonal tumour mutational burden and autochthonous tumours remained refractory to immunotherapy. However, by establishing clonal cell lines from these tumours we generated an immunogenic syngeneic transplantation model of KRAS mutant lung adenocarcinoma that was sensitive to immunotherapy. Unexpectedly, we found that anti-tumour immune responses were not directed against neoantigens but instead targeted derepressed endogenous retroviral antigens. The ability of KRASG12C inhibitors to cause regression of KRASG12C-expressing versions of these tumours was markedly potentiated by the adaptive immune system, providing a unique opportunity for the study of combinations of targeted and immunotherapies in immune-hot lung cancer.

sentences: A feedback loop between heterochromatin and the nucleopore complex controls germ-cell to oocyte transition during Drosophila oogenesis Germ cells differentiate into oocytes that become totipotent upon fertilization. How the highly specialized oocyte acquires this distinct cell fate is poorly understood. During Drosophila oogenesis, H3K9me3 histone methyltransferase SETDB1 translocates from the cytoplasm to the nucleus of germ cells concurrent with oocyte specification. Here, we discovered that nuclear SETDB1 is required to silence a cohort of differentiation-promoting genes by mediating their heterochromatinization. Intriguingly, SETDB1 is also required for the upregulation of 18 of the ~30 nucleoporins (Nups) that comprise the nucleopore complex (NPC). NPCs in turn anchor SETDB1-dependent heterochromatin at the nuclear periphery to maintain H3K9me3 and gene silencing in the egg chambers. Aberrant gene expression due to loss of SETDB1 or Nups results in loss of oocyte identity, cell death and sterility. Thus, a feedback loop between heterochromatin and NPCs promotes transcriptional reprogramming at the onset of oocyte specification that is critical to establish oocyte identity.

sentences: Heterogeneous recruitment abilities to RNA polymerases generate nonlinear scaling of gene expression level with cell volume While most genes expression levels are proportional to cell volumes, some genes exhibit nonlinear scaling between their expression levels and cell volume. Therefore, their mRNA and protein concentrations change as the cell volume increases, which often have crucial biological functions such as cell-cycle regulation. However, the biophysical mechanism underlying the nonlinear scaling between gene expression and cell volume is still unclear. In this work, we show that the nonlinear scaling is a direct consequence of the heterogeneous recruitment abilities of promoters to RNA polymerases based on a gene expression model at the whole-cell level. Those genes with weaker (stronger) recruitment abilities than the average ability spontaneously exhibit superlinear (sublinear) scaling with cell volume. Analysis of the promoter sequences and the nonlinear scaling of Saccharomyces cerevisiaes mRNA levels shows that motifs associated with transcription regulation are indeed enriched in genes exhibiting nonlinear scaling, in concert with our model.

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Differential timing for glucose assimilation in Prochlorococcus and coexistent microbial populations at the North Pacific Subtropical Gyre The marine cyanobacterium Prochlorococcus can utilize glucose as a source of carbon. However, the relative importance of inorganic and organic carbon assimilation and the timing of glucose assimilation are still poorly understood in these numerically dominant cyanobacteria. Here we investigated whole microbial community and group-specific primary production and glucose assimilation, using incubations with radioisotopes combined with flow cytometry cell sorting. We also studied changes in the microbial community structure in response to glucose enrichments and analyzed the transcription of Prochlorocccus genes involved in carbon metabolism and photosynthesis.

Our results showed a circadian rhythm for glucose assimilation in Prochlorococcus, with maximum assimilation during the midday and minimum at midnight, which was different compared with that of the total microbial community. This suggests that rhythms in glucose assimilation have been adapted in Prochlorococcus to couple the active transport to photosynthetic light reactions producing energy, and possibly to avoid competition from the rest of the microbial community. High-light Prochlorococcus strains showed most transcriptional changes upon glucose enrichment. Pathways involved in glucose metabolism as the pentose phosphate, the Entner-Dudoroff, glycolysis, respiration and glucose transport showed an increase in the transcript level. A few genes of the low-light strains showed opposite changes, suggesting that glucose assimilation has been subjected to diversification along the Prochlorococcus evolution.

sentences: Metabolic control of adult neural stem cell self-renewal by the mitochondrial protease YME1L The transition between quiescence and activation in neural stem and progenitor cells (NSPCs) is coupled to reversible changes in energy metabolism with key implications for life-long NSPC self-renewal and neurogenesis. How this metabolic plasticity is ensured between NSPC activity states is unclear. We found that a state-dependent rewiring of the mitochondrial proteome by the peptidase YME1L is required to preserve NSPC self-renewal in the adult brain. YME1L-mediated proteome rewiring regulates the rate of fatty acid oxidation (FAO) for replenishing Krebs cycle intermediates and dNTP precursors, which are required to sustain NSPC amplification. Yme1l deletion irreversibly shifts the metabolic profile of NSPCs away from a FAO-dependent state resulting in defective self-renewal, premature differentiation and NSPC pool depletion. Our results disclose an important role for YME1L in coordinating the switch between metabolic states of NSPCs and suggest that NSPC fate is regulated by compartmentalized changes in protein network dynamics.

sentences: Image processing and genome-wide association studies in sunflower identify loci associated with seed-coat characteristics Sunflower seeds (technically achenes) are characterized by a wide spectrum of sizes, shapes, and colors. These traits are genetically correlated with the branching plant architecture loci, which were introgressed into restorer lines to facilitate efficient hybrid production. To break this genetic correlation between branching and seed traits, high resolution mapping of the genes that regulate seed traits is necessary. Recent progress in genomics permits acquisition of comprehensive genotyping data for a large diversity panel, yet a major constraint for exploring the genetic basis of important phenotypes across large diversity panels is the ability to screen and characterize them efficiently. Here, we implement a cost-effective image analysis pipeline to phenotype seed characteristics in a large sunflower diversity panel comprised of 287 individuals that represents most of the genetic variation in cultivated sunflower. A genome-wide association analysis was performed for seed-coat size and shape traits and significant signals were identified around genes regulating phytohormone activity. In addition, significant seed-coat color QTLs were identified and candidate genes that effect pigmentation were detected including a phytomelanin regulating gene on chromosome 17. Finally, QTLs associated with the seed-coat striped pattern were identified and phytohormone regulating candidate genes were detected. The implementation of image analysis phenotyping for GWAS allowed efficient screening of a large diversity panel and identification of valuable genetic factors effecting seed characteristics at the finest resolution to date.

sentences: Subcellular dynamics and functional activity of the cleaved Na+ channel β1 subunit intracellular domain The voltage-gated Na+ channel {beta}1 subunit, encoded by SCN1B, regulates cell surface expression and gating of  subunits, and participates in cell adhesion. {beta}1 is cleaved by /{beta} and {gamma}-secretases, releasing an extracellular domain and intracellular domain (ICD) respectively. Abnormal SCN1B expression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on {beta}1 function in breast cancer cells. Using a series of GFP-tagged {beta}1 constructs, we show that {beta}1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal {beta}1-GFP levels occurred following {gamma}-secretase inhibition, implicating endosomes, and/or the preceding plasma membrane, as important sites for secretase processing. Using live-cell imaging, we report {beta}1-ICD-GFP accumulation in the nucleus. Furthermore, {beta}1-GFP and {beta}1ICD-GFP both increased Na+ current, whereas {beta}1STOP-GFP, which lacks the ICD, did not, thus highlighting that the {beta}1-ICD was necessary and sufficient to increase Na+ current measured at the plasma membrane. Importantly, although the endogenous Na+ current expressed in MDA-MB-231 cells is TTX-resistant (carried by Nav1.5), the Na+ current increased by {beta}1-GFP or {beta}1ICD-GFP was TTX-sensitive. Taken together, this work suggests that the {beta}1-ICD is a critical regulator of  subunit function. Our data further support the notion that {gamma}-secretase may play a key role in regulating {beta}1 function in breast cancer cells. This work thus highlights proteolytic processing of {beta}1 by secretase cleavage to be a relevant mechanism in diseases associated with abnormal {beta}1 function.

sentences: Examining the effectiveness of blanket curtailment strategies in reducing bat fatalities at terrestrial wind farms in North America Blanket curtailment of turbine operations during low wind conditions has become an accepted operational minimization tactic to reduce bat mortality at terrestrial wind facilities. Site-specific studies have demonstrated that operational curtailment effectively reduces impacts, but the exact nature of the relationship between increased cut-in speed and fatality reduction in bats remains unclear. To evaluate the efficacy of differing blanket curtailment regimes in reducing bat fatality, we examined data from turbine curtailment experiments in the United States and Canada in a meta-analysis framework. We tested multiple statistical models to explore possible linear and non-linear relationships between turbine cut-in speed and bat fatality reduction while controlling for control cut-in speed. Because the overall sample size for this meta-analysis was small (n = 36 control-treatment studies from 16 field sites from the American Wind Wildlife Information Center and a recent review), we conducted a power analysis to assess the number of control-impact curtailment studies that would be needed to understand the relationship between fatality rate and change in cut-in speed under different fatality reduction scenarios. We also identified the characteristics of individual field studies that may influence their power to detect fatality reduction due to curtailment. Using a response ratio approach, we found any curtailment strategy reduced fatality rates by 56% for studies included in this analysis (p < 0.001). However, we did not find strong evidence for linear (p =0 0.07) or non-linear (p > 0.11) associations between increasing cut-in speeds and fatality reduction. The power analyses showed that the power to detect effects in the meta-analysis was low if fatality reductions were less than 50%. Synthesizing across all analyses, we need more well-designed curtailment studies to determine the effect of increasing curtailment speed and the effect size is likely of a magnitude that we had limited power to detect.

sentences: Predicting behavior through dynamic modes in resting-state fMRI data Dynamic properties of resting-state functional connectivity (FC) provide rich information on brainbehavior relationships. Dynamic mode decomposition (DMD) has been used as a method to characterize FC dynamics. However, it remains unclear whether dynamic modes (DMs), spatial-temporal coherent patterns computed by DMD, provide information about individual behavioral differences. This study established a methodological approach to predict individual differences in behavior using DMs. Furthermore, we investigated the contribution of DMs within each of seven specific frequency bands (0-0.1,...,0.6-0.7 Hz) for prediction. To validate our approach, we confirmed whether each of 59 behavioral measures could be predicted by performing multivariate pattern analysis on a gram matrix, which was created using subject-specific DMs computed from resting-state functional magnetic resonance imaging (rs-fMRI) data of individuals. The prediction was successful, and DMD outperformed temporal independent component analysis, a conventional data decomposition method for extracting spatial activity patterns. Most of the behavioral measures that showed significant prediction accuracies in a permutation test were cognitive-behavioral measures. Our results suggested that DMs within frequency bands <0.2 Hz primarily contributed to prediction. In addition, we found that DMs <0.2 Hz had spatial structures similar to several common resting-state networks. We demonstrated the effectiveness of DMs, indicating that DMD is a key approach for extracting spatiotemporal features from rs-fMRI data.

sentences: Convergent evolution of polyploid genomes from across the eukaryotic tree of life By modeling the homoeologous gene losses that occurred in fifty genomes deriving from ten distinct polyploidy events, we show that the evolutionary forces acting on polyploids are remarkably similar, regardless of whether they occur in flowering plants, ciliates, fishes or yeasts. The models suggest these events were nearly all allopolyploidies, with two distinct progenitors contributing to the modern species. We show that many of the events show a relative rate of duplicate gene loss prior to the first post-polyploidy speciation that is significantly higher than in later phases of their evolution. The relatively low selective constraint seen for the single-copy genes these losses produced lead us to suggest that most of the purely selectively neutral duplicate gene losses occur in the immediate post-polyploid period. We also find ongoing and extensive reciprocal gene losses (RGL; alternative losses of duplicated ancestral genes) between these genomes. With the exception of a handful of closely related taxa, all of these polyploid organisms are separated from each other by tens to thousands of reciprocal gene losses. As a result, it is very unlikely that viable diploid hybrid species could form between these taxa, since matings between such hybrids would tend to produce offspring lacking essential genes. It is therefore possible that the relatively high frequency of recurrent polyploidies in some lineages may be due to the ability of new polyploidies to bypass RGL barriers.

sentences: Deciphering the differential physiological and molecular requirements for conidial anastomosis tube fusion and germ tube formation in Colletotrichum gloeosporioides. The conidia of a hemibiotrophic fungus Colletotrichum gloeosporioides can conventionally form germ tube (GT) and develop in to a fungal colony, while under certain conditions, they tend to get connected with each other through conidial anastomosis tube (CAT) so as to share the nutrients. CAT fusion is believed to be responsible for generation of genetic variations in few asexual fungi, which appears problematic for effective fungal disease management. The physiological and molecular mechanism underlying the GT versus CAT formation remained unexplored. In the present study, we have deciphered the decision switch responsible for GT formation versus CAT fusion in C. gloeosporioides. GT formation occurred at high frequency in the presence of nutrients, while CAT fusion was found to be higher in absence of nutrients. Younger conidia were found to form GT efficiently, whilst older conidia preferentially formed CAT. Whole transcriptome analysis of GT and CAT fusion revealed differential molecular requirements for these two processes. We identified 11050 and 9786 differentially expressed genes (DEGs) in GT and CAT, respectively. A total 1567 effector candidates were identified, of them 103 and 101 were uniquely secreted during GT and CAT formation respectively. Genes coding for cell wall degrading enzymes, germination, hyphal growth, host-fungus interaction and virulence were up-regulated during GT formation. Whilst, genes involved in stress response, cell wall remodelling, membrane transport, cytoskeleton, cell cycle, and cell rescue were highly up-regulated during CAT fusion. To conclude, the GT and CAT fusion were found to be mutually exclusive processes, requiring differential physiological conditions and sets of DEGs in C. gloeosporioides. This will help to understand the basic CAT biology in the genus Colletotrichum.

sentences: Dynamic and selective engrams emerge with memory consolidation Episodic memories are encoded by sparse populations of neurons activated during an experience.1 These neural ensembles constitute memory engrams that are both necessary and sufficient for inducing recall even long after memory acquisition.2 This suggests that following encoding, engrams are stabilized to reliably support memory retrieval. However, little is known about the temporal evolution of engrams over the course of memory consolidation or how it impacts mnemonic properties. Here we employed computational and experimental approaches to examine how the composition and selectivity of engrams change with memory consolidation. We modeled engram cells using a spiking recurrent neural network that yielded three testable predictions: memories transition from unselective to selective as neurons are removed from and added to the engram, inhibitory activity during recall is essential for memory selectivity, and inhibitory synaptic plasticity during memory consolidation is critical for engrams to become selective. Using the Cal-Light system to tag activated neurons in vivo with high spatiotemporal precision3 as well as optogenetic and chemogenetic techniques, we conducted contextual fear conditioning experiments that supported each of our models predictions. Our results reveal that engrams are dynamic even within hours of memory consolidation and that changes in engram composition mediated by inhibitory synaptic plasticity are crucial for the emergence of memory selectivity. These findings challenge classical theories of stable memory traces and point to a close link between engram state and memory expression.

sentences: Epigenetic clock and methylation studies in dogs DNA methylation profiles have been used to develop biomarkers of aging known as epigenetic clocks, which predict chronological age with remarkable accuracy and show promise for inferring health status as an indicator of biological age. Epigenetic clocks were first built to monitor human aging but the principles underpinning them appear to be evolutionarily conserved, as they have now been successfully developed for over 120 mammalian species. Here we describe reliable and highly accurate epigenetic clocks shown to apply to 51 domestic dog breeds. The methylation profiles were generated using a custom array with DNA sequences that are conserved across all mammalian species (HorvathMammalMethylChip40). Canine epigenetic clocks were constructed to estimate age and also predict mortality risk by estimating average time-to-death. We also present two highly accurate human-dog dual species epigenetic clocks (R=0.97), which may facilitate the ready translation from canine to human use (or vice versa) of anti-aging treatments being developed for longevity and preventive medicine. Finally, epigenome-wide association studies herein reveal individual methylation sites that may underlie the inverse relationship between breed weight and lifespan. Overall, we describe robust biomarkers to measure aging and potentially health status in canines.

sentences: Efficient tagging and purification of endogenous proteins for structural studies by single particle cryo-EM A major bottleneck in structural biology is producing biologically relevant samples at sufficient quantities. This is particularly true for large protein assemblies where conventional techniques of gene overexpression require substantial optimization, hampering structural studies and drug development efforts. Here we describe a method combining CRISPR/Cas gene editing and fluorescence cell sorting to rapidly tag and purify endogenous human proteins from cell lines, enabling structural analysis of native proteins that are properly folded and assembled. We applied this approach to study the human proteasome from HEK cells and rapidly determined structures of major proteasomal complexes. Structures of the PA28-20S complex reveal the native subunit stoichiometry of PA28 and a distinct functional state of the complex. The efficient strategy for tagging and extracting endogenous proteins described here will enable the structural study of many challenging targets and provide more biologically relevant samples for research and therapeutic development.

sentences: Pro-regenerative Extracellular Matrix Hydrogel Prevents and Mitigates Pathological Alterations of Pelvic Muscles Following Birth Injury Pelvic floor disorders, which include pelvic organ prolapse, and urinary and fecal incontinence, affect millions of women globally and represent a major public health concern. Pelvic floor muscle (PFM) dysfunction has been identified as one of the leading risk factors for the development of these morbid conditions. Even though childbirth, specifically vaginal delivery, has been long recognized as the most important potentially modifiable risk factor for PFM injury, the precise mechanisms of PFM dysfunction following childbirth remain elusive. In this study we demonstrate that PFMs undergo atrophy and severe fibrosis in parous women with symptomatic pelvic organ prolapse compared to age-matched nulliparous cadaveric donors without history of pelvic floor disorders. These pathological alterations are recapitulated in the pre-clinical rat model of simulated birth injury. The transcriptional signature of PFMs post-injury demonstrates a sustained inflammatory response, impairment in muscle anabolism, and persistent expression of extracellular matrix (ECM) remodeling genes. Next, we evaluated the administration of acellular injectable skeletal muscle extracellular matrix hydrogel for the prevention and mitigation of these pathological alterations. Treatment of PFMs with the biomaterial either at the time of birth injury or 4 weeks post-injury reduced muscle atrophy and mitigated fibrotic degeneration. By evaluating gene expression, we demonstrate that these changes are mainly driven by the hydrogel-induced modulation of the immune response and intramuscular fibrosis, as well as enhancement of the endogenous myogenesis. This work furthers our understanding of PFM birth injury and demonstrates proof-of-concept for a new pragmatic pro-regenerative biomaterial approach for treating injured PFMs.

sentences: Scalable and Automated CRISPR-Based Strain Engineering Using Droplet Microfluidics We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on chip. The microfluidic device contains a 10 x 10 element array, each element containing sets of electrodes for two electric field actuated operations-electrowetting for merging droplets to mix reagents and electroporation for transformation. It can perform up to 100 genetic modifications in parallel, providing a scalable platform for generating the large number of engineered strains required for combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the systems capabilities through CRISPR-based engineering of two test cases-1) disruption of the function of enzyme galactokinase (galK) in E. coli and 2) targeted engineering of glutamine synthetase gene (glnA) and blue-pigment synthetase (bpsA) enzyme to improve indigoidine production in E. coli.

sentences: Environment-dependence of the expression of mutational load and species' range limits Theoretical and empirical research on the causes of species range limits suggests the contribution of several intrinsic and extrinsic factors, with potentially complex interactions among them. An intrinsic factor proposed by recent theory is mutational load increasing towards range edges because of genetic drift. Furthermore, environmental quality may decline toward range edges and enhance the expression of load. Here we tested whether the expression of mutational load associated with range limits in the North American plant Arabidopsis lyrata is enhanced under stressful environmental conditions by comparing the performance of within- versus between-population crosses at common garden sites across the species distribution and beyond. Heterosis, reflecting the expression of load, increased with heightened estimates of genomic load and with environmental stress caused by warming, but the interaction was not significant. We conclude that range-edge populations suffer from a twofold genetic Allee effect caused by increased mutational load and stress-dependent load linked to general heterozygote deficiency, but no synergistic effect between them.

sentences: Distinct developmental mechanisms influence sexual dimorphisms in the milkweed bug Oncopeltus fasciatus Sexual dimorphism is common in animals. The most complete model of sex determination comes from Drosophila melanogaster, where the relative dosage of autosomes and X chromosomes leads indirectly to sex-specific transcripts of doublesex. Female Dsx interacts with a mediator complex protein encoded by intersex to activate female development. In males the transcription factor encoded by fruitless promotes male-specific behavior. The genetics of sex determination have been examined in a small number of other insects, yet several questions remain about the pleisomorphic state. Is doublesex required for female and male development? Is fruitless conserved in male behavior or morphology? Are other components such as intersex functionally conserved? To address these questions, we report expression and functional tests of doublesex, intersex and fruitless in the hemipteran Oncopeltus fasciatus, characterizing three sexual dimorphisms. doublesex prevents intersex phenotypes in all sexes and dimorphic traits in the milkweed bug. intersex and fruitless are expressed across the body, in females and males. fruitless and intersex also affect the genitalia of both sexes, but have effects limited to different dimorphic structures in different sexes. These results reveal roles for intersex and fruitless distinct from other insects, and demonstrate distinct development mechanisms in different sexually dimorphic structures.

sentences: Variation and variability in Drosophila grooming behavior Behavioral differences can be observed between species or populations (variation) or between individuals in a genetically similar population (variability). Here, we investigate genetic differences as a possible source of variation and variability in Drosophila grooming. Grooming confers survival and social benefits. Grooming features of five Drosophila species exposed to a dust irritant were analyzed. Aspects of grooming behavior, such as anterior to posterior progression, were conserved between and within species. However, significant differences in activity levels, proportion of time spent in different cleaning movements, and grooming syntax were identified between species. All species tested showed individual variability in the order and duration of action sequences. Genetic diversity was not found to correlate with grooming variability within a species: melanogaster flies bred to increase or decrease genetic heterogeneity exhibited similar variability in grooming syntax. Individual flies observed on consecutive days also showed grooming sequence variability. Standardization of sensory input using optogenetics reduced but did not eliminate this variability. In aggregate, these data suggest that sequence variability may be a conserved feature of grooming behavior itself. These results also demonstrate that large genetic differences result in distinguishable grooming phenotypes (variation), but that genetic heterogeneity within a population does not necessarily correspond to an increase in the range of grooming behavior (variability).

sentences: Probing effects of the SARS-CoV-2 E protein on membrane curvature and intracellular calcium SARS-CoV-2 contains four structural proteins in its genome. These proteins aid in the assembly and budding of new virions at the ER-Golgi intermediate compartment (ERGIC). Current fundamental research efforts largely focus on one of these proteins - the spike (S) protein. Since successful antiviral therapies are likely to target multiple viral components, there is considerable interest in understanding the biophysical role of its other structural proteins, in particular structural membrane proteins. Here, we have focused our efforts on the characterization of the full-length envelope (E) protein from SARS-CoV-2, combining experimental and computational approaches. Recombinant expression of the full-length E protein from SARS-CoV-2 reveals that this membrane protein is capable of independent multimerization, possibly as a tetrameric or smaller species. Fluorescence microscopy shows that the protein localizes intracellularly, and coarse-grained MD simulations indicate it causes bending of the surrounding lipid bilayer, corroborating a potential role for the E protein in viral budding. Although we did not find robust electrophysiological evidence of ion-channel activity, cells transfected with the E protein exhibited reduced intracellular Ca2+, which may further promote viral replication. However, our atomistic MD simulations revealed that previous NMR structures are relatively unstable, and result in models incapable of ion conduction. Our study highlights the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insights, and eventually permitting virtual drug screening.

sentences: Contribution Evenness: A functional redundancy metric sensitive to trait stability in microbial communities The concept of functional redundancy has received considerable attention in both the macroecology and microbial ecology literature. As a result, multiple metrics of functional redundancy have been proposed. These vary in how they weight trait levels, species abundance, functional richness, and species richness. Here we present a new functional redundancy metric tailored for community-aggregated traits, which are traits that are quantified at the community level and can be quantitatively partitioned among species. We call this metric Contribution Evenness (CE) because it measures how evenly species contribute to a community-aggregated trait. As CE is an evenness measurement, it ranges from 0 and 1, where 0 corresponds to a single species contributing to a community-aggregated trait and 1 corresponds to all species contributing equally. Using in silico simulations of species extinctions, we demonstrate that CE reflects the stability of an ecosystem function to species extinction, a hypothesized ecological consequence of functional redundancy. As a positive control and to illustrate how CE can be used with sequence data, we analyzed the functional redundancy of eight nitrogen-transforming pathways using 2,631 metagenome-assembled genomes from 47 marine sites. CE for marine nitrogen cycle marker genes was consistent with our qualitative understanding of which nitrogen pathways are most functionally redundant in the ocean. We found that, on average, the NH4+ assimilation pathway was the most functionally redundant (0.44 {+/-} 0.08) while dissimilatory nitrate reduction was the least redundant (0.005 {+/-} 0.005). As demonstrated here, CE provides a promising framework for measuring trait stability in microbiomes.

sentences: Caracterization of Am -A genomes chromosomes in diploid wheat, polyploid wheat and triticales by marker cytogenetic The distribution and the Caracterization of constitutive heterochromatin in A-Am genomes of diploid wheat (progenitor), polyploid wheat (hybrids) and triticales (primary and secondary) are analyzed and compared by C-bands. The Comparison of zones rich in highly repeated DNA sequences marked by C bands on the all chromosomes of Am - A genomes revealed an important structural heterogeneity. Four chromosomes of Triticum monococcum (1Am-3Am-4Am-5Am) are almost similar to their homologues in wheat (Triticum durum, Triticum aestivum) and triticale, by the presence or absence of C bands. Contrary to the chromosomes 2Am (rich in heterochromatin),6Am-7Am (absence of C bands) show a great differentiation compared to their homologues of Triticum durum and Triticum aestivum and x-Triticosecale Wittmack. In the triticales, A genome chromosomes are richer in heterochromatin compared to theirs homologous of polyploid wheats. This is explained by a "genome shock The confrontation of a C-bands genome (Triticum monococcum) with a C+ bands genome (durum wheat / or common wheat) produces an interspecific hybrid which at the sixth generation reveals C+ bands (triticales).The variations observed in our vegetal material indicated the existence of an intervarietal and interspecific heterochromatic polymorphism. The presence of B chromosomes in triticales, could be explained as a manifestation of their adaptation.

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Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory Genome remethylation is essential for mammalian development but specific reasons are unclear. Here we examined embryonic stem (ES) cell fate in the absence of de novo DNA methyltransferases. We observed that ES cells deficient for both Dnmt3a and Dnmt3b are rapidly eliminated from chimaeras. On further investigation we found that in vivo and in vitro the formative pluripotency transition is derailed towards production of trophoblast. This aberrant trajectory is associated with failure to suppress activation of Ascl2. Ascl2 encodes a bHLH transcription factor expressed in placenta. Misexpression of Ascl2 in ES cells provokes transdifferentiation to trophoblast-like cells. Conversely, Ascl2 deletion rescues formative transition of Dnmt3a/b mutants and improves contribution to chimaeric epiblast. Thus, de novo DNA methylation safeguards against ectopic activation of Ascl2. However, Dnmt3a/b-deficient cells remain defective in ongoing embryogenesis. We surmise that multiple developmental transitions may be secured by DNA methylation silencing potentially disruptive genes.

SIGNIFICANCE STATEMENTMammalian DNA is widely modified by methylation of cytosine residues. This modification is added to DNA during early development. If methylation is prevented, the embryo dies by mid-gestation with multiple abnormalities. In this study we found that stem cells lacking the DNA methylation enzymes do not differentiate efficiently into cell types of the embryo and are diverted into producing placental cells. This switch in cell fate is driven by a transcription factor, Ascl2, which should only be produced in placenta. In the absence of DNA methylation, the Ascl2 gene is mis-expressed. Removing Ascl2 redirects embryonic fate but not full differentiation potential, suggesting that methylation acts at multiple developmental transitions to restrict activation of disruptive genes.

sentences: Selfish bacteria are active throughout the water column of the ocean Heterotrophic bacteria use extracellular enzymes to hydrolyze high molecular weight (HMW) organic matter to low molecular weight (LMW) hydrolysis products that can be taken into the cell. These enzymes represent a considerable investment of carbon, nitrogen, and energy, yet the return on this investment is uncertain, since hydrolysis of a HMW substrate outside a cell yields LMW products that can be lost to diffusion and taken up by scavengers that do not produce extracellular enzymes1. However, an additional strategy of HMW organic matter utilization,  selfish uptake2, is used for polysaccharide degradation, and has recently been found to be widespread among bacterial communities in surface ocean waters3. During selfish uptake, polysaccharides are bound at the cell surface, initially hydrolyzed, and transported into the periplasmic space without loss of hydrolysis products2, thereby retaining hydrolysate for the selfish bacteria and reducing availability of LMW substrates to scavenging bacteria. Here we show that selfish bacteria are common not only in the sunlit upper ocean, where polysaccharides are freshly produced by phytoplankton, but also deeper in the oceanic water column, including in bottom waters at depths of more than 5,500 meters. Thus, the return on investment, and therefore also the supply of suitable polysaccharides, must be sufficient to maintain these organisms.

sentences: In-silico investigation of umami peptides with receptor T1R1/T1R3 for the discovering potential targets: A combined modeling approach Umami, providing amino acids/peptides for animal growth, represents one of the major attractive taste modalities. The biochemical and umami properties of peptide are both important for scientific research and food industry. In this study, we did the sequence analysis of 205 umami peptides with 2-18 amino acids, sought the active sites of umami peptides by quantum chemical simulations and investigated their recognition residues with receptor T1R1/T1R3 by molecular docking. The results showed the peptides with 2-3 amino acids accounting for 44% of the total umami peptides. Residues D and E are the key active sites no matter where they in peptides (N-terminal, C-terminal or middle), when umami peptides contain D/E residues. N69, D147, R151, A170, S172, S276 and R277 residues in T1R1 receptor were deem to the key residues binding umami peptides. Finally, a powerful decision rule for umami peptides was proposed to predict potential umami peptides, which was convenient, time saving and efficiently.

sentences: Directional Cell Migration Guided by a Strain Gradient Strain gradients, the percentage of the deformation changed across a continuous field by applying forces, have been observed in developing tissues due to the inherent heterogeneity of the mechanical properties. The directional movement of cells are essential for proper cell localization, and it is well-established that cells establish directional migration in responses to gradients of chemicals, rigidity, density of extracellular matrices, and topography of substrates. However, it is unclear whether strain gradients imposed on cells due to tissue growth are sufficient to drive directional cell migration. In this work, we develop a programmable uniaxial cell stretch device coupled with geometrical constraints to create controllable strain gradients on cells. We demonstrate that single rat embryonic fibroblasts (REFs) respond to very small strain gradients. In a gradient level of 4% per mm, 60%-70% of the REFs prefer to migrate towards the lower strain side in both the static and the 0.1 Hz cyclic stretch conditions. We confirm that such responses to strain gradient is distinct from durotaxis or haptotaxis. Moreover, by using the YFP-Paxillin reporter, we discover that the directional migration of the cells is initiated by increasing focal adhesion contact areas and higher rate of protrusion formation in the lower strain side of the cell. Together, in this work we establish strain gradient as a novel cue to regulate directional cell migration and may provide new insights in development and tissue repairs.

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A systematic characterization of intrinsically formed microglia-like cells during retinal organoid differentiation. Brain organoids differentiated from human induced pluripotent stem cells provide a unique opportunity to investigate the development, organization and connectivity of neurons in a complex cellular environment. However, organoids usually lack microglia, brain-resident immune cells which are both present in the early human embryonic brain and participate in neuronal circuit development.

Here, we find that microglia innately develop in unguided retinal organoid differentiation between week 3 and 4 in 2.5D culture and appear later in floating, non-pigmented, 3D-cystic compartments. We enriched for cystic structures using a low-dosed BMP4 application and performed mass spectrometry, thus defining the protein composition of microglia-containing compartments. We found that cystic compartments expressed both mesenchymal and epithelial markers with microglia enriched in the mesenchymal region. Interestingly, microglia-like cells started to express the border-associated macrophage marker CD163. The preferential localization of human microglia to a mesenchymal compartment provides insight into the behavior and migration of microglia. The model will ultimately allow detailed study of these enigmatic cells and how they enter and distribute within the human brain.

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Ecological stochasticity and phage induction diversify bacterioplankton communities at the microscale In many natural environments, microorganisms self-assemble around heterogeneously distributed resource patches. The growth and collapse of populations on resource patches can unfold within spatial ranges of a few hundred micrometers or less, making such microscale ecosystems hotspots of biological interactions and nutrient fluxes. Despite the potential importance of patch-level dynamics for the large-scale evolution and function of microbial communities, we have not yet been able to delineate the ecological processes that control natural populations at the microscale. Here, we addressed this challenge in the context of microbially-mediated degradation of particulate organic matter by characterizing the natural marine communities that assembled on over one thousand individual microscale chitin particles. Through shotgun metagenomics, we found significant variation in microscale community composition despite the similarity in initial species pools across replicates. Strikingly, a subset of particles was highly populated by rare chitin-degrading strains; we hypothesized that their conditional success reflected the impact of stochastic colonization and growth on community assembly. In contrast to the conserved functional structures that emerge in ecosystems at larger scales, this taxonomic variability translated to a wide range of predicted chitinolytic abilities and growth returns at the level of individual particles. We found that predation by temperate bacteriophages, especially of degrader strains, was a significant contributor to the variability in the bacterial compositions and yields observed across communities. Our study suggests that initial stochasticity in assembly states at the microscale, amplified through biotic interactions, may have significant consequences for the diversity and functionality of microbial communities at larger scales.

Significance StatementThe biogeochemical consequences of the degradation of particulate organic matter by microorganisms represent the cumulative effect of microbial activity on individual microscale resource patches. The ecological processes controlling community dynamics in these highly localized microenvironments remain poorly understood. Here, we find that complex marine communities growing on microscale resource particles diverge both taxonomically and functionally despite assembling under identical abiotic conditions from a common species pool. We show that this variability stems from bacteriophage predation and history-dependent factors in community assembly, which create stochastic dynamics that are spatially structured at the microscale. This microscale stochasticity may have significant consequences for the coexistence, evolution, and function of diverse bacterial and viral populations in the global ocean.

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The Main Themes and Experiences Shared with the Hashtag #BlackintheIvory: A Qualitative Analysis BackgroundThe movement #BlackintheIvory gave Black academics an opportunity to connect through a social media platform allowing them to share common experiences in the pursuit of higher education. Through the analysis of Twitter posts, using the hashtag #BlackintheIvory, this study investigates the main themes identified among Black scholars in academia and their shared experiences with teaching, mentoring, collegiality, identity, service, and racism.

MethodsUsing the Twitter API, we isolated all publicly available tweets, which can include text, images, and links to websites, posted with the hashtag #BlackintheIvory on the Twitter website (www.twitter.com) from the inception of the hashtag in June 2020 to the end of December 2020. To evaluate the tweets, we categorized the tweets inductively. Based on the content of the posts, we identified 6 themes: Teaching, Mentoring, Collegiality, Identity, Service, and Racism.

ResultsOur search yielded a total of 12,538 original posts, including tweets between inception in June 2020 to December 2020 from profiles made public (excluding modified tweets and duplicate tweets). We selected and analyzed the top retweeted 2500 tweets, which is 20% (2500/12538) of the total number of downloaded tweets. The greatest percentage of posts were about Teaching (881; 35%), followed by Service (441; 18%) and Racism (414; 17%). The remaining tweets were categorized as Collegiality (388; 15%), Identity (210; 8%), and Mentoring (166; 7%) of the total number of tweets from June-December 2020.

ConclusionThe experiences, perspectives, and narratives among the Black diaspora within #BlackintheIvory are not uniform. The commonality exists within the structural systemic racism which impacts Black academics within the ivory tower, this study is a resounding wake-up call for action.

FundingThis study was not subject to any funding

sentences: The emergence of a birth-dependent mutation rate in asexuals: causes and consequences AO_SCPLOWBSTRACTC_SCPLOWIn unicellular organisms such as bacteria and in most viruses, mutations mainly occur during reproduction. Thus, genotypes with a high birth rate should have a higher mutation rate. However, standard models of asexual adaptation such as the  replicator-mutator equation often neglect this generation-time effect. In this study, we investigate the emergence of a positive dependence between the birth rate and the mutation rate in models of asexual adaptation and the consequences of this dependence. We show that it emerges naturally at the population scale, based on a large population limit of a stochastic time-continuous individual-based model with elementary assumptions. We derive a reaction-diffusion framework that describes the evolutionary trajectories and steady states in the presence of this dependence. When this model is coupled with a phenotype to fitness landscape with two optima, one for birth, the other one for survival, a new trade-off arises in the population. Compared to the standard approach with a constant mutation rate, the symmetry between birth and survival is broken. Our analytical results and numerical simulations show that the trajectories of mean phenotype, mean fitness and the stationary phenotype distribution are in sharp contrast with those displayed for the standard model. The reason for this is that the usual weak selection limit does not hold in a complex landscape with several optima associated with different values of the birth rate. Here, we obtain trajectories of adaptation where the mean phenotype of the population is initially attracted by the birth optimum, but eventually converges to the survival optimum, following a hook-shaped curve which illustrates the antagonistic effects of mutation on adaptation.

sentences: Impaired in vitro Interferon-γ production in patients with visceral leishmaniasis is improved by inhibition of PD1/PDL-1 ligation Visceral leishmaniasis (VL) is a neglected tropical disease that causes substantial morbidity and mortality and is a growing health problem in Ethiopia, where this study took place. Most individuals infected with Leishmania donovani parasites will stay asymptomatic, but some develop VL that, if left untreated, is almost always fatal. This stage of the disease is associated with a profound immunosuppression, characterised by impaired production of Interferon{gamma} (IFN{gamma}), a cytokine that plays a key role in the control of Leishmania parasites, and high expression levels of an inhibitory receptor, programmed cell death 1 (PD1) on CD4+ T cells. Here, we tested the contribution of the interaction between the immune checkpoint PD1 and its ligand PDL-1 on the impaired production of IFN{gamma} in VL patients. Our results show that in the blood of VL patients, not only CD4+, but also CD8+ T cells express high levels of PD1 at the time of VL diagnosis. Next, we identified PDL-1 expression on different monocyte subsets and neutrophils and show that PDL-1 levels were significantly increased in VL patients. PD1/PDL-1 inhibition resulted in significantly increased production of IFN{gamma}, suggesting that therapy using immune checkpoint inhibitors might improve disease control in these patients.

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CRISPR-mediated knock-in of transgenes into the malaria vector Anopheles funestus The ability to introduce mutations, or transgenes, of choice to precise genomic locations has revolutionised our ability to understand how genes and organisms work.

In many mosquito species that are vectors of various human disease, the advent of CRISPR genome editing tools has shed light on basic aspects of their biology that are relevant to their efficiency as disease vectors. This allows a better understanding of how current control tools work and opens up the possibility of novel genetic control approaches, such as gene drives, that deliberately introduce genetic traits into populations. Yet for the Anopheles funestus mosquito, a significant vector of malaria in sub-Saharan Africa and indeed the dominant vector species in many countries, transgenesis has yet to be achieved.

We describe herein an optimised transformation system based on the germline delivery of CRISPR components that allows efficient cleavage of a previously validated genomic site and preferential repair of these cut sites via homology-directed repair (HDR), which allows introduction of exogenous template sequence, rather than end-joining repair. The rates of transformation achieved are sufficiently high that it should be able to introduce alleles of choice to a target locus, and recover these, without the need to include additional dominant marker genes. Moreover, the high rates of HDR observed suggest that gene drives, which employ an HDR-type mechanism to ensure their proliferation in the genome, may be well suited to work in An. funestus.

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Phosphoantigens are Molecular Glues that Promote Butyrophilin 3A1/2A1 Association Leading to Vγ9Vδ2 T Cell Activation Tumor cells and pathogen-infected cells are presented to human {gamma}{delta} T cells based on "inside-out" signaling in which metabolites called phosphoantigens (pAgs) inside target cells are recognized by the intracellular domain of a butyrophilin protein (BTN3A1), leading to an extracellular conformational change. Here, we report that pAgs function as molecular "glues" that initiate a heteromeric association between the intracellular domains of BTN3A1 and the structurally similar BTN2A1. Working with both exogenous and endogenous pAgs, we used x-ray crystallography, mutational studies, cellular assays, synthetic probe as well as molecular dynamics investigations to determine how pAgs glue intracellular BTN3A1 and BTN2A1 together for the "inside-out" signaling that triggers {gamma}{delta} T cell activation. This {gamma}{delta} T cell-specific mode of antigen sensing creates opportunities for the development of alternative immunotherapies against cancer and infectious diseases that do not involve {beta} T cells.

One Sentence SummaryThe responses of gamma-delta T cells to cancer cells or pathogens are initiated via the intracellular association of heteromeric butyrophilins that are glued together by isoprenoid metabolites.

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Distinct N and C cross-feeding networks in a synthetic mouse gut consortium The complex interactions between gut microbiome and host or pathogen colonization resistance cannot solely be understood from community composition. Missing are causal relationships such as metabolic interactions among species to better understand what shapes the microbiome. Here, we focused on metabolic niches generated and occupied by the Oligo-Mouse-Microbiota consortium, a synthetic community composed of 12 members that is increasingly used as a model for the mouse gut microbiome. Combining mono-cultures and spent medium experiments with untargeted metabolomics uncovered broad metabolic diversity in the consortium, constituting a dense cross-feeding network with more than 100 pairwise interactions. Quantitative analysis of the cross-feeding network revealed distinct C and N food webs that highlight the two Bacteroidetes consortium members B. caecimuris and M. intestinale as primary suppliers of carbon, and a more diverse group as nitrogen providers. Cross-fed metabolites were mainly carboxylic acids, amino acids, and the so far not reported nucleobases. In particular the dicarboxylic acids malate and fumarate provided a strong physiological benefit to consumers, presumably as anaerobic electron acceptors. Isotopic tracer experiments validated the fate of a subset of cross-fed metabolites, in particular the conversion of the most abundant cross-fed compound succinate to butyrate. Thus, we show that this consortium is tailored to produce the anti-inflammatory metabolite butyrate. Overall, we provide evidence for metabolic niches generated and occupied by OMM members that lays a metabolic foundation to facilitate understanding of the more complex in vivo behavior of this consortium in the mouse gut.

ImportanceThis article maps out the cross-feeding network amongst 10 members of a synthetic consortium that is increasingly used as the model mouse gut microbiota. Combining metabolomics with in vitro cultivations, two dense networks of carbon and nitrogen exchange are described. The vast majority of the about 100 interactions are synergistic in nature, in several cases providing distinct physiological benefits to the recipient species. These networks lay the ground work towards understanding gut community dynamics and host-gut microbe interactions.

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Screening of antimicrobial activity of aqueous and ethanolic extracts of some medicinal plants from Cameroon and assessment of their synergy with common antibiotics against multidrug-resistant uropathogenic bacteria. Background and aimThe Cameroonian flora abounds in plants with multidimensional therapeutic virtues which can play an important role in the fight against resistance to antibiotics and the search for new antimicrobials. This study aimed to screen the antimicrobial potential of aqueous and ethanolic extracts of thirteen (13) samples (Bark, leaf, seed) of eight (8) plants from Cameroon against 3 reference pathogens and to evaluate their synergy with conventional antibiotics against eleven (11) multiresistant uropathogenic (MRU) bacteria.

MethodBioactive compounds were extracted from leaves of Leucanthemum vulgare, Cymbopogon citratus (DC.) Stapf, Moringa oleifera Lam and Vernonia amygdalina Delile; barks of Cinchona officinalis and Enantia chlorantha Oliv; barks and seeds of Garcinia lucida Vesque and leaves and seeds of Azadirachta indica (Neem) using distilled water and ethanol as solvents. The extracts were tested against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538 and Candida albicans 10231 using the well diffusion method and the microdilution method. The synergistic effect was assessed (using disc diffusion method and the checkerboard method) against MRU bacteria namely Achromobacter xylosoxidans 4892, Citrobacter freundii 426, Enterococcus avium 1669, Escherichia coli 1449, Klebsiella oxytoca 3003, Kocuria rizophilia. 1542, Moraxella catarrhalis 4222, Morganella morganii 1543, Pseudomonas aeruginosa 3057, Staphylococcus aureus 1449 and Streptococcus agalactiae 3984.

ResultsWe found that distilled water extracted a more important mass of phytochemical compounds (7.9-21.2%) compared to ethanol (5.8-12.4%). Except C. officinalis and G. lucida leaves, the rest of extracts were active with inhibition diameters (ID) ranging from 5 to 36 mm. Both ethanolic (EE) and aqueous extract (AE) of E. chloranta bark (ECB) were the most active against all pathogens with the mean ID of 17 and 36 mm vs S. aureus ATCC 6538, 23 and 14 mm vs E. coli ATCC 25922 and 36 and 19 mm vs C. albicans ATCC 10231. Only the EE of E. chloranta bark (ECB) had a strong activity against all the microorganisms tested (MIC <2 mg / ml); L. vulgare leaves (LVL) and G. lucida seed (GLS) had moderate (average MIC of 8 mg/ml) while all other extracts showed very weak antimicrobial activity. In addition, the fractional inhibitory concentration (FIC) ranged from 0.125 to 0.750. No antagonism (FIC> 4) or indifference (1[≤] FIC[≤]4) was noted between the extracts and the antibiotics, but the best synergies were found with ECB which well-modulated Kanamycin (FIC = 0.125 against S. aureus and 0.250 against E. coli), nitrofurantoin (FIC = 0.250 against S. aureus and 0.188 against E. coli) and ampicillin (FIC = 0.125 against E. coli). Similarly, compared to other extracts, ECB, LVL and GLS also well-modulated ampicillin, ceftazidime, tetracycline, nitrofurantoin, and trimethoprim against all the above-mentioned resistant uropathogenic bacteria with important increase in fold area (IFA).

ConclusionThis study show that E. chlorantha bark, L. vulgare leaves G. lucida seed, have good antimicrobial activity against both bacteria (Gram positive and Gram negative) and fungi (C. albicans); and should be more investigated for their possible use to the fight against MDR and MRU microorganisms.

sentences: The absence of the autoimmune regulator gene (AIRE) impairs the three-dimensional structure of medullary thymic epithelial cell spheroids Besides controlling the expression of peripheral tissue antigens, the autoimmune regulator (AIRE) gene also regulates the expression of adhesion genes in medullary thymic epithelial cells (mTECs), an essential process for mTEC-thymocyte interaction for triggering the negative selection in the thymus. For these processes to occur, it is necessary that the medulla compartment forms an adequate three-dimensional (3D) architecture, preserving the thymic medulla. Previous studies have shown that AIRE knockout (KO) mice have a small and disorganized thymic medulla; however, whether Aire influences the mTEC-mTEC interaction in the maintenance of the 3D structure has been little explored. Considering that AIRE controls cell adhesion genes, we hypothesized that this gene affects 3D mTEC-mTEC interaction. To test this, we constructed an in vitro model system for mTEC spheroid formation, in which cells adhere to each other, establishing a 3D structure. The effect of Aire on mTEC-mTEC adhesion was evaluated by comparing AIRE wild type (AIREWT) versus Aire KO (AIRE-/-) mTECs. Considering the 3D spheroid model evaluated, we reported that the absence of AIRE disorganizes the 3D structure of mTEC spheroids, promotes a differential regulation of mTEC classical surface markers, and modulates genes encoding adhesion and other molecules.

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Amacrine cells shape retinal functions while dynamically preserving circuits for colour vision In vertebrate vision, the feature extracting circuits of the inner retina are driven by heavily pre-processed photoreceptor signals. For example, in larval zebrafish, outer retinal circuits serve to split "colour" from "greyscale" information across their four ancestral cone-photoreceptor types. How then can the inner retina simultaneously preserve such incoming spectral information despite the need to combine cone-signals to shape new greyscale functions?

To address this question, we imaged light-driven signals from the axon terminals of retinal bipolar cells in the presence and pharmacological absence of inhibition from amacrine cells. Surprisingly, this manipulation had no net effect on the inner retinal representation of colour-opponency, despite profound impacts on all tested greyscale functions such as the gain and kinetics of bipolar cell light responses. This  dynamic balance was achieved by amacrine cells driving opponency in some bipolar cells, while at the same time suppressing pre-existing opponency in others, such that the net change across the network was essentially zero. To do so, amacrine cells near-exclusively leveraged the On-channel, and correspondingly, a direct in vivo survey of amacrine cell functions revealed that all their colour-opponent responses were located in the On-layer. In contrast, Off-stratifying amacrine cells were largely achromatic. We conclude that complex interactions within the inner retina that underlie greyscale visual processing tasks are intricately balanced via the On-channel to not notably alter the pre-existing population representation of colour information.

sentences: Knockout of Anopheles stephensi immune gene LRIM1 by CRISPR-Cas9 reveals its unexpected role in reproduction and vector competence PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufactured using aseptically reared Anopheles stephensi mosquitoes. Immune response genes of Anopheles mosquitoes such as Leucin-Rich protein (LRIM1), inhibit Plasmodium SPZ development (sporogony) in mosquitoes by supporting melanization and phagocytosis of ookinetes. With the aim of increasing PfSPZ infection intensities, we generated an A. stephensi LRIM1 knockout line , {Delta}aslrim1 , by embryonic genome editing using CRISPR-Cas9. {Delta}aslrim1 mosquitoes had a significantly increased midgut bacterial load and an altered microbiome composition, including elimination of commensal acetic acid bacteria.  The alterations in the microbiome caused increased mosquito mortality and unexpectedly, significantly reduced sporogony. The survival rate of {Delta}aslrim1 and their ability to support PfSPZ development, were partially restored by antibiotic treatment of the mosquitoes, and fully restored to baseline when {Delta}aslrim1 mosquitoes were produced aseptically. Deletion of LRIM1 also affected reproductive capacity: oviposition, fecundity and male fertility were significantly compromised. Attenuation in fecundity was not associated with the altered microbiome. This work demonstrates that LRIM1s regulation of the microbiome has a major impact on vector competence and longevity of A. stephensi. Additionally, LRIM1 deletion identified an unexpected role for this gene in fecundity and reduction of sperm transfer by males.

sentences: Annual and 16-day rangeland production estimates for the western United States Rangeland production is a foundational ecosystem service and resource upon which livestock, wildlife, and people depend. Capitalizing on recent advancements in the use of remote sensing data across rangelands we provide estimates of herbaceous rangeland production from 1986-2019 at 16-day and annual time steps and 30m resolution across the western United States. A factorial comparison of this dataset and three national scale datasets is presented, and we highlight a multiple lines of evidence approach when using production estimates in decision-making. Herbaceous aboveground biomass at this scale and resolution provides critical information applicable for management and decision-making, particularly in the face of annual grass invasion and woody encroachment of rangeland systems. These readily available data remove analytical and technological barriers allowing immediate utilization for monitoring and management.

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Genotyping of structural variation using PacBio high-fidelity sequencing BackgroundStructural variations (SVs) pervade the genome and contribute substantially to the phenotypic diversity of species. However, most SVs were ineffectively assayed because of the complexity of plant genomes and the limitations of sequencing technologies. Recent advancement of third-generation sequencing technologies, particularly the PacBio high-fidelity (HiFi) sequencing, which generates both long and highly accurate reads, offers an unprecedented opportunity to characterize SVs and reveal their functionality. Since HiFi sequencing is new, it is crucial to evaluate HiFi reads in SV detection before applying the technology at scale.

ResultsWe sequenced wheat genomes using HiFi, then conducted a comprehensive evaluation of SV detection using mainstream long-read aligners and SV callers. The results showed the accuracy of SV discovery depends more on aligners rather than callers. For aligners, pbmm2 and NGMLR provided the most accurate results while detecting deletion and insertion, respectively. Likewise, cuteSV and SVIM achieved the best performance across all SV callers. We demonstrated that the combination of the aligners and callers mentioned above is optimal for SV detection. Furthermore, we evaluated the impact of sequencing depth on the accuracy of SV detection. The results showed that low-coverage HiFi sequencing is capable of generating high-quality SV genotyping.

ConclusionsThis study provides a robust benchmark of SV discovery with HiFi reads, showing the remarkable potential of long-read sequencing to investigate structural variations in plant genomes. The high accuracy SV discovery from low-coverage HiFi sequencing indicates that skim HiFi sequencing is an ideal approach to study structural variations at the population level.

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Duplication and overexpression of the genes encoding a beta 1,3-glucan synthase confer the intrinsic resistance to echinocandin in Mucor circinelloides Procedures such as solid organ transplants and cancer treatments can leave many patients in an immunocompromised state resulting in an increased susceptibility to opportunistic diseases including fungal infections. Mucormycosis infections are continually emerging and pose a serious threat to immunocompromised patients. Currently there has been a sharp increase in mucormycosis cases as a secondary infection in patients battling SARS-CoV-2 infections. Mucorales fungi are notorious for presenting resistance to most antifungal drugs. The absence of effective means to treat these infections results in mortality rates approaching 100% in cases of disseminated infection. One of the most effective antifungal drug classes currently available are echinocandins. Echinocandins seem to be efficacious in treatment of many other fungal infections. Unfortunately, susceptibility testing has found that echinocandins have no to little effect on Mucorales. In this study, we found that the model Mucorales Mucor circinelloides genome carries three copies of the genes encoding for the echinocandin {beta}-(1,3)-D-glucan synthase (fksA, fksB, and fksC). Interestingly, we revealed that exposing M. circinelloides to micafungin significantly increased the expression of the fksA and fksB genes when compared to an untreated control. We further uncovered that the serine/threonine phosphatase calcineurin is responsible for the overexpression of fksA and fksB as deletion of calcineurin results in a decrease in expression of all three fks genes and a lower minimal inhibitory concentration (MIC) to micafungin. Taken together, this study demonstrates that the fks gene duplication and overexpression by calcineurin contribute to the intrinsic resistance to echinocandins in Mucor.

IMPORTANCEThe recent emergence of mucormycosis cases in immunocompromised patients has become a more prevalent issue. The Mucorales fungi that cause these infections are known to be highly drug resistant, thus treatment options are limited and the mortalities of these types of infections remain unacceptably high. Echinocandins are one of the latest antifungal drugs developed to successfully treat fungal infections, but it remains unclear why Mucorales fungi are resistant to these treatments. In our study, we uncovered three copies of the genes (fks) encoding the drug target for echinocandins. Furthermore, we discovered that the serine-threonine phosphatase calcineurin is regulating the over-expression of these genes, which confers resistance. By inhibiting calcineurin we found that the expression of these drug targets decreases resulting in an increase in susceptibility to echinocandins both in vitro and in vivo

sentences: rDNA nascent transcripts promote a unique spatial organization during mouse early development During the first cell cycles of the early development, the chromatin of the embryo is highly reprogrammed alongside that embryonic genome starts its own transcription. The spatial organization of the genome is a major process that contributes to regulating gene transcription in time and space, however, it is poorly studied in the context of early embryos. To study the cause and effect link between transcription and spatial organization in embryos, we focused on the ribosomal genes, that are first silent and begin to transcribe during the 2-cell stage in mouse. We demonstrated that ribosomal sequences are spatially organized in a very peculiar manner from the 2-cell to the 16-cell stage with transcription and processing of ribosomal RNAs excluding mutually. Using drugs inhibiting the RNA polymerase I, we show that this organization, totally different from somatic cells, depends on an active transcription of ribosomal genes and induces a unique chromatin environment that favors major satellite sequences transcription after the 4-cell stage.

sentences: Subcellular mapping of the protein landscape of SARS-CoV-2 infected cells for target-centric drug repurposing The COVID-19 pandemic has resulted in millions of deaths and affected socioeconomic structure worldwide and the search for new antivirals and treatments are still ongoing. In the search for new drug target and to increase our understanding of the disease, we used large scale immunofluorescence to explore the host cell response to SARS-CoV-2 infection. Among the 602 host proteins studied in this host response screen, changes in abundance and subcellular localization were observed for 97 proteins, with 45 proteins showing increased abundance and 10 reduced abundances. 20 proteins displayed changed localization upon infection and an additional 22 proteins displayed altered abundance and localization, together contributing to diverse reshuffling of the host cell protein landscape. We then selected existing and approved small-molecule drugs (n =123) against our identified host response proteins and identified 3 compounds - elesclomol, crizotinib and rimcazole, that significantly reduced antiviral activity. Our study introduces a novel, targeted and systematic approach based on host protein profiling, to identify new targets for drug repurposing. The dataset of [~]75,000 immunofluorescence images from this study are published as a resource available for further studies.

sentences: Transcranial magnetic stimulation of right dorsolateral prefrontal cortex does not affect associative retrieval in healthy young or older adults We examined whether post-retrieval monitoring processes supporting memory performance are more resource limited in older adults relative to younger individuals. We predicted that older adults would be more susceptible to an experimental manipulation that reduced the neurocognitive resources available to support post-retrieval monitoring. Young and older adults received transcranial magnetic stimulation (TMS) to the right dorsolateral prefrontal cortex (DLPFC) or a vertex control site during an associative recognition task. The right DLPFC was selected as a TMS target because the region is held to be a key member of a network of regions engaged during retrieval monitoring and is readily accessible to administration of TMS. We predicted that TMS to the right DLPFC would lead to reduced associative recognition accuracy, and that this effect would be more prominent in older adults. The results did not support this prediction. Recognition accuracy was significantly reduced in older adults relative to their younger counterparts but the magnitude of this age difference was unaffected following TMS to the right DLPFC or vertex. These findings suggest that TMS to the right DLPFC was insufficient to deplete the neurocognitive resources necessary to support post-retrieval monitoring.

sentences: Cleavage of PGAM5 by the intramembrane protease PARL is governed by transmembrane helix dynamics and oligomeric state The intramembrane protease PARL is a crucial mitochondrial safeguard by cleaving the mitophagy regulators PINK1 and PGAM5. PGAM5 substrate determinates have not been rigorously investigated and it is unclear how uncoupling the mitochondrial membrane potential regulates its processing inversely to PINK1. Here we show that in PGAM5 several hydrophilic residues distant from the cleavage site serve as key determinant for PARL- catalyzed cleavage. NMR analysis indicates that a short N-terminal amphipathic helix, followed by a kink and a C-terminal helix harboring the scissile peptide bond, is key for a productive interaction with PARL. In difference to PINK1, PGAM5 is stably inserted into the inner mitochondrial membrane until uncoupling the membrane potential triggers its disassembly into monomers that are vulnerable to PARL-catalyzed processing. We suggest a model in which PGAM5 is a slowly processed substrate with PARL-catalyzed cleavage that is influenced by multiple hierarchical substrate features including a membrane-potential- dependent oligomeric switch.

sentences: Flow Cytometric Evaluation of Yeast-Bacterial Cell-Cell Interactions Synthetic cell-cell interaction systems can be useful for understanding multicellular communities or for screening binding molecules. We adapt a previously characterized set of synthetic cognate nanobody-antigen pairs to a yeast-bacteria coincubation format and use flow cytometry to evaluate cell-cell interactions mediated by binding between surface-displayed molecules. We further use fluorescence-activated cell sorting (FACS) to enrich for a specific yeast-displayed nanobody within a mixed yeast-display population. Finally, we demonstrate that this system supports characterization of a therapeutically relevant nanobody-antigen interaction: a previously discovered nanobody that binds to the intimin protein expressed on the surface of enterohemorrhagic E. coli. Overall, our findings indicate that the yeast-bacteria format supports efficient evaluation of ligand-target interactions. With further development, this format may facilitate systematic characterization and high throughput discovery of bacterial surface-binding molecules.

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Topical TMPRSS2 inhibition prevents SARS-CoV-2 infection in differentiated primary human airway cells BackgroundThere are limited effective prophylactic treatments for SARS-CoV-2 infection, and limited early treatment options. Viral cell entry requires spike protein binding to the ACE2 receptor and spike cleavage by TMPRSS2, a cell surface serine protease. Targeting of TMPRSS2 by either androgen blockade or direct inhibition is already in clinical trials in early SARS-CoV-2 infection.

MethodsThe likely initial cells of SARS-CoV-2 entry are the ciliated cells of the upper airway. We therefore used differentiated primary human airway epithelial cells maintained at the air-liquid interface (ALI) to test the impact of targeting TMPRSS2 on the prevention of SARS-CoV-2 infection.

ResultsWe first modelled the systemic delivery of compounds. Enzalutamide, an oral androgen receptor antagonist, had no impact on SARS-Cov-2 infection. By contrast, camostat mesylate, an orally available serine protease inhibitor, blocked SARS-CoV-2 entry. However, camostat is rapidly metabolised in the circulation in vivo, and systemic bioavailability after oral dosing is low. We therefore modelled local airway administration by applying camostat to the apical surface of the differentiated ALI cultures. We demonstrated that a brief exposure to topical camostat is effective at restricting SARS-CoV-2 viral infection.

ConclusionThese experiments demonstrate a potential therapeutic role for topical camostat for pre- or post-exposure prophylaxis of SARS-CoV-2, which can now be evaluated in a clinical trial.

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Cannabinoid 1/2 Receptor Activation Induces Strain-Dependent Behavioral and Neurochemical Changes in GAERS and NEC rats Childhood absence epilepsy (CAE) is characterized by absence seizures which are episodes of lack of consciousness accompanied by electrographic spike-wave discharges. About 60% of children and adolescents with ASs are affected by major neuropsychological comorbidities, including anxiety. Endocannabinoids and monoamines are likely involved in the pathophysiology of these CAE psychiatric comorbidities.

Here, we show that the synthetic cannabinoid receptor type 1/2 (CB1/2R) agonist WIN 55,212-2 (2 mg/kg, i.p.) has a strain-dependent effect on anxiety-like and motor behavior and cerebral monoaminergic levels in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and their non-epileptic control (NEC) rat strain. Using quantitative and Temporal-Pattern analysis, we found that WIN 55,212-2 did not affect the emotional status of GAERS but it was anxiolytic in NEC. Conversely, WIN 55,212-2 had a sedative effect in GAERS but was ineffective in NEC. Moreover, vehicle-treated GAERS are more motivated to explore by implementing more complex and articulated strategies. These behavioral changes correlate with the reduction of 5-HT in the hippocampus and substantia nigra (SN) and noradrenaline (NA) in the entopeduncular nucleus (EPN) in vehicle-treated GAERS compared to NEC rats which could contribute to their low anxiety status and hypermotility, respectively. On the other hand, the increased level of NA in the EPN and 5-HT in the SN is consistent with an activation of the basal ganglia output-mediated motor suppression observed in WIN 55,212-2-treated GAERS rats.

These data support the view of a strain-dependent alteration of the endocannabinoid system by adding evidence of lower emotional responsiveness and a basal ganglia hypersensitivity to cannabinoids in GAERS compared to NEC rats.

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Relative time constraints improve molecular dating Dating the tree of life is central to understanding the evolution of life on Earth. Molecular clocks calibrated with fossils represent the state of the art for inferring the ages of major groups. Yet, other information on the timing of species diversification can be used to date the tree of life. For example, horizontal gene transfer events and ancient coevolutionary relationships such as (endo)symbioses can imply temporal relationships between two nodes in a phylogeny (Davin et al. 2018). These alternative sources of temporal constraints can be particularly helpful when the geological record is sparse, e.g. for microorganisms, which represent the vast majority of extant and extinct biodiversity.

Here, we present a new method to combine fossil calibrations and relative age constraints to estimate chronograms. We provide an implementation of relative age constraints in RevBayes (Hohna et al. 2016) that can be combined in a modular manner with the wide range of molecular dating methods available in the software.

We use both realistic simulations and empirical datasets of 40 Cyanobacteria and 62 Archaea to evaluate our method. We show that the combination of relative age constraints with fossil calibrations significantly improves the estimation of node ages.

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A novel automated approach for improving standardization of the marble burying test enables quantification of burying bouts and activity characteristics The marble burying test is a commonly used paradigm to screen phenotypes in mouse models of neurodevelopmental and psychiatric disorders. The current methodological approach relies solely on reporting the number of buried marbles at the end of the test. By measuring the proxy of the behavior (buried marbles), rather than the behavior itself (burying bouts), many important characteristics regarding the temporal aspect of this assay are lost. Here we introduce a novel, automated method to quantify mouse behavior throughout the duration of the marble burying test with the focus on the burying bouts. Using open-source software packages, we trained a supervised machine learning algorithm (the "classifier") to distinguish burying behavior in freely moving mice. In order to confirm the classifiers accuracy and uncover the behavioral meaning of the marble burying test, we performed marble burying test in three mouse models: Ube3am-/p+ (Angelman Syndrome model), Shank2-/- (autism model), and Sapap3-/- (obsessive-compulsive disorder model) mice. The classifier scored burying behavior accurately and consistent with the literature in the Ube3am-/p+ mice, which showed decreased levels of burying compared to controls. Shank2-/- mice showed a similar pattern of decreased burying behavior, which was not found in Sapap3-/- mice. Tracking mouse behavior throughout the test enabled us to quantify activity characteristics, revealing hypoactivity in Ube3am-/p+ and hyperactivity in the Shank2-/- mice, indicating that mouse activity is unrelated to burying behavior. Together, we demonstrate that our classifier is an accurate method for the analysis of the marble burying test, providing more information than the currently used methods.

Significance StatementThe marble burying test is widely used in phenotyping neurodevelopmental and neuropsychiatric disorder mouse models. Currently, its analysis consists of manually scoring the number of buried marbles upon the completion of the assay. This approach is not standardized across laboratories, and leaves out important variables such as movement characteristics and information about the burying bouts. This leads to divergent interpretations of the marble burying test, ranging from anxiety to cognitive impairment. We introduce a method that reliably tracks mouse behavior throughout the experiment, classifies the duration and number of the burying bouts, and is generalizable across laboratories. Using machine learning for measuring the actual burying behavior standardizes this method, and provides rich information about the burying characteristics.

sentences: Depletion of CD206+ Tumour Macrophages via a Peptide-Targeted Star-Shaped Polyglutamate Inhibits Tumourigenesis and Metastatic Dissemination in Mouse Breast Cancer Models Although many studies have explored the depletion of tumour-associated macrophages (TAMs) as a therapeutic strategy for solid tumours, currently available compounds suffer from poor efficacy and dose-limiting side effects. Here, we developed a novel TAM-depleting agent ("OximUNO") that specifically targets CD206+ TAMs and demonstrated efficacy in triple negative breast cancer (TNBC) mouse models. OximUNO comprises a star-shaped polyglutamate (St-PGA) decorated with the CD206-targeting peptide mUNO that carries the chemotherapeutic drug doxorubicin (DOX). In TNBC models, a fluorescently labelled mUNO-decorated St-PGA homed to CD206+ TAMs within primary lesions and metastases. OximUNO exhibited no acute liver or kidney toxicity in vivo. Treatment with OximUNO reduced the progression of primary tumour lesions and pulmonary metastases, significantly diminished the number of CD206+ TAMs and increased the CD8/FOXP3 expression ratio (demonstrating immunostimulation). Our findings suggest the potential benefit of OximUNO as a TAM-depleting agent for TNBC treatment. Importantly, our studies also represent the first report of a peptide-targeted St-PGA as a targeted therapeutic nanoconjugate.

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ZIP9 is a Druggable Determinant of Sex Differences in Melanoma Melanoma and most other cancers occur more frequently, and have worse prognosis, in males compared with females. Though sex steroids are thought to be involved, classical androgen and estrogen receptors are not detectable in most melanomas. Here we show that testosterone promotes melanoma proliferation by activating ZIP9 (SLC39A9), a zinc transporter that is not intentionally targeted by available therapeutics, but is widely expressed in human melanoma. This testosterone activity requires zinc influx, MAPK activation and YAP1 nuclear translocation. We demonstrate that FDA approved inhibitors of the classical androgen receptor also inhibit ZIP9, and thereby antagonize the pro-tumorigenic effects of testosterone in melanoma. In male mice, androgen receptor inhibitors suppressed growth of ZIP9-expressing melanomas, but had no effect on isogenic melanomas lacking ZIP9, nor on melanomas in females. These data suggest that ZIP9 might be effectively targeted in melanoma and other cancers by repurposing androgen receptor inhibitors that are currently approved only for prostate cancer.

SignificanceMelanoma outcomes are worse in males than in females. Some of this difference is driven by testosterone signaling through ZIP9, a nonclassical testosterone receptor. Drugs that target AR can be repurposed to block ZIP9, and inhibit melanoma in males.

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Individual heterogeneity and its importance for metapopulation dynamics. Landscape connectedness shapes the exchange of individuals among patches, and hence metapopulation connectivity and dynamics. Connectedness, and its resulting effects on connectivity are therefore rightfully central in conservation biology. However, besides determining demographic fluxes of individuals between patches, connectedness also generates phenotypic sorting and thus impacts local and regional eco-evolutionary dynamics. Despite the central role of connectedness, its effects on individual phenotypic heterogeneity and spatial organization are so far neglected in theory and applications.

Through experimental metapopulations of Tetranychus urticae (two-spotted spider mite) with three levels of landscape connectedness and by regularly removing phenotypic structure in a subset of these populations, we tested how regional and local population dynamics are determined both by network connectedness and phenotypic spatial organization.

We find that the self-organizing phenotypic spatial structure increases local equilibrium population sizes and variability. It in contrast dampens the effects of imposed connectedness differences on population sizes and is therefore anticipated to improve metapopulation persistence. Contrary to theoretical expectations, the most locally connected patches within the network showed an overall reduced local population size, possibly originating from a faster depletion of resources from immigrants or transiting individuals.

This experiment shows how metapopulation dynamics can significantly deviate from theoretical expectations due to individual heterogeneity. This potential rescue effect stemming from phenotypical self-organization in space is a key point to consider for conservation actions, especially based on translocations.

sentences: An Explainable Deep Learning Approach for Multimodal Electrophysiology Classification In recent years, more biomedical studies have begun to use multimodal data to improve model performance. As such, there is a need for improved multimodal explainability methods. Many studies involving multimodal explainability have used ablation approaches. Ablation requires the modification of input data, which may create out-of-distribution samples and may not always offer a correct explanation. We propose using an alternative gradient-based feature attribution approach, called layer-wise relevance propagation (LRP), to help explain multimodal models. To demonstrate the feasibility of the approach, we selected automated sleep stage classification as our use-case and trained a 1-D convolutional neural network (CNN) with electroencephalogram (EEG), electrooculogram (EOG), and electromyogram (EMG) data. We applied LRP to explain the relative importance of each modality to the classification of different sleep stages. Our results showed that across all samples, EEG was most important, followed by EOG, and EMG. For individual sleep stages, EEG and EOG had higher relevance for classifying awake and non-rapid eye movement 1 (NREM1). EOG was most important for classifying REM, and EEG was most relevant for classifying NREM2-NREM3. Also, LRP gave consistent levels of importance to each modality for correctly classified samples across folds and inconsistent levels of importance for incorrectly classified samples. Our results demonstrate the additional insight that gradient-based approaches can provide relative to ablation methods and highlight their feasibility for explaining multimodal electrophysiology classifiers.

sentences: Essential role of translation factor eIF5a in cytokine production and cell cycle regulation in primary CD8 T lymphocytes Translational control adjusts protein production rapidly and facilitates local cellular responses to environmental conditions. Translation can be regulated through sequestration of mRNAs by regulatory proteins or RNAs, but also by the availability of ribosomes and translation factors which enable initiation and elongation of nascent polypeptides. Traditionally initiation of mRNA translation has been considered to be a major translational control point, however, control of peptide elongation can also play a role. Here we show that post-translational modification of the elongation factor, eIF5a, controls translation of subsets of proteins in naive T-cells upon activation. Sequencing of nascent polypeptides indicated that functional eIF5a was required for the production of proteins which regulate T-cell proliferation and effector function. Control of translation in multiple immune cell lineages is required to co-ordinate immune responses and these data illustrate that translational elongation can contribute to post-transcriptional regulons important for the control of inflammation.

sentences: Effective clustering for single cell sequencing cancer data Single cell sequencing (SCS) technologies provide a level of resolution that makes it indispensable for inferring from a sequenced tumor, evolutionary trees or phylogenies representing an accumulation of cancerous mutations. A drawback of SCS is elevated false negative and missing value rates, resulting in a large space of possible solutions, which in turn makes it difficult, sometimes infeasible using current approaches and tools. One possible solution is to reduce the size of an SCS instance -- usually represented as a matrix of presence, absence, and uncertainty of the mutations found in the different sequenced cells -- and to infer the tree from this reduced-size instance. In this work, we present a new clustering procedure aimed at clustering such categorical vector, or matrix data -- here representing SCS instances, called celluloid. We show that celluloid clusters mutations with high precision: never pairing too many mutations that are unrelated in the ground truth, but also obtains accurate results in terms of the phylogeny inferred downstream from the reduced instance produced by this method. We demonstrate the usefulness of a clustering step by applying the entire pipeline (clustering + inference method) to a real dataset, showing a significant reduction in the runtime, raising considerably the upper bound on the size of SCS instances which can be solved in practice. Our approach, celluloid: clustering single cell sequencing data around centroids is available at https://github.com/AlgoLab/celluloid/ under an MIT license, as well as on the Python Package Index (PyPI) at https://pypi.org/project/celluloid-clust/

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Coagulation disorders in patients with severe hemophagocytic lymphohistiocytosis BackgroundCoagulation disorders are common in patients with hemophagocytic lymphohistiocytosis (HLH), associated with an increased risk of bleeding and death. We aim to investigate coagulation disorders and their outcome implications in critically ill patients with HLH.

MethodsWe prospectively evaluated 47 critically ill patients with HLH (median age of 54 years [42-67]) between April 2015 and December 2018. Coagulation assessments were performed at day 1. Abnormal standard coagulation was defined as prothrombin time (PT) <50% and/or fibrinogen <2g/L. HLH aetiology was mostly ascribed to haematological malignancies (74% of patients).

ResultsCoagulation disorders and severe bleeding events were frequent, occurring in 30 (64%) and 11 (23%) patients respectively. At day 1, median fibrinogen level was 2{middle dot}65g/L [1.61-5.66]. Fibrinolytic activity was high as suggested by increased median levels of D-dimers, fibrin monomers, PAI-1 (plasminogen activator inhibitor) and tPA (tissue plasminogen activator). Forty-one (91%) patients had a decreased ADAMTS13 activity (A Disintegrin-like And Metalloproteinase with ThromboSpondin type 1 repeats, member 13). By multivariable analysis, the occurrence of a severe bleeding (OR 3.215 [1.194-8.653], p=0{middle dot}021) and SOFA score (Sepsis-Related Organ Failure Assessment) at day 1 (OR 1.305 per point [1.146-1.485], p<0{middle dot}001) were independently associated with hospital mortality. No early biological marker was associated with severe bleeding.

ConclusionsHyperfibrinolysis may be the primary mechanism responsible for hypofibrinogenemia and may also participate in ADAMTS13 degradation. Targeting the plasmin system appears as a promising approach in severe HLH-related coagulation disorders.

sentences: LIGHTHOUSE illuminates therapeutics for a variety of diseases including COVID-19 One of the bottlenecks in the application of basic research findings to patients is the enormous cost, time, and effort required for high-throughput screening of potential drugs for given therapeutic targets. Here we have developed LIGHTHOUSE, a graph-based deep learning approach for discovery of the hidden principles underlying the association of small-molecule compounds with target proteins. Without any 3D structural information for proteins or chemicals, LIGHTHOUSE estimates protein-compound scores that incorporate known evolutionary relations and available experimental data. It identified novel therapeutics for cancer, lifestyle-related disease, and bacterial infection. Moreover, LIGHTHOUSE predicted ethoxzolamide as a therapeutic for coronavirus disease 2019 (COVID-19), and this agent was indeed effective against alpha, beta, gamma, and delta variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that are rampant worldwide. We envision that LIGHTHOUSE will bring about a paradigm shift in translational medicine, providing a bridge from bench side to bedside.

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Global chromosome topology and the two-component systems in concerted manner regulate transcription in Streptomyces Bacterial gene expression is controlled at multiple levels, with chromosome supercoiling being one of the most global regulators. Global DNA supercoiling is maintained by the orchestrated action of topoisomerases. In Streptomyces, mycelial soil bacteria with a complex life cycle, topoisomerase I depletion led to elevated chromosome supercoiling, changed expression of significant fraction of genes, delayed growth and blocked sporulation. To identify supercoiling-induced sporulation regulators, we searched for S. coelicolor transposon mutants that were able to restore sporulation despite high chromosome supercoiling. We established that transposon insertion in genes encoding a novel two-component system named SatKR reversed the sporulation blockage resulting from topoisomerase I depletion. Transposition in satKR abolished the transcriptional induction of the genes within the so-called supercoiling-hypersensitive cluster (SHC). Moreover, we found that activated SatR also induced the same set of SHC genes under normal supercoiling conditions. We determined that the expression of genes in this region impacted S. coelicolor growth and sporulation. Interestingly, among the associated products is another two-component system (SitKR), indicating the potential for cascading regulatory effects driven by the SatKR and SitKR two-component systems. Thus, we demonstrated the concerted activity of chromosome supercoiling and a hierarchical two-component signalling system that impacts gene activity governing Streptomyces growth and sporulation.

IMPORTANCEStreptomyces, soil bacteria with complex life cycle, are the producers of a broad range of biologically active compounds (e.g. antibiotics). Streptomyces respond to various environmental signals using complex transcriptional regulation mechanism. Understanding regulation of their gene expression is crucial for Streptomyces application as industrial organisms. Here, based on extensive transcriptomics analyses, we describe the concerted regulation of genes crucial for growth and development by global DNA supercoiling and novel two-component system. Our data indicate that regulated genes encode growth and sporulation regulator. Thus, we demonstrate that Streptomyces link the global regulatory strategies to adjusts life cycle to unfavourable conditions.

sentences: Parasite-probiotic interactions in the gut: Bacillus sp. and Enterococcus faecium regulate type-2 inflammatory responses and modify the gut microbiota of pigs during helminth infection Dietary probiotics may enhance gut health by directly competing with pathogenic agents and through immunostimulatory effects. These properties are recognized in the context of bacterial and viral pathogens, but less is known about interactions with eukaryotic pathogens such as parasitic worms (helminths). In this study we investigated whether two probiotic mixtures (comprised of Bacillus amyloliquefaciens, B. subtilis, and Enterococcus faecium [BBE], or Lactobacillus rhamnosus LGG and Bifidobacterium animalis subspecies Lactis Bb12 [LB]) could modulate helminth infection kinetics as well as the gut microbiome and intestinal immune responses in pigs infected with the nodular worm Oesophagostomum dentatum. We observed that neither probiotic mixture influenced helminth infection levels. BBE, and to a lesser extent LB, changed the alpha- and beta-diversity indices of the colon and faecal microbiota, notably including an enrichment of faecal Bifidobacterium spp. by BBE. However, these effects were muted by concurrent O. dentatum infection. BBE (but not LB) significantly attenuated the O. dentatum-induced upregulation of genes involved in type-2 inflammation and restored normal lymphocyte ratios in the ileo-caecal lymph nodes that were altered by infection. Moreover, inflammatory cytokine release from blood mononuclear cells and intestinal lymphocytes was diminished by BBE. Collectively, our data suggest that selected probiotic mixtures can play a role in maintaining immune homeostasis during type 2-biased inflammation. In addition, potentially beneficial changes in the microbiome induced by dietary probiotics may be counteracted by helminths, highlighting the complex inter-relationships that potentially exist between probiotic bacteria and intestinal parasites.

sentences: Biochemical propensity mapping for structural and functional anatomy of importin α IBB domain Importin  has been described as a nuclear protein transport receptor that enables proteins synthesized in the cytoplasm to translocate into the nucleus. Besides its function in nuclear transport, an increasing number of studies have examined its non-nuclear transport functions. In both nuclear transport and non-nuclear transport, a functional domain called the IBB domain (importin {beta} binding domain) plays a key role in regulating importin  behavior, and is a common interacting domain for multiple binding partners. However, it is not yet fully understood how the IBB domain interacts with multiple binding partners, which leads to the switching of importin  function. In this study, we have distinguished the location and propensities of amino acids important for each function of the importin  IBB domain by mapping the biochemical/physicochemical propensities of evolutionarily conserved amino acids of the IBB domain onto the structure associated with each function. We found important residues that are universally conserved for IBB functions across organisms and families, in addition to those previously known, as well as residues that are presumed to be responsible for the differences in complex-forming ability between families and for functional switching.

sentences: Comparing Imaging Biomarkers of Cerebral Edema after TBI in Young Adult Male and Female Rats Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide. Cerebral edema following TBI is known to play a critical role in injury severity and prognosis. In the current study we used multimodal magnetic resonance imaging (MRI) to assess cerebral edema 24 hours after unilateral contusive TBI in male and female rats. We then directly quantified brain water content in the same subjects ex vivo. We found that both males and females had similarly elevated T2 values after TBI compared with sham controls. Apparent diffusion coefficient (ADC) was more variable than T2 and did not show significant injury effects in males or females. Brain water was elevated in male TBI rats compared with sham controls, but there was no difference between female TBI and sham groups. Notably, MRI biomarkers of edema were more closely correlated with brain water in male rats; female rats did not show any relationship between brain water and T2 or ADC. These observations raise questions about the interpretation of radiological findings traditionally interpreted as edema in female TBI patients. A better understanding of sex differences and similarities in the pathophysiology of post-traumatic edema is needed to help improve patient management and the development of effective treatment strategies for men and women.

sentences: Parameter inference on brain network models with unknown node dynamics and spatial heterogeneity Model-based data analysis of whole-brain dynamics links the observed data to model parameters in a network of neural masses. In recent years a special focus was placed on the role of regional variance of model parameters for the emergent activity. Such analyses however depend on the properties of the employed neural mass model, which is often obtained through a series of major simplifications or analogies. Here we propose a data-driven approach where the neural mass model needs not to be specified. Building on the recent progresses in identification of dynamical systems with neural networks, we propose a method to infer from the functional data both the neural mass model representing the regional dynamics as well as the region- and subject-specific parameters, while respecting the known network structure. We demonstrate on two synthetic data sets that our method is able to recover the original model parameters, and that the trained generative model produces dynamics resembling the training data both on the regional level and on the whole-brain level. We further apply the method to resting-state fMRI data from Human Connectome Project. We find that to achieve best fit, the model needs two dimensional state space and three regional parameters, one of which is strongly correlated with the map of genetic expression in human brain. The present approach opens a novel way to the analysis of resting-state fMRI with possible applications in understanding the changes of whole-brain dynamics during aging or in neurodegenerative diseases.

sentences: SLIMP: Supervised learning of metabolite-protein interactions from co-fractionation mass spectrometry data Metabolite-protein interactions affect and shape diverse cellular processes. Yet, despite advances, approaches for identifying metabolite-protein interactions at a genome-wide scale are lacking. Here we present an approach termed SLIMP that predicts metabolite-protein interactions using supervised machine learning on features engineered from metabolic and proteomic profiles from a co-fractionation mass spectrometry-based technique. By applying SLIMP with gold standards, assembled from public databases, along with metabolic and proteomic data sets from multiple conditions and growth stages we predicted over 9,000 and 20,000 metabolite-protein interactions for Saccharomyces cerevisiae and Arabidopsis thaliana, respectively. Extensive comparative analyses corroborated the quality of the predictions from SLIMP with respect to widely-used performance measures (e.g. F1-score exceeding 0.8). SLIMP predicted novel targets of 2, 3 cyclic nucleotides and dipeptides, which we analysed comparatively between the two organisms. Finally, predicted interactions for the dipeptide Tyr-Asp in Arabidopsis and the dipeptide Ser-Leu in yeast were independently validated, opening the possibility for future applications of supervised machine learning approaches in this area of systems biology.

sentences: Exploration of molecular mechanism underlying the self-flocculation of Zymomonas mobilis through comparative omics analyses and experimental validations for developing robust production systems Zymomonas mobilis metabolizes sugar through the Entner-Doudoroff pathway with less ATP generated for lower biomass accumulation and more substrate to product formation with improved yield, since ATP is dissipated predominately through growth for intracellular energy homeostasis, making it a platform to be engineered as microbial cell factories, particularly for producing bulk commodities with major cost from feedstock consumption. ZM401, a self-flocculating mutant, presents advantages for production including cost-effective biomass recovery through gravity sedimentation, self-immobilization within bioreactors for high cell density to improve productivity and enhanced tolerance to environmental stresses for high product titers, but molecular mechanism underlying this phenotype is largely unknown. In this work, we sequenced and assembled the genome of ZM401 to explore genetic basis for the self-flocculation of the bacterial cells through comparative genomic and transcriptomic analyses, molecular docking simulations for enzymes encoded by functional genes and their substrates/activators, and experimental validations. Our results demonstrated that the single nucleotide deletion in ZMO1082 disrupted its stop codon for the putative gene being fused with ZMO1083, which created an exciting gene encoding the subunit A of the bacterial cellulose synthase with unique function for synthesizing cellulose microfibrils to flocculate the bacterial cells, and the single nucleotide mutation in ZMO1055 compromised the function of bifunctional diguanylate cyclase/phosphodiesterase encoded by the gene on the degradation of c-di-GMP for its intracellular accumulation to activate the cellulose biosynthesis. These discoveries are significant not only for optimizing the self-flocculation of Z. mobilis, but also engineering other bacteria with the self-flocculating phenotype for robust production.

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Orthogonal control of gene expression in plants using synthetic promoters and CRISPR-based transcription factors BackgroundThe construction and application of synthetic genetic circuits is frequently improved if gene expression can be orthogonally controlled, relative to the host. In plants, orthogonality can be achieved via the use of CRISPR-based transcription factors that are programmed to act on natural or synthetic promoters. The construction of complex gene circuits can require multiple, orthogonal regulatory interactions, and this in turn requires that the full programmability of CRISPR elements be adapted to non-natural and non-standard promoters that have few constraints on their design. Therefore, we have developed synthetic promoter elements in which regions upstream of the minimal 35S CaMV promoter are designed from scratch to interact via programmed gRNAs with dCas9 fusions that allow activation of gene expression.

ResultsA panel of three, mutually orthogonal promoters that can be acted on by artificial gRNAs bound by CRISPR regulators were designed. Guide RNA expression targeting these promoters was in turn controlled by either Pol III (U6) or ethylene-inducible Pol II promoters, implementing for the first time a fully artificial Orthogonal Control System (OCS). Following demonstration of the complete orthogonality of the designs, the OCS was tied to cellular metabolism by putting gRNA expression under the control of an endogenous plant signaling molecule, ethylene. The ability to form complex circuitry was demonstrated via the ethylene-driven, ratiometric expression of fluorescent proteins in single plants.

ConclusionsThe design of synthetic promoters is highly generalizable to large tracts of sequence space, allowing Orthogonal Control Systems of increasing complexity to potentially be generated at will. The ability to tie in several different basal features of plant molecular biology (Pol II and Pol III promoters, ethylene regulation) to the OCS demonstrates multiple opportunities for engineering at the system level. Moreover, given the fungibility of the core 35S CaMV promoter elements, the derived synthetic promoters can potentially be utilized across a variety of plant species.

sentences: Perchlorate-Specific Proteomic Stress Responses of Debaryomyces hansenii Could Enable Microbial Survival in Martian Brines If life exists on Mars, it would face several challenges including the presence of perchlorates, which destabilize biomacromolecules by inducing chaotropic stress. However, little is known about perchlorate toxicity for microorganism on the cellular level. Here we present the first proteomic investigation on the perchlorate-specific stress responses of the halotolerant yeast Debaryomyces hansenii and compare these to generally known salt stress adaptations. We found that the responses to NaCl and NaClO4-induced stresses share many common metabolic features, e.g., signaling pathways, elevated energy metabolism, or osmolyte biosynthesis. However, several new perchlorate-specific stress responses could be identified, such as protein glycosylation and cell wall remodulations, presumably in order to stabilize protein structures and the cell envelope. These stress responses would also be relevant for life on Mars, which - given the environmental conditions - likely developed chaotropic defense strategies such as stabilized confirmations of biomacromolecules and the formation of cell clusters.

sentences: ARGONAUTE10 is required for cell fate specification and the control of formative cell divisions in the Arabidopsis root meristem A key question in plant biology is how oriented cell divisions are integrated with patterning mechanisms to generate organs with adequate cell type allocation. In the root vasculature, a miRNA gradient controls the abundance of HD-ZIP III transcription factors, which in turn control cell fate and spatially restrict vascular cell proliferation to specific cells. Here, we show that a functional miRNA gradient requires an opposing gradient of ARGONAUTE10, which sequesters miRNAs to protect HD-ZIP III transcripts from degradation. In the absence of ARGONAUTE10, xylem precursor cells undergo periclinal divisions that lead to continuous strands of differentiated xylem elements at ectopic positions. Notably, periclinal daughter cells maintain xylem identity even when they are located outside of the xylem axis, resulting in disrupted tissue boundaries. We further demonstrate that ARGONAUTE10 and HD-ZIP IIIs buffer cytokinin signalling to control formative cell divisions, providing a framework for integration of phytohormone and miRNA-mediated patterning.

sentences: Functional analysis of the polar amino acid in TatA transmembrane helix The twin-arginine translocation (Tat) pathway utilizes the proton-motive force (pmf) to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N-terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, a complete set of 19-amino-acid substitutions was examined. Unexpectedly, although being preferred overall, our experiments suggest that the polar amino acid is not necessary for a functional TatA. Hydrophobicity and helix stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophobicity, which was demonstrated to impact the Tat transport activity. Furthermore, a four-residue motif at the N-terminus of the TatA TMH was identified by sequence alignment. Using a biochemical approach, the N-terminal motif was found to be functionally significant, with evidence indicating a potential role in the preference for utilizing different pmf components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism.

sentences: Generation and analysis of context-specific genome-scale metabolic models derived from single-cell RNA-Seq data Single-cell RNA sequencing has the potential to unravel the differences in metabolism across cell types and cell states in both the healthy and diseased human body. The use of existing knowledge in the form of genome-scale metabolic models (GEMs) holds promise to strengthen such analyses, but the combined use of these two methods requires new computational methods. Here, we present a method for generating cell-type-specific genome-scale models from clusters of single-cell RNA-Seq profiles. Specifically, we developed a method to estimate the number of cells required to pool to obtain stable models, a bootstrapping strategy for estimating statistical inference, and a faster version of the tINIT algorithm for generating context-specific GEMs. In addition, we evaluated the effect of different RNA-Seq normalization methods on model topology and differences in models generated from single-cell and bulk RNA-Seq data. We applied our methods on data from mouse cortex neurons and cells from the tumor microenvironment of lung cancer and in both cases found that almost every cell subtype had a unique metabolic profile, emphasizing the need to study them separately rather than to build models from bulk RNA-Seq data. In addition, our approach was able to detect cancer-associated metabolic differences between cancer cells and healthy cells, showcasing its utility. With the ever-increasing availability of single-cell RNA-Seq datasets and continuously improved GEMs, their combination holds promise to become an important approach in the study of human metabolism.

sentences: Behavioral rebound following isolation of attention Stable sensory perception is achieved through balanced excitatory-inhibitory interactions of lateralized sensory processing. In real world experience, sensory processing is rarely equal across lateralized processing regions, resulting in continuous rebalancing. Using lateralized attention as a case study, we predicted rebalancing lateralized processing following prolonged spatial attention imbalance could cause a gain in attention the opposite direction. In neurotypical human adults, we isolated covert attention to one visual field with a 30-minute attention-demanding task and found an increase in attention in the opposite visual field after manipulation. We suggest a gain in lateralized attention in the previously unattended visual field is due to an overshoot through attention rebalancing. The offline post-manipulation effect is suggestive of long-term potentiation affecting behavior. Our finding of visual field specific attention rebound could be critical for the development of clinical rehabilitation for patients with a unilateral lesion and lateralized attention deficits. This proof-of-concept study initiates the examination of overshoot following the release of imbalance in other lateralized control and sensory domains, important in our basic understanding of lateralized processing.

sentences: Molecular characterization of a complex of Apoptosis Inducing Factor 1 (AIFM1) with cytochrome c oxidase of the mitochondrial respiratory chain Combining mass spectrometry based chemical cross-linking and complexome profiling, we analyzed the interactome of heart mitochondria. We focused on complexes of oxidative phosphorylation and found that dimeric apoptosis inducing factor 1 (AIFM1) forms a defined complex with ~10% of monomeric cytochrome c oxidase (COX), but hardly interacts with respiratory chain supercomplexes. Multiple AIFM1 inter-crosslinks engaging six different COX subunits provided structural restraints to build a detailed atomic model of the COX-AIFM12 complex. Application of two complementary proteomic approaches thus provided unexpected insight into the macromolecular organization of the mitochondrial complexome. Our structural model excludes direct electron transfer between AIFM1 and COX. Notably however, the binding site of cytochrome c remains accessible allowing formation of a ternary complex. The discovery of the previously overlooked COX-AIFM12 complex and clues provided by the structural model hint at a role of AIFM1 in OXPHOS biogenesis and in programmed cell death.

sentences: Subcortical Circuits Mediate Communication Between Primary Sensory Cortical Areas Integration of information across the senses is critical for perception and is a common property of neurons in the cerebral cortex, where it is thought to arise primarily from corticocortical connections. Much less is known about the role of subcortical circuits in shaping the multisensory properties of cortical neurons. We show that stimulation of the whiskers causes widespread suppression of sound-evoked activity in mouse primary auditory cortex (A1). This suppression depends on the primary somatosensory cortex (S1), and is implemented through a descending circuit that links S1, via the auditory midbrain, with thalamic neurons that project to A1. Furthermore, a direct pathway from S1 has a facilitatory effect on auditory responses in higher-order thalamic nuclei that project to other brain areas. Crossmodal corticofugal projections to the auditory midbrain and thalamus therefore play a pivotal role in integrating multisensory signals and in enabling communication between different sensory cortical areas.

sentences: Robust and efficient online auditory psychophysics with the right auditory hygiene Most human auditory psychophysics research has historically been conducted in carefully controlled environments with calibrated audio-equipment, and over potentially hours of repetitive testing with expert listeners. Here, we operationally define such conditions as having high  auditory hygiene. Hereof, conducting auditory psychophysical paradigms online presents a serious challenge, in that results may hinge on absolute sound presentation level, reliably estimated perceptual thresholds, and sustained motivation and attention. We introduce a set of procedures that address these challenges and facilitate auditory hygiene for online auditory psychophysics. First, we establish a simple means of setting sound presentation levels. Across a set of four level-setting conditions conducted in person, we demonstrate the stability and robustness of this amplitude setting procedure in open air and controlled settings. Second, we test participants tone-in-noise thresholds using widely adopted online experiment platforms and demonstrate that reliable threshold estimates can be derived online in approximately one minute of testing. Third, using these amplitude and threshold setting procedures to establish participant-specific stimulus conditions, we show that an online implementation of the classic probe-signal paradigm can be used to demonstrate frequency-selective attention on an individual participant basis, using a third of the trials used in recent in-lab experiments. Finally, we show how threshold and attentional measures relate to well-validated assays of online participants in-task motivation, fatigue, and confidence. This demonstrates the promise of online auditory psychophysics for asking new auditory neuroscience questions quickly, efficiently, and with more diverse samples. Code for the tests is publicly available through Pavlovia and Gorilla.

sentences: Differential regulation of developmental stages supports a linear model for C. elegans postembryonic development The repetitive nature of C. elegans postembryonic development is considered an oscillatory process, a concept that has gained traction from regulation by a circadian clock gene homolog. Nevertheless, each larval stage has a defined duration and entails specific events. We have measured the duration of each stage of development for over 2,500 larvae, under varied environmental conditions known to alter overall developmental rate. Our results show that distinct developmental stages respond differently to environmental perturbations, including changes in temperature, in food quantity and quality, and amount of insulin signaling. Furthermore, our high-resolution measurement of the effect of temperature on the stage-specific duration of development has unveiled novel features of temperature dependence in C. elegans postembryonic development. Altogether, our results support a model of linear progression of C. elegans development, with the duration of each stage determined by a unique program.

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A Kiosk Station for the Assessment of Multiple Cognitive Domains and Enrichment of Monkeys BackgroundNonhuman primates (NHPs) are self-motivated to perform cognitive tasks on touchscreens in their animal housing setting. To leverage this ability, fully integrated hardware and software solutions are needed, that work within housing and husbandry routines while also spanning cognitive task constructs of the Research Domain Criteria (RDoC).

New MethodWe describe a Kiosk Station (KS-1) that provides robust hardware and software solutions for running cognitive tasks in cage-housed NHPs. KS-1 consists of a frame for mounting flexibly on housing cages, a touchscreen animal interface with mounts for receptables, reward pumps and cameras, and a compact computer cabinet with an interface for controlling behavior. Behavioral control is achieved with a unity3D program that is virtual-reality capable, allowing semi-naturalistic visual tasks to assess multiple cognitive domains.

ResultsKS-1 is fully integrated into the regular housing routines of monkeys. A single person can operate multiple KS-1s. Monkeys engage with KS-1 at high motivation and cognitive performance levels at high intra-individual consistency.

Comparison with Existing MethodsKS-1 is optimized for flexible mounting onto standard apartment cage systems. KS-1 has a robust animal interface with options for gaze/reach monitoring. It has an integrated user interface for controlling multiple cognitive task using a common naturalistic object space designed to enhance task engagement. All custom KS-1 components are open-sourced.

ConclusionsKS-1 is a versatile tool for cognitive profiling and enrichment of cage-housed monkeys. It reliably measures multiple cognitive domains which promises to advance our understanding of animal cognition, inter-individual differences and underlying neurobiology in refined, ethologically meaningful behavioral foraging contexts.

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Microtubule dynamics and the evolution of mitochondrial populations in fission yeast cells: A kinetic Monte Carlo study Mitochondrial populations in cells are maintained by cycles of fission and fusion events. Perturbation of this balance has been observed in several diseases such as cancer and neurodegeneration. In fission yeast cells, the association of mitochondria with microtubules inhibits mitochondrial fission, [1] illustrating the intricate coupling between mitochondria and the dynamic population of microtubules within the cell. In order to understand this coupling, we carried out kinetic Monte Carlo (KMC) simulations to predict the evolution of mitochondrial size distributions for different cases; wild-type cells, cells with short and long microtubules, and cells without microtubules. Comparison are made with mitochondrial distributions reported in experiments with fission yeast cells. Using experimentally determined mitochondrial fission and fusion frequencies, simulations implemented without the coupling of microtubule dynamics predicted an increase in the mean number of mitochondria, equilibrating within 50 s. The mitochondrial length distribution in these models also showed a higher occurrence of shorter mitochondria, implying a greater tendency for fission, similar to the scenario observed in the absence of microtubules and cells with short microtubules. Interestingly, this resulted in overestimating the mean number of mitochondria and underestimating mitochondrial lengths in cells with wild-type and long microtubules. However, coupling mitochondrias fission and fusion events to the microtubule dynamics effectively captured the mitochondrial number and size distributions in wild-type and cells with long microtubules. Thus, the model provides greater physical insight into the temporal evolution of mitochondrial populations in different microtubule environments, allowing one to study both the short-time evolution as observed in the experiments (<5 minutes) as well as their transition towards a steady-state (>15 minutes). Our study illustrates the critical role of microtubules in mitochondrial dynamics and that coupling their growth and shrinkage dynamics is critical to predicting the evolution of mitochondrial populations within the cell.

Author summaryMitochondria are semi-autonomous organelles that undergo fission and fusion to facilitate quality control and exchange of mitochondrial mass within the cell. Impaired mitochondrial fusion and fission dynamics are associated with disease states such as cancer and neurodegeneration. Recent experiments in fission yeast cells revealed a reduction in mitochondrial fission events when mitochondria were bound to the microtubules and longer microtubules shifted the mitochondrial population to longer lengths. In a distinct departure from earlier reports [2-16], we develop a generic framework to study the evolution of the mitochondrial population in fission yeast cells to predict the observed mitochondrial population by coupling the microtubule and mitochondrial dynamics. Using kinetic Monte Carlo (KMC) simulations we predict the temporal evolution of mitochondria in both the mutated and wild-type states of microtubules in fission yeast cells. The mitochondrial population evolves due to multiple fission and fusion reactions occurring between mitochondrial species of various lengths. Several models with varying complexity have been developed to study mitochondrial evolution, and predictions of the mitochondrial populations agree well with experimental data on fission yeast cells without microtubules and cells with short, wild-type and long microtubules. These set of microtubule states are consistent with not only the microtubule dynamics typically observed in cells under different physiological stimuli such as mitosis and disease states but also the stable microtubule states obtained through post-translational modification of  and {beta} tubulin subunits of microtubules. Our study reveals that the temporal evolution of mitochondrial populations is an intrinsic function of the state of microtubules which modulates the fission and fusion frequencies to maintain mitochondrial homeostasis within cells.

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Gut microbial ecology of Xenopus tadpoles across life stages BackgroundThe microorganism world living in amphibians is still largely under-represented and under-studied in the literature. Among anuran amphibians, African clawed frogs of the Xenopus genus stand as well-characterized models with an in-depth knowledge of their developmental biological processes including their metamorphosis. In this study, we analyzed the succession of microbial communities and their activities across diverse body habitats of Xenopus tropicalis using different approaches including flow cytometry and 16s rDNA gene metabarcoding. We also evaluated the metabolic capacity of the premetamorphic tadpoles gut microbiome using metagenomic and metatranscriptomic sequencing.

ResultsWe analyzed the bacterial components of the Xenopus gut microbiota, the adult gut biogeography, the succession of communities during ontogeny, the impact of the alimentation in shaping the tadpoles gut bacterial communities and the transmission of skin and fecal bacteria to the eggs. We also identified the most active gut bacteria and their metabolic contribution to tadpole physiology including carbohydrate breakdown, nitrogen recycling, essential amino-acids and vitamin biosynthesis.

ConclusionsWe present a comprehensive new microbiome dataset of a laboratory amphibian model. Our data provide evidences that studies on the Xenopus tadpole model can shed light on the interactions between a vertebrate host and its microbiome. We interpret our findings in light of bile acids being key molecular components regulating the gut microbiome composition during amphibian development and metamorphosis. Further studies into the metabolic interactions between amphibian tadpoles and their microbiota during early development and metamorphosis should provide useful information on the evolution of host-microbiota interactions in vertebrates.

This article has been peer-reviewed and recommended by Peer Community in Genomics https://doi.org/10.24072/pci.genomics.100005

sentences: Fine-tuning of deep language models as a computational framework of modeling listeners' perspective during language comprehension Computational Deep Language Models (DLMs) have been shown to be effective in predicting neural responses during natural language processing. This study introduces a novel computational framework, based on the concept of fine-tuning (Hinton, 2007), for modeling differences in interpretation of narratives based on the listeners perspective (i.e. their prior knowledge, thoughts, and beliefs). We draw on an fMRI experiment conducted by Yeshurun et al. (2017), in which two groups of listeners were listening to the same narrative but with two different perspectives (cheating versus paranoia). We collected a dedicated dataset of ~3000 stories, and used it to create two modified (fine-tuned) versions of a pre-trained DLM, each representing the perspective of a different group of listeners. Information extracted from each of the two fine-tuned models was better fitted with neural responses of the corresponding group of listeners. Furthermore, we show that the degree of difference between the listeners interpretation of the story - as measured both neurally and behaviorally - can be approximated using the distances between the representations of the story extracted from these two fine-tuned models. These models-brain associations were expressed in many language-related brain areas, as well as in several higher-order areas related to the default-mode and the mentalizing networks, therefore implying that computational fine-tuning reliably captures relevant aspects of human language comprehension across different levels of cognitive processing.

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Angiomotin Counteracts the Negative Regulatory Effect of Host WWOX on Viral PPxY-Mediated Egress Filoviridae family members Ebola (EBOV) and Marburg (MARV) viruses and Arenaviridae family member Lassa virus (LASV) are emerging pathogens that can cause hemorrhagic fever and high rates of mortality in humans. A better understanding of the interplay between these viruses and the host will inform about the biology of these pathogens, and may lead to the identification of new targets for therapeutic development. Notably, expression of the filovirus VP40 and LASV Z matrix proteins alone drives assembly and egress of virus-like particles (VLPs). The conserved PPxY Late (L) domain motifs in the filovirus VP40 and LASV Z proteins play a key role in the budding process by mediating interactions with select host WW-domain containing proteins that then regulate virus egress and spread. To identify the full complement of host WW-domain interactors, we utilized WT and PPxY mutant peptides from EBOV and MARV VP40 and LASV Z proteins to screen an array of GST-WW-domain fusion proteins. We identified WW domain-containing oxidoreductase (WWOX) as a novel PPxY-dependent interactor, and we went on to show that full-length WWOX physically interacts with eVP40, mVP40 and LASV Z to negatively regulate egress of VLPs and of a live VSV/Ebola recombinant virus (M40). Interestingly, WWOX is a versatile host protein that regulates multiple signaling pathways and cellular processes via modular interactions between its WW-domains and PPxY motifs of select interacting partners, including host angiomotin (AMOT). Notably, we demonstrated recently that expression of endogenous AMOT not only positively regulates egress of VLPs, but also promotes egress and spread of live EBOV and MARV. Toward the mechanism of action, we show that the competitive and modular interplay among WWOX-AMOT-VP40/Z regulates VLP and M40 virus egress. Thus, WWOX is the newest member of an emerging group of host WW-domain interactors (e.g. BAG3; YAP/TAZ) that negatively regulate viral egress. These findings further highlight the complex interplay of virus-host PPxY/WW-domain interactions and their potential impact on the biology of both the virus and the host during infection.

Author SummaryFiloviruses (Ebola [EBOV] and Marburg [MARV]) and arenavirus (Lassa virus; LASV) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we identified host WW-domain containing protein WWOX as a novel interactor with VP40 and Z, and showed that WWOX inhibited budding of VP40/Z virus-like particles (VLPs) and live virus in a PPxY/WW-domain dependent manner. Our findings are important to the field as they expand the repertoire of host interactors found to regulate PPxY-mediated budding of RNA viruses, and further highlight the competitive interplay and modular virus-host interactions that impact both the virus lifecycle and the host cell.

sentences: Target-specific control of olfactory bulb periglomerular cells by GABAergic and cholinergic basal forebrain inputs The olfactory bulb (OB), the first relay for odor processing, receives dense GABAergic and cholinergic long-range projections from basal forebrain (BF) nuclei that provide information about the internal state and behavioral context of the animal. However, the targets, impact and dynamics of these afferents are still unclear. I studied how BF synaptic inputs modulate activity in diverse subtypes of periglomerular (PG) interneurons using optogenetic stimulation and loose cell-attached or whole-cell patch-clamp recording in OB slices from adult mice. GABAergic BF inputs potently blocked PG cells firing except in a minority of calretinin-expressing cells in which GABA release elicited spiking. Parallel cholinergic projections excited a previously overlooked PG cell subtype via synaptic activation of M1 muscarinic receptors. Low frequency stimulation of the cholinergic axons drove persistent firing in these PG cells thereby increasing tonic inhibition in principal neurons. Taken together, these findings suggest that modality-specific BF inputs can orchestrate inhibition in OB glomeruli using multiple, potentially independent, inhibitory or excitatory target-specific pathways.

sentences: Non-specific adhesive forces between filaments and membraneless organelles Membraneless organelles are liquid-like domains that form inside living cells by phase-separation. While standard physical models of their formation assume their surroundings to be a simple liquid, the cytoplasm is an active viscoelastic environment. To investigate potential coupling of phase separation with the cytoskeleton, we quantify structural correlations of stress granules and microtubules in a human-derived epithelial cell line. We find that microtubule networks are significantly perturbed in the vicinity of stress granules, and that large stress granules conform to the local pore-structure of the microtubule network. When microtubules are depolymerized by nocodazole, tubulin enrichment is localized near the surface of stress granules. We interpret these data using a thermodynamic model of partitioning of particles to the surface and bulk of droplets. This analysis shows that proteins generically have a non-specific affinity for droplet interfaces, which becomes most apparent when they weakly partition to the bulk of droplets and have a large molecular weight. In this framework, our data is consistent with a weak ([lsim] kbT) affinity of tubulin sub-units for stress granule interfaces. As microtubules polymerize their affinity for interfaces increases, providing sufficient adhesion to deform droplets and/or the network. We validate this basic physical phenomena in vitro through the interaction of a simple protein-RNA condensate with tubulin and microtubules.

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Histone demethylase UTX regulates glioblastoma progression through affecting periostin expression The histone H3K27 demethylase UTX participates in regulating multiple cancer types. However, less is known about the UTX function in glioblastoma (GBM). This study aims to define the effect of UTX on GBM. GEPIA2 database analysis showed that UTX expression was significantly increased in GBM and inversely correlated with survival. Knockdown UTX inhibited GBM cell proliferation, migration, and invasion while promoting apoptosis. Moreover, knockdown UTX also hampered tumor growth in the heterotopic xenograft model. RNA-seq combined with qRT-PCR and ChIP-qPCR were used to identify the target genes. The results showed that the UTX-mediated genes were strongly associated with tumor progression and the extracellular environment. Protein-protein interaction analysis suggested that periostin (POSTN) interacted with most of the other UTX-mediated genes. POSTN supplement abolished the effect of UTX knockdown in GBM cells. Furthermore, silencing UTX exhibited similar antitumor effect in patient-derived glioblastoma stem-like cells, while UTX functions were partially restored after exposing POSTN. Our findings may reveal a new insight into the onset of gliomagenesis and progression, providing a promising therapeutic strategy for GBM treatment.

Bullet PointsUTX correlates with survival in glioblastoma. Silencing UTX decreased the levels of H3K27 methylation in the POSTN gene, thereby suppressing POSTN expression. UTX-mediated POSTN expression is crucial for glioblastoma cell growth and tumorigenesis.

O_LIUTX expression is increased in GBM and negatively correlated with survival.
C_LIO_LIUTX knockdown influences proliferation and apoptosis in both GBM cells and GSCs.
C_LIO_LIThe antitumor effect of UTX knockdown is achieved by suppressing POSTN expression.
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sentences: Direct RNA sequencing of Respiratory Syncytial Virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing on an Oxford nanopore device. This yielded over 150,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We were also able to examine frequencies with which polycistronic readthrough mRNAs were generated and to assess the length of the polyadenylated tails for each group of transcripts. We show that there is a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not consistent. We show that the polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host cells polyadenylation machinery and broadly declined in length for most transcripts as infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails is much less for N, SH and G transcripts compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates.

sentences: Efemp1 modulates elastic fiber formation and mechanics of the extrahepatic bile duct EGF-Containing Fibulin Extracellular Matrix Protein 1 (EFEMP1, also called fibulin 3) is an extracellular matrix protein linked in a genome-wide association study to biliary atresia, a fibro-inflammatory disease of the neonatal extrahepatic bile duct. EFEMP1 is expressed in most tissues and Efemp1 null mice have decreased elastic fibers in visceral fascia; however, in contrast to other short fibulins (fibulins 4 and 5), EFEMP1 does not have a role in the development of large elastic fibers, and its overall function remains unclear. We demonstrated that EFEMP1 is expressed in the submucosa of both neonatal and adult mouse and human extrahepatic bile ducts and that, in adult Efemp1+/- mice, elastin organization into fibers is decreased. We used pressure myography, a technique developed to study the mechanics of the vasculature, to show that Efemp1+/- extrahepatic bile ducts are more compliant to luminal pressure, leading to increased circumferential stretch. We conclude that EFEMP1 has an important role in the formation of elastic fibers and mechanical properties of the extrahepatic bile duct. These data suggest that altered expression of EFEMP1 in the extrahepatic bile duct leads to an abnormal response to mechanical stress such as obstruction, potentially explaining the role of EFEMP1 in biliary atresia.

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Heat-treated virus inactivation rate depends strongly on treatment procedure: illustration with SARS-CoV-2 Decontamination helps limit environmental transmission of infectious agents. It is required for the safe re-use of contaminated medical, laboratory and personal protective equipment, and for the safe handling of biological samples. Heat treatment is a common decontamination method, notably used for viruses. We show that for liquid specimens (here, solution of SARS-CoV-2 in cell culture medium), virus inactivation rate under heat treatment at 70{degrees}C can vary by almost two orders of magnitude depending on the treatment procedure, from a half-life of 0.86 min (95% credible interval: [0.09, 1.77]) in closed vials in a heat block to 37.00 min ([12.65, 869.82]) in uncovered plates in a dry oven. These findings suggest a critical role of evaporation in virus inactivation via dry heat. Placing samples in open or uncovered containers may dramatically reduce the speed and efficacy of heat treatment for virus inactivation. Given these findings, we reviewed the literature temperature-dependent coronavirus stability and found that specimen containers, and whether they are closed, covered, or uncovered, are rarely reported in the scientific literature. Heat-treatment procedures must be fully specified when reporting experimental studies to facilitate result interpretation and reproducibility, and must be carefully considered when developing decontamination guidelines.

ImportanceHeat is a powerful weapon against most infectious agents. It is widely used for decontamination of medical, laboratory and personal protective equipment, and for biological samples. There are many methods of heat treatment, and methodological details can affect speed and efficacy of decontamination. We applied four different heat-treatment procedures to liquid specimens containing SARS-CoV-2. Our results show that the container used to store specimens during decontamination can substantially affect inactivation rate: for a given initial level of contamination, decontamination time can vary from a few minutes in closed vials to several hours in uncovered plates. Reviewing the literature, we found that container choices and heat treatment methods are only rarely reported explicitly in methods sections. Our study shows that careful consideration of heat-treatment procedure -- in particular the choice of specimen container, and whether it is covered -- can make results more consistent across studies, improve decontamination practice, and provide insight into the mechanisms of virus inactivation.

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Brain networks in human conscious visual perception Consciousness is not explained by a single mechanism, rather it involves multiple specialized neural systems overlapping in space and time. We hypothesize that synergistic, large-scale subcortical and cortical attention and signal processing networks encode conscious experiences. To identify brain activity in conscious perception without overt report, we classified visual stimuli as perceived or not using eye measurements. Report-independent event-related potentials and functional magnetic resonance imaging (fMRI) signals both occurred at early times after stimuli. Direct recordings revealed a novel thalamic awareness potential linked to conscious visual perception based on report. fMRI showed thalamic and cortical detection, arousal, attentional salience, task-positive, and default mode networks were involved independent of overt report. These findings identify a specific sequence of neural mechanisms in human conscious visual perception.

One-Sentence SummaryHuman conscious visual perception engages large-scale subcortical and cortical networks even without overt report.

sentences: No evidence for somatosensory attenuationduring action observation of self-touch The discovery of mirror neurons in the macaque brain in the 1990s triggered investigations on putative human mirror neurons and their potential functionality. The leading proposed function has been action understanding: accordingly, we understand the actions of others by  simulating them in our own motor system through a direct matching of the visual information to our own motor programs. Furthermore, it has been proposed that this simulation involves the prediction of the sensory consequences of the observed action, similar to the prediction of the sensory consequences of our executed actions. Here, we tested this proposal by quantifying somatosensory attenuation behaviorally during action observation. Somatosensory attenuation manifests during voluntary action and refers to the perception of self-generated touches as less intense than identical externally generated touches because the self-generated touches are predicted from the motor command. Therefore, we reasoned that if an observer simulates the observed action and, thus, he/she predicts its somatosensory consequences, then he/she should attenuate tactile stimuli simultaneously delivered to his/her corresponding body part. In three separate experiments, we found a systematic attenuation of touches during executed self-touch actions, but we found no evidence for attenuation when such actions were observed. Failure to observe somatosensory attenuation during observation of self-touch is not compatible with the hypothesis that the putative human mirror neuron system automatically simulates the observed action. In contrast, our findings emphasize a sharp distinction between the motor representations of self and others.

sentences: Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa. Single molecule RNA-FISH (smFISH) is a valuable tool for analysis of mRNA spatial patterning in fixed cells that is underutilized in filamentous fungi. A primary complication for fixed-cell imaging in filamentous fungi is the need for enzymatic cell wall permeabilization, which is compounded by considerable variability in cell wall composition between species. smFISH adds another layer of complexity due to a requirement for RNase free conditions. Here, we describe the cloning, expression, and purification of a chitinase suitable for supplementation of a commercially available RNase-free enzyme preparation for efficient permeabilization of the Neurospora cell wall. We further provide a method for smFISH in Neurospora which includes a tool for generating numerical data from images that can be used in downstream customized analysis protocols.

sentences: Candidatus Liberibacter asiaticus reduces callose and reactive oxygen species production in the phloem Huanglongbing (HLB) causes significant economic loss in citrus production worldwide. HLB is caused by Candidatus Liberibacter asiaticus (CLas), a gram-negative bacterium which inhabits the phloem exclusively. CLas infection results in accumulation of callose and reactive oxygen species in the phloem of infected plants, but little is known about the specific processes that take place during infection because of the sparse distribution of bacteria and the inaccessibility of the phloem inside the tree. In this study, we used the seed vasculatures, which accumulate a high number of CLas, as a model tissue to study CLas-host cellular interactions. In vasculature where CLas is abundant, sieve pore callose and H2O2 concentration were reduced compared to healthy seed vasculature. The expression of callose synthases (CalS) and respiratory burst oxidase homolog (RBOH) genes were downregulated in infected seeds compared to healthy ones. In leaves of HLB-infected plants, H2O2 concentration and CalS expression increased compared to uninfected leaves, but cells with CLas had lower levels of sieve plate callose compared to cells without CLas. Our results provide evidence that the bacteria manipulate cell metabolism to disable plant defenses and suggests that HLB disease is the result of a constant arms-race between the pathogen and a defense response, which is ultimately harmful to the host plant.

sentences: Molecular Basis of Antibiotic Self-Resistance in a Bee Larvae Pathogen Paenibacillus larvae, the causative agent of the devastating honey-bee disease American Foulbrood, produces the cationic polyketide-peptide hybrid paenilamicin that displays high antibacterial and antifungal activity. Its biosynthetic gene cluster contains a gene coding for the N-acetyltransferase PamZ. We show that PamZ acts as self-resistance factor in P. larvae by deactivation of paenilamicin. Using tandem MS, NMR spectroscopy and synthetic diastereomers, we identified the N-terminal amino group of the agmatinamic acid as the N-acetylation site. These findings highlight the pharmacophore region of paenilamicin, which we very recently identified as a new ribosome inhibitor. Here, we further elucidated the crystal structure of PamZ:acetyl-CoA complex at 1.34 [A] resolution. An unusual tandem-domain architecture provides a well-defined substrate-binding groove decorated with negatively-charged residues to specifically attract the cationic paenilamicin. Our results will help to understand the mode of action of paenilamicin and its role in pathogenicity of P. larvae to fight American Foulbrood.

sentences: Subjective confidence reflects representation of Bayesian probability in cortex What gives rise to the human sense of confidence? Here, we tested the Bayesian hypothesis that confidence is based on a probability distribution represented in neural population activity. We implemented several computational models of confidence, and tested their predictions using psychophysics and fMRI. Using a generative model-based fMRI decoding approach, we extracted probability distributions from neural population activity in human visual cortex. We found that subjective confidence tracks the shape of the decoded distribution. That is, when sensory evidence was more precise, as indicated by the decoded distribution, observers reported higher levels of confidence. We furthermore found that neural activity in the insula, anterior cingulate, and prefrontal cortex was linked to both the shape of the decoded distribution and reported confidence, in ways consistent with the Bayesian model. Altogether, our findings support recent statistical theories of confidence and suggest that probabilistic information guides the computation of ones sense of confidence.

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The assisting role of Polη in transcription facilitates formation of damage-induced cohesion The structural maintenance of chromosome (SMC) complex cohesin mediates sister chromatid cohesion established during replication, and damage-induced cohesion formed in response to DSBs post-replication. The translesion synthesis polymerase Pol{eta} is required for damage-induced cohesion through a hitherto unknown mechanism. Since Pol{eta} is functionally associated with transcription, and transcription triggers de novo cohesion in Schizosaccharomyces pombe, we hypothesized that transcription facilitates damage-induced cohesion in Saccharomyces cerevisiae. Here, we show dysregulated transcriptional profiles in Pol{eta}-depleted cells (rad30{Delta}), where genes involved in chromatin assembly and positive transcription regulation were downregulated. In addition, chromatin association of RNA polymerase II was reduced at promoters and coding regions in rad30{Delta} compared to WT cells, while occupancy of the H2A.Z variant (Htz1) at promoters was increased in rad30{Delta} cells. Perturbing histone exchange at promoters inactivated damage-induced cohesion, similarly to deletion of the RAD30 gene. Conversely, altering regulation of transcription elongation suppressed the deficient damage-induced cohesion in rad30{Delta} cells. These results indicate that Pol{eta} has an assisting role during the transcriptional process, which consecutively facilitates formation of damage-induced cohesion. This further suggests a potential linkage between regulation of transcription and formation of damage-induced cohesion after replication.

Author SummaryThe cohesin complex dynamically associates with chromosomes and holds sister chromatids together through cohesion established during replication. This ensures faithful chromosome segregation at anaphase. In budding yeast, DNA double strand breaks trigger sister chromatid cohesion even after replication. This so-called damage-induced cohesion is formed both close to the breaks, and genome-wide on undamaged chromosomes. The translesion synthesis polymerase eta (Pol{eta}) is specifically required for genome wide damage-induced cohesion. Although Pol{eta} is well characterized for its function in bypassing ultraviolet-induced DNA lesions, its mechanistic role in damage-induced cohesion is unclear. Here, we show that transcriptional regulation is perturbed in the absence of Pol{eta}. We propose that Pol{eta} could aid in chromatin association of RNA polymerase II through phosphorylation of the Pol{eta}-S14 residue, a non-canonical role of Pol{eta} which further facilitates formation of damage-induced cohesion genome wide. In addition, we observe the need of replication-independent nucleosome assembly/histone exchange for formation of damage-induced cohesion. This together provides new insight into formation of damage-induced cohesion after replication, which will be interesting to further explore.

sentences: Mapping lesion, structural disconnection, and functional disconnection to symptoms in semantic aphasia Patients with semantic aphasia have impaired control of semantic retrieval, often accompanied by executive dysfunction following left hemisphere stroke. Many but not all of these patients have damage to the left inferior frontal gyrus, important for semantic and cognitive control. Yet semantic and cognitive control networks are highly distributed, including posterior as well as anterior components. Accordingly, semantic aphasia might not only reflect local damage but also white matter structural and functional disconnection. Here we characterise the lesions and predicted patterns of structural and functional disconnection in individuals with semantic aphasia and relate these effects to semantic and executive impairment. Impaired semantic cognition was associated with infarction in distributed left- hemisphere regions, including in the left anterior inferior frontal and posterior temporal cortex. Lesions were associated with executive dysfunction within a set of adjacent but distinct left frontoparietal clusters. Performance on executive tasks was also associated with interhemispheric structural disconnection across the corpus callosum. In contrast, poor semantic cognition was associated with small left-lateralized structurally disconnected clusters, including in the left posterior temporal cortex. Little insight was gained from functional disconnection symptom mapping. These results demonstrate that while left- lateralized semantic and executive control regions are often damaged together in stroke aphasia, these deficits are associated with distinct patterns of structural disconnection, consistent with the bilateral nature of executive control and the left-lateralized yet distributed semantic control network.

sentences: Sex-specific association between urinary kisspeptin and pubertal development Kisspeptins are critical neuropeptides for puberty onset and progression, playing a pivotal role in the reactivation of the hypothalamus-pituitary-gonadal axis in late childhood. Despite their importance, little is known as to how kisspeptin peripheral concentrations are related to sexual maturation in humans, specially using non-invasive measures that allow more widespread testing. Using a cross-sectional design, we investigated whether peripheral kisspeptin concentrations, measured by enzyme-linked immunosorbent assays (ELISA) in two-hour retention midstream urine, are associated with developmental markers in 209 (120 girls) typically developing, 9 to 15-year-olds. Developmental variables were age, self-reported pubertal status using the Pubertal Development Scale, and saliva concentrations of hormones that indicate gonadal (testosterone) and adrenal (dehydroepiandrosterone sulfate) functioning. We found marked sexual differences in urine kisspeptins (controlled for body mass index and socioeconomic status). While concentrations were similar in both sexes until the age of around 12 years, in males there was a positive linear correlation with all developmental measures thereafter, while in girls, kisspeptin concentrations did not change. Of note, our results are in line with those of previous studies using more invasive methods (e.g. blood samples), which indicates that kisspeptin concentrations from two-hour retention midstream urine have potential in exploring sex-specific peripheral action of these peptides.

sentences: Dendritic signal integration in a Drosophila Mushroom Body Output Neuron (MBON) essential for learning and memory The ability to associate neutral stimuli with valence information and to store these associations as memories forms the basis for decision making. To determine the underlying computational principles, we build a realistic computational model of a central decision module within the Drosophila mushroom body (MB), the flys center for learning and memory. Our model combines the electron microscopy-based architecture of one MB output neuron (MBON-3), the synaptic connectivity of its 948 presynaptic Kenyon cells (KCs), and its in vivo membrane properties obtained from patch-clamp recordings. We show that this neuron is electrotonically compact and that synaptic input corresponding to simulated odor input robustly drives its spiking behavior. Therefore, sparse innervation by KCs can efficiently control and modulate MBON activity in response to learning with minimal requirements on the specificity of synaptic localization. This architecture allows efficient storage of large numbers of memories using the flexible stochastic connectivity of the circuit.

sentences: What happened when? Brain and behavioral responses to violated expectations about the structure and content of episodic memories Episodic memories are not static but can be modified on the basis of new experiences, potentially allowing us to make valid predictions in the face of an ever-changing environment. Recent research has identified prediction errors during retrieval as a possible trigger for such changes. In the present study, we used modified episodic cues to investigate whether different types of mnemonic prediction errors modulate brain activity and subsequent memory performance. Participants encoded different episodes which consisted of short toy stories. During a subsequent functional magnetic resonance imaging (fMRI) session, participants were then presented videos showing the original episodes, or modified versions thereof. In modified videos, either the order of two subsequent action steps was changed or an object was exchanged for another. Content modifications recruited inferior frontal, parietal, and temporo-occipital areas reflecting the processing of the new object information, while brain responses to structure modifications in right dorsal premotor and inferior frontal areas remained subthreshold. In a post-fMRI memory test, the participants tendency to accept modified episodes as originally encoded increased significantly when they had been presented modified versions already during the fMRI session. Our study provides valuable initial insights into the neural processing of different types of episodic prediction errors and their influence on subsequent memory.

sentences: Optimising expression quantitative trait locus mapping workflows for single-cell studies Single-cell RNA-sequencing (scRNA-seq) has enabled the unbiased, high-throughput quantification of gene expression specific to cell types and states. With the cost of scRNA-seq decreasing and techniques for sample multiplexing improving, population-scale scRNA-seq, and thus single-cell expression quantitative trait locus (sc-eQTL) mapping, is increasingly feasible. Mapping of sc-eQTL provides additional resolution to study the regulatory role of common genetic variants on gene expression across a plethora of cell types and states, and promises to improve our understanding of genetic regulation across tissues in both health and disease. While previously established methods for bulk eQTL mapping can, in principle, be applied to sc-eQTL mapping, there are a number of open questions about how best to process scRNA-seq data and adapt bulk methods to optimise sc-eQTL mapping. Here, we evaluate the role of different normalisation and aggregation strategies, covariate adjustment techniques, and multiple testing correction methods to establish best practice guidelines. We use both real and simulated datasets across single-cell technologies to systematically assess the impact of these different statistical approaches and provide recommendations for future single-cell eQTL studies that can yield up to twice as many eQTL discoveries as default approaches ported from bulk studies.

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Stonewall prevents expression of testis-enriched genes and binds to insulator elements in D. melanogaster Germline stem cells (GSCs) are the progenitor cells of the germline for the lifetime of an animal. In Drosophila, these cells reside in a cellular niche that is required for both their maintenance (self-renewal) and differentiation (asymmetric division resulting in a daughter cell that differs from the GSC). The stem cell-daughter cell transition is tightly regulated by a number of processes, including an array of proteins required for genome stability. The germline stem-cell maintenance factor Stonewall (Stwl) associates with heterochromatin, but its molecular function is poorly understood. We performed RNA-Seq on stwl mutant ovaries and found significant derepression of many transposon families but not heterochromatic genes. We also discovered that testis-enriched genes, including the differentiation factor bgcn and a large testis-specific cluster on chromosome 2, are upregulated or ectopically expressed in stwl mutant ovaries. Surprisingly, we also found that RNAi knockdown of stwl in somatic S2 cells results in ectopic expression of these genes.

Using parallel ChIP-Seq and RNA-Seq experiments in S2 cells, we discovered that Stwl binds upstream of transcription start sites and localizes to heterochromatic sequences. We also find that Stwl is enriched at repetitive sequences associated with telomeres. Finally, we identify Stwl binding motifs that are shared with known insulator binding proteins. We propose that Stwl affects gene regulation by binding insulators and establishing chromatin boundaries.

sentences: Goal-oriented predictive representations in the human hippocampus Recent work in cognitive and systems neuroscience has suggested that the hippocampus might support planning, imagination, and navigation by forming "cognitive maps" that capture the structure of physical spaces, tasks, and situations. Critically, navigation involves planning within a context and disambiguating similar contexts to reach a goal. We examined hippocampal activity patterns in humans during a goal-directed navigation task to examine how contextual and goal information are incorporated in the construction and execution of navigational plans. Results demonstrate that, during planning, the hippocampus carries a context-specific representation of a future goal. Importantly, this effect could not be explained by stimulus or spatial information alone. During navigation, we observed reinstatement of activity patterns in the hippocampus ahead of participants required actions, which was strongest for behaviorally relevant points in the sequence. These results suggest that, rather than simply representing overlapping associations, hippocampal activity patterns are powerfully shaped by context and goals.

sentences: Hippocampal-cortical encoding activity predicts the precision of episodic memory Our recollections of past experiences can vary both in the number of specific event details accessible from memory and the precision with which such details are reconstructed. Prior neuroimaging evidence suggests the success and precision of episodic recollection to rely on distinct neural substrates during memory retrieval. In contrast, the specific encoding mechanisms supporting later memory precision, and whether they differ from those underlying successful memory formation in general, are currently unknown. Here, we combined continuous measures of memory retrieval with model-based analyses of behavioural and neuroimaging data to tease apart the encoding correlates of successful memory formation and mnemonic precision. In the MRI scanner, participants encoded object-scene displays, and later reconstructed features of studied objects using a continuous scale. We observed overlapping encoding activity in inferior prefrontal and posterior perceptual regions to predict both which object features were later remembered versus forgotten, and the precision with which they were reconstructed from memory. In contrast, hippocampal encoding activity significantly predicted the precision, but not overall success, of subsequent memory retrieval. The current results identify a hippocampal-cortical encoding basis for episodic memory precision, and suggest a contribution of shared cortical encoding mechanisms to the formation of both accessible and precise memory representations.

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Segregation and integration of the functional connectome in neurodevelopmentally 'at risk' children Functional connectivity within and between Intrinsic Connectivity Networks (ICNs) transforms over development and supports high order cognitive functions. But how variable is this process, and does it diverge with altered cognitive developmental trajectories? We investigated age-related changes in integration and segregation within and between ICNs in neurodevelopmentally  at-risk children, identified by practitioners as experiencing cognitive difficulties in attention, learning, language, or memory. In our analysis we used performance on a battery of 10 cognitive tasks, alongside resting-state functional Magnetic Resonance Imaging in 175 at-risk children and 62 comparison children aged 5-16. We observed significant age-by-group interactions in functional connectivity between two network pairs. Integration between the ventral attention and visual networks and segregation of the limbic and fronto-parietal networks increased with age in our comparison sample, relative to at-risk children. Furthermore, functional connectivity between the ventral attention and visual networks in comparison children significantly mediated age-related improvements in executive function, compared to at-risk children. We conclude that integration between ICNs show divergent neurodevelopmental trends in the broad population of children experiencing cognitive difficulties, and that these differences in functional brain organisation may partly explain the pervasive cognitive difficulties within this group over childhood and adolescence.

Research HighlightsO_LIWe investigated functional brain organisation and its development in 175 children who experience neurodevelopmental difficulties in cognition and behaviour, relative to a comparison sample (n=62)
C_LIO_LIWe replicated common neurodevelopmental trends across the samples: functional connectivity increased within Intrinsic Connectivity Networks and the default-mode network increasingly segregated with age
C_LIO_LINeurodevelopmentally at-risk children also showed different age-related changes in functional connectivity between the ventral attention and visual networks and between the fronto-parietal and limbic networks
C_LIO_LIFurthermore, the integration between the ventral attention and visual networks in comparison children mediated age-related changes in cognition, relative to at-risk children
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Identification of Novel Genetic Susceptibility Loci for Thoracic and Abdominal Aortic Aneurysms Via Genome-Wide Association Study Using the UK Biobank Cohort BackgroundThoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA) are known to have a strong genetic component.

Methods and ResultsIn a genome-wide association study (GWAS) using the UK Biobank, we analyzed the genomes of 1,363 individuals with AAA compared to 27,260 age, ancestry, and sex-matched controls (1:20 case:control study design). A similar analysis was repeated for 435 individuals with TAA compared to 8,700 controls. Polymorphism with minor allele frequency (MAF) >0.5% were evaluated.

We identified novel loci near LINC01021, ATOH8 and JAK2 genes that achieved genome-wide significance for AAA (p-value <5x10-8), in addition to three known loci. For TAA, three novel loci in CTNNA3, FRMD6 and MBP achieved genome-wide significance. There was no overlap in the genes associated with AAAs and TAAs. Additionally, we identified a linkage group of high-frequency variants (MAFs [~]10%) encompassing FBN1, the causal gene for Marfan syndrome, which was associated with TAA. In Finngen PheWeb, this FBN1 haplotype was associated with aortic dissection. Finally, we found that baseline bradycardia was associated with TAA, but not AAA.

ConclusionsOur GWAS found that AAA and TAA were associated with distinct sets of genes, suggesting distinct underlying genetic architecture. We also found association between baseline bradycardia and TAA. These findings, including JAK2 association, offer plausible mechanistic and therapeutic insights. We also found a common FBN1 linkage group that is associated with TAA and aortic dissection in patients who do not have Marfan syndrome. These FBN1 variants suggest shared pathophysiology between Marfan disease and sporadic TAA.

Condensed AbstractIn genome-wide association study (GWAS) of thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA) using UK Biobank database, we found 3 novel loci associated with TAA, and 3 novel loci associated AAA. We also found significant association between baseline bradycardia and TAA. These findings, including JAK2 association, offer plausible mechanistic and therapeutic insights. Additionally, we identified a common FBN1 linkage group associated with TAA in patients who do not have Marfan syndrome. In the FinnGen cohort, this haplotype is associated with aortic dissection. These results suggest a shared pathophysiology between Marfan disease and sporadic TAA.

Study LimitationsAs with any GWAS study, the discovery of novel loci associated with aortopathies does not prove functional causality, and the findings described herein needs to be validated by analysis of other databases, ideally in a patient population of more diverse genetic origins than the UK Biobank. The use of the ICD10 codes to classify disease carriers and noncarriers in a population cohort may not be the most accurate assessment of prevalence of aortopathies. The association between baseline bradycardia and TAA does not take into account the concurrent use of medications that may impact heart rate.

HighlightsO_LIIdentification of 3 novel AAA-associated loci near LINC01021, ATOH8 and JAK2 genes.
C_LIO_LIIdentification of 3 novel TAA-associated loci near CTNNA3, FRMD6 and MBP genes.
C_LIO_LIIdentification of a linkage group of common FBN1 variants associated with non-syndromic TAA in the UK Biobank and with aortic dissection in the FinnGen cohort, strengthening the evidence for a shared pathophysiology between Marfan disease and nonsyndromic aortopathy.
C_LIO_LIAssociation between baseline bradycardia and TAA but not AAA.
C_LI

sentences: Possible Mechanism of Porosome-Mediated Exosome Release Supramolecular cup-shaped lipoprotein structures called porosomes embedded in the cell plasma membrane mediate fractional release of intra-vesicular contents from cells during secretion. The presence of porosomes, have been documented in many cell types including neurons, acinar cells of the exocrine pancreas, growth hormone secreting cells of the pituitary, and mouse insulin-secreting beta cell insulinomas called Min6. Electron micrographs of the nerve terminal in rat brain and of Min6 cells, suggest that besides the docking, fusion and content release of neurotransmitter containing synaptic vesicles and insulin containing granules, multi-vesicular bodies and exosome-like vesicles are released at the porosome complex. To test this hypothesis, the porosome-associated calcium transporting ATPase 1 [SPCA1] encoded by the ATP2C1 gene, was knocked-out in Min6 cells using CRISPR, to study glucose-stimulated insulin and exosome release. In agreement with electron micrographs, results from the study demonstrate a loss of both glucose-stimulated insulin and exosome release in the ATP2C1 knockout Min6 cells. These results further confirm the role of the porosome complex in insulin secretion and establishes a new paradigm in porosome-mediated exosome release in beta cells of the endocrine pancreas.

sentences: Over-expression Screen of Interferon-Stimulated Genes Identifies RARRES3 as a Restrictor of Toxoplasma gondii Infection Toxoplasma gondii is an important human pathogen infecting an estimated 1 in 3 people worldwide. The cytokine interferon gamma (IFN{gamma}) is induced during infection and is critical for restricting T. gondii growth in human cells. Growth restriction is presumed to be due to the induction interferon stimulated genes (ISGs) that are upregulated to protect the host from infection. Although there are hundreds of ISGs induced by IFN{gamma}, their individual roles in restricting parasite growth in human cells remain somewhat elusive. To address this deficiency, we screened a library of 414 IFN{gamma} induced ISGs to identify factors that impact T. gondii infection in human cells. In addition to IRF1, which likely acts through induction of numerous downstream genes, we identified RARRES3 as a single factor that restricts T. gondii infection by inducing premature egress of the parasite in multiple human cell lines. Overall, while we successfully identified a novel IFN{gamma} induced factor restricting T. gondii infection, the limited number of ISGs capable of restricting T. gondii infection when individually expressed suggests that IFN{gamma} mediated immunity to T. gondii infection is a complex, multifactorial process.

sentences: α-Helical peptidic scaffolds to target α-synuclein pathogenic species with high affinity and selectivity. -Synuclein aggregation is a key driver of neurodegeneration in Parkinsons disease and related syndromes. Accordingly, obtaining a molecule that targets -synuclein pathogenic assemblies with high affinity and selectivity is a long-pursued objective. Here, we have exploited the biophysical properties of toxic oligomers and amyloid fibrils to identify a family of -helical peptides that bind selectively to these -synuclein species with low nanomolar affinity, without interfering with the monomeric functional protein. This activity is translated into an unprecedented anti-aggregation potency and the ability to abrogate the oligomers toxicity. With a structure-function relationship in hand, we identified a human peptide expressed in the brain and in the gastrointestinal tract with exceptional binding, antiaggregation, and detoxifying properties, which suggests it might play a protective role against synucleinopathies. The chemical entities we describe here represent a new therapeutic paradigm and are promising tools to assist diagnosis by selectively detecting -synuclein pathogenic species in biofluids.

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Structural Covariance Networks in Post-Traumatic Stress Disorder: A Multisite ENIGMA-PGC Study IntroductionCortical thickness (CT) and surface area (SA) are established biomarkers of brain pathology in posttraumatic stress disorder (PTSD). Structural covariance networks (SCN) constructed from CT and SA may represent developmental associations, or unique interactions between brain regions, possibly influenced by a common causal antecedent. The ENIGMA-PGC PTSD Working Group aggregated PTSD and control subjects data from 29 cohorts in five countries (n=3439).

MethodsUsing Destrieux Atlas, we built SCNs and compared centrality measures between PTSD subjects and controls. Centrality is a graph theory measure derived using SCN.

ResultsNotable nodes with higher CT-based centrality in PTSD compared to controls were left fusiform gyrus, left superior temporal gyrus, and right inferior temporal gyrus. We found sex-based centrality differences in bilateral frontal lobe regions, left anterior cingulate, left superior occipital cortex and right ventromedial prefrontal cortex (vmPFC). Comorbid PTSD and MDD showed higher CT-based centrality in the right anterior cingulate gyrus, right parahippocampal gyrus and lower SA-based centrality in left insular gyrus.

ConclusionUnlike previous studies with smaller sample sizes ([≤]318), our study found differences in centrality measures using a sample size of 3439 subjects. This is the first cross-sectional study to examine SCN interactions with age, sex, and comorbid MDD. Although limited to group level inferences, centrality measures offer insights into a nodes relationship to the entire functional connectome unlike approaches like seed-based connectivity or independent component analysis. Nodes having higher centrality have greater structural or functional connections, lending them invaluable for translational treatments like neuromodulation.

sentences: Proximity labeling reveals spatial regulation of the anaphase-promoting complex/cyclosome by a microtubule adaptor The anaphase-promoting complex/cyclosome (APC/C) coordinates advancement through mitosis via temporally controlled polyubiquitination of effector proteins. Despite the long-appreciated spatial organization of key events in mitosis mediated largely by cytoskeletal networks, the spatial regulation of APC/C, the major mitotic E3 ligase, is poorly understood. Here, we describe a microtubule-resident protein, PLEKHA5, as an interactor of APC/C and spatial regulator of its activity in mitosis. PLEKHA5 knockdown delayed mitotic progression, causing accumulation of APC/C substrates dependent upon the PLEKHA5-APC/C interaction. To assess the spatial importance of this interaction, we developed microtubule-localized proximity biotinylation tools, which revealed that depletion of PLEKHA5 decreased the extent of APC/C association with the microtubule cytoskeleton. This decreased APC/C microtubule-localization in turn prevented efficient loading of APC/C with its co-activator CDC20, leading to defects in E3 ligase catalytic activity. We propose that PLEKHA5 functions as an adaptor of APC/C that promotes its subcellular localization to microtubules and facilitates its activation by CDC20, thus ensuring the timely turnover of key mitotic APC/C substrates and proper progression through mitosis.

sentences: Coral settlement and recruitment responses to reef fish foraging and trait diversity The process of coral recruitment is crucial to the healthy functioning of coral reef ecosystems, as well as recovery following disturbances. Fishes are key modulators of this process by feeding on algae and other benthic taxa that compete with corals for benthic space. However, foraging strategies within reef fish assemblages are highly diverse and the effect of foraging diversity on coral recruitment success remains poorly understood. Here, we test how the foraging traits of reef fishes affect coral settlement and juvenile success at Lizard Island, Great Barrier Reef. Using a multi-model inference approach incorporating six metrics of fish assemblage foraging diversity (foraging rates, trait richness, trait evenness, trait divergence, herbivore abundance, and benthic invertivore abundance), we found that herbivore abundance had positive effects on both coral settlement and recruitment success. However, foraging trait diversity had a negative effect on coral settlement but not on recruitment. Coral settlement was higher at sites with less trait diverse fish assemblages, specifically in trait divergence and richness. Moreover, these two trait diversity metrics were stronger predictors of coral settlement success compared to herbivore abundance. Our findings provide evidence that impacts mediated by fish foraging on coral juveniles can potentially be harmful during settlement, but the space-clearing effect overall remains advantageous. We show here that the variation of fish biodiversity across reefs can be a partial driver to spatially uneven patterns of coral recruitment and reef recovery.

sentences: Inter-specific Variability in Demographic Processes Affects Abundance-Occupancy Relationships Species with large local abundances tend to occupy more sites. One of the mechanisms proposed to explain this widely reported inter-specific relationship is a cross-scale hypothesis based on dynamics at the population level. Called the vital rates mechanism, it uses within-population demographic processes of population growth and density dependence to predict when inter-specific abundance-occupancy relationships can arise and when these relationships can weaken and even turn negative. Even though the vital rates mechanism is mathematically simple, its predictions has never been tested directly because of the difficulty estimating the demographic parameters involved. Here, using a recently introduced mark-recapture analysis method, we show that there is a weakly positive relationship between abundance and occupancy among 17 bird species. Our results are consistent with the predictions of the vital rate mechanism regarding the demographic processes that are expected to weaken this relationship. Specifically, we find that intrinsic growth rate and local abundance are weakly correlated; and density dependence strength shows considerable variation across species. Variability in density dependence strength is related to variability in species-level local average abundance and intrinsic growth rate; species with lower growth rate have higher abundance and are strongly regulated by density dependent processes, especially acting on survival rates. Species with higher growth rate, on average, have lower abundance and are more weakly regulated by density dependent processes especially acting on fecundity. More generally, our findings support a cross scale mechanism of macroecological abundance-occupancy relationship emerging from density-dependent dynamics at the population level.

sentences: A corollary discharge mediates saccade related inhibition of single units in mnemonic structures of the human brain Despite the critical link between visual exploration and memory, little is known about how single-unit activity (SUA) in the human mesial temporal lobe (MTL) is modulated by saccadic eye movements (SEMs). Here we characterize SEM associated SUA modulations, unit-by-unit, and contrast them to image onset, and to occipital lobe SUA. We reveal evidence for a corollary discharge (CD)-like modulatory signal that accompanies SEMs, inhibiting/exciting a unique population of broad/narrow spiking units, respectively, before and during SEMs, and with directional selectivity. These findings comport well with the timing, directional nature, and inhibitory circuit implementation of a CD. Additionally, by linking SUA to event-related potentials (ERPs), which are directionally modulated following SEMs, we recontextualize the ERP associated with SEM as a proxy for both the strength of inhibition and saccade direction, providing a mechanistic underpinning for the more commonly recorded SEM-related ERP in the human brain.

sentences: Optimized detection of somatic allelic imbalances specific for homologous recombination deficiency improves the prediction of clinical outcomes in ovarian cancer Homologous recombination DNA-repair deficiency (HRD) is a common driver of genomic instability and confers a therapeutic vulnerability in human cancer. Using a systematic pan- cancer characterization, we revealed distinct patterns of somatic allelic imbalances (AIs) in high-grade serous ovarian cancer (HGSC). Using machine learning on a multi-omics dataset, we generated an optimized algorithm to detect HRD in HGSC (ovaHRDscar). ovaHRDscar improved the prediction of clinical outcomes in three independent validation cohorts. Characterization of 98 spatiotemporally distinct tumor samples indicated ovary/adnex as the preferred site to assess HRD. Cancer-specific optimization improved the prediction of clinical outcomes also in triple-negative breast cancer (tnbcHRDscar). In conclusion, our systematic approach based on multi-omics data improves the detection of HRD with the premise to improve patient selection for HR-targeted therapies.

sentences: β-Catenin and canonical Wnts control two separate pattern formation systems in Hydra: Insights from mathematical modelling Formation of the body axes and the subsequent formation of the apical termini are fundamental steps during animal development. Nuclear {beta}-catenin and canonical Wnt3 have been identified as major players in both these processes in Hydra. Based on molecular knowledge of canonical Wnt signalling directly linking nuclear {beta}-catenin and Wnt3 activity, de novo axis formation and head formation have frequently been assumed to be part of the same pattern formation system. In this study, we investigated this relationship by combining new model simulations with available experimental results. We demonstrated that nuclear {beta}-catenin and Wnt3 likely contribute to two distinct de novo pattern formation systems in Hydra, organizing development and differentiation on two different spatial scales. In particular, the results suggested that nuclear {beta}-catenin acts at the scale of the whole body to control axis formation, whereas canonical Wnt3 signalling is involved in a downstream pathway responsible for small-scale patterning of the head. These results suggest that the underlying mechanisms may also be more complex than previously assumed in other animals where axis formation has been ascribed to canonical Wnt signalling.

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A Pan-Cancer Census of Dominant Tumor Immune Archetypes Cancers display significant heterogeneity with respect to tissue of origin, driver mutations and other features of the surrounding tissue. It is likely that persistent tumors differentially engage inherent patterns-here  Archetypes-of the immune system, to both benefit from a tumor immune microenvironment (TIME) and to disengage tumor-targeting. To discover dominant immune system archetypes, the Immunoprofiler Initiative (IPI) processed 364 individual tumors across 12 cancer types using standardized protocols. Computational clustering of flow cytometry and transcriptomic data obtained from cell sub compartments uncovered archetypes that exist across indications. These Immune composition-based archetypes differentiate tumors based upon unique immune and tumor gene-expression patterns. Archetypes discovered this way also tie closely to well-established classifications of tumor biology. The IPI resource provides a template for understanding cancer immunity as a collection of dominant patterns of immune infiltration and provides a rational path forward to learn how to modulate these patterns to improve therapy.

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sentences: Structural basis for activation and gating of IP3 receptors Calcium (Ca2+) is a universal and versatile cellular messenger used to regulate numerous cellular processes in response to external or internal stimuli. A pivotal component of the Ca2+ signaling toolbox in cells is the inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs), which mediate Ca2+ release from the endoplasmic reticulum (ER), controlling cytoplasmic and organellar Ca2+ concentrations1-3. IP3Rs are activated by IP3 and Ca2+, inhibited by Ca2+ at high concentrations, and potentiated by ATP1-3. However, the underlying molecular mechanisms are unclear due to the lack of structures in the active conformation. Here we report cryo-electron microscopy (cryo-EM) structures of human type-3 IP3R in multiple gating conformations; IP3-ATP bound pre-active states with closed channels, IP3-ATP-Ca2+ bound active state with an open channel, and IP3-ATP-Ca2+ bound inactive state with a closed channel. The structures demonstrate how IP3-induced conformational changes prime the receptor for activation by Ca2+, how Ca2+ binding leads to channel opening, and how ATP modulates the activity, providing insights into the long-sought questions regarding the molecular mechanism of the receptor activation and gating.

sentences: State and stimulus dependence reconcile motion computation and the Drosophila connectome Sensory systems dynamically optimize their processing properties in order to process a wide range of environmental and behavioral conditions. However, attempts to infer the function of these systems via modeling often treat system components as having static processing properties. This is particularly evident in the Drosophila motion detection circuit, where the core algorithm for motion detection is still debated, and where inputs to motion detecting neurons remain underdescribed. Using whole-cell patch clamp electrophysiology, we measured the state- and stimulus-dependent filtering properties of inputs to the OFF motion-detecting T5 cell in Drosophila. Simply summing these inputs within the framework of a connectomic-constrained model of the circuit demonstrates that changes in the shape of input temporal filters are sufficient to explain conflicting theories of T5 function. Therefore, with our measurements and our model, we reconcile motion computation with the anatomy of the circuit.

sentences: Idiosyncratic learning performance in flies generalizes across modalities Individuals vary in their innate behaviors, even when they have the same genome and have been reared in the same environment. The extent of individuality in plastic behaviors, like learning, is less well characterized. Also unknown is the extent to which intragenotypic differences in learning generalize: if an individual performs well in one assay, will it perform well in other assays? We investigated this using the fruit fly Drosophila melanogaster, an organism long-used to study the mechanistic basis of learning and memory. We found that isogenic flies, reared in identical lab conditions, and subject to classical conditioning that associated odorants with electric shock, exhibit clear individuality in their learning responses. Flies that performed well when an odor was paired with shock tended to perform well when other odors were paired with shock, or when the original odor was paired with bitter taste. Thus, individuality in learning performance appears to be prominent in isogenic animals reared identically, and individual differences in learning performance generalize across stimulus modalities. Establishing these results in flies opens up the possibility of studying the genetic and neural circuit basis of individual differences in learning in a highly suitable model organism.

sentences: Thalamic bursting and the role of timing and synchrony in thalamocortical signaling in the awake mouse The thalamus controls transmission of sensory signals from periphery to cortex, ultimately shaping perception. Despite this significant role, dynamic thalamic gating and the consequences for downstream cortical sensory representations have not been well studied in the awake brain. We optogenetically modulated the ventro-posterior medial thalamus in the vibrissa pathway of the awake mouse, and measured spiking activity in the thalamus, and activity in primary somatosensory cortex (S1) using extracellular electrophysiology and genetically encoded voltage imaging. Thalamic hyperpolarization significantly enhanced thalamic sensory-evoked bursting, yet surprisingly the S1 cortical response was not amplified, but instead timing precision was significantly increased, spatial activation more focused, and there was an increased synchronization of cortical inhibitory neurons. A thalamocortical network model implicates the modulation of precise timing of feedforward thalamic population spiking, presenting a highly sensitive, timing-based gating of sensory signaling to cortex.

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THE STABILIZATAION OF C-MYC BY THE NOVEL CELL CYCLE REGULATOR, SPY1, DECREASES EFFICACY OF BREAST CANCER TREATMENTS Purposec-Myc is frequently upregulated in breast cancers, however, targeting c-Myc has proven to be a challenge. Targeting of downstream mediators of c-Myc, such as the  cyclin-like cell cycle regulator Spy1, may be a viable therapeutic option in a subset of breast cancer subtypes.

MethodsMouse mammary tumour cells isolated from MMTV-Myc mice and human breast cancer cell lines were used to manipulate Spy1 levels followed by tamoxifen or chemotherapeutic treatment with a variety of endpoints. Patient samples from TNBC patients were obtained and constructed into a TMA and stained for c-Myc and Spy1 protein levels.

ResultsOver time, MMTV-Myc cells show a decreased response to tamoxifen treatment with increasing levels of Spy1 in the tamoxifen-resistant cells. shRNA against Spy1 re-establishes tamoxifen sensitivity. Spy1 was found to be highly elevated in human TNBC cell and patient samples, correlating to c-Myc protein levels. c-Myc was found to be stabilized by Spy1 and knocking down Spy1 in TNBC cells shows a significant increase in response to chemotherapy treatments.

ConclusionsUnderstanding the interplay between protein expression level and response to treatment is a critical factor in developing novel treatment options for breast cancer patients. These data have shown a connection between Spy1 and c-Myc protein levels in more aggressive breast cancer cells and patient samples. Furthermore, targeting c-Myc has proven difficult, these data suggest targeting Spy1 even when c-Myc is elevated can confer an advantage to current chemotherapies.

sentences: INCORPORATING A DYNAMIC GENE-BASED PROCESS MODULE INTO A CROP SIMULATION MODEL Dynamic crop simulation models are tools that predict plant phenotype grown in specific environments for genotypes using genotype-specific parameters (GSPs), often referred to as "genetic coefficients." These GSPs are estimated using phenotypic observations and may not represent "true" genetic information. Instead, estimating GSPs requires experiments to measure phenotypic responses when new cultivars are released. The goal of this study was to evaluate a new approach that incorporates a dynamic gene-based module for simulating time-to-flowering for common bean (Phaseolus vulgaris L.) into an existing dynamic crop model. A multi-environment study conducted in 2011 and 2012 included 187 recombinant inbred lines (RILs) from a bi-parental bean family to measure the effects of quantitative trait loci (QTL), environment (E), and QTLxE interactions across five sites. The dynamic mixed linear model from Vallejos et al. (2020) was modified in this study to create a dynamic module that was then integrated into the CSM-CROPGRO-Drybean model. This new hybrid crop model, with the gene-based flowering module replacing the original flowering component, requires allelic makeup of each genotype being simulated and daily E data. The hybrid model was compared to the original CSM model using the same E data and previously estimated GSPs to simulate time-to-flower. The integrated gene-based module simulated days of first flower agreed closely with observed values (root mean square error of 2.73 days and model efficiency of 0.90) across the five locations and 187 genotypes. The hybrid model with its gene-based module also described most of the G, E and GxE effects on time-to-flower and was able to predict final yield and other outputs simulated by the original CSM. These results provide the first evidence that dynamic crop simulation models can be transformed into gene-based models by replacing an existing process module with a gene-based module for simulating the same process.

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Acute thermal stress elicits interactions between gene expression and alternative splicing in a fish of conservation concern Transcriptomics provides a mechanistic understanding of an organisms response to environmental challenges such as increasing temperatures, which can provide key insights into the threats posed by thermal challenges associated with urbanization and climate change. Differential gene expression and alternative splicing are two elements of the transcriptomic stress response that may work in tandem, but relatively few studies have investigated these interactions in fishes of conservation concern. We studied the imperilled redside dace (Clinostomus elongatus) as thermal stress is hypothesised to be an important cause of population declines. We tested the hypothesis that gene expression-splicing interactions contribute to the thermal stress response. Wild fish exposed to acute thermal stress were compared with both handling controls and fish sampled directly from a river. Liver tissue was sampled to study the transcriptomic stress response. Thermally stressed fish showed a prominent transcriptional response (estimated with mRNA transcript abundance) related to transcription regulation and responses to unfolded proteins, and prominent alternatively spliced genes related to gene expression regulation and metabolism. One splicing factor, prpf38b, was upregulated in the thermally stressed group compared to the other treatments. This splicing factor may have a role in the Jun/AP-1 cellular stress response, a pathway with wide-ranging and context-dependent effects. Given large gene interaction networks and the context-dependent nature of transcriptional responses, our results highlight the importance of understanding interactions between gene expression and splicing for understanding transcriptomic responses to thermal stress. Our results also reveal transcriptional pathways that can inform conservation breeding, translocation, and reintroduction programs for redside dace and other imperilled species by identifying appropriate source populations.

SUMMARY STATEMENTGene expression and alternative splicing interact in response to thermal stress in an imperilled fish, with implications for conservation and mechanisms of thermal tolerance in vertebrate ectotherms.

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The human placenta dictates the phenotype of decidual macrophages. During human pregnancy, placenta-derived extravillous trophoblasts (EVT) invade the decidua and communicate with maternal immune cells. The decidua can be distinguished into basalis (decB) and parietalis (decP), the latter being unaffected by placentation. By defining a novel gating strategy, we report accumulation of myeloid cells in decB. We identified a decidua basalis-associated macrophage (decBAM) population with a differential transcriptome and secretome when compared to decidua parietalis-associated macrophages (decPAMs). decBAMs are CD11chi and efficient inducers of Tregs, proliferate in situ and secrete high levels of CXCL1, CXCL5, M-CSF, and IL-10. In contrast, decPAMs exert a dendritic cell-like, motile phenotype characterized by induced expression of HLA class II molecules, enhanced phagocytosis, and the ability to activate T cells. Strikingly, EVT-conditioned media are able to convert decPAMs into a decBAM phenotype. Cumulatively, these findings assign distinct macrophage phenotypes to decidual areas depending on placentation and further highlight a critical role for EVTs in the induction of pregnancy-tolerant macrophage polarization.

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A De Novo regulation design shows an effectiveness in altering plant secondary metabolism IntroductionTranscription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants.

ObjectivesWe selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering.

MethodsG-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism.

ResultsPAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P+T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P+T and PAP1 genotypes.

ConclusionG-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

sentences: Correcting Chimeric Crosstalk in Single Cell RNA-seq Experiments As part of the process of preparing sequencing libraries that include unique molecular identifiers (UMIs) such as many single cell RNA-seq (scRNA-seq) libraries, a diverse template must be amplified. During amplification, spurious chimeric molecules can be formed between molecules originating in different cells. While several computational and experimental strategies have been suggested to mitigate the impact of chimeric molecules, suitable approaches for scRNA-seq experiments do not exist. We demonstrate that chimeras become increasingly problematic as samples are sequenced deeply and propose both supervised and unsupervised computational solutions. These solutions are validated in the context of a deeply sequenced species mixing experiment, and, orthogonally, using replicate PCR amplifications of the same scRNA-seq library. Our code is publicly available at https://github.com/asncd/schimera.

sentences: The taxonomic and functional biogeographies of phytoplankton and zooplankton communities across boreal lakes AO_SCPLOWBSTRACTC_SCPLOWStrong trophic interactions link primary producers (phytoplankton) and consumers (zooplankton) in lakes. However, the influence of such interactions on the biogeographical distribution of the taxa and functional traits of planktonic organisms in lakes has never been explicitly tested. To better understand the spatial distribution of these two major aquatic groups, we related composition across boreal lakes (104 for zooplankton and 48 for phytoplankton) in relation to a common suite of environmental and spatial factors. We then directly tested the degree of coupling in their taxonomic and functional distributions across the subset of common lakes. Although phytoplankton composition responded mainly to properties related to water quality while zooplankton composition responded more strongly to lake morphometry, we found significant coupling between their spatial distributions at taxonomic and functional levels based on a Procrustes test. This coupling was not significant after removing the effect of environmental drivers (water quality and morphometry) on the spatial distributions of the two groups. This result suggests that top-down and bottom-up effects (e.g. nutrient concentration and predation) drove trophic interactions at the landscape level. We also found a significant effect of dispersal limitation on the distribution of taxa, which could explain why coupling was stronger for taxa than for traits at the scale of this study, with a turnover of species observed between regions, but no trait turnover. Our results indicate that landscape pelagic food web responses to anthropogenic changes in ecosystem parameters should be driven by a combination of top-down and bottom-factors for taxonomic composition, but with a relative resilience in functional trait composition of lake communities.

sentences: Long-term meditation training is associated with enhanced attention and stronger posterior cingulate - rostrolateral prefrontal cortex resting connectivity Mindfulness meditation has been shown to increase resting state functional connectivity (rsFC) between the posterior cingulate cortex (PCC) and dorsolateral prefrontal cortex (DLPFC), which is thought to reflect improvements in attention to the present moment. However, prior research in long-term meditation practitioners lacked quantitative measures of attention that would provide a more direct behavioral correlate and interpretational anchor for PCC-DLPFC connectivity and was inherently limited by small sample sizes. Moreover, whether mindfulness meditation primarily impacts brain function locally, or impacts the dynamics of large-scale brain networks, remained unclear. Here, we sought to replicate and extend prior findings of increased PCC - DLPFC rsFC in a sample of 40 long-term meditators (average practice= 3759 hours) who also completed a behavioral assay of attention. In addition, we tested a network-based framework of changes in inter-regional connectivity by examining network-level connectivity. We found that meditators had stronger PCC-rostrolateral prefrontal cortex (PFC) rsFC, lower connector hub strength across the default mode network (DMN) relative to other functional networks, and better attention to task, compared to 124 meditation-naive controls. Orienting attention positively correlated with PCC- rostrolateral PFC connectivity, and negatively correlated with DMN connector hub strength. These findings provide novel evidence that posterior cingulate - rostrolateral PFC rsFC may support attention orienting, consistent with a role for rostrolateral PFC in meta-cognitive background awareness that is a core component of mindfulness meditation training. Our results further demonstrate that long-term mindfulness meditation may improve attention and strengthen the underlying brain networks.

sentences: Effects of land-use and landscape drivers in the species richness and distribution of carnivores in Faragosa-Fura Landscape of Southern Rift Valley, Ethiopia Understanding the species richness and distribution of carnivores across anthropogenic land-use types in an area is an essential first step for biodiversity conservation and human-carnivore coexistence. However, quantitative data on carnivore species coexisting with humans in different land-use types remain largely missing. Thus, this paper investigated the effect of anthropogenic land-use and landscape drivers on carnivore species richness and distribution in the Faragosa-Fura Landscape, Gamo Zone, southern Ethiopia. To collect data, we employed the line transect method using three complementary field surveys techniques: sign survey, camera-trapping, and opportunistic sighting survey during wet and dry seasons in 2020 and 2021. We stratified the study landscape into five land-use types-forest, wetland, grassland, agricultural land, and settlement. The result proved the occurrence of 12 carnivore species belonging to six families, including vulnerable Felidae species - Panthera pardus. Family Felidae and Herpestidae were composed of a greater number of species, while Hyaenidae and Mustelidae were each represented by single species. Out of identified species, only two species (Panthera pardus and Crocuta crocuta) were large-sized, while the rest were medium and small-sized carnivores. Overall, the mean richness of the study area was 5.73{+/-}0.284(SE). The species richness was highest in the wetland (n = 12, mean = 7.67{+/-}0.494(SE)) and lowest in the settlement (n = 5, mean = 4.25{+/-}0.479(SE)). The regression analysis showed that most of the carnivores displayed a strong negative relationship with agriculture, roads, and settlement while displayed a strong positive relationship with wetland and forest. In general, out of 32 species recorded in Ethiopia, this study quantified 12 carnivore species that signify the area is an important area for wildlife conservation in Ethiopia. Further, the study concluded that the wetland is the most important habitat, particularly for larger-sized and habitat specialists while anthropogenic land-uses types adversely affecting species richness. Thus, a generic paradigm to reconcile land management and biodiversity conservation is highly important.

sentences: Generation of specialized blood vessels through transdifferentiation of lymphatic endothelial cells The lineage and developmental trajectory of a cell are key determinants of cellular identity. Yet, the functional relevance of deriving a specific cell type from ontologically distinct progenitors, remains an open question. In the case of the vascular system, blood and lymphatic vessels are composed of endothelial cells (ECs) that differentiate and diversify to cater the different physiological demands of each organ. While lymphatic vessels have been shown to originate from multiple cell sources, lymphatic ECs (LECs) themselves seem to have a unipotent cell fate. In this work we uncover a novel mechanism of blood vessel formation through transdifferentiation of LECs. Using advanced long-term reiterative imaging and lineage-tracing of ECs in zebrafish, from embryonic development through adulthood, we reveal a hitherto unknown process of LEC-to-BEC transdifferentiation, underlying vascularization of the anal fin (AF). Moreover, we demonstrate distinct functional implications for deriving AF vessels from either LECs or BECs, uncovering for the first time a clear link between cell ontogeny and functionality. Molecularly, we identify Sox17 as a negative regulator of lymphatic fate specification, whose specific expression in AF LECs suppresses its lymphatic cell fate. Finally, we show that akin to the developmental process, during adult AF regeneration the vasculature is re-derived from lymphatics, demonstrating that LECs in the mature fish retain both potency and plasticity for generating specialized blood vessels. Overall, our work highlights a novel mechanism of blood vessel formation through LEC trans-differentiation, and provides the first in vivo evidence for a link between cell ontogeny and functionality in ECs.

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Whole genome sequencing elucidates the species-wide diversity and evolution of fungicide resistance in the early blight pathogen Alternaria solani Early blight of potato is caused by the fungal pathogen Alternaria solani and is an increasing problem worldwide. The primary strategy to control the disease is applying fungicides such as succinate dehydrogenase inhibitors (SDHI). SDHI-resistant strains, showing reduced sensitivity to treatments, appeared in Germany in 2013, five years after the introduction of SDHIs. Two primary mutations in the Sdh complex (SdhB-H278Y and SdhC-H134R) have been frequently found throughout Europe. How these resistances arose and spread, and whether they are linked to other genomic features, remains unknown.

We performed whole-genome sequencing for A. solani isolates from potato fields across Europe (Germany, Sweden, Belgium, and Serbia) to better understand the pathogens genetic diversity in general and understand the development and spread of the genetic mutations that lead to SDHI resistance. We used ancestry analysis and phylogenetics to determine the genetic background of 48 isolates. The isolates can be grouped into 7 genotypes. These genotypes do not show a geographical pattern but appear spread throughout Europe. The Sdh mutations appear in different genetic backgrounds, suggesting they arose independently, and the observed admixtures might indicate a higher adaptive potential in the fungus than previously thought.

Our research gives insights into the genetic diversity of A. solani on a genome level. The mixed occurrence of different genotypes and apparent admixture in the populations indicate higher genomic complexity than anticipated. The conclusion that SDHI tolerance arose multiple times independently has important implications for future fungicide resistance management strategies. These should not solely focus on preventing the spread of isolates between locations but also on limiting population size and the selective pressure posed by fungicides in a given field to avoid the rise of new mutations in other genetic backgrounds.

sentences: Chemical mapping exposes the importance of active site interactions in governing the temperature dependence of enzyme turnover Uncovering the role of global protein dynamics in enzyme turnover is needed to fully understand enzyme catalysis. Recently, we have demonstrated that the heat capacity of catalysis, {Delta}CP{ddagger}, can reveal links between the protein free energy landscape, global protein dynamics and enzyme turnover, suggesting that subtle changes molecular interactions at the active site can affect long range protein dynamics and link to enzyme temperature activity. Here we use a model promiscuous enzyme (Glucose dehydrogenase from Sulfolobus Solfataricus) to chemically map how individual substrate interactions affect the temperature dependence of enzyme activity and the network of motions throughout the protein. Utilizing a combination of kinetics, REES spectroscopy and computational simulation we explore the complex relationship between enzyme-substrate interactions and the global dynamics of the protein. We find that changes in {Delta}CP{ddagger} and protein dynamics can be mapped to specific substrate-enzyme interactions. Our study reveals how subtle changes in substrate binding affect global changes in motion and flexibility extending throughout the protein.

sentences: A conserved PLK1 docking site in TopBP1 maintains genome integrity during mitosis TopBP1 is a large scaffold protein with multiple functions in genome integrity. We previously identified a novel role for TopBP1 during M phase by showing that TopBP1 reduces carry-over of DNA damage to daughter cells. This function emerges as a critical backup pathway in BRCA deficient cells, yet many aspects of TopBP1 regulation during mitosis are unclear. The mitotic kinase PLK1 has been reported to interact with TopBP1 but the functional relevance of this is unclear. Here, we identify and characterize a conserved PLK1 docking site in TopBP1. Endogenous deletion of the PLK1 docking site in TopBP1 results in increased number of mitotic TopBP1 foci, increased DNA damage in daughter cells, deficient mitotic DNA repair synthesis and increased frequency of binucleation. At the same time, cell cycle distribution and ATR activation are normal in cells with the PLK1 docking site deletion in TopBP1. Interestingly, mutation of this site in TopBP1 renders cells sensitive to PARP inhibitors but not to camptothecin hinting to different cellular effects of the two chemotherapeutics. Altogether, our data indicate that the PLK1-TopBP1 interaction is critical for the mitotic function of TopBP1.

sentences: Cellular perception of growth rate and the mechanistic origin of bacterial growth laws Bacteria organize many activities according to their grow rate via the ppGpp signaling system. Yet it is not clear how this signaling system "knows" how fast cells grow. Through quantitative experiments, we show that ppGpp responds inversely to the rate of translational elongation in E. coli. Together with its roles in inhibiting ribosome biogenesis and activity, ppGpp closes a key regulatory circuit that enables the cell to perceive and control the rate of its growth across conditions. The celebrated linear growth law relating the ribosome content and growth rate emerges as a consequence of keeping a supply of ribosome reserves while maintaining elongation rate in slow growth conditions. Further analysis suggests the elongation rate itself is detected by sensing the ratio of dwelling and translocating ribosomes, a strategy employed to collapse the complex, high-dimensional dynamics of the molecular processes underlying cell growth to perceive the physiological state of the whole.

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Low nitrogen availability inhibits the phosphorus starvation response in maize (Zea mays ssp. mays L.) BackgroundNitrogen (N) and phosphorus (P) are macronutrients essential for crop growth and productivity. In cultivated fields, N and P levels are rarely sufficient, contributing to the yield gap between realized and potential production. Fertilizer application increases nutrient availability, but not all farmers have access to fertilizers, nor are current rates of application sustainable or environmentally desirable. Transcriptomic studies of cereal crops have revealed dramatic responses to either low N or low P single stress treatments. In the field, however, levels of both N and P may be suboptimal. The interaction between N and P starvation responses remains to be fully characterized.

ResultsWe characterized growth and root and leaf transcriptomes of young maize plants under nutrient replete, low N, low P or combined low NP conditions. We identified 1,555 genes to respond to our nutrient treatments, in one or both tissues. A large group of genes, including many classical P starvation response genes, were regulated antagonistically between low N and P conditions. An additional experiment over a range of N availability indicated that a mild reduction in N levels was sufficient to repress the low P induction of P starvation genes. Although expression of P transporter genes was repressed under low N or low NP, we confirmed earlier reports of P hyper accumulation under N limitation.

ConclusionsTranscriptional responses to low N or P were distinct, with few genes responding in a similar way to the two single stress treatments. In combined NP stress, the low N response dominated, and the P starvation response was largely suppressed. A reduction in N availability was sufficient to repress the induction of P starvation associated genes. We conclude that activation of the transcriptional response to P starvation in maize is contingent on sufficient N availability.

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Early Patellofemoral Cartilage and Bone Degeneration in a Rat Model of Noninvasive Anterior Cruciate Ligament Rupture BackgroundAnterior cruciate ligament rupture (ACLR) is a well-known risk factor for the development of post-traumatic osteoarthritis (PTOA). While clinical and pre-clinical studies have characterized the onset and progression of PTOA in the tibiofemoral joint compartment, very little is known about degenerative changes in the patellofemoral compartment after ACL injury.

Hypothesis/PurposeTo evaluate the extent to which ACL rupture induces acute patellofemoral joint degeneration by quantifying articular cartilage morphology and remodeling of subchondral and trabecular bone microarchitecture in the patellofemoral compartment.

Study DesignDescriptive laboratory study.

MethodsAdult female Lewis rats were randomized to undergo either a non-surgical ACL rupture or a Sham procedure (n = 6 per group). Ex vivo contrast-enhanced micro-computed tomography ({micro}CT) and histological evaluation of the patellofemoral compartment were performed at 2-weeks post-injury, representing a timepoint of documented early PTOA in the tibiofemoral compartment in this model.

ResultsACL rupture causes osteophyte formation in the patella and mild degeneration in the superficial zone of articular cartilage (AC), including surface fibrillation, fissures, increased cellularity, and abnormal chondrocyte clustering at two weeks post-injury. Contrast-enhanced {micro}CT analysis demonstrates significant increases in AC thickness of patellar and trochlear cartilage. Loss of subchondral bone thickness, bone volume fraction, and tissue mineral density, as well as changes to trabecular microarchitecture in both the patella and trochlea, were indicative of catabolic bone remodeling.

ConclusionThese results demonstrate that the patellofemoral joint develops mild but evident degenerative changes in the acute time period following ACL rupture, extending the utility of this rat model to the study of degenerative patellofemoral changes following joint trauma.

Clinical RelevanceACL rupture causes mild degeneration and swelling of articular cartilage, coupled with catabolic bone remodeling in the patellofemoral compartment. Characterizing the pathophysiology of patellofemoral PTOA in its early stages may provide a better understanding of disease progression and provide opportunities for preventative therapeutic intervention.

sentences: Leishmania allelic selection during experimental sand fly infection correlates with mutational signatures of oxidative DNA damage Trypanosomatid pathogens are transmitted by blood-feeding insects, causing devastating human infections. Survival of these parasites in their vertebrate and invertebrate hosts relies on their capacity to differentiate into distinct stages that are the result of a co-evolutionary process. These stages show in addition important phenotypic shifts that often impacts infection, affecting for example parasite pathogenicity, tissue tropism, or drug susceptibility. Despite their clinical relevance, the evolutionary mechanisms that allow for the selection of such adaptive phenotypes remain only poorly investigated. Here we use Leishmania donovani as a trypanosomatid model pathogen to shed first light on parasite evolutionary adaptation during experimental sand fly infection. Applying a comparative genomics approach on hamster- isolated amastigotes and derived promastigotes before (input) and after (output) infection of Phlebotomus orientalis revealed a strong bottleneck effect on the parasite population as judged by principal component and phylogenetic analyses of input and output parasite DNA sequences. Despite random genetic drift caused by the bottleneck effect, our analyses revealed various genomic signals that seem under positive selection given their convergence between independent biological replicates. While no significant fluctuations in gene copy number were revealed between input and output parasites, convergent selection was observed for karyotype, haplotype and allelic changes during sand fly infection. Our analyses further uncovered signature mutations of oxidative DNA damage in the output parasite genomes, suggesting that Leishmania suffers from oxidative stress inside the insect digestive tract. Our results propose a new model of Leishmania genomic adaptation during sand fly infection, where oxidative DNA damage and DNA repair processes drive haplotype and allelic selection. The experimental and computational framework presented here provides a useful blueprint to assess evolutionary adaptation of other eukaryotic pathogens inside their insect vectors, such as Plasmodium spp, Trypanosoma brucei and Trypanosoma cruzi.

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Mechanism of a COVID-19 nanoparticle vaccine candidate that elicits a broadly neutralizing antibody response to SARS-CoV-2 variants Vaccines that induce potent neutralizing antibody (NAb) responses against emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential for combating the coronavirus disease 2019 (COVID-19) pandemic. We demonstrated that mouse plasma induced by self-assembling protein nanoparticles (SApNPs) that present 20 rationally designed S2G{Delta}HR2 spikes of the ancestral Wuhan-Hu-1 strain can neutralize the B.1.1.7, B.1.351, P.1, and B.1.617 variants with the same potency. The adjuvant effect on vaccine-induced immunity was investigated by testing 16 formulations for the multilayered I3-01v9 SApNP. Using single-cell sorting, monoclonal antibodies (mAbs) with diverse neutralization breadth and potency were isolated from mice immunized with the receptor binding domain (RBD), S2G{Delta}HR2 spike, and SApNP vaccines. The mechanism of vaccine-induced immunity was examined in mice. Compared with the soluble spike, the I3-01v9 SApNP showed 6-fold longer retention, 4-fold greater presentation on follicular dendritic cell dendrites, and 5-fold stronger germinal center reactions in lymph node follicles.

ONE-SENTENCE SUMMARYWith a well-defined mechanism, spike nanoparticle vaccines can effectively counter SARS-CoV-2 variants.

sentences: Precise characterization of somatic structural variations and mobile element insertions from paired long-read sequencing data with nanomonsv We introduce our novel software, nanomonsv, for detecting somatic structural variations (SVs) using tumor and matched control long-read sequencing data with a single-base resolution. Using paired long-read sequencing data from three cancer cell-lines and their matched lymphoblastoid lines, we demonstrate that our approach can identify not only somatic SVs that can be captured with short-read technologies but also novel ones especially those whose breakpoints are located in repeat regions. In addition, we have developed a workflow for classifying mobile element insertions while elucidating their in-depth properties such as 5' truncations, internal inversion as well as source sites in the case of LINE1 transductions. Finally, we identify complex SVs probably caused by replication mechanisms or telomere crisis by examining the co-occurrence of multiple somatic SVs in common supporting reads. In summary, our approaches applied to cancer long-read sequencing data can reveal various features of somatic SVs and will lead to further understanding of mutational processes and functional consequences of somatic SVs.

sentences: On the sparsity of fitness functions and implications for learning Fitness functions map biological sequences to a scalar property of interest. Accurate estimation of these functions yields biological insight and sets the foundation for model-based sequence design. However, the amount of fitness data available to learn these functions is typically small relative to the large combinatorial space of sequences; characterizing how much data is needed for accurate estimation remains an open problem. There is a growing body of evidence demonstrating that empirical fitness functions display substantial sparsity when represented in terms of epistatic interactions. Moreover, the theory of Compressed Sensing provides scaling laws for the number of samples required to exactly recover a sparse function. Motivated by these results, we develop a framework to study the sparsity of fitness functions sampled from a generalization of the NK model, a widely-used random field model of fitness functions. In particular, we present results that allow us to test the effect of the Generalized NK (GNK) models interpretable parameters--sequence length, alphabet size, and assumed interactions between sequence positions--on the sparsity of fitness functions sampled from the model and, consequently, the number of measurements required to exactly recover these functions. We validate our framework by demonstrating that GNK models with parameters set according to structural considerations can be used to accurately approximate the number of samples required to recover two empirical protein fitness functions and an RNA fitness function. In addition, we show that these GNK models identify important higher-order epistatic interactions in the empirical fitness functions using only structural information.

sentences: Diffusional variance-determined stochastic translation efficiency Full-transcriptome methods have brought versatile power to protein biosynthesis research, but remain difficult to apply for the quantification of absolute protein synthesis rates. Here we propose and, using modified translation complex profiling, confirm co-localisation of ribosomes on messenger(m)RNA resulting from their diffusional dynamics. We demonstrate that these stochastically co-localised ribosomes are linked with the translation initiation rate and provide a robust variable to model and quantify specific protein output from mRNA.

sentences: GABARAP membrane conjugation sequesters the FLCN-FNIP tumor suppressor complex to activate TFEB and lysosomal biogenesis Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here we show that during single membrane ATG8 conjugation (SMAC), Parkin-dependent mitophagy, and Salmonella-induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal homeostasis and capacity. GABARAP directly binds to a novel LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex with picomolar affinity and regulates its relocalization to these GABARAP-conjugated membrane compartments. This disrupts the regulation of RagC/D by the FLCN/FNIP GAP complex, resulting in impaired mTOR-dependent phosphorylation of TFEB without changing mTOR activity towards other substrates. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a universal molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.

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Large-scale tandem mass spectrum clustering using fast nearest neighbor searching RationaleAdvanced algorithmic solutions are necessary to process the ever increasing amounts of mass spectrometry data that is being generated. Here we describe the falcon spectrum clustering tool for efficient clustering of millions of MS/MS spectra.

Methodsfalcon succeeds in efficiently clustering large amounts of mass spectral data using advanced techniques for fast spectrum similarity searching. First, high-resolution spectra are binned and converted to low-dimensional vectors using feature hashing. Next, the spectrum vectors are used to construct nearest neighbor indexes for fast similarity searching. The nearest neighbor indexes are used to efficiently compute a sparse pairwise distance matrix without having to exhaustively perform all pairwise spectrum comparisons within the relevant precursor mass tolerance. Finally, density-based clustering is performed to group similar spectra into clusters.

ResultsSeveral state-of-the-art spectrum clustering tools were evaluated using a large draft human proteome dataset consisting of 25 million spectra, indicating that alternative tools produce clustering results with different characteristics. Notably, falcon generates larger highly pure clusters than alternative tools, leading to a larger reduction in data volume without the loss of relevant information for more efficient downstream processing.

Conclusionsfalcon is a highly efficient spectrum clustering tool. It is publicly available as open source under the permissive BSD license at https://github.com/bittremieux/falcon.

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Substance P and adenosine signaling pathways regulate exosomal sorting of miR-21 in colonic epithelial cells Background & AimsMicroRNAs (miRNAs) are transported in body fluids within exosomes, and exosomal-miRNA sorting is highly selective. In colonic epithelial cells (CECs), Substance P-neurokinin-1-receptor (SP/NK1R) signaling regulates miR-21 sorting into secreted exosomes. Here, we studied the molecular mechanisms driving miR-21 sorting into colonic epithelial exosomes (CEEs) under SP-stimulation.

MethodsWe performed studies with human colonic epithelial NCM460 cells overexpressing neurokinin-1-receptor (NCM460-NK1R) and in intestinal-epithelial-specific NK1R knockout mice. SP-regulated gene targets were validated by real-time polymerase chain reaction (RT-PCR) and immunoblotting. Small non-coding RNAs (sncRNAs) were isolated from NCM460-NK1R cells and secreted exosomes, and 3'-end adenylated and 3'-end uridylated fractions were separated. Cellular and exosomal sncRNA fractions were processed for miR-21 and miR-1307-3p expression and 3'-end adenylation to uridylation (A/U) ratios using RT-PCR. Pharmacological inhibition studies in NCM460-NK1R cells were performed in the presence of the Adenosine A2B receptor (ADORA2B) antagonist, PSB-1115, followed by RT-PCR and immunoblotting. Mass spectrometry validated in silico interacting partners of miR-21 in CECs.

ResultsIn CECs, miR-21 is predominantly polyuridylated under SP-stimulation and preferentially sorted to CEEs. SP/NK1R signaling activation upregulates extracellular adenosine (ADO) signaling via ADORA2B concomitant with reduced ADO uptake via epithelial-specific equilibrative nucleoside transporter-2 (ENT-2). Mass spectrometry and immunoblotting revealed upregulation of TUT7 in SP-stimulated CEEs. Knockdown of TUT7 decreased both TUT7 and miR-21 content in these exosomes. Pharmacological inhibition of ADORA2B in CECs resulted in decreased TUT7 and miR-21 in CEEs, regardless of SP stimulation.

ConclusionsSP/NK1R coupling in CECs activates ADORA2B and its downstream signaling cascade which mediates TUT7/miR-21 interaction and subsequent miR-21 polyuridylation and exosomal export.

SynopsisSubstance P-neurokinin-1 receptor signaling regulates terminal uridyl transferase7-mediated predominant 3-end polyuridylation and exosomal recruitment of miR-21 in human colonic epithelial NCM460 cells overexpressing NK-1R via activation of Adenosine A2B receptor and its downstream signaling cascade. Because Adenosine A2B receptor signaling is involved in IBD pathogenesis, it could therefore be exploited as a pharmacological target for the therapeutic benefits in IBD.

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Chemotherapy coupled to macrophage inhibition leads to enhanced T and B cell infiltration and durable triple negative breast cancer regression Immunosuppressive elements within the tumor microenvironment such as Tumor Associated Macrophages (TAMs) can present a barrier to successful anti-tumor responses by cytolytic T cells. We employed preclinical syngeneic p53 null mouse models of triple negative breast cancer (TNBC) to develop a treatment regimen that harnessed the immunostimulatory effects of low-dose cyclophosphamide coupled with the pharmacologic inhibition of TAMs using either a small molecule CSF1R inhibitor or an anti-CSF1R antibody. This therapeutic combination was used to successfully treat several highly aggressive TNBC murine mammary tumors and lung metastasis. Using this regimen and single cell RNA sequencing we characterized tumor infiltrating lymphocytes (TILs) including helper T cells and antigen-presenting B cells that were highly enriched in good responders to combination therapy. Using high dimensional imaging techniques, we identified the close spatial localization of B220+ CD86+ activated B cells and CD4+ T cells in tertiary lymphoid structures that were present up to 6 weeks post-treatment in one model that also exhibited long-term tumor regression post-treatment. We also characterized the transcriptional and metabolic heterogeneity of TAMS in these two closely related claudin-low/mesenchymal subtype tumor models with differential treatment responses. A murine TAM signature derived from the T12 model is highly expressed and conserved in human claudin-low breast cancers, and high expression of the T12 signature correlated with reduced overall survival. This T12 tumor TAM signature may help identify human claudin-low breast cancer patients that will benefit from the combination of cyclophosphamide and anti-CSF1R therapy. These studies illustrate the complexity of the tumor immune microenvironment and highlight different immune responses that result from rationale combinations of immunotherapy.

SignificanceA treatment regimen that harnessed the immunostimulatory effects of cyclophosphamide coupled with the inhibition of CSF1R was used to successfully treat several highly aggressive claudin-low TNBC murine mammary tumors and lung metastasis.

sentences: Biologically excretable AIE dots for visualizing through the marmosets intravitally: horizons in future clinical nanomedicine Superb reliability and biocompatibility equip aggregation-induced emission (AIE) dots with tremendous potential for fluorescence bioimaging. However, there is still a chronic lack of design instructions of excretable and bright AIE emitters. Here, we designed a kind of PEGylated AIE (OTPA-BBT) dots with strong absorption and extremely high NIR-II PLQY as 13.6%, and proposed the long-aliphatic-chain design blueprint contributing to their excretion from animal body. Assisted by the OTPA-BBT dots with bright fluorescence beyond 1100 nm and even 1500 nm (NIR-IIb), large-depth cerebral vasculature (beyond 600 m) as well as real-time blood flowing were monitored through-thinned-skull, and noninvasive NIR-IIb imaging with rich high-spatial-frequency information gave a precise presentation of gastrointestinal tract in marmosets. Importantly, after intravenous or oral administration, the definite excretion of OTPA-BBT dots from the body was demonstrated, which showed an influential evidence of bio-safety.

sentences: Detailed metabolic phenotyping of four tissue specific Cas9 transgenic mouse lines CRISPR/Cas9 technology has revolutionized gene editing and fast tracked our capacity to manipulate genes of interest for the benefit of both research and therapeutic applications. Whilst many advances have, and continue to be made in this area, perhaps the most utilized technology to date has been the generation of knockout cells, tissues and animals by taking advantage of Cas9 function to promote indels in precise locations in the genome. Whilst the advantages of this technology are many fold, some questions still remain regarding the effects that long term expression of foreign proteins such as Cas9, have on mammalian cell function. Several studies have proposed that chronic overexpression of Cas9, with or without its accompanying guide RNAs, may have deleterious effects on cell function and health. This is of particular concern when applying this technology in vivo, where chronic expression of Cas9 in tissues of interest may promote disease-like phenotypes and thus confound the investigation of the effects of the gene of interest. Although these concerns remain valid, no study to our knowledge has yet to demonstrate this directly. Thus, in this study we used the lox-stop-lox (LSL) spCas9 ROSA26 transgenic (Tg) mouse line to generate four tissue-specific Cas9-Tg models with expression in the heart, liver, skeletal muscle and adipose tissue. We performed comprehensive phenotyping of these mice up to 20-weeks of age and subsequently performed molecular analysis of their organs. We demonstrated that Cas9 expression in these tissues had no detrimental effect on whole body health of the animals, nor did it induce any tissue-specific effects on energy metabolism, liver health, inflammation, fibrosis, heart function or muscle mass. Thus, our data suggests that these models are suitable for studying the tissue specific effects of gene deletion using the LSL-Cas9-Tg model, and that phenotypes observed utilizing these models can be confidently interpreted as being gene specific, and not confounded by the chronic overexpression of Cas9.

sentences: SeqWho: Reliable, rapid determination of sequence file identity using k-mer frequencies With the vast improvements in sequencing technologies and increased number of protocols, sequencing is finding more applications to answer complex biological problems. Thus, the amount of publicly available sequencing data has tremendously increased in repositories such as SRA, EGA, and ENCODE. With any large online database, there is a critical need to accurately document study metadata, such as the source protocol and organism. In some cases, this metadata may not be systematically verified by the hosting sites and may result in a negative influence on future studies. Here we present SeqWho, a program designed to heuristically assess the quality of sequencing files and reliably classify the organism and protocol type. This is done in an alignment-free algorithm that leverages a Random Forest classifier to learn from native biases in k-mer frequencies and repeat sequence identities between different sequencing technologies and species. Here, we show that our method can accurately and rapidly distinguish between human and mouse, nine different sequencing technologies, and both together, 98.32%, 97.86%, and 96.38% of the time in high confidence calls respectively. This demonstrates that SeqWho is a powerful method for reliably checking the identity of the sequencing files used in any pipeline and illustrates the programs ability to leverage k-mer biases.

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Multi-omics and deep learning provide a multifaceted view of cancer Cancer is a complex disease with a large financial and healthcare burden on society. One hallmark of the disease is the uncontrolled growth and proliferation of malignant cells. Unlike Mendelian diseases which may be explained by a few genomic loci, a deeper molecular and mechanistic understanding of the development of cancer is needed. Such an endeavor requires the integration of tens of thousands of molecular features across multiple layers of information encoded in the cells. In practical terms, this implies integration of multi omics information from the genome, transcriptome, epigenome, proteome, metabolome, and even micro-environmental factors such as the microbiome. Finding mechanistic insights and biomarkers in such a high dimensional space is a challenging task. Therefore, efficient machine learning techniques are needed to reduce the dimensionality of the data while simultaneously discovering complex but meaningful biomarkers. These markers then can lead to testable hypotheses in research and clinical applications. In this study, we applied advanced deep learning methods to uncover multi-omic fingerprints that are associated with a wide range of clinical and molecular features of tumor samples. Using these fingerprints, we can accurately classify different cancer types, and their subtypes. Non-linear multi-omic fingerprints can uncover clinical features associated with patient survival and response to treatment, ranging from chemotherapy to immunotherapy. In addition, multi-omic fingerprints may be deconvoluted into a meaningful subset of genes and genomic alterations to support clinically relevant decisions.

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sentences: Discovery of small molecule inhibitors of the PTK7/β-catenin interaction targeting the Wnt signaling pathway in colorectal cancer Second cause of death due to cancer worldwide, colorectal cancer (CRC) is a major public health issue. The discovery of new therapeutic targets is thus essential. The pseudokinase PTK7 intervenes in the regulation of the Wnt/{beta}-catenin pathway signaling, in part, through a kinase-domain dependent interaction with the {beta}-catenin protein. PTK7 is overexpressed in CRC; an event associated with metastatic development and reduced survival of non-metastatic patient. In addition, numerous alterations have been identified in CRC inducing constitutive activation of Wnt/{beta}-catenin pathway signaling through {beta}-catenin accumulation. Thus, we thought that targeting PTK7/{beta}-catenin interaction could be of interest for future drug development. In this study, we have developed a NanoBRET screening assay recapitulating the interaction between PTK7 and {beta}-catenin to identify compounds able to disrupt this protein-protein interaction. A high-throughput screening allowed us to identify small molecule inhibitors targeting the Wnt pathway signaling and inducing anti-proliferative effect in vitro in CRC cells harboring {beta}-catenin or APC mutations downstream of PTK7. Thus, inhibition of the PTK7/{beta}-catenin interaction could represent, in the future, a new therapeutic strategy to inhibit cell growth dependent on Wnt signaling pathway. Moreover, despite a lack of enzymatic activity of its tyrosine kinase domain, targeting the PTK7 kinase domain-dependent functions appears to be of interest for further therapeutic development.

sentences: Homogeneity of antibody-drug conjugates critically impacts the therapeutic efficacy in brain tumors Glioblastoma multiforme (GBM) is characterized by aggressive growth and the poorest prognosis of all brain tumor types. Most therapies rarely provide clinically meaningful improvements in outcomes of patients with GBM. Antibody-drug conjugates (ADCs) are emerging chemotherapeutics with stunning success in cancer management. Although promising, clinical studies of three ADCs for treating GBM, including Depatux-M, have been discontinued because of safety concerns and limited therapeutic benefits. Here, we report that ADC homogeneity is a critical parameter to maximize the therapeutic potential in GBM therapy. We demonstrate that homogeneous conjugates generated using our linker show enhanced drug delivery to intracranial brain tumors. Notably, compared to heterogeneous ADCs, including a Depatux-M analog, our ADCs provide greatly improved antitumor effects and survival benefits in orthotopic brain tumor models, including a patient-derived xenograft model of GBM. Our findings warrant the future development of homogeneous ADCs as promising molecular entities toward cures for intractable brain tumors.

sentences: Acute inflammation, mediated by lung neutrophils, confers enhanced protection against Mycobacterium tuberculosis infection in mice Inflammation plays a crucial role in the control of Mycobacterium tuberculosis (M.tb) infection. In this study, we demonstrate that an inflammatory pulmonary environment at the time of infection mediated by liposaccharide (LPS) treatment in mice confers enhanced protection against M.tb for up to 6 months post infection. This transient protective inflammatory environment was associated with a neutrophil and monocyte/macrophage influx as well as increased inflammatory cytokines. In vitro infection of neutrophils from LPS treated mice demonstrated that LPS neutrophils exhibited increased recognition of M.tb, and had a greater innate capacity for killing M.tb. Finally, partial depletion of neutrophils in LPS treated mice showed an increase in M.tb burden, suggesting neutrophils conferred the enhanced protection observed in LPS treated mice. These results indicate a positive role of an inflammatory environment during initial M.tb infection, and suggests that acute inflammation at the time of M.tb infection can positively alter disease outcome.

sentences: microRNA profiling uncovers region-specific molecular correlates of threat responses in a marmoset model of anxiety Psychiatric diseases such as depression and anxiety are multifactorial conditions, highly prevalent in western societies. Human studies have identified a number of high-risk genetic variants for these diseases. Among them, polymorphisms in the promoter region of the serotonin transporter gene (SLC6A4) have attracted much attention. However, due to the paucity of experimental models, molecular alterations induced by these genetic variants and how they correlate to behavioral deficits have not been examined. Marmosets have emerged as a powerful model in translational neuroscience to investigate molecular underpinnings of complex behaviors. Here, we took advantage of naturally occurring genetic polymorphisms in marmoset SLC6A4 gene that have been linked to anxiety-like behaviors. Using FACS-sorted cells from different brain regions, we revealed that marmosets bearing different SLC6A4 variants exhibit distinct microRNAs signatures in a region of the prefrontal cortex whose activity has been consistently altered in patients with depression/anxiety. We also identified DCC, a gene previously linked to these diseases, as a downstream target of the dysregulated microRNAs. Significantly, we showed that levels of both microRNAs and DCC in this region were highly correlated to anxiety-like behaviors as well as to the response to citalopram, a selective serotonin re-uptake inhibitor and widely prescribed anti-depressant. Our findings establish links between genetic variants, molecular modifications in specific cortical regions and complex behavioral/pharmacological responses, providing new insights into gene-behavior relationships underlying human psychopathology.

sentences: SUMO conjugation regulates the activity of the Integrator complex RNA pol II transcribes snRNA genes in close proximity to Cajal bodies, subnuclear compartments that depend on the SUMO isopeptidase USPL1 for their assembly. We show here that overexpression of USPL1 alters snRNA 3-end cleavage, a process carried out by the Integrator complex. Beyond its role in snRNA biogenesis, this complex is responsible for regulating the expression of different non-coding and coding transcripts. We validated several subunits of the complex as SUMO conjugation substrates, and found that the SUMOylation of INTS11 subunit is regulated by USPL1. We defined Lys 381, Lys 462 and Lys 475 as bona fide SUMO attachment sites within INTS11 and observed that SUMOylation of this protein is required for efficient Integrator activity. Moreover, while an INTS11 SUMOylation deficient mutant is still capable of interacting with INTS4 and INTS9, its interaction with other subunits of the complex is affected. This mutant also shows a more cytoplasmatic localization than the wild type protein. These findings point to a regulatory role of SUMO conjugation on Integrator activity and suggest the involvement of INTS11 SUMOylation in the assembly of the complex. Furthermore, this work adds the Integrator-dependent RNA processing to the growing list of cellular processes regulated by SUMO conjugation.

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A simple, inexpensive and multi scale 3D fluorescent test sample for optical sectioning microscopies Fluorescence standards allow for quality control and for the comparison of data sets across instruments and laboratories in applications of quantitative fluorescence. For example, users of microscopy core facilities expect a homogenous and time-invariant illumination and a uniform detection sensitivity, which are prerequisites for quantitative imaging analysis, particle tracking or fluorometric pH or Ca2+-concentration measurements. Similarly, confirming the three-dimensional (3-D) resolution of optical sectioning micro-scopes prior to volumetric reconstructions calls for a regular calibration with a standardised point source. Typically, the test samples required for such calibration measurements are different ones, and they depend much on the very microscope technique used. Also, the ever-increasing choice among these techniques increases the demand for comparison and metrology across instruments. Here, we advocate and demonstrate the multiple uses of a surprisingly versatile and simple 3-D test sample that can complement existing and much more expensive calibration samples: simple commercial tissue paper labelled with a fluorescent highlighter pen. We provide relevant sample characteristics and show examples ranging from the sub-{micro}m to cm scale, acquired on epifluorescence, confocal, image scanning, two-photon (2P) and light-sheet microscopes.

Graphical abstractPyranine-labeled tissue paper, imaged upon 405-nm epifluorescence excitation through a 455LP LP dichroic and 465LP emission filter. Objective x20/NA0.25. Overlaid are the normalised absorbance (dashed) and emission spectra (through line), respectively. In the present work we show that this "primitive" and inexpensive three-dimensional (3-D) test sample is a surprisingly versatile and powerful tool for quality assessment, comparison across microscopes as well as routine metrology for optical sectioning techniques, both for research labs and imaging core facilities.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/441588v1_ufig1.gif" ALT="Figure 1">
View larger version (84K):
org.highwire.dtl.DTLVardef@13a11dforg.highwire.dtl.DTLVardef@16b818corg.highwire.dtl.DTLVardef@1061c64org.highwire.dtl.DTLVardef@32015a_HPS_FORMAT_FIGEXP  M_FIG C_FIG Research highlights- highlighter-pen marked tissue paper is a surprisingly powerful and versatile test sample for 3-D fluorescence microscopies
- standard tissue paper presents features ranging from 400 nm to centimetres
- our sample can simultaneously be used for testing intensity, field homogeneity, resolution, optical sectioning and image contrast
- it is easy to prepare, versatile, photostable and inexpensive

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Glutathione synthetase overexpression in Acidithiobacillus ferrooxidans improves halotolerance of iron oxidation Acidithiobacillus ferrooxidans are well-studied iron- and sulfur-oxidizing acidophilic chemolithoautotrophs that are exploited for their ability to participate in the bioleaching of metal sulfides. Here, we overexpressed the endogenous glutamate--cysteine ligase and glutathione synthetase genes in separate strains and found that glutathione synthetase overexpression increased intracellular glutathione levels. We explored the impact of pH on the halotolerance of iron oxidation in wild type and engineered cultures. The increase in glutathione allowed the modified cells to grow under salt concentrations and pH conditions that are fully inhibitory to wild type cells. These results indicate that glutathione overexpression can be used to increase halotolerance in A. ferrooxidans and would likely be a useful strategy on other acidophilic bacteria.

ImportanceThe use of acidophilic bacteria in the hydrometallurgical processing of sulfide ores can enable many benefits including the potential reduction of environmental impacts. The cells involved in bioleaching tend to have limited halotolerance, and increased halotolerance could enable several benefits, including a reduction in the need for fresh water resources. We show that the genetic modification of A. ferrooxidans for the overproduction of glutathione is a promising strategy to enable cells to resist the oxidative stress that can occur during growth in the presence of salt.

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Tree phyllospheres are a habitat for diverse populations of CO-oxidising bacteria BackgroundCarbon monoxide (CO) is a naturally occurring and ubiquitous trace gas in the atmosphere. As a product of combustion processes, it can reach concentrations in the mg/m3 range in urban areas, contributing to air pollution. Aerobic CO-degrading microorganisms have been identified previously and are thought to remove ~370 Tg of CO in soils and oceans per year. Based on the presence of genes encoding subunits of the enzyme carbon monoxide dehydrogenase in metagenomes, a large fraction of soil bacteria may have the potential for CO degradation. The activity and diversity of CO-degrading microorganisms in above ground habitats such as the phyllosphere has not been addressed, however, and their potential role in global CO cycling remains unknown.

ResultsMonitoring of CO-degradation in leaf washes of two common British trees, Ilex aquifolium and Crataegus monogyna, demonstrated CO uptake in all samples investigated. Leaf washes of I. aquifolium had significantly higher CO oxidation rates than those of C. monogyna. A diverse range of bacterial taxa were identified as candidate CO-oxidising taxa based on high-throughput sequencing and multivariate statistical analysis of 16S rRNA amplicon data, as well as functional diversity analysis based on coxL, the gene encoding the large subunit of CO-dehydrogenase. Candidate CO-oxidising taxa included a range of Rhizobiales and Burkholderiales, of which the Burkholderiales OTUs were abundant colonisers of the phyllosphere at the time of sampling, as indicated by 16S rRNA gene sequencing. In addition, an estimated 12.4% of leaf OTUs in samples of this study contained coxL homologues, based on their predicted genomes. We also mined data of publicly available phyllosphere metagenomes for genes encoding subunits of CO-dehydrogenase which indicated that, on average, 25% of phyllosphere bacteria contained CO-dehydrogenase gene homologues. A CO-oxidising Phyllobacteriaceae strain was isolated from phyllosphere samples which contains genes encoding both CODH as well as a RuBisCO.

ConclusionsThe phyllosphere, a vast microbial habitat, supports diverse and potentially abundant CO-oxidising bacteria. These findings identify tree phyllosphere bacteria as a potential sink for atmospheric CO and highlight the need for a more detailed assessment of phyllosphere microbial communities in the global cycle of CO.

sentences: A zebrafish reporter line reveals immune and neuronal expression of endogenous retrovirus. Endogenous retroviruses (ERVs) are fossils left in our genome from retrovirus infections of the past. Their sequences are part of every vertebrate genome and their random integrations are thought to have contributed to evolution. Although ERVs are mainly kept silenced by the host genome, they are found activated in multiple disease states such as auto-inflammatory disorders and neurological diseases. What makes defining their role in health and diseases challenging is the numerous copies in mammalian genomes and the lack of tools to study them. In this study, we identified 8 copies of the zebrafish endogenous retrovirus (zferv). We created and characterised the first in vivo ERV reporter line in any species. Using a combination of live imaging, flow cytometry and single cell RNA sequencing, we mapped zferv expression to early T cells and neurons. Thus, this new tool identified tissues expressing ERV in zebrafish, highlighting a potential role of ERV during brain development and strengthening the hypothesis that ERV play a role in immunity and neurological diseases. This transgenic line is therefore a suitable tool to study the function of ERV in health and diseases.

sentences: Genome annotation of Caenorhabditis briggsae by TEC-RED identifies new exons, paralogs, and conserved and novel operons The nematode Caenorhabditis briggsae is routinely used in comparative and evolutionary studies involving its well-known cousin C. elegans. The C. briggsae genome sequence has accelerated research by facilitating the generation of new resources, tools, and functional studies of genes. While substantial progress has been made in predicting genes and start sites, experimental evidence is still lacking in many cases. Here, we report an improved annotation of the C. briggsae genome using the Trans-spliced Exon Coupled RNA End Determination (TEC-RED) technique. In addition to identifying the 5 ends of expressed genes, we have discovered operons and paralogs. In summary, our analysis yielded 10,243 unique 5 end sequence tags with matches in the C. briggsae genome. Of these, 6,395 were found to represent 4,252 unique genes along with 362 paralogs and 52 previously unknown exons. These genes included 14 that are exclusively trans-spliced in C. briggsae when compared with C. elegans orthologs. A major contribution of this study is the identification of 493 operons, of which two-thirds are fully supported by tags. In addition, two SL1-type operons were discovered. Interestingly, comparisons with C. elegans showed that only 40% of operons are conserved. Of the remaining operons, 73 are novel, including 12 that entirely lack orthologs in C. elegans. Further analysis revealed that four of the 12 novel operons are conserved in C. nigoni. Altogether, the work described here has significantly advanced our understanding of the C. briggsae system and serves as a rich resource to aid biological studies involving this species.

sentences: Using landscape genomics to infer genomic regions involved in environmental adaptation of soybean genebank accessions Understanding how crops adapt to specific environmental conditions is becoming increasingly important in the face of accelerating climate change, but the genetics of local adaptation remain little understood for many crops. Landscape genomics can reveal patterns of genetic variation that indicate adaptive diversification during crop evolution and dispersal. Here, we examine genetic differentiation and association signatures with environmental gradients in soybean (Glycine max) germplasm groups from China that were inferred from the USDA Soybean Germplasm Collection (N = 17,019 accessions) based on population structure and passport information. We recover genes previously known to be involved in soybean environmental adaptation and report numerous new candidate genes in selection signatures implicated by genomic resources such as the genome annotation and gene expression datasets to function in flowering regulation, photoperiodism and stress reaction cascades. Linkage disequilibrium network analysis suggested functional relationships between genomic regions with selection signatures, consistent with the polygenic nature of environmental adaptation. We tested whether haplotypes associated with environmental adaptation in China were present in 843 North American and 160 European soybean cultivars and found that haplotypes in major genes for early maturity have been selected during breeding, but also that a large number of haplotypes exhibiting putative adaptive variation for cold regions at high latitudes are underrepresented in modern cultivars. Our results demonstrate the value of landscape genomics analysis in genebank germplasm as a starting point for the study of crop environmental adaptation and have the potential to inform future research efforts focused on improved soybean adaptation. Functional validation of candidate genes will support understanding of their adaptive roles and likely enable the transfer of beneficial adaptive variation into modern breeding germplasm.

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A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpus itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 required PTPN23 but not the retromer subunits. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.

Author SummaryVpu triggers the degradation or mis-localization of proteins important to the hosts immune response. Vpu acts as an adaptor, linking cellular protein targets to the ubiquitination and membrane trafficking machinery. Vpu has been localized to various cellular membrane systems. By fusing wild type Vpu and Vpu-mutants to the enzyme ascorbate peroxidase, we defined the cellular proteome in proximity to Vpu and correlated this with the proteins location. We found that wild type Vpu is proximal to ESCRT proteins, retromer complexes, and sorting and late endosomal proteins. Functionally, we found that the Vpu-mediated degradation of the innate defense protein BST-2 required PTPN23, an ALIX-like protein, consistent with our observation of Vpus presence at the limiting membranes of multi-vesicular bodies.

sentences: Hierarchical dinucleotide distribution in genome along evolution and its effect on chromatin packing Dinucleotide densities and their distribution patterns vary significantly among species. Previous studies revealed that CpG is susceptible to methylation, enriched at topologically associating domains (TADs) boundaries and its distribution along the genome correlates with chromatin compartmentalization. However, the multi-scale organizations of CpG in the linear genome, their role in chromatin organization, and how they change along the evolution are only partially understood. By comparing the CpG distribution at different genomic length scales, we quantify the difference between the CpG distributions of different species and evaluate how the hierarchical uneven CpG distribution appears in evolution. The clustering of species based on the CpG distribution is consistent with the phylogenetic tree. Interestingly, we found the CpG distribution and chromatin structure to be correlated in many different length scales, especially for mammals and avians, consistent with the mosaic CpG distribution in the genomes of these species.

sentences: Discovery of highly potent pancoronavirus fusion inhibitors that also effectively inhibit COVID-19 variants from the UK and South Africa We report here the discovery of several highly potent small molecules that showed low nM potency against SARS-CoV (IC50: as low as 13 nM), SARS-CoV-2 (IC50: as low as 23 nM), and MERS-CoV (IC50: as low as 76 nM) in pseudovirus based assays with excellent selectivity indices (SI: as high as > 5000) demonstrating their pancoronavirus inhibition. Some compounds also show 100% inhibition of CPE (IC100) at 1.25 {micro}M against an authentic SARS-CoV-2 (US_WA-1/2020). Furthermore, the most active inhibitors also potently inhibited variants of concerns (VOCs), such as the UK (B.1.1.7), South Africa (B.1.351), and Delta variant (B.1.617.2), originated in India. We confirmed that one of the potent inhibitors binds to the prefusion spike protein trimer of SARS-CoV-2 by SPR. Besides, we showed that they inhibit virus-mediated cell-cell fusion. The ADME data of one of the most active inhibitors, NBCoV1, show drug-like properties. In vivo PK of NBCoV1 in rats demonstrated excellent half-life (t1/2) of 11.3 h, mean resident time (MRT) of 14.2 h, and oral bioavailability. We expect the lead inhibitors to pave the way for further development to preclinical and clinical candidates.

sentences: Clock gene-independent daily regulation of haemoglobin oxidation in red blood cells Cellular circadian rhythms confer daily temporal organisation upon behaviour and physiology that is fundamental to human health and disease. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body. Being naturally anucleate, RBC circadian rhythms share key elements of post-translational, but not transcriptional, regulation with other cell types. The physiological function and developmental regulation of RBC circadian rhythms is poorly understood, however, partly due to the small number of appropriate techniques available. Here, we extend the RBC circadian toolkit with a novel biochemical assay for haemoglobin oxidation status, termed "Bloody Blotting". Our approach relies on a redox-sensitive covalent haem-haemoglobin linkage that forms during cell lysis. Formation of this linkage exhibits daily rhythms in vitro, which are unaffected by mutations that affect the timing of circadian rhythms in nucleated cells. In vivo, haemoglobin oxidation rhythms demonstrate daily variation in the oxygen-carrying and nitrite reductase capacity of the blood, and are seen in human subjects under controlled laboratory conditions as well as in freely-behaving humans. These results extend our molecular understanding of RBC circadian rhythms and suggest they serve an important physiological role in gas transport.

sentences: Trick or Treat? Pollinator attraction in Vanilla pompona (Orchidaceae) Natural pollination of species belonging to the pantropical orchid genus Vanilla remains poorly understood. Based on sporadic records, euglossine bees have been observed visiting flowers of Neotropical Vanilla species. Our research aimed at better understanding the pollinator attraction mechanism of the Neotropical species Vanilla pompona, a crop wild relative with valuable traits for vanilla crop improvement programs. Using video footage, we identified floral visitors and examined their behavior. The flowers of V. pompona attracted Eulaema cingulata males, which distinctively displayed two behaviors: floral scent collection and nectar search; with the latter leading to pollen removal. Morphological measurements of floral and visitor traits showed that other Eulaema species may also act as potential pollinators. Additionally, we recorded natural fruit set in three populations and over a period of two years, tested for nectar presence and analyzed floral fragrances through gas chromatography - mass spectrometry. We observed a low natural fruit set (2.42%) and did not detect nectar. Twenty floral volatile compounds were identified, with the dominant compound trans-carvone oxide previously found to attract Eulaema cingulata males. We hypothesize a dual attraction of Eulaema cingulata males to V. pompona flowers, based on floral fragrance reward as the primary long-distance attraction, and food deception for successful pollen removal. Further research confirming this hypothesis is recommended to develop appropriate conservation policies for Vanilla crop wild relatives, which are the primary reserves of this crops genetic variation.

sentences: The Neuroanatomy of Speech Processing: A Large-Scale Lesion Study The neural basis of language has been studied for centuries, yet the networks critically involved in simply identifying or understanding a spoken word remain elusive. Several functional-anatomical models of critical neural substrates of receptive speech have been proposed, including (1) auditory-related regions in the left mid-posterior superior temporal lobe, (2) motor-related regions in the left frontal lobe (in normal and/or noisy conditions), the left anterior superior temporal lobe, or (4) bilateral mid-posterior superior temporal areas. One difficulty in comparing these models is that they often focus on different aspects of the sound-to-meaning pathway and are supported by different types of stimuli and tasks. Two auditory tasks that are typically used in separate studies--syllable discrimination and word comprehension--often yield different conclusions. We assessed syllable discrimination (words and nonwords) and word comprehension (clear speech and with a noise masker) in 158 individuals with focal brain damage: left (n=113) or right (n=19) hemisphere stroke, left (n=18) or right (n=8) anterior temporal lobectomy, and 26 neurologically-intact controls. Discrimination and comprehension tasks are doubly dissociable both behaviorally and neurologically. In support of a bilateral model, clear speech comprehension was near ceiling in 95% of left stroke cases and right temporal damage impaired syllable discrimination. Lesion-symptom mapping analyses for the syllable discrimination and noisy word comprehension tasks each implicated most of the left superior temporal gyrus (STG). Comprehension but not discrimination tasks also implicated the left pMTG, while discrimination but not comprehension tasks also implicated more dorsal sensorimotor regions in posterior perisylvian cortex.

sentences: A drug repurposing screen identifies altiratinib as a selective inhibitor of a key regulatory splicing kinase and a potential therapeutic for toxoplasmosis and malaria Introductory paragraphThe apicomplexa comprise a large phylum of single-celled, obligate intracellular protozoa that infect humans and animals and cause severe parasitic diseases. Available therapeutics against these devastating diseases are limited by suboptimal efficacy and frequent side effects, as well as the emergence and spread of resistance. Here, we use a drug repurposing strategy and identify altiratinib, a compound originally developed to treat glioblastoma, as a promising drug candidate with broad spectrum activity against apicomplexans. Altiratinib is parasiticidal and blocks the development of intracellular zoites in the nanomolar range and with a high selectivity index. We have identified TgPRP4K of T. gondii as the primary target of altiratinib by genetic target deconvolution, highlighting key residues within the kinase catalytic site that, when mutated, confer resistance to the drug. We have further elucidated the molecular basis of the inhibitory mechanism and species selectivity of altiratinib for TgPRP4K as well as for its P. falciparum counterpart PfCLK3. Our data also point to structural features critical for binding of the other PfCLK3 inhibitor, TCMDC-135051. Consistent with the role of this kinase family in splicing in a broad spectrum of eukaryotes, we have shown that altiratinib causes global disruption of splicing, primarily through intron retention in both T. gondii and P. falciparum. Thus, our data establish parasitic PRP4K/CLK3 as a promising pan-apicomplexan target whose repertoire of inhibitors can be expanded by the addition of altiratinib.

sentences: Endothelial transmigration hotspots limit vascular leakage through heterogeneous expression of ICAM1 Upon inflammation, leukocytes leave the circulation by crossing the endothelial monolayer at specific transmigration  hotspot regions. Although these regions support leukocyte transmigration, their functionality is not clear. We found that endothelial hotspots function to limit vascular leakage during transmigration events. Using the photo-convertible probe mEos4b, we traced back and identified original endothelial transmigration hotspots. Using this method, we show that the heterogeneous distribution of ICAM-1 determines the location of the transmigration hotspot. Interestingly, loss of ICAM-1 heterogeneity either by CRISPR/Cas9-induced knockout of ICAM-1 or equalizing the distribution of ICAM-1 in all endothelial cells results in loss of TEM hotspots but not necessarily in reduced TEM events. Functionally, loss of endothelial hotspots results in increased vascular leakage during TEM. Mechanistically, we demonstrate that the 3 extracellular Ig-like domains of ICAM-1 are crucial for hotspot recognition. However, the intracellular tail of ICAM-1 and the 4th Ig-like dimerization domain are not involved, indicating that intracellular signalling or ICAM-1 dimerization is not required for hotspot recognition. Together, we discovered that hotspots function to limit vascular leakage during inflammation-induced extravasation.

sentences: Decoding representations of discriminatory and hedonic information during appetitive and aversive touch Emotion is typically understood to be an internal subjective experience originating in the brain. Yet in the somatosensory system hedonic information is coded by mechanoreceptors at the point of sensory contact before it reaches the central nervous system. It remains unknown, however, how these distinct peripheral channels for tactile hedonic information contribute to representations of interoceptive states relative to exteroceptive experience. In this fMRI study we applied representational similarity analyses with pattern component modeling, a technique that deconstructs representational states into a weighted set of distinct predefined constructs, to dissociate how discriminatory vs. hedonic tactile information, carried by A- and C-/CT-fibers respectively, contributes to population code representations in the human brain. Results demonstrated that information about appetitive and aversive tactile sensation is represented separately from non-hedonic tactile information across cortical structures. Specifically, although hedonic touch originates as a peripheral signal, labeled at the point of contact, representations in somatosensory cortices are guided by experiences of non-hedonic touch, By contrast, representations in regions associated with interoception and affect encode signals of hedonic touch. This provides evidence of complex tactile encoding that involves both external-exteroceptive and internal-interoceptive dimensions. Importantly, hedonic touch contributes to representations of internal state as well as those of externally generated stimulation.

sentences: SCON - A Short Conditional intrON for conditional knockout with one-step zygote injection The generation of conditional alleles using CRISPR technology is still challenging. Here, we introduce a Short Conditional intrON (SCON, 189 bp) that enables rapid generation of conditional alleles via one-step zygote injection. SCON has conditional intronic function in various vertebrate species and its target insertion is as simple as CRISPR/Cas9-mediated gene tagging.

sentences: Detection of Chronic Wasting Disease prions in fetal tissues of free-ranging white-tailed deer The transmission of chronic wasting disease (CWD) has largely been attributed to contact with infectious prions shed in excretions (saliva, urine, feces, blood) by direct animal-to-animal exposure or indirect contact with the environment. Less-well studied has been the role mother-to-offspring transmission may play in the facile transmission of CWD. We asked whether such extensive spread may also be due to mother-to-offspring transmission, perhaps before birth. We thereby focused on a population of white-tailed deer from West Virginia, USA, in which CWD has been detected. Fetal tissues, ranging from 113 to 158 days of gestation, were harvested from the uteri of CWD+ dams in the asymptomatic phase of infection. Using serial protein misfolding amplification (sPMCA), we detected evidence of prion seeds in 6 of 14 in utero harvested fetuses, with earliest detection at 113 gestational days. This is the first report of CWD detection in free ranging white-tailed deer fetal tissues. Further investigation within cervid populations across North America will help define the role and impact of mother-to-offspring vertical transmission of CWD.

sentences: Efficient evolution of human antibodies from general protein language models and sequence information alone Natural evolution must explore a vast landscape of possible sequences for desirable yet rare mutations, suggesting that learning from natural evolutionary strategies could accelerate artificial evolution. Here, we report that deep learning algorithms known as protein language models can evolve human antibodies with high efficiency, despite providing the models with no information about the target antigen, binding specificity, or protein structure, and also requiring no additional task-specific finetuning or supervision. We performed language-model-guided affinity maturation of seven diverse antibodies, screening 20 or fewer variants of each antibody across only two rounds of evolution. Our evolutionary campaigns improved the binding affinities of four clinically relevant antibodies up to 7-fold and three unmatured antibodies up to 160-fold across diverse viral antigens, with many designs also demonstrating improved thermostability and viral neutralization activity. Notably, our algorithm requires only a single wildtype sequence and computes recommended amino acid changes in less than a second. Moreover, the same models that improve antibody binding also guide efficient evolution across diverse protein families and selection pressures, indicating that these results generalize to many natural settings. Contrary to prevailing notions of evolution as difficult and resource-intensive, our results suggest that when constrained to a narrow manifold of evolutionary plausibility, evolution can become much easier, which we refer to as the "efficient manifold hypothesis."

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Resistance to chemical carcinogenesis induction via a dampened inflammatory response in naked mole-rats Naked mole-rats (NMRs) have a very low spontaneous carcinogenesis rate, which has prompted scientists to study their cancer resistance mechanisms in order to provide clues for human cancer prevention. Although cancer resistance in NMRs has been intensively investigated at the cellular level, it is still unknown how strongly resistant NMR individuals are to carcinogenesis and how NMR tissues respond to experimental carcinogenesis induction. Here, we show that NMRs exhibit extraordinary resistance against potent chemical carcinogenesis induction through a dampened inflammatory response. Although carcinogenic insults damaged skin cells of both NMRs and mice, NMR skin showed markedly lower immune cell infiltration and reduced induction of inflammatory genes. NMRs harbor loss-of-function mutations in receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) genes, which are essential for necroptosis, a type of necrotic cell death that activates strong inflammation. A necroptosis-inducing stimulus did not increase death of NMR cells. After carcinogenic insults, leakage of the HMGB1, a marker of necrotic cell death, was not increased in NMR skin. In mice, inhibition or knockout of RIPK3 reduced immune cell infiltration and delayed the onset of chemical carcinogenesis. Therefore, necroptosis deficiency may serve as a cancer resistance mechanism via attenuating the inflammatory response in NMRs. Our study sheds light on the importance of a dampened inflammatory response as a non-cell-autonomous cancer resistance mechanism in NMRs. Further in vivo study of the unusual tissue immune system and carcinogenesis resistance of NMRs may lead to the development of new strategies to prevent carcinogenesis in humans.

Significance StatementIn contrast with intensive studies of cancer resistance mechanisms in naked mole-rats (NMRs) at the cellular level, little is known about how NMR individuals respond to carcinogenesis induction, despite the fact that cell-to-cell interactions in tissues regulate carcinogenesis in vivo. Here, we demonstrate that NMRs are remarkably resistant to chemical carcinogenesis induction and characteristically have attenuated tissue inflammatory responses to carcinogenic insults. NMRs have loss-of-function mutations in RIPK3 and MLKL genes and thus cannot activate necroptosis, a type of inflammation-inducing cell death. RIPK3 inhibition in mice reduced immune cell infiltration in response to carcinogenic insults and delayed the onset of chemical-induced carcinogenesis. Our results highlight the importance of studies on dampened tissue inflammatory responses to understand cancer resistance of NMRs.

sentences: Compartmap enables inference of higher-order chromatin structure in individual cells from scRNA-seq and scATAC-seq Single-cell profiling of chromatin structure remains a challenge due to cost, throughput, and resolution. We introduce compartmap to reconstruct higher-order chromatin domains in individual cells from transcriptomic (RNAseq) and epigenomic (ATACseq) assays. In cell lines and primary human samples, compartmap infers higher-order chromatin structure comparable to specialized chromatin capture methods, and identifies clinically relevant structural alterations in single cells. This provides a common lens to integrate transcriptional and epigenomic results, linking higher-order chromatin architecture to gene regulation and to clinically relevant phenotypes in individual cells.

sentences: Adaptive evolution of Moniliophthora PR-1 proteins towards its pathogenic lifestyle Moniliophthora perniciosa and Moniliophthora roreri are hemibiotrophic fungi that harbor a large number of Pathogenesis-Related 1 genes, many of which are induced in the biotrophic interaction with Theobroma cacao. Here, we provide evidence that the evolution of PR-1 in Moniliophthora was adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Phylogenetic analysis revealed conserved PR-1 genes, shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity in Moniliophthora, as well as in recent specialization cases within both species. PR-1 families in Moniliophthora with higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host-pathogen arm race. Furthermore, we show that the highly diversified MpPR-1 genes are not induced by two phytoalexins, suggesting detoxification might not be their main function as proposed before.

sentences: Species-resolved sequencing of low-biomass microbiomes by 2bRAD-M Microbiome samples with low microbial biomass or severe DNA degradation remain challenging for amplicon-based (e.g., 16S/18S-rRNA) or whole-metagenome sequencing (WMS) approaches. Here, we introduce 2bRAD-M, a highly reduced and cost-effective metagenome-sequencing strategy which only sequences ~1% of metagenome and can simultaneously produce species-level bacterial, archaeal, and fungal profiles for low-biomass and highly degraded samples. For mock communities, 2bRAD-M can accurately generate species-level taxonomic profiles for otherwise hard-to-sequence samples with (i) low biomass of merely 1 pg of total DNA, (ii) high host DNA contamination (99%), and (iii) severely fragmented DNA (50-bp) from degraded samples. Tests of 2bRAD-M on stool, skin and environment-surface samples deliver successful reconstruction of comprehensive, high-resolution microbial profiles with agreement across 16S-rRNA, WMS and existing literature. In addition, it enables microbial profiling in formalin-fixed paraffin-embedded (FFPE) cervical tissue samples which were recalcitrant to conventional approaches due to the low amount and heavy degradation of microbial DNA, and discriminated healthy tissue, pre-invasive cancer and invasive cancer via species-level microbial profiles with 91.1% accuracy. Therefore, 2bRAD-M greatly expands the reach of microbiome sequencing.

sentences: An early-morning flowering trait in rice helps retain grain yield under heat stress field conditions at flowering stage Early-morning flowering (EMF) trait is supposed to be effective in retaining grain yield due to mitigation of heat-induced spikelet sterility at flowering in rice. This study evaluated (i) phenotypic differences between a near-isogenic line carrying a QTL for EMF trait, designated as IR64+qEMF3, and a recurrent parent, IR64, under wide variation in climates and (ii) whether an EMF trait can retain grain yield under heat stress at flowering. IR64+qEMF3 had significant earlier flower opening time (FOT) in diverse environmental conditions including temperate, subtropical, and tropical regions. Under normal temperatures at flowering, IR64+qEMF3 had similar grain yield to IR64 with some significant changes in agronomic traits and yield components. Field trials in heat-vulnerable regions of central Myanmar for seven crop seasons showed that higher percentage of filled grains contributed to the significantly higher grain yield in IR64+qEMF3 among yield components when plants were exposed to daily maximum air temperatures around 36.5 {degrees}C or higher. Lower spikelet sterility in IR64+qEMF3 was attributed to the earlier FOT during cooler early morning hours. This is the first field study that clearly demonstrates the advantage of the EMF trait for retaining grain yield by stabilizing percentage of fertile grains under heat stress at flowering.

sentences: The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin Summary paragraphPoly-(ADP-ribose) polymerase inhibitors (PARPi) elicit anti-tumour activity in homologous recombination defective cancers by promoting cytotoxic, chromatin-bound, "trapped" PARP1. How cells process trapped PARP1 remains unclear. By exploiting wild-type or trapping-resistant PARP1 transgenes combined with either a rapid immunoprecipitation mass-spectrometry of endogenous proteins (RIME)-based approach, or PARP1 Apex2-proximity labelling linked to mass-spectrometry, we generated proteomic profiles of trapped and non-trapped PARP1 complexes. This combined approach identified an interaction between trapped PARP1 and the ubiquitin-regulated p97 ATPase (aka VCP). Subsequent experiments demonstrated that upon trapping, PARP1 is SUMOylated by the SUMO-ligase PIAS4 and subsequently ubiquitinated by the SUMO-targeted E3-ubiquitin ligase, RNF4, events that promote p97 recruitment and p97 ATPase-mediated removal of trapped-PARP1 from chromatin. Consistent with this, small molecule p97 complex inhibitors, including a metabolite of the clinically-used drug disulfiram (CuET) that acts as a p97 sequestration agent, prolong PARP1 trapping and thus enhance PARPi-induced cytotoxicity in homologous recombination-defective tumour cells and patient-derived tumour organoids. Taken together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 from chromatin and the response of homologous recombination defective tumour cells to PARPi.

sentences: Comparative host-pathogen dynamics of Snake Fungal Disease in sympatric species of water snakes (Nerodia) The ascomycete fungus Ophidiomyces ophiodiicola (Oo) is the causative agent of ophidiomycosis (Snake Fungal Disease), which has been detected globally. However, surveillance efforts in the central U.S., specifically Texas, have been minimal. The threatened and rare Brazos water snake (Nerodia harteri harteri) is one of the most range restricted snakes in the U.S. and is sympatric with two wide-ranging congeners, N. erythrogaster transversa and N. rhombifer, in north central Texas; thus, providing an opportunity to test comparative host-pathogen dynamics in this system. To accomplish this, we surveyed a portion of the Brazos river drainage (~400 river km) over 29 months and tested 150 Nerodia spp. for the presence of Oo via quantitative PCR and recorded any potential signs of Oo infection. We found Oo was distributed across the entire range of N. h. harteri, Oo prevalence was 46 % overall, and there was a significant association between Oo occurrence and signs of infection in our sample. Models indicated adults had a higher probability of Oo infection than juveniles and subadults, and adult N. h. harteri had a higher probability of infection than adult N. rhombifer but not higher than adult N. e. transversa. High Oo prevalence estimates (94.4%) in adult N. h. harteri has implications for their conservation and management owing to their patchy distribution, comparatively low genetic diversity, and threats from anthropogenic habitat modification.

sentences: Simulation scalability of large brain neuronal networks thanks to time asynchrony We present a new algorithm to efficiently simulate random models of large neural networks satisfying the property of time asynchrony. The model parameters (average firing rate, number of neurons, synaptic connection probability, and postsynaptic duration) are of the order of magnitude of a small mammalian brain, or of human brain areas. Through the use of activity tracking and procedural connectivity (dynamical regeneration of synapses), both computational and memory complexities of this algorithm are proved to be theoretically linear with the number of neurons. These results are experimentally validated by sequential simulations of millions of neurons and billions of synapses running in few minutes using a single thread of an equivalent desktop computer.

sentences: Copy number-aware deconvolution of tumor-normal DNA methylation profiles Aberrant methylation is a hallmark of cancer, but bulk tumor data is confounded by admixed normal cells and copy number changes. Here, we introduce Copy number-Aware Methylation Deconvolution Analysis of Cancers (CAMDAC; https://github.com/VanLoo-lab/CAMDAC), which outputs tumor purity, allele-specific copy number and deconvolved methylation estimates. We apply CAMDAC to 122 multi-region samples from 38 TRACERx non-small cell lung cancers profiled by reduced representation bisulfite sequencing. CAMDAC copy number profiles parallel those derived from genome sequencing and highlight widespread chromosomal instability. Deconvolved polymorphism-independent methylation rates enable unbiased tumor-normal and tumor-tumor differential methylation calling. Read-phasing validates CAMDAC methylation rates and directly links genotype and epitype. We show increased epigenetic instability in adenocarcinoma vs. squamous cell carcinoma, frequent hypermethylation at sites carrying somatic mutations, and parallel copy number losses and methylation changes at imprinted loci. Unlike bulk methylomes, CAMDAC profiles recapitulate tumor phylogenies and evidence distinct patterns of epigenetic heterogeneity in lung cancer.

sentences: Can you affect me? The influence of vitality forms on action perception and motor response During the interaction with others, action, speech, and touches can communicate positive, neutral, or negative attitudes. Offering an apple can be gentle or rude, a caress can be kind or rushed. These subtle aspects of social communication have been named vitality forms by Daniel Stern. Although they characterize all human interactions, to date it is not clear whether vitality forms expressed by an agent affect the action perception and the motor response of the receiver. To this purpose, we carried out a psychophysics study aiming to investigate how perceiving different vitality forms can influence cognitive and motor tasks performed by participants. In particular, participants were stimulated with requests made through a physical contact or vocally and conveying rude or gentle vitality forms, and then they were asked to estimate the end of a passing action observed in a monitor (action estimation task) or to perform an action in front of it (action execution task) with the intention to pass an object to the other person presented in the video. Results showed that the perception of different vitality forms influences both the estimated duration of the action and the motor response of participants, suggesting how these forms of communication can positively or negatively affect our behavior.

sentences: The oligomannose N-glycans 3D architecture and its response to the FcγRIIIa structural landscape Oligomannoses are evolutionarily the oldest class of N-glycans, where the arms of the common pentasaccharide unit, i.e. Man 1-6)-[Man(1-3)]-Man{beta}(1-4)-GlcNAc{beta}(1-4)-GlcNAc{beta}1-Asn, are functionalized exclusively with branched arrangements of mannose (Man) monosaccharide units. In mammalian species oligomannose N-glycans can have up to 9 Man, meanwhile structures can grow to over 200 units in yeast mannan. The highly dynamic nature, branching complexity and 3D structure of oligomannoses have been recently highlighted for their roles in immune escape and infectivity of enveloped viruses, such as HIV-1 and SARS-CoV2. The architectural features that allow these N-glycans to perform their functions is yet unclear, due to their intrinsically disordered nature that hinders their structural characterization. In this work we will discuss the results of over 54 {micro}s of cumulative sampling by molecular dynamics (MD) simulations of differently processed, free (not protein-linked) oligomannose N-glycans common in vertebrates. We then discuss the effects of a protein surface on their structural equilibria based on over 4 {micro}s cumulative MD sampling of the fully glycosylated CD16a Fc gamma receptor (Fc{gamma}RIIIa), where the type of glycosylation is known to modulate its binding affinity for IgG1s, regulating the antibody-dependent cellular cytotoxicity (ADCC). Our results show that the proteins structural constraints shift the oligomannoses conformational ensemble to promote conformers that satisfy the steric requirements and hydrogen bonding networks demanded by the proteins surface landscape. More importantly, we find that the protein does not actively distort the N-glycans into structures not populated in the unlinked forms in solution. Ultimately, the highly populated conformations of the Man5 linked glycans support experimental evidence of high levels of hybrid complex forms at N45 and show a specific presentation of the arms at N162, which may be involved in mediating binding affinity to the IgG1 Fc.

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Eliminating accidental deviations to minimize generalization error and maximize replicability: applications in connectomics and genomics Replicability, the ability to replicate scientific findings, is a prerequisite for scientific discovery and clinical utility. Troublingly, we are in the midst of a replicability crisis. A key to replicability is that multiple measurements of the same item (e.g., experimental sample or clinical participant) under fixed experimental constraints are relatively similar to one another. Thus, statistics that quantify the relative contributions of accidental deviations--such as measurement error--as compared to systematic deviations--such as individual differences--are critical. We demonstrate that existing replicability statistics, such as intra-class correlation coefficient and fingerprinting, fail to adequately differentiate between accidental and systematic deviations in very simple settings. We therefore propose a novel statistic, discriminability, which quantifies the degree to which an individuals samples are relatively similar to one another, without restricting the data to be univariate, Gaussian, or even Euclidean. Using this statistic, we introduce the possibility of optimizing experimental design via increasing discriminability and prove that optimizing discriminability improves performance bounds in subsequent inference tasks. In extensive simulated and real datasets (focusing on brain imaging and demonstrating on genomics), only optimizing data discriminability improves performance on all subsequent inference tasks for each dataset. We therefore suggest that designing experiments and analyses to optimize discriminability may be a crucial step in solving the replicability crisis, and more generally, mitigating accidental measurement error.

Author SummaryIn recent decades, the size and complexity of data has grown exponentially. Unfortunately, the increased scale of modern datasets brings many new challenges. At present, we are in the midst of a replicability crisis, in which scientific discoveries fail to replicate to new datasets. Difficulties in the measurement procedure and measurement processing pipelines coupled with the influx of complex high-resolution measurements, we believe, are at the core of the replicability crisis. If measurements themselves are not replicable, what hope can we have that we will be able to use the measurements for replicable scientific findings? We introduce the "discriminability" statistic, which quantifies how discriminable measurements are from one another, without limitations on the structure of the underlying measurements. We prove that discriminable strategies tend to be strategies which provide better accuracy on downstream scientific questions. We demonstrate the utility of discriminability over competing approaches in this context on two disparate datasets from both neuroimaging and genomics. Together, we believe these results suggest the value of designing experimental protocols and analysis procedures which optimize the discriminability.

sentences: Modeling miRNA-driven post-transcriptional regulation by using exon-intron split analysis (EISA) in pigs The contribution of microRNAs (miRNAs) to mRNA regulation has often been explored by post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes of mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc-Gottingen minipigs. Exon-intron split analysis (EISA) of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of upregulated miRNAs also in the fed state. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, EISA provides a valuable view about the regulation of gene expression, complementary to canonical differential expression analyses.

sentences: Metabolomic signature of the maternal microbiota in the fetus The maternal microbiota affects the development of the offspring by microbial metabolites translocating to the fetus. We investigated samples of placenta, fetal intestine and brain from germ-free (GF) and specific pathogen free (SPF) mouse dams by non-targeted metabolic profiling. One hundred one annotated metabolites and altogether 3680 molecular features were present in significantly different amounts in the placenta and/or fetal organs of GF and SPF mice. The concentrations of more than half of the annotated and differentially expressed metabolites were lower in the GF organs, suggesting their microbial origin or a metabolic response of the host to the presence of gut microbiota. The clearest separation was observed in the placenta. Metabolites that were detected in lower amounts in the fetal organs in the GF mice included 5-aminovaleric acid betaine, trimethylamine N-oxide, catechol-O-sulphate, hippuric and pipecolic acid. Derivatives of the amino acid tryptophan, such as kynurenine, 3-indolepropionic acid and hydroxyindoleacetic acid, were also decreased in the absence of microbiota. Several metabolites had higher levels in the GF mice. These could be precursors of microbial metabolites or indicators of host metabolic response to the absence of gut microbiota. Ninety-nine molecular features were only detected in the SPF mice, suggesting the existence of yet unidentified microbially modified metabolites that potentially influence fetal development.

sentences: Multisensory integration of social signals by a pathway from the basal amygdala to the auditory cortex in maternal mice Social encounters are inherently multimodal events, yet how and where social cues of distinct modalities merge and interact in the brain is poorly understood. For example, when their pups wander away from the nest, mother mice use a combination of vocal and olfactory signals emitted by the pups to locate and retrieve them. Previous work revealed the emergence of multisensory interactions in the auditory cortex (AC) of both dams and virgins who co-habitate with pups ( surrogates). Here we identify a neural pathway that integrates information about odors with responses to sound. We found that a scattered population of glutamatergic neurons in the basal amygdala (BA) projects to the AC and responds to odors, including the smell of pups. These neurons also exhibit increased activity when the surrogate female is searching for pups. Finally, we show that selective optogenetic activation of BA-AC neurons modulates responses to pup calls, and that this modulation switches from predominantly suppressive to predominantly excitatory after maternal experience. This supports an underappreciated role for the amygdala in directly shaping sensory representations in an experience-dependent manner. We propose that the BA-AC pathway integrates olfaction and audition to facilitate maternal care, and speculate that it may carry valence information to the AC.

sentences: EukProt: a database of genome-scale predicted proteins across the diversity of eukaryotes EukProt is a database of published and publicly available predicted protein sets selected to represent the breadth of eukaryotic diversity, currently including 993 species from all major supergroups as well as orphan taxa. The goal of the database is to provide a single, convenient resource for gene-based research across the spectrum of eukaryotic life, such as phylogenomics and gene family evolution. Each species is placed within the UniEuk taxonomic framework in order to facilitate downstream analyses, and each data set is associated with a unique, persistent identifier to facilitate comparison and replication among analyses. The database is regularly updated, and all versions will be permanently stored and made available via FigShare. The current version has a number of updates, notably  The Comparative Set (TCS), a reduced taxonomic set with high estimated completeness while maintaining a substantial phylogenetic breadth, which comprises 196 predicted proteomes. We invite the community to provide suggestions for new data sets and new annotation features to be included in subsequent versions, with the goal of building a collaborative resource that will promote research to understand eukaryotic diversity and diversification.

sentences: Two step aggregation kinetics of Amyloid-β proteins from fractal analysis Self-aggregation in proteins has long been studied and modeled due to its ubiquity and importance in many biological contexts. Several models propose a two step aggregation mechanism, consisting of linear growth of fibrils and branch formation. Single molecule imaging techniques such as total internal reflection fluorescence (TIRF) microscopy can provide direct evidence of such mechanisms, however, analyzing such large datasets is challenging. In this paper, we analyze for the first time, images of growing amyloid fibrils obtained from TIRF microscopy using the techniques of fractal geometry, which provides a natural framework to disentangle the two types of growth mechanisms at play. We find that after an initial linear growth phase, identified by a plateau in the average fractal dimension with time, the occurrence of branching events leads to a further increase in the fractal dimension with a final saturation value {approx} 2. We also simulate the aggregation process using the identified linear growth and secondary nucleation mechanisms, using an event driven algorithm. We theoretically model this system using a set of coupled nonlinear differential equations describing a mean field model for branching and linear growth, which we use to characterize the growth process observed in simulations as well as experiments. Finally, we provide estimates for the parameter regimes that govern the two step aggregation process observed in experiments.

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Rapid adaptation and range expansion in response to agriculture over the last two centuries North America has seen a massive increase in cropland use since 1800, accompanied more recently by the intensification of agricultural practices. Through genome analysis of present-day and historical samples spanning environments over the last two centuries, we studied the impact of these changes in farming on the extent and tempo of evolution in the native common waterhemp (Amaranthus tuberculatus), a now pervasive agricultural weed. Modern agriculture has imposed strengths of selection rarely observed in the wild (0.027-0.10), with striking shifts in allele frequency trajectories since agricultural intensification in the 1960s. An evolutionary response to this extreme selection was facilitated by a concurrent human-mediated range shift. By reshaping genome-wide diversity and variation for fitness, agriculture has driven the success of this 21st-century weed.

One Sentence SummaryModern agriculture has dramatically shaped the evolution of a native plant into an agricultural weed through imposing strengths of selection rarely observed in the wild.

sentences: Inferring kinetic rate constants from single-molecule FRET trajectories - a blind benchmark of kinetic analysis tools Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We tested them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

sentences: Development and validation of Arc nanobodies: new tools for probing Arc dynamics and function Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N-and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc and analysis of capsid domain specific functions.

sentences: Single voxel autocorrelation uncovers gradients of temporal dynamics in the hippocampus and entorhinal cortex during rest and navigation During navigation, information at multiple scales needs to be integrated. Single- unit recordings in rodents suggest that gradients of temporal dynamics in the hippocampus and entorhinal cortex support this integration. In humans, gradients of representation are observed, such that granularity of information represented increases along the long axis of the hippocampus. The neural underpinnings of this gradient in humans, however, are still unknown. Current research is limited by coarse fMRI analysis techniques that obscure the activity of individual voxels, preventing investigation of how moment-to-moment changes in brain signal are organized and how they are related to behavior. Here, we measured the signal stability of single voxels over time to uncover previously unappreciated gradients of temporal dynamics in the hippocampus and entorhinal cortex. Using our novel, single voxel autocorrelation technique, we show a medial-lateral hippocampal gradient, as well as a continuous autocorrelation gradient along the anterolateral- posteromedial entorhinal extent. Importantly, we show that autocorrelation in the anterior-medial hippocampus was modulated by navigational difficulty, providing the first evidence that changes in signal stability in single voxels are relevant for behavior. This work opens the door for future research on how temporal gradients within these structures support the integration of information for goal- directed behavior.

sentences: Genetic, metabolic, and molecular insights into the diverse outcomes of diet-induced obesity in mice Overweight and obesity are increasingly common public health issues worldwide, leading to a wide range of diseases from metabolic syndrome to steatohepatitis and cardiovascular diseases. While the increase in the prevalence of obesity is partly attributable to changes in lifestyle (i.e. increased sedentarity and changes in eating behaviour), the metabolic and clinical impacts of these obesogenic conditions varies between sexes and genetic backgrounds. The conception of personalised treatments of obesity and its complications require a thorough understanding of the diversity of responses to conditions such as high-fat diet intake. By analysing nine genetically diverse mouse strains, we show that much like humans, mice respond to high-fat diet in a genetic- and sex-dependent manner. Physiological and molecular responses to high-fat diet are associated with expression of genes involved in immunity and mitochondrial function. Finally, we find that mitochondrial function may explain part of the diversity of physiological responses. By exploring the complex interactions between genetics and metabolic phenotypes via gene expression and molecular traits, we shed light on the importance of genetic background and sex in determining metabolic outcomes. In addition to providing the community with an extensive resource for optimizing future experiments, our work serves as an exemplary design for more generalizable translational studies.

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SAILER: Scalable and Accurate Invariant Representation Learning for Single-Cell ATAC-Seq Processing and Integration MotivationSingle-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) provides new opportunities to dissect epigenomic heterogeneity and elucidate transcriptional regulatory mechanisms. However, computational modelling of scATAC-seq data is challenging due to its high dimension, extreme sparsity, complex dependencies, and high sensitivity to confounding factors from various sources.

ResultsHere we propose a new deep generative model framework, named SAILER, for analysing scATAC-seq data. SAILER aims to learn a low-dimensional nonlinear latent representation of each cell that defines its intrinsic chromatin state, invariant to extrinsic confounding factors like read depth and batch effects. SAILER adopts the conventional encoder-decoder framework to learn the latent representation but imposes additional constraints to ensure the independence of the learned representations from the confounding factors. Experimental results on both simulated and real scATAC-seq datasets demonstrate that SAILER learns better and biologically more meaningful representations of cells than other methods. Its noise-free cell embeddings bring in significant benefits in downstream analyses: Clustering and imputation based on SAILER result in 6.9% and 18.5% improvements over existing methods, respectively. Moreover, because no matrix factorization is involved, SAILER can easily scale to process millions of cells. We implemented SAILER into a software package, freely available to all for large-scale scATAC-seq data analysis.

AvailabilityThe software is publicly available at https://github.com/uci-cbcl/SAILER

Contactjingz31@uci.edu and xhx@uci.edu

sentences: Lesion environments direct transplanted neural progenitors towards a wound repair astroglial phenotype Neural progenitor cells (NPC) represent potential cell transplantation therapies for CNS injuries. To understand how lesion environments influence transplanted NPC fate in vivo, we derived NPC expressing a ribosomal protein-hemagglutinin tag (RiboTag) for transcriptional profiling of transplanted NPC. Here, we show that NPC grafted into uninjured CNS generate cells that are transcriptionally similar to healthy astrocytes and oligodendrocyte lineages. In striking contrast, NPC transplanted into serum-exposed CNS lesions after stroke or spinal cord injury generate cells that share transcriptional, morphological and functional features with newly proliferated host astroglia that restrict inflammation and fibrosis and thereby protect adjacent neural tissue. Our findings reveal overlapping differentiation potentials of grafted NPC and proliferating host astrocytes; and show that in the absence of other interventions, non-cell autonomous cues in CNS lesions direct the differentiation of grafted NPC predominantly towards a naturally occurring neuroprotective wound repair astroglial phenotype.

sentences: Mapping of individual sensory nerve axons from digits to spinal cord with the Transparent Embedding Solvent System Understanding the connections and projections of neurons has been a fundamental issue for neuroscience. Although strategies have been developed to map the projection of individual axons within the mouse brain, high resolution mapping of individual peripheral nerve axons in peripheral organs or spinal cord has never been achieved. Here, we designed the Transparent Embedding Solvent System (TESOS) method and developed a technical pipeline for imaging, reconstructing and analyzing large samples containing various tissue types at sub-micron resolution. The mouse whole body was reconstructed at micron-scale resolution. We were able to image, reconstruct and analyze the complete axonal projection of individual sensory neurons within an intact mouse paw or spinal cord at sub-micron resolution. Furtherly, we imaged and reconstructed the entire projection course of individual sensory neurons from adult mouse digits to the spinal cord. The TESOS method provides an efficient tool for micron-scale connectome mapping of the peripheral nervous system.

sentences: Poly(rC)-binding protein 1 limits hepatitis C virus assembly and secretion The hepatitis C virus (HCV) co-opts numerous cellular elements - including proteins, lipids, and microRNAs - to complete its viral life cycle. The cellular RNA-binding protein, poly(rC)-binding protein 1 (PCBP1), was previously reported to bind to the 5 untranslated region (UTR) of the HCV genome; however, its importance in the viral life cycle has remained unclear. Herein, we sought to clarify the role of PCBP1 in the HCV life cycle. Using the HCV cell culture (HCVcc) system, we found that knockdown of endogenous PCBP1 resulted in an overall decrease in viral RNA accumulation, yet resulted in an increase in extracellular viral titers. To dissect PCBP1s specific role in the HCV life cycle, we carried out assays for viral entry, translation, genome stability, RNA replication, as well as virion assembly and secretion. We found that PCBP1 knockdown did not directly affect viral entry, translation, RNA stability, or RNA replication, but resulted in an overall increase in infectious particle secretion. This increase in virion secretion was evident even when viral RNA synthesis was inhibited, and blocking virus secretion could partially restore the viral RNA accumulation decreased by PCBP1 knockdown. We therefore propose a model where endogenous PCBP1 normally limits virion assembly and secretion, which increases viral RNA accumulation in infected cells by preventing the departure of viral genomes packaged into virions. Overall, our findings improve our understanding of how cellular RNA-binding proteins influence viral genomic RNA utilization during the HCV life cycle.

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Effective infection with dengue virus in experimental neonate and adult mice through intranasal route As a member of the family Flaviviridae, the dengue virus is transmitted to humans through mosquito vectors. The virus causes dengue fever with flu-like symptoms, life-threatening dengue hemorrhagic fever, and dengue shock syndrome that affects four billion people in 128 countries. It can also be transmitted through atypical routes, such as needle stick injury, vertical transmission, and the receipt of contaminated blood or organs. Although sporadic cases cannot be classified as atypical transmission routes and raise respiratory exposure concerns, the risks remain unclear. Here, we analyzed the respiratory infectivity of the dengue virus in BALB/c suckling and A6 adult mice using the dengue virus serotype 2 and intranasal inoculation. The results showed that the clinical symptoms of intranasally infected mice included excitement, emaciation, malaise, and death, and histopathological changes and viremia dynamics were observed in multiple tissues, including the brain, liver, and spleen. Notably, the DENV serotype 2 showed clear brain tropism after intranasal infection, and viral loads peaked at seven days post-inoculation. Furthermore, the virus was isolated from mouse brains, and the sequence homology between the viral stock and dengue virus isolates was 99.99%. Similar results were observed in adult IFN-/{beta} receptor-deficient mice. Taken together, our results suggest that the DENV serotype 2 can infect suckling mice and adult immune-deficient mice via the nasal route. The results of our study broaden our perception of atypical dengue transmission routes and may help explain dengue virus infections that occur without the presence of mosquito vectors.

ImportanceDengue is transmitted mainly via mosquito vectors. Flu-like dengue fever or life-threatening severe dengue threatens four billion people in 128 countries. Non-vector dengue cases have occurred occasionally since 1990. These cases cannot be classified as atypical transmission routes and are suspected to have arisen via sexual or respiratory transmission. Recently, the sexual transmission of dengue has been confirmed in humans. The respiratory infectivity of other members of the Flaviviridae family, such as Zika and Japanese encephalitis viruses, has been determined under experimental conditions. The potential for respiratory infection with the dengue virus has not yet been confirmed. In this study, we analyzed the respiratory infectivity of the dengue virus in suckling mice and adult immunodeficient mice via intranasal inoculation, thereby exploring the potential for respiratory transmission in cases with non-vector dengue infection, especially for patients who were exposed to dengue virus contaminants.

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Effects of different environmental intervention durations on the intestinal mucosal barrier and the brain-gut axis in rats with colorectal cancer ObjectivesEnriched environment (EE) is a promising strategy to protect the intestinal mucosal barrier and regulate brain-gut peptide expression. However, the optimal enriched environment intervention duration is unknown. Here, different EE intervention durations were applied to assess the optimal intervention duration in rats with colorectal cancer.

MethodsWe used a rat model of 1,2-dimethylhydrazine-induced colorectal cancer. Rats were housed in an EE for 0, 2, 4 or 8 weeks. The intestinal mucosa and serum TNF-, IL-6, IL-10, DAO, ATP, CRF, and occludin levels and bacterial translocation (BT) were measured, and the intestinal mucosa morphology was evaluated.

ResultsEight-week EE intervention was more beneficial to the intestinal mucosal mechanical barrier than 2-week or 4-week intervention (P<0.05). There was a significant different between the 4-week and 8-week groups on BT (P=0.049). However, which intervention duration had the greatest advantages in intestinal mucosa and serum inflammatory factor regulation was not determined. There were no significant differences in the effects of different EE intervention durations on BT or brain intestinal peptide levels among the other groups (P>0.05).

ConclusionsThe effect of an 8-week environmental intervention duration on the intestinal mucosal barrier was better than that of 2-week and 4-week durations overall, but the effect of different environmental intervention durations on brain-gut peptide levels was not obvious. In the future, we can further explore the molecular biological mechanism of the effect of different EE intervention durations on the intestinal mucosal barrier and analyze the effect of an EE on other brain-gut peptides.

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Rare and population-specific functional variation across pig lines BackgroundIt is expected that functional, mainly missense and loss-of-function (LOF), and regulatory variants are responsible for phenotypic differences among breeds, genetic lines, and varieties of livestock and crop species that have undergone diverse selection histories. However, there is still limited knowledge about the existing missense and LOF variation in livestock commercial populations, in particular regarding population-specific variation and how it can affect applications such as across-breed genomic prediction.

MethodsWe re-sequenced the whole genome of 7,848 individuals from nine commercial pig breeding lines (average sequencing coverage: 4.1x) and imputed whole-genome genotypes for 440,610 pedigree-related individuals. The called variants were categorized according to predicted functional annotation (from LOF to intergenic) and prevalence level (number of lines in which the variant segregated; from private to widespread). Variants in each category were examined in terms of distribution along the genome, minor allele frequency, Wrights fixation index (FST), individual load, and association to production traits.

ResultsOf the 46 million called variants, 28% were private (called in only one line) and 21% were widespread (called in all nine lines). Genomic regions with low recombination rate were enriched with private variants. Low-prevalence variants (called in one or a few lines only) were enriched for lower allele frequencies, lower FST, and putatively functional and regulatory roles (including loss-of-function and deleterious missense variants). Only a small subset of low-prevalence variants was found at intermediate allele frequencies and had large estimated effects on production traits. Individuals on average carried less private deleterious missense alleles than expected compared to other predicted consequence types. A small subset of low-prevalence variants with intermediate allele frequencies and higher FST were detected as significantly associated to the production traits and explained small fractions of phenotypic variance (up to 3.2%). These associations were tagged by other more widespread variants, including intergenic variants.

ConclusionsMost low-prevalence variants are kept at very low allele frequency and only a small subset contributed detectable fractions of phenotypic variance. Not accounting for low-prevalence variants is therefore unlikely to hinder across-breed analyses, in particular for genomic prediction of breeding values using reference populations of a different genetic background.

sentences: Knockdown of astrocytic monocarboxylate transporter 4 (MCT4) in the motor cortex leads to loss of dendritic spines and a deficit in motor learning Monocarboxylate transporters (MCTs) shuttle molecules, including L-lactate, involved in metabolism and cell signaling of the central nervous system. Astrocyte-specific MCT4 is a key component of the astrocyte-neuron lactate shuttle (ANLS) and is important for neuroplasticity and learning of the hippocampus. However, the importance of astrocyte-specific MCT4 in neuroplasticity of the M1 primary motor cortex remains unknown. In this study, we investigated astrocyte-specific MCT4 in motor learning and neuroplasticity of the M1 primary motor cortex using a cell-type specific shRNA knockdown of MCT4. Knockdown of astrocyte-specific MCT4 resulted in impaired motor performance and learning on the accelerating rotarod. In addition, MCT4 knockdown was associated with a reduction of neuronal dendritic spine density and spine width and decreased protein expression of PSD95 and Arc. Using near-infrared-conjugated 2-deoxyglucose uptake as a surrogate marker for neuronal activity, MCT4 knockdown was also associated with decreased neuronal activity in the M1 primary motor cortex and associated motor regions including the dorsal striatum and ventral thalamus. Our study supports a potential role for astrocyte-specific MCT4 and the ANLS in the neuroplasticity of the M1 primary motor cortex. Targeting MCT4 may serve to enhance neuroplasticity and motor repair in several neurological disorders, including Parkinsons disease and stroke.

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Adaptive anti-tumor immunity is orchestrated by a population of CCL5-producing tissue-resident NK cells T cell-directed cancer immunotherapy often fails to generate lasting tumor control. Harnessing additional effectors of the immune response against tumors may strengthen the clinical benefit of immunotherapies. Here, we demonstrate that therapeutic targeting of the IFN{gamma}-IL-12 pathway relies on the ability of a population of tissue-resident NK (trNK) cells to orchestrate an anti-tumor microenvironment. Particularly, utilizing an engineered adenoviral platform, we show that paracrine IL-12 enhances functional DC-CD8 T cell interactions to generate adaptive anti-tumor immunity. This effect depends on the abundance of trNK cells and specifically their capacity to produce the cDC1-chemoattractant CCL5. Failure to respond to IL-12 and other IFN{gamma}-inducing therapies such as immune checkpoint blockade in tumors with low trNK cell infiltration could be overcome by intra-tumoral delivery of CCL5. Our findings reveal a novel barrier for T cell-focused therapies and offer mechanistic insights into how T cell-NK cell-DC crosstalk can be enhanced to promote anti-tumor immunity and overcome resistance.

SignificanceWe identified the lack of CCL5-producing, tissue-resident NK (trNK) cells as a barrier to T cell-focused therapies. While IL-12 induces anti-tumoral DC-T cell crosstalk in trNK cellrich tumors, resistance to IL-12 or anti-PD-1 in trNK cellpoor tumors can be overcome by the additional delivery of CCL5.

sentences: Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli Bacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of {beta}-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3[->]3 cross-links compared to 4[->]3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify amongst eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4[->]3 (MepM) and 3[->]3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4[->]3 and 3[->]3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4[->]3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of {beta}-lactams.

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Radical pairs may explain reactive oxygen species-mediated effects of hypomagnetic field on neurogenesis Exposures to a hypomagnetic field can affect biological processes. Recently, it has been observed that hypomagnetic field exposure can adversely affect adult hippocampal neurogenesis and hippocampus-dependent cognition in mice. In the same study, the role of reactive oxygen species (ROS) in hypomagnetic field effects has been demonstrated. However, the mechanistic reasons behind this effect are not clear. This study proposes a radical pair mechanism based on a flavin-superoxide radical pair to explain the modulation of ROS production and the attenuation of adult hippocampal neurogenesis in a hypomagnetic field. The results of our calculations favor a singlet-born radical pair over a triplet-born radical pair. Our model predicts hypomagnetic field effects on the triplet/singlet yield of comparable strength as the effects observed in experimental studies on adult hippocampal neurogenesis. Our predictions are also in qualitative agreement with experimental results on superoxide concentration and other observed ROS effects. We also predict the effects of applied magnetic fields and oxygen isotopic substitution on adult hippocampal neurogenesis. Our findings strengthen the idea that nature might harness quantum resources in the context of the brain.

Author summaryExposure to magnetic fields influences many neurobiological processes. The formation of new neurons (neurogenesis) in the hippocampal region of the adult brain plays a crucial role in learning and memory. It can be adversely affected by shielding the earths magnetic field, and this effect is intimately related to ROS concentration. In this study, we have developed a quantum mechanical model to explain this magnetic field dependence of adult hippocampal neurogenesis. Our model is also consistent with the observed ROS effects.

sentences: Design, synthesis, and characterization of mG2P026 as a high contrast PET imaging ligand for metabotropic glutamate receptor 2 An array of triazolopyridines based on JNJ-46356479 (6) were synthesized as potential PET imaging ligands for metabotropic glutamate receptor 2 (mGluR2) in the brain. The selected candidates 8-11 featured an enhanced positive allosteric modulator (PAM) activity (37-fold max.) and an apparent mGluR2 agonist activity (25-fold max.) compared to compound 6. Radiolabeling of compounds 8 and 9 (also named mG2P026) was achieved via the Cu(I)-mediated radiofluorination in the automated TRACERLabTM FXF-N platform. Both [18F]8 and [18F]9 were obtained with satisfactory radiochemical yields (> 5%, non-decay corrected), high molar activity (> 180 GBq/mol), and excellent chemical and radiochemical purities (> 98%). Preliminary characterization of [18F]8 and [18F]9 in rats confirmed their excellent brain permeability with [18F]9 showing better brain heterogeneity and favorable binding kinetics. Pretreatment with different classes of PAMs enhanced the radioactivity uptake for both [18F]8 and [18F]9 at the regions of interest by 20.3-40.9% and 16.7-81.6%, respectively, due to their pharmacological effects. Further evaluation of [18F]9 in a nonhuman primate confirmed its superior brain heterogeneity in mapping mGlu2 receptors and its higher specific binding than [18F]6. Pretreatment with 0.5 mg/kg BINA led (2) to an enhanced brain uptake of [18F]9 by 3% in high tracer uptake regions that was consistent with the rat studies. Therefore, [18F]9 has the potential to be translated for human studies.

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Organization of DNA replication origin firing in Xenopus egg extracts : the role of intra-S checkpoint During cell division, the duplication of the genome starts at multiple positions called replication origins. Origin firing requires the interaction of rate-limiting factors with potential origins during the S(ynthesis)-phase of the cell cycle. Origins fire as synchronous clusters which is proposed to be regulated by the intra-S checkpoint. By modelling the unchallenged, the checkpoint-inhibited and the checkpoint protein Chk1 over-expressed replication pattern of single DNA molecules from Xenopus sperm chromatin replicated in egg extracts, we demonstrate that the quantitative modelling of data requires: 1) a segmentation of the genome into regions of low and high probability of origin firing; 2) that regions with high probability of origin firing escape intra-S checkpoint regulation and 3) the variability of the rate of DNA synthesis close to replication forks is a necessary ingredient that should be taken in to account in order to describe the dynamic of replication origin firing. This model implies that the observed origin clustering emerges from the apparent synchrony of origin firing in regions with high probability of origin firing and challenge the assumption that the intra-S checkpoint is the main regulator of origin clustering.

Author summaryDNA replication is one of the fundamental cell functions. The genome of eukaryotic organisms is duplicated from multiple positions named replication origins. Single molecule experiments allow to visualise the dynamics of spatio-temporal patterns created during replication process. The dynamic of replication process is regulated by checkpoints. However, the influence and the role of checkpoint regulation in the dynamics of spatio-temporal patterns of DNA replication is not understood. In this work we build a minimal, process-based and data rooted numerical model that allows to decipher the impact of checkpoint regulation on the dynamics of spatio-temporal pattern of DNA replication.

sentences: CG2AT2: An Enhanced Fragment-based approach for Serial Multi-scale Molecular Dynamics simulations Coarse-grained molecular dynamics provides a means for simulating the assembly and interactions of macromolecular complexes at a reduced level of representation, thereby allowing both longer timescale and larger sized simulations. Here, we describe an enhanced fragment-based protocol for converting macromolecular complexes from coarse-grained to atomistic resolution, for further refinement and analysis. While the focus is upon systems that comprise an integral membrane protein embedded in a phospholipid bilayer, the technique is also suitable for e.g. membrane-anchored and soluble protein/nucleotide complexes. Overall, this provides a method for generating an accurate and well equilibrated atomic-level description of a macromolecular complex. The approach is evaluated using a diverse test set of eleven system configurations of vary size and complexity. Simulations are assessed in terms of protein stereochemistry, conformational drift, lipid/protein interactions, and lipid dynamics.

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SkewDB: A comprehensive database of GC and 10 other skews for over 28,000 chromosomes and plasmids GC skew denotes the relative excess of G nucleotides over C nucleotides on the leading versus the lagging replication strand of eubacteria. While the effect is small, typically around 2.5%, it is robust and pervasive. GC skew and the analogous TA skew are a localized deviation from Chargaffs second parity rule, which states that G and C, and T and A occur with (mostly) equal frequency even within a strand.

Most bacteria also show the analogous TA skew. Different phyla show different kinds of skew and differing relations between TA and GC skew.

This article introduces an open access database (https://skewdb.org) of GC and 10 other skews for over 28,000 chromosomes and plasmids. Further details like codon bias, strand bias, strand lengths and taxonomic data are also included. The SkewDB database can be used to generate or verify hypotheses. Since the origins of both the second parity rule, as well as GC skew itself, are not yet satisfactorily explained, such a database may enhance our understanding of microbial DNA.

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Vapor cannabis exposure generationally affects male reproductive functions in mice This study was performed to examine whether vapor exposure to cannabis plant matter negatively impacts male reproductive functions and testis development in mice. Adult CD-1 male mice (F0) were exposed to air (control) or 200 mg of vaporized cannabis plant matter 3x/day over a 10 day period. Subsequently, F0 males were bred with drug naive CD-1 females to generate F1 males, and F1 offspring were used to generate F2 males. Cannabis vapor exposure decreased sperm count and/or motility in F0 and F1 males and disrupted the progression of germ cell development, as morphometric analyses exhibited an abnormal distribution of the stages of spermatogenesis in F0 males. Although plasma levels of testosterone were not affected by cannabis exposure in any ages or generations of males, dysregulated steroidogenic enzymes, Cyp11a1 and Cyp19a1, were observed in F0 testis. In the neonatal testis from F1 males, while apoptosis was not altered, DNA damage and DNMT1, but not DNMT3A and DNMT3B, were increased in germ cells following cannabis exposure. In contrast, the alterations of DNA damage and DNMT1 expression were not observed in F2 neonatal males. These results suggest that cannabis vapor exposure generationally affects male reproductive functions, probably due to disruption of spermatogenesis in the developing testis.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/457271v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1a6782borg.highwire.dtl.DTLVardef@1b5e42org.highwire.dtl.DTLVardef@c94d78org.highwire.dtl.DTLVardef@1fd269d_HPS_FORMAT_FIGEXP  M_FIG C_FIG HIGHLIGHTSO_LICannabis vapor exposure to adult males impairs sperm counts, motility and spermatogenesis.
C_LIO_LICannabis vapor exposure to F0 males causes reduced sperm counts in F1, but not in F2 males.
C_LIO_LIPlasma testosterone levels are not affected in F0, F1 and F2 males by cannabis exposure.
C_LIO_LICannabis increases DNA damage and the expression of DNA methyltransferase (DNMT) 1 in the F1 neonatal testis.
C_LI

sentences: LanA (Language Atlas): A probabilistic atlas for the language network based on fMRI data from >800 individuals Two analytic traditions characterize fMRI language research. One relies on averaging activations voxel-wise across individuals. This approach has limitations: because of inter-individual variability in the locations of language areas, a location in a common brain space cannot be meaningfully linked to function. An alternative approach relies on identifying language areas in each individual using a functional  localizer. Because of its greater sensitivity, functional resolution, and interpretability, functional localization is gaining popularity, but it is not always feasible, and cannot be applied retroactively to past studies. We provide a solution for bridging these currently disjoint approaches in the form of a probabilistic functional atlas created from fMRI data for an extensively validated language localizer in 806 individuals. This atlas enables estimating the probability that any given location in a common brain space belongs to the language network, and thus can help interpret group-level peaks and meta-analyses of such peaks, and lesion locations in patient investigations. More meaningful comparisons of findings across studies should increase robustness and replicability in language research.

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Mitochondrial antiviral-signalling protein is a client of the BAG6 protein quality control complex The heterotrimeric BAG6 complex coordinates the direct handover of newly synthesised tail-anchored (TA) membrane proteins from an SGTA-bound preloading complex to the endoplasmic reticulum (ER) delivery component TRC40. In contrast, defective precursors, including aberrant TA proteins, form a stable complex with this cytosolic protein quality control factor, enabling such clients to be either productively re-routed or selectively degraded. We identify the mitochondrial TA protein MAVS (mitochondrial antiviral-signalling protein) as an endogenous client of both SGTA and the BAG6 complex. Our data suggest that the BAG6 complex binds to a cytosolic pool of MAVS before its misinsertion into the ER membrane, from where it can subsequently be removed via ATP13A1-mediated dislocation. This BAG6- associated fraction of MAVS is dynamic and responds to the activation of an innate immune response, suggesting that BAG6 may modulate the pool of MAVS that is available for coordinating the cellular response to viral infection.

SUMMARY STATEMENTMitochondrial antiviral-signalling (MAVS) protein is a favoured client of the cytosolic BAG6 complex. We discuss how this dynamic interaction may modulate MAVS biogenesis at signalling membranes.

sentences: Protein-primed RNA synthesis in SARS-CoVs and structural basis for inhibition by AT-527 How viruses from the Coronaviridae family initiate viral RNA synthesis is unknown. Here we show that the SARS-CoV-1 and -2 Nidovirus RdRp-Associated Nucleotidyltransferase (NiRAN) domain on nsp12 uridylates the viral cofactor nsp8, forming a UMP-Nsp8 covalent intermediate that subsequently primes RNA synthesis from a poly(A) template; a protein-priming mechanism reminiscent of Picornaviridae enzymes. In parallel, the RdRp active site of nsp12 synthesizes a pppGpU primer, which primes (-)ssRNA synthesis at the precise genome-poly(A) junction. The guanosine analogue 5-triphosphate AT-9010 (prodrug: AT-527) tightly binds to the NiRAN and inhibits both nsp8-labeling and the initiation of RNA synthesis. A 2.98 [A] resolution Cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-(nsp8)2 /RNA/NTP quaternary complex shows AT-9010 simultaneously binds to both NiRAN and RdRp active site of nsp12, blocking their respective activities. AT-527 is currently in phase II clinical trials, and is a potent inhibitor of SARS-CoV-1 and -2, representing a promising drug for COVID-19 treatment.

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Myeloperoxidase-induced fibrinogen unfolding and clotting Fibrinogen is a major protein of blood coagulation system and is a promising component of biomaterials and protein matrixes. Conformational changes of fibrinogen underlie the important mechanism of thrombin mediated fibrinogen clotting but also may induce the loss of its biological activity and (amyloid) aggregation. Understanding and controlling of fibrinogen unfolding is important for the development of fibrinogen based materials with tunable properties. We have discovered that myeloperoxidase induces denaturation of fibrinogen molecules followed by fibrinogen clotting, which is not thrombin-dependent. This is the first example of ATP-independent, non-targeted protein-induced protein denaturation. The morphological structure of unfolded fibrinogen molecules and "non-conventional" fibrinogen clots has been characterized using high-resolution atomic force microscopy and scanning electron microscopy techniques. Circular dichroism (CD) spectroscopy has shown no significant changes of the secondary structure of the fibrinogen clots. The absorbance spectrophotometry has demonstrated that the kinetics of myeloperoxidase induced fibrinogen clotting strongly decays with growth of ionic strength indicating a major role of the Debye screening effect in regulating of this process. The obtained results provide with the novel concepts of protein unfolding and open new insights into fibrinogen clotting. Moreover, they give new possibilities in biotechnological and biomedical applications, e.g., for regulation of fibrinogen clotting and platelet adhesion and for the development of fibrinogen-based matrices.

The organization of a protein molecule is characterized by different hierarchical levels such as primary, secondary, tertiary and quaternary structure. Protein unfolding or denaturation, i.e. its transformation to a lower order structure (and loss of a higher order structure), is a biologically and biotechnologically relevant process. Protein unfolding is a prerequisite for an alternative folding pathway including amyloid aggregation 1,2. Unfolded proteins may be used in development of protein films and coatings with special properties such as enhanced mechanical stability 3-5, resistance to protein adsorption or platelet adhesion 6,7 and other advantages 8. Unfolding of a protein molecule may lead to the loss of its biological function 9 that has important consequences in biosensor 10,11 and pharmaceutical applications 12.

sentences: Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling Ribosome biogenesis is an essential process that requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. In particular, maturation of the peptidyl transferase center (PTC), the catalytic core of the ribosome, is mediated by universally conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial ribosomal large subunit (mtLSU) using a combination of endogenous complex purification, in vitro reconstitution and cryo-electron microscopy (cryo-EM). Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Subsequent addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch by releasing MTERF4-NSUN4 and GTPBP5 accompanied by the progression to a near-mature PTC state. In addition, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results define the molecular basis of dynamic GTPase-mediated PTC maturation during mitochondrial ribosome biogenesis and provide a framework for understanding step-wise progression of PTC folding as a critical quality control checkpoint in all translation systems.

sentences: Heterogeneous but not random: Cargo degradation in phagosomes is kinetically coupled to 1 their intracellular mobility Immune cells degrade internalized pathogens in phagosomes through sequential biochemical changes. The degradation must be fast enough for effective infection control. The presumption is that each phagosome degrades cargos autonomously with a distinct but stochastic kinetic rate. Here we report that the degradation kinetics of individual phagosomes is not stochastic but coupled to their intracellular motility. By engineering RotSensors that are optically anisotropic, magnetic responsive, and fluorogenic in response to degradation activities in phagosomes, we monitored cargo degradation kinetics in single phagosomes simultaneously with their translational and rotational dynamics. We show that phagosomes that move faster centripetally are more likely to encounter and fuse with lysosomes, thereby acidifying faster and degrading cargos more efficiently. The degradation rates increase nearly linearly with the translational and rotational velocities of phagosomes. Our results indicate that the centripetal motion of phagosomes functions as a clock for controlling the progression of cargo degradation.

sentences: Genome-wide identification, characterization, and comparative analysis of NLR resistance genes in Coffea spp. The largest family of disease resistance genes in plants are nucleotide-binding site leucine-rich repeat genes (NLRs). The products of these genes are responsible for recognizing avirulence proteins (Avr) of phytopathogens and triggering specific defense responses. Identifying NLRs in plant genomes with standard gene annotation software is challenging due to their multidomain nature, sequence diversity, and clustered genomic distribution. We present the results of a genome-wide scan and comparative analysis of NLR loci in three coffee species (Coffea canephora, Coffea eugenioides and their interspecific hybrid Coffea arabica). A total of 1311 non-redundant NLR loci were identified in C. arabica, 927 in C. canephora, and 1079 in C. eugenioides, of which 809, 562, and 695 are complete loci, respectively. The NLR-annotator tool used in this study showed extremely high sensitivities and specificities (over 99%) and increased the detection of putative NLRs in the reference coffee genomes. The NLRs loci in coffee are distributed among all chromosomes and are organized mostly in clusters. The C. arabica genome presented a smaller number of NLR loci when compared to the sum of the parental genomes (C. canephora, and C. eugenioides). There are orthologous NLRs (orthogroups) shared between coffee, tomato, potato, and reference NLRs and those that are shared only among coffee species, which provides clues about the functionality and evolutionary history of these orthogroups. Phylogenetic analysis demonstrated orthologous NLRs shared between C. arabica and the parental genomes and those that were possibly lost. The NLR family members in coffee are subdivided into two main groups: TIR-NLR (TNL) and non-TNL. The non-TNLs seem to represent a repertoire of resistance genes that are important in coffee. These results will support functional studies and contribute to a more precise use of these genes for breeding disease-resistant coffee cultivars.

sentences: Molecular evolutionary dynamics of energy limited microorganisms Microorganisms have the unique ability to survive extended periods of time in environments with extremely low levels of exploitable energy. To determine the extent that energy limitation affects microbial evolution, we examined the molecular evolutionary dynamics of a phylogenetically diverse set of taxa over the course of 1,000-days. We found that periodic exposure to energy limitation affected the rate of molecular evolution, the accumulation of genetic diversity, and the rate of extinction. We then determined the degree that energy limitation affected the spectrum of mutations as well as the direction of evolution at the gene level. Our results suggest that the initial depletion of energy altered the direction and rate of molecular evolution within each taxon, though after the initial depletion the rate and direction did not substantially change. However, this consistent pattern became diminished when comparisons were performed across phylogenetically distant taxa, suggesting that while the dynamics of molecular evolution under energy limitation are highly generalizable across the microbial tree of life, the targets of adaptation are specific to a given taxon.

sentences: Oncogenic RAS instructs morphological transformation of human epithelia via differential tissue mechanics. The RAS proto-oncogene is a critical regulator of cell state, morphology and mechanics, and plays a key role in cancer progression. Here, by using a human epithelial model in vitro, we ask how morpho-mechanical changes driven by oncogenic RAS activation at the level of individual cells are collectively integrated to drive changes in tissue behaviour. We found that the uniform oncogenic expression of HRAS.V12 in confined epithelial monolayers causes reproducible changes in the structure and organization of the tissue, which acquires a transitory bilayered morphology. RAS-driven bilayering associates with reproducible layer-specific differences in cell-cell contractility and cell-matrix forces. These drive the initially flat tissues to form three-dimensional structures mimicking some of the behaviours seen in human cancers. Our findings establish a physical mechanism of cellular collectives through which uniform expression of RAS can be interpreted differently in different places of the same tissue to regulate its physiological and pathological morphology.

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Prevalence of heterotrophic methylmercury detoxifying bacteria across oceanic regions Microbial reduction of inorganic divalent mercury (Hg2+) and methylmercury (MeHg) demethylation is performed by the mer operon, specifically by merA and merB genes respectively, but little is known about the mercury tolerance capacity of marine microorganisms and its prevalence in the global ocean. Here, we explored the distribution of these genes in 290 marine heterotrophic bacteria (Alteromonas and Marinobacter spp.) isolated from different oceanographic regions and depths, and assessed their tolerance to diverse concentrations of Hg2+ and MeHg. About 25% of the isolates presented merA and only 8.9% presented both merAB genes, including the strain ISS312 that exhibited the highest tolerance capacity and a degradation efficiency of 98.2% in 24 h. Fragment recruitment analyses of ISS312 genome against microbial metagenomes indicated an extensive distribution across the global bathypelagic ocean. Our findings highlighted that mercury resistance genes are widely distributed in a non-highly polluted environment such as the pelagic marine environment, and that degradation of the neurotoxic MeHg can be performed through the ocean water column by some heterotrophic bacteria at high efficiency with important implications in the biogeochemical cycle of mercury and potentially for the environment and human health.

TeaserActive mercury resistance genes detected in marine cultured bacteria are widely distributed in the ocean including the bathypelagic zone.

sentences: High interaction valency ensures cohesion and persistence of a microtubule +TIP body at the plus-end of a single specialized microtubule in yeast Microtubule plus-end tracking proteins (+TIPs) control microtubule specialization and are as such essential notably during eukaryotic cell division. Here, we investigated interactions and functions of the budding yeast Kar9 network consisting of the core +TIPs components Kar9 (functional homologue of APC, MACF, and SLAIN), Bim1 (orthologue of EB1), and Bik1 (orthologue of CLIP-170). Our data indicate that a redundant, multivalent web of interactions links the three +TIPs together to form a "Kar9 body" at the tip of a single cytoplasmic microtubule. They further suggest that this body is a liquid-like condensate, allowing it to persist on both growing and shrinking microtubule tips, and functions as a mechanical coupling device between microtubules and actin cables during mitosis. Our study underlines the power of dissecting the web of low-affinity interactions driving liquid-liquid phase separation of proteins in order to demonstrate the importance and establish the functional roles of condensation processes in vivo.

sentences: Sugar transporter Slc37a2 regulates bone metabolism via a dynamic tubular lysosomal network in osteoclasts Osteoclasts are giant bone-digesting cells that harbour specialized lysosome-related organelles termed secretory lysosomes (SLs). SLs store cathepsin K and serve as a membrane precursor to the ruffled border, the osteoclasts  resorptive apparatus. Yet, the molecular composition and spatiotemporal organization of SLs remains incompletely understood. Here, using organelle-resolution proteomics, we identify member a2 of the solute carrier 37 family (Slc37a2) as a SL sugar transporter. We demonstrate that Slc37a2 localizes to the SL limiting membrane and that these organelles adopt a hitherto unnoticed but dynamic tubular network in living osteoclasts that is required for bone digestion. Accordingly, mice lacking Slc37a2 accrue high bone mass owing to uncoupled bone metabolism and disturbances in SL export of monosaccharide sugars, a prerequisite for SL delivery to the ruffled border. Thus, Slc37a2 is a physiological component of the osteoclasts unique secretory organelle and a potential therapeutic target for metabolic bone diseases.

sentences: Maturation of prefrontal input to dorsal raphe increases behavioral persistence in mice The ability to persist towards a desired objective is a fundamental aspect of behavioral control whose impairment is implicated in several behavioral disorders. One of the prominent features of behavioral persistence is that its maturation occurs relatively late in development. This is presumed to echo the developmental time course of a corresponding circuit within late-maturing parts of the brain, such as the prefrontal cortex, but the specific identity of the responsible circuits is unknown. Here, we describe the maturation of the projection from layer 5 neurons of the prefrontal cortex to the dorsal raphe nucleus in mice. We show using pathway-specific optogenetic stimulation that this connection undergoes a dramatic increase in synaptic potency between postnatal weeks 3 and 8, corresponding to the transition from juvenile to adult. We then show that this period corresponds to an increase in the behavioral persistence that mice exhibit in a foraging task. Finally, we use genetic targeting to selectively ablate this pathway in adulthood and show that mice revert to a behavioral phenotype similar to juveniles. These results suggest that the prefrontal to dorsal raphe pathway is a critical anatomical and functional substrate of the development and manifestation of behavioral control.

sentences: Regulation of protein complex partners as a compensatory mechanism in aneuploid tumors Aneuploidy, a state of chromosome imbalance, is a hallmark of human tumors, but its role in cancer still remains to be fully elucidated. To understand the consequences of whole chromosome-level aneuploidies on the proteome, we integrated aneuploidy, transcriptomic and proteomic data from hundreds of TCGA/CPTAC tumor samples. We found a surprisingly large number of expression changes happened on other, non-aneuploid chromosomes. Moreover, we identified an association between those changes and co-complex members of proteins from aneuploid chromosomes. This co-abundance association is tightly regulated for aggregation-prone aneuploid proteins and those involved in a smaller number of complexes. On the other hand, we observe that complexes of the cellular core machinery are under functional selection to maintain their stoichiometric balance in aneuploid tumors. Ultimately, we provide evidence that those compensatory and functional maintenance mechanisms are established through post-transcriptional control and that the degree of success of a tumor to deal with aneuploidy-induced stoichiometric imbalance impacts the activation of cellular protein degradation programs and patient survival.

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Ecological and evolutionary drivers of phenotypic and genetic variation in the European crabapple (Malus sylvestris (L.) Mill.), a wild relative of the cultivated apple O_LICharacterizing the phenotypic and genetic variation among populations of crop wild relatives help understanding the ecological and evolutionary processes involved in population divergence, and better harness their diversity to mitigate the impact of climate change on crops. We assessed genetic and phenotypic diversity of the European crabapple, Malus sylvestris, a main contributor to the cultivated apple genome (Malus domestica), and investigated for ecological divergence.
C_LIO_LIWe assessed variation in growth rate and traits related to carbon uptake between seedlings measured in a common garden, and related it to the genetic ancestry of the seedlings, assessed using 13 microsatellite loci and Bayesian clustering method. The occurrence of patterns of isolation-by-distance, -by-climate and -by-adaptation that might have caused genetic and phenotypic differentiation among M. sylvestris populations was also tested.
C_LIO_LISeedlings belonged to seven M. sylvestris populations in Europe, with 11.6% of seedlings introgressed by M. domestica. Significant trait variation among M. sylvestris populations was observed, which for some was of moderate to high heritability. Lack of association between trait and genetic divergence suggests that this significant phenotypic variation is not adaptive, but strong association between genetic variation and the climate during the last glacial maximum suggests local adaptation of M. sylvestris to past climates.
C_LIO_LIThis study provides an insight into the ecological and evolutionary drivers of phenotypic and genetic differentiation among populations of a wild apple species and relative of cultivated apples, which is a starting point for future breeding programs.
C_LI

Societal impact StatementApple is a major fruit crop worldwide and a model species for understanding the evolutionary processes underlying perennial crop domestication. Several wild species have contributed to the genetic make-up of the cultivated apple, yet phenotypic and genetic diversity data across their natural distribution is lacking. This study revealed phenotypic variation between populations of the European crabapple, and showed that both geography, and surprisingly, past but not current climate, shaped its genetic structure. We provide a starting point for harnessing wild apple diversity for apple breeding programs to mitigate the impact of climate change on this perennial crop.

sentences: The PH/MyTH4/FERMmolecule MAX-1 inhibits UNC-5 activity in regulation of VD growth cone protrusion in Caenorhabditis elegans UNC-6/Netrin is a secreted conserved guidance cue that regulates dorsal-ventral axon guidance of C. elegans and in the vertebral spinal cord. In the polarity/protrusion model of VD growth cone guidance away from ventrally-expressed UNC-6 (repulsion), UNC-6 first polarizes the growth cone via the UNC-5 receptor such that filopodial protrusions are biased dorsally. UNC-6 then regulates a balance of protrusion in the growth cone based upon this polarity. UNC-5 inhibits protrusion ventrally, and the UNC-6 receptor UNC-40/DCC stimulates protrusion dorsally, resulting in net dorsal growth cone outgrowth. UNC-5 inhibits protrusion through the flavin monooxygenases FMO-1, 4, and 5 and possible actin destabilization, and inhibits pro-protrusive microtubule entry into the growth cone utilizing UNC-33/CRMP. The PH/MyTH4/FERM myosin-like protein was previously shown to act with UNC-5 in VD axon guidance utilizing axon guidance endpoint analysis. Here, we analyzed the effects of MAX-1 on VD growth cone morphology during outgrowth. We found that max-1 mutant growth cones were smaller and less protrusive than wild-type, the opposite of the unc-5 mutant phenotype. Furthermore, genetic interactions suggest that MAX-1 might normally inhibit UNC-5 activity, such that in a max-1 mutant growth cone, UNC-5 is overactive. Our results, combined with previous studies suggesting that MAX-1 might regulate UNC-5 levels in the cell or plasma membrane localization, suggest that MAX-1 attenuates UNC-5 signaling by regulating UNC-5 stability or trafficking. In summary, in the context of growth cone protrusion, MAX-1 inhibits UNC-5, demonstrating the mechanistic insight that can be gained by analyzing growth cones during outgrowth in addition to axon guidance endpoint analysis.

sentences: moRphology - dEep Learning Imaging Cells (RELIC) - to Differentiate Between Normal and Pathological Kidney Exfoliated Cells Chronic kidney disease (CKD) is characterised by progressive loss of kidney function leading to kidney failure. Significant kidney damage can occur before symptoms are detected. Currently, kidney tissue biopsy is the gold standard for evaluation of renal damage and CKD severity. This study explores how to precisely quantify morphology characteristics of kidney cells exfoliated into urine, with a view to establish a future urine-based non-invasive diagnostic for CKD. We report the development of a novel deep learning method, which was able to discover a RELIC (moRphology dEep Learning Imaging Cells) signature that can differentiate between kidney cells exfoliated in human urine of and CKD patients with varying degree of kidney damage and non-CKD controls. Exfoliated proximal tubule cells (PTCs) originating from kidneys were isolated from the urine of patients with different levels of kidney damage using previously published methods. An advanced combination of artificial intelligence techniques, deep learning, swarm intelligence, and discriminative analysis was used to discover a RELIC signature in brightfield microscopy images of exfoliated PTCs. Kidney damage in the study subjects was characterised by assessing kidney tissues obtained through a nephrectomy or kidney biopsy. A special deep learning algorithm was developed and trained to create a predictive tool. The algorithm was then used to analyse data from patients with normal and fibrotic kidneys. Data were then classified according to different groups (healthy or fibrosis) and clustering of the training and validation cells was determined for model validation. We developed a novel deep learning method, to obtain RELIC signatures and identify specific deep morphological features which can be used to differentiate urinary PTC cells shed by people with CKD (confirmed by tissue histology obtained from an invasive kidney biopsy) from those without CKD, with a discriminatory accuracy of 82%. We identified a RELIC signature which can be used on a collection of bright field images of exfoliated urinary PTCs to create a predictive tool and differentiate between normal and pathological kidney cells. This study, for the first time, provides a proof of concept that urinary exfoliated tubule cells in patients with kidney fibrosis and healthy controls differ in appearance (morphology) as observed under a basic brightfield microscope. The results suggest that morphological signatures of exfoliated PTCs have the potential to serve as a non-invasive marker of kidney fibrosis.

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Tourmaline: a workflow for rapid and reproducible amplicon sequence analysis using QIIME 2 and Snakemake BackgroundAmplicon sequencing (metabarcoding) is a common method to survey diversity of environmental communities whereby a single genetic locus is amplified and sequenced from the DNA of whole or partial organisms, organismal traces (e.g., skin, mucus, feces), or microbes in an environmental sample. Several software packages exist for analyzing amplicon data, among which QIIME 2 has emerged as a popular option because of its broad functionality, plugin architecture, provenance tracking, and interactive visualizations. However, each new analysis requires the user to keep track of input and output file names, parameters, and commands; this lack of automation and standardization is inefficient and creates barriers to meta-analysis and sharing of results.

FindingsWe developed Tourmaline, a Python-based workflow that implements QIIME 2 and is built using the Snakemake workflow management system. Starting from a configuration file that defines parameters and input files--a reference database, a sample metadata file, and a manifest or archive of FASTQ sequences--it uses QIIME 2 to run either the DADA2 or Deblur denoising algorithm, assigns taxonomy to the resulting representative sequences, performs analyses of taxonomic, alpha, and beta diversity, and generates an HTML report summarizing and linking to the output files. Features include support for multiple cores, automatic determination of trimming parameters using quality scores, representative sequence filtering (taxonomy, length, abundance, prevalence, or ID), support for multiple taxonomic classification and sequence alignment methods, outlier detection, and automated initialization of a new analysis using previous settings. The workflow runs natively on Linux and macOS or via a Docker container. We ran Tourmaline on a 16S rRNA amplicon dataset from Lake Erie surface water, showing its utility for parameter optimization and the ability to easily view interactive visualizations through the HTML report, QIIME 2 viewer, and R- and Python-based Jupyter notebooks.

ConclusionsAutomated workflows like Tourmaline enable rapid analysis of environmental and biomedical amplicon data, decreasing the time from data generation to actionable results. Tourmaline is available for download at github.com/aomlomics/tourmaline.

sentences: Nanomechanics combined with HDX reveal allosteric drug binding sites of CFTR NBD1 Cystic fibrosis is most frequently caused by the deletion of F508 ({Delta}F508) in CFTRs nucleotide binding domain 1 (NBD1), compromising CFTR folding, stability and domain assembly. The limitation of developing a successful therapy is due to the lack of molecules that synergistically facilitate folding by targeting distinct structural defects of {Delta}F508-CFTR. To improve drug efficacy by targeting the {Delta}F508-NBD1 folding and stability, and to study potential {Delta}F508-NBD1 allosteric corrector binding sites at the atomic level, we combined molecular dynamics (MD) simulations, atomic force spectroscopy (AFM) and hydrogen-deuterium exchange (HDX) experiments. These methods allowed us to describe unfolding intermediates and forces acting during NBD1 mechanical unfolding and to elucidate the stabilization mechanism of {Delta}F508-NBD1 by 5-bromoindole-3-acetic acid (BIA). An NBD1 region, including the -subdomain, was identified as a potentially important participant of the first folding steps, characterized by non-native interactions of F508, thus destabilized in the deletion mutant. The instability was counteracted by the low-potency corrector BIA, increasing the mechanical resistance of the {Delta}F508-NBD1 -subdomain, which was confirmed as a binding site by computational modeling and HDX experiments. Our results underline the complementarity of computational and experimental methods and provide a possible strategy to improve folding correctors.

sentences: Experimental infection of mink with SARS-COV-2 Omicron (BA.1) variant leads to symptomatic disease with lung pathology and transmission We report an experimental infection of American mink with SARS-CoV-2 Omicron variant and show that minks remain virus RNA positive for days, develop clinical signs and histopathological changes, and transmit the virus to uninfected recipients warranting further studies and preparedness.

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ACME: an Affinity-based Cas9 Mediated Enrichment method for targeted nanopore sequencing Targeted sequencing significantly improves accuracy and coverage and aids in providing the depth necessary to detect rare alleles in a heterogenous population of cells. Until the introduction of nanopore Cas9 Targeted-Sequencing (nCATS), a lack of efficient long-read compatible targeting techniques made it difficult to study specific regions of interest on long-read platforms. Existing nCATS-based strategies are currently limited by the per molecule target lengths capturable (<30kb), requiring several Cas9 guides to tile across larger regions of interest, ultimately reducing the number of targets that can be surveyed per reaction. Also, longer read lengths help reduce mapping errors, making it more likely that complex structural rearrangements can be resolved. Absence of a background reduction step in nCATS also increases the competition between non-target and target fragments in the sequencing pool for pore occupancy, decreasing the overall percentage of on-target reads. To address this, we introduce ACME - an Affinity-based Cas9-Mediated Enrichment method - that helps reduce background reads, increasing on-target coverage and size of target regions that can be spanned with single reads to 100kb.

ACME uses a HisTag-based isolation and pulldown of Cas-9 bound non-target reads, reducing the background noise in sequencing. We designed a panel of guide RNAs targeting 10 genes to enrich for specific regions of the cancer genome and tested them in two breast cell lines - MCF 10A and SK-BR-3. These gene targets spanned different size ranges (10kb to 150kb) allowing us to identify the largest target sizes that could be optimally captured by single molecules spanning the entire region. When compared with using just nCATS, the ACME method for background reduction increased the overall coverage across the entire length of all targets by 2-fold to 25-fold. By using ACME to eliminate smaller competing non-targets from the sequencing library, we saw a 3- to 7-fold increase in the number of reads spanning 100% of the gene targets when compared to nCATS. For one of our larger targets, BRCA2, we observed >60-fold target enrichment, close to 70x coverage, and 3-20 reads spanning the entire 95kb target. We observed an increase in enrichment, depth, and number of whole gene spanning reads for other genes on the panel as well across both cell lines, with enrichment as high as 4000-fold for some genes. Furthermore, ACME identified all SVs previously called within our targets by ONT and PacBio whole genome sequencing and performed on par with these platforms for SNP detection when compared with Illumina short-read whole genome sequencing.

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Tripartite networks show that keystone species can multitask Keystone species are disproportionately important for ecosystem functioning (1,2). However, while all species engage in multiple interaction types with other species, the importance of keystone species is often defined based on a single dimension of their Eltonian niche (3), that is, one type of interaction (e.g., keystone predator). Therefore, it remains unclear whether the importance of keystone species is unidimensional or if it extends across interaction types. We conducted a meta-analysis of tripartite interaction networks to examine whether species importance in one dimension of their niche is mirrored in other niche dimensions, and whether this is associated with interaction outcome, intimacy, or species richness. We show that keystone species importance is positively associated across multiple ecological niche dimensions, independently of species abundance, and find no evidence that multidimensionality of keystone species is influenced by the explanatory variables. We propose that the role of keystone species extends across multiple ecological niche dimensions, with important implications for ecosystem resilience and conservation.

Significance StatementKeystone species are often identified by focusing on a single type of interaction (e.g., predation, pollination, herbivory) which contrasts with the multiple roles that species play in biological communities. We conducted a meta-analysis of 18 tripartite interaction networks to explore if keystonness is correlated across the multiple dimensions of species Eltonian niches. Our results suggest that species importance tends to span across multiple interaction types, independently from abundance, which can be key to understand community resilience and collapse in face of multiple threats.

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Lupus Gut Microbiota Transplants Cause Autoimmunity and Inflammation BackgroundThe etiology of systemic lupus erythematosus (SLE) is multifactorial. Recently, growing evidence suggests that the microbiota plays a role in SLE, yet whether gut microbiota participates in the development of SLE remains largely unknown. To investigate this issue, we carried out 16s rDNA sequencing analyses in a cohort of 18 female un-treated active SLE patients and 7 female healthy controls, and performed fecal microbiota transplantation from patients and healthy controls to germ-free mice.

ResultsCompared to the healthy controls, we found no significant different microbial diversity but some significantly different species in SLE patients including Turicibacter genus and other 5 species. Fecal transfer from SLE patients to germ free (GF) C57BL/6 mice caused GF mice to develop a series of lupus-like phenotyptic features, which including an increased serum autoimmune antibodies, and imbalanced cytokines, altered distribution of immune cells in mucosal and peripheral immune response, and upregulated expression of genes related to SLE in recipient mice that received SLE fecal microbiota transplantation (FMT). Moreover, the metabolism of histidine was significantly altered in GF mice treated with SLE patient feces, as compared to those which received healthy fecal transplants.

ConclusionsOverall, our results describe a causal role of aberrant gut microbiota in contributing to the pathogenesis of SLE. The interplay of gut microbial and histidine metabolism may be one of the mechanisms intertwined with autoimmune activation in SLE.

sentences: Inhibiting SARS-CoV-2 infection in vitro by suppressing its receptor, angiotensin-converting enzyme 2, via aryl-hydrocarbon receptor signal Since understanding molecular mechanisms of SARS-CoV-2 infection is extremely important for developing effective therapies against COVID-19, we focused on the internalization mechanism of SARS-CoV-2 via ACE2. Although cigarette smoke is generally believed to be harmful to the pathogenesis of COVID-19, cigarette smoke extract (CSE) treatments were surprisingly found to suppress the expression of ACE2 in HepG2 cells. We thus tried to clarify the mechanism of CSE effects on expression of ACE2 in mammalian cells. Because RNA-seq analysis suggested that suppressive effects on ACE2 might be inversely correlated with induction of the genes regulated by aryl hydrocarbon receptor (AHR), the AHR agonists 6-formylindolo(3,2-b)carbazole (FICZ) and omeprazole (OMP) were tested to assess whether those treatments affected ACE2 expression. Both FICZ and OMP clearly suppressed ACE2 expression in a dose-dependent manner along with inducing CYP1A1. Knock-down experiments indicated a reduction of ACE2 by FICZ treatment in an AHR-dependent manner. Finally, treatments of AHR agonists inhibited SARS-CoV-2 infection into Vero E6 cells as determined with immunoblotting analyses detecting SARS-CoV-2 specific nucleocapsid protein. We here demonstrate that treatment with AHR agonists, including CSE, FICZ, and OMP, decreases expression of ACE2 via AHR activation, resulting in suppression of SARS-CoV-2 infection in mammalian cells.

sentences: NNeurite: artificial neuronal networks for the unsupervised extraction of axonal and dendritic time-lapse signals Fluorescence microscopy of Ca2+ transients in small neurites of the behaving mouse provides an unprecedented view of the micrometer-scale mechanisms supporting neuronal communication and computation, and therefore opens the way to understanding their role in cognition. However, the exploitation of this growing and precious experimental data is impeded by the scarcity of methods dedicated to the analysis of images of neurites activity in vivo. We present NNeurite, a set of mathematical and computational techniques specialized for the analysis of time-lapse microscopy images of neurite activity in small behaving animals. Starting from noisy and unstable microscopy images containing an unknown number of small neurites, NNeurite simultaneously aligns images, denoises signals and extracts the location and the temporal activity of the sources of Ca2+ transients. At the core of NNeurite is a novel artificial neuronal network (NN) which we have specifically designed to solve the non-negative matrix factorization (NMF) problem modeling source separation in fluorescence microscopy images. For the first time, we have embedded non-rigid image alignment in the NMF optimization procedure, hence allowing to stabilize images based on the transient and weak neurite signals. NNeurite processing is free of any human intervention as NN training is unsupervised and the unknown number of Ca2+ sources is automatically obtained by the NN-based computation of a low-dimensional representation of time-lapse images. Importantly, the spatial shapes of the sources of Ca2+ fluorescence are not constrained in NNeurite, which allowed to automatically extract the micrometer-scale details of dendritic and axonal branches, such dendritic spines and synaptic boutons, in the cortex of behaving mice. We provide NNeurite as a free and open-source library to support the efforts of the community in advancing in vivo microscopy of neurite activity.

sentences: Natural variation in the control of flowering and shoot architecture in diploid Fragaria species In perennial fruit and berry crops of the Rosaceae family, flower initiation occurs in late summer or autumn after downregulation of a strong repressor TERMINAL FLOWER1 (TFL1) and flowering and fruiting takes place the following growing season. Rosaceous fruit trees typically form two types of axillary shoots, short flower-bearing shoots called spurs and long shoots that are respectively analogous to branch crowns and stolons in strawberry. However, regulation of flowering and shoot architecture differs between species and environmental and endogenous controlling mechanisms have just started to emerge. In woodland strawberry (Fragaria vesca L.), long days maintain vegetative meristems and promote stolon formation by activating TFL1 and GIBBERELLIN 20-OXIDASE4 (GA20ox4), respectively, while silencing of these factors by short days and cool temperatures induces flowering and branch crown formation. We characterized flowering responses of 14 accessions of seven diploid Fragaria species native to diverse habitats in the northern hemisphere, and selected two species with contrasting environmental responses, F. bucharica Losinsk. and F. nilgerrensis Schlecht. ex J. Gay for detailed studies together with F. vesca. Similar to F. vesca, F. bucharica was induced to flower in short days at 18{degrees}C and regardless of photoperiod at 11{degrees}C after silencing of TFL1. F. nilgerrensis maintained higher TFL1 expression level and likely required cooler temperatures or longer exposure to inductive treatments to flower. We also found that high expression of GA20ox4 was associated with stolon formation in all three species, and its downregulation by short days and cool temperature caused branch crown formation in F. vesca and F. nilgerrensis, although the latter did not flower. F. bucharica, in contrast, rarely formed branch crowns, regardless of flowering or GA20ox4 expression level. Our findings highlighted diploid Fragaria species as a rich source of genetic variation controlling flowering and plant architecture, with potential applications in breeding of Rosaceous crops.

sentences: Chemoenzymatic Generation of Phospholipid Membranes Mediated by Type I Fatty Acid Synthase The de novo formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce in vitro. As such, the reconstitution of phospholipid membrane synthesis from simple metabolic building blocks remains a challenge. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a non-canonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. To our knowledge, this is the first example of reconstituting phospholipid membrane formation directly from acetyl-CoA and malonyl-CoA precursors. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles.

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Geology and discovery record of the Trinil Pithecanthropus erectus site, Java The scientific utility of Eugene Dubois Pithecanthropus erectus (P.e.) Skullcap (Trinil 1), Femur I (Trinil 3) and associated paleontological specimens has been impaired for over a century by questions about their provenience. Firsthand accounts and contemporaneous field photographs, presented here, extensively document the site geology and discovery history.

The P.e. specimens and numerous-other fossils were unearthed in 1891-1893 from small excavations dug into a flat-lying bonebed exposed near the seasonal low-water level of the Solo River along its incised left embankment. Dubois on-site supervisors specified that the two P.e. fossils came from a [~]0.2-m-thick bonebed subunit traced at a single elevation for [~]12m from the 1891 Skullcap pit ([~]30m2) to the 1892 Femur-discovery excavation and across an enlarged 1892-1893 trench ([~]170m2). The depositional co-occurrence of the finds is supported by key documentation: the supervisors letters to Dubois about Femur I; his initial reporting to the Indies government; 1892-1893 accounts about expanding excavation of the Femur I stratum; Dubois 1891-1893 government submissions and 1894-1896 publications; confirmation by the Selenka Expedition in 1907-1908; Dubois annotations on unpublished site photographs; and a letter he wrote the year he died. Field studies in the 1930s to 1970s confirmed the essential aspects of the site geology.

The bonebed of 1891-1893 contained fossils referable to the extinct Trinil fauna species Axis lydekkeri, Duboisia santeng and Stegodon trigonocephalus. The Selenka Expedition excavations had a similar assemblage in the same stratigraphic position which they named the Hauptknochenschicht. The bonebed was thin bioclast-rich gravelly volcaniclastic sandstone with taphonomic and sedimentary features indicating an unusual origin. Bioclasts range from proboscidean craniums and logs to rat teeth, freshwater mollusc shells and leaves. The terrestrial-vertebrate skeletal elements are overwhelmingly disarticulated and frequently broken. Their surfaces are little-abraded by fluvial transport. The bone fossilization is quite uniform. More than one-hundred ungulate individuals perished. No evidence has been found of hominin- or terrestrial-carnivore involvement. The bioclasts varied in density from place-to-place and vertically, and were matrix supported in the bonebed. No substantial internal depositional hiatus was reported. In combination with Trinils paleogeographic context, these features implicate a catastrophic mortality of ungulates in a population aggregation along the floodplain of a perennial paleo-river, followed by lahar-flood transport and deposition of gravel-size lithic- and biotic-materials.

Trinil provides evidence favoring a broad archaic-hominin presence in southern Sundaland. The Trinil fauna is a lynch-pin in a long-lasting paleobiogeographic association between H. erectus and certain lineages of large bovids, cervids, proboscideans, rhinoceros, suids and tiger. The bonebeds paleogeographic setting exemplifies the stratovolcanic drainages that H. erectus occupied for >0.8 million years in Java, including the watershed of a marine delta [~]150km east of Trinil, a volcanic island [~]100km north of Trinil, and areas to its west for 500km (where species associated with Trinil H. erectus occur). In the Java Sea (Sunda Shelf), seismic data image immense Pleistocene river- and coastal-terranes which archaic hominins and other large-mammals, like those at Trinil, might have inhabited.

sentences: HippUnfold: Automated hippocampal unfolding, morphometry, and subfield segmentation Like neocortical structures, the archicortical hippocampus differs in its folding patterns across individuals. Here, we present an automated and robust BIDS-App, HippUnfold, for defining and indexing individual-specific hippocampal folding in MRI, analogous to popular tools used in neocortical reconstruction. Such tailoring is critical for inter-individual alignment, with topology serving as the basis for homology. This topological framework enables qualitatively new analyses of morphological and laminar structure in the hippocampus or its subfields. It is critical for refining current neuroimaging analyses at a meso- as well as micro-scale. HippUnfold uses state-of-the-art deep learning combined with previously developed topological constraints to generate uniquely folded surfaces to fit a given subjects hippocampal conformation. It is designed to work with commonly employed sub-millimetric MRI acquisitions, with possible extension to microscopic resolution. In this paper we describe the power of HippUnfold in feature extraction, and highlight its unique value compared to several extant hippocampal subfield analysis methods.

sentences: A world of viruses nested within parasites: Unraveling viral diversity within parasitic flatworms (Platyhelminthes) Because parasites have an inextricable relationship with their host, they have the potential to serve as viral reservoirs or facilitate virus host-shifts. Yet, little is known about viruses infecting parasitic hosts except for blood-feeding arthropods that are well-known vectors of zoonotic viruses. Herein we uncover viruses of flatworms (Phylum Platyhelminthes, group Neodermata) that specialize in parasitizing vertebrates and their ancestral free-living relatives. We discovered 115 novel viral sequences, including 1 in Macrostomorpha, 5 in Polycladida, 44 in Tricladida, 1 in Monogenea, 15 in Cestoda and 49 in Trematoda, through data mining. The majority of newly identified viruses constitute novel families or genera. Phylogenetic analyses show that the virome of flatworms changed dramatically during the transition of Neodermatans to a parasitic lifestyle. Most Neodermatan viruses seem to co-diversify with their host, with the exception of rhabdoviruses which may switch host more often, based on phylogenetic relationships. Neodermatan rhabodviruses also have an ancestral position to vertebrate-associated viruses, including Lyssaviruses, suggesting that vertebrate rhabdoviruses emerged from a flatworm rhabdovirus in a parasitized host. This study reveals an extensive diversity of viruses in Platyhelminthes and highlights the need to evaluate the role of viral infection in flatworm-associated diseases.

sentences: C. elegans PEZO-1 is a Mechanosensitive Ion Channel Involved in Food Sensation PIEZO channels are force sensors essential for physiological processes, including baroreception and proprioception. The Caenorhabditis elegans genome encodes an ortholog gene of the Piezo family, pezo-1, which is expressed in several tissues, including the pharynx. This myogenic pump is an essential component of the C. elegans alimentary canal, whose contraction and relaxation are modulated by mechanical stimulation elicited by food content. Whether pezo-1 encodes a mechanosensitive ion channel and contributes to pharyngeal function remain unknown. Here, we leverage genome editing, genetics, microfluidics, and electropharyngeogram recording to establish that pezo-1 is expressed in the pharynx, including in a proprioceptive-like neuron, and regulates pharyngeal function. Knockout (KO) and gain-of-function (GOF) mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, sensing osmolarity, and food mechanical properties. Using pressure-clamp experiments in primary C. elegans embryo cultures, we determine that pezo-1 KO cells do not display mechanosensitive currents, whereas cells expressing wild-type or GOF PEZO-1 exhibit mechanosensitivity. Moreover, infecting the Spodoptera frugiperda cell line with a baculovirus containing the G-isoform of pezo-1 (among the longest isoforms) demonstrates that pezo-1 encodes a mechanosensitive channel. Our findings reveal that pezo-1 is a mechanosensitive ion channel that regulates food sensation in worms.

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Isotopic signatures induced by upwelling tag regional fish populations in Lake Tanganyika Lake Tanganyikas pelagic fish sustain the second largest inland fishery in Africa and are under pressure from heavy fishing and global warming related increases in stratification. Only little is known about whether basin-scale hydrodynamics - including a more stratified north and an upwelling-driven south - induce ecological and genetic differences among populations of highly mobile, pelagic fish inhabiting these different areas. Here, we examine whether the basin-scale dynamics leave distinct isotopic imprints in the pelagic fish of Lake Tanganyika, which may reveal differences in habitat, diet, or lipid content. We conducted two lake-wide campaigns during different seasons and collected physical, nutrient, chlorophyll, phytoplankton and zooplankton data. Additionally, we analyzed the pelagic fish - the clupeids Stolothrissa tanganicae, Limnothrissa miodon and four Lates species - for their isotopic and elemental carbon (C) and nitrogen (N) compositions. The {delta}13C values were significantly higher in the productive south after the upwelling/mixing period across all trophic levels, implying that the fish have regional foraging grounds, and thus record these latitudinal isotope gradients. By combining our isotope data with genetics, we demonstrate that the fish form regional populations on a seasonal to multiannual time scale. Based on {delta}15N and C:N ratios, we found no strong evidence for varying diets or lipid contents between those regional populations.

Additional analyses revealed that isotopic variations between specimens from the same location are not linked to genetic differences. We suggest that the development of basinscale ecological differences in response to the prevailing hydrodynamic regimes may be inhibited by lake-wide gene flow on the long term. Our findings show that the pelagic fish species are genetically adapted to the whole lake, but they form regional populations on short time scales. This implies that sustainable management strategies may adopt basin-scale fishing quotas.

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Mapping language and non-language cognitive deficits in post-stroke anomic aphasia While language impairment is the defining symptom of aphasia, the co-occurrence of non-language cognitive deficits and their importance in predicting rehabilitation and recovery outcomes is well documented. Despite this, people with aphasia (PWA) are rarely tested on assessments of higher order cognitive functions, making it difficult for studies to associate these functions with a consistent lesion correlate. Contrary to classic models of speech and language, cumulative evidence shows that Brocas area and surrounding regions in the left inferior frontal cortex (LIFC) are involved in, but not specific to, speech production - suggesting that these regions may be involved in higher-level cognitive functions that support language production. A better understanding of language processing in the context of other domain general cognitive functions is essential for improving aphasia treatments.

This study aimed to explore the brain-behaviour relationships between tests of individual cognitive skill and language abilities in people with post-stroke aphasia, with a focus on language production deficits and their associated lesion correlates. We predicted our analysis would reveal a latent (non-language specific) cognitive component, that would be driven by damage to LIFC.

We analysed the behavioural and neural correlates of an extensive battery of language and non-language cognitive tests in a sample of thirty-six adults with long-term speech production deficits from post-stroke aphasia. All participants were anomic, with relatively intact speech comprehension and no apraxia of speech. The behavioural variables were analysed using Principal Component Analysis and their neural correlates were estimated using Voxel-Based Correlational Morphology. A significant number of anomic adults showed impaired performance on tests of non-language specific cognitive function. The variance underlying behavioural performance was best captured by four orthogonal components, two higher-order cognitive components (executive functions and verbal working memory) and two linguistic processing components (phonology and semantics). Brain-behaviour relationships revealed separable neural correlates for each component in line with previous studies and an executive functions correlate in the left inferior frontal cortex (LIFC).

Our findings suggest that in adults with chronic post-stroke language production deficits (anomia), higher-level cognitive functions explain more of the variance in language function than classical models of the condition imply. Additionally, lesions to the LIFC, including Brocas area, were associated with executive (dys)function, independent of language abilities, suggesting that lesions to this area are associated with non-language specific higher-level cognitive functions that support speech production. These findings support contemporary models of speech production that place language processing within the context of domain-general perception, action and conceptual knowledge.

sentences: NanoTox: Development of a parsimonious in silico model for toxicity assessment of metal-oxide nanoparticles using physicochemical features Metal-oxide nanoparticles find widespread applications in mundane life today, and cost-effective evaluation of their cytotoxicity and ecotoxicity is essential for sustainable progress. Machine learning models use existing experimental data, and learn the relationship of various features to nanoparticle cytotoxicity to generate predictive models. In this work, we adopted a principled approach to this problem by formulating a feature space based on intrinsic and extrinsic physico-chemical properties, but exclusive of any in vitro characteristics such as cell line, cell type, and assay method. A minimal set of features was developed by applying variance inflation analysis to the correlation structure of the feature space. Using a balanced dataset, a mapping was then obtained from the normalized feature space to the toxicity class using various hyperparameter-tuned machine learning models. Evaluation on an unseen test set yielded > 96% balanced accuracy for both the random forest model, and neural network with one hidden layer model. The obtained cytotoxicity models are parsimonious, with intelligible inputs, and include an applicability check. Interpretability investigations of the models yielded the key predictor variables of metal-oxide nanoparticle cytotoxicity. Our models could be applied on new, untested oxides, using a majority-voting ensemble classifier, NanoTox, that incorporates the neural network, random forest, support vector machine, and logistic regression models. NanoTox is the very first predictive nanotoxicology pipeline made freely available under the GNU General Public License (https://github.com/NanoTox).

sentences: Ddp1 cooperates with Ppx1 to counter a stress response initiated by non-vacuolar polyphosphate In diverse cells from bacterial to mammalian species, inorganic phosphate is stored in long chains called polyphosphates (polyP). These near universal polymers, ranging from 3 to thousands of phosphate moieties in length, are associated with molecular functions including energy homeostasis, protein folding, and cell signaling. In many cell types, polyphosphate is concentrated in subcellular compartments or organelles. In the budding yeast S. cerevisiae, polyP synthesis by the membrane-bound VTC complex is coupled to its translocation into the lumen of the vacuole, a lysosome-related organelle, where it is stored at high concentrations. In contrast, ectopic expression of bacterial polyphosphate kinase, PPK, results in the toxic accumulation of polyP outside of the vacuole. In this study, we used label-free mass spectrometry to investigate the mechanisms underlying this toxicity. We find that PPK expression results in the activation of a stress response mediated in part by the Hog1 and Yak1 kinases, and Msn2/Msn4 transcription factors. This response is countered by the combined action of the Ddp1 and Ppx1 polyphosphatases that function together to counter polyP accumulation and downstream toxicity. In contrast, ectopic expression of previously proposed mammalian polyphosphatases did not impact PPK-mediated toxicity in this model, suggesting either that these enzymes do not function directly as polyphosphatases in vivo or that they require co-factors unique to higher eukaryotes. Our work provides a mechanistic explanation for why polyP accumulation outside of lysosome-related organelles is toxic. Further, it serves as a resource for exploring how polyP may impact conserved biological processes at a molecular level.

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Sleep Disruption Selectively Weakens Reactivated Memories A widely accepted view in memory research is that recently stored information can be reactivated during sleep, leading to memory strengthening. Two recent studies have shown this effect can be reversed in participants with highly disrupted sleep. To test whether weakening of reactivated memories can result directly from sleep disruption, in this experiment we varied the intensity of memory reactivation cues, such that some produced sleep arousals. Prior to sleep, participants (local community members) learned the locations of 75 objects, each accompanied by a sound naturally related to that object. Location recall was tested before and after sleep, and a subset of the sounds were presented during sleep. Reactivation with arousal weakened memories, unlike the improvement typically found. We conclude that reactivated memories can be selectively weakened during sleep, and that memory reactivation may strengthen or weaken memories depending on additional factors such as concurrent sleep disruption.

Statement of RelevanceThe results of this study have implications for both human health and basic psychology. Sleep disorders like apnea are associated with memory problems; our results suggest a possible mechanism where frequent arousal may disrupt the naturally occurring reactivation of memory in sleep. These results also highlight the importance of avoiding brief sleep disruption for good sleep hygiene. Finally, we raise the possibility that reactivation with sleep disruption could be used therapeutically to weaken distressing memories.

Our observation that memories can also be either weakened or strengthened by sleep reactivation has implications for understanding the mechanisms of memory consolidation. In particular, we suggest that sleep memory reactivation may be a reconsolidation-like process with memory restabilization required after reactivation. Our findings also suggest avenues for future experiments; such as using sleep disruption to study the time course of memory reactivation.

sentences: An overlapped natural antisense lncRNA FCER1A-AS controls anaphylactic reaction by promoting FcϵRIα expression Functioned as -subunit of the high-affinity immunoglobulin E receptor (Fc{varepsilon} RI), Fc{varepsilon} RI plays a central role in the pathogenesis of Ig-E-mediated allergy and other IgE-related disorders. Fc{varepsilon} RI is normally expressed only in limited spectrum of cells like basophils and mast cells, but the mechanism of controlling Fc{varepsilon} RI expression in these cells is less well understood. In this study, we found fully overlapped natural antisense transcript (NATs) of Fc{varepsilon} RI (FCER1A-AS) is co-expressed with cognate sense transcript (FCER1A-S) in IL-3 induced Fc{varepsilon} RI-expressing cells or in high Fc{varepsilon} RI-expressing cell line MC/9. When FCER1A-AS is selectively knocked down by CRISPR/RfxCas13d (CasRx) approach in MC/9, expression of mRNA and proteins of FCER1A-S is also markedly decreased. Furthermore, deficiency of FCER1A-AS along with lack of FCER1A-S expression is found in two different lines of gene-targeted mice in which FCER1A locus is disturbed at different sites. More importantly, the FCER1A-AS-deficient homozygous mice display similarly diminished anaphylactic reaction as FCER1A gene knockout mice. Thus, we uncovered in this investigation that the expression of FCER1A-S is positively regulated by co-expressed fully overlapped antisense transcript.

sentences: The emergence of highly fit SARS-CoV-2 variants accelerated by recombination The SARS-CoV-2 pandemic recently entered an alarming new phase with the emergence of the variants of concern (VOC) and understanding their biology is paramount to predicting future ones. Current efforts mainly focus on mutations in the spike glycoprotein (S), but changes in other regions of the viral proteome are likely key. We analyzed more than 900,000 SARS-CoV-2 genomes with a computational systems biology approach including a haplotype network and protein structural analyses to reveal lineage-defining mutations and their critical functional attributes. Our results indicate that increased transmission is promoted by epistasis, i.e., combinations of mutations in S and other viral proteins. Mutations in the non-S proteins involve immune-antagonism and replication performance, suggesting convergent evolution. Furthermore, adaptive mutations appear in geographically disparate locations, suggesting that either independent, repeat mutation events or recombination among different strains are generating VOC. We demonstrate that recombination is a stronger hypothesis, and may be accelerating the emergence of VOC by bringing together cooperative mutations. This emphasizes the importance of a global response to stop the COVID-19 pandemic.

sentences: Spike-based symbolic computations on bit strings and numbers The brain uses recurrent spiking neural networks for higher cognitive functions such as symbolic computations, in particular, mathematical computations. We review the current state of research on spike-based symbolic computations of this type. In addition, we present new results which show that surprisingly small spiking neural networks can perform symbolic computations on bit sequences and numbers and even learn such computations using a biologically plausible learning rule. The resulting networks operate in a rather low firing rate regime, where they could not simply emulate artificial neural networks by encoding continuous values through firing rates. Thus, we propose here a new paradigm for symbolic computation in neural networks that provides concrete hypotheses about the organization of symbolic computations in the brain. The employed spike-based network models are the basis for drastically more energy-efficient computer hardware - neuromorphic hardware. Hence, our results can be seen as creating a bridge from symbolic artificial intelligence to energy-efficient implementation in spike-based neuromorphic hardware.

sentences: TRIM24 controls induction of latent HIV-1 by stimulating transcriptional elongation The conserved HIV-1 LTR cis elements RBE1/3 bind the factor RBF2, consisting of USF1/2 and TFII-I, and are essential for reactivation of HIV-1 by T cell signaling. We determined that TFII-I recruits the tripartite motif protein TRIM24 to the LTR, and this interaction is required for efficient reactivation of HIV-1 expression in response to T cell signaling, similar to the effect of TFII-I depletion. Knockout of TRIM24 did not affect recruitment of RNA Pol II to the LTR promoter, but inhibited transcriptional elongation, an effect that was associated with decreased RNA Pol II CTD S2 phosphorylation and impaired recruitment of CDK9 to the LTR. These results demonstrate that TFII-I promotes transcriptional elongation in response to T cell activation through recruitment of the co-factor TRIM24, which is necessary for efficient recruitment of the elongation factor P-TEFb.

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The toll-like receptor 7 agonist imiquimod increases ethanol self-administration and induces expression of toll-like receptor related genes BackgroundThere is growing evidence that immune signaling may be involved in both the causes and consequences of alcohol abuse. Toll-like receptor (TLR) expression is increased by alcohol consumption and is implicated in AUD, and specifically TLR7 may play an important role in ethanol consumption.

MethodsWe administered the TLR7-specific agonist imiquimod in male and female Long-Evans rats to determine 1) gene expression changes in brain regions involved in alcohol reinforcement, the nucleus accumbens core and anterior insular cortex, in rats with and without an alcohol history, and 2) whether TLR7 activation could modulate operant alcohol self-administration.

ResultsInterferon regulatory factor 7 (IRF7) was dramatically increased in both sexes at both 2 and 24 h post-injection regardless of alcohol history, while TLR3 and 7 gene expression changes were region- and sex-specific. The pro-inflammatory cytokine TNF was increased 24h post-injection in rats with an alcohol self-administration history but this effect did not persist after four injections, suggesting molecular tolerance. In both males and females, ethanol consumption was increased 24 h after imiquimod injections with sex-specific differences: in females this effect emerged following the first injection but in males this increase did not occur until the third injection, suggesting sex differences in adaptation to repeated TLR7 activation. Notably, imiquimod reliably induced weight loss, indicating that sickness behavior persisted across repeated injections.

ConclusionThese findings show that TLR7 activation can modulate alcohol drinking in an operant self-administration paradigm, and suggest that TLR7 and IRF7 signaling pathways may be a viable druggable target for treatment of AUD.

sentences: A panel of single nucleotide polymorphism (SNP) markers identifies potential duplicates in cassava (Manihot esculenta Crantz) varieties from Cote dIvoire Accurate identification of varieties is paramount to optimizing efficiencies in the management and conservation of genetic resources. A relatively inexpensive, rapid methodology is required to identify putative duplicates from any collection, when morphological traits give insufficient discrimination. Here we select a panel of 36 SNPs, visualized using the Kompetitive Allele-specific PCR (KASP) system. We used a panel of 95 cassava genotypes from Cote dIvoire to identify varieties that are not duplicates and few potential duplicates which could be put forward for further verification. The genetic variability and population structure of the germplasm is also described. 36 SNPs were polymorphic across the panel of 95 varieties with polymorphic information contents ranging from 0.23 to 0.37. Using these SNPs, we were able to identify 66 unique genotypes from the panel of 95 genotypes, discriminate three sets of known duplicates and identify 11 sets of unknown putative duplicates which can be subjected to further verification using higher density genotyping. As expected in an outcrossing species, both expected heterozygosity (0.46) and observed heterozygosity (0.48) were high with an analysis of molecular variance (AMOVA) indicating that the majority of variation was within individuals. Three statistical approaches i.e., hierarchical ascending clustering, Bayesian analysis and discriminant analysis of principal components were used and all revealed low genetic differentiation between sub-populations, a conclusion that was supported by the low value of the fixation index (0.05). This panel of SNPs can be used to enhance cost-effectiveness and efficiency of germplasm conservation and enhance quality control at various stages in the breeding process through varietal tracking.

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A low-cost, long-term underwater camera trap network coupled with deep residual learning image analysis AO_SCPLOWBSTRACTC_SCPLOWO_LIUnderstanding long-term trends in marine ecosystems requires accurate and repeatable counts of fishes and other aquatic organisms on spatial and temporal scales that are difficult or impossible to achieve with diver-based surveys. Long-term, spatially distributed cameras, like those used in terrestrial camera trapping, have not been successfully applied in marine systems due to limitations of the aquatic environment.
C_LIO_LIHere, we develop methodology for a system of low-cost, long-term camera traps (Dispersed Environment Aquatic Cameras), deployable over large spatial scales in remote marine environments. We use machine learning to classify the large volume of images collected by the cameras. We present a case study of these combined techniques use by addressing fish movement and feeding behavior related to grazing halos, a well-documented benthic pattern in shallow tropical reefscapes.
C_LIO_LICameras proved able to function continuously underwater at deployed depths (up to 7 m, with later versions deployed to 40 m) with no maintenance or monitoring for over five months, and collected time-lapse images during daylight hours for a total of over 100,000 images. Our ResNet-50-based deep learning model achieved 92.5% overall accuracy in sorting images with and without fish, and diver surveys revealed that the camera images accurately represented local fish communities.
C_LIO_LIThe cameras and machine learning classification represent the first successful method for broad-scale underwater camera trap deployment, and our case study demonstrates the cameras potential for addressing questions of marine animal behavior, distributions, and large-scale spatial patterns.
C_LI

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Determinants shaping the nanoscale architecture of the mouse rod outer segment The unique membrane organization of the rod outer segment (ROS), the specialized sensory cilium of rod photoreceptor cells, provides the foundation for phototransduction, the initial step in vision. ROS architecture is characterized by a stack of identically shaped and tightly packed membrane disks loaded with the visual receptor rhodopsin. A wide range of genetic aberrations compromise ROS ultrastructure, impairing photoreceptor viability and function. Yet, the structural basis giving rise to the remarkable long-range order of ROS membrane stacks and the molecular mechanisms underlying genetically inherited diseases remain elusive. Here, cryo-electron tomography (cryo-ET) performed on native ROS at molecular resolution provides insights into key structural determinants of ROS membrane architecture.

Our data reveal the existence of two molecular connectors/spacers which likely contribute to the nanometer-scale precise stacking of the ROS disks. We further show that the extreme radius of curvature at the disk rims is enforced by a continuous supramolecular assembly composed of peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) tetramers. We suggest that, together these molecular assemblies constitute the structural basis of the highly specialized ROS functional architecture. Cryo-ET therefore provides novel quantitative and structural information on the molecular architecture in ROS and insights into possible mechanisms underlying pathologies of certain PRPH2 mutations leading to blindness.

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Single-nucleus transcriptomes reveal functional and evolutionary properties of cell types in the Drosophila accessory gland Many traits responsible for male reproduction evolve quickly, including gene expression phenotypes in germline and somatic male reproductive tissues. Rapid male evolution in polyandrous species is thought to be driven by competition among males for fertilizations and conflicts between male and female fitness interests that manifest in post-copulatory phenotypes. In Drosophila, seminal fluid proteins secreted by three major cell types of the male accessory gland and ejaculatory duct are required for female sperm storage and use, and influence female post-copulatory traits. Recent work has shown that these cell types have overlapping but distinct effects on female post-copulatory biology, yet relatively little is known about their evolutionary properties. Here we use single-nucleus RNA-Seq of the accessory gland and ejaculatory duct from Drosophila melanogaster and two closely related species to comprehensively describe the cell diversity of these tissues and their transcriptome evolution for the first time. We find that seminal fluid transcripts are strongly partitioned across the major cell types, and expression of many other genes additionally define each cell type. We also report previously undocumented diversity in main cells. Transcriptome divergence was found to be heterogeneous across cell types and lineages, revealing a complex evolutionary process. Furthermore, protein adaptation varied across cell types, with potential consequences for our understanding of selection on male post-copulatory traits.

SIGNIFICANCE STATEMENTRapid evolution of male traits may result from competition among males or antagonistic interactions between the sexes over control of reproduction. In animals with internal fertilization, interactions may occur in the female reproductive tract. Drosophila seminal fluid proteins, which are secreted by three major cell types of the male accessory gland and ejaculatory duct, are required for female sperm storage and use, and influence female behavior and physiology. These cell types have distinct effects on females, yet relatively little is known about their evolutionary properties. Here we characterize diversity and transcriptome evolution of seminal fluid-producing tissues at the cell level. These data reveal new functional properties of these cells and complex evolutionary patterns that vary across cell types and lineages.

sentences: Dynamical Systems Models for Plasma Dilution Recent experiments provide evidence that diluting the blood plasma restores the plasma environment to a more youthful level at least partially restoring the health of organs and tissues throughout the body. We propose that a dynamical-systems model representing the plasma constituents could support the optimization process and help determine the appropriate dilution level, frequency and any simultaneous plasma infusions to achieve the most favorable outcome. We use a combination of a gradient descent, a simulated annealing and a genetic algorithm to find a population of models that fit illustrative data. We analyze this population and present distributions of the model parameters and include a collection of plots of the dilution process for illustrative models. We then consider modifications of the dilution in order to illustrate what predictions might be possible had we more data to disambiguate the model.

sentences: Dual TCR-alpha expression on MAIT cells as a potential confounder of TCR interpretation Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are highly abundant in human blood and tissues. Most MAIT cells have an invariant T cell receptor (TCR)  chain that uses TRAV1-2 joined to TRAJ33/20/12 and recognize metabolites from bacterial riboflavin synthesis bound to the antigen-presenting molecule, MR1. Recently, our attempts to identify alternative MR1-presented antigens led to the discovery of rare MR1-restricted T cells with non-TRAV1-2 TCRs. Because altered antigen specificity is likely to lead to altered affinity for the most potent known antigen, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), we performed bulk TCR and {beta} chain sequencing, and single cell-based paired TCR sequencing, on T cells that bound the MR1-5-OP-RU tetramer, but with differing intensities. Bulk sequencing showed that use of V genes other than TRAV1-2 was enriched among MR1-5-OP-RU tetramerlow cells. Whereas we initially interpreted these as diverse MR1-restricted TCRs, single cell TCR sequencing revealed that cells expressing atypical TCR chains also co-expressed an invariant MAIT TCR chain. Transfection of each non-TRAV1-2 TCR chain with the TCR{beta} chain from the same cell demonstrated that the non-TRAV1-2 TCR did not bind the MR1-5-OP-RU tetramer. Thus, dual TCR chain expression in human T cells and competition for the endogenous {beta} chain explains the existence of some MR1-5-OP-RU tetramerlow T cells. The discovery of simultaneous expression of canonical and non-canonical TCRs on the same T cell means that claims of roles for non-TRAV1-2 TCR in MR1 response must be validated by TCR transfer-based confirmation of antigen specificity.

sentences: An Association Test of the Spatial Distribution of Rare Missense Variants within Protein Structures Improves Statistical Power of Sequencing Studies Over 90% of variants are rare, and 50% of them are singletons in the Alzheimers Disease Sequencing Project Whole Exome Sequencing (ADSP WES) data. However, either single variant tests or unit-based tests are limited in the statistical power to detect the association between rare variants and phenotypes. To best utilize rare variants and investigate their biological effect, we exam their association with phenotypes in the context of protein. We developed a protein structure-based approach, POKEMON (Protein Optimized Kernel Evaluation of Missense Nucleotides), which evaluates rare missense variants based on their spatial distribution on the protein rather than allele frequency. The hypothesis behind this is that the three-dimensional spatial distribution of variants within a protein structure provides functional context and improves the power of association tests. POKEMON identified four candidate genes from the ADSP WES data, namely two known Alzheimers disease (AD) genes (TREM2 and SORL) and two novel genes (DUSP18 and CSF1R). For known AD genes, the signal from the spatial cluster is stable even if we exclude known AD risk variants, indicating the presence of additional low frequency risk variants within these genes. DUSP18 has a cluster of variants primarily shared by case subjects around the ligand-binding domain, and this cluster is further validated in a replication dataset with a larger sample size. POKEMON is an open-source tool available at https://github.com/bushlab-genomics/POKEMON.

sentences: Structure and dynamics of the SARS-CoV-2 envelope protein monomer Coronaviruses, especially SARS-CoV-2, present an ongoing threat for human wellbeing. Consequently, elucidation of molecular determinants of their function and interaction with host is an important task. Whereas some of the coronaviral proteins are extensively characterized, others remain understudied. Here, we use molecular dynamics simulations to analyze the structure and dynamics of the SARS-CoV-2 envelope (E) protein (a viroporin) in the monomeric form. The protein consists of the hydrophobic -helical transmembrane domain (TMD) and amphiphilic -helices H2 and H3, connected by flexible linkers. We show that TMD has a preferable orientation in the membrane, while H2 and H3 reside at the membrane surface. Orientation of H2 is strongly influenced by palmitoylation of cysteines Cys40, Cys43 and Cys44. Glycosylation of Asn66 affects the orientation of H3. We also observe that the E protein both generates and senses the membrane curvature, preferably localizing with the C-terminus at the convex regions of the membrane. This may be favorable for assembly of the E protein oligomers, whereas induction of curvature may facilitate budding of the viral particles. The presented results may be helpful for better understanding of the function of coronaviral E protein and viroporins in general, and for overcoming the ongoing SARS-CoV-2 pandemic.

sentences: Microenvironmental factors in cell segregation and heterogeneity in breast cancer development We analyzed a quantitative model that describes the epigenetic dynamics during the growth and evolution of an avascular tumor. A gene regulatory network (GRN) formed by a set of ten genes that are believed to play an important role in breast cancer development was kinetically coupled to the microenvironmental agents: glucose, estrogens and oxygen. The dynamics of spontaneous mutations was described by a Yule-Furry master equation whose solution represents the probability that a given cell in the tissue undergoes a certain number of mutations at a given time. We assumed that mutations rate is modified by nutrients spatial gradients. The tumor mass was grown by means of a cellular automata supplemented with a set of reaction diffusion equations that described the transport of the microenvironmental agents. By analyzing the epigenetic states space described by the GRN dynamics, we found three attractors that were identified with the cellular epigenetic states: normal, precancer and cancer. For two-dimensional (2D) and three-dimensional (3D) tumors we calculated the spatial distributions of the following quantities: (i) number of mutations, (ii) mutations of each gene and, (iii) phenotypes. Using estrogens as the principal microenvironmental agent that regulates cells proliferation process, we obtained the tumor shapes for different values of the estrogen consumption and supply rates. It was found that he majority of mutations occurred in cells that were located close to the 2D tumor perimeter or close to the 3D tumor surface. Also It was found that the occurrence of different phenotypes in the tumor are controlled by the levels of estrogen concentration since they can change the individual cell threshold and gene expression levels. All the results were consistently observed for 2D and 3D tumors.

sentences: Common and lifestyle-specific traits of mycorrhiza-associated metabolite alterations in plant roots reflects strategies of root-mycorrhizal interactions Convergent patterns in morphological and genetic traits of mycorrhizas have been well-documented and reflect common selection forces that define mycorrhizas. However, generalizable patterns of mycorrhiza-associated chemical alterations, which are immediately linked to plant and fungal strategies for successful symbiosis, have yet to be emerged. Comparing root metabolomes of phylogenetically-diverse plants inoculated by mycorrhizal fungi across two major lifestyles (arbuscular- vs. ecto-mycorrhizas), our study uncovers metabolite changes unique to each mycorrhizal lifestyle and those common across plant-mycorrhizal combinations irrespective of lifestyles. Arbuscular and ecto- mycorrhizal colonized roots accumulated different sets of carbohydrates, indicating unique carbon partitioning strategies: particularly, arbuscular mycorrhizal roots accumulated cyclic polyols inaccessible for symbionts, suggesting tighter regulation of plants in carbon partitioning. Mycorrhizas also altered specialized metabolism, featuring frequent increases of flavan-3-ols and decreases of flavanols irrespective of mycorrhizal lifestyles, suggesting tactical reconfiguration of specialized metabolites to facilitate/contain symbiosis. Our data show for the first time, to our knowledge, that part of the root metabolite alterations by mycorrhizas were relatively common across plant-mycorrhizal systems, highlighting their potentially critical regulatory and evolutionary role for successful symbiosis. This commonality appears robust to phylogenetic diversity of host plants and thus may be widespread in land plants. Our findings offer future research venues to elucidate the finer roles of these common traits of mycorrhiza-associated metabolite alterations and thus help to eventually develop a comprehensive understanding of this omnipresent plant-fungus partnership.

sentences: High-throughput molecular dynamics-based alchemical free energy calculations for predicting the binding free energy change associated with the common mutations in the spike receptor-binding domain of SARS-CoV-2 The ongoing pandemic caused by SARS-CoV-2 has gone through various phases. From the initial outbreak the virus has mutated several times, with some lineages showing even stronger infectivity and faster spread than the original virus. Among all the variants, beta, gamma, delta and the latest (omicron) are currently classified as variants of concern (VOC) while the remaining are labelled either as variants of interest (VOI) or variants under monitoring (VUM). In this work, we have focused on the mutations observed in important variants, particularly at the receptor-binding domain (RBD) of the spike protein that is responsible for the interactions with the host ACE2 receptor and binding of antibodies. Studying these mutations is particularly important for understanding the viral infectivity, spread of the disease and for tracking the escape routes of this virus from antibodies. Molecular dynamics (MD) based alchemical free energy calculations have been shown to be very accurate in predicting the free energy change due to a mutation that could have a deleterious or a stabilising effect on the protein itself or its binding affinity to another protein. Here, we investigated the significance of six commonly observed spike RBD mutations on the stability of the spike protein binding to ACE2 by free energy calculations using high throughput MD simulations. For comparison, we also used other (rigorous and non-rigorous) binding free energy prediction methods and compared our results with the experimental data if available. The alchemical free energy-based method consistently predicted the free-energy changes with an accuracy close to {+/-}1.0 kcal/mol when compared with the available experimental values. As per our simulation data the most significant mutations responsible for stabilising the spike RBD interactions with human ACE2 are N501Y and L452R.

sentences: Opsonization by non-neutralizing antibodies can confer protection to SARS-CoV-2 despite Spike-dependent modulation of phagocytosis Spike-specific antibodies are central to effective COVID19 immunity. Research efforts have focused on antibodies that neutralize the ACE2-Spike interaction but not on non-neutralizing antibodies. Antibody-dependent phagocytosis is an immune mechanism enhanced by opsonization, where typically, more bound antibodies trigger a stronger phagocyte response. Here, we show that Spike-specific antibodies, dependent on concentration, can either enhance or reduce Spike-bead phagocytosis by monocytes independently of the antibody neutralization potential. Surprisingly, we find that both convalescent patient plasma and patient-derived monoclonal antibodies lead to maximum opsonization already at low levels of bound antibodies and is reduced as antibody binding to Spike protein increases. Moreover, we show that this Spike-dependent modulation of opsonization seems to affect the outcome in an experimental SARS-CoV-2 infection model. These results suggest that the levels of anti-Spike antibodies could influence monocyte-mediated immune functions and propose that non-neutralizing antibodies could confer protection to SARS-CoV-2 infection by mediating phago-cytosis.

sentences: Global determinants of the distribution of insect genetic diversity Understanding global patterns of genetic diversity (GD) is essential to describe, monitor, and preserve the processes giving rise to life on Earth. To date, efforts to map macrogenetic patterns have been restricted to vertebrate groups that comprise a small fraction of Earths biodiversity. Here, we construct the first global map of predicted insect genetic diversity. We calculate the global distribution of GD mean (GDM) and evenness (GDE) of insect assemblages, identify the global environmental correlates of insect GD, and make predictions for undersampled regions. Based on the largest and most species-rich single-locus genetic dataset assembled to date, we find that both GD metrics follow a bimodal latitudinal gradient, where GDM and GDE correlate with contemporary climate variation. Our models explain 1/4 and 1/3 of the observed variation in GDM and GDE in insects, respectively, making an important step towards describing global biodiversity patterns in the most diverse animal taxon.

sentences: Plasma membrane localization of paralogous leucine permeases Bap2 and Bap3 is regulated by Bul1 Timeliness in expression and degradation of the nutrient permeases is crucial for any organism. In Saccharomyces cerevisiae, post translational regulation of nutrient permeases such as trafficking and turnover are poorly understood. We found that loss of a leucine permease BAP2, but not other permeases lead to severe growth retardation when the carbon source is glucose or galactose but not glycerol and lactate. Leucine prototrophy suppressed the retardation, showing BAP2 and LEU2 are synthetically lethal. We discovered that loss of BUL1, an arrestin involved in trafficking of diverse permeases suppressed this lethality. The suppression required another leucine permease, BAP3. Our results suggest that BUL1 downregulate permeases BAP2 and BAP3 present in plasma membrane through Rsp5 dependent endocytosis. We speculate that by regulating leucine import BUL1 regulates the activity of TORC1.

sentences: Late-acting self-incompatible system, preferential allogamy and delayed selfing in the heteromorphic invasive populations of Ludwigia grandiflora subsp. hexapetala AO_SCPLOWBSTRACTC_SCPLOWBreeding system influences local population genetic structure, effective size, offspring fitness and functional variation. Determining the respective importance of self- and cross-fertilization in hermaphroditic flowering plants is thus important to understand their ecology and evolution. The worldwide invasive species, Ludwigia grandiflora subsp. hexapetala (Lgh) presents two floral morphs: one self-compatible short-styled morph (S-morph) and one self-incompatible long-styled morph (L-morph). In this study, we identified the breeding systems of western European experimental and natural populations of Lgh by comparing structural characteristics of pollen and style, by studying self- and cross-pollen tube elongations and the viability of the resulting seeds and seedlings in both floral morphs. Our results showed no differences in pollen shape and stigma surfaces no matter the floral morph. In the self-incompatible L-morph flowers, self-pollen tubes were stopped tardily, in the ovarian area, and were unable to fertilize the ovules. This first formal identification of a late-acting, prezygotic self-incompatible system (LSI) in Ludwigia genus contributes a case of LSI in an additional family within the Myrtales order. In the self-compatible S-morph flowers, self-pollen always succeeded to self-fertilize the ovules that nearly all developed into viable seedlings. However, cross-pollen tubes always elongated faster than self-pollen tubes in both morphs. S-morph individuals may thus advantage preferential allogamy over selfing when cross-pollen is available despite its self-compatibility. As expected in late-acting self-incompatible systems, L-morph flowers authorised 0.2{per thousand} of selfed seeds during the uppermost flowering season, that increased to 1{per thousand} at the end of the flowering season. Such delayed selfing resulted in a significant quantity of viable floating seeds. They may contribute to the local regeneration, seed bank and propagation of the L-morph, which may contribute to explain its invasion success worldwide. Management plans of Lgh would gain to consider the breeding systems we identified.

sentences: Enhancing oscillations in intracranial electrophysiological recordings with data-driven spatial filters In invasive electrophysiological recordings, a variety of neuronal oscillations can be detected across the cortex, with overlap in space and time. This overlap complicates measurement of neuronal oscillations using standard referencing schemes, like common average or bipolar referencing. Here, we illustrate the effects of spatial mixing on measuring neuronal oscillations in invasive electrophysiological recordings and demonstrate the benefits of using data-driven referencing schemes in order to improve measurement of neuronal oscillations. We discuss referencing as the application of a spatial filter. Spatio-spectral decomposition is used to estimate data-driven spatial filters, a computationally fast method which specifically enhances signal-to-noise ratio for oscillations in a frequency band of interest. We show that application of these data-driven spatial filters has benefits for data exploration, investigation of temporal dynamics and assessment of peak frequencies of neuronal oscillations. We demonstrate multiple use cases, exploring between-participant variability in presence of oscillations, spatial spread and waveform shape of different rhythms as well as narrowband noise removal with the aid of spatial filters. We find high between-participant variability in the presence of neural oscillations, a large variation in spatial spread of individual rhythms and many non-sinusoidal rhythms across the cortex. Improved measurement of cortical rhythms will yield better conditions for establishing links between cortical activity and behavior, as well as bridging scales between the invasive intracranial measurements and noninvasive macroscale scalp measurements.

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SPT6 loss Permits the Transdifferentiation of Keratinocytes into anIntestinal Fate that Recapitulates Barretts Metaplasia Transient depletion of the transcription elongation factor SPT6 in the keratinocyte has been recently shown to inhibit epidermal differentiation and stratification; instead, they transdifferentiate into a gut-like lineage. We show here that this phenomenon of transdifferentiation recapitulates Barretts metaplasia, the only human pathophysiologic condition in which a stratified squamous epithelium that is injured due to chronic acid reflux is trans-committed into an intestinal fate. The evidence we present here not only lend support to the notion that the keratinocytes are the cell of origin of Barretts metaplasia, but also provide mechanistic insights linking transient acid exposure, downregulation of SPT6, stalled transcription of the master regulator of epidermal fate TP63, loss of epidermal fate and metaplastic progression. Because Barretts metaplasia in the esophagus (BE) is a pre-neoplastic condition with no preclinical human models, these findings have a profound impact on the modeling Barretts metaplasia-in-a-dish.

GRAPHIC ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/442930v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@8ce08aorg.highwire.dtl.DTLVardef@e98c83org.highwire.dtl.DTLVardef@107f521org.highwire.dtl.DTLVardef@16e7523_HPS_FORMAT_FIGEXP  M_FIG C_FIG HIGHLIGHTSO_LIKeratinocytes transdifferentiate into the gut lineage upon depletion of SPT6
C_LIO_LISuch transdifferentiation recapitulates Barretts metaplasia, not the healthy gut
C_LIO_LIAcid downregulates SPT6, which derails the expression and functions of TP63
C_LIO_LISuch downregulation precedes the metaplasia-dysplasia-neoplasia cascade
C_LI

sentences: In vivo Monitoring of Glucose using Ultrasound-induced Resonance in Implantable Smart Hydrogel Microstructures A novel glucose sensor is presented that uses smart hydrogels as a biocompatible implantable sensing element, which completely eliminates the need for any implanted electronics and uses an external conventional medical-grade ultrasound transducer for readout. The readout mechanism makes use of resonance absorption of ultrasound waves in glucose-sensitive hydrogels. Changes in in vivo glucose concentration in the interstitial tissue lead to swelling and de-swelling of the gels which in turn lead to changes in resonance behavior. The hydrogels are designed and shaped such as to exhibit specific mechanical resonance frequencies while remaining sonolucent to other frequencies. Thus, they allow conventional and continued ultrasound imaging, while yielding a sensing signal at specific frequencies that is correlated with glucose concentration. The resonance frequencies can be tuned by changing the shape and mechanical properties of the gel structures, such as to allow for multiple, co-located implanted hydrogels with different sensing characteristics or targets to be employed and read out, without interference, using the same ultrasound transducer, by simply toggling frequencies. The fact that there is no need for any implantable electronics, also opens the path towards future use of biodegradable hydrogels, thus creating a platform that allows injection of sensors that do not need to be retrieved when they reach the end of their useful lifespan.

sentences: Mutant alleles differentially shape cattle complex traits and fitness Classical mutant alleles (MAs), with large effects on phenotype, tend to be deleterious to traits and fitness. Is this the case for mutations with small effects? We infer MAs for 8 million sequence variants in 113k cattle and quantify the effects of MA on 37 complex traits. Heterozygosity for variants at genomic sites conserved across 100 vertebrates increase fertility, stature, and milk production, positively associating these traits with fitness. MAs decrease stature and fat and protein concentration in milk, but increase gestation length and somatic cell count in milk (the latter indicative of mastitis). However, the allele frequency of MAs that decrease fat and protein concentration and stature and increase gestation length and somatic cell count is lower than the allele frequency of MAs with the opposite effect. These results suggest bias in the direction of effect of mutation (e.g. towards reduced protein in milk), but selection operating to reduce the frequency of these MAs. Taken together, our results imply two classes of genomic sites subject to long-term selection: sites conserved across vertebrates show hybrid vigour while sites subject to less long-term selection show a bias in mutation towards alleles that are selected against.

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Deep neural network analysis employing diffusion basis spectrum imaging metrics as classifiers improves prostate cancer detection and grading Structural and cellular complexity of prostatic histopathology limits the accuracy of noninvasive detection and grading of prostate cancer (PCa). We addressed this limitation by employing a novel diffusion basis spectrum imaging (DBSI) to derive structurally-specific diffusion fingerprints reflecting various underlying prostatic structural and cellular components. We further developed diffusion histology imaging (DHI) by combining DBSI-derived structural fingerprints with a deep neural network (DNN) algorithm to more accurately classify different histopathological features and predict tumor grade in PCa. We examined 243 patients suspected with PCa using in vivo DBSI. The in vivo DBSI-derived diffusion metrics detected coexisting prostatic pathologies distinguishing inflammation, PCa, and benign prostatic hyperplasia. DHI distinguished PCa from benign peripheral and transition zone tissues with over 95% sensitivity and specificity. DHI also demonstrated over 90% sensitivity and specificity for Gleason score noninvasively. We present DHI as a novel diagnostic tool capable of noninvasive detection and grading of PCa.

One sentence summaryDiffusion histology imaging noninvasively and accurately detects and grades prostate cancer.

sentences: A chromosome-level genome assembly of the European Beech (Fagus sylvativa) reveals anomalies for organelle DNA integration, repeat content and distribution of SNPs The European Beech is the dominant climax tree in most regions of Central Europe and valued for its ecological versatility and hardwood timber. Even though a draft genome has been published recently, higher resolution is required for studying aspects of genome architecture and recombination. Here we present a chromosome-level assembly of the more than 300 year-old reference individual, Bhaga, from the Kellerwald-Edersee National Park (Germany). Its nuclear genome of 541 Mb was resolved into 12 chromosomes varying in length between 28 Mb and 73 Mb. Multiple nuclear insertions of parts of the chloroplast genome were observed, with one region on chromosome 11 spanning more than 2 Mb of the genome in which fragments up to 54,784 bp long and covering the whole chloroplast genome were inserted randomly. Unlike in Arabidopsis thaliana, ribosomal cistrons are present in Fagus sylvatica only in four major regions, in line with FISH studies. On most assembled chromosomes, telomeric repeats were found at both ends, while centromeric repeats were found to be scattered throughout the genome apart from their main occurrence per chromosome. The genome- wide distribution of SNPs was evaluated using a second individual from Jamy Nature Reserve (Poland). SNPs, repeat elements and duplicated genes were unevenly distributed in the genomes, with one major anomaly on chromosome 4. The genome presented here adds to the available highly resolved plant genomes and we hope it will serve as a valuable basis for future research on genome architecture and for understanding the past and future of European Beech populations in a changing climate.

sentences: Systematic dissection of a complex gut bacterial community Efforts to model the gut microbiome have yielded important insights into the mechanisms of interspecies interactions, the impact of priority effects on ecosystem dynamics, and the role of diet and nutrient availability in determining community composition. However, the model communities studied to date have been defined or complex but not both, limiting their utility. Here, we construct a defined community of 104 bacterial strains composed of the most common taxa from the human gut microbiota. By propagating this community in growth media missing one amino acid at a time, we show that branched-chain amino acids have an outsize impact on community structure and identify a pathway in Clostridium sporogenes for generating ATP from arginine. We constructed and propagated the complete set of single-strain dropout communities, revealing a sparse network of strain-strain interactions including a novel interaction between C. sporogenes and Lactococcus lactis driven by metabolism. This work forms a foundation for studying strain-strain and strain-nutrient interactions in highly complex defined communities, and it provides a starting point for interrogating the rules of synthetic ecology at the 100+ strain scale.

sentences: Use of high-refractive index hydrogels and tissue clearing for large biological sample imaging Recent advances in tissue clearing and light sheet fluorescence microscopy have improved insights into and understanding of tissue morphology and disease pathology by imaging large samples without the requirement of histological sectioning. However, sample handling and conservation of sample integrity during lengthy staining and acquisition protocols remains a challenge. This study overcomes these challenges with acrylamide hydrogels synthesised to match the refractive index of solutions typically utilised in aqueous tissue clearing protocols. These hydrogels have a high-water content (82.0{+/-}3.7% by weight). The gels are stable over time and FITC-IgG readily permeated into, and effluxed out of them. Whilst the gels deformed and/or swelled over time in some commonly used solutions, this was overcome by using a previously described custom refractive index matched solution. To validate their use, CUBIC cleared mouse tissues and whole embryos were embedded in hydrogels, stained using fluorescent small molecule dyes, labels and antibodies and successfully imaged using light sheet fluorescence microscopy. In conclusion, the high-water content, high refractive index hydrogels described in this study have a broad applicability to research that delves into pathophysiological processes by stabilising and protecting large and fragile samples.

sentences: New insights from 33,813 publicly available metagenome-assembled-genomes (MAGs) assembled from the rumen microbiome Recent rumen microbiome metagenomics papers1,2 have published hundreds of metagenome-assembled genomes (MAGs), comparing them to 4,941 MAGs published by Stewart et al3 in order to define novelty. However, there are many more publicly available MAGs from ruminants. In this paper, for the first time, all available resources are combined, catalogued and de-replicated to define putative species-level bins. As well as providing new insights into the constitution of the rumen microbiome, including an updated estimate of the number of microbial species in the rumen, this work demonstrates that a lack of community-adopted standards for the release and annotation of MAGs hinders progress in microbial ecology and metagenomics.

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Collagen Deposition in Diabetic Kidney Disease Boosts Intercellular Signaling: A Mathematical Model Diabetic kidney disease is a health burden that is becoming more prevalent in the US and worldwide. The limited options for treating and preventing diabetic kidney disease are in part due to gaps in our understanding of the progression of diabetic kidney damage and its impacts on cellular function. An important cellular function in the kidney glomerulus is intercellular communication via the release and uptake of soluble cytokines and growth factors. In diabetic kidney disease, excess collagen deposition alters the mesangial matrix properties, which, we hypothesize, diminishes the intercellular signaling between key glomerular cells. To test our hypothesis, we utilized established mathematical models of transport to study the impact of pathological deposition on the ability of cells to communicate via intercellular signaling. Our analysis reveals that pathological collagen deposition can enhance the signaling range of the glomerular cells rather than diminishing it.

SIGNIFICANCEThe incidence of diabetes is expected to rise to over 600 million by the year 2040, one third of whom are expected to develop diabetic kidney disease. Our lack of understanding of how diabetic kidney disease progresses and affects cellular and tissue function has led to our inability to mitigate the rapidly rising burden of diabetic kidney disease. A hallmark of diabetic kidney damage is collagen deposition, yet its impact on cellular and tissue function is still not well understood. The elucidation of the impact of collagen deposition on cellular and tissue function will enable the identification of mechanisms that exacerbate the progression of diabetic kidney damage and thus provide novel avenues for preventing or slowing the progression of diabetic kidney damage.

sentences: Activation of Histone 3 Lysine 9 methyl writing and reading capabilities within the G9a-GLP heterodimer Unique among metazoan repressive histone methyltransferases, G9a and GLP, which chiefly target histone 3 lysine 9 (H3K9), require dimerization for productive H3K9 mono (me1)- and dimethylation (me2) in vivo. Intriguingly, even though each enzyme can independently methylate H3K9, the predominant active form in vivo is a heterodimer of G9a and GLP. How dimerization influences the central H3K9 methyl binding ("reading") and deposition ("writing") activity of G9a and GLP, and why heterodimerization is essential in vivo remains opaque. Here, we examine the H3K9me "reading" and "writing" activities of defined, recombinantly produced homo- and heterodimers of G9a and GLP. We find that both reading and writing are significantly enhanced in the heterodimer. Compared to the homodimers, the heterodimer has higher recognition of H3K9me2, and a striking [~] 10-fold increased turnover rate for nucleosomal substrates under multiple turnover conditions, which is not evident on histone tail peptide substrates. Crosslinking Mass Spectrometry suggests that differences between the homodimers and the unique activity of the heterodimer may be encoded in altered ground state conformations, as each dimer displays different domain contacts. Our results indicate that heterodimerization may be required to relieve autoinhibition of H3K9me reading and chromatin methylation evident in G9a and GLP homodimers. Relieving this inhibition may be particularly important in early differentiation when large tracts of H3K9me2 are deposited by G9a-GLP, which may require a more active form of the enzyme.

sentences: Pre-determined diversity in resistant fates emerges from homogenous cells after anti-cancer drug treatment Even amongst genetically identical cancer cells, therapy resistance often only emerges from a very small subset of those cells. Much effort has gone into uncovering the molecular differences in rare individual cells in the initial population that may allow certain cells to become therapy resistant; however, comparatively little is known about variability in the resistant outcomes themselves. Here, we develop and apply FateMap, a framework that combines DNA barcoding with single-cell RNA sequencing to reveal the fates of hundreds of thousands of clones exposed to anti-cancer therapies. We show that resistant clones emerging from single-cell-derived cancer cells adopt molecularly, morphologically, and functionally distinct fate types. These different resistant types are largely predetermined by molecular differences between cells before addition of drug and not by extrinsic cell-specific microenvironmental factors. Changes in dose and kind of drug can, however, switch the resistant fate type of an initial cell, even resulting in the generation and elimination of certain fate types. Diversity in resistant fates was observed across several single-cell-derived cancer cell lines and types treated with a variety of drugs. Cell fate diversity as a result of variability in intrinsic cell states may be a generic feature of response to external cues.

sentences: New Music System Reveals Spectral Contribution to Statistical Learning 1Knowledge of speech and music depends upon the ability to perceive relationships between sounds in order to form a stable mental representation of statistical structure. Although evidence exists for the learning of musical scale structure from the statistical properties of sound events, little research has been able to observe how specific acoustic features contribute to statistical learning independent of the effects of long-term exposure. Here, using a new musical system, we show that spectral content is an important cue for acquiring musical scale structure. In two experiments, participants completed probe-tone ratings before and after a half-hour period of exposure to melodies in a novel musical scale with a predefined statistical structure. In Experiment 1, participants were randomly assigned to either a no-exposure control group, or to exposure groups who heard pure tone or complex tone sequences. In Experiment 2, participants were randomly assigned to exposure groups who heard complex tones constructed with odd harmonics or even harmonics. Learning outcome was assessed by correlating pre/post-exposure ratings and the statistical structure of tones within the exposure period. Spectral information significantly affected sensitivity to statistical structure: participants were able to learn after exposure to all tested timbres, but did best at learning with timbres with odd harmonics, which were congruent with scale structure. Results show that spectral amplitude distribution is a useful cue for statistical learning, and suggest that musical scale structure might be acquired through exposure to spectral distribution in sounds.

sentences: Water-triggered, irreversible conformational change of SARS-CoV-2 main protease on passing from the solid state to aqueous solution The main protease from SARS-CoV-2 is a homodimer. Yet, a recent 0.1 ms long molecular dynamics simulation shows that it readily undergoes a symmetry breaking event on passing from the solid state to the aqueous solution. As a result, the subunits present distinct conformations of the binding pocket. By analysing this long time simulation, here we uncover a previously unrecognised role of water molecules in triggering the transition. Interestingly, each subunit presents a different collection of long-lived water molecules. Enhanced sampling methods performed here, along with machine learning approaches, further establish that the transition to the asymmetric state is essentially irreversible.

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Strobemers: an alternative to k-mers for sequence comparison K-mer-based methods are widely used in bioinformatics for various types of sequence comparison. However, a single mutation will mutate k consecutive k-mers and makes most k-mer based applications for sequence comparison sensitive to variable mutation rates. Many techniques have been studied to overcome this sensitivity, e.g., spaced k-mers and k-mer permutation techniques, but these techniques do not handle indels well. For indels, pairs or groups of small k-mers are commonly used, but these methods first produce k-mer matches, and only in a second step, a pairing or grouping of k-mers is performed. Such techniques produce many redundant k-mer matches due to the size of k.

Here, we propose strobemers as an alternative to k-mers for sequence comparison. Intuitively, strobemers consist of linked minimizers. We use simulated data to show that strobemers provide more evenly distributed sequence matches and are less sensitive to different mutation rates than k-mers and spaced k-mers. Strobemers also produce a higher match coverage across sequences. We further implement a proof-of-concept sequence matching tool StrobeMap, and use synthetic and biological Oxford Nanopore sequencing data to show the utility of using strobemers for sequence comparison in different contexts such as sequence clustering and alignment scenarios. A reference implementation of our tool StrobeMap together with code for analyses is available at https://github.com/ksahlin/strobemers.

sentences: Natural selection and the advantage of recombination The ubiquity of sex and recombination in nature is widely viewed as enigmatic, despite an abundance of limited-scope explanations. Natural selection, it seems, should amplify well-matched combinations of genes. Recombination would break up these well-matched combinations and should thus be suppressed. We show, to the contrary, that natural selection amplifies poorly-matched gene combinations and creates negative associations in the process. Recombination breaks up these poorly-matched combinations, neutralizes the negative associations, and should thus be passively and universally favored.

sentences: Reprogrammed iBlastoids contain amnion-like cells but not trophectoderm With the advancement of human stem cell cultures and interest in understanding human embryogenesis, human blastocyst-like structures, or human blastoids, are being developed. Thus far, two different strategies have been taken, generating blastoids from naive human pluripotent stem cells (hPSC)1-3 or through reprogramming of adult human somatic cells4. All these studies utilize comparative transcriptome analyses to authenticate blastoid cells and identify their counterparts in the human blastocyst5,6. However, the validity of comparative transcriptome analysis is critically hinged on including relevant reference data, not only those from targeted cell types but also from potential alternative cell lineages. Thus, we sought to reevaluate the single-cell transcriptome data from the blastoids based on a more comprehensive cellular reference, which includes data from in vitro cultured human6,7, non-human primate (NHP, Cynomolgus macaque) blastocysts8, a human stem cell-based post-implantation amniotic sac embryoid (PASE) model9, and an in vivo gastrulation-stage human embryo specimen10, using four different analysis strategies. Our analyses unequivocally support that blastoids developed by reprogramming adult human somatic cells largely fail to generate cells with a transcriptome profile consistent with the human blastocyst trophectoderm. Instead, cells identified as trophectoderm-like have a transcriptional profile more similar to the amniotic ectoderm in the gastrulating human and NHP embryos. To facilitate cell lineage identifications in human embryo models, we further identified a set of human amniotic ectoderm and trophectoderm markers that could be utilized to distinguish these two lineages. We further built a neural-network based online prediction tool, which accurately discerns the full cellular composition of blastoids.

sentences: Assessing temporal and geographic contacts across the Adriatic Sea through the analysis of genome-wide data from Southern Italy Southern Italy was characterised by a complex prehistory that started with different Palaeolithic cultures, later followed by the Neolithic transition and the demic dispersal from the Pontic-Caspian Steppe during the Bronze Age. Archaeological and historical evidence points to demic and cultural influences between Southern Italians and the Balkans, starting with the initial Palaeolithic occupation until historical and modern times. To shed light on the dynamics of these contacts, we analysed a genome-wide SNP dataset of more than 700 individuals from the South Mediterranean area (102 from Southern Italy), combined with ancient DNA from neighbouring areas. Our findings revealed high affinities of South-Eastern Italians with modern Eastern Peloponnesians, and a closer affinity of ancient Greek genomes with those from specific regions of South Italy than modern Greek genomes. The higher similarity could be associated with the presence of a Bronze Age component ultimately originating from the Caucasus and characterised by high frequencies of Iranian and Anatolian Neolithic ancestries. Furthermore, to reveal possible signals of natural selection, we looked for extremely differentiated allele frequencies among Northern and Southern Italy, uncovering putatively adapted SNPs in genes involved in alcohol metabolism, nevi features and immunological traits, such as ALDH2, NID1 and CBLB.

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Protein Domain-Based Prediction of Compound-Target Interactions and Experimental Validation on LIM Kinases Predictive approaches such as virtual screening have been used in drug discovery with the objective of reducing developmental time and costs. Current machine learning and network- based approaches have issues related to generalization, usability, or model interpretability, especially due to the complexity of target proteins structure/function, and bias in system training datasets. Here, we propose a new computational method "DRUIDom" to predict bio- interactions between drug candidate compounds and target proteins by utilizing the domain modularity of proteins, to overcome problems associated with current approaches. DRUIDom is composed of two methodological steps. First, ligands/compounds are statistically mapped to structural domains of their target proteins, with the aim of identifying physical or functional interactions. As such, other proteins containing the mapped domain or domain pair become new candidate targets for the corresponding compounds. Next, a million-scale dataset of small molecule compounds, including the ones mapped to domains in the previous step, are clustered based on their molecular similarities, and their domain associations are propagated to other compounds within the same clusters. Experimentally verified bioactivity data points, obtained from public databases, are meticulously filtered to construct datasets of active/interacting and inactive/non-interacting compound-target pairs ([~]2.9M data points), and used as training data for calculating parameters of compound- domain mappings, which led to 27,032 high-confidence associations between 250 domains and 8,165 compounds, and a finalized output of [~]5 million new compound-protein interactions. DRUIDom is experimentally validated by syntheses and bioactivity analyses of compounds predicted to target LIM-kinase proteins, which play critical roles in the regulation of cell motility, cell cycle progression, and differentiation through actin filament dynamics. We showed that LIMK-inhibitor-2 and its derivatives significantly block the cancer cell migration through inhibition of LIMK phosphorylation and the downstream protein cofilin. One of the derivative compounds (LIMKi-2d) was identified as a promising candidate due to its action on resistant Mahlavu liver cancer cells. The results demonstrated that DRUIDom can be exploited to identify drug candidate compounds for intended targets and to predict new target proteins based on the defined compound-domain relationships. The datasets, results, and the source code of DRUIDom are fully-available at: https://github.com/cansyl/DRUIDom.

Author SummaryDrug development comprises several interlinked steps from designing drug candidate molecules to running clinical trials, with the aim to bring a new drug to market. A critical yet costly and labor-intensive stage is drug discovery, in which drug candidate molecules that specifically interact with the intended biomolecular target (mostly proteins) are identified. Lately, data-centric computational methods have been proposed to aid experimental procedures in drug discovery. These methods have the ability to rapidly assess large molecule libraries and reduce the time and cost of the process; however, most of them suffer from problems related to producing reliable biologically relevant results, preventing them from gaining real-world usage. Here, we have developed a new method called DRUIDom to predict unknown interactions between drugs/drug candidate compounds and biological targets by utilizing the modular structure of proteins. For this, we identify the domains, i.e., the evolutionary and functional building blocks of proteins, where these potential drug compounds can bind, and utilize this information along with protein domain annotations to predict new drug targets. We have tested the biological relevance of DRUIDom on selected proteins that play critical roles in the progression of numerous types of cancer. Cell-based experimental results indicated that predicted inhibitors are effective even on drug-resistant cancer cells. Our results suggest that DRUIDom produces novel and biologically relevant results that can be directly used in the early steps of the drug discovery process.

sentences: A Proposal for Finite But Unbounded Human Lifetimes Close analysis of an extensive data set combined with independent evidence prompts our proposal to view human lifetimes as individually finite but collectively unbounded. We formulate a model incorporating this idea whose predictions agree very well with the observed data. In the model, human lifetimes are theoretically unbounded, but the probability of an individual living to an extreme age is negligible, so lifetimes are effectively limited. Our model incorporates a mortality hazard rate plateau and a late-life mortality deceleration effect in conjunction with a newly observed advanced age mortality acceleration. This reconciles many previously observed effects. The model is temporally stable: consistent with observation, parameters do not change over time. As an application, assuming no major medical advances, we predict the emergence of many individuals living past 120, but due to accelerating mortality find it unlikely that any will subsequently survive to an age of 125.

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Suitability of GRK antibodies for individual detection and quantification of GRK isoforms in western blots G protein-coupled receptors (GPCRs) are regulated by GPCR kinases (GRKs) which phosphorylate intracellular domains of the active receptor. This leads to the recruitment of arrestins resulting in desensitization and internalization of the GPCR. Aside from acting on GPCRs, GRKs regulate a variety of membrane, cytosolic, and nuclear proteins not only via phosphorylation but also by acting as scaffold. This multifunctionality is also reflected by their diverse roles in pathological conditions like cancer, influenza infection, malaria, and metabolic disease.

Reliable tools to study GRKs are the key to specify their role in complex cellular signaling networks. Thus, we examined the specificity of eight commercially available antibodies targeting the four ubiquitously expressed GRK2, GRK3, GRK5, and GRK6 in western blot analysis. We thereby identified one antibody that did not recognize its antigen, as well as antibodies that showed unspecific signals or cross reactivity. Thus, we strongly recommend testing any antibody with exogenously expressed proteins to clearly confirm identity of the obtained western blot results.

Utilizing the most suitable antibodies we established the western blot-based, cost-effective, simple tag-guided analysis of relative protein abundance (STARPA). This method allows comparison of protein levels obtained by immunoblotting with different antibodies. Furthermore, we applied STARPA to determine GRK protein levels in five commonly used cell lines revealing differential isoform expression.

sentences: Twisting DNA by Salt The structure and properties of DNA depend on the environment, in particular the ion atmosphere. Here, we investigate how DNA twist -one of the central properties of DNA- changes with concentration and identity of the surrounding ions. To resolve how cations influence the helical twist, we combine single-molecule magnetic tweezer experiments and extensive all- atom molecular dynamics simulations. Two interconnected trends are observed for monovalent alkali and divalent alkaline earth cations. First, DNA twist increases monotonously with increasing concentration for all ions investigated. Second, for a given salt concentration, DNA twist strongly depends on cation identity. At 100 mM concentration, DNA twist increases as Ba2+ < Na+ < K+ < Rb+ < Li+ {approx} Cs+ < Sr2+ < Mg2+ < Ca2+. Our molecular dynamics simulations reveal that preferential binding of the cations to the DNA backbone or the nucleobases has opposing effects on DNA twist and provides the microscopic explanation of the observed ion specificity. However, the simulations also reveal shortcomings of existing force field parameters for Cs+ and Sr2+. The comprehensive view gained from our combined approach provides a foundation for understanding and predicting cation-induced structural changes both in nature and in DNA nanotechnology.

sentences: Function of the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE is evolutionarily conserved in embryophytes The plant phenylpropanoid pathway generates a major class of specialized metabolites and precursors of essential extracellular polymers that initially appeared upon plant terrestrialization. Despite its evolutionary significance, little is known about the complexity and function of this major metabolic pathway in extant bryophytes, which represent the non-vascular stage of embryophyte evolution. Here, we report that the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE (HCT) gene that plays a critical function in the phenylpropanoid pathway during seed plant development, is functionally conserved in Physcomitrium patens (Physcomitrella), in the moss lineage of bryophytes. Phylogenetic analysis indicates that bonafide HCT function emerged in the progenitor of embryophytes. In vitro enzyme assays, moss phenolic pathway reconstitution in yeast and in planta gene inactivation coupled to targeted metabolic profiling, collectively indicate that P. patens HCT (PpHCT), similar to tracheophyte HCT orthologs, uses shikimate as a native acyl acceptor to produce a p-coumaroyl-5-O-shikimate intermediate. Phenotypic and metabolic analyses of loss-of-function mutants show that PpHCT is necessary for the production of caffeate derivatives, including previously reported caffeoyl-threonate esters, and for the formation of an intact cuticle. Deep conservation of HCT function in embryophytes is further suggested by the ability of HCT genes from P. patens and the liverwort Marchantia polymorpha to complement an Arabidopsis thaliana CRISPR/Cas9 hct mutant, and by the presence of phenolic esters of shikimate in representative species of the three bryophyte lineages.

sentences: Theobroma grandiflorum breeding optimization based on repeatability, stability and adaptability information The cultivation of Theobroma grandiflorum in the Brazilian Amazon is mainly conducted by family farmers who use a range of different management strategies. Thus, breeding programs of the species must address the challenge of developing cultivars that are adapted to and stable in a variety of cultivation environments. In this context, this study aimed to estimate the optimum number of harvests for genetic selection of T. grandiflorum progenies and identify the most promising ones in terms of productivity, stability, and adaptability. The trials were implemented in three environments, using a randomized complete block design, with 25 full-sib progenies, five replications, and three plants per plot. The traits mean number of fruits/plant, mean fruit production/plant, and rate of infection with witches broom (Moniliophthora perniciosa) were evaluated over 11 harvests. The Restricted Maximum Likelihood/Best Linear Unbiased Prediction (REML/BLUP) mixed model method was used to estimate genetic parameters and predict genetic values, which were then applied to assess stability and adaptability. The results show that there is genetic variability among the studied T. grandiflorum progenies and that accurate genetic selection aiming at recombination is effective after three harvests, for recombination, or eleven harvests for identification of recommended progenies. Six progenies were selected that met the requirements for productivity, stability, and adaptability to different cultivation environments. These results can be used to optimize and advance T. grandiflorum breeding programs.

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Epigenetic and Transcriptional Dysregulation in CD4+ T cells of Patients with Atopic Dermatitis Atopic dermatitis (AD) is one of the most common skin disorders in children. Disease etiology involves genetic and environmental factors, with the 29 independent AD risk loci enriched for risk allele-dependent gene expression in the skin and CD4+ T cell compartments. We investigated epigenetic mechanisms that may account for genetic susceptibility in CD4+ T cells. To understand gene regulatory activity differences in peripheral blood T cells in AD, we measured chromatin accessibility (ATAC-seq), NFKB1 binding (ChIP-seq), and gene expression (RNA-seq) in stimulated CD4+ T cells from subjects with active moderate-to-severe AD and age-matched, non-allergic controls. Open chromatin regions in stimulated CD4+ T cells were highly enriched for AD genetic risk variants, with almost half of AD risk loci overlapping with AD-dependent ATAC-seq peaks. AD-specific open chromatin regions were strongly enriched for NF{kappa}B DNA binding motifs. ChIP-seq identified hundreds of NFKB1-occupied genomic loci that were AD-specific or Control-specific. As expected, the AD-specific ChIP-seq peaks were strongly enriched for NF{kappa}B DNA binding motifs. Surprisingly, Control-specific NKFB1 ChIP-seq peaks were not enriched for NFKB1 motifs, instead containing motifs for other classes of human TFs, suggesting a mechanism involving altered indirect NFKB1 binding. Using DNA sequencing data, we identified 63 instances of genotype-dependent chromatin accessibility at 36 AD risk variants (30% of AD risk loci) that could lead to genotype-dependent expression at these loci. We propose that CD4+ T cells respond to stimulation in an AD-specific manner, resulting in disease and genotype-dependent chromatin accessibility involving NFKB binding.

AUTHOR SUMMARYStimulated CD4+ T cells from patients with atopic dermatitis have disease-dependent regulation of how gene expression is regulated. This regulation is disease dependent and the way the DNA is accessible and the transcription factor NFKB1 binds is enriched for genetic risk variants. Clinically, the CD4+ T cells in the peripheral blood of patients with AD respond to stimulation in a disease and genotype-dependent manner.

sentences: The evolution of Sox gene repertoires and regulation of segmentation in arachnids The Sox family of transcription factors regulate many processes during metazoan development, including stem cell maintenance and nervous system specification. Characterising the repertoires and roles of these genes can therefore provide important insights into animal evolution and development. We further characterised the Sox repertoires of several arachnid species with and without an ancestral whole genome duplication (WGD), and compared their expression between the spider Parasteatoda tepidariorum and the harvestman Phalangium opilio. We also found that most Sox families have been retained as ohnologs after WGD and evidence for potential subfunctionalisation and/or neofunctionalization events. Our results also suggest that Sox21b-1 likely regulated segmentation ancestrally in arachnids, playing a similar role to the closely related SoxB gene, Dichaete, in insects. We previously showed that Sox21b-1 is required for the simultaneous formation of prosomal segments and sequential addition of opisthosomal segments in P. tepidariorum. We studied the expression and function of Sox21b-1 further in this spider and found that while this gene regulates the generation of both prosomal and opisthosomal segments, it plays different roles in the formation of these tagmata reflecting their contrasting modes of segmentation and deployment of gene regulatory networks with different architectures.

sentences: A Scalable, Open-Source Implementation of a Large-Scale Mechanistic Model for Single Cell Proliferation and Death Signaling Mechanistic models of how single cells respond to different perturbagens can help integrate disparate big data sets or predict response to varied drug combinations. However, the construction and simulation of such models have proved challenging. Our lab previously constructed one of the largest mechanistic models for single mammalian cell regulation of proliferation and death (774 species, 141 genes, 8 ligands, 2400 reactions). However, this, as many other large-scale models, was written using licensed software (MATLAB) with intricate programming structure, impeding alteration, expansion, and sharing. Here, we generated a new foundation for this model, which includes a python-based creation and simulation pipeline converting a few structured text files into an SBML-compatible format. This new open-source model (named SPARCED) is high-performance- and cloud-computing compatible and enables the study of virtual cell population responses at the single-cell level. We applied this new model to a subset of the LINCS MCF10A Data Cube, which observed that IFN{gamma} acts as an anti-proliferative factor, but the reasons why were unknown. After expanding the SPARCED model with an IFN{gamma} signaling module (to 950 species, 150 genes, 9 ligands, 2500 reactions), we ran stochastic single-cell simulations for two different putative crosstalk mechanisms and looked at the number of cycling cells in each case. Our model-based analysis suggested, and experiments support that these observations are better explained by IFN{gamma}-induced SOCS1 expression sequestering activated EGF receptors, thereby downregulating AKT activity, as opposed to direct IFN{gamma}-induced upregulation of p21 expression. This work forms a foundation for increased mechanistic model-based data integration on a single-cell level, an important building block for clinically predictive mechanistic models.

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Hexose translocation mediated by SlSWEET5b is required for pollen maturation in Solanum lycopersicum Pollen fertility is critical for successful fertilization and, accordingly, for crop yield. While sugar unloading affects growth and development of all types of sink organs, the molecular nature for sugar import to tomato pollen is poorly understood. However, SWEET transporters have been proposed to function in pollen development. Here, qRT-PCR revealed that SlSWEET5b was markedly expressed in flowers when compared to the remaining tomato SlSWEETs; particularly, in the stamens of maturing flower buds undergoing mitosis. Distinct accumulation of SlSWEET5b-GUS fusion proteins was present in mature flower buds, especially in anther vascular and inner cells, symplasmic isolated pollen cells and styles. The demonstration that GFP fusion proteins located to the plasma membrane support the idea that the SlSWEET5b carrier functions in apoplasmic sugar translocation during pollen maturation. Such function is in line with data from yeast complementation experiments and radiotracer uptakes, showing that SlSWEET5b operates as a low affinity hexose-specific passive facilitator, with a KM of ~36 mM. Most importantly, RNAi-mediated suppression of SlSWEET5b expression resulted in shrunken nucleus-less pollen cells, impaired germination and low seed yield. Interestingly, stamens from SlSWEET5b-silenced tomato mutants contained significantly lower amounts of sucrose and increased invertase activity, pointing to reduced carbon supply and perturbed sucrose homeostasis in this tissue. Taken together, our findings reveal an essential role of SlSWEET5b in mediating apoplasmic hexose import into phloem unloading cells and into developing pollen cells to support pollen mitosis and maturation in tomato flowers.

One-sentence SummaryPlasma-membrane-localized SlSWEET5b facilitates a sequential hexose flux, from phloem to anther cells and from anther locule to pollen, to support pollen maturation and fertility in tomato flowers.

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Probabilistic quotient's work and pharmacokinetics' contribution: countering size effect in metabolic time series measurements Metabolomic time course analyses of biofluids are highly relevant for clinical diagnostics. However, many sampling methods suffer from unknown sample sizes commonly known as size effects. This prevents absolute quantification of biomarkers. Recently, several mathematical post acquisition normalization methods have been developed to overcome these problems either by exploiting already known pharmacokinetic information or by statistical means.

Here we present an improved normalization method, MIX, that combines the advantages of both approaches. It couples two normalization terms, one based on a pharmacokinetic model (PKM) and the other representing a popular statistical approach, probabilistic quotient normalization (PQN), in a single model.

To test the performance of MIX, we generated synthetic data closely resembling real finger sweat metabolome measurements. We show that MIX normalization successfully tackles key weaknesses of the individual strategies: it (i) reduces the risk of overfitting with PKM, and (ii) contrary to PQN, it allows to compute sample volumes. Finally, we validate MIX by using real finger sweat as well as blood plasma metabolome data and demonstrate that MIX allows to better and more robustly correct for size effects.

In conclusion, the MIX method improves the reliability and robustness of quantitative biomarker detection in finger sweat and other biofluids, paving the way for quantitative biomarker discovery and hypothesis generation from metabolomic time course data.

sentences: Time scale separation of information processing between sensory and associative regions A hierarchy of local timescales with a back (sensory)-to-front (prefrontal) gradient reflects brain region specialization. However, cognitive processes emerge from the coordinated activity across regions, and the corresponding timescales should refer to the interactions rather than to regional activity. Using edgewise connectivity on magnetoencephalography signals, we demonstrate a reverse front-to-back gradient when non-local interactions are prominent. Thus, the timescales are dynamic and reconfigure between back-to-front and front-to-back patterns.

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Metagenomic identification of viral sequences in laboratory reagents 2.Metagenomic next-generation sequencing has transformed the discovery and diagnosis of infectious disease, with the power to characterize the complete  infectome (bacteria, viruses, fungi, parasites) of an individual host organism. However, the identification of novel pathogens has been complicated by widespread microbial contamination in commonly used laboratory reagents. Using total RNA sequencing ("metatranscriptomics") we documented the presence of contaminant viral sequences in multiple libraries of  blank negative control sequencing libraries that comprise a sterile water and reagent mix. Accordingly, we identified 14 viral sequences in 7 negative control sequencing libraries. As in previous studies, several circular replication-associated protein encoding (CRESS) DNA virus-like sequences were recovered in the blank libraries, as well as contaminating sequences from the RNA virus families Totiviridae, Tombusviridae and Lentiviridae. These data suggest that the contamination of common laboratory reagents is likely widespread and can comprise a wide variety of viruses.

3. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

1.5 RepositoriesThe viral genome sequence data generated in this study has been deposited in the NCBI database under accession numbers MZ824225-MZ824237. Sequence reads are available at the public Sequence Read Archive (SRA) database with accession SRX6803604 and under the BioProject accession PRJNA735051 reference numbers SRR14737466-71 and BioSample numbers SAMN20355437-40.

sentences: Identification of enhancer regulatory elements that direct epicardial gene expression during zebrafish heart regeneration The epicardium is a mesothelial tissue layer that envelops the heart. Cardiac injury activates dynamic gene expression programs in epicardial tissue, which in the case of zebrafish enables subsequent regeneration through paracrine and vascularizing effects. To identify tissue regeneration enhancer elements (TREEs) that control injury-induced epicardial gene expression during heart regeneration, we profiled transcriptomes and chromatin accessibility in epicardial cells purified from regenerating zebrafish hearts. We identified hundreds of candidate TREEs, defined by increased chromatin accessibility of non-coding elements near genes with increased expression during regeneration. Several of these candidate TREEs were incorporated into stable transgenic lines, with 5 of 6 elements directing injury-induced epicardial expression but not ontogenetic epicardial expression in hearts of larval animals. Whereas two independent TREEs linked to the gene gnai3 showed similar functional features of gene regulation in transgenic lines, two independent ncam1a-linked TREEs directed distinct spatiotemporal domains of epicardial gene expression. Thus, multiple TREEs linked to a regeneration gene can possess either matching or complementary regulatory controls. Our study provides a new resource and principles for understanding the regulation of epicardial genetic programs during heart regeneration.

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Microscope-Cockpit: Python-based bespoke microscopy for bio-medical science We have developed "Microscope-Cockpit" (Cockpit), a highly adaptable open source user-friendly Python-based GUI environment for precision control of both simple and elaborate bespoke microscope systems. The user environment allows next-generation near-instantaneous navigation of the entire slide landscape for efficient selection of specimens of interest and automated acquisition without the use of eyepieces. Cockpit uses "Python-Microscope" (Microscope) for high-performance coordinated control of a wide range of hardware devices using open source software. Microscope also controls complex hardware devices such as deformable mirrors for aberration correction and spatial light modulators for structured illumination via abstracted device models. We demonstrate the advantages of the Cockpit platform using several bespoke microscopes, including a simple widefield system and a complex system with adaptive optics and structured illumination. A key strength of Cockpit is its use of Python, which means that any microscope built with Cockpit is ready for future customisation by simply adding new libraries, for example machine learning algorithms to enable automated microscopy decision making while imaging.

HighlightsO_LIUser-friendly setup and use for simple to complex bespoke microscopes.
C_LIO_LIFacilitates collaborations between biomedical scientists and microscope technologists.
C_LIO_LITouchscreen for near-instantaneous navigation of specimen landscape.
C_LIO_LIUses Python-Microscope, for abstracted open source hardware device control.
C_LIO_LIWell-suited for user training of AI-algorithms for automated microscopy.
C_LI

sentences: Different brain systems support the aversive and appetitive sides of human pain-avoidance learning Both unexpected pain and unexpected pain absence can drive avoidance learning, but whether they do so via shared or separate neural and neurochemical systems is largely unknown. To address this issue, we combined an instrumental pain-avoidance learning task with computational modeling, functional magnetic resonance imaging (fMRI) and pharmacological manipulations of the dopaminergic (100 mg levodopa) and opioidergic (50 mg naltrexone) systems (N=83). Computational modeling provided evidence that untreated participants learned more from received than avoided pain. Our dopamine and opioid manipulations negated this learning asymmetry by selectively increasing learning rates for avoided pain. Furthermore, our fMRI analyses revealed that pain prediction errors were encoded in subcortical and limbic brain regions, whereas no-pain prediction errors were encoded in frontal and parietal cortical regions. However, we found no effects of our pharmacological manipulations on the neural encoding of prediction errors. Together, our results suggest that human pain-avoidance learning is supported by separate threat- and safety-learning systems, and that dopamine and endogenous opioids specifically regulate learning from successfully avoided pain.

sentences: Finding structure in time: Humans, machines, and language The emergence of artificial neural networks (ANNs) that seem capable of emulating the remarkable human capacity for language has raised fundamental questions about complex cognition in humans and machines. This debate has taken place, however, in the context of limited empirical evidence about how the internal operations of ANNs relate to dynamic processes in the human brain as listeners understand language. Using Representational Similarity Analysis, we conducted a set of in-depth comparisons between these two types of systems, focusing on a core aspect of human language - the building of a structured and meaningful interpretation as each word in a sentence is heard in sequence. We extracted incremental structural measures of unfolding sentences from a deep language ANN and tested these against the spatiotemporally resolved brain activity recorded by electro/magnetoencephalography when human participants were listening to the same sentences. These uniquely neurocomputationally specific comparisons revealed strong behavioral and neural alignments between humans and ANNs in the use of multi-dimensional probabilistic constraints to build word-by-word structural interpretations, suggesting important commonalities in basic computational strategies for finding structure in time.

sentences: A rapid ecological assessment for necrophagous flies (Diptera, Calyptratae) in a mosaic landscape of the Colombian Andes A necrophagous flies ensemble (Diptera, Calyptratae) was rapidly assessed in four coverages of an anthropogenic landscape in the east range of the Colombian Andes. Ninetyseven individuals belonging to eight species were collected in only two hours of sampling. The highest diversity values and the occurrence of asynanthropic flies in the forest under conservation management may reflect a positively natural restoration process in the area assessed. Grassland, rural and urban coverages were similarly low in diversity and composition. A brief discussion about the flies bionomy and their environment association is offered. Necrophagous flies ensembles by coverage behave as an indicator of the anthropic impact on the landscape.

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Spatial Patterns of Microbial Diversity, Composition and Community Structure in Fe-Mn Deposits and Associated Sediments in the Atlantic and Pacific Oceans Mining of deep-sea Fe-Mn deposits will remove crusts and nodules in large areas from the seafloor. The growth of a few millimeters of these minerals by Fe and Mn oxides precipitation takes millions of years, and yet little is known about their microbiome. Besides being key elements of the biogeochemical cycles and essential links of food and energy to deep-sea trophic webs, microbes have been identified to affect manganese oxide formation. Hence, polymetallic crusts and nodules may present unique habitats that deserve better understanding. In this study, we determined the composition and diversity of Bacteria and Archaea in deep-sea Fe-Mn crusts, nodules, and associated sediments from two oceanic elevations in the Atlantic Ocean, the Tropic Seamount in the northeast and the Rio Grande Rise (RGR) in the southwest. Sequencing of the 16S rRNA gene was performed using the Illumina MiSeq platform and statistical analyses using environmental data were performed in R. Additionally, we included public domain environmental DNA data of Fe-Mn crusts, nodules, and associated sediments from Clarion-Clipperton Zone and Takuyo-Daigo Seamount in the Pacific Ocean to compare microbial diversity in Fe-Mn deposits from different ocean basins. Our results indicated that Atlantic seamounts harbor an unusual and unknown Fe-Mn deposit microbiome with lower diversity and richness compared to deposits from Pacific areas. Crusts and nodules from Atlantic seamounts revealed the presence of unique taxa (Alteromonadales, Nitrospira, and Magnetospiraceae) and a higher relative abundance of sequences related to potential metal-cycling bacteria, such as Betaproteobacteriales and Pseudomonadales. The microbial beta-diversity from Atlantic seamounts was clearly grouped into microhabitats according to crusts, nodules, and sediments geochemical composition. Furthermore, community structure analysis using principal coordinate analysis also showed that the microbial communities of all seamounts were significantly divided into ocean basins and sampling areas. Despite the time scale of million years for these deposits to grow, a combination of environmental settings (temperature, salinity, depth, substrate geochemistry, nutrient, and organic matter availability) played a significant role in shaping the crusts and nodules microbiome, which was distinct between the Atlantic and Pacific Fe-Mn deposits. Our results suggest that the microbial community inhabiting Fe-Mn deposits participate in biogeochemical reactions indispensable to deep-sea ecosystems, which implies that understanding the microbial community is of utmost importance for any baseline environmental study in areas of potential deep-sea mining.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/485154v1_ufig1.gif" ALT="Figure 1">
View larger version (49K):
org.highwire.dtl.DTLVardef@1214ccorg.highwire.dtl.DTLVardef@1139796org.highwire.dtl.DTLVardef@144f967org.highwire.dtl.DTLVardef@151f069_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIAtlantic deposits showed lower diversity and richness compared to Pacific deposits
C_LIO_LIFe-Mn crusts and nodules are a potential specific ecological niche
C_LIO_LIAtlantic Fe-Mn deposits harbor an unusual and unknown microbiome
C_LIO_LITemperature, salinity, depth, and substrate geochemistry at Atlantic Fe-Mn deposits may drive community composition
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sentences: Identification of differentially distributed gene expression and distinct sets of cancer-related genes identified by changes in expression mean and variability There is increasing evidence that changes in the variability or overall distribution of gene expression are important both in normal biology and in diseases, particularly cancer. Genes whose expression differs in variability or distribution without a difference in mean are ignored by traditional differential expression-based analyses. Using a Bayesian hierarchical model that provides tests for both differential variability and differential distribution for bulk RNA-seq data, we report here an investigation into differential variability and distribution in cancer. Analysis of eight paired tumour-normal datasets from The Cancer Genome Atlas confirms that differential variability and distribution are able to identify cancer-related genes. We further demonstrate that differential variability identifies cancer-related genes that are missed by differential expression analysis, and that differential expression and differential variability identify functionally distinct sets of genes. These results suggest that differential variability analysis may provide insights into genetic aspects of cancer that would not be revealed by differential expression, and that differential distribution analysis may allow for more comprehensive identification of cancer-related genes than analyses based on changes in mean or variability alone.

sentences: Root-specific reduction of cytokinin perception enhances shoot growth with an alteration of trans-zeatin distribution in Arabidopsis thaliana Compelling evidence demonstrates that root-derived cytokinins (CKs) contribute to shoot growth via long-distance transport; therefore, we hypothesized that an increase in root-derived CKs enhances shoot growth. To demonstrate this, we grafted Arabidopsis Col-0 (WT) scion onto rootstock originated from WT or a double-knockout line of CK receptors AHK2 and AHK3 (ahk23) because the knockout line over accumulates CKs in the body due to feedback homeostasis regulation. The grafted plants (scion/rootstock: WT/WT and WT/ahk23) were grown in vermiculite pots or solid media under high and low nitrogen regimes for vegetative growth and biochemical analysis. The root-specific deficiency of AHK2 and AHK3 increased root concentrations of trans-zeatin (tZ)-type and N6-({Delta}2-isopentenyl) adenine (iP)-type CKs, induced CK biosynthesis genes, and repressed CK degradation genes in the root. Shoot growth, shoot concentrations of tZ-type CKs, and shoot expression of CK-inducible marker genes were consistently larger in the WT/ahk23 plants than in the WT/WT plants. Moreover, the root-specific deficiency of AHK2 and AHK3 enhanced shoot growth in the WT scion more strongly than in the ahk23 scion. Given that tZ-type CKs are predominantly produced from iP-type CKs in the root and xylem-mobile, it is concluded that the root-specific reduction of CK perception would enhance shoot growth by increasing the amount of root-derived tZ-type CKs and their perception by the shoot. This study will present a novel approach to improve plant growth and productivity.

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Immune Regulation of Intestinal Stem Cell Proliferation and Differentiation in Drosophila. Intestinal progenitor cells integrate signals from their niche, and from the gut lumen, to divide and differentiate at a rate that maintains an epithelial barrier to microbial invasion of the host interior. Despite the importance of evolutionarily conserved innate immune defenses to maintain stable host-microbiota relationships, we know little about specific contributions of stem cell immunity to gut homeostasis. We used the Drosophila model to determine the consequences of compromised intestinal stem cell immune activity for epithelial homeostasis. We showed that loss of stem cell immunity greatly impacted growth and renewal in the adult gut. In particular, we noticed that inhibition of stem cell immunity impeded key growth and differentiation events in the progenitor cell compartment leading to a gradual loss of stem cell numbers with age, and an impaired differentiation of mature enteroendocrine cells. Our results highlight the importance of immune signaling in the stem cell population for epithelial function in the adult gut.

HIGHLIGHTSO_LIThe TNFR-like Immune Deficiency (IMD) pathway is active in Drosophila intestinal progenitor cells.
C_LIO_LIInhibition of IMD in progenitors impairs progenitor cell proliferation.
C_LIO_LIBlocking progenitor cell IMD negatively affects generation of mature epithelial cells.
C_LI

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metapredict: a fast, accurate, and easy-to-use cross-platform predictor of consensus disorder Intrinsically disordered proteins and protein regions make up a substantial fraction of many proteomes where they play a wide variety of essential roles. A critical first step in understanding the role of disordered protein regions in biological function is to identify those disordered regions correctly. Computational methods for disorder prediction have emerged as a core set of tools to guide experiments, interpret results, and develop hypotheses. Given the multiple different predictors available, consensus scores have emerged as a popular approach to mitigate biases or limitations of any single method. Consensus scores integrate the outcome of multiple independent disorder predictors and provide a per-residue value that reflects the number of tools that predict a residue to be disordered. Although consensus scores help mitigate the inherent problems of using any single disorder predictor, they are computationally expensive to generate. They also necessitate the installation of multiple different software tools, which can be prohibitively difficult. To address this challenge, we developed a deep-learning-based predictor of consensus disorder scores. Our predictor, metapredict, utilizes a bidirectional recurrent neural network trained on the consensus disorder scores from 12 proteomes. By benchmarking metapredict using two orthogonal approaches, we found that metapredict is among the most accurate disorder predictors currently available. Metapredict is also remarkably fast, enabling proteome-scale disorder prediction in minutes. Importantly, metapredict is fully open source and is distributed as a Python package, a collection of command-line tools, and a web server, maximizing the potential practical utility of the predictor. We believe metapredict offers a convenient, accessible, accurate, and high-performance predictor for single-proteins and proteomes alike.

Statement of SignificanceIntrinsically disordered regions are found across all kingdoms of life where they play a variety of essential roles. Being able to accurately and quickly identify disordered regions in proteins using just the amino acid sequence is critical for the appropriate design and interpretation of experiments. Despite this, performing large-scale disorder prediction on thousands of sequences is challenging using extant disorder predictors due to various difficulties including general installation and computational requirements. We have developed an accurate, high-performance and easy-to-use predictor of protein disorder and structure. Our predictor, metapredict, was designed for both proteome-scale analysis and individual sequence predictions alike. Metapredict is implemented as a collection of local tools and an online web server, and is appropriate for both seasoned computational biologists and novices alike.

sentences: Development of a machine learning model to estimate biotic ligand model-based predicted no-effect concentrations for copper in freshwater The copper biotic ligand model (BLM) has been used for environmental risk assessment by taking into account the bioavailability of copper in freshwater. However, the BLM-based environmental risk of copper has been assessed only in Europe and North America, with monitoring datasets containing all of the BLM input variables. For other areas, it is necessary to apply surrogate tools with reduced data requirements to estimate the BLM-based predicted no-effect concentration (PNEC) from commonly available monitoring datasets. To develop an optimized PNEC estimation model based on an available monitoring dataset, an initial model that considers all BLM variables, a second model that requires variables excluding alkalinity, and a third model using electrical conductivity as a surrogate of the major cations and alkalinity have been proposed. Furthermore, deep neural network (DNN) models have been used to predict the nonlinear relationships between the PNEC (outcome variable) and the required input variables (explanatory variables). The predictive capacity of DNN models in this study was compared with the results of other existing PNEC estimation tools using a look-up table and multiple linear and multivariate polynomial regression methods. Three DNN models, using different input variables, provided better predictions of the copper PNECs compared with the existing tools for four test datasets, i.e., Korean, United States, Swedish, and Belgian freshwaters. The adjusted r2 values in all DNN models were higher than 0.95 in the test datasets, except for the Swedish dataset (adjusted r2 > 0.87). Consequently, the most applicable model among the three DNN models could be selected according to the data availability in the collected monitoring database. Because the most simplified DNN model required only three water quality variables (pH, dissolved organic carbon, and electrical conductivity) as input variables, it is expected that the copper BLM-based risk assessment can be applied to monitoring datasets worldwide.

sentences: Understanding SARS-CoV-2 budding through molecular dynamics simulations of M and E protein complexes SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratorys refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics which inhibit coronavirus budding.

sentences: Bile Salts and N-Acetyl-D-Glucosamine induce transcription of the vieSAB Operon in Vibrio cholerae El Tor The vieSAB operon regulates motility, biofilm formation and cholera toxin production in the classical biotype of V. cholerae. Classical biotype mutants lacking the cyclic-di-GMP phosphodiesterase and response regulator vieA or its cognate sensor kinase vieS fail to effectively colonize the infant mouse small intestine, are less motile than the wild-type strain, and are hyper-biofilm formers. However, deletion of vieA or vieS in the currently-circulating El Tor biotype does not have a demonstrable effect on these phenotypes. To begin to understand the role of vieSAB in the El Tor biotype, we studied its regulation and the effect of vieA expression on bacterial physiology, separating the functional roles of VieAs enzymatic and DNA-binding activities. We identify bile salts and N-acetylglucosamine, which can be found at high concentration in the small intestine, as stimulating expression of vieSAB during in vitro growth. We show that, in contrast to many response regulators, VieA does not regulate its own expression. Together, these data indicate that V. cholerae upregulates vieSAB in response to small-intestinal signals, allowing VieA to alter the physiology of the cell through its transcriptional regulation and cyclic-di-GMP signaling activities in order to adapt to the human host.

sentences: Exosomal MFGE8 from high glucose induced endothelial cells is involved in calcification/senescence of vascular smooth muscle cells Vascular calcification/aging is a crucial feature of diabetic macro vasculopathy, resulting in serious cardiovascular diseases. The calcification/senescence of vascular smooth muscle cells (VSMCs) induced by hyperglycemia can cause diabetic vascular calcification/aging. However, the mechanism of VSMCs calcification/senescence involved in diabetic vascular calcification/aging remains unknown. The purpose of this study was to determine how the high glucose (HG) information in circulating blood is transmitted from vascular endothelial cells (ECs) to VSMCs, which are not contacted with blood directly. Exosomes have attracted much attention for their vital roles in regulating cell-to-cell communication. In this study, we found that milk fat globule epidermal growth factor 8 (MFGE8) was enriched in high glucose induced human umbilical vein endothelial cell exosomes (HG-HUVEC-Exo) and regulate VSMCs calcification/senescence, characterized by up-regulated expressions of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (Runx2), as well as the increased mineralized nodules and senescence-associated {beta}-galactosidase (SA-{beta}-gal) positive cells. Upstream mechanism studies showed that sirtuin1 (SIRT1) was involved in VSMCs calcification/senescence by affecting the expression of MFGE8. We also found that inflammatory response mediated by IL-1{beta}, IL-6, and IL-8 was closely associated with MFGE8 and played a key role in regulating HG-HUVEC-Exo-induced VSMCs calcification/senescence. These findings provide a new insight into the mechanism of exosomal MFGE8 as a potential preventive and therapeutic target for the intervention of diabetic vascular calcification/aging.

sentences: Petascale neural circuit reconstruction: automated methods 3D electron microscopy (EM) has been successful at mapping invertebrate nervous systems, but the approach has been limited to small chunks of mammalian brains. To scale up to larger volumes, we have built a computational pipeline for processing petascale image datasets acquired by serial section EM, a popular form of 3D EM. The pipeline employs convolutional nets to compute the nonsmooth transformations required to align images of serial sections containing numerous cracks and folds, detect neuronal boundaries, label voxels as axon, dendrite, soma, and other semantic categories, and detect synapses and assign them to presynaptic and postsynaptic segments. The output of neuronal boundary detection is segmented by mean affinity agglomeration with semantic and size constraints. Pipeline operations are implemented by leveraging distributed and cloud computing. Intermediate results of the pipeline are held in cloud storage, and can be effortlessly viewed as images, which aids debugging. We applied the pipeline to create an automated reconstruction of an EM image volume spanning four visual cortical areas of a mouse brain. Code for the pipeline is publicly available, as is the reconstructed volume.

sentences: UVC inactivation of pathogenic samples suitable for cryoEM analysis Cryo-electron microscopy has become an essential tool to understand structure and function of biological samples, from individual proteins to whole cells. Especially for pathogens, such as disease-causing bacteria and viruses, insights gained by cryo-EM can aid in developing cures. However, due to the biosafety restrictions of human pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the delicate ultrastructure of the sample. Alternatively, researchers use in vitro or ex vivo models, which are non-pathogenic but lack the complexity of the pathogen of interest. Here we show that ultraviolet-C (UVC) radiation at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of two model organisms, a pathogenic bacterium (Vibrio cholerae) and a virus-like particle (the ICP1 bacteriophage). We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography (cryo-ET) followed by sub-tomogram averaging (STA), and single particle analysis (SPA). Additionally, we applied the UVC irradiation to the protein apoferritin (ApoF), which is a widely used test sample for high resolution SPA studies. The UVC-treated ApoF sample resulted in a 2.1 [A] structure that did not reveal any discernable structural damage. Together, these results show that the UVC irradiation dose that effectively inactivates cryo-EM samples does not negatively impact their structure. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.

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The use of Ipratropium and Tiotropium as novel agents to reduce inflammation in in vitro macrophage models. BackgroundChronic Obstructive Pulmonary Disease (COPD) affects an estimated 330 million individuals worldwide. Approximately, 3 million individuals died of COPD in 2012 and it is predicted that COPD would be the third leading factor for deaths worldwide by 2020. In United Kingdom nearly one million individuals suffer from COPD.

PurposeThere are no effective pharmacotherapies available for COPD. it is only managed by using bronchodilators and inhaled corticosteroids mostly. However, cardiovascular effects are associated with these drugs. Most importantly, there is an unmet need of COPD treatment worldwide. Our research aim was to identify Ipratropium and Tiotropium as novel anti-inflammatory agents in in vitro macrophage models.

AimsTo investigate the LPS stimulated pro-inflammatory cytokines IL-6 and TNF-  levels in THP-1 cells. To investigate whether the drugs Ipratropium and Tiotropium are capable of decreasing LPS-induced inflammation in THP-1 cells.

MaterialsHuman monocytic cell line THP-1 cells, Rosewell Park Memorial Institute RPMI 1640 with Glutamax I, 1% Penicillin Streptomycin (PenStrep) and 10% foetal bovine serum (FBS), Lipopolysaccharide 10l/ml, 0.05% Tween20, 0.4% Trypan blue, Reagent diluent (10% Bovine Serum Albumin in PBS), Budesonide Fenoterol, Ipratropium and Tiotropium. Human IL-6 DuoSet ELISA, Human TNF- ELISA, TMB ELISA Substrate solution and Stop solution.

MethodsTHP-1 cells were cultured and challenges with LPS to stimulate the IL-6 and TNF- cytokines. The cells were treated with Budesonide, Fenoterol, Ipratropium and Tiotropium. ELISA was performed to determine the concentrations of cytokines.

ResultsThe results suggested that Ipratropium and Tiotropium reduce IL-6 and TNF-  concentrations in the cells. However, Budesonide and Fenoterol were found to reduce cytokines more effectively than Ipratropium and Tiotropium. The data was considered significant only when P <0.05.

ConclusionsThe anti-inflammatory or cytokine reducing properties of Ipratropium and Tiotropium were acknowledged. The research hypothesis was found to be true. Budesonide and Fenoterol substantially reduce cytokine levels. The receptor interactions of Ipratropium and Tiotropium may be responsible for their duration of action. Overall, Ipratropium and Tiotropium display the characteristics of novel anti-inflammatories.

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Inference of an integrative network for Rheumatoid Arthritis combining data-driven machine learning approaches and a state-of-the-art mechanistic disease map Rheumatoid arthritis (RA) is a multifactorial, complex autoimmune disease that involves various genetic, environmental and epigenetic factors. Systems biology approaches provide the means to study complex diseases through the integration of different layers of biological information. Combining multiple data types can help compensate for missing or conflicting information and limit the possibility of false positives. In this work, we aim to unravel mechanisms governing the regulation of key transcription factors in RA and derive patient-specific models to gain more insights into the disease heterogeneity and the response to treatment.

We first make use of publicly available transcriptomic datasets (peripheral blood) relative to RA and machine learning to create an RA specific transcription factor (TF) co-regulatory network. The TF cooperativity network is subsequently enriched in signalling cascades and upstream regulators using a state-of-the-art, RA-specific molecular map. The integrative network is then used as a template to analyze patients data regarding their response to anti-TNF treatment and identify master regulators and upstream cascades affected by the treatment. We use the Boolean formalism to simulate in silico subparts of the integrated network and identify combinations and conditions that can switch on or off the identified TFs, mimicking the effects of single and combined perturbations.

sentences: BRAIN-BASED AUTHENTICATION: TOWARDS A SCALABLE, COMMERCIAL GRADE SOLUTION USING NONINVASIVE BRAIN SIGNALS. Here we report on a field test where we asked if it is feasible to deliver a scalable, commercial-grade solution for brain-based authentication given currently available head wearables. In this study, forty-nine (49) participants completed multiple sessions in their natural home environment over a single week. Participants used an off-the-shelf brain signal measuring headband to record their own brain activity while completing various tasks. Recording sessions were self-operated by the participants and unsupervised by any expert or technician to simulate real world use cases, while also contrasting common research approaches to this topic that rely on data from controlled laboratory conditions. Although brain signals have a non-stationary, complex nature, when participants watched rapidly presented images, our authentication system was able to successfully construct a unique and robust "brain ID" for each participant. Based on this brain ID, we developed a simplified brain-based authentication method that captures distinguishable information with reliable, commercial-grade performance from participants at their own homes. We conclude that noninvasively measured brain signals are ideal for use in biometric authentication systems, especially in environments where head wearables such as headphones or AR/VR devices are used as these devices offer a natural form factor for capturing participant brain ID continuously.

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Operational and biochemical aspects of co-digestion (co-AD) from sugarcane vinasse, filter cake and deacetylation liquor This work performed co-AD from the vinasse and filter cake (from 1G ethanol production) and deacetylation liquor (from the pre-treatment of sugarcane straw for 2G ethanol production) in a semi-Continuous Stirred Tank Reactor (s-CSTR) aiming to provide optimum operational parameters for continuous CH4 production. Using filter cake as co-substrate may allow the reactor to operate throughout the year, as it is available in the sugarcane off-season, unlike vinasse. A comparison was made from the microbial community of the seed sludge and the reactor sludge when CH4 production stabilized. Lactate, butyrate and propionate fermentation routes were denoted at the start-up of the s-CSTR, characterizing the acidogenic phase: the Oxidation-Reduction Potential (ORP) values ranged from -800 to -100 mV. Once the methanogenesis was initiated, alkalizing addition was no longer needed as its demand by the microrganisms was supplied by the alkali-characteriscs of the deacetylation liquor. The gradual increase of the applied Organic Load Rates (OLR) allowed stabilization of the methanogenesis from 3.20 gVS L-1 day-1: the highest CH4 yield (230 NmLCH4 gSV-1) and average organic matter removal efficiency (83% {+/-} 13) was achieved at ORL of 4.16 gVS L-1day-1. The microbial community changed along the reactor operation, presenting different metabolic routes mainly due to the used lignocellulosic substrates. Bacteria from the syntrophic acetate oxidation (SAO) process coupled to hydrogenotrophic methanogenesis were predominant ([~] 2% Methanoculleus) during the CH4 production stability. The overall results are useful as preliminary drivers in terms of visualizing the co-AD process in a sugarcane biorefinery integrated to scale.

KeypoitnsIntegration of 1G2G sugarcane ethanol biorefinery from co-digestion of its residues Biogas production from vinasse, filter cake and deacetylation liquor in a semi-CSTR Lignicellulosic substrates affected the biochemical routes and microbial community Biomol confirmed the stablismenht of thermophilic community from mesophilic sludge

sentences: The unfolded protein response triggers the immune deficiency pathway in ticks The insect immune deficiency (IMD) pathway is a defense mechanism that senses and responds to Gram negative bacteria. Ticks lack genes encoding upstream components that initiate the IMD pathway. Despite this deficiency, core signaling molecules are present and functionally restrict tick-borne pathogens. The molecular events preceding activation remain undefined. Here, we show that the Unfolded Protein Response (UPR) initiates the IMD network in Ixodes scapularis ticks. The endoplasmic reticulum (ER) stress receptor, IRE1, is phosphorylated in response to tick-borne bacteria, but does not splice the mRNA encoding XBP1. Instead, through protein modeling and reciprocal pulldowns, we show that Ixodes IRE1 complexes with TRAF2. Disrupting IRE1-TRAF2 signaling blocks IMD pathway activation and diminishes the production of reactive oxygen species. Through in vitro, in vivo, and ex vivo techniques we demonstrate that the UPR-IMD pathway circuitry limits the Lyme disease-causing spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale (anaplasmosis). Altogether, our study uncovers a novel linkage between the UPR and the IMD pathway in ticks.

sentences: A human monoclonal antibody potently pan-neutralizes SARS-CoV-2 VOCs by targeting RBD invariant sites The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel corona virus disease (COVID-19). The neutralizing monoclonal antibodies (mAbs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are among the most promising strategies to prevent and treat COVID-19. However, SARS-CoV-2 variants of concern (VOCs) profoundly reduced the efficacies of most of mAbs and vaccines approved for clinical use. Herein, we demonstrated mAb 35B5 efficiently neutralizes both wild-type (WT) SARS-CoV-2 and VOCs, including B.1.617.2 (delta) variant, in vitro and in vivo. Cryo-electron microscopy (cryo-EM) revealed that 35B5 neutralizes SARS-CoV-2 by targeting a unique epitope that avoids the prevailing mutation sites on RBD identified in circulating VOCs, providing the molecular basis for its pan-neutralizing efficacy. The 35B5-binding epitope could also be exploited for the rational design of a universal SARS-CoV-2 vaccine.

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A novel hidden Markov approach to studying dynamic functional connectivity states in human neuroimaging IntroductionHidden Markov models are a popular choice to extract and examine recurring patterns of activity or functional connectivity in neuroimaging data, both in terms of spatial patterns and their temporal progression. Although many diverse hidden Markov models have been applied to neuroimaging data, most have defined states based on activity levels (intensity-based states) rather than patterns of functional connectivity between brain areas (connectivity-based states), which is problematic if we want to understand connectivity dynamics: intensity-based states are unlikely to provide useful information about dynamic connectivity patterns.

MethodsWe addressed this problem by introducing a new hidden Markov model that defines states based on full functional connectivity profiles among brain regions. We empirically explored the behavior of this new model in comparison to existing approaches based on intensity-based or summed functional connectivity states using the HCP unrelated 100 functional magnetic resonance imaging "resting state" dataset.

ResultsOur  full functional connectivity model discovered connectivity states with more distinguishable patterns than previous approaches, and recovered simulated connectivity-based states more faithfully.

DiscussionThus, if our goal is to extract and interpret connectivity states in neuroimaging data, our new model outperforms previous methods which miss crucial information about the evolution of functional connectivity in the brain.

Impact statementHidden Markov models can be used to investigate brain states noninvasively. Previous models "recover" connectivity from intensity-based hidden states, or from connectivity  summed across nodes. Here we introduce a novel connectivity-based hidden Markov model and show how it can reveal true connectivity hidden states under minimal assumptions.

sentences: An age-depth model and revised stratigraphy of vertebrate-bearing units in Natural Trap Cave, Wyoming Almost a half-century ago excavations at Natural Trap Cave (NTC) began to yield evidence of the steppe paleoecology along the western slope of the Bighorn Mountains in north central Wyoming. The first decade of fieldwork led to the discovery of a diverse fauna that existed at the end of the Last Glacial Maximum. Stratigraphic deposits below the entrance of the cave were studied soon after excavations began, but never formally published. Although stratigraphy, taphonomy, and depositional circumstances were briefly discussed over the following years, little has been done to correlate the numerous stratigraphic schemes used by various authors. In this study, four stratigraphic sections were measured and analysed to establish an easily modifiable lithostratigraphic system of nomenclature. We provide the first correlations of all stratigraphic nomenclature used throughout excavations at NTC to facilitate comparisons with current and previous collections and publications. By leveraging more than 100 radioisotopic dates we developed an age-depth model and chronostratigraphic framework to further interrogate spatiotemporal relationships between strata, paleoenvironmental proxies, and fossil assemblages. Deposition is shown to be discontinuous; sediment accumulation in the study area is restricted to the buildup through peak penultimate and Last Glacial maxima. More recent (<10 ka) Holocene deposits unconformably cover the eroded surface of underlying Pleistocene strata. There is active reworking of sediments with transport and deposition of reactivated sediments within the Lower Chamber. We note that the two hiatuses coincide with interglacial periods and may reflect changing depositional circumstances within the cave such as extended periods of non-deposition, erosion, or bypass (possibly leading to deposition in the Lower Chamber). Contrary to previous reports, we demonstrate that it is unlikely a prominent snow cone existed or contributed to the pattern of sediment and fossil distribution within the study area, furthermore, we do not observe a continuous Pleistocene-Holocene transition in the study area. Further stratigraphic work will be needed to better understand the interrelationship between Main and Lower chamber deposits and the evolution of sediment accumulation in NTC.

sentences: Conjugation of palbociclib with MHI-148 has an increased cytotoxic effect for breast cancer cells and an altered mechanism of action The CDK4/6 inhibitor palbociclib, combined with endocrine therapy, has been shown to be effective in postmenopausal women with estrogen receptor positive, HER2-negative advanced or metastatic breast cancer. However, palbociclib is not as effective in the highly aggressive triple-negative breast cancer that lacks sensitivity to chemotherapy or endocrine therapy. We hypothesized that conjugation of the near-infrared dye MHI-148 with palbociclib can produce a potential theranostic in triple-negative as well as estrogen receptor positive breast cancer cells. In our study, the conjugate was found to have enhanced activity in all mammalian cell lines tested in vitro. However, the conjugate was cytotoxic and did not induce G1 cell cycle arrest in breast cancer cells suggesting the mechanism of action differed from the parent compound palbociclib. The study highlights the importance of investigating the mechanism of conjugates of near-infrared dyes to therapeutic compounds as conjugation can potentially result in a change of mechanism or target, with an enhanced cytotoxic effect in this case.

sentences: Cognitive process modeling addresses context independence violations in the ABCD Study stop-signal task The Adolescent Brain Cognitive Development (ABCD) Study is a longitudinal neuroimaging study of unprecedented scale that is in the process of following over 11,000 youth from middle childhood though age 20. However, a design feature of the studys stop-signal task violates "context independence", an assumption critical to current non-parametric methods for estimating stop-signal reaction time (SSRT), a key measure of inhibitory ability in the study. This has led some experts to call for the task to be changed and for previously collected data to be used with caution. We present a formal cognitive process model, the BEESTS-ABCD model, that provides a mechanistic explanation for the impact of this design feature, describes key behavioral trends in the ABCD data, and allows biases in SSRT estimates resulting from context independence violations to be quantified. We use the model to demonstrate that, although non-parametric SSRT estimates generally preserve the rank ordering of participants SSRT values, failing to account for context independence violations can lead to erroneous inferences in several realistic scenarios. Nonetheless, as the BEESTS-ABCD model can be used to accurately recover estimates of SSRT and other mechanistic parameters of interest from ABCD data, the impact of such violations can be effectively mitigated.

sentences: Encoding and decoding analysis of music perception using intracranial EEG Music perception engages multiple brain regions, however the neural dynamics of this core human experience remains elusive. We applied predictive models to intracranial EEG data from 29 patients listening to a Pink Floyd song. We investigated the relationship between the song spectrogram and the elicited high-frequency activity (70-150Hz), a marker of local neural activity. Encoding models characterized the spectrotemporal receptive fields (STRFs) of each electrode and decoding models estimated the population-level song representation. Both methods confirmed a crucial role of the right superior temporal gyri (STG) in music perception. A component analysis on STRF coefficients highlighted overlapping neural populations tuned to specific musical elements (vocals, lead guitar, rhythm). An ablation analysis on decoding models revealed the presence of unique musical information concentrated in the right STG and more spatially distributed in the left hemisphere. Lastly, we provided the first song reconstruction decoded from human neural activity.

sentences: m6A-mediated Cell-cell Communication Controls Planarian Regeneration Regeneration is the regrowth of damaged tissues or organs, a vital mechanism responding to damages from primitive organisms to higher mammals. Planarian possesses active whole-body regenerative capability owning to its vast reservoir of adult stem cells, neoblasts, thus provides an ideal model to delineate the underlying mechanisms for regeneration. N6-methyladenosine (m6A) regulates stem cell renewal and differentiation. However, how m6A controls regeneration at whole-organism level remains largely unknown. Here, we demonstrate that the depletion of m6A methyltransferase regulatory subunit wtap abolishes planarian regeneration, through regulating cell-cell communication and cell cycle. scRNA-Seq analysis unveils that the wtap knockdown induces a unique type of neural progenitor-like cells (NP-like cells), characterized by specific expression of the cell-cell communication ligand grn. Intriguingly, the depletion of m6A-modified transcripts grn/cdk9 (or cdk7) axis rescues the defective regeneration of planarian without wtap. Overall, our study reveals an indispensable role of m6A-dependent cell-cell communication essential for whole-organism regeneration.

sentences: Hydrogel-based slow release of a receptor-binding domain subunit vaccine elicits neutralizing antibody responses against SARS-CoV-2 The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID-19. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, we found that clinically-relevant adjuvants Alum, AddaVax, and CpG/Alum were unable to elicit neutralizing responses following a prime-boost immunization. Here we show that sustained delivery of an RBD subunit vaccine comprising CpG/Alum adjuvant in an injectable polymer-nanoparticle (PNP) hydrogel elicited potent anti-RBD and anti-spike antibody titers, providing broader protection against SARS-CoV-2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically-relevant adjuvant systems. Notably, a SARS-CoV-2 spike-pseudotyped lentivirus neutralization assay revealed that hydrogel-based vaccines elicited potent neutralizing responses when bolus vaccines did not. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.

sentences: Prenatal murine skeletogenesis partially recovers from absent skeletal muscle as development progresses Skeletal muscle contractions are critical for normal growth and morphogenesis of the skeleton, but it is unclear how the detrimental effects of absent muscle on the bones and joints change over time. Joint size, shape and cavitation, and rudiment length and mineralisation were assessed in multiple rudiments at two developmental stages (Theiler Stage (TS)24 and TS27) in the splotch-delayed "muscleless limb" mouse model and littermate controls. As development progressed, the effects of absent muscle on all parameters except for cavitation become less severe. All major joints in muscleless limbs were qualitatively and quantitatively abnormal in shape at TS24, while, by TS27, most muscleless joint shapes were normal, or nearly normal. In contrast, any joints which were fused at TS24 did not cavitate by TS27. Therefore, recovery in joint shape over development occurred despite absent cavitation. Mineralisation showed the most pronounced changes between TS24 and TS27 in the muscleless limbs. At TS24, all muscleless rudiments studied had less mineralisation than the controls, while at TS27, muscleless limb rudiments had either the same or significantly more mineralisation than controls of the same age. We conclude that the effects of absent muscle on prenatal murine skeletogenesis are most pronounced in early skeletal development and reduce in severity prior to birth. Understanding how mammalian bones and joints continue to develop in an environment without muscle contractions, but with mechanical stimulation due to the movement of the mother, provides important insights into conditions affecting human babies such as developmental dysplasia of the hip and arthrogryposis.

sentences: Mediator is broadly recruited to gene promoters via a Tail-independent mechanism Mediator (MED) is a conserved factor with important roles in both basal and activated transcription. It is believed that MED plays a direct role in transcriptional regulation at most genes by functionally bridging enhancers and promoters. Here, we investigate the genome-wide roles of yeast MED by rapid depletion of its activator-binding domain (Tail) and monitoring changes in nascent transcription. We find that MED Tail and activator-mediated MED recruitment regulate only a small subset of genes. At most genes, MED bypasses the UAS and is directly recruited to promoters to facilitate transcription initiation. Our results define three classes of genes that differ in PIC assembly pathways and the requirements for MED Tail, SAGA, TFIID and BET factors Bdf1/2. We also find that the depletion of the MED middle module subunit Med7 mimics inactivation of Tail, suggesting a functional link. Our combined results have broad implications for the roles of MED, other coactivators, and mechanisms of transcriptional regulation at different gene classes.

sentences: Genome-wide analysis identifies Homothorax and Extradenticle as regulators of insulin in Drosophila Insulin-Producing cells Drosophila Insulin-Producing Cells (IPCs) are the main production site of the Drosophila Insulin-like peptides or Dilps which have key roles in regulating growth, development, reproduction, lifespan and metabolism. To better understand the signalling pathways and transcriptional networks that are active in the IPCs we queried publicly available transcriptome data of over 180 highly inbred fly lines for dilp expression and used this as the input for a Genome-wide association study (GWAS). This resulted in the identification of variants in 125 genes that were associated with variation in dilp expression. The function of 57 of these genes in the IPCs was tested using an RNAi-based approach. We found that IPC-specific depletion of most genes resulted in differences in expression of one or more of the dilps. We then elaborated further on homothorax, one of the candidate genes with the strongest effect on dilp expression. We found that Homothorax and its binding partner Extradenticle are involved in regulating dilp2, -3 and -5 expression and that genetic depletion of both transcription factors leads to phenotypes associated with reduced insulin signalling. Furthermore, we provide evidence that other transcription factors involved in eye development are also functional in the IPCs. In conclusion, we showed that this GWAS approach using gene expression levels as input identified genetic regulators implicated in IPC function and dilp expression.

sentences: Engineering Gac/Rsm signaling cascade for optogenetic induction of pathogenicity switch in Pseudomonas aeruginosa Bacterial pathogens operate by tightly controlling the pathogenicity to facilitate invasion and survival in host. While small molecule inducers can be designed to modulate pathogenicity to perform studies of pathogen-host interaction, these approaches, due to the diffusion property of chemicals, may have unintended, or pleiotropic effects that can impose limitations on their use. By contrast, light provides superior spatial and temporal resolution. Here, using optogenetics we reengineered GacS of the opportunistic pathogen Pseudomonas aeruginosa, signal transduction protein of the global Gac/Rsm cascade which is of central importance for regulation of infection factors. The resultant protein YGS24 displayed significant light-dependent activity of GacS kinases in Pseudomonas aeruginosa. When introduced in Caenorhabditis elegans host systems, YGS24 stimulated the pathogenicity of PAO1 in BHI and of PA14 in SK medium progressively upon blue-light exposure. This optogenetic system provides an accessible way to spatiotemporally control bacterial pathogenicity in defined host even specific tissues to develop new pathogenesis systems, which may in turn expedite development of innovative therapeutics.

sentences: Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel As the first potassium channel with a X-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related in particular to the allosteric coupling between its gates remain open. The many currently available X-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state NMR (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible X-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs. fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.

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Phase-locking patterns underlying effective communication in exact firing rate models of neural networks Macroscopic oscillations in the brain have been observed to be involved in many cognitive tasks but their role is not completely understood. One of the suggested functions of the oscillations is to dynamically modulate communication between neural circuits. The Communication Through Coherence (CTC) theory establishes that oscillations reflect rhythmic changes in excitability of the neuronal populations. Thus, populations need to be properly phase-locked so that input volleys arrive at the peaks of excitability of the receiving population to communicate effectively. Here, we present a modeling study to explore synchronization between neuronal circuits connected with unidirectional projections. We consider an Excitatory-Inhibitory (E-I) network of quadratic integrate-and-fire neurons modeling a Pyramidal-Interneuronal Network Gamma (PING) rhythm. The network receives an external periodic input from either one or two sources, simulating the inputs from other oscillating neural groups. We use recently developed mean-field models which provide an exact description of the macroscopic activity of the spiking network. This low-dimensional mean field model allows us to use tools from bifurcation theory to identify the phase-locked states between the input and the target population as a function of the amplitude, frequency and coherence of the inputs. We identify the conditions for optimal phaselocking and selective communication. We find that inputs with high coherence can entrain the network for a wider range of frequencies. Besides, faster oscillatory inputs than the intrinsic network gamma cycle show more effective communication than inputs with similar frequency. Our analysis further shows that the entrainment of the network by inputs with higher frequency is more robust to distractors, thus giving them an advantage to entrain the network. Finally, we show that pulsatile inputs can switch between attended inputs in selective attention.

Author summaryOscillations are ubiquitous in the brain and are involved in several cognitive tasks but their role is not completely understood. The Communication Through Coherence theory proposes that background oscillations in the brain regulate the information flow between neural populations. The oscillators that are properly phase-locked so that inputs arrive at the peaks of excitability of the receiving population communicate effectively. In this paper, we study the emerging phase-locking patterns of a network generating PING oscillations under external periodic forcing, simulating the oscillatory input from other neural groups. We identify the conditions for optimal phase-locking and selective communication. Namely, we find that inputs with higher frequency and coherence have an adavantage to entrain the network and we quantify how robust are to distractors. Furthermore, we show how selective attention can be implemented by means of phase locking and we show that pulsatile inputs can switch between attended inputs.

sentences: Polygenic Scores for Cognitive Abilities and their Association with Different Aspects of General Intelligence - a Deep Phenotyping Approach Intelligence is a highly polygenic trait and genome-wide association studies (GWAS) have identified thousands of DNA variants contributing with small effects. Polygenic scores (PGS) can aggregate those effects for trait prediction in independent samples. As large-scale light-phenotyping GWAS operationalized intelligence as performance in rather superficial tests, the question arises which intelligence facets are actually captured. We used deep-phenotyping to investigate the molecular determinants of individual differences in cognitive ability. We therefore studied the association between PGS of intelligence (IQ-PGS), cognitive performance (CP-PGS) and educational attainment (EA-PGS) with a wide range of intelligence facets in a sample of 557 healthy adults. IQ-PGS, CP-PGS and EA-PGS had the highest incremental R2s for general (2.71%; 4.27%; 2.06%), verbal (3.30%; 4.64%; 1.61%) and numerical intelligence (3.06%; 3.24%; 1.26%) and the weakest for non-verbal intelligence (0.89%; 1.47%; 0.70%) and memory (0.80%; 1.06%; 0.67%). These results indicate that PGS derived from light-phenotyping GWAS do not reflect different facets of intelligence equally well, and thus should not be interpreted as genetic indicators of intelligence per se. The findings refine our understanding of how PGS are related to other traits or life outcomes.

sentences: Sex-specific transcript diversity is regulated by a maternal pioneer factor in early Drosophila embryos Maternally deposited factors play a crucial role in initiating zygotic transcription. However, the mechanisms by which maternal factors regulate early zygotic transcript diversity through alternative splicing remain unclear. Furthermore, how early in development widespread sex-specific transcript diversity occurs is not known. We show that widespread sex-specific transcript diversity occurs much earlier than previously thought and present a new pipeline called time2splice to quantify splicing changes over time. Using the powerful Drosophila model, we define several mechanisms by which a maternal factor regulates sex-specific zygotic transcriptome diversity: 1) GA-binding pioneer factor CLAMP (Chromatin linked adapter for MSL proteins) binds to the gene bodies of sex-specifically spliced genes to alter the recruitment of spliceosome components; 2) CLAMP modulates chromatin accessibility of an alternatively-spliced exon within sxl, the master regulator of sex determination; 3) CLAMP regulates the distribution of the spliceosome component Maleless (MLE) to prevent aberrant sex-specific splicing. Overall, we provide key insight into how maternal factors influence sex-specific transcript diversity.

sentences: ROS-Degradable Polythioketal Urethane Foam Dressings to Promote Porcine Skin Wound Repair Impaired skin healing and progression into chronic wounds is a prevalent and growing medical problem. Porous, resorbable biomaterials can be used as temporary substrates placed into skin defects to support cell infiltration, neo-tissue formation, and remodeling of nonhealing wounds. Naturally-derived biomaterials have promising healing benefits, but their low mechanical properties and exuberant costs limit their performance and use. Synthetic materials can be affordably manufactured and tuned across a broader range of physiochemical properties, but opportunities remain for tailoring them for ideal host immune and regenerative responses. Polyesters are the most clinically-tested class of synthetic biomaterials, but their hydrolysis releases acidic degradation products that can cause autocatalytic degradation processes that are poorly controlled and are not tied to cellular or other biologic activities. Here, we systemically explored a series of ROS-degradable polythioketal (PTK) urethane (UR) foams with varied hydrophilicity as an alternative class of synthetic biomaterials for wound healing. It was found that the most hydrophilic PTK- UR variant, which had 7 ethylene glycol (EG7) repeats flanking each side of each thioketal bond, had the highest ROS reactivity of the PTK-URs tested. In an in vivo porcine excisional skin wound healing model, hydrophilic EG7 PTK-UR foams more effectively promoted tissue integration, ECM deposition, and re- epithelialization of full-thickness skin wound compared to more hydrophobic PTK-UR variants. Resolution of type 1 inflammation and lower foreign body response to scaffold remnants was also observed for EG7 versus more hydrophobic PTK-UR scaffolds. Finally, porcine wound healing studies showed that EG7 PTK-UR foams had similar wound healing response to a collagen-based clinical gold standard product, Integra Bilayer Wound Matrix (BWM), while outperforming polyester UR foam-based NovoSorb Biodegradable Temporizing Matrix (BTM) with respect to increased ECM production, vascularization, and biomaterial-associated immune phenotype. In sum, PTK-UR foams warrant further development toward a new class of synthetic biomaterial foams for skin wound healing applications.

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Linking rhizosphere processes across scales: Opinion PurposeSimultaneously interacting small-scale rhizosphere processes determine emergent plant-scale behaviour, including growth, transpiration, nutrient uptake, soil carbon storage and transformation by microorganisms. Current advances in modelling and experimental methods open the path to unravel and link those processes.

MethodsWe present a series of examples of state-of-the art simulations addressing this multi-scale, multi-process problem from a modelling point of view, as well as from the point of view of integrating newly available rhizosphere data and images.

ResultsEach example includes a model that links scales and experimental data to set-up simulations that explain and predict spatial and temporal distribution of rhizodeposition as driven by root architecture development, soil structure, presence of root hairs, soil water content and distribution of soil water. Furthermore, two models explicitly simulate the impact of the rhizodeposits on plant nutrient uptake and soil microbial activity, respectively. This exemplifies the currently available state of the art modelling tools in this field: image-based modelling, pore-scale modelling, continuum scale modelling and functional-structural plant modelling. We further show how to link the pore scale to the continuum scale by homogenisation or by deriving effective physical parameters like viscosity from nano-scale chemical properties.

ConclusionModelling allows to integrate and make use of new experimental data across different rhizosphere processes (and thus across different disciplines) and scales. Described models are tools to test hypotheses and consequently improve our mechanistic understanding of how rhizosphere processes impact plant-scale behaviour. Linking multiple scales and processes is the logical next step for future research.

sentences: Secreted ORF8 is a pathogenic cause of severe Covid-19 and potentially targetable with select NLRP3 inhibitors Despite extensive research, the specific factor associated with SARS-CoV-2 infection that mediates the life-threatening inflammatory cytokine response in patients with severe Covid-19 remains unidentified. Herein we demonstrate that the virus-encoded Open Reading Frame 8 (ORF8) protein is abundantly secreted as a glycoprotein in vitro and in patients with newly diagnosed Covid-19. ORF8 specifically binds to the NOD-like receptor family pyrin domain-containing 3 (NLRP3) in CD14+/CD16+ monocytes to induce an inflammasomal cytokine response. The levels of ORF8 protein in the blood correlate with disease mortality in patients with acute infection, and the disease trajectory in patients with severe Covid-19. Furthermore, in vitro the ORF8-induced inflammasome response can be readily inhibited by the select NLRP3 inhibitor MCC950. Our results identify the pathogenic cause and mechanism of severe disease, and a potential new treatment of severe Covid-19.

sentences: HuluFISH non-denaturing in situ detection of genomic DNA opened by CRISPR-Cas9 Nickase and Exonuclease DNA fluorescence in situ hybridization (FISH) has been widely used in diagnosis and genetic research. Traditional Bacterial artificial chromosome (BAC) or oligonucleotide based probe to detect DNA in situ is only effective when the target is relatively large, usually over 150Kb DNA fragments. And it involves heat denaturation steps to open the DNA for in situ hybridization. The heat process can affect the fine structure of nuclei. Here we reported a novel method based on Cas9 nickase and exonuclease digestion of double strand DNA and permanently mark the DNA in single strand state for FISH. With this novel design, we detected non-repetitive genomic loci as small as 2Kb.

sentences: Rare CNVs and phenome-wide profiling: a tale of brain-structural divergence and phenotypical convergence Copy number variations (CNVs) are rare genomic deletions and duplications that can exert profound effects on brain and behavior. Previous reports of pleiotropy in CNVs imply that they converge on shared mechanisms at some level of pathway cascades, from genes to large-scale neural circuits to the phenome. However, studies to date have primarily examined single CNV loci in small clinical cohorts. It remains unknown how distinct CNVs escalate the risk for the same developmental and psychiatric disorders. Here, we quantitatively dissect the impact on brain organization and behavioral differentiation across eight key CNVs. In 534 clinical CNV carriers from multiple sites, we explored CNV-specific brain morphology patterns. We extensively annotated these CNV-associated patterns with deep phenotyping assays through the UK Biobank resource. Although the eight CNVs cause disparate brain changes, they are tied to similar phenotypic profiles across [~]1000 lifestyle indicators. Our population-level investigation established brain structural divergences and phenotypical convergences of CNVs, with direct relevance to major brain disorders.

sentences: Light-sheet mesoscopy with the Mesolens provides fast sub-cellular resolution imaging throughout large tissue volumes. Rapid imaging of large biological tissue specimens such as ultrathick sections of mouse brain cannot easily be performed with a standard microscope. Optical mesoscopy offers a solution, but thus far imaging has been too slow to be useful for routine use. We have developed two different illuminators for light-sheet mesoscopy with the Mesolens and we demonstrate their use in high-speed optical mesoscale imaging of large tissue specimens. The first light-sheet approach uses Gaussian optics and is straightforward to implement. It provides excellent lateral resolution and high-speed imaging, but the axial resolution is poor. The second light-sheet is a more complex Airy light-sheet that provides sub-cellular resolution in three dimensions that is comparable in quality to point-scanning confocal mesoscopy, but the light-sheet method of illuminating the specimen reduces the imaging time by a factor of 14. This creates new possibilities for high-content, higher-throughput optical bioimaging at the mesoscale.

sentences: A DNA Replication-Independent Function of the pre-Replication Complex during Cell Invasion in C. elegans Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of C. elegans orthologs of genes over-expressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC functions cell-autonomously in the G1-arrested AC to promote invasion, independently of its role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC acts in the non-proliferating AC as a transcriptional regulator that facilitates the switch to an invasive phenotype.

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Cyclic, condition-independent activity in primary motor cortex predicts corrective movement behavior Reaching movements are known to have large condition-independent neural activity and cyclic neural dynamics. A new precision center-out task was performed by rhesus macaques to test the hypothesis that cyclic, condition-independent neural activity in the primary motor cortex (M1) occurs not only during initial reaching movements but also during subsequent corrective movements. Corrective movements were observed to be discrete with time courses and bell-shaped speed profiles similar to the initial movements. Condition-independent cyclic neural trajectories were similar and repeated for initial and each additional corrective submovement. The phase of the cyclic condition-independent neural activity predicted the time of peak movement speed more accurately than regression of instantaneous firing rate, even when the subject made multiple corrective movements. Rather than being controlled as continuations of the initial reach, a discrete cycle of motor cortex activity encodes each corrective submovement.

Significance StatementDuring a precision center-out task, initial and subsequent corrective movements occur as discrete submovements with bell-shaped speed profiles. A cycle of condition-independent activity in primary motor cortex neuron populations corresponds to each submovement, such that the phase of this cyclic activity predicts the time of peak speeds--both initial and corrective. These submovements accompanied by cyclic neural activity offer important clues into how we successfully execute precise, corrective reaching movements and may have implications for optimizing control of brain-computer interfaces.

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Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases Mycobacterium tuberculosis adenylyl cyclase (AC) Rv1625c / Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signaling is well established, the function of their transmembrane (TM) regions remains unknown. Here we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 [A] resolution. The TM helices 1-5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands.

One-Sentence SummaryStructure of M. tuberculosis membrane adenylyl cyclase Cya

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Faster SARS-CoV-2 sequence validation and annotation for GenBank using VADR BackgroundIn 2020 and 2021, more than 1.5 million SARS-CoV-2 sequences were submitted to GenBank. The initial version (v1.0) of the VADR (Viral Annotation DefineR) software package that GenBank uses to automatically validate and annotate incoming viral sequences is too slow and memory intensive to process many thousands of SARS-CoV-2 sequences in a reasonable amount of time. Additionally, long stretches of ambiguous N nucleotides, which are common in many SARS-CoV-2 sequences, prevent VADR from accurate validation and annotation.

ResultsVADR has been updated to more accurately and rapidly annotate SARS-CoV-2 sequences. Stretches of consecutive Ns are now identified and temporarily replaced with expected nucleotides to facilitate processing, and the slowest steps have been overhauled using blastn and glsearch, increasing speed, reducing the memory requirement from 64Gb to 2Gb per thread, and allowing simple, coarse-grained parallelization on multiple processors per host.

ConclusionVADR is now nearly 1000 times faster than it was in early 2020 for processing SARS-CoV-2 sequences submitted to GenBank. It has been used to screen and annotate more than 1.5 million SARS-CoV-2 sequences since June 2020, and it is now efficient enough to cope with the current rate of hundreds of thousands of submitted sequences per month. Version 1.4.1 is freely available (https://github.com/ncbi/vadr) for local installation and use.

sentences: Quantitative analysis of surface wave patterns of Min proteins The Min protein system is arguably the best-studied model systems for biological pattern formation. It exhibits pole-to-pole oscillations in E. coli bacteria as well as a variety of surface wave patterns in in vitro reconstitutions. Such Min surface wave patterns pose particular challenges to quantification as they are typically only semi-periodic and non-stationary. Here, we present a methodology for quantitatively analyzing such Min patterns, aiming for reproducibility, user-independence, and easy usage. After introducing pattern-feature definitions and image-processing concepts, we present an analysis pipeline where we use autocorrelation analysis to extract global parameters such as the average spatial wavelength and oscillation period. Subsequently, we describe a method that uses flow-field analysis to extract local properties such as the wave propagation velocity. We provide descriptions on how to practically implement these quantification tools and provide Python code that can directly be used to perform analysis of Min patterns.

sentences: The Rise of West Nile Virus in Europe: investigatingthe combined effects of climate, land use and economic changes West Nile Virus (WNV) has recently emerged as a major public health concern in Europe, its recent expansion also coincided with some remarkable socio-economic and environmental changes, including an economic crisis and some of the warmest temperatures on record. Here we empirically investigate the drivers of this phenomenon at a European wide scale by constructing and analyzing a unique spatial-temporal data-set, that includes data on climate, land-use, the economy, and government spending on environmental related sectors. Drivers and risk factors of WNV were identified by building a conceptual framework, and relationships were tested using a Generalized Additive Model (GAM), which could capture complex non-linear relationships and also account for the spatial and temporal auto-correlation. Some of the key risk factors identified in our conceptual framework, such as a higher percentage of wetlands and arable land, climate factors (higher summer rainfall and higher summer temperatures) were positive predictors of WNV infections. Interestingly, winter temperatures of between 2 {degrees}C and 6 {degrees}C were among some of the strongest predictors of annual WNV infections, one possible explanation for this result is that successful overwintering of infected adult mosquitoes (likely Culex pipiens) is key to the intensity of outbreaks for a given year. Furthermore, lower surface water extent over the summer is also associated with more more intense outbreaks, suggesting that drought, which is known to induce positive changes in WNV prevalence in mosquitoes is also contributing to the upward trend in WNV cases in affected regions. Our indicators representing the economic crisis were also strong predictors of WNV infections, suggesting there is an association between austerity and cuts to key sectors, which could have benefited vector species and the virus during this crucial period. These results, taken in the context of recent winter warming due to climate change, and more frequent droughts, may offer an explanation of why the virus has become so prevalent in Europe.

sentences: A cell atlas of chromatin accessibility across 25 adult human tissues Current catalogs of regulatory sequences in the human genome are still incomplete and lack cell type resolution. To profile the activity of human gene regulatory elements in diverse cell types and tissues in the human body, we applied single cell chromatin accessibility assays to 25 distinct human tissue types from multiple donors. The resulting chromatin maps comprising [~]500,000 nuclei revealed the status of open chromatin for over 750,000 candidate cis-regulatory elements (cCREs) in 54 distinct cell types. We further delineated cell type-specific and tissue-context dependent gene regulatory programs, and developmental stage specificity by comparing with a recent human fetal chromatin accessibility atlas. We finally used these chromatin maps to interpret the noncoding variants associated with complex human traits and diseases. This rich resource provides a foundation for the analysis of gene regulatory programs in human cell types across tissues and organ systems.

sentences: A base-line cellular antiviral state is maintained by cGAS and its most frequent naturally occurring variant rs610913 Upon recognition of aberrantly located DNA, the innate immune sensor cGAS activates STING/IRF-3-driven antiviral responses. Here we characterized the ability of a specific variant of the cGAS-encoding gene MB21D1, rs610913, to alter cGAS-mediated DNA sensing and viral infection. rs610913 is a frequent G>T polymorphism resulting in a P261H exchange in the cGAS protein. Data from the International Collaboration for the Genomics of HIV suggested that rs610913 nominally associates with HIV-1 acquisition in vivo. Molecular modeling of cGAS(P261H) hinted towards the possibility for an additional binding site for a potential cellular co-factor in cGAS dimers. However, cGAS(WT) or cGAS(P261H)-reconstituted THP-1 cGAS KO cells shared steady-state expression of interferon-stimulated genes (ISGs), as opposed to cells expressing the enzymatically inactive cGAS(G212A/S213A). Accordingly, cGAS(WT) and cGAS(P261H) cells were less susceptible to lentiviral transduction and infection with HIV-1, HSV-1, and Chikungunya virus as compared to cGAS KO- or cGAS(G212A/S213A) cells. Upon DNA challenge, innate immune activation appeared to be mildly reduced upon expression of cGAS(P261H) compared to cGAS(WT). Finally, DNA challenge of PBMCs from donors homozygously expressing rs610913 provoked a trend towards a slightly reduced type I IFN response as compared to PBMCs from GG donors. Taken together, the steady-state activity of cGAS maintains a base-line antiviral state rendering cells more refractory to ISG-sensitive viral infections. Even though rs610913 failed to grossly differ phenotypically from the wild-type gene, its expression potentially results in a slightly altered susceptibility to viral infections in vivo.

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Towards a More General Understanding of the Algorithmic Utility of Recurrent Connections Lateral and recurrent connections are ubiquitous in biological neural circuits. Yet while the strong computational abilities of feedforward networks have been extensively studied, our understanding of the role and advantages of recurrent computations that might explain their prevalence remains an important open challenge. Foundational studies by Minsky and Roelfsema argued that computations that require propagation of global information for local computation to take place would likely particularly benefit from the sequential, parallel nature of processing in recurrent networks. Such "tag propagation" algorithms perform repeated, local propagation of information and were originally introduced in the context of detecting connectedness, a task that is challenging for feedforward networks. Here, we advance the understanding of the utility of lateral and recurrent computation by first performing a large-scale empirical study of neural architectures for the computation of connectedness to explore feedforward solutions more fully and establish robustly the importance of recurrent architectures. In addition, we highlight a tradeoff between computation time and performance and demonstrate hybrid feedforward/recurrent models that perform well even in the presence of varying computational time limitations. We then generalize tag propagation architectures to multiple, interacting propagating tags, and demonstrate that these are efficient computational substrates for more general computations of connectedness by introducing and solving an abstracted biologically inspired decision-making task. Our work thus clarifies and expands the set of computational tasks that can be solved efficiently by recurrent computation, yielding hypotheses for structure in population activity that may be present in such tasks.

Author SummaryIn striking contrast to the majority of current-day artificial neural network research which primarily uses feedforward architectures, biological brains make extensive use of lateral and recurrent connections, raising the possibility that this difference makes a fundamental contribution to the gap in computational power between real neural circuits and artificial neural networks. Thus, despite the challenge of making effective comparisons between different network architectures, developing a more detailed understanding of the computational role played by such connections is a pressing need. Here, we leverage the computational capabilities of large-scale machine learning to robustly explore how differences in architectures affect a networks ability to learn tasks that require propagation of global information. We first focus on the task of determining whether two pixels are connected in an image which has an elegant and efficient recurrent solution: propagate a connected label or tag along paths. Inspired by this solution, we show that it can be generalized in many ways, including propagating multiple tags at once and changing the computation performed on the result of the propagation. Strikingly, this simple expansion of the tag propagation network is sufficient to solve a crucial abstraction to temporal connectedness at the core of many decision-making problems, which we illustrate for a an abstracted competitive foraging task Our results shed light on the set of computational tasks that can be solved efficiently by recurrent computation and how these solutions may relate to the structure of neural activity.

sentences: Effects of carbon to nitrogen ratio (C:N) on water quality and growth performance of Litopenaeus vannamei (Boone, 1931) in the biofloc system with a salinity of 5% This study aimed to investigate the effects of carbon to nitrogen ratio (C:N) on the water quality and shrimp growth performance during the grow-out culture of Litopenaeus vannamei in the biofloc system under a low salinity condition. Three biofloc treatments with an C:N (contained in the inputted feed and carbon source with the assumption that 75% of the feed nitrogen is excreted) of 8:1 (CN8), 16:1 (CN16) and 24:1 (CN24), respectively, were designed to stocking shrimp juveniles (~ 0.8 g) at a density of 270 individuals m-3, for a 63-days culture experiment at a salinity of about 5{per thousand}. Results showed that in CN8 treatment, the levels of pH (6.9{+/-}0.1), carbonate alkalinity (104.0{+/-}2.8mg L-1 CaCO3), biofloc volume (4.8{+/-}0.9mL L-1) and TSS (327.4{+/-}24.4mg L-1) were significantly lower than those in the other two treatments ([≥]7.6{+/-}0.3, [≥]157.6{+/-}21.6mg L-1 CaCO3, [≥]24.1{+/-}3.7mL L-1 and [≥]508.1{+/-}32.3mg L-1, P<0.05); whereas the levels of TAN (7.1{+/-}0.9mg L-1), nitrite (14.0{+/-}3.6mg L-1) and nitrate (77.0{+/-}5.0mg L-1) were significantly higher than those in the other treatments ([≤]2.0{+/-}0.6mg L-1, [≤]4.9{+/-}3.1mg L-1 and [≤]14.7{+/-}5.9mg L-1, P<0.05). The zootechnical parameters of shrimp were not significantly different between three treatments (P>0.05), except that the survival rates in CN16 treatment (96.8{+/-}2.0%) and CN24 treatment (93.7{+/-}4.2%) were significantly higher than that of CN8 treatment (81.5{+/-}6.4%, P<0.05). The results indicated that an inputted C:N higher than 16:1 was suitable for the biofloc system with a low salinity of 5{per thousand}, with an optimal inferred C:N range of 18.5-21.0:1 for water quality and growth performance.

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Dynamic evolution of MLKL in primate and poxvirus genomes indicates necroptosis is a critical, not auxiliary, countermeasure during infection Pathogen infection triggers host innate defenses which can lead to the activation of regulated cell death (RCD) pathways such as apoptosis. Given a key role in immunity, apoptotic effectors are often counteracted by pathogen-encoded antagonists. Mounting evidence indicates that programmed necrosis, which is mediated by the RIPK3/MLKL axis and termed necroptosis, evolved as a countermeasure to pathogen-mediated inhibition of apoptotic signaling. However, whether this emerging inflammatory RCD pathway functions primarily as a "back-up" or fundamental response remains inconclusive. We hypothesized that if necroptosis is an instrumental defense, then its effectors should display specific signatures associated with pathogen conflict that are rare in combination: rapid evolution, viral homolog hereafter virolog, and induction by cytokines (e.g. interferons). Our rapid evolution analysis across the necroptosis pathway revealed: 1) strong signatures of positive selection for RIPK3 and MLKL in primate genomes and to a lesser extent DAI/ZBP1, 2) elevated rates of amino acid substitution on multiple surfaces including the RIPK3/MLKL binding interface and 3) evidence supporting a means of activating RIPK3 independent of homotypic RHIM domain interactions. Interestingly, a poxvirus MLKL homolog has recently been identified that acts as a RIPK3 pseudosubstrate. Our findings indicate that poxvirus MLKLs are also subject to similar but distinct volatile patterns of evolution comparable to host necroptotic factors. Specifically, viral MLKLs have undergone numerous gains and losses in poxvirus evolution with some species harboring three distinct copies. Furthermore, we confirm that MLKL can be induced by cytokines like interferon gamma. In summary, MLKL displays all three hallmarks of pivotal immune factors of which only OAS1, but not other factors like cGAS, APOBEC3G, or PKR, exhibits. These data support the hypothesis that over evolutionary time, necroptosis has served as a key battleground during infection and is therefore, not an auxiliary response.

SummaryRegulated cell death (RCD), such as apoptosis, is a common host defense against invading pathogens. Necroptosis, an inflammatory RCD pathway, is thought to have emerged as an auxiliary response when other cell death pathways are suppressed by pathogens during infection. In our analyses, we have identified genetic changes in host and viral factors associated with necroptosis that display signatures of adaptation and may have served as evolutionary countermeasures to shape infection outcomes. Consistent with repeated targeting by pathogen-encoded inhibitors, we found robust signatures of rapid evolution for the essential catalysts of necroptosis, RIPK3 and MLKL. Notably, an evolutionary signature specific to RIPK3 for a domain shared with other necroptotic factors suggests an undefined means to trigger this host defense pathway. In contrast, poxviruses appear to circumvent this pathway by constantly altering the number and nature of factors they deploy to suppress necroptosis including a mimic of MLKL, which was stolen from infected cells. Collectively, our findings provide new insights into host and viral genetics that may influence infection outcomes and the factors shaping the ability of pathogens to infect and spread to new species. Furthermore, these data support the notion that necroptosis is a fundamental, not auxiliary, host response during infection.

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Genotype-specific benefits and risks of a mutualist association during natural epidemics O_LIWhile pathogenic and mutualistic microbes are ubiquitous across ecosystems and often co-occur within hosts, how they interact to determine patterns of disease in genetically diverse host populations is unknown.
C_LIO_LITo test whether microbial mutualists provide protection against pathogens, and whether this is influenced by host genotype, we conducted a field experiment in three naturally-occurring epidemics of a fungal pathogen, Podosphaera plantaginis, infecting a host plant, Plantago lanceolata, in the [A]land Islands, Finland. In each population, we collected epidemiological data on experimental plants, originating from six allopatric populations, which had been inoculated with a mixture of mutualistic mycorrhizal fungi or a non-mycorrhizal control.
C_LIO_LIInoculation with mycorrhizal fungi introduced benefits to host from every population, such as increased host growth and reduced disease severity. Host genotypes that were more susceptible to the pathogen received stronger protective effects from mycorrhizal fungi. However, inoculation with mycorrhizal fungi also increased host infection risk.
C_LIO_LIOur results show that mycorrhizal fungi introduce growth and defensive benefits, and shift infection risks in host populations under pathogen attack. Understanding how mutualists alter host susceptibility to disease will be important for predicting infection outcomes in ecological communities and in agriculture.
C_LI

Plain Language SummaryBeneficial,  mycorrhizal fungi in roots help plants grow and can protect them from disease caused by other, pathogenic microbes. This study shows that mycorrhizal fungi influence patterns of plant disease during pathogen outbreaks in a natural landscape.

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The non-genomic vitamin D pathway links β-amyloid to autophagic apoptosis in Alzheimer's disease Vitamin D is an important hormonal molecule, which exerts genomic and non-genomic actions in maintaining brain development and adult brain health. Many epidemiological studies have associated vitamin D deficiency with Alzheimers disease (AD). Nevertheless, the underlying signaling pathway through which this occurs remains to be characterized. We were intrigued to find that although vitamin D levels are significantly low in AD patients, their hippocampal vitamin D receptor (VDR) levels are inversely increased in the cytosol of the brain cells, and colocalized with A{beta}42 plaques, gliosis and autophagosomes, suggesting that a non-genomic form of VDR is implicated in AD. Mechanistically, A{beta}42 induces the conversion of nuclear heterodimer of VDR/RXR heterodimer into a cytoplasmic VDR/p53 heterodimer. The cytosolic VDR/p53 complex mediates the A{beta}42-induced autophagic apoptosis. Reduction of p53 activity in AD mice reverses the VDR/RXR formation and rescues AD brain pathologies and cognitive impairment. In line with the impaired genomic VDR pathway, the transgenic AD mice fed a vitamin D sufficient diet exhibit lower plasma vitamin D levels since early disease phases, raising the possibility that vitamin D deficiency may actually be an early manifestation of AD. Despite the deficiency of vitamin D in AD mice, vitamin D supplementation not only has no benefit but lead to exacerbated A{beta}42 depositions and cognitive impairment. Together, these data indicate that the impaired genomic vitamin D pathway links A{beta}42 to induce autophagic apoptosis, and suggest that VDR/p53 pathway could be targeted for the treatment of AD.

Significance StatementVitamin D exerts a genomic action for neuroprotection through VDR/RXR transcriptional complex. Thus, insufficient vitamin D has been linked to AD, but the signaling pathway involved remains unclear. Surprisingly, we find that the genomic action of VDR/RXR to be compromised and converted into a non-genomic VDR/p53 complex in promoting AD neurodegeneration. The cytosolic VDR/p53 complex contribute to autophagy-induced neuronal apoptosis. The VDR/RXR pathway can be a new therapeutic target for AD because targeting VDR/p53 ameliorates AD. Importantly, we provide evidence that vitamin D deficiency might be an early AD manifestation, and vitamin D supplementation exacerbates AD. This work uncovers a non-genomic VDR action in promoting AD and suggests a potential aggravating effect of vitamin D supplementation on AD.

sentences: A systematic analysis of diet-induced nephroprotection reveals overlapping and conserved changes in cysteine catabolism Caloric Restriction (CR) extends lifespan and augments cellular stress-resistance from yeast to primates, making CR an attractive strategy for organ protection in the clinic. Translation of CR to patients is complex, due to problems regarding adherence, feasibility and safety concerns in frail patients. Novel tailored dietary regimens, which modulate the dietary composition of macro- and micronutrients rather than reducing calorie intake promise similar protective effects and increased translatability. However, a direct head-to-head comparison to identify the most potent approach for organ protection as well as overlapping metabolic consequences has not been performed. We systematically analyzed six dietary preconditioning protocols - fasting mimicking diet (FMD), ketogenic diet (KD), dietary restriction of branched chained amino acids (BCAA), two dietary regimens restricting sulfur-containing amino acids (SR80/100) and CR - in a rodent model of renal ischemia-reperfusion injury (IRI) to quantify diet-induced resilience in kidneys. Of the administered diets, FMD, SR80/100 and CR efficiently protect from kidney damage after IRI. Interestingly, these approaches show overlapping changes in oxidative and hydrogen sulfide (H2S)-dependent cysteine catabolism as a potential common mechanism of organ protection. Importantly, these metabolic changes can be recapitulated in patients preconditioned by a diet limiting sulfur-containing amino acids indicating conserved diet-induced mechanisms of stress-resistance that may ultimately lead to clinical application.

sentences: Investigating a trade-off between the quality of nest grown feathers and pace of development in an altricial bird. Life history theory provides a framework for understanding how trade-offs generate negative trait associations. Among nestling birds, developmental rate, risk of predation, and lifespan covary, but some associations are only found within species while others are only observed between species. A recent comparative study suggests that allocation trade-offs may be alleviated by disinvestment in ephemeral traits, such as nest-grown feathers, that are quickly replaced. However, direct resource allocation trade-offs cannot be inferred from inter-specific trait-associations without complementary intra-specific studies. Here, we asked whether there is evidence for a within-species allocation trade-off between feather quality and developmental speed in tree swallows (Tachycineta bicolor). Consistent with the idea that ephemeral traits are deprioritized, nest-grown feathers had lower barb density than adult feathers. However, despite substantial variation in fledging age among nestlings, there was no evidence for a negative association between developmental pace and feather quality. Furthermore, accounting for differences in resource availability by considering provisioning rate and a nest predation treatment did not reveal a trade-off that was masked by variation in resources. Our results are most consistent with the idea that the inter-specific association between development and feather quality arises from adaptive specialization, rather than from a direct allocation trade-off.

sentences: A neuronal prospect theory model in the brain reward circuitry Prospect theory, arguably the most prominent theory of choice, is an obvious candidate for neural valuation models. How the activity of individual neurons, a possible computational unit, reflects prospect theory remains unknown. Here, we show with theoretical accuracy equivalent to that of human neuroimaging studies that single-neuron activity in four core reward-related cortical and subcortical regions represents the subjective valuation of risky gambles in monkeys. The activity of individual neurons in monkeys passively viewing a lottery reflects the desirability of probabilistic rewards, parameterized as a multiplicative combination of a utility and probability weighting functions in the prospect theory framework. The diverse patterns of valuation signals were not localized but distributed throughout most parts of the reward circuitry. A network model aggregating these signals reliably reconstructed risk preferences and subjective probability perceptions revealed by the animals choices. Thus, distributed neural coding explains the computation of subjective valuations under risk.

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Analytical kinetic model of native tandem promoters in E. coli Closely spaced promoters in tandem formation are abundant in bacteria. We investigated the evolutionary conservation, biological functions, and the RNA and single-cell protein expression of genes regulated by tandem promoters in E. coli. We also studied the sequence (distance between transcription start sites  dTSS, pause sequences, and distances from oriC) and potential influence of the input transcription factors of these promoters. From this, we propose an analytical model of gene expression based on measured expression dynamics, where RNAP-promoter occupancy times and dTSS are the key regulators of transcription interference due to TSS occlusion by RNAP at one of the promoters (when dTSS [≤] 35 bp) and RNAP occupancy of the downstream promoter (when dTSS > 35 bp). Occlusion and downstream promoter occupancy are modeled as linear functions of occupancy time, while the influence of dTSS is implemented by a continuous step function, fit to in vivo data on mean single-cell protein numbers of 30 natural genes controlled by tandem promoters. The best-fitting step is at 35 bp, matching the length of DNA occupied by RNAP in the open complex formation. This model accurately predicts the squared coefficient of variation and skewness of the natural single-cell protein numbers as a function of dTSS. Additional predictions suggest that promoters in tandem formation can cover a wide range of transcription dynamics within realistic intervals of parameter values. By accurately capturing the dynamics of these promoters, this model can be helpful to predict the dynamics of new promoters and contribute to the expansion of the repertoire of expression dynamics available to synthetic genetic constructs.

Author SummaryTandem promoters are common in nature, but investigations on their dynamics have so far largely relied on synthetic constructs. Thus, their regulation and potentially unique dynamics remain unexplored. We first performed a comprehensive exploration of the conservation of genes regulated by these promoters in E. coli and the properties of their input transcription factors. We then measured protein and RNA levels expressed by 30 Escherichia coli tandem promoters, to establish an analytical model of the expression dynamics of genes controlled by such promoters. We show that start site occlusion and downstream RNAP occupancy can be realistically captured by a model with RNAP binding affinity, the time length of open complex formation, and the nucleotide distance between transcription start sites. This study contributes to a better understanding of the unique dynamics tandem promoters can bring to the dynamics of gene networks and will assist in their use in synthetic genetic circuits.

sentences: Recruitment of KRAS downstream target ARL4C to membrane protrusions accelerates pancreatic cancer cell invasion. Pancreatic cancer (PC) has a high mortality rate due to metastasis. Whereas KRAS is mutated in most PC patients, controlling KRAS or its downstream effectors has not been succeeded clinically. ARL4C is a small G protein whose expression is induced by the Wnt and EGF-RAS pathways. In the present study, we found that ARL4C is frequently overexpressed in PC patients and showed that its unique localization to membrane protrusions is required for cancer cell invasion. IQGAP1 was identified as a novel interacting protein for ARL4C. ARL4C recruited IQGAP1 and its downstream effector, MMP14, to membrane protrusions. Specific localization of ARL4C, IQGAP1, and MMP14 was the active site of invasion, which induced degradation of the extracellular matrix. Moreover, subcutaneously injected antisense oligonucleotide (ASO) against ARL4C into tumor-bearing mice suppressed metastasis of PC. These results suggest that ARL4C-IQGAP1-MMP14 signaling is activated at membrane protrusions of PC cells.

sentences: Understanding conformational dynamics from macromolecular crystal diffuse scattering All macromolecular crystals contain some extent of disorder. The diffraction from such crystals contains diffuse scattering in addition to Bragg peaks and this scattering contains information about correlated displacements in the constituent molecules. While much work has been performed recently in decoding the dynamics of the crystalline ordering, the goal of understanding the internal dynamics of the molecules within a unit cell has been out-of-reach. In this article, we propose a general framework to extract the internal conformational modes of a macromolecule from diffuse scattering data. We combine insights on the distribution of diffuse scattering from short- and long-range disorder with a Bayesian global optimization algorithm to obtain the best fitting internal motion modes to the data. To illustrate the efficacy of the method, we apply it to a publicly available dataset from triclinic lysozyme. Our mostly parameter-free approach can enable the recovery of a much richer, dynamic structure from macromolecular crystallography.

sentences: Coiled coils 9-to-5: Rational de novo design of α-helical barrels with tunable oligomeric states The rational design of linear peptides that assemble controllably and predictably in water is challenging. Sequences must encode unique target structures and avoid alternative states. However, the stabilizing and discriminating non-covalent forces available are weak in water. Nonetheless, for -helical coiled-coil assemblies considerable progress has been made in rational de novo design. In these, sequence repeats of nominally hydrophobic (h) and polar (p) residues, hpphppp, direct the assembly of amphipathic helices into dimeric to tetrameric bundles. Expanding this pattern to hpphhph can produce larger -helical barrels. Here, we show that pentamers to nonamers are achieved simply by varying the residue at one of these h sites. In L/I-K-E-I-A-x-Z repeats, decreasing the size of Z from threonine to serine to alanine to glycine gives progressively larger oligomers. X-ray crystal structures of the resulting -helical barrels rationalize this: side chains at Z point directly into the helical interfaces, and smaller residues allow closer helix contacts and larger assemblies.

sentences: Remodeling and activation mechanisms of outer arm dyneins revealed by cryo-electron microscopy Cilia are thin microtubule-based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs conformational changes caused by ATP hydrolysis. Here, we report the native ODA complex structure attaching to the doublet microtubule by cryoelectron microscopy. The structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with molecular dynamics simulations, we present a model of how the attachment of the ODA to the doublet microtubule induces remodeling and activation of the ODA complex.

sentences: Structural variants are a major source of gene expression differences in humans and often affect multiple nearby genes Structural variants (SVs) are an important source of human genome diversity but their functional effects are not well understood. We mapped 61,668 SVs in 613 individuals with deep genome sequencing data from the GTEx project and measured their effects on gene expression. We estimate that common SVs are causal at 2.66% of eQTLs, which is a 10.5-fold enrichment relative to their abundance in the genome and consistent with prior work using smaller sample sizes. Duplications and deletions were the most impactful variant types, whereas the contribution of mobile element insertions was surprisingly small (0.12% of eQTLs, 1.9-fold enriched). Multi-tissue analysis of expression effects revealed that gene-altering SVs show significantly more constitutive effects than other variant types, with 62.09% of coding SV-eQTLs active in all tissues with known eQTL activity compared to 23.08% of coding SNV- and indel-eQTLs, whereas noncoding SVs, SNVs and indels show broadly similar patterns. We also identified 539 rare SVs associated with nearby gene expression outliers. Of these, 62.34% are noncoding SVs that show strong effects on gene expression yet modest enrichment at known regulatory elements, demonstrating that rare noncoding SVs are a major source of gene expression differences but remain difficult to predict from current annotations. Remarkably, both common and rare noncoding SVs often show strong regional effects on the expression of multiple genes: SV-eQTLs affect an average of 1.82 nearby genes compared to 1.09 genes affected by SNV- and indel-eQTLs, and 21.34% of rare expression-altering SVs show strong effects on 2-9 different genes. We also observe significant effects on gene expression extending 1 Mb from the SV. This provides a mechanism by which individual noncoding SVs may have strong and/or pleiotropic effects on phenotypic variation and disease.

sentences: Free Energy Landscape of RNA Binding Dynamics in Start Codon Recognition by Eukaryotic Ribosomal Pre-Initiation Complex Specific interaction between the start codon, 5-AUG-3, and the anticodon, 5-CAU-3, ensures accurate initiation of translation. Recent studies show that several near-cognate start codons (e.g. GUG and CUG) can play a role in initiating translation in eukaryotes. However, the mechanism allowing initiation through mismatched base-pairs at the ribosomal decoding site is still unclear at an atomic level. In this work, we propose an extended simulation-based method to evaluate free energy profiles, through computing the distance between each base-pair of the triplet interactions (d1, d2 and d3) involved in recognition of start codons in eukaryotic translation pre-initiation complex. Our method provides not only the free energy penalty ({Delta}{Delta}G) for mismatched start codons relative to the AUG start codon, but also the preferred pathways of transitions between bound and unbound states, which has not been described by previous studies. To verify the method, the binding dynamics of cognate (AUG) and near-cognate start codons (CUG and GUG) were simulated. Evaluated free energy profiles agree with experimentally observed changes in initiation frequencies from respective codons. This work proposes for the first time how a G:U mismatch at the first position of codon (GUG)-anticodon base-pairs destabilizes the accommodation in the initiating eukaryotic ribosome and how initiation at a CUG codon is nearly as strong as, or sometimes stronger than, that at a GUG codon. Our method is expected to be applied to study the affinity changes for various mismatched base-pairs.

sentences: Microglial ablation does not affect opioid-induced hyperalgesia in rodents Opioids are the frontline analgesics in pain management. However, chronic use of opioid analgesics causes paradoxical pain that contributes to the decrease of their efficacy in pain control and the escalation of dose in long-term management of pain. The underling pathogenic mechanism is not well understood. Microglia have been commonly thought to play a critical role in the expression of opioid-induced hyperalgesia (OIH) in animal models. We performed microglial ablation experiments using either a genetic (CD11b-diphtheria toxin receptor transgenic mouse) or pharmacological (colony-stimulating factor 1 receptor inhibitor PLX5622) approaches. Surprisingly, ablating microglia using these specific and effective approaches did not cause detectable impairment in the expression of hyperalgesia induced by morphine. We confirmed this conclusion with behavioral test of mechanical and thermal hyperalgesia, in male and female mice, and with different species (mouse and rat). These findings raise caution about the widely assumed contribution of microglia to the development of OIH.

sentences: Three Mutations Convert the Selectivity of a Protein Sensor From Nicotinic Agonists to S-Methadone For Use in Cells, Organelles, and Biofluids We report a reagentless, intensity-based S-methadone fluorescent sensor, iS-methadoneSnFR, consisting of a circularly permuted GFP inserted within the sequence of a mutated bacterial periplasmic binding protein (PBP). We evolved a previously reported nicotine-binding PBP to become a selective S-methadone-binding sensor, via three mutations in the PBPs second shell and hinge regions. iS-methadoneSnFR displays the necessary sensitivity, kinetics, and selectivity - notably enantioselectivity against R-methadone - for biological applications. Robust iS-methadoneSnFR responses in human sweat and saliva and mouse serum enable diagnostic uses. Expression and imaging in mammalian cells demonstrate that S-methadone enters at least two organelles and undergoes acid trapping in the Golgi apparatus, where opioid receptors can signal. This work shows a straightforward strategy in adapting existing PBPs to serve real-time applications ranging from subcellular to personal pharmacokinetics.

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Understanding the mechanism of monoADP ribosylation in OsSRT1 and its linkage to the DNA repair system under stress conditions. The role of sirtuins in plants are slowly unraveling. There are only reports of H3K9Ac deacetylation by OsSRT1. Here our studies shade light on its dual enzyme capability with preference for mono ADP ribosylation over deacetylation. OsSRT1 can specifically transfer the single ADP ribose group on its substrates in an enzymatic manner. This mono ADPr effect is not well known in plants, more so for deacetylases. The products of this reaction (NAM and ADP ribose) have immense negative effect on this enzyme suggesting a tighter regulation. Resveratrol, a natural plant polyphenol proves to be a strong activator of this enzyme at 150 M concentration. Under different abiotic stress conditions, we could link this ADP ribosylase activity to the DNA repair pathway by activating the enzyme PARP1. Metal stress in plants also influences these enzyme activities.

HighlightsO_LIOsSRT1 can transfer a single moiety of ADP-ribose on itself as well as other nuclear proteins like histones H3 and H2A.
C_LIO_LINAM, ADP-ribose and certain metal ions negatively regulate this ADP-ribose transfer.
C_LIO_LIADPr of OsPARP1 and OsPARP2 links OsSRT1 to DNA damage repair pathways.
C_LIO_LIOsSRT1 positively regulates the activity of OsPARP1 by ADP ribosylating it.
C_LIO_LIOn plants exposure to H2O2 (oxidative stress) and Arsenic toxicity, there is a link between the increased activity of the players of DNA damage repair system and overexpression of OsSRT1.
C_LI

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Age-Related Changes in the Rhesus Macaque Eye PurposeTo assess age-related changes in the rhesus macaque eye and evaluate them to corresponding human age-related eye disease.

MethodsData from eye exams and imaging tests including intraocular pressure (IOP), lens thickness, axial length, and retinal optical coherence tomography (OCT) images were evaluated from 142 individuals and statistically analyzed for age-related changes. Quantitative autofluorescence (qAF) was measured as was the presence of macular lesions as related to age.

ResultsAges of the 142 rhesus macaques ranged from 0.7 to 29 years (mean=16.4 years, stdev=7.5 years). Anterior segment measurements such as IOP, lens thickness, and axial length were acquired. Advanced retinal imaging in the form of optical coherence tomography and qAF were obtained. Quantitative assessments were made and variations by age groups were analyzed to compare with established age-related changes in human eyes. Quantitative analysis of data revealed age-related increase in intraocular pressure, ocular biometry (lens thickness and axial length), and presence of macular lesions. Age-related changes in thicknesses of retinal layers on OCT were observed and quantified. Age was correlated with increased qAF.

ConclusionsThe rhesus macaque has age-related ocular changes similar to humans. IOP increases with age while retinal ganglion cell layer thickness decreases. Macular lesions develop in some aged animals. Our findings support the concept that rhesus macaques may be useful for the study of important age-related diseases such as glaucoma, macular diseases, and cone disorders, and for development of therapies for these diseases.

sentences: DAZL regulates mRNA deadenylation independently of translation in germ cells Aberrant gene expression during gametogenesis is one of the factors underlying infertility, which affects roughly 15% of couples worldwide. Deleted-in-Azoospermia-Like (DAZL), a member of the DAZ-gene family, encodes an mRNA-specific regulator of translation which is essential for gametogenesis in both sexes. In this study we show that DAZL controls gene expression in oocytes by regulating the length of the mRNA poly(A) tail, a major determinant of temporal and amplitudinal gene regulation in germ cells, in which gene expression is regulated entirely post-transcriptionally. We show that DAZL does not induce polyadenylation but that binding of DAZL efficiently inhibits mRNA deadenylation induced by oocyte maturation. We reveal that this activity depends on DAZL-mediated recruitment of poly(A)-binding protein, PABP, to the mRNA. Although DAZL also activates mRNA translation via PABP recruitment, mechanistic analysis revealed that neither translation nor translational activation are required for DAZL to stabilise the poly(A) tail, suggesting two mutually independent posttranscriptional roles for the DAZL-PABP complex. We show that recruited PABP must maintain its ability to bind RNA, leading to a model in which DAZL recruits PABP and/or stabilises PABP binding to poly(A) thereby preventing access of deadenylases. These results indicate that the role of DAZL in regulating germ-cell mRNA fate is more complex than previously thought and inform on the poorly understood links between mRNA translation and deadenylation, showing that they can be mechanistically separable.

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Quality control and processing of nascent RNA profiling data Nascent RNA profiling is growing in popularity; however, there is no standard analysis pipeline to uniformly process the data and assess quality. Here, we introduce PEPPRO, a comprehensive, scalable work-flow for GRO-seq, PRO-seq, and ChRO-seq data. PEPPRO produces uniformly processed output files for downstream analysis and assesses adapter abundance, RNA integrity, library complexity, nascent RNA purity, and run-on efficiency. PEPPRO is restartable and fault-tolerant, records copious logs, and provides a web-based project report. PEPPRO can be run locally or using cluster, providing a portable first step for genomic nascent RNA analysis.

AvailabilityBSD2-licensed code and documentation: https://peppro.databio.org.

sentences: Optimum growth temperature declines with body size within fish species According to the temperature-size rule, warming of aquatic ecosystems is generally predicted to increase individual growth rates but reduce asymptotic body sizes of ectotherms. However, we lack a comprehensive understanding of how growth and key processes affecting it, such as consumption and metabolism, depend on both temperature and body mass within species. This limits our ability to inform growth models, link experimental data to observed growth patterns, and advance mechanistic food web models. To examine the combined effects of body size and temperature on individual growth, as well as the link between maximum consumption, metabolism and body growth, we conducted a systematic review and compiled experimental data on fishes from 52 studies that combined body mass and temperature treatments. By fitting hierarchical models accounting for variation between species, we estimated how maximum consumption and metabolic rate scale jointly with temperature and body mass within species. We found that whole-organism maximum consumption increases more slowly with body mass than metabolism, and is unimodal over the full temperature range, which leads to the prediction that optimum growth temperatures decline with body size. Using an independent dataset, we confirmed this negative relationship between optimum growth temperature and body size. Small individuals of a given population may therefore exhibit increased growth with initial warming, whereas larger conspecifics could be the first to experience negative impacts of warming on growth. These findings help advance mechanistic models of individual growth and food web dynamics and improve our understanding of how climate warming affects the growth and size structure of aquatic ectotherms.

sentences: Time-lapse imaging of bacterial morphology facilitates classification of antimicrobial agents Antimicrobial resistance is a major threat to human health. Basic knowledge of antimicrobial mechanism of action (MoA) is imperative for patient care and for identification of novel antimicrobials. However, the process of antimicrobial MoA identification is relatively laborious. Here, we developed a simple, quantitative time-lapse fluorescence imaging method, Dynamic Bacterial Morphology Imaging (DBMI), to facilitate this process. It uses a membrane dye and a nucleoid dye to track the morphological changes of single Bacillus subtilis cells in response to antimicrobials for up to 60 min. DBMI of bacterial cells facilitated assignment of the MoAs of 14 distinct, known antimicrobial compounds to the five main classes. Using this method, we found that the poorly studied antimicrobial, harzianic acid, a secondary metabolite that we purified from the fungal culture of Oidiodendron flavum, targets the cell envelope. We conclude that DBMI is a simple method, which facilitates rapid classification of the MoA of antimicrobials in functionally distinct classes.

sentences: Induction of Sis1 promotes fitness but not feedback in the heat shock response Previously, we combined modeling and experiments to demonstrate that the heat shock response (HSR) functions as a negative feedback loop in which undefined chaperone clients activate the HSR by sequestering Hsp70, and subsequent induction of Hsp70 deactivates the response (Zheng et al., 2016; Krakowiak et al., 2018). Here, we formally define newly synthesized proteins (NSPs) as a major class of HSR activators and determine the role of Sis1, a co-chaperone of Hsp70, in HSR regulation. We develop and experimentally validate a new mathematical model that incorporates NSPs and Sis1. Unexpectedly, genetic decoupling and pulse-labeling experiments reveal that Sis1 induction promotes fitness during prolonged stress rather than providing negative feedback to the HSR. These results support an overall model in which NSPs signal the HSR by sequestering Sis1 and Hsp70, while induction of Hsp70 - but not Sis1 - attenuates the response.

sentences: Reading instruction causes changes in category-selective visual cortex Education sculpts specialized neural circuits for skills like reading that are critical to success in modern society but were not anticipated by the selective pressures of evolution. Does the emergence of brain regions that selectively process novel visual stimuli like words occur at the expense of cortical representations of other stimulus categories like faces and objects? To answer this question we designed a randomized controlled trial where pre-literate children (five years of age) were randomly assigned to intervention programs that either taught reading skills (Letter Intervention) or oral language comprehension skills (Language Intervention). Magnetoencephalography (MEG) data collected before and after the interventions revealed that being assigned to the Letter versus Language Intervention induced different patterns of changes in category selective responses in high-level visual cortex. We found moderate support for the notion that words compete with other objects for cortical territory as children become attuned to this new class of visual stimuli. How these changes play out over a longer timescale is still unclear but, based on these data, we can surmise that high-level visual cortex undergoes rapid changes as children enter school and begin establishing new skills like literacy.

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Crowding changes appearance systematically in peripheral, amblyopic, and developing vision Visual crowding is the disruptive effect of clutter on object recognition. Although most prominent in adult peripheral vision, crowding also disrupts foveal vision in typically-developing children and those with strabismic amblyopia. Do these crowding effects share the same mechanism? Here we exploit observations that crowded errors in peripheral vision are not random: target objects appear either averaged with the flankers (assimilation), or replaced by them (substitution). If amblyopic and developmental crowding share the same mechanism then their errors should be similarly systematic. We tested foveal vision in children aged 3-8 years with typical vision or strabismic amblyopia, and peripheral vision in typical adults. The perceptual effects of crowding were measured by requiring observers to adjust a reference stimulus to match the perceived orientation of a target  Vac-Man element. When the target was surrounded by flankers that differed by {+/-}30{degrees}, all three groups (adults and children with typical or amblyopic vision) reported orientations between the target and flankers (assimilation). Errors were reduced with {+/-}90{degrees} differences, but primarily matched the flanker orientation (substitution) when they did occur. A population pooling model of crowding successfully simulated this pattern of errors in all three groups. We conclude that the perceptual effects of amblyopic and developing crowding are systematic and resemble the near periphery in adults, suggesting a common underlying mechanism.

PrecisCrowding strongly disrupts peripheral vision, as well as the foveal vision of children with typical vision and amblyopia. We show that typically developing and amblyopic children make the same crowded errors as adults in the visual periphery, consistent with a common mechanism in all three cases.

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Dynamics of visual perceptual decision-making in freely behaving mice Studying the temporal dynamics of perceptual decisions offers key insights into the cognitive processes contributing to it. Conducting such investigation in a genetically tractable animal model can facilitate the subsequent unpacking of the mechanistic basis of different stages in perceptual dynamics. Here, we investigated the time course as well as fundamental psychophysical constants governing visual perceptual decision-making in freely behaving mice. We did so by analyzing response accuracy against reaction time (i.e., conditional accuracy), in a series of 2-AFC orientation discrimination tasks in which we varied target size, luminance, duration, and presence of a foil. Our results quantified two distinct stages in the time course of mouse visual decision-making - a  sensory encoding stage, in which conditional accuracy exhibits a classic tradeoff with response speed, and a subsequent  short term memory-dependent stage in which conditional accuracy exhibits a classic asymptotic decay following stimulus offset. We estimated the duration of visual sensory encoding as 200-320 ms across tasks, the lower bound of the duration of short-term memory as ~1700 ms, and the briefest duration of visual stimulus input that is informative as [≤]50 ms. Separately, by varying stimulus onset delay, we demonstrated that the conditional accuracy function and RT distribution can be independently modulated, and found that the duration for which mice naturally withhold from responding is a quantitative metric of impulsivity. Taken together, our results establish a quantitative foundation for investigating the neural circuit bases of visual decision dynamics in mice.

SIGNIFICANCE STATEMENTThis study presents a quantitative breakdown of the time course of visual decision-making in mice during naturalistic behavior. It demonstrates parallel stages in mouse visual perceptual decision dynamics to those in humans, estimates their durations, and shows that mice are able to discriminate well under challenging visual conditions - with stimuli that are brief, low luminance, and small. These results set the stage for investigating the neural bases of visual perceptual decision dynamics and their dysfunction in mice.

sentences: Single-cell mosaicism analysis reveals cell-type-specific somatic mutational burden in Alzheimer's Dementia Despite significant advances in identifying genetic drivers of neurodegenerative disorders, the majority of affected individuals lack molecular genetic diagnosis, with somatic mutations proposed as one potential contributor to increased risk. Here, we report the first cell-type-specific map of somatic mosaicism in Alzheimers Dementia (AlzD), using 4,014 cells from prefrontal cortex samples of 19 AlzD and 17 non-AlzD individuals. We integrate full-transcript single-nucleus RNA-seq (SMART-Seq) with matched individual-level whole-genome sequencing to jointly infer mutational events and the cell-type in which they occurred. AlzD individuals show increased mutational burden, localized in excitatory neurons, oligodendrocytes, astrocytes and disease-associated "senescent" cells. High-mutational-burden cells showed mutational enrichment and similar single-cell expression profiles in AlzD cases versus non-AlzD individuals, indicating cellular-level genotype-to-phenotype correlation. Somatic mutations are specifically enriched for known AlzD genes, and implicate biologically meaningful cell-type specific processes, including: neuronal energy regulation, endocytic trafficking (NEFM), lipid metabolism (CNP, CRYAB), proteostasis (USP34), cytoskeleton, and microtubule dynamics (MACF1).

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Mechanical demands of the two-handed hardstyle kettlebell swing in novice older adults: an exploratory profile BackgroundUnderstanding the force profile of an exercise increases clinical confidence when assessing the benefits and potential risks of a prescribed exercise. This exploratory study presents the force profile of the hardstyle kettlebell swing in novice older adults and compares peak force with kettlebell deadlifts. These data will help inform healthcare providers and coaches who are considering prescribing kettlebell exercises for older adults.

MethodsThirty-five community-dwelling males and females (59-79 years) were recruited, from applicants to participate in the BELL trial. Two-handed hardstyle swings were performed with 8-16 kg. Deadlifts were performed with 8-24 kg and 8-32 kg for females and males, respectively. Ground reaction force was obtained from a floor-mounted force platform. Pairwise comparisons of peak force, forward force, rate of force development, swing cadence, sex, and kettlebell mass, were investigated for the kettlebell swing, with representative force-time curves described. Pairwise comparisons of peak force, sex and kettlebell mass were investigated for the deadlift, with comparisons of peak force, kettlebell mass, and sex, between swings and deadlifts.

ResultsFor kettlebells up to 16 kg, paired samples T-tests show a large exercise effect ({delta} > 1.4) with peak force higher for swings than deadlifts. Data shows: (i) higher peak force during swings than deadlifts ({delta} = 1.77), reaching 4.5 (1.0) N.kg-1, (ii) peak force during an 8 kg swing was greater than a 32kg deadlift, (iii) negligible difference in normalised peak force between males and females performing kettlebell swings, but a moderately large effect size during deadlifts (males > females, {delta} = 0.69), (iv) mean rate of force development of 19.9 (4.7) N.s-1.kg-1 with a very weak, positive correlation with kettlebell mass (y = 14.4 + 0.32x), and trivial effect of sex, (v) mean forward force equal to 5.5% of vertical force during swings, increasing from 3.8 (1.6) % with 8 kg to 7.1 (2.6) % with 16 kg.

ConclusionDuring kettlebell swings, there is negligible difference in normalised net peak force between novice males and females using the same absolute loads. Where ground reaction force is a therapeutic target, kettlebell swings with an 8 kg kettlebell could have similar effects to much heavier deadlifts (>24 kg). Kettlebell swings performed with lighter loads, could provider similar therapeutic value to much heavier deadlifts, and may be a more appealing, affordable, and convenient option for older adults.

sentences: Assessment of local potato cultivars found in Cis-Himalayan region of West Bengal through morphology and biochemical profiling Potato is a major global food crop grown for tubers (underground storage stems) that are high in carbohydrates, minerals, and vitamins. The presence of anthocyanins in tuber skin and flesh can have a significant impact on customer preferences. Potatoes are also high in resistant starches, which have a variety of health benefits, including enhanced fullness, cholesterol reduction, and a reduced risk of colon cancer etc. In West Bengal, diabetes is prevalent. Similarly, according to ICMR studies, colon cancer affects 8.9% of all cancer patients in West Bengal cancer which is caused by ill eating habits or the consumption of foods that are low in antioxidants. 5-10% of colon cancers are genetically caused, while the remainder are caused by poor eating habits or the consumption of foods that are low in antioxidants. To address these issues, one strategy is to eat foods with a low glycemic index and high antioxidant content as a staple food. The tuber tissues of the potato (S. tuberosum L.) accumulate various quantities of anthocyanins, which are commonly consumed around the world. Anthocyanins are pigments that range in colour from red to purple and are found throughout the plant kingdom. Anthocyanins are powerful antioxidants that are water soluble. The red skin potato is well-known among the general public. The epidermal layer contains a high quantity of red anthocyanins, which causes the skin to be red. With this background the present study has been undertaken to address the following objectives morphological (quantitative and qualitative traits) and biochemical characterization of local potato cultivars and identification of most stable genotypes based on the anthocyanin content and tuber yield of local potato cultivars. For our recent research 14 local potato cultivar from North Bengal were collected and evaluated for DUS characterization showed wide range of variability with respect to different phenotypic variants of ten characters. Overall predominant DUS characters of local potato cultivars found in North Bengal are medium sized apical length of sprout, short stem height, compact foliage structure, open leaf structure, ovate lanceolate type leaflet shape, purple leaf sprout predominant colour, spherical leaf sprout shape, medium intensity of anthicyanin coloraton at the base, light intensity of anthicyanin coloraton at the tip and weak nature of light sprout pubescence base. The results of the present investigation suggest that local potato cultivars collected from the northern part of West Bengal showed a high level of genetic variation. The differences between genotypes were highly significant at the 1% and 5% alpha level for all characters like tuber yield, length and weight of tuber, length of sprout, chlorophyll content, canopy temperature, and number of tuber per plant, according to the analysis of variance. Three quantitative traits namely tuber yield, tuber breadth, tuber length played major role in the genetic variance. Anthocyanin content had more contribution to diversify the local potato cultures according to biochemical characterization AMMI analysis suggested that Jalpai from CoochBehar is the most stable potato cultivars in respect to anthocyanin content and tuber yield per plant.

sentences: BMSC-Derived Exosomal MiRNAs Can Modulate Bone Restoration in Diabetic Rats with Femoral Defects The exosomal miRNAs of BMSCs participate in hyperglycemia induced poor healing of bone defects. Here, we demonstrate that exosomes derived from BMSCs harvested from diabetes mellitus(DM) rats suppressed bone formation when administered to normal rats with bone defects. Using high-throughput sequencing analysis of microRNA molecules, high miR-140-3p levels were expressed in exosomes released by N-BMSCs. Using TargetScan software and luciferase activity assays, plxnb1 was identified as the downstream molecular target of exosomal miR-140-3p that regulated osteogenesis. Transplantation of exosomes that overexpressed miR-140-3p into DM rats promoted the restoration of bone defects. Furthermore, miR-140-3p significantly promoted the differentiation of DM-BMSCs into osteoblasts and inhibited the expression p-RohA and p-ROCK in the plexin B1 signaling pathway. Taken together, these observations suggest that DM decreases the levels of exosomal miR-140-3p, which impedes bone formation and the differentiation of BMSCs. MiR-140-3p may represent a potential therapeutic target for DM related to abnormal bone metabolism.

sentences: High rates of spontaneous chromosomal duplications are compensated by translational regulation in a photosynthetic unicellular eukaryote While duplications have long been recognized as a fundamental process driving major evolutionary innovations, direct estimations of spontaneous chromosome duplication rates are scarce. Here, we provide the first estimations of spontaneous whole chromosome duplication rates in four green algae and in one diatom species from mutation accumulation (MA) experiments. The spontaneous whole duplication events are 7 to 70 times less frequent than spontaneous point mutations per cell division. Comparative transcriptome analyses between the control versus a MA line with a whole chromosome duplication indicate a [~]1.94-fold higher relative level of mRNAs in genes located within the duplicated regions. However, comparative analyses of the translation rate of mRNAs demonstrate that the excess of mRNAs is compensated by a proportional decrease to [~]0.67 in translation rates of genes located on duplicated chromosomes. This provides evidence that translation regulation mechanisms orchestrate dosage compensation in duplicated chromosomes. We further investigate the role of the poly(A) tail length in tempering mRNA translation and suggest that poly(A) tail length is regulated and shorter for transcripts linked to the chromosome duplication. These results point out the existence of a post-transcriptional mechanism co-ordinating the translation of hundreds of transcripts from genes located on duplicated regions in eukaryotes.

sentences: Rejuveinix Mitigates Sepsis-Associated Oxidative Stress in the Brain of Mice: Clinical Impact Potential in COVID-19 and Nervous System Disorders Here, we demonstrate that our anti-sepsis and COVID-19 drug candidate Rejuveinix (RJX) substantially improves the survival outcome in the LPS-GalN animal model of sepsis and multi-organ failure. One hundred (100) percent (%) of untreated control mice remained alive throughout the experiment. By comparison, 100% of LPS-GalN injected mice died at a median of 4.6 hours. In contrast to the invariably fatal treatment outcome of vehicle-treated control mice, 40% of mice treated with RJX (n=25) remained alive with a 2.4-fold longer median time survival time of 10.9 hours (Log-rank X2=20.60, P<0.0001). Notably, RJX increased the tissue levels of antioxidant enzymes SOD, CAT, and GSH-Px, and reduced oxidative stress in the brain. These findings demonstrate the clinical impact potential of RJX as a neuroprotective COVID-19 and sepsis drug candidate.

sentences: Amino acid variability, tradeoffs and optimality in human diet While the quality of fat (e.g. saturated/unsaturated) and carbohydrate (e.g. whole grain/simple sugars) intake has been of great interest, less attention has been made to the type of protein and resulting amino acid intake profiles in human diets. Studies at the molecular level however demonstrate that dietary amino acid intake produces substantial effects on health and disease such as cancer by modulating metabolism. How these effects may manifest in human food consumption and dietary patterns is unknown. We developed a series of algorithms to map, characterize and model the landscape of amino acid content in human food, dietary patterns, and individual consumption including relations to health status, covering over 2,000 foods, ten dietary patterns, and over 30,000 dietary records. We found that the type of amino acids contained in foods and human consumption is highly dynamic with variability far exceeding that of fat and carbohydrate. Some amino acids positively associate with diseases such as obesity while others contained in the same food negatively link to disease. Using linear programming and machine learning, we show that these health trade-offs among can be accounted to satisfy biochemical constraints in food and human eating patterns to construct a Pareto front in dietary practice, a means of achieving optimality in the face of tradeoffs that are commonly considered in economic and evolutionary theories. Thus this study may enable the design of human protein quality intake guidelines based on a quantitative framework.

sentences: The Integrity of the Speciation Core Complex is necessary for centromeric binding and reproductive isolation in Drosophila Postzygotic isolation by genomic conflict is a major cause for the formation of species. Despite its importance, the molecular mechanisms that result in the lethality of interspecies hybrids are still largely unclear. The genus Drosophila, which contains over 1600 different species, is one of the best characterized model systems to study these questions. We showed in the past that the expression levels of the two hybrid incompatibility factors Hmr and Lhr diverged in the two closely related Drosophila species, D. melanogaster and D. simulans, resulting in an increased level of both proteins in interspecies hybrids. This overexpression leads to mitotic defects, a misregulation in the expression of transposable elements and a decreased fertility. In this work, we describe a distinct six subunit Speciation Core Complex (SCC) containing HMR and LHR and analyse the effect of Hmr mutations on complex function and integrity. Our experiments suggest that HMR acts as a bridging factor between centromeric chromatin and pericentromeric heterochromatin, which is required for both its physiological function and its ability to cause hybrid male lethality.

sentences: Guest-host supramolecular assembly of injectable hydrogel fibers for cell encapsulation The fibrous architecture of the extracellular matrix (ECM) is recognized as an integral regulator of cell function. However, there is an unmet need to develop mechanically robust biomaterials mimicking nanofibrous tissue topography that are also injectable to enable minimally invasive delivery. In this study we have developed a fibrous hydrogel composed of supramolecularly-assembled hyaluronic acid (HA) nanofibers that exhibits mechanical integrity, shear-thinning, rapid self-healing, and cytocompatibility. HA was modified with methacrylates to permit fiber photocrosslinking following electrospinning and either  guest adamantane or  host {beta}-cyclodextrin groups to guide supramolecular fibrous hydrogel assembly. Analysis of fibrous hydrogel rheological properties showed that the mixed guest-host fibrous hydrogel was more mechanically robust (6.6 {+/-} 2.0 kPa, storage modulus (G')) than unmixed guest hydrogel fibers (1.0 {+/-} 0.1 kPa, G') or host hydrogel fibers (1.1 {+/-} 0.1 kPa, G') separately. The reversible nature of the guest-host supramolecular interactions also allowed for shear-thinning and self-healing behavior as demonstrated by cyclic deformation testing. Human mesenchymal stromal cells (hMSCs) encapsulated in fibrous hydrogels demonstrated satisfactory viability following injection and after seven days of culture (> 85%). Encapsulated hMSCs were more spread and elongated when cultured in viscoelastic guest-host hydrogels compared to non-fibrous elastic controls, with hMSCs also showing significantly decreased circularity in fibrous guest-host hydrogels compared to non-fibrous guest-host hydrogels. Together, these data highlight the potential of this injectable fibrous hydrogel platform for cell and tissue engineering applications requiring minimally invasive delivery.

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Discovery of aryl aminothiazole γ-secretase modulators with novel effects on amyloid β-peptide production A series of analogs based on a prototype aryl aminothiazole {gamma}-secretase modulator (GSM) were synthesized and tested for their effects on the profile of 37-to-42-residue amyloid {beta}-peptides (A{beta}), generated through processive proteolysis of precursor protein substrate by {gamma}-secretase. Certain substitutions on the terminal aryl D ring resulted in an altered profile of A{beta} production compared to that seen with the parent molecule. Small structural changes led to concentration-dependent increases in A{beta}37 and A{beta}38 production without parallel decreases in their precursors A{beta}40 and A{beta}42, respectively. The new compounds therefore apparently also stimulate carboxypeptidase trimming of A{beta} peptides [≥]43 residues, providing novel chemical tools for mechanistic studies of processive proteolysis by {gamma}-secretase.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: Heterogeneity in the size of the apical surface of cortical progenitors The apical surface of epithelial cells is highly specialized, it is important for morphogenetic processes that are essential to shape organs and tissues and it plays a role in morphogen and growth factor signaling. Apical progenitors in the mammalian neocortex are pseudoepithelial cells whose apical surface lines the ventricle. Whether changes in their apical surface sizes are important for cortical morphogenesis and/or other aspects of neocortex development after neurulation has not been thoroughly addressed. Here we show that apical progenitors are heterogeneous with respect to their apical surface area. In Efnb1 mutants, the size of the apical surface is modified and this correlates with discrete alterations of tissue organization without impacting proliferation or differentiation. Altogether, our data reveal heterogeneity in apical progenitors AS area in the developing neocortex and shows a role for Ephrin B1 in controlling AS size. Our study also indicates that changes in AS size does not have strong repercussion on apical progenitor behavior.

sentences: The effects of sequence length and composition of random sequence peptides on the growth of E. coli cells We study the potential for the de novo evolution of genes from random nucleotide sequences using libraries of E. coli expressing random sequence peptides. We assess the effects of such peptides on cell growth by monitoring frequency changes of individual clones in a complex library through four serial passages. Using a new analysis pipeline that allows to trace peptides of all lengths, we find that over half of the peptides have consistent effects on cell growth. Across nine different experiments, around 16 % of clones increase in frequency and 36 % decrease, with some variation between individual experiments. Shorter peptides (8-20 residues), are more likely to increase in frequency, longer ones are more likely to decrease. GC content, amino acid composition, intrinsic disorder and aggregation propensity show slightly different patterns between peptide groups. Sequences that increase in frequency tend to be more disordered with lower aggregation propensity. This coincides with the observation that young genes with more disordered structures are better tolerated in genomes. Our data indicate that random sequences can be a source of evolutionary innovation, since a large fraction of them are well tolerated by the cells or can provide a growth advantage.

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Analysis of the H-Ras mobility pattern in vivo shows cellular heterogeneity inside epidermal tissue Over the last two decades, developments in single-molecule microscopy (SMM) have enabled imaging and tracking of individual, fluorescently labelled proteins in biological systems, and most of these studies have focused on the analysis of protein mobility patterns inside cultured cells. In the present study, SMM was applied in vivo, using the zebrafish embryo model. We studied the protein dynamics of the membrane protein H-Ras, mutants of this protein, and its membrane-anchoring domain, C10H-Ras, in epidermal cells of living two-day-old embryos, using a total internal reflection fluorescence microscopy (TIRFM) setup. For all proteins studied, our results consistently confirm the presence of a fast- and a slow-diffusing subpopulations of molecules, which both confine to microdomains within the plasma membrane. Although the mobility patterns of H-Ras, mutants of this proteins and its membrane-anchoring domain were remarkably similar, the constitutively active H-Ras mutant, H-RasV12, exhibited significantly higher diffusion rates than the wild-type H-Ras and its inactive mutant, H-RasN17. Ultimately, we characterized variability in our data obtained using the zebrafish embryo model and demonstrated that differences between cells within the same embryo are the largest source of variation in our data. Our findings are in line with a model in which the cellular architecture of individual cells within a tissue determine the mobility of H-Ras proteins anchored in the plasma membrane cytoplasmic leaflet. Thereby, our results underline the growing importance of performing SMM imaging in vivo in order to better understand factors influencing the protein dynamics in an intact living organism.

SUMMARY STATEMENTBy application of single-molecule microscopy to living zebrafish embryos, factors altering the in vivo dynamics of H-Ras proteins in epidermal cells were analyzed.

sentences: Multi-Atlas Image Soft Segmentation via Computation of the Expected Label Value The use of multiple atlases is common in medical image segmentation. This typically requires deformable registration of the atlases (or the average atlas) to the new image, which is computationally expensive and susceptible to entrapment in local optima. We propose to instead consider the probability of all possible atlas-to-image transformations and compute the expected label value (ELV), thereby not relying merely on the transformation deemed "optimal" by the registration method. Moreover, we do so without actually performing deformable registration, thus avoiding the associated computational costs. We evaluate our ELV computation approach by applying it to brain, liver, and pancreas segmentation on datasets of magnetic resonance and computed tomography images.

sentences: Increasing discovery rates in preclinical research through optimised statistical decision criteria The success of scientific discovery in preclinical drug development is based on the different roles of exploration and confirmation. Exploration requires sensitivity while confirmation needs to be specific. Based on published empirical data, we simulated two preclinical research trajectories to examine their effectiveness to detect true effects while keeping animal numbers low. We compare a conventional p-value based approach with a method based on an a priori determined smallest effect size of interest (SESOI). Using a SESOI increases transition rates from exploration to confirmation and results in higher detection rates across the trajectory. Specificity in the SESOI trajectory increases if the prior probability of true effects is low. We conclude that employing a SESOI is superior to a p-value based approach. Based on our findings, we propose a reconsideration of planning and conducting preclinical experiments, especially when the prior probability of true hypotheses is low.

sentences: Transcriptional regulation of photoprotection in dark-to-light transition - more than just a matter of excess light energy In nature, photosynthetic organisms are exposed to different light spectra and intensities depending on the time of day and atmospheric and environmental conditions. When photosynthetic cells absorb excess light, they induce non-photochemical quenching to avoid photo-damage and trigger expression of  photoprotective genes. In this work, we used the green alga Chlamydomonas reinhardtii to assess the impact of light intensity, light quality, wavelength, photosynthetic electron transport and CO2 on induction of the  photoprotective genes (LHCSR1, LHCSR3 and PSBS) during dark-to-light transitions. Induction (mRNA accumulation) occurred at very low light intensity, was independently modulated by blue and UV-B radiation through specific photoreceptors, and only LHCSR3 was strongly controlled by CO2 levels through a putative enhancer function of CIA5, a transcription factor that controls genes of the carbon concentrating mechanism. We propose a model that integrates inputs of independent signaling pathways and how they may help the cells anticipate diel conditions and survive in a dynamic light environment.

sentences: Alpha variant versus D614G strain in the Syrian hamster model Late 2020, SARS-CoV-2 Alpha variant from lineage B.1.1.7 emerged in United Kingdom and gradually replaced the G614 strains initially involved in the global spread of the pandemic. In this study, we used a Syrian hamster model to compare a clinical strain of Alpha variant with an ancestral G614 strain. The Alpha variant succeeded to infect animals and to induce a pathology that mimics COVID-19. However, both strains replicated to almost the same level and induced a comparable disease and immune response. A slight fitness advantage was noted for the G614 strain during competition and transmission experiments. These data do not corroborate the epidemiological situation observed during the first half of 2021 in humans nor reports that showed a more rapid replication of Alpha variant in human reconstituted bronchial epithelium.

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Mechanosensitivity of nucleocytoplasmic transport Mechanical force controls fundamental cellular processes in health and disease, and increasing evidence shows that the nucleus both experiences and senses applied forces. Here we show that nuclear forces differentially control passive and facilitated nucleocytoplasmic transport, setting the rules for the mechanosensitivity of shuttling proteins. We demonstrate that nuclear force increases permeability across nuclear pore complexes, with a dependence on molecular weight that is stronger for passive than facilitated diffusion. Due to this differential effect, force leads to the translocation into or out of the nucleus of cargoes within a given range of molecular weight and affinity for nuclear transport receptors. Further, we show that the mechanosensitivity of several transcriptional regulators can be both explained by this mechanism, and engineered exogenously by introducing appropriate nuclear localization signals. Our work sets a novel framework to understand mechanically induced signalling, with potential general applicability across signalling pathways and pathophysiological scenarios.

One sentence summaryForce application to the nucleus leads to nuclear accumulation of proteins by differentially affecting passive versus facilitated nucleocytoplasmic transport.

sentences: A multifaceted ecological assessment reveals the invasion of the freshwater red macroalga Montagnia macrospora (Batrachospermales, Rhodophyta) in Taiwan Invasive freshwater macroalgae are rarely described. Recently, a freshwater red alga, Montagnia macrospora, was introduced from South America to East Asia via the global aquarium trade. The earliest occurrence record of this alga in Taiwan is dated 2005. To determine whether M. macrospora has become an invasive species in Taiwan and to understand its traits that facilitated its invasion, we took a total-evidence approach that combines examination of ecological background and population genetic analysis. Our island-wide survey showed that M. macrospora alga was widespread in the field across Taiwan, where the climate greatly differs from that of South America. Our population genetic analysis revealed that the cox2-3 sequences of all the specimens of M. macrospora from Taiwan were identical, consistent with the hypothesis that the alga expanded through asexual reproduction. Moreover, during our long-term ecological assessments and field surveys, we observed that M. macrospora is an ecological generalist that can self-sustain for a decade and bloom. Taken together, our data suggest that M. macrospora has successfully invaded the freshwater ecosystems in Taiwan due to its ability to disperse asexually and to grow under broad environmental conditions. We hope that our study brings attention to invasive freshwater algae, which have been overlooked in conservation planning and management.

sentences: HistoClean: Open-source Software for Histological Image Pre-processing and Augmentation to Improve Development of Robust Convolutional Neural Networks The growth of digital pathology over the past decade has opened new research pathways and insights in cancer prediction and prognosis. In particular, there has been a surge in deep learning and computer vision techniques to analyse digital images. Common practice in this area is to use image pre-processing and augmentation to prevent bias and overfitting, creating a more robust deep learning model. Herein we introduce HistoClean; user-friendly, graphical user interface that brings together multiple image processing modules into one easy to use toolkit. In this study, we utilise HistoClean to pre-process images for a simple convolutional neural network used to detect stromal maturity, improving the accuracy of the model at a tile, region of interest, and patient level. HistoClean is free and open-source and can be downloaded from the Github repository here: https://github.com/HistoCleanQUB/HistoClean.

sentences: 300 years of change for native fish species in the upper Danube River Basin - historical flow alterations versus future climate change River ecosystems belong to the most threatened ecosystems on Earth. Historical anthropogenic alterations have, and future climate change will further affect river ecosystems and the species therein. While many studies assess the potential effects of expected future changes on species, little is known about the severity of these changes compared to historical alterations. Here, we used a unique 300-year time series of hydrological and climate data to assess the vulnerability of 48 native fish species in the upper Danube River Basin. We calculated species-specific vulnerability estimates relative to the reference period (1970-2000) for the periods 1800-1830, 1900-1930, and 2070-2100, including two Representative Concentration Pathways (RCP 4.5 and 8.5) and identified the environmental drivers of vulnerability estimates. Models showed that future changes under RCP 4.5 would result in moderate species vulnerability compared to historical conditions, while under RCP 8.5, the vulnerability for all species increased substantially. In addition, species vulnerability was mainly driven by hydrology in the past and is likely to be driven by temperature in the future. Our results show that future climate change would alter environmental conditions for riverine fish species at a similar magnitude as historical anthropogenic hydrological river alterations have. Shedding light on such long-term historical and possible future anthropogenic alterations provides valuable insights for prioritising conservation actions for riverine fish species.

sentences: ASXL3 controls cortical neuron fate specification through extrinsic self-renewal pathways During corticogenesis, transcription plasticity is fundamental to the restriction of neural progenitor cell (NPC) multipotency and production of cortical neuron heterogeneity. Human and mouse genetic studies have highlighted the role of Polycomb transcriptional regulation in this process. ASXL3, which encodes a component of the Polycomb repressive deubiquitination (PR-DUB) complex, has been identified as a high confidence autism spectrum disorder (ASD) risk gene. Genetic inactivation of Asxl3, in a mouse model that carries a clinically relevant ASXL3 frameshift (Asxl3fs) variant, disrupts lateral expansion of NPCs and delays cortical neuron differentiation. Single-cell RNA sequencing analysis implicates Notch signaling, which alters the composition of excitatory neurons and fidelity of cortical layer deposition. Our data provides a new link between extrinsic signaling cues and intrinsic epigenetic regulation that together control the timing of cell fate programs. Furthermore, transcriptomic analysis revealed dysregulation of other known ASD risk genes indicating that a convergent developmental pathway is affected. Collectively our work provides important insights about developmental mechanisms that contribute to ASD neuropathology.

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Rabies virus with a destabilization domain added to its nucleoprotein spreads between neurons only if the domain is removed Monosynaptic tracing using rabies virus is an important technique in neuroscience, allowing brain-wide labeling of neurons directly presynaptic to a targeted neuronal population. A 2017 article reported development of a noncytotoxic version - a major advance - based on attenuating the rabies virus by addition of a destabilization domain to the C-terminus of a viral protein. However, this modification did not appear to hinder the ability of the virus to spread between neurons. We analyzed two viruses provided by the authors and show here that both were mutants that had lost the intended modification, explaining the papers paradoxical results. We then made a virus that actually did have the intended modification and found that it did not spread under the conditions described in the original paper - namely, without an exogenous protease being expressed in order to remove the destabilization domain - but that it did spread, albeit with relatively low efficiency, if the protease was supplied. We conclude that the new approach is not robust but that it may become a viable technique given further optimization and validation.

SIGNIFICANCE STATEMENTRabies virus, which spreads between synaptically connected neurons, has been one of the primary tools used by neuroscientists to reveal the organization of the brain. A new modification to rabies virus was recently reported to allow the mapping of connected neurons without adverse effects on the cells health, unlike earlier versions. Here we show that the conclusions of that study were probably incorrect and based on having used viruses that had lost the intended modification because of mutations. We also show that a rabies virus that does retain the intended modification does not spread between neurons under the conditions reported previously; however, it does spread between neurons under different conditions, suggesting that the approach may be successful if refined further.

sentences: Transferrin receptor 1-mediated iron uptake regulates bone mass in mice via osteoclast mitochondria and cytoskeleton Increased intracellular iron spurs mitochondrial biogenesis and respiration to satisfy high-energy demand during osteoclast differentiation and bone-resorbing activities. Transferrin receptor 1 (TFR1) mediates cellular iron uptake through endocytosis of iron-loaded transferrin and its expression increases during osteoclast differentiation. Nonetheless, the precise functions of TFR1 and TFR1-mediated iron uptake in osteoclast biology and skeletal homeostasis remain incompletely understood. To investigate the role of TFR1 in osteoclast lineage cells, we conditionally deleted Tfr1 gene in myeloid precursors or mature osteoclasts by crossing Tfr1- floxed mice with LysM-Cre and Ctsk-Cre mice, respectively. Skeletal phenotyping by {micro}CT and histology unveiled that loss of Tfr1 in osteoclast progenitor cells resulted in a three-fold increase in trabecular bone mass in the long bones of 10-week old female but not male mice. Although high trabecular bone volume in long bones was seen in both male and female mice with deletion of Tfr1 in mature osteoclasts, this phenotype was more pronounced in female knockout mice. Mechanistically, disruption of Tfr1 expression attenuated mitochondrial metabolism and cytoskeletal organization in mature osteoclasts, leading to decreased bone resorption with no impact on osteoclastogenesis. These results indicate that Tfr1-mediated iron uptake is specifically required for osteoclast function and is indispensable for bone remodeling.

sentences: Comparing divergence landscapes from reduced-representation and whole-genome re-sequencing in the yellow-rumped warbler (Setophaga coronata) species complex Researchers seeking to generate genomic data for non-model organisms are faced with a number of trade-offs when deciding which method to use. The selection of reduced representation approaches versus whole genome re-sequencing will ultimately affect the marker density, sequencing depth, and the number of individuals that can multiplexed. These factors can affect researchers ability to accurately characterize certain genomic features, such as landscapes of divergence--how FST varies across the genomes. To provide insight into the effect of sequencing method on the estimation of divergence landscapes, we applied an identical bioinformatic pipeline to three generations of sequencing data (GBS, ddRAD, and WGS) produced for the same system, the yellow-rumped warbler species complex. We compare divergence landscapes generated using each method for the myrtle warbler (Setophaga coronata coronata) and the Audubons warbler (S. c. auduboni), and for Audubons warblers with deeply divergent mtDNA resulting from mitochondrial introgression. We found that most high-FST peaks were not detected in the ddRAD dataset, and that while both GBS and WGS were able to identify the presence of large peaks, WGS was superior at a finer scale. Comparing Audubons warblers with divergent mitochondrial haplotypes, only WGS allowed us to identify small (10-20kb) regions of elevated differentiation, one of which contained the nuclear-encoded mitochondrial gene NDUFAF3. We calculated the cost per base pair for each method and found it was comparable between GBS and WGS, but significantly higher for ddRAD. These comparisons highlight the advantages of WGS over reduced representation methods when characterizing landscapes of divergence.

sentences: Enumeration of Citrus endophytic bacterial communities based on illumine metagenomics technique Citrus is a valuable crop in Pakistan because it is rich in vitamin C and antioxidants. Huanglongbing (HLB) has an influence on citrus production around the world caused by a bacterium "Candidatus liberibacter asiaticus" (CLas), africanus and americanus. The structure and diversity of bacterial species in various ecosystems can be quickly examined using NGS. This approach is considerably quicker and more precise than outdated methods. Healthy or citrus greening infected leaf samples of Grapefruit, Citrus aurantifolia, and Citrus reticulata Blanco was used for diversity analysis. In this study high throughput, NGS technique was used to access the population of both cultivable and non-cultivable bacterial endophytes from citrus leaves, by using PCR amplicons of 16S rDNA sequences (V5-V7 regions) with Illumina Hi seq. As a result, a total number of 68,722 sequences were produced from the test samples. According to the NGS-based diversity classification, the most common genera of exploited bacterial endophytes were Proteobacteria, Firmicutes, Bacteroides, Cyanobacteria, and Actinobacteria. Citrus aurantifolia and Citrus paradisi showed almost equal diversity, whereas Citrus reticulata Blanco had a higher proportion of Proteobacteria and Cyanobacteria in their leaves. To determine alpha diversity (AD), additional data was analyzed using statistical indices such as Shannon, Chao1, and Simpson. According to the inverse Simpson diversity index, the abundance of the microbial population in six different citrus samples was 0.48, 0.567, and 0.163, respectively. The metagenomics of microbiota in plant tissues was successfully recorded by NGS technology, which can help us learn more about the interactions between plants and microbes. This research is the first step toward a better understanding of 16SrRNA-based metagenomics from citrus in Pakistan using Illumina (Hi seq) Technology.

sentences: Efficient Inhibition of SARS-CoV-2 Using Chimeric Antisense Oligonucleotides through RNase L Activation There is an urgent need for effective antiviral drugs to alleviate the current COVID-19 pandemic. Here, we rationally designed and developed chimeric antisense oligonucleotides to degrade envelope and spike RNAs of SARS-CoV-2. Each oligonucleotide comprises a 3 antisense sequence for target recognition and a 5-phosphorylated 2-5 poly(A)4 for guided ribonuclease L (RNase L) activation. Since RNase L can potently cleave single strand RNA during innate antiviral response, the improved degradation efficiency of chimeric oligonucleotides was twice as much as classic antisense oligonucleotides in Vero cells, for both SARS-CoV-2 RNA targets. In pseudovirus infection models, one of chimeric oligonucleotides targeting spike RNA achieved potent and broad-spectrum inhibition of both SARS-CoV-2 and its recently reported N501Y and/or {Delta}H69/{Delta}V70 mutants. These results showed that the constructed chimeric oligonucleotides could efficiently degrade pathogenic RNA of SARS-CoV-2 facilitated by immune activation, showing promising potentials as antiviral nucleic acid drugs for COVID-19.

sentences: TreeKnit: Inferring Ancestral Reassortment Graphs of influenza viruses When two influenza viruses co-infect the same cell, they can exchange genome segments in a process known as reassortment. Reassortment is an important source of genetic diversity and is known to have been involved in the emergence of most pandemic influenza strains. However, because of the difficulty in identifying reassortments events from viral sequence data, little is known about its role in the evolution of the seasonal influenza viruses. Here we introduce TreeKnit, a method that infers ancestral reassortment graphs (ARG) from two segment trees. It is based on topological differences between trees, and proceeds in a greedy fashion by finding regions that are compatible in the two trees. Using simulated genealogies with reassortments, we show that TreeKnit performs well in a wide range of settings and that it is as accurate as a more principled bayesian method, while being orders of magnitude faster. Finally, we show that it is possible to use the inferred ARG to better resolve segment trees and to construct more informative visualizations of reassortments.

sentences: Co-occupancy analysis reveals novel transcriptional synergies for axon growth Transcription factors (TFs) act as powerful levers to regulate neural physiology and can be targeted to improve cellular responses to injury or disease. Because TFs often depend on cooperative activity, a major challenge is to identify and deploy optimal sets. Here we developed a novel bioinformatics pipeline, centered on TF co-occupancy of regulatory DNA, and used it to predict factors that potentiate the effects of pro-regenerative Klf6. High content screens of neurite outgrowth identified cooperative activity by 12 candidates, and systematic testing in an animal model of corticospinal tract (CST) damage substantiated three novel instances of pairwise cooperation. Combined Klf6 and Nr5a2 drove the strongest growth, and transcriptional profiling of CST neurons identified Klf6/Nr5a2-responsive gene networks involved in macromolecule biosynthesis and DNA repair. These data identify novel TF combinations that promote enhanced CST growth, clarify the transcriptional correlates, and provide a bioinformatics roadmap to detect TF synergy.

sentences: Gating the channel pore of ionotropic glutamate receptors with bacterialsubstrate binding proteins Tetrameric ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the mammalian central nervous system and are involved in learning, memory formation, and pathological processes. Based on structural and sequence similarities of the ligand-binding and channel domains of iGluR subunits to bacterial binding proteins and potassium channels, iGluRs are thought to have originally arisen from their fusion. Here we report the functional coupling of the bacterial ectoine binding protein EhuB to the channel pore-forming transmembrane domains of the bacterial GluR0 receptor by stabilization of dimeric binding domains. Insertion of a disulfide bridge in the dimer interface abolished desensitization of the channel current analogous to mammalian iGluRs. These results demonstrate the functional compatibility of bacterial binding proteins to the gate of the channel pore of an iGluR. Moreover, our results highlight the modular structure and crucial role of binding domain dimerization in the functional evolution of iGluRs.

sentences: Effects of Perinatal Fluoxetine Exposure on Novelty-induced Social and Non-Social Investigation Behaviors in a Seminatural Environment Selective serotonin reuptake inhibitors (SSRIs) are increasingly prescribed as medication for various affective disorders during pregnancy. SSRIs cross the placenta and affect serotonergic neurotransmission in the fetus, but the neurobehavioral consequences for the offspring remain largely unclear. Recent rodent research has linked perinatal SSRI exposure to alterations in both social and non-social aspects of behavior. However, this research has mainly focused on behavior within simplified environments. The current study investigates the effects of perinatal SSRI exposure on social and non-social investigation behaviors of adult rat offspring upon introduction to a novel seminatural environment with unknown conspecifics. During the perinatal period (gestational day 1 until postnatal day 21), rat dams received daily treatment with either an SSRI (fluoxetine, 10 mg/kg) or vehicle. Adult male and female offspring were observed within the first hour after introduction to a seminatural environment. The results showed that perinatal fluoxetine exposure altered aspects of non-social investigation behaviors, while not altering social investigation behaviors. More specifically both fluoxetine exposed males and females spent more total time on locomotor activity than controls. Furthermore, fluoxetine exposed females spent less time exploring objects and specific elements in the environment. The data suggest that perinatal exposure to SSRIs leads to a quicker, less detailed investigation strategy in novel environments, and that the alteration is mostly pronounced in females.

sentences: Tree of mutually exclusive oncogenes Identifying cancer driver genes and their interrelations is critical in understanding cancer progression mechanisms. In this paper, we introduce ToMExO, a probabilistic method to infer cancer driver genes and how they affect each other, using cross-sectional data from cohorts of tumors. We model cancer progression dynamics using a tree with sets of driver genes in the nodes. This model explains the temporal orders among driver mutations and their mutual exclusivity patterns. We introduce a dynamic programming procedure for the likelihood calculation and build an MCMC inference algorithm. Together with our engineered MCMC moves, our efficient likelihood calculations enable us to work with datasets having hundreds of genes and thousands of tumors in the datasets. We demonstrate our methods performance on several synthetic datasets covering various scenarios for cancer progression dynamics. We then present the analyses of several biological datasets using the ToMExO method and validate the results using a set of method-independent metrics.

sentences: Behavioral and postural analyses establish sleep-like states for mosquitoes that can impact host landing and blood feeding Sleep is an evolutionarily conserved process that has been described in different animal systems. For insects, sleep characterization has been primarily achieved using behavioral and electrophysiological correlates in a few systems. Sleep in mosquitoes, which are important vectors of disease-causing pathogens, has not been directly examined. This is surprising as circadian rhythms, which have been well studied in mosquitoes, influence sleep in other systems. In this study, we characterized sleep in mosquitoes using body posture analysis and behavioral correlates, and quantified the effect of sleep deprivation on sleep rebound, host landing and blood-feeding propensity. Body and appendage position metrics revealed a clear distinction between the posture of mosquitoes in their putative sleep and awake states for multiple species, which correlate with a reduction in responsiveness to host cues. Sleep assessment informed by these posture analyses indicated significantly more sleep during periods of low activity. Nighttime and daytime sleep deprivation resulting from the delivery of vibration stimuli induced sleep rebound in the subsequent phase in day and night active mosquitoes, respectively. Lastly, sleep deprivation suppressed host landing in both laboratory and field settings and also impaired blood feeding of a human host when mosquitoes would normally be active. These results suggest that quantifiable sleep states occur in mosquitoes, and highlight the potential epidemiological importance of mosquito sleep.

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Immune microenvironment subtypes and association with tumor cell mutations and antigen expression in follicular lymphoma. Follicular lymphoma (FL) is a B-cell lymphoma with a complex tumor microenvironment that is rich in non-malignant immune cells. We applied single-cell RNA-sequencing to characterize the diverse tumor and immune cell populations of FL and identified major phenotypic subsets of FL T-cells including a novel cytotoxic CD4 T-cell population. Their relative proportions of T-cells defined four major FL subtypes, characterized by differential representation or relative depletion of distinct T-cell subsets. By integrating exome sequencing, we observed that somatic mutations are associated with, but not definitive for, reduced antigen presentation on FL cells. In turn, expression of MHC class II genes by FL cells was associated with significant differences in the proportions and targetable immunophenotypic characteristics. This provides a classification framework of the FL microenvironment, their association with FL genotypes and antigen presentation, and informs different potential immunotherapeutic strategies based upon tumor cell MHC class II expression.

Statement of significanceWe have characterized the FL-infiltrating T-cells, identified cytotoxic CD4 T-cells as an important component, showed that the abundance of these T-cell populations is associated with tumor-cell-intrinsic characteristics, and identified sets of targetable immune checkpoints on T-cells that differed between FLs with normal versus low antigen presentation.

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Quantitative single-cell transcriptome-based ranking of engineered AAVs in human retinal explants Gene therapy is a rapidly developing field, and adeno-associated virus (AAV) is a leading viral vector candidate for therapeutic gene delivery. Newly engineered AAVs with improved abilities are now entering the clinic. It has proven challenging, however, to predict the translational potential of gene therapies developed in animal models, due to cross-species differences. Human retinal explants are the only available model of fully developed human retinal tissue, and are thus important for the validation of candidate AAV vectors. In this study, we evaluated 18 wildtype and engineered AAV capsids in human retinal explants using a recently developed single-cell RNA-Seq AAV engineering pipeline (scAAVengr). Human retinal explants retained the same major cell types as fresh retina, with similar expression of cell-specific markers, except for a cone population with altered expression of cone-specific genes. The efficiency and tropism of AAVs in human explants were quantified, with single-cell resolution. The top performing serotypes, K91, K912, and 7m8, were further validated in non-human primate and human retinal explants. Together, this study provides detailed information about the transcriptome profiles of retinal explants, and quantifies the infectivity of leading AAV serotypes in human retina, accelerating the translation of retinal gene therapies to the clinic.

Graphic abstract

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sentences: Exposure to the gut microbiota from cigarette smoke-exposed mice exacerbates cigarette smoke extract-induced inflammation in zebrafish larvae Cigarette smoke (CS)-induced inflammation leads to a range of diseases including chronic obstructive pulmonary disease and cancer. The gut microbiota is a major modifying environmental factor that determine the severity of cigarette smoke-induced pathology. Microbiomes and metabolites from CS-exposed mice exacerbate lung inflammation via the gut-lung axis of shared mucosal immunity in mice but these systems are expensive to establish and analyse. Zebrafish embryos and larvae have been used to model the effects of cigarette smoking on a range of physiological processes and offer an amenable platform for screening modifiers of cigarette smoke-induced pathologies with key features of low cost and rapid visual readouts. Here we exposed zebrafish larvae to cigarette smoke extract (CSE) and characterised a CSE-induced leukocytic inflammatory phenotype with increased neutrophilic and macrophage inflammation in the gut. The CSE-induced phenotype was exacerbated by co-exposure to microbiota from the faeces of CS-exposed mice, but not control mice. Microbiota could be recovered from the gut of zebrafish and studied in isolation in a screening setting. This demonstrates the utility of the zebrafish-CSE exposure platform for identifying environmental modifiers of cigarette smoking-associated pathology and demonstrates that the CS-exposed mouse gut microbiota potentiates the inflammatory effects of CSE across host species.

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Spike-timing-dependent plasticity rewards synchrony rather than causality Spike-timing-dependent plasticity (STDP) is a candidate mechanism for information storage in the brain, but the whole-cell recordings required for the experimental induction of STDP are typically limited to one hour. This mismatch of time scales is a long-standing weakness in synaptic theories of memory. Here we use spectrally separated optogenetic stimulation to fire precisely timed action potentials (spikes) in CA3 and CA1 pyramidal cells. Twenty minutes after optogenetic induction of STDP (oSTDP), we observed timing-dependent depression (tLTD) and timing-dependent potentiation (tLTP), depending on the sequence of spiking. As oSTDP does not require electrodes, we could also assess the strength of these paired connections three days later. At this late time point, late tLTP was observed for both causal (CA3 before CA1) and anti-causal (CA1 before CA3) timing, but not for asynchronous activity patterns ({Delta}t = 50 ms). Blocking activity after induction of oSTDP prevented stable potentiation. Our results confirm that neurons wire together if they fire together, but suggest that synaptic depression after anti-causal activation (tLTD) is a transient phenomenon.

HighlightsO_LIOptogenetic induction of spike-timing-dependent plasticity at Schaffer collateral synapses
C_LIO_LICausal pairing induces potentiation whereas anti-causal pairing induces depression during patch-clamp recordings.
C_LIO_LIThree days after optogenetic induction, the consequence of STDP is potentiation (tLTP) irrespective of spiking order.
C_LIO_LILate tLTP requires ongoing activity in the days following oSTDP.
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Graphical Abstract

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sentences: Putting pesticides on the map for pollinator research and conservation Wild and managed pollinators are essential to food production and the function of natural ecosystems; however, their populations are threatened by multiple stressors including pesticide use. Because pollinator species can travel hundreds to thousands of meters to forage, recent research has stressed the importance of evaluating pollinator decline at the landscape scale. However, scientists and conservationists ability to do this has been limited by a lack of accessible data on pesticide use at relevant spatial scales and in toxicological units meaningful to pollinators. Here, we synthesize information from several large, publicly available datasets on pesticide use patterns, land use, and toxicity to generate novel datasets describing pesticide use by active ingredient (kg, 1997-2017) and aggregate insecticide load (kg and honey bee lethal doses, 1997-2014) for state-crop combinations in the contiguous U.S. Furthermore, by linking pesticide datasets with land-use data in the contiguous United States, we describe a method to map pesticide indicators at spatial scales relevant to pollinator research and conservation.

sentences: Antiviral and Neuroprotective Abilities of Influenza Virus Infection in Tractable Brain Organoids Human pluripotent stem cell (hPSC)-derived brain organoids offer an unprecedented opportunity for various applications as an in vitro model, such as modeling virus infection and drug screening. In this study, we present an experimental brain organoid platform for modeling infection with multiple viruses (e.g., influenza virus or enterovirus). Brain organoids challenged by influenza viruses (H1N1-WSN and H3N2-HKT68) had decreased overall organoid size, similar to ZIKA virus infection, while enteroviruses (EV68 and EV71) infected brain organoids displayed the opposite result. Then, we studied the molecular events in WSN-infected organoids, and we found that WSN could widely infect multiple cell types, and preferentially infected MAP2+ neurons compared to SOX2+ neural stem cells (NSCs) and GFAP+ astrocytes in brain organoids, and induced apoptosis of NSCs and neurons, but not astrocytes. The inflammatory responses in organoids observed to occur (Tumor necrosis factor alpha, interferon gamma, and interleukin 6) after WSN infection may further facilitate brain damage. Furthermore, transcriptional profiling revealed several upregulated genes (CSAG3 and OAS2) and downregulated genes (CDC20B, KCNJ13, OTX2-AS1, CROCC2, and F5) after WSN infection for 24 hpi and 96 hpi, implicating antiviral drugs development responses to WSN. Finally, we explored neurotrophic factors (e.g., BDNF, GDNF, and NT3) and PYC-12 as antiviral and neuroprotective reagents, which could significantly suppress virus infection, apoptosis, and inflammatory responses. Collectively, we established a tractable experimental model system to investigate the impact and mechanism of virus infection on human brain development, and provide a platform for rapidly screening therapeutic compounds, advancing the development of antiviral strategies.

sentences: Probabilistic Evaluation of Null Models for West Nile Virus in the United States Null models provide a useful baseline for the development of new models. A variety of options for null models exist. These options have become more sophisticated with the advent of probabilistic modeling approaches. Here, we evaluate 10 different null models for West Nile virus, a primarily mosquito-borne disease introduced to the United States in 1999. The Historical Null was significantly better than all models other than the Negative Binomial. We recommend the use of either of these models as a baseline when developing new models to predict spatial and temporal dynamics of West Nile virus at the county-annual scale. We expect these results to be scale-dependent, and a future direction is to examine performance of null models at finer spatial and temporal scales.

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Simulation of receptor triggering by kinetic segregation shows role of oligomers and close-contacts The activation of T cells, key players of the immune system, involves local evacuation of phosphatase CD45 from a region of the T cells surface, segregating it from the T cell receptor. What drives this evacuation? In the presence of antigen, what ensures evacuation happens in the sub-second timescales necessary to initiate signaling? In the absence of antigen, what mechanisms ensure evacuation does not happen spontaneously, which could cause signaling errors? Phenomena known to influence spatial organization of CD45 or similar surface molecules include diffusive motion in the lipid bilayer, oligomerization reactions, and mechanical compression against a nearby surface, such as that of the cell presenting antigen. Computer simulations can investigate hypothesized spatiotemporal mechanisms of T cell signaling. The challenge to computational studies of evacuation is that the base process, spontaneous evacuation by simple diffusion, is in the extreme rare event limit, meaning direct stochastic simulation is unfeasible. Here we combine particle-based spatial stochastic simulation with the Weighted Ensemble method for rare events to compute the mean first-passage time for cell surface availability by surface reorganization of CD45. We confirm mathematical estimates that, at physiological concentrations, spontaneous evacuation is extremely rare, roughly 300 years. We find that dimerization decreases the time required for evacuation. A weak bi-molecular interaction (dissociation constant estimate 460 microMolar) is sufficient for an order of magnitude reduction of spontaneous evacuation times, and oligomerization to hexamers reduces times to below 1 second. This introduces a mechanism whereby CD45 oligomerization could be accessible to an engineered therapeutic. For large regions of close-contact, such as those induced by large microvilli, molecular size and compressibility imply a nonzero re-entry probability 60%, decreasing evacuation times. Simulations show that these reduced evacuation times are still unrealistically long, suggesting that a yet-to-be-described mechanism, besides compressional exclusion at a close contact, drives evacuation.

Statement of SignificanceIn the immune system, T cells sensing pathogens depends on a process called T cell receptor triggering. In this process, proteins on the cell surface undergo reorganization, including local depletion of large membrane proteins from the area surrounding the T cell receptor. Computer simulations of protein dynamics provide a means to investigate phenomena in greater detail than that afforded by experiments. However, even simulations present challenges, because tracking the motion and interactions of individual molecules is computationally expensive. Combining a rare event algorithm with spatial simulations, we show that biochemical and mechanical properties drastically affect depletion timescales, and thus receptor triggering. Quantitative understanding of these timescales will constrain hypothesized mechanistic models and could suggest new strategies for T cell engineering.

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Generic solving of physiologically-based kinetic models in support of next generation risk assessment due to chemicals O_FIG O_LINKSMALLFIG WIDTH=175 HEIGHT=200 SRC="FIGDIR/small/490045v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@a56a22org.highwire.dtl.DTLVardef@1db11cdorg.highwire.dtl.DTLVardef@16dbe2corg.highwire.dtl.DTLVardef@1e04ad2_HPS_FORMAT_FIGEXP  M_FIG C_FIG Increase the confidence in using in vitro and in silico data to aid the chemical risk assessment process is one, if not the most, important challenge currently facing regulatory authorities. A particularly crucial concern is to fully take advantage of scientifically valid physiologically-based kinetic (PBK) models. Nevertheless, risk assessors remain still unwilling in employing PBK models within their daily work. Indeed, PBK models are not often included in current official guidance documents. In addition, most users have limited a limited experience in using modelling in general. So, the complexity of PBK models, together with a lack to evaluation methods of their performances, certainly contributes to their under-use.

This paper proposes an innovative and unified modelling framework, in both the writing of PBK equations as matrix ordinary differential equations (ODE), and in its exact solving simply expressed with matrix products. This generic PBK solution allows to consider as many as state-variables as needed to quantify chemical absorption, distribution, metabolism and excretion processes within living organisms when exposed to chemical substances. This generic PBK model makes possible any compartmentalisation of the model to be considered, as well as all appropriate inter-connections between compartments and with the external medium.

We first introduce our PBK modelling framework, with all intermediate steps from the matrix ODE until the exact solution. Then we apply this framework to bioaccumulation testing, before illustrating its concrete use through complementary case studies in terms of species, compound and model complexity. Upon our findings, we finally propose to consider this approach to eventually be part of a future revision of the current regulation.

sentences: Coherent natural scene structure facilitates the extraction of task-relevant object information in visual cortex Looking for objects within complex natural environments is a task everybody performs multiple times each day. In this study, we explore how the brain uses the typical composition of real-world environments to efficiently solve this task. We recorded fMRI activity while participants performed two different categorization tasks on natural scenes. In the object task, they indicated whether the scene contained a person or a car, while in the scene task, they indicated whether the scene depicted an urban or a rural environment. Critically, each scene was presented in an "intact" way, preserving its coherent structure, or in a "jumbled" way, with information swapped across quadrants. In both tasks, participants categorization was more accurate and faster for intact scenes. These behavioral benefits were accompanied by stronger responses to intact than to jumbled scenes across high-level visual cortex. To track the amount of object information in visual cortex, we correlated multi-voxel response patterns during the two categorization tasks with response patterns evoked by people and cars in isolation. We found that object information in object- and body-selective cortex was enhanced when the object was embedded in an intact, rather than a jumbled scene. However, this enhancement was only found in the object task: When participants instead categorized the scenes, object information did not differ between intact and jumbled scenes. Together, these results indicate that coherent scene structure facilitates the extraction of object information in a task-dependent way, suggesting that interactions between the object and scene processing pathways adaptively support behavioral goals.

sentences: A synthetic membrane shaper for controlled liposome deformation Shape defines the structure and function of cellular membranes. In cell division, the cell membrane deforms into a  dumbbell shape, while organelles such as the autophagosome exhibit  stomatocyte shapes. Bottom-up in vitro reconstitution of protein machineries that stabilize or resolve the membrane necks in such deformed liposome structures is of considerable interest to characterize their function. Here we develop a DNA-nanotechnology-based approach that we call Synthetic Membrane Shaper (SMS), where cholesterol-linked DNA structures attach to the liposome membrane to reproducibly generate high yields of stomatocytes and dumbbells. In silico simulations confirm the shape-stabilizing role of the SMS. We show that the SMS is fully compatible with protein reconstitution by assembling bacterial divisome proteins (DynaminA, FtsZ:ZipA) at the catenoidal neck of these membrane structures. The SMS approach provides a general tool for studying protein binding to complex membrane geometries that will greatly benefit synthetic cell research.

sentences: Ramp-shaped neural tuning supports graded population-level representation of the object-to-scene continuum We can easily perceive the spatial scale depicted in a picture, regardless of whether it is a small space (e.g., a close-up view of a chair) or a much larger space (e.g., an entire class room). How does the human visual system encode this continuous dimension? Here, we investigated the underlying neural coding of depicted spatial scale, by examining the voxel tuning and topographic organization of brain responses. We created naturalistic yet carefully-controlled stimuli by constructing virtual indoor environments, and rendered a series of snapshots to smoothly sample between a close-up view of the central object and far-scale view of the full environment (object-to-scene continuum). Human brain responses were measured to each position using functional magnetic resonance imaging. We did not find evidence for a smooth topographic mapping for the object-to-scene continuum on the cortex. Instead, we observed large swaths of cortex with opposing ramp-shaped profiles, with highest responses to one end of the object-to-scene continuum or the other, and a small region showing a weak tuning to intermediate scale views. Importantly, when we considered the multi-voxel patterns of the entire ventral occipito-temporal cortex, we found smooth and linear representation of the object-to-scene continuum. Thus, our results together suggest that depicted spatial scale is coded parametrically in large-scale population codes across the entire ventral occipito-temporal cortex.

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Phertilizer: growing a clonal tree from ultra-low coverage single-cell DNA sequencing data of tumors MotivationEmerging ultra-low coverage single-cell DNA sequencing (scDNA-seq) technologies have enabled high resolution evolutionary studies of copy number aberrations (CNAs) within tumors. While these sequencing technologies are well suited for identifying CNAs due to the uniformity of sequencing coverage, the sparsity of coverage poses challenges for the study of single-nucleotide variants (SNVs). In order to maximize the utility of increasingly available ultra-low coverage scDNA-seq data and obtain a comprehensive understanding of tumor evolution, it is important to simultaneously analyze the evolution of SNVs alongside CNAs from the same set of tumor cells.

ResultsWe present Phertilizer, a method to infer a clonal tree, capturing both SNV and CNA evolution, from ultra-low coverage single-cell DNA sequencing of a tumor. Based on a probabilistic model, our method recursively partitions the data by identifying key evolutionary events in the history of the tumor. We demonstrate the performance of Phertilizer on simulated data as well as on two real datasets, finding that Phertilizer effectively utilizes the copy-number signal inherent in the data to more accurately uncover clonal structure and genotypes compared to previous methods.

Availabilityhttps://github.com/elkebir-group/phertilizer

Contact{idoia,melkebir}@illinois.edu

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: Susceptibility monitoring and the molecular characterization of resistance of Spodoptera frugiperda (Lepidoptera: Noctuidae) to lambda-cyhalothrin and chlorpyrifos Spodoptera frugiperda (J. E. Smith) is a serious and widespread agricultural pest with several records of resistance to different insecticides and Bt proteins, including the neurotoxic insecticides chlorpyrifos (organophosphate) and lambda-cyhalothrin (pyrethroid). In this study, we (i) characterized and monitored the susceptibility of field populations of S. frugiperda to chlorpyrifos (194 populations) and lambda-cyhalothrin (197 populations) collected from major maize-growing regions of Brazil from 2003 to 2016, and (ii) compared gene expression levels of chlorpyrifos- and lambda-cyhalothrin-resistant strains to a susceptible reference strain (Sf-ss) of S. frugiperda. Laboratory-guided assays to monitor larval susceptibility detected average survival ranging from 29.3% to 36.0% to chlorpyrifos, and 23.1% to 68.0% to lambda-cyhalothrin at diagnostic concentration, based on LC99 of the susceptible reference strain of each insecticide. The resistance ratio of the chlorpyrifos-resistant strain (Clo-rr) was 25.4-fold and of the lambda-cyhalothrin-resistant strain (Lam-rr) was 217-fold. Differential gene expression analyses between resistant vs susceptible strains identified 1,098 differentially expressed genes (DEGs) between Clo-rr and Sf-ss, and 303 DEGs between Lam-rr and Sf-ss. Functional analyses of the DEGs revealed the up-regulation of several detoxification enzymes, mainly cytochrome P450 belonging to the CYP3 and CYP6 clans. Genes associated with regulatory processes, such as the forkhead box O (FoxO) were also up-regulated. Our data points that the resistance mechanisms of Clo-rr and Lam-rr strains of S. frugiperda to chlorpyrifos and lambda-cyhalothrin are mainly mediated by enzyme detoxification.

sentences: Effector loss drives adaptation of Pseudomonas syringae pv. actinidiae to Actinidia arguta A pandemic isolate of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars of Actinidia chinensis. In contrast, A. arguta (kiwiberry) is resistant to Psa3. This resistance is mediated via effector-triggered immunity, as demonstrated by induction of the hypersensitive response in infected A. arguta leaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease in A. arguta have been isolated and analyzed, revealing a 49 kb deletion in the exchangeable effector locus (EEL). This natural EEL-mutant isolate and strains with synthetic knockouts of the EEL were more virulent in A. arguta plantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growth in planta for knockouts of four effectors - AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a - suggesting a resistance response in A. arguta. Hypersensitive response (HR) assays indicate that three of these effectors trigger a host species-specific HR. A Psa3 strain with all four effectors knocked out escaped host recognition, but a cumulative increase in bacterial pathogenicity and virulence was not observed. These avirulence effectors can be used in turn to identify the first cognate resistance genes in Actinidia for breeding durable resistance into future kiwifruit cultivars.

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Genomic and phenotypic characterization of finger millet indicates a complex diversification history Advances in sequencing technologies mean that insights into crop diversification aiding future breeding can now be explored in crops beyond major staples. For the first time, we use a genome assembly of finger millet, an allotetraploid orphan crop, to analyze DArTseq single nucleotide polymorphisms (SNPs) at the sub-genome level. A set of 8,778 SNPs and 13 agronomic traits characterizing a broad panel of 423 landrace accessions from Africa and Asia suggested the crop has undergone complex, context-specific diversification consistent with a long domestication history. Both Principal Component Analysis and Discriminant Analysis of Principal Components of SNPs indicated four groups of accessions that coincided with the principal geographic areas of finger millet cultivation. East Africa, the considered origin of the crop, appeared the least genetically diverse. A Principal Component Analysis of phenotypic data also indicated clear geographic differentiation, but different relationships among geographic areas than genomic data. Neighbour-joining trees of sub-genomes A and B showed different features which further supported the crops complex evolutionary history. Our genome-wide association study indicated only a small number of significant marker-trait associations. We applied then clustering to marker effects from a ridge regression model for each trait which revealed two clusters of different trait complexity, with days to flowering and threshing percentage among simple traits, and finger length and grain yield among more complex traits. Our study provides comprehensive new knowledge on the distribution of genomic and phenotypic variation in finger millet, supporting future breeding intra- and inter-regionally across its major cultivation range.

Core ideasO_LI8,778 SNPs and 13 agronomic traits characterized a panel of 423 finger millet landraces.
C_LIO_LI4 clusters of accessions coincided with major geographic areas of finger millet cultivation.
C_LIO_LIA comparison of phenotypic and genomic data indicated a complex diversification history.
C_LIO_LIThis was confirmed by the analysis of allotetraploid finger millets separate sub-genomes.
C_LIO_LIComprehensive new knowledge for intra- and inter-regional breeding is provided.
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sentences: Stress-induced phase separation of ERES components into Sec bodies precedes ER exit inhibition in mammalian cells. Phase separation of ER-exit-sites (ERES) components into membraneless compartments, the Sec bodies, occurs in Drosophila cells upon specific cellular stressors, i.e., salt stress and amino acid starvation, and their formation is linked to the inhibition of the early secretory pathway. Here, we show Sec bodies also form in secretory mammalian INS-1 cells upon the same stress. These reversible and membraneless structures are positive for ERES components, including both isoforms of Sec16 (A and B) and COPII subunits. We find that Sec16A, but not Sec16B, is a driver for Sec body formation. We show that the coalescence of ERES components into Sec bodies occurs by fusion, in line with their liquid-droplet properties. Lastly, we demonstrate that stress-induced ER-exit inhibition is a consequence of the significant coalescence of Sec16A into Sec bodies, leading to its depletion from ERES that become non-functional. Stress relief causes an immediate dissolution of Sec bodies and the concomitant restoration of protein exit from the ER. We propose a model in which dynamic conversion between ERES and Sec body assembly, driven by Sec16A, regulates protein exit from the ER during stress and upon stress relief in mammalian cells, thus providing a conserved pro-survival mechanism in response to stress.

sentences: Identification of large offspring syndrome during pregnancy through ultrasonography and maternal blood transcriptome analyses The use of assisted reproductive technologies (ART) in cattle can result in large/abnormal offspring syndrome (LOS/AOS) which is characterized by macrosomia. LOS can cause dystocia and lead to the death of dam and calf. Currently, no test exists to identify LOS pregnancies. We hypothesized that fetal ultrasonography and/or maternal blood markers are useful to identify LOS. Bovine fetuses were generated by artificial insemination (control) or ART. Fetal ultrasonographies were taken on gestation day 55 (D55) and fetal collections performed on D56 or D105 (gestation in cattle {approx}280 days). ART fetuses weighing [≥]97 percentile of the control weight were considered LOS. Ultrasonography results show that the product of six D55 measurements can be used to identify extreme cases of LOS. To determine whether maternal blood can be used to identify LOS, leukocyte mRNA from 23 females was sequenced. Unsupervised hierarchical clustering grouped the transcriptomes of the two females carrying the two largest LOS fetuses. Comparison of the leukocyte transcriptomes of these two females to the transcriptome of all other females identified several misregulated transcripts on gestation D55 and D105 with LOC783838 and PCDH1 being misregulated at both time-points. Together our data suggest that LOS is identifiable during pregnancy in cattle.

sentences: A re-analysis of the data in Sharkey et al.'s (2021) minimalist revision reveals that BINs do not deserve names, but BOLD Systems needs a stronger commitment to open science Halting biodiversity decline is one of the most critical challenges for humanity, but monitoring biodiversity is hampered by taxonomic impediments. One impediment is the large number of undescribed species (here called "dark taxon impediment") while another is caused by the large number of superficial species descriptions which can only be resolved by consulting type specimens ("superficial description impediment"). Recently, Sharkey et al. (2021) proposed to address the dark taxon impediment for Costa Rican braconid wasps by describing 403 species based on barcode clusters ("BINs") computed by BOLD Systems. More than 99% of the BINs (387 of 390) are converted into species by assigning binominal names (e.g., BIN "BOLD:ACM9419" becomes Bracon federicomatarritai) and adding a minimal diagnosis (usually consisting only of a consensus barcode). We here show that many of Sharkey et al.s species are unstable when the underlying data are analyzed using different species delimitation algorithms. Add the insufficiently informative diagnoses, and many of these species will become the next "superficial description impediment" for braconid taxonomy because they will have to be tested and redescribed after obtaining sufficient evidence for confidently delimiting species. We furthermore show that Sharkey et al.s approach of using consensus barcodes as diagnoses is not functional because it cannot be consistently applied. Lastly, we reiterate that COI alone is not suitable for delimiting and describing species and voice concerns over Sharkey et al.s uncritical use of BINs because they are calculated by a proprietary algorithm (RESL) that uses a mixture of public and private data. We urge authors, reviewers, and editors to maintain high standards in taxonomy by only publishing new species that are rigorously delimited with open-access tools and supported by publicly available evidence.

sentences: The Core Human Fecal Metabolome Summary ParagraphAmong the biomolecules at the center of human health and molecular biology is a system of molecules that defines the human phenotype known as the metabolome. Through an untargeted metabolomic analysis of fecal samples from human individuals from Africa and the Americas--the birthplace and the last continental expansion of our species, respectively--we present the characterization of the core human fecal metabolome. The majority of detected metabolite features were ubiquitous across populations, despite any geographic, dietary, or behavioral differences. Such shared metabolite features included hyocholic acid and cholesterol. However, any characterization of the core human fecal metabolome is insufficient without exploring the influence of industrialization. Here, we show chemical differences along an industrialization gradient, where the degree of industrialization correlates with metabolomic changes. We identified differential metabolite features like leucyl-leucine dipeptides and urobilin as major metabolic correlates of these behavioral shifts. Our results indicate that industrialization significantly influences the human fecal metabolome, but diverse human lifestyles and behavior still maintain a core human fecal metabolome. This study represents the first characterization of the core human fecal metabolome through untargeted analyses of populations along an industrialization gradient.

sentences: TXN, a Xanthohumol Derivative, Significantly Attenuates High-Fat Diet Induced Hepatic Steatosis In Vivo by Antagonizing PPARγ We previously reported xanthohumol (XN), and its synthetic derivative tetrahydro-XN (TXN) attenuates high-fat diet (HFD) induced obesity and metabolic syndrome in C57BL/6J mice. The objective of the current study was to determine the effect of XN and TXN on lipid accumulation in the liver. Non-supplemented mice were unable to adapt their caloric intake to 60% HFD, resulting in obesity and hepatic steatosis; however, TXN reduced weight gain and decreased hepatic steatosis. Liver transcriptomics indicated TXN might antagonize lipogenic PPAR{gamma} actions in vivo. XN and TXN inhibited rosiglitazone-induced 3T3-L1 cell differentiation concomitant with decreased expression of lipogenesis-related genes. A PPAR{gamma} competitive binding assay showed XN and TXN bind to PPAR{gamma} with an IC50 similar to pioglitazone and 8-10 times stronger than oleate. Molecular docking simulations demonstrated XN and TXN bind in the PPAR{gamma} ligand-binding domain pocket. Our findings are consistent with XN and TXN acting as antagonists of PPAR{gamma}.

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The Diameter Factor of Aligned Electrospinning Membranes Facilitates Cutaneous Wound Healing by Promoting Epithelialization Topographical properties (such as pattern and diameter) of biomaterials play important roles in influencing cell activities and manipulating the related immune response during the skin wound healing. We prepared aligned electrospinning membranes with different fiber diameters including 319{+/-}100 nm (A300), 588{+/-}132 nm (A600) and 1048{+/-}130 nm (A1000). The A300 membranes significantly improved cell proliferation and spreading, and facilitated skin wound healing (epithelization and vascularization) with the regeneration of hair follicles. The transcriptome revealed the underlying molecular mechanism that A300 could promote macrophages transformation to reparative phenotypes with highly expressed IL10 and TGF{beta}, subsequently promoting epidermal cell migration via secreting matrix metalloproteinases 12 (MMP12). All the results indicate the A300 to be a potential candidate for guided skin regeneration applications.

Graphic abstract

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sentences: Genomic signatures of hybridization on the neo-X chromosome of Rumex hastatulus Natural hybrid zones provide opportunities for studies of the evolution of reproductive isolation in wild populations. Although several recent investigations have found that the formation of neo-sex chromosomes is associated with reproductive isolation, the mechanisms remain unclear in most cases. Here, we assess the contemporary structure of gene flow in the contact zone between largely allopatric cytotypes of the dioecious plant Rumex hastatulus, a species in which there is evidence of sex chromosome turn-over. Males to the west of the Mississippi river, USA, have an X and a single Y chromosome, whereas populations to the east of the river have undergone a chromosomal rearrangement giving rise to a larger X and two Y chromosomes. Using reduced-representation sequencing, we provide evidence that hybrids form readily and survive multiple backcross generations in the field, demonstrating the potential for ongoing gene flow between the cytotypes. At the scale of chromosomes, cline analysis of each chromosome separately captured no signals of difference in cline shape between chromosomes. However, when comparing SNPs, principal component regression revealed a significant increase in the contribution of individual SNPs to inter-cytotype differentiation on the neo-X, but no correlation with recombination rate. Cline analysis revealed that the only SNPs with significantly shallower clines than the genome-average were located on the neo-X. Our data are consistent with a role for the neo-sex chromosome in reproductive isolation between R. hastatulus cytotypes. Our investigation highlights the importance of studying plant hybrid zones in species with sex chromosomes for understanding mechanisms of reproductive isolation and for understanding the role of gene flow in governing the spread of the neo-X chromosomes.

sentences: Plasticity of cold hardiness in the eastern spruce budworm, Choristoneura fumiferana High latitude insect populations must cope with extreme conditions, particularly cold temperatures. Insects use a variety of cold hardiness mechanisms to withstand this temperature stress, and these can drive geographic distributions through overwintering mortality. The degree of cold hardiness can be altered by two evolved responses: phenotypic plasticity and local adaptation. Phenotypic plasticity can occur within or between generations (transgenerational plasticity; TGP), and local adaptation can evolve through directional selection in response to regional climatic differences. We used the eastern spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae) as a model to explore the role that variable winter temperatures play in inducing two aspects of plasticity in cold hardiness: TGP and local adaptation in phenotypic plasticity. This species is one of the most destructive boreal forest pests in North America, therefore accurately predicting overwintering survival is essential for effective management. While we found no evidence of TGP in cold hardiness, there was a long-term fitness cost to larvae that experienced repeated cold exposures. We also found evidence of local adaptation in both seasonal and short-term plasticity of cold hardiness. These findings provide evidence for the importance of phenotypic plasticity and local adaptation when modelling species distributions.

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Tbx1, a 22q11.2-encoded gene, is a link between alterations in fimbria myelination and cognitive speed in mice Copy number variants (CNVs) have provided a reliable entry point to identify structural correlates of atypical cognitive development. Hemizygous deletion of human chromosome 22q11.2 is associated with impaired cognitive function; however, the mechanisms by which numerous genes encoded in this CNV contribute to cognitive deficits via diverse structural alterations in the brain remain unclear. This study aimed to determine the cellular basis of the link between alterations in brain structure and cognitive functions in a mouse model. The heterozygosity of Tbx1, a 22q11.2 gene, altered the composition of myelinated axons in the fimbria, reduced oligodendrocyte production capacity, and slowed the acquisition of spatial memory and cognitive flexibility. Our findings provide a cellular basis for specific cognitive dysfunctions that occur in patients with loss-of-function TBX1 variants and 22q11.2 hemizygous deletion.

TeaserA risk gene for autism alters myelin composition in the hippocampal connection and slows cognitive speed.

sentences: Transcriptomic analysis revealed reactive oxygen species scavenging mechanisms associated with ferrous iron toxicity in aromatic Keteki Joha rice Lowland acidic soils with water-logged regions are often affected by ferrous iron (Fe2+) toxicity, a major yield-limiting factor of rice production. The Reactive Oxygen Species (ROS) was hypothesized to be crucial under severe Fe2+ toxicity conditions. However, molecular mechanisms and associated ROS homeostasis genes are still not well-explored. In this study, a comparative RNA-Seq based transcriptome analysis was conducted to understand the Fe2+ toxicity tolerance mechanism in aromatic Keteki Joha. About 69 Fe homeostasis related genes and their homologs were identified, where most of the genes were downregulated. Differentially expressed genes (DEGs) are associated with biological processes-response to stress, stimulus and abiotic stimulus. DEGs involved in the Biosynthesis of amino acids, RNA degradation, Glutathione metabolism etc. were induced, whereas, Phenylpropanoid biosynthesis, Photosynthesis, and Fatty acid elongation were inhibited. The Mitochondrial iron transporter (OsMIT), Vacuolar Iron Transporter 2 (OsVIT2), Ferritin (OsFER), Vacuolar Mugineic Acid Transporter (OsVMT), Phenolic Efflux Zero1 (OsPEZ1), Root Meander Curling (OsRMC), Nicotianamine synthase (OsNAS3), etc. were upregulated in different tissues suggesting the importance of Fe retention and sequestration for detoxification. However, several antioxidants, ROS scavenging genes and abiotic stress-responsive transcription factors indicate ROS homeostasis as one of the most important defense mechanisms under severe Fe2+ toxicity. The CAT, GSH, APX, MDHAR, DHAR, GR were upregulated. Moreover, abiotic stress-responsive transcription factors NAC, MYB, ARF, bZIP, WRKY, C2H2-ZFP were also upregulated. Accordingly, ROS homeostasis has been proposed to be an important defense mechanism under such conditions. Thus, our results will enrich the knowledge of understanding Fe-homeostasis in rice.

sentences: Using mass Spectrometry to investigate fluorescent compounds in squirrel fur While an array of taxa are capable of producing fluorescent pigments, fluorescence in mammals is a novel and poorly understood phenomenon. We believe that a first step towards understanding the potential adaptive functions of fluorescence in mammals is to develop an understanding of fluorescent compounds, or fluorophores, that are present in fluorescent tissue. Here we use Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) of flying squirrel fur known to fluoresce under ultraviolet (UV) light to identify potentially fluorescent compounds in squirrel fur. All of the potentially fluorescent compounds we identified were either present in non-fluorescent fur or were not present in all species of fluorescent flying squirrel. Therefore, we consider that the compounds responsible for fluorescence in flying squirrels may also be present in non-fluorescent mammal fur. Some factor currently unexplained likely leads to excitation of fluorophores in flying squirrel fur. A recently suggested hypothesis that fluorescence in mammals is widely caused by porphyrins is consistent with our findings.

sentences: The adolescent pattern of sleep spindle development revealed by HD-EEG polysomnography Sleep spindles are developmentally relevant cortical oscillatory patterns; however, they have mostly been studied by considering the entire spindle frequency range (11 to 15 Hz) without a distinction between the functionally and topographically different slow and fast spindles, using relatively few electrodes and analysing wide age-ranges. Here, we employ HD-EEG polysomnography in three age-groups between 12 to 20 years of age, with an equal distribution between the two genders, and analyse the adolescent developmental pattern of the four major parameters of slow and fast sleep spindles. Most of our findings corroborate those very few previous studies that also make a distinction between slow and fast spindles in their developmental analysis. We find spindle frequency increasing with age, although spindle density change is not obvious in our study. We confirm the declining tendencies for amplitude and duration, although within narrower, more specific age-windows than previously. Spindle frequency seems to be higher in females in the oldest age-group. Based on the pattern of our findings, we suggest that HD-EEG, specifically targeting slow and fast spindle ranges and relatively narrow age-ranges would advance the understanding of both adolescent development and the functional relevance of sleep spindles in general.

sentences: Reduced interferon antagonism but similar drug sensitivity in Omicron variant compared to Delta variant SARS-CoV-2 isolates The SARS-CoV-2 Omicron variant is currently causing a large number of infections in many countries. A number of antiviral agents are approved or in clinical testing for the treatment of COVID-19. Despite the high number of mutations in the Omicron variant, we here show that Omicron isolates display similar sensitivity to eight of the most important anti-SARS-CoV-2 drugs and drug candidates (including remdesivir, molnupiravir, and PF-07321332, the active compound in paxlovid), which is of timely relevance for the treatment of the increasing number of Omicron patients. Most importantly, we also found that the Omicron variant displays a reduced capability of antagonising the host cell interferon response. This provides a potential mechanistic explanation for the clinically observed reduced pathogenicity of Omicron variant viruses compared to Delta variant viruses.

sentences: Transcriptomic Analysis of Human Sensory Neurons in Painful Diabetic Neuropathy Reveals Inflammation and Neuronal Loss Pathological sensations caused by peripheral painful neuropathy occurring in Type 2 diabetes mellitus (T2DM) are often described as  sharp and  burning and are commonly spontaneous in origin. Proposed etiologies implicate dysfunction of nociceptive sensory neurons in dorsal root ganglia (DRG) induced by generation of reactive oxygen species, microvascular defects, and ongoing axonal degeneration and regeneration. To investigate the molecular mechanisms contributing to diabetic pain, DRGs were acquired postmortem from patients who had been experiencing painful diabetic peripheral neuropathy (DPN) and subjected to transcriptome analyses to identify genes contributing to pathological processes and neuropathic pain. DPN occurs in distal extremities resulting in the characteristic "glove and stocking" pattern. Accordingly, the L4 and L5 DRGs, which contain the perikarya of primary afferent neurons innervating the foot, were analyzed from five DPN patients and compared with seven controls. Transcriptom e analyses identified 844 differentially expressed genes. We observed increases in levels of inflammation-associated genes from macrophages in DPN patients that may contribute to increased pain hypersensitivity and, conversely, there were frequent decreases in neuronally-related genes. The elevated inflammatory gene profile and the accompanying downregulation of multiple neuronal genes provide new insights into intraganglionic pathology and mechanisms causing neuropathic pain in DPN patients with T2DM.

sentences: Schizophrenia-linked protein tSNARE1 regulates endolysosomal trafficking in cortical neurons TSNARE1, which encodes the protein tSNARE1, is a high-confidence gene candidate for schizophrenia risk, but nothing is known about its cellular or physiological function. We identified the major gene products of TSNARE1 and their cytoplasmic localization and function in endolysosomal trafficking in murine cortical neurons. We validated four primary isoforms of TSNARE1 expressed in human brain, all of which encode a syntaxin-like Qa SNARE domain. RNA-sequencing data from adult and fetal human brain suggested that the majority of tSNARE1 lacks a transmembrane domain that is thought to be necessary for membrane fusion. Live-cell imaging demonstrated that brain tSNARE1 isoforms localized to compartments of the endosomal network. The most abundant brain isoform, tSNARE1c, localized most frequently to Rab7+ late endosomal compartments. Expression of either tSNARE1b or tSNARE1c, which differ only in their inclusion or exclusion of a Myb-like domain, delayed the trafficking of known endosomal cargo, Neep21, into late endosomal and lysosomal compartments. These data suggest that tSNARE1 regulates endosomal trafficking in cortical neurons, likely through negatively regulating endocytic trafficking or maturation of early endosomes to late endosomes.

sentences: Computational identification and characterization of antigenic properties of Rv3899c of Mycobacterium tuberculosis and its interaction with Human leukocyte antigen (HLA) Tuberculosis (TB) is the second largest infectious disease that kills 1.2 million people annually worldwide. A rise in drug-resistant TB cases demands continued efforts towards the discovery & development of drugs and vaccines. In the recent past, though novel drugs have been added to the current TB regimen [1], research on new vaccine candidates needs a greater thrust. Secretory proteins of H37Rv are frequently studied for their antigenicity and their scope as protein subunit vaccines decrees further analysis. In this study, Rv3899c of H37Rv emerges as a potential vaccine candidate on its evaluation by several bioinformatics tools. It is a non-toxin, secretory protein with an  immunoglobulin-like fold which does not show similarity with a human protein. We found Rv3899c homologs in several mycobacterial species and its antigenic score (0.54) to compare well with the known immunogens such as ESAT-6 (0.56) and Rv1860 (0.52). Structural examination of Rv3899c predicted ten antigenic peptides, an accessibility profile of the antigenic determinants constituting B-cell epitope rich regions and a low Abundance of Antigenic Regions (AAR) value. Significantly, our study shows ESX-2 secretion system proteins and antigenic PE/PPE proteins of H37Rv as the interacting partners of Rv3899c. Further, molecular docking predicted Rv3899c to interact with human leukocyte antigen HLA-DRB1*04:01 through its antigenically conserved motif (RAAEQQRLQRIVDAVARQEPRISWAAGLRDDGTT). Interestingly, the binding affinity was observed to increase on citrullation of its Arg1 residue. Taken together, the computational characterization and predictive information suggest Rv3899c to be a promising TB vaccine candidate, which should be examined and validated experimentally.

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Defining marine bacterioplankton community assembly rules by contrasting the importance of environmental determinants and biotic interactions Bacterioplankton communities play major roles in governing marine productivity and biogeochemical cycling, yet what drives the relative influence of the types of deterministic ecological processes which result in diversity patterns remains unclear. Here we examine how differing deterministic processes (environmental factors and biotic interactions) drive temporal dynamics of bacterioplankton diversity at three different oceanographic time-series locations, spanning 15 degrees of latitude, which are each characterized by different environmental conditions and varying degrees of seasonality. Monthly surface samples, collected over a period of 5.5 years, were analyzed using 16S rRNA amplicon sequencing. The high and mid-latitude sites of Maria Island and Port Hacking were characterized by high and intermediate levels of environmental heterogeneity respectively, with both alpha (local) diversity (72 % and 24 % of total variation) and beta diversity (32 % and 30 %) patterns within bacterioplankton assemblages primarily explained by environmental determinants, including day length, ammonium, and mixed layer depth. In contrast, at North Stradbroke Island, a sub-tropical location where environmental conditions are less seasonally variable, interspecific interactions were of increased importance in structuring bacterioplankton diversity (alpha diversity: 33 %; beta diversity: 26 %) with environment only contributing 11 and 13 % to predicting diversity, respectively. Our results demonstrate that bacterioplankton diversity is the result of both deterministic environmental and biotic processes and that the importance of these different deterministic processes varies, potential in response to environmental heterogeneity.

ImportanceMarine bacterioplankton drives important biological processes, including the cycling of key nutrients or fixing atmospheric carbon. Therefore, to predict future climate scenarios its critical to model these communities accurately. Processes that drive bacterioplankton diversity patterns in the oceans however remain unresolved, with most studies focusing on deterministic environmental drivers, ie temperature or available inorganic nutrients. Biotic deterministic processes including interactions among individuals are also important for structuring diversity patterns, however, this is rarely included to predict bacterioplankton communities. We develop an approach for determining the relative contribution of environmental and potential biotic interactions that structure marine bacterioplankton at three series at different latitudes. Environmental factors best predicted temporal trends in bacterioplankton diversity at the two high latitude time series, while biotic influence was most apparent at the low latitude time series. Our results suggest environmental heterogeneity is an important attribute driving the contribution of varying deterministic influence of bacterioplankton diversity.

sentences: The MLR COMPASS-like complex is required to regulate triglyceride depletion and stress response in the Drosophila fat body MLR COMPASS-like complexes are highly conserved histone modifiers and enhancer regulators recruited by multiple transcription factors during differentiation and development, including lineage-determining factors, nuclear receptors, and developmental signaling pathway effectors. While the essential functions of MLR complexes during differentiation-associated transcriptional reprogramming has been well-explored, roles that these complexes play during reprogramming events in terminally differentiated cells remain understudied. We determined that the Drosophila MLR complex is required in fat body adipocytes for proper regulation of the triglyceride depletion rate during non-feeding periods, including metamorphosis and adult starvation. Transcriptome analysis revealed that the complex plays a critical role in controlling stress-related reprogramming in these cells, suggesting that the metabolic phenotypes are indirect effects of dysregulated stress transcription. Furthermore, our evidence suggests that the complex interacts with Foxo and Relish (Nf-{kappa}b) pathways and is required for proper expression of their targets. This investigation further elucidates the necessary functions of MLR complexes in regulating transcriptional reprogramming in terminally differentiated cells as well as suggests novel binding partners. Apparent roles for these complexes in the proper regulation of stress-induced transcription implies new mechanisms involved in cancer and other human diseases associated with MLR subunit mutation.

sentences: Reanalysis of double EphA3 knockin maps in mouse shows the importance of chemoaffinity in topographic map formation Double visual maps in the EphA3 knockin mouse have been used extensively to investigate the mechanisms for the formation of ordered nerve connections. Here, EphA3+ and EphA3-retinal ganglion cells, forming a mosaic, each make a separate map on the superior colliculus. We constructed ordered 2D maps from EphA3 knockin Fourier-based intrinsic imaging data. Over both EphA3+/- and EphA3+/+ genotypes, the extent and location in the visual field of each projection of a double map vary considerably. Aided by computer modelling results, we suggest that all EphA3 knockin maps are examples of a single type, with the amount of EphA3 in EphA3+ retinal cells varying between animals. Previous analysis of the same dataset suggested that EphA3+/- maps represent an unstable transition between single (wild type) and double (EphA3+/+) maps, reflecting a balance between molecular and electrical signalling. In contrast, our analysis highlights the importance of molecular-based cues in establishing the retinotopic map.

sentences: TGFβ signalling is required to maintain pluripotency of human naïve pluripotent stem cells The signalling pathways that maintain primed human pluripotent stem cells (hPSCs) have been well characterised, revealing a critical role for TGF{beta}/Activin/Nodal signalling. In contrast, the signalling requirements of naive human pluripotency have not been fully established. Here, we demonstrate that TGF{beta} signalling is required to maintain naive hPSCs. The downstream effector proteins - SMAD2/3 - bind common sites in naive and primed hPSCs, including shared pluripotency genes. In naive hPSCs, SMAD2/3 additionally bind to active regulatory regions near to naive pluripotency genes. Inhibiting TGF{beta} signalling in naive hPSCs causes the downregulation of SMAD2/3-target genes and pluripotency exit. Single-cell analyses reveal that naive and primed hPSCs follow different transcriptional trajectories after inhibition of TGF{beta} signalling. Primed hPSCs differentiate into neuroectoderm cells, whereas naive hPSCs transition into trophectoderm. These results establish that there is a continuum for TGF{beta} pathway function in human pluripotency spanning a developmental window from naive to primed states.

sentences: cDNA-detector: Detection and removal of cDNA contamination in DNA sequencing libraries Exogenous cDNA introduced into an experimental system, either intentionally or accidentally, can appear as added read coverage over that gene in next-generation sequencing libraries derived from this system. If not properly recognized and managed, this cross-contamination with exogenous signal can lead to incorrect interpretation of research results. Yet, this problem is not routinely addressed in current sequence processing pipelines. Here, we present cDNA-detector, a computational tool to identify and remove exogenous cDNA contamination in DNA sequencing experiments. We apply cDNA-detector to several highly-cited public databases (TCGA, ENCODE, NCBI SRA) and show that contaminant genes appear in sequencing experiments where they lead to incorrect coverage peak calls. Our findings highlight the importance of sensitive detection and removal of contaminant cDNA from NGS libraries before downstream analysis.

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24-nt phasiRNAs move from tapetal to meiotic cells in maize anthers O_LIIn maize, 24-nt phased, secondary small interfering RNAs (phasiRNAs) are abundant in meiotic stage anthers, but their distribution and functions are not precisely known.
C_LIO_LIUsing laser capture microdissection, we analyzed tapetal cells, meiocytes, and other somatic cells at several stages of anther development to establish the timing of 24-PHAS precursor transcripts and the 24-nt phasiRNA products.
C_LIO_LIBy integrating RNA and small RNA profiling plus single-molecule and small RNA FISH (smFISH or sRNA-FISH) spatial detection, we demonstrate that the tapetum is the primary site of 24-PHAS precursor and Dcl5 transcripts and the resulting 24-nt phasiRNAs. Interestingly, 24-nt phasiRNAs accumulate in all cell types, with the highest levels in meiocytes, followed by tapetum.
C_LIO_LIOur data support the conclusion that 24-nt phasiRNAs are mobile from tapetum to meiocytes and to other somatic cells. We discuss possible roles for 24-nt phasiRNAs in anther cell types.
C_LI

sentences: ZEB2 transduces HIF1α dependent regulation of Transglutaminase 2 in glomerular podocytes Glomerular podocytes are instrumental in ensuring glomerular permselectivity and regulating the integrity of glomerular biology. However, podocytes are vulnerable to various noxious stimuli such as hypoxia, and podocyte injury presented with glomerulosclerosis and impaired kidney function. The mechanism of hypoxia-induced podocyte injury vis-a-vis glomerulosclerosis has remained enigmatic. Hypoxia inducible factor 1 (HIF1) that transduces hypoxic adaptations, induces Transglutaminase 2 (TG2), a calcium dependent enzyme that catalyzes intramolecular {varepsilon}-({gamma}-glutamyl) lysine cross-links of extracellular matrix (ECM) proteins. In this study, we investigated the mechanism of regulation of TG2 by HIF1. Stabilization of HIF1 by FG4592 (Roxadustat) and physiological hypoxia, resulted in elevated expression of ZEB2 (zinc-finger E-box-homeobox 2) and its downstream target TRPC6 (transient receptor potential channel 6). ZEB2 transcriptionally activates TG2 expression, whereas, via TRPC6, it induces calcium influx, inturn it increases the TG2 activity. Blocking the TRPC6 action or suppressing its expression only partially attenuated FG4592 induced TG2 activity, whereas suppression of ZEB2 expression significantly abolished TG2 activity. This study demonstrates that stabilization of HIF1 stimulates both TG2 expression and activity, whereas abrogation of HIF1 by metformin prevented HIF1 regulated TG2 and consequent glomerular injury.

sentences: Human genetic diversity modifies therapeutic gene editing off-target potential CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to predict and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and {beta}-thalassemia. We find that the top predicted off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF)=4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels ([~]9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. This report illustrates how genetic variation may modify the genomic outcomes of therapeutic gene editing and provides a simple tool for comprehensive off-target assessment.

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Molecular mechanisms of Plasmodium development in male and female Anopheles mosquitoes Vector competence influences the ability of Anopheles mosquitoes to transmit Plasmodium parasites. The aim of this study was to determine the competence of male and female Anopheles mosquitoes to support the development of Plasmodium parasites. Male and female A. stephensi (STE2 strain) were infected with in vitro-cultured P. berghei (ANKA strain) ookinetes. We found that the number of oocysts produced was higher in males than in females. RNA-seq analysis of male and female mosquitoes injected with P. berghei ookinetes showed that predominantly genes of unknown function changed in expression levels in response to ookinete infection; however, further studies are required to elucidate their functions. Moreover, male mosquitoes were injected with in vitro-cultured P. falciparum (3D7 strain) gametocytes or zygotes. The development of P. falciparum in males was detected using nested polymerase chain reaction. We found the DNA content was higher on day 15 than on day 0, indicating that P. falciparum developed in the mosquito hemocoel. This study revealed promising new mechanisms underlying the interactions between Plasmodium and mosquitoes.

Author SummaryAnopheles mosquitoes transmit Plasmodium parasites that cause malaria disease. Vector competence is the ability of vectors to support pathogen development. The aim of this study was to determine the competence of male and female mosquitoes for Plasmodium parasites. Oocysts were formed in both male and female mosquitoes injected with ookinetes from in vitro-cultured Plasmodium berghei, which causes malaria in mice. The number of oocysts was higher in males, indicating that the competence in males was higher than that in females. RNA-seq analysis showed that the expression of genes of unknown function was highly variable in males and females. The genes defining the competence factors need further study, but the results indicate novel genes may be discovered. Furthermore, the development of Plasmodium falciparum, which causes the most severe malaria in humans, in male mosquitoes injected with in vitro-cultured gametocytes or zygotes was detected. As male mosquitoes do not suck blood, this method may allow us to experiment with Plasmodium more safely.

sentences: Selection and structural characterisation of anti-TREM2 scFvs that reduce levels of shed ectodomain Single point mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration including Alzheimers disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signalling cascades necessary for neuroprotection. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterised. Here, we present phage display generated scFv antibody binders to human TREM2 ectodomain. Cocrystal structures revealed the binding of two scFvs to an epitope on the globular TREM2 domain distal to the putative ligand-binding site. Enhanced functional activity was observed for oligomeric scFv species which inhibited the production of soluble TREM2 in a HEK293 cell model. We hope that detailed characterisation of their epitopes and properties will facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.

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An Infant Sleep Electroencephalographic Marker of Thalamocortical Connectivity Predicts Behavioral Outcome in Late Infancy Infancy represents a critical period during which thalamocortical brain connections develop and mature. Deviations in the maturation of thalamocortical connectivity are linked to neurodevelopmental disorders. There is a lack of early biomarkers to detect and localize neuromaturational deviations, which can be overcome with mapping through high-density electroencephalography (hdEEG) assessed in sleep. Specifically, slow waves and spindles in non-rapid eye movement (NREM) sleep are generated by the thalamocortical system, and their characteristics, slow wave slope and spindle density, are closely related to neuroplasticity and learning. Recent studies further suggest that information processing during sleep underlying sleep-dependent learning is promoted by the temporal coupling of slow waves and spindles, yet slow wave-spindle coupling remains unexplored in infancy. Thus, we evaluated three potential biomarkers: 1) slow wave slope, 2) spindle density, and 3) the temporal coupling of slow waves with spindles. We use hdEEG to first examine the occurrence and spatial distribution of these three EEG features in healthy infants and second to evaluate a predictive relationship with later behavioral outcomes. We report four key findings: First, infants EEG features appear locally: slow wave slope is maximal in occipital and frontal areas, whereas spindle density is most pronounced frontocentrally. Second, slow waves and spindles are temporally coupled in infancy, with maximal coupling strength in the occipital areas of the brain. Third, slow wave slope, spindle density, and slow wave-spindle coupling are not associated with concurrent behavioral status (6 months). Fourth, spindle density in central and frontocentral regions at age 6 months predicts later behavioral outcomes at 12 and 24 months. Neither slow wave slope nor slow wave-spindle coupling predict behavioral development. Our results propose spindle density as an early EEG biomarker for identifying thalamocortical maturation, which can potentially be used for early diagnosis of neurodevelopmental disorders in infants. These findings are complemented by our companion paper that demonstrates the linkage of spindle density to infant nighttime movement, framing the possible role of spindles in sensorimotor microcircuitry development. Together, our studies suggest that early sleep habits, thalamocortical maturation, and behavioral outcome are closely interwoven. A crucial next step will be to evaluate whether early therapeutic interventions may be effective to reverse deviations in identified individuals at risk.

HighlightsO_LISlow waves and spindles occur in a temporally coupled manner in infancy
C_LIO_LISlow wave slope, spindle density, and slow wave-spindle coupling are not related to concurrent behavioral development
C_LIO_LISpindle density at 6 months predicts behavioral status at 12 and 24 months
C_LIO_LISlow wave slope and slow wave-spindle coupling are not predictive of behavioral development
C_LI

sentences: CoCoA: Conditional Correlation Models with Association Size SO_SCPLOWUMMARYC_SCPLOWMany scientific questions can be formulated as hypotheses about conditional correlations. For instance, in tests of cognitive and physical performance, the trade-off between speed and accuracy motivates study of the two variables together. A natural question is whether speed-accuracy coupling depends on other variables, such as sustained attention. Classical regression techniques, which posit models in terms of covariates and outcomes, are insufficient to investigate the effect of a third variable on the symmetric relationship between speed and accuracy. In response, we propose CoCoA (Conditional Correlation Model with Association Size), a likelihood-based statistical framework to estimate the conditional correlation between speed and accuracy as a function of additional variables. We propose novel measures of the association size, which are analogous to effect sizes on the correlation scale, while adjusting for confound variables. In simulation studies, we compare likelihood-based estimators of conditional correlation to semi-parametric estimators adapted from genome association studies, and find that the former achieves lower bias and variance under both ideal settings and model assumption misspecification. Using neurocognitive data from the Philadelphia Neurodevelopmental Cohort, we demonstrate that greater sustained attention is associated with stronger speed-accuracy coupling in a complex reasoning task while controlling for age. By highlighting conditional correlations as the outcome of interest, our model provides complementary insights to traditional regression modelling and partitioned correlation analyses.

sentences: Melanism in a wild sifaka population: Darker where cold and fragmented Pigmentation is one of the most striking examples of diversity in the natural world. Mainly, pelage (hair/fur) pigmentation provides a substrate for selection (i.e., crypsis, signaling, thermoregulation) and is capable of rapid change. Thus, this trait may be the one potential early signal of adaptation (or maladaptation) in wild primate populations. However, most of our hypotheses on the forces responsible for primate pelage pigmentation are based solely on macro-evolutionary studies. Here, we characterize pelage color and pattern variation within a population of wild primates, diademed sifakas (Propithecus diadema), exhibiting striking diversity in coloration (melanic to tri-colored). Our approach jointly assesses climate and pelage variation across the region. We score pelage using a semi-quantitative methodology. We then test if pelage variation is associated with climatic or demographic factors (i.e., sex-class, age-class) across the Tsinjoarivo forest, Madagascar. We find darker bodies and less complex faces occur in colder and more fragmented forests. We explore three hypotheses that may explain this phenotypic pattern: isolation by distance, an environmental gradient, or unique local adaptation. Importantly, each scenario signals the need for enhanced conservation of diademed sifakas in the Tsinjoarivo forest. More studies on primate pigmentation in wild populations will be needed to contextualize if this pattern is exceptional or typical. It is likely that in other primate populations pigmentation may also foretell of adaptation or environmental mismatch.

sentences: DLPacker: Deep Learning for Prediction of Amino Acid Side Chain Conformations in Proteins AO_SCPLOWBSTRACTC_SCPLOWPrediction of side chain conformations of amino acids in proteins (also termed  packing) is an important and challenging part of protein structure prediction with many interesting applications in protein design. A variety of methods for packing have been developed but more accurate ones are still needed. Machine learning (ML) methods have recently become a powerful tool for solving various problems in diverse areas of science, including structural biology. In this work we evaluate the potential of Deep Neural Networks (DNNs) for prediction of amino acid side chain conformations. We formulate the problem as image-to-image transformation and train a U-net style DNN to solve the problem. We show that our method outperforms other physics-based methods by a significant margin: reconstruction RMSDs for most amino acids are about 20% smaller compared to SCWRL4 and Rosetta Packer with RMSDs for bulky hydrophobic amino acids Phe, Tyr and Trp being up to 50% smaller.

sentences: OzTracs: Optical Osmolality Reporters Engineered from Mechanosensitive Ion Channels Interactions between physical forces and membrane proteins underpin many forms of environmental sensation and acclimation. Microbes survive sudden osmotic stresses with the help of mechanically gated ion channels and osmolyte transporters. Plant mechanosensitive ion channels have been shown to function in defense signaling. Here, we engineered genetically encoded osmolality sensors (OzTracs) by fusing green fluorescent protein spectral variants to the mechanosensitive ion channels MscL from E. coli or MSL10 from A. thaliana. When expressed in yeast cells, OzTrac sensors reported osmolality changes as a proportional change in emission ratio of the two fluorescent protein domains. Live-cell imaging revealed accumulation of fluorescent sensors in internal aggregates presumably derived from the endomembrane system. Thus, OzTrac sensors serve as osmolality-dependent reporters through an indirect mechanism, such as effects on molecular crowding or fluorophore solvation.

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The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs involved in Chloroplast Development The Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7 or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL and their barley homologs and to the function of the putative cTPs of CIA2 and CIL.

HIGHLIGHTThe nucleus-localized CCT domain proteins CIA2 and CIL in Arabidopsis and the homologous chloroplast-localized HvCMF3 and HvCMF7 in barley retained partially overlapping functions in chloroplast development.

sentences: Huntingtin turnover: Modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550 Huntingtons disease (HD) is a neurodegerative disorder caused by an inherited unstable HTT CAG repeat that expands further, thereby eliciting a disease process that may be initiated by polyglutamine-expanded huntingtin or a short polyglutamine-product. Phosphorylation of selected candidate residues is reported to mediate polyglutamine-fragment degradation and toxicity. Here to support the discovery of phospho-sites involved in the life-cycle of (full-length) huntingtin, we employed mass spectrometry-based phosphoproteomics to systematically identify sites in purified huntingtin and in the endogenous protein, by proteomic and phospho-proteomic analyses of members of an HD neuronal progenitor cell panel. Our results bring total huntingtin phospho-sites to 95, with more located in the N-HEAT domain relative to numbers in the Bridge and C-HEAT domains. Moreover, phosphorylation of C-HEAT Ser2550 by cAMP-dependent protein kinase (PKA), the top hit in kinase activity screens, was found to hasten huntingtin degradation, such that levels of the catalytic subunit (PRKACA) were inversely related to huntingtin levels. Taken together these findings highlight categories of phospho-sites that merit further study and provide a phospho-site kinase pair (pSer2550-PKA) with which to investigate the biological processes that regulate huntingtin degradation and thereby influence the steady state levels of huntingtin in HD cells.

sentences: The origins and molecular evolution of SARS-CoV-2 lineage B.1.1.7 in the UK The first SARS-CoV-2 variant of concern (VOC) to be designated was lineage B.1.1.7, later labelled by the World Health Organisation (WHO) as Alpha. Originating in early Autumn but discovered in December 2020, it spread rapidly and caused large waves of infections worldwide. The Alpha variant is notable for being defined by a long ancestral phylogenetic branch with an increased evolutionary rate, along which only two sequences have been sampled. Alpha genomes comprise a well-supported monophyletic clade within which the evolutionary rate is more typical of SARS-CoV-2. The Alpha epidemic continued to grow despite the continued restrictions on social mixing across the UK, and the imposition of new restrictions, in particular the English national lockdown in November 2020. While these interventions succeeded in reducing the absolute number of cases, the impact of these non-pharmaceutical interventions was predominantly to drive the decline of the SARS-CoV-2 lineages which preceded Alpha. We investigate the only two sampled sequences that fall on the branch ancestral to Alpha. We find that one is likely to be a true intermediate sequence, providing information about the order of mutational events that led to Alpha. We explore alternate hypotheses that can explain how Alpha acquired a large number of mutations yet remained largely unobserved in a region of high genomic surveillance: an under-sampled geographical location, a non-human animal population, or a chronically-infected individual. We conclude that the last hypothesis provides the best explanation of the observed behaviour and dynamics of the variant, although we find that the individual need not be immunocompromised, as persistently-infected immunocompetent hosts also display a higher within-host rate of evolution. Finally, we compare the ancestral branches and mutation profiles of other VOCs to each other, and identify that Delta appears to be an outlier both in terms of the genomic locations of its defining mutations, and its lack of rapid evolutionary rate on the ancestral branch. As new variants, such as Omicron, continue to evolve (potentially through similar mechanisms) it remains important to investigate the origins of other variants to identify ways to potentially disrupt their evolution and emergence.

sentences: Oligodendrocytes depend on MCL-1 to prevent spontaneous apoptosis and white matter degeneration Neurologic disorders often disproportionately affect specific brain regions, and different apoptotic mechanisms may contribute to white matter pathology in leukodystrophies or gray matter pathology in poliodystrophies. We previously showed that neural progenitors that generate cerebellar gray matter depend on the anti-apoptotic protein BCL-xL. Conditional deletion of Bcl-xL in these progenitors produces spontaneous apoptosis and cerebellar hypoplasia, while similar conditional deletion of Mcl-1 produces no phenotype. Here, we show that, in contrast, postnatal oligodendrocytes depend on MCL-1. We found that brain-wide Mcl-1 deletion caused apoptosis specifically in mature oligodendrocytes while sparing astrocytes and oligodendrocyte precursors, resulting in impaired myelination and progressive white matter degeneration. Disabling apoptosis through co-deletion of Bax or Bak rescued white matter degeneration, implicating the intrinsic apoptotic pathway in Mcl-1-dependence. Bax and Bak co-deletions rescued different aspects of the Mcl-1-deleted phenotype, demonstrating their discrete roles in white matter stability. MCL-1 protein abundance was reduced in eif2b5-mutant mouse model of the leukodystrophy vanishing white matter disease (VWMD), suggesting the potential for MCL-1 deficiency to contribute to clinical neurologic disease. Our data show that oligodendrocytes require MCL-1 to suppress apoptosis, implicate MCL-1 deficiency in white matter pathology, and suggest apoptosis inhibition as a leukodystrophy therapy.

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Profile of basal cell carcinoma mutations and copy number alterations - focus on gene-associated noncoding variants Basal cell carcinoma (BCC) of the skin is the most common cancer in humans, characterized by the highest mutation rate among cancers, and is mostly driven by mutations in genes involved in the hedgehog pathway. To date, almost all BCC genetic studies have focused exclusively on protein-coding sequences; therefore, the impact of noncoding variants on the BCC genome is unrecognized. In this study, with the use of whole-exome sequencing of 27 tumor/normal pairs of BCC samples, we performed an analysis of somatic mutations in both protein-coding sequences and gene-associated noncoding regions, including 5UTRs, 3UTRs, and exon-adjacent intron sequences. Separately, in each region, we performed hotspot identification, mutation enrichment analysis, and cancer driver identification with OncodriveFML. Additionally, we performed a whole-genome copy number alteration analysis with GISTIC2. Of the >80,000 identified mutations, [~]50% were localized in noncoding regions. The results of the analysis generally corroborated the previous findings regarding genes mutated in coding sequences, including PTCH1, TP53, and MYCN, but more importantly showed that mutations were also clustered in specific noncoding regions, including hotspots. Some of the genes specifically mutated in noncoding regions were identified as highly potent cancer drivers, of which BAD had a mutation hotspot in the 3UTR, DHODH had a mutation hotspot in the Kozak sequence in the 5UTR, and CHCHD2 frequently showed mutations in the 5UTR. All of these genes are functionally implicated in cancer-related processes (e.g., apoptosis, mitochondrial metabolism, and de novo pyrimidine synthesis) or the pathogenesis of UV radiation-induced cancers. We also found that the identified BAD and CHCHD2 mutations frequently occur in melanoma but not in other cancers via The Cancer Genome Atlas analysis. Finally, we identified frequent deletion of chr9q, encompassing PTCH1, and unreported frequent copy number gain of chr9p, encompassing the genes encoding the immune checkpoint ligands PD-L1 and PD-L2. In conclusion, this study is the first systematic analysis of coding and noncoding mutations in BCC and provides a strong basis for further analyses of the variants in BCC and cancer in general.

Author summaryThe study is the first systematic analysis of both coding and noncoding mutations in basal cell carcinoma (BCC), the most common and the most highly mutated human cancer. Noncoding mutations accounted for [~]50% ([~]40K mutations) of all mutations detected by the standard WES approach. Among the genes frequently mutated in noncoding regions are: BAD with a hotspot in the 3UTR, DHODH with a hotspot in the Kozak sequence, and CHCHD2 with mutations in 5UTR, all genes functionally related to cell metabolism and apoptosis. Analysis of copy number alterations showed frequent chr9q deletion, encompassing PTCH1 (the key BCC tumor suppressor), and frequent copy number gain of chr9p, encompassing the genes of the immune checkpoint proteins PD-L1 and PD-L2.

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Disentangling environmental effects in microbial association networks BackgroundEcological interactions among microorganisms are fundamental for ecosystem function, yet they are mostly unknown or poorly understood. High-throughput-omics can indicate microbial interactions through associations across time and space, which can be represented as association networks. Associations could result from either ecological interactions between microorganisms, or from environmental selection, where the associations are environmentally-driven. Therefore, before downstream analysis and interpretation, we need to distinguish the nature of the association, particularly if it is due to environmental selection or not.

ResultsWe present EnDED (Environmentally-Driven Edge Detection), an implementation of four approaches as well as their combination to predict which links between microorganisms in an association network are environmentally-driven. The four approaches are Sign Pattern, Overlap, Interaction Information, and Data Processing Inequality. We tested EnDED on networks from simulated data of 50 microorganisms. The networks contained on average 50 nodes and 1087 edges, of which 60 were true interactions but 1026 false associations (i.e. environmentally-driven or due to chance). Applying each method individually, we detected a moderate to high number of environmentally-driven edges--87% Sign Pattern and Overlap, 67% Interaction Information, and 44% Data Processing Inequality. Combining these methods in an intersection approach resulted in retaining more interactions, both true and false (32% of environmentally-driven associations). After validation with the simulated datasets, we applied EnDED on a marine microbial network inferred from 10 years of monthly observations of microbial-plankton abundance. The intersection combination predicted that 8.3% of the associations were environmentally-driven, while individual methods predicted 24.8% (Data Processing Inequality), 25.7% (Interaction Information), and up to 84.6% (Sign Pattern as well as Overlap). The fraction of environmentally-driven edges among negative microbial associations in the real network increased rapidly with the number of environmental factors.

ConclusionsTo reach accurate hypotheses about ecological interactions, it is important to determine, quantify, and remove environmentally-driven associations in marine microbial association networks. For that, EnDED offers up to four individual methods as well as their combination. However, especially for the intersection combination, we suggest using EnDED with other strategies to reduce the number of false associations and consequently the number of potential interaction hypotheses.

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Simulated hemiparesis increases optimal spatiotemporal gait asymmetry but not metabolic cost Several neuromuscular impairments, such as weakness (hemiparesis), occur after an individual has a stroke, and these impairments primarily affect one side of the body more than the other. Predictive musculoskeletal modeling presents an opportunity to investigate how a specific impairment affects gait performance post-stroke. Therefore, our aim was to use to predictive simulation to quantify the spatiotemporal asymmetries and changes to metabolic cost that emerge when muscle strength is unilaterally reduced. We also determined how forced spatiotemporal symmetry affects metabolic cost. We modified a 2-D musculoskeletal model by uniformly reducing the peak isometric muscle force in all left-limb muscles. We then solved optimal control simulations of walking across a range of speeds by minimizing the sum of the cubed muscle excitations across all muscles. Lastly, we ran additional optimizations to test if reducing spatiotemporal asymmetry would result in an increase in metabolic cost. Our results showed that the magnitude and direction of effort-optimal spatiotemporal asymmetries depends on both the gait speed and level of weakness. Also, the optimal metabolic cost of transport was 1.25 m/s for the symmetrical and 20% weakness models but slower (1.00 m/s) for the 40% and 60% weakness models, suggesting that hemiparesis can account for a portion of the slower gait speed seen in people post-stroke. Adding spatiotemporal asymmetry to the cost function resulted in small increases (~4%) in metabolic cost. Overall, our results indicate that spatiotemporal asymmetry may be optimal for people post-stroke, who have asymmetrical neuromuscular impairments. Additionally, the effect of speed and level of weakness on spatiotemporal asymmetry may explain the well-known heterogenous distribution of spatiotemporal asymmetries observed in the clinic. Future work could extend our results by testing the effects of other impairments on optimal gait strategies, and therefore build a more comprehensive understanding of the gait patterns in people post-stroke.

Author SummaryA stroke causes damage to the brain. This typically results in several changes to the nervous and muscular (neuromuscular) system that change how people post-stroke tend to walk and perform other tasks. Individuals post-stroke tend to walk with an asymmetrical motion and expend more energy while walking than other age-matched individuals. We still do not understand how each specific change to the neuromuscular system is linked with changes in walking patterns, in part because it is difficult to test one individual change at a time in people. Instead, we can use a mathematical model of the musculoskeletal system that represents the individual changes to the muscular system that occur in people post-stroke. In this study, we modeled how a common change in people post-stroke (muscle weakness) can impact walking patterns. We found that the level of weakness and the walking speed affect the asymmetrical walking patterns of our models, but do not change the total energy cost. Overall, our study is one step towards better understanding how neuromuscular changes in people post-stroke affects walking patterns. This knowledge could be applied to identify rehabilitation strategies that are most likely to improve walking in people post-stroke.

sentences: Towards a general framework for modeling large-scale biophysical neuronal networks: a full-scale computational model of the rat dentate gyrus Large-scale computational models of the brain are necessary to accurately represent anatomical and functional variability in neuronal biophysics across brain regions and also to capture and study local and global interactions between neuronal populations on a behaviorally-relevant temporal scale. We present the methodology behind and an initial implementation of a novel open-source computational framework for construction, simulation, and analysis of models consisting of millions of neurons on high-performance computing systems, based on the NEURON and CoreNEURON simulators (Carnevale and Hines, 2006, Kumbhar et al., 2019). This framework uses the HDF5 data format and software library (HDF Group, 2021) and includes a data format for storing morphological, synaptic, and connectivity information of large neuronal network models, and an accompanying open-source software library that provides efficient, scalable parallel storage and MPI-based data movement capabilities. We outline our approaches for constructing detailed large-scale biophysical models with topographical connectivity and input stimuli, and present simulation results obtained with a full-scale model of the dentate gyrus constructed with our framework. The model generates sparse and spatially selective population activity that fits well with in-vivo experimental data. Moreover, our approach is fully general and can be applied to modeling other regions of the hippocampal formation in order to rapidly evaluate specific hypotheses about large-scale neural architectural features.

sentences: The Epstein-Barr virus ubiquitin deconjugase BPLF1 regulates the activity of Topoisomerase II during virus replication Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 selectively inhibited the ubiquitination of TOP2 following treatment with topoisomerase poisons, interacted with TOP2 and TOP2{beta} in co-immunoprecipitation and in vitro pull-down, stabilized Etoposide-trapped TOP2 cleavage complexes (TOP2cc) and promoted TOP2 SUMOylation, which halted the DNA-damage response and reduced Etoposide toxicity. Induction of the productive virus cycle promoted the accumulation of TOP2{beta}cc, enhanced TOP2{beta} SUMOylation, and reduced Etoposide toxicity in lymphoblastoid cell lines carrying recombinant EBV encoding the active enzyme. Attenuation of this phenotype upon expression of a catalytic mutant BPLF1-C61A impaired viral DNA synthesis and virus release. These findings highlight a previously unrecognized function of BPLF1 in promoting non-proteolytic pathways for TOP2cc debulking that favor cell survival and virus production.

sentences: Multilocus adaptation to vaccination Pathogen adaptation to public health interventions, such as vaccination, may take tortuous routes and involve multiple mutations at distinct locations in the pathogen genome, acting on distinct phenotypic traits. Despite its importance for public health, how these multilocus adaptations jointly evolve is poorly understood. Here we consider the joint evolution of two adaptations: the pathogens ability to escape the vaccine-induced immune response and adjustments to the pathogens virulence and transmissi-bility. We elucidate the role played by epistasis and recombination, with an emphasis on the different protective effects of vaccination. We show that vaccines reducing transmission and/or increasing clearance generate positive epistasis between the vaccine-escape and virulence alleles, favouring strains that carry both mutations, whereas vaccines reducing virulence mortality generate negative epistasis, favouring strains that carry either mutation, but not both. High rates of recombination can affect these predictions. If epistasis is positive, frequent recombination can lead to the sequential fixation of the two mutations and prevent the transient build-up of more virulent escape strains. If epistasis is negative, frequent recombination between loci can create an evolutionary bistability, such that whichever adaptation is more accessible tends to be favoured in the long-term. Our work provides a timely alternative to the variant-centered perspective on pathogen adaptation and captures the effect of different types of vaccines on the interference between multiple adaptive mutations.

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CD72-semaphorin3A axis: a possible new player in immune regulation Semaphorin3A (sema3A) inhibits the activity of B and T cells in autoimmune diseases such as Systemic Lupus Erythematosus (SLE). We have now found that CD72 functions as a novel sema3A binding and signal-transducing receptor. These functions of CD72 are independent of the known sema3A receptor neuropilin-1 (NRP-1). We find that sema3A induces the phosphorylation of CD72 on tyrosine residues and the association of CD72 with SHP-1 and SHP-2. In contrast, sema4D/CD100 inhibits these functions. sema3A signals mediated by CD72, inhibit the phosphorylation of STAT-4 and HDAC-1 and induce the phosphorylation of p38-MAPK and PKC-theta in B-cells derived B-lymphoblastoid (BLCL) cells lacking NRP-1 expression, and in primary B-cells isolated from either healthy donors or SLE (Systemic Lupus Erythematosus) patients. We have also generated a modified truncated sema3A (T-sema3A) which cannot signal via NRP-1 yet still activates inhibitory CD72 signaling. We propose that T-sema3A may have potential as a possible therapeutic for autoimmune diseases such as SLE.

One Sentence SummaryCD72 found as a novel sema3A receptor transduces inhibitory signals in Bcells. A modified sema3A can be used to treat autoimmunity.

sentences: Impact of an Urban Sanitation Intervention on Enteric Pathogen Detection in Soils Environmental fecal contamination is common in many low-income cities, contributing to a high burden of enteric infections and associated negative sequelae. To evaluate the impact of a shared onsite sanitation intervention in Maputo, Mozambique on enteric pathogens in the domestic environment, we collected 179 soil samples at shared latrine entrances from intervention (n= 49) and control (n= 51) compounds during baseline (pre-intervention) and after 24 months (post-intervention) as part of the Maputo Sanitation Trial. We tested soils for the presence of nucleic acids associated with 20 enteric pathogens using a multiplex reverse transcription qPCR platform. We detected at least one pathogen-associated target in 91% (163/179) of soils and a median of 3 (IQR=1.5, 5) pathogens. Using a difference-in-difference analysis and adjusting for compound population, visibly wet soil, sun exposure, wealth, temperature, animal presence, and visible feces, we estimate the intervention reduced the probability of [≥]1 pathogen detected by 15% (adjusted prevalence ratio, aPR=0.85; 95% CI: 0.70, 1.0) and the total number of pathogens detected by 35% (aPR =0.65; 0.44, 0.95) in soil 24 months following the intervention. These results suggest that the intervention reduced the presence of some fecal contamination in the domestic environment, but pathogen detection remained prevalent 24-months following the introduction of new latrines.

sentences: Genomic patterns of transcription-replication interactions in mouse primary B cells Conflicts between transcription and replication machinery are a potent source of replication stress and genome stability; however, no technique currently exists to identify endogenous genomic locations prone to transcription-replication interactions. Here, we report a novel method to identify genomic loci prone to transcription-replication interactions termed transcription-replication immunoprecipitation on nascent DNA sequencing, TRIPn-Seq. TRIPn-Seq employs the sequential immunoprecipitation of RNA polymerase 2 phosphorylated at serine 5 (RNAP2s5) followed by enrichment of nascent DNA previously labeled with bromodeoxyuridine. Using TRIPn-Seq, we mapped 1,009 unique transcription-replication interactions (TRIs) in mouse primary B cells characterized by a bimodal pattern of RNAP2s5, bidirectional transcription, an enrichment of RNA:DNA hybrids, and a high probability of forming G-quadruplexes. While TRIs themselves map to early replicating regions, they exhibit enhanced Replication Protein A association and replication fork termination, marks of replication stress. TRIs colocalize with double-strand DNA breaks, are enriched for deletions, and accumulate mutations in tumors. We propose that replication stress at TRIs induces mutations potentially contributing to age-related disease, as well as tumor formation and development.

sentences: Body representation plasticity is altered in Developmental Coordination Disorder Developmental Coordination Disorder (DCD) is a pathological condition characterized by impaired motor skills. Current theories advance that a deficit of the internal models is mainly responsible for DCD childrens altered behavior. Yet, accurate movement execution requires not only correct movement planning, but also integration of sensory feedback into body representation for action (Body Schema) to update the state of the body. Here we advance and test the hypothesis that the plasticity of this body representation is altered in DCD. To probe Body Schema (BS) plasticity, we submitted a well-established tool-use paradigm to seventeen DCD children, required to reach for an object with their hand before and after tool use, and compared their movement kinematics to that of a control group of Typically Developing (TD) peers. We also asked both groups to provide explicit estimates of their arm length to probe plasticity of their Body Image (BI). Results revealed that DCD children explicitly judged their arm shorter after tool use, showing changes in their BI comparable to their TD peers. Unlike them, though, DCD did not update their implicit BS estimate: kinematics showed that tool use affected their peak amplitudes, but not their latencies. Remarkably, the kinematics of tool use showed that the motor control of the tool was comparable between groups, both improving with practice, confirming that motor learning abilities are preserved in DCD. This study thus brings evidence in favor of an alternative theoretical account of the DCD etiology. Our findings point to a deficit in the plasticity of the body representation used to plan and execute movements. Though not mutually exclusive, this widens the theoretical perspective under which DCD should be considered: DCD may not be limited to a problem affecting the internal models and their motor functions, but may concern the state of the effector they have to use.

sentences: LncRNA XIST knockdown suppresses cell proliferation and promotes apoptosis in diabetic cataract via miR-34a/SMAD2 axis Emerging evidence has manifested that long non-coding RNAs (lncRNAs) played critical roles in diabetes. The present research aimed to investigate the role and mechanism of XIST on proliferation, migration and apoptosis in diabetic cataract (DC). In the present study, lens epithelial cells (SRA01/04) were treated by high glucose (HG). The levels of XIST, miR-34a and SMAD2 were examined by RT-qPCR. MTT, transwell, wound healing and TUNEL assays were employed to examine cell proliferation, invasion, migration and apoptosis. The interaction between miR-34a and XIST or SMAD2 was verified by luciferase reporter assay. It was found that XIST expression was increased and miR-34a level was decreased in DC tissues and HG-induced SRA01/04 cells. XIST knockdown or miR-34a addition attenuated cell proliferation and migration, and induced apoptosis in SRA01/04 cells under HG. XIST targeted miR-34a and regulated DC progression via miR-34a. SMAD2 was a target gene of miR-34a and was positively modulated by XIST. SMAD2 addition accelerated cell proliferation, migration and inhibited the apoptosis in HG-stimulated SRA01/04 cells, which were abrogated by XIST depletion. In conclusion, XIST facilitated the proliferation, migration and invasion, and inhibited the apoptosis via miR-34a/SMAD2 axis in DC.

sentences: A fluorescent nanosensor paint reveals the heterogeneity of dopamine release from neurons at individual release sites The neurotransmitter dopamine is released from discrete axonal structures called varicosities. Its release is essential in behaviour and is critically implicated in prevalent neuropsychiatric diseases. Existing dopamine detection methods are not able to detect and distinguish discrete dopamine release events from multiple varicosities. This prevents an understanding of how dopamine release is regulated across populations of discrete varicosities. Using a near infrared fluorescent (980 nm) dopamine nanosensor  paint (AndromeDA), we show that action potential-evoked dopamine release is highly heterogeneous across release sites and also requires molecular priming. Using AndromeDA, we visualize dopamine release at up to 100 dopaminergic varicosities simultaneously within a single imaging field with high temporal resolution (15 images/s). We find that  hotspots of dopamine release are highly heterogeneous and are detected at only ~17% of all varicosities. In neurons lacking Munc13 proteins, which prime synaptic vesicles, dopamine release is abolished during electrical stimulation, demonstrating that dopamine release requires vesicle priming. In summary, AndromeDA reveals the spatiotemporal organization of dopamine release.

sentences: PEN receptor GPR83 in anxiety-like behaviors: differential regulation in global vs amygdalar knockdown Anxiety disorders are prevalent across the United States and result in a large personal and societal burden. Currently, numerous therapeutic and pharmaceutical treatment options exist. However, drugs to classical receptor targets have shown limited efficacy and often come with unpleasant side effects, highlighting the need to identify novel targets involved in the etiology and treatment of anxiety disorders. GPR83, a recently deorphanized receptor activated by the abundant neuropeptide PEN, has also been identified as a glucocorticoid regulated receptor (and named GIR) suggesting that this receptor may be involved in stress-responses that underlie anxiety. Consistent with this, GPR83 null mice have been found to be resistant to stress-induced anxiety. However, studies examining the role of GPR83 within specific brain regions or potential sex differences have been lacking. In this study, we investigate anxiety-related behaviors in male and female mice with global knockout and following local GPR83 knockdown in female mice. We find that a global knockdown of GPR83 has minimal impact on anxiety-like behaviors in female mice and a decrease in anxiety-related behaviors in male mice. In contrast, a local GPR83 knockdown in the basolateral amygdala leads to more anxiety-related behaviors in female mice. Local GPR83 knockdown in the central amygdala or nucleus accumbens showed no significant effect on anxiety-related behaviors. Finally, dexamethasone administration leads to a significant decrease in receptor expression in the amygdala and nucleus accumbens of female mice. Together, our studies uncover a significant, but divergent role for GPR83 in different brain regions in the regulation of anxiety-related behaviors, which is furthermore dependent on sex.

sentences: Robust inference of expression state in bulk and single-cell RNA-Seq using curated intergenic regions RNA-Seq is a powerful technique to provide quantitative information on gene expression. While many applications focus on estimated expression levels, it is also important to determine which genes are actively transcribed, and which are not. The problem can be viewed as simply setting a biologically meaningful threshold for calling a gene expressed. We propose to define this threshold per sample relative to the background level for non-expressed genomic features, inferred by the amount of reads mapped to intergenic regions of the genome. To this aim, we first define a stringent set of reference intergenic regions, based on available bulk RNA-Seq libraries for each species. We provide predefined regions selected for different animal species with varying genome annotation quality through the Bgee database. We then call genes expressed if their level of expression is significantly higher than the background noise. This approach can be applied to bulk as well as single-cell RNA-Seq, on a single library as well as on a combination of libraries over one condition. We show that the estimated proportion of expressed genes is biologically meaningful and stable between libraries originating from the same tissue, in both model and non-model organisms.

sentences: Using the UniFrac metric on Whole Genome Shotgun data The UniFrac metric has proven useful in revealing diversity across metagenomic communities. Due to the phylogeny-based nature of this measurement, UniFrac has historically only been applied to 16S rRNA data. Simultaneously, Whole Genome Shotgun (WGS) metagenomics has been increasingly widely employed and proven to provide more information than 16S data, but a UniFrac-like diversity metric suitable for WGS data has not previously been developed. The main obstacle for UniFrac to be applied directly to WGS data is the absence of phylogenetic distances in the taxonomic relationship derived from WGS data. In this study, we demonstrate a method to overcome this intrinsic difference and compute the UniFrac metric on WGS data by assigning branch lengths to the taxonomic tree obtained from input taxonomic profiles. We conduct a series of experiments to demonstrate that this WGSUniFrac method is comparably robust to traditional 16S UniFrac and is not highly sensitive to branch lengths assignments, be they data-derived or model-prescribed. Code implementing a prototype of WGSUniFrac along with paper reproducible are available at https://github.com/KoslickiLab/WGSUniFrac.

sentences: Single-cell multiome of the human retina and deep learning nominate causal variants in complex eye diseases Genome-wide association studies (GWAS) of eye disorders have identified hundreds of genetic variants associated with ocular disease. However, the vast majority of these variants are noncoding, making it challenging to interpret their function. Here, we present a joint single-cell atlas of gene expression and chromatin accessibility of the adult human retina with >50,000 cells, which we used to analyze noncoding single-nucleotide polymorphisms (SNPs) implicated by GWAS of age-related macular degeneration, glaucoma, diabetic retinopathy, myopia, and type 2 macular telangiectasia. We integrate this atlas with a HiChIP enhancer connectome, expression quantitative trait loci (eQTL) data, and base-resolution deep learning models to predict noncoding SNPs with causal roles in eye disease, assess SNP impact on transcription factor binding, and define their known and novel target genes. Our efforts nominate pathogenic SNP-target gene interactions for multiple vision disorders and provide a potentially powerful resource for interpreting noncoding variation in the eye.

sentences: Development, validation, and application of the ribosome separation and reconstitution system for protein translation in vitro The conventional view regarding regulation of gene expression is based on transcription control. However, a growing number of recent studies has revealed the important additional impact of translational regulation. Eukaryotic translational machinery appears to be capable of reprogramming mRNA translation to generate proteins required to maintain a healthy cellular proteostasis under particular physiological conditions or to adapt to stress. Although the mechanisms of such remarkable regulation are beginning to emerge, recent studies have identified the ribosome as one of the major constituents of translation-dependent control of gene expression that is especially important during stress. Built of RNA and proteins, ribosomes are susceptible to environmental and intracellular stresses. How stress-modified ribosomes regulate translation and whether they play a role in stress-induced gene expression remain largely elusive. This knowledge gap is likely due to the lack of an appropriate experimental system. Canonical approaches based on exposing cells or cell-free extracts to stressors provide inconclusive results due to off-target effects of modifying agents. Here we describe a robust and simple in vitro assay that allows separation of yeast ribosomes from other translational machinery constituents, followed by reconstitution of the translation reaction. This ribosome separation and reconstitution assay (RSR) is highly advantageous, as it allows modification of ribosomes without compromising other key translational components, followed by supplementing the ribosomes back into translation reactions containing undamaged, translationally-competent yeast lysate. Besides addressing the impact of ribosome-derived stress on translation, RSR can also be used to characterize mutated ribosomes and ribosomes devoid of associated factors.

sentences: Structural and biochemical basis of FANCI-FANCD2 interdependent ubiquitination The Fanconi Anaemia pathway operates for the repair of interstrand crosslinks and the maintenance of genomic stability upon replication stalling. Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step in this pathway. Evidence suggests that FANCD2 ubiquitination precedes FANCI ubiquitination, and that both the FANCD2-ubiquitinated (ID2Ub) and the di-monoubiquitinated (IUbD2Ub) complex clamp on DNA. However, FANCD2 is deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (IUbD2). Here, we present a 4.1 [A] cryo-EM structure of IUbD2 complex bound to double-stranded DNA. We show that this complex, like ID2Ub and IUbD2Ub, is also in the closed ID2 conformation and clamps on DNA. While the target lysine of FANCD2 (K561) is partially buried in the non-ubiquitinated ID2-DNA complex, it becomes fully exposed in the IUbD2-DNA structure, and thus can be ubiquitinated at a faster rate. The IUbD2-DNA complex cannot easily revert to the non-ubiquitinated ID2 state, due to USP1-UAF1-resistance, conferred by the presence of DNA and FANCD2. ID2Ub-DNA, on the other hand, can be efficiently deubiquitinated by USP1-UAF1, unless further ubiquitination on FANCI occurs. FANCI ubiquitination also progresses at a faster rate in ID2Ub-DNA over ID2-DNA complex, and results in partial DNA-dependent protection from FANCD2 deubiquitination. Taken together, our results suggest that, while FANCD2 ubiquitination promotes FANCI ubiquitination, FANCI ubiquitination in turn maintains FANCD2 ubiquitination by two mechanisms: it prevents excessive FANCD2 deubiquitination within an IUbD2Ub-DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, IUbD2 complex.

sentences: Identification of CD73 as the antigen of an antigen-unknown monoclonal antibody established by exosome immunization, and its antibody-drug conjugate exerts an antitumor effect on glioblastoma cell lines Development of antibodies against the native structure of membrane proteins with multiple transmembrane domains is challenging because it is difficult to prepare antigens with native structures. Previously, we successfully developed a monoclonal antibody against multi-pass membrane protein TMEM180 by exosome immunization in rats. This approach yielded antibod-ies that recognized cancer-specific antigens on the exosome. In this study, we performed im-munoprecipitation using magnetic beads to identify the antigen of one of the rat antibody clones, 0614, as CD73. We then converted antibody 0614 to human chimeric antibody 0614-5 and found that the humanized antibody did not inhibit CD73 enzymatic activity. Glioblastoma (GB) was the cancer type with the highest expression of CD73 in the tumor relative to healthy tissue. An antibody-drug conjugate (ADC) of 0614-5 exerted an antitumor effect on GB cell lines according to expression of CD73. The 0614-5-ADC could be used to treat cancers with high CD73 expression. In addition, our strategy could be used to determine the antigen of any antibody produced by exosome immunization.

sentences: Mapping vegetation types in Antarctic Peninsula and South Shetlands islands using Sentinel-2 images and Google Earth Engine cloud computing The Antarctic vegetation maps are usually made using very high-resolution images collected by orbital sensors or unmanned aerial vehicles, generating isolated maps with information valid only for the time of image acquisition. In the context of global environmental change, mapping the current Antarctic vegetation distribution on a regular basis is necessary for a better understanding of the changes in this fragile environment. This work aimed to generate validated vegetation maps for the North Antarctic Peninsula and South Shetlands Islands based on Sentinel-2 images using cloud processing. Sentinel-2 imagery level 1C, acquired between 2016 and 2021 (January-April), were used. Land pixels were masked with the minimum value composite image for the "water vapor" band. The NDVI maximum value composite image was sliced, and its classes were associated with the occurrence of algae (0.15 - 0.20), lichens (0.20 - 0.50), and mosses (0.50 - 0.80). The vegetation map was validated by comparing it with those from the literature. The present study showed that Sentinel-2 images allow building a vegetation type distribution map for Antarctica Peninsula and South Shetlands Islands.

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Two microbiota subtypes identified in Irritable Bowel Syndrome with distinct responses to the low FODMAP diet ObjectiveReducing FODMAPs can be clinically beneficial in IBS but the mechanism is poorly understood. We aimed to detect microbial signatures that might predict response to the low FODMAP diet and assess whether microbiota compositional and functional shifts could provide insights into its mode of action.

DesignWe used metagenomics to determine high-resolution taxonomic and functional profiles of the stool microbiota from IBS cases and household controls (n=56 pairs) on their usual diet. Clinical response and microbiota changes were studied in 41 pairs after 4 weeks on a low FODMAP diet.

ResultsUnsupervised analysis of baseline IBS cases pre-diet identified two distinct microbiota profiles, which we refer to as IBSP (pathogenic-like) and IBSH (health-like) subtypes. IBSP microbiomes were enriched in Firmicutes and genes for amino acid and carbohydrate metabolism, but depleted in Bacteroidetes species. IBSH microbiomes were similar to controls. On the low FODMAP diet IBSH and control microbiota were unaffected, but the IBSP signature shifted towards a health-associated microbiome with an increase in Bacteroidetes (p=0.009), a decrease in Firmicutes species (p=0.004) and normalization of primary metabolic genes. The clinical response to the low FODMAP diet was greater in IBSP subjects compared to IBSH (p = 0.02).

Conclusion50% of IBS cases manifested a  pathogenic gut microbial signature. This shifted towards the healthy profile on the low FODMAP diet; and IBSP cases showed an enhanced clinical responsiveness to the dietary therapy. The effectiveness of FODMAP exclusion in IBSP may result from the alterations in gut microbiota and metabolites produced. Microbiota signatures could be useful as biomarkers to guide IBS treatment; and investigating IBSP species and metabolic pathways might yield insights regarding IBS pathogenic mechanisms.

O_TEXTBOXSignificance of this studyO_ST_ABSWhat is already known on this subject?C_ST_ABSO_LIIBS subjects often respond to a low FODMAP diet.
C_LIO_LIThe gut microbiota has been implicated in IBS.
C_LIO_LIThe microbiota in IBS subjects may change with diet.
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What are the new findings?O_LIWe were able to stratify patients with IBS according to their gut microbiota species and metabolic gene signatures.
C_LIO_LIWe identified a distinct gut microbiota subtype with an enhanced clinical response to a low FODMAP diet compared to other IBS subjects.
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How might it impact on clinical practice in the foreseeable future?O_LIThe potential development of a microbiota signature as a biomarker to manage IBS cases with a low FODMAP diet recommendation.
C_LIO_LIIf the bacteria represented in the IBSP subtype are shown to play a pathogenic role in IBS, perhaps through the metabolic activity this provides a target for new therapies and an intermediate phenotype by which to assess them.
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C_TEXTBOX

sentences: Geodesic-based distance reveals non-linear topological features in neural activity from mouse visual cortex An increasingly popular approach to the analysis of neural data is to treat activity patterns as being constrained to and sampled from a manifold, which can be characterized by its topology. The persistent homology method identifies the type and number of holes in the manifold thereby yielding functional information about the coding and dynamic properties of the underlying neural network. In this work we give examples of highly non-linear manifolds in which the persistent homology algorithm fails when it uses the Euclidean distance which does not always yield a good approximation of the true distance distribution of a point cloud sampled from a manifold. To deal with this issue we propose a simple strategy for the estimation of the geodesic distance which is a better approximation of the true distance distribution and can be used to successfully identify highly non-linear features with persistent homology. To document the utility of our method we model a circular manifold, based on orthogonal sinusoidal basis functions and compare how the chosen metric determines the performance of the persistent homology algorithm. Furthermore we discuss the robustness of our method across different manifold properties and point out strategies for interpreting its results as well as some possible pitfalls of its application. Finally we apply this analysis to neural data coming from the Visual Coding - Neuropixels dataset recorded in mouse visual cortex after stimulation with drifting gratings at the Allen Institute. We find that different manifolds with a non-trivial topology can be seen across regions and stimulus properties. Finally, we discuss what these manifolds say about visual computation and how they depend on stimulus parameters.

sentences: Rational design yields molecular insights on leaf binding of the anchor peptide Macaque Histatin 1In times of a constantly growing world population and increasing demand for food, sustainable agriculture is crucial. To reduce the amount of applied nutrients, herbicides, and fungicides, the rainfastness of plant protection agents is of pivotal importance. As a result of protective agent wash-off, plant protection is lost, and soils and groundwater are severely polluted. To date, rainfastness of plant protection products is achieved by adding polymeric adjuvants to the agrochemicals. However, polymeric adjuvants will be regarded as microplastics in the future, and environmentally friendly alternatives are needed. Anchor peptides (APs) are promising biobased and biodegradable adhesion promoters. While the adhesion of anchor peptides to artificial surfaces, such as polymers, has already been investigated in theory and experimentally, exploiting the adhesion to biological surfaces remains challenging. The complex nature and composition of biological surfaces such as plant leaf and fruit surfaces complicate the generation of accurate models. Here, we present the first detailed three-layered atomistic model of the surface of apple leaves and use it to compute free energy profiles of the adhesion and desorption of APs to and from that surface. Our model is validated by a novel fluorescence-based MTP assay that mimicks these complex processes and allows quantifying them. For the AP Macaque Histatin, we demonstrate that aromatic and positively charged amino acids are essential for binding to the waxy apple leaf surface. The established protocols should generally be applicable for tailoring the binding properties of APs to biological interfaces.

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Molecular evidence for sweeping discontinuity between peracarid (Crustacea) fauna of Macaronesian islands and nearby continental coasts: over fifty candidate endemic species Oceanic islands are recognized evolutionary hotspots for terrestrial organisms, but little is known about their impact on marine organisms evolution and biogeography. The volcanic archipelagos of Macaronesia occupy a vast and complex region which is particularly suitable to investigate marine island biogeography.

In this study, we used mitochondrial DNA sequences to investigate the genetic diferentiation between the populations from Webbnesia (i.e. Madeira, Selvagens and Canaries) and adjacent coasts, of 23 intertidal peracarid species. All species had unexpectedly high intraspecific genetic distances, reaching more than 20% in some cases. Between 79 and 95 Molecular Operational Taxonomic Units (MOTUs) were found in these species. Webbnesia populations displayed an impressive genetic diversity and high endemicity, with 83% of the MOTUs being private to these islands, particularly La Palma and Madeira. Network analyses suggested higher similarity between Webbnesia and Azores than with adjacent continental coasts.

These results reveal an unanticipated and sweeping biogeographic discontinuity of peracaridean fauna between Webbnesia and the Iberian Peninsula, raising suspicion about the possible occurrence of identical patterns in other groups of marine invertebrates in the region. We emphasize the unique genetic heritage hosted by these islands, underlining the need to consider the fine scale endemicity in marine conservation efforts.

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Reconstructing contact network structure andcross-immunity patterns from multiple infection histories Interactions within a population shape the spread of infectious diseases but contact patterns between individuals are difficult to access. We hypothesised that key properties of these patterns can be inferred from multiple infection data in longitudinal follow-ups. We developed a simulator for epidemics with multiple infections on networks and analysed the resulting individual infection time series by introducing the concept of infection barcodes. We find that, depending on infection multiplicity and network sampling, infection barcode summary statistics can recover network properties such as degree distribution. Furthermore, we show that by mining infection barcodes for multiple infection patterns, one can detect immunological interference between pathogens (i.e. the fact that past infections in a host condition future probability of infection). The combination of individual-based simulations and barcode analysis of infection histories opens promising perspectives to infer and validate transmission networks and immunological interference for infectious diseases from longitudinal cohort data.

Author summaryInfectious disease dynamics are constrained both by between-host contacts and pathogen interactions within a host. Furthermore, multiple parasites circulate such that hosts are infected (sometimes simultaneously) by a variety of strains or species. We hypothesise that multiple infection history can inform us about the networks on which parasites are transmitted, but also on within-host interactions such as immunological interference. We developed a simulator for multiple infections on networks. By combining intuitive novel metrics for multiple infection events and established tools from computational data analysis, we show that similarity in infection history between two hosts correlates with their proximity in the contact network. By analysing pathogens co-occurrence patterns within hosts, we also recover immunological interference at the population level. The demonstrated robustness of our results in terms of observability, network clustering, and pathogen diversity opens new perspectives to extract host contact and between-pathogen immunity information from longitudinal cohort data.

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Platelet proteome analysis reveals an early hyperactive phenotype in SARS-CoV-2-infected humanized ACE2 mice Coronavirus disease-2019 (COVID-19) provokes a hypercoagulable state with increased incidence of thromboembolism and mortality. Platelets are major effectors of thrombosis and hemostasis. Suitable animal models are needed to better understand COVID-19-associated coagulopathy (CAC) and underlying platelet phenotypes. Here, we assessed K18-hACE2 mice undergoing a standardized SARS-CoV-2 infection protocol to study dynamic platelet responses via mass spectrometry-based proteomics. In total, we found significant changes in >1,200 proteins. Strikingly, protein alterations occurred rapidly by 2 days post-infection (dpi) and preceded outward clinical signs of severe disease. Pathway enrichment analysis of 2dpi platelet proteomes revealed that SARS-CoV-2 infection upregulated complement-coagulation networks (F2, F12, CFH, CD55/CD59), platelet activation-adhesion-degranulation proteins (PF4, SELP, PECAM1, HRG, PLG, vWF), and chemokines (CCL8, CXCL5, CXCL12). When mice started to lose weight at 4dpi, pattern recognition receptor signaling (RIG-I/MDA5, CASP8, MAPK3), and interferon pathways (IFIT1/IFIT3, STAT1) were predominant. Interestingly, SARS-CoV-2 spike protein in the lungs was observed by immunohistochemistry, but in platelets was undetected by proteomics. Similar to patients, K18-hACE2 mice during SARS-CoV-2 infection developed progressive lymphohistiocytic interstitial pneumonia with platelet aggregates in the lungs and kidneys. In conclusion, this model recapitulates activation of coagulation, complement, and interferon responses in circulating platelets, providing valuable insight into platelet pathology during COVID-19.

Key PointsO_LISARS-CoV-2-infected humanized ACE2 mice recapitulate platelet reprogramming towards activation-degranulation-aggregation.
C_LIO_LIComplement/coagulation pathways are dominant in platelets at 2 days post-infection (dpi), while interferon signaling is dominant at 4dpi.
C_LI

sentences: Valproic acid induces p19Arf-mediated cellular senescence to cause neurodevelopmental defects Timely induction of cellular senescence is important during embryonic development and tissue repair, while aberrant activation contributes to many age-related diseases. However, whether abnormal induction of senescence in the embryo contributes to developmental defects remains unclear. To explore this, we investigated the clinically significant model of Valproic acid- (VPA) induced developmental defects. VPA is a widely prescribed drug to treat epilepsy, bipolar disorder and migraine. If taken during pregnancy however, exposure to the developing embryo can cause birth defects, cognitive impairment and Autism-Spectrum Disorder. How VPA causes these developmental defects remains unknown. We used embryonic mice and human organoids to model key features of drug exposure, including exencephaly, microcephaly and spinal defects. In the malformed tissues, in which neurogenesis is defective, we find pronounced induction of cellular senescence in the neuroepithelial cells. Critically, through genetic and functional studies, we identified p19Arf as the instrumental mediator of senescence and microcephaly, but surprisingly, not exencephaly and spinal defects. Together, these findings uncover how misregulated senescence in neuroepithelial cells can contribute to developmental defects.

sentences: Beta Turn Propensity and a Model Polymer Scaling Exponent Identify Disordered Proteins that Phase Separate The complex cellular milieu can spontaneously de-mix in a process controlled in part by proteins that are intrinsically disordered (ID). A proteins propensity to de-mix is thought to be driven by the preference for protein-protein rather than protein-solvent interactions. The hydrodynamic size of monomeric proteins, as quantified by the polymer scaling exponent (v), is driven by a similar balance. We hypothesize that mean v, as predicted by the protein sequence, will be smaller for proteins with a strong propensity to de-mix. To test this hypothesis, we analyzed protein databases containing subsets that are either folded, disordered, or disordered and known to spontaneously phase separate. We find that the phase separating disordered proteins, on average, have lower calculated values of v compared to their non-phase separating counterparts. Moreover, these proteins have a higher sequence-predicted propensity for {beta}-turns. Using a simple, surface areabased model, we propose a physical mechanism for this difference: transient {beta}-turn structures reduce the desolvation penalty of forming a protein-rich phase and increase exposure of atoms involved in {pi}/sp2 electronic interactions. By this mechanism, {beta}-turns act as energetically favored nucleation points, which may explain the increased propensity for turns in ID regions (IDRs) that are utilized biologically for phase separation. Phase separating IDRs, non-phase separating IDRs, and folded regions could be distinguished by combining v and {beta}-turn propensity, and we propose a new algorithm, ParSe (partition sequence), for predicting phase separating protein regions. ParSe is able to accurately identify folded, disordered, and phase-separating protein regions from the primary sequence.

sentences: The pyruvate dehydrogenase complex regulates mitophagic selectivity independently of its catalytic activity The mitophagic degradation of mitochondrial matrix proteins is selective, reflecting a pre-engulfment sorting step. This selectivity is regulated through phosphorylation of mitochondrial matrix proteins by the matrix kinases Pkp1 and Pkp2, which in turn appear to be regulated by the phosphatase Aup1/Ptc6. However, these proteins also regulate the phosphorylation and catalytic activity of the yeast pyruvate dehydrogenase complex. To understand the relationship between these two functions, we tested the effect of deleting PDA1 on mitophagic selectivity. We report that pda1{Delta} cells show a mitophagy selectivity phenotype nearly identical to that of the pkp1{Delta} pkp2{Delta} double mutant. However, this is not due to a role for pyruvate dehydrogenase enzymatic activity in regulating mitophagy. Rather, our data suggest a novel mechanism wherein the pyruvate dehydrogenase complex directly regulates its cognate kinases and phosphatases, independent of its catalytic activity, to determine the phosphorylation state of mitochondrial matrix proteins in response to metabolic cues.

sentences: 13C tracer analysis identifies extensive recycling of endogenous CO2 in vivo 13C tracing analysis is increasingly used to monitor cellular metabolism in vivo and in intact cells, but data interpretation is still the key element to unveil the complexity of metabolic activities. We have performed [U-13C]-glucose and [U-13C]-glutamine tracing in sarcoma-bearing mice (in vivo) and in cancer cell lines (in vitro). 13C enrichment of metabolites in cultured cells and tissues was determined by liquid chromatography coupled with high-resolution mass spectrometer (LC-HRMS). As expected, citrate M+2 or M+4 is the dominant mass isotopologue in vitro. However, citrate M+1 was unexpectedly the dominant isotopologue in mice receiving [U-13C]-glucose or [U-13C]-glutamine infusion. One plausible explanation is that 13CO2 produced from the oxidation of 13C tracers in vitro is negligible due to the dilution of HCO3 - supplemented to cell culture when sodium bicarbonante is used and diffusible volume of CO2 in the culture incubator, while endogenous 13CO2 in vivo is substantial and is fixed into the TCA cycle, purine, and serine, resulting in M+1 isotopologues. A time course study shows the generation of high abundance citrate M+1 early in plasma, which may serve as a potent non-invasive biomarker of tissue pyruvate carboxylase activity. Altogether, our results show that recycling of endogenous CO2 is substantial in vivo and provides important insights into the experimental design and data interpretation of 13C tracing assays.

sentences: Alpha oscillations link action to cognition: An oculomotor account of the brain's dominant rhythm Power modulations in alpha oscillations (8-14Hz) have been associated with most human cognitive functions and psychopathological conditions studied. These reports are often inconsistent with the prevailing view of a specific relationship of alpha oscillations to attention and working memory (WM). We propose that conceptualizing the role of alpha oscillations in oculomotor control resolves this inconsistency. This proposition is based on a review of results across species (human Npooled=295, one non-human primate, honey bee N=5), experimental conditions (rest, attention, and working memory), and recording techniques (EEG, ECOG, eye-tracking, and MEG) that encourage the following relationships between alpha oscillations and eye-movement control: (i) saccade initiation prompts power decrease in brain circuits associated with saccadic control; (ii) the direction of a saccade is consistent with alpha lateralization, both during task and resting conditions; (iii) the phase of alpha activity informs saccade occurrence and biases miniature eye movements during fixation (e.g. fixational tremor); and (iv) oculomotor action differentiates WM load. A new theory on how alpha oscillations link oculomotor action to cognition is proposed. Generalizing across tasks and species: low oculomotor activity is associated with high alpha power and vice versa. Alpha oscillations regulate how long to look at a given target and how fast to saccade to a next. By ensuring steady gaze position, any potential input outside foveal vision is "suppressed".

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Old players in new posts: the role of P53, ATM and DNAPK in DNA damage-related ubiquitylation-dependent removal of S2P RNAPII DNA double-strand breaks are the most deleterious lesions for the cells, therefore understanding the macromolecular interactions in the DNA repair-related mechanisms is essential. DNA damage triggers transcription silencing at the damage site, leading to the removal of the elongating RNA polymerase II (S2P RNAPII) from this locus, which provides accessibility for the repair factors to the lesion. Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNAPK) are the two main regulatory kinases of homologous recombination and non-homologous end joining, respectively. Although these kinases are involved in the activation of different repair pathways, they have common target proteins, such as P53. We previously demonstrated that following transcription block, P53 plays a pivotal role in transcription elongation process by interacting with S2P RNAPII. In the current study, we reveal that P53, ATM and DNAPK are involved in the fine-tune regulation of the ubiquitin-proteasome system-related degradation of S2P RNAPII. However, they act differently in this process: P53 delays the removal of S2P RNAPII, while ATM and DNAPK participate in the activation of members of E3 ligase complexes involved in the ubiquitylation of S2P RNAPII. We also demonstrate that WW domain-containing protein 2 (WWP2) and Cullin-3 (CUL3) are interaction partners of S2P RNAPII, thus forming a complex with the transcribing RNAPII complex.

Simple SummaryTo ensure the proper repair following DNA double-strand breaks, the eviction of the arrested elongating RNA polymerase II (S2P RNAPII) is required. Here, we report an emerging role of P53, Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNAPK) in the ubiquitin-proteasome system-dependent removal of S2P RNAPII. We also identified interactions between S2P RNAPII and WW domain-containing protein 2 (WWP2) or Cullin-3 (CUL3) (members of E3 ligase complexes), which are involved in the ubiquitylation of S2P RNAPII following DNA damage. Furthermore, the RNAPII-E3 ligase complex interactions are mediated by P53, ATM and DNAPK, which suggests potential participation of all three proteins in the effective resolution of transcription block at the damage site. Altogether, our results provide a better comprehension of the molecular background of transcription elongation block-related DNA repair processes and highlight an indispensable function of P53, ATM and DNAPK in these mechanisms.

sentences: A structural dynamic explanation for observed escape of SARS-CoV-2 BA.2 variant mutation S371L/F The SARS-CoV-2 Omicron sub-variants BA.1 and BA.2 have become the dominant variants worldwide due to enhanced transmissibility and immune evasion. In response to the rise of BA.1 and BA.2, two recent studies by Liu et al. and Iketani et al. provide a detailed analysis of loss of therapeutic antibody potency through evaluation of escape by pseudotyped viruses harboring BA.1 and BA.2 receptor binding domain (RBD) point mutations. Surprisingly, Liu et al. and Iketani et al. observed a profoundly broad escape effect for the individual mutations S371L and S371F. This result cannot be explained by known escape mechanisms of the SARS-CoV-2 RBD, and conflicts with existing computational and experimental escape measurements for S371 mutations performed on monomeric RBD. Through an examination of these conflicting datasets and a structural analysis of the antibodies assayed by Liu et al. and Iketani et al., we propose a mechanism to explain S371L/F escape according to a perturbation of spike trimer conformational dynamics that has not yet been described for any SARS-CoV-2 escape mutation. The proposed mechanism is relevant to Omicron and future variant surveillance as well as therapeutic antibody design.

sentences: Structural basis of anti-SARS-CoV-2 activity of HCQ: specific binding to N protein to disrupt its interaction with nucleic acids and LLPS Great efforts have led to successfully developing the spike-based vaccines but challenges still exist to completely terminate the SARS-CoV-2 pandemic. SARS-CoV-2 nucleocapsid (N) protein plays the essential roles in almost all key steps of the viral life cycle, thus representing a top drug target. Almost all key functions of N protein including liquid-liquid phase separation (LLPS) depend on its capacity in interacting with nucleic acids. Therefore, only the variants with their N proteins functional in binding nucleic acids might survive and spread in evolution and indeed, the residues critical for binding nucleic acids are highly conserved. Very recently, hydroxychloroquine (HCQ) was shown to prevent the transmission in a large-scale clinical study in Singapore but so far, no specific SARS-CoV-2 protein was experimentally identified to be targeted by HCQ. Here by NMR, we unambiguously decode that HCQ specifically binds NTD and CTD of SARS-CoV-2 N protein with Kd of 112.1 and 57.1 M respectively to inhibit their interaction with nucleic acid, as well as to disrupt LLPS essential for the viral life cycle. Most importantly, HCQ-binding residues are identical in SARS-CoV-2 variants and therefore HCQ is likely effective to them all. The results not only provide a structural basis for the anti-SARS-CoV-2 activity of HCQ, but also renders HCQ to be the first known drug capable of targeting LLPS. Furthermore, the unique structure of the HCQ-CTD complex decodes a promising strategy for further design of better anti-SARS-CoV-2 drugs from HCQ. Therefore, HCQ is a promising candidate to help terminate the pandemic.

sentences: Simultaneous inhibition of human CD4 and 4-1BB biogenesis suppresses cytotoxic T lymphocyte proliferation The small molecule cyclotriazadisulfonamide (CADA) down-modulates the human CD4 receptor, an important factor in T cell activation. Here, we addressed the immunosuppressive potential of CADA using in vitro activation models. CADA inhibited lymphocyte proliferation in a mixed lymphocyte reaction, and when human PBMCs were stimulated with CD3/CD28 beads or phytohemagglutinin. The immunosuppressive effect of CADA involved both CD4+ and CD8+ T cells but was, surprisingly, most prominent in the CD8+ T cell subpopulation where it inhibited cell-mediated lympholysis. We discovered a direct down-modulatory effect of CADA on 4-1BB (CD137) expression, a survival factor for activated CD8+ T cells. More specifically, CADA blocked 4-1BB protein biosynthesis by inhibition of its co-translational translocation across the ER membrane in a signal peptide-dependent way. This study demonstrates that CADA, as potent down-modulator of human CD4 and 4-1BB, has promising in vitro immunomodulatory characteristics for future in vivo exploration as immunosuppressive drug.

sentences: Probing single cell fermentation flux and intercellular exchange networks via pH-microenvironment sensing and inverse modeling The homeostatic control of their environment is an essential task of living cells. It has been hypothesized that when microenvironmental pH inhomogeneities are induced by high cellular metabolic activity, diffusing protons act as signaling molecules, driving the establishment of cross-feeding networks sustained by the cell-to-cell shuttling of overflow products such as lactate. Despite their fundamental role, the extent and dynamics of such networks is largely unknown due to the lack of methods in single cell flux analysis. In this study we provide direct experimental characterization of such exchange networks. We devise a method to quantify single cell fermentation fluxes over time by integrating high-resolution pH microenvironment sensing via ratiometric nanofibers with constraint-based inverse modeling. We apply our method to cell cultures with mixed populations of cancer cells and fibroblasts. We find that the proton trafficking underlying bulk acidification is strongly heterogeneous, with maximal single cell fluxes exceeding typical values by up to 3 orders of magnitude. In addition, a crossover in time from a networked phase sustained by densely connected "hubs" (corresponding to cells with high activity) to a sparse phase dominated by isolated dipolar motifs (i.e. by pair-wise cell-to-cell exchanges) is uncovered, which parallels the time course of bulk acidification. Our method promises to shed light on issues ranging from the homeostatic function of proton exchange to the metabolic coupling of cells with different energetic demands, and paves the way for real-time non-invasive single cell metabolic flux analysis.

sentences: Real-time conversion of tissue-scale mechanical forces into an interdigitated growth pattern The leaf epidermis is a dynamic biomechanical shell that integrates growth across spatial scales to influence organ morphology. Pavement cells, the fundamental unit of this tissue, morph irreversibly into highly lobed cells that drive planar leaf expansion. Here we define how tissue-scale cell wall tensile forces and the microtubule-cellulose synthase systems pattern interdigitated growth in real-time. A morphologically potent subset of cortical microtubules span the periclinal and anticlinal cell faces to pattern cellulose fibers that generate a patch of anisotropic wall. The result is local polarized growth that is mechanically coupled to the adjacent cell via a pectin-rich middle lamella, and this drives lobe formation. Finite element pavement cell models revealed cell wall tensile stress as an upstream patterning element that links cell- and tissue-scale biomechanical parameters to interdigitated growth. Cell lobing in leaves is evolutionarily conserved, occurs in multiple cell types, and is associated with important agronomic traits. Our general mechanistic models of lobe formation provide a foundation to analyze the cellular basis of leaf morphology and function.

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The molecular appearance of native TRPM7 channel complexes identified by high-resolution proteomics The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed membrane protein consisting of ion channel and protein kinase domains. TRPM7 plays a fundamental role in the cellular uptake of divalent cations such as Zn2+, Mg2+ and Ca2+, and thus shapes cellular excitability, plasticity and metabolic activity. The molecular appearance and operation of TRPM7 channel complexes in native tissues have remained unresolved. Here, we investigated the subunit composition of endogenous TRPM7 channels in rodent brain by multi-epitope affinity purification and high-resolution quantitative MS analysis. We found that native TRPM7 channels are high molecular-weight multi-protein complexes that contain the putative metal transporter proteins CNNM1-4 and a small G-protein ARL15. Heterologous reconstitution experiments confirmed the formation of TRPM7/CNNM/ARL15 ternary complexes and indicated that ARL15 effectively and specifically impacts TRPM7 channel activity. These results open up new avenues towards a mechanistic understanding of the cellular regulation and function of TRPM7 channels.

Impact StatementHigh-resolution proteomics in conjunction with biochemical and electrophysiological experiments revealed that the channel-kinase TRPM7 in the rodent brain forms macromolecular complexes containing the metal transporters CNNM1-4 and a small G protein ARL15.

sentences: The Oncoprotein BCL6 Enables Cancer Cells to Evade Genotoxic Stress Genotoxic agents remain the mainstay of cancer treatment. Unfortunately, the clinical benefits are often countered by a rapid tumor adaptive response. Here, we report that the oncoprotein B cell lymphoma 6 (BCL6) is a core component that confers tumor adaptive resistance to genotoxic stress. Multiple genotoxic agents promoted BCL6 transactivation, which was positively correlated with a weakened therapeutic efficacy and a worse clinical outcome. Mechanistically, we discovered that treatment with the genotoxic agent etoposide led to the transcriptional reprogramming of multiple pro-inflammatory cytokines, among which the interferon- and interferon-{gamma} responses were selectively and substantially enriched in resistant cells. Our results further revealed that the activation of interferon/signal transducer and activator of transcription 1 axis directly upregulated BCL6 expression. The increased expression of BCL6 further repressed the tumor suppressor PTEN and consequently enabled resistant cancer cell survival. Accordingly, targeted inhibition of BCL6 remarkably enhanced etoposide-triggered DNA damage and apoptosis both in vitro and in vivo. Our findings highlight the importance of BCL6 signaling in conquering tumor tolerance to genotoxic stress, further establishing a rationale for a combined approach with genotoxic agents and BCL6-targeted therapy.

sentences: Deep mutational scanning and machine learning reveal structural and molecular rules governing allosteric hotspots in homologous proteins A fundamental question in protein science is where allosteric hotspots - residues critical for allosteric signaling - are located, and what properties differentiate them. We carried out deep mutational scanning (DMS) of four homologous bacterial allosteric transcription factors (aTF) to identify hotspots and built a machine learning model with this data to glean the structural and molecular properties of allosteric hotspots. We found hotspots to be distributed protein-wide rather than being restricted to "pathways" linking allosteric and active sites as is commonly assumed. Despite structural homology, the location of hotspots was not superimposable across the aTFs. However, common signatures emerged when comparing hotspots coincident with long-range interactions, suggesting that the allosteric mechanism is conserved among the homologs despite differences in molecular details. Machine learning with our large DMS datasets revealed that global structural and dynamic properties to be a strong predictor of whether a residue is a hotspot than local and physicochemical properties. Furthermore, a model trained on one protein can predict hotspots in a homolog. In summary, the overall allosteric mechanism is embedded in the structural fold of the aTF family, but the finer, molecular details are sequence-specific.

sentences: Dataset-adaptive minimizer order reduces memory usage in k-mer counting The rapid, continuous growth of deep sequencing experiments requires development and improvement of many bioinformatics applications for analysis of large sequencing datasets, including k-mer counting and assembly. Several applications reduce RAM usage by binning sequences. Binning is done by employing minimizer schemes, which rely on a specific order of the minimizers. It has been demonstrated that the choice of the order has a major impact on the performance of the applications. Here we introduce a method for tailoring the order to the dataset. Our method repeatedly samples the dataset and modifies the order so as to flatten the k-mer load distribution across minimizers. We integrated our method into Gerbil, a state-of-the-art memory efficient k-mer counter, and were able to reduce its memory footprint by 30% - 50% for large k, with only minor increase in runtime. Our tests also showed that the orders produced by our method produced superior results when transferred across datasets from the same species, with little or no order change. This enables memory reduction with essentially no increase in runtime.

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Base editing in bovine embryos reveals a species-specific role of SOX2 in regulation of pluripotency The emergence of the first three lineages during development are orchestrated by a network of transcription factors, which are best characterized in mice. However, the role and regulation of these factors are not completely conserved in other mammals, including human and cattle. Here, we establish a gene inactivation system by introducing premature codon with cytosine base editor in bovine embryos with a robust efficiency. Of interest, SOX2 is universally localized in early blastocysts but gradually restricted into the inner cell mass in cattle. SOX2 knockout results in a failure of the establishment of pluripotency. Indeed, OCT4 level is significantly reduced and NANOG was barely detectable. Furthermore, the formation of primitive endoderm is compromised with few SOX17 positive cells. Single embryo RNA-seq reveals a dysregulation of 2074 genes, among which 90% are up-regulated in SOX2-null blastocysts. Intriguingly, more than a dozen lineage-specific genes, including OCT4 and NANOG, are down-regulated. Moreover, SOX2 expression is sustained in the trophectoderm in absence of CDX2 in bovine late blastocysts. Overall, we propose that SOX2 is dispensable for OCT4 and NANOG expression and disappearance of SOX2 in the trophectoderm depends on CDX2 in cattle, which are all in sharp contrast with results in mice.

SignificanceThe first and second cell fate decisions of a new life are important for subsequent embryonic and plancental development. These events are finely controlled by a network of transcriptional factors, which are extensively characterized in mice. Species-specific roles of these proteins are emerging in mammals. Here, we develop a gene loss-of-function system by using cytosine base editors in bovine embryos. We find that expression pattern, functional roles, and regulation of SOX2 are all different between mouse and bovine embryos. Remarkbly, SOX2 is required for OCT4 and NANOG, two well established pluripoteny genes. Furthermore, CDX2 is required to shut down SOX2 in the trophectoderm. Given similar expression pattern of SOX2 between human and bovine blastocysts, bovine embryos represents a putative model to investigate human pluripotency regulation in vivo.

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Rapid Cortical Adaptation and the Role of Thalamic Synchrony During Wakefulness Rapid sensory adaptation is observed across all sensory systems, and strongly shapes sensory percepts in complex sensory environments. Yet despite its ubiquity and likely necessity for survival, the mechanistic basis is poorly understood. A wide range of primarily in-vitro and anesthetized studies have demonstrated the emergence of adaptation at the level of primary sensory cortex, with only modest signatures in earlier stages of processing. The nature of rapid adaptation and how it shapes sensory representations during wakefulness, and thus the potential role in perceptual adaptation, is underexplored, as are the mechanisms that underlie this phenomenon. To address these knowledge gaps, we recorded spiking activity in primary somatosensory cortex (S1) and the upstream ventral posteromedial (VPm) thalamic nucleus in the vibrissa pathway of awake male and female mice, and quantified responses to whisker stimuli delivered in isolation and embedded in an adapting sensory background. We found that cortical sensory responses were indeed adapted by persistent sensory stimulation; putative excitatory neurons were profoundly adapted, and inhibitory neurons only modestly so. Further optogenetic manipulation experiments and network modeling suggest this largely reflects adaptive changes in synchronous thalamic firing combined with robust engagement of feedforward inhibition, with little contribution from synaptic depression. Taken together, these results suggest that cortical adaptation in the regime explored here results from changes in the timing of thalamic input, and the way in which this differentially impacts cortical excitation and feedforward inhibition, pointing to a prominent role of thalamic gating in rapid adaptation of primary sensory cortex.

Significance StatementRapid adaptation of sensory activity strongly shapes representations of sensory inputs across all sensory pathways over the timescale of seconds, and has profound effects on sensory perception. Despite its ubiquity and theoretical role in the efficient encoding of complex sensory environments, the mechanistic basis is poorly understood, particularly during wakefulness. In this study in the vibrissa pathway of awake mice, we show that cortical representations of sensory inputs are strongly shaped by rapid adaptation, and that this is mediated primarily by adaptive gating of the thalamic inputs to primary sensory cortex and the differential way in which these inputs engage cortical sub-populations of neurons.

sentences: Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na+/H+ antiporter NHA2 The human Na+/H+ antiporter NHA2 (SLC9B2) transports Na+ or Li+ across the plasma membrane in exchange for protons, and is implicated in various pathologies. It is a 537 amino acids protein with an 82 residues long hydrophilic cytoplasmic N-terminus followed by a transmembrane part comprising 14 transmembrane helices. We optimized the functional expression of HsNHA2 in the plasma membrane of a salt-sensitive Saccharomyces cerevisiae strain and characterized a set of mutated or truncated versions of HsNHA2 in terms of their substrate specificity, transport activity, localization and protein stability. We identified a highly conserved proline 246, located in the core of the protein, as being crucial for ion selectivity. The replacement of P246 with serine or threonine resulted in antiporters with altered substrate specificity and increased resistance to the HsNHA2-specific inhibitor phloretin that were not only highly active at an acidic pH of 4.0 (like the native antiporter), but also at neutral pH. We also experimentally confirmed the importance of a putative salt bridge between E215 and R432 for antiporter function and structural integrity. Truncations of the first 50 - 70 residues of the N-terminus doubled the transport activity of HsNHA2, whilst changes in the charge at positions E47, E56, K57, or K58 decreased the antiporters transport activity. Thus, the hydrophilic N-terminal part of the protein appears to allosterically autoinhibit its cation transport. Our data also show this in vivo approach to be useful for a rapid screening of SNPs effect on HsNHA2 activity.

sentences: Coiled Coil Crosslinked Alginate Hydrogels Dampen Macrophage-Driven Inflammation Alginate hydrogels are widely used for tissue engineering and regenerative medicine due to their excellent biocompatibility. A facile and commonly used strategy to crosslink alginate is the addition of Ca2+ that leads to hydrogelation. However, extracellular Ca2+ is a secondary messenger in activating inflammasome pathways following physical injury or pathogenic insult leading to persistent inflammation and scaffold rejection. Here we present graft copolymers of charge complementary heterodimeric coiled coil (CC) peptides and alginate that undergo supramolecular self-assembly to form Ca2+ free alginate hydrogels. The formation of heterodimeric CCs was confirmed using circular dichroism spectroscopy and scanning electron microscopy revealed a significant difference in pore size between Ca2+ and CC crosslinked gels. The resulting hydrogels were self-supporting and display shear-thinning and shear-recovery properties. In response to lipopolysaccharide (LPS) stimulation, peritoneal macrophages and bone marrow derived dendritic cells cultured in the CC crosslinked gels exhibited a 10-fold reduction in secretion of the proinflammatory cytokine IL-1{beta} compared to Ca2+ crosslinked gels. A similar respose was also observed in vivo upon peritoneal delivery of Ca2+ or CC crosslinked gels. Analysis of peritoneal lavage showed that macrophages in mice injected with Ca2+ crosslinked gels display a more inflammatory phenotype compared to macrophages from mice injected with CC crosslinked gels. These results suggest that CC peptides by virtue of their tunable sequence-structure-function relationship and mild gelation conditions are promising alternative crosslinkers for alginate and other biopolymer scaffolds used in tissue engineering.

sentences: Global warming readiness: Feasibility of enhanced biological phosphorus removal from wastewater at 35oC Recent research has shown enhanced biological phosphorus removal (EBPR) from municipal wastewater at warmer temperatures around 30{degrees}C to be stable in both laboratory-scale reactors and full-scale treatment plants. In the context of a changing climate, the feasibility of EBPR at even higher temperatures is of interest. We operated two lab-scale EBPR sequencing batch reactors with alternating anaerobic and aerobic phases for over 300 days at 30{degrees}C and 35{degrees}C, respectively, and followed the dynamics of the communities of phosphorus accumulating organisms (PAOs) and competing glycogen accumulating organisms (GAOs) using a combination of 16S rRNA gene metabarcoding, quantitative PCR and fluorescent in-situ hybridization analyses. Stable and nearly complete P removal was achieved at 30{degrees}C; similarly, long term P removal was stable at 35{degrees}C with effluent PO43--P concentrations < 0.5 mg/L on half of all monitored days. Diverse and abundant Ca. Accumulibacter amplicon sequence variants were closely related to those found in temperate environments, suggesting that EBPR at this temperature does not require a highly specialized PAO community. The slow-feeding strategy used effectively limited the carbon uptake rates of GAOs, allowing PAOs to outcompete GAOs at both temperatures. Candidatus Competibacter was the main GAO, along with cluster III Defluviicoccus members. These organisms withstood the slow-feeding regime, suggesting that their bioenergetic characteristics of carbon uptake differ from those of their tetrad-forming relatives. This specific lineage of GAOs warrants further study to establish how complete P removal can be maintained. Comparative cycle studies at two temperatures for each reactor revealed higher activity of Ca. Accumulibacter when the temperature was increased from 30{degrees}C to 35{degrees}C, suggesting that the stress was a result of the higher carbon (and/or P) metabolic rates of PAOs and GAOs, the resultant carbon deficiency, and additional community competition. An increase in the TOC to PO43--P ratio (from 25:1 to 40:1) effectively eased the carbon deficiency and benefited the proliferation of PAOs. In general, the slow-feeding strategy and sufficiently high carbon input benefited a high and stable EBPR at elevated temperature and represent basic conditions for full-scale applications.

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Dual Inhibition of Cathepsin L and 3CL-Pro by GC-376 Constrains SARS Cov2 Infection Including Omicron Variant Recurrent waves of SARS CoV2 infections remain a major global health concern. Emergence of highly infectious variants with reduced sensitivity to neutralization by vaccines and monoclonal antibodies (mAb) necessitates a deeper understanding of factors involved in SARS CoV2 infections and identification of drug candidates to halt infection. Here, we determined the primacy of endosomal protease cathepsin-L in mediating SARS CoV2 entry and screened a library of well-annotated bioactive compounds for potent cathepsin-L inhibitory activity. Whilst the potent cathepsin-L inhibitors were capable of inhibiting SARS CoV2 entry and cytopathic effect (CPE) in less susceptible cell lines such as human ACE2 expressing 293T cells, these drugs failed to inhibit SARS CoV2 in highly susceptible cell lines such as human TMPRSS2 or human-ACE2-TMPRSS2 overexpressing Vero E6 cells. Only drugs with dual inhibitory effect on both host cathepsin-L and virus 3CL-Protease enzymes such as Z-FA-FMK and GC-376 were capable of inhibiting prototypic (USA-WA1/2020, Lineage A) SARS CoV2 induced CPE in highly susceptible cell lines. Moreover, these drugs inhibited delta (Lineage-B.1.617.2) and omicron (Lineage-B.1.1.529) infection with equal potency showing that the newer mutations harbored in these variants did not affect the mechanism of action of these drugs such as cathepsin-L or 3CL-Pro inhibition. Moreover, our early evidence that 3CL-Pro inhibition can effectively inhibit omicron-induced CPE in highly susceptible cell lines suggests that the recently FDA-approved oral drug, a 3CL-Pro inhibitor which is a combination of nirmatrelvir/ritonavir (Paxlovid) could be effective against omicron variant which shows reduced sensitivity to vaccines and mAb.

ImportanceWe report that cathepsin-L and 3CL-Pro as major targets for designing antivirals against SARS CoV2. Dual inhibition of cathepsin-L and 3CL-Pro by GC-376 renders it effective in inhibiting SARS CoV2-induced cytopathic effect in highly susceptible cell lines. Moreover, this candidate drug is equally effective against prototypic SARS CoV2 lineage A and emerging variants such as delta and omicron which show reduced sensitivity to vaccines and monoclonal antibodies. Given the recent wave of SARS CoV2 omicron variant infection around the world, and 3CL-Pro inhibitor nirmatrelvir is one of the components of the FDA-approved Paxlovid, our findings are timely, important and should be of broad interest.

sentences: Heparin-derived theranostic nanoprobes overcome the blood-brain barrier and target glioma in murine model The poor permeability of theranostic agents across the blood-brain-barrier (BBB) significantly hampers the development of new treatment modalities for neurological diseases. We have discovered a new biomimetic nanocarrier using heparin (HP) that effectively passes the BBB and targets glioblastoma. Specifically, we designed HP coated gold nanoparticles (HP-AuNPs) that were labeled with three different imaging modalities namely, fluorescein (FITC-HP-AuNP), radioisotope 68Gallium (68Ga-HP-AuNPs), and MRI active gadolinium (Gd-HP-AuNPs). The systemic infusion of FITC-HP-AuNPs in three different mouse strains (C57BL/6JRj, FVB, and NMRI-nude) displayed excellent penetration and revealed uniform distribution of fluorescent particles in the brain parenchyma (69-86%) with some accumulation in neurons (8-18%) and microglia (4-10%). Tail-vein administration of radiolabeled 68Ga-HP-AuNPs in healthy rats also showed 68Ga-HP-AuNP inside the brain parenchyma and in areas containing cerebrospinal fluid, such as the lateral ventricles, the cerebellum, and brain stem. Finally, tail-vein administration of Gd-HP-AuNPs (that display [~]3 fold higher relaxivity than that of commercial Gd-DTPA) in an orthotopic glioblastoma (U87MG xenograft) model in nude mice demonstrated enrichment of T1-contrast at the intracranial tumor with a gradual increase in the contrast in the tumor region between 1h-3h. We believe, our finding offers the untapped potential of HP-derived-NPs to deliver cargo molecules for treating neurological disorders.

sentences: A model of preferential pairing between epithelial and dendritic cells in thymic antigen transfer Medullary thymic epithelial cells (mTECs) which produce and present self-antigens are essential for the establishment of central tolerance. Since mTEC numbers are limited, their function is complemented by thymic dendritic cells (DCs), which transfer mTEC-produced self-antigens via cooperative antigen transfer (CAT). While CAT is required for effective T cell selection, many aspects remain enigmatic. Given the recently described heterogeneity of mTECs and DCs, it is unclear whether the antigen acquisition from a particular TEC subset is mediated by preferential pairing with specific subset of DCs. Using several relevant Cre-based mouse models controlling the expression of fluorescent proteins, we found that in regards to CAT, each subset of thymic DCs preferentially targets distinct mTEC subset(s) and importantly, XCR1+ activated DCs represented the most potent subset in CAT. Interestingly, one thymic DC can acquire antigen repetitively and of these, monocyte-derived DCs (moDC) were determined to be the most efficient in repetitive CAT. moDCs also represented the most potent DC subset in the acquisition of antigen from other DCs. These findings suggest a preferential pairing model for the distribution of mTEC-derived antigens among distinct populations of thymic DCs.

sentences: Development of a Novel PROTAC using the Nucleic Acid Aptamer as a Targeting Ligand for Tumor Selective Degradation of Nucleolin PROteolysis TArgeting Chimeras (PROTACs) induce targeted protein degradation by hijacking the intracellular ubiquitin proteasome system, thus emerging as a new strategy for drug development. However, most PROTACs generated lack cell-type selectivity and have poorly soluble in water. To address this drawback, we developed a novel PROTAC ZL216 using aptamer AS1411 as a targeting ligand of nucleolin to conjugate with a small molecule ligand of E3 ligase VHL, which shows high aqueous solubility and serum stability. Based on the differential expression of nucleolin on the cell surface, ZL216 could bind to and internalize into breast cancer cells, but not normal breast cells. Furthermore, we revealed that ZL216 promoted the formation of a nucleolin-ZL216-VHL ternary complex in breast cancer cells and potently induced nucleolin degradation in vitro and in vivo. As a result, ZL216 inhibited the proliferation and migration of breast cancer cells. These studies demonstrate that in addition to peptides and small molecule compounds, nuclei acid aptamers can also be used to generate PROTACs, which broadens the toolbox constructing PROTACs and provides a promising strategy for development of tumor-selective PROTACs.

sentences: Systems analysis reveals ageing-related perturbations in retinoids and sex hormones in Alzheimer's and Parkinson's diseases Neurodegenerative diseases (NDDs), including Alzheimers (AD) and Parkinsons diseases (PD), are complex heterogeneous diseases with highly variable patient responses to treatment. Due to the growing evidence for ageing-related clinical and pathological commonalities between AD and PD, these diseases have recently been studied in tandem. In this study, we analyse transcriptomic data from AD and PD patients, and stratify these patients into three subclasses with distinct gene expression and metabolic profiles. Through integrating transcriptomic data with a genome-scale metabolic model and validating our findings by network exploration and co-analysis using a zebrafish ageing model, we identify retinoids as a key ageing-related feature in all subclasses of AD and PD. We also demonstrate that the dysregulation of androgen metabolism by three different independent mechanisms is a source of heterogeneity in AD and PD. Taken together, our work highlights the need for stratification of AD/PD patients and development of personalised and precision medicine approaches based on the detailed characterisation of these subclasses.

sentences: RAID: Regression Analysis based Inductive DNA microarray for Precise Read-Across Chemical structure-based read-across represents a promising method for chemical toxicity evaluation without the need for animal testing; however, a chemical structure is not necessarily related to toxicity. Therefore, in vitro studies were often used for read-across reliability refinement; however, their external validity has been hindered by the gap between in vitro and in vivo conditions. Thus, we developed a virtual DNA microarray, Regression Analysis based Inductive DNA microarray (RAID), which quantitatively predicts in vivo gene expression profiles based on the chemical structure and/or in vitro transcriptome data. For each gene, elastic-net models were constructed using chemical descriptors and in vitro transcriptome data to predict in vivo data from in vitro data (in vitro to in vivo extrapolation; IVIVE). In feature selection, useful genes for assessing the quantitative structure activity relationship (QSAR) and IVIVE were identified. Predicted transcriptome data derived from the RAID system reflected the in vivo gene expression profiles of characteristic hepatotoxic substances. Moreover, gene ontology and pathway analyses indicated that xenobiotic response and metabolic activation via nuclear receptors are related to those gene expressions. The identified IVIVE-related genes were associated with fatty acid-, xenobiotic-, and drug metabolism, indicating that in vitro studies were effective in evaluating these key events. Furthermore, validation studies revealed that chemical substances associated with these key events could be detected as hepatotoxic biosimilar substances. These results indicate that the RAID system could represent an alternative screening test for repeated-dose toxicity test and toxicogenomic analyses. Our technology provides a critical solution to IVIVE-based read-across by considering the mode of action and chemical structures.

sentences: GEDI: an R package for integration of transcriptomic data from multiple high-throughput platforms Transcriptomic data is often expensive and difficult to generate in large cohorts in comparison to genomic data and therefore is often important to integrate multiple transcriptomic datasets from both microarray and next generation sequencing (NGS) based transcriptomic data across similar experiments or clinical trials to improve analytical power and discovery of novel transcripts and genes. However, transcriptomic data integration presents a few challenges including re-annotation and batch effect removal. We developed the Gene Expression Data Integration (GEDI) R package to enable transcriptomic data integration by combining already existing R packages. With just four functions, the GEDI R package makes constructing a transcriptomic data integration pipeline straightforward. Together, the functions overcome the complications in transcriptomic data integration by automatically re-annotating the data and removing the batch effect. The removal of the batch effect is verified with Principal Component Analysis and the data integration is verified using a logistic regression model with forward stepwise feature selection. To demonstrate the functionalities of the GEDI package, we integrated five bovine endometrial transcriptomic datasets from the NCBI Gene Expression Omnibus. The datasets included Affymetrix, Agilent and RNA-sequencing data. Furthermore, we compared the GEDI package to already existing tools and found that GEDI is the only tool that provides a full transcriptomic data integration pipeline including verification of both batch effect removal and data integration.

sentences: Ontogenetic Color Switching in Lizards as a by-Product of Guanine Cell Development Many animals undergo dramatic changes in colour during development1,2. Changes in predation risk during ontogeny are associated with spectacular switches in defensive colours, typically involving the replacement of skin or the production of new pigment cells3. Ontogenetic colour systems are ideal models for understanding the evolution and formation mechanisms of animal colour which remain largely enigmatic2. We show that defensive colour switching in lizards arises by reorganization of a single photonic system, as an incidental by-product of chromatophore maturation. The defensive blue tail colour of hatchling A. beershebensis lizards is produced by light scattering from premature guanine crystals in underdeveloped iridophore cells. Camouflaged adult tail colours emerge upon reorganization of the guanine crystals into a photonic reflector during chromatophore maturation. The substituent guanine crystals form by the attachment of individual nanoscopic plates, which coalesce during growth to form single crystals. Our results show that the blue colour of hatchlings is a fortuitous, but necessary, precursor to the development of adult colour. Striking functional colours in animals can thus arise not as distinct evolutionary innovations but via exploitation of the timing of naturally occurring changes in chromatophore cell development.

sentences: The autophagy initiating kinase ULK1 is required for pancreatic cancer cell growth and survival Amongst cancer subtypes, pancreatic ductal adenocarcinoma (PDA) has been demonstrated to be most sensitive to autophagy inhibition, which may be due to unique metabolic rewiring in these cells. The serine/threonine kinase ULK1 forms the catalytic center of a complex mediating the first biochemical step of autophagy. ULK1 directly recieves signals from mTORC1 and AMPK to trigger autophagy under stress and nutrient poor conditions. Studies in genetic engineered mouse models of cancer have revealed that deletion of core downstream autophagy genes (ATG5, ATG7) at the time of tumor iniation leads to a profound block in tumor progression leading to the development of autophagy inhibitors as cancer therapeutics. However, most preclinical studies and all clinical studies have relied on non-specific lysomotropic agents such as chloroquine and its derivatives, whose toxicity and off-target issues preclude further clinical development and which do not represent the impact of solely biochemically disrupting the autophagy pathway. Furthermore, druggable targets in the core autophagy pathway are quite limited, with ULK1 and ULK2 representing the only protein kinases in the pathway. Here we explore the genetic requirement for ULK1 and ULK2 in human PDA cancer cell lines and xenografts, and take advantage of new small molecule ULK1 inhibitors to demonstrate that ULK inhibition can overcome autophagy induction triggered by PDA therapeutics including chemotherapy and MEK inhibition. Finally we show that ULK inhibitors increase MHC Class I in PDA cells, suggestion a potential therapeutic avenue for such agents in combination with checkpoint immunotherapy.

sentences: Functional binding dynamics relevant to the evolution of zoonotic spillovers in endemic and emergent Betacoronavirus strains Comparative functional analysis of the dynamic interactions between various Betacoronavirus mutant strains and broadly utilized target proteins such as ACE2 and CD26, is crucial for a more complete understanding of zoonotic spillovers of viruses that cause diseases such as COVID-19. Here, we employ machine learning to replicated sets of nanosecond scale GPU accelerated molecular dynamics simulations to statistically compare and classify atom motions of these target proteins in both the presence and absence of different endemic and emergent strains of the viral receptor binding domain (RBD) of the S spike glycoprotein. Machine learning was used to identify functional binding dynamics that are evolutionarily conserved from bat CoV-HKU4 to human endemic/emergent strains. Conserved dynamics regions of ACE2 involve both the N-terminal helices, as well as a region of more transient dynamics encompassing K353, Q325 and a novel motif AAQPFLL 386-92 that appears to coordinate their dynamic interactions with the viral RBD at N501. We also demonstrate that the functional evolution of Betacoronavirus zoonotic spillovers involving ACE2 interaction dynamics are likely pre-adapted from two precise and stable binding sites involving the viral bat progenitor strains interaction with CD26 at SAMLI 291-5 and SS 333-334. Our analyses further indicate that the human endemic strains hCoV-HKU1 and hCoV-OC43 have evolved more stable N-terminal helix interactions through enhancement of an interfacing loop region on the viral RBD, whereas the highly transmissible SARS-CoV-2 variants (B.1.1.7, B.1.351 and P.1) have evolved more stable viral binding via more focused interactions between the viral N501 and ACE2 K353 alone.

sentences: Adipocyte Tribbles1 Regulates Plasma Adiponectin and Plasma Lipids in Mice Multiple GWAS have identified SNPs in the 8q24 locus near the TRIB1 gene that significantly associate with plasma lipids and coronary artery disease. While subsequent studies have uncovered roles for hepatic and myeloid Trib1 in contributing to either plasma lipids or atherosclerosis, the causal tissue for these GWAS associations remains unclear. The same 8q24 SNPs significantly associate with plasma adiponectin levels in humans as well, suggesting a role for TRIB1 in adipose tissue. Here, we report that adipocyte-specific Trib1 knockout mice (Trib1_ASKO) have increased plasma adiponectin levels and decreased plasma cholesterol and triglycerides. We demonstrate that loss of Trib1 increases adipocyte production and secretion of adiponectin independent of the known TRIB1 function of regulating proteasomal degradation. RNA-seq analysis of adipocytes and livers from Trib1_ASKO mice suggests that alterations in adipocyte function underlie the plasma lipid changes observed in these mice. Secretomics and RNA-seq analysis revealed that Trib1_ASKO mice have increased production of Lpl and decreased production of Angptl4 in adipose tissue, and fluorescent substrate assays confirm an increase in adipose tissue Lpl activity, which likely underlies the observed triglyceride phenotype. In summary, we demonstrate here a novel role for adipocyte Trib1 in regulating plasma adiponectin, total cholesterol, and triglycerides in mice, confirming previous genetic associations observed in humans and providing a novel avenue through which Trib1 regulates plasma lipids and coronary artery disease.

sentences: Changes in wheat rhizosphere microbiota in response to chemical inputs, plant genotype and phenotypic plasticity Since modern wheat varieties are grown with chemical inputs, we ignore if changes observed in rhizosphere microorganisms between ancient and modern varieties are due to i) breeding-induced changes in plant genotype, ii) modifications of the environment via synthetic chemical inputs, or (iii) phenotypic plasticity, defined as the interaction between the genotype and the environment. In the field, we evaluated the effects of various wheat varieties (modern and ancient) grown with or without chemical inputs (N-fertilizer, fungicide and herbicide together) in a crossed factorial design. We analysed rhizosphere bacteria and fungi by amplicons sequencing and mycorrhizal association by microscopic observations. When considered independently of plant genotype, chemical inputs were responsible for an increase in dominance for bacteria and decrease in evenness for bacteria and fungi. Independently of inputs, modern varieties had richer and more even bacterial communities compared to ancient varieties. Phenotypic plasticity had a significant effect: bacterial and fungal diversity decreased when inputs were applied in ancient varieties but not in modern ones. Mycorrhiza were more abundant in modern than ancient varieties, and less abundant when using chemical inputs. Although neglected, phenotypic plasticity is important to understand the evolution of plant-microbiota associations and a relevant target in breeding programs.

sentences: Novel bisubstrate inhibitors for protein N-terminal acetyltransferase D Protein N-terminal acetyltransferase D (NatD, NAA40) that specifically acetylates the alpha-N-terminus of histone H4 and H2A has been implicated in various diseases, but no inhibitor has been reported for this important enzyme. Based on the acetyl transfer mechanism of NatD, we designed and prepared a series of highly potent NatD bisubstrate inhibitors by covalently linking coenzyme A to different peptide substrates via an acetyl or propionyl spacer. The most potent bisubstrate inhibitor displayed an apparent Ki value of 1.0 nM. Biochemical studies indicated that bisubstrate inhibitor is competitive to the peptide substrate and noncompetitive to the cofactor, suggesting NatD undergoes an ordered Bi-Bi mechanism. We also demonstrated that these inhibitors are highly specific towards NatD, displaying about 1,000-fold selectivity over other closely related acetyltransferases. High-resolution crystal structures of NatD bound to two of these inhibitors revealed the molecular basis for their selectivity and inhibition mechanism, providing a rational path for future inhibitor development.

sentences: Transient late gestation prenatal hypoxic insult results in functional deficits but not gross neuroanatomic deficits in mice Intrauterine hypoxia is a common cause of brain injury in children resulting in a broad spectrum of long-term neurodevelopmental sequela, including life-long disabilities that can occur even in the absence of severe neuroanatomic damage. Postnatal hypoxia-ischemia rodent models are commonly used to understand the effects of ischemia and transient hypoxia on the developing brain. Postnatal models, however, have some limitations. First, they do not test the impact of placental pathologies on outcomes from hypoxia. Second, they primarily recapitulate severe injury because they provoke substantial cell death, which is not seen in children with mild hypoxic injury. Lastly, they do not model preterm hypoxic injury. Prenatal models of hypoxia in mice may allow us to address some of these limitations to expand our understanding of developmental brain injury. The published rodent models of prenatal hypoxia employ multiple days of hypoxic exposure or complicated surgical procedures, making these models challenging to perform consistently in mice. Furthermore, large animal models suggest that transient prenatal hypoxia without ischemia is sufficient to lead to significant functional impairment to the developing brain. However, these large animal studies are resource-intensive and not readily amenable to mechanistic molecular studies. Therefore, here we characterized the effect of late gestation (embryonic day 17.5) transient prenatal hypoxia (5% inspired oxygen) on long-term anatomical and neurodevelopmental outcomes in mice. Late gestation transient prenatal hypoxia increased hypoxia-inducible factor 1 alpha protein levels (a marker of hypoxic exposure) in the fetal brain. Hypoxia exposure predisposed animals to decreased weight at postnatal day 2, which normalized by day 8. However, hypoxia did not affect gestational age at birth, litter size at birth, or pup survival. No differences in fetal brain cell death or long-term gray or white matter changes resulted from hypoxia. Animals exposed to prenatal hypoxia did have several long-term functional consequences, including sex-dichotomous changes. Hypoxia exposure was associated with a decreased seizure threshold and abnormalities in hindlimb strength and repetitive behaviors in males and females. Males exposed to hypoxia had increased anxiety-related deficits, whereas females had deficits in social interaction. Neither sex developed any motor or visual learning deficits. This study demonstrates that late gestation transient prenatal hypoxia in mice is a simple, clinically relevant paradigm for studying putative environmental and genetic modulators of the long-term effects of hypoxia on the developing brain.

sentences: Monitoring spore dispersal and early infections of  Diplocarpon coronariae causing apple blotch using selected spore traps and a new qPCR method Apple blotch (AB) is a major disease of apples in Asia and recently also emerging in Europe and the USA. It is caused by the fungus Diplocarpon coronariae (Dc) (formerly: Marssonina coronaria; teleomorph: Diplocarpon mali) and leads to severe defoliation of apple trees in late summer and thus to reduced yield and fruit quality. To develop effective crop protection strategies, a sound knowledge of the pathogens biology is crucial. However, especially data on the early phase of disease development is scarce, and no data on spore dispersal for Europe is available. In this study, we assessed different spore traps for their capacity to capture Dc spores, and we developed a highly sensitive TaqMan qPCR method to quantify Dc conidia in spore trap samples. With these tools, we monitored the temporal and spatial spore dispersal and disease progress in spring and early summer in an extensively managed apple orchard in Switzerland in 2019 and 2020. Our results show that Dc overwinters in leaf litter and that spore dispersal and primary infections occur already in late April and beginning of May. We provide the first results on early-season spore dispersal of Dc, which, combined with the observed disease progress, helps to understand the disease dynamics and improve disease forecast models. Using the new qPCR method, we finally detected Dc in buds, on bark and on fruit mummies, suggesting that these apple organs may serve as additional overwintering habitats for the fungus.

sentences: Phylogenetic and population structure analyses uncover pervasive misclassification and help assessing the biosafety of Pseudomonas alloputida for biotechnological applications The Pseudomonas putida group comprises strains with biotechnological and clinical relevance. P. alloputida was proposed as a new species and highlighted the misclassification of P. putida. Nevertheless, the population structure of P. alloputida remained unexplored. We retrieved 11,025 Pseudomonas genomes and used P. alloputida Kh7T to delineate the species. The P. alloputida population structure comprises at least 7 clonal complexes (CCs). Clinical isolates are mainly found in CC4 and acquired resistance genes are present at low frequency in plasmids. Virulence profiles support the potential of CC7 members to outcompete other plant or human pathogens through a type VI secretion system. Finally, we found that horizontal gene transfer had an important role in shaping the ability of P. alloputida to bioremediate aromatic compounds such as toluene. Our results provide the grounds to understand P. alloputida genetic diversity and safety for environmental applications.

sentences: Expert annotation and life-cycle transcriptomics of transcription factors in rust fungi (Pucciniales) highlight the role of cold shock proteins in dormancy exit Fungi of the order Pucciniales are obligate plant biotrophs causing rust diseases. They exhibit a complex life cycle with the production of up to five spore types, infection of two unrelated hosts and an overwintering stage. Transcription factors (TFs) are key regulators of gene expression in eukaryote cells. In order to better understand genetic programs expressed during major transitions of the rust life cycle, we surveyed the complement of TFs in fungal genomes with an emphasis on Pucciniales. We found that despite their large gene numbers, rust genomes have a reduced repertoire of TFs compared to other fungi. The proportions of C2H2 and Zinc cluster - two of the most represented TF families in fungi-indicate differences in their evolutionary relationships in Pucciniales and other fungal taxa. The cold shock protein (CSP) family showed a striking expansion in Pucciniomycotina with specific duplications in the order Pucciniales. The survey of expression profiles collected by transcriptomics along the life cycle of the poplar rust fungus revealed TF genes related to major biological transitions, e.g. response to environmental cues and host infection. Particularly, poplar rust CSPs were strongly expressed in basidia produced after the overwintering stage suggesting a possible role in dormancy exit. Expression during transition from dormant telia to basidia confirmed the specific expression of the three poplar rust CSP genes. Their heterologous expression in yeast improved cell growth after cold stress exposure, strengthening their implication in dormancy exit. This study addresses for the first time TF involved in developmental transition in the rust life cycle opening perspectives to further explore molecular regulation in the biology of the Pucciniales.

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New distribution data and phylogenetic approach reveal bioregionalization of the western Palearctic ants AimBiogeographic regionalization has fascinated biogeographers and ecologists for centuries and is endued with new vitality by evolutionary perspectives. However, progress is scant for most insect groups due to shortfalls in distribution and phylogenetic information, namely Wallacean and Darwinian shortfalls respectively. Here, we used the western Palearctic ants as the case to tackle these shortfalls and test their biogeographic structure through novel distribution data and phylogenetic approaches.

LocationWestern Palearctic realm.

TaxonAnts (Formicidae).

MethodsFirstly, we developed a refined database integrating the occurrences of 747 ant species across 207 regions of the western Palearctic realm, based on newly expert-validated records derived from the existing global ant biodiversity informatics. Using range estimates for these species derived from polygons and species distribution modelling, we produced species assemblages in 50 x 50 km grid cells. We calculated taxonomic and phylogenetic turnover of ant assemblages, performing hierarchical clustering analysis using the Simpson dissimilarity index to delineate biogeographic structure.

ResultsAt both the regional list- and grid assemblage-levels, the Mediterranean has higher turnover and more biogeographic regions than northern Europe, both taxonomically and phylogenetically. Delineations based on grid assemblages detected more detailed biogeographic transitions, while those based on regional lists showed stronger insularity in biogeographic structure. The phylogenetic regionalization suggested closer but varied affinities between assemblages in comparison to the taxonomic approach.

Main conclusionsHere, we integrated expert-validated regional lists, species distribution modelling, and a recent phylogeny to tackle Wallacean and Darwinian shortfalls for an important insect group by developing a next-generation map of biogeographic regionalization for the western Palearctic ants. The results of this study suggest strong constraints from geographic barriers and potential effects of climatic history on ant distributions and evolutionary history, and also provide baseline spatial information for future investigations of regional insect distributions.

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Performance Determinants of Unsupervised Clustering Methods for Microbiome Data BackgroundIn microbiome data analysis, unsupervised clustering is often used to identify naturally occurring clusters, which can then be assessed for associations with characteristics of interest. In this work, we systematically compared beta diversity and clustering methods commonly used in microbiome analyses. We applied these to four published datasets where highly distinct microbiome profiles could be seen between sample groups.

ResultsAlthough no single method outperformed the others consistently, we did identify key scenarios where certain methods can underperform. Specifically, the Bray Curtis metric resulted in poor clustering in a dataset where high-abundance OTUs were relatively rare. In contrast, the unweighted UniFrac metric clustered poorly when used on a dataset with a high prevalence of low-abundance OTUs. To test our proposition, we systematically modified properties of the poorly performing datasets and found that this approach resulted in improved Bray Curtis and unweighted UniFrac performance. Based on these observations, we rationally combined the Bray Curtis metric and the unweighted UniFrac metrics and found that this new beta diversity metric showed high performance across all datasets. We also evaluated our findings by examining a clinical dataset where clusters are less separated.

ConclusionsOur systematic evaluation of clustering performance in these five datasets demonstrates that there is no existing clustering method that universally performs best across all datasets. We propose a combined metric of Bray Curtis and unweighted UniFrac that capitalizes on the complementary strengths of the two metrics.

sentences: Microchannel patterning strategies for in vitro structural connectivity modulation of neural networks Compartmentalized microfluidic chips have demonstrated tremendous potential to create in vitro minimalistic environments for the reproduction of the neural circuitry of the brain. Although the protocol for seeding neural soma in these devices is well known and has been widely used in myriad studies, the accurate control of the number of neurites passing through the microchannels remains challenging. However, the regulation of axonal density among different groups of neurons is still a requirement to assess the inherent structural connectivity between neuronal populations. In this work, we report the effect of microchannel patterning strategies on the modulation of neuronal connectivity by applying dimensional modifications on microchannel-connected microfluidic chambers. Our results show that those strategies can modulate the direction and the number of neuronal projections of passage, therefore regulating the strength of the structural connections between two populations of neurons. With this approach, we provide innovative microfluidic design rules for the engineering of in vitro physiologically relevant neural networks.

sentences: Sex-specific body mass ageing trajectories in adult Asian elephants In species with marked sexual dimorphism and where one sex undergoes stronger intrasexual competition, that sex is expected to age earlier or quicker. Here, we utilise a unique, longitudinal dataset of a semi-captive population of Asian elephants (Elephas maximus), a species with marked male-biased intrasexual competition, with males being larger and living shorter, and test the hypothesis that males show earlier and/or faster body mass ageing than females. We show sex-specific body mass ageing trajectories: adult males gained weight up to the age of 48 years old, followed by a decrease in body mass until natural death. In contrast, adult females gained body mass with age until a body mass decline in the last year of life. Our study shows that sex-specific life-histories shape ageing patterns, consistent with the predictions of the classical theory of ageing.

sentences: Automatic and unbiased segmentation and quantification of myofibers in skeletal muscle Skeletal muscle has the remarkable ability to regenerate. However, with age and disease muscle strength and function decline. Myofiber size, which is affected by injury and disease, is a critical measurement to assess muscle health. Here, we test and apply Cellpose, a recently developed deep learning algorithm, to automatically segment myofibers within murine skeletal muscle. We first show that tissue fixation is necessary to preserve cellular structures such as primary cilia, small cellular antennae, and adipocyte lipid droplets. However, fixation generates heterogeneous myofiber labeling, which impedes intensity-based segmentation. We demonstrate that Cellpose efficiently delineates thousands of individual myofibers outlined by a variety of markers, even within fixed tissue with highly uneven myofiber staining. We created a novel ImageJ plugin (LabelsToRois) that allows processing multiple Cellpose segmentation images in batch. The plugin also contains a semi-automatic erosion function to correct for the area bias introduced by the different stainings, identifying myofibers as accurately as human experts. We successfully applied our segmentation pipeline to uncover myofiber size differences between two different muscle injury models, cardiotoxin and glycerol. Thus, Cellpose combined with LabelsToRois allows for fast, unbiased, and reproducible myofiber quantification for a variety of staining and fixation conditions.

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Sleep and circadian phenotype in people without cone-mediated vision BackgroundLight exposure entrains the circadian clock through the intrinsically photosensitive retinal ganglion cells, which sense light in addition to the cones and rods. In congenital achromatopsia (ACHM; prevalence 1:30-50,000), the cone system is non-functional, resulting in severe light avoidance and photophobia at daytime light levels. How this condition affects circadian and neuroendocrine responses to light is not known.

MethodsIn genetically confirmed ACHM patients (n=7; age 30-72 years; 6 women, 1 male), we examined survey-assessed sleep/circadian phenotype (PSQI, ESS, MEQ, MCTQ), self-reported visual function (NEI-VFQ-25), sensitivity to light (VLSQ-8) and use of spectral filters that modify chronic light exposure. In all but one patient, we measured rest-activity cycles using actigraphy over 3 weeks and measured the melatonin phase angle of entrainment using the dim-light melatonin onset (DLMO).

ResultsACHM patients experience a severely attenuated light-dark cycle due to severe light sensitivity and habitual use of filters to reduce retinal illumination. In aggregate, both MEQ and MCTQ indicated a tendency to late chronotype. We found regular rest-activity patterns in all patients and normal phase angles of entrainment in participants with a measurable DLMO.

ConclusionsOur results reveal that a functional cone system and exposure to daytime light intensities are not necessary for regular behavioural and hormonal entrainment, even when survey-assessed sleep and circadian phenotype indicated a tendency for a late chronotype and sleep problems in our ACHM cohort. Our results can be explained by an adaptation mechanism in circadian photoreception which adjusts to the range of habitual light exposures.

sentences: A technique for resolution assessment in blind-SIM experiments Super resolution techniques are an excellent alternative to wide field microscopy, providing high resolution also in (typically fragile) biological sample. Among the various super resolution techniques, Structured Illumination Microscopy (SIM) improve resolution by employing multiple illumination patterns to be deconvolved with a dedicated software. In the case of blind SIM techniques, unknown patterns, such as speckles, are used, thus providing super resolved images, nearly unaffected by aberrations with a simplified experimental setup. Scattering Assisted Imaging, a special blind SIM technique, exploits an illumination PSF (speckle grains size), smaller than the collection PSF (defined by the collection objectives), to surpass the typical SIM resolution enhancement. However, if SAI is used, it is very difficult to extract the resolution enhancement from a priori considerations. In this paper we propose a protocol and experimental setup for the resolution measurement, demonstrating the resolution enhancement for different collection PSF values.

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The phosphatase Bph and peptidyl-prolyl isomerase PrsA are required for gelatinase expression and activity in Enterococcus faecalis Enterococcus faecalis is a common commensal bacterium in the gastrointestinal tract as well as a frequent nosocomial pathogen. The secreted metalloprotease gelatinase (GelE) is an important E. faecalis virulence factor that contributes to numerous cellular activities such as autolysis, biofilm formation, and biofilm-associated antibiotic resistance. Expression of gelE has been extensively studied and is regulated by the Fsr quorum-sensing system. Here, we identify two additional factors regulating gelatinase expression and activity in E. faecalis OG1RF. The Bph phosphatase is required for expression of gelE in an Fsr-dependent manner. Additionally, the membrane-anchored protein foldase PrsA is required for GelE activity, but not fsr or gelE gene expression. Disrupting prsA also leads to increased antibiotic sensitivity in biofilms independent of the loss of GelE activity. Together, our results expand the model for gelatinase production in E. faecalis, which has important implications for fundamental studies of GelE function in Enterococcus and also E. faecalis pathogenesis.

ImportanceIn Enterococcus faecalis, gelatinase (GelE) is a virulence factor that is also important for biofilm formation and interactions with other microbes as well as the host immune system. The long-standing model for GelE production is that the Fsr quorum sensing system positively regulates expression of gelE. Here, we update that model by identifying two additional factors that contribute to gelatinase production. The biofilm-associated Bph phosphatase regulates the expression of gelE through Fsr, and the peptidyl-prolyl isomerase PrsA is required for production of active GelE through an Fsr-independent mechanism. This provides important insight into how regulatory networks outside of the fsr locus coordinate expression of gelatinase.

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Plasma donation induces a protein expression profile shift in circulating human blood Commercial plasma donation yields a change in protein concentration similar to that observed in therapeutic plasma exchange (TPE) in humans. The post-donation expression profile shows an increase in many proteins after even a single donation, and the effect is enhanced with additional donations. Unlike TPE, human plasma donation returns saline without albumin to the donor, thus eliminating supplemental albumin as a contributing factor. The observed fold change falls outside the variation expected by natural circadian expression changes.

N=6 humans (four donors, one monthly control and one circadian test). Seventeen antibody tests were performed, measuring the concentration of 507 proteins for each test.

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The unbiased estimation of r2 between two sets of noisy neural responses The Pearson correlation coefficient squared, r2, is often used in the analysis of neural data to estimate the relationship between neural tuning curves. Yet this metric is biased by trial-to-trial variability: as trial-to-trial variability increases, measured correlation decreases. Major lines of research are confounded by this bias, including the study of invariance of neural tuning across conditions and the similarity of tuning across neurons. To address this, we extend the estimator, [Formula], developed for estimating model-to-neuron correlation to the neuron-to-neuron case. We compare the estimator to a prior method developed by Spearman, commonly used in other fields but widely overlooked in neuroscience, and find that our method has less bias. We then apply our estimator to the study of two forms of invariance and demonstrate how it avoids drastic confounds introduced by trial-to-trial variability.

Significant StatementQuantifying the similarity between two sets of averaged neural responses is fundamental to the analysis of neural data. A ubiquitous metric of similarity, the correlation coefficient, is attenuated by trial-to-trial variability that depends on a variety of irrelevant factors. Spearman recognized this problem and proposed corrected methods that have been extended over a century. We show this method has large asymptotic biases and derive a novel estimator to overcome this. Despite the frequent use of the correlation coefficient in neuroscience, consensus on how to address this fundamental statistical issue has not been reached. We both explicate this issue in a neuroscience setting while at the same time making major strides in addressing it.

sentences: Modular safe-harbor Transgene Insertion (MosTI) for targeted single-copy and extrachromosomal array integration in C. elegans. Efficient and reproducible transgenesis facilitates and accelerates research using genetic model organisms. Here we describe a modular safe harbor transgene insertion (MosTI) for use in C. elegans which improves targeted insertion of single-copy transgenes by homology directed repair and targeted integration of extrachromosomal arrays by non-homologous end-joining. MosTI allows easy conversion between selection markers at insertion site and a collection of universal targeting vectors with commonly used promoters and fluorophores. Insertions are targeted at three permissive safe-harbor intergenic locations and transgenes are reproducibly expressed in somatic and germ cells. Chromosomal integration is mediated by CRISPR/Cas9, and positive selection is based on a set of split markers (unc-119, hygroR, and gfp) where only animals with chromosomal insertions are rescued, resistant to antibiotics, or fluorescent, respectively. Single-copy insertion is efficient using either constitutive or heat-shock inducible Cas9 expression (25 - 75%) and insertions can be generated from a multiplexed injection mix. Extrachromosomal array integration is also efficient (7 - 44%) at MosTI landing sites or at the endogenous unc-119 locus. We use short-read sequencing to estimate the plasmid copy numbers for eight integrated arrays (6 to 37 copies) and long-read Nanopore sequencing to determine the structure and size (5.4 Mb) of one array. Using universal targeting vectors, standardized insertion strains, and optimized protocols, it is possible to construct complex transgenic strains which should facilitate the study of increasingly complex biological problems in C. elegans.

sentences: Visualization of bacterial type 3 secretion system components down the molecular level by MINFLUX nanoscopy Type 3 secretion systems (T3SS) are essential virulence factors of numerous bacterial pathogens and inject effector proteins into host cells. The needle-like T3SS machinery consists of more than 20 components, has a length of around 100 nm and a diameter of up to 30 nm according to EM studies. Its intrabacterial components are highly dynamic and in permanent exchange with other bacterial structures. Therefore, a temporally and spatially resolved visualization of the T3SS using fluorescence microscopy techniques has been challenging. In the present study, novel labeling strategies were combined with super-resolution microscopy such as STED, STORM and MINFLUX. MINFLUX nanoscopy allowed to visualize components of the T3SS machinery such as the dynamic sorting platform component YscL and the extrabacterial pore protein YopD at unprecedented resolutions. The presented results represent the basis for an in depth investigation of T3SS structure and function and therefore gain new insights into the infection process of human pathogens in order to develop novel treatment and prevention strategies.

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An efficient multi-scale CNN model with intrinsic feature integration for motor imagery EEG subject classification in brain-machine interfaces ObjectiveElectroencephalogram (EEG) based motor imagery (MI) classification is an important aspect in brain-machine interfaces (BMIs) which bridges between neural system and computer devices decoding brain signals into recognizable machine commands. However, the MI classification task is challenging due to inherent complex properties, inter-subject variability, and low signal-to-noise ratio (SNR) of EEG signals. To overcome the above-mentioned issues, the current work proposes an efficient multi-scale convolutional neural network (MS-CNN).

ApproachIn the framework, discriminant user-specific features have been extracted and integrated to improve the accuracy and performance of the CNN classifier. Additionally, different data augmentation methods have been implemented to further improve the accuracy and robustness of the model.

Main resultsThe model achieves an average classification accuracy of 93.74% and Cohens kappa-coefficient of 0.92 on the BCI competition IV2b dataset outperforming several baseline and current state-of-the-art EEG-based MI classification models.

SignificanceThe proposed algorithm effectively addresses the shortcoming of existing CNN-based EEG-MI classification models and significantly improves the classification accuracy.

sentences: TransCRISPR - sgRNA design tool for CRISPR/Cas9 experiments targeting transcription factor motifs Transcription factors (TFs) regulate gene expression via binding to specific sequence motifs in promoters or enhancers. They are involved in various biological processes and their aberrant action may lead to multiple pathological states. To better understand the function of a given TF, it is necessary to identify its crucial binding sites and target genes, which is not a trivial task. CRISPR/Cas9 has been recently successfully applied in the study of TFs. Here, we present an online tool, transCRISPR, created to search for TF binding motifs in the user-provided query and design optimal sgRNAs targeting them. Users can obtain sgRNAs for chosen TFs motifs, for up to tens of thousands of target regions in the human or mouse genome, either for the Cas9 or dCas9 system. TransCRISPR provides user-friendly tables and visualizations, summarizing features of identified motifs and designed sgRNAs such as genomic localization, quality scores, closest transcription start sites, and others. TransCRISPR is available at https://transcrispr.igcz.poznan.pl/transcrispr/.

sentences: Quantifying the dynamics of viral recombination during free virus and cell-to-cell transmission in HIV-1 infection Recombination has been shown to contribute to HIV-1 evolution in vivo, but the underlying dynamics are extremely complex, depending on the nature of the fitness landscapes and of epistatic interactions. A less well-studied determinant of recombinant evolution is the mode of virus transmission in the cell population. HIV-1 can spread by free virus transmission, resulting largely in singly infected cells, and also by direct cell-to-cell transmission, resulting in the simultaneous infection of cells with multiple viruses. We investigate the contribution of these two transmission pathways to recombinant evolution, by applying mathematical models to in vitro experimental data on the growth of fluorescent reporter viruses under static conditions (where both transmission pathways operate), and under gentle shaking conditions, where cell-to-cell transmission is largely inhibited. The parameterized mathematical models are then used to extrapolate the viral evolutionary dynamics beyond the experimental settings. Assuming a fixed basic reproductive ratio of the virus (independent of transmission pathway), we find that recombinant evolution is fastest if virus spread is driven only by cell-to-cell transmission, and slows down if both transmission pathways operate. Recombinant evolution is slowest if all virus spread occurs through free virus transmission. This is due to cell-to-cell transmission (i) increasing infection multiplicity, (ii) promoting the co-transmission of different virus strains from cell to cell, and (iii) increasing the rate at which point mutations are generated as a result of more reverse transcription events. This work further resulted in the estimation of various parameters that characterize these evolutionary processes. For example, we estimate that during cell-to-cell transmission, an average of 3 viruses successfully integrated into the target cell, which can significantly raise the infection multiplicity compared to free virus transmission. In general, our study points towards the importance of infection multiplicity and cell-to-cell transmission for HIV-evolution.

sentences: SPIKE-TRIGGERED INTRASPINAL MICROSTIMULATION IMPROVES MOTOR PERFORMANCE IN AN AMBULATORY RAT MODEL OF SPINAL CORD INJURY The purpose of this study was to determine if spike-triggered intraspinal microstimulation (ISMS) results in improved motor performance in an ambulatory rat model of spinal cord injury (SCI). Experiments were carried out in adult male Sprague Dawley rats with 175 kdyn moderate T8 contusion injury. Rats were randomly assigned to one of two groups: Control or Activity Dependent Stimulation (ADS) therapy. Four weeks post-SCI, all rats were implanted with a recording electrode in the left hindlimb motor cortex and a fine-wire, custom-made stimulating electrode in the contralateral lumbar spinal cord. Intracortical and intraspinal microstimulation were used to find sites of similar hip representation areas, which were paired together for ADS therapy. In the ADS therapy group, spike-stimulus conditioning was administered for 4 hours/day, 4 days/week, for 4 weeks via a tethered cable in a testing chamber. During therapy sessions, single-unit spikes were discriminated in real time in the hindlimb motor cortex and used to trigger stimulation in the spinal cord ventral horn. The optimal stimulus intensity (50% ISMS movement threshold) and spike-stimulus delay (10ms) determined in preliminary anesthetized preparations were used during ADS. Control rats were similarly implanted with electrodes but did not receive stimulation therapy. Motor performances of each rat were evaluated before SCI contusion, once a week post-SCI for four weeks (prior to electrode implantation), and once a week post-conditioning for four weeks. Behavioral testing included BBB scoring, Ledged Beam walking, Horizontal Ladder walking, treadmill kinematics via the DigiGait and TreadScan system, and open field walking using OptiTrack kinematic analysis. BBB scores were significantly improved in ADS rats compared to Control rats after 1 week of therapy. In the ADS therapy rats, BBB scores were significantly improved after two weeks of ADS therapy when compared to pre-therapy. Foot fault scores on the Horizontal Ladder were significantly lower in ADS rats compared to pre-therapy ADS and Control rats after 1 week of therapy and returned to pre-injury measures after three weeks of ADS therapy. The Ledged Beam test and kinematic analysis using the DigiGait and TreadScan system showed deficits after SCI in both ADS and Control rats but there were no significant differences between groups after 4 weeks of ADS therapy. These results show that activity dependent stimulation after spinal cord injury using spike-triggered ISMS enhances behavioral recovery of locomotor function as measured by the BBB score and the Horizontal Ladder task.

sentences: Partial response electromyography as a markerof the individual stopping latency Response inhibition is among the core constructs of cognitive control. It is notoriously difficult to quantify from overt behavior, since the outcome of successful inhibition is the lack of a behavioral response. Currently, the most common measure of action stopping, and by proxy response inhibition, is the model-based stop signal reaction time (SSRT) derived from the stop signal task. Recently, partial response electromyography (prEMG) has been introduced as a complementary physiological measure to capture individual stopping latencies. PrEMG refers to muscle activity initiated by the go signal that plummets after the stop signal before its accumulation to a full response. Whereas neither the SSRT nor the prEMG is an unambiguous marker for neural processes underlying response inhibition, our analysis indicates that the prEMG peak latency is better suited to investigate brain mechanisms of action stopping. This study is a methodological resource with a comprehensive overview of the psychometric properties of the prEMG in a stop signal task, and further provides practical tips for data collection and analysis.

sentences: Mitochondrial and redox modifications in early stages of Huntington disease Defects in mitochondrial function and mitochondrial-related redox deregulation have been attributed to Huntingtons disease (HD), a genetic neurodegenerative disorder largely affecting the striatum. However, whether these changes occur in early stages of the disease and can be detected in vivo is still unclear. Thus, in the present study, we analyzed changes in mitochondrial function and overreduced states associated with production of reactive oxygen species (ROS) at early stages and along disease progression. Studies were performed in vivo in human brain using positron emission tomography (PET) using [64Cu]-ATSM and ex vivo in human skin fibroblasts of premanifest and prodromal (Pre-M) and manifest HD patients; in vivo brain [64Cu]-ATSM PET and isolated mitochondria derived from striatum and cortex were also analyzed in YAC128 transgenic mouse at pre-symptomatic (3 month-old, mo) and symptomatic (6 to 12 mo) stages. Oxygen consumption rates were assessed by Seahorse analysis, hydrogen peroxide levels were determined using fluorescent probes and mitochondrial morphology by transmission electron microscopy in human skin fibroblasts and mouse striatal and cortical isolated mitochondria. Pre-M HD carriers exhibited enhanced whole-brain (with exception of caudate) [64Cu]-ATSM labelling, correlating with CAG repeat number. Fibroblasts from Pre-M showed enhanced basal and maximal respiration, proton (H+) leak and increased hydrogen peroxide levels, the later progressing to manifest HD; mitochondria from fibroblasts of Pre-M HD carriers also showed reduced roundness, while higher number of mitochondrial DNA copies correlated with maximal respiratory capacity. In vivo animal PET analysis showed increased accumulation of [64Cu]-ATSM in YAC128 mouse striatum. Pre-symptomatic YAC128 mouse striatal isolated mitochondria exhibited a rise in basal and maximal mitochondrial respiration and in ATP production, along with increased complex II and III activities; mouse HD mitochondria also showed enhanced mitochondrial hydrogen peroxide levels and roundness, as revealed by brain ultrastructure analysis, and defects in Ca2+ handling, supporting increased striatal susceptibility in YAC128 mouse brain. Data demonstrate both human and mouse mitochondrial overactivity and altered morphology at early HD stages, facilitating redox unbalance, the latter extending over manifest disease stages.

sentences: Manual Annotation of Genes within Drosophila Species: the Genomics Education Partnership protocol Annotating the genomes of multiple organisms allows us to study their genes as well as the evolution of those genes. While many eukaryotic genome assemblies already include computational gene predictions, these predictions can benefit from review and refinement through manual gene annotation. The Genomics Education Partnership (GEP; thegep.org) has developed an annotation protocol for protein-coding genes that enables undergraduate students and other researchers to create high-quality gene annotations that can be utilized in subsequent scientific investigations. For example, this protocol has been utilized by the GEP faculty to engage undergraduate students in the comparative annotation of genes involved in the insulin signaling pathway in 28 Drosophila species, using D. melanogaster as the informant genome. Students construct gene models using multiple lines of computational and experimental evidence including expression data (e.g., RNA-Seq), sequence similarity (e.g., BLAST, multiple sequence alignments), and computational gene predictions. For quality control, each gene is annotated by at least two students working independently, followed by reconciliation of the submitted gene models by a more experienced student. This article provides an overview of the annotation protocol and describes how discrepancies in student submitted gene models are resolved to produce a final, high-quality gene set suitable for subsequent analyses. This annotation protocol can be adapted to other scientific questions (e.g., expansion of the Drosophila Muller F element) and other species (e.g., parasitoid wasps) to provide additional opportunities for undergraduate students to participate in genomics research. These student annotation efforts can substantially improve the quality of gene annotations in publicly available genomic databases.

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Understanding the role of memory re-activation and cross-reactivity in the defense against SARS-CoV-2 Recent efforts in understanding the course and severity of SARS-CoV-2 infections have highlighted both potential beneficial as well as detrimental effects of cross-reactive antibodies derived from memory immunity. Specifically, due to a significant degree of sequence similarity between SARS-CoV-2 and other members of the coronavirus family, memory B-cells that emerged from previous infections with endemic human coronaviruses (HCoVs) could be re-activated upon encountering the newly emerged SARS-CoV-2, thus prompting the production of cross-reactive antibodies. Understanding the affinity and concentration of these potentially cross-reactive antibodies to the new SARS-CoV-2 antigens is therefore particularly important when assessing both existing immunity against common HCoVs and adverse effects like antibody-dependent enhancement (ADE) in COVID-19. However, these two fundamental parameters cannot easily be deconvoluted by surface-based assays like enzyme-linked immunosorbent assays (ELISAs) which are routinely used to assess cross-reactivity.

Here, we have used microfluidic antibody-affinity profiling (MAAP) to quantitatively evaluate the humoral immune response in COVID-19 convalescent patients by determining both antibody affinity and concentration against spike antigens of SARS-CoV-2 directly in nine convalescent COVID-19 patient and three pre-pandemic sera that were seropositive for common HCoVs. All 12 sera contained low concentrations of high affinity antibodies against spike antigens of HCoV-NL63 and HCoV-HKU1, indicative of past exposure to these pathogens, while the affinity against the SARS-CoV-2 spike protein was lower. These results suggest that cross-reactivity as a consequence of memory re-activation upon an acute SARS-CoV-2 infection may not be a significant factor in generating immunity against SARS-CoV-2.

sentences: Automated verification, assembly, and extension of GBM stem cell network model with knowledge from literature and data Signaling network models are usually assembled from information in literature and expert knowledge or inferred from data. The goal of modeling is to gain mechanistic understanding of key signaling pathways and provide predictions on how perturbations affect large-scale processes such as disease progression. For glioblastoma multiforme (GBM), this task is critical, given the lack of effective treatments and pace of disease progression. Both manual and automated assembly of signaling networks from data or literature have drawbacks. Existing GBM networks, as well as networks assembled using state-of-the-art machine reading, fall short when judged by the quality and quantity of information, as well as certain attributes of the overall network structure. The contributions of this work are two-fold. First, we propose an automated methodology for verification of signaling networks. Next, we discuss automation of network assembly and extension that relies on methods and resources used for network verification, thus, implicitly including verification in these processes. In addition to these methods, we also present, and verify a comprehensive GBM network assembled with a hybrid of manual and automated methods. Finally, we demonstrate that, while an automated network assembly is fast, such networks still lack precision and realistic network topology.

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Pinch2 is a novel regulator of myelination in the Central Nervous System The extensive morphological changes of oligodendrocytes during axon ensheathment and myelination involve assembly of the Ilk-Parvin-Pinch (IPP) heterotrimeric complex of proteins to relay essential mechanical and biochemical signals between integrins and the actin cytoskeleton. Binding of Pinch 1 and 2 isoforms to Ilk is mutually exclusive and allows the formation of distinct IPP complexes with specific signaling properties. Using tissue-specific conditional gene ablation in mice, we reveal an essential role for Pinch2 during central nervous system myelination. Unlike Pinch1-gene ablation, loss of Pinch2 in oligodendrocytes results in hypermyelination and in the formation of pathological myelin outfoldings in white matter regions. These structural changes concurred with inhibition of Rho GTPases RhoA and Cdc42 activities and phenocopied aspects of myelin pathology observed in corresponding mouse mutants. We propose a dual role for Pinch2 in preventing excess of myelin wraps through RhoA-dependent control of membrane growth and in fostering myelin stability via Cdc42-dependent organization of cytoskeletal septins. Together, these findings indicate that IPP-containing Pinch2 is a novel critical cell-autonomous molecular hub ensuring synchronous control of key signaling networks during developmental myelination.

Summary statementPinch proteins are core components of a ternary protein complex comprising Ilk and Parvin (IPP). This work identifies Pinch2 as key regulator of the formation and maturation of CNS myelin.

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A comprehensive comparison on cell type composition inference for spatial transcriptomics data Spatial transcriptomic (ST) technologies allow researchers to examine high-quality RNA-sequencing data along with maintained two-dimensional positional information as well as a co-registered histology image. A popular use of ST omics data is to provide insights about tissue structure and spatially unique features. However, due to the technical nature unique to most ST data, the resolution varies from a diameter of 2-10m to 50-100m instead of single-cell resolution, which brings uncertainty into cell number and cell mixture within each ST spot. Motivated by the important role for spatial arrangement of cell types within a tissue in physiology and disease pathogenesis, several ST deconvolution methods have been developed and are being used to explore gene expression variation and identification of spatial domains. The aim of this work is to review state-of-the-art methods for ST deconvolution, while comparing their strengths and weaknesses. Specifically, we use four real datasets to examine the performance of eight methods across different tissues and technological platforms.

Key PointsO_LICell mixture inference is a critical step in the analysis of spatial transcriptomics (ST) data to prevent downstream analysis suffering from confounding factors at the spot level.
C_LIO_LIExisting ST deconvolution methods can be classified into three groups: probabilistic-based, non-negative matrix factorization and non-negative least squares based, and other deep learning framework-based methods.
C_LIO_LIWe compared eight ST deconvolution methods by using two single cell level resolution datasets and two spot level resolution ST datasets. We provided practical guidelines for the choice of method under different scenarios as well as the optimal subsets of genes to use for each method.
C_LI

sentences: Single cell Iso-Sequencing enables rapid genome annotation for scRNAseq analysis Single cell RNA sequencing (scRNAseq) is a powerful technique that continues to expand across various biological applications. However, incomplete 3' UTR annotations in less developed or non-model systems can impede single cell analysis resulting in genes that are partially or completely uncounted. Performing scRNAseq with incomplete 3' UTR annotations can impede the identification of cell identities and gene expression patterns and lead to erroneous biological inferences. We demonstrate that performing single cell isoform sequencing (ScISOr-Seq) in tandem with scRNAseq can rapidly improve 3' UTR annotations. Using threespine stickleback fish (Gasterosteus aculeatus), we show that gene models resulting from a minimal embryonic ScISOr-Seq dataset retained 26.1% greater scRNAseq reads than gene models from Ensembl alone. Furthermore, pooling our ScISOr-Seq isoforms with a previously published adult bulk Iso-Seq dataset from stickleback, and merging the annotation with the Ensembl gene models, resulted in a marginal improvement (+0.8%) over the ScISOr-Seq only dataset. In addition, isoforms identified by ScISOr-Seq included thousands of new splicing variants. The improved gene models obtained using ScISOr-Seq lead to successful identification of cell types and increased the reads identified of many genes in our scRNAseq stickleback dataset. Our work illuminates ScISOr-Seq as a cost-effective and efficient mechanism to rapidly annotate genomes for scRNAseq.

sentences: Exhaustive Cross-linking Search with Protein Feedback Improving the sensitivity of protein-protein interaction detection and protein structure probing is a principal challenge in cross-linking mass spectrometry (XL-MS) data analysis. In this paper, we propose an exhaustive cross-linking search method with protein feedback (ECL-PF) for cleavable XL-MS data analysis. ECL-PF adopts an optimized /{beta} mass detection scheme and establishes protein-peptide association during the identification of cross-linked peptides. Existing major scoring functions can all benefit from the ECL-PF workflow to a great extent. In comparisons using synthetic datasets and hybrid simulated datasets, ECL-PF achieved three-fold higher sensitivity over standard techniques. In experiments using real datasets, it also identified 91.6% more cross-link spectrum matches and 52.6% more unique cross-links.

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A new prognostic model for pancreatic cancer based on pyroptosis-related genes Pancreatic cancer is one of the most common malignant tumors of the digestive tract. It is known as the "king of cancer" in the field of cancer, and is one of the worst prognosis malignant tumors. Pyroptosis is a kind of programmed cell death, which can promote the inflammatory response of cells. Studies have shown that the effect of pyroptosis-related genes in cancer is significant. However, the role of pyroptosis in pancreatic cancer is not clear. The aim of this study is to establish a prognostic model based on pyroptosis. The gene expression and clinical data of pancreatic cancer patients were obtained from TCGA and verified in GEO. The differential expression of 33 pyroptosis-related genes in pancreatic cancer and normal tissues was analyzed, of which 6 genes were up-regulated and 12 genes were down regulated. Then, it was analyzed that pyroptosis-related genes were mainly enriched in the defense against bacteria and pyroptosis pathways. A concise and reliable model is established by lasso-cox regression analysis. Km curve shows that there are differences between high-risk group and low-risk group. And the nomogram has reliable prediction ability. In conclusion, pyroptosis an important role in pancreatic cancer, which can be used for the prediction of pancreatic cancer and provide a new perspective for the treatment of pancreatic cancer.

AUTHOR APPROVALSThe authors have seen and approved the manuscript, and that it hasnt been accepted or published elsewhere.

COMPETING INTERESTSThe authors declare no competing interests.

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From climate warming to accelerated cellular ageing: an experimental study in wild birds Climate change is increasing both the average ambient temperature and the frequency and severity of heat waves. While direct mortality induced by heat waves is increasingly reported, sub-lethal effects are also likely to impact wild populations. We hypothesized that accelerated ageing could be a cost of being exposed to higher ambient temperature, especially in early-life when thermoregulatory capacities are not fully developed. We tested this hypothesis in wild great tit (Parus major) by experimentally increasing nest box temperature by ca. 2{degrees}C during postnatal growth and measuring telomere length, a biomarker of cellular ageing predictive of survival prospects in many bird species. While increasing early-life temperature does not affect growth or survival to fledging, it accelerates telomere shortening and reduces medium-term survival from 34% to 19%. Heat-induced telomere shortening was not explained by oxidative stress, but more likely by an increase in energy demand (i.e. higher thyroid hormones levels, increased expression of glucocorticoid receptor, increased mitochondrial density) leading to a reduction in telomere maintenance mechanisms (i.e. decrease in the gene expression of telomerase and protective shelterin). Our results thus suggest that climate warming can affect ageing rate in wild birds, with potential impact on population dynamics and persistence.

Significance statementStressful environmental conditions are known to accelerate cellular ageing, especially when experienced early in life. One unexplored avenue through which climate warming might affect wild animal populations is accelerated ageing. Here we show that increasing nest temperature by ca. 2{degrees}C during postnatal growth in a wild bird species can impact numerous physiological pathways and medium-term survival. Notably, artificially warming nests accelerates the shortening of telomeres, which are the protective end-caps of chromosomes considered as a hallmark of ageing. We thus suggest that warm ambient temperatures might accelerate ageing in wild animals, which can potentially impact population dynamics and extinction risk in the face of climate change.

sentences: piggyBac-system-mediated genomic integration of large variant libraries for deep mutational scanning in mammalian cells There is a need of introducing large variant libraries into mammalian cells to facilitate deep mutational scanning (DMS). In this study, we develop an approach to integrate in-vitro ligation dsDNA variant library into cultured mammalian cell genome via the piggyBac transposon system. T4 DNA ligase was employed to produce dsDNA ligation products containing variant library sequence flanked by piggyBac inverted terminal repeat sequences (ITRs) at both ends. The ligation products were then cotransfected with a piggyBac transposase expression vector into mammalian cells for library integration. The clone formation assay results showed that about 29,000 resistance clones were generated from a single well of 6-well-plate transfected HEK293T cells. As a proof of principle of using the strategy for DMS, a GFP chromophore random variant library was integrated into the HEK293T cell genome. By FACS selection, we enriched GFP-positive cells. The next-generation sequencing (NGS) results showed that the GFP-chromophore amino acid sequences were restored after selection.

sentences: Two mosquito odorant receptors with reciprocal specificity mediated by a single amino acid residue Insects perceive the environment with a repertoire of promiscuous and narrowly tuned odorant receptors (ORs). The southern house mosquito, Culex quinquefasciatus, utilizes two ORs, CquiOR10 and CquiOR2, which are narrowly tuned and well conserved among mosquito species. They detect the oviposition attractants skatole and indole, respectively, with reciprocal specificity. We envisioned that by reverting specificity with chimeric receptors, we could gain a better insight into the specificity determinants of insect ORs. We swapped the seven transmembrane (TM) domains of CquiOR10 and CquiOR2 and identified TM2 as a specificity determinant. With additional mutations, we showed that CquiOR10A73L behaved like CquiOR2. Conversely, CquiOR2L74A recapitulated CquiOR10 specificity. Next, we generated structural models of CquiOR10 and CquiOR10A73L using RoseTTAFold and AlphaFold and docked skatole and indole using RosettaLigand. These modeling studies suggested space-filling constraints around A73, although this residue in CquiOR10 did not directly form contacts with skatole or indole. Consistent with this hypothesis, CquiOR10 mutants with a bulkier residue (Ile, Val) were insensitive to skatole and indole, whereas CquiOR10A73G retained the specificity to skatole and showed a higher sensitivity than the wild-type receptor CquiOR10. On the other hand, Leu to Gly mutation of the indole receptor CquiOR2 reverted the specificity to skatole. Lastly, CquiOR10 was insensitive to 3-ethylindole, whereas CquiOR10A73G responded to the three ligands with 3-ethylindole eliciting a slightly higher response than indole. We then concluded that the specificity of these receptors is mediated by a single amino acid substitution leading to dilation or contraction of the binding pocket.

sentences: Molecular Mechanism of Hyperactivation Conferred by a Truncated TRPA1 Disease Mutant Suggests New Gating Insights The wasabi receptor, TRPA1, is a non-selective homotetrameric cation channel expressed in primary sensory neurons of the pain pathway, where it is activated by diverse chemical irritants. A direct role for TRPA1 in human health has been highlighted by the discovery of genetic variants associated with severe pain disorders. One such TRPA1 mutant was identified in a father-son pair with cramp fasciculation syndrome (CFS) and neuronal hyperexcitability-hypersensitivity symptoms that may be caused by aberrant channel activity, though the mechanism of action for this mutant is unknown. Here, we show the CFS-associated R919* TRPA1 mutant is functionally inactive when expressed alone in heterologous cells, which is not surprising since it lacks the 201 C-terminal amino acids that house critical channel gating machinery including the pore-lining transmembrane helix. Interestingly, the R919* mutant confers enhanced agonist sensitivity when co-expressed with wild type (WT) TRPA1. This channel hyperactivation mechanism is conserved in distant TRPA1 species orthologues and can be recapitulated in the capsaicin receptor, TRPV1. Using a combination of ratiometric calcium imaging, immunostaining, surface biotinylation, pulldown assays, fluorescence size exclusion chromatography, and proximity biotinylation assays, we show that the R919* mutant co-assembles with WT subunits into heteromeric channels. Within these heteromers, we postulate that R919* TRPA1 subunits contribute to hyperactivation by lowering energetic barriers to channel activation contributed by the missing regions. Additionally, we show heteromer activation can originate from the R919* TRPA1 subunits, which suggests an unexpected role for the ankyrin repeat and coiled coil domains in concerted channel gating. Our results demonstrate the R919* TRPA1 mutant confers gain-of-function thereby expanding the physiological impact of nonsense mutations, reveals a novel and genetically tractable mechanism for selective channel sensitization that may be broadly applicable to other receptors, and uncovers new gating insights that may explain the molecular mechanism of temperature sensing by some TRPA1 orthologues.

sentences: Feature-based attention enables robust, long-lasting location transfer in human perceptual learning Visual perceptual learning (VPL) is typically specific to the trained location and feature. However, the degree of specificity depends upon particular training protocols. Manipulating covert spatial attention during training facilitates learning transfer to other locations. Here we investigated whether feature-based attention (FBA), which enhances the representation of particular features throughout the visual field, facilitates VPL transfer, and how long such an effect would last. To do so, we implemented a novel task in which observers discriminated a stimulus orientation relative to two reference angles presented simultaneously before each block. We found that training with FBA enabled remarkable location transfer, reminiscent of its global effect across the visual field, but preserved orientation specificity in VPL. Critically, both the perceptual improvement and location transfer persisted after one year. Our results reveal robust, long-lasting benefits induced by FBA in VPL, and have translational implications for improving generalization of training protocols in visual rehabilitation.

sentences: Empirical prediction of variant-associated cryptic-donors with 87% sensitivity and 95% specificity Predicting which cryptic-donors may be activated by a genetic variant is notoriously difficult. Through analysis of 5,145 cryptic-donors activated by 4,811 variants (versus 86,963 decoy-donors not used; any GT or GC), we define an empirical method predicting cryptic-donor activation with 87% sensitivity and 95% specificity. Strength (according to four algorithms) and proximity to the authentic-donor appear important determinants of cryptic-donor activation. However, other factors such as auxiliary splicing elements, which are difficult to identify, play an important role and are likely responsible for current prediction inaccuracies. We find that the most frequent mis-splicing events at each exon-intron junction, mined from 40,233 RNA-sequencing samples, predict with remarkable accuracy which cryptic-donor will be activated in rare disease. Aggregate RNA-Sequencing splice-junction data provides an accurate, evidence-based method to predict variant-activated cryptic-donors in genetic disorders, assisting pathology consideration of possible consequences of a variant for the encoded protein and RNA diagnostic testing strategies.

sentences: Combined impacts of climate and land use change and the future of Neotropical bat biodiversity Forecasting the effects of global change on biodiversity is necessary to anticipate the threats operating at different scales in space and time. Climate change may create unsuitable environmental conditions, forcing species to move to persist. However, land-use changes create barriers that limit the access of some species to future available habitats. Here, we project the impacts of climate and land-use change on 228 Neotropical bat species by forecasting changes in environmental suitability, while accounting for the effect of habitat type specialization and simulating dispersal across suitable patches. We also identify the most vulnerable ecoregions and those that may offer future stable refugia. We further investigate potential functional changes by analysing the response of different trophic guilds. We found that the range contraction of habitat specialists, especially frugivores, was more frequent and stronger under all simulated scenarios. Projected changes differ markedly across ecoregions. While the Amazon region is likely to undergo high turnover rates in bat composition, the Andean grassland, Cerrado and Chaco might experience the greatest losses. The expansion of habitat generalists, which forage in open areas and commonly establish large colonies in manmade structures, coupled with the range contraction of habitat specialists is projected to homogenize bat communities across the Neotropics. Overall, dispersal will likely be the key for the future of Neotropical bat diversity. Therefore, safeguarding the refugia highlighted here, by expanding and connecting the existing network of protected areas, for example, may allow species to move in response to global change.

sentences: Active Cell Divisions Generate Exotic Fourfold Orientationally Ordered Phase in Living Tissue AO_SCPLOWBSTRACTC_SCPLOWMorphogenesis, the process through which genes generate form, establishes tissue scale order as a template for constructing the complex shapes of the body plan. The extensive growth required to build these ordered substrates is fuelled by cell proliferation, which, naively, should destroy order. The active mechanisms that couple cellular and physical processes to generate and maintain global order, thereby reconciling this seeming contradiction, remain elusive. Using live imaging and tissue cartography, we quantitatively analyze the dynamics of fourfold tissue ordering in the crustacean Parhyale hawaiensis. We show that cell divisions are the main drivers of tissue flow leading to a fourfold orientationally ordered phase. Waves of anisotropic cell proliferation propagate across the embryo with precise choreography, such that defects introduced into the nascent lattice by cell divisions are healed by subsequent divisions through active defect climb. Orchestrating cell proliferation rates and orientations enables cell divisions to organize, rather than fluidize, the tissue. The result is a robust, active mechanism for generating global orientational order in a non-equilibrium system that sets the stage for the subsequent development of shape and form.

sentences: Prolonged development of long-term potentiation at lateral entorhinal cortex synapses onto adult-born neurons Critical period plasticity at adult-born neuron synapses is widely believed to contribute to the learning and memory functions of the hippocampus. Experience regulates circuit integration and for a transient interval, until cells are ~6 weeks old, new neurons display enhanced long-term potentiation (LTP) at afferent and efferent synapses. Since neurogenesis declines substantially with age, this raises questions about the extent of lasting plasticity offered by adult-born neurons. Notably, however, the hippocampus receives sensory information from two major cortical pathways. Broadly speaking, the medial entorhinal cortex conveys spatial information to the hippocampus via the medial perforant path (MPP), and the lateral entorhinal cortex, via the lateral perforant path (LPP), codes for the cues and items that make experiences unique. While enhanced critical period plasticity at MPP synapses is relatively well characterized, no studies have examined long-term plasticity at LPP synapses onto adult-born neurons, even though the lateral entorhinal cortex is uniquely vulnerable to aging and Alzheimers pathology. We therefore investigated LTP at LPP inputs both within (4-6 weeks) and beyond (8+ weeks) the traditional critical period. At immature stages, adult-born neurons did not undergo significant LTP at LPP synapses, and often displayed long-term depression after theta burst stimulation. However, over the course of 3-4 months, adult-born neurons displayed increasingly greater amounts of LTP. Analyses of short-term plasticity point towards a presynaptic mechanism, where transmitter release probability declines as cells mature, providing a greater dynamic range for strengthening synapses. Collectively, our findings identify a novel form of new neuron plasticity that develops over an extended interval, and may therefore be relevant for maintaining cognitive function in aging.

sentences: A δ-cell subpopulation with a pro-β-cell identity confers efficient age-independent recovery in a zebrafish model of diabetes Restoring damaged {beta}-cells in diabetic patients by harnessing the plasticity of other pancreatic cells raises the questions of the efficiency of the process and of the functionality of the new Insulin-expressing cells. To overcome the weak regenerative capacity of mammals, we used regeneration-prone zebrafish to study {beta}-cells arising following destruction. We show that most new insulin cells differ from the original {beta}-cells as they are Somatostatin+ Insulin+, but are nevertheless functional and normalize glycemia. These bihormonal cells are transcriptionally close to a subset of {delta}-cells in normal islets characterized by the expression of somatostatin 1.1 (sst1.1), the {beta}-cell genes pdx1, slc2a2 and gck, and the machinery for glucose-induced Insulin secretion. {beta}-cell destruction triggers massive sst1.1 {delta}-cell conversion to bihormonal cells. Our work shows that their pro- {beta}-cell identity predisposes this zebrafish {delta}-cell subpopulation to efficient age-independent neogenesis of Insulin-producing cells and provides clues to restoring functional {beta}-cells in mammalian diabetes models.

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TMEM106B is increased in Multiple Sclerosis plaques, and deletion causes accumulation of lipid after demyelination During inflammatory, demyelinating diseases such as multiple sclerosis (MS) axonal damage is prevalent early in the disease course. Axonal damage includes swellings, defects in transport, and failure to clear damaged intracellular proteins, all of which affect recovery and compromise the integrity of neurons and remyelination. Autophagy and the clearance of damaged cell components by the proteasome are important for the maintenance of normal cellular turnover; and the restoration of cellular homeostasis. The gradual accumulation of insoluble proteins in the brain is known to impair recovery from several neurodegenerative diseases. In this study, we used mass spectrometry to identify insoluble proteins within high-speed, mercaptoethanol/sarcosyl-insoluble pellets from purified white matter plaques isolated from the brains of individuals with MS. We determined that insoluble transmembrane protein106B (TMEM106B), expressed in neurons and glia and normally lysosomal-associated, is increased in MS plaques relative to normal-appearing white matter from individuals with Alzheimers disease and non-neurologic controls. We found that decreased TMEM106B protein in mice results in significant axonal damage and lipid droplet accumulation in the spinal cord following chronic myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. When TMEM106Bt/t mice were treated with cuprizone to experimentally induce demyelination, a significant increase in lipid deposition was observed in the corpus callosum of TMEM106Bt/t mice post-cuprizone withdrawal. Our study shows that the brain and spinal cord from challenged TMEM106Bt/t mice accumulate OilRedO+/Perilipin2+ lipid droplets. We postulate that increased insolubility of TMEM106B in MS plaques limits debris clearance by the lysosome which over time contributes to failed remyelination and axonal defects.

Abbreviated AbstractTransmembrane protein106B (TMEM106B), a lysosome-associated protein, is significantly less soluble in multiple sclerosis plaques than in white matter controls. Decreased TMEM106B produces significant axonal damage and lipid accumulation in mouse models of demyelinating diseases. TMEM106B insolubility and likely loss of function may limit lysosome transport and contribute to CNS pathology.

sentences: Prediction Under Uncertainty: Dissociating Sensory from Cognitive Expectations in Highly Uncertain Musical Contexts Predictive models in the brain rely on the continuous extraction of regularities from the environment. These models are thought to be updated by novel information, as reflected in prediction error responses such as the mismatch negativity (MMN). However, although in real life individuals often face situations in which uncertainty prevails, it remains unclear whether and how predictive models emerge in high-uncertainty contexts. Recent research suggests that uncertainty affects the magnitude of MMN responses in the context of music listening. However, musical predictions are typically studied with MMN stimulation paradigms based on Western tonal music, which are characterized by relatively high predictability. Hence, we developed an MMN paradigm to investigate how the high uncertainty of atonal music modulates predictive processes as indexed by the MMN and behavior. Using MEG in a group of 20 subjects without musical training, we demonstrate that the magnetic MMN in response to pitch, intensity, timbre, and location deviants is evoked in both tonal and atonal melodies, with no significant differences between conditions. In contrast, in a separate behavioral experiment involving 39 non-musicians, participants detected pitch deviants more accurately and rated confidence higher in the tonal than in the atonal musical context. These results indicate that contextual tonal uncertainty modulates processing stages in which conscious awareness is involved, although deviants robustly elicit low-level pre-attentive responses such as the MMN. The achievement of robust MMN responses, despite high tonal uncertainty, is relevant for future studies comparing groups of listeners MMN responses to increasingly ecological music stimuli.

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A cell-ECM mechanism for connecting the ipsilateral eye to the brain Information about features in the visual world are parsed by circuits in the retina and are then transmitted to the brain by distinct subtypes of retinal ganglion cells (RGCs). Axons from RGC subtypes are stratified in retinorecipient brain nuclei, such as the superior colliculus (SC), to provide a segregated relay of parallel and feature-specific visual streams. Here, we sought to identify the molecular mechanisms that direct the stereotyped laminar targeting of these axons. We focused on ipsilateral-projecting subtypes of RGCs (ipsiRGCs) whose axons target a deep SC sublamina. We identified an extracellular glycoprotein, Nephronectin (NPNT), whose expression is restricted to this ipsiRGC-targeted sublamina. SC-derived NPNT and integrin receptors generated by ipsiRGCs are both required for the targeting of ipsiRGC axons to the deep sublamina of SC. Thus, a cell-extracellular matrix (ECM) recognition mechanism specifies precise laminar targeting of ipsiRGC axons and the assembly of eye-specific parallel visual pathways.

Significance StatementDistinct features of the visual world are transmitted from the retina to the brain through anatomically segregated circuits. Despite this being an organizing principle of visual pathways in mammals, we lack an understanding of the signaling mechanisms guiding axons of different types of retinal neurons into segregated layers of brain regions. We explore this question by identifying how axons from the ipsilateral retina innervate a specific lamina of the superior colliculus. Our studies reveal a unique cell-extracellular matrix (ECM) recognition mechanism that specifies precise targeting of these axons to the superior colliculus. Loss of this mechanism not only resulted in the absence of this eye-specific visual circuit, but it led to an impairment of innate predatory visual behavior as well.

sentences: Nuclear Translocation of Vitellogenin in the Honey Bee (Apis mellifera) Vitellogenin (Vg) is a conserved protein used by nearly all oviparous animals to produce eggs. It is also pleiotropic and performs functions in oxidative stress resistance, immunity, and, in honey bees, behavioral development of the worker caste. It has remained enigmatic how Vg affects multiple traits. Here, we asked whether Vg enters the nucleus and acts via DNA-binding. We used immunohistology, cell fractionation and cell culturation to show that a structural subunit of honey bee Vg translocates into cell nuclei. We then demonstrated Vg-DNA binding theoretically and empirically with prediction software and chromatin immunoprecipitation with sequencing (ChIP-seq), finding binding sites at genes influencing immunity and behavior. Finally, we investigated the immunological and enzymatic conditions affecting Vg cleavage and nuclear translocation, and constructed a 3D structural model. Our data are the first to show Vg in the nucleus and suggests a new fundamental regulatory role for this ubiquitous protein.

sentences: Marker-free coselection for successive rounds of prime editing in human cells Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, editing efficiency remains a key challenge in a broad range of cell types. We designed a robust coselection strategy to perform successive rounds of genetic changes in human cells using CRISPR-Cas nucleases and prime editors. Through coediting, the recovery of cells modified at a target gene of interest is coupled to selection for dominant alleles of the ubiquitous and essential sodium/potassium pump (Na+/K+ ATPase). The method improves prime editing efficiency 1.5-10-fold in K562 and HeLa cells, reaching up to 83% of desired alleles in cell populations, for previously optimized prime editing guide RNAs (pegRNAs). Using pegRNAs designed de novo, we readily engineered homozygous cell lines with cancer-associated or drug resistant MTOR mutations displaying altered mTORC1 signaling. Our approach streamlines the production of cell lines with multiple genetic modifications to create cellular models for biological research and lays the foundation for the development of cell-type specific coselection strategies.

sentences: Controlling pallidal oscillations in real-time in Parkinson's disease using evoked interference deep brain stimulation (eiDBS): proof of concept in the human Approaches to control basal ganglia neural activity in real-time are needed to clarify the causal role of 8-35 Hz ("beta band") oscillatory dynamics in the manifestation of Parkinsons disease (PD) motor signs. Here, we show that resonant beta oscillations evoked by electrical pulse with precise amplitude and timing can be used to predictably suppress or amplify spontaneous beta band activity in the internal segment of the globus pallidus (GPi) in the human. Using this approach, referred to as closed-loop evoked interference deep brain stimulation (eiDBS), we could suppress or amplify frequencyspecific (16-22 Hz) neural activity in a PD patient. Our results highlight the utility of eiDBS to characterize the role of oscillatory dynamics in PD and other brain conditions, and to develop personalized neuromodulation systems.

sentences: A Hierarchical Process Model Links Behavioral Aging and Lifespan in C. elegans Individuals who remain vigorous longer tend to live longer, supporting the design of predictive behavioral biomarkers of aging. In C. elegans, the timing of age-associated vigorous movement cessation (VMC) and lifespan correlate strongly between individuals. However, many genetic and pharmaceutical interventions that alter aging produce disproportional effects on VMC and lifespan, appearing to "uncouple" the rate of behavioral aging and lifespan. To study the causal structure underlying such uncoupling, we developed a high-throughput, automated imaging platform to quantify behavioral aging and lifespan at an unprecedented scale. Our method reveals an inverse correlation between each individuals vigorous movement span and their remaining lifespan. Robust across many lifespan-altering interventions including a new RNA-polymerase II auxin-inducible degron system, our data shows that individual C. elegans experience at least two distinct but coupled physical declines--one governing VMC and the other governing lifespan. Through simulations and modeling, we clarify the causal relationship between these two "biological ages" and highlight a crucial but often untested assumption in conventional aging biomarker research: predictive biomarkers may not always report on the same biological age as that which determines long-term health outcomes.

sentences: Direct detection of the myosin super-relaxed state and interacting heads motif in solution We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to detect the interacting-heads motif (IHM) of {beta}-cardiac myosin in solution. Evidence for the IHM has been observed by several structural techniques, and it has been proposed to be the structural basis for the super-relaxed state (SRX), a low-ATPase state of myosin that has been observed biochemically in skinned muscle fibers using fluorescent ATP. It has been proposed that the disruption of this state, by mutation or chemical modification, is a major cause of heart disease, so drugs are being developed to stabilize it. The goal of the present study is to determine directly and quantitatively the correlation between the measured fractions of myosin in the IHM state and the SRX state under the same conditions in solution. We used TR-FRET to measure the distance between the two heads of bovine cardiac myosin, and found that there are two distinct populations, one of which is observable by FRET at a center distance of 2.0 nm, and the other is not detected, implying a distance greater than 4 nm. Under the same conditions, we also measured the fraction of heads in the SRX state using fluorescent nucleotide and stopped-flow kinetics. We found that, in the absence of crosslinking, the population of SRX exceeded that of IHM. In particular, the stabilizing effect of mavacamten was much greater on SRX (55% increase) than on IHM (4% increase). We conclude that the SRX and IHM states are related, but they are not identical.

sentences: Effects of co-incubation of LPS-stimulated RAW 264.7 macrophages on leptin production by 3T3-L1 adipocytes: A method for co-incubating distinct adipose tissue cell lines. Adipose tissue is a major endocrine organ capable of releasing inflammatory adipokines. Inflammatory adipokine release is linked to the changes occurring in adipose tissue in the overfed state, where tissue remodeling results in hypertrophic adipocytes that recruit monocytes to infiltrate the tissue and take on an inflammatory phenotype. As the proportion of inflammatory macrophages increases there is a concurrent increase in release of macrophage-specific inflammatory mediators, further contributing to the inflamed state and setting an inflammatory loop between the macrophages and adipocytes. Although most inflammatory adipokines are released by macrophages, adipocytes can also release immunomodulatory adipokines, such as leptin. The objective of this research was to determine if co-incubation of activated macrophages with mature adipocytes, using Transwell inserts, affected leptin release by mature adipocytes. We also examined if there were differences in the amount of cell-secreted products quantified in cell-conditioned media collected from macrophage-containing (Transwell insert) and adipocyte-containing (well) compartments. Mature adipocytes (differentiated 3T3-L1 murine fibroblasts) were co-incubated with control and lipopolysaccharide-stimulated (0.01 g/ml) murine macrophages (RAW264.7), and nitric oxide, interleukin-6, and leptin levels were quantified in the cell-conditioned media from the two compartments. Activation status of the macrophages did not affect leptin release by the adipocytes. We observed higher amounts of leptin in wells compared to Transwells. Nitric oxide and interleukin-6 levels were similar between Transwells and wells, suggesting that these adipokines are traveling through the Transwell inserts and reaching equilibrium between the two compartments. There was a weak negative relationship between nitric oxide release by macrophages and leptin release by adipocytes. Our results suggest that co-incubating activated macrophages and adipocytes using Transwell inserts can result in distinct microenvironments in the different cellular compartments and that separate sampling of these compartments is required to detect the subtle signaling dynamics that exist between these cells.

sentences: MorphVAE: Generating Neural Morphologies from 3D-Walks using a Variational Autoencoder with Spherical Latent Space For the past century, the anatomy of a neuron has been considered one of its defining features: The shape of a neurons dendrites and axon fundamentally determines what other neurons it can connect to. These neurites have been described using mathematical tools e.g. in the context of cell type classification, but generative models of these structures have only rarely been proposed and are often computationally inefficient. Here we propose MO_SCPLOWORPHC_SCPLOWVAE, a sequence-to-sequence variational autoencoder with spherical latent space as a generative model for neural morphologies. The model operates on walks within the tree structure of a neuron and can incorporate expert annotations on a subset of the data using semi-supervised learning. We develop our model on artificially generated toy data and evaluate its performance on dendrites of excitatory cells and axons of inhibitory cells of mouse motor cortex (M1) and dendrites of retinal ganglion cells. We show that the learned latent feature space allows for better cell type discrimination than other commonly used features. By sampling new walks from the latent space we can easily construct new morphologies with a specified degree of similarity to their reference neuron, providing an efficient generative model for neural morphologies.

sentences: A novel SARS-CoV-2 related coronavirus in bats from Cambodia Knowledge of the origin and reservoir of the coronavirus responsible for the ongoing COVID-19 pandemic is still fragmentary. To date, the closest relatives to SARS-CoV-2 have been detected in Rhinolophus bats sampled in the Yunnan province, China. Here we describe the identification of SARS-CoV-2 related coronaviruses in two Rhinolophus shameli bats sampled in Cambodia in 2010. Metagenomic sequencing identified nearly identical viruses sharing 92.6% nucleotide identity with SARS-CoV-2. Most genomic regions are closely related to SARS-CoV-2, with the exception of a small region corresponding to the spike N terminal domain. The discovery of these viruses in a bat species not found in China indicates that SARS-CoV-2 related viruses have a much wider geographic distribution than previously understood, and suggests that Southeast Asia represents a key area to consider in the ongoing search for the origins of SARS-CoV-2, and in future surveillance for coronaviruses.

sentences: Assessing chromatin relocalization in 3D using the patient rule induction method Three dimensional (3D) genome architecture is critical for numerous cellular processes, including transcription, while certain conformation-driven structural alterations are frequently oncogenic. Inferring 3D chromatin configurations has been advanced by the emergence of chromatin conformation capture assays, notably Hi-C, and attendant 3D reconstruction algorithms. These have enhanced understanding of chromatin spatial organization and afforded numerous downstream biological insights. Until recently, comparisons of 3D reconstructions between conditions and/or cell types were limited to prescribed structural features. However, multiMDS, a pioneering approach developed by Rieber and Mahony (2019) that performs joint reconstruction and alignment, enables quantification of all locus-specific differences between paired Hi-C data sets. By subsequently mapping these differences to the linear (1D) genome the identification of relocalization regions is facilitated through use of peak calling in conjunction with continuous wavelet transformation. Here, we seek to refine this approach by performing the search for significant relocalization regions in terms of the 3D structures themselves, thereby retaining the benefits of 3D reconstruction and avoiding limitations associated with the 1D perspective. The search for (extreme) relocalization regions is conducted using the patient rule induction method (PRIM). Considerations surrounding orienting structures with respect to compartmental and principal component axes are discussed, as are approaches to inference and reconstruction accuracy assessment. Illustration makes recourse to comparisons between four different cell types.

sentences: A heterogeneous drug tolerant persister state in BRAF-mutant melanoma is characterized by ion channel dysregulation and susceptibility to ferroptosis There is increasing interest in cancer cell subpopulations that can withstanding treatment via non-genetic mechanisms, such as tumor cell plasticity and adaptation. These cell populations may be comprised of cells with diverse phenotypes, e.g., quiescent or slow cycling. Such populations have been broadly termed "drug-tolerant persisters" (DTPs) and may be responsible for minimal residual disease following anticancer treatment and acquired resistance. Understanding molecular mechanisms that drive emergence of DTPs could lead to new strategies to improve therapeutic outcomes. Recently, we reported that BRAF-mutant melanoma cells under prolonged BRAF inhibition enter a DTP state with balanced cell death and division, which we termed "idling." Here, we apply single cell barcoding to show that idling DTP populations emerge via cell state transitions, rather than selection of a few pre-existing drug-tolerant clones. Within the time frame of our experiments, DTPs exhibit varying proportions of fast- and slow-cycling cells within each lineage, suggesting that entry into the DTP state is a stochastic process. Furthermore, single-cell transcriptomics and bulk epigenomics reveal common gene expression and ontology signatures in DTP lineages that are consistent with rebalancing of ion channels. Calcium flux experiments uncover a reduction of divalent cation reserves in intracellular organelles, likely leading to endoplasmic reticulum stress. Accordingly, idling DTPs are more prone to ferroptotic cell death, as indicated by increased sensitivity to inhibition of glutathione peroxidase 4 (GPX4), which prevents removal of toxic lipid peroxides. In summary, we propose that ion channel homeostasis is a central process underlying idling DTP emergence in BRAF-mutated melanoma. Future studies will investigate translational aspects of this insight.

sentences: Revisiting tolerance to ocean acidification: insights from a new framework combining physiological and molecular tipping points of Pacific oyster Studies on the impact of ocean acidification on marine organisms involve exposing organisms to future acidification scenarios as projected for open ocean, which has limited relevance for coastal calcifiers. Characterization of reaction norms across a range of pH and identification of tipping points beyond which detrimental effects are observed has been limited and focus on only a few macro-physiological traits. Here we filled this knowledge gap by developing a framework to analyze the broad macro-physiological and molecular responses over a wide pH range of juvenile Pacific oyster, a model species for which the tolerance threshold to acidification remains unknown. We identify low tipping points for physiological traits at pH 7.3-6.9 that coincide with a major reshuffling in membrane lipids and transcriptome. In contrast, shell parameters exhibit effects with pH drop well before tipping points, likely impacting animal fitness. These findings were made possible by the development of an innovative methodology to synthesize and identify the main patterns of variations in large -omic datasets, fit them to pH and identify molecular tipping-points. We propose the application of our framework broadly to the assessment of effects of global change on other organisms.

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Rhamnan Sulfate Reduces Atherosclerotic Plaque Formation and Vascular Inflammation ObjectiveWhile lipid-lowering drugs have become a mainstay of clinical therapy these treatments only slow the progression of the disease and can have side effects. Thus, new treatment options are needed to supplement the effects of lipid lowering therapy for treating atherosclerosis. We examined the use of an inexpensive and widely available marine polysaccharide rhamnan sulfate as an oral therapeutic for limiting vascular inflammation and atherosclerosis.

Methods and ResultsWe found rhamnan sulfate enhanced the barrier function of endothelial cells, preventing the deposition of LDL and maintaining barrier function even in the presence of glycocalyx-degrading enzymes. Rhamnan sulfate was also found to bind directly to FGF-2, PDGF-BB and NF-{kappa}B subunits with high affinity. In addition, rhamnan sulfate was a potent inhibitor of NF-{kappa}B pathway activation in endothelial cells by TNF-. We treated ApoE-/- mice with a high fat diet for 4 weeks and then an addition 9 weeks of high fat diet with or without rhamnan sulfate. Rhamnan sulfate reduced vascular inflammation and atherosclerosis in both sexes of ApoE-/- mice but had a stronger therapeutic effect in female mice. Oral consumption of rhamnan sulfate induced a significant decrease in cholesterol plasma levels in female mice but not in male mice.

ConclusionsRhamnan sulfate has beneficial effects in reducing inflammation, binding growth factors and NF-{kappa}B, enhancing endothelial barrier function and reducing atherosclerotic plaque formation in ApoE-/- mice.

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Illusory tactile movement crosses arms and legs and is coded in external space Humans often misjudge where on the body a touch occurred. Theoretical accounts have ascribed such misperceptions to local interactions in peripheral and primary somatosensory neurons, positing that spatial-perceptual mechanisms adhere to limb boundaries and skin layout. Yet, perception often reflects integration of sensory signals with prior experience. On their trajectories, objects often touch multiple limbs; therefore, body-environment interactions should manifest in perceptual mechanisms that reflect external space.

Here, we demonstrate that humans perceived the cutaneous rabbit illusion - the percept of multiple identical stimuli as hopping across the skin - along the Euclidian trajectory between stimuli on two body parts and regularly mislocalized stimuli from one limb to the other. A Bayesian model based on Euclidian, as opposed to anatomical, distance faithfully reproduced key aspects of participants localization behavior.

Our results suggest that prior experience of touch in space critically shapes tactile spatial perception and illusions beyond anatomical organization.

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Genome-guided discovery of natural products through multiplexed low coverage whole-genome sequencing of soil Actinomycetes on Oxford Nanopore Flongle Genome-mining is an important tool for discovery of new natural products; however, the number of publicly available genomes for natural product-rich microbes such as Actinomycetes, relative to human pathogens with smaller genomes, is small. To obtain contiguous DNA assemblies and identify large (ca. 10 to greater than 100 Kb) biosynthetic gene clusters (BGCs) with high-GC (>70%) and -repeat content, it is necessary to use long-read sequencing methods when sequencing Actinomycete genomes. One of the hurdles to long-read sequencing is the higher cost.

In the current study, we assessed Flongle, a recently launched platform by Oxford Nanopore Technologies, as a low-cost DNA sequencing option to obtain contiguous DNA assemblies and analyze BGCs. To make the workflow more cost-effective, we multiplexed up to four samples in a single Flongle sequencing experiment while expecting low-sequencing coverage per sample. We hypothesized that contiguous DNA assemblies might enable analysis of BGCs even at low sequencing depth. To assess the value of these assemblies, we collected high-resolution mass-spectrometry data and conducted a multi-omics analysis to connect BGCs to secondary metabolites.

In total, we assembled genomes for 20 distinct strains across seven sequencing experiments. In each experiment, 50% of the bases were in reads longer than 10 Kb, which facilitated the assembly of reads into contigs with an average N50 value of 3.5 Mb. The programs antiSMASH and PRISM predicted 629 and 295 BGCs, respectively. We connected BGCs to metabolites for N,N-dimethyl cyclic-ditryptophan, a novel lassopeptide and three known Actinomycete-associated siderophores, namely mirubactin, heterobactin and salinichelin.

ImportanceShort-read sequencing of GC-rich genomes such as Actinomycetes results in a fragmented genome assembly and truncated biosynthetic gene clusters (often 10 to >100 Kb long), which hinders our ability to understand the biosynthetic potential of a given strain and predict the molecules that can be produced. The current study demonstrates that contiguous DNA assemblies, suitable for analysis of BGCs, can be obtained through low-coverage, multiplexed sequencing on Flongle, which provides a new low-cost workflow ($30-40 per strain) for sequencing Actinomycete strain libraries.

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SALARECON connects the Atlantic salmon genome to growth and feed efficiency Atlantic salmon (Salmo salar) is the most valuable farmed fish globally and there is much interest in optimizing its genetics and rearing conditions for growth and feed efficiency. Marine feed ingredients must be replaced to meet global demand, with challenges for fish health and sustainability. Metabolic models can address this by connecting genomes to metabolism, which converts nutrients in the feed to energy and biomass, but such models are currently not available for major aquaculture species such as salmon. We present SALARECON, a model focusing on energy, amino acid, and nucleotide metabolism that links the Atlantic salmon genome to metabolic fluxes and growth. It performs well in standardized tests and captures expected metabolic (in)capabilities. We show that it can explain observed hypoxic growth in terms of metabolic fluxes and apply it to aquaculture by simulating growth with commercial feed ingredients. Predicted limiting amino acids and feed efficiencies agree with data, and the model suggests that marine feed efficiency can be achieved by supplementing a few amino acids to plant- and insect-based feeds. SALARECON is a high-quality model that makes it possible to simulate Atlantic salmon metabolism and growth. It can be used to explain Atlantic salmon physiology and address key challenges in aquaculture such as development of sustainable feeds.

Author summaryAtlantic salmon aquaculture generates billions of euros annually, but faces challenges of sustainability. Salmon are carnivores by nature, and fish oil and fish meal have become scarce resources in fish feed production. Novel, sustainable feedstuffs are being trialed hand in hand with studies of the genetics of growth and feed efficiency. This calls for a mathematical-biological framework to integrate data with understanding of the effects of novel feeds on salmon physiology and its interplay with genetics. We have developed the SALARECON model of the core salmon metabolic reaction network, linking its genome to metabolic fluxes and growth. Computational analyses show good agreement with observed growth, amino acid limitations, and feed efficiencies, illustrating the potential for in silico studies of potential feed mixtures. In particular, in silico screening of possible diets will enable more efficient animal experiments with improved knowledge gain. We have adopted best practices for test-driven development, virtual experiments to assay metabolic capabilities, revision control, and FAIR data and model management. This facilitates fast, collaborative, reliable development of the model for future applications in sustainable production biology.

sentences: The evolution of trait correlations constrains phenotypic adaptation to high CO2 in a eukaryotic alga Microbes form the base of food webs and drive biogeochemical cycling. Predicting the effects of microbial evolution on global elemental cycles remains a significant challenge due to the sheer number of interacting environmental and trait combinations. Here we present an approach for integrating multivariate trait data into a predictive model of trait evolution. We investigated the outcome of thousands of possible adaptive walks parameterized using empirical evolution data from the alga Chlamydomonas exposed to high CO2. We found that the direction of historical bias (existing trait correlations) influenced both the rate of adaptation and the evolved phenotypes (trait combinations). Critically, we use fitness landscapes derived directly from empirical trait values to capture known evolutionary phenomena. This work demonstrates that ecological models need to represent both changes in traits and changes in the correlation between traits in order to accurately capture phytoplankton evolution and predict future shifts in elemental cycling.

sentences: Effective spatio-temporal regimes for wound treatment by way of macrophage polarization: A mathematical model Wound healing consists of a sequence of biological processes often grouped into different stages. Interventions applied to accelerate normal wound healing must take into consideration timing with respect to wound healing stages in order to maximize treatment effectiveness. Macrophage polarization from M1 to M2 represents a transition from the inflammatory to proliferation stage of wound healing. Accelerating this transition may be an effective way to accelerate wound healing; however, it must be induced at the appropriate time. We search for an optimal spatio-temporal regime to apply wound healing treatment in a mathematical model of wound healing. In this work we show that to maximize effectiveness, treatment must not be applied too early or too late. We also show that effective spatial distribution of treatment depends on the heterogenity of the wound surface. In conclusion, this research provides a possible optimal regime of therapy that focuses on macrophage activity and a hypothesis of treatment outcome to be tested experimentally in future. Finding best regimes for treatment application is a first step towards development of intelligent algorithms of wound treatment that minimize healing time.

sentences: Adult neuromarkers of sustained attention and working memory predict inter- and intra-individual differences in these processes in youth Sustained attention (SA) and working memory (WM) are critical processes, but the brain networks supporting these abilities in development are unknown. We characterized the functional brain architecture of SA and WM in 9-11-year-old children and adults. First, we found that adult network predictors of SA generalized to predict individual differences and fluctuations in SA in youth. A WM network model predicted WM performance both across and within children--and captured individual differences in later recognition memory--but underperformed in youth relative to adults. We next characterized functional connections differentially related to SA and WM in youth compared to adults. Results revealed two network configurations: a dominant architecture predicting performance in both age groups and a secondary architecture, more prominent for WM than SA, predicting performance in one. Thus, functional connectivity predicts SA and WM in youth, with networks predicting WM changing more from preadolescence to adulthood than those predicting SA.

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Live imaging of the co-translational recruitment of XBP1 mRNA to the ER and its processing by diffuse, non-polarized IRE1a. Endoplasmic reticulum (ER) to nucleus homeostatic signalling, known as the unfolded protein response (UPR), relies on the non-canonical splicing of XBP1 mRNA. The molecular switch that initiates splicing is the oligomerization of the ER stress sensor and UPR endonuclease IRE1. While IRE1 can form large clusters that have been proposed to function as XBP1 processing centers on the ER, the actual oligomeric state of active IRE1 complexes as well as the targeting mechanism that recruits XBP1 to IRE1 oligomers, remain unknown.

Here, we used a single molecule imaging approach to directly monitor the recruitment of individual XBP1 transcripts to the ER surface. We confirmed that stable ER association of unspliced XBP1 mRNA is established through HR2-dependent targeting and relies on active translation. In addition, we show that IRE1-catalyzed splicing mobilizes XBP1 mRNA from the ER membrane in response to ER stress. Surprisingly, we find that XBP1 transcripts are not recruited into large IRE1 clusters, which only assemble upon overexpression of fluorescently-tagged IRE1 during ER stress. Our findings support a model where ribosome-engaged, ER-poised XBP1 mRNA is processed by functional IRE1 assemblies that are homogenously distributed throughout the ER membrane.

sentences: Monophyly of Diverse Bigyromonadea and their Impact on Phylogenomic Relationships Within Stramenopiles Stramenopiles are a diverse but relatively well-studied eukaryotic supergroup with considerable genomic information available (Sibbald and Archibald, 2017). Nevertheless, the relationships between major stramenopile subgroups remain unresolved, in part due to a lack of data from small nanoflagellates that make up a lot of the genetic diversity of the group. This is most obvious in Bigyromonadea, which is one of four major stramenopile subgroups but represented by a single transcriptome. To examine the diversity of Bigyromonadea and how the lack of data affects the tree, we generated transcriptomes from seven novel bigyromonada species described in this study: Develocauda condao, Develocanicus komovi, Develocanicus vyazemskyi, Cubaremonas variflagellatum, Pirsonia chemainus, Feodosia pseudopoda, and Koktebelia satura. Both maximum likelihood and Bayesian phylogenomic trees based on a 247 gene-matrix recovered a monophyletic Bigyromonadea that includes two diverse subgroups, Developea and Pirsoniales, that were not previously related based on single gene trees. Maximum likelihood analyses show Bigyromonadea related to oomycetes, whereas Bayesian analyses and topology testing were inconclusive. We observed similarities between the novel bigyromonad species and motile zoospores of oomycetes in morphology and the ability to self- aggregate. Rare formation of pseudopods and fused cells were also observed, traits that are also found in members of labyrinthulomycetes, another osmotrophic stramenopiles. Furthermore, we report the first case of eukaryovory in the flagellated stages of Pirsoniales. These analyses reveal new diversity of Bigyromonadea, and altogether suggest their monophyly with oomycetes, collectively known as Pseudofungi, is the most likely topology of the stramenopile tree.

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Nitric oxide-induced tyrosine nitration of TrkB impairs BDNF signaling and restrains neuronal plasticity Nitric oxide has been long recognized as an important modulator of neural plasticity, but characterization of the molecular mechanisms involved - specially the guanylyl cyclase-independent ones - has been challenging. There is evidence that NO could modify BDNF-TRKB signaling, a key mediator of neuronal plasticity. However, the mechanism underlying the interplay of NO and TRKB remains unclear. Here we show that nitric oxide induces nitration of the tyrosine 816 in the TRKB receptor in vivo and in vitro, and that post-translational modification inhibits TRKB phosphorylation and binding of phospholipase C{gamma}1 (PLC{gamma}1) to this same tyrosine residue. Additionally, nitration triggers clathrin-dependent endocytosis of TRKB through the adaptor protein AP2M and ubiquitination, thereby increasing translocation of TRKB away from the neuronal surface and directing it towards lysosomal degradation. Accordingly, inhibition of nitric oxide increases TRKB phosphorylation and TRKB-dependent neurite branching in neuronal cultures. In vivo, chronic inhibition of neuronal nitric oxide synthase (nNOS) dramatically reduced TRKB nitration and facilitated TRKB signaling in the primary visual cortex, and promoted a shift in ocular dominance upon monocular deprivation in the visual cortex - an indicator of increased plasticity. Altogether, our data describe and characterize a new molecular brake on plasticity, namely nitration of TRKB receptors.

Significance statementWe described the nitration of TRKB receptors at the tyrosine residue 816 as a new post-translational modification (PTM) that restrains the signaling of the neurotrophic factor BDNF in neurons. This new PTM leads to endocytosis and degradation of the TRKB receptors. Intriguingly, this mechanism is tonically active under physiological conditions in vivo, and it is important for restricting ocular dominance plasticity in the visual cortex. This mechanism directly links two major systems involved in brain plasticity, BDNF/TRKB and nitric oxide. Our data provides a model for how NO production from nNOS can compromise TRKB function, and for the effects of nNOS inhibitors promoting plasticity.

sentences: Development of Tribo-Electroceutical Fabrics for Potential Application in Self-sanitizing Personal Protective Equipment (PPE) Attachment of microbial bodies including coronavirus on the surface of personal protective equipment (PPE) is found to be potential threat of spreading infection. Here, we report the development of a novel tribo-electroceutical fabric (TECF) consisting of commonly available materials namely Nylon, and Silicone Rubber (SR) for the fabrication of protective gloves on Nitrile platform, as a model wearable PPE. A small triboelectric device (2 cm x 2 cm) consisting of SR and Nylon on Nitrile can generate more than 20 volt transient or 41 {micro}W output power, which is capable of charging a capacitor up to 65 V in only [~]50 sec. The novelty of the present work relies on the TECF led anti-microbial activity through the generation of an electric current in saline water. The fabrication of TECF based functional prototype gloves can generate hypochlorite ions through the formation of electrolysed water upon rubbing them with saline water. Further a computational modelling has been employed to reveal the optimum structure and mechanistic pathway of anti-microbial hypochlorite generation. Detailed anti-microbial assays have been performed to establish effectiveness of such TECF based gloves to reduce the risk from life threatening pathogen spreading. The present work provides the rationale to consider the studied TECF, or other material with comparable properties, as material of choice for the development of self-sanitizing PPE in the fight against microbial infections including COVID-19.

sentences: Particle localization using local gradients and its application to nanometer stabilization of a microscope Accurate localization of single particles plays an increasingly important role in a range of biological techniques, including single molecule tracking and localization-based superresolution microscopy. Such techniques require fast and accurate particle localization algorithms as well as nanometer-scale stability of the microscope. Here, we present a universal method for three-dimensional localization of single labeled and unlabeled particles based on local gradient calculation of microscopy images. The method outperforms current techniques in high noise conditions, and it is capable of nanometer accuracy localization of nano- and micro-particles with sub-ms calculation time. By localizing a fixed particle as fiducial mark and running a feedback loop, we demonstrate its applicability for active drift correction in sensitive nanomechanical measurements such as optical trapping and superresolution imaging. A multiplatform open software package comprising a set of tools for local gradient calculation in brightfield and fluorescence microscopy is shared to the scientific community.

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Effects of electroacupuncture on the expression of hypothalamic neuropeptide Y and ghrelin in pubertal rats with polycystic ovary syndrome BackgroundPolycystic ovary syndrome often starts in puberty, and its pathogenesis is not clear. This study aimed to explore the pathogenesis of pubertal polycystic ovary syndrome (PCOS) and assess the therapeutic effect of electroacupuncture on pubertal PCOS.

MethodsDihydrotestosterone (DHT) was used to induce rat models of pubertal PCOS. pubertal rats with PCOS were randomly divided into a model group (M), an electroacupuncture group (EA), and a sham acupuncture group (SA). Age-matched normal rats were regarded as normal controls (N). Rats were treated with EA or SA five times a week for 25 minutes during their 6th-7th week. At the end of the experiment, we observed any changes in ovarian morphology; detected levels of metabolic indices in serum, the hypothalamus and pancreas.

ResultsEA significantly improved estrous cycle disorders and the ovarian polycystic morphology in pubertal rats with PCOS, but SA only improved disorders of the estrous cycle. The serum levels of insulin, neuropeptide Y(NPY) and fasting blood glucose(FBG) increased significantly, while the serum levels of ghrelin(GHRL) decreased in the model group. After treatment with EA, the levels of NPY and FBG went into decrease, whereas the levels of GHRL and insulin increased. There was few differences in the hypothalamic expression of galanin (GAL), galanin-like peptide (GALP) and ghrelin receptor(GHSR) between the four groups. The upregulation of NPY mRNA and neuropeptide Y2 receptor(NPY2R) mRNA and the downregulation of GHRL protein and mRNA in the hypothalamus, and the increased expression of NPY and NPY2R as well as the decreased expression of GHRL in the arcuate nucleus (ARC) can be rescued by EA. But, surprisingly, SA seem to make no difference to the levels of FBG and insulin, and the protein expression of ghrelin in the hypothalamus and ARC. Co-expression of kisspeptin and GHSR, and co-expression of gonadotrophin releasing hormone(GnRH) and NPY2R were observed in ARC. No differences were found between groups in protein of GAL, GALP and GHRL expression in the pancreas. Neither EA nor SA can attenuate the upregulated kisspeptin protein expression in the pancreas of PCOS model rats.

ConclusionsEA and SA improved the symptoms of pubertal PCOS rats, and the mechanism might be associated with regulating hypothalamic NPY and ghrelin levels.

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Comparing statistical and mechanistic models to identify the drivers of mortality within a rear-edge beech population AO_SCPLOWBSTRACTC_SCPLOWSince several studies have been reporting an increase in the decline of forests, a major issue in ecology is to better understand and predict tree mortality. The interactions between the different factors and the physiological processes giving rise tree mortality, as well as the inter-individual variability in mortality risk, still need to be better assessed.

This study investigates mortality in a rear-edge population of European beech (Fagus sylvatica L.) using a combination of statistical and process-based modelling approaches. Based on a survey of 4323 adult beeches since 2002 within a natural reserve, we first used statistical models to quantify the effects of competition, tree growth, size, defoliation and fungi presence on mortality. Secondly, we used an ecophysiological process-based model (PBM) to separate out the different mechanisms giving rise to temporal and inter-individual variations in mortality by simulating depletion of carbon stocks, loss of hydraulic conductance and damage due to late frosts in response to climate.

The combination of all these simulated processes was associated with the temporal variations in the population mortality rate. The individual probability of mortality decreased with increasing mean growth, and increased with increasing crown defoliation, earliness of budburst, fungi presence and increasing competition, in the statistical model. Moreover, the interaction between tree size and defoliation was significant, indicating a stronger increase in mortality associated to defoliation in smaller than larger trees. Finally, the PBM predicted a higher conductance loss together with a higher level of carbon reserves for trees with earlier budburst, while the ability to defoliate the crown was found to limit the impact of hydraulic stress at the expense of the accumulation of carbon reserves.

We discuss the convergences and divergences obtained between statistical and process-based approaches and we highlight the importance of combining them to characterize the different processes underlying mortality, and the factors modulating individual vulnerability to mortality.

sentences: Sex-limited diversification of the eye in Heliconius butterflies Butterflies have evolved an immense diversity in eye organization to support a range of vision-based behaviors including courtship, oviposition, and foraging. This diversity has been surveyed extensively across the butterfly phylogeny, and here we take a complementary approach to characterize the eye within a group of closely related Heliconius butterflies. Using a combination of immunostaining for different opsins and eyeshine for determining the distribution of light-filtering screening pigments, we identified several sexually dimorphic features of eye organization where male eyes varied and female eyes did not. Ultraviolet (UV) sensitive photoreceptors varied in which of two UV opsins were expressed, including co-expression of both within single photoreceptors, and these differences were consistent with a role in courtship and conspecific identification. Additional differences across species and sex included the distribution of three ommatidial types defined by the expression pattern of UV and blue opsins, the distribution of a red screening pigment, and which ommatidial types expressed the red screening pigment. We hypothesize that female eyes are optimized for a dimorphic behavior such as oviposition, while male eyes adapt to other selective pressures such as the local light environment.

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The MYB33, MYB65, and MYB101 transcription factors affect Arabidopsis and potatoresponses to drought by regulating the ABA signaling pathway AimsDrought is a climate threat limiting crop production. Potato is one of the four most important food crops worldwide and is sensitive to water shortage. The CBP80 gene was shown to affect plant response to drought by regulating the level of microRNA159, and, consequently, the levels of the MYB33 and MYB101 transcription factors. (TF) Our studies aimed to show whether indeed the level of MYB33, MYB65, and MYB101 TFs affects plant response to water shortage.

MethodsArabidopsis transgenic plants exhibiting downregulation and Arabidopsis and potato transgenic plants exhibiting overexpression of selected MYB TFs were obtained. Plants response to drought was mainly measured using relative water content (RWC) and stomata closure upon exogenous ABA.

ResultsThree MYB TFs studied are involved in plant response to drought. When downregulated in Arabidopsis, the MYB33, MYB65 and MYB101 genes cause stomatal hyposensitivity to ABA, leading to reduced tolerance to drought. Transgenic Arabidopsis and potato plants overexpressing a mutated version of these genes with changed miR159 recognition site, show hypersensitivity to ABA and relatively high tolerance to drought conditions.

ConclusionsThe MYB33, MYB65, and MYB101 genes are good be potential targets for innovative breeding to obtain crops with relatively high tolerance to drought.

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Gfastats: conversion, evaluation and manipulation of genome sequences using assembly graphs MotivationWith the current pace at which reference genomes are being produced, the availability of tools that can reliably and efficiently generate genome assembly summary statistics has become critical. Additionally, with the emergence of new algorithms and data types, tools that can improve the quality of existing assemblies through automated and manual curation are required.

ResultsWe sought to address both these needs by developing gfastats, as part of the Vertebrate Genomes Project (VGP) effort to generate high-quality reference genomes at scale. Gfastats is a standalone tool to compute assembly summary statistics and manipulate assembly sequences in fasta, fastq, or gfa [.gz] format. Gfastats stores assembly sequences internally in a gfa-like format. This feature allows gfastats to seamlessly convert fast* to and from gfa [.gz] files. Gfastats can also build an assembly graph that can in turn be used to manipulate the underlying sequences following instructions provided by the user, while simultaneously generating key metrics for the new sequences.

Availability and implementationGfastats is implemented in C++. Precompiled releases (Linux, MacOS, Windows) and commented source code for gfastats are available under MIT license at https://github.com/vgl-hub/gfastats. Examples of how to run gfastats are provided in the Github. Gfastats is also available in Bioconda, in Galaxy (https://assembly.usegalaxy.eu) and as a MultiQC module [1] (https://github.com/ewels/MultiQC). An automated test workflow is available to ensure consistency of software updates.

Supplementary informationSupplementary data are available at Bioinformatics online.

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Penguin: A Tool for Predicting Pseudouridine Sites in Direct RNA Nanopore Sequencing Data Pseudouridine is one of the most abundant RNA modifications, occurring when uridines are catalyzed by Pseudouridine synthase proteins. It plays an important role in many biological processes and also has an importance in drug development. Recently, the single-molecule sequencing techniques such as the direct RNA sequencing platform offered by Oxford Nanopore technologies enable direct detection of RNA modifications on the molecule that is being sequenced, but to our knowledge this technology has not been used to identify RNA Pseudouridine sites. To this end, in this paper, we address this limitation by introducing a tool called Penguin that integrates several developed machine learning (ML) models (i.e., predictors) to identify RNA Pseudouridine sites in Nanopore direct RNA sequencing reads. Penguin extracts a set of features from the raw signal measured by the Oxford Nanopore and the corresponding basecalled k-mer. Those features are used to train the predictors included in Penguin, which in turn, is able to predict whether the signal is modified by the presence of Pseudouridine sites. We have included various predictors in Penguin including Support vector machine (SVM), Random Forest (RF), and Neural network (NN). The results on the two benchmark data sets show that Penguin is able to identify Pseudouridine sites with a high accuracy of 93.38% and 92.61% using SVM in random split testing and independent validation testing respectively. Thus, Penguin outperforms the existing Pseudouridine predictors in the literature that achieved an accuracy of 76.0 at most with an independent validation testing. A GitHub of the tool is accessible at https://github.com/Janga-Lab/Penguin.

HIGHLIGHTSO_LIPenguin integrates several developed ML learning models (i.e., predictors) to identify RNA {Psi} sites in Nanopore direct RNA sequencing reads.
C_LIO_LIThe pipeline of penguin automates the data preprocessing including Nanopore direct RNA reads alignment using Minimap2, and Nanopore signal extraction using Nanopolish, feature extraction from raw Nanopore signal for training ML predictors integrated in its platform, and the prediction of RNA {Psi} sites with those predictors.
C_LIO_LIPenguin can predict {Psi} sites with a performance that outperforms the performance of the state-of-the-art research methods existing in the literature.
C_LIO_LIPenguin platform can be adopted to be used for predicting other/various types of RNA modification.
C_LIO_LIThere are 6137606 U-mers samples predicted by penguin best ML model (SVM) as {Psi} ones from a total of 67491289 U-mers samples in the complete Hek293 cell line with 556813 unique genomic location of {Psi}.
C_LIO_LIThere are 1193192 U-mers samples predicted by penguin best ML model (SVM) as {Psi} ones from a total of 229637931 U-mers samples in the complete Hela cell line with 39384 unique genomic locations of {Psi}.
C_LIO_LIThere is a small fraction of 0.01% (6482 unique genomic locations) of {Psi} that are common (overlapped) between both Hek293 and Hela cell lines.
C_LIO_LIThe extend of {Psi} modification (the number of U-mers samples predicted as {Psi} samples to the total number of U-mer samples in the complete RNA sequence of the cell line) in RNA sequence of Hek293 cell line is much greater than its counterpart for Hela cell line (9% for Hek293 versus 0. 5 % for Hela cell line).
C_LI

sentences: The dual function monoclonal antibodies VIR-7831 and VIR-7832 demonstrate potent in vitro and in vivo activity against SARS-CoV-2 Sotrovimab (VIR-7831) and VIR-7832 are dual action monoclonal antibodies (mAbs) targeting the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sotrovimab and VIR-7832 were derived from a parent antibody (S309) isolated from memory B cells of a 2003 severe acute respiratory syndrome coronavirus (SARS-CoV) survivor. Both mAbs contain an "LS" mutation in the Fc region to prolong serum half-life. In addition, VIR-7832 encodes an Fc GAALIE mutation that has been shown previously to evoke CD8+ T-cells in the context of an in vivo viral respiratory infection. Sotrovimab and VIR-7832 neutralize wild-type and variant pseudotyped viruses and authentic virus in vitro. In addition, they retain activity against monoclonal antibody resistance mutations conferring reduced susceptibility to previously authorized mAbs. The sotrovimab/VIR-7832 epitope continues to be highly conserved among circulating sequences consistent with the high barrier to resistance observed in vitro. Furthermore, both mAbs can recruit effector mechanisms in vitro that may contribute to clinical efficacy via elimination of infected host cells. In vitro studies with these mAbs demonstrated no enhancement of infection. In a Syrian Golden hamster proof-of concept wildtype SARS-CoV-2 infection model, animals treated with sotrovimab had less weight loss, and significantly decreased total viral load and infectious virus levels in the lung compared to a control mAb. Taken together, these data indicate that sotrovimab and VIR-7832 are key agents in the fight against COVID-19.

sentences: Polaramycin B, and not physical interaction, is the signal that rewires fungal metabolism in the Streptomyces - Aspergillus interaction. Co-culturing the bacterium Streptomyces rapamycinicus and the ascomycete Aspergillus nidulans has previously been shown to trigger the production of orsellinic acid and its derivates in the fungal cells. Based on these studies it was assumed that direct physical contact between fungal and bacterial cells is a prerequisite for the metabolic reaction of the fungus that involves an amino acid starvation response and concomitant activating chromatin modifications at the affected biosynthetic gene clusters (BGCs). Here we show that not physical contact, but a guanidine containing macrolide, named Polaramycin B, triggers the response. This metabolite is produced constitutively by the bacterium and production does not need stimulation by a fungal co-culture. Polaramycin B freely diffuses in an aqueous environment and provokes the production of orsellinic acid above a certain concentration. In addition to orsellinic acid, the production of several other secondary metabolites was triggered by Polaramycin B. Our genome-wide transcriptome analysis showed that Polaramycin B treatment causes down-regulation of fungal genes necessary for membrane stability as well as genes involved in general metabolism and growth. A compensatory genetic response can be observed in the fungus that included up-regulation of BGCs and genes necessary for ribosome biogenesis, translation and membrane stability. Our work discovered a specific chemical communication in which Polaramycin B, an antifungal metabolite constitutively produced by different Streptomycete species, leads to the production of fungal defence chemicals and to the up-regulation of genes necessary to compensate for the cellular damage caused by Polaramycin B.

sentences: Reconstruction of dynamic regulatory networks reveals signaling-induced topology changes associated with germ layer specification Elucidating regulatory relationships between transcription factors (TFs) and target genes is fundamental to understanding how cells control their identity and behavior. Computational gene regulatory network (GRN) reconstruction methods aim to map this control by inferring relationships from transcriptomic data. Unfortunately, existing methods are imprecise, may be computationally burdensome, and do not uncover how networks transition from one topology to another. Here we present Epoch, a computational network reconstruction tool that leverages single cell transcriptomics to infer dynamic network structures. Epoch performs favorably when benchmarked using data derived from in vivo, in vitro, and in silico sources. To illustrate the usefulness of Epoch, we applied it to identify the dynamic networks underpinning directed differentiation of mouse embryonic stem cells (ESC) guided by multiple primitive streak induction treatments. Our analysis demonstrates that modulating signaling pathways drives topological network changes that shape cell fate potential. We also find that Peg3 is a central contributor to the rewiring of the pluripotency network to favor mesoderm specification. By integrating signaling pathways with GRN structures, we traced how Wnt activation and PI3K suppression govern mesoderm and endoderm specification, respectively. Finally, we compare the networks established in in vitro directed differentiation of ESCs to those in in vivo gastrulation and mesoderm specification. The methods presented here are available in the R package Epoch, and provide a foundation for future work in understanding the biological implications of dynamic regulatory structures.

sentences: Heritable and climatic sources of variation in juvenile tree growth in an Austrian common garden experiment of Central European Norway spruce populations We leveraged publicly available data on juvenile tree height of 299 Central European Norway spruce populations grown in a common garden experiment across 24 diverse trial locations in Austria and weather data from the trial locations and population provenances to parse the heritable and climatic components of juvenile tree height variation. Principal component analysis of geospatial and weather variables demonstrated high interannual variation among trial environments, largely driven by differences in precipitation, and separation of population provenances based on altitude, temperature, and snowfall. Tree height was highly heritable and modeling the covariance between populations and trial environments based on climatic data led to more stable estimation of heritability and population x environment variance. Climatic similarity among population provenances was highly predictive of population x environment estimates for tree height.

sentences: An amino residue that guides the correct photoassembly the water-oxidation complex but is not required for high affinity Mn2+ binding The assembly of the Mn4O5Ca cluster of the photosystem II (PSII) starts from the initial binding and photooxidation of the first Mn2+ at a high affinity site (HAS). Recent cryo-EM apo-PSII structures reveal an altered geometry of amino ligands in this region and suggest the involvement of D1-Glu189 ligand in the formation of the HAS. We now find that Gln and Lys substitution mutants photoactivate with reduced quantum efficiency compared to the wild-type. However, the affinity of Mn2+ at the HAS in D1-E189K was very similar to the wild-type (~2.2 M). Thus, we conclude that D1-E189 does not form the HAS (~2.9 M) and that the reduced quantum efficiency of photoactivation in D1-E189K cannot be ascribed to the initial photooxidation of Mn2+ at the HAS. Besides reduced quantum efficiency, the D1-E189K mutant exhibits a large fraction of centers that fail to recover activity during photoactivation starting early in the assembly phase, becoming recalcitrant to further assembly. Fluorescence relaxation kinetics indicate on the presence of an alternative route for the charge recombination in Mn-depleted samples in all studied mutants and exclude damage to the photochemical reaction center as the cause for the recalcitrant centers failing to assemble and show that dark incubation of cells reverses some of the inactivation. This reversibility would explain the ability of these mutants to accumulate a significant fraction of active PSII during extended periods of cell growth. The failed recovery in the fraction of inactive centers appears to a reversible mis-assembly involving the accumulation of photooxidized, but non-catalytic high valence Mn at the donor side of photosystem II, and that a reductive mechanism exists for restoration of assembly capacity at sites incurring mis-assembly. Given the established role of Ca2+ in preventing misassembled Mn, we conclude that D1-E189K mutant impairs the ligation of Ca2+ at its effector site in all PSII centers that consequently leads to the mis-assembly resulting in accumulation of non-catalytic Mn at the donor side of PSII. Our data indicate that D1-E189 is not functionally involved in Mn2+ oxidation\binding at the HAS but rather involved in Ca2+ ligation and steps following the initial Mn2+ photooxidation.

sentences: Obesity affects mitochondrial metabolism and proliferative potential of endometrial progenitor cells The study aimed to investigate the influence of obesity on cellular features of equine endometrial progenitor cells (Eca EPCs), including viability, proliferation capacity, mitochondrial metabolism, and oxidative homeostasis. Eca EPCs derived from non-obese (Non-OB) and obese (OB) mares were characterized by cellular phenotype and multipotency. Obesity-induced changes in the activity of Eca EPCs include the decline of their proliferative activity, clonogenic potential, mitochondrial metabolism and enhanced oxidative stress. Eca EPCs isolated from obese mares were characterized by an increased occurrence of early apoptosis, loss of mitochondrial dynamics, and senescence-associated phenotype. Attenuated metabolism of Eca EPCs OB was related to increased expression of pro-apoptotic markers (CASP9, BAX, P53, P21), enhanced expression of OPN, PI3K and AKT, simultaneously with decreased signaling stabilizing cellular homeostasis (including mitofusin, SIRT1, FOXP3). Obesity alters functional features and the self-renewal potential of endometrial progenitor cells. The impaired cytophysiology of progenitor cells from obese endometrium predict lower regenerative capacity if used as autologous transplants.

sentences: Centrally expressed Cav3.2 T-type calcium channel is critical for the initiation and maintenance of neuropathic pain Cav3.2 T-type calcium channel is a major molecular actor of neuropathic pain in peripheral sensory neurons, but its involvement at the supra-spinal level is almost unknown. In the Anterior Pretectum (APT), a hub of connectivity of the somatosensory system involved in pain perception, we show that Cav3.2 channels are expressed in a sub-population of GABAergic neurons co-expressing parvalbumin (PV). In these PV-expressing neurons, Cav3.2 channels contribute to a high frequency bursting activity, which is increased in the spared nerve injury model of neuropathy. Specific deletion of Cav3.2 channels in APT neurons reduced both the initiation and maintenance of mechanical and cold allodynia. These data are a direct demonstration that centrally expressed Cav3.2 channels also play a fundamental role in pain pathophysiology.

sentences: Target recognition in tandem WW domains: complex structures for parallel and antiparallel ligand orientation in h-FBP21 tandem WW Protein-protein interactions often rely on specialized recognition domains, such as WW domains, which bind to specific proline-rich sequences. The specificity of these protein-protein interactions can be increased by tandem repeats, i.e. two WW domains connected by a linker. With a flexible linker, the WW domains can move freely with respect to each other. Additionally, the tandem WW domains can bind in two different orientations to their target sequences. This makes the elucidation of complex structures of tandem WW domains extremely challenging. Here, we identify and characterize two complex structures of the tandem WW domain of human formin-binding protein 21 and a peptide sequence from its natural binding partner, the core-splicing protein SmB/B. The two structures differ in the ligand orientation, and consequently also in the relative orientation of the two WW domains. We analyze and probe the interactions in the complexes by molecular simulations and NMR experiments. The workflow to identify the complex structures uses molecular simulations, density-based clustering and peptide docking. It is designed to systematically generate possible complex structures for repeats of recognition domains. These structures will help us to understand the synergistic and multivalency effects that generate the astonishing versatility and specificity of protein-protein interactions.

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Delineating the genetic regulation of cannabinoid biosynthesis during female flower development in Cannabis sativa O_LICannabinoids are predominantly produced in the glandular trichomes on cannabis female flowers. There is little known on how cannabinoid biosynthesis is regulated during female flower development. We aim to understand the rate-limiting step(s) in the cannabinoid biosynthetic pathway.
C_LIO_LIWe investigated the transcript levels of cannabinoid biosynthetic genes as well as cannabinoid contents during 7 weeks of female flower development. We demonstrated that the enzymatic steps for producing CBG, which involve genes GPPS, PT and OAC, could be rate limit cannabinoid biosynthesis. Our findings further suggest that cannabinoid synthases, CBDAS and THCAS in a hemp and medical marijuana variety respectively, are not critical for cannabinoid biosynthesis. The cannabinoid biosynthetic genes are generally upregulated during flower maturation, which indicate glandular trichome development.
C_LIO_LIMeJA can potentially increase cannabinoid production. We propose that biweekly application of 100 M MeJA staring from flower initiation would be efficacious for promoting cannabinoid biosynthesis.
C_LIO_LIOur findings suggest that the step of CBG production could rate limit the terminal cannabinoid biosynthesis. In addition, different cannabis varieties demonstrated discrete transcriptional regulation of cannabinoid biosynthetic genes.
C_LI

sentences: Consensus clustering for Bayesian mixture models Cluster analysis is an integral part of precision medicine and systems biology, used to define groups of patients or biomolecules. Consensus clustering is an ensemble approach that is widely used in these areas, which combines the output from multiple runs of a non-deterministic clustering algorithm. Here we consider the application of consensus clustering to a broad class of heuristic clustering algorithms that can be derived from Bayesian mixture models (and extensions thereof) by adopting an early stopping criterion when performing sampling-based inference for these models. While the resulting approach is non-Bayesian, it inherits the usual benefits of consensus clustering, particularly in terms of computational scalability and providing assessments of clustering stability/robustness. In simulation studies, we show that our approach can successfully uncover the target clustering structure, while also exploring different plausible clusterings of the data. We show that, when a parallel computation environment is available, our approach offers significant reductions in runtime compared to performing sampling-based Bayesian inference for the underlying model, while retaining many of the practical benefits of the Bayesian approach, such as exploring different numbers of clusters. We propose a heuristic to decide upon ensemble size and the early stopping criterion, and then apply consensus clustering to a clustering algorithm derived from a Bayesian integrative clustering method. We use the resulting approach to perform an integrative analysis of three 'omics datasets for budding yeast and find clusters of co-expressed genes with shared regulatory proteins. We validate these clusters using data external to the analysis. These clusters can help assign likely function to understudied genes, for example GAS3 clusters with histones active in S-phase, suggesting a role in DNA replication. Our approach can be used as a wrapper for essentially any existing sampling-based Bayesian clustering implementation, and enables meaningful clustering analyses to be performed using such implementations, even when computational Bayesian inference is not feasible, e.g. due to poor exploration of the target density (often as a result of increasing numbers of features) or a limited computational budget that does not along sufficient samples to drawn from a single chain. This enables researchers to straightforwardly extend the applicability of existing software to much larger datasets, including implementations of sophisticated models such as those that jointly model multiple datasets.

sentences: Molecular determinants of phase separation for Drosophila DNA replication licensing factors Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. We have shown that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit specific partner proteins. How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Using D. melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6- hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive self-assembly and condensate specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and specificity a priori.

sentences: Exclusion of the fittest predicts microbial community diversity in fluctuating environments Microorganisms live in environments that inevitably fluctuate between mild and harsh conditions. As harsh conditions may cause extinctions, the rate at which fluctuations occur can shape microbial communities and their diversity, but we still lack an intuition on how. Here, we build a mathematical model describing two microbial species living in an environment where substrate supplies randomly switch between abundant and scarce. We then vary the rate of switching as well as different properties of the interacting species, and measure the probability of the weaker species driving the stronger one extinct. We find that this probability increases with the strength of demographic noise under harsh conditions and peaks at either low, high, or intermediate switching rates depending on both species ability to withstand the harsh environment. This complex relationship shows why finding patterns between environmental fluctuations and diversity has historically been difficult. In parameter ranges where the fittest species was most likely to be excluded, however, the beta diversity in larger communities also peaked. In sum, how environmental fluctuations affect interactions between a few species pairs predicts their effect on the beta diversity of the whole community.

sentences: Microgravity enhances the phenotype of Arabidopsis zigzag-1 and reduces the Wortmannin-induced vacuole fusion in root cells The spaceflight environment of the International Space Station poses a multitude of stresses on plant growth including reduced gravity. Plants exposed to microgravity and other conditions on the ISS display root skewing, changes in gene expression and protein abundance that may result in changes in cell wall composition, antioxidant accumulation and modification of growth anisotropy. Systematic studies that address the effects of microgravity on cellular organelles are lacking but altered numbers and sizes of vacuoles have been detected in previous flights. The prominent size of plant vacuoles makes them ideal models to study organelle dynamics in space. Here, we used Arabidopsis zigzag1 (zig-1) as a sensitized genotype to study the effect of microgravity on plant vacuole fusion. Wortmannin was used to induce vacuole fusion in seedlings and a formaldehyde-based fixation protocol was developed to visualize plant vacuole morphology after sample return, using confocal microscopy. Our results indicate that microgravity enhances the zig-1 phenotype by reducing hypocotyl growth and vacuole fusion in some cells. This study demonstrates the feasibility of chemical inhibitor treatments for plant cell biology experiments in space.

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Unexpected short- and long-term effects of chronic adolescent HU-210 exposure on emotional behavior Chronic adolescent cannabinoid receptor agonist exposure has been shown to lead to persistent increases in depressive-like behaviors. This has been a key obstacle to the development of cannabinoid-based therapeutics. However, most of the published work has been performed with only three compounds, namely {Delta}9-tetrahydrocannabinol, CP55,940 and WIN55,212-2. Hypothesizing that different compounds may lead to distinct outcomes, we herein used the highly potent CB1R/CB2R full agonist HU-210, and first aimed at replicating cannabinoid-induced long-lasting effects, by exposing adolescent female Sprague-Dawley rats to increasing doses of HU-210, for 11 days and testing them at adulthood, after a 30-day drug washout. Surprisingly, HU-210 did not significantly impact adult anxious- or depressive-like behaviors. We then tested whether chronic adolescent HU-210 treatment resulted in short-term (24h) alterations in depressive-like behavior. Remarkably, HU-210 treatment simultaneously induced marked antidepressant- and prodepressant-like responses, in the modified forced swim (mFST) and sucrose preference tests (SPT), respectively. Hypothesizing that mFST results were a misleading artifact of HU-210-induced behavioral hyperreactivity to stress, we assessed plasmatic noradrenaline and corticosterone levels, under basal conditions and following an acute swim-stress episode. Notably, we found that while HU-210 did not alter basal noradrenaline or corticosterone levels, it greatly augmented the stress-induced increase in both.

Our results show that, contrary to previously studied cannabinoid receptor agonists, HU-210 does not induce persisting depressive-like alterations, despite inducing marked short-term increases in stress-induced reactivity. By showing that not all cannabinoid receptor agonists may induce long-term negative effects, these results hold significant relevance for the development of cannabinoid-based therapeutics.

sentences: Sonic hedgehog is basolaterally sorted from the TGN and transcytosed to the apical domain involving Dispatched-1 at Rab11-ARE Hedgehog (Hh) secretion from apical and/or basolateral domains occurs in different epithelial cells impacting development and tissue homeostasis. Palmitoylation and cholestyrolation attach Hh proteins to membranes and Dispatched-1 (Disp-1) promotes their release. How these lipidated proteins are handled by the complex secretory and endocytic pathways of polarized epithelial cells remains unknown. We show that MDCK cells address newly synthesized sonic hedgehog (Shh) from the TGN to the basolateral cell surface and then to the apical domain through a transcytosis pathway that includes Rab11-apical recycling endosomes (Rab11-ARE). Both palmitoylation and cholestyrolation contribute to this sorting behavior, otherwise Shh lacking these lipid modifications is unpolarized. Disp-1 mediates first basolateral secretion from the TGN and then transcytosis from the Rab11-ARE. At steady state, Shh predominates apically and can be basolaterally transcytosed. This complex Shh trafficking provides several steps for regulation and variation in different epithelia, subordinating the apical to the basolateral secretion.

sentences: Genome-wide survey of odorant-binding proteins in the dwarf honey bee Apis florea Odorant binding proteins (OBPs) in insects bind to volatile chemical cue and help in their binding to odorant receptors. The odor coding hypothesis states that OBPs may bind with specificity to certain volatiles and aid the insect in various behaviours. Honeybees are eusocial insects with complex behaviour that requires olfactory inputs. Here, we have identified and annotated odorant binding proteins from the genome of the dwarf honey bee, Apis florea using an exhaustive homology-based bioinformatic pipeline and analyzed the evolutionary relationships between the OBP subfamilies. Our study suggests that Minus-C subfamily may have diverged from the Classic subfamily of odorant binding proteins in insects.

sentences: Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remained unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1s lumenal domain. Phosphorylation of IRE1s kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans- autophosphorylation, which in turn drives IRE1s RNase activity.

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3CAC: improving the classification of phages and plasmids from metagenomic assemblies using assembly graphs MotivationBacteriophages and plasmids usually coexist with their host bacteria in microbial communities and play important roles in microbial evolution. Accurately identifying sequence contigs as phages, plasmids, and bacterial chromosomes in mixed metagenomic assemblies is critical for further unravelling their functions. Many classification tools have been developed for identifying either phages or plasmids in metagenomic assemblies. However, only two classifiers, PPR-Meta and viralVerify, were proposed to simultaneously identify phages and plasmids in mixed metagenomic assemblies. Due to the very high fraction of chromosome contigs in the assemblies, both tools achieve high precision in the classification of chromosomes but perform poorly in classifying phages and plasmids. Short contigs in these assemblies are often wrongly classified or classified as uncertain.

ResultsHere we present 3CAC, a new three-class classifier that improves the precision of phage and plasmid classification. 3CAC starts with an initial three-class classification generated by existing classifiers and improves the classification of short contigs and contigs with low confidence classification by using proximity in the assembly graph. Evaluation on simulated metagenomes and on real human gut microbiome samples showed that 3CAC outperformed PPR-Meta and viralVerify in both precision and recall, and increased F1-score by 10-60 percentage points.

AvailabilityThe 3CAC software is available on https://github.com/Shamir-Lab/3CAC.

Contactrshamir@tau.ac.il

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: Effects of cryo-EM cooling on structural ensembles Structure determination by cryo electron microscopy (cryo-EM) provides information on structural heterogeneity and ensembles at atomic resolution. To obtain cryo-EM images of macromolecules, the samples are first rapidly cooled down to cryogenic temperatures. To what extent the structural ensemble is perturbed during cooling is currently unknown. Here, to quantify the effects of cooling, we combined continuum model calculations of the temperature drop, molecular dynamics simulations of a ribosome complex before and during cooling with kinetic models. Our results suggest that three effects markedly contribute to the narrowing of the structural ensembles: thermal contraction, reduced thermal motion within local potential wells, and the equilibration into lower free-energy conformations by overcoming separating free-energy barriers. During cooling, barrier heights below 10 kJ/mol were found to be over-come, which is expected to reduce B-factors in ensembles imaged by cryo-EM. Our approach now enables the quantification of the heterogeneity of room-temperature ensembles from cryo-EM structures.

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Floral evolution and pollinator diversification in Hedychium J.Koenig (Zingiberaceae): one of Mr. Darwin's tropical fantasies PREMISEHedychium J.Koenig (ginger lilies: Zingiberaceae) is endemic to the Indo-Malayan Realm (IMR) and is known for its fragrant flowers. Two different pollination syndromes characterize the genus: diurnal or bird pollination and nocturnal or moth pollination systems. To date, no attempt has been undertaken to understand the evolution of floral traits in this genus.

METHODSWe estimated ancestral character-states, phylogenetic signals, and character correlations for thirteen discrete and eight continuous floral traits representing 75% species diversity of Hedychium. Diversification rate estimation analyses were also employed to understand trait-dependent diversification in the genus.

RESULTSInflorescence structure, cincinnus capacity, and curvature of floral tubes revealed strong phylogenetic dependence, whereas number of open flowers per inflorescence per day, color of the labellum, and exertion of the stigma characterized higher ecological effects. Diversification rate estimations suggested that the labellum width, floral tube length, and labellum color played a major role in the evolutionary diversification of Hedychium.

CONCLUSIONSWe identified bract type and cincinnus capacity as synapomorphies for Hedychium, while the island-specific clade III was characterized by slender cylindrical inflorescence, coiling of floral tubes, and longer bract to calyx ratio. The circum-Himalayan clade IV is the most speciose, derived, and with most variable floral traits. Although floral color and size lacked any association with pollinator-specific traits (moth and bird pollination), pale colored flowers were most common in the early diverging clades (clade I, II-el., and II-de.), indicating their ancestral nature, when compared to brightly colored flowers.

sentences: Erythroid differentiation in mouse erythroleukemia cells is driven via actin filament-tropomodulin3-tropomyosin networks Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament assembly and organization during terminal ED have revealed essential roles for the pointed-end actin filament capping proteins, tropomodulins (Tmod1 and Tmod3). Additionally, tropomyosin (Tpm) binding to Tmods is a key feature promoting Tmod-mediated actin filament capping. Global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To test a cell autonomous function for Tmod3 and further decipher its biochemical function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability and increased apoptosis. In Mel ds19 cells, both Tpms and actin were preferentially associated with the Triton-X 100 insoluble cytoskeleton during ED, indicating Tpm-coated actin filament assembly during ED. While loss of Tmod3 did not lead to a change in total actin levels, it led to a severe reduction in the proportion of Tpms and actin associated with the Triton-X 100 insoluble cytoskeleton during ED. We conclude that Tmod3-regulation of actin cytoskeleton assembly via Tpms is integral to morphological maturation and cell survival during normal erythroid terminal differentiation.

sentences: Deep Learning-Based Brain Transcriptomic Signatures Associated with the Neuropathological and Clinical Severity of Alzheimer's Disease Brain tissue gene expression from donors with and without Alzheimers disease (AD) have been used to help inform the molecular changes associated with the development and potential treatment of this disorder. Here, we use a deep learning method to analyze RNA-seq data from 1,114 brain donors from the AMP-AD consortium to characterize post-mortem brain transcriptome signatures associated with amyloid-{beta} plaque, tau neurofibrillary tangles, and clinical severity in multiple AD dementia populations. Starting from the cross-sectional data in the ROSMAP cohort (n = 634), a deep learning framework was built to obtain a trajectory that mirrors AD progression. A severity index (SI) was defined to quantitatively measure the progression based on the trajectory. Network analysis was then carried out to identify key gene (index gene) modules present in the model underlying the progression. Within this dataset, SIs were found to be very closely correlated with all AD neuropathology biomarkers (R [~] 0.5, p < 1e-11) and global cognitive function (R = -0.68, p < 2.2e-16). We then applied the model to additional transcriptomic datasets from different brain regions (MAYO, n = 266; MSBB, n = 214), and observed that the model remained significantly predictive (p < 1e-3) of neuropathology and clinical severity. The index genes that significantly contributed to the model were integrated with AD co-expression regulatory networks, resolving four discrete gene modules that are implicated in vascular and metabolic dysfunction in different cell types respectively. Our work demonstrates the generalizability of this signature to frontal and temporal cortex measurements and additional brain donors with AD, other age-related neurological disorders and controls; and revealed the transcriptomic network modules contribute to neuropathological and clinical disease severity. This study illustrates the promise of using deep learning methods to analyze heterogeneous omics data and discover potentially targetable molecular networks that can inform the development, treatment and prevention of neurodegenerative diseases like AD.

sentences: Ultrafast and Reproducible Proteomics from Small Amounts of Heart Tissue Enabled by Azo and timsTOF Pro Global bottom-up mass spectrometry (MS)-based proteomics is widely used for protein identification and quantification to achieve a comprehensive understanding of the composition, structure, and function of the proteome. However, traditional sample preparation methods are time-consuming, typically including overnight tryptic digestion, extensive sample clean-up to remove MS-incompatible surfactants, and offline sample fractionation to reduce proteome complexity prior to online liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Thus, there is a need for a fast, robust, and reproducible method for protein identification and quantification from complex proteomes. Herein, we developed an ultrafast bottom-up proteomics method enabled by Azo, a photocleavable, MS-compatible surfactant that effectively solubilizes proteins and promotes rapid tryptic digestion, combined with the Bruker timsTOF Pro, which enables deeper proteome coverage through trapped ion mobility spectrometry (TIMS) and parallel accumulation-serial fragmentation (PASEF) of peptides. We applied this method to analyze the complex human cardiac proteome and identified nearly 4,000 protein groups from as little as 1 mg of human heart tissue in a single one-dimensional LC-TIMS-MS/MS run with high reproducibility. Overall, we anticipate this ultrafast, robust, and reproducible bottom-up method empowered by both Azo and the timsTOF Pro will be generally applicable and greatly accelerate the throughput of large-scale quantitative proteomic studies. Raw data are available via the MassIVE repository with identifier MSV000087476.

sentences: Whole-brain dynamics of human sensorimotor adaptation Humans vary greatly in their motor learning abilities, yet little is known about the neural processes that underlie this variability. We identified distinct profiles of human sensorimotor adaptation that emerged across two days of learning, linking these profiles to the dynamics of whole-brain functional networks early on the first day, when cognitive strategies toward sensorimotor adaptation are believed to be most prominent. During early learning, greater recruitment of a network of higher-order brain regions, involving prefrontal and anterior temporal cortex, was associated with faster learning. At the same time, greater integration of this  cognitive network with a sensorimotor network was associated with slower learning, consistent with the notion that cognitive strategies toward adaptation operate in parallel with implicit learning processes of the sensorimotor system. On the second day, greater recruitment of a network that included the hippocampus was associated with faster re-learning, consistent with the notion that savings involves declarative memory systems. Together, these findings provide novel evidence for the role of higher-order brain systems in driving individual differences in adaptation.

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Contributions of h- and Na+/K+ pump currents to the generation of episodic and continuous rhythmic activities Developing spinal motor networks produce a diverse array of outputs, including episodic and continuous patterns of rhythmic activity. Variation in excitability state and neuromodulatory tone can facilitate transitions between episodic and continuous rhythms; however, the intrinsic mechanisms that govern these rhythms and their transitions are poorly understood. Here, we tested the capacity of a single central pattern generator (CPG) circuit with tunable properties to generate multiple outputs. To address this, we deployed a computational model composed of an inhibitory half-centre oscillator (HCO). Following predictions of our computational model, we tested the contributions of key properties to the generation of an episodic rhythm produced by isolated spinal cords of the newborn mouse. The model recapitulates the diverse state-dependent rhythms evoked by dopamine. In the model, episodic bursting depended predominantly on the endogenous oscillatory properties of neurons, with Na+/K+ ATPase pump (IPump) and hyperpolarization-activated currents (Ih) playing key roles. Modulation of either IPumpMax or Ih produced transitions between episodic and continuous rhythms and silence. As IPump increased, the episode duration and period increased along with a reduction in interepisode interval. Increasing Ih increased the episode period along with an increase in episode duration. Pharmacological manipulations of Ih with ZD7288 and IPump with ouabain or monensin in isolated spinal cords produced findings consistent with the model. Our modelling and experimental results highlight key roles of Ih and IPump in producing episodic rhythms and provide insight into mechanisms that permit a single CPG to produce multiple patterns of rhythmicity.

Significance statementThe ability of a single CPG to produce and transition between multiple rhythmic patterns of activity is poorly understood. We deployed a complementary computational half-centre oscillator model and an isolated spinal cord experimental preparation to identify key currents whose interaction produced episodic and continuous rhythmic activity. Together, our experimental and modelling approaches suggest mechanisms in spinal networks that govern diverse rhythms and transitions between them. This work sheds light on the ability of a single CPG to produce episodic bouts observed in behavioural and pathological contexts.

sentences: Colour Ambiguity In Real Scenes And The Role Of Perceptual Organisation In this study we show a reproduction of the dress-ambiguity phenomenon in a real scene and we report quantitative measurements of the corresponding colour perceptions. The original, real dress, known from #thedress-illusion, was illuminated by combined short- and longwavelength broadband lights from two slide projectors. Test subjects viewing the dress reported to perceive the dress fabric and lace colours as blue & black, white & gold or light blue & brown; their corresponding perceptual matches were distributed along the blue/yellow cardinal axis, and exhibited a variability comparable to the ambiguity of the dress photograph. It is particularly noteworthy that the colour ambiguity emerged despite explicit knowledge of the observers about the direction of the light source. Manipulating the background of the real dress (change in chromaticity and luminance, or masking) revealed significant differences between the perceptual groups regarding lightness and colour of the dress. Our findings suggest that observer specific differences in the perceptual organisation of the visual scene are responsible for the colour ambiguity observed for the real dress; in particular, we conclude that colour computations of white & gold viewers focused onto the local region of the dress, whereas the colour processes of blue & black and light-blue & brown viewers were strongly influenced by contextual computations including the background. Our segmentation hypothesis extends existing explanations for the dress ambiguity and proposes image based (in the case of the real scene) and high level (in the case of the photograph) neural processes which control the spatial reach of contextual colour computations. The relation between the ambiguity in our real scene and the dress photograph is discussed.

sentences: Beyond coral-algal regimes: high taxonomic resolution surveys and trait-based analyses reveal multiple benthic regimes As coral reef communities change and reorganise in response to anthropogenic and climate disturbances, there is a growing need of detecting and understanding the different emerging species regimes and their contribution to key ecosystem processes. Using a case study on coral reefs at the epicentre of tropical marine biodiversity (North Sulawesi), we explored how application of different biodiversity approaches (i.e. use of major taxonomic categories, high taxonomic resolution categories and trait-based approaches) affects the detection of distinct fish and benthic community assemblages. Our results show that using major categories (family level or above) to study coral reef communities fails to identify distinct regimes. We also show that for detection of different benthic regimes, especially communities dominated by non-coral organisms, monitoring of only scleractinian coral communities is insufficient, and that all types of benthic organisms (e.g. sponges, ascidians, soft corals, algae etc.) need to be considered. We have implemented for the first time, the use of a trait-based approach to study the functional diversity of whole coral reef benthic assemblages, which allowed us to detect five different community regimes, only one of which was dominated by scleractinian corals. We circumvented the challenge that for some benthic groups (e.g. sponges, ascidians or some soft corals) visual identification up to the species level is not possible, by identifying and categorising traits that can be applied to groups of similar organisms instead of specific species. Furthermore, by the parallel study of benthic and fish communities we provide new insights into key processes and functions that might dominate or be compromised in the different community regimes.

sentences: Novel methodology for localizing and studying the insect dorsal rim area morphology in 2D and 3D 1.Polarized light-based navigation in insects is facilitated by a specialised polarisation-sensitive part of the eye known as the dorsal rim area (DRA). Existing methods to study the anatomy of the DRA are in most cases destructive and very time consuming. Here, we present a novel method that allows for DRA localization using 3D volumetric images acquired through micro-computed tomography in combination with 2D photographs. We used the method on size polymorphic buff-tailed bumblebees, Bombus terrestris. We found that the size of the DRA can be easily acquired from photographs of the dorsal part of the eye. An allometric analysis of the size of the DRA in relation to body size in B. terrestris showed that the DRA region increases with body size but not at the same rate. Using our method, we localised the DRA in a 3D volume rendering of the eye using 2D photos localization of an individuals DRA onto the volumetric image stack of the head allowed for individual-level descriptions of the ommatidial structures (lens, crystalline cones, rhabdoms) to be performed on three different eye regions (DRA, non-DRA, proximate to DRA). The only feature distinctly unique to the DRA ommatidia was the smaller dimension of crystalline cones in comparison to other regions. Using our novel methodology we provide the first individual-level description of DRA ommatidial features and a comparison of how the DRA area varies with body size in bumblebees.

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Clock time: a foreign measure to brain dynamics Human thought is highly flexible and dynamic, achieved by evolving patterns of brain activity across groups of cells. Neuroscience aims to understand cognition in the brain by analysing these intricate patterns. Here, we argue that this goal is impeded by the time format of our data - clock time. The brain is a system with its own dynamics and regime of time, with no intrinsic concern for the human-invented second. A more appropriate time format is cycles of brain oscillations, which coordinate neural firing and are widely implicated in cognition. These brain dynamics do not obey clock time - they start out of tune with clock time and drift apart even further as oscillations unpredictably slow down, speed up, and undergo abrupt changes. Since oscillations clock cognition, their dynamics should critically inform our analysis. We describe brain time warping as a new method to transform data in accordance with brain dynamics, which sets the time axis to cycles of clocking oscillations (a native unit) rather than milliseconds (a foreign unit). We also introduce the Brain Time Toolbox, a software library that implements brain time warping for electrophysiology data and tests whether it reveals information patterns in line with how the brain uses them.

Glossary

O_TBL View this table:
org.highwire.dtl.DTLVardef@d81c2org.highwire.dtl.DTLVardef@c1ff2corg.highwire.dtl.DTLVardef@57dfa4org.highwire.dtl.DTLVardef@11b8c43org.highwire.dtl.DTLVardef@195a419_HPS_FORMAT_FIGEXP  M_TBL C_TBL

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ortho2align: a sensitive approach for searching for orthologues of novel lncRNAs BackgroundMany novel long noncoding RNAs have been discovered in recent years due to advances in high-throughput sequencing experiments. Finding orthologues of these novel lncRNAs might facilitate clarification of their functional role in living organisms. However, lncRNAs exhibit low sequence conservation, so specific methods for enhancing the signal-to-noise ratio were developed. Nevertheless, current methods such as transcriptomes comparison approaches or searches for conserved secondary structures are not applicable to novel lncRNAs dy design.

ResultsWe present ortho2align -- a versatile sensitive synteny-based lncRNA orthologue search tool with statistical assessment of sequence conservation. This tool allows control of the specificity of the search process and optional annotation of found orthologues. ortho2align shows similar performance in terms of sensitivity and resource usage as the state-of-the-art method for aligning orthologous lncRNAs but also enables scientists to predict unannotated orthologous sequences for lncRNAs in question. Using ortho2align, we predicted orthologues of three distinct classes of novel human lncRNAs in six Vertebrata species to estimate their degree of conservation.

ConclusionsBeing designed for the discovery of unannotated orthologues of novel lncRNAs in distant species, ortho2align is a versatile tool applicable to any genomic regions, especially weakly conserved ones. A small amount of input files makes ortho2align easy to use in orthology studies as a single tool or in bundle with other steps that researchers will consider sensible.

sentences: Periodic attention operates faster during more complex visual search Attention has been found to sample visual information periodically, in a wide range of frequencies below 20 Hz. This periodicity may be supported by brain oscillations at corresponding frequencies. We propose that part of the discrepancy in periodic frequencies observed in the literature is due to differences in attentional demands, resulting from heterogeneity in tasks performed. To test this hypothesis, we used visual search and manipulated task complexity, i.e., target discriminability (high, medium, low) and number of distractors (set size), while electro-encephalography was simultaneously recorded. We replicated previous results showing that the phase of pre-stimulus low-frequency oscillations predicts search performance. Crucially, such effects were observed at increasing frequencies within the theta-alpha range (6-18 Hz) for decreasing target discriminability. In medium and low discriminability conditions, correct responses were further associated with higher post-stimulus phase-locking than incorrect ones, in increasing frequency and latency. Finally, the larger the set size, the later the post-stimulus effect peaked. Together, these results suggest that increased complexity (lower discriminability or larger set size) requires more attentional cycles to perform the task, partially explaining discrepancies between reports of attentional sampling. Low-frequency oscillations structure the temporal dynamics of neural activity and aid top-down, attentional control for efficient visual processing.

sentences: Reproducible stool metagenomic biomarkers linked to the melanoma immunotherapy positive outcome The human gut microbiome plays an important role both in humans health and disease. Recent studies have shown the undeniable influence of gut microbiota composition on cancer immunotherapy efficacy. However, these researches show a lack of consensus in defining reproducible metagenomic markers for a positive immunotherapy outcome. Accordingly, extended published data re-analysis may help reveal clearer associations between the composition of the gut microbiota and treatment response. In this study, we analyzed 358 stool metagenomes from 5 studies published earlier: 210 metagenomes from melanoma patients with positive immunotherapy outcome, 148 metagenomes from melanoma patients with negative immunotherapy outcome. The biomarkers were selected by the group comparison of patients stool samples with different treatment responses (47 responders vs 55 non-responders, 102 metagenomes). Selected biomarkers were verified using the available data describing the influence of the fecal microbiota transplantation on melanoma immunotherapy outcomes (9 donors, 6 responders, 19 non-responders, 256 metagenomes). According to our analysis, the resulting cross-study reproducible taxonomic biomarkers correspond to 12 Firmicutes, 4 Bacteroidetes, and 3 Actinobacteria. 140 gene groups were identified as reproducible functional biomarkers, including those potentially involved in production of immune-stimulating molecules and metabolites. In addition, we ranked taxonomic biomarkers by the number of functional biomarkers found in their metagenomic context. In other words, we predicted a list of the potential "most beneficial" bacteria for a positive response to melanoma immunotherapy. The obtained results can be used to make recommendations for the gut microbiota correction in cancer immunotherapy, and the resulting list of biomarkers can be considered for potential diagnostic ways for predicting melanoma immunotherapy outcome. Another important point is the functional biomarkers of positive immunotherapy outcome are distributed in different bacterial species that can explain the lack of consensus of defining melanoma immunotherapy beneficial species between different studies.

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Uncertainty in population estimates: a meta-analysis for petrels O_LIPopulation estimates are commonly generated and used in conservation science. All estimates carry inherent uncertainty, but little attention has been given to when and how this uncertainty limits their use. This requires an understanding of the specific purposes for which population estimates are intended, an assessment of the level of uncertainty each purpose can tolerate, and information on current uncertainty.
C_LIO_LIWe conducted a review and meta-analysis for a widespread group of seabirds, the petrels, to better understand how and why population estimates are being used. Globally petrels are highly threatened, and aspects of their ecology make them difficult to survey, introducing high levels of uncertainty into population estimates.
C_LIO_LIWe found that by far the most common intended use of population estimates was to inform status and trend assessments, while less common uses were trialling methods to improve estimates, and assessing threat impacts and conservation outcomes.
C_LIO_LIThe mean coefficient of variation for published estimates was 0.17 (SD = 0.14), with no evidence that uncertainty has been reduced through time. As a consequence of this high uncertainty, when we simulated declines equivalent to thresholds commonly used to trigger management, only 5% of studies could detect significant differences between population estimates collected 10 years apart for populations declining at a rate of 30% over three generations.
C_LIO_LIReporting of uncertainty was variable with no dispersion statistics reported with 38% of population estimates and most not reporting key underlying parameters: nest numbers/density and nest occupancy. We also found no correlation between population estimates and either island size, body size or species threat status--potential predictors of uncertainty.
C_LIO_LISynthesis and applications--Key recommendations for managers are to be mindful of uncertainty in past population estimates if aiming to collect contemporary estimates for comparison, to report uncertainty clearly for new estimates, and to give careful consideration to whether a proposed estimate is likely to achieve the requisite level of certainty for the investment in its generation to be warranted. We recommend a practitioner-based Value of Information assessment to confirm where there is value in reducing uncertainty.
C_LI

sentences: NEDD4 Regulates Ubiquitination and Stability of the Cell adhesion Molecule IGPR-1 via Lysosomal Pathway The cell adhesion molecule immunoglobulin and proline-rich receptor-1 (IGPR-1) regulates various critical cellular processes including, cell-cell adhesion, mechanosensing and autophagy. However, the molecular mechanisms governing IGPR-1 cell surface expression levels remains unknown. In the present study, we used an in vitro ubiquitination assay and identified ubiquitin E3 ligase NEDD4 and the ubiquitin conjugating enzyme UbcH6 involved in the ubiquitination of IGPR-1. In vitro GST-pulldown and in vivo co-immunoprecipitation assays demonstrated that NEDD4 binds to IGPR-1. Over-expression of wild-type NEDD4 downregulated IGPR-1 and deletion of WW domains (1-4) of NEDD4 revoked its effects on IGPR-1. Similarly, knockdown of NEDD4 increased IGPR-1 levels in A375 melanoma cells. Furthermore, deletion of 57 amino acids encompassing polyproline rich (PPR) motif on the C-terminus of IGPR-1 nullified the binding of NEDD4 with IGPR-1. Moreover, we demonstrate that NEDD4 promotes K48- and K63-dependent polyubiquitination of IGPR-1. The NEDD4-mediated polyubiquitination of IGPR-1 stimulated lysosomal degradation of IGPR-1 as the treatment of cells with the lysosomal inhibitors, bafilomycine and ammonium chloride increased IGPR-1 levels in the HEK-293 cells ectopically expressing IGPR-1 and in multiple human skin melanoma cell lines. Hence, these findings suggest that ubiquitin E3 ligase NEDD4 is a key regulator of IGPR-1 with a significant implication in the therapeutic targeting of IGPR-1.

sentences: UBP12 and UBP13 deubiquitinases destabilize the CRY2 blue-light receptor to regulate growth All organisms undergo growth, which is precisely controlled by exogenous and endogenous signals. Unchecked growth often leads to neoplasia and other developmental defects, severely affecting an organisms fitness. Light is a vital exogenous signal sensed by cryptochrome (CRY) blue light receptors to modulate growth and the circadian clock in plants and animals. Yet, how CRYs interpret light quantity to regulate growth in plants remains poorly understood. We show that UBP12 and UBP13 deubiquitinases physically interact with CRY2 in light. UBP12/13 negatively regulated CRY2 protein levels by promoting its ubiquitination and turnover to fine-tune growth. Unexpectedly, the destabilization of CRY2 by UBP12/13 is contrary to the general view that deubiquitinases stabilize proteins by preventing their degradation. Growth and development were explicitly affected in blue light when UBP12/13 was disrupted or overexpressed, indicating their role alongside CRY2. UBP12/13 also interacted and stabilized COP1, which is partially required for the turnover of CRY2. Despite decades of studies on deubiquitinases, the knowledge on how they are regulated is limited. Our study offers an insight into how exogenous signals and their receptors regulate deubiquitinase activity by protein-protein interaction. Altogether, our results provide a new module of cryptochromes and deubiquitinases in sensing and interpreting light cues to control growth at the most appropriate time.

sentences: The mechanism of RNA capping by SARS-CoV-2 The SARS-CoV-2 RNA genome contains a 5-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made is not completely understood. Here, we reconstitute the SARS-CoV-2 7MeGpppA2-O-Me-RNA cap using virally encoded non-structural proteins (nsps). We show that the kinase-like NiRAN domain5 of nsp12 transfers RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers RNA to GDP, forming the cap core structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N-terminus of nsp9 and the kinase-like active site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.

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Taxonomy-aware, sequence similarity ranking reliably predicts phage-host relationships BackgroundSimilar regions in virus and host genomes provide strong evidence for phage-host interaction, and BLAST is one of the leading tools to predict prokaryotic hosts from phage sequences. However, BLAST-based host prediction has three major limitations: (i) top-scoring sequences do not always point to the actual host, (ii) mosaic phage genomes may match to many, typically related, bacteria, and (iii) phage and host sequences may diverge beyond the point where their relationship can be detected by a BLAST alignment.

ResultsWe created an extension to BLAST, named Phirbo, that improves host prediction quality beyond what is obtainable from standard BLAST searches. The tool harnesses information concerning sequence similarity and bacteria relatedness to predict phage-host interactions. Phirbo was evaluated on three benchmark sets of known phage-host pairs, and it improved precision and recall by 11-40 percentage points over currently available, state-of-the-art, alignment-based, alignment-free, and machine learning host prediction tools. Moreover, the discriminatory power of Phirbo for the recognition of phage-host relationships surpassed the results of other tools by at least 10 percentage points (Area Under the Curve = 0.95), yielding a mean host prediction accuracy of 57% and 68% at the genus and family levels respectively, and drops by 12 percentage points when using only a fraction of phage genome sequences (3 kb). Finally, we provide insights into a repertoire of protein and ncRNA genes that are shared between phages and hosts and may be prone to horizontal transfer during infection.

ConclusionsOur results suggest that Phirbo is a simple and effective tool for predicting phage host relationships.

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A VLP-based vaccine targeting ANGPTL3 lowers plasma triglycerides in mice Elevated triglycerides (TGs) are an important risk factor for the development of coronary heart disease (CHD) and in acute pancreatitis. Angiopoietin-like proteins 3 (ANGPTL3) and 4 (ANGPTL4) are critical regulators of TG metabolism that function by inhibiting the activity of lipoprotein lipase (LPL), which is responsible for hydrolyzing triglycerides in lipoproteins into free fatty acids. Interestingly, individuals with loss-of-function mutations in ANGPTL3 and ANGPTL4 have low plasma TG levels, have a reduced risk of CHD, and are otherwise healthy. Consequently, interventions targeting ANGPTL3 and ANGPTL4 have emerged as promising new approaches for reducing elevated TGs. Here, we developed virus-like particle (VLP) based vaccines that target the LPL binding domains of ANGPTL3 and ANGPTL4. ANGPTL3 VLPs and ANGPTL4 VLPs are highly immunogenic in mice and vaccination with ANGPTL3 VLPs, but not ANGPTL4 VLPs, was associated with reduced steady state levels of TGs. Immunization with ANGPTL3 VLPs rapidly cleared circulating TG levels following an oil gavage challenge and enhanced plasma LPL activity. These data indicate that targeting ANGPTL3 by active vaccination is potential alternative to other ANGPTL3-inhibiting therapies.

HighlightsO_LIANGPTL3 and ANGPTL4 are mediators of lipoprotein metabolism that inhibit lipoprotein lipase (LPL) activity.
C_LIO_LIVaccination using virus-like particles (VLPs) targeting ANGPTL3 and ANGPTL4 elicits high-titer IgG antibody responses.
C_LIO_LIImmunization with ANGPTL3 VLPs lowers steady-state plasma triglycerides and enhances LPL activity.
C_LI

sentences: The composition and function of Enterococcus faecalismembrane vesicles Membrane vesicles (MVs) contribute to various biological processes in bacteria, including virulence factor delivery, host immune evasion, and cross-species communication. MVs are frequently being discharged from the surface of both Gram-negative and Gram-positive bacteria during growth. In some Gram-positive bacteria, genes affecting MV biogenesis have been identified, but the mechanism of MV formation is unknown. In Enterococcus faecalis, a causative agent of life-threatening bacteraemia and endocarditis, neither mechanisms of MV formation nor their role in virulence has been examined. Since MVs of many bacterial species are implicated in host-pathogen interactions, biofilm formation, horizontal gene transfer, and virulence factor secretion in other species, we sought to identify, describe, and functionally characterize MVs from E. faecalis. Here we show that E. faecalis releases MVs that possess unique lipid and protein profiles, distinct from the intact cell membrane, and are enriched in lipoproteins. MVs of E. faecalis are specifically enriched in unsaturated lipids that might provide membrane flexibility to enable MV formation, providing the first insights into the mechanism of MV formation in this Gram-positive organism.

sentences: Acetaldehyde makes a distinct mutation signature in single-stranded DNA Acetaldehyde (AA), a by-product of ethanol metabolism, is acutely toxic due to its ability to react with various biological molecules including DNA and proteins, which can greatly impede key processes such as replication and transcription and lead to DNA damage. As such AA is classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC). Previous in vitro studies have shown that AA generates bulky adducts on DNA, with signature guanine-centered (GG[->]TT) mutations. However, due to its weak mutagenicity, short chemical half-life, and the absence of powerful genetic assays, there is considerable variability in reporting the genotoxic effects of AA in vivo. Here, we used an established yeast genetic reporter system and demonstrate that AA is highly mutagenic and makes strand-biased mutations on guanines (G[->]T) at a high frequency on single stranded DNA (ssDNA). We further demonstrate that AA-derived mutations occur through lesion bypass on ssDNA by the translesion polymerase Pol{zeta}. Finally, we describe a unique mutation signature for AA, which we then identify in several whole-genome and -exome sequenced cancers, particularly those associated with alcohol consumption.

sentences: Ecdysone coordinates plastic growth with robust pattern in the developing wing Animals develop in unpredictable, variable environments. In response to environmental change some aspects of development adjust to generate plastic phenotypes. Other aspects of development, however, are buffered against environmental change to produce robust phenotypes. How organ development is coordinated to accommodate both plastic and robust developmental responses is poorly understood. Here, we demonstrate that the steroid hormone ecdysone coordinates both plasticity of organ size and robustness of organ pattern in the developing wings of the fruit fly Drosophila melanogaster. Using fed and starved larvae that lack prothoracic glands, which synthesise ecdysone, we show that nutrition regulates growth both via ecdysone and via an ecdysone-independent mechanism, while nutrition regulates patterning only via ecdysone. We then demonstrate that growth shows a graded response to ecdysone concentration, while patterning shows a threshold response. Collectively, these data support a model where nutritionally-regulated ecdysone fluctuations confer plasticity by regulating disc growth in response to basal ecdysone levels, and confers robustness by initiating patterning only once ecdysone peaks exceeds a threshold concentration. This could represent a generalizable mechanism through which hormones coordinate plastic growth with robust patterning in the face of environmental change.

sentences: Fine-mapping from summary data with the "Sum of Single Effects" model In recent work, Wang et al introduced the "Sum of Single Effects" (SuSiE) model, and showed that it provides a simple and efficient approach to fine-mapping genetic variants from individual-level data. Here we present new methods for fitting the SuSiE model to summary data, for example to single-SNP z-scores from an association study and linkage disequilibrium (LD) values estimated from a suitable reference panel. To develop these new methods, we first describe a simple, generic strategy for extending any individual-level data method to deal with summary data. The key idea is to replace the usual regression likelihood with an analogous likelihood based on summary data. We show that existing fine-mapping methods such as FINEMAP and CAVIAR also (implicitly) use this strategy, but in different ways, and so this provides a common framework for understanding different methods for fine-mapping. We investigate other common practical issues in fine-mapping with summary data, including problems caused by inconsistencies between the z-scores and LD estimates, and we develop diagnostics to identify these inconsistencies. We also present a new refinement procedure that improves model fits in some data sets, and hence improves overall reliability of the SuSiE fine-mapping results. Detailed evaluations of fine-mapping methods in a range of simulated data sets show that SuSiE applied to summary data is competitive, in both speed and accuracy, with the best available fine-mapping methods for summary data.

sentences: Whole genome sequencing revealed genetic diversity and selection of Guangxi indigenous chickens Guangxi chickens play an important role in promoting the high-quality development of the broiler industry in China, but their value and potential are yet to be discovered. To determine the genetic diversity and population structure of Guangxi indigenous chicken, we analyzed the whole genomes of 185 chicken from 8 phenotypically and geographically representative Guangxi chicken breeds, together with 12 RJFt, 12 BRA and 12 WL genomes available from previous studies. Calculation of heterozygosity (Hp), nucleotide diversity ({pi}), and LD level indicated that Guangxi populations were characterized by higher genetic diversity and lower differentiation than RJFt and commercial breeds except HGFC. Population structure analysis also confirmed the introgression from commercial broiler breeds. Each population clustered together while the overall differentiation was small, MA has the richest genetic diversity among all varieties. Selective sweep analysis revealed BCO2, EDN3 and other candidate genes have received strong selection in local breeds, these also provided novel breeding visual and data basis for future breeding.

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Dissecting the Pyrenophora tritici-repentis (tan spot of wheat) pangenome We sequenced the genome of a global collection (40 isolates) of the fungus Pyrenophora tritici-repentis (Ptr), a major foliar pathogen of wheat and model for the evolution of necrotrophic pathogens. Ptr exhibited an open-pangenome, with 43% of genes in the core set and 57% defined as accessory (present in only a subset of isolates), of which 56% were singleton genes (present in only one isolate). A clear distinction between pathogenic and non-pathogenic genomes was observed in size, gene content, and phylogenetic relatedness. Chromosomal rearrangements and structural organization, specifically around the effector coding genes, were explored further using the annotated genomes of two isolates sequenced by PacBio RS II and Illumina HiSeq. The Ptr genome exhibited major chromosomal rearrangements, including chromosomal fusion, translocation, and segment duplications. An intraspecies translocation of ToxA, the necrosis-inducing effector-coding gene, was facilitated within Ptr via a 143 kb  Starship transposon (dubbed  Horizon). Additionally, ToxB, the gene encoding the chlorosis-inducing effector, was clustered as three copies on a 294 kb transposable element in a ToxB-producing isolate. ToxB and its carrying transposon were missing from the ToxB non-coding reference isolate, but the homolog toxb and the transposon were both present in another non-coding isolate. The Ptr genome also appears to exhibit a  one-compartment organization, but may still possess a  two-speed genome that is facilitated by copy-number variation as reported in other fungal pathosystems.

IMPORTANCEPtr is one of the most destructive wheat pathogens worldwide. Its genome is a mosaic of present and absent effectors, and serves as a model for examining the evolutionary processes behind the acquisition of virulence in necrotrophs and disease emergence. In this work, we took advantage of a diverse collection of pathogenic Ptr isolates with different global origins and applied short- and long-read sequencing technologies to dissect the Ptr genome. This study provides comprehensive insights into the Ptr genome and highlights its structural organization as an open pangenome with  one-compartment. In addition, we identified the potential involvement of transposable elements in genome expansion and the movement of virulence factors. The ability of effector-coding genes to shuffle across chromosomes on large transposons was illustrated by the intraspecies translocation of ToxA and the multi-copy ToxB. In terms of gene contents, the Ptr genome exhibits a large percentage of orphan genes, particularly in non-pathogenic or weakly-virulent isolates.

sentences: The role of antifreeze glycopeptides (AFGP) and polyvinyl alcohol/polyglycerol (X/Z-1000) cocktails as ice modulators during partial freezing of rat livers. The current liver organ shortage has pushed the field of liver transplantation to develop new methods to prolong the preservation time of livers from the current clinical standard of static cold storage. Our approach, termed partial freezing, aims to induce a thermodynamically stable frozen state at deeper storage temperatures (-10{degrees}C to -15{degrees}C) than can be achieved with supercooling, while simultaneously maintaining a sufficient unfrozen fraction to limit dehydration and ice damage. This research first demonstrated that partially frozen glycerol treated rat livers were functionally similar after thawing from either -10 or -15{degrees}C with respect to subnormothermic machine perfusion metrics and histology. Next, we assessed the effect of adding either of two ice modulators, antifreeze glycoprotein (AFGP) and a polyvinyl alcohol/polyglycerol combination (X/Z-1000), on the viability and structural integrity of partially frozen rat livers compared to glycerol-only control livers. Results showed that AFGP livers had high levels of ATP and the least edema but suffered from significant endothelial cell damage. X/Z-1000 livers had the highest levels of ATP and energy charge (EC) but also demonstrated endothelial damage and post-thaw edema. Glycerol-only control livers exhibited the least DNA damage on Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining but also had the lowest levels of ATP and EC. Further research is necessary to optimize the ideal ice modulator cocktail for our partial-freezing protocol. Modifications to cryoprotective agent (CPA) combinations, as well as improvements to machine perfusion CPA loading and unloading, can help improve the viability of these partially frozen organs.

sentences: iPSC-based modeling of THD recapitulates disease phenotypes and reveals neuronal malformation Tyrosine hydroxylase deficiency (THD) is a rare genetic disorder leading to dopaminergic depletion and early-onset parkinsonism. Affected children present with either a severe form that does not respond to L-Dopa treatment (THD-B), or a milder L-Dopa responsive form (THD-A). We generated induced pluripotent stem cells (iPSCs) from THD patients that were differentiated into dopaminergic neurons (DAn) and compared with control-DAn from healthy individuals and gene-corrected isogenic controls. Consistent with patients, THD iPSC-DAn displayed lower levels of DA metabolites and reduced TH expression, when compared to controls. Moreover, THD iPSC-DAn showed abnormal morphology, including reduced total neurite length and either an abnormal TH proximodistal gradient (THDA), or neurite arborization defects (THDB). Treatment of THD-iPSC-DAn with L-Dopa rescued the neuronal defects and disease phenotype only in THDA-DAn. Interestingly, L-Dopa treatment at the stage of neuronal precursors could prevent the alterations in THDB-iPSC-DAn, thus suggesting the existence of a critical developmental window in THD. Our iPSC-based model recapitulates THD disease phenotypes and response to treatment, representing a promising tool for investigating pathogenic mechanisms, drug screening, and personalized management.

sentences: Reducing Movement Synchronization to Increase Interest Improves Interpersonal Liking Synchronization has been identified as a key aspect of achieving mutual goals and enhancing social bonding. While synchronization could be maximized by increasing the predictability of an interaction, such predictability is in tension with individuals level of interest, which is tied to the interactions complexity and novelty. In this study, we tested the interplay between synchronization and interest. We asked 104 female dyads to play the Mirror Game, in which they were instructed to move their hands as coordinately as possible, and then report how much they liked each other. Utilizing information theory and video processing tools, we found that a combination of movement synchronization and complexity explained liking almost two times better than movement synchronization alone. Moreover, we found that people initiated novel and challenging interactions, even though they paid a price - being less synchronized. Examining the interactions dynamics, we found that people who liked each other moved in a more synchronized, complex, and novel manner during most of the interaction. This suggests that in addition to synchronization, maintaining interest may be critical for positive social bonding. Thus, we propose a new framework in which balancing synchronization and interest, rather than merely maximizing synchronization, optimizes the interaction quality.

sentences: Evolutionarily conserved genetic interactions between nphp-4 and bbs-5 mutations exacerbate ciliopathy phenotypes Primary cilia are sensory and signaling hubs with a protein composition that is distinct from the rest of the cell due to the barrier function of the transition zone (TZ) at the base of the cilium. Protein transport across the TZ is mediated in part by the BBSome, and mutations disrupting TZ and BBSome proteins cause human ciliopathy syndromes. Ciliopathies have phenotypic variability even among patients with identical genetic variants, suggesting a role for modifier loci. To identify potential ciliopathy modifiers, we performed a mutagenesis screen on nphp-4 mutant C. elegans and uncovered a novel allele of bbs-5. Nphp-4;bbs-5 double mutant worms have phenotypes not observed in either individual mutant strain. To test whether this genetic interaction is conserved, we also analyzed zebrafish and mice mutants. While Nphp4 mutant zebrafish appeared overtly normal, Bbs5 mutants exhibited scoliosis. When combined, Nphp4;Bbs5 double mutant zebrafish did not exhibit synergistic effects, but the lack of a phenotype in Nphp4 mutants makes interpreting these data difficult. In contrast, viable Nphp4;Bbs5 double mutant mice were not obtained and there were fewer mice than expected carrying three mutant alleles. Additionally, postnatal loss of Bbs5 in mice using a conditional allele compromised survival when combined with a Nphp4 allele. As cilia are formed in the double mutant mice, the exacerbated phenotype is likely a consequence of disrupted ciliary signaling. Collectively, these data support an evolutionarily conserved genetic interaction between Bbs5 and Nphp4 alleles that may contribute to the variability in ciliopathy phenotypes.

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Wheat Panache - a pangenome graph database representing presence/absence variation across 16 bread wheat genomes Bread wheat is one of humanitys most important staple crops, characterized by a large and complex genome with a high level of gene presence/absence variation between cultivars, hampering genomic approaches for crop improvement. With the growing global population and the increasing impact of climate change on crop yield, there is an urgent need to apply genomic approaches to accelerate wheat breeding. With recent advances in DNA sequencing technology, a growing number of high-quality reference genomes are becoming available, reflecting the genetic content of a diverse range of cultivars. However, information on the presence or absence of genomic regions has been hard to visualize and interrogate due to the size of these genomes and the lack of suitable bioinformatics tools. To address this limitation, we have produced a wheat pangenome graph maintained within an online database to facilitate interrogation and comparison of wheat cultivar genomes. The database allows users to visualize regions of the pangenome to assess presence/absence variation between bread wheat genomes.

Database URL: http://www.appliedbioinformatics.com.au/wheat_panache

sentences: Three simple steps to improve the interpretability of EEG-SVM studies Research in machine-learning classification of electroencephalography (EEG) data offers important perspectives for the diagnosis and prognosis of a wide variety of neurological and psychiatric conditions, but the clinical adoption of such systems remains low. We propose here that much of the difficulties translating EEG-machine learning research to the clinic result from consistent inaccuracies in their technical reporting, which severely impair the interpretability of their often-high claims of performance. Taking example from a major class of machine-learning algorithms used in EEG research, the support-vector machine (SVM), we highlight three important aspects of model development (normalization, hyperparameter optimization and cross-validation) and show that, while these 3 aspects can make or break the performance of the system, they are left entirely undocumented in a shockingly vast majority of the research literature. Providing a more systematic description of these aspects of model development constitute three simple steps to improve the interpretability of EEG-SVM research and, in fine, its clinical adoption.

sentences: Heptanol-mediated phase separation determines phase preference of molecules in live cell membranes Plasma membranes contain diverse nanoscale assemblies of lipid and protein domains. Specific localization of lipids and proteins in these domains is often essential for membrane function and integrity. Due to the nanoscale size and dynamic nature of membrane domains, identification of molecules residing in domains either is not possible with modern imaging techniques or requires advanced methods with high spatiotemporal resolution. Such methods need expensive equipment making these approaches inaccessible and thus difficult to implement at large scale. Here, we present a novel membrane fluidizer-induced clustering (MFIC) approach to identify the phase-preference of molecules in intact cell membranes. Experiments in phase-separated bilayers and live cells on molecules with known phase preference demonstrate that heptanol hyperfluidizes the membrane and stabilizes phase separation in cell membranes. The domain stabilization results in a transition of nano-to micron-sized clusters of associated molecules and allows identification of molecules localized in domains using routine microscopy techniques. This assay can be carried out on both genetically and extrinsically labelled molecules in live cell membranes, does not require any invasive sample preparation and can be carried out in 10-15 minutes. This inexpensive and easy to implement assay can be conducted at large-scale and will allow easy identification of molecules partitioning into domains.

sentences: Label-free imaging to track reprogramming of human somatic cells The process of reprogramming patient samples to human induced pluripotent stem cells (iPSCs) is stochastic, asynchronous, and inefficient leading to a heterogeneous population of cells. Here, we track the reprogramming status of single patient-derived cells during reprogramming with label-free live-cell imaging of cellular metabolism and nuclear morphometry to identify high-quality iPSCs. Erythroid progenitor cells (EPCs) isolated from human peripheral blood showed distinct patterns of autofluorescence lifetime for the reduced form of nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) during reprogramming. Random forest models classified starting EPCs, partially-reprogrammed intermediate cells, and iPSCs with [~]95% accuracy. Reprogramming trajectories resolved at the single cell level indicated significant reprogramming heterogeneity along different branches of cell state. This combination of micropatterning, autofluorescence imaging, and machine learning provides a unique non-destructive method to assess the quality of iPSCs in real-time for various applications in regenerative medicine, cell therapy biomanufacturing, and disease modeling.

sentences: Multi-ancestry fine-mapping improves precision to identify causal genes in transcriptome-wide association studies Transcriptome-wide association studies (TWAS) are a powerful approach to identify genes whose expression associates with complex disease risk. However, non-causal genes can exhibit association signals due to confounding by linkage disequilibrium patterns (LD) and eQTL pleiotropy at genomic risk regions which necessitates fine-mapping of TWAS signals. Here, we present MA-FOCUS, a multi-ancestry framework for the improved identification of genes underlying traits of interest. We demonstrate that by leveraging differences in ancestry-specific patterns of LD and eQTL signals, MA-FOCUS consistently outperforms single-ancestry fine-mapping approaches with equivalent total sample size across multiple metrics. We perform 15 blood trait TWAS using genome-wide summary statistics (average NEA=511k, NAA=13k) and lymphoblastoid cell line eQTL data from cohorts of primarily European and African continental ancestries. We recapitulate evidence demonstrating shared genetic architectures for eQTL and blood traits between the two ancestry groups and observe that gene-level effects correlate 20% more strongly across ancestries compared with SNP-level effects. We perform fine-mapping using MA-FOCUS and find evidence that genes at TWAS risk regions are more likely to be shared across ancestries rather than ancestry-specific. Using multiple lines of evidence to validate our findings, we find gene sets produced by MA-FOCUS are more enriched in hematopoietic categories compared to alternative approaches (P = 1.73 x 10-16). Our work demonstrates that including, and appropriately accounting for, genetic diversity can drive deeper insights into the genetic architecture of complex traits.

sentences: Flagellar dynamics reveal fluctuations and kinetic limit in the Escherichia coli chemotaxis network Biochemical signaling networks allow living cells to adapt to a changing environment, but these networks must cope with unavoidable number fluctuations ("noise") in their molecular constituents. Escherichia coli chemotaxis, by which bacteria modulate their random run/tumble swimming pattern to navigate their environment, is a paradigm for the role of noise in cell signaling. The key signaling protein, CheY, when activated by (reversible) phosphorylation, causes a switch in the rotational direction of the flagellar motors propelling the cell, leading to tumbling. CheY-P concentration, [CheY-P], is thus a measure of the chemotaxis networks output, and temporal fluctuations in [CheY-P] provide a proxy for network noise. Here we quantify the fluctuations in [CheY-P] from the switching dynamics of individual flagella, observed using time-resolved fluorescence microscopy of optically trapped E. coli cells. This approach reveals [CheY-P] fluctuations at steady state, which may play a critical role in driving flagellar switching and cell tumbling. A stochastic theoretical model, inspired by work on gene expression noise, points to CheY activation occurring in bursts, driving the [CheY-P] fluctuations. When the network is stimulated chemically to higher activity, we observe a dramatic decrease in [CheY-P] fluctuations. Our stochastic model shows that an intrinsic kinetic ceiling on network activity places an upper limit on [CheY-P], which when encountered suppresses its fluctuations. This limit may also prevent cells from tumbling unproductively in steep gradients.

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A Simple Parametric Representation of the Hodgkin-Huxley Model The Hodgkin-Huxley model, decades after its first presentation, is still a reference model in neuroscience as it has successfully reproduced the electrophysiological activity of many organisms. The primary signal in the model represents the membrane potential of a neuron. A parametric and simple representation of this signal is presented in this paper.

The new proposal is an adapted Frequency Modulated Mobius multicomponent model defined as a flexible decomposition in waves that describe the signal morphology. A specific feature of the new model is that the parameters are subject to interpretable restrictions.

A broad simulation experiment is conducted to show the new model accurately represents the simulated Hodgkin-Huxley signal. Moreover, the model potential to predict the neurons relevant characteristics, described with parameters of the Hodgkin Huxley model, is shown using different Machine Learning methods. The proposed model is also validated with real data from Squid Giant Axons. The comparison of the parameter configuration between the simulated and real data demonstrated the flexibility of the model as well as interesting differences.

Author summaryAlejandro Rodriguez-Collado. I received the double degree in Statistics and Computer Engineering and the Masters degree in Business Intelligence and Big Data from the Universidad de Valladolid in 2019 and 2020, respectively. I work as researcher and Professor for the Department of Statistics and Operational Research at the Universidad de Valladolid. My main research interests include oscillatory signal processing, neuroscience, multivariate data analysis and supervised learning.

Cristina Rueda. I received the BS degree in mathematics from the Universidad de Valladolid in 1987 and the PhD degree in statistical science from the Universidad de Valladolid in 1989. I am currently Professor in the Department of Statistics and Operational Research at the Universidad de Valladolid. My main research interests include statistical inference methods under restrictions, circular data, computational biology, and statistical methods for signal analysis.

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LuxT controls specific quorum-sensing-regulated behaviors in Vibrionaceae spp. via repression of qrr1, encoding a small regulatory RNA Quorum sensing (QS) is a process of chemical communication bacteria use to transition between individual and collective behaviors. QS depends on the production, release, and synchronous response to signaling molecules called autoinducers (AIs). The marine bacterium Vibrio harveyi monitors AIs using a signal transduction pathway that relies on five small regulatory RNAs (called Qrr1-5) that post-transcriptionally control target genes. Curiously, the small RNAs function redundantly making it difficult to understand the necessity for five of them. Here, we identify LuxT as a transcriptional repressor of qrr1. LuxT does not regulate qrr2-5, demonstrating that qrr genes can be independently controlled to drive unique downstream QS gene expression patterns. LuxT reinforces its control over the same genes it regulates indirectly via repression of qrr1, through a second transcriptional control mechanism. Genes dually regulated by LuxT specify public goods including an aerolysin-type pore-forming toxin. Phylogenetic analyses reveal that LuxT is conserved among Vibrionaceae and sequence comparisons predict that LuxT represses qrr1 in additional species. The present findings reveal that redundant QS regulatory RNAs can carry out both shared and unique functions to endow bacteria with plasticity in their output behaviors.

Author SummaryBacteria communicate and count their cell numbers using a process called quorum sensing (QS). In response to changes in cell density, QS bacteria alternate between acting as individuals and participating in collective behaviors. Vibrio harveyi is used as a model organism to understand QS-mediated communication. Five small RNAs lie at the heart of the V. harveyi QS system, and they regulate the target genes that underlie the QS response. The small RNAs function redundantly making it difficult to understand why V. harveyi requires five of them. Here, we discover a regulator, called LuxT, that exclusively represses the gene encoding one of the QS small RNAs. LuxT regulation of one QS small RNA enables unique control of a specific subset of QS target genes. LuxT is broadly conserved among Vibrionaceae. Our findings show how redundant regulatory components can possess both common and unique roles that provide bacteria with plasticity in their behaviors.

sentences: SUMO monoclonal antibodies vary in sensitivity, specificity, and ability to detect SUMO conjugate types. Monoclonal antibodies (MAb) to members of the Small Ubiquitin-like modifier (SUMO) family are essential tools in the study of cellular SUMOylation. However, many reagents are poorly validated, and the choice of which antibody to use for which detection format is without an evidence base. Here we test twenty-four anti-SUMO monoclonal antibodies for sensitivity and specificity to SUMO1-4 in monomeric and polymeric states in dot-blots, immunoblots, immunofluorescence and immunoprecipitation. We find substantial variability between SUMO MAbs for different conjugation states, detection of increased SUMOylation in response to thirteen different stress agents and as enrichment reagents for analysis of SUMOylated RanGAP1 or KAP1. All four anti-SUMO4 monoclonal antibodies tested cross-reacted with SUMO2/3, and several SUMO2/3 monoclonal antibodies cross-reacted with SUMO4. ~10% of tested monoclonal antibodies produced specific results across multiple applications.

sentences: SARS-CoV2 envelop proteins reshape the serological responses of COVID-19 patients The SARS-CoV-2 pandemic has elicited a unique international mobilization of the scientific community to better understand this coronavirus and its associated disease and to develop efficient tools to combat infection. Similar to other coronavirae, SARS-CoV-2 hijacks the host cell complex secretory machinery to produce properly folded viral proteins that will compose the nascent virions; including Spike, Envelope and Membrane proteins, the most exposed membrane viral proteins to the host immune system. Antibody response is part of the anti-viral immune arsenal that infected patients develop to fight viral particles in the body. Herein, we investigate the immunogenic potential of Spike (S), Envelope (E) and Membrane (M) proteins using a human cell-based system to mimic membrane insertion and N-glycosylation. We show that both S and M proteins elicit the production of specific IgG, IgM and IgA in SARS-CoV-2 infected patients. Elevated Ig responses were observed in COVID+ patients with moderate and severe forms of the disease. Finally, when SARS-CoV-2 Spike D614 and G614 variants were compared, reduced Ig binding was observed with the Spike G614 variant. Altogether, this study underlines the needs for including topological features in envelop proteins to better characterize the serological status of COVID+ patients, points towards an unexpected immune response against the M protein and shows that our assay could represent a powerful tool to test humoral responses against actively evolving SARS-CoV-2 variants and vaccine effectiveness.

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Conserved FimK truncation coincides with increased expression of type 3 fimbriae and cultured bladder epithelial cell association in Klebsiella quasipneumoniae Klebsiella spp. commonly cause both uncomplicated urinary tract infection (UTI) and recurrent UTI (rUTI). Klebsiella quasipneumoniae, a relatively newly defined species of Klebsiella, has been shown to be metabolically distinct from Klebsiella pneumoniae, but its urovirulence mechanisms have not been defined. K. pneumoniae uses both type 1 and type 3 fimbriae to attach to host epithelial cells. The type 1 fimbrial operon is well-conserved between Escherichia coli and K. pneumoniae with the exception of fimK, which is unique to Klebsiella spp. FimK contains an N-terminal DNA binding domain and a C-terminal phosphodiesterase (PDE) domain that has been hypothesized to cross-regulate type 3 fimbriae expression via modulation of cellular levels of cyclic di-GMP. Here, we find that a conserved premature stop codon in K. quasipneumoniae fimK results in truncation of the C-terminal PDE domain and that the bladder epithelial cell association and invasion of uropathogenic K. quasipneumoniae strain KqPF9 is dependent on type 3 but not type 1 fimbriae. Further, we show that basal expression of both type 1 and type 3 fimbrial operons as well as bladder epithelial cell association is elevated in KqPF9 relative to uropathogenic K. pneumoniae TOP52 and that complementation of KqPF9{Delta}fimK with the TOP52 fimK allele reduced type 3 fimbrial expression and bladder epithelial cell attachment. Taken together these data suggest that the C-terminal PDE of FimK can modulate type 3 fimbrial expression in K. pneumoniae and its absence in K. quasipneumoniae may lead to a loss of type 3 fimbrial cross-regulation.

ImportanceK. quasipneumoniae is often indicated as the cause of many opportunistic infections including urinary tract infection (UTI), which affects >50% of women worldwide. However, virulence mechanisms of K. quasipneumoniae remain uninvestigated. Prior to this work, K. quasipneumoniae and K. pneumoniae had only been distinguished phenotypically by metabolic differences. This work contributes to the understanding of K. quasipneumoniae virulence phenotypes by evaluating the contribution of type 1 and type 3 fimbriae, which are critical colonization factors encoded by all Klebsiella spp., to K. quasipneumoniae bladder epithelial cell attachment. We identify clear phenotypic differences in both bladder epithelial cell attachment between uropathogenic K. pneumoniae and K. quasipneumoniae. Importantly, we find that a structural difference in the fimbrial regulatory gene fimK may contribute to differential co-regulation of type 1 and type 3 fimbriae between the two Klebsiella species.

sentences: EOMES is responsible for WNT memory and can substitute for WNT in mesendoderm specification Embryogenesis is guided by a limited set of signaling pathways dynamically expressed in different places. How a context dependent signaling response is generated has been a central question of developmental biology, which can now be addressed with in vitro models of human embryos that are derived from embryonic stem cells (hESCs) called gastruloids. Our previous work demonstrated that during early self-organization of gastruloids, cells chronicle signaling hierarchy. Only cells that have been exposed (primed) by WNT signaling can respond to subsequent Activin exposure and differentiate to mesendodermal (ME) fates. Here, we show that WNT priming does not alter SMAD2 binding nor its chromatin opening, but rather, acts by inducing the expression of the SMAD2 co-factor, EOMES. Expression of EOMES is sufficient to replace WNT upstream of Activin-mediated ME differentiation, thus unveiling the mechanistic basis for priming and cellular memory in early development.

sentences: Cytotoxic T-cell-based vaccine against SARS-CoV2: a hybrid immunoinformatic approach This paper presents an alternative vaccination platform that provides long-term cellular immune protection mediated by cytotoxic T-cells. The immune response via cellular immunity creates superior resistance to viral mutations, which are currently the greatest threat to the global vaccination campaign. Furthermore, we also propose a safer, more facile and physiologically appropriate immunization method using either intra-nasal or oral administration. The underlying technology is an adaptation of synthetic long peptides (SLPs) previously used in cancer immunotherapy. SLPs comprising HLA class I and class II epitopes are used to stimulate antigen cross-presentation and canonical class II presentation by dendritic cells. The result is a cytotoxic T cell-mediated prompt and specific immune response against the virus-infected epithelia and a rapid and robust virus clearance. Peptides isolated from COVID-19 convalescent patients were screened for the best HLA population coverage and were tested for toxicity and allergenicity. 3D peptide folding followed by molecular docking studies provided positive results, suggesting a favourable antigen presentation.

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Tissue-wide Effects Overrule Cell-intrinsic Gene Function in Cortical Projection Neuron Migration The mammalian neocortex is composed of diverse neuronal and glial cell classes that broadly arrange in six distinct laminae. Cortical layers emerge during development and defects in the developmental programs that orchestrate cortical lamination are associated with neurodevelopmental diseases. The developmental principle of cortical layer formation is based on concerted radial projection neuron migration, from their birthplace to their final target position. Radial migration occurs in defined sequential steps that are regulated by a large array of signaling pathways. However, based on genetic loss-of-function experiments, most studies have thus far focused on the role of cell-autonomous gene function. Yet, cortical neuron migration in situ is a complex process and migrating neurons traverse along diverse cellular compartments and environments. The role of tissue-wide properties and genetic state in radial neuron migration is however not well understood. Here, we utilized Mosaic Analysis with Double Markers (MADM) technology to either sparsely or globally delete gene function followed by quantitative single cell phenotyping. The MADM-based gene ablation paradigms in combination with computational modeling demonstrated that global tissue-wide effects predominate cell-autonomous gene function albeit in a gene-specific manner. Our results thus suggest that the genetic landscape in a tissue critically impacts the overall migration phenotype of individual cortical projection neurons. In a broader context our findings imply that global tissue-wide effects represent an essential component of the underlying etiology associated with focal malformations of cortical development (FMCD) in particular, and neurological diseases in general.

LAY SUMMARYThe assembly of the mammalian brain is a complex process and depends on tightly regulated developmental and genetic programs. The coordinated process of nerve cell (neuron) migration is essential to home neurons into their correct position which is vital for normal brain development. Disturbance of the neuron migration process at any point of development leads to severe brain malformations which result in devastating disease. To date, studies have mainly focused on cell-intrinsic gene functions controlling neuronal migration. Therefore, very little is known about the possible contribution of the cellular surrounding and tissue-wide effects. The scale and nature of such global tissue-wide effects remain completely unclear. We thus established genetic platforms based on Mosaic Analysis with Double Markers (MADM) to visualize and quantify tissue-wide effects in a defined genetic context and at single cell resolution. We found a critical predominant role of the genetic landscape and state of cellular environment affecting overall neuron migration properties. In a broader context our results suggest that global tissue-wide effects are a major component of the underlying etiology of neurological diseases such as focal malformations of cortical development (FMCD).

sentences: Phase separated ribosome nascent chain complexes paused in translation are capable to continue expression of proteins playing role in genotoxic stress response upon DNA damage EDTA- and RNase-resistant ribonucleoprotein complexes of arrested ribosomes with protruding nascent polypeptide chains have recently been described in yeast and human cells. These complexes have been termed assemblysomes, a type of soluble condensates distinct from other known granules. Here, we use bioinformatics to identify additional proteins that likely form assemblysomes during translation. We characterize soluble condensates of the DNA helicase Sgs1, one such identified protein and a key player in the repair of DNA double-strand breaks in yeast. We show that paused ribosome-associated nascent chains of Sgs1 in condensates are able to resume translation upon UV irradiation, consistent with the return of mRNA to the ribosome pool. By extending our studies to human cell lines, we found that EDTA-resistant pellets of ribosomes from the human prostate cancer cell line DU145 are sensitive to treatment with 1,6-hexanediol, which is known to dissolve liquid-liquid phase-separated condensates. In addition, transmission electron microscopy shows that 1,6-hexanediol dissolves ring ribosomal structures from the cytoplasm of radioresistant A549 cells while making the cells more sensitive to X-rays. These results suggest that the stress response is based on a conserved mechanism involving the regulated return of phase-separated paused ribosome-nascent chain complexes to translating ribosomes.

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Pregnancy and weaning regulate human maternal liver size and function BACKGROUNDDuring pregnancy, the rodent liver undergoes hepatocyte proliferation and increases in size, followed by weaning-induced involution via hepatocyte cell death and stromal remodeling, creating a pro-metastatic niche. These data suggest a mechanism for increased liver metastasis in postpartum breast cancer patients.

OBJECTIVESInvestigate if the human liver changes in size and function during pregnancy and weaning.

METHODSAbdominal imaging was obtained in healthy women at early and late pregnancy, and post-wean. During pregnancy time points, endogenous glucose production was measured and fasting blood taken to measure bile acids.

RESULTSIndependent of weight gain, most womens livers increased in size with pregnancy, returning to baseline post-wean. Putative roles for bile acids in liver growth were observed in pregnant women and rodents.

CONCLUSIONSThe human liver is regulated by reproductive state with growth during pregnancy and volume loss post-wean. These findings may have broad implications for sex-specific liver diseases and cancer.

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A single oral immunization with a replication-competent adenovirus-vectored vaccine protects mice from influenza respiratory infection The development of effective and flexible vaccine platforms is a major public health challenge as recently highlighted by the COVID-19 pandemic. Adenoviruses (AdVs) are easy to produce and have a good safety and efficacy profile when administered orally as demonstrated by the long-term use of oral AdV 4 and 7 vaccines in the US military. These viruses therefore appear to be the ideal backbone for the development of oral replicative vector vaccines. However, research on these vaccines is limited by the ineffective replication of human AdVs in laboratory animals. The use of mouse AdV type 1 (MAV-1) in its natural host allows infection to be studied under replicative conditions. Here, we orally vaccinated mice with MAV-1 vectors expressing the full length or the "headless" hemagglutinin (HA) of influenza to assess the protection conferred against an intranasal challenge of influenza. We showed that while the headless HA vector did not generate a significant humoral or cellular immune response to influenza, a single oral immunisation with the full-length HA vaccine generated influenza-specific and neutralizing antibodies and completely protected the mice against clinical signs and viral replication.

ImportanceGiven the constant threat of pandemics and the need for annual vaccination against influenza and possibly emerging agents such as SARS-CoV-2, new types of vaccines that are easier to produce and administer and therefore more widely accepted are a critical public health need. Here, using a relevant animal model, we have shown that replicative oral AdV vaccine vectors can help make vaccination against major respiratory diseases more available, better accepted and therefore more effective. These results could be of major importance in the coming years in the fight against emerging diseases such as COVID-19.

sentences: Optimization of the precursor supply for an enhanced FK-506 production in Streptomyces tsukubaensis Tacrolimus (FK-506) is a macrolide widely used as immunosuppressant to prevent transplant rejection. Synthetic production of FK-506 is not efficient and costly, whereas the biosynthesis of FK-506 is complex and the level produced by the wild type strain, Streptomyces tsukubaensis, is very low. We therefore engineered FK-506 biosynthesis and the supply of the precursor L-lysine to generate strains with improved FK-506 yield. To increase FK-506 production, first the intracellular supply of the essential precursor lysine was improved in the native host S. tsukubaensis by engineering the lysine biosynthetic pathway. Therefore, a feedback deregulated aspartate kinase AskSt* of S. tsukubaensis was generated by site directed mutagenesis. Whereas overexpression of AskSt* resulted only in a 17% increase in FK-506 yield, heterologous overexpression of a feedback deregulated AskCg* from Corynebacterium glutamicum was proven to be more efficient. Combined overexpression of AskCg* and DapASt, showed a strong enhancement of the intracellular lysine pool following increase in the yield by approximately 73% compared to the wild type. Lysine is coverted into the FK-506 building block pipecolate by the lysine cyclodeaminase FkbL. Construction of a {Delta}fkbL mutant led to a complete abolishment of the FK-506 production, confirming the indispensability of this enzyme for FK-506 production. Chemical complementation of the {Delta}fkbL mutant by feeding pipecolic acid and genetic complementation with fkbL as well as with other lysine cyclodeaminase genes (pipAf, pipASt, originating from Actinoplanes friuliensis and Streptomyces pristinaespiralis, respectively) completely restored FK-506 production. Subsequently, FK-506 production was enchanced by heterologous overexpression of PipAf and PipASp in S. tsukubaensis. This resulted in a yield increase by 65% compared to the WT in the presence of PipAf from A. friuliensis. For further rational yield improvement, the crystal structure of PipAf from A. friuliensis was determined at 1.3 [A] resolution with the cofactor NADH bound and at 1.4 [A] with its substrate lysine. Based on the structure the Ile91 residue was replaced by Val91 in PipAf, which resulted in an overall increase of FK-506 production by approx. 100% compared to the WT.

sentences: ICOR: Improving codon optimization with recurrent neural networks In protein sequences--as there are 61 sense codons but only 20 standard amino acids--most amino acids are encoded by more than one codon. Although such synonymous codons do not alter the encoded amino acid sequence, their selection can dramatically affect the expression of the resulting protein. Codon optimization of synthetic DNA sequences is important for heterologous expression. However, existing solutions are primarily based on choosing high-frequency codons only, neglecting the important effects of rare codons. In this paper, we propose a novel recurrent-neural-network based codon optimization tool, ICOR, that aims to learn codon usage bias on a genomic dataset of Escherichia coli. We compile a dataset of over 7,000 non-redundant, high-expression, robust genes which are used for deep learning. The model uses a bidirectional long short-term memory-based architecture, allowing for the sequential context of codon usage in genes to be learned. Our tool can predict synonymous codons for synthetic genes toward optimal expression in Escherichia coli. We demonstrate that sequential context achieved via RNN may yield codon selection that is more similar to the host genome, therefore improving protein expression more than frequency-based approaches. ICOR is evaluated on 1,481 Escherichia coli genes as well as a benchmark set of 40 select DNA sequences whose heterologous expression has been previously characterized. ICORs performance across five metrics is compared to that of five different codon optimization techniques. The codon adaptation index -- a metric indicative of high real-world expression -- was utilized as the primary benchmark in this study. ICOR is shown to improve the codon adaptation index by 41.69% and 17.25% compared to the original and Genscripts GenSmart-optimized sequences, respectively. Our tool is provided as an open-source software package that includes the benchmark set of sequences used in this study.

sentences: Clipper: p-value-free FDR control on high-throughput data from two conditions High-throughput biological data analysis commonly involves identifying features such as genes, genomic regions, and proteins, whose values differ between two conditions, from numerous features measured simultaneously. The most widely-used criterion to ensure the analysis reliability is the false discovery rate (FDR), which is primarily controlled based on p-values. However, obtaining valid p-values relies on either reasonable assumptions of data distribution or large numbers of replicates under both conditions. Clipper is a general statistical framework for FDR control without relying on p-values or specific data distributions. Clipper outperforms existing methods for a broad range of applications in high-throughput data analysis.

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Biceps femoris long head sarcomere and fascicle length adaptations after three weeks of eccentric exercise training PurposeEccentric exercise is widely used to increase muscle fascicle lengths and thus decrease the risk of muscle strain injuries. However, the mechanisms behind this protection are still unknown. The aim of this study was to determine whether Biceps femoris long head (BFlh) fascicle length increases in response to three weeks of eccentric exercise training are the result of addition of in-series sarcomeres within muscle fibres.

MethodsTen recreationally active participants (age: 27 {+/-} 3 years, mass: 70 {+/-} 14 kg, height: 174 {+/-} 9 cm) completed three weeks of Nordic hamstring exercise (NHE) training. We collected in vivo sarcomere and muscle fascicle images of the BFlh in two regions (central and distal), utilising microendoscopy and 3D ultrasonography. These images allowed us to estimate sarcomere length, sarcomere number and fascicle lengths before and after the training intervention.

ResultsEccentric knee flexion strength increased after the training (15%, P < 0.001, {eta}p2= 0.75). Further, we found a significant increase in fascicle (21%, P < 0.001, {eta}p2 = 0.81) and sarcomere (17%, P < 0.001, {eta}p2 = 0.9) lengths in the distal but not in the central portion of the muscle. The estimated number of in series sarcomeres did not change in either region.

ConclusionFascicle length adaptations appear to be heterogeneous in the BFlf in response to three weeks of NHE training. An increase in sarcomere length, rather than the addition of sarcomeres in series, appears to be underlying this adaptation. The mechanism driving regional increases in fascicle and sarcomere length remain unknown, but we speculate it may be driven by regional changes in the passive tension of muscle or connective tissue adaptations.

sentences: Comparative Analysis of non-coding small-RNAs in Pseudomonas aeruginosa Keratitis Strains with Different Antibiotic Susceptibility Pseudomonas aeruginosa, is a gram-negative bacterium causes opportunistic or nosocomial infections in immunocompromised individuals. In recent years, a steady increase in human corneal infections of P. aeruginosa has been reported with increased multi-drug resistance (MDR) or extensively drug resistance (XDR). Several non-coding sRNAs, has been identified to regulate various physiological processes in P. aeruginosa, including biofilm formation, quorum sensing. However, the regulatory mechanism of sRNAs in MDR/XDR pathways of P. aeruginosa keratitis strains is not yet studied. In this study, we identified bacterial sRNAs in publicly available P. aeruginosa keratitis genomes and investigated their regulatory role in MDR/XDR pathways using bioinformatic analysis. Totally, 46 P. aeruginosa keratitis strains from different geographical regions were included. Of 46, Eight (30%) out of Twenty-seven and Nine (52) out of Nineteen P. aeruginosa strains from India and Australia were identified as not-MDR. Whereas, 10 (38%) Indian and 9 (47%) Australian strains were identified as MDR. Eight Indian strains were identified as XDR. Out of 46 strains, 23 (50%) carried ExoU, 21(45%) carried ExoS and two (5%) strains carried both ExoU and ExoS, exotoxins for their virulence. The sRNA, SPA0021 was identified in 18 MDR/XDR and 6 not-MDR strains along with UCBPP-PA14. Interestingly, majority of the imipenem resistant P. aeruginosa keratitis strains from the present study was found to be carried SPA0023 sRNA (18 out of 30 strains). The outer membrane porin protein OprD, identified as binding target of SPA0023. Negative regulation or inactivation of OprD, reported in increased imipenem resistance in P. aeruginosa. Mutation analysis revealed that SPA0023 carrying P. aeruginosa keratitis strains contains a lesser number of amino acid changes in OprD protein than other strains. These findings indicate, imipenem resistance in SPA0023 carried strains might arose from the negative regulation or inhibition of OprD by SPA0023. However, functional studies are warranted with large number of P. aeruginosa keratitis strains to confirm the negative regulation of OprD by SPA0023 and imipenem resistance.

sentences: Steroid-resistant nephrotic syndrome associated MYO1E mutations have differential effects on myosin 1e localization, dynamics, and activity Myo1e is a non-muscle motor protein enriched in the podocyte foot processes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Here, we set out to differentiate between the pathogenic and neutral MYO1E variants identified in SRNS patients by exome sequencing. Based on protein sequence conservation and structural predictions, two mutations in the motor domain, T119I and D388H, were selected for this study. EGFP-tagged Myo1e constructs were delivered into the Myo1e-KO podocytes via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculoviral expression system and used to measure Myo1e ATPase and motor activity in vitro. Both mutants were expressed as full-length proteins in the Myo1e-KO podocytes. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs) in podocytes. In contrast, the D388H variant localization was similar to the WT. Surprisingly, the dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting that this mutation also affects Myo1e activity. The ATPase activity and the ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting the findings from cell-based experiments. The experimental pipeline developed in this study allowed us to determine that the T119I and D388H mutations appear to be pathogenic and gain additional knowledge in the Myo1e role in podocytes. This workflow can be applied to the future characterization of novel MYO1E variants associated with SRNS.

sentences: Small and equipped: the rich repertoire of antibiotic resistance genes in Candidate Phyla Radiation genomes Microbes belonging to Candidate Phyla Radiation (CPR) have joined the tree of life as a new unique branch, thanks to the intensive application of metagenomics and advances of sequencing technologies. Despite their ultra-small size, reduced genome and metabolic pathways which mainly depend on symbiotic/exo-parasitic relationship with their bacterial host, CPR microbes are abundant and ubiquitous in almost all environments and are consequently survivors in highly competitive circumstances within microbial communities. They have been eventually identified by 16S rRNA analysis and represent more than 26% of microbial diversity. CPR microbes were able to survive in this context, although their defence mechanisms and phenotypic characteristic remain, however, poorly explored. Here, we conducted a thorough in-silico analysis on 4,062 CPR genomes to test whether these ultrasmall microorganisms might encode for antibiotic resistance (AR)-like enzymes. We used an adapted AR screening criteria with an exhaustive consensus database and complementary steps conferring their resistance functions. We conclude by reporting the surprising discovery of rich reservoir of divergent AR-like genes (n= 30,545 HITs, mean=7.5 HITs/genome [0-41] encoding for 89 AR enzymes, distributed across the 13 CPR phyla, and associated with 14 different chemical classes of antimicrobials. However, most HITs found (93.6%) were linked to glycopeptide, beta-lactams, macrolide-lincosamide-streptogramin, tetracycline and aminoglycoside resistance. Moreover, a distinct AR profile was discerned between the microgenomates group and Candidatus Parcubacteria, and between each of them and other CPR phyla. CPR cells seem to be active players during microbial competitive interactions and are well-equipped for the microbial combat in different habitats, supporting their natural survival/persistence and continued existence.

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Molecular Basis for the Interaction of Catalase with D-Penicillamine : Rationalization of some of its Deleterious Effects D-penicillamine (D-Pen) is a sulfur compound used in the management of rheumatoid arthritis, Wilsons disease (WD), and alcohol dependence. Many side effects are associated with its use, particularly after long-term treatment. However, the molecular bases for such side effects are poorly understood. Based on the well-known oxidase activity of hemoproteins, and the participation of catalase in cellular H2O2 redox signaling, we posit that D-Pen could inactivate catalase, thus disturbing H2O2 levels. Herein, we report on the molecular bases that could partly explain the side effects associated with this drug compound, and we demonstrate that it induces the formation of compound II, a temporarily inactive state of the enzyme, through two distinct mechanisms. Initially, D-Pen reacts with native catalase and/or iron metal ions, used to mimic non heme iron overload observed in long-term treated WD patients, to generate thiyl radicals. These partake into a futile redox cycling, thus producing superoxide radical anions O2*- and hydrogen peroxide H2O2. Then, either H2O2 unexpectedly reacts with native CAT-Fe(II) to produce compound II, or both aforementioned reactive oxygen species intervene into compound II generation through compound I formation then reduction. These findings support evidence that D-Pen could perturb H2O2 redox homeostasis through transient but recurring catalase inactivation, which may in part rationalize some deleterious effects observed with this therapeutic agent, as discussed.

Graphical abstract

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sentences: Sibling rivalry among the ZBTB transcription factor family: homo vs. heterodimers The BTB domain is an oligomerization domain found in over 200 proteins encoded in the human genome. In the family of BTB domain and Zinc Finger-containing (ZBTB) transcription factors, 49 members share the same protein architecture. The N-terminal BTB domain is structurally conserved among the family members and serves as the dimerization site while the C-terminal zinc finger motifs mediate DNA binding. The available BTB domain structures from this family reveal a natural inclination for homodimerization. In this study we investigated the potential for heterodimer formation in the cellular environment. We selected five BTB homodimers and four heterodimer structures. We performed in vitro binding assays with fluorescent protein-BTB domain fusions to assess dimer formation. We tested the binding of several BTB pairs, and we were able to confirm the heterodimeric physical interaction between the BTB domains of PATZ1 and PATZ2, previously reported only in an interactome mapping experiment. We also found this pair to be co-expressed in several immune system cell types. Finally, we used the available structures of BTB domain dimers and newly constructed models in extended molecular dynamics simulations (500 ns) to understand the energetic determinants of homo and heterodimer formation. We conclude that heterodimer formation, although frequently described as less preferred than homodimers, is a possible mechanism to increase the combinatorial specificity of this transcription factor family.

sentences: Perivascular cells support folliculogenesis in the developing ovary Granulosa cells, supporting cells of the ovary, are essential for ovarian differentiation by providing a nurturing environment for oogenesis. Sufficient numbers of granulosa cells are vital for establishment of follicles and the oocyte reserve; therefore, identifying the cellular source from which granulosa cells are derived is critical for understanding basic ovarian biology. One cell type that has received little attention in this field is the perivascular cell. Here we use lineage tracing and organ culture techniques in mice to identify ovarian Nestin+ perivascular cells as multipotent progenitors that contribute to granulosa, thecal, and pericyte lineages. Maintenance of these progenitors was dependent on vascular-mesenchymal Notch signaling. Ablation of postnatal Nestin+ cells resulted in a disruption of granulosa cell specification and an increased incidence of polyovular ovarian follicles, thus uncovering key roles for vasculature in ovarian differentiation. These findings may provide new insights into the origins of female gonad dysgenesis and infertility.

sentences: Learning attention-controllable border-ownership for objectness inference and binding Human visual systems can parse a scene composed of novel objects and infer their surfaces and occlusion relationships without relying on object-specific shapes or textures. Perceptual grouping can bind together spatially disjoint entities to unite them as one object even when the object is entirely novel, and bind other perceptual properties like color and texture to that object using object-based attention. Border-ownership assignment, the assignment of perceived occlusion boundaries to specific perceived surfaces, is an intermediate representation in the mammalian visual system that facilitates this perceptual grouping. Since objects in a scene can be entirely novel, inferring border ownership requires integrating global figural information, while dynamically postulating what the figure is, a chicken-and egg process that is complicated further by missing or conflicting local evidence regarding the presence of boundaries. Based on neuroscience observations, we introduce a model - the cloned Markov random field (CMRF)- that can learn attention-controllable representations for border-ownership. Higher-order contour representations that distinguish border-ownerships emerge as part of learning in this model. When tested with a cluttered scene of novel 2D objects with noisy contour-only evidence, the CMRF model is able to perceptually group them, despite clutter and missing edges. Moreover, the CMRF is able to use occlusion cues to bind disconnected surface elements of novel objects into coherent objects, and able to use top-down attention to assign border ownership to overlapping objects. Our work is a step towards dynamic binding of surface elements into objects, a capability that is crucial for intelligent agents to interact with the world and to form entity-based abstractions.

sentences: Neurogenesis-mediated forgetting of complex paired-associates memories The hippocampus is a critical structure involved in many forms of learning and memory. It is also one of the only regions in the adult mammalian brain that continues to generate new neurons throughout adulthood. This process of adult neurogenesis may increase the plasticity of the hippocampus which could be beneficial for learning but has also been demonstrated to decrease the stability of previously acquired memories. Here we test whether increased production of new neurons following the formation of a gradually acquired paired-associates task will result in forgetting of this type of memory. We trained mice in a touchscreen-based object/location task and then increased neurogenesis using voluntary exercise. Our results indicate that mice with increased neurogenesis show poor recall of the previously established memory. When subsequently exposed to a reversal task we also show that mice with increased neurogenesis require fewer correction trials to acquire the new task contingencies. This suggests that prior forgetting reduces perseveration on the now outdated memory. Together our results add to a growing body of literature which indicates the important role of adult neurogenesis in destabilizing previously acquired memories to allow for flexible encoding of new memories.

sentences: Rapidly Increasing SARS-CoV-2 Neutralization by Intravenous Immunoglobulins Produced from Plasma Collected During the 2020 Pandemic Immunoglobulin (IG) lots (N=176) released since March 2020 were tested for SARS-CoV-2 neutralizing antibodies, with first positive results for September 2020 lots, mean = 1.8 IU/ml, 46% of lots positive. From there, values steadily increased, in correlation with the cumulative COVID-19 incidence, to reach a mean of 36.7 IU/ml and 93% of lots positive by January 2021. Extrapolating the correlation, IGs could reach an anti-SARS-CoV-2 potency of ~400 IU/ml by July 2021. At that stage, prophylactic IG treatment for primary/secondary immunodeficiency could contain similar doses of anti-SARS-CoV-2 as convalescent plasma which is used for treatment of COVID-19.

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Photoinduced isomerization sampling of retinal in bacteriorhodopsin Photoisomerization of retinoids inside a confined protein pocket represents a critical chemical event in many important biological processes from animal vision, non-visual light effects, to bacterial light sensing and harvesting. Light driven proton pumping in bacteriorhodopsin entails exquisite electronic and conformational reconfigurations during its photocycle. However, it has been a major challenge to delineate transient molecular events preceding and following the photoisomerization of the retinal from noisy electron density maps when varying populations of intermediates coexist and evolve as a function of time. Here I report several distinct early photoproducts deconvoluted from the recently observed mixtures in time-resolved serial crystallography. This deconvolution substantially improves the quality of the electron density maps hence demonstrates that the all-trans retinal undergoes extensive isomerization sampling before it proceeds to the productive 13-cis configuration. Upon light absorption, the chromophore attempts to perform trans-to-cis isomerization at every double bond coupled with the stalled anti-to-syn rotations at multiple single bonds along its polyene chain. Such isomerization sampling pushes all seven transmembrane helices to bend outward, resulting in a transient expansion of the retinal binding pocket, and later, a contraction due to recoiling. These ultrafast responses observed at the atomic resolution support that the productive photoreaction in bacteriorhodopsin is initiated by light-induced charge separation in the prosthetic chromophore yet governed by stereoselectivity of its protein pocket. The method of a numerical resolution of concurrent events from mixed observations is also generally applicable.

Significance StatementPhotoisomerization of retinal is a critical rearrangement reaction in many important biological processes from animal vision, non-visual light effects, to bacterial light sensing and harvesting. It has been a major challenge to visualize rapid molecular events preceding and following photoisomerization so that many protein functions depending on such reaction remain vaguely understood. Here I report a direct observation of the stereoselectivity of bacteriorhodopsin hence delineate the structural mechanism of isomerization. Upon a light-induced charge separation, the retinal in a straight conformation attempts to perform double bond isomerization and single bond rotation everywhere along its polyene chain before it proceeds to the productive configuration. This observation improves our understanding on how a non-specific attraction force could drive a specific isomerization.

sentences: Electrophysiological signatures of hierarchical learning Human perception and learning is thought to rely on a hierarchical generative model that is continuously updated via precision-weighted prediction errors (pwPEs). However, the neural basis of such cognitive process and how it unfolds during decision making, remain poorly understood. To investigate this question, we combined a hierarchical Bayesian model (i.e., Hierarchical Gaussian Filter, HGF) with electrophysiological (EEG) recording, while participants performed a probabilistic reversal learning task in alternatingly stable and volatile environments. Behaviorally, the HGF fitted significantly better than two control, non-hierarchical, models. Neurally, low-level and high-level pwPEs were independently encoded by the P300 component. Low-level pwPEs were reflected in the theta (4-8 Hz) frequency band, but high-level pwPEs were not. Furthermore, the expressions of high-level pwPEs were stronger for participants with better HGF fit. These results indicate that the brain employs hierarchical learning, and encodes both low- and high-level learning signals separately and adaptively.

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Functional disruption of Transferrin expression alters reproductive physiologyin Anopheles culicifacies Iron metabolism is crucial to maintain optimal physiological homeostasis of every organism and any alteration of the iron concentration (i.e. deficit or excess) can have adverse consequences. Transferrins are glycoproteins that play important role in iron transportation and have been widely characterized in vertebrates, and insects, but poorly studied in blood-feeding mosquitoes. Here, we characterized a 2102 bp long transcript AcTrf1a encoding putative transferrin homolog protein from mosquito An. culicifacies. A detailed in silico analysis predicts AcTrf1a (ACUA023913-RA) encodes 624 amino acid (aa) long polypeptide that carries transferrin domain. AcTrf1a also showed a putative N-linked glycosylation site, a characteristic feature of most of the mammalian transferrins and certain non-blood feeding insects. Structure modelling prediction confers the presence of an iron binding site at the N-terminal lobe of the transferrin. Our spatial and temporal expression analysis under altered pathophysiological conditions showed that AcTrf1a abundantly express in the fat-body, ovary, and its response is significantly altered (enhanced) after blood meal uptake, and exogenous bacterial challenge. Additionally, a non-heme iron supplementation of FeCl3 at 1 mM concentration not only augmented the AcTrf1a transcript expression in fat-body, also enhanced the reproductive fecundity of gravid adult female mosquitoes. RNAi mediated knockdown of AcTrf1a causes a significant reduction in the egg laying/fecundity, confirmed important role of transferrin in oocyte maturation. Further detailed characterization may help to select this transcript as a unique target to impair the mosquito reproductive outcome.

HighlightsO_LIInsect transferrins are mostly glycoprotein of about 60-80 kDa molecular weight, involved in myriad physiological events and serve as a major iron transport protein.
C_LIO_LIHere, we identified and characterized a 2102 bp long transcript encoding putative transferrin homolog of 624 aa long peptide, carrying only one fully functional transferrin domain at N-terminal from An. culicifacies.
C_LIO_LISpatial and temporal expression analysis of AcTrf1a highlights an enriched expression in fat-body and ovary during vitellogenesis.
C_LIO_LIIron supplementation and dsRNA mediated knockdown experiments together confer that AcTrf1a may have key role in the iron homeostasis regulation during oogenesis, and egg maturation in the gravid female mosquitoes.
C_LI

Graphical abstract

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Mosquito acquires iron either from blood meal or iron supplementation in sugar meal. Fat-body derived transferrin proceed towards the gut surface, load iron in its N-terminal iron-binding pocket and deliver iron to ovary. This blood meal iron is required by adult female for completion of gonotrophic cycle. (a) limited iron availability in sugar meal does not support the ovary development and hence no oogenesis; (b) when sugar meal is replaced by blood meal upregulation of transferrin protein results in rapid iron transport to various organs including ovary results in healthy ovarian growth; (c) RNAi mediated knockdown of this transporter protein transferrin in fat-body followed by blood meal, may cause reduced iron transport to ovary and consequently declines in oocyte load.

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An Intrinsically Disordered Peptide Tag that Confers an Unusual Solubility to Aggregation-Prone Proteins There is a high demand for the production of recombinant proteins in Escherichia coli for biotechnological applications but their production is still limited by their insolubility. Fusion tags have been successfully used to enhance the solubility of aggregation-prone proteins; however, smaller and more powerful tags are desired for increasing the yield and quality of target proteins. Herein, NEXT tag, a 53 amino acid-length solubility enhancer, is described. The NEXT tag showed outstanding ability to improve both in vivo and in vitro solubilities with minimal effect on passenger proteins. The C-terminal region of the tag was mostly responsible for in vitro solubility, while the N-terminal region was essential for in vivo soluble expression. The NEXT tag appeared to be intrinsically disordered and seemed to exclude neighboring molecules and prevent protein aggregation by acting as an entropic bristle. This novel peptide tag should have general use as a fusion partner to increase the yield and quality of difficult-to-express proteins.

IMPORTANCEProduction of recombinant protein in Escherichia coli still suffers from the insolubility problem. Conventional solubility enhancers with large sizes represented by maltose-binding protein (MBP) have remained as the first-choice tags, however, the success in the soluble expression of tagged protein is largely unpredictable. In addition, the large tags can negatively affect the function of target proteins. In this work, NEXT tag, an intrinsically disordered peptide, was introduced as a small but powerful alternative to MBP. The NEXT tag could significantly improve both expression level and solubility of target proteins including a thermostable carbonic anhydrase and a polyethylene terephthalate (PET)-degrading enzyme that are remarkable enzymes for environmental bioremediation.

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Toolkit for Oscillatory Real-time Tracking and Estimation (TORTE) BackgroundClosing the loop between brain activity and behavior is one of the most active areas of development in neuroscience. There is particular interest in developing closed-loop control of neural oscillations. Many studies report correlations between oscillations and functional processes. Oscillation-informed closed-loop experiments might determine whether these relationships are causal and would provide important mechanistic insights which may lead to new therapeutic tools. These closed-loop perturbations require accurate estimates of oscillatory phase and amplitude, which are challenging to compute in real time.

New MethodWe developed an easy to implement, fast and accurate Toolkit for Oscillatory Real-time Tracking and Estimation (TORTE). TORTE operates with the open-source Open Ephys GUI (OEGUI) system, making it immediately compatible with a wide range of acquisition systems and experimental preparations.

ResultsTORTE efficiently extracts oscillatory phase and amplitude from a target signal and includes a variety of options to trigger closed-loop perturbations. Implementing these tools into existing experiments is easy and adds minimal latency to existing protocols.

Comparison with Existing MethodsMost labs use in-house lab-specific approaches, limiting replication and extension of their experiments by other groups. Accuracy of the extracted analytic signal and accuracy of oscillation-informed perturbations with TORTE match presented results by these groups. However, TORTE provides access to these tools in a flexible, easy to use toolkit without requiring proprietary software.

ConclusionWe hope that that the availability of a high-quality, open-source, and broadly applicable toolkit will increase the number of labs able to perform oscillatory closed-loop experiments, and will improve the replicability of protocols and data across labs.

HighlightsO_LITORTE provides a toolkit to investigate closed loop oscillation-informed experiments.
C_LIO_LIThe toolkit is versatile and open-source promoting replicability across scientists.
C_LIO_LIThe analytic signal algorithm within TORTE preforms equally to existing algorithms.
C_LI

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Zika virus induces mitotic catastrophe in human neural progenitors by triggering unscheduled mitotic entry in the presence of DNA damage while functionally depleting nuclear PNKP Vertical transmission of Zika virus (ZIKV) leads with high frequency to congenital ZIKV syndrome (CZS), whose worst outcome is microcephaly. However, the mechanisms of congenital ZIKV neurodevelopmental pathologies, including direct cytotoxicity to neural progenitor cells (NPC), placental insufficiency, and immune responses, remain incompletely understood. At the cellular level, microcephaly typically results from death or insufficient proliferation of NPC or cortical neurons. NPCs replicate fast, requiring efficient DNA damage responses to ensure genome stability. Like congenital ZIKV infection, mutations in the polynucleotide 5-kinase 3-phosphatase (PNKP) gene, which encodes a critical DNA damage repair enzyme, results in recessive syndromes often characterized by congenital microcephaly with seizures (MCSZ). We thus tested whether there were any links between ZIKV and PNKP.

Here we show that two PNKP phosphatase inhibitors or PNKP knockout inhibited ZIKV replication. PNKP relocalized from the nucleus to the cytoplasm in infected cells, co-localizing with the marker of ZIKV replication factories (RF) NS1 and resulting in functional nuclear PNKP depletion. Although infected NPC accumulated DNA damage, they failed to activate the DNA damage checkpoint kinases Chk1 and Chk2. ZIKV also induced activation of cytoplasmic CycA/CDK1 complexes, which trigger unscheduled mitotic entry. Inhibition of CDK1 activity inhibited ZIKV replication and the formation of RF, supporting a role of cytoplasmic CycA/CDK1 in RF morphogenesis. In brief, ZIKV infection induces mitotic catastrophe resulting from unscheduled mitotic entry in the presence of DNA damage. PNKP and CycA/CDK1 are thus host factors participating in ZIKV replication in NPC, and pathogenesis to neural progenitor cells.

SignificanceThe 2015-2017 Zika virus (ZIKV) outbreak in Brazil and subsequent international epidemic revealed the strong association between ZIKV infection and congenital malformations, mostly neurodevelopmental defects up to microcephaly. The scale and global expansion of the epidemic, the new ZIKV outbreaks (Kerala state, India, 2021), and the potential burden of future ones pose a serious ongoing risk. However, the cellular and molecular mechanisms resulting in microcephaly remain incompletely understood. Here we show that ZIKV infection of neuronal progenitor cells results in cytoplasmic sequestration of an essential DNA repair protein itself associated with microcephaly, with the consequent accumulation of DNA damage, together with an unscheduled activation of cytoplasmic CDK1/Cyclin A complexes in the presence of DNA damage. These alterations result in mitotic catastrophe of neuronal progenitors, which would lead to a depletion of cortical neurons during development.

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MutaFrame - an interpretative visualization framework for deleteriousness prediction of missense variants in the human exome MotivationHigh-throughput experiments are generating ever increasing amounts of various -omics data, so shedding new light on the link between human disorders, their genetic causes, and the related impact on protein behavior and structure. While numerous bioinformatics tools now exist that predict which variants in the human exome cause diseases, few tools predict the reasons why they might do so. Yet, understanding the impact of variants at the molecular level is a prerequisite for the rational development of targeted drugs or personalized therapies.

ResultsWe present the updated MutaFrame webserver, which aims to meet this need. It offers two deleteriousness prediction softwares, DEOGEN2 and SNPMuSiC, and is designed for bioinformaticians and medical researchers who want to gain insights into the origins of monogenic diseases. It contains information at two levels for each human protein: its amino acid sequence and its 3-dimensional structure; we used the experimental structures whenever available, and modeled structures otherwise. MutaFrame also includes higher-level information, such as protein essentiality and protein-protein interactions. It has a user-friendly interface for the interpretation of results and a convenient visualization system for protein structures, in which the variant positions introduced by the user and other structural information are shown. In this way, MutaFrame aids our understanding of the pathogenic processes caused by single-site mutations and their molecular and contextual interpretation.

AvailabilityMutaframe webserver at http://mutaframe.com

sentences: Local activation of focal adhesion kinase orchestrates the positioning of presynaptic scaffold proteins and Ca2+ channel function to control glucose dependent insulin secretion. A developing understanding suggests that spatial compartmentalisation of components the glucose stimulus-secretion pathway in pancreatic {beta} cells are critical in controlling insulin secretion. To investigate the mechanisms, we have developed live-cell sub-cellular imaging methods using the organotypic pancreatic slice. We demonstrate that the organotypic pancreatic slice, when compared with isolated islets, preserves intact {beta} cell structure, and enhances glucose dependent Ca2+ responses and insulin secretion. Using the slice technique, we have discovered the essential role of local activation of integrins and the downstream component, focal adhesion kinase, in regulating {beta} cells. Integrins and focal adhesion kinase are exclusively activated at the {beta} cell capillary interface and using in situ and in vitro models we show their activation both positions presynaptic scaffold proteins, like ELKS and liprin, and regulates glucose dependent Ca2+ responses and insulin secretion. We conclude that focal adhesion kinase orchestrates the final steps of glucose dependent insulin secretion within the restricted domain where {beta} cells contact the islet capillaries.

sentences: CbpD crystal structure adds intrigue to substrate-specificity motifs in chitin-active lytic polysaccharide monooxygenases Pseudomonas aeruginosa secretes diverse proteins via its Type 2 Secretion System, including a 39 KDa Chitin-Binding Protein, CbpD. CbpD was recently shown to be a lytic polysaccharide monooxygenase active on chitin, and to contribute substantially to virulence. To-date no structure of this virulence factor has been reported. Its first two domains are homologous to those found in the crystal structure of Vibrio cholerae GbpA, while the third domain is homologous to the NMR structure of the Cellvibrio japonicus CjLPMO10A CBM73 domain. We report the 3.0 [A] resolution crystal structure of CbpD solved by molecular replacement, which required ab initio models of each CbpD domain generated by the artificial intelligence deep learning structure prediction algorithm RoseTTAFold. The structure of CbpD confirms previously postulated chitin-specific motifs in the AA10 domain while challenging the deterministic effects of other postulated substrate specificity motifs. Additionally, the structure of CbpD shows that post translational modifications occur on the chitin binding surface. Moreover, the structure raises interesting possibilities about how Type 2 Secretion System substrates may interact with the secretion machinery and demonstrates the utility of new artificial intelligence protein structure prediction algorithms in making challenging structural targets tractable.

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Paired Associates Learning is Disrupted After Unilateral Parietal Lobe Controlled Cortical Impact in Rats: A Trial-by-Trial Behavioral Analysis Approximately 60-70 million people suffer from a traumatic brain injury (TBI) each year. As animal models continue to be paramount in understanding and treating cognitive impairment following TBI, the necessity of testing intervention strategies in clinically relevant settings cannot be ignored. This study used a unilateral parietal lobe controlled cortical impact (CCI) model of TBI and tested rats on a touchscreen-based associative learning task, Paired Associates Learning (PAL). In humans, PAL has been used to assess cognitive deficits in stimulus-location association in a multitude of disease states, including TBI. To date, the extent to which a rat model of TBI produces deficits in PAL has not yet been reported, although the usage of PAL will be important for understanding the clinical consequences of cognitive impairment post-injury and throughout intervention treatment. This study details the behavioral and histological consequences of the CCI injury model and closes a translational research gap between basic and clinical TBI research.

HIGHLIGHTSO_LIPAL performance declines in a rat model of TBI.
C_LIO_LIResponse-driven bias in PAL becomes elevated after TBI.
C_LIO_LIInflammatory microglial response in the thalamus correlates with PAL deficit.
C_LI

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Protein prediction for trait mapping in diverse populations Genetically regulated gene expression has helped elucidate the biological mechanisms underlying complex traits. Improved high-throughput technology allows similar interrogation of the genetically regulated proteome for understanding complex trait mechanisms. Here, we used the Trans-omics for Precision Medicine (TOPMed) Multi-omics pilot study, which comprises data from Multi-Ethnic Study of Atherosclerosis (MESA), to optimize genetic predictors of the plasma proteome for genetically regulated proteome-wide association studies (PWAS) in diverse populations. We built predictive models for protein abundances using data collected in TOPMed MESA, for which we have measured 1,305 proteins by a SOMAscan assay. We compared predictive models built via elastic net regression to models integrating posterior inclusion probabilities estimated by fine-mapping SNPs prior to elastic net. In order to investigate the transferability of predictive models across ancestries, we built protein prediction models in all four of the TOPMed MESA populations, African American (n=183), Chinese (n=71), European (n=416), and Hispanic/Latino (n=301), as well as in all populations combined. As expected, fine-mapping produced more significant protein prediction models, especially in African ancestries populations, potentially increasing opportunity for discovery. When we tested our TOPMed MESA models in the independent European INTERVAL study, fine-mapping improved cross-ancestries prediction for some proteins. Using GWAS summary statistics from the Population Architecture using Genomics and Epidemiology (PAGE) study, which comprises ~50,000 Hispanic/Latinos, African Americans, Asians, Native Hawaiians, and Native Americans, we applied S-PrediXcan to perform PWAS for 28 complex traits. The most protein-trait associations were discovered, colocalized, and replicated in large independent GWAS using proteome prediction model training populations with similar ancestries to PAGE. At current training population sample sizes, performance between baseline and fine-mapped protein prediction models in PWAS was similar, highlighting the utility of elastic net. Our predictive models in diverse populations are publicly available for use in proteome mapping methods at https://doi.org/10.5281/zenodo.4837328.

Author summaryGene regulation is a critical mechanism underlying complex traits. Transcriptome-wide association studies (TWAS) have helped elucidate potential mechanisms because each association connects a gene rather than a variant to the complex trait. Like genome-wide association studies (GWAS), most TWAS are still conducted exclusively in populations of European ancestry, which misses the opportunity to test the full spectrum of human genetic variation for associations with complex traits. Here, move beyond the transcriptome and because protein measurement assays are growing to allow interrogation of the proteome, we use data from TOPMed MESA to develop genetic predictors of protein abundance in diverse ancestry populations. We compare model-building strategies with the goal of providing the best resource for protein association discovery with available data. We demonstrate how these prediction models can be used to perform proteome-wide association studies (PWAS) in diverse populations. We show the most protein-trait associations were discovered, colocalized, and replicated in independent cohorts using proteome prediction model training populations with similar ancestries to individuals in the GWAS. We shared our protein prediction models and performance statistics publicly to facilitate future proteome mapping studies in diverse populations.

sentences: Lipid-mediated Association of the Slg1 Transmembrane Domains in Yeast Plasma Membranes Clustering of transmembrane proteins underlies a multitude of fundamental biological processes at the plasma membrane (PM) such as receptor activation, lateral domain formation and mechanotransduction. The self-association of the respective transmembrane domains (TMD) has also been suggested to be responsible for the micron-scaled patterns seen for integral membrane proteins in the budding yeast plasma membrane. However, the underlying interplay between local lipid composition and TMD identity is still not mechanistically understood. In this work we combined coarse-grained molecular dynamics (MD) simulations of simplified bilayer systems with high resolution live-cell microscopy to analyze the distribution of a representative helical yeast TMD from the PM sensor Slg1 within different lipid environments. In our simulations we specifically evaluated the effects of acyl chain saturation and anionic lipids head groups on the association of two TMDs. We found that weak lipid-protein interactions significantly affect the configuration of TMD dimers and the free energy of association. Increased amounts of unsaturated phospholipids strongly reduced helix-helix interaction, while the presence of anionic phosphatidylserine (PS) hardly affected dimer formation. We could experimentally confirm this surprising lack of effect of PS using the network factor, a mesoscopic measure of PM pattern formation in yeast cells. Simulations also showed that formation of TMD dimers in turn increased the order parameter of the surrounding lipids and induced long-range perturbations in lipid organization. In summary, our results shed new light on the mechanisms for lipid-mediated dimerization of TMDs in complex lipid mixtures.

sentences: In search of a core cellular network with single cell transcriptome data in the fruit fly Along with specialized functions, cells of multicellular organisms also perform essential functions. Whether diverse cells do this by using the same set of genes, interacting in a fixed coordinated fashion, remains a central question in biology. Single-cell RNA-sequencing (scRNA-seq) measures gene expression of individual cells, enabling researchers to discover gene expression patterns that contribute to the diversity of cell functions. Current efforts focus primarily on identifying differentially expressed genes. However, patterns of co-expression between genes are more indicative of biological processes than are the expression of individual genes. Here, we constructed cell type-specific gene co-expression networks using the fly brain transcriptome atlas to search for a core cellular network. We detected a set of highly coordinated genes preserved across cell types and defined this set as the core network. This network was further validated through two additional datasets profiling fly brains or heads. This core has a small size and shows dense connectivity. Modules within this core are enriched for basic cellular functions and gene members of these modules have distinct evolutionary signatures. Overall, we demonstrated that a core cellular network exists in diverse cell types of fly brains and this core exhibits unique topological, structural, functional and evolutionary properties.

sentences: The record of Deinotheriidae from the Miocene of the Swiss Jura Mountains (Jura Canton, Switzerland) The Miocene sands of the Swiss Jura Mountains, long exploited in quarries for the construction industry, have yielded abundant fossil remains of large mammals. Among Deinotheriidae (Proboscidea), two species, Prodeinotherium bavaricum and Deinotherium giganteum, had previously been identified in the Delemont valley, but never described. A third species, Deinotherium levius, from the locality of Charmoille in Ajoie, is reported herein for the first time in Switzerland. These occurrences are dated from the middle to the late Miocene, correlating to the European Mammal biozones MN5 to MN9. The study is completed by a discussion on the palaeobiogeography of deinotheres at the European scale.

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The C-terminal domain of Hsp70 is responsible for paralog-specific regulation of ribonucleotide reductase The Hsp70 family of molecular chaperones is well-conserved and expressed in all organisms. In budding yeast, cells express four highly similar cytosolic Hsp70s Ssa1, 2, 3 and 4 which arose from gene duplication. Ssa1 and 2 are constitutively expressed while Ssa3 and 4 are induced upon heat shock. Recent evidence suggests that despite their amino acid similarity, these Ssas have unique roles in the cell. Here we examine the relative importance of Ssa1-4 in the regulation of the enzyme ribonucleotide reductase (RNR). We demonstrate that cells expressing either Ssa3 or Ssa4 as their sole Ssa are compromised for their resistance to DNA damaging agents and activation of DDR transcription. In addition, we show that the steady state levels and stability of RNR small subunits Rnr2 and Rnr4 are reduced in Ssa3 or Ssa4-expressing cells, a result of decreased Ssa-RNR interaction. Interaction between the Hsp70 co-chaperone Ydj1 and RNR is correspondingly decreased in cells only expressing Ssa3 and 4. Through studies of Ssa2/4 domain swap chimeras, we determined that the C-terminal domain of Ssas are the source of this functional specificity. Taking together, our work suggests a distinct role for Ssa paralogs in regulating DNA replication mediated by C-terminus sequence variation.

Author SummaryCells require molecular chaperones to fold proteins into their active conformation. A major mystery however is why cells express so many highly-related and apparently redundant Hsp70 paralogs. We examined the role of four Hsp70 paralogs in budding yeast (Ssa1, 2, 3 and 4) on the activity of the ribonucleotide reductase (RNR complex). Importantly, we demonstrate there is selectivity of RNR subunits for Ssa1 and Ssa2 subunits, which is dictated by the co-chaperone Ydj1. Taken together, our work provides new insight into the functional specificity of Hsp70 paralogs using a native client protein.

sentences: Collateral Responses to Classical Cytotoxic Chemotherapies are Heterogeneous and Sensitivities are Sparse Chemotherapy resistance is a major obstacle to curing cancer patients. Combination drug regimens have shown promise as a method to overcome resistance; however, to date only some cancers have been cured with this method. Collateral sensitivity - the phenomenon whereby resistance to one drug is co-occurrent with sensitivity to a second drug - has been gaining traction as a promising new concept to guide rational design of combination regimens. Here we survey collateral responses to acquisition of resistance to four classical chemotherapy agents. Although collateral sensitivities have been documented for antibiotics and targeted cancer therapies, we did not observe collateral sensitivities to any of the cytotoxic agents we studied. Interestingly, we did observe heterogeneity in the phenotypic response to acquisition of resistance to each drug, suggesting the existence of multiple different states of resistance for each drug. Surprisingly, this phenotypic heterogeneity was unrelated to transcriptomic heterogeneity in the resistant cell lines. These features of phenotypic and transcriptomic heterogeneity must be taken into account in future studies of treated tumor subclones and in design of chemotherapy combinations.

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A subset of gut leukocytes have telomerase-dependent 'hyper-long' telomeres and require telomerase for function in zebrafish Telomerase, the enzyme capable of elongating telomeres, is usually restricted in human somatic cells, which contributes to progressive telomere shortening with cell-division and ageing. Using the zebrafish model, we show that subsets gut immune cells have telomerase-dependent "hyper-long" telomeres, which we identified as being predominantly macrophages and dendritics (mpeg1.1+ and cd45+mhcII+). Notably, mpeg+ macrophages have much longer telomeres in the gut than in their haematopoietic tissue of origin, suggesting that there is a gut-specific modulation of telomerase in these cells.

Moreover, we show that a subset of gut mpeg+ cells express telomerase (tert) in young WT zebrafish, but that the relative proportion of these cells decreases with ageing. Importantly, this is accompanied by telomere shortening and DNA damage responses with ageing and a telomerase-dependent decrease in expression of autophagy and immune activation markers. Finally, these telomerase-dependent molecular alterations are accompanied by impaired phagocytosis of E.coli and increased gut permeability in vivo. Together, our data show that limiting levels of telomerase lead to changes in gut immunity and gut permeability, which, together, are likely contributors to local and systemic tissue degeneration and increased susceptibility to infection with ageing.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/478480v1_ufig1.gif" ALT="Figure 1">
View larger version (21K):
org.highwire.dtl.DTLVardef@141a6caorg.highwire.dtl.DTLVardef@12d24baorg.highwire.dtl.DTLVardef@c64267org.highwire.dtl.DTLVardef@319449_HPS_FORMAT_FIGEXP  M_FIG C_FIG Our data show that limiting levels of telomerase contribute to alterations in gut immunity, namely increased apoptosis, decreased autophagy and immune activation of mpeg+ macrophages in the gut. This is accompanied by a decreased ability to clear pathogens and increased gut permeability. Together, these are likely contributors to local and systemic tissue degeneration and susceptibility to infection with ageing.

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Using Deep Learning to Predict Human Decisions and Cognitive Models to Explain Deep Learning Models Deep neural networks (DNN) models have the potential to provide new insights in the study of human decision making, due to their high capacity and data-driven design. While these models may be able to go beyond theory-driven models in predicting human behaviour, their opaque nature limits their ability to explain how an operation is carried out. This explainability problem remains unresolved. Here we demonstrate the use of a DNN model as an exploratory tool to identify predictable and consistent human behaviour in value-based decision making beyond the scope of theory-driven models. We then propose using theory-driven models to characterise the operation of the DNN model. We trained a DNN model to predict human decisions in a four-armed bandit task. We found that this model was more accurate than a reinforcement-learning reward-oriented model geared towards choosing the most rewarding option. This disparity in accuracy was more pronounced during times when the expected reward from all options was similar, i.e., no unambiguous good option. To investigate this disparity, we introduced a reward-oblivious model, which was trained to predict human decisions without information about the rewards obtained from each option. This model captured decision-sequence patterns made by participants (e.g., a-b-c-d). In a series of experimental offline simulations of all models we found that the general model was in line with a reward-oriented models predictions when one option was clearly better than the others.

However, when options expected rewards were similar to each other, it was in-line with the reward-oblivious models pattern completion predictions. These results indicate the contribution of predictable but task-irrelevant decision patterns to human decisions, especially when task-relevant choices are not immediately apparent. Importantly, we demonstrate how theory-driven cognitive models can be used to characterise the operation of DNNs, making them a useful explanatory tool in scientific investigation.

Author SummaryDeep neural networks (DNN) models are an extremely useful tool across multiple domains, and specifically for performing tasks that mimic and predict human behaviour. However, due to their opaque nature and high level of complexity, their ability to explain human behaviour is limited. Here we used DNN models to uncover hitherto overlooked aspects of human decision making, i.e., their reliance on predictable patterns for exploration. For this purpose, we trained a DNN model to predict human choices in a decision-making task. We then characterised this data-driven model using explicit, theory-driven cognitive models, in a set of offline experimental simulations. This relationship between explicit and data-driven approaches, where high-capacity models are used to explore beyond the scope of established models and theory-driven models are used to explain and characterise these new grounds, make DNN models a powerful scientific tool.

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Aerosol Formation During Processing of Potentially Infectious Samples on Roche Immunochemistry Analyzers (cobas e analyzers) and in an End-to-End Laboratory Workflow to Model SARS-CoV-2 Infection Risk for Laboratory Operators BackgroundThis study assessed formation of potentially infectious aerosols during processing of infectious samples in a real-world laboratory setting, which could then be applied in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

MethodsThis two-part study assessed aerosol formation when using cobas e analyzers only and in an end-to-end laboratory workflow. To estimate aerosol formation, recombinant hepatitis B surface antigen (HBsAg) was used as a surrogate marker for infectious virus particles to evaluate the potential risk of SARS-CoV-2 infection to laboratory operators. Using the HBsAg model, air sampling was performed at different positions around the cobas e analyzers and in four scenarios reflecting critical handling and/or transport locations in an end-to-end laboratory workflow. Aerosol formation of HBsAg was quantified using the Elecsys(R) HBsAg II quant II assay. The model was then applied to a SARS-CoV-2 context using SARS-CoV-2 infection-specific parameters to calculate viral RNA copies.

ResultsFollowing application to SARS-CoV-2, the mean HBsAg uptake per hour when recalculated into viral RNA copies was 1.9 viral RNA copies across the cobas e analyzers and 0.87 viral RNA copies across all tested scenarios in an end-to-end laboratory workflow. This corresponds to a maximum aspiration rate of <16 viral RNA copies during an 8-hour shift when using cobas e analyzers and/or in an end-to-end laboratory workflow.

ConclusionsThe low production of marker-containing aerosol when using cobas e analyzers and in an end-to-end laboratory workflow is consistent with a remote risk of laboratory-acquired SARS-CoV-2 infection for laboratory operators.

SummaryThis study investigated the formation of potentially infectious aerosols during processing of infectious samples in a model using hepatitis B surface antigen (HBsAg) as a marker for infectious virus particles. The risk to laboratory operators of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was then inferred. Air sampling was performed around cobas e analyzers and in an end-to-end laboratory workflow, after which HBsAg was quantified and applied to SARS-CoV-2 using SARS-CoV-2 infection-specific parameters. The maximum aspiration rate of <16 viral RNA copies/8-hour shift, when applied to a SARS-CoV-2 context, poses a remote risk of SARS-CoV-2 infection to laboratory operators.

sentences: Does the rainbow trout ovarian fluid guide the spermatozoon on its way to the egg? Fertilization of freshwater fish occurs in an environment that may negatively affect the gametes; therefore, the specific mechanisms triggering the encounters of gametes would be highly expedient. The egg and ovarian fluid (OF) are likely the primary sources of these triggers in fish that we confirmed here for rainbow trout Oncorhynchus mykiss. The ovarian fluid significantly affected the spermatozoa performance: it supported high velocity for a more extended period and changed the motility pattern from tumbling in water to straightforward moving in the ovarian fluid. Rainbow trout OF induced a trapping chemotaxis-like effect on activated male gametes, and this effect depended on the properties of the activating media. The interaction of the spermatozoa with the attracting agents was accompanied by their "turn-and-run" behavior involving asymmetric flagellar beating and Ca2+ concentration bursts in the bent flagella segment, characteristic of the chemotactic response. Collectively, the ovarian fluid creates the optimal environment for rainbow trout spermatozoa performance, being an effective promoter of fertilization. The individual peculiarities of the egg (ovarian fluid) - sperm interaction in rainbow trout reflect the specific features of the spawning process in this species.

sentences: Biomechanical and biochemical assessment of YB-1 expression in melanoma cells Malignant melanoma is the most lethal form of skin cancer; its incidence has increased over the last five decades. Y-box binding protein 1 (YB-1) plays a prominent role in mediating metastatic behavior by promoting epithelial-to-mesenchymal transition (EMT) processes. Migratory melanoma cells exhibit two major phenotypes: elongated mesenchymal or rounded amoeboid. The actomyosin cytoskeleton is key in both phenotypes, but intermediate filaments also undergo a significant rearrangement process, switching from cytokeratin-rich to vimentin and nestin-rich network. In this study, we aimed to investigate to what extent YB-1 impacts the biomechanical (cell stiffness) and biochemical aspects of melanoma cells and their cytoskeleton. To this end, we subjected A375 YB-1 knock-out and parental cells to atomic force microscopy investigations (stiffness determination), immunolabelling, and proteome analysis. We found that YB-1 expressing cells were significantly stiffer compared to the corresponding YB-1 knock-out cell line. Proteomic analysis revealed that expression of YB-1 results in a strong co-expression of nestin, vimentin, fascin-1, and septin-9. In the YB-1 knock-out nestin was completely depleted, but zyxin was strongly upregulated. Collectively, our results showed that YB-1 knock-out acquires some characteristics of mesenchymal phenotype but lacks important markers of malignancy and invasiveness such as nestin or vimentin. We posit that there is an association of YB-1 expression with an amoeboid phenotype, which would explain the increased migratory capacity.

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Assessing heterogeneity in spatial data using the HTA index with applications to spatial transcriptomics and imaging MotivationTumour heterogeneity is being increasingly recognised as an important characteristic of cancer and as a determinant of prognosis and treatment outcome. Emerging spatial transcriptomics data hold the potential to further our understanding of tumour heterogeneity and its implications. However, existing statistical tools are not sufficiently powerful to capture heterogeneity in the complex setting of spatial molecular biology.

ResultsWe provide a statistical solution, the HeTerogeneity Average index (HTA), specifically designed to handle the multivariate nature of spatial transcriptomics. We prove that HTA has an approximately normal distribution, therefore lending itself to efficient statistical assessment and inference. We first demonstrate that HTA accurately reflects the level of heterogeneity in simulated data. We then use HTA to analyse heterogeneity in two cancer spatial transcriptomics datasets: spatial RNA sequencing by 10x Genomics and spatial transcriptomics inferred from H&E. Finally, we demonstrate that HTA also applies to 3D spatial data using brain MRI. In spatial RNA sequencing we use a known combination of molecular traits to assert that HTA aligns with the expected outcome for this combination. We also show that HTA captures immune-cell infiltration at multiple resolutions. In digital pathology we show how HTA can be used in survival analysis and demonstrate that high levels of heterogeneity may be linked to poor survival. In brain MRI we show that HTA differentiates between normal ageing, Alzheimers disease and two tumours. HTA also extends beyond molecular biology and medical imaging, and can be applied to many domains, including GIS.

Availability<attached; publicly available upon acceptance>.

Contactlevyalona@gmail.com zohar.yakhini@gmail.com

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: Assessment of the suitability of marker fragments DNA sequences of the internal transcribed spacer region (ITS), beta-tubulin gene (TUB) and elongation factor (EF1) genes for diagnostics of Fusarium tricinctum species complex in Russian in grain products By results of laboratory researches from plant samples of grain crops species Fusarium avenaceum, Fusarium tricinctum and Fusarium acuminatum related to Fusarium tricinctum species complex were isolated and identified, based on cultural and morphological characteristics, molecular-genetic analysis of 3 DNA fragments was performed: internal transcribed spacer (ITS) site, beta-tubulin gene site (TUB), elongation factor gene site (EF1) showed insignificant intraspecific and interspecific variability at ITS and EF1 sites, which may negatively affect the reliability of target species identification, TUB site was more effective for distinguishing and identifying the studied species, due to significant intraspecific and interspecific variability

sentences: First report of Lactoria cornuta from the west coast of India, Evidence of the role of tropical cyclone Ockhi India is known for its rich biodiversity and is fortunate to have several endemic species from different classes of vertebrates. India is home to 7.5% of the global fish diversity, with 91 endemic species of ray finned fishes, the actinopterygians. Some fish species have never been reported until recently, one such example is the long-horned Cowfish (Ostracidae), best known for inhabiting only certain areas of the Indo-Pacific oceans. However, this fish has been reported from Bay of Bengal, Costs of Goa and Lakshadweep. This is the first time this fish has been found on the Coasts of Karnataka, west coast of India. Given its morphology, the migration seems highly improbable since it can only endure lethargic swimming. In this article, we have explored the role of other external forces that could have contributed to its range extension in the east and west coast of India. In this paper we are exploring the role of Super Cyclones as a vital force in determining the expansion of its range. The fact of collection longhorn cowfish for the first time from the coast of Karwar coincides with the passage of Cyclone Ockhi on the west coast of India this suggests the possible role of super cyclone in its range extension. A year later, another specimen was recovered from a location very close to the first, which indicates the successful establishment of Lacturia cornuta in its new environment. Related morphometric and meristic studies of our specimens are consistent with previous studies reported in the Bay of Bengal.

sentences: Transcriptome innovations in primates revealed by single-molecule long-read sequencing Transcriptomic diversity greatly contributes to the fundamentals of disease, lineage-specific biology, and environmental adaptation. However, much of the actual isoform repertoire contributing to shaping primate evolution remains unknown. Here, we combined deep long- and short-read sequencing complemented with mass spectrometry proteomics in a panel of lymphoblastoid cell lines (LCLs) from human, three other great apes, and rhesus macaque, producing the largest full-length isoform catalog in primates to date. Our transcriptomes reveal thousands of novel transcripts, some of them under active translation, expanding and completing the repertoire of primate gene models. Our comparative analyses unveil hundreds of transcriptomic innovations and isoform usage changes related to immune function and immunological disorders. The confluence of these innovations with signals of positive selection and their limited impact in the proteome points to changes in alternative splicing in genes involved in immune response as an important target of recent regulatory divergence in primates.

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Repeated birth injuries lead to pelvic floor muscle dysfunction and impairment in regeneration ObjectivesChildbirth is a key risk factor for pelvic floor muscle (PFM) injury and dysfunction, and subsequent pelvic floor disorders (PFDs). Multiparity further exacerbates these risks. Using the pre-clinical rat model of simulated birth injury (SBI), we previously identified that an SBI leads to PFM atrophy and fibrosis. We hypothesized that multiple SBIs further overwhelm PFM regenerative capacity, leading to functionally relevant pathological alterations long-term.

Study DesignRats underwent SBI and were allowed to recover for 8 weeks to undergo another SBI. Animals were sacrificed at acute, subacute, and long-term time points post-second injury (N=3-6/time point), and pubocaudalis (PCa) was harvested to assess ex vivo muscle function, histomorphological properties and gene expression.

ResultsAcutely following the 1st SBI, PCa force was decreased relative to controls. At 4 weeks, PCa force was recovered and remained unchanged at 8 weeks. Similarly, lower PCa force was observed immediately after repeated SBI. In contrast to functional recovery after 1st SBI, PCa force remained lower at 4 weeks post-2nd SBI and continued to be decreased even after 12 weeks after repeated injury. Fiber size was smaller at the long-term time points after 2nd SBI compared to controls and single SBI groups. As opposed to the resolution of centralized nuclei at 8 weeks post-1st SBI, regenerating myofibers persisted even at 12 weeks post-2nd SBI. In contrast to the peak of collagen content at 4 weeks post-1st SBI, this parameter raised progressively over 12 weeks after repeated SBIs. Prolonged inflammatory response, impairment in muscle anabolism, and sustained expression of ECM remodeling genes were observed after repeated SBIs.

ConclusionsRepeated birth injuries delay PFM regeneration and impair function in the pre-clinical rat model.

sentences: IntroSpect: motif-guided immunopeptidome database building tool to improve the sensitivity of HLA binding peptide identification Although database search tools originally developed for shotgun proteome have been widely used in immunopeptidomic mass spectrometry identifications, they have been reported to achieve undesirably low sensitivities and/or high false positive rates as a result of the hugely inflated search space caused by the lack of specific enzymic digestions in immunopeptidome. To overcome such a problem, we have developed a motif-guided immunopeptidome database building tool named IntroSpect, which is designed to first learn the peptide motifs from high confidence hits in the initial search and then build a targeted database for refined search. Evaluated on three representative HLA class I datasets, IntroSpect can improve the sensitivity by an average of 80% comparing to conventional searches with unspecific digestions while maintaining a very high accuracy ([~]96%) as confirmed by synthetic validation experiments. A distinct advantage of IntroSpect is that it does not depend on any external HLA data so that it performs equally well on both well-studied and poorly-studied HLA types, unlike a previously developed method SpectMHC. We have also designed IntroSpect to keep a global FDR that can be conveniently controlled, similar to conventional database search engines. Finally, we demonstrate the practical value of IntroSpect by discovering neoantigens from MS data directly. IntroSpect is freely available at https://github.com/BGI2016/IntroSpect.

sentences: Multifunctional role of GPCR signaling in epithelial tube formation Epithelial tube formation requires Rho1-dependent actomyosin contractility to generate the cellular forces that drive cell shape changes and rearrangement. Rho1 signaling is activated by G protein-coupled receptor (GPCR) signaling at the cell surface. During Drosophila embryonic salivary gland (SG) invagination, the GPCR ligand Folded gastrulation (Fog) activates Rho1 signaling to drive apical constriction. The SG receptor that transduces the Fog signal into Rho1-dependent myosin activation has not been identified. Here, we reveal that the Smog GPCR transduces Fog signal to regulate Rho kinase accumulation and myosin activation in the apicomedial region of cells to control apical constriction during SG invagination. We also report on unexpected Fog-independent roles for Smog in maintaining epithelial integrity and organizing cortical actin. Our data supports a model wherein Smog regulates distinct myosin pools and actin cytoskeleton in a ligand-dependent manner during epithelial tube formation.

sentences: Chitin and cuticle proteins form the cuticular layer in the spinning duct of silk-spinning arthropods Chitin is found in the exoskeleton and peritrophic matrix of arthropods, but recent studies have also identified chitin in the spinning duct of silk-spinning arthropods. Here, we report the presence and function of chitin and cuticle proteins ASSCP1 and ASSCP2 in the spinning duct of silkworms. We show that chitin and these proteins are co-located in the cuticular layer of the spinning duct. Ultrastructural analysis indicates that the cuticular layer has a multilayer structure by layered stacking of the chitin laminae. After knocking down ASSCP1 and ASSCP2, the fine structure of this layer was disrupted, which had negative impacts on the mechanical properties of silk. This work clarifies the function of chitin in the spinning duct of silk-spinning arthropods. Chitin and cuticle proteins are the main components of the hard and rigid cuticular layer, providing the shearing stress during silk fibrillogenesis and regulating the final mechanical properties of silk.

sentences: A community-based approach to image analysis of cells, tissues and tumors Emerging multiplexed imaging platforms provide an unprecedented view of an increasing number of molecular markers at subcellular resolution and the dynamic evolution of tumor cellular composition. As such, they are capable of elucidating cell-to-cell interactions within the tumor microenvironment that impact clinical outcome and therapeutic response. However, the rapid development of these platforms has far outpaced the computational methods for processing and analyzing the data they generate. While being technologically disparate, all imaging assays share many computational requirements for post-collection data processing. We convened a workshop to characterize these shared computational challenges and a follow-up hackathon to implement solutions for a selected subset of them. Here, we delineate these areas that reflect major axes of research within the field, including image registration, segmentation of cells and subcellular structures, and identification of cell types from their morphology. We further describe the logistical organization of these events, believing our lessons learned can aid others in uniting the imaging community around self-identified topics of mutual interest, in designing and implementing operational procedures to address those topics and in mitigating issues inherent in image analysis (e.g., sharing exemplar images of large datasets and disseminating baseline solutions to hackathon challenges through open-source code repositories).

sentences: Light-dependent synthesis of a nucleotide second messenger controls bacterial motility Nucleotide second messengers are universally crucial factors for the signal transduction of various organisms. In prokaryotes, cyclic nucleotide messengers are involved in the bacterial life cycle and function, such as virulence, biofilm formation, and others mainly via gene regulation. Here we show that the swimming motility of a soil bacterium is rapidly modulated by cyclic adenosine monophosphate (cAMP) synthesized upon light exposure. Analysis of a loss-of-photoresponsivity mutant obtained by transposon random mutagenesis determined the novel sensory gene, and its expression in Escherichia coli through codon optimization revealed the light-dependent synthesis of cAMP. GFP labeling showed the localization of the photoresponsive enzyme at the cell poles where flagellar motors reside. The present findings highlight the new role of cAMP that rapidly controls the flagella-dependent bacterial motility and the global distribution of the discovered photoactivated cyclase among diverse microbial species.

sentences: Compared with SARS-CoV2 wild type's spike protein, the SARS-CoV2 omicron's receptor binding motif has adopted a more SARS-CoV1 and/or bat/civet-like structure. Our study focuses on free energy calculations of SARS-CoV2 spike protein receptor binding motives (RBMs) from wild type and variants-of-concern with particular emphasis on currently emerging SARS- CoV2 omicron variants of concern (VOC). Our computational free energy analysis underlines the occurrence of positive selection processes that specify omicron host adaption and bring changes on the molecular level into context with clinically relevant observations. Our free energy calculations studies regarding the interaction of omicrons RBM with human ACE2 shows weaker binding to ACE2 than alphas, deltas, or wild types RBM. Thus, less virus is predicted to be generated in time per infected cell. Our mutant analyses predict with focus on omicron variants a reduced spike-protein binding to ACE2-receptor protein possibly enhancing viral fitness / transmissibility and resulting in a delayed induction of danger signals as trade-off. Finally, more virus is produced but less per cell accompanied with delayed Covid-19 immunogenicity and pathogenicity. Regarding the latter, more virus is assumed to be required to initiate inflammatory immune responses.

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Cation and anion channelrhodopsins: Sequence motifs and taxonomic distribution Cation and anion channelrhodopsins (CCRs and ACRs, respectively) primarily from two algal species, Chlamydomonas reinhardtii and Guillardia theta, have become widely used as optogenetic tools to control cell membrane potential with light. We mined algal and other protist polynucleotide sequencing projects and metagenomic samples to identify 75 channelrhodopsin homologs from three channelrhodopsin families, including one revealed in dinoflagellates in this study. We carried out electrophysiological analysis of 33 natural channelrhodopsin variants from different phylogenetic lineages and 10 metagenomic homologs in search of sequence determinants of ion selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins. Our results show that association of a reduced number of glutamates near the conductance path with anion selectivity depends on a wider protein context, because prasinophyte homologs with the identical glutamate pattern as in cryptophyte ACRs are cation-selective. Desensitization is also broadly context-dependent, as in one branch of stramenopile ACRs and their metagenomic homologs its extent roughly correlates with phylogenetic relationship of their sequences. Regarding spectral tuning, two prasinophyte CCRs exhibit red-shifted spectra to 585 nm, although their retinal-binding pockets do not match those of previously known similarly red-shifted channelrhodopsins. In cryptophyte ACRs we identified three specific residue positions in the retinal-binding pocket that define the wavelength of their spectral maxima. Lastly, we found that dinoflagellate rhodopsins with a TCP motif in the third transmembrane helix and a metagenomic homolog exhibit channel activity.

IMPORTANCEChannelrhodopsins are widely used in neuroscience and cardiology as research tools and are considered as prospective therapeutics, but their natural diversity and mechanisms remain poorly characterized. Genomic and metagenomic sequencing projects are producing an ever-increasing wealth of data, whereas biophysical characterization of the encoded proteins lags behind. In this study we used manual and automated patch clamp recording of representative members of four channelrhodopsin families including a family that we report in this study in dinoflagellates. Our results contribute to a better understanding of molecular determinants of ionic selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins.

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Quantifying the impact of ecological memory on the dynamics of interacting communities Ecological memory refers to the influence of past events on the response of an ecosystem to exogenous or endogenous changes. Memory has been widely recognized as a key contributor to the dynamics of ecosystems and other complex systems, yet quantitative community models often ignore memory and its implications.

Recent modeling studies have shown how interactions between community members can lead to the emergence of resilience and multistability under environmental perturbations. We demonstrate how memory can be introduced in such models using the framework of fractional calculus. We study how the outcomes of a well-characterized interaction model are affected by gradual increases in ecological memory under varying initial conditions, perturbations, and stochasticity.

Our results highlight the implications of memory on several key aspects of community dynamics. In general, memory introduces inertia into the dynamics. This favors species coexistence under perturbation, enhances system resistance to state shifts, mitigates hysteresis, and can affect system resilience both ways depending on the time scale considered. Memory also promotes long transient dynamics, such as long-standing oscillations and delayed regime shifts, and contributes to the emergence and persistence of alternative stable states. Our study highlights the fundamental role of memory on ecological communities, and provides quantitative tools to introduce it in ecological models and analyse its impact under varying conditions.

Author summaryAn ecosystem is said to exhibit ecological memory when its future states do not only depend on its current state but also on its initial state and trajectory. Memory may arise through various mechanisms as organisms adapt to their environment, modify it, and accumulate biotic and abiotic material. It may also emerge from phenotypic heterogeneity at the population level. Despite its commonness in nature, ecological memory and its potential influence on ecosystem dynamics have been so far overlooked in many applied contexts. Here, we use modeling to investigate how memory can influence the dynamics, composition, and stability landscape of ecological communities. We incorporate long-term memory effects into a multi-species model recently introduced to investigate alternative stable states in microbial communities. We assess the impact of memory on key aspects of model behavior and validate our findings using a model parameterized by empirical data from the human gut microbiota. Our approach for modeling memory and studying its implications has the potential to improve our understanding of microbial community dynamics and ultimately our ability to predict, manipulate, and experimentally design microbial ecosystems. It could also be applied more broadly in the study of systems composed of interacting components.

sentences: The context dependent effects of host competence, competition, and the pathogen transmission mode on disease prevalence Biodiversity in communities is changing globally, including the gain and loss of host species in host-pathogen communities. Increased host diversity can cause infection prevalence in a focal host to increase (amplification) or decrease (dilution). However, it is unclear what general rules govern the context dependent effects, in part because theories for pathogens with different transmission modes have developed largely independently. Using a two-host model, we explore how the pathogen transmission mode and characteristics of a second host (disease competence and competitive ability) influence disease prevalence in a focal host. Our work shows how the theories for pathogens with environmental transmission, density-dependent direct transmission, and frequency-dependent direct transmission can be unified. Our work also identifies general rules about how host and pathogen characteristics affect amplification/dilution. For example, higher competence hosts promote amplification, unless they are strong interspecific competitors; strong interspecific competitors promote dilution, unless they are large sources of new infections; and dilution occurs under frequency-dependent direct transmission more than density-dependent direct transmission, unless interspecific host competition is sufficiently strong. Our work helps explain how the characteristics of the pathogen and a second host affect disease prevalence in a focal host.

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3D atlas of the pituitary gland of the model fish medaka In vertebrates, the anterior pituitary plays a crucial role in regulating several essential physiological processes via the secretion of at least seven peptide hormones by different endocrine cell types. Comparative and comprehensive knowledge of the spatial distribution of those endocrine cell types is required to better understand their role during the animal life. Using medaka as the model and several combinations of multi-color fluorescence in situ hybridization, we present the first 3D atlas revealing the gland-wide distribution of seven endocrine cell populations: lactotropes, thyrotropes, Lh and Fsh gonadotropes, somatotropes, and pomca-expressing cells (corticotropes and melanotropes) in the anterior pituitary of a teleost fish. By combining in situ hybridization and immunofluorescence techniques, we deciphered the location of corticotropes and melanotropes within the pomca-expressing cell population. The 3D localization approach reveals sexual dimorphism of tshba- and pomca-expressing cells in the adult medaka pituitary. Finally, we show the existence of bi-hormonal cells co-expressing lhb-fshb, fshb-tshba and lhb-sl using single-cell transcriptomics analysis and in situ hybridization. This study offers a solid basis for future comparative studies of the teleost pituitary and its developmental plasticity.

Highlights- We offer the first 3D atlas of a teleost pituitary, which presents a valuable resource to the endocrinology and model fish community.
- The atlas reveals the 3D spatial distribution of the seven endocrine cell types and blood vessels in the juvenile/adult male and female pituitary.
- Gene expression for tshba and pomca, as well as the population size of cells expressing these genes, displays obvious sexual dimorphism in the adult medaka pituitary.
- Multi-color in situ hybridization and single cell RNA-seq reveal the existence of bi-hormonal cells, co-expressing lhb-fshb, fshb-tshba, lhb-sl, and a few multi-hormonal cells.
- An online version of the atlas is available at https://www.nmbu.no/go/mpg-atlas.

sentences: Combined application of biochar with nitrogen fertilizer  improves soil quality and reduces soil respiration whilst sustaining wheat grain yield in a semiarid environment This study was conducted to investigate the effect of biochar, straw and N fertilizer on soil properties, soil respiration and grain yield of spring wheat (Triticum aestivum L.) in semi-arid Western Loess Plateau of northwestern China. The two carbon sources (straw and biochar) were applied alone or combined with nitrogen fertilizer (urea, 46% nitrogen [N]), whilst the soil without carbon is made up of nitrogen fertilizer applied at 0, 50 and 100 kg N/ha. The experiment was arranged in a randomized complete block design with three replicates and was conducted in 2014, 2015 and 2016 cropping season. Results showed that the greatest grain yields were found with 100 kg N ha-1 fertilization rate under biochar, straw and soils without carbon, but the greatest effect occurred on the biochar amended soils. Biochar amendment produced the greatest grain yield at 1906 kg ha-1, followed by straw treated soils at 1643 kg ha-1, and soils without carbon the lowest at 1553 kg ha-1. This results is supported by the fact that, biochar amended soils (at 0-10 cm) increased soil organic C by 17.14% and 21.65% compared to straw treated soils and soils without carbon respectively. Seasonal soil respirations were between 19.05% and 23.67% lower in BN100 compared with SN50 and CN100. Soil respiration reduced with increasing N fertilization rates under all treatments, but the greatest effect occurred on biochar plots. Biochar amended soils decreased carbon emission by 26.80% and 9.54% compared to straw treated soils and soils without carbon amendment respectively. Increased grain yield and the decreased carbon emission in BN100 translated into greater carbon emission efficiency (2.88 kg kg-1) which was significantly different compared with the other treatments. Combined application of biochar with 100 kg N ha-1 in rainfed spring wheat was a suitable agricultural practice.

sentences: Strong and tunable anti-CRISPR/Cas9 activity of AcrIIA4 in plants This study describes the strong anti-CRISPR activity of the bacterial AcrIIA4 protein in Nicotiana benthamiana, a model plant used as molecular farming platform. The results demonstrate that AcrIIA4 abolishes site-directed mutagenesis in leaves when transiently co-expressed with CRISPR/Cas9. We also show that AcrIIA4 represses CRISPR/dCas9-based transcriptional activation (CRISPRa) of both reporter and endogenous genes in a highly efficient, dose-dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin-regulated activation of a downstream reporter gene. The strong anti-Cas9 activity of AcrIIA4 reported here opens new possibilities for customized control of gene editing and gene expression in plants.

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A specific relationship between musical sophistication and auditory working memory Musical engagement may be associated with better listening skills, such as the perception of and working memory for notes, in addition to the appreciation of musical rules. The nature and extent of this association is controversial. In this study we assessed the relationship between musical engagement and both sound perception and working memory.

We developed a task to measure auditory perception and working memory for sound using a behavioural measure for both, precision. We measured the correlation between these tasks and musical sophistication based on a validated measure (the Goldsmiths Musical Sophistication Index) that can be applied to populations of both musicians and non-musicians. The data show that musical sophistication accounts for 21% of the variance in the precision of working memory for frequency in an analysis that accounts for age and non-verbal intelligence. Musical sophistication was not significantly associated with the precision of working memory for amplitude modulation rate or with the precision of perception of either acoustic feature.

The work supports a specific association between musical sophistication and working memory for sound frequency.

sentences: A ribosomal protein variant that confers macrolide resistance differentially regulates acid resistance, catabolism, and biofilm formation related genes in Escherichia coli Mutational changes in bacterial ribosomes that confer antibiotic resistance decrease cell fitness. Determining the genetic factors that interconnect antibiotic resistance and cell fitness is critical in the fight against bacterial infections. Here, we describe gene expression and phenotypic changes presented in Escherichia coli cells carrying an uL22(K90D) mutant ribosomal protein, which showed growth defects and resistance to macrolide antibiotics. Ribosome profiling analyses revealed reduced expression of operons involved in catabolism, electron transportation, indole production, and lysine-decarboxylase acid resistance. In general, ribosome occupancy was increased at rare codons while translation initiation of proximal genes in several of the affected operons was substantially reduced. Decline of the activity of these genes was accompanied by increased expression of macrolide multidrug efflux pumps, the glutamate-decarboxylase regulon, and the autoinducer-2 metabolic regulon. In concordance with these changes, uL22(K90D) mutant cells grew better in acidic conditions and generated more biofilm in static cultures than their parental strain. Our work provides new insights on how mutations in ribosomal proteins induce the acquisition of macrolide and pH resistance, and increase the ability to generate biofilms.

sentences: Immune function of the serosa in hemimetabolous insect eggs Insects comprise more than a million species and many authors have attempted to explain this success by evolutionary innovations. A much overlooked evolutionary novelty of insects is the serosa, an extraembryonic epithelium around the yolk and embryo. We have shown previously that this epithelium provides innate immune protection to eggs of the beetle Tribolium castaneum. It remained elusive, however, whether this immune competence evolved in the Tribolium lineage or is ancestral to all insects. Here, we expand our studies to two hemimetabolous insects, the bug Oncopeltus fasciatus and the swarming grasshopper Locusta migratoria. For Oncopeltus, RNA sequencing reveals an extensive response upon infection, including the massive upregulation of antimicrobial peptides (AMPs). We demonstrate antimicrobial activity of these peptides using in vitro bacterial growth assays, and describe two novel AMP families called Serosins and Ovicins. For both insects, qPCRs show immune competence of the eggs when the serosa is present, and in situ hybridizations demonstrate that immune gene expression is localized in the serosa. This first evidence from hemimetabolous insect eggs suggests that immune competence is an ancestral property of the serosa. The evolutionary origin of the serosa with its immune function might have facilitated the spectacular radiation of the insects.

sentences: The dynamic interplay of PIP2 and ATP in the regulation of the KATP channel ATP-sensitive potassium (KATP) channels couple the intracellular ATP concentration to insulin secretion. KATP channel activity is inhibited by ATP binding to the Kir6.2 tetramer and activated by phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we use molecular dynamics (MD) simulation, electrophysiology and fluorescence spectroscopy to show that ATP and PIP2 occupy different binding pockets that share a single amino acid residue, K39. When both ligands are present, K39 shows a greater preference to co-ordinate with PIP2 than ATP. A neonatal diabetes mutation at K39 (K39R) increases the number of hydrogen bonds formed between K39 and PIP2, reducing ATP inhibition. We also find direct effects on nucleotide binding from mutating E179, a residue proposed to interact with PIP2. Our work suggests PIP2 and ATP interact allosterically to regulate KATP channel activity.

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Efficient inactivation of African swine fever virus by a highly complexed iodine combined with compound organic acids African swine fever (ASF) is a highly contagious disease with high morbidity and mortality caused by African swine fever virus (ASFV). Cleaning and disinfection remain one of the most effective biosecurity measures to prevent and control the spread of ASFV. In this study, we evaluated the inactivation effects of highly complexed iodine (HPCI) combined with compound organic acids (COAs) against ASFV under different conditions. The results showed that the inactivation rates of the disinfectants on the reporter ASFV increased in dose- and time-dependent manners, the best inactivation effects were obtained when the compatibility ratio of HPCI and COAs was 5:1 at 25{degrees}C. Furthermore, there were no significant differences by comparing the efficacy of HPCI combined with COAs (HPCI+COAs) in inactivating wild-type ASFV and the reporter ASFV (P > 0.05). ASFV of 104.0 TCID50/mL was completely inactivated by 0.13% HPCI (0.0065% effective iodine), 0.06% COAs or 0.13% HPCI+COAs (approximately 0.0054% effective iodine), respectively, while 106.0 TCID50/mL ASFV was completely inactivated by 1.00% HPCI (0.05% effective iodine), 0.50% COAs or 1.00% HPCI+COAs (0.042% effective iodine), respectively. Therefore, HPCI+COAs had synergistic effects to inactivate ASFV. This study demonstrated that HPCI+COAs could rapidly and efficiently inactivate ASFV and represent an effective compound disinfectant for the control of ASF.

IMPORTANCEAfrican swine fever (ASF) is a highly contagious disease with high morbidity and mortality caused by African swine fever virus (ASFV). Due to the lack of commercial vaccines and treatment available for ASF, effective disinfectants and the proper use of them are extremely essential to inactivate ASFV. The significance of this research is in searching for an ideal disinfectant that not only has the advantages of low toxicity and non-pollution but also can inactivate ASFV rapidly and efficiently. In this study, we proved that HPCI+COAs not only exhibited low cytotoxicity, but also could completely inactivate ASFV within 5 min at 4{degrees}C, 25{degrees}C and 37{degrees}C. In addition, HPCI+COAs had synergistic effects on inactivated ASFV. Thus, HPCI +COAs could be used as an effective disinfectant for the control of ASF.

sentences: Variant Polycomb complexes in Drosophila consistent with ancient functional diversity Polycomb group (PcG) mutants were first identified in Drosophila based on their failure to maintain proper Hox gene repression during development. The proteins encoded by the corresponding fly genes mainly assemble into one of two discrete Polycomb Repressive Complexes: PRC1 or PRC2. However, biochemical analyses in mammals have revealed alternative forms of PRC2, and multiple distinct types of non-canonical or variant PRC1. Through a series of proteomic analyses, we identify analogous PRC2 and variant PRC1 complexes in Drosophila, as well as a broader repertoire of interactions implicated in early development. Our data provide strong support for the ancient diversity of PcG complexes, and a framework for future analysis in a longstanding and versatile genetic system.

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Chasing the cardiomyopathy modifier genes: Integrative computational analysis interweave the missing links in cardiomyopathy diseasome BackgroundCardiomyopathies are genetic disorders resulting in an abnormal heart phenotype due to intrinsic cardiac muscle malfunction, which leads to heart failure. These are chronic disorders contributing more than 36% of heart failure in the patients. Cardiomyopathies also show co-morbidity with other diseases that frequently affect the elderly. Apart from this, many drugs used for treating other unrelated diseases cause cardio-toxicity and lead to cardiomyopathies. Unravelling the cross-talk between them is essential for useful characterization, subtyping, and better treatment of the cardiomyopathies.

ResultsIn this work, we applied a systems biology-based analysis to explore the associations between cardiomyopathies and other morbidities. We built a cardiomyopathy disease-disease network based on the combined biological data relevant to cardiomyopathies. We made use of publicly available genetic, non-coding RNA, drug and PPI datasets related to cardiomyopathies and network science tools to identify new modifiers genes that may have a role in cardiomyopathies. These modifier genes are further enriched using mouse phenotype data. We highlight key modifier genes that are potentially associated with cardiomyopathies. Many predicted genes link cardiomyopathies to neoplasms, metabolic and neurological disorders, some of these have been detailed as case studies in this analysis.

ConclusionsTraditional one drug-one target-one disease outlook alone in drug discovery is challenging. We hereby develop an integrative approach based on primary cardiomyopathy target genes to discover new targets. These can provide clues in drug repurposing, possible off-targets and hidden genetic overlap between cardiomyopathies and other morbidities. We report potential modifier genes like NOTCH4 and SIRT1 that may be investigated for their role in cardiomyopathy and other diseases.

sentences: The Gut Microbiome of Exudivorous Marmosets in the Wild and Captivity Mammalian captive dietary specialists like folivores are prone to gastrointestinal distress and primate dietary specialists suffer the greatest gut microbiome diversity losses in captivity compared to the wild. Marmosets represent another group of dietary specialists, exudivores that eat plant exudates, but whose microbiome remains relatively less studied. The common occurrence of gastrointestinal distress in captive marmosets prompted us to study the Callithrix gut microbiome composition and predictive function through bacterial 16S ribosomal RNA V4 region sequencing. We sampled 59 wild and non-wild Callithrix across four species and their hybrids. Host environment had a stronger effect on the gut microbiome than host taxon. Wild Callithrix gut microbiomes were enriched for Bifidobacterium, which process host-indigestible carbohydrates. Captive marmoset guts were enriched for Enterobacteriaceae, a family containing pathogenic bacteria. While gut microbiome function was similar across marmosets, Enterobacteriaceae seem to carry out most functional activities in captive host guts. More diverse bacterial taxa seem to perform gut functions in wild marmosets, with Bifidobacterium being important for carbohydrate metabolism. Non-wild marmosets showed gut microbiome composition aspects seen in human gastrointestinal diseases. Thus, captivity may perturb the exudivore gut microbiome, which raises implications for captive exudivore welfare and calls for husbandry modifications.

sentences: Antibody and T cell memory immune response after two doses of the BNT162b2 mRNA vaccine in older adults with and without prior SARS-CoV-2 infection We quantified S1-specific IgG, neutralizing antibody titers, specific IFN{gamma} secreting T cells and functionality of specific CD4+ and CD8+ T cells in 130 young adults (median age 44.0 years) and 106 older residents living in a long-term care facility (86.5 years) after 2 doses of BNT162b2. Three months after the first injection, humoral and cellular memory responses were dramatically impaired in the 54 COVID-19-naive older compared to the 121 COVID-19-naive younger adults. Notably, older participants neutralizing antibodies, detected in 76.5% (versus 100% in young adults, P < 0.0001), were ten times lower than the youngers antibody titers (P < 0.0001). Antibody and T cell responses were greater among the 52 COVID-19-recovered than among the 54 COVID-19-naive older adults (P < 0.0001). Our study shows that 2 doses of BNT162b2 does not guarantee long-term protection against SARS-CoV-2 in the older. An additional dose should be considered to boost their specific memory response.

sentences: Here be dragons: important spatial uncertainty driven by climate data in forecasted distribution of an endangered insular reptile The effect of future climate change is poorly documented in the tropics, especially in mountainous areas. Yet, species living in these environments are predicted to be strongly affected. Newly available high-resolution environmental data and statistical methods enable the development of forecasting models. Nevertheless, the uncertainty related to climate models can be strong, which can lead to ineffective conservation actions. Predicted studies aimed at providing conservation guidelines often account for a range of future climate predictions (climate scenarios and global circulation models). However, very few studies considered potential differences related to baseline climate data and/or did not account for spatial information (overlap) in uncertainty assessments. We modelled the environmental suitability for Phelsuma borbonica, an endangered reptile native to Reunion Island. Using two metrics of species range change (difference in overall suitability and spatial overlap), we quantified the uncertainty related to the modelling technique (n = 10), sample bias correction, climate change scenario, global circulation models (GCM) and baseline climate (CHELSA versus Worldclim). Uncertainty was mainly driven by GCMs when considering overall suitability, while for spatial overlap the uncertainty related to baseline climate became more important than that of GCMs. The uncertainty driven by sample bias correction and variable selection was much higher when assessed based on spatial overlap. The modelling technique was a strong driver of uncertainty in both cases. We eventually provide a consensus ensemble prediction map of the environmental suitability of P. borbonica to identify the areas predicted to be the most suitable in the future with the highest certainty. Predictive studies aimed at identifying priority areas for conservation in the face of climate change need to account for a wide panel of modelling techniques, GCMs and baseline climate data. We recommend the use of multiple approaches, including spatial overlap, when assessing uncertainty in species distribution models.

sentences: Learning Sparse Log-Ratios for High-Throughput Sequencing Data The automatic discovery of sparse biomarkers that are associated with an outcome of interest is a central goal of bioinformatics. In the context of high-throughput sequencing (HTS) data, and compositional data (CoDa) more generally, an important class of biomarkers are the log-ratios between the input variables. However, identifying predictive log-ratio biomarkers from HTS data is a combinatorial optimization problem, which is computationally challenging. Existing methods are slow to run and scale poorly with the dimension of the input, which has limited their application to low- and moderate-dimensional metagenomic datasets. Building on recent advances from the field of deep learning, we present CoDaCoRe, a novel learning algorithm that identifies sparse, interpretable, and predictive log-ratio biomarkers. Our algorithm exploits a continuous relaxation to approximate the underlying combinatorial optimization problem. This relaxation can then be optimized efficiently using the modern ML toolbox, in particular, gradient descent. As a result, CoDaCoRe runs several orders of magnitude faster than competing methods, all while achieving state-of-the-art performance in terms of predictive accuracy and sparsity. We verify the outperformance of CoDaCoRe across a wide range of microbiome, metabolite, and microRNA benchmark datasets, as well as a particularly high-dimensional dataset that is outright computationally intractable for existing sparse log-ratio selection methods.1

sentences: Vitronectin acts as a key regulator of adhesion and migration in human umbilical cord-derived MSCs under different stress conditions Mesenchymal stem cell (MSC)-based therapy gets compromised as adverse microenvironmental conditions like nutrient deprivation, ischemia, hypoxia at the target site affect migration, engraftment and viability, of MSCs post transplantation. To improve the treatment efficacy, it is critical to identify factors involved in regulating migration and adhesion of MSCs under such microenvironmental stress conditions. In our study, we observed that human Whartons jelly-derived MSCs (WJ-MSCs) exhibited increase in cell spread area and adhesion, with reduction in cellular migration under serum starvation stress. The changes in adhesion and migration characteristics were accompanied by formation of large number of super mature focal adhesions along with extensive stress fibres and altered ECM gene expression with notable induction in vitronectin (VTN) expression. NF-{kappa}{beta} was found to be a positive regulator of VTN expression while ERK pathway regulated it negatively. Molecular and phenotypic comparison studies with inhibition of these signalling pathways or knocking down of VTN under serum starvation established the correlation between increase in VTN expression and increased cellular adhesion with corresponding reduction in cell migration. VTN knockdown also resulted in reduction of super mature focal adhesions and extensive stress fibres, which were formed under serum starvation. Additionally, VTN induction was not detected in hypoxia-treated WJ-MSCs, and the MSCs showed no significant change in the adhesion or migration properties under hypoxia. However, when VTN expression was induced under hypoxia by ERK pathway inhibition, similar increase in cell spread area and adhesion were observed. Our study thus highlights VTN as a key player which is induced under serum starvation stress and possibly regulates the adhesion and migration properties of WJ-MSCs via focal adhesion signalling.

sentences: Drug Sensitivity Prediction From Cell Line-Based Pharmacogenomics Data: Guidelines for Developing Machine Learning Models The goal of precision oncology is to tailor treatment for patients individually using the genomic profile of their tumors. Pharmacogenomics datasets such as cancer cell lines are among the most valuable resources for drug sensitivity prediction, a crucial task of precision oncology. Machine learning methods have been employed to predict drug sensitivity based on the multiple omics data available for large panels of cancer cell lines. However, there are no comprehensive guidelines on how to properly train and validate such machine learning models for drug sensitivity prediction. In this paper, we introduce a set of guidelines for different aspects of training gene expression-based predictors using cell line datasets. These guidelines provide extensive analysis of the generalization of drug sensitivity predictors, and challenge many current practices in the community including the choice of training dataset and measure of drug sensitivity. Application of these guidelines in future studies will enable the development of more robust preclinical biomarkers.

sentences: The loss of enzymatic activity of the PHARC associated lipase ABHD12 results in increased phagocytosis that causes neuroinflammation Phagocytosis is an important evolutionary conserved process, essential for clearing pathogens and cellular debris in higher organisms, including humans. This well-orchestrated innate immunological response is intricately regulated by numerous cellular factors, important amongst which, are the immunomodulatory lysophosphatidylserines (lyso-PSs) and the pro-apoptotic oxidized phosphatidylserines (PSs) signaling lipids. Interestingly, in mammals, both these signaling lipids are physiologically regulated by the lipase ABHD12, mutations of which, cause the human neurological disorder PHARC. Despite the biomedical significance of this lipase, detailed mechanistic studies and the specific contribution of ABHD12 to innate processes like phagocytosis remain poorly understood. Here, by immunohistochemical and immunofluorescence approaches, using the murine model of PHARC, we show, that upon an inflammatory stimulus, activated microglial cells in the cerebellum of mice deficient in ABHD12 have an amoeboid morphology, increased soma size, and display heightened phagocytosis activity. We also report that upon an inflammatory stimulus, cerebellar levels of ABHD12 increase to possibly metabolize the heightened oxidized PS levels, temper phagocytosis and in turn control neuroinflammation during oxidative stress. Next, to complement these findings, using biochemical approaches in cultured microglial cells, we show that the pharmacological inhibition and/or genetic deletion of ABHD12 results in increased phagocytic uptake in a fluorescent bead uptake assay. Together, our studies provide compelling evidence that ABHD12 plays an important role in regulating phagocytosis in cerebellar microglial cells, and provides a possible explanation, as to why human PHARC subjects display neuroinflammation and atrophy in the cerebellum.

sentences: Local Connectivity and Synaptic Dynamics in Mouse and Human Neocortex To elucidate cortical microcircuit structure and synaptic properties we present a unique, extensive, and public synaptic physiology dataset and analysis platform. Through its application, we reveal principles that relate cell type to synapse properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Despite these associations, synaptic properties are heterogeneous in most subclass to subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, while the strength axis accounts for significant heterogeneity within the subclass. In human cortex, excitatory to excitatory synapse dynamics are distinct from those in mouse and short-term plasticity varies with depth across layers 2 and 3. With a novel connectivity analysis that enables fair comparisons between circuit elements, we find that intralaminar connection probability among cell subclasses exhibits a strong layer dependence.These and other findings combined with the analysis platform create new opportunities for the neuroscience community to advance our understanding of cortical microcircuits.

sentences: Suppression of aggregate and amyloid formation by a novel intrinsically disordered region in metazoan Hsp110 chaperones Molecular chaperones maintain protein homeostasis (proteostasis) by ensuring the proper folding of polypeptides. Loss of proteostasis has been linked to the onset of numerous neurodegenerative disorders including Alzheimers, Parkinsons, and Huntingtons disease. Hsp110 is related to the canonical Hsp70 class of protein folding molecular chaperones and interacts with Hsp70 as a nucleotide exchange factor (NEF), promoting rapid cycling of ADP for ATP. In addition to its NEF activity, Hsp110 possesses an Hsp70-like substrate binding domain (SBD) whose biological roles remain undefined. Previous work in Drosophila melanogaster has shown that loss of the sole Hsp110 gene (Hsc70cb) accelerates the aggregation of polyglutamine (polyQ)-expanded human Huntingtin, while its overexpression protects against polyQ-mediated neuronal cell death. We hypothesize that in addition to its role as an Hsp70 NEF, Drosophila Hsp110 may function in the fly as a protective protein "holdase", preventing the aggregation of unfolded polypeptides via the SBD-{beta} subdomain. Using an in vitro protein aggregation assay we demonstrate for the first time that Drosophila Hsp110 effectively prevents aggregation of the model substrate citrate synthase. We also report the discovery of a redundant and heretofore unknown potent holdase capacity in a 138 amino-acid region of Hsp110 carboxyl-terminal to both SBD-{beta} and SBD- (henceforth called the C-terminal extension). This sequence is highly conserved in metazoan Hsp110 genes, completely absent from fungal representatives, including Saccharomyces cerevisiae SSE1, and is computationally predicted to contain an intrinsically disordered region (IDR). We demonstrate that this IDR sequence within the human Hsp110s, Apg-1 and Hsp105, inhibits the formation of amyloid A{beta}-42 and -synuclein fibrils in vitro but cannot mediate fibril disassembly. Together these findings demonstrate the existence of a second independent, passive holdase property of metazoan Hsp110 chaperones capable of suppressing both general protein aggregation and amyloidogenesis and raise the possibility of exploitation of this IDR for therapeutic benefit in combating neurodegenerative disease.

sentences: Estradiol mediates stress-susceptibility in the male brain In susceptible populations, stress is a major risk factor for the development of mental disorders, including depression. Estradiol, often considered a female hormone, is distributed in the male brain via aromatization of testosterone. The role of estrogen receptors (ERs) in male stress susceptibility and depression is not well understood. We found that absence of ER{beta} is associated with susceptibility to stress in male mice and that activity of ER{beta}-projecting neurons from the basolateral amygdala to nucleus accumbens is reduced in hypogonadal mice subjected to stress, while activation of this circuit reverses stress-induced maladaptive behaviors. We identified that absence of estradiol, but not testosterone per se, underlies stress susceptibility and that brain-selective delivery of estradiol prevents the development of depression-related behaviors. Our findings provide evidence for an estrogen-based mechanism underlying stress susceptibility and offer an unexpected therapeutic strategy for treating depression in males.

sentences: Anti-prothrombin autoantibodies enriched after infection with SARS-CoV-2 and influenced by strength of antibody response against SARS-CoV-2 proteins Antiphospholipid antibodies (aPL), assumed to cause antiphospholipid syndrome (APS), are notorious for their heterogeneity and detect phospholipids and phospholipid-binding proteins. The persistent presence of Lupus anticoagulant and/or aPL against cardiolipin and/or {beta}2 glycoprotein I have been shown to be independent risk factors for vascular thrombosis and pregnancy morbidity in APS. aPL production is thought to be triggered by - among other factors - viral infections, though infection-associated aPL have mostly been considered non-pathogenic. Recently, the potential pathogenicity of infection-associated aPL has gained momentum since an increasing number of patients infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been described with coagulation abnormalities and hyperinflammation, together with the presence of aPL. Here, we present data from a multicentric, mixed-severity study including three cohorts of individuals who contracted SARS-CoV-2 as well as non-infected blood donors. We simultaneously measured 10 different criteria and non-criteria aPL (IgM and IgG) by using a line immunoassay. Further, IgG antibody response against three SARS-CoV-2 proteins was investigated using tripartite automated blood immunoassay technology. Our analyses revealed that selected non-criteria aPL were enriched concomitant to or after an infection with SARS-CoV-2. Linear mixed-effects models suggest an association of aPL to prothrombin (PT) with the strength of the antibody response against SARS-CoV-2 and that it is further influenced by SARS-CoV-2 disease severity and sex of the individuals. In conclusion, our study is the first to report an association between disease severity, anti-SARS-CoV-2 immunoreactivity and aPL against PT in patients with SARS-CoV-2.

sentences: Authentication of In Situ Measurements for Thoracic Aortic Aneurysms in Mice Aortic diameter is a standard parameter for defining disease severity of thoracic aortic aneurysms. In mouse studies, aortic diameters can be measured in situ directly, but this approach has a potential confounder of underestimation due to the absence of physiological arterial blood pressure. In the present study, we developed an in situ approach for authentic aortic measurements. Thoracic aortic aneurysms were induced by beta-aminopropionitrile (BAPN, 0.5% wt/vol) administration in 4-week-old male C57BL/6J mice. Ultrasonography was performed to examine aortic dimensions, and mice with thoracic aortic dilatations were terminated subsequently. After saline perfusion through the left ventricle, periaortic tissues were removed to expose thoracic aortas. Optimal cutting temperature (OCT) compound was injected via the left ventricle to maintain aortic patency. In situ aortic images were captured pre- and post-OCT injection. In mice with severe thoracic aortic aneurysms, smaller aortic diameters were observed prior to OCT injection compared to ultrasound measurements, while aortic diameters in situ after OCT were comparable to diameters measured using ultrasound. A telemetry system demonstrated that maximal luminal pressures during 150 ul of OCT injection were 90 mmHg. Immunostaining for CD31 revealed that endothelial cells were preserved in the intima after OCT injection. These results indicate that OCT injection does not cause aortic damages due to excess pressures. In conclusion, in situ imaging with OCT injection provides authentic aortic measurements without overt aortic damage in mice with thoracic aortic aneurysms.

sentences: Nitrate-regulated growth processes involve activation of gibberellin pathway Nitrate, one of the main nitrogen (N) sources for crops, acts as a nutrient and key signaling molecule coordinating gene expression, metabolism and various growth processes throughout the plant life cycle. It is widely accepted that nitrate-triggered developmental programs cooperate with hormone synthesis and transport, to finely adapt plant architecture to N availability. Here, we report that nitrate, acting through its signaling pathway, promotes growth in Arabidopsis and wheat, in part by modulating the accumulation of gibberellin (GA)-regulated DELLA growth repressors. We show that nitrate reduces the abundance of DELLAs by increasing GA contents through activation of GA metabolism gene expression. Consistently, the growth restraint conferred by nitrate deficiency is partially rescued in global-DELLA mutant that lacks all DELLAs. At the cellular level, we show that nitrate enhances both cell proliferation and elongation in a DELLA-dependent and -independent manner, respectively. Our findings establish a connection between nitrate and GA signaling pathways that allow plants to adapt their growth to nitrate availability.

sentences: Group dynamics and habitat use of the Giant Otter, Pteronura brasiliensis (Zimmermann, 1780), in seasonally flooded forest in the Araguaia River, Central Brazil: A 10-years study. We carried out monthly surveys of the giant otter population between 2010 and 2020 in a study area comprised of 1,500 hectares of igapo flooded forest with oxbow lakes in the Cantao region of central Brazil. We recorded 16-32 resident adults in the study area each year, distributed in 4-8 groups. Resident groups exhibited extensive home range overlap, with each group using several lakes and larger lakes used in rotation by up to six groups. Dens and campsites were also shared by multiple groups, but lakes were used by only one group at a time, and encounters between groups were very rare. 24 adult otters were observed to join an existing group. Some individuals changed groups multiple times. Resident adult turnover was high. Each year an average of 36% of resident adults were new immigrants, and 72% of groups left the area within two years. Resident groups had, on average, one litter every three years, and annual cub production showed high variability and a negative correlation to the number of new immigrants in the area. No pairs of giant otters reproduced successfully during the study. Groups of three otters formed through the recruitment of an adult individual by an existing pair and reproduced as successfully as larger groups. Group dynamics and territorial behavior in the Cantao flooded forest ecosystem, where optimal giant otter habitat is continuous in all directions, were found to be different from that reported in areas composed of patchy (isolated oxbow lakes) or linear (rivers) habitat. This suggest that giant otter social and territorial behavior is plastic and adapts to the spatial characteristics of the habitat.

sentences: Constitutively active STAT5b feminizes mouse liver gene expression STAT5 is an essential transcriptional regulator of the sex-biased actions of growth hormone (GH) in the liver. Delivery of constitutively active STAT5 (STAT5CA) to male mouse liver using an engineered adeno-associated virus with high tropism for the liver is shown to induce widespread feminization of the liver, with extensive induction of female-biased genes and repression of male-biased genes, largely mimicking results obtained when male mice are given GH as a continuous infusion. Many of the gene expression changes observed were associated with STAT5 binding to liver chromatin, supporting the proposed direct role of persistently active STAT5 in continuous GH-induced liver feminization. The feminizing effects of STAT5CA were dose-dependent; moreover, at higher levels, overexpression of STAT5CA resulted in some histopathology not seen in continuous GH-infused male liver, including hepatocyte hyperplasia and distorted liver architecture. These findings establish that the persistent activation of STAT5 by GH that characterizes female liver is by itself sufficient to account for the female-biased expression of a majority of female-biased genes. Moreover, histological changes seen when STAT5CA is overexpressed highlight the importance of carefully evaluating such effects before considering such STAT5 derivatives for therapeutic use in treating liver disease.

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Caspase inhibition mitigates tau cleavage and neurotoxicity in iPSC-induced neurons with the V337M MAPT mutation Tau post-translational modifications (PTMs) are associated with progressive tau accumulation and neuronal loss in tauopathies, including forms of frontotemporal lobar degeneration (FTLD) and Alzheimers disease (AD). Proteolytic cleavage of tau by active caspases, including caspase-6, represents an underexplored tau PTM implicated in tau pathology. Caspase-cleaved tau is toxic and prone to self-aggregation in experimental models. To elucidate the presence and temporal course of caspase activation, tau cleavage, and neuronal death, we generated two neoepitope monoclonal antibodies (mAbs) against caspase-6 tau proteolytic sites and cortical neurons from induced pluripotent stem cells (iPSCs) with the frontotemporal dementia (FTD)-causing V337M MAPT mutation. FTLD V337M MAPT and AD postmortem brains showed positivity for both cleaved tau mAbs as well as active caspase-6. Relative to isogenic wild-type MAPT controls, V337M MAPT neurons showed a time-dependent increase in pathogenic tau in the form of tau oligomers, caspase-cleaved tau, and p-tau. Accumulation of toxic tau species in 3-month V337M MAPT neurons also increased vulnerability to stress, which was pharmacologically rescued by caspase inhibition. We propose a model in which time-dependent accumulation of caspase-cleaved tau in V337M MAPT neurons promotes neurotoxicity that is reversed by caspase-6 inhibition. Caspase-cleaved tau may be a biomarker of tauopathy, and caspases could be viable targets for therapeutic intervention against tau pathogenesis in FTLD and other tauopathies.

SignificanceThe mechanisms leading to tau pathology in frontotemporal dementia (FTD) and Alzheimers disease (AD) remain elusive. Experimental studies in AD demonstrate that tau cleavage by active caspase-6 contributes to tau pathology since cleaved tau may be toxic and prone to self-aggregation. Yet, the role of caspase-cleaved tau in promoting toxicity and cell death is unclear. Here, we generated two neoepitope monoclonal antibodies against caspase-6 tau and evaluated tau cleavage in postmortem human brains, iPSC-induced cortical neurons with the FTD-causing V337M MAPT mutation, and isogenic wild-type MAPT controls. Our results demonstrate a time-dependent accumulation of caspase-cleaved tau and increased neurotoxicity in the mutant iNs that is reversed by caspase-6 inhibition. Caspases could be viable therapeutic targets against tau pathology in tauopathies.

sentences: Polymorphic SNPs, short tandem repeats and structural variants are responsible for differential gene expression across C57BL/6 and C57BL/10 substrains Mouse substrains are an invaluable model for understanding disease. We compared C57BL/6J, which is the most commonly used inbred mouse strain, with 8 C57BL/6 and 5 C57BL/10 closely related inbred substrains. Whole genome sequencing and RNA-sequencing analysis yielded 352,631 SNPs, 109,096 INDELs, 150,344 short tandem repeats (STRs), 3,425 structural variants (SVs) and 2,826 differentially expressed genes (DEGenes) among these 14 strains. 312,981 SNPs (89%) distinguished the B6 and B10 lineages. These SNPs were clustered into 28 short segments that are likely due to introgressed haplotypes rather than new mutations. Outside of these introgressed regions, we identified 53 SVs, protein-truncating SNPs and frameshifting INDELs that were associated with DEGenes. Our results can be used for both forward and reverse genetic approaches, and illustrate how introgression and mutational processes give rise to differences among these widely used inbred substrains.

sentences: Evolution of the quorum sensing regulon in cooperating populations of Pseudomonas aeruginosa The bacterium Pseudomonas aeruginosa is an opportunistic pathogen and it thrives in many different saprophytic habitats. In this bacterium acyl-homoserine lactone quorum sensing (QS) can activate expression of over 100 genes, many of which code for extracellular products. P. aeruginosa has become a model for studies of cell-cell communication and coordination of cooperative activities. We hypothesized that long-term growth of bacteria under conditions where only limited QS-controlled functions were required would result in a reduction in the size of the QS-controlled regulon. To test this hypothesis, we grew P. aeruginosa for about 1000 generations in a condition in which expression of QS-activated genes is required for growth. We compared the QS regulons of populations after about 35 generations to those after about 1000 generations in two independent lineages by using quorum quenching and RNA-seq technology. In one evolved lineage the number of QS-activated genes identified was reduced by about 70% and in the other by about 45%. Our results lend important insights about the variations in the number of QS-activated genes reported for different bacterial strains and, more broadly, about the environmental histories of P. aeruginosa.

sentences: Selective binding and transport of protocadherin 15 isoforms by stereocilia unconventional myosins in a heterologous expression system During hair cell development, the mechanoelectrical transduction (MET) apparatus is assembled at the stereocilia tips, where it coexists with the stereocilia actin regulatory machinery. While the myosin-based tipward transport of actin regulatory proteins is well studied, isoform complexity and built-in redundancies in the MET apparatus have limited our understanding of how MET components are transported. We used a heterologous expression system to elucidate the myosin selective transport of isoforms of protocadherin 15 (PCDH15), the protein that mechanically gates the MET apparatus. We show that MYO7A selectively transports the CD3 isoform while MYO3A and MYO3B transports the CD2 isoform. Furthermore, MYO15A showed an insignificant role in the transport of PCDH15, and none of the myosins tested transport PCDH15-CD1. Our data suggest an important role for MYO3A, MYO3B, and MYO7A in the MET apparatus formation and highlight the intricate nature of MET and actin regulation during development and functional maturation of the stereocilia bundle.

sentences: Resolving the 3D rotational and translational dynamics of single molecules using radially and azimuthally polarized fluorescence We report a radially and azimuthally polarized (raPol) microscope for high detection and estimation performance in single-molecule orientation-localization microscopy (SMOLM). With 5000 photons detected from Nile red (NR) transiently bound within supported lipid bilayers (SLBs), raPol SMOLM achieves 2.9 nm localization precision, 1.5{degrees} orientation precision, and 0.17 sr precision in estimating rotational wobble. Within DPPC SLBs, SMOLM imaging reveals the existence of randomly oriented binding pockets that prevent NR from freely exploring all orientations. Treating the SLBs with cholesterol-loaded methyl-{beta}-cyclodextrin (M{beta}CD-chol) causes NRs orientational diffusion to be dramatically reduced, but curiously, NRs median lateral displacements drastically increase from 20.8 nm to 75.5 nm (200 ms time lag). These jump diffusion events overwhelmingly originate from cholesterol-rich nanodomains within the SLB. These detailed measurements of single-molecule rotational and translational dynamics are made possible by raPols high measurement precision and are not detectable in standard SMLM.

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Intranasal gene therapy to prevent infection by SARS-CoV-2 variants SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. This manuscript demonstrates an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity-matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by pre-emergent members, ACE2-dependent CoVs that are poised for zoonosis.

Author summarySARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. This manuscript demonstrates an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity-matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by pre-emergent members, ACE2-dependent CoVs that are poised for zoonosis.

sentences: Neural language processing across time, space, frequency and age: MEG-MVPA classification of intertrial phase coherence Language is a key part of human cognition. Whereas many neurocognitive abilities decline with age, for language the picture is much less clear and how exactly language processing changes with aging is still unknown. To investigate this, we employed magnetoencephalography (MEG) and recorded neuromagnetic brain responses to auditory linguistic stimuli in healthy participants of younger and older age using a passive task-free paradigm and a range of different linguistic stimulus contrasts, which enabled us to assess neural language processes at multiple levels (lexical, semantic, morphosyntactic). By using machine learning-based classification algorithms to scrutinise intertrial phase coherence of MEG responses in source space, we found significant differences between younger and older participants across several frequency bands and for all tested processing types, which shows multiple changes in the brains neurolinguistic circuits which may be due to both healthy aging in general and compensatory processes in particular.

sentences: Single-cell genomic analysis of triple-negative breast cancer fibroblasts uncovers evolutionarily conserved features and potential therapeutic targets To explore cancer associated fibroblasts (CAFs) in triple-negative breast cancers (TNBC), we performed scRNA-seq analysis of fibroblasts from murine and human TNBCs. We observed three distinct CAF subtypes in mouse TNBC: two that are intermingled and adjacent to tumor cells, and one that is more distal. We present evidence that progression of CAFs from normal resident fibroblasts/pericytes involves upregulation of their Pdgf and Tgfb receptors along with reciprocal ligand upregulation in other cells within the tumor microenvironment. Additionally, extracellular matrix, glycolytic, and mitochondrial respiratory genes are strongly upregulated in all CAFs. Activation of extracellular matrix genes specifically in CAFs and not in normal fibroblasts provides numerous targets for CAF-based therapeutics, many of which are conserved in CAFs from human TNBC. In contrast, the subtype structure of CAFs was less conserved, which along with their transcriptional heterogeneity suggests that molecular targeting of CAFs is more practical than targeting CAF subtypes.

sentences: m6A-TSHub: unveiling the context-specific m6A methylation and m6A-affecting mutations in 23 human tissues As the most pervasive epigenetic marker present on mRNA and lncRNA, N6-methyladenosine (m6A) RNA methylation has been shown to participate in essential biological processes. Recent studies revealed the distinct patterns of m6A methylome across human tissues, and a major challenge remains in elucidating the tissue-specific presence and circuitry of m6A methylation. We present here a comprehensive online platform m6A-TSHub for unveiling the context-specific m6A methylation and genetic mutations that potentially regulate m6A epigenetic mark. m6A-TSHub consists of four core components, including (1) m6A-TSDB: a comprehensive database of 184,554 functionally annotated m6A sites derived from 23 human tissues and 499,369 m6A sites from 25 tumor conditions, respectively; (2) m6A-TSFinder: a web server for high-accuracy prediction of m6A methylation sites within a specific tissue from RNA sequences, which was constructed using multi-instance deep neural networks with gated attention; (3) m6A-TSVar: a web server for assessing the impact of genetic variants on tissue-specific m6A RNA modification; and (4) m6A-CAVar: a database of 587,983 TCGA cancer mutations (derived from 27 cancer types) that were predicted to affect m6A modifications in the primary tissue of cancers. The database should make a useful resource for studying the m6A methylome and genetic factor of epitranscriptome disturbance in a specific tissue (or cancer type). m6A-TSHub is accessible at: www.xjtlu.edu.cn/biologicalsciences/m6ats.

sentences: Which Index Should I Use? A Comparison of Indices for Enclosure Use Studies Enclosure use assessments have gained popularity as one of the tools for animal welfare assessments and Post Occupancy Evaluations. There are now a plethora of studies and enclosure use indices available in published literature, and identification of the most appropriate index for each research question is often challenging. The benefits and limitations of four different enclosure use indices; Original and Modified Spread of Participation Index, Entropy, and Electivity Index were compared. Three artificial data sets were developed to represent the challenges commonly found in animal exhibits, and these indices were applied to these contrived enclosure settings. Three of the indices (Original SPI, Modified SPI, and Entropy) were used to assess a single measure of enclosure use variability. When zones within an exhibit were comparable in size, all three indices performed similarly. However, with less equal zone sizes, Modified SPI outperformed Original SPI and Entropy, suggesting that the Modified formula was more useful for assessing overall enclosure use variability under such conditions. Electivity Index assessed the use of individual zones, rather than the variability of use across the entire exhibit, and therefore could not be compared directly to the other three indices. This index is therefore most valuable for assessing individual resources, especially after exhibit modifications.

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Involvement of prelimbic cortex neurons and related circuits in the acquisition of cooperative learning by pairs of rats Social behaviors such as cooperation are crucial for mammals. A deeper knowledge of the neuronal mechanisms underlying cooperation can be beneficial for people suffering from pathologies with impaired social behavior. Our aim was to study the brain activity when two animals synchronize their behavior to obtain a mutual reinforcement. In a previous work, we showed that the activity of the prelimbic cortex (PrL) was enhanced during cooperation in rats, especially in the ones leading most cooperative trials (leader rats). In this study, we investigated the specific cell type/s in the PrL contributing to cooperative behaviors. To this end, we collected rats brains at key moments of the learning process to analyze the levels of c-FOS expression in the main cellular groups of the PrL (glutamatergic cells containing D1 and D2 receptors and interneurons). Leader rats showed increased c-FOS activity in cells expressing D1 receptors during cooperation. In addition, we analyzed the levels of anxiety, dominance, and locomotor behavior, finding that leader rats are in general less anxious and less dominant than followers. We also recorded local field potentials (LFPs) from the PrL, the nucleus accumbens septi (NAc), and the basolateral amygdala (BLA). Spectral analysis showed that delta activity in PrL and NAc increased when rats cooperated, while BLA activity in delta and theta bands decreased considerably during cooperation. The PrL and NAc also increased their connectivity in the high theta band during cooperation. Thus, the present work identifies the specific PrL cell types engaged in this behavior, as well as its connectivity with subcortical brain regions (BLA, NAc) during cooperation.

Significance StatementBrain mechanisms underlying cooperative behaviors remain unknown. The present study identified specific neuronal types from the PrL cortex engaged in the acquisition of a cooperative task, as well as their connectivity with subcortical projection sites, such as the NAc and the BLA during cooperation. Rats leading the cooperation trials (designated leaders) presented an increased activation of D1-containing neurons in the PrL during cooperation. The PrL and NAc electrical activity increased when rats were cooperating, while the BLA activity increased before cooperation. The PrL and NAc showed increased functional connectivity at the moment of cooperation on the platform, whereas during the individual phase the highest connectivity was found before the animals climbed individually onto the platform.

sentences: Genetic diversity of the pathogenic banana Fusarium wilt in northern Viet Nam Fusarium is one of the most important fungal genera of plant pathogens that affect the cultivation of a wide range of crops. Agricultural losses caused by Fusarium oxysporum f. sp. cubense (Foc) have a direct effect on the income, subsistence and nourishment of thousands of smallholder farmers worldwide. In addition, also commercial growers are strongly affected. For Viet Nam, predictions on the impact of Foc for the future are dramatic with an estimated loss in banana production area of 8% within the next 5 years and up to 71% within the next 25 years. In the current study we applied a combined morphological - molecular approach to assess the taxonomic identity and phylogenetic position of the different Foc isolates that were collected in northern Viet Nam. In addition, we aim to estimate the proportion of the different Fusarium races that are infecting bananas in northern Viet Nam. The morphology of the isolates was investigated by growing the collected Fusarium isolates on four distinct nutritious media (PDA, SNA, CLA, and OMA). Molecular phylogenetic relationships were inferred by sequencing partial rpb1, rpb2 and tef1a genes and adding the obtained sequences into a large phylogenetic framework. The present study showed that Foc Race 1 is the most common strain in northern Viet Nam, causing 74% of all the infections. A more in-depth molecular characterization shows that of the Foc Race 1 infections, 92% are caused by Fusarium tardichlamydosporum and 8% by F. duoseptatum. Compared to Foc Race 1, Foc TR4 (represented by F. odoratissimum) account for only 10.5% of the Fusarium wilting in northern Viet Nam demonstrating that Foc TR4 is not yet a dominant strain in the region. Foc Race 2 infections of Vietnamese bananas also account for 10.5% of the Fusarium wilting in northern Viet Nam. One of the isolates cultured from diseased bananas collected in northern Viet Nam was phylogenetically not positioned within the F. oxysporum species complex (FOSC), but in contrast fell within the Fusarium fujikuroi species complex (FFSC). As a result, it is possible that a new pathogen for bananas has been found. Besides being present on several ABB  Tay banana, Foc Race 1 was also found as pathogen on wild Musa lutea, showing the importance of wild bananas as possible sink for Foc.

sentences: Metabolic dysfunction induced by high-fat diet modulates hematopoietic stem and myeloid progenitor cells in brown adipose tissue of mice Brown adipose tissue (BAT) may be an important metabolic regulator of whole-body glucose. While important roles have been ascribed to macrophages in regulating metabolic functions in BAT, little known is known of the roles of other immune cells subsets, particularly dendritic cells (DCs). Eating a high fat diet may compromise the development of hematopoietic stem and progenitor cells (HSPC) - which give rise to DCs - in bone marrow, with less known of its effects in BAT. We have previously demonstrated that ongoing exposure to low-dose ultraviolet radiation (UVR) significantly reduced the  whitening effect of eating a high-fat diet upon interscapular (i)BAT of mice. Here, we examined whether this observation may be linked to changes in the phenotype of HSPC and myeloid-derived immune cells in iBAT and bone marrow of mice using 12-colour flow cytometry. Many HSPC subsets declined in both iBAT and bone marrow with increasing metabolic dysfunction. Conversely, with rising adiposity and metabolic dysfunction, conventional (c)DCs increased in both of these tissues. When compared to low-fat diet, consumption of high-fat diet significantly reduced proportions of myeloid, common myeloid and megakaryocyte-erythrocyte progenitors in iBAT, and short-term hematopoietic stem cells in bone marrow. In mice fed a high-fat diet, exposure to low-dose UVR significantly reduced proportions of cDCs in iBAT, independently of nitric oxide release from irradiated skin (blocked using the scavenger, cPTIO), but did not significantly modify HSPC subsets in either tissue. Further studies are needed to determine whether changes in these cell populations contribute towards metabolic dysfunction.

sentences: Improved Ultrasound Localization Microscopy based on Microbubble Uncoupling via Transmit Excitation (MUTE) Ultrasound localization microscopy (ULM) demonstrates great potential for visualization of tissue microvasculature at depth with high spatial resolution. The success of ULM heavily depends on the robust localization of isolated microbubbles (MBs), which can be challenging in vivo especially within larger vessels where MBs can overlap and cluster close together. While MB dilution alleviates the issue of MB overlap to a certain extent, it drastically increases the data acquisition time needed for MBs to populate the microvasculature, which is already on the order of several minutes using recommended MB concentrations. Inspired by optical super-resolution imaging based on stimulated emission depletion (STED), here we propose a novel ULM imaging sequence based on microbubble uncoupling via transmit excitation (MUTE). MUTE "silences" MB signals by creating acoustic nulls to facilitate MB separation, which leads to robust localization of MBs especially under high concentrations. The efficiency of localization accomplished via the proposed technique was first evaluated in simulation studies with conventional ULM as a benchmark. Then an in vivo study based on the chorioallantoic membrane (CAM) of chicken embryos showed that MUTE could reduce the data acquisition time by half thanks to the enhanced MB separation and localization. Finally, the performance of MUTE was validated in an in vivo mouse brain study. These results demonstrate the high MB localization efficacy of MUTE-ULM, which contributes to a reduced data acquisition time and improved temporal resolution for ULM.

sentences: Optimized detection of allelic imbalances specific for homologous recombination deficiency improves the prediction of clinical outcomes in cancer Homologous recombination DNA-repair deficiency (HRD) is a common driver of genomic instability and confers a therapeutic vulnerability in human cancer. Using a systematic pan- cancer characterization, we revealed distinct patterns of somatic allelic imbalances (AIs) in high-grade serous ovarian cancer (HGSC). Using machine learning on a multi-omics dataset, we generated an optimized algorithm to detect HRD in HGSC (ovaHRDscar). ovaHRDscar improved the prediction of clinical outcomes in three independent validation cohorts. Characterization of 98 spatiotemporally distinct tumor samples indicated ovary/adnex as the preferred site to assess HRD. Cancer-specific optimization improved the prediction of clinical outcomes also in triple-negative breast cancer (tnbcHRDscar). In conclusion, our systematic approach based on multi-omics data improves the detection of HRD with the premise to improve patient selection for HR-targeted therapies.

sentences: Novel trans-translation-associated gene regulation revealed by prophage excision-triggered switching of ribosome rescue pathway Escherichia coli has multiple pathways to release a nonproductive ribosome complex stalled at the 3 end of nonstop mRNAs: SsrA RNA-mediated trans-translation and stop codon-independent termination by ArfA/RF2 or ArfB (YaeJ). The arfA mRNA lacks a stop codon and thus its expression is repressed by trans-translation. Therefore, ArfA is considered to complement the ribosome rescue activity of trans-translation, but the situations in which ArfA is expressed to rescue the nonproductive complexes have not been elucidated. Here, we demonstrated that the excision of the CP4-57 prophage adjacent to the E. coli ssrA gene leads to the inactivation of SsrA RNA and switches the primary rescue pathway from trans-translation to the ArfA/RF2 pathway. A comparative quantitative proteomic analysis revealed that the switching of the rescue pathway rearranges not only the proteome landscape in E. coli cells but also the phenotype, such as motility. Among the proteins with significantly increased abundance in the ssrA-inactivated cells, we found ZntR, whose mRNA is transcribed together as the upstream part of the nonstop arfA mRNA. Further analysis revealed that the translation of the nonstop ORF of arfA triggered the repression of the upstream zntR ORF, via trans-translation-coupled exonucleolytic (RUTEX) degradation by a polynucleotide phosphorylase. These results provide a novel example of trans-translation-dependent regulation, and shed new light on the physiological roles of prophages in gene expression.

sentences: 14-3-3ζ constrains insulin secretion by regulating mitochondrial function in pancreatic β-cells While critical for neurotransmitter synthesis in the brain, members of the 14-3-3 protein family are often assumed to have redundant, over-lapping roles due to their high sequence homology and ubiquitous expression. Despite this assumption, various mammalian 14-3-3 isoforms have now been implicated in regulating cellular and organismal metabolism; however, these functions were primarily observed in cell lines or from systemic knockout mouse models. To date, we have begun to define the contributions of 14-3-3{zeta} in adipocytes, but whether 14-3-3{zeta} has additional metabolic roles in other cell types, such as the pancreatic {beta}-cell, is unclear. We previously documented a pro-survival role of 14-3-3{zeta} in MIN6 insulinoma cells, as depletion of 14-3-3{zeta} induced cell death, but paradoxically, whole-body deletion of 14-3-3{zeta} in mice resulted in significantly enlarged {beta}-cell area with no effects on insulin secretion. To better understand the role of 14-3-3{zeta} in {beta}-cells, we generated {beta}-cell-specific 14-3-3{zeta} knockout ({beta}14-3-3{zeta}KO) mice, and while no differences in {beta}-cell mass were observed, {beta}14-3-3{zeta}KO mice displayed potentiated insulin secretion due to enhanced mitochondrial function and ATP synthesis. Deletion of 14-3-3{zeta} led to profound changes to the {beta}-cell transcriptome, where pathways associated with mitochondrial respiration and oxidative phosphorylation were upregulated. Acute treatment of mouse islets and human islets with pan-14-3-3 inhibitors recapitulated the potentiation in glucose-stimulated insulin secretion (GSIS) and mitochondrial function, suggesting that 14-3-3{zeta} is a critical isoform in {beta}-cells that regulates GSIS. In dysfunctional db/db islets and islets from type 2 diabetic donors, expression of Ywhaz/YWHAZ, the gene encoding 14-3-3{zeta}, was inversely associated with insulin secretory capacity, and pan-14-3-3 protein inhibition was capable of enhancing GSIS and mitochondrial function. Taken together, this study demonstrates important regulatory functions of 14-3-3{zeta} and its related isoforms in insulin secretion and mitochondrial function in {beta}-cells. A deeper understanding of how 14-3-3{zeta} influences {beta}-cell function will further advance our knowledge of how insulin secretion from {beta}-cells is regulated.

sentences: Rational Exploration of Fold Atlas for Human Solute Carrier Proteins Solute carrier (SLC) superfamily is the largest group responsible for transmembrane transport of substances in human cells. It includes more than 400 members which are organized into 65 families according to their physiological function and sequence similarity. Different families of solute transporters can adopt the same or different folds that determine the working mechanism and reflect the evolutionary relationship between SLC members. Analysis of structural data in literatures shows that there are 13 different folds in the solute carrier superfamily covering 40 families and 343 members. To further study their working mechanism, we systematically explored the SLC superfamily to look for more folds. Based on our results, at least three new folds were found for SLC superfamily, and one was experimentally verified in the SLC44 family. Our work has laid a foundation and provided important insights for the systematic and comprehensive study on the structure and function of solute carriers.

sentences: Unexpected post-glacial colonisation route explains the white colour of barn owls (Tyto alba) from the British Isles The climate fluctuations of the Quaternary shaped the movement of species in and out of glacial refugia. In Europe, the majority of species followed one of the described traditional postglacial recolonization routes from the southern peninsulas towards the north. Like most organisms, barn owls are assumed to have colonized the British Isles by crossing over Doggerland, a land bridge that connected Britain to northern Europe. However, while they are dark rufous in northern Europe, barn owls in the British Isles are conspicuously white, a contrast that could suggest selective forces are at play on the islands. However, analysis of known candidate genes involved in colouration found no signature of selection. Instead, using whole genome sequences and species distribution modelling, we found that owls colonised the British Isles soon after the last glaciation, directly from a white coloured refugium in the Iberian Peninsula, before colonising northern Europe. They would have followed a yet unknown post-glacial colonization route to the Isles over a westwards path of suitable habitat in now submerged land in the Bay of Biscay, thus not crossing Doggerland. As such, they inherited the white colour of their Iberian founders and maintained it through low gene flow with the mainland that prevents the import of rufous alleles. Thus, we contend that neutral processes likely explain this contrasting white colour compared to continental owls. With the barn owl being a top predator, we expect future research will show this unanticipated route was used by other species from its paleo community.

sentences: A small molecule probe elucidates the role of mitochondrial translocase TIMM44 in PINK1/Parkin regulated mitophagy Neurodegenerative diseases have been linked to a dysfunctional mitochondrial quality control system that is partially maintained by proteins PINK1 and Parkin. Whereas mitophagy pathways are becoming well-characterized, less is known about the molecular mechanisms of PINK1 trafficking in mitochondria. Accordingly, we have used a small molecule probe (MitoBloCK-10/MB-10) that modulates the activity of TIMM44, an essential component of the protein associated motor (PAM) complex for the mitochondrial inner membrane (TIM23) translocase, to characterize PINK1 import. MB-10 did not inhibit the import or degradation of PINK1 in energized mitochondria. However, when mitophagy was induced by the addition of an uncoupler or respiratory inhibitor, MB-10 treatment altered PINK1 trafficking by inhibiting association with the TOM complex and impairing Parkin recruitment and subsequent mitophagy. MB-10 analogs that did not inhibit TIMM44 activity failed to impair mitophagy, thereby assigning specificity to MB-10. Because PINK1 undergoes lateral release from the TIM23 translocon to interact with inner membrane (IM) modulators, our studies support that TIMM44 may be a key regulator and that the PAM complex has a central role in regulating PINK1-dependent mitophagy. Our studies also provide a probe for dissecting PINK1/Parkin events for mitochondria as well as studying PINK1-dependent mitophagy in cell and animal models.

sentences: Intermittent Fasting Activates AMP-Kinase to Restructure Right Ventricular Lipid Metabolism and Microtubules in Rodent Pulmonary Arterial Hypertension Intermittent fasting (IF) extends lifespan via pleotropic mechanisms, but one important molecular mediator of the beneficial effects of IF is AMP-kinase (AMPK). AMPK enhances lipid metabolism and modulates microtubule dynamics. Dysregulation of these two molecular pathways causes right ventricular (RV) failure in pulmonary arterial hypertension (PAH). In rodent PAH, IF activates RV AMPK, which restores mitochondrial and peroxisomal density and restructures mitochondrial and peroxisomal lipid metabolism protein regulation. In addition, IF increases electron transport chain (ETC) protein abundance and activity in the RV. Echocardiographic and hemodynamic measures of RV function are positively associated with fatty acid oxidation and ETC protein levels in correlational heatmapping analyses. IF also combats heightened microtubule density, which normalizes t-tubule structure. In summation, we demonstrate IF-mediated AMPK signaling counteracts two key molecular drivers of RV failure. Thus, IF may be a novel treatment approach for RV dysfunction, a currently untreatable and lethal consequence of PAH.

sentences: The gut bacterial natural product colibactin triggers induction of latent viruses in diverse bacteria Colibactin is a chemically unstable small molecule genotoxin produced by multiple different bacteria, including members of the human gut microbiome.1,2 While the biological activity of colibactin has been extensively investigated in mammalian systems,3 little is known about its effects on other microorganisms. Here, we discover that colibactin targets bacteria carrying prophages, inducing lytic development via the bacterial SOS response. DNA, added exogenously, protects bacteria from colibactin, as does expressing a colibactin resistance protein (ClbS) in non-colibactin-producing cells. The prophage-inducing effects we observe apply broadly across taxonomically diverse phage-bacteria systems. Finally, we identify bacteria that possess colibactin resistance genes but lack colibactin biosynthetic genes. Many of these bacteria are infected with predicted prophages, and we show that the expression of their ClbS homologs provides immunity from colibactin-triggered induction. Our study reveals a mechanism by which colibactin production could impact microbiomes and highlights an underappreciated role for microbial natural products in influencing population-level events such as phage outbreaks.

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FishPhyloMaker: An R package to generate phylogenies for ray-finned fishes Phylogenies summarize evolutionary information that is essential in the investigation of ecological and evolutionary causes of diversity patterns. They allow investigating hypotheses from trait evolution to the relationship between evolutionary diversity and ecosystem functioning. However, obtaining a comprehensive phylogenetic hypothesis can be difficult for some groups, especially those with a high number of species, that is the case for fishes, particularly tropical ones. The lack of species in phylogenetic hypotheses, called Darwinian shortfalls, can hinder ecological and evolutionary studies involving this group. To tackle this problem, we developed FishPhyloMaker, an R package that facilitates the generation of phylogenetic trees through a reliable and reproducible procedure, even for a large number of species. The package adopts well-known rules of insertion based on cladistic hierarchy, allowing its use by specialists and non-specialists in fish systematics. We tested the reliability of our algorithm in maintaining important properties of phylogenetic distances running a sensitivity analysis. We also exemplified the use of the FishPhyloMaker package by constructing complete phylogenies for fishes inhabiting the four richest freshwater ecoregions of the world. Furthermore, we proposed a new method to calculate Darwinian shortfalls and mapped this information for the major freshwater drainages of the world. FishPhyloMaker will expand the range of evolutionary and ecological questions that can be addressed using ray-finned fishes as study models, mainly in the field of community phylogenetics, by providing an easy and reliable way to obtain comprehensive phylogenies. Further, FishPhyloMaker presents the potential to be extended to other taxonomic groups that suffer from the same difficulty in the obtention of comprehensive phylogenetic hypothesis.

HighlightsO_LIWe provide the first automated procedure to check species names, construct phylogenetic trees and calculate Darwinian shortfalls for ray-finned fishes (Actinopterygii) by the R package FishPhyloMaker.
C_LIO_LIThis package provides functions to assemble phylogenies through a fast, reliable, and reproducible method, allowing its use and replicability by specialists and non-specialists in fish systematics.
C_LIO_LIThe package also provides an interactive procedure that gives more flexibility to the user when compared with other existing tools that construct phylogenetic trees for other highly speciose groups.
C_LIO_LIThe package includes a new method to compute Darwinian shortfalls for ray-finned fishes, but the rationale of the provided algorithm can be extended in future studies to be used in other groups of organisms
C_LI

sentences: Optimal control methods for nonlinear parameter estimation in biophysical neuron models Functional forms of biophysically-realistic neuron models are constrained by neurobiological and anatomical considerations, such as cell morphologies and the presence of known ion channels. Despite these constraints, neurons models still contain unknown static parameters which must be inferred from experiment. This inference task is most readily cast into the framework of state-space models, which systematically takes into account partial observability and measurement noise. Inferring only dynamical state variables such as membrane voltages is a well-studied problem, and has been approached with a wide range of techniques beginning with the well-known Kalman filter. Inferring both states and fixed parameters, on the other hand, is less straightforward. Here, we develop a method for joint parameter and state inference that combines traditional state space modeling with chaotic synchronization and optimal control. Our methods are tailored particularly to situations with considerable measurement noise, sparse observability, very nonlinear or chaotic dynamics, and highly uninformed priors. We illustrate our approach both in a canonical chaotic model and in a phenomenological neuron model, showing that many unknown parameters can be uncovered reliably and accurately from short and noisy observed time traces. Our method holds promise for estimation in larger-scale systems, given ongoing improvements in calcium reporters and genetically-encoded voltage indicators.

sentences: Small size of recorded neuronal structures confines the accuracy in direct axonal voltage measurements Patch-clamp instruments including amplifier circuits and pipettes affect the recorded voltage signals. We hypothesized that realistic and complete in silico representation of recording instruments together with detailed morphology and biophysics of small recorded structures will precisely reveal signal distortions and provides a tool that predicts native signals from distorted voltage recordings. Therefore, we built a model that was verified by small axonal recordings. The model accurately recreated actual action potential measurements with typical recording artefacts and predicted the native electrical behavior. The simulations verified that recording instruments substantially filter voltage recordings. Moreover, we revealed that instrumentation directly interferes with local signal generation depending on the size of the recorded structures, which complicates the interpretation of recordings from smaller structures, such as axons. However, our model offers a straightforward approach that predicts the native waveforms of fast voltage signals and the underlying conductances even from the smallest neuronal structures.

sentences: ILC3s select for RORγt+ Tregs and establish tolerance to intestinal microbiota Microbial colonization of the mammalian intestine elicits inflammatory or tolerogenic T cell responses, but the mechanisms controlling these distinct outcomes remain poorly understood and accumulating evidence indicates that aberrant immunity to intestinal microbiota is causally associated with infectious, inflammatory, and malignant diseases1-8. Here, we define a critical pathway controlling the fate of inflammatory versus tolerogenic T cells that are specific for the microbiota and express the transcription factor ROR{gamma}t. We profiled all ROR{gamma}t+ immune cells at single cell resolution from the intestine-draining lymph nodes of mice and reveal a dominant presence of Tregs and lymphoid tissue-induced (LTi)-like group 3 innate lymphoid cells (ILC3s), which co-localize at interfollicular regions. These ILC3s have interconverting potential with ROR{gamma}t+ extrathymic Aire-expressing cells, abundantly express major histocompatibility complex class II, and are necessary and sufficient to promote microbiota-specific ROR{gamma}t+ Tregs and prevent their expansion as inflammatory T helper (Th)17 cells. This occurs through ILC3-mediated antigen-presentation, interleukin-2 gradients, and v integrin. Finally, single-cell analyses demonstrate that ILC3 and ROR{gamma}t+ Treg interactions are impaired in inflammatory bowel disease. Our results define a novel paradigm whereby ILC3s positively select for antigen specific ROR{gamma}t+ Tregs, and against Th17 cells, to establish immune tolerance to the microbiota and intestinal health.

sentences: γδ T cells are effectors of immune checkpoint blockade in mismatch repair-deficient colon cancers with antigen presentation defects DNA mismatch repair deficient (MMR-d) cancers present an abundance of neoantigens that likely underlies their exceptional responsiveness to immune checkpoint blockade (ICB)1,2. However, MMR-d colon cancers that evade CD8+ T cells through loss of Human Leukocyte Antigen (HLA) class I-mediated antigen presentation3-6, frequently remain responsive to ICB7 suggesting the involvement of other immune effector cells. Here, we demonstrate that HLA class I-negative MMR-d cancers are highly infiltrated by {gamma}{delta} T cells. These {gamma}{delta} T cells are mainly composed of V{delta}1 and V{delta}3 subsets, and express high levels of PD-1, activation markers including cytotoxic molecules, and a broad repertoire of killer-cell immunoglobulin-like receptors (KIRs). In vitro, PD-1+ {gamma}{delta} T cells, isolated from MMR-d colon cancers, exhibited a cytolytic response towards HLA class I-negative MMR-d colon cancer cell lines and {beta}2-microglobulin (B2M)-knockout patient-derived tumor organoids (PDTOs), which was enhanced as compared to antigen presentation-proficient cells. This response was diminished after blocking the interaction between NKG2D and its ligands. By comparing paired tumor samples of MMR-d colorectal cancer patients obtained before and after dual PD-1 and CTLA-4 blockade, we found that ICB profoundly increased the intratumoral frequency of {gamma}{delta} T cells in HLA class I-negative cancers. Taken together, these data indicate that {gamma}{delta} T cells contribute to the response to ICB therapy in patients with HLA class I-negative, MMR-d colon cancers, and illustrate the potential of {gamma}{delta} T cells in cancer immunotherapy.

sentences: SARS-CoV-2 spike glycoprotein S1 induces neuroinflammation in BV-2 microglia In addition to respiratory complications produced by SARS-CoV-2, accumulating evidence suggests that some neurological symptoms are associated with the disease caused by this coronavirus. In this study, we investigated the effects of the SARS-CoV-2 spike protein S1 stimulation on neuroinflammation in BV-2 microglia. Analyses of culture supernatants revealed an increase in the production of TNF, IL-6, IL-1{beta} and iNOS/NO. S1 also increased protein levels of phospho-p65 and phospho-I{kappa}B, as well as enhancing DNA binding and transcriptional activity of NF-{kappa}B. These effects of the protein were blocked in the presence of BAY11-7082 (1 M). Exposure of S1 to BV-2 microglia also increased the protein levels of NLRP3 inflammasome and enhanced caspase-1 activity. Increased protein levels of p38 MAPK was observed in BV-2 microglia stimulated with the spike protein S1 (100 ng/mL), an action that was reduced in the presence of SKF 86002 (1 M). Results of immunofluorescence microscopy showed an increase in TLR4 protein expression in S1-stimulated BV-2 microglia. Furthermore, pharmacological inhibition with TAK 242 (1 M) and transfection with TLR4 siRNA resulted in significant reduction in TNF and IL-6 production in S1-stimulated BV-2 microglia. These results have provided the first evidence demonstrating S1-induced neuroinflammation in BV-2 microglia. We propose that induction of neuroinflammation by this protein in the microglia is mediated through activation of NF-{kappa}B and p38 MAPK, possibly as a result of TLR4 activation. These results contribute to our understanding of some of the mechanisms involved in CNS pathologies of SARS-CoV-2.

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INPP4B promotes leukemia by restricting leukemic stem cell differentiation through regulation of lysosomal functions Signaling pathways that control vital features of leukemic stem cells including multipotency, self-renewal, clonal expansion and quiescence remain unclear. Emerging studies illustrate critical roles for lysosomes in hematopoietic and leukemic stem cell fate. By investigating consequences of INPP4B alterations in AML, we have discovered its role in driving leukemic  stemness. We observed that INPP4B is highly expressed leukemic stem cell populations and Inpp4b-deficeint leukemias demonstrate increased disease latency, reduced leukemia initiating potential which is associated with a differentiated leukemic phenotype. Molecular analyses show that Inpp4b-deficient leukemias have compromised lysosomal gene expression, lysosomal content, and lysosomal activity. Our discovery of a novel pathway linking INPP4B, lysosomal biogenesis and leukemic stemness, provides a mechanism to explain the association of high INPP4B expression with poor AML prognosis, and highlights novel patient stratification strategies and LSC-specific leukemic therapies.

Key PointsOur findings highlight a novel pathway linking INPP4B, lysosomal function and leukemic stemness that explains the prognostic role of INPP4B in AML.
Our data reveal the utility of INPP4B as a biomarker of aggressive AML and provide a rationale to explore INPP4B and its associated function in lysosome biology as novel strategies to target LSC and AML

sentences: Prdm16 and Notch functionally and physically interact during artery development Proper vascular function requires correct arteriovenous (AV) endothelial cell (EC) differentiation. While Notch and its effector Hey2 favor arterial specification, transcription factor (TF) Coup-TFII inhibits Notch activity to induce venous identity. TFs competing with Coup-TFII to orchestrate arterial specification upstream of Notch remain largely unknown. Transcriptional profiling of human and mouse ECs and whole mount in situ hybridization in zebrafish embryos revealed that the TF Prdm16 is exclusively expressed by arterial ECs throughout development, independent of species and hemodynamic factors. Accordingly, prdm16 deficiency in zebrafish perturbed AV specification and caused aberrant shunts between the arterial and venous circulation in an EC- autonomous manner. Prdm16 regulated arterial Notch activity both in vitro and in vivo and simultaneous knockdown of notch aggravated the vascular defects observed in prdm16 morphant zebrafish. In vitro studies revealed that Prdm16 did not alter the levels of the transcriptionally active, intracellular domain of the Notch1 receptor (N1ICD). Rather, it boosted Notch signaling by physically and functionally interacting with both N1ICD and the Notch downstream effector Hey2. Together, these data demonstrate the hitherto unexplored role for Prdm16 during arterial development, by its dual activity on arterial canonical Notch during embryogenesis.

sentences: Stimulus degradation impairs performance in a rodent continuous performance test Sustained attention is a core cognitive domain that is often disrupted in neuropsychiatric disorders. Continuous performance tests (CPTs) are the most common clinical assay of sustained attention. In CPTs, participants produce a behavioral response to target stimuli and refrain from responding to non-target stimuli. Performance in CPTs is measured as the ability to discriminate between targets and non-targets. Rodent versions of CPTs (rCPT) have been developed and validated with both anatomical and pharmacological studies, providing a translational platform for understanding the neurobiology of sustained attention. In human studies, using degraded stimuli (decreased contrast) in CPTs impairs performance and patients with schizophrenia experience a larger decrease in performance compared to healthy controls. In this study, we tested multiple levels of stimulus degradation in a touchscreen version of the CPT in mice. We found that stimulus degradation significantly decreased performance in both males and females. The changes in performance consisted of a decrease in stimulus discrimination, measured as d, and increases in hit reaction time and reaction time variability. These findings are in line with the effects of stimulus degradation in human studies. Overall, female mice demonstrated a more liberal response strategy than males, but response strategy was not affected by stimulus degradation. These data extend the utility of the mouse CPT by demonstrating that stimulus degradation produces equivalent behavioral responses in mice and humans. Therefore, the degraded stimuli rCPT has high translational value as a preclinical assay of sustained attention.

sentences: Detecting expressed cancer somatic mutations from single-cell RNA sequencing data Identifying expressed somatic mutations directly from single-cell RNA sequencing (scRNA-seq) data is challenging but highly valuable. Computational methods have been attempted but no reliable methods have been reported to identify somatic mutations with high fidelity. We present RESA -- Recurrently Expressed SNV Analysis, a computational framework that identifies expressed somatic mutations from scRNA-seq data with high precision. We test RESA in multiple cancer cell line datasets, where RESA demonstrates average area under the curve (AUC) of 0.9 on independently held out test sets, and achieves average precision of 0.71 when evaluated by bulk whole exome, which is substantially higher than previous approaches. In addition, RESA detects a median of 201 mutations per cell, 50 times more than what was reported in experimental technologies with simultaneous expression and mutation profiling. Furthermore, applying RESA to scRNA-seq from a melanoma patient, we demonstrate that RESA recovers the known BRAF driver mutation of the sample and melanoma dominating mutational signatures, identifies mutation associated expression signatures, reveals nondriver perturbed and stage specific cancer hallmarks, and unveils the complex relationship between genomic and transcriptomic intratumor heterogeneity. Therefore, RESA could provide novel views in the study of intratumor heterogeneity and relate genetic alterations to transcriptional changes at single cell level.

sentences: Single cell profiling of T and B cell repertoires following SARS-CoV-2 mRNA vaccine mRNA based vaccines for SARS-CoV-2 have shown exceptional clinical efficacy providing robust protection against severe disease. However, our understanding of transcriptional and repertoire changes following full vaccination remains incomplete. We used single-cell RNA sequencing and functional assays to compare humoral and cellular responses to two doses of mRNA vaccine with responses observed in convalescent individuals with asymptomatic disease. Our analyses revealed enrichment of spike-specific B cells, activated CD4 T cells, and robust antigen-specific polyfunctional CD4 T cell responses in all vaccinees. On the other hand, CD8 T cell responses were both weak and variable. Interestingly, clonally expanded CD8 T cells were observed in every vaccinee, as observed following natural infection. TCR gene usage, however, was variable, reflecting the diversity of repertoires and MHC polymorphism in the human population. Natural infection induced expansion of larger CD8 T cell clones occupied distinct clusters, likely due to the recognition of a broader set of viral epitopes presented by the virus not seen in the mRNA vaccine. Our study highlights a coordinated adaptive immune response where early CD4 T cell responses facilitate the development of the B cell response and substantial expansion of effector CD8 T cells, together capable of contributing to future recall responses.

sentences: Interrogating theoretical models of neural computation with emergent property inference 1A cornerstone of theoretical neuroscience is the circuit model: a system of equations that captures a hypothesized neural mechanism. Such models are valuable when they give rise to an experimentally observed phenomenon - whether behavioral or a pattern of neural activity - and thus can offer insights into neural computation. The operation of these circuits, like all models, critically depends on the choice of model parameters. A key step is then to identify the model parameters consistent with observed phenomena: to solve the inverse problem. In this work, we present a novel technique, emergent property inference (EPI), that brings the modern probabilistic modeling toolkit to theoretical neuroscience. When theorizing circuit models, theoreticians predominantly focus on reproducing computational properties rather than a particular dataset. Our method uses deep neural networks to learn parameter distributions with these computational properties. This methodology is introduced through a motivational example inferring conductance parameters in a circuit model of the stomatogastric ganglion. Then, with recurrent neural networks of increasing size, we show that EPI allows precise control over the behavior of inferred parameters, and that EPI scales better in parameter dimension than alternative techniques. In the remainder of this work, we present novel theoretical findings gained through the examination of complex parametric structure captured by EPI. In a model of primary visual cortex, we discovered how connectivity with multiple inhibitory subtypes shapes variability in the excitatory population. Finally, in a model of superior colliculus, we identified and characterized two distinct regimes of connectivity that facilitate switching between opposite tasks amidst interleaved trials, characterized each regime via insights afforded by EPI, and found conditions where these circuit models reproduce results from optogenetic silencing experiments. Beyond its scientific contribution, this work illustrates the variety of analyses possible once deep learning is harnessed towards solving theoretical inverse problems.

sentences: Laminar Mechanisms of Saccadic Suppression in Primate Visual Cortex Saccades are a ubiquitous and crucial component of our visual system, allowing for the efficient deployment of the fovea and its accompanying neural resources. Initiation of a saccade is known to cause saccadic suppression, a temporary reduction in visual sensitivity1, 2 and visual cortical firing rates3-6. While saccadic suppression has been well characterized at the level of perception and single neurons, relatively little is known about the visual cortical networks governing this phenomenon. Here we examine the effects of saccadic suppression on distinct neural subpopulations within visual area V4. We find cortical layer- and cell type-specific differences in the magnitude and timing of peri-saccadic modulation. Neurons in the input layer show changes in firing rate and inter-neuronal correlations prior to saccade onset, indicating that this layer receives information about impending saccades. Putative inhibitory interneurons in the input layer elevate their firing rate during saccades, suggesting they play a role in suppressing the activity of other cortical subpopulations. A computational model of this circuit recapitulates our empirical observations and demonstrates that an input layer-targeting pathway can initiate saccadic suppression by enhancing local inhibitory activity. Collectively, our results provide a mechanistic understanding of how eye movement signaling interacts with cortical circuitry to enforce visual stability.

sentences: All-Atom MD Simulations of the HBV Capsid Complexed with AT130 Reveal Secondary and Tertiary Structural Changes and Mechanisms of Allostery The hepatitis B virus (HBV) capsid is an attractive drug target, relevant to combating viral hepatitis as a major public health concern. Among small molecules known to interfere with capsid assembly, the phenylpropenamides, including AT130, represent an important anti-viral paradigm based on disrupting the timing of genome encapsulation. Crystallographic studies of AT130-bound complexes have been essential in explaining the effects of the small molecule on HBV capsid structure; however, computational examination reveals that key changes attributed to AT130 were erroneous, likely a consequence of interpreting poor resolution arising from a highly flexible protein. Here, all-atom molecular dynamics simulations of an intact AT130-bound HBV capsid reveal that, rather than damaging spike helicity, AT130 enhances the capsids ability to recover it. A new conformational state is identified, which can lead to dramatic opening of the intradimer interface and disruption of communication within the spike tip. A novel salt bridge is also discovered, which can mediate contact between the spike tip and fulcrum even in closed conformations, revealing a mechanism of direct communication across these domains. Combined with dynamical network analysis, results describe a connection between the intra- and interdimer interfaces and enable mapping of allostery traversing the entire capsid protein dimer.

sentences: CpG enrichment as a vaccine strategy for influenza A virus Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. However, such recoding typically hinders virus growth. A potential solution to this is CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), leading to viral RNA degradation and/or type I interferon stimulation and consequently reduced viral yields. Therefore, in principle, removing the ZAP CpG sensor from a virus propagation system will reverse attenuation of a CpG-enriched virus. Using influenza A viruses (IAVs) engineered for increased CpG content in different genome regions, we show that ZAP mediated virus attenuation is dependent on the viral segment to which CpGs are added. In ZAP-expressing human cells, CpG introduction invariably impaired transcript and protein production, but ZAP-sensitivity did not convey type I interferon activation. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Furthermore, the ZAP-sensitive virus was fully replication competent in MDCK cells and in embryonated hens eggs, both of which are used to propagate live attenuated influenza vaccines. Thus, ZAP-sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform for live attenuated vaccine development.

sentences: The Genetic Basis of Differential Autodiploidization in Evolving Yeast Populations Spontaneous whole-genome duplication, or autodiploidization, is a common route to adaptation in experimental evolution of haploid budding yeast populations. The rate at which autodiploids fix in these populations appears to vary across strain backgrounds, but the genetic basis of these differences remains poorly characterized. Here we show that the frequency of autodiploidization differs dramatically between two closely related laboratory strains of Saccharomyces cerevisiae, BY4741 and W303. To investigate the genetic basis of this difference, we crossed these strains to generate hundreds of unique F1 segregants and tested the tendency of each segregant to autodiplodize across hundreds of generations of laboratory evolution. We find that variants in the SSD1 gene are the primary genetic determinant of differences in autodiploidization. We then used multiple laboratory and wild strains of S. cerevisiae to show that clonal populations of strains with a functional copy of SSD1 autodiploidize more frequently in evolution experiments, while knocking out this gene or replacing it with the W303 allele reduces autodiploidization propensity across all genetic backgrounds tested. These results suggest a potential strategy for modifying rates of spontaneous whole-genome duplications in laboratory evolution experiments in haploid budding yeast. They may also have relevance to other settings in which eukaryotic genome stability plays an important role, such as biomanufacturing and the treatment of pathogenic fungal diseases and cancers.

sentences: Comparative genome analysis revealed gene inversions, boundary expansion and contraction, and gene loss in Stemona sessilifolia (Miq.) Miq. chloroplast genome Stemona sessilifolia (Miq.) Miq., commonly known as Baibu, is one of the most popular herbal medicines in Asia. In Chinese Pharmacopoeia, Baibu has multiple authentic sources, and there are many homonym herbs sold as Baibu in the herbal medicine market. The existence of the counterfeits of Baibu brings challenges to its identification. To assist the accurate identification of Baibu, we sequenced and analyzed the complete chloroplast genome of Stemona sessilifolia using next-generation sequencing technology. The genome was 154,039 bp in length, possessing a typical quadripartite structure consisting of a pair of inverted repeats (IRs: 27,094 bp) separating by a large single copy (LSC: 81,950 bp) and a small single copy (SSC: 17,901 bp). A total of 112 unique genes were identified, including 80 protein-coding, 28 transfer RNA, and four ribosomal RNA genes. Besides, 45 tandem, 27 forward, 23 palindromic, and 72 simple sequence repeats were detected in the genome by repeat analysis. Compared with its counterfeits (Asparagus officinalis and Carludovica palmate), we found that IR expansion and SSC contraction events of Stemona sessilifolia resulted in two copies of the rpl22 gene in the IR regions and partial duplication of the ndhF gene in the SSC region. Secondly, an approximately 3-kb-long inversion was identified in the LSC region, leading to the petA and cemA gene presented in the complementary strand of the chloroplast DNA molecule. Comparative analysis revealed some highly variable regions, including trnF-GAA_ndhJ, atpB_rbcL, rps15_ycf1, trnG-UCC_trnR-UCU, ndhF_rpl32. Finally, gene loss events were investigated in the context of phylogenetic relationships. In summary, the complete plastome of Stemona sessilifolia will provide valuable information for the molecular identification of Baibu and assist in elucidating the evolution of Stemona sessilifolia.

sentences: Subgenomic SARS-CoV-2 replicon and reporter replicon cell lines enable ultrahigh throughput antiviral screening and mechanistic studies with antivirals, viral mutations or host factors that affect COVID-19 replication Replicon-based technologies were used to develop reagents and assays for advanced drug discovery efforts against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and for examining all facets of the SARS-CoV-2 replication cycle at reduced biocontainment level. Specifically: a) 21 replicons were cloned in bacterial artificial chromosomes (BACs) and delivered as transfectable plasmid DNA or transcribed RNA in various cell types. Replicons carrying mutations that affect the activity or antiviral susceptibility of SARS-CoV-2 enzymes were used to establish utility for mechanistic studies while reducing the community risks associated with gain-of-function studies in fully infectious virus. b) A BHK-21 stable cell line harboring SARS-CoV-2 replicon was generated and characterized in robust high/ultra-high throughput assays of antiviral efficacy with orthogonal SARS-CoV-2 replication reporter genes (Nano luciferase and enhanced green fluorescent protein-eGFP); the estimated antiviral potencies in the fully infectious SARS-CoV-2 system and in the transient or stable replicon systems were similar. HEK293 and Calu1 stable cell lines expressing SARS-CoV-2 replicon have also been prepared. Finally, c) we generated trans-encapsidated replicons by co-expression with SARS-CoV-2 structural proteins, thus producing single-round infectious SARS-CoV-2 virus-like particles that are able to transduce susceptible cell types and have expanded utility to enable study of virion assembly and entry into target cells. Hence, these SARS-CoV-2 replicon-based reagents include a novel approach to replicon-harboring cell line generation and are valuable tools that can be used at lower biosafety level (BSL2) for drug discovery efforts, characterization of SARS-CoV-2 and variant evolution in the COVID-19 pandemic, mechanisms of inhibition and resistance, and studies on the role of SARS-CoV-2 genes and host dependency factors.

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Weak Task Synchronization of Default Mode Network in Task Based Paradigms Default mode network (DMN) are the areas of the brain that get deactivated upon the presentation of externally focused, attention demanding tasks. Interestingly, these areas are consistently deactivated irrespective of the nature of the task. Recent evidence shows that the behavior of the DMN may be more nuanced than what was earlier believed. We wanted to investigate the dynamics of DMN further and utilized novel statistical approaches to study the same. We analysed four publicly available datasets (total 223 subjects) across seven tasks related to different cognitive modalities and found out that there is a large amount of variability in the presumed task-negative network across subjects, trials and regions. We designed a method called Inter Trial Temporal Synchronization Analysis (IT-TSA) and Inter Subject TSA (IS-TSA) to analyse variability across trials and subjects respectively. We found out that all task-negative regions and even weak task-positive regions have low synchronization across trials and subjects. We hypothesize that the DMN variability might be more due to neural differences rather than structural or HRF variations. We also investigated the use of non GLM technique like the Inter Subject Correlation (ISC) and found that it may only be suitable to study tightly synchronized task-positive regions. Our study challenges the understanding of DMN as a task-negative region and advances the findings that DMN acts as an active component associated with a unique set of self-referential, autobiographical processes which are deactivated differentially and non linearly across trials and subjects in the presence of extrinsic processes.

HighlightsO_LIDefault Mode Network is weakly synchronized across trials and subjects in task paradigms.
C_LIO_LIInter Subject Correlation is not suited to detect task-based activations in DMN.
C_LIO_LISynchronization Analysis reveals temporal structure in fMRI data.
C_LI

sentences: Compartmentalization and persistence of dominant (regulatory) T cell clones indicates antigen skewing in juvenile idiopathic arthritis Autoimmune inflammation is characterized by tissue infiltration and expansion of antigen-specific T cells. Although this inflammation is often limited to specific target tissues, it remains yet to be explored whether distinct affected sites are infiltrated with the same, persistent T cell clones. Here we performed CyTOF analysis and T cell receptor (TCR) sequencing to study immune cell composition and (hyper-)expansion of circulating and joint-derived Tregs and non-Tregs in Juvenile Idiopathic Arthritis (JIA). We studied different joints affected at the same time, as well as over the course of relapsing-remitting disease. We found that the composition and functional characteristics of immune infiltrates are strikingly similar between joints within one patient, and observed a strong overlap between dominant T cell clones, especially Treg, of which some could also be detected in circulation and persisted over the course of relapsing remitting disease. Moreover, these T cell clones were characterized by a high degree of sequence similarity, indicating the presence of TCR clusters responding to the same antigens. These data suggest that in localized autoimmune disease there is auto-antigen driven expansion of both Teffector and Treg clones, that are highly persistent and are (re)circulating. These dominant clones might represent interesting therapeutic targets.

sentences: Mate choice enhances post-zygotic barriers to gene flow via ancestry bundling Hybridization and subsequent genetic introgression are now known to be common features of the histories of many species, including our own. Following hybridization, selection often purges introgressed DNA genome-wide. While mate choice can prevent hybridization in the first place, it is also known to play an important role in post-zygotic selection against hybrids, and thus the purging of introgressed DNA. However, this role is usually thought of as a direct one: a mating preference for conspecifics reduces the sexual fitness of hybrids, reducing the transmission of introgressed ancestry. Here, we explore a second, indirect role of mate choice as a barrier to gene flow. Under assortative mating, parents covary in their ancestry, causing ancestry to be "bundled" in their offspring and later generations. This bundling effect increases ancestry variance in the population, enhancing the efficiency with which post-zygotic selection purges introgressed DNA. Using whole-genome simulations, we show that the bundling effect can comprise a substantial portion of mate choices overall effect as a post-zygotic barrier to gene flow, and that it is driven by ancestry covariances between and within maternally and paternally inherited genomes. We derive a simple method for estimating the impact of the bundling effect from standard measures of assortative mating. Applying this method to data from a diverse set of hybrid zones, we find that the bundling effect increases the purging of introgressed DNA by between 1.2-fold (in a baboon system with weak assortative mating) and 14-fold (in a swordtail system with strong assortative mating). Thus, the bundling effect of mate choice contributes substantially to the genetic isolation of species.

sentences: Mapping the microbial diversity and natural resistome of north Antarctica soils The rising of multiresistant bacterial pathogens is currently one of the most critical threats to global health, demanding a better understanding of the origin and spread of antibiotic resistance. In this regard, the resistome hosted by the microbiota from natural and remote environments remains poorly explored. Moreover, little is known about the availability of antimicrobial resistance genes (ARGs) from these environments to be disseminated through horizontal transfer, potentially mediating the rise of novel resistance factors among clinically relevant pathogens. In this context, the North Antarctica soils are attractive ecosystems to study due to the presence of a microbiota naturally adapted to thrive in harsh conditions, including potential factors to resist natural toxic substances. In this work, we evaluated the antibiotic resistance of bacteria isolated from soils collected in humanized and non-intervened areas of North Antarctica. We identified resistance to a wide array of antibiotics, with isolates harboring up to 10 simultaneous resistances, mainly native Pseudomonas. Genomic analysis revealed the presence of a wide array of genes encoding efflux pumps but the lack of genes explaining some of the resistance phenotypes, suggesting novel uncharacterized mechanisms. Also, using 16S rRNA amplicon and shotgun metagenome sequencing, we explored the microbial diversity in the sampled soils and evaluated the presence of ARGs and their host microbiota. High microbial diversity was found in all the sites, with Proteobacteria, Bacteroidota, Acidobacteriota, and Verrucomicrobiota being the most abundant Phyla, while Candidatus Udaeobacter, RB41, Polaromonas, and Ferruginibacter the most abundant genera. We identified hundreds of genes potentially conferring resistance to more than 15 drug classes, both by short reads analyses and ARG detection among assembled contigs and MAGs obtained combining short and long-read sequence data. Polaromonas, Pseudomonas, Streptomyces, Variovorax, Bhurkolderia, and Gemmatimonas were the main host taxa of the identified ARGs. Part of these ARGs was found inside predicted plasmids, including a putative OXA-like beta-lactamase from Polaromonas harboring the key conserved residues of this kind of enzyme and a conserved predicted protein structure. All this evidence indicates that microbial communities from North Antarctica soil have a highly diverse natural resistome, part of it located inside mobile genetic elements, which would act as a source of novel ARGs.

sentences: Epigenomic signature of the progeroid Cockayne syndrome exposes distinct and common features with physiological ageing Cockayne syndrome (CS) and UV-sensitivity syndrome (UVSS) are rare genetic disorders caused by mutation of the DNA repair and chromatin remodelling proteins CSA or CSB, but only CS patients display a progeroid and neurodegenerative phenotype. As epigenetic modifications constitute a well-established hallmark of ageing, we characterized genome-wide DNA methylation (DNAm) of fibroblasts from CS versus UVSS patients and healthy donors. The analysis of differentially methylated positions and regions revealed a CS-specific epigenetic signature, enriched in developmental transcription factors, transmembrane transporters, and cell adhesion factors. The CS-specific signature compared to DNAm changes in other progeroid diseases and regular ageing, identifyied commonalities and differences in epigenetic remodelling. CS shares DNAm changes with normal ageing more than other progeroid diseases do, and according to the methylation clock CS samples show up to 13-fold accelerated ageing. Thus, CS is characterized by a specific epigenomic signature that partially overlaps with and exacerbates DNAm changes occurring in physiological aging. Our results unveil new genes and pathways that are potentially relevant for the progeroid/degenerative CS phenotype.

sentences: In vivo physiology of foveal retinal ganglion cells in Macaca fascicularis The primate fovea is specialized for high-resolution spatial and chromatic vision, possessing a unique topography characterized by a pit in which the inner retina and associated vasculature have been excavated, the highest density of cones anywhere in the retina, and a disproportionately large representation in visual cortex. The crucial visual properties conferred by the fovea are mediated by retinal ganglion cells (RGCs) the somas of which lie at the margin of the foveal pit. Microelectrode recordings of these centermost retinal ganglion cells have been challenging due to the fragility of the fovea in the excised retina. This has been overcome in this study by combining high resolution fluorescence adaptive optics ophthalmoscopy with calcium imaging to optically record functional responses of foveal RGCs to visual stimuli in the living eye. Here we use this approach to study the chromatic responses of RGCs to uniform fields modulated in different directions in color space and achromatic responses with monochromatic drifting gratings. We repeated these recordings in many cells in each of three macaca fasicularis primates over weeks or months. We find that most of the L/M chromatically opponent cells serving the most central cones respond to high spatial frequencies, reflecting strong surround inhibition that sacrifices sensitivity at low frequencies in favor of the transmission of fine detail in the retinal image. In addition, we find the L/M cells vary in spatial frequency bandwidths, suggesting that L/M chromatic opponency, often considered largely due to input from single cones, has more diverse receptive field structure than previously thought. By characterizing the functional properties of retinal ganglion cells in vivo through adaptive optics, we provide a lower bound on the response characteristics of these cells in situ and their role in vision processing.

sentences: An assembly-free method of phylogeny reconstruction using short-read sequences from pooled samples without barcodes A current strategy for obtaining haplotype information from several individuals involves short-read sequencing of pooled amplicons, where fragments from each individual is identified by a unique DNA barcode. In this paper, we report a new method to recover the phylogeny of haplotypes from short-read sequences obtained using pooled amplicons from a mixture of individuals, without barcoding. The method, AFPhyloMix, accepts an alignment of the mixture of reads against a reference sequence, obtains the single-nucleotide-polymorphisms (SNP) patterns along the alignment, and constructs the phylogenetic tree according to the SNP patterns. AFPhyloMix adopts a Bayesian model of inference to estimates the phylogeny of the haplotypes and their relative frequencies, given that the number of haplotypes is known. In our simulations, AFPhyloMix achieved at least 80% accuracy at recovering the phylogenies and frequencies of the constituent haplotypes, for mixtures with up to 15 haplotypes. AFPhyloMix also worked well on a real data set of kangaroo mitochondrial DNA sequences.

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Food web aggregation: effects on key positions Providing standard definitions of what should be considered as a node in food webs is still an unsolved problem. Especially for comparative and predictive food web modelling, a more systematic understanding is needed for the effects of trophic aggregation procedures. Aggregation is unavoidable during data management. Therefore, it is crucial to know whether food web properties are conserved during this process.

Here, we study how different aggregation methods change the positional importance of species in food webs. In particular, we investigated the effects of various aggregation algorithms on 24 indices of importance. Our work was carried out on 76 aquatic food webs coming from the Ecopath with Ecosim database (EcoBase). We considered six main types of aggregation, according to the way that the nodes were clustered. These were (i) hierarchical clustering based on the Jaccard index, (ii) hierarchical clustering based on the regular equivalence index (REGE), (iii) maximisation of directed modularity, (iv) maximisation of modularity according to modules in which species fed on the same preys, (v) maximisation of modularity according to modules in which species are fed upon by the same predators, and (vi) clustering through the group model.

Hierarchical clustering based on the Jaccard index and REGE index outperformed the other four methods on maintaining the relative importance of species for all the indices of importance (except for the contrastatus index (s') and betweenness centrality (BC)). The choice between these two methods should follow our research question and the importance index we are interested in studying. The other four aggregation methods change more the centrality of species, especially the one based on maximising directed modularity. When using these aggregation algorithms, one has to keep in mind that the network will not only be smaller but also provides different information.

sentences: A resource for generating and manipulating human microglial states in vitro Microglia have emerged as key players in the pathogenesis of neurodegenerative conditions such as Alzheimers disease (AD). In response to CNS stimuli, these cells adopt distinct transcriptional and functional subtypes known as states. However, an understanding of the function of these states has been elusive, especially in human microglia, due to lack of tools to model and manipulate this cell-type. Here, we provide a platform for modeling human microglia transcriptional states in vitro. Using single-cell RNA sequencing, we found that exposure of human stem-cell differentiated microglia (iMGLs) to brain-related challenges generated extensive transcriptional diversity which mapped to gene signatures identified in human brain microglia. We identified two in vitro transcriptional clusters that were analogous to human and mouse disease-associated microglia (DAMs), a state enriched in neurodegenerative disease contexts. To facilitate scalable functional analyses, we established a lentiviral approach enabling broad and highly efficient genetic transduction of microglia in vitro. Using this new technology, we demonstrated that MITF (Melanocyte Inducing Transcription Factor), an AD-enriched transcription factor in microglia, drives both a disease-associated transcriptional signature and a highly phagocytic state. Finally, we confirmed these results across iMGLs differentiated from multiple iPSC lines demonstrating the broad utility of this platform. Together, these tools provide a comprehensive resource that enables the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

sentences: Placental gene expression-based cell type deconvolution: Cell proportions drive preeclampsia gene expression differences The placenta mediates adverse pregnancy outcomes, including preeclampsia, which is characterized by gestational hypertension and proteinuria. Placental cell type heterogeneity in preeclampsia is not well-understood and limits mechanistic interpretation of bulk gene expression measures. We generated single-cell RNA-sequencing samples for integration with existing data to create the largest deconvolution reference of 19 fetal and 8 maternal cell types from placental villous tissue at term (n=15,532 cells). We deconvoluted eight published microarray case-control studies of preeclampsia (n=330). Deconvolution revealed excess extravillous trophoblasts and fewer mesenchymal cells. Adjustment for cellular composition reduced preeclampsia-associated differentially expressed genes (FDR<0.05) from 1,224 to 0, whereas pathway alterations exhibiting a metabolic adaptation to hypoxia were robust to cell type adjustment. Cellular composition explained 35.1% of the association between preeclampsia and FLT1 overexpression. Our findings indicate substantial placental cellular heterogeneity in preeclampsia that predicts previously observed bulk gene expression differences. Our deconvolution reference lays the groundwork for cellular heterogeneity-aware investigation into placental dysfunction and adverse birth outcomes.

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Impact of the pear psyllid Cacopsylla pyri host instar on the behavior and fitness of the parasitoid Trechnites insidious O_LIPear is one of the most important fruit crops of temperate regions. The control of its mains pest, Cacopsylla pyri, is still largely based on the use of chemical pesticides, with all that this implies in terms of negative effects on the environment and health.
C_LIO_LIWithin the context of integrated pest management, innovative and ecologically sustainable strategies must be developed for. Although Trechnites insidiosus is the most abundant parasitoid of C. pyri, its biology and its potential as a control agent have been little studied.
C_LIO_LIIn this paper, we conducted experiments to evaluate the behavior of the specialist parasitoid T. insidiosus when exposed to different larval instars of the pear psyllid C. pyri, and to assess the quality of the next generation of parasitoids.
C_LIO_LIWe found that although T. insidiosus accepts all host instars for oviposition, the third and fourth instars were the most suitable host in terms of behavioral acceptance and progeny development.
C_LIO_LIOur study is a first step for further studies on the interaction between psyllids and parasitoids and provides evidence that the specialist parasitoid T. insidiosus is a promising candidate for biological control strategies of the pear psyllid C. pyri.
C_LI

sentences: Can the Insect Path Integration Memory be a Bump Attractor? Many animal species are able to return to their nest after a foraging excursion without using familiar visual cues to guide them. They accomplish this by using a navigation competence known as path integration, which is vital in environments that do not have prominent visual features. To perform path integration, an animal maintains an estimate of the distance and direction to its origin as it moves. The distance and direction information needs to be maintained in memory for the duration of the trip so that the animal can return to its nest. However, the neural substrate of this memory remains unclear. A common hypothesis is that the information is maintained in a bump attractors state. We test the bump attractor hypothesis and find that its predictions do not match behavioural data from ants, thus falsifying the bump attractor hypothesis and raising the need for alternative models of path integration memory.

sentences: Neonatal multi-modal cortical profiles predict 18-month developmental outcomes Developmental delays in infanthood often persist, turning into life-long difficulties, and coming at great cost for the individual and community. By examining the developing brain and its relation to developmental outcomes we can start to elucidate how the emergence of brain circuits is manifested in variability of infant motor, cognitive and behavioural capacities. In this study, we examined if cortical structural covariance at birth, indexing coordinated development, is related to later infant behaviour. We included 193 healthy term-born infants from the Developing Human Connectome Project (dHCP). An individual cortical connectivity matrix derived from morphological and microstructural features was computed for each subject (morphometric similarity networks, MSNs) and was used as input for the prediction of behavioural scores at 18 months using Connectome-Based Predictive Modeling (CPM). Neonatal MSNs successfully predicted social-emotional performance. Predictive edges were distributed between and within known functional cortical divisions with a specific important role for primary and posterior cortical regions. These results reveal that multi-modal neonatal cortical profiles showing coordinated maturation are related to developmental outcomes and that network organization at birth provides an early infrastructure for future functional skills.

sentences: Dynamic Network Curvature Analysis of RNA-Seq Data in Sarcoma In this work, we utilized network features of cancer gene interactomes to cluster pediatric sarcoma tumors and identify candidate therapeutic targets in an unsupervised manner. RNA-Seq data were mapped to protein-level interactomes to construct weighted networks for mathematical analysis. We employed a geometric approach centered on a discrete notion of curvature, which provides a measure of the functional association between genes in the context of their connectivity. Specifically, we adopted a recently proposed dynamic extension of graph curvature to extract features of the non-Euclidean, multiscale structure of genomic networks. We propose a hierarchical clustering approach to reveal preferential gene clustering according to their geometric cooperation which captured the characteristic EWSR1-FLI1 fusion in Ewing sarcoma. We also performed in silico edge perturbations to assess systemic response to simulated interventions quantified by changes in curvature. These results demonstrate that geometric network-based features can be useful for identifying non-trivial gene associations in an agnostic manner.

sentences: Organizing memories for generalization in complementary learning systems Our ability to remember the past is essential for guiding our future behavior. Psychological and neurobiological features of declarative memories are known to transform over time in a process known as systems consolidation. While many theories have sought to explain the time-varying role of hippocampal and neocortical brain areas, the computational principles that govern these transformations remain unclear. Here we propose a theory of systems consolidation in which hippocampal-cortical interactions serve to optimize generalizations that guide future adaptive behavior. We use mathematical analysis of neural network models to characterize fundamental performance tradeoffs in systems consolidation, revealing that memory components should be organized according to their predictability. The theory shows that multiple interacting memory systems can outperform just one, normatively unifying diverse experimental observations and making novel experimental predictions. Our results suggest that the psychological taxonomy and neurobiological organization of declarative memories reflect a system optimized for behaving well in an uncertain future.

sentences: Single-cell RNA sequencing uncovers the excitatory/inhibitory synaptic unbalance in the retrosplenial cortex after peripheral nerve injury Nerve injury in the somatosensory pathway may induce maladaptive changes at the transcriptional or protein level, contributing to the development and maintenance of neuropathic pain. In contrast to the retrosplenial cortex (RSC), which processes nociceptive information and exhibits structural and molecular changes after nerve injury, detailed transcriptional changes in the RSC are not yet known. Here we confirm the involvement of the RSC in regulating pain sensation and observe that the same peripheral stimulation activates more retrosplenial neurons after nerve injury; reducing the activities of CaMKII+ splenial cells relieves peripheral pain hypersensitivity after nerve injury. Using a single-cell RNA sequencing (scRNA-seq) approach, we identified cell-type-specific gene expression changes after nerve injury, and the gene set enrichment analysis results revealed suppressed ion homeostasis in CaMKII+ neurons. Furthermore, examination of the expression of genes encoding ligand-gated ion channels showed a decrease in Gabar1a but an increase in Gria1 in CaMKII+ neurons; consistently, we confirmed the unbalanced excitatory/inhibitory synaptic transmission by using the electrophysiological recording approach. Moreover, micro-infusion of 1-Naphthyl acetyl spermine in the RSC to reduce excitatory synaptic transmission alleviated peripheral pain hypersensitivity. Our data confirm the involvement of the RSC in pain regulation and provide information on cell type-dependent transcriptomic changes after nerve injury, which will contribute to the understanding of the mechanisms mediating neuropathic pain.

sentences:
Spd-2 Gene Duplication Suggests Cell-Type Specific Assembly Mechanisms of Pericentriolar Material Centrosomes are multi-protein complexes that function as the major microtubule organizing center (MTOC) for the cell. While centrosomes play tissue-specific MTOC functions, little is known about how particular centrosome proteins are regulated across cell types to achieve these different functions. To investigate this cell type-specific diversity, we searched for gene duplications of centrosome genes in the Drosophila lineage with the aim of identifying centrosome gene duplications where each copy evolved for specialized functions. Through in depth functional analysis of a Spd-2 gene duplication in the Willistoni group, we discovered differences in the regulation of PCM in somatic and male germline cells. The parental gene, Spd-2A, is expressed in somatic cells, where it can function to organize pericentriolar material (PCM) and the mitotic spindle in larval brain neuroblasts. Spd-2A is absent during male meiosis, and even when ectopically expressed in spermatocytes it fails to rescue PCM and spindle organization. In contrast, the new gene duplicate, Spd-2B, is expressed specifically in spermatocytes. During male meiosis, Spd-2B localizes to centrosomes, organizes PCM and spindles, and is sufficient for proper male fertility. Experiments using chimeric transgenes reveal that differences in the C-terminal tails of Spd-2A and Spd-2B are responsible for these functional changes. Thus, Spd-2A and Spd-2B have evolved complementary functions by specializing for distinct subsets of cells. Together, our results demonstrate that somatic cells and male germline cells have fundamentally different requirements for PCM, suggesting that PCM proteins such as Spd-2 is differentially regulated across cell types to satisfy distinct requirements.

HighlightsO_LISpd-2 gene duplication in the Willistoni group is rapidly evolving
C_LIO_LIParent gene Spd-2A is expressed in somatic cells, whereas the gene duplicate Spd-2B is expressed in spermatocytes
C_LIO_LISpd-2A organizes pericentriolar material during somatic cell mitosis
C_LIO_LISpd-2B is specialized for pericentriolar material organization during male meiosis
C_LI

sentences: Visual imagery vividness declines across the lifespan The capacity to elicit vivid visual mental images varies within an extensive range across individuals between hyper- and aphantasia. It is not clear, however, whether imagery vividness is constant across the lifespan or changes during development and later in life. Without enforcing the constraints of strict experimental procedures and representativity across the entire population, our purpose was to take a first look at the self-reported level of imagery vividness and determine the relative proportion of aphantasic/hyperphantasic participants in different age-groups. Relying on the frequently used Vividness of Visual Mental Imagery questionnaire, we collected data on a random sample of 2252 participants between the ages of 12 to 60 years. We found a novel developmental pattern describing a declining ability to elicit vivid visual mental images in the group averages of different age-groups from adolescence to middle age. This effect involves both a decreasing proportion of individuals with very vivid imagery and an increasing proportion of individuals with weak imagery as maturation (assessed by boneage estimations in adolescents) and aging progresses. This finding may help to shed light on yet unknown developmental mechanisms of our internal, stimulus-independent processes, and might also help to determine genetic, maturational, and age-dependent factors in the extreme cases of hyper- and aphantasia.

sentences: Effects of transcranial alternating current stimulation on spiking activity in computational models of single neocortical neurons Neural oscillations are a key mechanism for information transfer in brain circuits. Rhythmic fluctuations of local field potentials control spike timing through cyclic membrane de- and hyperpolarization. Transcranial alternating current stimulation (tACS) is a non-invasive neuromodulation method which can directly interact with brain oscillatory activity by imposing an oscillating electric field on neurons. Despite its increasing use, the basic mechanisms of tACS are still not fully understood. Here, we investigate in a computational study the effects of tACS on morphologically realistic neurons with ongoing spiking activity. We characterize the membrane polarization as a function of electric field strength and subsequent effects on spiking activity in a set of 25 neurons from different neocortical layers. We find that tACS does not affect the firing rate of investigated neurons for electric field strengths applicable to human studies. However, we find that the applied electric fields entrain the spiking activity of large pyramidal neurons at < 1mV/mm field strengths. Our model results are in line with recent experimental studies and can provide a mechanistic framework to understand the effects of oscillating electric fields on single neuron activity. They highlight the importance of neuron morphology in responsiveness to electrical stimulation and suggest that large pyramidal neurons are most likely the prime target for tACS.

sentences: Heightened condition dependent expression of structural colouration in the faces, but not wings, of male and female flies Structurally coloured sexual signals are a conspicuous and widespread class of ornament used in mate choice, though the extent to which they encode information on the quality of their bearers is not fully resolved. Theory predicts that signalling traits under strong sexual selection as  honest indicators should evolve to be more developmentally integrated and exaggerated than nonsexual traits, thereby leading to heightened condition dependence. Here we test this prediction through examination of the sexually dimorphic faces and wings of the cursorial fly Lispe cana. Males and females possess structural UV-white and golden faces, respectively, and males present their faces and wings to females during close-range, ground-based courtship displays, thereby creating the opportunity for mutual inspection. Across a field-collected sample of individuals, we found that the appearance of the faces of both sexes scaled positively with individual condition, though along separate axes. Males in better condition expressed brighter faces as modelled according to conspecific flies, whereas condition scaled with facial saturation in females. We found no such relationships for their wing interference pattern nor abdomens, with the latter included as a nonsexual control. Our results suggest that the structurally coloured faces, but not the iridescent wings, of male and female Lispe cana are reliable guides to individual quality and support the broader potential for structural colours as honest signals. They also highlight the potential for mutual mate choice in this system, while arguing for one of several alternate signalling roles for wing interferences patterns among the myriad taxa which bear them.

sentences: Alterations to the Gut Microbiota of a Wild Juvenile Passerine in an Urban Mosaic Urbanisation is a major anthropogenic perturbation that progressively alters multiple environmental parameters, thereby presenting novel evolutionary challenges to wild populations. Symbiotic microorganisms residing in the gastrointestinal tracts (gut) of vertebrates have well-established mutual connections with the physiology of their hosts and respond quickly to environmental alterations. However, the impacts of anthropogenic changes and urbanisation on gut microbiota remain poorly understood, especially in early development. To address this knowledge gap, we investigated the gut microbiota in juvenile great tits (Parus major) in an urban mosaic. First, we compared the microbiota of nestlings reared in artificial nest boxes or natural cavities. Next, we analysed microbiota variations employing two distinct urbanisation frameworks, (i) the classical urban/rural dichotomy and (ii) gradual changes in the amount of impervious surface area (ISA) in the urban space and identified the environmental variables that predicted the changes in the microbial communities. Alpha diversity was influenced by cavity type and by the amount of impervious surface surrounding the breeding location. Alpha diversity was also positively correlated with the tree cover density. Beta diversity differed between urban and rural sites, and these alterations covaried with sound pollution, the distance to the city centre and the distance to the closest road. Overall, the microbial communities reflect and are possibly influenced by the heterogeneous environmental modifications that are typical of the urban space. Strikingly, the choice of framework used to define the urban space can influence the outcomes of studies investigating animal-microbe symbiosis. Our results open new perspectives from which the potential function of microbial symbionts in the adaptation of their hosts to anthropogenic stress can be investigated.

sentences: Structural variant selection for high-altitude adaptation using single-molecule long-read sequencing Structural variants (SVs) can be important drivers of human adaptation with strong effects, but previous studies have focused primarily on common variants with weak effects. Here, we used large-scale single-molecule long-read sequencing of 320 Tibetan and Han samples, to show that SVs are key drivers of selection under high-altitude adaptation. We expand the landscape of global SVs, apply robust models of selection and population differentiation combining SVs, SNPs and InDels, and use epigenomic analyses to predict driver enhancers, target genes, upstream regulators, and biological functions, which we validate using enhancer reporter and DNA pull-down assays. We reveal diverse Tibetan-specific SVs affecting the cis- and trans-regulatory circuitry of diverse biological functions, including hypoxia response, energy metabolism, lung function, etc. Our study greatly expands the global SV landscape, reveals the central role of gene-regulatory circuitry rewiring in human adaptation, and illustrates the diverse functional roles that SVs can play in human biology.

sentences: Cortical Depth-Dependent Modeling of Visual Hemodynamic Responses A physiologically based three-dimensional (3D) hemodynamic model is used to predict the experimentally observed blood oxygen level dependent (BOLD) responses versus the cortical depth induced by visual stimuli. Prior 2D approximations are relaxed in order to analyze 3D blood flow dynamics as a function of cortical depth. Comparison of the predictions with experimental data for typical stimuli demonstrates that the full 3D model matches at least as well as previous approaches while requiring significantly fewer assumptions and model parameters (e.g., there is no more need to define depth-specific parameter values for spatial spreading, peak amplitude, and hemodynamic velocity).

sentences: FITTING OF HYPERELASTIC CONSTITUTIVE MODELS IN DIFFERENT SHEEP HEART REGIONS BASED ON BIAXIAL MECHANICAL PROPERTIES Heart failure remains one of the leading causes of death especially among people over the age of 60 years worldwide. To develop effective therapy and suitable replacement materials for the heart muscle it is necessary to understand its biomechanical behaviour under load. This paper investigates the passive mechanical response of the sheep myocardia excised from three different regions of the heart. Due to the relatively higher cost and huge ethical demands in acquisition and testing of real animal heart models, this paper evaluates the fitting performances of five different constitutive models on the myocardial tissue responses. Ten sheep were sacrificed, and their hearts excised and transported within 3h to the testing biomechanical laboratory. The upper sections of the hearts above the short axes were carefully dissected out. Tissues were dissected from the mid-sections of the left ventricle, mid-wall and right ventricle for each heart. The epicardia and endocardia were then carefully sliced off each tissue to leave the myocardia. Stress-strain curves were calculated, filtered and resampled. The results show that Choi-Vito model was found to provide the best fit to the LV, the polynomial (Anisotropic) model to RV, the Four-Fiber Family model to RV, Holzapfel (2000) to RV, Holzapfel (2005) to RV and the Fung model to LV.

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Air leaks: stapling affects porcine lungs biomechanics During thoracic operations, surgical staplers resect cancerous tumors and seal the spared lung. However, post-operative air leaks are undesirable clinical consequences: staple legs wound lung tissue. Subsequent to this trauma, air leaks from lung tissue into the pleural space. This affects the lungs physiology and patients recovery.

The objective is to biomechanically and visually characterize porcine lung tissue with and without staples in order to gain knowledge on air leakage following pulmonary resection. Therefore, a syringe pump filled with air inflates and deflates eleven porcine lungs cyclically without exceeding 10 cmH2O of pressure. Cameras capture stereo-images of the deformed lung surface at regular intervals while a microcontroller simultaneously records the alveolar pressure and the volume of air pumped. The raw images are then used to compute tri-dimensional displacements and strains with the Digital Image Correlation method (DIC).

Air bubbles originated at staple holes of inner row from exposed porcine lung tissue due to torn pleural on costal surface. Compared during inflation, left upper or lower lobe resections have similar compliance (slope of the pressure vs volume curve), which are 9% lower than healthy lung compliance. However, lower lobes statistically burst at lower pressures than upper lobes (p-value<0.046) in ex vivo conditions confirming previous clinical in vivo studies. In parallel, the lung deformed mostly in the vicinity of staple holes and presented maximum shear strain near the observed leak location. To conclude, a novel technique DIC provided concrete evidence of the post-operative air leaks biomechanics. Further studies could investigate causal relationships between the mechanical parameters and the development of an air leak.

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Oncogenic ZMYND11-MBTD1 fusion protein anchors the NuA4/TIP60 histone acetyltransferase complex to the coding region of active genes A chromosomal translocation found in cannibalistic acute myeloid leukemia (AML) leads to an in-frame fusion of the transcription elongation repressor ZMYND11 to MBTD1, a subunit of the NuA4/TIP60 histone acetyltransferase (HAT) complex. In contrast to the NuA4/TIP60 complex, ZMYND11 is linked to repression of actively transcribed genes through recognition of H3.3K36me3. To understand the abnormal molecular events that expression of this ZMYND11-MBTD1 fusion protein can create, we performed its biochemical and functional characterization in comparison to each individual fusion partner. ZMYND11-MBTD1 is stably incorporated into the endogenous NuA4/TIP60 complex but does not bring any additional interactors as the fusion lacks the MYND domain of ZMYND11. Nevertheless, this truncated ZMYND11 moiety in the fusion leads to mislocalization of the NuA4/TIP60 complex on the body of genes normally bound by ZMYND11 in the genome, in a PWWP-H3.3K36me3 interaction-dependent manner. This can be correlated to increased chromatin acetylation and altered gene transcription, most notably on the MYC oncogene, and alternative splicing. Importantly, expression of ZMYND11-MBTD1, but not the individual fusion partners, during embryonic stem cell differentiation, leads to decreased expression of specific differentiation markers, while favoring Myc-driven pluripotency. It also favors self-renewal of hematopoietic stem/progenitor cells. Altogether, these results indicate that the ZMYND11-MBTD1 fusion protein functions primarily by mistargeting the NuA4/TIP60 complex to the body of genes, altering normal transcription of specific genes, likely driving oncogenesis in part through the Myc regulatory network.

Highlights-A recurrent chromosomal translocation detected in cannibalistic acute myeloid leukemia leads to the production of a ZMYND11-MBTD1 fusion protein.
-The ZMYND11-MBTD1 fusion protein is stably incorporated into the endogenous NuA4/TIP60 complex.
-ZMYND11-MBTD1 leads to mistargeting of NuA4/TIP60 activity to the coding region of ZMYND11-target genes, altering gene expression and splicing.
-ZMYND11-MBTD1 binds the MYC gene leading to its upregulation, favoring growth and pluripotency while inhibiting differentiation markers.

sentences: The Fatty Acid Methyl Ester (FAME) profile of Phytophthora agathidicida and its potential use as diagnostic tool. Phytophthora diseases cause devastation to crops and native ecosystems worldwide. In New Zealand, Phytophthora agathidicida is threatening the survival of kauri, an endemic, culturally and ecologically important tree species. The current method for detecting P. agathidicida is a soil bating assay that is time-consuming and requires high levels of expertise to assess, thus limiting the analytical sample throughput. Here, we characterized the fatty acid methyl ester (FAME) profile of P. agathidicida. We also compared it with the FAME profile of P. cinnamomi and assessed the efficacy of FAME analysis as a diagnostic tool for detecting the pathogen in soil samples. In FAME analysis, the total fatty acid content is isolated from a sample and converted to FAMEs for analysis, a process that takes less than a day. Unique fatty acid acyl chains can serve as biomarkers for specific organisms. We detected 12 fatty acids in P. agathidicida, two of which (20:4{omega}6 and 20:5{omega}3) show promise as potential Phytophthora specific biomarkers. Collectively, these findings advance our fundamental understanding of P. agathidicida biology and provide a promising technique to increase the rate of sample processing and the speed of pathogen detection for P. agathidicida in soil.

sentences: Early neural activity changes associated with visual conscious perception The neural mechanisms of visual conscious perception have been investigated for decades. However, the spatiotemporal dynamics associated with the earliest neural responses following consciously perceived stimuli are still poorly understood. Using a dataset of intracranial EEG recordings, the current study aims to investigate the neural activity changes associated with the earliest stages of visual conscious perception. Subjects (N=10, 1,693 grey matter electrode contacts) completed a continuous performance task in which individual letters were presented in series and subjects were asked to press a button when they saw a target letter. Broadband gamma power (40-115Hz) dynamics were analyzed in comparison to baseline prior to stimulus and contrasted for target trials with button presses and non-target trials without button presses. Regardless of event type, we observed early gamma power changes within 30-150 ms from stimulus onset in a network including increases in bilateral occipital, fusiform, frontal (including frontal eye fields), and medial temporal cortex, increases in left lateral parietal-temporal cortex, and decreases in the right anterior medial occipital cortex. No significant differences were observed between target and non-target stimuli until >150 ms post-stimulus, when we saw greater gamma power increases in left motor and premotor areas, suggesting a possible role of these later signals in perceptual decision making and/or motor responses with the right hand. The early gamma power findings suggest a broadly distributed cortical visual detection network that is engaged at early times tens of milliseconds after signal transduction from the retina.

sentences: An optimized FM-index library for nucleotide and amino acid search Pattern matching is a key step in a variety of biological sequence analysis pipelines. The FM-index is a compressed data structure for pattern matching, with search run time that is independent of the length of the database text. We present AvxWindowedFMindex (AWFM-index), an open-source, thread-parallel FM-index library written in C that is optimized for indexing nucleotide and amino acid sequences. AWFM-index is easy to incorporate into bioinformatics software and is able to perform exact match count and locate queries ~2-4x faster than SeqAn3s FM-index implementation for nucleotide search, and ~2-6x faster for amino acid search in a single-threaded context. This performance is due to (i) a new approach to storing FM-index data in a strided bit-vector format that enables extremely efficient computation of the FM-index occurrence function via AVX2 bitwise instructions, and (ii) inclusion of a cache-efficient lookup table for partial k-mer searches. AWFM-index also trivially parallelizes to multiple threads with good scaling, and enables efficient on-disk storage of the memory-intensive suffix array. The open-source library is available for download at https://github.com/TravisWheelerLab/AvxWindowFmIndex.

sentences: TRIOBP promotes bidirectional radial stiffness gradients within the organ of Corti Hearing depends on complex mechanical properties of the inner ear sensory epithelium. Yet, the individual contributions of different cell types to the stiffness spectrum of the sensory epithelium have not been thoroughly investigated. Using sub-100 nanometer spatial resolution PeakForce Tapping Atomic Force Microscopy (PFT-AFM), we mapped the Youngs modulus (stiffness) of the apical surface of different cells of freshly-dissected cochlear epithelium from wild-type mice and mice lacking the F-actin bundling protein TRIOBP-5 or TRIOBP-4 and TRIOBP-5. Variants of the genes encoding human and mouse TRIOBP are associated with deafness. We show that TRIOBP deficiency affects formation of supporting cell apical phalangeal microfilaments and bundled cortical F-actin of hair cell cuticular plates, softening the apical surface of the sensory epithelium. Unexpectedly, high-resolution PFT-AFM-mapping also revealed previously unrecognized reticular lamina radial stiffness gradients of opposite orientations in wild-type supporting and hair cells. Deafness-associated TRIOBP deficiencies significantly modified these bidirectional radial stiffness gradients.

sentences: Accurate prediction of functional states of cis-regulatory modules reveals the common epigenetic rules in humans and mice We proposed a two-step approach for predicting active cis-regulatory modules (CRMs) in a cell/tissue type. We first predict a map of CRM loci in the genome using all available transcription factor binding data in the organism, and then predict functional states of all the putative CRMs in any cell/tissue type using few epigenetic marks. We have recently developed a pipeline dePCRM2 for the first step, and now presented machine-learning methods for the second step. Our approach substantially outperforms existing methods. Our results suggest common epigenetic rules for defining functional states of CRMs in various cell/tissue types in humans and mice.

sentences: mRNA-1273 and Ad26.COV2.S vaccines protect against the B.1.621 variant of SARS-CoV-2 Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, viral variants with greater transmissibility or immune evasion properties have arisen, which could jeopardize recently deployed vaccine and antibody-based countermeasures. Here, we evaluated in mice and hamsters the efficacy of preclinical non-GMP Moderna mRNA vaccine (mRNA-1273) and the Johnson & Johnson recombinant adenoviral-vectored vaccine (Ad26.COV2.S) against the B.1.621 (Mu) South American variant of SARS-CoV-2, which contains spike mutations T95I, Y144S, Y145N, R346K, E484K, N501Y, D614G, P681H, and D950N. Immunization of 129S2 and K18-human ACE2 transgenic mice with mRNA-1273 vaccine protected against weight loss, lung infection, and lung pathology after challenge with B.1.621 or WA1/2020 N501Y/D614G SARS-CoV-2 strain. Similarly, immunization of 129S2 mice and Syrian hamsters with a high dose of Ad26.COV2.S reduced lung infection after B.1.621 virus challenge. Thus, immunity induced by mRNA-1273 or Ad26.COV2.S vaccines can protect against the B.1.621 variant of SARS-CoV-2 in multiple animal models.

sentences: Presynaptic Stochasticity Improves Energy Efficiency and Alleviates the Stability-Plasticity Dilemma When an action potential arrives at a synapse there is a large probability that no neurotransmitter is released. Surprisingly, simple computational models suggest that these synaptic failures enable information processing at lower metabolic costs. However, these models only consider information transmission at single synapses ignoring the remainder of the neural network as well as its overall computational goal. Here, we investigate how synaptic failures affect the energy efficiency of models of entire neural networks that solve a goal-driven task. We find that presynaptic stochasticity and plasticity improve energy efficiency and show that the network allocates most energy to a sparse subset of important synapses. We demonstrate that stabilising these synapses helps to alleviate the stability-plasticity dilemma, thus connecting a presynaptic notion of importance to a computational role in lifelong learning. Overall, our findings present a set of hypotheses for how presynaptic plasticity and stochasticity contribute to sparsity, energy efficiency and improved trade-offs in the stability-plasticity dilemma.

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SARS-CoV-2 causes human BBB injury and neuroinflammation indirectly in a linked organ chip platform COVID-19 is a multi-system disease affecting many organs outside of the lungs, and patients generally develop varying degrees of neurological symptoms. Whereas, the pathogenesis underlying these neurological manifestations remains elusive. Although in vitro models and animal models are widely used in studies of SARS-CoV-2 infection, human organ models that can reflect the pathological alterations in a multi-organ context are still lacking. In this study, we propose a new strategy to probe the effects of SARS-CoV-2 on human brains in a linked alveolus-BBB organ chip platform. The new multi-organ platform allows to recapitulate the essential features of human alveolar-capillary barrier and blood-brain barrier in a microfluidic condition by co-culturing the organ-specific cells. The results reveal direct SARS-CoV-2 exposure has no obvious effects on BBB chip alone. While, infusion of endothelial medium from infected alveolus chips can cause BBB dysfunction and neuroinflammation on the linked chip platform, including brain endothelium disruption, glial cell activation and inflammatory cytokines release. These new findings suggest that SARS-CoV-2 could induce neuropathological alterations, which might not result from direct viral infection through hematogenous route, but rather likely from systemic inflammation following lung infection. This work provides a new strategy to study the virus-host interaction and neuropathology at an organ-organ context, which is not easily obtained by other in vitro models. This will facilitate to understand the neurological pathogenesis in SARS-CoV-2 and accelerate the development of new therapeutics.

SUMMARYO_LIA linked human alveolus-BBB chip platform is established to explore the influences of SARS-CoV-2 on human brains in an organ-organ context.
C_LIO_LISARS-CoV-2 infection could induce BBB injury and neuroinflammation.
C_LIO_LIThe neuropathological changes are caused by SARS-CoV-2 indirectly, which might be mediated by systemic inflammation following lung infection, but probably not by direct viral neuroinvasion.
C_LI

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Stimulation related artifacts and a multipurpose template-based offline removal solution for a novel sensing-enabled deep brain stimulation device BackgroundThe Medtronic "Percept" is the first FDA approved deep brain stimulation (DBS) device with sensing capabilities during active stimulation. Its real-world signal recording properties have yet to be fully described.

ObjectiveThis study details sources of artifact (and potential mitigations) in local field potential (LFP) signals collected by the Percept, and assesses the potential impact of artifact on the future development of adaptive DBS (aDBS) using this device.

MethodsLFP signals were collected from seven subjects in both experimental and clinical settings. The presence of artifacts and their effect on the spectral content of neural signals were evaluated in both the stimulation ON and OFF states using three distinct offline artifact removal techniques.

ResultsTemplate subtraction successfully removed multiple sources of artifact, including 1) electrocardiogram (ECG), 2) non-physiologic polyphasic artifacts, and 3) ramping related artifacts seen when changing stimulation amplitudes. ECG removal from stimulation ON (at 0 mA) signals recovered the spectral shape seen when OFF stimulation (averaged difference in normalized power in theta, alpha, and beta bands [≤] 3.5%). ECG removal using singular value decomposition was similarly successful, though required subjective researcher input. QRS interpolation produced similar recovery of beta-band signal, but resulted in residual low-frequency artifact.

ConclusionsArtifacts present when stimulation is enabled notably affected the spectral properties of sensed signals using the Percept. Multiple discrete artifacts could be successfully removed offline using an automated template subtraction method. The presence of unrejected artifact likely influences online power estimates, with the potential to affect aDBS algorithm performance.

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Divergent Acyl Carrier Protein Mediates Mitochondrial Fe-S Cluster Biosynthesis in Malaria Parasites Plasmodium falciparum malaria parasites are early-diverging eukaryotes with many unusual metabolic adaptations. Understanding these adaptations will give insight into parasite evolution and unveil new parasite-specific drug targets. Most eukaryotic cells retain a mitochondrial fatty acid synthesis (FASII) pathway whose acyl carrier protein (mACP) and 4-phosphopantetheine (Ppant) prosthetic group provide a soluble scaffold for acyl chain synthesis. In yeast and humans, mACP also functions to biochemically couple FASII activity to electron transport chain (ETC) assembly and Fe-S cluster biogenesis. In contrast to most eukaryotes, the Plasmodium mitochondrion lacks FASII enzymes yet curiously retains a divergent mACP lacking a Ppant group. We report that ligand-dependent knockdown of mACP is lethal to parasites, indicating an essential FASII-independent function. Decyl-ubiquinone rescues parasites temporarily from death, suggesting a dominant dysfunction of the mitochondrial ETC followed by broader cellular defects. Biochemical studies reveal that Plasmodium mACP binds and stabilizes the Isd11-Nfs1 complex required for Fe-S cluster biosynthesis, despite lacking the Ppant group required for this association in other eukaryotes, and knockdown of parasite mACP causes loss of both Nfs1 and the Rieske Fe-S protein in ETC Complex III. This work reveals that Plasmodium parasites have evolved to decouple mitochondrial Fe-S cluster biogenesis from FASII activity, and this adaptation is a shared metabolic feature of other Apicomplexan pathogens, including Toxoplasma and Babesia. This discovery also highlights the ancient, fundamental role of ACP in mitochondrial Fe-S cluster biogenesis and unveils an evolutionary driving force to retain this interaction with ACP independent of its eponymous function in FASII.

Significance StatementPlasmodium malaria parasites are single-celled eukaryotes that evolved unusual metabolic adaptations. Parasites require a mitochondrion for blood-stage viability, but essential functions beyond the electron transport chain are sparsely understood. Unlike yeast and human cells, the Plasmodium mitochondrion lacks fatty acid synthesis enzymes but retains a divergent acyl carrier protein (mACP) incapable of tethering acyl groups. Nevertheless, mACP is essential for parasite viability by binding and stabilizing the core mitochondrial Fe-S cluster biogenesis complex via a divergent molecular interface lacking an acyl-pantetheine group that contrasts with other eukaryotes. This discovery unveils an essential metabolic adaptation in Plasmodium and other human parasites that decouples mitochondrial Fe-S cluster biogenesis from fatty acid synthesis and evolved at or near the emergence of Apicomplexan parasitism.

sentences: Dynamic plasticity within the EMT spectrum, rather than static mesenchymal traits, drives tumor heterogeneity and metastatic progression of breast cancers The Epithelial-to-Mesenchymal Transition (EMT) is a developmental cellular program frequently coopted by cancer cells1 and is a key contributor to both heterogeneity in solid tumors2-4 and later stage chemo-resistance and metastasis5,6. Rather than being a switch from an epithelial to a mesenchymal state, increasing evidence points to the existence of intermediate EMT states, wherein cells co-express both epithelial and mesenchymal traits7-13. Multiple stable intermediate EMT states possessing unique characteristics exist across the EMT spectrum7,8,14,15, contributing to the complex heterogeneity of tumors to promote metastasis16. While much work has been carried out identifying and characterizing EMT-inducing transcription factors17-20, the transcriptional and epigenetic networks responsible for the stability and maintenance of the midpoints along the EMT spectrum are poorly defined. In addition, there are currently no approaches to identifying and quantifying intermediate EMT subpopulations within patient tumors to evaluate their prognostic significance. Using clonally isolated derivatives of the SUM149PT breast cancer cell line, we systematically interrogate how each EMT state independently contributes to heterogeneity and influences metastatic progression, uncovering the role of RUNX2 in stabilizing certain intermediate states. Using SUM149PT-derived tumors as a training set, we develop an entropy-based model to quantify phenotypic heterogeneity and EMT status. Remarkably, the majority of cell states captured in the SUM149PT model are represented in a selection of patient tumors, laying the foundation for quantification of epithelial-mesenchymal heterogeneity and understanding the role of the intermediate EMT state in tumor progression.

sentences: A Wake-Up Call: Determination of Antibiotics Residue Level in Raw Meat in Abattoir and Selected Slaughterhouses in Five Local Government in Kano State, Nigeria. The frequent use of antibiotics may result in drug residues that can be discovered at varying quantities in animal products such as milk or meat. The presence of pharmaceutical residues in food above the MRLs has been perceived globally by various persons. Antibiotics residues are present in food, which can endanger human health by causing antibiotic sensitivity, allergic reactions, microflora imbalance, bacterial resistance to antibiotics in microorganisms, and financial loss to the food industry. Farmers around the world utilize them on a sporadic basis for both preventative and curative purposes. This study assessed the antibiotics residues in raw meat sold in 6 slaughter houses in Kano States. The study is a descriptive cross-sectional study involving six (6) major slaughter house in Kano state. Muscle, Kidney and liver samples were collected from each slaughterhouse. The antibiotic residues in the meat samples were analysed using high performance liquid chromatography (HPLC) for tetracycline, ciprofloxacin and oxytetracycline residue results were presented in charts and tables. Out of a total of 18 beef samples analyzed during this study, 15 (83%) of the total samples had detectable levels of tetracycline residues from which 6(33.3%) had tetracycline residues at violative levels above the WHO/FAO maximum residue limits (MRLs), out of those 18 beef samples analyzed during this study, 6(33%) of the total samples had detectable levels of oxytetracycline residues from which 3(17%) had oxytetracycline residues at violative levels above the WHO/FAO maximum residue limits (MRLs) and out of those 18 beef samples analyzed during this study, 12(67%) of the total samples had detectable levels of ciprofloxacin, all levels are below the WHO/FAO maximum residue limits (MRLs). This high level of tetracycline and oxytetracycline residues in greater proportion of meat destined for human consumption at violative levels could be as a result of the indiscriminate use and misuse of veterinary drugs as commonly practiced among livestock producers and marketers without observing withdrawal period prior to slaughter. These results indicate that consumers may be predisposed to health hazards and hinder international meat trade from Nigeria. Regulatory authorities should therefore ensure compliance with good agricultural practices including withdrawal period of drugs used for treatment of food animals.

sentences: A new murine Rpl5 (uL18) mutation provides a unique model of variably penetrant Diamond Blackfan Anemia Ribosome dysfunction is implicated in multiple abnormal developmental and disease states in humans. Heterozygous germline mutations in genes encoding ribosomal proteins (RPs) are found in the majority of individuals with Diamond Blackfan anemia (DBA) while somatic mutations have been implicated in a variety of cancers and other disorders. Ribosomal protein-deficient animal models show variable phenotypes and penetrance, similar to human DBA patients. The spontaneous anemia remission observed in some DBA patients occurs via unknown mechanism(s) and has not been previously described in animal models. Here we characterized a novel ENU mouse mutant (Skax23m1Jus) with growth and skeletal defects, cardiac malformations and increased mortality. Following genetic mapping and whole exome sequencing, we identified an intronic Rpl5 mutation, which segregated with all affected mice. This mutation was associated with decreased ribosome generation, consistent with Rpl5 haploinsufficiency. Rpl5Skax23-Jus mutant animals had a profound delay in erythroid maturation and increased mortality at embryonic day E12.5, which improved by E14.5. Surviving mutant animals had a macrocytic anemia at birth as well as evidence of ventricular septal defect (VSD). Surviving adult and aged mice exhibited no hematopoietic defect or VSD. We propose that this novel Rpl5Skax23-Jus mutant mouse will be useful to study the factors influencing the variable penetrance and anemia remission that are observed in DBA.

sentences: Individual Variability in the Innate Functional Organization of the Human Brain The adult brain is organized into distinct functional networks, forming the basis of information processing and determining individual differences in behavior. Is this network organization genetically determined and present at birth? And what is the individual variability in this organization in neonates? Here, we use unsupervised learning to uncover intrinsic functional brain organization using resting-state connectivity from a large cohort of neonates (Developing Human Connectome Project). We identified a set of symmetric, hierarchical, and replicable networks: sensorimotor, visual, default mode, ventral attention, and high-level vision. We quantified individual variability across neonates, and found the most individual variability in the ventral attention networks. Crucially, the variability of these networks were not driven by SNR differences or differences from adult networks (Yeo et al., 2011). Finally, differential gene expression provided a potential explanation for the emergence of these distinct networks and identified potential genes of interest for future developmental and individual variability research. Overall, we found neonatal connectomes (even at the voxel-level) can reveal broad individual- specific information processing units. The presence of individual differences in neonates and the framework for personalized parcellations demonstrated here has the potential to improve prediction of behavior and future outcomes from neonatal and infant brain data.

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HIV-linked gut dysbiosis associates with cytokine production capacity in viral-suppressed people living with HIV People living with HIV (PLHIV) are exposed to chronic immune dysregulation, even when virus replication is suppressed by antiretroviral therapy (ART). Given the emerging role of the gut microbiome in immunity, we hypothesized that the gut microbiome may be related to the cytokine production capacity of PLHIV. To test this hypothesis, we collected metagenomic data from 143 ART-treated PLHIV and assessed the ex vivo production capacity of eight different cytokines (IL-1{beta}, IL-6, IL-1Ra, IL-10, IL17, IL22, TNF and IFN-{gamma}) in response to different stimuli. We also characterized CD4+ T cell-counts, HIV reservoir and other clinical parameters. Compared to 190 age- and sex-matched controls and a second independent control cohort, PLHIV showed microbial dysbiosis that was correlated with viral reservoir levels, cytokine production capacity and sexual behavior. Notably, we identified two genetically different P. copri strains that were enriched in either PLHIV or healthy controls. The control-enriched strain was negatively associated with IL-10, IL-6 and TNF production, independent of age, sex and sexual behavior, and positively associated with CD4+ T cell-level, whereas the PLHIV-enriched strain showed no associations. Our findings suggest that modulating the gut microbiome may be a strategy to modulate immune response in PLHIV.

Novel PointsO_LIWe identified compositional and functional changes in the gut microbiome of PLHIV that were strongly related to sexual behavior.
C_LIO_LIHIV-associated bacterial changes are negatively associated with HIV reservoir. The relative abundance of Firmicutes bacterium CAG 95 and Prevotella sp CAG 5226 both show a negative association with CD4+ T cell-associated HIV-1 DNA.
C_LIO_LIPrevotella copri and Bacteroides vulgatus show association with PBMC production capacity of IL-1{beta} and IL-10 that is independent of age, sex, BMI and sexual behavior.
C_LIO_LIWe observed two genetically different P. copri strains that are enriched in PLHIV and healthy individuals, respectively.
C_LIO_LIThe control-related P. copri strain specifically shows a negative association with IL-10, IL-6 and TNF production and a positive association with CD4+ T cell-level. This suggests it plays a potential protective role in chronic inflammation, which may be related to enrichment of a specific epitope peptide.
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sentences: Influence of selected plant growth stimulators in enhancing germinability and germination parameters of Zea mays L. under microgravity conditions simulated by a two-dimensional clinostat The earth has become increasingly overcrowded as a result of rapid urbanization and population growth, predicting that its carrying capacity could be overstretched. As a result, it is important to test the possibilities of growing plants under space exploration conditions, especially gravitational balance. Since microgravity impedes plant development, to what extent can plant growth stimulators reverse or enhance this trend? A total of 12 maize seeds were weighed and placed sideways in petri dish and inoculated with plant growth stimulators such as indole acetic acid (IAA), gibberellic acid (GA), and ascorbate (AA) and the clinorotated at different rates (0.5, 1.0 and 2.0 rpm), while control seeds were just placed on a table. Results showed that at 72 hrs, the maize seeds under microgravity showed reduced germination percentage with increasing clinorotation rates as against the control. But when stimulated with IAA, GA and AA, improved germination percentage was observed as against the control even under microgravity condition. The seedling dry weight, germination time and other germination parameters also showed similar improvements. Comparatively, the three growth stimulators showed no major variations in their ability to improve germination percentage under microgravitational impact. However, IAA showed more improvement on seedling vigor as against others, while GA showed more effect on the peak time and rate of germination. This research confirmed the possibilities of improving germinability of maize seeds under space exploration condition.

sentences: Periplasm homeostatic regulation maintains spatial constraints essential for cell envelope processes and cell viability The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics and a synthetic lethal screen we show that lengthening Lpp to the upper limit does not change the spatial constraint, but rather impacts the load-bearing capacity across the outer membrane. Our findings demonstrate E. coli expressing elongated Lpp homeostatically counteracts periplasmic enlargement with a combination of tilting Lpp and reducing Lpp abundance. By genetic screening we identified all of the genes in E. coli that become essential in order to enact this homeostasis, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to enact this homeostasis. We observed increased levels of factors determining cell stiffness, decrease membrane integrity, increase membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling and protein translocation

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Combination of High Dose Rate Radiations (10X FFF/2400 MU/min/10 MV X-rays) and Paclitaxel Selectively Eliminates Melanoma Cells PurposeMelanoma is one of the most aggressive cancer with 1.6% of total cancer deaths in United States. In recent years treatment options for metastatic melanoma have been improved by the FDA approval of new therapeutic agents. However, these inhibitors based therapies are non-specific and have severe toxicities including hyperkeratosis, photosensitivity, hepatitis, arthralgia and fatigue. The aim of this study is to determine the synthetic lethal effect (paclitaxel and radiations) on melanoma cells and reduce the total radiation doses by increasing the dose rates up to 2400 MU/min.

MethodsWe previously reported a radiation treatment (10 MV x-rays, 10X-FFF, dose rate 2400MU/min, low total dose 0.5 Gy) that kills melanoma cells with 80% survival of normal HEM in vitro. In this study we extended the radiation cycle up to four and include paclitaxel treatment to study the synthetic lethal effect on melanoma and two additional normal primary cells, HDF and HEK. Cells were treated with paclitaxel prior to radiations of dose rate of 400 and 2400 MU/min with total radiation dose of only 0.5 Gy. To study induction of apoptosis and cell death, mitochondria respiration assay, DNA damage assay and colony formation assay were performed.

ResultsFour days of consequent radiation treatment with paclitaxel significantly reduces the survival of melanoma cells by inducing of apoptosis and mitochondrial damages. After treatment, excessive DNA damage in melanoma cells leads to increase in expression of pro-apoptotic genes (Casp3) and decrease in expression of DNA repair gene (PARP1) and anti-apoptotic gene (Bcl2) to activate apoptosis pathway. Combination of paclitaxel and radiations reduces the survival of melanoma cells colonies when compared to radiation alone.

ConclusionOur study indicates radiations with paclitaxel has potential synthetic lethal effect on melanoma cells and can be develop as therapy for melanoma without having toxicities or harmful effects to normal primary skin cells.

sentences: A transcriptomic census reveals that Rbfox contributes to a broad but selective recapitulation of peripheral tissue splicing patterns in the thymus Thymic epithelial cells (TEC) control the selection of a T-cell repertoire reactive to pathogens but tolerant of self. While this process is known to involve the promiscuous expression of virtually the entire protein-coding gene repertoire the extent to which TEC recapitulate peripheral isoforms, and the mechanisms by which they do so, have remained largely unknown. We performed the first assembly-based transcriptomic census of transcript structures and splicing factor (SF) expression in mouse medullary TEC (mTEC) and 21 peripheral tissues. Mature mTEC expressed 60.1% of all protein-coding transcripts, more than was detected in any of the peripheral tissues. However, for genes with tissue-restricted expression, we found that mTEC produced fewer isoforms than did the relevant peripheral tissues. Analysis of exon inclusion revealed an absence of brain-specific micro-exons in mTEC. We did not find unusual numbers of novel transcripts in TEC and show that Aire, the facilitator of promiscuous gene expression, promotes usage of long transcripts but has only a limited impact on alternative splicing in mTEC. Comprehensive assessment of SF expression in mTEC identified a small set of non-promiscuously expressed SF genes amongst which we confirmed RBFOX to be present with AIRE in mTEC nuclei. Using a conditional loss of function approach, we show that Rbfox2 promotes mTEC development and regulates the alternative splicing of promiscuously expressed genes. These data indicate that TEC recommission a small number of peripheral SFs, including members of the Rbfox family, to generate a broad but selective representation of the peripheral splice isoform repertoire.

sentences: Identification of enhancer-like elements defines regulatory networks active in planarian adult stem cells Planarians have become an established model system to study regeneration and stem cells, but the regulatory elements in the genome remain almost entirely undescribed. Here, by integrating epigenetic and expression data we use multiple sources of evidence to identify enhancer elements active in the adult stem cell populations that drive regeneration. We have used ChIP-seq data to identify regions with histone modifications consistent with enhancer identity and activity, and ATAC-seq data to identify accessible chromatin. Overlapping these signals allowed for the identification of a set of high confidence candidate enhancers predicted to be active in planarian adult stem cells. These enhancers are enriched for conserved transcription factor (TF) binding sites for TFs and TF families expressed in planarian adult stem cells. Foot-printing analyses provided further evidence that these potential TF binding sites are occupied in adult stem cells. We integrated these analyses to build testable hypotheses for the regulatory function of transcription factors in stem cells, both with respect to how pluripotency might be regulated, and to how lineage differentiation programs are controlled. Our work identifies active enhancers regulating adult stem cells and regenerative mechanisms.

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Cis-Regulatory Hubs: a Relevant 3D Model to Study the Genetics of Complex Diseases with an Application to Schizophrenia The 3-dimensional (3D) conformation of the chromatin creates complex networks of noncoding regulatory regions (distal elements) and promoters impacting gene regulation. Despite the importance of the role of noncoding regions in complex diseases, little is known about their interplay within regulatory hubs and implication in multigenic diseases like schizophrenia. Here we show that cis-regulatory hubs (CRHs) in neurons highlight functional interactions between distal elements and promoters, providing a model to explain epigenetic mechanisms involved in complex diseases. CRHs represent a new 3D model, where distal elements interact to create a complex network of active genes. In a disease context, CRHs highlighted strong enrichments in schizophrenia-associated genes, schizophrenia-associated SNPs and schizophrenia heritability compared to equivalent structures. Finally, CRHs exhibit larger proportions of genes differentially expressed in schizophrenia compared to promoter-distal element pairs or TADs. CRHs thus capture causal regulatory processes improving the understanding of complex disease etiology such as schizophrenia. These multiple lines of genetic and statistical evidence support CRHs as 3D models to study dysregulation of gene expression in complex diseases more generally.

Summary BlurbGenes and their regulatory elements are organized within 3D networks in neurons which model functional structures and explain schizophrenia genetic etiology.

sentences: RIG-I uses its intrinsically disordered CARDs-Helicase linker in RNA proofreading functions The innate immune receptor RIG-I provides the first line of defense against viral infections. Viral RNAs are recognized by the C-terminal domain (CTD) of RIG-I, but the RNA must engage the helicase domain to release the signaling domain CARDs from their autoinhibitory CARD2:Hel2i interactions. Because the helicase lacks RNA specificity, there must be mechanisms to proofread RNAs entering the helicase domain. Although such mechanisms are crucial in preventing aberrant immune responses by non-specific RNAs, they remain largely unknown. This study reveals a previously unknown proofreading mechanism that RIG-I uses to ensure the helicase engages RNAs chosen explicitly by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs-Helicase Linker (CHL), which uses its negatively charged regions to electrostatically antagonize incoming RNAs. In addition to RNA gating, CHL is essential in stabilizing the CARD2:Hel2i interface. The CHL and CARD2:Hel2i interface work together, establishing a tunable gating mechanism that allows CTD-chosen RNAs to bind into the helicase while blocking non-specific RNAs. With its critical regulatory functions, CHL represents a novel target for RIG-I-based therapeutics.

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A connectivity signature for glioblastoma Tumor cell extensions called tumor microtubes (TMs) in glioma resemble neurites during neurodevelopment and connect glioma cells to a network that has considerable relevance for tumor progression and therapy resistance. The determination of interconnectivity in individual tumors has been challenging and the impact of tumor cell connectivity on patient survival remained unresolved so far. Here, a connectivity signature from single-cell RNA-sequenced (scRNA-Seq) xenografted primary glioblastoma (GB) cells was established and clinically validated. Thirty-four of 40 connectivity genes were related to neurogenesis, neural tube development or glioma progression, including the TM-network-relevant GAP43 gene. Astrocytic-like and mesenchymal-like GB cells had the highest connectivity signature scores in scRNA-Seq data of patient-derived xenografts and patient samples. In 230 human GBs, high connectivity correlated with the mesenchymal expression subtype, TP53 wildtype, and with dismal patient survival. CHI3L1 was identified as a robust molecular marker of connectivity. Thus, the connectivity signature allows novel insights into brain tumor biology, provides a proof-of-principle that tumor cell connectivity is relevant for patients prognosis, and serves as a robust biomarker that can be used for future clinical trials.

Statement of significanceIntegration of GB cells into functional networks drives tumor progression and resistance. Here, we established and validated a novel connectivity gene expression signature of single GB cells and whole tumors that can be easily applied to clinical and preclinical samples. It is shown that connectivity is determining prognosis combining molecular, functional and clinical insights into the disease.

sentences: Genome-wide analyses reveal a strong association between LEPR gene variants and body fat reserves in ewes Among the adaptive capacities of animals, the management of energetic body reserves (BR) through the BR mobilization and accretion processes (BR dynamics, BRD) has become an increasingly valuable attribute for livestock sustainability, allowing animals to cope with more variable environments. BRD has previously been reported to be heritable in ruminants. In the present study, we conducted genome-wide studies (GWAS) in sheep to determine genetic variants associated with BRD. BR levels and BR changes over time were obtained through body condition score measurements at eight physiological stages throughout each productive cycle in Romane ewes (n=1034) and were used as phenotypes for GWAS. After quality controls and imputation, 48,513 single nucleotide polymorphisms (SNP) were included in the GWAS. Among the QTLs identified, a major QTL associated with BR levels during pregnancy and lactation was identified on chromosome 1. In this region, several significant SNPs mapped to the leptin receptor gene (LEPR), among which one SNP mapped to the coding sequence. The point mutation induces the p.P1019S substitution in the cytoplasmic domain, close to tyrosine phosphorylation sites. The frequency of the SNP associated with increased BR levels was 32%, and the LEPR genotype explained up to 5% of the variance of the trait. These results provide strong evidence for involvement of LEPR in the regulation of BRD in sheep and highlight it as a major candidate for improving adaptive capacities.

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Allocation of visuospatial attention indexes evidence accumulation for reach decisions Visuospatial attention is a prerequisite for the performance of visually guided movements: Perceptual discrimination is regularly enhanced at target locations prior to movement initiation. It is known that this attentional prioritization evolves over the time of movement preparation; however, it is not clear whether this build-up simply reflects a time requirement of attention formation or whether, instead, attention build-up reflects the emergence of the movement decision. To address this question, we combined behavioral experiments, psychophysics, and computational decision-making models to characterize the time course of attention build-up during motor preparation. Participants (n = 46, 29 female) executed center-out reaches to one of two potential target locations and reported the identity of a visual discrimination target that occurred concurrently at one of various time-points during movement preparation and execution. Visual discrimination increased simultaneously at the two potential target locations but was modulated by the experiment-wide probability that a given location would become the final goal. Attention increased further for the location that was then designated as the final goal location, with a time course closely related to movement initiation. A sequential sampling model of decision-making predicted key temporal characteristics of attentional allocation. Together, these findings provide evidence that visuospatial attentional prioritization during motor preparation does not simply reflect that a spatial location has been selected as movement goal, but rather indexes the time-extended, cumulative decision that leads to selection, hence constituting a link between perceptual and motor aspects of sensorimotor decisions.

Significance statementWhen humans perform a goal-directed movement such as a reach, attention shifts towards the goal location already before movement initiation, indicating that motor goal selection relies on the use of attention. Here, we demonstrate that key temporal aspects of visuospatial attention are predicted by a well-known computational model of decision-making. These findings suggest that visual attention signals much more than simply that a motor goal has been selected: instead, the time-course of emergent, visuospatial attention reflects the time-extended, cumulative decision that leads to goal selection, offering a window onto the tight link of perceptual and motor aspects in sensorimotor decision-making.

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Dynamic causal modelling shows a prominent role of local inhibition in alpha power modulation in higher visual cortex During resting-state EEG recordings, alpha activity is more prominent over the posterior cortex in eyes-closed (EC) conditions compared to eyes-open (EO). In this study, we characterized the difference in spectra between EO and EC conditions using dynamic causal modelling. Specifically, we investigated the role of intrinsic and extrinsic connectivity--within the visual cortex--in generating EC-EO alpha power differences over posterior electrodes. The primary visual cortex (V1) and the bilateral middle temporal visual areas (V5) were equipped with bidirectional extrinsic connections using a canonical microcircuit. The states of four intrinsically coupled subpopulations--within each occipital source--were also modelled. Using Bayesian model selection, we tested whether modulations of the intrinsic connections in V1, V5 or extrinsic connections (or a combination thereof) provided the best evidence for the data. In addition, using parametric empirical Bayes (PEB), we estimated group averages under the winning model. Bayesian model selection showed that the winning model contained both extrinsic connectivity modulations, as well as intrinsic connectivity modulations in all sources. The PEB analysis revealed increased extrinsic connectivity during EC. Overall, we found a reduction in the inhibitory intrinsic connections during EC. The results suggest that the intrinsic modulations in V5 played the most important role in producing EC-EO alpha differences, suggesting an intrinsic disinhibition in higher order visual cortex, during EC resting state.

Author summaryOne of the strongest signals that can be measured using EEG are so called alpha rhythms. These are neural oscillations that fall within the 8-12Hz frequency range. Alpha rhythms are most prominent when the eyes are closed and are seen at the electrodes placed at the back of the head. In this study, we studied the mechanism of alpha rhythms changes when going from eyes-open to an eyes-closed state. We used a biologically plausible model including different neural populations. We focused on modelling connections within and between different neural sources of the visual cortex and how they are modulated when going from eyes-open to an eyes closed state. We found evidence that inhibitory neurons play an important role in alpha rhythms.

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A Substantia Innominata-midbrain Circuit Controls a General Aggressive Response While aggressive behaviors are universal and essential for survival,  uncontrollable and abnormal aggressive behaviors in animals or humans may have severe adverse consequences or social costs. Neural circuits regulating specific forms of aggression under defined conditions have been described, but how brain circuits govern a general aggressive response remains unknown. Here, we found that posterior substantia innominata (pSI) neurons responded to several aggression-provoking cues with the graded activity of differential dynamics, predicting the aggressive state and the topography of aggression in mice. Activation of pSI neurons projecting to the periaqueductal gray (PAG) increased aggressive arousal and robustly initiated/promoted all the types of aggressive behavior examined in an activity level-dependent manner. Inactivation of the pSI circuit largely blocked diverse aggressive behaviors but not mating. By encoding a general aggressive response, the pSI-PAG circuit universally drives multiple aggressive behaviors and may provide a potential target for alleviating human pathological aggression.

In BriefZhu et al. find that the posterior substantia innominata neurons universally drive aggressive behaviors by representing aggressive state and topography of aggression in mice.

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C_LIO_LIThe pSI-PAG circuit promotes arousal and elicits thirteen aggressive behaviors
C_LIO_LIThe pSI controls various aggressive behaviors in an activity level-dependent manner
C_LIO_LIInactivation of the pSI circuit blocks diverse aggressive behaviors but not mating
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sentences: Immune stimulation alters chemical profiles of Tribolium castaneum I.Rapid recognition of disease cues is essential for preventing pathogenic infections and for disease management in group-living animals. Healthy individuals across taxa can detect illness in other conspecifics and adjust their responses to limit further infections of themselves and the group. However, little is known about potential changes in chemical phenotypes due to disease, which may mediate these responses. We here asked whether individual immune experience resulting from wounding or the injection of heat-killed bacteria of Bacillus thuringiensis (i.e., immune priming) leads to changes in the chemical profiles of adult red flour beetles (Tribolium castaneum). This group-living insect species is a well-studied example for both immune priming as a form of innate immune memory and niche construction via  external immunity, i.e., the secretion of quinone-containing antimicrobials into the flour. Upon interaction with wounded conspecifics, naive beetles were previously found to not only up-regulate immunity, but moreover reduce gene expression of the evolutionary capacitor HSP90, an effect that has the potential to enhance adaptability. We here used gas chromatography-flame-ionisation detection (GC-FID) to study the composition of stink gland secretions and cuticular hydrocarbons (CHCs) of immune-primed and wounded beetles compared to controls. The overall profiles as well as target compounds of the stink gland secretions showed transient, slight changes after these treatments, particularly in wounded females. Priming and wounding led to pronounced changes in CHC profiles with increases in the proportion of methyl-branched alkanes. Furthermore, we found sex-specific differences, that were particularly pronounced in the CHCs, although the changes due to immune stimulation were overall similar in both sexes. We suggest that CHCs are potential candidates for the transfer of information related to individual immunological experience into the group.

sentences: Scorepochs: a computer-aided scoring tool for resting-state M/EEG epochs M/EEG resting-state analysis often requires the definition of the epoch length and the criteria to select which epochs to include in the subsequent steps. However, the effects of epoch selection remain scarcely investigated and the procedure used to (visually) inspect, label, and remove bad epochs is often not documented, thus hindering the reproducibility of the reported results. In this study, we present Scorepochs, a simple and freely available tool for the automatic scoring of resting-state M/EEG epochs that aims to provide an objective method to aid M/EEG experts during epoch selection procedure. We tested our approach on a freely available EEG dataset containing recordings from 109 subjects using the BCI2000 64-channel system.

sentences: Host-mediated, cross-generational intraspecific competition in a herbivore species AO_SCPLOWBSTRACTC_SCPLOWConspecific insect herbivores co-occurring on the same host plant interact both directly through interference competition and indirectly through exploitative competition, plant-mediated interactions and enemy-mediated interactions. However, the situation is less clear when the interactions between conspecific insect herbivores are separated in time within the same growing season, as it is the case for multivoltine species. We hypothesized that early season herbivory would result in reduced egg laying and reduced performance of the next generation of herbivores on previously attacked plants. We tested this hypothesis in a choice experiment with box tree moth females (Cydalima perspectalis Walker, Lepidoptera: Crambidae). These females were exposed to box trees (Buxus sempervirens L., Buxaceae) that were either undamaged or attacked by conspecific larvae earlier in the season. We then compared the performance of the next generation larvae on previously damaged vs undamaged plants. Previous herbivory had no effect on oviposition behaviour, but the weight of next generation larvae was significantly lower in previously damaged plants. There was a negative correlation between the number of egg clutches laid on plants by the first generation and the performance of the next generation larvae. Overall, our findings reveal that early season herbivory reduces the performance of conspecific individuals on the same host plant later in the growing season, and that this time-lagged intraspecific competition results from a mismatch between the oviposition preference of females and the performance of its offspring.

sentences: Global Patterns in Culturable Soil Yeast Diversity Yeasts, broadly defined as unicellular fungi, fulfill essential roles in soil ecosystems as decomposers and nutrition sources for fellow soil-dwellers. Broad-scale investigations of soil yeasts pose a methodological challenge as metagenomics are of limited use on this group of fungi. Here we characterize global soil yeast diversity using fungal DNA barcoding on 1473 yeasts cultured from 3826 soil samples obtained from nine countries in six continents. We identify mean annual precipitation and international air travel as two significant predictors of soil yeast community structure and composition worldwide. Anthropogenic influences on soil yeast communities, directly via travel and indirectly via altered rainfall patterns resulting from climate change, are concerning as we found common infectious yeasts frequently distributed in soil in several countries. Our discovery of 41 putative novel species highlights the need to revise the current estimate of ~1500 recognized yeast species. Our findings demonstrate the continued need for culture-based studies to advance our knowledge of environmental yeast diversity.

sentences: In silico evidence of superantigenic features in ORF8 protein from COVID-19 Very early on COVID-19 pandemic outbreak, it was noted that the some of the virus-induced clinical conditions resembled features of toxaemia caused by the toxic shock syndrome toxin type 1, which is a soluble superantigen produced by Staphylococcus aureus. Among all SARS proteins, the ORF8 protein from SARS-2 virus is significantly different from other known SARS-like coronaviruses, and therefore could exhibit unique pathogenic properties. We assess if ORF8 protein bears super antigenic features using in silico tools. We show that ORF8 has properties of an extracellular soluble protein and shares a significant degree of amino acid sequence identity with toxic shock syndrome toxin. Besides, docking and binding affinity analyses between monomeric and homodimeric ORF-8 with V{beta} 2.1 and TRBV11-2 reveal strong interaction and high binding affinity. ORF8-TRBV11-2 strong interaction can contribute to the observed clonal expansion of that chain during COVID-19-associated multisystem inflammatory syndrome. Taken together, the evidence presented here supports the hypothesis that ORF8 protein from SARS-2 bears super antigenic properties.

sentences: Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM): a method for detection of antimicrobial resistance in environmental samples Effective monitoring of antibiotic resistance genes and their dissemination in environmental ecosystems has been hindered by the cost and efficiency of methods available for the task. We developed a method entitled the Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM), a system implementing high-throughput sequencing to simultaneously sequence thousands of antibiotic resistant genes representing a full-spectrum of antibiotic resistance classes commonly seen in environmental systems. In this study, we demonstrated DARTE-QM by screening 662 antibiotic resistance genes within environmental samples originated from manure, soil, and animal feces, in addition to a mock-community used as a control to test performance. DARTE-QM offers a new approach to studying antibiotic resistance in environmental microbiomes, showing advantages in efficiency and the ability to scale for many samples. This method provides a means of data acquisition that will alleviate the obstacles that many researchers in this area currently face.

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Mena regulates the LINC complex to control actin-nuclear lamina associations, trans-nuclear membrane signalling and cancer gene expression Interactions between cells and the extracellular matrix, mediated by integrin adhesion complexes (IACs), play key roles in cancer progression and metastasis. We investigated systems-level changes in the integrin adhesome during metastatic progression of a patient-derived cutaneous squamous cell carcinoma (cSCC), and found that the actin regulatory protein Mena is enriched in IACs in metastatic cSCC cells. Mena is connected within a subnetwork of actin-binding proteins to the LINC complex component nesprin-2, with which it interacts and co-localises at the nuclear envelope of metastatic cells. Moreover, Mena potentiates the interactions of nesprin-2 with the actin cytoskeleton and the nuclear lamina. CRISPR-mediated Mena depletion causes altered nuclear morphology, reduces tyrosine phosphorylation of the nuclear membrane protein emerin and downregulates expression of the immunomodulatory gene PTX3 via the recruitment of its enhancer to the nuclear periphery. We have uncovered an unexpected novel role for Mena at the nuclear membrane, where it controls the LINC complex, nuclear architecture, chromatin repositioning and cancer gene expression. This is the first description of an adhesion protein regulating gene transcription via direct signalling across the nuclear envelope.

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sentences: A novel multi-functional role for the MCM8/9 helicase complex in maintaining fork integrity during replication stress The minichromosome maintenance (MCM) 8/9 helicase is a AAA+ complex involved in DNA replication-associated repair. Despite high sequence homology to the MCM2-7 helicase, an active role for MCM8/9 has remained elusive. We interrogated fork progression in cells lacking MCM8 or 9 and find there is a functional partitioning. Loss of MCM8 or 9 slows overall replication speed and increases markers of genomic damage and fork instability, further compounded upon treatment with hydroxyurea. MCM8/9 acts upstream and antagonizes the recruitment of BRCA1 in nontreated conditions. However, upon treatment with fork stalling agents, MCM9 recruits Rad51 to protect and remodel persistently stalled forks. The helicase function of MCM8/9 aids in normal replication fork progression, but upon excessive stalling, MCM8/9 directs additional stabilizers to protect forks from degradation. This evidence defines novel multifunctional roles for MCM8/9 in promoting normal replication fork progression and promoting genome integrity following stress.

sentences: Integrated resource for functional and structural connectivity of the marmoset brain Comprehensive integration of structural and functional connectivity data is required for the accurate modeling of brain functions. While resources for studying structural connectivity of non-human primate brains already exist, their integration with functional connectivity data has remained unavailable. Here we present a comprehensive resource that integrates the largest awake non-human primate resting-state fMRI available to date (39 marmoset monkeys, 710 runs, 12117 mins) with previously published cellular-level neuronal tracing (52 marmoset monkeys, 143 injections), and multi-resolution diffusion MRI datasets. The combination of these data allowed us to: (1) map the fine-detailed functional brain networks and cortical parcellations; (2) develop a deep-learning-based parcellation generator that preserves the topographical organization of functional connectivity and reflect individual variabilities, and (3) investigate the structural basis underlying functional connectivity by computational modeling. This resource will enable modeling structure-function relationships and facilitate future comparative and translational studies of primate brains.

sentences: Water stress and nitrogen supply affect floral traits and pollination in the white mustard, <Sinapis alba> (Brassicaceae) Changes in environmental conditions are likely to have a complex effect on the growth of plants, their phenology, plant-pollinator interactions, and reproductive success. The current world is facing an ongoing climate change along with other human-induced environmental changes. Most research has focused on the impact of increasing temperature as a major driving force for climate change, but other factors may have important impacts on plant traits and pollination too and these effects may vary between different seasons. In addition, it is likely that the effects of multiple environmental factors, such as increasing temperature, water stress, and nitrogen enrichment are not independent. Therefore, we tested the impact of three key factors - season, water, and nitrogen supply - on plant traits, pollination, and seed production in Sinapis alba (Brassicaceae). Plants were grown in three seasons with two levels of water and nitrogen supply in a factorial design. We collected data on multiple vegetative and floral traits and assessed the response of pollinators in the field. Additionally, we evaluated the effect of growing conditions on seed set in plants exposed to pollinators and in hand-pollinated plants. Our results show that water stress impaired vegetative growth, decreased flower production, reduced visitation by pollinators and seed set, while nitrogen availability played an important role in nectar production. Seasonality modulated the effect of water and nitrogen availability on vegetative and floral traits and strongly affected flowering phenology and flower production. We demonstrated that changes in water and nitrogen availability alter plant vegetative and floral traits, which impacts flower visitation and consequently plant reproduction. We conclude that ongoing environmental changes such as altered precipitation regimes and nitrogen enrichment may thus affect plant-pollinator interactions with negative consequences for the reproduction of wild plants and insect-pollinated crops.

sentences: Structural basis of actin filament assembly and aging The dynamic turnover of actin filaments (F-actin) controls cellular motility in eukaryotes and is coupled to changes in the F-actin nucleotide state. It remains unclear how F-actin hydrolyzes ATP and subsequently undergoes subtle conformational rearrangements that ultimately lead to filament depolymerization by actin-binding proteins. Here, we present cryo-EM structures of F-actin in all nucleotide states, polymerized in the presence of Mg2+ or Ca2+, at resolutions ([~]2.2 [A]) that allow for the visualization of hundreds of water molecules. The structures reveal that the G- to F-actin transition induces the relocation of water molecules in the nucleotide binding pocket, activating one of them for the nucleophilic attack of ATP. Unexpectedly, the back door for the subsequent release of inorganic phosphate (Pi) is closed in all structures, indicating that the F-actin conformation that allows for Pi release occurs transiently. The small changes in the nucleotide-binding pocket after ATP hydrolysis and Pi release are sensed by a key amino acid, amplified and transmitted to the filament periphery. Furthermore, differences in the positions of waters in the nucleotide binding pocket explain why Ca2+-actin exhibits slower polymerization rates than Mg2+-actin. Our work elucidates the solvent-driven rearrangements that govern actin filament assembly and aging and lays the foundation for the rational design of drugs and small molecules for imaging and therapeutic applications.

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Degradation of visible autumn icons and conservation opportunities: trends in deciduous forest loss in the contiguous US Temperate deciduous forests are one of the most visible biomes on Earth because of their autumn aesthetics and because they harbor some of the most heavily populated regions. Their ability to attract visitors may increase opportunities for people to experience nature, which has been linked to greater conservation action. Identifying regions with high leaf-peeping opportunities and regions where color has been lost to landscape conversion may help to inform these connections. We use spatial overlay analyses to quantify temperate deciduous forest coverage, disturbance, and protections in each U.S. ecoregion. We evaluated recent (1984-2016) and predicted (2016-2050) disturbance under extreme future scenarios. Almost all ecoregions saw a decline in deciduous forest cover between 1985 and 2016. Some ecoregions with the greatest opportunities for leaf-peeping are also underrepresented in the protected areas network and vulnerable to additional losses. Under economic-growth forecasting scenarios, losses are predicted to continue. However, environmentally focused scenarios suggest there is still opportunity to reverse deciduous forest loss in some ecoregions. Differences in forest loss between predictions scenarios emphasize the importance of human approaches in securing environmental stability. Increasing public exposure to temperate forests may help ensure conservation of more natural areas and preserve the quantity and quality of autumn forest viewing.

Key PointsO_LITemperate deciduous forests aesthetics attract visitors to experience nature, but degradation and loss can hinder connections.
C_LIO_LIUS ecoregions with the greatest leaf-peeping opportunities are underrepresented in the protected areas and vulnerable to additional losses.
C_LIO_LIDifferences in predictions scenarios emphasize the importance of conservation action, which may be linked to human connections with nature.
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sentences: A combined computational fluid dynamics and MRI Arterial Spin Labeling modeling strategy to quantify patient-specific cerebral hemodynamics in cerebrovascular occlusive disease Cerebral hemodynamics in the presence of cerebrovascular occlusive disease (CVOD) are influenced by the anatomy of the intracranial arteries, the degree of stenosis, the patency of collateral pathways, and the condition of the cerebral microvasculature. Accurate characterization of cerebral hemodynamics is a challenging problem. In this work, we present a strategy to quantify cerebral hemodynamics using computational fluid dynamics (CFD) in combination with arterial spin labeling MRI (ASL). First, we calibrated patient-specific CFD outflow boundary conditions using ASL-derived flow splits in the Circle of Willis. Following, we validated the calibrated CFD model by evaluating the fractional blood supply from the main neck arteries to the vascular territories using Lagrangian particle tracking and comparing the results against vessel-selective ASL (VS-ASL). Finally, cerebral hemodynamics were assessed in two patients with CVOD and a healthy control subject. We demonstrated that the calibrated CFD model accurately reproduced the fractional blood supply to the vascular territories, as obtained from VS-ASL. The two patients revealed significant differences in pressure drop over the stenosis, collateral flow, and resistance of the distal vasculature, despite similar degrees of clinical stenosis severity. Our results demonstrated the advantages of a patient-specific CFD analysis for assessing the hemodynamic impact of stenosis.

sentences: Defining Moults in Migratory Birds: A Sequence-based Approach Two broad nomenclatures have emerged to describe moult strategies in birds, the "life-cycle" system which describes moults relative to present-day breeding and other life-history events and the Humphrey-Parkes (H-P) system which reflects the evolution of moults along ancestral lineages. Using either system, challenges have arisen defining strategies in migratory species with more than one moult per year. When all or part of two moults occur in non-breeding areas they may fail to be recognized as two moults or have been discriminated temporally, whether feathers are replaced in fall, winter, or spring. But in some cases feather replacement can span the non-breeding period, and this has resulted in an inability to identify inserted moults and to compare moult strategies between species. Furthermore, recent analyses on factors influencing the extent of the postjuvenile or preformative moults have either confined this moult to the summer grounds or presumed that it can be suspended and resumed on winter grounds, which has lead to quite divergent results. Evolutionarily, the timing, extent, and location of moults are very plastic whereas the sequence in which feathers are replaced is comparatively fixed. As, such, I propose taking an evolutionary approach to define moults on the basis of feather-replacement sequences as opposed to timing or location of replacement, including strategies in which moults can overlap temporally. I provide examples illustrating the functionality of a sequence-based definition in three migratory North American passerines that can undergo feather replacement twice in non breeding areas, and I demonstrate how this system can effectively apply to moults in many other passerine and non-passerine species. I recommend that authors studying the evolutionary drivers of moult strategies in migratory birds adopt a sequence-based approach or carefully consider replacement strategies both prior to and following autumn migration.

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Effective holistic characterization of small molecule effects using heterogeneous biological networks Drug discovery is the practice of identifying chemical entities with activities useful for treating human diseases. Despite substantial advancements in biotechnologies such as assays for detecting promising hits and computational methods for identifying targets to pursue, the success rate of novel therapeutics in the clinic is rapidly declining. The two most common reasons for drug attrition in clinical trials are efficacy, in that the drug is not capable of treating the disease, and safety, in which the compound is too toxic to patients and does not outweigh any purported benefits. Here we describe the use of an integrated human interactome network to describe the complete picture of drug behavior in biological systems and lead to more promising therapeutic candidate generation. The Computational Analysis of Novel Drug Opportunities (CANDO) platform for shotgun multiscale drug discovery, repurposing, and design was enhanced to include drugs/compounds, proteins, diseases/indications, Gene Ontology entities, and drug side effects, accompanied with various known associations between them, and interactomic signatures were generated for all compounds using the node2vec algorithm for the purpose of relating them in the context of diseases for which they are approved. The results indicate there is significant biological information available in the side effect profile of a drug and they can be used to better relate drugs in terms of their therapeutic behavior. Side effects were also predicted for all compounds in the platform; known drug-side effect associations were recovered at rates ten times higher than what can be expected with random chance. The network architecture was also investigated in the context of protein pathways, where drug distance to various pathways served as the features for input to a random forest machine learning model in the context of indications they are associated to treat, with interesting results highlighted for mental disorders and cancer metastasis. This pipeline highlights the ability of CANDO to accurately relate drugs in a multitarget interactomic context, and paves the way for predicting novel putative drug candidates using the information gleaned from not only their side effect profiles, but their impacts on protein pathways as well.

Significance StatementOur study outlines the capability of our drug discovery, repurposing, and design platform known as CANDO to more accurately describe small molecule therapeutic behavior in biological systems via the use of heterogeneous networks. This includes the ability to predict indications as well as side effects for compounds without clinical data, ultimately advancing the science of drug discovery.

sentences: MPI8 is Potent Against SARS-CoV-2 by Inhibiting Dually and Selectively the SARS-CoV-2 Main Protease and the Host Cathepsin L A number of inhibitors have been developed for the SARS-CoV-2 main protease (MPro) as potential COVID-19 medications but little is known about their selectivity. Using enzymatic assays, we characterized inhibition of TMPRSS2, furin, and cathepsins B/K/L by more than a dozen of previously developed MPro inhibitors including MPI1-9, GC376, 11a, 10-1, 10-2, and 10- 3. MPI1-9, GC376 and 11a all contain an aldehyde for the formation of a reversible covalent hemiacetal adduct with the MPro active site cysteine and 10-1, 10-2 and 10-3 contain a labile ester to exchange with the MPro active site cysteine for the formation of a thioester. Our data revealed that all these inhibitors are inert toward TMPRSS2 and furin. Diaryl esters also showed low inhibition of cathepsins. However, all aldehyde inhibitors displayed high potency in inhibiting three cathepsins. Their determined IC50 values vary from 4.1 to 380 nM for cathepsin B, 0.079 to 2.3 nM for cathepsin L, and 0.35 to 180 nM for cathepsin K. All aldehyde inhibitors showed similar inhibition levels toward cathepsin L. A cellular analysis indicated high potency of MPI5 and MPI8 in inhibiting lysosomal activity, which is probably attributed to their inhibition of cathepsins. Among all aldehyde inhibitors, MPI8 shows the best selectivity toward cathepsin L. With respect to cathepsins B and K, the selective indices are 192 and 150, respectively. MPI8 is the most potent compound among all aldehyde inhibitors in cellular MPro inhibition potency and anti-SARS-CoV-2 activity in Vero E6 cells. Cathepsin L has been demonstrated to play a critical role in the SARS-CoV-2 cell entry. By selectively inhibiting both SARS-CoV-2 MPro and the host cathepsin L, MPI8 potentiates dual inhibition effects to synergize its overall antiviral potency and efficacy. Due to its high selectivity toward cathepsin L that reduces potential toxicity toward host cells and high cellular and antiviral potency, we urge serious consideration of MPI8 for preclinical and clinical investigations for treating COVID-19.

sentences: SDC3 acts as a timekeeper of myogenic differentiation by regulating the insulin/AKT/mTOR axis in muscle stem cell progeny Muscle stem cells (MuSCs) are indispensable for muscle regeneration. A multitude of extracellular stimuli direct MuSC fate decisions from quiescent progenitors to differentiated myocytes. The activity of these signals is modulated by coreceptors such as syndecan-3 (SDC3). We investigated the global landscape of SDC3-mediated regulation of myogenesis using a phosphoproteomics approach which revealed, with the precision level of individual phosphosites, the large-scale extent of SDC3-mediated regulation of signal transduction in MuSCs. We then focused on INSR/AKT/mTOR as a key pathway regulated by SDC3 during myogenesis and mechanistically dissected SDC3-mediated inhibition of insulin signaling in MuSCs. SDC3 interacts with INSR limiting insulin signal transduction via AKT/mTOR. Both knockdown of INSR and inhibition of AKT rescue Sdc3-/- MuSC differentiation to wild type levels. Since SDC3 is rapidly downregulated at the onset of differentiation, our study suggests that SDC3 acts a timekeeper to restrain proliferating MuSC response to insulin and prevent premature differentiation.

sentences: Extracellular Matrix Levels Modulate Outgrowths Dynamics in Ovarian Cancer. Ovarian carcinoma (OC) form outgrowths that extend from the outer surface of an afflicted organ into the peritoneum. OC outgrowth formation is poorly understood because there is a limited availability of OC cell culture models to examine the behavior of cell assemblies that form outgrowths. Prompted by immuno-chemical evaluation of extracellular matrix (ECM) components, laminin {gamma}1 and collagens, in human tissues representing untreated and chemotherapy-recovered OC, we developed laminin and collagen-rich ECM-reconstituted cell culture models amenable to studies of cell assemblies that can form outgrowths. We demonstrate that ECM promotes outgrowth formation in fallopian tube non-ciliated epithelial cells (FNE) expressing mutant p53R175H and various OC cell lines. Outgrowths were initiated by cell assemblies that had undergone outward translocation and, upon mechanical detachment, could intercalate into mesothelial cell monolayers. Electron microcopy, optical coherence tomography (OCT) and small-amplitude oscillatory shear experiments revealed that elevating ECM levels increased ECM fibrous network thickness and led to high shear elasticity of ECM environment. These physical characteristics were associated with suppression of outgrowths. Culture environment with low ECM content mimicked viscoelasticity and fibrous networks of ascites and supported cell proliferation, cell translocation and outgrowth formation. These results highlight the importance of ECM microenvironments in modulating OC growth and could provide additional explanation of why primary and recurrent ovarian tumors form outgrowths that protrude into the peritoneal cavity containing ascites.

sentences: Probing behavior of the corn leafhopper Dalbulus maidis on susceptible and resistant maize hybrids The corn leafhopper Dalbulus maidis is the main vector of the pathogens that cause corn stunt, a major disease of maize in the Americas. As host resistance is an efficient tool to control diseases, the findings of a previous report showed that some corn hybrids are resistant to D. maidis. In this work, we assessed the probing behavior of D. maidis on susceptible and resistant corn hybrids using EPG (Electrical Penetration Graph) technology. Fifteen-day-old females were monitored for 20 hours, with access to hybrids DK390, DK670, DK79-10, and DK72-10. Hybrids DK390 and DK72-10 showed resistance to D. maidis in phloem, since insects feeding on these hybrids presented more salivation events in phloem without subsequent ingestion, which are seen as failed attempts to ingest. A reduction of the total duration of phloem ingestion was observed, and accordingly of the time spent by insects with access to these hybrids on xylem ingestion. The hybrid DK390 also had mesophyll resistance, seen as less probing time and a higher number of probes of short duration. These findings support and are consistent with previous research, providing useful information to characterize maize hybrids resistant to D. maidis, and so to corn stunt.

sentences: Brain network dynamics codify heterogeneity in seizure propagation Dynamic functional brain connectivity facilitates adaptive cognition and behavior. Abnormal alterations within such connectivity could result in disrupted functions observed across various neurological conditions. As one of the most common neurological disorders, epilepsy is defined by the seemingly random occurrence of spontaneous seizures. A central but unresolved question concerns the mechanisms by which extraordinarily diverse dynamics of seizures emerge. Here, we apply a graph-theoretical approach to assess dynamic reconfigurations in the functional brain connectivity before, during, and after seizures that display heterogeneous propagation patterns despite sharing similar origins. We demonstrate unique reconfigurations in globally-defined network properties preceding seizure onset that predict propagation patterns of impending seizures, and in locally-defined network properties that differentiate post-onset dynamics. These results characterize quantitative network features underlying the heterogeneity of seizure dynamics and the accompanying clinical manifestations. Decoding these network properties could improve personalized preventative treatment strategies for epilepsy as well as other neurological disorders.

sentences: Molecular dynamics simulation study of effects of key mutations in SARS-CoV-2 on protein structures SARS-CoV-2 has been spreading rapidly since 2019 and has produced large-scale mutations in the genomes. The mutation in genes may lead to changes in protein structure, which would have a great impact on the epidemiological characteristics. In this study, we selected the key mutations of SARS-CoV-2 from a real-time monitoring tool, including D614G, A222V, N501Y, T716I, S982A, D1118H of spike (S) protein, and performed molecular dynamics (MD) simulations on single-site mutant D614G, double-site mutant D614G&A222V and penta-site mutant N501Y&D614G&T716I&S982A&D1118H to investigate their effects on protein structure and stability using molecular dynamics (MD) simulations. The results suggested that D614G improved the stability of S protein, while D614G&A222V and N501Y&D614G&T716I&S982A&D1118H showed an increased solvent accessible surface area and they might enhance the ability of protein to react with the outside environment. Our findings could complement the mechanistic link between genotype--phenotype--epidemiological characteristics in the study of SARS-CoV-2. We also found no significant difference between the antigenicity of S protein and the mutants through Ellipro, which may reference for vaccine development and application.

sentences: Metabolic reprogramming of cytotoxic T lymphocytes by high glucose enhances pancreatic beta cell apoptosis via TRAIL Cytotoxic T lymphocytes (CTLs) are involved in development of diabetes. However, the impact of excessive glucose on CTL-mediated antigen-independent killing remains elusive. Here, we report that TNF-related apoptosis inducing ligand (TRAIL) is substantially up- regulated in CTLs in environments with high glucose (HG) both in vitro and in vivo. The PI3K- Akt-NF{kappa}B axis and non-mitochondrial reactive oxygen species are essential in HG-induced TRAIL upregulation in CTLs. TRAILhigh CTLs induce apoptosis of pancreatic beta cell line 1.4E7. Metformin and Vitamin D synergistically reduce HG-enhanced expression of TRAIL in CTLs and coherently protect 1.4E7 cells from TRAIL-mediated apoptosis. Notably, in patients with diabetes, correlation between Vitamin D concentrations in plasma and glucose levels is linked to HG-enhanced TRAIL expression on CTLs. Microarray data reveal that OXCT2, an important enzyme in ketone body catabolism, is a promising target in response to vitamin D. Our work not only reveals a novel mechanism of CTL involvement in progression of diabetes, but also establishes CTLs as a target for combined metformin and vitamin D therapy to protect pancreatic beta cells of diabetic patients.

sentences: Reinforcement Learning Based Approach for Ligand Pose Prediction Identification of the potential binding site and the correct ligand pose are two crucial steps among the various steps in protein ligand interaction for a novel or known target. Currently most of the deep learning methods work on protein ligand pocket datasets for various predictions. In this study, we propose a reinforcement learning (RL) based method for predicting the optimized ligand pose where the RL agent also identifies the binding site based on its training. In order to apply various reinforcement learning techniques, we suggest a novel approach to represent the protein ligand complex using graph CNN which would help utilize both atomic and spatial features. To the best of our knowledge, this is the first time an RL based approach has been put forward for predicting optimized ligand pose.

sentences: β-catenin perturbations control differentiation programs in mouse embryonic stem cells The Wnt/{beta}-catenin pathway is involved in development, cancer and embryonic stem cell (ESC) maintenance; its dual role in stem cell self-renewal and differentiation is still controversial. Here, by applying an in vitro system enabling inducible gene expression control, we report that moderate induction of transcriptionally active exogenous {beta}- catenin in {beta}-catenin null mouse ESCs promotes epiblast-like cell (EpiLC) derivation in vitro. Instead, in wild type cells moderate chemical pre-activation of the Wnt/{beta}-catenin pathway promotes EpiLC in vitro derivation. Finally, we suggest that moderate {beta}- catenin levels in {beta}-catenin null mouse ESCs favour early stem cell commitment towards mesoderm if the exogenous protein is induced only in the  ground state of pluripotency condition, or endoderm if the induction is maintained during the differentiation. Overall, our results confirm previous findings about the role of {beta}-catenin in pluripotency and differentiation, while indicating a role for its doses in promoting specific differentiation programs.

sentences: SARS-CoV-2 B.1.617.2 Delta variant replication, sensitivity to neutralising antibodies and vaccine breakthrough The SARS-CoV-2 B.1.617.2 (Delta) variant was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha). In vitro, B.1.617.2 is 6-fold less sensitive to serum neutralising antibodies from recovered individuals, and 8-fold less sensitive to vaccine-elicited antibodies as compared to wild type Wuhan-1 bearing D614G. Serum neutralising titres against B.1.617.2 were lower in ChAdOx-1 versus BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies against the receptor binding domain (RBD) and N-terminal domain (NTD), in particular to the clinically approved bamlavinimab and imdevimab monoclonal antibodies. B.1.617.2 demonstrated higher replication efficiency in both airway organoid and human airway epithelial systems as compared to B.1.1.7, associated with B.1.617.2 spike being in a predominantly cleaved state compared to B.1.1.7. Additionally we observed that B.1.617.2 had higher replication and spike mediated entry as compared to B.1.617.1, potentially explaining B.1.617.2 dominance. In an analysis of over 130 SARS-CoV-2 infected healthcare workers across three centres in India during a period of mixed lineage circulation, we observed substantially reduced ChAdOx-1 vaccine efficacy against B.1.617.2 relative to non-B.1.617.2. Compromised vaccine efficacy against the highly fit and immune evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.

sentences: Exploring Extreme Signaling Failures in Intracellular Molecular Networks Developing novel methods for the analysis of intracellular signaling networks is essential for understanding interconnected biological processes that underlie complex human disorders. A fundamental goal of this research is to quantify the vulnerability of a signaling network to the dysfunction of one or multiple molecules, when the dysfunction is defined as an incorrect response to the input signals. In this study, we propose an efficient algorithm to identify the extreme signaling failures that can induce the most detrimental impact on the physiological function of a molecular network. The algorithm basically finds the molecules, or groups of molecules, with the maximum vulnerability, i.e., the highest probability of causing the network failure, when they are dysfunctional. We propose another algorithm that efficiently accounts for signaling feedbacks in this analysis. The algorithms are tested on two experimentally verified ERBB and T cell signaling networks. Surprisingly, results reveal that as the number of concurrently dysfunctional molecules increases, the maximum vulnerability values quickly reach to a plateau following an initial increase. This suggests the specificity of vulnerable molecule (s) involved, as a specific number of faulty molecules cause the most detrimental damage to the function of the network. Increasing a random number of simultaneously faulty molecules does not further deteriorate the function of the network. Such a group of specific molecules whose dysfunction causes the extreme signaling failures can better elucidate the molecular mechanisms underlying the pathogenesis of complex trait disorders, and can offer new insights for the development of novel therapeutics.

sentences: Sensitive detection and structural characterisation of UV-induced cross-links in protein-RNA complexes using CLIR-MS Cross-linking coupled with mass spectrometry is an increasingly popular methodology for elucidating structural information from biological complexes. Whilst protein-protein cross-linking workflows are widely used and well characterised, adoption of protein-RNA cross-linking workflows for structural studies is less widespread, and data produced from such experiments remains less well understood. The cross-linking of stable isotope labelled RNA coupled to mass spectrometry (CLIR-MS) workflow uses isotope labelled RNA to simultaneously confirm that peptides are cross-linked to RNA and aid cross-link localisation in an RNA sequence. For broader application of CLIR-MS as part of the structural analysis of ribonucleoproteins, the method must be sensitive, robust, and its reaction products need to be well characterised. We enhanced our previously published workflow, improving coverage and sensitivity. We used it to infer common properties of protein-RNA cross-links such as cross-linking distance, and to assess the impact of substitution of uracil with 4-thio-uracil in structural proteomics experiments. We profiled the compositional diversity of RNA-derived peptide modifications, and subsequently defined a more inclusive data analysis approach which more than doubles the number of cross-link spectrum matches compared with our past work. We defined distance restraints from these cross-links, and with the aid of visualisation software, demonstrated that on their own they provide sufficient information to localise an RNA chain to the correct position on the surface of a protein. We applied our enhanced workflow and understanding to characterise the binding interface of several protein-RNA complexes containing classical and uncommon RNA binding domains. The enhanced sensitivity and understanding demonstrated here underpin a wider adoption of protein-RNA cross-linking in structural biology.

sentences: Mechanisms of lactic acid gustatory attraction in Drosophila Sour has been studied almost exclusively as an aversive taste modality. Yet, recent work in Drosophila demonstrates that specific carboxylic acids are attractive at ecologically relevant concentrations. Here, we demonstrate that lactic acid is an appetitive and energetic tastant, which stimulates feeding through activation of sweet gustatory receptor neurons (GRNs). This activation displays distinct, mechanistically separable, stimulus onset and removal phases. Ionotropic receptor 25a (IR25a) primarily mediates the onset response, which shows specificity for the lactate anion and drives feeding initiation. Conversely, sweet gustatory receptors (Gr64a-f) mediate a non-specific removal response to low pH that primarily impacts ingestion. While mutations in either receptor family have marginal impacts on feeding, lactic acid attraction is completely abolished in combined mutants. Thus, specific components of lactic acid are detected through two classes of receptors to activate a single set of sensory neurons in physiologically distinct ways, ultimately leading to robust behavioural attraction.

sentences: Multi-timescale biological learning algorithms train spiking neuronal network motor control Biological learning operates at multiple interlocking timescales, from long evolutionary stretches down to the relatively short timespan of an individuals life. While each process has been simulated individually as a learning algorithm in spiking neuronal networks (SNNs), the integration of the two is limited. In this study, we first train SNNs separately using individual model learning using spike-timing dependent reinforcement learning (STDP-RL) and evolutionary (EVOL) learning algorithms to solve the CartPole reinforcement learning (RL) control problem. We then develop an interleaved algorithm inspired by biological evolution that combines the EVOL and STDP-RL learning in sequence (EVOL+STDP-RL). This sequential algorithm implements the Baldwin Effect, by allowing learning during a models lifetime to indirectly influence future model generations produced by EVOL. We evaluate performance of each algorithm after training and through the creation of sensory/motor action maps that delineate the networks transformation of sensory inputs into higher-order representations and motor decisions. Compared to STDP-RL and EVOL algorithms operating on their own, our EVOL+STDP-RL interleaved training paradigm enhanced performance robustness and shortened the required training to reach optimal performance. The different model strategies were revealed through analysis of sensory/motor mappings. Our modeling opens up new capabilities for SNNs in RL and could serve as a testbed for neurobiologists aiming to understand multi-timescale learning mechanisms and dynamics in neuronal circuits.

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Estimating the timing of multiple admixture events using 3-locus Linkage Disequilibrium Estimating admixture histories is crucial for understanding the genetic diversity we see in present-day populations. Allele frequency or phylogeny-based methods are excellent for inferring the existence of admixture or its proportions. However, to estimate admixture times, spatial information from admixed chromosomes of local ancestry or the decay of admixture linkage disequilibrium (ALD) is used. One popular method, implemented in the programs ALDER and ROLLOFF, uses two-locus ALD to infer the time of a single admixture event, but is only able to estimate the time of the most recent admixture event based on this summary statistic. To address this limitation, we derive analytical expressions for the expected ALD in a three-locus system and provide a new statistical method based on these results that is able to resolve more complicated admixture histories. Using simulations, we evaluate the performance of this method on a range of different admixture histories. As an example, we apply the method to the Colombian and Mexican samples from the 1000 Genomes project. The implementation of our method is available at https://github.com/Genomics-HSE/LaNeta.

Author summaryWe establish a theoretical framework to model 3-locus admixture linkage disequilibrium of an admixed population taking into account the effects of genetic drift, migration and recombination. The theory is used to develop a method for estimating the times of multiple admixtures events. We demonstrate the accuracy of the method on simulated data and we apply it to previously published data from Mexican and Columbian populations to explore the complex history of American populations in the post-Columbian period.

sentences: Heterogeneous non-canonical nucleosomes predominate in yeast cells in situ Nuclear processes depend heavily on the organization of chromatin, whose subunits are cylinder-shaped complexes called nucleosomes. A subset of mammalian nucleosomes in situ resemble the canonical structure determined in vitro 24 years ago. The structure of nucleosomes in situ is otherwise poorly understood. Here we use cryo-ET and 3-D classification analysis to study the structure of yeast nucleosomes both in vitro and in situ. We show that the class averages of GFP-tagged yeast nucleosomes in vitro resemble canonical nucleosomes, with additional GFP densities. In contrast, none of the class averages of nucleosome-like particles in situ (inside cells) resemble canonical nucleosomes. The heterogeneous nature of the in situ class averages suggests that the intranuclear environment favors multiple conformations. Using the structural observations here and the results of previous genomics and biochemical studies, we propose a model in which the average yeast nucleosomes DNA is partially detached in situ.

sentences: Regulation of let-7 microRNAs by a two-step turnover pathway is of critical pathophysiological significance in humans MicroRNAs are critical regulators of diverse developmental and physiological processes, and they themselves get regulated both at the level of biogenesis and turnover. We demonstrate that the ribonuclease XRN2 can degrade the mature forms of certain let-7 family members in multiple human cancer cell lines, without affecting their precursors. XRN2 depletion results in a reduction in the expression of a number of oncogenes, and diminishes the proliferative and metastatic potential of cancer cells. The clinical relevance of these observations is also verified in tumour transcriptomics data from public RNA-sequencing datasets, where XRN2 mRNA expression is inversely correlated with the levels of a large number of miRNAs, including let-7 members, and high XRN2 mRNA levels are associated with poor survival in hepatocellular carcinoma, lung adenocarcinoma, and glioblastoma. We demonstrate that the miRNA is released by an as-yet unidentified proteinaceous  miRNA release factor from the grasp of Argonaute before its degradation. Our analyses of the patient-derived transcriptomics data also show that XRN2, via its regulation of let-7, affects multiple pathways in a consistent manner across epithelial and glial cell lineages, and thus, is of critical pathophysiological significance.

sentences: Genome Methylation Predicts Age and Longevity of Bats Exceptionally long-lived species, including many bats, rarely show overt signs of aging, making it difficult to determine why species differ in lifespan. Here, we use DNA methylation (DNAm) profiles from 712 known-age bats, representing 26 species, to identify epigenetic changes associated with age and longevity. We demonstrate that DNAm accurately predicts chronological age. Across species, longevity is negatively associated with the rate of DNAm change at age-associated sites. Furthermore, analysis of several bat genomes reveals that hypermethylated age- and longevity-associated sites are disproportionately located in promoter regions of key transcription factors (TF) and enriched for histone and chromatin features associated with transcriptional regulation. Predicted TF binding site motifs and enrichment analyses indicate that age-related methylation change is influenced by developmental processes, while longevity-related DNAm change is associated with innate immunity or tumorigenesis genes, suggesting that bat longevity results from augmented immune response and cancer suppression.

sentences: Injectable Nanoparticle-Based Hydrogels Enable the Safe and Effective Deployment of Immunostimulatory CD40 Agonist Antibodies When properly deployed, the immune system can eliminate deadly pathogens, eradicate metastatic cancers, and provide long-lasting protection from diverse diseases. Unfortunately, realizing these remarkable capabilities is inherently risky as disruption to immune homeostasis can elicit dangerous complications or autoimmune disorders. While current research is continuously expanding the arsenal of potent immunotherapeutics, there is a technological gap when it comes to controlling when, where, and how long these drugs act on the body. Here, we explore the ability of a slow-releasing injectable hydrogel depot to reduce the problematic dose-limiting toxicities of immunostimulatory CD40 agonist (CD40a) while maintaining their potent anti-cancer efficacy. We leverage a previously described polymer-nanoparticle (PNP) hydrogel system that exhibits shear-thinning and yield-stress properties that we hypothesized would improve locoregional delivery of the CD40a immunotherapy. Using PET imaging, we demonstrate that prolonged hydrogel-based delivery redistributes CD40a exposure to the tumor and the tumor draining lymph node (TdLN), thereby reducing weight loss, hepatotoxicity, and cytokine storm associated with standard treatment. Moreover, CD40a-loaded hydrogels mediate improved local cytokine induction in the TdLN and improve treatment efficacy in the B16F10 melanoma model. PNP hydrogels, therefore, represent a facile, drug-agnostic method to ameliorate immune-related adverse effects and explore locoregional delivery of immunostimulatory drugs.

sentences: Characterization of SdGA, a cold-adapted glucoamylase from Saccharophagus degradans We investigated the structural and functional properties of SdGA, a glucoamylase (GA) from Saccharophagus degradans, a marine bacterium which degrades different complex polysaccharides at high rate. SdGA is composed mainly by a N-terminal GH15_N domain linked to a C-terminal catalytic domain (CD) found in the GH15 family of glycosylhydrolases with an overall structure similar to other bacterial GAs. The protein was expressed in Escherichia coli cells, purified and its biochemical properties were investigated. Although SdGA has a maximum activity at 39{degrees}C and pH 6.0, it also shows high activity in a wide range, from low to mild temperatures, like cold-adapted enzymes. Furthermore, SdGA has a higher content of flexible residues and a larger CD due to various amino acid insertions compared to other thermostable GAs. We propose that this novel SdGA, is a cold-adapted enzyme that might be suitable for use in different industrial processes that require enzymes which act at low or medium temperatures.

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Removal of KCNQ2 from Parvalbumin-expressing Interneurons Improves Anti-Seizure Efficacy of Retigabine Anti-seizure drug (ASD) targets are widely expressed in both excitatory and inhibitory neurons. It remains unknown if the action of an ASD upon inhibitory neurons could counteract its beneficial effects on excitatory neurons (or vice versa), thereby reducing the efficacy of the ASD. Here, we examine whether the efficacy of the ASD retigabine (RTG) is altered after removal of the Kv7 potassium channel subunit KCNQ2, one of its drug targets, from parvalbumin-expressing interneurons (PV-INs). Parvalbumin-Cre (PV-Cre) mice were crossed with Kcnq2-floxed (Kcnq2fl/fl) mice to conditionally delete Kcnq2 from PV-INs. In these conditional knockout mice (cKO, PV-Kcnq2fl/fl), RTG (10 mg/kg, i.p.) significantly delayed the onset of either picrotoxin (PTX, 10 mg/kg, i.p)- or kainic acid (KA, 30mg/kg, i.p.)-induced convulsive seizures compared to vehicle, while RTG was not effective in wild-type littermates (WT). Immunostaining for KCNQ2 and KCNQ3 revealed that both subunits were enriched at axon initial segments (AISs) of hippocampal CA1 PV-INs, and their specific expression was selectively abolished in cKO mice. Accordingly, the M-currents recorded from CA1 PV-INs and their sensitivity to RTG were significantly reduced in cKO mice. While the ability of RTG to suppress CA1 excitatory neurons in hippocampal slices was unchanged in cKO mice, its suppressive effect on the spike activity of CA1 PV-INs was significantly reduced compared with WT mice. In addition, the RTG-induced suppression on intrinsic membrane excitability of PV-INs in WT mice was significantly reduced in cKO mice. These findings suggest that preventing RTG from suppressing PV-INs improves its anticonvulsant effect.

Significance StatementOne out of three patients with epilepsy remain resistant to anti-seizure drugs (ASD). Therefore, strategies that can enhance the efficacy of current ASDs or aid in the development of novel ASDs are in high demand. Here we show that by sparing a subset of inhibitory neurons, the ASD retigabine becomes more efficacious. These findings indicate that developing the ASDs that have relatively greater effects on excitatory over inhibitory neurons may be an effective strategy to improve the efficacy of ASDs.

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CHEK2 SIGNALING IS THE KEY REGULATOR OF OOCYTE SURVIVAL AFTER CHEMOTHERAPY Radiation and chemotherapy can damage the primordial follicle reserve in female cancer patients leading to ovarian failure and infertility. Preservation of ovarian function requires treatment strategies that prevent loss of immature oocytes in primordial follicles during cancer therapy. Checkpoint kinase 2 (CHEK2) inhibition prevents loss of primordial oocytes caused by DNA damage and thus is a promising target for ovoprotective treatment against genotoxic agents. To determine which cancer treatments could benefit from ovoprotective activity of CHEK2 inhibition we investigated oocyte survival in Chek2-/- mice exposed to different chemotherapy drugs. Here, we show that loss of CHEK2 function prevents elimination of primordial oocytes damaged by cisplatin, cyclophosphamide, mafosfamide, doxorubicin, and etoposide, suggesting it could be used to reduce ovarian damage caused by wide range of drugs. Using genetic knockouts we reveal a critical role for TRP53 in oocyte response to chemotherapy drugs and show that both targets of CHEK2--TAp63 and TRP53-- are activated by cisplatin and cyclophosphamide. Furthermore, we show that checkpoint kinase inhibitor and radiation- and chemotherapy sensitizer AZD7762 reduces oocyte elimination after radiation and chemotherapy treatments, despite its cytotoxic effect on ovarian somatic cells. Altogether, these findings demonstrate the role for CHEK2 as the master regulator of primordial oocyte survival or death and credential its targeting for ovoprotective treatments.

SIGNIFICANCEChemotherapy and radiation are ovotoxic and increase the risk of premature ovarian insufficiency and infertility in women cancer survivors. Development of treatment strategies preserving ovarian function and ensuring future reproductive health of female cancer patients depends on better understanding of the mechanisms underlying ovarian toxicity caused by different chemotherapy treatments. Preservation of long-term ovarian function can only be achieved by preventing the loss of immature oocytes in primordial follicles during toxic cancer therapies. Checkpoint kinase 2 (CHEK2) inhibition is an attractive strategy for protecting ovarian reserve with a potential additional benefit of sensitizing cancer cells to radiation and chemotherapy. Using a genetic approach, we show that blocking CHEK2 function is sufficient to prevent elimination of primordial oocytes damaged by chemotherapy drugs such as cisplatin, cyclophosphamide, mafosfamide, doxorubicin and etoposide. Many chemotherapy drugs are used in combination (e.g. cyclophosphamide with doxorubicin), thus the protective effect of CHEK2 inhibition is likely to be beneficial for a broad spectrum of patient treatments.

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Gene prioritization based on random walks with restarts and absorbing states, to define gene sets regulating drug pharmacodynamics from single-cell analyses Prioritizing genes for their role in drug sensitivity, is an important step in understanding drugs mechanisms of action and discovering new molecular targets for co-treatment. To formalize this problem, we consider two sets of genes X and P respectively composing the predictive gene signature of sensitivity to a drug and the genes involved in its mechanism of action, as well as a protein interaction network (PPIN) containing the products of X and P as nodes. We introduce Genetrank, a method to prioritize the genes in X for their likelihood to regulate the genes in P.

Genetrank uses asymmetric random walks with restarts, absorbing states, and a suitable renormalization scheme. Using novel so-called saturation indices, we show that the conjunction of absorbing states and renormalization yields an exploration of the PPIN which is much more progressive than that afforded by random walks with restarts only. Using MINT as underlying network, we apply Genetrank to a predictive gene signature of cancer cells sensitivity to tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL), performed in single-cells. Our ranking provides biological insights on drug sensitivity and a gene set considerably enriched in genes regulating TRAIL pharmacodynamics when compared to the most significant differentially expressed genes obtained from a statistical analysis framework alone. We also introduce gene expression radars, a visualization tool to assess all pairwise interactions at a glance.

Genetrank is made available in the Structural Bioinformatics Library (https://sbl.inria.fr/doc/Genetrank-user-manual.html). It should prove useful for mining gene sets in conjunction with a signaling pathway, whenever other approaches yield relatively large sets of genes.

sentences: Towards rainy Arctic winters: experimental icing impacts tundra plant productivity and reproduction The Arctic is warming rapidly, and the rate of warming is twice as fast in winter as in summer. More frequent extreme warm spells and associated rain-on-snow events in winter can dramatically alter snowpack conditions, ultimately encapsulating the tundra vegetation in  basal ice for several months. As most tundra vegetation studies have focused on summer warming, potential icing effects on phenology, productivity, and reproduction remain largely unexplored. We addressed this gap by performing a pioneer field experiment in high Arctic Svalbard, simulating extreme rain-on-snow and resultant basal ice ( icing) in five consecutive winters and assessing vascular plant responses throughout each subsequent growing season. We also tested whether icing responses were modified by experimentally increased summer temperatures. Icing alone delayed early vegetative and reproductive phenology of the dominant dwarf shrub, Salix polaris, but with clear evidence for a  catch-up in terms of improved community-level primary production later in the growing season. This compensatory growth occurred at the expense of flower production and delayed seed maturation. Both the phenological delay and allocation trade-offs were associated with icing-induced lags in thawing out and subsequent warming up of the soil, crucial in regulating plant nutrient availability and acquisition. However, experimental summer warming modified icing effects by advancing plant phenology, thus increasing primary productivity already early in the growing season, and partially offsetting negative icing effects on reproduction. Consequently, both winter and summer warming must be considered simultaneously in order to predict climate change responses of Arctic tundra vegetation. Our findings demonstrate that more frequent extreme warm spells in winter have the potential to shape key aspects of high Arctic plant communities. As we are heading towards rain-dominated Arctic winters, this calls for similar experiments in coordinated circumpolar monitoring programs across tundra plant communities.

sentences: Citrullination of glucokinase linked to autoimmune diabetes Inflammation, including reactive oxygen species and inflammatory cytokines in tissue microenvironments amplify the appearance of various post-translational modifications (PTMs) of self-proteins. Previously, a number of PTMs have been identified as autoimmune biomarkers in the initiation and progression of Type 1 diabetes (T1D). Herein, we have identified the citrullination of glucokinase (GK) as a result of inflammation, triggering autoimmunity and affecting its biological functions. Glucokinase is predominantly expressed in hepatocytes to regulate glycogen synthesis, and in pancreatic beta cells, where it acts as a glucose sensor to initiate glycolysis and insulin signaling. Herein, we demonstrate that glucokinase is citrullinated in inflamed non-obese diabetic (NOD) islets as well as in human GK. Autoantibodies against both native and citrullinated GK arise in both spontaneous human T1D and murine models. Likewise, autoreactive CD4+ T cells to both native and citrullinated glucokinase epitopes are present in the circulation of T1D patients. Finally, citrullination alters GK biologic activity and suppresses glucose-stimulated insulin secretion. Our studies define glucokinase as a T1D biomarker, providing new insights into altering glucose metabolism, creating neoautoantigens, and better define the broad impact of PTMs on the tissue pathology of T1D.

sentences: Template-based assembly of proteomic short reads for de novo antibody sequencing and repertoire profiling Antibodies can target a vast molecular diversity of antigens. This is achieved by generating a complementary diversity of antibody sequences though somatic recombination and hypermutation. A full understanding of the antibody repertoire in health and disease therefore requires dedicated de novo sequencing methods. Next generation cDNA sequencing methods have laid the foundation of our current understanding of the antibody repertoire, but these methods share one major limitation in that they target the antibody-producing B-cells, rather than the functional secreted product in bodily fluids. Mass spectrometry-based methods offer an opportunity to bridge this gap between antibody repertoire profiling and bulk serological assays, as they can access antibody sequence information straight from the secreted polypeptide products. In a step to meeting the challenge of MS-based antibody sequencing, we present a fast and simple software tool (Stitch) to map proteomic short reads to user-defined templates with dedicated features for both monoclonal antibody sequencing and profiling of polyclonal antibody repertoires. We demonstrate the use of Stitch by fully reconstructing 2 monoclonal antibody sequences with >98% accuracy (including I/L assignment); sequencing a Fab from patient serum isolated by reversed-phase LC fractionation against a high background of homologous antibody sequences; sequencing antibody light chains from urine of multiple-myeloma patients; and profiling the IgG repertoire in sera from patients hospitalized with COVID-19. We demonstrate that Stitch assembles a comprehensive overview of the antibody sequences that are represented in the dataset and provides an important first step towards analyzing polyclonal antibodies and repertoire profiling.

sentences: RORβ modulates a gene program that is protective against articular cartilage damage Osteoarthritis (OA) is the most prevalent chronic joint disease which increases in frequency with age eventually impacting most people over the age of 65. OA is the leading cause of disability and impaired mobility, yet the pathogenesis of OA remains unclear. Treatments have focused mainly on pain relief and reducing joint swelling. Currently there are no effective treatments to slow the progression of the disease and to prevent irreversible loss of cartilage. Here we demonstrate that stable expression of ROR{beta} in cultured cells results in alteration of a gene program that is supportive of chondrogenesis and is protective against development of OA. Specifically, we determined that ROR{beta} regulates the balance of FGFRs signaling on FGFR1/FGFR3 that ERK1/2-MAPK signaling was suppressed by FGFR1(cartilage destruction) and AKT signaling was enhanced by FGFR3 (cartilage protection). These results suggest a critical role for ROR{beta} in chondrogenesis and suggest that identification of mechanisms that control the expression of ROR{beta} in chondrocytes could lead to the development of disease modifying therapies for the treatment of OA.

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Cortical-subcortical structural connections support transcranial magnetic stimulation engagement of the amygdala The amygdala processes valenced stimuli, influences affective states, and exhibits aberrant activity across anxiety disorders, depression, and PTSD. Interventions that modulate amygdala activity hold promise for treating transdiagnostic affective symptoms. We investigated (N=45) whether transcranial magnetic stimulation (TMS) elicits indirect changes in amygdala activity when applied to ventrolateral prefrontal cortex (vlPFC), a region important for affect regulation. Harnessing in-scanner interleaved TMS/functional MRI (fMRI), we reveal that vlPFC neurostimulation evoked acute, dose-dependent modulations of amygdala fMRI BOLD signal. Larger TMS-evoked changes in amygdala fMRI signal were associated with higher fiber density in a vlPFC-amygdala white matter pathway, suggesting this pathway facilitated stimulation-induced communication between cortex and subcortex. This work provides evidence of amygdala engagement by TMS, highlighting stimulation of vlPFC-amygdala circuits as a candidate treatment for affective psychopathology. More broadly, it indicates that targeting cortical-subcortical connections may enhance the impact of TMS on subcortical neural activity and, by extension, subcortex-subserved behaviors.

TeaserIndividualized, connectivity-guided transcranial magnetic stimulation modulates the amygdala, demonstrating therapeutic potential.

sentences: Core and indicative bacterial and fungal taxa define characteristic soil communities of arable land, grassland, and forest Soil microbial diversity has major influences on ecosystem functions and services. However, due to its complexity and uneven distribution of abundant and rare taxa, quantification of soil microbial diversity remains challenging and thereby impeding its integration into long-term monitoring programs. Using metabarcoding, we analyzed soil bacterial and fungal communities over five years at thirty long-term soil monitoring sites from the three land-use types, arable land, permanent grassland, and forest. Unlike soil microbial biomass and alpha-diversity, microbial community compositions and structures were site- and land-use-specific with CAP reclassification success rates of 100%. The temporally stable site core communities included 38.5% of bacterial and 33.1% of fungal OTUs covering 95.9% and 93.2% of relative abundances. We characterized bacterial and fungal core communities and their land-use associations at the family-level. In general, fungal families revealed stronger land-use type associations as compared to bacteria. This is likely due to a stronger vegetation effect on fungal core taxa, while bacterial core taxa were stronger related to soil properties. The assessment of core communities can be used to form cultivation-independent reference lists of microbial taxa, which may facilitate the development of microbial indicators for soil quality and the use of soil microbiota for long-term soil biomonitoring.

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New SARS-CoV-2 lineages could evade CD8+ T-cells response BackgroundMany SARS-CoV-2 variants of concern have emerged since the Covid-19 outburst, notably the lineages detected in the UK, South Africa, and Brazil. Their increased transmissibility and higher viral load put them in the spotlight. Much has been investigated on the ability of those new variants to evade antibody recognition. However, not enough attention has been given to pre-existing and induced SARS-CoV-2-specific CD8+ T cell responses during the natural course of infection by new lineages.

MethodsIn this work, we investigated the SARS-CoV-2-specific CD8+ T cell epitopes from the main variants of concern and the potential of associated mutations to trigger or hinder CD8+ T-cells response. We also estimated the populations coverage of these different lineages, considering peptide binding predictions to class I HLA alleles from 29 countries to investigate differences in the fraction of individuals expected to respond to a given epitope set from new and previous lineages.

ResultsWe observed a lower populational coverage for 20B/S.484K (P.2 lineage) in contrast to an increased coverage found for 20H/501Y.V2 (B.1.351 Lineage) and 20J/501Y.V3 (P.1 lineage) compared to a reference lineage. Moreover, mutations such as Spike N501Y and Nucleocapsid T205I were predicted to have an overall higher affinity through HLA-I than the reference sequence.

ConclusionsIn summary, the data in this work provided evidence for the existence of potentially immunogenic and conserved epitopes across new SARS-CoV-2 variants, but also highlights the reduced populationals coverage for the Brazilian lineage P.2, suggesting its potential to evade from CD8+ T-cell responses. Our results also may guide efforts to characterize and validate relevant peptides to trigger CD8+ T-cell responses, and design new universal T-cell-inducing vaccine candidates that minimize detrimental effects of viral diversification and at the same time induce responses to a broad human population.

sentences: Translation rescue by targeting Ppp1r15a upstream open reading frame in vivo The eIF2 initiation complex is central to maintaining a functional translation machinery. Extreme stress such as life-threatening sepsis exposes vulnerabilities in this tightly regulated system, resulting in an imbalance between the opposing actions of kinases and phosphatases on the main regulatory subunit eIF2. Here, we report that translation shutdown is a hallmark of established sepsis-induced kidney injury brought about by excessive eIF2 phosphorylation and sustained by blunted expression of the counterregulatory phosphatase subunit Ppp1r15a. We determined that the blunted Ppp1r15a expression persists because of the presence of an upstream open reading frame (uORF). Overcoming this barrier with genetic approaches enabled the derepression of Ppp1r15a, salvaged translation and improved kidney function in an endotoxemia model. We also found that the loss of this uORF has broad effects on the composition and phosphorylation status of the immunopeptidome that extended beyond the eIF2 axis. Collectively, our findings define the breath and potency of the highly conserved Ppp1r15a uORF and provide a paradigm for the design of uORF-based translation rheostat strategies. The ability to accurately control the dynamics of translation during sepsis will open new paths for the development of therapies at codon level precision.

sentences: Local and collective transitions in sparsely-interacting ecological communities Interactions in natural communities can be highly heterogeneous, with any given species interacting appreciably with only some of the others, a situation commonly represented by sparse interaction networks. We study the consequences of sparse competitive interactions, in a theoretical model of a community assembled from a species pool. We find that communities can be in a number of different regimes, depending on the interaction strength. When interactions are strong, the network of coexisting species breaks up into small subgraphs, while for weaker interactions these graphs are larger and more complex, eventually encompassing all species. This process is driven by emergence of new allowed subgraphs as interaction strength decreases, leading to sharp changes in diversity and other community properties, and at weaker interactions to two distinct collective transitions: a percolation transition, and a transition between having a unique equilibrium and having multiple alternative equilibria. Understanding community structure is thus made up of two parts: first, finding which subgraphs are allowed at a given interaction strength, and secondly, a discrete problem of matching these structures over the entire community. In a shift from the focus of many previous theories, these different regimes can be traversed by modifying the interaction strength alone, without need for heterogeneity in either interaction strengths or the number of competitors per species.

sentences: A New Likelihood-based Test for Natural Selection We present a new statistic for testing for neutral evolution from allele frequency data summarised as a site frequency spectrum, which we call the relative likelihood neutrality test or{rho} . Classical methods of testing for natural selection, such as Tajimas D and its relatives, require the null model to have constant population size over time and therefore can confound demographic change with natural selection.{rho} can directly incorporate a null hypothesis reflecting general demographic histories. It has a natural Bayesian interpretation as an approximation to the log-probability of the null model, given the data. We use simulations to show that{rho} has greater power than Tajimas D to detect departure from neutrality for a range of scenarios of positive and negative selection. We also show how{rho} can be adapted to account for sequencing error. Application to the ACKR1 (FYO) gene in humans supported previous studies inferring positive selection in sub-Saharan populations which were based on inter-population comparisons. However, we did not find the signal of selection to be maximal in the region of the FY*O or Duffy-null allele in these populations. We also applied{rho} to investigate in greater detail a region on the 2q11.1 band of the human genome that has previously been identified as showing evidence of selection. This was done for a range of populations: for the European populations we incorporated a demographic history with a bottleneck corresponding to the putative out of Africa event. We were able to localise signals of selection to some specific regions and genes. Overall, we suggest that{rho} will be a useful tool for identifying genomic regions that may be subject to natural selection.

sentences: Kdm6a Deficiency Restricted to Mouse Hematopoietic Cells Causes an Age- and Sex-dependent Myelodysplastic Syndrome-Like Phenotype AbstractKdm6a/Utx, a gene on the X chromosome, encodes a histone K27me3 demethylase that has an orthologue on the Y chromosome (Uty)[1]. We previously identified inactivating mutations of Kdm6a in approximately 50% of mouse acute promyelocytic leukemia samples; however, somatic mutations of KDM6A are more rare in human AML samples, ranging in frequency from 2-15% in different series of patients, where their role in pathogenesis is not yet clear. In this study, we show that female Kdm6aflox/flox mice (with allele inactivation initiated by Vav1-Cre in hematopoietic stem and progenitor cells (HSPCs) have a sex-specific phenotype that emerges with aging, with features resembling a myelodysplastic syndrome (MDS). Female Kdm6a-knockout (KO) mice have an age-dependent expansion of their HSPCs with aberrant self-renewal, but they did not differentiate normally into downstream progeny. These mice became mildly anemic and thrombocytopenic, but did not develop overt leukemia, or die from these cytopenias. ChIP-seq and ATAC-seq studies showed only minor changes in H3K27me3, H3K27ac, H3K4me, H3K4me3 and chromatin accessibility between Kdm6a-WT and Kdm6a-KO mice. Utilizing scRNA-seq, Kdm6a loss was linked to the transcriptional repression of genes that mediate hematopoietic cell fate determination. These data demonstrate that Kdm6a plays an important role in normal hematopoiesis, and that its inactivation may contribute to AML pathogenesis by altering the epigenetic state of HSPCs.

sentences: Differential neutralization and inhibition of SARS-CoV-2 variants by antibodies elicited by COVID-19 mRNA vaccines IntroductoryThe evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of many new variant lineages that have exacerbated the COVID-19 pandemic. Some of those variants were designated as variants of concern/interest (VOC/VOI) by national or international authorities based on many factors including their potential impact on vaccines. To ascertain and rank the risk of VOCs and VOIs, we analyzed their ability to escape from vaccine-induced antibodies. The variants showed differential reductions in neutralization and replication titers by post-vaccination sera. Although the Omicron variant showed the most escape from neutralization, sera collected after a third dose of vaccine (booster sera) retained moderate neutralizing activity against that variant. Therefore, vaccination remains the most effective strategy to combat the COVID-19 pandemic.

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Glue genes are subjected to diverse selective forces during Drosophila development Molecular evolutionary studies usually focus on genes with clear roles in adult fitness or on developmental genes expressed at multiple time points during the life of the organism. Here, we examine the evolutionary dynamics of Drosophila glue genes, a set of eight genes tasked with a singular primary function during a specific developmental stage: the production of glue that allows animal pupa to attach to a substrate for several days during metamorphosis. Using phenotypic assays and available data from transcriptomics, PacBio genomes, and genetic variation from global populations, we explore the selective forces acting on the glue genes within the cosmopolitan D. melanogaster species and its five closely related species, D. simulans, D. sechellia, D. mauritiana, D. yakuba, and D. teissieri. We observe a three-fold difference in glue adhesion between the least and the most adhesive D. melanogaster strain, indicating a strong genetic component to phenotypic variation. These eight glue genes are among the most highly expressed genes in salivary glands yet they display no notable codon bias. New copies of Sgs3 and Sgs7 are found in D. yakuba and D. teissieri with the Sgs3 coding sequence evolving rapidly after duplication in the D. yakuba branch. Multiple sites along the various glue genes appear to be constrained. Our population genetics analysis in D. melanogaster suggests signs of local adaptive evolution for Sgs3, Sgs5 and Sgs5bis and traces of selective sweeps for Sgs1, Sgs3, Sgs7 and Sgs8. Our work shows that stage-specific genes can be subjected to various dynamic evolutionary forces. (249 words)

Significance statementDrosophila larvae produce a glue to stick themselves to a substrate for several days during metamorphosis. Here we observe wide variation in stickiness among Drosophila melanogaster strains and we analyze the molecular evolution of eight glue genes. We find several recent gene duplications and heterogenous rates of evolution among these genes.

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Temporal context actively shapes EEG signatures of time perception Our subjective perception of time is optimized to temporal regularities in the environment. This is illustrated by the central tendency effect: when estimating a range of intervals, short intervals are overestimated whereas long intervals are underestimated to reduce the overall estimation error. Most models of interval timing ascribe this effect to the weighting of the current interval with previous memory traces after the interval has been perceived. Alternatively, the perception of the duration could already be flexibly tuned to its temporal context. We investigated this hypothesis using an interval reproduction task in which human participants (both sexes) reproduced a shorter and longer interval range. As expected, reproductions were biased towards the subjective mean of each presented range. EEG analyses showed that temporal context indeed affected neural dynamics during the perception phase. Specifically, longer previous durations decreased CNV and P2 amplitude and increased beta power. In addition, multivariate pattern analysis showed that it is possible to decode context from the transient EEG signal quickly after both onset and offset of the perception phase. Together, these results suggest that temporal context creates dynamic expectations which actively affect the perception of duration.

Significance StatementThe subjective sense of duration does not arise in isolation, but is informed by previous experiences. This is demonstrated by abundant evidence showing that the production of duration estimates is biased towards previously experienced time intervals. However, it is yet unknown whether this temporal context actively affects perception or only asserts its influence in later, post-perceptual stages as proposed by most current formal models of this task. Using an interval reproduction task, we show that EEG signatures flexibly adapt to the temporal context during perceptual encoding. Furthermore, interval history can be decoded from the transient EEG signal even when the current duration was identical. Thus, our results demonstrate that context actively influences perception.

sentences: A stochastic model of hippocampal synaptic plasticity with geometrical readout of enzyme dynamics Discovering the rules of synaptic plasticity is an important step for understanding brain learning. Existing plasticity models are either 1) top-down and interpretable, but not flexible enough to account for experimental data, or 2) bottom-up and biologically realistic, but too intricate to interpret and hard to fit to data. To avoid the shortcomings of these approaches, we present a new plasticity rule based on a geometrical readout mechanism that flexibly maps synaptic enzyme dynamics to predict plasticity outcomes. We apply this readout to a multi-timescale model of hippocampal synaptic plasticity induction that includes electrical dynamics, calcium, CaMKII and calcineurin, and accurate representation of intrinsic noise sources. Using a single set of model parameters, we demonstrate the robustness of this plasticity rule by reproducing nine published ex vivo experiments covering various spike-timing and frequency-dependent plasticity induction protocols, animal ages, and experimental conditions. Our model also predicts that in vivo-like spike timing irregularity strongly shapes plasticity outcome. This geometrical readout modelling approach can be readily applied to other excitatory or inhibitory synapses to discover their synaptic plasticity rules.

sentences: Biofilm Growth Under Elastic Confinement Bacteria often form surface-bound communities, embedded in a self-produced extracellular matrix, called biofilms. Quantitative studies of their growth have typically focused on unconfined expansion above solid or semi-solid surfaces, leading to exponential radial growth. This geometry does not accurately reflect the natural or biomedical contexts in which biofilms grow in confined spaces. Here we consider one of the simplest confined geometries: a biofilm growing laterally in the space between a solid surface and an overlying elastic sheet. A poroelastic framework is utilised to derive the radial growth rate of the biofilm; it reveals an additional self-similar expansion regime, governed by the stiffness of the matrix, leading to a finite maximum radius, consistent with our experimental observations of growing Bacillus subtilis biofilms confined by PDMS.

sentences: Opposing effects of competitive exclusion determine invasion outcomes and structure ant assemblages Understanding how species phenotypic differences affect competition is key to explaining community assembly and predicting biodiversity responses. Many studies overlook the variable effects that species trait differences can have on the direction of competitive exclusion, which reverses depending on the specific mechanism at play. We performed a comprehensive trait-based study of an ant invasion integrating morphological, dietary, physiological and behavioral analyses. We found that trait differences between invasive and resident species were not only associated with niche differences which promoted the coexistence of dissimilar species, but also competitive ability differences which acted in the opposite fashion. Furthermore, competition along separate trait axes led to complex and contrasting patterns in the invaded assemblages, where species were at once similar (clustered) in some traits but also dissimilar (overdispersed) in others. Our results reveal that different aspects of phenotype may distinctly modulate the effect of competition in structuring ecological communities and functional diversity.

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Enterococcus faecalis persists and replicates within epithelial cells in vitro and in vivo during wound infection Enterococcus faecalis is a frequent opportunistic pathogen of wounds, whose infections are associated with biofilm formation, persistence, and recalcitrance toward treatment. We have previously shown that E. faecalis wound infection persists for at least 7 days. Here we report that viable E. faecalis are present within both immune and non-immune cells at the wound site up to 5 days after infection, raising the prospect that intracellular persistence contributes to chronic E. faecalis infection. Using an in vitro keratinocyte infection model, we show that a subpopulation of E. faecalis becomes internalized via macropinocytosis into single membrane-bound compartments, where they can survive and replicate. These intracellular E. faecalis can persist in late endosomes up to 72 hours after infection in the absence of colocalization with the lysosomal protease cathepsin D or apparent fusion with the lysosome, suggesting that E. faecalis blocks endosomal maturation. Indeed, intracellular E. faecalis infection results in a marked reduction in Rab7 expression, a small GTPase required for endosome-lysosome fusion. Finally, we demonstrate that intracellular E. faecalis derived from infected keratinocytes are significantly more efficient in reinfecting new keratinocytes. Together, these data suggest that intracellular proliferation of E. faecalis may contribute to its persistence in the face of a robust immune response, providing a primed reservoir of bacteria for subsequent reinfection.

Author SummaryEnterococcus faecalis is often isolated from chronic wounds. Prior to this study, E. faecalis has been observed within different cell types, suggesting that it can successfully colonize intracellular spaces. However, to date, little is known about the mechanisms E. faecalis use to survive intracellularly. Here, we describe key features of the intracellular lifestyle of E. faecalis. We show that E. faecalis exists in an intracellular state within immune cells and non-immune cells during mammalian wound infection. We show that E. faecalis can survive and replicate inside keratinocytes, and intracellularly replicating E. faecalis are primed to more efficiently cause reinfection, potentially contributing to chronic or persistent infections. In order to establish this intracellular lifestyle, E. faecalis is taken up by keratinocytes via macropinocytosis, whereupon it manipulates the endosomal pathway and expression of trafficking molecules required for endo-lysosomal fusion, enabling E. faecalis to avoid lysosomal degradation and consequent death. These results advance our understanding of E. faecalis pathogenesis, demonstrating mechanistically how this classic extracellular pathogen can co-opt host cells for intracellular persistence, and highlight the heterogeneity of mechanisms bacteria can use to avoid host-mediated killing in order to cause disease.

sentences: Flexible neural coding in sensory, parietal, and frontal cortices during goal-directed virtual navigation We do not understand how neural nodes operate within the recurrent action-perception loops that characterize naturalistic self-environment interactions, nor how brain networks reconfigure during changing computational demands. Here, we record local field potentials (LFPs) and spiking activity simultaneously from the dorsomedial superior temporal area (MSTd), parietal area 7a, and dorsolateral prefrontal cortex (dlPFC) as monkeys navigate in virtual reality to "catch fireflies". This task requires animals to actively sample from a closed-loop visual environment while concurrently computing latent variables: the evolving distance and angle to a memorized firefly. We observed mixed selectivity in all areas, with even a traditionally sensory area (MSTd) tracking latent variables. Strikingly, global encoding profiles and unit-to-unit coupling suggested a functional subnetwork between MSTd and dlPFC, and not between these are 7a, as anatomy would suggest. When sensory evidence was rendered scarce, lateral connectivity through neuron-to-neuron coupling within MSTd strengthened but its pattern remains fixed, while neuronal coupling adaptively remapped within 7a and dlPFC. The larger the remapping in 7a/dlPFC and the greater the stability within MSTd, the less was behavior impacted by loss of sensory evidence. These results highlight the distributed nature of neural coding during closed-loop action- perception naturalistic behaviors and suggest internal models may be housed in the pattern of fine-grain lateral connectivity within parietal and frontal cortices.

sentences: Optofluidic Fiber Component for Separation and counting of Micron-Sized Particles An all-fiber separation component capable of sorting and counting micron-sized particles based on size is presented. A sequence of silica fiber capillaries with various diameters and longitudinal cavities were used to fabricate the component for separation and detection in an uninterrupted flow. Fluorescence microparticles of 1 m and 10 m sizes are mixed in a visco-elastic fluid and infused into the all-fiber separation component. Elasto-inertial forces focus the larger particle to the center of the silica capillary, while the smaller microparticles exit from a side capillary. Analysis of the separated particles at the output showed a separation efficiency of 100% for the 10 m and 97% for the 1 m particles. In addition, the counting of the larger particles is demonstrated in the same flow. The separated 10 m particles are further routed through another all-fiber component for counting. A counting speed of ~1400 particles/min and with the variation in amplitude of 10% is achived. A combination of separation and counting can be powerful tool may find several applications in biology and medicine, such as separation and analysis of exosomes, bacteria, and blood cell sub-populations.

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A population-level statistic for assessing Mendelian behavior of genotyping-by-sequencing data from highly duplicated genomes BackgroundGiven the economic and environmental importance of allopolyploids and other species with highly duplicated genomes, there is a need for methods to distinguish paralogs, i.e. duplicate sequences within a genome, from Mendelian loci, i.e. single copy sequences that pair at meiosis. The ratio of observed to expected heterozygosity is an effective tool for filtering loci but requires genotyping to be performed first at a high computational cost, whereas counting the number of sequence tags detected per genotype is computationally quick but very ineffective in inbred or polyploid populations. Therefore, new methods are needed for filtering paralogs.

ResultsWe introduce a novel statistic, Hind/HE, that uses the probability that two reads sampled from a genotype will belong to different alleles, instead of observed heterozygosity. The expected value of Hind/HE is the same across all loci in a dataset, regardless of read depth or allele frequency. In contrast to methods based on observed heterozygosity, it can be estimated and used for filtering loci prior to genotype calling. In addition to filtering paralogs, it can be used to filter loci with null alleles or high overdispersion, and identify individuals with unexpected ploidy and hybrid status. We demonstrate that the statistic is useful at read depths as low as five to 10, well below the depth needed for accurate genotype calling in polyploid and outcrossing species.

ConclusionsOur methodology for estimating Hind/HE across loci and individuals, as well as determining reasonable thresholds for filtering loci, is implemented in polyRAD v1.6, available at https://github.com/lvclark/polyRAD. In large sequencing datasets, we anticipate that the ability to filter markers and identify problematic individuals prior to genotype calling will save researchers considerable computational time.

sentences: Single-cell dissection of live human hearts in ischemic heart disease and heart failure reveals cell-type-specific driver genes and pathways Ischemic heart disease is the single most common cause of death worldwide with an annual death rate of over 9 million people. Genome-wide association studies have uncovered over 200 genetic loci underlying the disease, providing a deeper understanding of the causal mechanisms leading to it. However, in order to understand ischemic heart disease at the cellular and molecular level, it is necessary to identify the cell-type-specific circuits enabling dissection of driver variants, genes, and signaling pathways in normal and diseased tissues. Here, we provide the first detailed single-cell dissection of the cell types and disease-associated gene expression changes in the living human heart, using cardiac biopsies collected during open-heart surgery from control, ischemic heart disease, and ischemic and non-ischemic heart failure patients. We identify 84 cell types/states, grouped in 12 major cell types. We define markers for each cell type, providing the first extensive reference set for the live human heart. These major cell types include cardiovascular cells (cardiomyocytes, endothelial cells, fibroblasts), rarer cell types (B lymphocytes, neurons, Schwann cells), and rich populations of previously understudied layer-specific epicardial and endocardial cells. In addition, we reveal substantial differences in disease-associated gene expression at the cell subtype level, revealing t arterial pericytes as having a central role in the pathogenesis of ischemic heart disease and heart failure. Our results demonstrate the importance of high-resolution cellular subtype mapping in gaining mechanistic insight into human cardiovascular disease.

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In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center at the Channel Entrance Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as  minimized or  expanded based on whether assembly requires only a core set of signature subunits or additional system-specific components. The prototypical  minimized systems mediating Agrobacterium tumefaciens T-DNA transfer and conjugative transfer of plasmids pKM101 and R388 are built from 12 subunits generically named VirB1-VirB11 and VirD4. In this study, we visualized the pKM101-encoded T4SS in the native context of the bacterial cell envelope by in situ cryoelectron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SSR388, a large substructure of which was previously purified and analyzed by negative-stain electron microscopy (nsEM). The IMC of the in situ T4SSpKM101 machine is highly symmetrical and exhibits 6-fold symmetry, dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three  expanded T4SSs previously visualized by in situ CryoET, but strikingly differs from the IMC of the purified T4SSR388 whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Together, our findings support a unified architectural model for all T4SSs assembled in vivo regardless of their classification as  minimized or  expanded: the signature VirB4-like ATPases invariably are arranged as central hexamers of dimers at the entrances to the T4SS channels.

SignificanceBacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryoelectron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope. The inner membrane complex (IMC) of the in situ T4SS differs remarkably from that of a closely-related T4SS analyzed in vitro by single particle electron microscopy. Our findings underscore the importance of comparative in vitro and in vivo analyses of the T4SS nanomachines, and firmly support a unified model in which the signature VirB4 ATPases of the T4SS superfamily function as a central hexamer of dimers to regulate substrate entry into and passage through T4SS channels.

sentences: Adversarial interspecies relationships facilitate population suppression by gene drive in spatially explicit models Suppression gene drives are designed to bias their inheritance and increase in frequency in a population, disrupting an essential gene in the process. When the frequency is high enough, the population will be unable to reproduce above the replacement level and could be eliminated. CRISPR suppression drives based on the homing mechanism have already seen success in the laboratory, particularly in malaria mosquitoes. However, several models predict that the use of these drives in realistic populations with spatial structure may not achieve complete success. This is due to the ability of wild-type individuals to escape the drive and reach empty areas with reduced competition, allowing them to achieve high reproductive success and leading to extinction-recolonization cycles across the landscape. Here, we extend our continuous space gene drive framework to include two competing species or predator-prey species pairs. We find in both discrete-generation and mosquito-specific models that the presence of a competing species or predator can greatly facilitate drive-based suppression, even for drives with modest efficiency. However, the presence of a competing species also substantially increases the frequency of outcomes in which the drive is lost before suppression is achieved. These results are robust in models with seasonal population fluctuations. We also found that suppression can be somewhat more difficult if targeting a predator with strong predator-prey interactions. Our results illustrate the difficulty of predicting outcomes of interventions that could substantially affect the populations of interacting species in complex ecosystems. However, our results are also potentially promising for the prospects of less powerful gene drives in achieving successful elimination of target pest populations.

sentences: Lentiviral vector optimization enhances the expression and cytotoxicity of chimeric antigen receptors Adoptive chimeric antigen receptor (CAR)-modified T or NK cells (CAR-T/NK) have emerged as a novel form of disease treatment. Lentiviral vectors (LVs) are commonly employed to engineer T/NK cells for the efficient expression of CARs. This study reported for the first time the influence of single-promoter and dual-promoter LVs on the CAR expression and cytotoxicity of engineered NK cells. Our results demonstrated that the selected CAR exhibits both a higher expression level and a higher coexpression concordance with the GFP reporter in HEK-293T or NK92 cells by utilizing the optimized single-promoter pCDHsp rather than the original dual-promoter pCDHdp. After puromycin selection, the pCDHsp produces robust CAR expression and enhanced in vitro cytotoxicity of engineered NK cells. Therefore, infection with a single-promoter pCDHsp lentivector is recommended to prepare CAR-engineered cells. This research will help to optimize the production of CAR-NK cells and improve their functional activity, to provide CAR-NK cell products with better and more uniform quality.

sentences: Fatigue Metabolites of Large Yellow Croaker and Construction of Swimming Model A trend in large yellow croaker (Larimichthys crocea) aquaculture is to establish new production sites that are suitable for extreme weather conditions. However, continuous and strong currents can harm fish welfare. To determine the location of the net cage, it is necessary to assess the swimming ability of large yellow croaker. Currently, our research on large yellow croakers is focusing on behavior analysis. This article investigates the effect of swimming large yellow croakers on metabolites in the body by examining the preferred speed of the group and the endurance swimming ability of single-tailed fish. We evaluated the factors that influence the large yellow croakers swimming fatigue by quantifying the content of metabolites and constructed the endurance swimming model using those results. Various results showed large yellow croaker populations tend to grow in low-velocity environments, and this matches their traditional habitat. The samples were taken at different swimming times at a flow rate of 0.35 m/s. According to the results of the metabolite content determination, blood glucose levels is closely related to swimming ability in large yellow croakers. The content of liver glycogen, which regulates blood glucose concentration, decreases in a certain linear relationship. The endurance swimming model of large yellow croaker was constructed according to the changes of liver glycogen content. The goals of this article are to provide a deeper understanding of the physiological characteristics of large yellow croaker swimming, and to provide a reference for choosing fishing and cage sites for large yellow croaker.

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SIX1 is a master regulator of the Rhabdomyosarcoma undifferentiated state Rhabdomyosarcoma (RMS) is a pediatric skeletal muscle sarcoma characterized by the expression of the myogenic-lineage transcription factors (TF) MYOD1 and MYOG. Despite high expression of these TFs, RMS cells fail to terminally differentiate, suggesting the presence of factors that alter their function. Here, we demonstrate that the developmental TF, SIX1, is highly expressed in RMS and is critical to maintain a muscle progenitor-like state. SIX1 loss induces terminal differentiation of RMS cells into myotube-like cells and dramatically impedes tumor growth in vivo. We show that SIX1 maintains the RMS undifferentiated state by controlling enhancer activity and MYOD1 occupancy at loci more permissive to tumor growth over terminal muscle differentiation. Finally, we demonstrate that a gene signature derived from SIX1 loss correlates with differentiation status in RMS and predicts RMS progression in human disease. Our findings demonstrate a master regulatory role for SIX1 in the repression of RMS differentiation via genome-wide alterations in MYOD1-mediated transcription.

HighlightsO_LISIX1 prevents differentiation in RMS while it promotes differentiation during normal development
C_LIO_LIFN-RMS are highly dependent on SIX1 for growth in both zebrafish and mouse xenograft models
C_LIO_LILoss of SIX1 alters the transcriptional landscape of RMS cells, inducing a growth to differentiation switch
C_LIO_LISIX1 knockdown in FN-RMS causes reduced super enhancer-based activity at stem-related genes and enhanced MYOD1 binding to differentiation loci, resulting in the activation of a myogenic differentiation program
C_LIO_LIA gene signature derived from SIX1 loss strongly correlates with myogenic differentiation status and is predictive of advanced RMS.
C_LI

sentences: Functional-Hybrid Modeling through automated adaptive symbolic regression for interpretable mathematical expressions Mathematical models used for the representation of (bio)-chemical processes can be grouped into two broad paradigms: white-box or mechanistic models, completely based on knowledge or black-box data-driven models based on patterns observed in data. However, in the past two-decade, hybrid modeling that explores the synergy between the two paradigms has emerged as a pragmatic compromise. The data-driven part of these have been largely based on conventional machine learning algorithm (e.g., artificial neural network, support vector regression), which prevents interpretability of the finally learnt model by the domain-experts. In this work we present a novel hybrid modeling framework, the Functional-Hybrid model, that uses the ranked domain-specific functional beliefs together with symbolic regression to develop dynamic models. We demonstrate the successful implementation of these hybrid models for four benchmark systems and a microbial fermentation reactor, all of which are systems of (bio)chemical relevance. We also demonstrate that compared to a similar implementation with the conventional ANN, the performance of Functional-Hybrid model is at least two times better in interpolation and extrapolation. Additionally, the proposed framework can learn the dynamics in 50% lower number of experiments. This improved performance can be attributed to the structure imposed by the functional transformations introduced in the Functional-Hybrid model.

sentences: Traditional use of Cissampelos pareira L. for hormone disorder and fever provides molecular links of ESR1 modulation to viral inhibition Bioactive fractions or compounds obtained from medicinal plants have been used for the treatment of multiple diseases. This effect could be due to common pathways underlying these conditions that are targeted by such medicines. In this study, we explored the molecular basis of action of one such herbal formulation Cissampelos pareira, used for the treatment of female hormone disorders and fever. Genome-wide expression studies on MCF7 cell lines treated with Cipa extract were carried out using Affymetrix arrays. Transcriptome analysis revealed a downregulation of signatures of estrogen response governed by estrogen receptor  (ER). Molecular docking analysis identified 38 constituent molecules in Cipa that potentially bind ({Delta}G< -7.5) with ER at the same site as estrogen. Cipa transcriptome signatures show high positive connectivity (https://clue.io/) scores with protein translation inhibitors such as emetine (score: 99.61) and knockdown signatures of genes linked to the antiviral response such as ribosomal protein RPL7 (score: 99.92), which is also an ER coactivator. Cipa exhibits antiviral activity in dengue infected MCF7 cells that is decreased upon ESR1 (estrogen receptor 1) gene knockdown. This approach reveals a novel pathway involving ESR1-RPL7 axis that could be a potential target in dengue viral infection.

sentences: Loop competition and extrusion model predicts CTCF interaction specificity Three-dimensional chromatin looping interactions play an important role in constraining enhancer-promoter interactions and mediating transcriptional gene regulation. CTCF is thought to play a critical role in the formation of these loops, but the specificity of which CTCF binding events form loops and which do not is difficult to predict. Loops often have convergent CTCF binding site motif orientation, but this constraint alone is only weakly predictive of genome-wide interaction data. Here we present an easily interpretable and simple mathematical model of CTCF mediated loop formation which is consistent with Cohesin extrusion and can predict ChIA-PET CTCF looping interaction measurements with high accuracy. Competition between overlapping loops is a critical determinant of loop specificity. We show that this model is consistent with observed chromatin interaction frequency changes induced by CTCF binding site deletion, inversion, and mutation, and is also consistent with observed constraints on validated enhancer-promoter interactions.

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Spontaneous emergence of music detectors in a deep neural network trained for natural sound recognition Music exists in almost every society, has universal acoustic features, and is processed by distinct neural circuits in humans even with no experience of musical training. These characteristics suggest an innateness of the sense of music in our brain, but it is unclear how this innateness emerges and what functions it has. Here, using an artificial deep neural network that models the auditory information processing of the brain, we show that units tuned to music can spontaneously emerge by learning natural sound detection, even without learning music. By simulating the responses of network units to 35,487 natural sounds in 527 categories, we found that various subclasses of music are strongly clustered in the embedding space, and that this clustering arises from the music-selective response of the network units. The music-selective units encoded the temporal structure of music in multiple timescales, following the population-level response characteristics observed in the brain. We confirmed that the process of generalization is critical for the emergence of music-selectivity and that music-selectivity can work as a functional basis for the generalization of natural sound, thereby elucidating its origin. These findings suggest that our sense of music can be innate, universally shaped by evolutionary adaptation to process natural sound.

One-sentence summaryMusic-selectivity can arise spontaneously in deep neural networks trained for natural sound detection without learning music.

sentences: MraZ is a transcriptional inhibitor of cell division in Bacillus subtilis The bacterial division and cell wall (dcw) cluster is a highly conserved region of the genome which encodes several essential cell division factors including the central divisome protein FtsZ. Understanding the regulation of this region is key to our overall understanding of the division process. mraZ is found at the 5 end of the dcw cluster and previous studies have described MraZ as a sequence-specific DNA binding protein. In this article, we investigate MraZ to elucidate its role in Bacillus subtilis. Through our investigation, we demonstrate that increased levels of MraZ result in lethal filamentation due to repression of its own operon (mraZ-mraW-ftsL-pbpB). We observe rescue of filamentation upon decoupling ftsL expression, but not other genes in the operon, from MraZ control. Furthermore, through timelapse microscopy we were able to identify that overexpression of mraZ, results in de-condensation of the FtsZ ring (Z-ring). This is likely due to depletion of FtsL, and thus, we believe the precise role of FtsL is likely in Z-ring maturation and promotion of subsequent treadmilling. Our data suggests that regulation of the mra operon may be an alternative way for cells to quickly arrest cytokinesis potentially during entry into stationary phase and in the event of DNA replication arrest.

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Crucial Roles of Carotenoids as Bacterial Endogenous Defense System for Bacterial Radioresistance of Deinococcus radiodurans BackgroundThe central dogma of radiation biology is that the cytotoxic and mutagenic effects of radiation are principally the result of biological macromolecules (DNA, RNA, polysaccharides, proteins and lipids) damage caused during the course of irradiation. Previous studies on the radioresistance of the extremely radiation-resistant red-carotenoid-pigmented bacterium Deinococcus radiodurans have been focused on DNA repair system. However, relative little is known about the biochemical basis of the extraordinary recovery capacity of D. radiodurans for ionizing radiation and can include hundreds of DNA double-strand breaks.

Methodology/Principal FindingsHere we identified a novel carotenoid in D. radiodurans as ((all-E)-1-hydroxy-3,4-didehydro-1,2,2,3-quahydro-{beta},{psi}-carotene-4-one-1) and characterized the intracellular distribution of carotenoids. Following exposure to ionizing radiation, the wild-type parent of D. radiodurans survived much higher compared with radiation-sensitive colorless mutants screened by 60Co irradiation and by knockout of crtB or crtI genes for the carotenoid biosynthesis. Electron paramagnetic resonance spectroscopy analysis showed that two carotenoid can effectively scavenge radicals including superoxide anion (O2.-) and hydroxyl radical (.OH). Consistent results showing the substantial antioxidant abilities of the two carotenoids were obtained by UV-induction of bacteriophage {lambda} method in vivo, and the detectable reaction potentials of two carotenoids with nitric oxide (NO) evaluated by ultraviolet-visible near-infrared ray (UV/vis/NIR) spectra assay.

Conclusions/SignificanceOur findings support the idea that the degree of bacterial radioresistance of D. radiodurans is partly related to the levels of carotenoids in the cells. Carotenoids, as an important member of the structural and non-structural components of the cells, are endogenous scavengers of reactive oxygen species and can effectively quench radicals generated during irradiation. The current work suggests that carotenoids play a previously unrecognized protection role in the endogenous defense System (EDS) that facilitates the recovery from radiation injury. And we propose a complete model to elucidate the mechanisms of extreme radioresistance in this remarkable bacterium.

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Wavelet characterization of spatial pattern in allele frequency Characterizing spatial patterns in allele frequencies is fundamental to evolutionary biology because such patterns can inform on underlying processes. However, the spatial scales at which changing selection, gene flow, and drift act are often unknown. Many of these processes can operate inconsistently across space (causing non-stationary patterns). We present a wavelet approach to characterize spatial pattern in genotype that helps solve these problems. We show how our approach can characterize spatial patterns in ancestry at multiple spatial scales, i.e. a multi-locus wavelet genetic dissimilarity. We also develop wavelet tests of spatial differentiation in allele frequency and quantitative trait loci (QTL). With simulation we illustrate these methods under a variety of scenarios. We apply our approach to natural populations of Arabidopsis thaliana and traditional varieties of Sorghum bicolor to characterize population structure and locally-adapted loci across scales. We find, for example, that Arabidopsis flowering time QTL show significantly elevated scaled wavelet variance at [~] 300 - 1300 km scales. Wavelet transforms of population genetic data offer a flexible way to reveal geographic patterns and underlying processes.

Author summaryBiologists can learn about evolutionary processes by studying spatial/geographic changes in the genotype of organisms in nature. However, many previous approaches to measure spatial genetic patterns have been limited by forcing individual samples into bins of discrete size and location, hindering our ability to learn about evolution. Here we present a new continuous approach to spatial genetics that allows us to resolve patterns that change in space and opposing patterns that occur at different spatial scales.

sentences: Brassica napus genes Rlm4 and Rlm7, conferring resistance to Leptosphaeria maculans, are alleles of the Rlm9 wall-associated kinase-like resistance locus Brassica napus (canola/rapeseed) race specific resistance genes against blackleg disease, caused by the ascomycete fungus Leptosphaeria maculans, have been commonly used in canola breeding. To date; LepR3, Rlm2 and Rlm9 R genes against L. maculans have been cloned from B. napus. LepR3 and Rlm2 are Receptor Like Proteins (RLP) and the recently reported Rlm9 is a Wall Associated Kinase-Like (WAKL) protein. Rlm9 located on chromosome A07 is closely linked with Rlm3, Rlm4, RLm7 genes. Recognition of AvrLm5-9 and AvrLm3 by their corresponding Rlm9 and Rlm3 proteins is masked in the presence of AvrLm4-7. Here we report cloning of Rlm4 and Rlm7 by generating genome sequence of the doubled haploid (DH) B. napus cv Topas DH16516 introgression lines Topas-Rlm4 and Topas-Rlm7. Candidate Rlm4 and Rlm7 genes were identified form the genome sequence and gene structures were determined by mapping RNA-sequence reads, generated from infected cotyledon tissues, to the genome of Topas-Rlm4 and Topas-Rlm7. Rlm4 and Rlm7 genomic constructs with their native promoters were transferred into the blackleg susceptible B. napus cv Westar N-o-1. Complementation of resistance response in the transgenic Westar:Rlm4 and Westar:Rlm7 that were inoculated with L. maculans transgenic isolates 2367:AvrRlm4-7 or 2367:AvrLm7 confirmed the function of Rlm4 and Rlm7 genes. Wild type L. maculans isolate 2367 that does not contain AvrLm4-7 or AvrLm7, and transgenic 2367:AvrLm3 and 2367:AvrLm5-9 did not induce resistance proving the specificity of Rlm4 and Rlm7 response. Rlm4 and Rlm7 alleles are also allelic to Rlm9. Rlm4 and Rlm7 genes encode WAKL proteins. Comparison of highly homologous sequences of Rlm4 and Rlm7 with each other and with the sequence of additional alleles, using whole genome sequencing of additional 128 lines, identified a limited number of point mutation located within the predicted extracellular receptor domains.

sentences: Biosynthesis of linear protein nanoarrays using the flagellar axoneme Applications in biotechnology and synthetic biology often make use of soluble proteins, but there are many potential advantages to anchoring enzymes to a stable substrate, including stability and the possibility for substrate channeling. To avoid the necessity of protein purification and chemical immobilization, there has been growing interest in bio-assembly of protein-containing nanoparticles, exploiting the self-assembly of viral capsid proteins or other proteins that form polyhedral structures. But these nanoparticle are limited in size which constrains the packaging and the accessibility of the proteins. The axoneme, the insoluble protein core of the eukaryotic flagellum or cilium, is a highly ordered protein structure that can be several microns in length, orders of magnitude larger than other types of nanoparticles. We show that when proteins of interest are fused to specific axonemal proteins and expressed in living cells, they become incorporated into linear arrays which have the advantages of high protein loading capacity, high stability, and single-step purification with retention of biomass. The arrays can be isolated as membrane enclosed vesicle or as exposed protein arrays. The approach is demonstrated for both fluorescent proteins and enzymes, and in the latter case it is found that incorporation into axoneme arrays provides increased stability for the enzyme.

sentences: PICASSO: Ultra-multiplexed fluorescence imaging of biomolecules through single-round imaging and blind source unmixing Ultra-multiplexed fluorescence imaging of biomolecules is essential to studying heterogeneous biological systems. However, this is challenging due to fluorophores spectral overlap and variation of the emission spectra. Here, we propose a strategy termed PICASSO, which enables more than 15-colour multiplexed imaging of thick tissue slices through a single imaging process and blind unmixing without reference spectra measurement. We show that PICASSO can be used to achieve a high multiplexing capability in diverse applications, such as 3D protein imaging, expansion microscopy, tissue clearing, imaging of clinical specimens, and cyclic immunofluorescence imaging. PICASSO only requires an equal number of images as the number of fluorophores, enabling such a high level of multiplexed imaging even with bandpass filter-based microscopy. As such, PICASSO would be a useful tool for the study of cancer, the immune system, and the brain, as well as for the diagnosis of cancer, as it enables ultra-multiplexed imaging of diverse specimens with minimum instrumental requirements and experimental processes.

sentences: Cis-Regulatory Element Hijacking by Structural Variants Overshadows Higher-Order Topological Changes in Primary Prostate Cancer Prostate cancer is a heterogeneous disease whose progression is linked to genome instability. However the impact of this instability on the three-dimensional chromatin organization and how this drives progression is unclear. Using primary benign and tumour tissue, we find a high concordance in the higher-order three-dimensional genome organization across normal and prostate cancer cells. This concordance argues for constraints to the topology of prostate tumour genomes. Nonetheless, we identify changes to focal chromatin interactions and show how structural variants can induce these changes to guide cis-regulatory element hijacking. Such events result in opposing differential expression on genes found at antipodes of rearrangements. Collectively, our results argue that cis-regulatory element hijacking from structural variant-induced altered focal chromatin interactions overshadows higher-order topological changes in the development of primary prostate cancer.

sentences: Autophagy receptor NDP52 alters DNA conformation to modulate RNA Polymerase II transcription NDP52 is an autophagy receptor involved in the recognition and degradation of invading pathogens and damaged organelles. Although NDP52 was first identified in the nucleus and is expressed throughout the cell, to date, there is no clear nuclear function for NDP52. Here, we use a multidisciplinary approach to characterise the biochemical properties and nuclear roles of NDP52. We found that NDP52 clusters with RNA Polymerase II (RNAPII) at transcription initiation sites and that its overexpression promotes the formation of additional transcriptional clusters. We also show that depletion of NDP52 impacts overall gene-expression levels in two model mammalian cells, and that transcription inhibition affects the spatial organisation and molecular dynamics of NDP52 in the nucleus. This directly links NDP52 to a role in RNAPII-dependent transcription. Furthermore, we also show that NDP52 binds specifically and with high affinity to double-stranded DNA (dsDNA) and that this interaction leads to changes in DNA structure in vitro. This, together with our proteomics data indicating enrichment for interactions with nucleosome remodelling proteins and DNA structure regulators, suggests a possible function for NDP52 in chromatin regulation. Overall, here we uncover novel nuclear roles for NDP52 in gene expression and DNA structure regulation.

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Environmental selection and epistasis in an empirical phenotype-environment-fitness landscape Fitness landscapes, mappings of genotype/phenotype to their effects on fitness, are invaluable concepts in evolutionary biochemistry. Though widely discussed, measurements of phenotype-fitness landscapes in proteins remain scarce. Here, we quantify all single mutational effects on fitness and phenotype of VIM-2 {beta}-lactamase across a 64-fold range of ampicillin concentrations. We then construct a phenotype-fitness landscape that takes variations in environmental selection pressure into account. We found that a simple, empirical landscape accurately models the ~39,000 mutational data points, suggesting the evolution of VIM-2 can be predicted based on the selection environment. Our landscape provides new quantitative knowledge on the evolution of the {beta}-lactamases and proteins in general, particularly their evolutionary dynamics under sub-inhibitory antibiotic concentrations, as well as the mechanisms and environmental dependence of nonspecific epistasis.

One Sentence SummaryAn empirical fitness landscape discloses the environmental dependence of mutational effects in VIM-2 {beta}-lactamase.

sentences: A Comparison of Persistence of SARS-CoV-2 Variants on Stainless Steel The survival of newer variants of SARS-CoV-2 on a representative surface has been compared to the established UK circulating isolate to determine whether enhanced environmental stability could play a part in their increased transmissibility. Stainless-steel coupons were inoculated with liquid cultures of the three variants, with coupons recovered over seven days and processed for recoverable viable virus using plaque assay. After drying, there was no significant difference in inactivation rates between variants. Indicating there is no increased environmental persistence from the new variants.

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Dietary and serum tyrosine, white matter microstructure and inter-individual variability in executive functions in overweight adults: relation to sex/gender and age Tyrosine (tyr), the precursor of the neurotransmitter dopamine, is known to modulate cognitive functions including executive attention. Tyr supplementation is suggested to influence dopamine-modulated cognitive performance. However, results are inconclusive, regarding the presence or strength and also the direction of the association between tyr and cognitive function. This pre-registered cross-sectional analysis investigates whether diet-associated serum tyr relates to executive attention performance, and whether this relationship is moderated by differences in white matter microstructure. 59 healthy, overweight, young to middle-aged adults (20F, 28.3 {+/-} 6.6 years, BMI: 27.3 {+/-} 1.5 kg/m2) drawn from a longitudinal study reported dietary habits, donated blood and completed diffusion-weighted brain magnetic resonance imaging and the attention network test. Main analyses were performed using linear regressions and non-parametric voxel-wise inference testing.

Confirmatory analyses did neither support an association between dietary and serum tyr nor a relationship between relative serum tyr/large neutral amino acids (LNAA) levels or white matter microstructure and executive attention performance. However, exploratory analyses revealed higher tyr intake, higher serum tyr and better executive attention performance in the male sex/gender group. In addition, older age was associated with higher dietary tyr intake and lower fractional anisotropy in a widespread cluster across the brain. Finally, a positive association between relative serum tyr/LNAA and executive attention performance was found in the male sex/gender group when accounting for age effects.

Our analysis advances the field of dopamine-modulated cognitive functions by revealing sex/gender and age differences which might be diet-related. Longitudinal or intervention studies and larger sample sizes are needed to provide more reliable evidence for links between tyr and executive attention.

sentences: Luminosity thresholds of colored surfaces are determined by their heuristic upper-limit luminances in the visual system We typically have a fairly good idea whether a given object is self-luminous or illuminated, but it is not fully understood how we make this judgement. This study aimed to identify determinants of the luminosity threshold, a luminance level at which a surface begins to appear self-luminous. We specifically tested a hypothesis that our visual system knows the maximum luminance level that a surface can reach under the physical constraint that a surface cannot reflect more light than any incident light and applies this prior to determine the luminosity thresholds. Observers were presented with a 2-degree circular test field surrounded by numerous overlapping colored circles and luminosity thresholds were measured as a function of (i) the chromaticity of the test field, (ii) the shape of surrounding color distribution and (iii) the color of the illuminant of the surrounding colors. We found that the luminosity thresholds peaked around the chromaticity of test illuminants and decreased as the purity of the test chromaticity increased. However, the loci of luminosity thresholds across chromaticities were nearly invariant to the shape of the surrounding color distribution and generally resembled the loci drawn from theoretical upper-limit luminances and upper-limit luminance boundaries of real objects. These trends were particularly evident for illuminants on the black-body locus and did not hold well under atypical illuminants such as magenta or green. These results support the idea that our visual system empirically internalizes the gamut of surface colors under natural illuminants and a given object appears self-luminous when its luminance exceeds this internalized upper-limit luminance.

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Linked machine learning classifiers improve species classification of fungi when using error-prone long-reads on extendedmetabarcodes BackgroundThe increased usage of error-prone long-read sequencing for metabarcoding of fungi has not been matched with adequate public databases and concomitant analysis approaches. We address this gap and present a proof-of-concept study for classifying fungal taxa using linked machine learning classifiers. We demonstrate the capability of linked machine learning classifiers to accurately classify species and strains using real-world and simulated fungal ribosomal DNA datasets, including plant and human pathogens. We benchmark our new approach in comparison to current alignment and k-mer based methods based on synthetic mock communities. We also assess real world applications of species identification in complex unlabelled datasets.

ResultsOur machine learning approach assigned individual nanopore long-read amplicon sequences to fungal species with high recall rates and low false positive rates. Importantly, our approach successfully distinguished between closely-related species and strains when individual read errors were higher than the genetic distance between individual taxa, which the alignment and k-mer methods could not do. The machine learning approach showed an ability to identify key species with high recall rates, even in complex samples of unknown species composition.

ConclusionsA proof of concept machine learning approach using a tree-descent approach on a decision tree of classifiers can identify known taxa with high accuracy, and precisely detect known target species from complex samples with high recall rates. We propose this approach is suitable for detecting the known knowns of pathogens or invasive species in any environment of mostly unknown composition, including agriculture and wild ecosystems.

sentences: Multi-omic analysis along the gut-brain axis points to a functional architecture of autism Autism is a highly heritable neurodevelopmental disorder characterized by heterogeneous cognitive, behavioral and communication impairments. Disruption of the gut-brain axis (GBA) has been implicated in autism, with dozens of cross-sectional microbiome and other omic studies revealing autism-specific profiles along the GBA albeit with little agreement in composition or magnitude. To explore the functional architecture of autism, we developed an age and sex-matched Bayesian differential ranking algorithm that identified autism-specific profiles across 10 cross-sectional microbiome datasets and 15 other omic datasets, including dietary patterns, metabolomics, cytokine profiles, and human brain expression profiles. The analysis uncovered a highly significant, functional architecture along the GBA that encapsulated the overall heterogeneity of autism phenotypes. This architecture was determined by autism-specific amino acid, carbohydrate and lipid metabolism profiles predominantly encoded by microbial species in the genera Prevotella, Enterococcus, Bifidobacterium, and Desulfovibrio, and was mirrored in brain-associated gene expression profiles and restrictive dietary patterns in individuals with autism. Pro-inflammatory cytokine profiling and virome association analysis further supported the existence of an autism-specific architecture associated with particular microbial genera. Re-analysis of a longitudinal intervention study in autism recapitulated the cross-sectional profiles, and showed a strong association between temporal changes in microbiome composition and autism symptoms. Further elucidation of the functional architecture of autism, including of the role the microbiome plays in it, will require deep, multi-omic longitudinal intervention studies on well-defined stratified cohorts to support causal and mechanistic inference.

sentences: Soil microbial diversity impacts plant microbiomes more than herbivory Interactions between plants and microbiomes play a key role in ecosystem functioning and are of broad interest due to their influence on nutrient cycling and plant protection. However, we do not yet have a complete understanding of how plant microbiomes are assembled. Here, we tested and quantified the effect of different factors driving the diversity and composition of plant-associated microbial communities. We manipulated soil microbial diversity (high or low diversity), plant species (Solanum tuberosum or Solanum vernei), and herbivory (presence or absence of a phloem-feeding insect Macrosiphum euphorbiae), and found that soil microbial diversity influenced the herbivore-associated microbiome composition, but also plant species and herbivory influenced the soil microbiome composition. We quantified the relative strength of these effects and demonstrated that the initial soil microbiome diversity explained the most variation in plant- and herbivore-associated microbial communities. Our findings strongly suggest that soil microbial community diversity is a driver of the composition of multiple associated microbiomes (plant and insect), and this has implications for the importance of management of soil microbiomes in multiple systems.

sentences: Rapid gene evolution in an ancient post-transcriptional and translationalregulatory system compensates for meiotic X chromosomal inactivation It is conventionally assumed that conserved pathways evolve slowly with little participation of gene evolution. Nevertheless, it has been recently observed that young genes can take over fundamental functions in essential biological processes, for example, development and reproduction. It is unclear how newly duplicated genes are integrated into ancestral networks and reshape the conserved pathways of important functions. Here, we investigated origination and function of two autosomal genes that evolved recently in Drosophila: Poseidon and Zeus, which were created by RNA-based duplications from the X-linked CAF40, a subunit of the conserved CCR4-NOT deadenylase complex involved in post-transcriptional and translational regulation. Knockdown and knockout assays show that the two genes quickly evolved critically important functions in viability and male fertility. Moreover, our transcriptome analysis demonstrates that the three genes have a broad and distinct effect in the expression of hundreds of genes, with almost half of the differentially expressed genes being perturbed exclusively by one paralog, but not the others. Co-immunoprecipitation and tethering assays show that the CAF40 paralog Poseidon maintains the ability to interact with the CCR4-NOT deadenylase complex and might act in post-transcriptional mRNA regulation. The rapid gene evolution in the ancient post-transcriptional and translational regulatory system may be driven by evolution of sex chromosomes to compensate for the meiotic X chromosomal inactivation (MXCI) in Drosophila.

sentences: Eusociality and the evolution of aging in superorganisms Eusocial insects - ants, bees, wasps and termites - are being recognized as model organisms to unravel the evolutionary paradox of aging for two reasons: (1) queens (and kings, in termites) of social insects outlive similar sized solitary insects by up to several orders of magnitude; (2) all eusocial taxa show a divergence of long queen and shorter worker lifespans, despite their shared genomes and even under risk-free laboratory environments. Traditionally, these observations have been explained by invoking classical evolutionary aging theory: well-protected inside their nests, queens are much less exposed to external hazards than foraging workers, and this provides natural selection the opportunity to favor queens that perform well at advanced ages. Although quite plausible, these verbal arguments have not been backed up by mathematical analysis. Here, for the first time, we provide quantitative models for the evolution of caste-specific aging patterns. We show that caste-specific mortality risks are in general neither sufficient nor necessary to explain the evolutionary divergence in lifespan between queens and workers and the extraordinary queen lifespans. Reproductive monopolization and the delayed production of sexual offspring in highly social colonies lead natural selection to inherently favor queens that live much longer than workers, even when exposed to the same external hazards. Factors that reduce a colonys reproductive skew, such as polygyny and worker reproduction, tend to reduce the evolutionary divergence in lifespan between queens and workers. Caste-specific extrinsic hazards also affect lifespan divergence but to a much smaller extent than reproductive monopolization.

sentences: An End-to-End Deep Learning Sound Coding Strategy for Cochlear Implants Cochlear implant (CI) users struggle to understand speech in noisy conditions. To address this problem, we propose a deep learning speech denoising sound coding strategy that estimates the CI electric stimulation patterns out of the raw audio data captured by the micro-phone, performing end-to-end CI processing. To estimate the relative denoising performance differences between various approaches, we compared this technique to a classic Wiener filter and to a convTasNet. Speech enhancement performance was assessed by means of signal-to-noise-ratio improvement and the short-time objective speech intelligibility measure. Additionally, 5 CI users were evaluated for speech intelligibility in noise to assess the potential benefits of each algorithm. Our results show that the proposed method is capable of replacing a CI sound coding strategy while preserving its general use for every listener and performing speech enhancement in noisy environments, without sacrificing algorithmic latency.

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Investigation of the Effects of N-Linked Glycans on the Stability of the Spike Protein in SARS-CoV-2 by Molecular Dynamics Simulations We perform all-atom molecular dynamics simulations to study the effects of the N-linked glycans on the stability of the spike glycoprotein in SARS-CoV-2. After a 100 ns of simulation on the spike proteins without and with the N-linked glycans, we found that the presence of glycans increases the local stability in their vicinity; even though their effect on the full structure is negligible.

Graphical Abstract

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Better tired than lost: turtle ant trail networks favor coherence over shortest edges Creating a routing backbone is a fundamental problem in both biology and engineering. The routing backbone of the trail networks of arboreal turtle ants (Cephalotes goniodontus) connects many nests and food sources using trail pheromone deposited by ants as they walk. Unlike species that forage on the ground, the trail networks of arboreal ants are constrained by the vegetation. We examined what objectives the trail networks meet by comparing the observed ant trail networks with networks of random, hypothetical trail networks in the same surrounding vegetation and with trails optimized for four objectives: minimizing path length, minimizing average edge length, minimizing number of nodes, and minimizing opportunities to get lost. The ants trails minimized path length by minimizing the number of nodes traversed rather than choosing short edges. In addition, the ants trails reduced the opportunity for ants to get lost at each node, favoring nodes with 3D configurations most likely to be reinforced by pheromone. Thus, rather than finding the shortest edges, turtle ant trail networks take advantage of natural variation in the environment to favor coherence, keeping the ants together on the trails.

Author SummaryWe investigated the trail networks of arboreal turtle ants in the canopy of the tropical forest, to ask what characterizes the colonys choice of foraging paths within the vegetation. We monitored day to day changes in the junctions and edges of trail networks of colonies in the dry forest of western Mexico. We compared the paths used by the ants to simulated random paths in the surrounding vegetation. We found that the paths of turtle ants prioritize coherence, keeping ants together on the trail, over minimizing the average edge length. The choice of paths reduces the number of junctions in the trail where ants could get lost, and favors junctions with a physical configuration that makes it likely that successive ants will reinforce the same path. Our work suggests that design principles that emphasize keeping information flow constrained to streamlined, coherent trails may be useful in human-designed distributed routing and transport networks or robot swarms.

sentences: Single-cell anatomical analysis of human hippocampus and entorhinal cortex uncovers early-stage molecular pathology in Alzheimer's disease The human hippocampal formation plays a central role in Alzheimers disease (AD) progression, cognitive traits, and the onset of dementia; yet its molecular states in AD remain uncharacterized. Here, we report a comprehensive single-cell transcriptomic dissection of the human hippocampus and entorhinal cortex across 489,558 cells from 65 individuals with varying stages of AD pathology. We transcriptionally characterize major brain cell types and neuronal classes, including 17 glutamatergic and 8 GABAergic neuron subpopulations. Combining evidence from human and mouse tissue-microdissection, neuronal cell isolation and spatial transcriptomics, we show that single-cell expression patterns capture fine-resolution neuronal anatomical topography. By stratifying subjects into early and late pathology groups, we uncover stage-dependent and cell-type specific transcriptional modules altered during AD progression. These include early-stage cell-type specific dysregulation of cellular and cholesterol metabolism, late-stage neuron-glia alterations in neurotransmission, and late-stage signatures of cellular stress, apoptosis, and DNA damage broadly shared across cell types. Late-stage signatures show signs of convergence in hippocampal and cortical cells, while early changes diverge; highlighting the relevance of characterizing molecular pathology across brain regions and AD progression. Finally, we characterize neuron subregion-specific responses to AD pathology and show that CA1 pyramidal neurons are the most transcriptionally altered while CA3 and dentate gyrus granule neurons the least. Our study provides a valuable resource to extend cell type-specific studies of AD to clinically relevant brain regions affected early by pathology in disease progression.

sentences: Functional coupling between TRPV4 channel and TMEM16F modulates human trophoblast fusion TMEM16F, a Ca2+-activated phospholipid scramblase (CaPLSase), is critical for placental trophoblast syncytialization, HIV infection, and SARS-CoV2-mediated syncytialization. How TMEM16F is activated during cell fusion is unclear. Here, we used trophoblasts as a model for cell fusion and demonstrated that Ca2+ influx through Ca2+ permeable transient receptor potential vanilloid channel TRPV4 is critical for TMEM16F activation and subsequent human trophoblast fusion. GSK1016790A, a TRPV4 specific agonist, robustly activates TMEM16F in trophoblasts. Patch-clamp electrophysiology demonstrated that TRPV4 and TMEM16F are functionally coupled within Ca2+ microdomains in human trophoblasts. Pharmacological inhibition or gene silencing of TRPV4 hindered TMEM16F activation and subsequent trophoblast syncytialization. Our study uncovers the functional expression of TRPV4 and a physiological activation mechanism of TMEM16F in human trophoblasts, thus providing us with novel strategies to regulate CaPLSase activity as a critical checkpoint of physiologically- and disease-relevant cell fusion events.

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Life histories as mosaics: plastic and genetic components differ among traits that underpin life-history strategies Life-history variation reflects phenotypic variation across suites of traits. Differences among life-history strategies result from genetic differentiation, phenotypic plasticity, and genotype-by-environment interactions. If the relative strength and direction of these components differed among traits underlying a strategy, life histories might not evolve as a cohesive unit.

We tested this hypothesis on the high- and low-predation ecotypes of Trinidadian guppies, defined by distinct life-history strategies. Using common garden experiments, we assessed how strongly 36 traits were determined by ancestral habitat (i.e., ecotype) or food availability, a key environmental difference between ecotypes. Our dataset was large (1178 individuals) and included six putatively independent origins of the derived ecotype.

Traits could be confidently assigned to four groups, defined by highly significant effects of only food (13 traits), only habitat (6), both (6), or neither (11), revealing substantial variation among traits in levels of genetic and environmental control. Ecotype-food (i.e., genotype-by-environment) interactions were negligible. The directions of plastic and genetic effects were usually aligned.

This suggests that life histories are mosaics with unequal rates of phenotypic and evolutionary change. Broadly speaking of "life-history evolution" masks a complex interplay of genes and environment on the multiple traits that underpin life-history strategies.

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From synaptic activity to human in vivo quantification of neurotransmitter dynamics: a neural modelling approach Understanding the role of neurotransmitters glutamate and GABA during normal and abnormal brain function and under external stimulation in humans are critical neuroscientific and clinical goals. The recent development of functional 1H-Magnetic resonance spectroscopy (fMRS) has allowed us to study neuro-transmitter activity in vivo for the first time. However, the physiological basis of the observed fMRS signal remains unclear. It has been proposed that fMRS detects shifts in metabolite concentrations as they move from presynaptic vesicles, where they are largely invisible, to extracellular and cytosolic pools, where they are visible.

Here we bridge the gap between neural dynamics and fMRS by developing a mean-field model to link the neurotransmitter dynamics at the microscopic-level to the macroscopic-level imaging measurements. GABA and glutamate are described as cycling between three metabolic pools: in the vesicles; active in the cleft; or undergoing recycling in the astrocytic or neuronal cytosol. We interrogate the model by applying a current to manipulate the mean membrane potential and firing rate of the neural populations.

We find that by disregarding the contribution from the vesicular pool, our model predicts activity-dependent changes in the MRS signal, which are consistent with reported empirical findings. Further, we show that current magnitude and direction has a selective effect on the GABA/glutamate-MRS signal: inhibitory stimulation leads to reduction of both metabolites, whereas excitatory stimulation leads to increased glutamate and decreased GABA. In doing so, we link neural dynamics and fMRS and provide a mechanistic account for the activity-dependent change in the observed MRS signal.

Key Points SummaryO_LIThe recent development of functional 1H-Magnetic resonance spectroscopy (fMRS) has allowed us to study neurotransmitter activity in vivo for the first time in humans. However, the physiological basis of the observed fMRS signal is unclear.
C_LIO_LIIt has been proposed that fMRS detects shifts in metabolite concentrations as they move from presynaptic vesicles, where they are largely invisible to MRS, to extracellular and cytosolic pools, where they are visible to MRS.
C_LIO_LIWe test this hypothesis using a mean field model which links the neural dynamics of neurotransmitters at the microscopic-level to the macroscopic-level imaging measurements obtained in experimental studies.
C_LIO_LIBy disregarding activity in the vesicular pool, our model can generate activity-dependent changes in the MRS signal in response to stimulation which are consistent with experimental findings in the literature.
C_LIO_LIWe provide a mechanistic account for the activity-dependent change in observed neurotransmitter concentrations using MRS.
C_LI

sentences: Exploiting noise, nonlinearity, and feedback to differentially control multiple synthetic cells with a single optogenetic input Synthetic biology seeks to develop modular bio-circuits that combine to produce complex, controllable behaviors. These designs are often subject to noisy fluctuations and uncertainties, and most modern synthetic biology design processes have focused to create robust components to mitigate the noise of gene expression and reduce the heterogeneity of single-cell responses. However, deeper understanding of noise can achieve control goals that would otherwise be impossible. We explore how an "Optogenetic Maxwell Demon" could selectively amplify noise to control multiple cells using single-input-multiple-output (SIMO) feedback. Using data-constrained stochastic model simulations and theory, we show how an appropriately selected stochastic SIMO controller can drive multiple different cells to different user-specified configurations irrespective of initial condition. We explore how controllability depends on cells regulatory structures, the amount of information available to the controller, and the accuracy of the model used. Our results suggest that gene regulation noise, when combined with optogenetic feedback and non-linear biochemical auto-regulation, can achieve synergy to enable precise control of complex stochastic processes.

sentences: Single-molecule mechanical unfolding kinetics of unmodified Saccharomyces cerevisiae tRNAPhe: a hint to the tRNA chaperone-tRNA interaction mechanism The biological activity of tRNA is closely related to its mechanical folding properties. Although previous studies focused on the folding and unfolding mechanism of tRNA, its kinetics are largely unknown. In this study, combining optical tweezers and molecule dynamics simulations, we characterized the mechanical folding and unfolding processes of a single unmodified Saccharomyces cerevisiae tRNAphe. We identified the intermediates and pathways for tRNA mechanical folding and unfolding in the presence of Mg2+, discovering that the folding/unfolding kinetics of D stem-loop and T stem-loop but not the anti-codon stem-loop significantly affected by their upstream and downstream structures. The cooperative unfolding of the tRNA in the presence of Mg2+ lead to a large hysteresis between the folding and unfolding pathway, and such hysteresis and unfolding cooperativity are significantly reduced by lowering the Mg2+ concentration or mutating the nucleotides forming the  elbow structure. Moreover, both steered molecular dynamics simulation and optical tweezers experiment results support that, formation of tertiary interactions in the elbow region increases energy barriers of the mechanical unfolding pathway, including those in between intermediates, and determines the overall unfolding cooperativity. Our studies may shed light on the detailed tRNA chaperone mechanism of TruB and TrmA.

sentences: The neuroinflammatory interleukin-12 signaling pathway drives Alzheimer's disease-like pathology by perturbing oligodendrocyte survival and neuronal homeostasis Alzheimers disease (AD) is characterized by deposition of pathological amyloid-{beta} (A{beta}) and tau protein aggregates and involves chronic neuroinflammation, ultimately leading to neurodegeneration and cognitive decline. Central in AD-related neuroinflammation is the proinflammatory interleukin-12 (IL-12)/IL-23 signaling pathway whose inhibition has been shown to attenuate pathology and cognitive defects in AD-like mice. In order to explore which cell types are involved in this neuroinflammatory cascade, we used single-nuclei RNA sequencing in AD-like APPPS1 mice lacking or harboring IL-12/IL-23 signaling. We found Il12b transcripts encoding the common p40 subunit of IL-12/IL-23 signaling to be expressed preferentially, but not exclusively, in microglia in an AD-specific manner. In contrast, transcripts for the other subunits of the IL-12 signaling pathway were expressed constitutively in neurons and oligodendrocytes irrespective of AD pathology, while transcripts for IL-23 were almost undetectable. Notably, genetic ablation of IL-12/IL-23 signaling did not affect the inflammatory gene expression profile of the AD-specific disease associated microglia (DAM), but reversed the loss of mature myelin-producing oligodendrocytes and alterations in neuronal homeostasis in APPPS1 mice. Taken together, our results reveal that IL-12, but not IL-23 is the main driver of AD-specific IL-12/IL-23 neuroinflammation, which alters neuronal and oligodendrocyte functions. Given that drugs targeting IL-12 already exist, our data may foster first clinical trials in AD subjects using this novel neuroimmune target.

sentences: Neural dynamics between anterior insular cortex and right supramarginal gyrus dissociate genuine affect sharing from automatic responses to pretended pain Empathy for pain engages both shared affective responses and self-other distinction. In this study, we addressed the highly debated question of whether neural responses previously linked to affect sharing could result from the perception of salient affective displays. Moreover, we investigated how affect sharing and self-other distinction interact to determine our response to a pain that is either perceived as genuine or pretended (while in fact both were acted for reasons of experimental control). We found stronger activations in regions associated with affect sharing (anterior insula, aIns, and anterior mid-cingulate cortex, aMCC) as well as with affective self-other distinction (right supramarginal gyrus, rSMG), in participants watching video clips of genuine vs. pretended facial expressions of pain. Using dynamic causal modeling (DCM), we then assessed the neural dynamics between the right aIns and rSMG in these two conditions. This revealed a reduced inhibitory effect on the aIns to rSMG connection for genuine compared to pretended pain. For genuine pain only, brain-to-behavior regression analyses highlighted a linkage between this inhibitory effect on the one hand, and pain ratings as well as empathic traits on the other. These findings imply that if the pain of others is genuine and thus calls for an appropriate empathic response, neural responses in the aIns indeed seem related to affect sharing and self-other distinction is engaged to avoid empathic over-arousal. In contrast, if others merely pretend to be in pain, the perceptual salience of their painful expression results in neural responses that are down-regulated to avoid inappropriate affect sharing and social support.

sentences: Goldilocks calcium and the mitochondrial respiratory chain: too much, too little, just right Calcium (Ca2+) is a key regulator in diverse intracellular signaling pathways, and has long been implicated in metabolic control and mitochondrial function. Mitochondria can actively take up large amounts of Ca2+, thereby acting as important intracellular Ca2+ buffers and affecting cytosolic Ca2+ transients. Excessive mitochondrial matrix Ca2+ is known to be deleterious due to opening of the mitochondrial permeability transition pore (mPTP) and consequent membrane potential dissipation, leading to mitochondrial swelling, rupture and cell death. But moderate Ca2+ within the organelle can directly or indirectly activate mitochondrial matrix enzymes, possibly impacting on ATP production. However, in vitro studies involving the regulation of mitochondrial enzymes by Ca2+ may not uncover its full effects on oxidative phosphorylation. Here, we aimed to determine if extra or intramitochondrial Ca2+ modulate oxidative phosphorylation in mouse liver mitochondria and intact hepatocytes. We found that isolated mitochondria present increased respiratory control ratios (a measure of oxidative phosphorylation efficiency) when incubated with low and medium Ca2+ concentrations in the presence of complex I-linked substrates pyruvate plus malate and -ketoglutarate, respectively, but not complex II-linked succinate. In intact hepatocytes, both low and high cytosolic Ca2+ led to decreased respiratory rates, while ideal rates were present under physiological conditions. High Ca2+ decreased mitochondrial respiration in cells in a substrate-dependent manner, mediated by mPTP. Overall, our results uncover a Goldilocks effect of Ca2+ on liver mitochondria, with specific "just right" concentrations that activate oxidative phosphorylation.

sentences: Efficient multi-gene expression in cell-free droplet microreactors Cell-free transcription and translation systems promise to accelerate and simplify the engineering of proteins, biological circuits and metabolic pathways. Their encapsulation on microfluidic platforms can generate millions of cell-free reactions in picoliter volume droplets. However, current methods struggle to create DNA diversity between droplets while also reaching sufficient protein expression levels. In particular, efficient multi-gene expression has remained elusive. We here demonstrate that co-encapsulation of DNA-coated beads with a defined cell-free system allows high protein expression while also supporting genetic diversity between individual droplets. We optimize DNA loading on commercially available microbeads through direct binding as well as through the sequential coupling of up to three genes via a solid-phase Golden Gate assembly or BxB1 integrase-based recombineering. Encapsulation with an off-the-shelf microfluidics device allows for single or multiple protein expression from a single DNA-coated bead per 14 pL droplet. We envision that this approach will help to scale up and parallelize the rapid prototyping of more complex biological systems.

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Using a Homeogram to Detect Sleep in Free-living Animals O_LISleep is appreciated as a behavior critical to homeostasis, performance, and fitness. Yet, most of what we know about sleep comes from humans or controlled laboratory experiments. Assessing sleep in wild animals is challenging, as it is often hidden from view, and electrophysiological recordings that define sleep states are difficult to obtain. Accelerometers have offered great insight regarding gross movement, although ambiguous quiescent states like sleep have been largely ignored, limiting our understanding of this ubiquitous behavior.
C_LIO_LIWe developed a broadly applicable sleep detection method called a homeogram that can be applied to accelerometer data collected from wild animals. We applied our methodology to detect sleep in free-ranging North American red squirrels (Tamiasciurus hudsonicus) in a region that experiences drastic seasonal shifts in light, temperature, and behavioral demands.
C_LIO_LIOur method characterized sleep in a manner consistent with limited existing studies and expanded those observations to provide evidence that red squirrels apply unique sleep strategies to cope with changing environments.
C_LIO_LIApplying our analytical strategy to accelerometer data from other species may open new possibilities to investigate sleep patterns for researchers studying wild animals.
C_LI

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Brief stimuli cast a long-term trace in visual cortex Visual processing is strongly influenced by the recent stimulus history - a phenomenon termed adaptation. Prominent theories cast adaptation as a consequence of optimized encoding of visual information, by exploiting the temporal statistics of the world. However, this would require the visual system to track the history of individual briefly experienced events, within a stream of visual input, to build up statistical representations over longer timescales. Here, using an openly available dataset from the Allen Brain Observatory, we show that neurons in the early visual cortex of the mouse indeed maintain long-term traces of individual past stimuli that persist despite the presentation of several intervening stimuli, leading to long-term and stimulus-specific adaptation over dozens of seconds. Long-term adaptation was selectively expressed in cortical, but not in thalamic neurons, which only showed short-term adaptation. Early visual cortex thus maintains concurrent stimulusspecific memory traces of past input, enabling the visual system to build up a statistical representation of the world to optimize the encoding of new information in a changing environment.

Significance StatementIn the natural world, previous sensory input is predictive of current input over multi-second timescales. The visual system could exploit these predictabilities by adapting current visual processing to the long-term history of visual input. However, it is unclear whether the visual system can track the history of individual briefly experienced images, within a stream of input, to build up statistical representations over such long timescales. Here, we show that neurons in early visual cortex of the mouse brain exhibit remarkably long-term adaptation to brief stimuli, persisting over dozens of seconds, and despite the presentation of several intervening stimuli. The visual cortex thus maintains long-term traces of individual briefly experienced past images, enabling the formation of statistical representations over extended timescales.

sentences: Improved prediction of protein-protein interactions using AlphaFold2 Predicting the structure of interacting protein chains is a fundamental step towards understanding protein function. Unfortunately, no computational method can produce accurate structures of protein complexes. AlphaFold2, has shown unprecedented levels of accuracy in modelling single chain protein structures. Here, we apply AlphaFold2 for the prediction of heterodimeric protein complexes. We find that the AlphaFold2 protocol together with optimized multiple sequence alignments, generate models with acceptable quality (DockQ[≥]0.23) for 63% of the dimers. From the predicted interfaces we create a simple function to predict the DockQ score which distinguishes acceptable from incorrect models as well as interacting from non-interacting proteins with state-of-art accuracy. We find that, using the predicted DockQ scores, we can identify 51% of all interacting pairs at 1% FPR. The protocol can be found at: https://gitlab.com/ElofssonLab/FoldDock.

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Shedding Light on the Black Box of a Neural Network Used to Detect Prostate Cancer in Whole Slide Images by Occlusion-Based Explainability Diagnostic histopathology is facing increasing demands due to aging populations and expanding healthcare programs. Semi-automated diagnostic systems employing deep learning methods are one approach to alleviate this pressure, with promising results for many routine diagnostic procedures. However, one major issue with deep learning approaches is their lack of interpretability--after adequate training they perform their assigned tasks admirably, but do not explain how they reach their conclusions. Knowledge of how a given method performs its task with high sensitivity and specificity would be advantageous to understand the key features responsible for diagnosis, and should in turn allow fine-tuning of deep learning approaches.

This paper presents a deep learning-based system for carcinoma detection in whole slide images of prostate core biopsies, achieving state-of-the-art performance; 100% area under curve and sensitivity of 0.978 for 8 detected false positives on average per slide.

Furthermore, we investigated various methods to extract the key features used by the neural network for classification. Of these, the technique called occlusion, adapted to whole slide images, analyzes the sensitivity of the detection system to changes in the input images. This technique produces heatmaps indicating which parts of the image have the strongest impact on the systems output that a histopathologist can examine to identify the networks reasoning behind a given classification. Reassuringly, the heatmaps identified several prevailing histomorphological features characterizing carcinoma, e.g. single-layered epithelium, presence of small lumina, and hyperchromatic nuclei with halos.

A convincing finding was the recognition of their mimickers in non-neoplastic tissue. The results show that the neural network approach to recognize prostatic cancer is similar to that taken by a human pathologist at medium optical resolution. The use of explain-ability heatmaps provides added value for automated digital pathology to analyze and fine-tune deep learning systems, and improves trust in computer-based decisions.

sentences: Maternal transfer and long-term population effects of PCBs in Baltic grey seals using a new toxicokinetic-toxicodynamic population model Empirical evidence has shown that historical exposure of polychlorinated biphenyls (PCBs) to Baltic grey seals not only severely affected individual fitness, but also population growth rates and most likely caused the retarded recovery rate of the depleted population for decades. We construct a new model which we term a toxicokinetic-toxicodynamic (TKTD) population model to quantify these effects. The toxicokinetic sub model describes in detail the bioaccumulation, elimination, and vertical transfer from mother to offspring of PCBs, and was linked to a toxicodynamic model for estimation of PCB-related damage, hazard, and stress impacts on fertility and survival rates. Both sub-models were then linked to a Leslie matrix population model to calculate changes in population growth rate and age structure given different rates of PCB exposure. Toxicodynamic model parameters related to reproductive organ lesions were calibrated using published historical data on observed pregnancy rates in Baltic grey seal females. Our model showed that increased PCB exposure caused reduced fertility, decreased vertical transfer, and increased biomagnification. Compared to empirical data, the TKTD population model described well the age-specific bioaccumulation pattern of PCBs in Baltic grey seals, and thus, the toxicokinetic parameters, deduced from literature, are believed to be reliable. The model also captured well the general effects of PCBs on historical population growth rates. The developed model can be used to perform population viability analyses of Baltic grey seals with multiple stressors, also including by-catches and different hunting regimes. The model can also be extended to other marine mammals and other contaminants than PCB by adjustments of model parameter values and thus provides a test bed in silico for new substances.

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Evaluation of maternal-infant dyad inflammatory cytokines in pregnancies affected by maternal SARS-CoV-2 infection in early and late gestation. ObjectiveSARS-CoV-2 infection induces significant inflammatory cytokine production in adults, but infant cytokine signatures in pregnancies affected by maternal SARS-CoV-2 are less well characterized. We aimed to evaluate cytokine profiles of mothers and their infants following COVID-19 in pregnancy.

Study DesignSerum samples at delivery from 31 mother-infant dyads with maternal SARS-CoV-2 infection in pregnancy (COVID) were examined in comparison to 29 control dyads (Control). Samples were evaluated using a 13-plex cytokine assay.

ResultsIn comparison with controls, interleukin (IL)-6 and interferon gamma-induced protein 10 (IP-10) were higher in COVID maternal and infant samples (p<0.05) and IL-8 uniquely elevated in COVID infant samples (p<0.05). Significant elevations in IL-6, IP-10 and IL-8 were found among both early (1st/2nd Trimester) and late (3rd Trimester) maternal SARS-CoV-2 infections.

ConclusionsMaternal SARS-CoV-2 infections throughout gestation are associated with increased maternal and infant inflammatory cytokines at birth with potential to impact long-term infant health.

sentences: Degradable endovascular neural interface for minimally invasive neural recording and stimulation Neural recording and stimulation have been widely used to mitigate traumatic injuries, neurodegenerative diseases or mental disorders. Most neural interfaces commonly require invasive surgery, potentially entailing both transient and permanent complications. A promising strategy designed to overcome these risks involves exploiting the cerebrovascular system as an access route to the neural tissue. Here we present a novel endovascular neural interface for neural recording and stimulation, fully polymeric and degradable. This concept might allow for better integration of the device in the body, reduced inflammatory reaction, the possibility of replacing the implant after degradation, and avoiding removal surgeries. The vasculatures strategic distribution and the use of soft polymers for the devices fabrication will permit targeting both the brain vasculature and the peripheral system. Therefore, this novel endovascular neural interface will broaden the range of applications from neurological diseases and mental disorders to bioelectronics medicine.

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SAR11 Cells Rely on Enzyme Multifunctionality to Transport and Metabolize a Range of Polyamine Compounds In the ocean surface layer and cell culture, the polyamine transport protein PotD of SAR11 bacteria is often one of the most abundant proteins detected. Polyamines are organic cations produced by all living organisms and are thought to be an important component of dissolved organic matter (DOM) produced in planktonic ecosystems. We hypothesized that SAR11 cells transport and metabolize multiple polyamines and use them as sources of carbon and nitrogen. Metabolic footprinting and fingerprinting were used to measure the uptake of five polyamine compounds (putrescine, cadaverine, agmatine, norspermidine, and spermidine) in two SAR11 strains that represent the majority of SAR11 cells in the surface ocean environment, Ca. Pelagibacter st. HTCC7211 and C. P. ubique st. HTCC1062. Both strains transported all five polyamines and concentrated them to micromolar or millimolar intracellular concentrations. Both strains could use most of the polyamines to meet their nitrogen requirements, but we did not find evidence of use as carbon sources. We propose potABCD transports cadaverine, agmatine, and norspermidine, in addition to its usual substrates of spermidine and putrescine, and that spermidine synthase, speE, is reversible, catalyzing the breakdown of spermidine and norspermidine, in addition to its usual biosynthetic role. These findings provide support for the hypothesis that enzyme multifunctionality enables streamlined cells in planktonic ecosystems to increase the range of DOM compounds they oxidize.

ImportanceGenome streamlining in SAR11 bacterioplankton has resulted in a small repertoire of genes, yet paradoxically they consume a substantial fraction of primary production in the oceans. Enzyme multifunctionality is hypothesized to be an adaptation that increases the range of organic compounds oxidized by cells in environments where selection favors genome minimization. We provide experimental support for this hypothesis by demonstrating that SAR11 cells use multiple polyamine compounds and propose that a small set of multifunctional genes catalyze this metabolism. We also report polyamine uptake rates can exceed metabolism, resulting in high intracellular concentrations of these nitrogen-rich compounds and an increase in cell size. Increases in cytoplasmic solute concentrations during transient episodes of high nutrient exposure has previously been observed in SAR11 cells and may be a feature of their strategy for maximizing the share of labile DOM acquired when in competition with other cell types.

sentences: Genome-wide association analyses of individual differences in quantitatively assessed reading- and language-related skills in up to 34,000 people The use of spoken and written language is a capacity that is unique to humans. Individual differences in reading- and language-related skills are influenced by genetic variation, with twin-based heritability estimates of 30-80%, depending on the trait. The relevant genetic architecture is complex, heterogeneous, and multifactorial, and yet to be investigated with well-powered studies. Here, we present a multicohort genome-wide association study (GWAS) of five traits assessed individually using psychometric measures: word reading, nonword reading, spelling, phoneme awareness, and nonword repetition, with total sample sizes ranging from 13,633 to 33,959 participants aged 5-26 years (12,411 to 27,180 for those with European ancestry, defined by principal component analyses). We identified a genome-wide significant association with word reading (rs11208009, p=1.098 x 10-8) independent of known loci associated with intelligence or educational attainment. All five reading-/language-related traits had robust SNP-heritability estimates (0.13-0.26), and genetic correlations between them were modest to high. Using genomic structural equation modelling, we found evidence for a shared genetic factor explaining the majority of variation in word and nonword reading, spelling, and phoneme awareness, which only partially overlapped with genetic variation contributing to nonword repetition, intelligence and educational attainment. A multivariate GWAS was performed to jointly analyse word and nonword reading, spelling, and phoneme awareness, maximizing power for follow-up investigation. Genetic correlation analysis of multivariate GWAS results with neuroimaging traits identified association with cortical surface area of the banks of the left superior temporal sulcus, a brain region with known links to processing of spoken and written language. Analysis of evolutionary annotations on the lineage that led to modern humans showed enriched heritability in regions depleted of Neanderthal variants. Together, these results provide new avenues for deciphering the biological underpinnings of these uniquely human traits.

sentences: Computational Assessment of Different Structural Models for Claudin-5 Complexes in Blood-Brain-Barrier Tight-Junctions The blood-brain barrier (BBB) strictly regulates the exchange of ions and molecules between the blood and the central nervous system. Tight junctions (TJs) are multimeric structures that control the transport through the paracellular spaces between adjacent brain endothelial cells of the BBB. Claudin-5 (Cldn5) proteins are essential for the TJ formation and assemble into multi-protein complexes via cis-interactions within the same cell membrane and trans-interactions across two contiguous cells. Despite the relevant biological function of Cldn5 proteins and their role as targets of brain drug delivery strategies, the molecular details of their assembly within TJs are still unclear. Two different structural models have been recently introduced, in which Cldn5 dimers belonging to opposite cells join to generate paracellular pores. However, a comparison of these models in terms of ionic transport features is still lacking. In this work, we used molecular dynamics simulations and free energy (FE) calculations to assess the two Cldn5 pore models and investigate the thermodynamic properties of water and physiological ions permeating through them. Despite different FE profiles, both structures present single/multiple FE barriers to ionic permeation, while being permissive to water flux. These results reveal that both models are compatible with the physiological role of Cldn5 TJ strands. By identifying the protein-protein surface at the core of TJ Cldn5 assemblies, our computational investigation provides a basis for the rational design of synthetic peptides and other molecules capable of opening paracellular pores in the BBB.

sentences: Autism-associated mutation in Hevin/Sparcl1 induces endoplasmic reticulum stress through structural instability Hevin is a secreted extracellular matrix protein that is encoded by SPARCL1 gene. Recent studies show that Hevin plays an important role in regulating synaptogenesis and synaptic plasticity. Mutations in SPARCL1 gene increase the risk of autism spectrum disorder (ASD). However, the molecular basis of how mutations in SPARCL1 increase the risk of ASD has not been fully understood. In this study, we show that one of SPARCL1 mutations associated with ASD impairs normal Hevin secretion. We identified Hevin mutants lacking the EF-hand motif through analyzing ASD-related mice with vulnerable spliceosome functions. Hevin deletion mutants accumulate in the ER, leading to the activation of unfolded protein responses. We also found that a single amino acid substitution of Trp647 with Arg in the EF-hand motif associated with a familial case of ASD causes a similar phenotype with the EF-hand deletion mutant. Importantly, molecular dynamics (MD) simulation revealed that this single amino acid substitution triggers exposure of hydrophobic amino acid to the surface, increasing the binding of Hevin with a molecular chaperon, BIP. Taken together, these data suggest that the integrity of EF-hand motif in Hevin is crucial for proper folding and ASD-related mutation impairs an export of Hevin from the endoplasmic reticulum (ER). Our data provide a novel mechanism linking a point mutation in SPARCL1 gene to the molecular and cellular characteristics involved in ASD.

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Reducing indoor particle exposure using mobile air purifiers - experimental and numerical analysis Aerosol particles are one of the main routes of transmission of COVID-19. Mobile air purifiers are used to reduce the risk of infection indoors. We focus on an air purifier which generates a defined volumetric air flow through a highly efficient filter material. We investigate the transport of aerosol particles from an infected dummy equipped with an aerosol generator to receiving thermal dummies. For analysis, we use up to 12 optical particle counters to monitor the particle concentration with high spatial resolution. Based on the measurement data, a computational fluid dynamics (CFD) model is set up and validated. The experimental and numerical methods are used to investigate how the risk of infection suggested by the particle exposure in an exemplary lecture hall can be reduced by a clever choice of orientation of the air purifier. The particle concentration at head height deviates by 13 % for variations of location and orientation. Finally, CFD simulation was used to monitor the particle fates. The steady simulation results fit quite well to the experimental findings and provide additional information about particle path and for assessing comfort level due to air flow.

Practical implicationsDifferent installation locations and operating conditions of the air purifier are evaluated and the use of thermal dummies mimics the conditions of practical use cases. The measurement results show the integral particle mass over time in the "faces of the dummies", representing the potentially inhaled particle load of persons present in the room. At an air change per hour of 5, the cumulated PM1 mass at head level was reduced by 75 %, independently of the location of the infected dummy, compared to the "natural decay" case showing that filtration is an effective means of reducing aerosol particle concentrations. It turns out that obstructing the outlet stream of the air purifier may be particularly advantageous.

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Constructive Connectomics: how neuronal axons get from here to there using gene-expression maps derived from their family trees During brain development, billions of axons must navigate over multiple spatial scales to reach specific neuronal targets, and so build the processing circuits that generate the intelligent behavior of animals. However, the limited information capacity of the zygotic genome puts a strong constraint on how, and which, axonal routes can be encoded. We propose and validate a mechanism of development that can provide an efficient encoding of this global wiring task. The key principle, confirmed through simulation, is that basic constraints on mitoses of neural stem cells--that mitotic daughters have similar gene expression to their parent and do not stray far from one another--induce a global hierarchical map of nested regions, each marked by the expression profile of its common progenitor population. Thus, a traversal of the lineal hierarchy generates a systematic sequence of expression profiles that traces a staged route, which growth cones can follow to their remote targets. We have analyzed gene expression data of developing and adult mouse brains published by the Allen Institute for Brain Science, and found them consistent with our simulations: gene expression indeed partitions the brain into a global spatial hierarchy of nested contiguous regions that is stable at least from embryonic day 11.5 to postnatal day 56. We use this experimental data to demonstrate that our axonal guidance algorithm is able to robustly extend arbors over long distances to specific targets, and that these connections result in a qualitatively plausible connectome. We conclude that, paradoxically, cell division may be the key to uniting the neurons of the brain.

Author SummaryThe embryological development of each brain installs an essentially identical communication network between its cells that is roughly as complex as that between the billions of people living on Earth. Although vast scientific resources are currently applied to identifying the final pattern of connections, the connectome, there has until now been relatively little effort to answer the fundamental question of how this complex network across billions of neurons realized through the mitotic elaboration of the initial embryonic cell. The problem is sharpened by the constraints that construction of the network is limited by the information budget of the initial genome, and that it has no pre-existing address space for placing neurons and guiding axons. We explain how Biology can solve this problem by using the family tree of neurons to install a global space of molecular addresses, which axons can use to navigate from their source neuron to its relatives. We provide experimental evidence for this familial address space in gene expression patterns of the developing mouse brain, and demonstrate through simulation that the experimentally observed address space indeed supports global navigation to produce a qualitatively plausible default connectome.

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mTOR-activating mutations in RRAGD cause kidney tubulopathy and cardiomyopathy (KICA) syndrome BackgroundOver the last decaces, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, {+/-}20% of all tubulopathy patients remain without genetic diagnosis. Here, we explore a large multicentric patient cohort with a novel inherited salt-losing tubulopathy, hypomagnesemia and dilated cardiomyopathy (DCM).

MethodsWhole exome and genome sequencings were performed with various subsequent functional analyses of identified RRAGD variants in vitro.

ResultsIn 8 children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt-wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients additionally suffered from DCM requiring heart transplantation in 3 of them. An additional dominant variant in RRAGD was simultaneously identified in eight members of a large family with a similar renal phenotype. RRAGD encodes GTPase RagD mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron include the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro,

ConclusionsOur findings establish a novel disease phenotype combining kidney tubulopathy and cardiomyopathy (KICA) caused by an activation of mTOR signaling suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.

SIGNIFICANCE STATEMENTHere, we report on heterozygous variants in RRAGD in patients with profound hypomagnesemia, renal salt wasting, nephrocalcinosis, and dilated cardiomyopathy. The identified RagD variants induce a constitutive activation of mTOR signaling in vitro. These findings not only establish a novel monogenic disorder of the kidney tubule, but demonstrate the essential role of mTOR signaling for distal tubular electrolyte handling and cardiac function.

sentences: Yeast biopanning for detecting antibody binding to site-specific phosphorylations in tau The detection of phosphorylated tau (p-tau) levels in clinical samples is of extreme importance for the detection of Alzheimers Disease (AD) as well as other neurodegenerative diseases. Recent reports show that detecting low levels of p-tau in plasma can be used as a reliable biomarker for detecting AD prior to the onset of memory loss. The ability to detect such low levels of p-tau is dependent on antibodies specific to the post translationally modified protein. However, the need for reliable phospho-site specific antibodies persists due to a lack of approaches for identifying monoclonal antibodies and characterizing non-specific binding. Here, we report a novel approach using the principles of yeast biopanning to create a robust platform that uses synthetic peptides as target antigens. Using peptides as antigens enables screening antibodies against defined post-translational modification sites, particularly for targeting intrinsically disordered proteins such as the human tau protein. To readily assess yeast binding and distinguish non-specific binding, we developed bi-directional expression vectors that allow antibody fragment surface display and intracellular fluorescent protein expression. We show that our platform can specifically and robustly detect a specific site within the p-tau target peptide when compared against non-phosphorylated controls. By improving biopanning parameters, we enabled phospho-specific capture of yeast cells displaying single-chain variable region fragments (scFvs) against p-tau with a wide range of affinities (KD = 0.2 to 60 nM). These results demonstrate that yeast biopanning can robustly capture yeast cells based on phospho-site specific antibody binding, opening doors for facile identification of high-quality monoclonal antibodies.

sentences: Comprehensive analysis of pyroptosis-associated in molecular classification, immunity and prognostic of glioma Integrative analysis was performed in the Chinese Glioma Genome Atlas and The Cancer Genome Atlas to describe the pyroptosis-associated molecular classification and prognostic signature in glioma. Pyroptosis-related genes were used for consensus clustering and to develop a prognostic signature. The immune statuses, molecular alterations and clinical features of differentially expressed genes were analyzed among different subclasses and risk groups. A lncRNA-miRNA-mRNA network was built, and drug sensitivity analysis was used to identify small molecular drugs for the identified genes. Glioma can be divided into two subclasses using 30 pyroptosis-related genes. Cluster 1 displayed high immune signatures and poor prognosis as well as high immune-related function scores. A prognostic signature based on 15 pyroptosis-related genes of the CGGA cohort can predict the overall survival of glioma and was well validated in the TCGA cohort. Cluster 1 had higher risk scores. The high-risk group had high immune cell and function scores and low DNA methylation of pyroptosis-related genes. The differences in pyroptosis-related gene mutations and somatic copy numbers were significant between the high-risk and low-risk groups. The ceRNA regulatory network uncovered the regulatory patterns of different risk groups in glioma. Nine pairs of target genes and drugs were identified. In vitro, CASP8 promotes the progression of glioma cells. Pyroptosis-related genes can reflect the molecular biological and clinical features of glioma subclasses. The established prognostic signature can predict prognosis and distinguish molecular alterations in glioma patients. Our comprehensive analyses provide valuable guidelines for improving glioma patient management and individualized therapy.

sentences: Structural Ramifications of Spike Protein D614G Mutation in SARS-CoV-2 A single mutation from aspartate to glycine at position 614 has dominated all circulating variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). D614G mutation induces structural changes in the Spike (S) protein that strengthen the virus infectivity. Here, we use molecular dynamics simulations to dissect the effects of mutation and 630-loop rigidification on wild-type structure. The introduction of mutation with ordered 630-loop induces structural changes toward S-G614 Cryo-EM structure. An ordered 630-loop weakens the stabilizing interactions of the anionic D614, suggesting its disorder in wild-type. The mutation allosterically alters the receptor binding domain (RBD) forming an asymmetric and mobile Down conformation, which facilitate Up transition. The loss of D614_K854 salt-bridge upon mutation, generally stabilize S-protein protomer, including the fusion peptide proximal region that mediates membrane fusion. Understanding of the molecular basis of D614G is crucial as it dominates in all variants of concern including Delta and Omicron.

sentences: CovidExpress: an interactive portal for intuitive investigation on SARS-CoV-2 related transcriptomes Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans could cause coronavirus disease 2019 (COVID-19). Since its first discovery in Dec 2019, SARS-CoV-2 has become a global pandemic and caused 3.3 million direct/indirect deaths (2021 May). Amongst the scientific communitys response to COVID-19, data sharing has emerged as an essential aspect of the combat against SARS-CoV-2. Despite the ever-growing studies about SARS-CoV-2 and COVID-19, to date, only a few databases were curated to enable access to gene expression data. Furthermore, these databases curated only a small set of data and do not provide easy access for investigators without computational skills to perform analyses. To fill this gap and advance open-access to the growing gene expression data on this deadly virus, we collected about 1,500 human bulk RNA-seq datasets from publicly available resources, developed a database and visualization tool, named CovidExpress (https://stjudecab.github.io/covidexpress). This open access database will allow research investigators to examine the gene expression in various tissues, cell lines, and their response to SARS-CoV-2 under different experimental conditions, accelerating the understanding of the etiology of this disease to inform the drug and vaccine development. Our integrative analysis of this big dataset highlights a set of commonly regulated genes in SARS-CoV-2 infected lung and Rhinovirus infected nasal tissues, including OASL that were under-studied in COVID-19 related reports. Our results also suggested a potential FURIN positive feedback loop that might explain the evolutional advantage of SARS-CoV-2.

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Evaluating Extraction Methods to Study Canine Urine Microbiota The urinary microbiota is the collection of microbes present in urine that play a role in host health. Studies of urine microbiota have traditionally relied upon culturing methods aimed at identifying pathogens. However, recent culture-free sequencing studies of the urine microbiota have determined that a diverse array of microbes are present in health and disease. To study these microbes and their potential role in diseases like bladder cancer or interstitial cystitis, consistent extraction and detection of microbial DNA from urine is critical. However, urine is a low biomass substrate, requiring sensitive methods to capture DNA and making the risk of contamination high. To address this challenge, we collected urine samples from ten healthy dogs and extracted DNA from each sample using five different commercially available extraction methods. Extraction methods were compared based on total and bacterial DNA concentrations and microbial community composition and diversity assessed through 16S rRNA gene sequencing. Significant differences in the urinary microbiota were observed by dog and sex but not extraction method. The Bacteremia kit yielded the highest total DNA concentrations (Kruskal-Wallis, p = 0.165, not significant) and the highest bacterial DNA concentrations (Kruskal-Wallis, p = 0.044). Bacteremia also extracted bacterial DNA from the greatest number of samples. Taken together, these results suggest that the Bacteremia kit is an effective option for studying the urine microbiota. This work lays the foundation to study the urine microbiome in a wide range of urogenital diseases in dogs and other species.

HighlightsO_LICanine urine microbiota differed by sex and dog but not extraction method.
C_LIO_LIQiagen Bacteremia kit yielded the highest bacterial DNA concentrations from urine.
C_LIO_LIThe Bacteremia kit extracted bacterial DNA from the greatest number of samples.
C_LIO_LIAbsolute abundance of Sphingomonas species increased in female dog urine.
C_LIO_LIPasteurellaceae bacterium canine oral taxon 272 increased in male dog urine.
C_LI

sentences: Molecular characterization of Mycobacterium ulcerans DNA gyrase and identification of mutations reduced susceptibility against quinolones in vitro Buruli ulcer disease is a neglected necrotizing and disabling cutaneous tropical illness caused by Mycobacterium ulcerans (Mul). Fluoroquinolone (FQ), used in the treatment of this disease, has been known to act by inhibiting the enzymatic activities of DNA gyrase; however, the detailed molecular basis of these characteristics and the FQ resistance mechanisms in Mul remains unknown. This study investigated the detailed molecular mechanism of Mul DNA gyrase and the contribution of FQ resistance in vitro using recombinant proteins from the Mul subsp. shinshuense and Agy99 strains with reduced sensitivity to FQs. The IC50 of FQs against Ala91Vla and Asp95Gly mutants of Mul shinshuense and Agy99 GyrA subunits were 3.7- to 42.0-fold higher than those against wild-type enzyme. Similarly, the CC25 was 10- to 210-fold higher than those for the WT enzyme. Furthermore, the interaction between the amino acid residues of WT/mutant Mul DNA gyrase and FQ side chains was assessed via molecular docking studies. This is the first detailed study showing the contribution of Mul DNA GyrA subunit mutations to reduce the susceptibility against FQs.

sentences: Predicting proprioceptive cortical anatomy and neural coding with topographic autoencoders Proprioception is one of the least understood senses yet fundamental for the control of movement. Even basic questions of how limb pose is represented in the somatosensory cortex are unclear. We developed a variational autoencoder with topographic lateral connectivity (topo-VAE) to compute a putative cortical map from a large set of natural movement data. Although not fitted to neural data, our model reproduces two sets of observations from monkey centre-out reaching: 1. The shape and velocity dependence of proprioceptive receptive fields in hand-centered coordinates despite the model having no knowledge of arm kinematics or hand coordinate systems. 2. The distribution of neuronal preferred directions (PDs) recorded from multi-electrode arrays. The model makes several testable predictions: 1. Encoding across the cortex has a blob-and-pinwheel-type geometry PDs. 2. Few neurons will encode just a single joint. Topo-VAE provides a principled basis for understanding of sensorimotor representations, and the theoretical basis of neural manifolds, with applications to the restoration of sensory feedback in brain-computer interfaces and the control of humanoid robots.

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Combinatorial patterns of gene expression changes contribute to variable expressivity of the developmental delay-associated 16p12.1 deletion BackgroundRecent studies have suggested that individual variants do not sufficiently explain the variable expressivity of phenotypes observed in complex disorders. For example, the 16p12.1 deletion is associated with developmental delay and neuropsychiatric features in affected individuals, but is inherited in >90% of cases from a mildly-affected parent. While children with the deletion are more likely to carry additional "second-hit" variants than their parents, the mechanisms for how these variants contribute to phenotypic variability are unknown.

MethodsWe performed detailed clinical assessments, whole-genome sequencing, and RNA sequencing of lymphoblastoid cell lines for 32 individuals in five large families with multiple members carrying the 16p12.1 deletion. We identified contributions of the 16p12.1 deletion and "second-hit" variants towards a range of expression changes in deletion carriers and their family members, including differential expression, outlier expression, alternative splicing, allele-specific expression, and expression-quantitative trait loci analyses.

ResultsWe found that the deletion dysregulates multiple autism and brain development genes such as FOXP1, ANK3, and MEF2. Carrier children also showed an average of 5,323 gene expression changes compared with one or both parents, which matched with 33/39 observed developmental phenotypes. We identified significant enrichments for 13/25 classes of "second-hit" variants in genes with expression changes, where 4/25 variant classes were only enriched when inherited from the non-carrier parent, including loss-of-function SNVs and large duplications. In 11 instances, including for ZEB2 and SYNJ1, gene expression was synergistically altered by both the deletion and inherited "second-hits" in carrier children. Finally, brain-specific interaction network analysis showed strong connectivity between genes carrying "second-hits" and genes with transcriptome alterations in deletion carriers.

ConclusionsOur results suggest a potential mechanism for how "second-hit" variants modulate expressivity of complex disorders such as the 16p12.1 deletion through transcriptomic perturbation of gene networks important for early development. Our work further shows that family-based assessments of transcriptome data are highly relevant towards understanding the genetic mechanisms associated with complex disorders.

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Evolution under low antibiotic concentrations: a risk for the selection of Pseudomonas aeruginosa multidrug resistant mutants in nature BACKGROUNDAntibiotic pollution of non-clinical environments might have a relevant impact on human health if resistant pathogens are selected. However, this potential risk is often overlooked, since drug concentrations in nature are usually below their minimal inhibitory concentrations (MICs). Albeit, antibiotic resistant bacteria can be selected even at sub-MIC concentrations, in a range that is dubbed the sub-MIC selective window, which depends on both the antibiotic and the pathogen.

OBJECTIVESDetermine the sub-MIC selective windows of seven antibiotics of clinical relevance in the opportunistic pathogen Pseudomonas aeruginosa and evaluate the risk for selecting resistant mutants in nature, based on published data about the amount of antimicrobials detected in natural environments.

METHODSWe conducted evolution experiments of P. aeruginosa PA14 in presence of sub-MIC concentrations of ceftazidime, amikacin, levofloxacin, ciprofloxacin, tetracycline, polymyxin B or imipenem, and measured drug susceptibility of the evolved populations.

RESULTSSub-MIC selective window of quinolones was the largest, and the ones of polymyxin B and imipenem, the narrowest. Clinically relevant multidrug resistant (MDR) mutants (presenting MICs above EUCAST clinical breakpoints) arose within the sub-MIC selective windows of the majority of antibiotics tested, being these phenotypes probably mediated by efflux pumps' activity.

DISCUSSIONOur data show that the concentration of antibiotics reported in aquatic ecosystems -colonizable by P. aeruginosa- are, in occasions, higher than the ones able to select MDR mutants. This finding has implications for understanding the role of different ecosystems and conditions in the emergence of antibiotic resistance from a One-Health point of view. Further, it highlights the importance of delineating the sub-MIC selective windows for drugs of clinical value in pathogens with environmental niches, in order to evaluate the health risks due to antibiotic pollution of natural ecosystems and ultimately tackle antibiotic resistance.

sentences: Macrophages in the reticuloendothelial system inhibit the propagation phase of mouse apolipoprotein A-II amyloidosis Amyloidosis refers to a group of degenerative diseases that are characterized by the deposition of misfolded protein fibrils in various organs. Deposited amyloid may be removed by a phagocyte-dependent innate immune system; however, the precise mechanisms during disease progression remain unclear. We herein investigated the properties of macrophages that contribute to amyloid degradation and disease progression using transmissible apolipoprotein A-II amyloidosis model mice. Intravenously injected AApoAII amyloid was efficiently engulfed by reticuloendothelial macrophages in the liver and spleen and disappeared by 24 h. While cultured murine macrophages degraded AApoAII via the endosomal-lysosomal pathway, AApoAII fibrils reduced cell viability and phagocytic capacity. Furthermore, the depletion of reticuloendothelial macrophages prior to the induction of AApoAII markedly increased hepatic and splenic AApoAII deposition. These results highlight the physiological role of reticuloendothelial macrophages against inter-individual amyloid propagation and suggest the maintenance of phagocytic integrity as a therapeutic strategy to inhibit disease progression.

sentences: SARS-CoV-2 Delta variant pathogenesis and host response in Syrian hamsters B.1.617 lineage is becoming a dominant SARS-CoV-2 lineage worldwide and was the dominant lineage reported in second COVID-19 wave in India, which necessitated studying the properties of the variant. We evaluated the pathogenicity and virus shedding of B.1.617.2 (Delta) and B.1.617.3 lineage of SARS-CoV-2 and compared with that of B.1, an early virus isolate with D614G mutation in Syrian hamster model. Viral load, antibody response and lung disease were studied. No significant difference in the virus shedding pattern was observed among these variants studied. A significantly high SARS-CoV-2 sub genomic RNA could be detected in the respiratory tract of hamsters infected with Delta variant for 14 days. Delta variant induced lung disease of moderate severity in 40% of infected animals. The neutralizing capability of the B.1, Delta and B.1.617.3 variant infected animals were found significantly lower with the B.1.351 (Beta variant). The findings of the study support the attributed disease severity and the increased transmission potential of the Delta variant.

sentences: MetaBinner: a high-performance and stand-alone ensemble binning method to recover individual genomes from complex microbial communities Binning is an essential procedure during metagenomic data analysis. However, the available individual binning methods usually do not simultaneously fully use different features or biological information. Furthermore, it is challenging to integrate multiple binning results efficiently and effectively. Therefore, we developed an ensemble binner, MetaBinner, which generates component results with multiple types of features and utilizes single-copy gene (SCG) information for k-means initialization. It then utilizes a two-step ensemble strategy based on SCGs to integrate the component results. Extensive experimental results over three large-scale simulated datasets and one real-world dataset demonstrate that MetaBinner outperforms other state-of-the-art individual binners and ensemble binners. MetaBinner is freely available at https://github.com/ziyewang/MetaBinner.

sentences: Cross-generational heritability analysis of physiological traits in Porites astreoides across an inshore-offshore gradient in the Lower Florida Keys Estimating the heritable genetic variation in fitness-related traits is key to projecting the adaptive evolution of organisms in response to a changing environment. While heritability studies on reef-building corals to date support adaptive capacity, little is known about the dynamics of trait heritability across life stages in which distinct selective pressures can have long-lasting effects both within and across generations. In this study, we obtained heritability estimates for energetic and thermal stress response traits in larval, recruit, and adult Porites astreoides from two populations in the Lower Florida Keys. To induce bleaching phenotypes among individual families, larvae were exposed to a 4-day thermal stress at 32 {degrees}C, whereas adults and recruits received the same treatment for 22 days. Origin-dependent tolerance was observed in two life stages where offshore recruits lost more symbiont cells under heat than inshore recruits compared to their respective controls and heat-treated offshore adults suffered a greater loss in total protein content. Surprisingly, larvae appeared to be largely insensitive to heat regardless of origin. Broad sense heritability (H2) estimates varied greatly among traits and life stages, which may reflect changes in the relative importance of genetic and environmental variation throughout development. Notably, more than 80% of the variation in larval chlorophyll a concentration was attributed to genetic factors. The overall moderate to high H2 estimates measured here suggest these corals have considerable potential to adapt to environmental change.

sentences: Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone receptor 1 Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to Gs and increased activation of Gq compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.

sentences: Exploring Energy Landscapes of Intrinsically Disordered Proteins: Insights into Functional Mechanisms Intrinsically disordered proteins (IDPs) lack a rigid 3D structure and populate a polymorphic ensemble of conformations. Because of the lack of a reference conformation, their energy landscape representation in terms of reaction coordinates presents a daunting challenge. Here, our newly developed Energy Landscape Visualization Method (ELViM), a reaction coordinate-free approach, shows its prime application to explore frustrated energy landscapes of an intrinsically disordered protein, Prostate-Associated Gene 4 (PAGE4). PAGE4 is a transcriptional coactivator that potentiates the oncogene c-Jun. Two kinases, namely HIPK1 and CLK2, phosphorylate PAGE4 generating variants phosphorylated at different serine/threonine residues (HIPK1-PAGE4 and CLK2-PAGE4, respectively) with opposing functions. While HIPK1-PAGE4 predominantly phosphorylates Thr51 and potentiates c-Jun, CLK2-PAGE4 hyper-phosphorylates PAGE4 and attenuates transactivation. To understand the underlying mechanisms of conformational diversity among different phosphoforms, we have analyzed their atomistic trajectories simulated using AWSEM forcefield and the energy landscapes were elucidated using ELViM. This method allows us to identify and compare the population distributions of different conformational ensembles of PAGE4 phosphoforms using the same effective phase space. The results reveal a predominant conformational ensemble with an extended C-terminal segment of WT PAGE4, which exposes a functional residue Thr51, implying its potential of undertaking a fly-casting mechanism while binding to its cognate partner. In contrast, for HIPK1-PAGE4, a compact conformational ensemble enhances its population sequestering phosphorylated-Thr51. This clearly explains the experimentally observed weaker affinity of HIPK1-PAGE4 for c-Jun. ELViM appears as a powerful tool especially to analyze the highly-frustrated energy landscape representation of IDPs where appropriate reaction coordinates are hard to apprehend.

sentences: BRAF inhibitor resistance confers increased sensitivity to mitotic inhibitors Single agent and combination therapy with BRAFV600E/K and MEK inhibitors have remarkable efficacy against melanoma tumors with activating BRAF mutations, but in most cases resistance eventually develops. The purpose of this study is to uncover pharmacological vulnerabilities of BRAFi-resistant melanoma cells, with the goal of identifying new therapeutic options for patients whose tumors have developed resistance to BRAFi/MEKi therapy. We screened a well-annotated compound library against a panel of isogenic pairs of parental and BRAFi-resistant melanoma cell lines to identify classes of compounds that selectively target BRAFi-resistant cells over their BRAFi-sensitive counterparts. Two distinct patterns of increased sensitivity to classes of pharmacological inhibitors emerged. In two cell line pairs, BRAFi resistance conferred increased sensitivity to compounds that share the property of cell cycle arrest at M-phase, including inhibitors of aurora kinase (AURK), polo-like kinase (PLK), tubulin, and kinesin. Live cell microscopy used to track mitosis in real time revealed that parental, but not BRAFi-resistant, melanoma cells were able to exit from compound-induced mitotic arrest through mitotic slippage, thus escaping death. Consistent with the key role of Cyclin B1 levels in regulating mitosis at the spindle checkpoint, in arrested cells we found higher Cyclin B1 levels in parental over BRAFi-resistant melanoma cells, suggesting that altered Cyclin B1 expression levels may explain why these BRAFi resistant cells have gained increased vulnerability to mitotic inhibitors. Another BRAFi-resistant cell line showed increased sensitivity to Chk1/2 inhibitors, possibly due to an accumulation of DNA damage, resulting in mitotic failure. This study shows that BRAFi-resistance in melanoma cells confers vulnerability to pharmacological disruption of mitosis and suggests a targeted synthetic lethal approach to treat BRAF-mutant melanomas that have become resistant to BRAF/MEK-directed therapies.

sentences: The evolution of fluoroquinolone resistance in Salmonella under exposure to sub-inhibitory concentration of enrofloxacin The evolution of resistance in Salmonella to fluoroquinolones (FQs) under a broad range of sub-inhibitory concentrations (sub-MICs) has not been systematically studied. This study investigated the mechanism of resistance development in Salmonella enterica serovar Enteritidis (S. Enteritidis) under sub-MICs of 1/128xMIC to 1/2xMIC of enrofloxacin (ENR), a widely used veterinary FQ. It was shown that the resistance rate and resistance level of S. Enteritidis varied with the increase of ENR concentration and duration of selection. qRT-PCR results demonstrated that the expression of outer membrane porin (OMP) genes, ompF, ompC and ompD, were down-regulated first to rapidly adapt and develop resistance of [≤] 4xMIC, and as the resistance level increased ([≥]8xMIC), the up-regulated expression of efflux pump genes, acrB, emrB amd mdfA, along with mutations in quinolone resistance-determining region (QRDR) gradually played a decisive role. Cytohubba analysis based on transcriptomic profiles demonstrated that purB, purC, purD, purF, purH, purK, purL, purM, purN and purT were the hub genes for the FQs resistance.  de novo IMP biosynthetic process, purine ribonucleoside monophosphate biosynthetic process and purine ribonucleotide biosynthetic process were the top three biological processes screened by MCODE. This study first described the dynamics of FQ resistance evolution in Salmonella under a long-term selection of sub-MICs of ENR in vitro. In addition, this work offers greater insight into the transcriptome changes of S. Enteritidis under the selection of ENR and provides a framework for FQs resistance of Salmonella for further studies.

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Cell-Type-Specific Decrease of the Intrinsic Excitability of Motor Cortical Pyramidal Neurons in Parkinsonism The hypokinetic motor symptoms of Parkinsons disease (PD) are closely linked with a decreased motor cortical output as a consequence of elevated basal ganglia inhibition. However, whether and how the loss of dopamine alters the cellular properties of motor cortical neurons in PD remains undefined. We induced parkinsonism in adult C57BL6 mice of both sexes by injecting neurotoxin, 6-hydroxydopamine, into the medial forebrain bundle. By using ex vivo patch-clamp recording and retrograde tracing approach, we found that the intrinsic excitability of pyramidal tract neurons (PTNs) in the motor cortical layer 5b was greatly decreased in parkinsonism; but the intratelencephalic neurons (ITNs) were not affected. The cell-type-specific intrinsic adaptations were associated with a depolarized threshold and broadened width of action potentials in PTNs. Moreover, the loss of midbrain dopaminergic neurons impaired the capability of M1 PTNs to sustain high-frequency firing, which could underlie their abnormal pattern of activity in the parkinsonian state. We also showed that the decreased excitability in parkinsonism was caused by an impaired function of both persistent sodium channels and the large conductance, Ca2+-activated K+ channels. Acute activation of dopaminergic receptors failed to rescue the impaired intrinsic excitability of M1 PTNs in parkinsonian mice. Altogether, our data demonstrated a cell-type-specific decrease of the excitability of M1 pyramidal neurons in parkinsonism. Thus, intrinsic adaptations in the motor cortex, together with pathological basal ganglia inhibition, underlie the decreased motor cortical output in parkinsonian state and exacerbate parkinsonian motor deficits.

Significance statementThe degeneration of midbrain dopaminergic neurons in Parkinsons disease remodels the connectivity and function of cortico-basal ganglia-thalamocortical network. However, whether and how dopaminergic degeneration and the associated basal ganglia dysfunction alter motor cortical circuitry remain undefined. We found that pyramidal neurons in the layer 5b of the primary motor cortex (M1) exhibit distinct adaptations in response to the loss of midbrain dopaminergic neurons, depending on their long-range projections. Besides the decreased thalamocortical synaptic excitation as proposed by the classical model of Parkinsons pathophysiology, these results, for the first time, show novel cellular and molecular mechanisms underlying the abnormal motor cortical output in parkinsonism.

sentences: Photolabile curcumin with physically and molecularly produced light for attenuating of neurotoxicity of amyloid beta species Over the past decades, classical drug development approaches for Alzheimers disease have yielded limited success, and this futileness has prompted scientists to seek non-classical approaches. In this report, we demonstrated that, with irradiation of LED light or with molecularly generated light (dubbed as "molecular light") from chemiluminescence probe ADLumin-4, photolabile CRANAD-147 could cause changes of properties, structures (sequences) and neurotoxicity of amyloid beta (A{beta}) species in vitro. We further demonstrated that, with the assistance from molecular chemiluminescence imaging, the combination of CRANAD-147/LED or CRANAD-147/ADLumin-4 (molecular light) could slow down the accumulation of A{beta}s in transgenic 5xFAD mice in vivo. Due to the unlimited capacity of tissue penetration of molecular light in vivo, phototherapy with the combination of photolabile A{beta} ligand and molecular light has great potential as an alternative approach for AD drug discovery.

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MUCOSECRETORY LUNG DISEASE: DIFFERENT ASSEMBLIES OF JAG1 AND JAG2 DETERMINE TRACHEOBRONCHIAL CELL FATE Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. These cell types are derived from tracheobronchial stem/progenitor cells via a Notch dependent mechanism. Although specific arrays of Notch receptors regulate cell fate determination, the function of the ligands Jagged1 (JAG1) and Jagged2 (JAG2) is unclear. This study used primary human bronchial air-liquid- interface cultures, gamma secretase inhibition, and neutralizing antibodies to show: 1) JAG1 and JAG2 were necessary for secretory progenitor cell fate determination; 2) JAG2 suppressed squamous differentiation; and 3) pausing of the ciliated cell differentiation process after Notch inhibition. Histological, cell fractionation, cell surface biotinylation, and ubiquitination analyses demonstrated that all cells were JAG1 positive but that little JAG1 was present on the cell surface. In contrast, JAG2 was expressed in a positive-negative pattern and was abundant on the cell surface. Glycogen synthase kinase 3 (GSK3) and tankyrase inhibition studies showed that GSK3 regulated JAG2 trafficking, and that this mechanism was WNT-independent. Collectively, these data indicate that variation in JAG2 trafficking creates regions of high, medium, and low ligand expression. Thus, distinct assemblies of JAG1 and JAG2 may regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells.

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sentences: Circuit motifs and graph properties of connectome development in C. elegans Network science is a powerful tool that can be used to better explore the complex structure of brain networks. Leveraging graph and motif analysis tools, we interrogate C. elegans connectomes across multiple developmental time points and compare the resulting graph characteristics and substructures over time. We show the evolution of the networks and highlight stable invariants and patterns as well as those that grow or decay unexpectedly, providing a substrate for additional analysis.

sentences: ER-localized phosphatidylethanolamine synthase plays a conserved role in lipid droplet formation The asymmetric distribution of phospholipids in membranes is a fundamental principle of cellular compartmentalization and organization. Phosphatidylethanolamine (PE), a nonbilayer phospholipid that contributes to organelle shape and function, is synthesized at several subcellular localizations via semi-redundant pathways. Previously, we demonstrated in the yeast Saccharomyces cerevisiae that the PE synthase Psd1, which primarily operates on the mitochondrial inner membrane, is additionally targeted to the endoplasmic reticulum (ER). While ER-localized Psd1 is required to support cellular growth in the absence of redundant pathways, its physiological function at the ER is unclear. We now demonstrate that ER-localized Psd1 sub-localizes on the ER to lipid droplet (LD) attachment sites and further show it is specifically required for normal LD formation. We also find that the role of PSD enzymes in LD formation is conserved in other organisms. Thus, we have identified PSD enzymes as novel regulators of LDs and demonstrate that both mitochondria and LDs in yeast are organized and shaped by the spatial positioning of a single PE synthesis enzyme.

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Neuroprotective mechanisms of red clover and soy isoflavones in Parkinson's disease models Parkinsons disease (PD) is a neurodegenerative disorder characterized by nigrostriatal degeneration and the spreading of aggregated forms of the presynaptic protein -synuclein (aSyn) throughout the brain. PD patients are currently only treated with symptomatic therapies, and strategies to slow or stop the progressive neurodegeneration underlying the diseases motor and cognitive symptoms are greatly needed. The time between the first neurobiochemical alterations and the initial presentation of symptoms is thought to span several years, and early neuroprotective dietary interventions could delay the disease onset or slow PD progression. In this study, we characterized the neuroprotective effects of isoflavones, a class of dietary polyphenols found in soy products and in the medicinal plant red clover (Trifolium pratense). We found that isoflavone-rich extracts and individual isoflavones rescued the loss of dopaminergic neurons and the shortening of neurites in primary mesencephalic cultures exposed to two PD-related insults, the environmental toxin rotenone and an adenovirus encoding the A53T aSyn mutant. The extracts and individual isoflavones also activated the Nrf2-mediated antioxidant response in astrocytes via a mechanism involving inhibition of the ubiquitin-proteasome system, and they alleviated deficits in mitochondrial respiration. Furthermore, an isoflavone-enriched soy extract reduced motor dysfunction exhibited by rats lesioned with the PD-related neurotoxin 6-OHDA. These findings suggest that plant-derived isoflavones could serve as dietary supplements to delay PD onset in at-risk individuals and mitigate neurodegeneration in the brains of patients.

Graphical Abstract

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sentences: Multiscale Modeling of Hepatitis B Virus Capsid Assembly and its Dimorphism Hepatitis B Virus (HBV) is an endemic, chronic virus that leads to 800,000 deaths per year. Central to the HBV lifecycle, the viral core has a protein capsid assembled from many copies of a single protein. The capsid protein adopts different (quasi-equivalent) conformations to form icosahedral capsids containing 180 or 240 proteins, T=3 or T=4 respectively in Caspar-Klug nomenclature. HBV capsid assembly has become an important target for new antivirals; nonetheless the assembly pathways and mechanisms that control HBV dimorphism remain unclear. We describe computer simulations of HBV assembly, using a coarse-grained model that has parameters learned from all-atom molecular dynamics simulations of a complete HBV capsid, and yet is computationally tractable. Dynamical simulations with the resulting model reproduce experimental observations of HBV assembly pathways and products. By constructing Markov state models and employing transition path theory, we identify pathways leading to T=3, T=4, and other experimentally observed capsid morphologies. The analysis identifies factors that control this polymorphism, in particular, the conformational free energy landscape of the capsid proteins and their interactions.

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Early flowering in oilseed-type Brassica rapa plants results from nonsense-mediated mRNA decay (NMD) of BrFLC2 Many Brassica species require vernalization (long-term winter-like cooling) for transition to the reproductive stage. In the past several decades, scientific efforts have been made to discern the molecular mechanisms underlying vernalization in many species. Thus, to identify the key regulators required for vernalization in Brassica rapa L., we constructed a linkage map composed of 7,833 single nucleotide polymorphism (SNP) markers using the late-flowering Chinese cabbage (B. rapa L. ssp. pekinensis) inbred line  Chiifu and the early-flowering yellow sarson (B. rapa L. ssp. trilocularis (Roxb.)) line  LP08 and identified a single major QTL on the upper-arm of the chromosome A02. In addition, we compared the transcriptomes of the lines  Chiifu and  LP08 at five vernalization time points, including both non-vernalized and post-vernalization conditions. We observed that BrFLC2 was significantly downregulated in the early flowering  LP08 and had two deletion sites around the BrFLC2 genomic region compared with the BrFLC2 genomic region in  Chiifu. In the present study, we also demonstrate that early flowering in  LP08 line is attributed to the low expression of BrFLC2, which is caused by nonsense-mediated mRNA decay (NMD). Therefore, this study provides a better understanding of the molecular mechanisms underlying floral transition in B. rapa.

One sentence summaryNMD-mediated degradation of BrFLC2 mRNA transcripts is the main cause of rapid flowering of oilseed-type B. rapa  LP08 plants.

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Exploration of BAY 11-7082 as a novel antibiotic Exposure of the Gram-negative pathogen Pseudomonas aeruginosa to sub-inhibitory concentrations of antibiotics increases formation of biofilms. We exploited this phenotype to identify molecules with potential antimicrobial activity in a biofilm-based high-throughput screen. The anti-inflammatory compound BAY 11-7082 induced dose-dependent biofilm stimulation, indicative of antibacterial activity. We confirmed that BAY 11-7082 inhibits growth of P. aeruginosa and other priority pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). We synthesized 27 structural analogues, including a series based on the related scaffold 3-(phenylsulfonyl)-2-pyrazinecarbonitrile (PSPC), 10 of which displayed increased anti-Staphylococcal activity. Because the parent molecule inhibits the NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome, we measured the ability of select analogues to reduce IL-1{beta} production in mammalian macrophages, identifying minor differences in the structure-activity relationship for the anti-inflammatory and antibacterial properties of this scaffold. Although we could evolve stably resistant MRSA mutants with cross resistance to BAY 11-7082 and PSPC, their lack of shared mutations suggested that the two molecules could have multiple targets. Finally, we showed that BAY 11-7082 and its analogues potentiate the activity of penicillin G against MRSA, suggesting that this scaffold may serve as an interesting starting point for the development of antibiotic adjuvants.

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Open chromatin analysis in Trypanosoma cruzi life forms highlights critical differences in genomic compartments and developmental regulation at tDNA loci BackgroundGenomic organization and gene expression regulation in trypanosomes are remarkable because protein-coding genes are organized into codirectional gene clusters with unrelated functions. Moreover, there is no dedicated promoter for each gene, resulting in polycistronic gene transcription, with posttranscriptional control playing a major role. Nonetheless, these parasites harbor epigenetic modifications at critical regulatory genome features that dynamically change among parasite stages, which are not fully understood.

ResultsHere, we investigated the impact of chromatin changes in a scenario commanded by posttranscriptional control exploring the parasite Trypanosoma cruzi and its differentiation program using genome-wide approaches supported by transmission electron microscopy. The integration of FAIRE and MNase-seq data, two complementary epigenomic approaches, enabled us to identify differences in T. cruzi genome compartments, putative transcriptional start regions and virulence factors. In addition, we also detected developmental chromatin regulation at tRNA loci (tDNA), which seems to be linked to the translation regulatory mechanism required for parasite differentiation. Strikingly, a positive correlation was observed between active chromatin and steady-state transcription levels.

ConclusionTaken together, our results indicate that chromatin changes reflect the unusual gene expression regulation of trypanosomes and the differences among parasite developmental stages, even in the context of a lack of canonical transcriptional control of protein-coding genes.

sentences: Unexpected arabinosylation after humanization of plant protein N-glycosylation As biopharmaceuticals, recombinant proteins have become indispensable tools in medicine. An increasing demand, not only in quantity but also in diversity, drives the constant development and improvement of production platforms. The N-glycosylation pattern on biopharmaceuticals plays an important role in activity, serum half-life and immunogenicity. Therefore, production platforms with tailored protein N-glycosylation are of great interest. Plant-based systems have already demonstrated their potential to produce pharmaceutically relevant recombinant proteins, although their N-glycan patterns differ from those in humans. Plants have shown great plasticity towards the manipulation of their glycosylation machinery, and some have already been glyco-engineered in order to avoid the attachment of plant-typical, putatively immunogenic sugar residues. This resulted in complex-type N-glycans with a core structure identical to the human one. Compared to humans, plants lack the ability to elongate these N-glycans with {beta}1,4-linked galactoses and terminal sialic acids. However, these modifications, which require the activity of several mammalian enzymes, have already been achieved for Nicotiana benthamiana and the moss Physcomitrella. Here, we present the first step towards sialylation of recombinant glycoproteins in Physcomitrella, human {beta}1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric {beta}1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss 1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human {beta}1,4-galactosyltransferase. Stable FTGT expression led to the desired {beta}1,4-galactosylation. However, additional pentoses of unknown identity were also observed. The nature of these pentoses was subsequently determined by Western blot and enzymatic digestion followed by mass spectrometric analysis and resulted in their identification as -linked arabinoses. Since a pentosylation of {beta}1,4-galactosylated N-glycans was reported earlier, e.g. on recombinant human erythropoietin produced in glyco-engineered Nicotiana tabacum, this phenomenon is of a more general importance for plant-based production platforms. Arabinoses, which are absent in humans, may prevent the full humanization of plant-derived products. Therefore, the identification of these pentoses as arabinoses is important as it creates the basis for their abolishment to ensure the production of safe biopharmaceuticals in plant-based systems.

sentences: Dietary palmitic acid induces trained immunity that controls inflammation and infection Trained immunity is an innate immune memory response that is induced by primary microbial or sterile stimuli that sensitizes monocytes and macrophages to a secondary pathogenic challenge, reprogramming the host response to infection and inflammatory disease. Nutritional components, such as dietary fatty acids, can act as inflammatory stimuli, but it is unknown if they can act as the primary stimuli in the context of innate immune memory. Here we find mice fed diets enriched in saturated fatty acids (SFAs) confer a hyper-inflammatory response to systemic lipopolysaccharide (LPS) and increased mortality, independent of diet-induced microbiome and glycemic modulation. Ex vivo, we show monocytes and splenocytes from mice fed enriched SFAs do not have altered baseline inflammation, but enhanced responses to a secondary inflammatory challenge. Lipidomics identified enhanced free palmitic acid (PA) and PA-associated lipids in SFA-fed mice serum. We found pre-treatment with physiologically relevant concentrations of PA alone reprograms macrophages to induce a hyper-inflammatory response to secondary challenge with LPS. This response was found to be dependent on the synthesis of ceramide, and reversible when treated with oleic acid, a mono-unsaturated FA that depletes intracellular ceramide. In vivo, we found systemic PA confers enhanced inflammation and mortality during an acute septic response to systemic LPS, which was not reversible for up to 7 days post-PA-exposure. While PA-treatment is harmful for acute septic shock outcome, we find PA exposure enhanced clearance of Candida albicans in RAG-/- mice. These are the first data to implicate enriched dietary SFAs, and specifically PA, in the induction of long-lived innate immune memory that is detrimental during an acute septic response, but beneficial for clearance of pathogens.

sentences: Oligodendrocyte origin and development in the zebrafish visual system Oligodendrocytes are the myelinating cells in the central nervous system (CNS). Their developmental origin and specification are well known in birds and mammals but remains unclear in fish. To fulfill this gap, we have studied their early progression during zebrafish visual morphogenesis using the transgenic line Olig2:GFP. We have tracked the Olig2+ cells in the visual system from 48 hours post fertilization (hpf) until 11 days post fertilization (dpf). We have also analyzed the differential expression of the Sox2 and Sox10 transcription factors in this cellular line. Oligodendrocyte progenitor cells (OPCs) originate at 48hpf in regions close to the preoptic area, near to the ventral hypothalamus. Then, at 5 dpf, they migrate to the optic chiasm, where they invade the optic nerve, extending towards the retina. While OPCs in the retina also express Sox2, in the the optic tectum they express Sox10. Within the optic nerve tract, they express both. We have also observed that these Olig2:GFP line do not colocalize with the expression of Mbpa, a myelin marker, but are intimately intertwined. Our data matches with other animal models, where OPCs are specified in the preoptic area and migrate to the optic nerve through the optic chiasm. We revealed that oligodendrocyte is a complex population of cells expressing different transcription factors.

sentences: sciCAN: Single-cell chromatin accessibility and gene expression data integration via Cycle-consistent Adversarial Network The booming single-cell technologies bring a surge of high dimensional data that come from different sources and represent cellular systems from different views. With advances in single-cell technologies, integrating single-cell data across modalities arises as a new computational challenge and gains more and more attention within the community. Here, we present a novel adversarial approach, sciCAN, to integrate single-cell chromatin accessibility and gene expression data in an unsupervised manner. We benchmarked sciCAN with 3 state-of-the-art (SOTA) methods in 5 scATAC-seq/scRNA-seq datasets, and we demonstrated that our method dealt with data integration with better balance of mutual transferring between modalities than the other 3 SOTA methods. We further applied sciCAN to 10X Multiome data and confirmed the integrated representation preserves information of the hematopoietic hierarchy. Finally, we investigated CRSIPR-perturbed single-cell K562 ATAC-seq and RNA-seq data to identify cells with related responses to different perturbations in these different modalities.

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NFE2L1-mediated proteasome function protects from ferroptosis ObjectiveFerroptosis continues to emerge as a novel modality of cell death with important therapeutic implications for a variety of diseases, most notably cancer and degenerative diseases. While susceptibility, initiation, and execution of ferroptosis have been linked to reprogramming of cellular lipid metabolism, imbalances in iron-redox homeostasis, and aberrant mitochondrial respiration, the detailed mechanisms of ferroptosis are still insufficiently well understood.

Methods and ResultsHere we show that diminished proteasome function is a new mechanistic feature of ferroptosis. The transcription factor nuclear factor erythroid-2, like-1 (NFE2L1) protects from ferroptosis by sustaining proteasomal activity. In cellular systems, loss of NFE2L1 reduced cellular viability after the induction of both chemically and genetically induced ferroptosis, which was linked to the regulation of proteasomal activity under these conditions. Importantly, this was reproduced in a Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD) patient-derived cell line carrying mutated glutathione peroxidase-4 (GPX4), a critical regulator of ferroptosis. Also, reduced proteasomal activity was associated with ferroptosis in Gpx4-deficient mice. In a mouse model for genetic Nfe2l1 deficiency, we observed brown adipose tissue (BAT) involution, hyperubiquitination of ferroptosis regulators, including the GPX4 pathway, and other hallmarks of ferroptosis.

ConclusionOur data highlight the relevance of the NFE2L1-proteasome pathway in ferroptosis. Manipulation of NFE2L1 activity might enhance ferroptosis-inducing cancer therapies as well as protect from aberrant ferroptosis in neurodegeneration, general metabolism, and beyond.

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HighlightsO_LIProteasome function is diminished during ferroptosis
C_LIO_LINFE2L1-mediated proteasomal activity protects from ferroptosis
C_LIO_LIThe ubiquitination of the GPX4-glutathione pathway is implicated in Nfe2l1 deficiency
C_LIO_LINFE2L1 deficiency in brown fat is associated with hallmarks of ferroptosis
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sentences: Isolation and characterization of barley (Hordeum vulgare) extracellular vesicles and their role in RNAi-based crop protection The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce gene silencing of pathogenic targets was groundbreaking. However, future field applications will require fundamental mechanistic knowledge on dsRNA uptake, processing, and its transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. Here, we examined the role of EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to the target fungus Fusarium graminearum. We found barley EVs with 156 nm in size containing predominantly 21 and 19 nucleotide (nt) siRNAs starting with a 5-terminal Adenine. Notably, barley EVs contain less siRNA compared to EVs isolated from transgenic HIGS Arabidopsis plants. Together our results further underpin mechanistic differences between HIGS and SIGS applications and a minor role of EVs in SIGS.

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Local brain-state dependency of effective connectivity: evidence from TMS-EEG BackgroundSpontaneous cortical oscillations have been shown to modulate cortical responses to transcranial magnetic stimulation (TMS). If not controlled for, they might increase variability in responses and mask meaningful changes in the signals of interest when studying the brain with TMS combined with electroencephalography (TMS-EEG). To address this challenge in future closed-loop stimulation paradigms, we need to understand how spontaneous oscillations affect TMS-evoked responses.

ObjectiveTo describe the effect of the pre-stimulus phase of cortical mu (8-13 Hz) and beta (13-30 Hz) oscillations on TMS-induced effective connectivity patterns.

MethodsWe applied TMS to the left primary motor cortex and right pre-supplementary motor area of three subjects while recording EEG. We classified trials off-line into positive- and negative-phase classes according to the mu and beta rhythms. We calculated differences in the global mean-field amplitude (GMFA) and compared the cortical spreading of the TMS-evoked activity between the two classes.

ResultsPhase had significant effects on the GMFA in 11 out of 12 datasets (3 subjects x 2 stimulation sites x 2 frequency bands). Seven of the datasets showed significant differences in the time range 15-50 ms, nine in 50-150 ms, and eight after 150 ms post-stimulus. Source estimates showed complex spatial differences between the classes in the cortical spreading of the TMS-evoked activity.

ConclusionsTMS-evoked effective connectivity appears to depend on the phase of local cortical oscillations at the stimulated site. This may be crucial for efficient design of future brain-state-dependent and closed-loop stimulation paradigms.

sentences: Drivers of genetic differentiation and recent evolutionary history of an Eurasian wild pea Genetic diversity a major determinant for the capacity of species to persist and adapt to their environments. Unraveling the factors affecting genetic differentiation is crucial to understand how genetic diversity is shaped and species may react to changing environments. We employed genotyping by sequencing to test the influence of climate, space, latitude, altitude and land cover on genetic differentiation in a collection of 81 wild pea samples (Pisum sativum ssp. elatius) from across its distribution range from western Europe to central Asia. We also attempted to elucidate the species recent evolutionary history and its effect on the current distribution of genetic diversity. Association of single SNPs with climate variables were analyses to test for signatures of local adaptation. Genetic variation was geographically structured into six distinct genetic cluster. Two of which were associated with a taxonomic group (Pisum sativum ssp. humile) that according to some researchers does not qualify for a sub-species rank due to its alleged lack of genetic distinctness from other conspecific groups. The effect of the tested factors influencing genetic differentiation were rather variable among genetic clusters. The climate predictors were most important in all clusters. Land use was more important in clusters from areas strongly influenced by human land use, especially by agriculture. We found a statistically significant association of 3,623 SNPs (2.4 % of all SNPs) with one of the environmental predictors. Most of them were correlated with latitude followed by temperature, precipitation and altitude. Estimation of SNP effects of the candidates resulted in a missense to silent ratio of 0.45, suggesting many of the observed candidates SNPs may alter the encoded amino acid sequence. Wild peas went through a genetic bottleneck during the last glacial period followed by population recovery. Probably associated with this population recovery, we detected a range expansion, which may have led to an eastward range expansion of the European cluster to Turkey and thereof southwards and eastwards. Overall, the interplay of several environmental factors and the recent evolutionary history affected the distribution of genetic diversity in wild peas where each subpopulations were differently affected by those factors and processes.

sentences: Metagenomics Study of Bacterial Communities from Yamuna River water of city of Taj, Agra Yamuna river water of Agra city is heavily contaminated with toxic pollutants including heavy metal that causes severe damage to ecological and social aspects. At present, the direct use of river water for the purpose of drinking causes severe hazards due to anthropogenic activities causing environmental pollution in rivers. In present study, Yamuna River water was collected from three different sites of Agra city. Various physico-chemical parameters were estimated by following the standard methods of APHA and the concentration of heavy metals were measured using Atomic Absorption Spectrophotometer (AAS). In case of physico-chemical parameters characterization, the obtained values were extremely above the permissible limits. On account of the research work; the Yamuna river water samples contains heavy metal concentrations (Cadmium, Chromium, Copper, Lead and Nickel) above the desirable and admissible levels except for Zinc. During analysis of non-culturable bacteria a substantial bacterial diversity was observed in the Yamuna river water samples collected from different sites. The water samples were subjected to metagenomic analysis using Illumina platform which revealed that Proteobacteria (phylum), Betaproteobacteria (class), Burkholderiales (order), Comamonadaceae (family), Hydrogenophaga (genus) and Chloroflexi bacterium OLB 14 (species) were found as the most dominant bacterial taxonomic abundance in the river water samples. The presence of such bacterial communities in water indicates the availability of pollutants and suggests the futuristic use in the field of bioremediation.

sentences: A robust receptive field code for optic flow detection and decomposition during self-motion The perception of optic flow is essential for any visually guided behaviour of a moving animal. To mechanistically predict behaviour and understand the emergence of self-motion perception in vertebrate brains, it is essential to systematically characterize the motion receptive fields (RFs) of optic flow processing neurons. Here, we present the fine-scale RFs of thousands of motion-sensitive neurons studied in the diencephalon and the midbrain of zebrafish. We found neurons that serve as linear filters and robustly encode directional and speed information of translation-induced optic flow. These neurons are topographically arranged in pretectum according to translation direction. The unambiguous encoding of translation enables the decomposition of translational and rotational self-motion information from mixed optic flow. In behavioural experiments, we successfully demonstrated the predicted decomposition in the optokinetic and optomotor responses. Together, our study reveals the algorithm and the neural implementation for self-motion estimation in a vertebrate visual system.

sentences: A sequence-based global map of regulatory activity for deciphering human genetics Sequence is at the basis of how the genome shapes chromatin organization, regulates gene expression, and impacts traits and diseases. Epigenomic profiling efforts have enabled large-scale identification of regulatory elements, yet we still lack a sequence-based map to systematically identify regulatory activities from any sequence, which is necessary for predicting the effects of any variant on these activities. We address this challenge with Sei, a new framework for integrating human genetics data with sequence information to discover the regulatory basis of traits and diseases. Our framework systematically learns a vocabulary for the regulatory activities of sequences, which we call sequence classes, using a new deep learning model that predicts a compendium of 21,907 chromatin profiles across >1,300 cell lines and tissues, the most comprehensive to-date. Sequence classes allow for a global view of sequence and variant effects by quantifying diverse regulatory activities, such as loss or gain of cell-type-specific enhancer function. We show that sequence class predictions are supported by experimental data, including tissue-specific gene expression, expression QTLs, and evolutionary constraints based on population allele frequencies. Finally, we applied our framework to human genetics data. Sequence classes uniquely provide a non-overlapping partitioning of GWAS heritability by tissue-specific regulatory activity categories, which we use to characterize the regulatory architecture of 47 traits and diseases from UK Biobank. Furthermore, the predicted loss or gain of sequence class activities suggest specific mechanistic hypotheses for individual regulatory pathogenic mutations. We provide this framework as a resource to further elucidate the sequence basis of human health and disease.

sentences: β-catenin and canonical Wnts in Hydra pattern formation: Insights from mathematical modelling into two distinct systems Formation of the body axes and the subsequent formation of the apical termini are fundamental steps during animal development. Nuclear {beta}-catenin and canonical Wnt3 have been identified as major players in both these processes in Hydra. Based on molecular knowledge of canonical Wnt signalling directly linking nuclear {beta}-catenin and Wnt3 activity, de novo axis formation and head formation have frequently been assumed to be part of the same pattern formation system. In this study, we investigated this relationship by combining new model simulations with available experimental results. We demonstrated that nuclear {beta}-catenin and Wnt3 likely contribute to two distinct de novo pattern formation systems in Hydra, organizing development and differentiation on two different spatial scales. In particular, the results suggested that nuclear {beta}-catenin acts at the scale of the whole body to control axis formation, whereas canonical Wnt3 signalling is involved in a downstream pathway responsible for small-scale patterning of the head. These results suggest that the underlying mechanisms may also be more complex than previously assumed in other animals where axis formation has been ascribed to canonical Wnt signalling.

sentences: SEX-SPECIFIC TRAJECTORIES OF REELIN-DEPENDENT MATURATION OF DEEP LAYER PREFRONTAL NEURONS Throughout early adulthood, the anatomical and functional maturation of PFC circuitry continues under the influence of multiple extrinsic and intrinsic factors, most notably electrical activity, and molecular cues. We previously showed that the extracellular matrix protein reelin orchestrates the structural and functional maturation of deep layers medial PFC (mPFC) pyramidal neurons. Additionally, we reported that reelin haploinsufficiency is associated to prefrontal disruptions of long-term memory retention thereby illustrating the eminent role of reelin in cognitive maturation of the PFC. Prefrontal maturation follows a sex-specific developmental pattern, supporting the existence of sexual differences in the morphology-functional properties PFC. Here, we interrogated the role of reelin in the functional maturation of excitatory networks in the mPFC. The developmental trajectory of reelins expression and deep layer pyramidal neurons synaptic plasticity was tracked in the mPFC of male and female mice, from the juvenile period to adulthood. To assess the role of reelin in both sexes, wild-type and heterozygous reeler mice (HRM) were compared. The results show that the maturational profile of reelin expression in the mPFC is sex-dependent and that the developmental trajectory of long-term potentiation is different between wild-type males and females. These data demonstrate reelins influence on prefrontal synapses is sex and period specific.

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Molecular-enriched functional connectivity in the human brain using multiband multi-echo simultaneous ASL/BOLD fMRI Receptor-Enriched Analysis of functional Connectivity by Targets (REACT) is a novel analytical strategy that enriches functional connectivity (FC) information from functional MRI (fMRI) with molecular information on the neurotransmitter distribution density in the human brain, providing a biological basis to the FC analysis. So far, this integrative approach has been used in blood oxygen level-dependent (BOLD) fMRI studies only, providing new insights into the brain mechanisms underlying specific disorders and its response to pharmacological challenges. In this study, we demonstrate that the application of REACT can be further extended to arterial spin labelling (ASL) fMRI. Some of the advantages of this extension include the combination of neurotransmitter specific information provided by molecular imaging with a quantitative marker of neuronal activity, the suitability of ASL for pharmacological MRI (phMRI) studies assessing drug effects on baseline brain function, and the possibility to acquire images that are not affected by susceptibility artifacts in the regions linked to major neurotransmitter systems.

In this work, we tested the feasibility of applying REACT to resting state ASL fMRI and compared the molecular-enriched FC maps derived from ASL data with those derived from BOLD data. We applied REACT to high-resolution, whole-brain simultaneous ASL/BOLD resting-state fMRI data of 29 healthy subjects and estimated the ASL- and BOLD-based FC maps related to six molecular systems, including the transporters of dopamine, noradrenaline, serotonin and vesicular acetylcholine, and the GABA-A and mGlu5 receptors. We then compared the ASL and BOLD FC maps in terms of spatial similarity, using the Dice Similarity Index and the voxel-wise spatial correlation. On a data subsample (N=19) we also evaluated the test-retest reproducibility of each modality using the regional intraclass correlation coefficient, and compared the two modalities.

Our results showed robust spatial patterns of molecular-enriched functional connectivity for both modalities, moderate to high similarity between BOLD- and ASL-derived FC maps and mixed results in terms of reproducibility (i.e., none of the modalities outperformed the other). Overall, our findings show that the ASL signal is as informative as BOLD in detecting functional circuits associated with specific molecular pathways, and that the two modalities may provide complementary information related to these circuits.

Considering the more direct link of ASL imaging with neuronal acrivity compared to BOLD and its suitability for phMRI studies, this new integrative approach could become a valuable asset in clinical studies investigating functional alterations in patients with brain disorders, or in pharmacological studies investigating the effects of new or existing compounds on the brain.

sentences: Phylogroup-specific variation shapes pangenome dynamics in Pseudomonas aeruginosa Pseudomonas aeruginosa is a human opportunistic pathogen consisting of three phylogroups (hereafter named A, B, and C) of unevenly distributed size. Here, we assessed phylogroup-specific evolutionary dynamics in a collection of publicly available and newly sequenced genomes of P. aeruginosa. We explored to what extent antimicrobial resistance (AMR) genes, defence systems, and virulence genes vary in their distribution across regions of genome plasticity (RGPs) and "masked" (RGP-free) genomes, and to what extent this variation differs among the phylogroups. We found that members of phylogroup B possess larger genomes, contribute a comparatively larger number of gene families to the pangenome, but show lower abundance of CRISPR-Cas systems. Furthermore, AMR and defence systems are pervasive in RGPs and integrative and conjugative/mobilizable elements (ICEs/IMEs) from phylogroups A and B, and the abundance of these two types of cargo genes is often significantly correlated. Moreover, multiple inter- and intra-phylogroup interaction events occur at the accessory genome content level, suggesting that recombination events are frequent. Finally, we provide here a panel of phylogenetically diverse P. aeruginosa strains that can be used as a reference set for future functional analyses. Altogether, our results highlight distinct pangenome characteristics of the three phylogroups of P. aeruginosa, that are possibly influenced by variation in the abundance of CRISPR-Cas systems and that are shaped by the differential distribution of AMR and other defence systems.

sentences: High School Science Fair: Ethnicity Trends in Student Participation and Experience In this paper, we report ethnicity trends in student participation and experience in high school science and engineering fair (SEFs). SEF participation showed significant ethnic diversity. For survey students, the approximate distribution was Asian-32%; Black-11%; Hispanic-20%; White-33%; Other-3%. Comparing the SEF level at which students competed from school to district to region to state levels, we observed that black students made up only 4.5% of the students who participated in SEF beyond the school level, whereas students from other ethnic groups were more equally represented at all levels. The lower percentage of Black students resulted from a combination of lower overall participation in SEF and lower percentage of those students who did participate to advance to SEFs beyond the school level. Students who advanced to SEFs beyond the school level frequently received help from scientists, coaching for the interview, and were not required to participate in SEF. Black students received the least help from scientists, were least likely to receive coaching for the interview, and were most likely to be required to participate in SEF. They also were most likely to receive no help from parents, teachers, or scientists. Asian and Hispanic students (63.8% and 56.8%) indicated a greater interest in careers in science and engineering (S&E) compared to Black and White students (43.7% & 50.7%). In addition to career interest, the most important experiences that correlated with students who indicated that SEF increased their interests in S&E were getting help from the internet, books and magazines; getting help fine tuning the report; and overcoming obstacles by doing more background research, making a timeline, and perseverance. Black students did not report a positive effect of any of these strategies but experienced time pressure as more of an obstacle than did other students. Our findings identify a wide range of student experiences associated with positive SEF outcomes that could be enhanced for all students but especially Black students. More involvement of scientists in helping students who participate in SEFs would be particularly valuable.

sentences: Transcription Factor Activation Profiles (TFAP) identify compounds promoting differentiation of Acute Myeloid Leukemia cell lines Repurposing of drugs for new therapeutic use has received considerable attention for its potential to reduce time and cost of drug development. Here we present a new strategy to identify chemicals that are likely to induce differentiation of leukemic cells. As Acute Myeloid Leukemia (AML) is the result of a block in myeloid differentiation, finding new drugs that are capable of inducing blast terminal maturation is considered a valuable strategy. We used data from the Connectivity Map (CMap) to identify drugs that could be repositioned for their potential to activate transcription factors that mediate myeloid differentiation. Compounds promoting the activation of transcription factors that play a positive role in myeloid differentiation were considered candidate pro-differentiation drugs. This approach yielded a list of chemicals ranked according to the potential to activate transcription factors that induce differentiation of leukemic progenitor cells. Drugs that are already used in differentiation therapy, such as for instance all-trans retinoic acid (ATRA) are in the top positions of this ranked list. To validate our strategy, we tested the in vitro differentiation potential of 22 candidate compounds using the HL-60 human cell line as a myeloid differentiation model. Ten out of 22 compounds, ranking high in the inferred list, were confirmed to induce significant differentiation of HL-60. Some of these compounds are known to trigger the DNA damage response, thus identifying this process as a target to modulate myeloid differentiation. These results underscore the potential of our approach to accelerate the drug discovery process. The method that we have developed is highly versatile and it can be adapted to different drug repurposing projects.

sentences: Neurons in the Nucleus Tractus Solitarius are Selective to the Orientation of Gastric Electrical Stimulation Gastric electrical stimulation (GES) is a bioelectric intervention for gastroparesis, obesity, and other functional gastrointestinal disorders. In a potential mechanism of action, GES activates the nerve endings of vagal afferent neurons and induces the vago-vagal reflex through the nucleus tractus solitarius (NTS) in the brainstem. However, it is unclear where and how to stimulate in order to optimize the vagal afferent responses. To address this question with electrophysiology in rats, we applied mild electrical currents to two serosal targets on the distal forestomach with dense distributions of vagal intramuscular arrays that innervated the circular and longitudinal smooth muscle layers. During stimulation, we recorded single and multi-unit responses from gastric neurons in NTS and evaluated how the recorded responses depended on the stimulus orientation and amplitude. We found that NTS responses were highly selective to the stimulus orientation for a range of stimulus amplitudes. The strongest responses were observed when the applied current flowed in the same direction as the intramuscular arrays in parallel with the underlying smooth muscle fibers. Our results suggest that gastric neurons in NTS may encode the orientation-specific activity of gastric smooth muscles relayed by vagal afferent neurons. This finding suggests that the orientation of GES is critical to effective engagement of vagal afferents and should be considered in light of the structural phenotypes of vagal terminals in the stomach.

sentences: Redox-dependent Structure and Dynamics of Macrophage Migration Inhibitory Factor Reveal Sites of Latent Allostery Macrophage migration inhibitory factor (MIF) is a multifunctional immunoregulatory protein that is a key player in the innate immune response. Given its overexpression at sites of inflammation in a wide range of diseases marked by increasingly oxidative cellular environment, a comprehensive structural understanding of how cellular redox conditions may impact the structure and function of MIF is necessary. We used solution NMR spectroscopy and mass spectrometry to investigate structural and dynamic signatures of MIF under varied solution redox conditions. Our results indicate that the MIF structure is modified and becomes increasingly dynamic in an oxidative environment, which may be a means to alter the MIF functional response in a redox-dependent manner. We identified latent allosteric sites within MIF that are redox-sensitive and mutational analysis reveals that loss of redox-responsive residues attenuates activation of the coreceptor CD74. Leveraging sites of redox-sensitivity therefore reveals an avenue to modulate MIF function in its "disease state" via structure-based drug design.

sentences: Theta oscillations in frontal cortex differentially modulate accuracy and speed in flexible stimulus- and action-based learning Flexible reward learning relies on frontal cortex, with substantial evidence indicating the anterior cingulate cortex (ACC) and orbitofrontal cortex (OFC) play important roles. Recent studies in both rat and macaque suggest theta oscillations (5-10 Hz) may be a spectral signature that coordinates this learning. However, causal network-level interactions between ACC and OFC in flexible learning remain unclear. We investigated the learning of stimulus-outcome (S-O) and action-outcome (A-O) associations using a combination of simultaneous in-vivo electrophysiology in dorsal ACC and ventral OFC, partnered with bilateral inhibitory DREADDs in ACC. In freely-behaving male and female rats and using a within-subject design, we examined accuracy and speed of response across distinct and precisely-defined trial epochs including correct choice, incorrect choice, and reward collection. We report significant modulation of accuracy by theta power in both ACC and OFC. Both ACC and OFC theta oscillations consistently signaled accuracy in the initial discrimination phase, whereas it was OFC theta alone in the reversal phases of both S-O and A-O learning. Theta power in either region was not correlated with deliberation speed. Indeed, theta modulation of accuracy was dissociable from its involvement in speed of response which was affected by ACC inhibition, most prominently in A-O learning. Results are discussed with reference to the nonhuman primate literature, where there is by contrast more reported specialization of OFC and ACC in flexible learning of stimuli and actions. The present results also point to a specific role of OFC theta in signaling a reversal of either kind.

sentences: The Digital Brain Bank, an open access platform for post-mortem datasets Post-mortem MRI provides the opportunity to acquire high-resolution datasets to investigate neuroanatomy, and validate the origins of image contrast through microscopy comparisons. We introduce the Digital Brain Bank (open.win.ox.ac.uk/DigitalBrainBank), a data release platform providing open access to curated, multimodal post-mortem neuroimaging datasets. Datasets span three themes - Digital Neuroanatomist: datasets for detailed neuroanatomical investigations; Digital Brain Zoo: datasets for comparative neuroanatomy; Digital Pathologist: datasets for neuropathology investigations. The first Digital Brain Bank release includes twenty one distinctive whole-brain diffusion MRI datasets for structural connectivity investigations, alongside microscopy and complementary MRI modalities. This includes one of the highest-resolution whole-brain human diffusion MRI datasets ever acquired, whole-brain diffusion MRI in fourteen non-human primate species, and one of the largest post-mortem whole-brain cohort imaging studies in neurodegeneration. The Digital Brain Bank is the culmination of our labs investment into post-mortem MRI methodology and MRI-microscopy analysis techniques. This manuscript provides a detailed overview of our work with post-mortem imaging to date, including the development of diffusion MRI methods to image large post-mortem samples, including whole, human brains. Taken together, the Digital Brain Bank provides crossscale, cross-species datasets facilitating the incorporation of post-mortem data into neuroimaging studies.

sentences: Multiplex spatial systems analysis of local nanodose drug responses predicts effective treatment combinations of immunotherapies and targeted agents in mammary carcinoma Better methods are needed to identify effective combinations of immunotherapies with chemotherapies and targeted anti-cancer agents. Here we present a Multiplex Implantable Microdevice Assay (MIMA) system for rapid in vivo assessment of the effects of multiple, spatially separate anticancer drugs directly in the complex tumor microenvironment. In prototypic experiments, olaparib, lenvatinib, palbociclib, venetoclax, panobinostat, doxorubicin, and paclitaxel and combinations thereof were administered simultaneously to murine mammary tumor models. Quantitative multiplex immunohistochemistry and spatial systems analyses of each local drug response defined cellular relations of fibroblasts, endothelial cells, immune lineages, immunogenic cell death, tumor proliferation and/or cancer stem cells that were used to predict effective drug combinations. A predicted combination of panobinostat, venetoclax and anti-CD40 showed long-term anti-tumor efficacy in multiple mouse models with no observable toxicity when administered systemically. Future MIMA use promises to design effective drug combinations for tumor cell control and immune activation on a personalized basis.

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overlappingCGM: Automatic detection and analysis of overlapping co-expressed gene modules BackgroundWhen it comes to the co-expressed gene module detection, its typical challenges consist of overlap between identified modules and local co-expression in a subset of biological samples. The nature of module detection is the use of unsupervised clustering approaches and algorithms. Those methods are advanced undoubtedly, but the selection of a certain clustering method for sample- and gene-clustering tasks is separate, in which the latter task is often more complicated.

ResultsThis study presented an R-package, Overlapping CoExpressed gene Module (oCEM), armed with the decomposition methods to solve the challenges above. We also developed a novel auxiliary statistical approach to select the optimal number of principal components using a permutation procedure. We showed that oCEM outperformed state-of-the-art techniques in the ability to detect biologically relevant modules additionally.

ConclusionsoCEM helped non-technical users easily perform complicated statistical analyses and then gain robust results. oCEM and its applications, along with example data, were freely provided at https://github.com/huynguyen250896/oCEM.

sentences: Spinal cord synaptic plasticity by GlyRβ release from receptor fields and syndapin-dependent uptake Glycine receptor-mediated inhibitory neurotransmission is key for spinal cord function. Recent observations suggested that by largely elusive mechanisms also glycinergic synapses display synaptic plasticity. We here identify syndapin I as critical player. Interestingly, syndapin I cooperates but in part also competes with gephyrin. Syndapin I deficiency led to fragmentation of glycine receptor fields, more disperse receptors and increased receptor mobility. Kainate treatment highlighted syndapin Is importance even more. Our analyses unveiled that PKC-mediated S403 phosphorylation-mediated glycine receptor {beta} decoupling from gephyrin scaffolds simultaneously promoted syndapin I association. In line, kainate-treated syndapin I KO spinal cords showed even more severe receptor field fragmentation. Furthermore, syndapin I deficiency completely disrupted kainate-induced glycine receptor internalization. Together, this unveiled important mechanisms controlling the number and organization of glycine receptor fields at inhibitory postsynapses during both steady-state and kainate-induced synaptic rearrangement - principles organizing and fine-tuning synaptic efficacy of inhibitory synapses in the spinal cord.

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Pareto-optimal trade-off for phenotypic switching of populations in a stochastic environment Finding optimal survival strategies of living systems embedded in fluctuating environments generally involves a balance between phenotypic diversification and sensing. If we neglect sensing mechanisms, it is known that slow, resp. fast, environmental transitions favor a regime of heterogeneous, resp. homogeneous, phenotypic response.

We focus here on the simplest non-trivial case, i.e. two randomly switching phenotypes subjected to two stochastically switching environments. The optimal asymptotic (long term) growth rate of this model was studied elsewhere; we further expand these results by discussing finite time growth rate fluctuations. An exact asymptotic expression for the variance, alongside with approximations valid in different regimes, are tested numerically in details. Our simulations of the dynamics suggest a close connection between this variance and the extinction probability, understood as risk for the population. Motivated by an earlier trade-off analysis between average capital growth rate and risk in Kellys gambling model, we study the trade-off between the average growth rate and the variance in the present model. Despite considerable differences between the two models, we find similar optimal trade-off curves (Pareto fronts), suggesting that our conclusions are robust, and broadly applicable in various fields ranging from biology/ecology to economics.

sentences: Ca2+ Mediates HIF-dependent Upregulation of Aquaporin 1 in Pulmonary Arterial Smooth Muscle Cells Prolonged exposure to hypoxia causes structural remodeling and sustained contraction of the pulmonary vasculature, resulting in the development of pulmonary hypertension. Both pulmonary arterial smooth muscle cell (PASMC) proliferation and migration contribute to the vascular remodeling. We previously showed that the protein expression of aquaporin 1 (AQP1), a membrane water channel protein, is elevated in PASMCs during following in vivo or in vitro exposure to hypoxia. Studies in other cell types suggest that AQP1 is a direct transcriptional target of hypoxia inducible factor (HIF)-1. Moreover, we and others have shown that an increase in intracellular calcium concentration ([Ca2+]i) is a hallmark of hypoxic exposure in PASMCs. Thus, we wanted to determine whether HIF regulates AQP1 in PASMCs and, if so, whether the process occurred via transcriptional regulation or was Ca2+-dependent. PASMCs were exposed to hypoxia, incubated with DMOG, which inhibits HIF protein degradation or infected with constitutively active forms of HIF-1 or HIF-2. Hypoxia, DMOG and HIF1/2 produced a time-dependent increase in AQP1 protein, but not mRNA. Interestingly, incubation with increasing HIF1/2 levels and DMOG increased [Ca2+]i in PASMCs, and this elevation was prevented by the voltage-gated Ca2+ channel inhibitor, verapamil (VER) and nonselective cation channel inhibitor SKF96365 (SKF). VER and SKF also blocked upregulation of AQP1 protein by DMOG or HIF1/2, but had no effect on expression of GLUT1, a canonical HIF transcriptional target. Silencing of AQP1 abrogated increases in PASMC migration and proliferation induced by HIF1/2, suggesting induction of AQP1 protein by HIF1/2 has a functional outcome in these cells. Thus, our results show that contrary to reports in other cell types, in PASMCs, AQP1 does not appear to be a direct target for HIF transcriptional regulation. Instead, AQP1 protein may be upregulated by a mechanism involving HIF-dependent increases in [Ca2+]i.

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Comparing protein-protein interaction networks of SARS-CoV-2 and (H1N1) influenza using topological features BackgroundSARS-CoV-2 pandemic first emerged in late 2019 in China. It has since infected more than 183 million individuals and caused about 4 million deaths globally. A protein-protein interaction network (PPIN) and its analysis can provide insight into the behavior of cells and lead to advance the procedure of drug discovery. The identification of essential proteins is crucial to understand for cellular survival. There are many centrality measures to detect influential nodes in complex networks. Since SARS-CoV-2 and (H1N1) influenza PPINs pose 553 common proteins. Analyzing influential proteins and comparing these networks together can be an effective step helping biologists in drug design.

ResultsWe used 21 centrality measures on SARS-CoV-2 and (H1N1) influenza PPINs to identify essential proteins. PCA-based dimensionality reduction was applied on normalized centrality values. Some measures demonstrated a high level of contribution in comparison to others in both PPINs, like Barycenter, Decay, Diffusion degree, Closeness (Freeman), Closeness (Latora), Lin, Radiality, and Residual. Using validation measures, the appropriate clustering method was chosen for centrality measures. We also investigated some graph theory-based properties like the power law, exponential distribution, and robustness.

ConclusionsThrough analysis and comparison, both networks exhibited remarkable experimental results. The network diameters were equal and in terms of heterogeneity, SARS-CoV-2 PPIN tends to be more heterogeneous. Both networks under study display a typical power-law degree distribution. Dimensionality reduction and unsupervised learning methods were so effective to reveal appropriate centrality measures.

sentences: Genetic architecture of dispersal and local adaptation drives accelerating range expansions Contemporary evolution has the potential to significantly alter biotic responses to global change, including range expansion dynamics and biological invasions. However, predictive models often make highly simplifying assumptions about the genetic architecture underlying relevant traits. This can be problematic since genetic architecture defines evolvability, that is, evolutionary rates, and higher order evolutionary processes, which determine whether evolution will be able to keep up with environmental change or not. Therefore, we here study the impact of the genetic architecture of dispersal and local adaptation, two central traits of high relevance for range expansion dynamics, on the speed and variability of range expansions into an environmental gradient, such as temperature. In our theoretical model we assume that dispersal and local adaptation traits result from the products of two non-interacting gene-regulatory networks (GRNs). We compare our model to simpler quantitative genetics models and show that in the GRN model, range expansions are accelerated, faster and more variable. Increased variability implies that these evolutionary changes reduce predictability. We further find that acceleration in the GRN model is primarily driven by an increase in the rate of local adaptation to novel habitats which results from greater sensitivity to mutation (decreased robustness) and increased gene expression. Our results highlight how processes at microscopic scales, here, within genomes, can impact the predictions of large scale, macroscopic phenomena, such as range expansions, by modulating the rate of evolution.

sentences: In-silico identification of Tyr232 in AMPKα2 as a dephosphorylation site for the protein tyrosine phosphatase PTP-PEST The AMP-activated protein kinase (AMPK) is known to be activated by the protein tyrosine phosphatase non-receptor type 12 (PTP-PEST) under hypoxic conditions. This activation is mediated by tyrosine dephosphorylation of the AMPK subunit. However, the identity of the phosphotyrosine residues remains unknown. In this study we first predicted the structure of the complex of the AMPK2 subunit and PTP-PEST catalytic domain using bioinformatics tools and further confirm the stability of the complex using molecular dynamics simulations. Evaluation of the protein-protein interfaces indicates that residue Tyr232 is the most likely site of dephosphorylation on AMPK2. In addition, we explored the effect of phosphorylation of PTP-PEST residue Tyr64 on the stability of the complex. The phosphorylation of Tyr64, an interface residue, enhances the stability of the complex via the rearrangement of a network of electrostatic interactions in conjunction with conformational changes in the catalytic WPD loop. Our findings present a plausible structural basis of AMPK regulation mediated by PTP-PEST and shows how phosphorylation of PTP-PEST could be involved in its activation.

sentences: Rapid nuclear deadenylation of mammalian messenger RNA Poly(A) tails protect RNAs from degradation and their deadenylation rates determine RNA stability. Although poly(A) tails are generated in the nucleus, deadenylation of tails has mostly been investigated within the cytoplasm. Here, we combined long-read sequencing with metabolic labeling, splicing inhibition, and cell fractionation experiments to quantify, separately, the genesis and trimming of nuclear and cytoplasmic tails in vitro and in vivo. We present evidence for genome-wide, nuclear synthesis of tails longer than 200 nt, which are rapidly shortened within minutes after transcription. Our data show that rapid deadenylation is a nuclear process, and that different classes of transcripts and even transcript isoforms have distinct nuclear tail lengths. For example, many long-noncoding RNAs escape rapid nuclear deadenylation. Modelling deadenylation dynamics predicts nuclear deadenylation about 10 times faster than cytoplasmic deadenylation. In summary, our data suggest that nuclear deadenylation is a key mechanism for regulating mRNA stability, abundance, and subcellular localization.

sentences: R7 photoreceptor axon targeting requires matching levels of lost and found expression in R7 and its synaptic partners As neural circuits form, growing processes select the correct synaptic partners through interactions between cell surface proteins. The presence of such proteins on two neuronal processes may lead to either adhesion or repulsion; however, the consequences of mismatched expression have rarely been explored. Here we show that the Drosophila CUB-LDL protein Lost and found (Loaf) is required in the UV-sensitive R7 photoreceptor for normal axon targeting only when Loaf is also present in its synaptic partners. Although targeting occurs normally in loaf mutant animals, removing loaf from photoreceptors or expressing it in their postsynaptic neurons Tm5a/b or Dm9 in a loaf mutant causes mistargeting of R7 axons. Loaf localizes primarily to intracellular vesicles including endosomes. We propose that Loaf regulates the trafficking or function of one or more cell surface proteins, and an excess of these proteins on the synaptic partners of R7 prevents the formation of stable connections.

sentences: Membrane-assisted assembly and selective autophagy of enteroviruses Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. It is unknown how virions assemble around these newly synthesized genomes and how they are then loaded into autophagic membranes for release through secretory autophagy. Here, we use cryo-electron tomography of infected cells to show that poliovirus assembles directly on replication membranes. Pharmacological untethering of capsids from membranes abrogates RNA encapsidation. Our data directly visualize a membrane-bound half-capsid as a prominent virion assembly intermediate. Assembly progression past this intermediate depends on the class III phosphatidylinositol 3-kinase VPS34, a key host-cell autophagy factor. On the other hand, the canonical autophagy initiator ULK1 is shown to restrict virion production since its inhibition leads to increased accumulation of virions in vast intracellular arrays, followed by an increased vesicular release at later time points. Finally, we identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from other types of autophagosome contents. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle.

sentences: Compositional analysis of ALS-linked stress granule-like structures reveals factors and cellular pathways dysregulated by mutant FUS under stress Formation of cytoplasmic RNA-protein structures called stress granules (SGs) is a highly conserved cellular response to stress. Abnormal metabolism of SGs may contribute to the pathogenesis of (neuro)degenerative diseases such as amyotrophic lateral sclerosis (ALS). Many SG proteins are affected by mutations causative of these conditions, including fused in sarcoma (FUS). Mutant FUS variants have high affinity to SGs and also spontaneously form de novo cytoplasmic RNA granules. Mutant FUS-containing assemblies (mFAs), often called "pathological SGs", are proposed to play a role in ALS-FUS pathogenesis. However, global structural differences between mFAs and physiological SGs remain largely unknown, therefore it is unclear whether and how mFAs may affect cellular stress responses. Here we used affinity purification to characterise the protein and RNA composition of normal SGs and mFAs purified from stressed cells. Comparison of the SG and mFA proteomes revealed that proteasome subunits and certain nucleocytoplasmic transport factors are depleted from mFAs, whereas translation elongation, mRNA surveillance and splicing factors as well as mitochondrial proteins are enriched in mFAs, as compared to SGs. Validation experiments for a hit from our analysis, a splicing factor hnRNPA3, confirmed its RNA-dependent sequestration into mFAs in cells and into pathological FUS inclusions in a FUS transgenic mouse model. Furthermore, silencing of the Drosophila hnRNPA3 ortholog dramatically enhanced FUS toxicity in transgenic flies. Comparative transcriptomic analysis of SGs and mFAs revealed that mFAs recruit a significantly less diverse spectrum of RNAs, including reduced recruitment of transcripts encoding proteins involved in protein translation, DNA damage response, and apoptotic signalling. However mFAs abnormally sequester certain mRNAs encoding proteins involved in stress signalling cascades. Overall, our study establishes molecular differences between physiological SGs and mFAs and identifies the spectrum of proteins, RNAs and respective cellular pathways affected by mFAs in stressed cells. In conclusion, we show that mFAs are compositionally distinct from SGs and that they cannot fully substitute for SG functions while gaining novel, potentially toxic functions in cellular stress response. Results of our study support a pathogenic role for stress-induced cytoplasmic FUS assemblies in ALS-FUS.

sentences: Gene networks under circadian control exhibit diurnal organization in primate organs Mammalian organs are individually controlled by autonomous circadian clocks. At the molecular level, this process is defined by the cyclical co-expression of both core transcription factors and off-target genes across time. While interactions between these molecular clocks are likely necessary for proper homeostasis, these features remain undefined. Here, we utilize integrative analysis of a baboon diurnal transcriptome atlas to characterize the properties of gene networks under circadian control. We found that 53.4% (8,120) of baboon genes are rhythmically expressed body-wide. In addition, >30% of gene-gene interactions exhibit periodic co-expression patterns, with core circadian genes more cyclically co-expressed than others. Moreover, two basic network modes were observed at the systems level: daytime and nighttime mode. Daytime networks were enriched for genes involved in metabolism, while nighttime networks were enriched for genes associated with growth and cellular signaling. A substantial number of diseases only form significant disease modules at either daytime or nighttime. In addition, we found that 216 of 313 genes encoding products that interact with SARS-CoV-2 are rhythmically expressed throughout the body. Importantly, more than 80% of SARS-CoV-2 related genes enriched modules are rhythmically expressed, and have significant network proximities with circadian regulators. Our data suggest that synchronization amongst circadian gene networks is necessary for proper homeostatic functions and circadian regulators have close interactions with SARS-CoV-2 infection.

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Bacterial Cytological Profiling Identifies Rhodanine-containing PAINS Analogs as Specific Inhibitors of E. coli Thymidylate Kinase in Vivo In this study, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules inhibiting the growth of E. coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite their structural similarity to related PAINS. A genetic analysis of resistant mutants revealed that thymidylate kinase (an enzyme essential for DNA synthesis) is an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by assays of in vitro activity as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the lowest IC50 in vitro and MIC in vivo displayed the greatest specificity for the inhibition of DNA replication in E. coli, despite containing a rhodamine moiety. While its generally thought that PAINS cannot be developed as antibiotics, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics, and it showcases novel inhibitors of E. coli thymidylate kinase.

ImportanceWe demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotics in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and non-specific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.

sentences: The effects of antibiotic combination treatments on Pseudomonas aeruginosa tolerance evolution and coexistence with Stenotrophomonas maltophilia Pseudomonas aeruginosa bacterium is a common pathogen of Cystic Fibrosis (CF) patients due to its ability to evolve resistance to antibiotics during treatments. While P. aeruginosa resistance evolution is well characterised in monocultures, it is less well understood in polymicrobial CF infections. Here, we investigated how exposure to ciprofloxacin, colistin, or tobramycin antibiotics, administered at sub-MIC doses alone and in combination, shaped the tolerance evolution of P. aeruginosa (PAO1 lab and clinical CF LESB58 strains) in the absence and presence of a commonly co-occurring species, Stenotrophomonas maltophilia. Increases in antibiotic tolerances were primarily driven by the presence of that antibiotic in the treatment. We observed a reciprocal cross-tolerance between ciprofloxacin and tobramycin, and when combined these antibiotics selected increased MICs for all antibiotics. Though the presence of S. maltophilia did not affect the tolerance or the MIC evolution, it drove P. aeruginosa into extinction more frequently in the presence of tobramycin due to its relatively greater innate tobramycin tolerance. In contrast, P. aeruginosa dominated and drove S. maltophilia extinct in most other treatments. Together, our findings suggest that besides driving high-level antibiotic tolerance evolution, sub-MIC antibiotic exposure can alter competitive bacterial interactions, leading to target pathogen extinctions in multi-species communities.

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A critical role for E2-p53 interaction during the human papillomavirus 16 life cycle Human papillomaviruses (HPV) are causative agents in ano-genital and oral cancers; HPV16 is the most prevalent type detected in human cancers. The HPV16 E6 protein targets p53 for proteasomal degradation to facilitate proliferation of the HPV16 infected cell. However, in HPV16 immortalized cells E6 is predominantly spliced (E6*) and unable to degrade p53. Here we demonstrate that human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16), and HPV16 positive oropharyngeal cancers, retain significant expression of p53. In addition, p53 levels can be increased in HPV16+ head and neck cancer cell lines following treatment with cisplatin. Introduction of full-length E6 into HFK+HPV16 resulted in attenuation of cellular growth (in hTERT immortalized HFK, E6 expression promoted enhanced proliferation). An understudied interaction is that between E2 and p53 and we investigated whether this was important for the viral life cycle. We generated mutant genomes with E2 unable to interact with p53 resulting in profound phenotypes in primary HFK. The mutant induced hyper-proliferation, but an ultimate arrest of cell growth; {beta}-galactosidase staining demonstrated increased senescence, and COMET assays showed increased DNA damage compared with HFK+HPV16 wild type cells. There was failure of the viral life cycle in organotypic rafts with the mutant HFK resulting in premature differentiation and reduced proliferation. The results indicate that the E2-p53 interaction is critical during the HPV16 life cycle, and that disruption of this interaction has anti-viral potential. We discuss potential mechanisms to explain these phenotypes.

ImportanceHuman papillomaviruses are causative agents in around 5% of all cancers. There are currently no antivirals available to combat these infections and cancers, therefore it remains a priority to enhance our understanding of the HPV life cycle. Here we demonstrate that an interaction between the viral replication/transcription/segregation factor E2 and the tumor suppressor p53 is critical for the HPV16 life cycle. HPV16 immortalized cells retain significant expression of p53, and the critical role for the E2-p53 interaction demonstrates why this is the case. If the E2-p53 interaction is disrupted then HPV16 immortalized cells fail to proliferate, have enhanced DNA damage and senescence, and there is premature differentiation during the viral life cycle. Results suggest that targeting the E2-p53 interaction would have therapeutic benefits, potentially attenuating the spread of HPV16.

sentences: Revision and annotation of DNA barcode records for marine invertebrates: report of the 8th iBOL conference hackathon The accuracy of the identification of unknown specimens using DNA barcoding and metabarcoding relies on reference libraries containing records with reliable taxonomy and sequence quality. A rampant growth in barcode data led to a stringent need for data curation, especially in taxonomically difficult groups such as marine invertebrates. A major effort in curating marine barcode data deposited in the Barcode of Life Data Systems (BOLD) has been undertaken during the 8th International Barcode of Life Conference (Trondheim, Norway, 2019). For practical reasons, only major taxonomic groups were reviewed and annotated (crustaceans, echinoderms, molluscs, and polychaetes). The congruence of Linnean names with Barcode Index Numbers (BINs) was investigated, and the records deemed uncertain were annotated with four tags: a) MIS-ID (misidentified, mislabeled or contaminated records), b) AMBIG (ambiguous records unresolved with the current data), c) COMPLEX (species occurring in multiple BINs), and d) SHARE (barcodes shared between species). A total of 83,712 specimen records corresponding to 7,576 species were reviewed and 39% of the species were tagged (7% MIS-ID, 17% AMBIG, 14% COMPLEX, and 1% SHARE). High percentages (>50%) of AMBIG tags were recorded in gastropods, whereas COMPLEX tags dominated in crustaceans and polychaetes. This high proportion of tagged species reflects either flaws in the barcoding workflow (e.g., misidentification, cross -contamination) or taxonomic difficulties (e.g., synonyms, undescribed species). Although data curation is crucial for barcode applications, such manual efforts of reviewing large datasets are not sustainable and the implementation of automated solutions to the furthest possible extent is hi ghly desirable.

sentences: Antagonistic modules, SIB1 and LSD1, regulate photosynthesis-associated nuclear genes via GOLDEN2-LIKE transcription factors in Arabidopsis GOLDEN2-LIKE (GLK) transcription factors drive the expression of photosynthesis-associated nuclear genes (PhANGs), indispensable for chloroplast biogenesis. We previously demonstrated that the salicylic acid (SA)-induced SIGMA FACTOR-BINDING PROTEIN 1 (SIB1), a transcription coregulator and positive regulator of cell death, interacts with GLK1 and GLK2 to reinforce their activities. The SIB1-GLK interaction raises the level of light-harvesting antenna proteins in photosystem II, aggravating photoinhibition and singlet oxygen (1O2) burst. 1O2 then contributes to SA-induced cell death via EXECUTER 1 (EX1, 1O2 sensor protein)-mediated retrograde signaling upon reaching a critical level. We now reveal that LESION-SIMULATING DISEASE 1 (LSD1), a transcription coregulator and negative regulator of SA-primed cell death, interacts with GLK1/2 to repress their activities. Consistently, the overexpression of LSD1 represses GLK target genes including PhANGs, whereas the loss of LSD1 increases their expression. Remarkably, LSD1 overexpression inhibits chloroplast biogenesis, resembling the characteristic glk1glk2 double mutant phenotype. The subsequent chromatin immunoprecipitation analysis coupled with quantitative PCR further reveals that LSD1 inhibits the DNA-binding activity of GLK1 towards its target promoters. The SA-induced nuclear-targeted SIB1 appears to counteractively interact with GLK1/2, leading to the activation of EX1-mediated 1O2 signaling. Taken together, we provide a working model that SIB1 and LSD1, mutually exclusive SA-signaling components, antagonistically regulate GLK1/2 to fine-tune the expression of PhANGs, thereby modulating 1O2 homeostasis and related stress responses.

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Estimating Ectopic Beat Probability with Simplified Statistical Models that Account for Experimental Uncertainty Ectopic beats (EBs) are cellular arrhythmias that can trigger lethal arrhythmias. Simulations using biophysically-detailed cardiac myocyte models can reveal how model parameters influence the probability of these cellular arrhythmias, however such analyses can pose a huge computational burden. Here, we develop a simplified approach in which logistic regression models (LRMs) are used to define a mapping between the parameters of complex cell models and the probability of EBs (P(EB)). As an example, in this study, we build an LRM for P(EB) as a function of diastolic cytosolic Ca2+ concentration ([Ca2+]i), sarcoplasmic reticulum (SR) Ca2+ load, and kinetic parameters of the inward rectifier K+ current (IK1) and ryanodine receptor (RyR). This approach, which we refer to as arrhythmia sensitivity analysis, allows for evaluation of the relationship between these arrhythmic event probabilities and their associated parameters. This LRM is also used to demonstrate how uncertainties in experimentally measured values determine the uncertainty in P(EB). In a study of the role of [Ca2+]SR uncertainty, we show a special property of the uncertainty in P(EB), where with increasing [Ca2+]SR uncertainty, P(EB) uncertainty first increases and then decreases. Lastly, we demonstrate that IK1 suppression, at the level that occurs in heart failure myocytes, increases P(EB).

Author summaryAn ectopic beat is an abnormal cellular electrical event which can trigger dangerous arrhythmias in the heart. Complex biophysical models of the cardiac myocyte can be used to reveal how cell properties affect the probability of ectopic beats. However, such analyses can pose a huge computational burden. We develop a simplified approach that enables a highly complex biophysical model to be reduced to a rather simple statistical model from which the functional relationship between myocyte model parameters and the probability of an ectopic beat is determined. We refer to this approach as arrhythmia sensitivity analysis. Given the efficiency of our approach, we also use it to demonstrate how uncertainties in experimentally measured myocyte model parameters determine the uncertainty in ectopic beat probability. We find that, with increasing model parameter uncertainty, the uncertainty in probability of ectopic beat first increases and then decreases. In general, our approach can efficiently analyze the relationship between cardiac myocyte parameters and the probability of ectopic beats and can be used to study how uncertainty of these cardiac myocyte parameters influences the ectopic beat probability.

sentences: Design of an Integrated Microvascularised Human Skin-on-a-Chip Tissue Equivalent Model Tissue engineered skin constructs have been under development since the 1980s as a replacement for human skin tissues and animal models for therapeutics and cosmetic testing. These have evolved from simple single cell-assays to increasingly complex models with integrated dermal equivalents and multiple cell types including a dermis, epidermis and vasculature. The development of micro-engineered platforms and biomaterials has enabled scientists to better recreate and capture the tissue microenvironment in vitro, including the vascularization of tissue models and their integration into microfluidic chips. However, to date, microvascularised human skin equivalents in a microfluidic context have not been reported. Here we present the design of a novel skin-on-a-chip model integrating human derived primary and immortalized cells in a full thickness skin equivalent. The model is housed in a microfluidic device, in which a microvasculature was previously established. We characterize the impact of our chip design on the quality of the microvascular networks formed and evidence that this enables the formation of more homogenous networks. We developed a methodology to harvest tissues from embedded chips, after 14 days of culture, and characterize the impact of culture conditions and vascularization (including with pericyte co-cultures) on the stratification of the epidermis in the resulting skin equivalents. Our results indicate that vascularization enhances stratification and differentiation (thickness, architecture and expression of terminal differentiation markers such as involucrin and transglutaminase 1), allowing formation of more mature skin equivalents in microfluidic chips. The skin-on-a-chip tissue equivalents developed, thanks to their realistic microvasculature, may find application for the testing efficacy and safety of therapeutics delivered systemically, in a human context.

sentences: Interaural Time Difference Tuning in the Rat Inferior Colliculus is Predictive of Behavioral Sensitivity Recent studies have shown that rats are a useful model for binaural cochlear implant (CI) research, with behavioral sensitivity to interaural time differences (ITDs) of CI stimuli which are better than those of human patients. Here, we characterize ITD tuning in the rat inferior colliculus (IC) and explore whether quality of tuning can predict behavioral performance. We recorded IC responses to stimuli of varying pulse rates and envelope types and quantified both mutual information (MI) and neural d' as measures of ITD sensitivity. Neural d' values paralleled behavioral ones, declining with increasing click rates or when envelopes changed from rectangular to Hanning windows. While MI values increased with experience, neural d' did not. However, neural d' values correlated much better with behavioral performance than MI. Thus, neural d' appears to be a particularly well suited to predicting how stimulus parameters will impact behavioral performance.

sentences: ZCCHC8 is required for the degradation of pervasive transcripts originating from multiple genomic regulatory features The vast majority of mammalian genomes are transcribed as non-coding RNA in what is referred to as "pervasive transcription." Recent studies have uncovered various families of non-coding RNA transcribed upstream of transcription start sites. In particular, highly unstable promoter upstream transcripts known as PROMPTs have been shown to be targeted for exosomal degradation by the nuclear exosome targeting complex (NEXT) consisting of the RNA helicase MTR4, the zinc-knuckle scaffold ZCCHC8, and the RNA binding protein RBM7. Here, we report that in addition to its known RNA substrates, ZCCHC8 is required for the targeted degradation of pervasive transcripts produced at CTCF binding sites, open chromatin regions, promoters, promoter flanking regions, and transcription factor binding sites. Additionally, we report that a significant number of RIKEN cDNAs and predicted genes display the hallmarks of PROMPTs and are also substrates for ZCCHC8 and/or NEXT complex regulation suggesting these are unlikely to be functional genes. Our results suggest that ZCCHC8 and/or the NEXT complex may play a larger role in the global regulation of pervasive transcription than previously reported.

sentences: Both consumptive and non-consumptive effects of predators impact mosquito populations and have implications for disease transmission Predator-prey interactions influence prey traits through both consumptive and non-consumptive effects, and variation in these traits can shape vector-borne disease dynamics. Meta-analysis methods were employed to generate predation effect sizes by different categories of predators and mosquito prey. This analysis showed that multiple families of aquatic predators are effective in consumptively reducing mosquito survival, and that the survival of Aedes, Anopheles, and Culex mosquitoes is negatively impacted by consumptive effects of predators. Mosquito larval size was found to play a more important role in explaining the heterogeneity of consumptive effects from predators than mosquito genus. Mosquito survival and body size were reduced by non-consumptive effects of predators, but development time was not significantly impacted. In addition, Culex vectors demonstrated predator avoidance behavior during oviposition. The results of this meta-analysis suggest that predators limit disease transmission by reducing both vector survival and vector size, and that associations between drought and human West Nile virus cases could be driven by the vector behavior of predator avoidance during oviposition. These findings are likely to be useful to infectious disease modelers who rely on vector traits as predictors of transmission.

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Glacier-fed stream biofilms harbour diverse resistomes and biosynthetic gene clusters BackgroundAntimicrobial resistance (AMR) is a universal phenomenon whose origins lay in natural ecological interactions such as competition within niches, within and between micro- to higher-order organisms. However, the ecological and evolutionary processes shaping AMR need to be better understood in view of better antimicrobial stewardship. Resolving antibiotic biosynthetic pathways, including biosynthetic gene clusters (BGCs), and corresponding antimicrobial resistance genes (ARGs) may therefore help in understanding the inherent mechanisms. However, to study these phenomena, it is crucial to examine the origins of AMR in pristine environments with limited anthropogenic influences. In this context, epilithic biofilms residing in glacier-fed streams (GFSs) are an excellent model system to study diverse, intra- and inter-domain, ecological crosstalk.

ResultsWe assessed the resistomes of epilithic biofilms from GFSs across the Southern Alps (New Zealand) and the Caucasus (Russia) and observed that both bacteria and eukaryotes encoded twenty-nine distinct AMR categories. Of these, beta-lactam, aminoglycoside, and multidrug resistance were both abundant and taxonomically distributed in most of the bacterial and eukaryotic phyla. AMR-encoding phyla included Bacteroidota and Proteobacteria among the bacteria, alongside Ochrophyta (algae) among the eukaryotes. Additionally, BGCs involved in the production of antibacterial compounds were identified across all phyla in the epilithic biofilms. Furthermore, we found that several bacterial genera (Flavobacterium, Polaromonas, etc.) including representatives of the superphylum Patescibacteria encode both ARGs and BGCs within close proximity of each other, thereby demonstrating their capacity to simultaneously influence and compete within the microbial community.

ConclusionsOur findings highlight the presence and abundance of AMR in epilithic biofilms within GFSs. Additionally, we identify their role in the complex intra- and inter-domain competition and the underlying mechanisms influencing microbial survival in GFS epilithic biofilms. We demonstrate that eukaryotes may serve as AMR reservoirs owing to their potential for encoding ARGs. We also find that the taxonomic affiliation of the AMR and the BGCs are congruent. Importantly, our findings allow for understanding how naturally occurring BGCs and AMR contribute to the epilithic biofilms mode of life in GFSs. Importantly, these observations may be generalizable and potentially extended to other environments which may be more or less impacted by human activity.

sentences: Cardiac ventricular myosin and slow skeletal myosin exhibit dissimilar chemo-mechanical properties despite the same myosin heavy chain isoform. The myosin II motors are ATP-powered, force-generating machines driving cardiac and muscle contraction. Myosin II heavy chain isoform-beta ({beta}-MyHC) is primarily expressed in the ventricular myocardium and slow-twitch muscle fibers, such as in M. soleus. M. soleus-derived myosin II (SolM-II) is often used as an alternative to the ventricular {beta}-cardiac myosin ({beta}M-II); however, the direct assessment of detailed biochemical and mechanical features of the native myosins is limited. By employing the optical trapping method, we examined the mechanochemical properties of the native myosins isolated from rabbit heart ventricle and M. soleus muscles at the single-molecule level. Contrary to previous reports, the purified motors from the two tissue sources, despite the same MyHC isoform, displayed distinct motile and ATPase kinetic properties. {beta}M-II was [~]threefold faster in the actin filament-gliding assay than SolM-II. The maximum acto-myosin (AM) detachment rate derived in single-molecule assays was [~]threefold higher in {beta}M-II. The stroke size for both myosins was comparable. The stiffness of the  AM rigor cross-bridge was also similar for both the motor forms. The stiffness of {beta}M-II was found to be determined by the nucleotide state of the actin-bound myosin. Our analysis revealed distinct kinetic differences, i.e., a higher AM detachment rate for the {beta}M-II, corresponding to the ADP release rates from the cross-bridge, thus elucidating the observed differences in the motility driven by {beta}M-II and SolM-II. These studies have important implications for the future choice of tissue sources to gain insights into cardiomyopathies

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Compartmentalized Cell Envelope Biosynthesis in Mycobacterium tuberculosis The intracellular membrane domain (IMD) is a metabolically active and laterally discrete membrane domain initially discovered in Mycobacterium smegmatis. The IMD correlates both temporally and spatially with the polar cell envelope elongation in M. smegmatis. Whether or not a similar membrane domain exists in pathogenic species remains unknown. Here we show that the IMD is a conserved membrane structure found in Mycobacterium tuberculosis. We used two independent approaches, density gradient fractionation of membrane domains and visualization of IMD-associated proteins through fluorescence microscopy, to determine the characteristics of the plasma membrane compartmentalization in M. tuberculosis. Proteomic analysis revealed that the IMD is enriched in metabolic enzymes that are involved in the synthesis of conserved cell envelope components such as peptidoglycan, arabinogalactan, and phosphatidylinositol mannosides. Using a fluorescent protein fusion of IMD-associated proteins, we demonstrated that this domain is concentrated in the polar region of the rod-shaped cells, where active cell envelope biosynthesis is taking place. Proteomic analysis further revealed the enrichment of enzymes involved in synthesis of phthiocerol dimycocerosates and phenolic glycolipids in the IMD. We validated the IMD association of two enzymes, 1,3-fucosyltransferase and fucosyl 4- O-methyltransferase, which are involved in the final maturation steps of phenolic glycolipid biosynthesis. Taken together, these data indicate that functional compartmentalization of membrane is an evolutionarily conserved feature found in both M. tuberculosis and M. smegmatis, and M. tuberculosis utilizes this membrane location for the synthesis of its surface- exposed lipid virulence factors.

IMPORTANCEM. tuberculosis remains an important public health threat, with more than one million deaths every year. The pathogens ability to survive in the human host for decades highlights the importance of understanding how this bacterium regulates and coordinates its metabolism, cell envelope elongation, and growth. The IMD is a membrane structure that associates with the subpolar growth zone of actively growing mycobacteria, but its existence is only known in a non- pathogenic model, M. smegmatis. Here, we demonstrated the presence of the IMD in M. tuberculosis, making the IMD an evolutionarily conserved plasma membrane compartment in mycobacteria. Furthermore, our study revealed that the IMD is the factory for synthesizing phenolic glycolipids, virulence factors produced by slow-growing pathogenic species.

sentences: Craniofacial cartilage organoids from human embryonic stem cells via a neural crest cell intermediate Severe birth defects or major injuries to the face require surgical reconstruction and rehabilitation. The ability to make bona fide craniofacial cartilage - cartilage of the head and face - from patient-derived induced pluripotent stem cells (iPSCs) to repair these birth defects and injuries has tremendous translational applications, but is not yet possible. The neural crest is the normal developmental pathway for craniofacial cartilage, however, the knowledge of cell signaling pathways that drive neural crest differentiation into craniofacial chondrocytes is limited. Here we describe a differentiation protocol that generated self-organizing craniofacial cartilage organoids from human embryonic stem cells (hESCs) and IPSCs through a neural crest stem cell (NCSC) intermediate. Histological staining of cartilage organoids revealed tissue architecture typical of hyaline cartilage. Organoids were composed of rounded aggregates of glassy, gray matrix that contained scattered small nuclei in lacunae. Mass spectrometry shows that the organoids express robust levels of cartilage markers including aggrecan, perlecan, proteoglycans, and many collagens. Organoids expressed markers indicative of neural crest lineage, as well as growth factors that are candidates for chondrocyte differentiation factors. The data suggest that chondrocyte differentiation is initiated by autocrine loops driven by a combination of secreted growth factors that bind to chondrocyte receptors. Craniofacial cartilage organoids were continuously cultured for one year, reaching up to one centimeter in diameter. The ability to grow craniofacial cartilage from NCSCs provides insights into the cell signaling mechanisms of differentiation into craniofacial cartilage, which lays the groundwork for understanding mechanistic origins of congenital craniofacial anomalies and repairing cartilaginous structures of the head and face.

sentences: Genetic tools for the stable overexpression of circular RNAs Circular RNAs (circRNAs) are a class of non-coding RNAs that feature a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study gene functions have significant limitations when studying circRNAs. Overexpression studies in particular suffer from the lack of efficient genetic tools. While mammalian expression plasmids enable transient overexpression of circRNAs in cultured cells, most cell biological studies require long-term ectopic expression. Here we report the development and characterization of genetic tools enabling stable circRNA overexpression in vitro and in vivo. We demonstrated that circRNA expression constructs can be delivered to cultured cells via transposons, whereas lentiviral vectors have limited utility for the delivery of circRNA constructs. We further showed that circRNA transposons can be supplied to mouse livers via hydrodynamic tail vein injection, resulting in ectopic circRNA expression in a hepatocellular carcinoma mouse model. Furthermore, we generated genetically engineered mice harboring circRNA expression constructs. We demonstrate that this approach enables constitutive, global circRNA overexpression as well as inducible circRNA expression directed specifically to melanocytes in a melanoma mouse model. Overall, these tools expand the genetic toolkit available for the functional characterization of circRNAs of interest.

sentences: Tree canopy influences ground level atmospheric electrical and biogeochemical variability Static electric fields in the atmosphere are increasingly recognized to interact with various organisms over several levels of biological organization. Recently, a link between atmospheric electrical variations and biogeochemical processes has been established in the context of open fields, yet biological structures like trees produce substantial alterations in atmospheric electric properties. Here, we assess whether these structural changes affect the dynamics of both biogenic and abiotic electrical landscapes and their relation to geochemical processes. To this end, we theoretically assess how trees alter their surrounding electric fields and empirically compare the temporal dynamics of atmospheric potential gradients, positive ions in the near-ground level atmosphere and soil electrochemical properties in an open field and under a tree. The developed model of electric fields around trees provides insight into the extent to which trees shield underlying electric landscape, revealing that a substantial increase in atmospheric potential gradient only marginally affects the electric field under the canopy. We further show that soil electrochemical properties are tied to temporal dynamics of positive ions in the near-ground level atmosphere, and that the presence of a tree reduces the temporal variability in both ground level positive ions concentrations and soil redox potential. This suggests that a tree can have a stabilizing effect on drivers of temporal variability in atmospheric electricity and soil electro-chemistry, thereby likely indirectly influencing soil microorganisms and processes as well as electro-sensitive organisms that perceive and utilize atmospheric electric fields.

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Local Metabolism of Ethanol to Acetic Acid/Acetate in the Central Nucleus of Amygdala Elicits Sympathoexcitatory Responses through Activation of NMDAR in Sprague Dawley Rats AimBinge alcohol consumption elicits robust sympathoexcitation and excitatory neuronal output. However, the central mechanism that mediates these effects remains elusive. We investigated the effects of ethanol metabolism within the central nucleus of the amygdala (CeA) on sympathoexcitation, and elucidated the role of acetate in these excitatory responses.

MethodsIn vivo arterial blood pressure, heart rate and sympathetic nerve activity responses to CeA microinjected ethanol or acetate with appropriate inhibitors/antagonists were tracked. In vitro whole-cell electrophysiology recording responses to acetate in CeA neurons with axon projecting to the rostral ventrolateral medulla (CeA-RVLM) were investigated, and cytosolic calcium responses in primary neuronal cultures were quantified.

ResultsWe demonstrate that in Sprague Dawley rats, local brain metabolism of ethanol in the CeA to acetic acid/acetate elicits sympathoexcitatory responses in vivo through activation of NMDAR. Aldehyde dehydrogenase inhibition using cyanamide and NMDAR antagonism using AP5 or memantine blunted these effects. Whole-cell patch-clamp recordings in brain slices containing autonomic CeA-RVLM neurons revealed a dose-dependent increase in neuronal excitability in response to acetate. The NMDAR antagonists AP5 and memantine suppressed the acetate-induced increase in CeA-RVLM neuronal excitability, as well as the direct activation of NMDAR-mediated inward currents by acetate in brain slices. We observed that acetate increased cytosolic Ca2+ in a time-dependent manner in primary neuronal cell cultures. The acetate enhancement of calcium signaling was abolished by memantine.

ConclusionThese findings suggest that within the CeA, ethanol is sympathoexcitatory through local brain metabolism, which generates acetic acid/acetate leading to activation of NMDAR.

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The giant Staphylococcus epidermidis surface protein Embp binds selectively to fibrillated fibronectin through a Velcro-like mechanism Staphylococcus epidermidis causes some of the most hard-to-treat clinical infections by forming biofilms: Multicellular communities of bacteria encased in a protective matrix, supporting immune evasion and tolerance against antibiotics. Biofilms occur most commonly on medical implants, and a key event in implant colonization is the robust adherence to the surface, facilitated by interactions between bacterial surface proteins and host matrix components. S. epidermidis is equipped with a giant adhesive protein, Embp, which facilitates bacterial interactions with surface-deposited, but not soluble fibronectin. The structural basis behind this selective binding process has remained obscure. Using a suite of single-cell and single-molecule analysis techniques, we show that S. epidermidis is capable of such distinction because Embp binds specifically to fibrillated fibronectin on surfaces, while ignoring globular fibronectin in solution. S. epidermidis adherence is critically dependent on multi-valent interactions involving 50 fibronectin-binding repeats of Embp. This unusual, Velcro-like interaction proved critical for colonization of surfaces under high flow, making this newly identified attachment mechanism particularly relevant for colonization of intravascular devices, such as prosthetic heart valves or vascular grafts. Other biofilm-forming pathogens, such as Staphylococcus aureus, express homologs of Embp and likely deploy the same mechanism for surface colonization. Our results may open for a novel direction in efforts to combat devastating, biofilm-associated infections, as the development of implant materials that steer the conformation of adsorbed proteins is a much more manageable task than avoiding protein adsorption altogether.

Graphical abstractFibronectin exists in two different conformations in the body. It circulates in the bodily fluids in globular conformation, however, it become fibrillated once adsorbed to an implant surface. S. epidermidis possess a giant 1 MDa receptor known as Embp bind specifically to fibrillated Fn but not to the globular Fn.

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sentences: Analysis of correlation and trail coefficients for componentperformance s in nine experimental tomato lines The objective of the research was to analyze the main variables related to tomato yield, and guide the selection of materials for the INIAF vegetable improvement program. The experiment was carried out in the open field using nine tomato lines on the grounds of the National Vegetable Seed Production Center, during the 2017-2018 agricultural campaign. A randomized complete block experimental design was used, with three repetitions and 10 plants per experimental unit. For the analysis of the data, the variable yield was considered as dependent and the variables number of flowers per inflorescence, number of clusters per plant, number of fruits per plant, weight of fruit, equatorial and polar diameter as independent variables. Analysis of variance, phenotypic correlations and path coefficients were performed. The performance of the L015 line was 80. 79 t ha-1 higher than the L014, L019 and Rio Grande lines. The fruit yield had a significant correlation with the weight of fruit per plant followed by the polar diameter, equatorial diameter, number of fruits per plant and weight of fruit. The analysis of path coefficients showed that the number of fruits per plant had the highest direct positive effect on the fruit yield, fruit weight and equatorial diameter that have a significant correlation and a direct effect on the fruit yield, emerged as the components with coefficients of 0.96 and 0.52 respectively. These characters may be relevant within the selection criteria in the development of new varieties. number of fruits per plant and weight of fruit. The analysis of path coefficients showed that the number of fruits per plant had the highest direct positive effect on the fruit yield, fruit weight and equatorial diameter that have a significant correlation and a direct effect on the fruit yield, emerged as the components with coefficients of 0.96 and 0.52 respectively. These characters may be relevant within the selection criteria in the development of new varieties. number of fruits per plant and weight of fruit. The analysis of path coefficients showed that the number of fruits per plant had the highest direct positive effect on the fruit yield, fruit weight and equatorial diameter that have a significant correlation and a direct effect on the fruit yield, emerged as the components with coefficients of 0.96 and 0.52 respectively. These characters may be relevant within the selection criteria in the development of new varieties.

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Transient beta activity and connectivity during sustained motor behaviour Neural oscillations are thought to play a central role in orchestrating activity states between distant neural populations. In humans, long-range neural connectivity has been particularly well characterised for 13-30 Hz beta activity which becomes phase coupled between the motor cortex and the contralateral muscle during isometric contraction. Based on this and related observations, beta activity and connectivity have been linked to sustaining stable cognitive and motor states - or the  status quo - in the brain. Recently, however, beta activity has been shown to be short-lived, as opposed to sustained - though so far this has been reported for regional beta activity in tasks without sustained motor demands. Here, we measured magnetoencephalography (MEG) and electromyography (EMG) in 18 human participants performing an isometric-contraction (gripping) task designed to yield sustained behavioural output. If cortico-muscular beta connectivity is directly responsible for sustaining a stable motor state, then beta activity should be (or become) sustained in this context. In contrast, we found that beta activity and connectivity with the downstream muscle were transient, even when participants engaged in sustained gripping. Moreover, we found that sustained motor requirements did not prolong beta-event duration in comparison to rest. These findings suggest that long-range neural synchronisation may entail short  bursts of frequency-specific connectivity, even when task demands - and behaviour - are sustained.

HighlightsO_LITrial-average 13-30 Hz beta activity and connectivity with the muscle appear sustained during stable motor behaviour
C_LIO_LISingle-trial beta activity and connectivity are short-lived, even when motor behaviour is sustained
C_LIO_LISustained task demands do not prolong beta-event duration in comparison to resting state
C_LI

sentences: Audiovisual task switching rapidly modulates sound encoding in mouse auditory cortex In everyday behavior, sensory systems are in constant competition for attentional resources, but the cellular and circuit-level mechanisms of modality-selective attention remain largely uninvestigated. We conducted translaminar recordings in mouse auditory cortex (AC) during an audiovisual (AV) attention shifting task. Attending to sound elements in an AV stream reduced both pre-stimulus and stimulus-evoked spiking activity, primarily in deep layer neurons. Despite reduced spiking, stimulus decoder accuracy was preserved, suggesting improved sound encoding efficiency. Similarly, task-irrelevant probe stimuli during intertrial intervals evoked fewer spikes without impairing stimulus encoding, indicating that these attention influences generalized beyond training stimuli. Importantly, these spiking reductions predicted trial-to-trial behavioral accuracy during auditory attention, but not visual attention. Together, these findings suggest auditory attention facilitates sound discrimination by filtering sound-irrelevant spiking in AC, and that the deepest cortical layers may serve as a hub for integrating extramodal contextual information.

sentences: Macromolecular crowding and supersaturation protect hemodialysis patients from the onset of dialysis-related amyloidosis Dialysis-related amyloidosis (DRA), a serious complication among long-term hemodialysis patients, is caused by amyloid fibrils of {beta}2-microglobulin ({beta}2m). Although high serum {beta}2m levels and a long dialysis vintage are the primary and secondary risk factors for the onset of DRA, respectively, patients with these do not always develop DRA, indicating that there are additional risk factors. To clarify these unknown factors, we investigated the effects of human sera on {beta}2m amyloid fibril formation. Although sera markedly inhibited amyloid fibril formation, the inhibitory effects were weaker for sera collected from dialysis patients than for control sera. When sera collected before and after maintenance dialysis treatments were compared, the latter inhibited amyloid fibril formation more than the former. These results indicate that, although the inhibitory effects of sera were deteriorated in long-term dialysis patients, they were ameliorated by maintenance dialysis treatments in the short term. Maintenance dialysis decreases the body weight by 5% and consequently increases the concentrations of serum components. Among these components, we found that serum albumin prevented amyloid fibril formation based on macromolecular crowding effects, and that the decreased serum albumin concentration in dialysis patients is a tertiary risk factor for the onset of DRA. The model was constructed assuming accumulative effects of three risk factors and may be useful for predicting the onset of DRA. Furthermore, the model suggested the importance of monitoring temporary and accumulated risks to prevent the development of amyloidoses in general, which occurs based on supersaturation-limited amyloid fibril formation in a crowded milieu.

sentences: Model guided design of enhanced bi-stable controllers to effectively switch cellular states 1Bi-stable gene regulatory motifs are found in a wide variety of natural gene regulatory networks and typically effect transcriptional switching between stable phenotypic states in cells. In synthetic gene regulatory circuits, these architectures can be used to dynamically switch between distinct metabolic states for metabolic engineering and therapeutic applications. Though it has been over two decades since the first synthetic bi-stable switch was developed, the lack of modularity and predictability of these motifs in varying environments has limited widespread application, especially since the factors that affect switching characteristics are still unclear. In this work, we develop a mathematical model describing a bi-stable gene regulatory element using mass action kinetics. Using this model and a newly developed dynamical modeling and continuity analysis framework, we identify the changes in switching characteristics shown by the bi-stable motif over a range of biologically relevant model parameter values. Interestingly, there appears to be a trade-off between the robustness of the motif - the parameter ranges over which it retains bi-stable function, and the speed at which it effects a phenotypic change. Further, using E. coli as a model host, we constructed a large library of transcriptional switches that show a wide range of switching speeds, to experimentally demonstrate the presence of this trade-off. The presence of this trade-off has significant implications on the design of transcriptional switches for diverse applications and could potentially explain the circuit architecture of natural transcriptional switches as well. Additionally, we anticipate that our diverse library of bi-stable switches will be valuable to effect phenotypic changes with differing switching speed requirements for metabolic engineering applications.

sentences: Detection of low numbers of bacterial cells in pharmaceutical drug product using Raman Spectroscopy and PLS-DA multivariate analysis. Sterility testing is a laborious and slow process to detect contaminants present in drug products. Raman spectroscopy is a promising label-free tool to detect microorganisms and thus gaining relevance as future alternative culture-free method for sterility testing in pharmaceutical industry. However, reaching detection limits similar to standard procedures while keeping a high accuracy remains challenging, due to weak bacterial Raman signal. In this work, we show a new non-invasive approach focusing on detect different bacteria in concentrations below 100 CFU/ml within drug product containers using Raman spectroscopy and multivariate data analysis. Even though Raman spectra form drug product with and without bacteria are similar, a partial least squared discriminant analysis (PLS-DA) model shows great performance to distinguish samples with bacteria contaminants in limits below 10 CFU/ml. We use spiked samples with bacteria spores for independent validation achieving a detection accuracy of 99%. Our results indicate a great potential of this rapid, and cost-effective approach to be use in quality control of pharmaceutical industry.

sentences: A human stem cell resource to decipher the biochemical and cellular basis of neurodevelopmental defects in Lowe Syndrome Human brain development is a complex process where multiple cellular and developmental events are co-ordinated to generate normal structure and function. Alteration in any of these events can impact brain development, manifesting clinically as neurodevelopmental disorders. Human genetic disorders of lipid metabolism often present with features of altered brain function. Lowe syndrome (LS), is a X-linked recessive disease with features of altered brain function. LS results from mutations in OCRL1 that encodes a phosphoinositide 5-phosphatase enzyme. However, the cellular mechanisms by which loss of OCRL1 leads to brain defects remain unknown. Human brain development involves several cellular and developmental features not conserved in other species and understanding such mechanisms remains a challenge. Rodent models of LS have been generated, but failed to recapitulate features of the human disease. Here we describe the generation of human stem cell lines from LS patients. Further, we present biochemical characterization of lipid metabolism in patient cell lines and demonstrate their use as a "disease-in-a-dish" model for understanding the mechanism by which loss of OCRL1 leads to altered cellular and physiological brain development.

sentences: Genetic influences on hub connectivity of the human connectome Brain network hubs are both highly connected and highly inter-connected, forming a critical communication backbone for coherent neural dynamics. The mechanisms driving this organization are poorly understood. Using diffusion-weighted imaging in twins, we identify a major role for genes, showing that they preferentially influence connectivity strength between network hubs of the human connectome. Using transcriptomic atlas data, we show that connected hubs demonstrate tight coupling of transcriptional activity related to metabolic and cytoarchitectonic similarity. Finally, comparing over thirteen generative models of network growth, we show that purely stochastic processes cannot explain the precise wiring patterns of hubs, and that model performance can be improved by incorporating genetic constraints. Our findings indicate that genes play a strong and preferential role in shaping the functionally valuable, metabolically costly connections between connectome hubs.

sentences: Plasma microbiome in COVID-19 subjects: an indicator of gut barrier defects and dysbiosis The gut is a well-established route of infection and target for viral damage by SARS-CoV-2. This is supported by the clinical observation that about half of COVID-19 patients exhibit gastrointestinal (GI) symptoms. We asked whether the analysis of plasma could provide insight into gut barrier dysfunction in patients with COVID-19 infection. Plasma samples of COVID-19 patients (n=30) and healthy control (n=16) were collected during hospitalization. Plasma microbiome was analyzed using 16S rRNA sequencing, metatranscriptomic analysis, and gut permeability markers including FABP-2, PGN and LPS in both patient cohorts. Almost 65% (9 out 14) COVID-19 patients showed abnormal presence of gut microbes in their bloodstream. Plasma samples contained predominately Proteobacteria, Firmicutes, and Actinobacteria. The abundance of gram-negative bacteria (Acinetobacter, Nitrospirillum, Cupriavidus, Pseudomonas, Aquabacterium, Burkholderia, Caballeronia, Parabhurkholderia, Bravibacterium, and Sphingomonas) was higher than the gram-positive bacteria (Staphylococcus and Lactobacillus) in COVID-19 subjects. The levels of plasma gut permeability markers FABP2 (1282{+/-}199.6 vs 838.1{+/-}91.33; p=0.0757), PGN (34.64{+/-}3.178 vs 17.53{+/-}2.12; p<0.0001), and LPS (405.5{+/-}48.37 vs 249.6{+/-}17.06; p=0.0049) were higher in COVID-19 patients compared to healthy subjects. These findings support that the intestine may represent a source for bacteremia and may contribute to worsening COVID-19 outcomes. Therapies targeting the gut and prevention of gut barrier defects may represent a strategy to improve outcomes in COVID-19 patients.

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Neural evidence for narrative-based processing of evidence and bias in juror decision making Efforts to explain complex human decisions have focused on competing theories emphasizing utility and narrative mechanisms. These are difficult to distinguish using behavior alone. Both narrative and utility theories have been proposed to explain juror decisions, which are among the most consequential complex decisions made in a modern society. Here, we use patterns of brain activation derived from large neuroimaging databases to look for signatures of the cognitive processes associated with different models of juror decision making. We asked jury-eligible subjects to rate the strength of a series of criminal cases while recording the resulting patterns of brain activation. We identified cognitive processes associated with the cumulative evidence presented. Evidence accumulation correlated with multiple narrative processes, including reading and recall. Of the cognitive processes traditionally viewed as components of utility, activation patterns associated with uncertainty, but not value, were more active with stronger evidence. Independent of utility and narrative, activations linked to reasoning and relational logic also correlated with increasing evidence. Formal comparison of narrative and utility models supported a central role for narrative in integrating evidence in complex decisions.

Significance StatementThe last decade has seen a sharply increased interest in narrative as a central cognitive process in human decision making and as an important factor in the evolution of human societies. However, the roles of narrative versus utility models of decision making remain hotly debated. While available models frequently produce similar behavioral predictions, they rely on different cognitive processes and so their roles can be separated using the right neural tests. Here, we use brain imaging during mock juror decisions to show that cognitive processes associated with narrative, and to a lesser extent utility, were engaged while subjects evaluated evidence. These results support a role for narrative in complex decisions.

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Spatio-temporal heterogeneity in hippocampal metabolism in control and epilepsy conditions. The hippocampuss dorsal and ventral parts are involved in different operative circuits, which functions vary in time during the night and day cycle. These functions are altered in epilepsy. Since energy production is tailored to function, we hypothesized that energy production would be space- and time-dependent in the hippocampus and that such organizing principle would be modified in epilepsy. Using metabolic imaging and metabolite sensing ex vivo, we show that the ventral hippocampus favors aerobic glycolysis over oxidative phosphorylation as compared to the dorsal part in the morning in control mice. In the afternoon, aerobic glycolysis is decreased, and oxidative phosphorylation increased. In the dorsal hippocampus, the metabolic activity varies less between these two times but is weaker than in the ventral. Thus, the energy metabolism is different along the dorsoventral axis and changes as a function of time in control mice. In an experimental model of epilepsy, we find a large alteration of such spatio-temporal organization. In addition to a general hypometabolic state, the dorsoventral difference disappears in the morning, when seizure probability is low. In the afternoon, when seizure probability is high, the aerobic glycolysis is enhanced in both parts, the increase being stronger in the ventral area. We suggest that energy metabolism is tailored to the functions performed by brain networks, which vary over time. In pathological conditions, the alterations of these general rules may contribute to network dysfunctions.

SIGNIFICANCE STATEMENTThe dorsal part of the hippocampus is involved in spatial learning and memory processes, while the ventral is implicated in motivational and emotional behavior. These functions change during the night and day cycle, and they are altered in epilepsy. Here we show that energy production (glycolysis versus oxidative phosphorylation) varies along the dorsoventral axis in a circadian manner ex vivo in control mice. These rules are altered in experimental epilepsy. Thus, energy production may be tailored to the function performed by hippocampal subdivisions and to the time of the day. Alterations in epilepsy may contribute to seizure generation and cognitive deficits.

sentences: Characterization of mouse Bmp5 regulatory injury element in zebrafish wound models Many key signaling molecules used to build tissues during embryonic development are re-activated at injury sites to stimulate tissue regeneration and repair. Bone morphogenetic proteins provide a classic example, but the mechanisms that lead to reactivation of BMPs following injury are still unknown. Previous studies have mapped a large "injury response element" (IRE) in the mouse Bmp5 gene that drives gene expression following bone fractures and other types of injury. Here we show that the large mouse IRE region is also activated in both zebrafish tail resection and mechanosensory hair cell injury models. Using the ability to test multiple constructs and image temporal and spatial dynamics following injury responses, we have narrowed the original size of the mouse IRE region by over 100 fold and identified a small 142 bp minimal enhancer that is rapidly induced in both mesenchymal and epithelial tissues after injury. These studies identify a small sequence that responds to evolutionarily conserved local signals in wounded tissues and suggest candidate pathways that contribute to BMP reactivation after injury.

sentences: The PAIR-R24M Dataset for Multi-animal 3D Pose Estimation Understanding the biological basis of social and collective behaviors in animals is a key goal of the life sciences, and may yield important insights for engineering intelligent multi-agent systems. A critical step in interrogating the mechanisms underlying social behaviors is a precise readout of the 3D pose of interacting animals. While approaches for multi-animal pose estimation are beginning to emerge, they remain challenging to compare due to the lack of standardized training and benchmark datasets. Here we introduce the PAIR-R24M (Paired Acquisition of Interacting oRganisms - Rat) dataset for multi-animal 3D pose estimation, which contains 24.3 million frames of RGB video and 3D ground-truth motion capture of dyadic interactions in laboratory rats. PAIR-R24M contains data from 18 distinct pairs of rats and 24 different viewpoints. We annotated the data with 11 behavioral labels and 3 interaction categories to facilitate benchmarking in rare but challenging behaviors. To establish a baseline for markerless multi-animal 3D pose estimation, we developed a multi-animal extension of DANNCE, a recently published network for 3D pose estimation in freely behaving laboratory animals. As the first large multi-animal 3D pose estimation dataset, PAIR-R24M will help advance 3D animal tracking approaches and aid in elucidating the neural basis of social behaviors.

sentences: HIV-1 preintegration complex preferentially integrates the viral DNA into nucleosomes containing trimethylated histone 3-lysine 36 modification HIV-1 DNA integration into the host chromosomes is carried out by the preintegration complex (PIC). The PIC contains the viral DNA, virally encoded integrase enzyme and other critical viral/host factors. The PIC-associated viral DNA is preferentially integrated into gene bodies of actively transcribing genes. Here, we identify a biochemical mechanism underlying the preference of PIC-mediated viral DNA integration (PIC-VDI). Specifically, we observed that the PIC-VDI into human chromatin is preferred over the genomic DNA. Surprisingly, nucleosome core particles without any histone modifications were not preferred for PIC-VDI when compared to the analogous naked DNA. However, PIC-VDI was markedly enhanced with nucleosomes containing the trimethylated histone 3 lysine 36 (H3K36me3), an epigenetic mark linked to HIV-1 DNA integration preference. Interestingly, we observed that nucleosomes with flanking linker DNA promoted PIC-VDI in the presence of LEDGF/p75. We also discovered that nucleosomes with linker DNA and H3K36me3 served as the optimal substrate for PIC-VDI. Mapping of the integration sites within these substrates identified preference of specific regions of the nucleosome core DNA for integration. Finally, we provide biochemical and genetic evidence that histone H1 protein, that condenses the chromatin, negatively regulates HIV-1 DNA integration, consistent with the integration preference for open chromatin structure. Collectively, these results identify the role of specific chromatin marks that drive HIV-1 integration preference and define the optimal substrate requirement for efficient DNA integration by the PIC.

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A recombinant ACE2 Triple Decoy that traps and neutralizes SARS-CoV-2 shows enhanced affinity for highly transmissible SARS-CoV-2 variants The highly-transmissible SARS-CoV-2 variants now replacing the first wave strain pose an increased threat to human health by their ability, in some instances, to escape existing humoral protection conferred by previous infection, neutralizing antibodies, and possibly vaccination. Thus, other therapeutic options are necessary. One such therapeutic option that leverages SARS-CoV-2 initiation of infection by binding of its spike receptor binding domain (S RBD) to surface-expressed host cell angiotensin-converting enzyme 2 (ACE2) is an ACE2  decoy that would trap the virus by competitive binding and thus inhibit propagation of infection. Here, we used Molecular Dynamic (MD) simulations to predict ACE2 mutations that might increase its affinity for S RBD and screened these candidates for binding affinity in vitro. A double mutant ACE2(T27Y/H34A)-IgG1FC fusion protein was found to have very high affinity for S RBD and to show greater neutralization of SARS-CoV-2 in a live virus assay as compared to wild type ACE2. We further modified the double mutant ACE2 decoy by addition of an H374N mutation to inhibit ACE2 enzymatic activity while maintaining high S RBD affinity. We then confirmed the potential efficacy of our ACE2(T27Y/H34A/H374N)-IgG1FC Triple Decoy against S RBD expressing variant-associated E484K, K417N, N501Y, and L452R mutations and found that our ACE2 Triple Decoy not only maintains its high affinity for S RBD expressing these mutations, but shows enhanced affinity for S RBD expressing the N501Y or L452R mutations and the highest affinity for S RBD expressing both the E484K and N501Y mutations. The ACE2 Triple Decoy also demonstrates the ability to compete with wild type ACE2 in the cPass surrogate virus neutralization in the presence of S RBD with these mutations. Additional MD simulation of ACE2 WT and decoy interactions with S RBD WT or B.1.351 variant sequence S RBD provides insight into the enhanced affinity of the ACE2 decoy for S RBD and reveals its potential as a tool to predict affinity and inform therapeutic design. The ACE2 Triple Decoy is now undergoing continued assessment, including expression by a human adenovirus serotype 5 (hAd5) construct to facilitate delivery in vivo.

Summary sentenceAn ACE2(N27Y/H34A/H374N)-IgG1FC fusion protein decoy sustains high affinity to all SARS-CoV-2 spike receptor binding domain (RBD) protein variants tested, shows enhanced affinity for the N501Y and L452R variants, and the highest affinity for combined N501Y and E484K variants.

sentences: NMPylation and de-NMPylation of SARS-CoV-2 Nsp9 by the NiRAN domain Nsp12, the catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), contains two active sites that catalyze nucleotidyl-monophosphate (NMP) transfer (NMPylation). RNA synthesis is mediated by the RdRp active site that is conserved among all RNA viruses and has been a focus of mechanistic studies and drug discovery. The second active site resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. Both catalytic reactions are essential for viral replication, but the mechanism and targets of NiRAN are poorly characterized. One recent study showed that NiRAN transfers NMP to the first residue of RNA-binding protein Nsp9. Another study reported a structure of SARS-CoV-2 replicase with an extended Nsp9 in the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 Nsp12 efficiently and reversibly NMPylates the native but not the extended Nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas a substitution of a catalytic RdRp Asp residue does not. NMPylation is inhibited by nucleotide analogs, pyrophosphate, and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We hypothesize that Nsp9 remodels both active sites of Nsp12 to support initiation of RNA synthesis by RdRp and subsequent capping of the product RNA by the NiRAN domain.

sentences: Insight into Polyproline II Helical Bundle Stability and Folding from NMR Spectroscopic Characterization of the Snow Flea Antifreeze Protein Denatured State The use of PPII helices in protein design is currently hindered by limitations in our understanding of their conformational stability and folding. Recent studies of the snow flea antifreeze protein (sfAFP), a useful model system composed of six PPII helices, suggested that a low denatured state entropy contributes to folding thermodynamics. To get atomic level information on the conformational ensemble and entropy of the reduced denatured state of sfAFP, we have analyzed its chemical shifts and {1H}-15N relaxation parameters by NMR spectroscopy at three experimental conditions. No significant populations of preferred secondary structure were detected. The stiffening of certain N-terminal residues at neutral versus acidic pH leads us to suggest that favorable charge-charge interactions could bias the conformational ensemble to favor the formation of the two disulfide bonds during nascent folding. Despite a high content of flexible glycine residues, the mobility of the sfAFP denatured ensemble is similar for denatured /{beta} proteins both on fast ps/ns as well as slower s/ms timescales. These results are in line with a conformational entropy in the denatured ensemble resembling that of typical proteins and suggest that new structures based on PPII helical bundles should be amenable to protein design.

sentences: Development of spontaneous firing of fusiform neurons from the dorsal cochlear nucleus of mice occurs after hearing onset. The dorsal cochlear nucleus (DCN) in the auditory brainstem integrates auditory and somatosensory information. Mature fusiform neurons express two qualitative intrinsic states in equal proportions: quiet, with no spontaneous regular action potential firing, or active, with regular spontaneous action potential firing. However, how these firing states and other electrophysiological properties of fusiform neurons develop during early postnatal days to adulthood is not known. Thus, we recorded fusiform neurons from mice from P4 to P21 and analyzed their electrophysiological properties. In the pre-hearing phase (P4-P13), we found that fusiform neurons are mostly quiet, with the active state emerging after hearing onset at P14. Subthreshold properties present more variations before hearing onset, while action potential properties vary more after P14, developing bigger, shorter, and faster action potentials. Interestingly, the activity threshold is more depolarized in pre-hearing cells suggesting that persistent sodium current (INaP) increases its expression after hearing. In fact, INaP increases its expression after hearing, accordingly with the development of active neurons. Thus, we suggest that the post-hearing expression of INaP creates the active state of the fusiform neuron. At the same time, other changes refine the passive membrane properties and increase the speed of action potential firing of fusiform neurons.

sentences: A novel class of sulfur-containing aminolipids widespread in marine roseobacters Marine roseobacter group bacteria are numerically abundant and ecologically important players in ocean ecosystems. These bacteria are capable of modifying their membrane lipid composition in response to environmental change. Remarkably, a variety of lipids are produced in these bacteria, including phosphorus-containing glycerophospholipids and several amino acid-containing aminolipids such as ornithine lipids and glutamine lipids. Here, we present the identification and characterization of a novel sulfur-containing aminolipid (SAL) in roseobacters. Using high resolution accurate mass spectrometry, a SAL was found in the lipid extract of Ruegeria pomeroyi DSS-3 and Phaeobacter inhibens DSM 17395. Using comparative genomics, transposon mutagenesis and targeted gene knockout, we identified a gene encoding a putative lyso-lipid acyltransferase, designated SalA, which is essential for the biosynthesis of this SAL. Multiple sequence analysis and structural modelling suggest that SalA is a novel member of the lysophosphatidic acid acyltransferase (LPAAT) family, the prototype of which is the PlsC acyltransferase responsible for the biosynthesis of the phospholipid phosphatidic acid. SAL appears to play a key role in biofilm formation in roseobacters. SalA is widely distributed in Tara Oceans metagenomes and actively expressed in Tara Oceans metatranscriptomes. Our results raise the importance of sulfur-containing membrane aminolipids in marine bacteria.

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Independent population coding of the past and the present in prefrontal cortex during learning Medial prefrontal cortex (mPfC) activity represents information about the state of the world, including present behaviour, such as decisions, and the immediate past, such as short-term memory. Unknown is whether information about different states of the world are represented in the same mPfC neural population and, if so, how they are kept distinct. To address this, we analyse here mPfC population activity of rats learning rules in a Y-maze, with self-initiated choice trials to an arm-end followed by a self-paced return during the inter-trial interval (ITI). We find that trial and ITI population activity from the same population fall into different low-dimensional subspaces. These subspaces encode different states of the world: multiple features of the task can be decoded from both trial and ITI activity, but the decoding axes for the same feature are roughly orthogonal between the two task phases, and the decodings are predominantly of features of the present during the trial but features of the preceding trial during the ITI. These subspace distinctions are carried forward into sleep, where population activity is preferentially reactivated in post-training sleep, but differently for activity from the trial and ITI subspaces. Our results suggest that the problem of interference when representing different states of the world is solved in mPfC by population activity occupying different subspaces for the world states, which can be independently decoded by downstream targets and independently addressed by upstream inputs.

Significance statementActivity in the medial prefrontal cortex plays a roles in representing the current and past states of the world. We show that during a maze task the activity of a single population in medial prefrontal cortex represents at least two different states of the world. These representations were sequential and sufficiently distinct that a downstream population could separately read out either state from that activity. Moreover, the activity representing different states is differently reactivated in sleep. Different world states can thus be represented in the same medial prefrontal cortex population, but in such a way that prevents potentially catastrophic interference between them.

sentences: Magnetic Nanoprobes for Spatio-Mechanical Manipulation in Single Cells Magnetic nanoparticles (MNPs) are widely known as valuable agents for biomedical applications. Yet, for their successful application within cells they need to fulfill a variety of demands such as monodispersity, biocompatibility or sufficient magnetic response. Given these prerequisites, MNPs may be used for remote, non-invasive manipulation, where their spatial redistribution or force response in a magnetic field provides a fine-tunable stimulus to a cell. Here, we investigate the properties of two different MNPs and their suitability for spatio-mechanical manipulations: semisynthetic magnetoferritin nanoparticles and fully synthetic  nanoflower-shaped iron-oxide nanoparticles. Next to characterizing their structure, surface potential and magnetic response, we monitor the MNP performance in a living cell environment using fluorescence microscopy and confirm their biocompatibility. We then demonstrate their capability to spatially redistribute and to respond to magnetic force gradients inside a cell. Our remote manipulation assays present these tailored magnetic materials as suitable agents for applications in magnetogenetics, biomedicine or nanomaterial research.

sentences: A systematic analysis of Trypanosoma brucei chromatin factors identifies novel protein interaction networks associated with sites of transcription initiation and termination Nucleosomes composed of histones are the fundamental units around which DNA is wrapped to form chromatin. Transcriptionally active euchromatin or repressive heterochromatin is regulated in part by the addition or removal of histone post-translational modifications (PTMs) by  writer and  eraser enzymes, respectively. Nucleosomal PTMs are recognised by a variety of  reader proteins which alter gene expression accordingly. The histone tails of the evolutionarily divergent eukaryotic parasite Trypanosoma brucei have atypical sequences and PTMs distinct from those often considered universally conserved. Here we identify 68 predicted readers, writers and erasers of histone acetylation and methylation encoded in the T. brucei genome and, by epitope tagging, systemically localize 63 of them in the parasites bloodstream form. ChIP-seq demonstrated that fifteen candidate proteins associate with regions of RNAPII transcription initiation. Eight other proteins exhibit a distinct distribution with specific peaks at a subset of RNAPII transcription termination regions marked by RNAPIII-transcribed tRNA and snRNA genes. Proteomic analyses identified distinct protein interaction networks comprising known chromatin regulators and novel trypanosome-specific components. Notably, several SET-domain and Bromo-domain protein networks suggest parallels to RNAPII promoter-associated complexes in conventional eukaryotes. Further, we identify likely components of TbSWR1 and TbNuA4 complexes whose enrichment coincides with the SWR1-C exchange substrate H2A.Z at RNAPII transcriptional start regions. The systematic approach employed provides detail of the composition and organization of the chromatin regulatory machinery in Trypanosoma brucei and establishes a route to explore divergence from eukaryotic norms in an evolutionarily ancient but experimentally accessible eukaryote.

sentences: Tempo and mode of gene expression evolution in the brain across Primates Primate evolution has led to a remarkable diversity of behavioral specializations and pronounced brain size variation among species 1,2. Gene expression provides a promising opportunity for studying the molecular basis of brain evolution, but it has been explored in very few primate species to date e.g. 3,4. To understand the landscape of gene expression evolution across the primate lineage, we generated and analyzed RNA-Seq data from four brain regions in an unprecedented eighteen species. Here we show a remarkable level of variation in gene expression among hominid species, including humans and chimpanzees, despite their relatively recent divergence time from other primates. We found that individual genes display a wide range of expression dynamics across evolutionary time reflective of the diverse selection pressures acting on genes within primate brain tissue. Using our sample that represents an unprecedented 190-fold difference in primate brain size, we identified genes with variation in expression most correlated with brain size and found several with signals of positive selection in their regulatory regions. Our study extensively broadens the context of what is known about the molecular evolution of the brain across primates and identifies novel candidate genes for study of genetic regulation of brain development and evolution.

sentences: Chimeric chromosome landscapes of human somatic cell cultures show dependence on stress and regulation of genomic repeats by CGGBP1. Genomes of somatic cells in culture are prone to spontaneous mutations due to errors in replication and DNA repair. Some of these errors, such as chromosomal fusions, are not rectifiable and subject to selection or elimination in growing cultures. Somatic cell cultures are thus expected to generate background levels of potentially stable chromosomal chimeras. A description of the landscape of such spontaneously generated chromosomal chimeras in cultured cells will help us understand the factors affecting somatic mosaicism. Here we show that short homology-associated non-homologous chromosomal chimeras occur in normal human fibroblasts and HEK293T cells at genomic repeats. The occurrence of chromosomal chimeras is enhanced by heat stress and depletion of a repeat regulatory protein CGGBP1. We also present evidence of homologous chromosomal chimeras between allelic copies in repeat-rich DNA obtained by methylcytosine immunoprecipitation. The formation of homologous chromosomal chimeras at Alu and L1 repeats increases upon depletion of CGGBP1. Our data are derived from de novo sequencing from three different cell lines under different experimental conditions and our chromosomal chimera detection pipeline is applicable to long read as well as short read sequencing platforms. These findings present significant information about the generation, sensitivity and regulation of somatic mosaicism in human cell cultures.

sentences: THINGSvision: a Python toolbox for streamlining the extraction of activations from deep neural networks Over the past decade, deep neural network (DNN) models have received a lot of attention due to their near-human object classification performance and their excellent prediction of signals recorded from biological visual systems. To better understand the function of these networks and relate them to hypotheses about brain activity and behavior, researchers need to extract the activations to images across different DNN layers. The abundance of different DNN variants, however, can often be unwieldy, and the task of extracting DNN activations from different layers may be non-trivial and error-prone for someone without a strong computational background. Thus, researchers in the fields of cognitive science and computational neuroscience would benefit from a library or package that supports a user in the extraction task. THINGSvision is a new Python module that aims at closing this gap by providing a simple and unified tool for extracting layer activations for a wide range of pretrained and randomly-initialized neural network architectures, even for users with little to no programming experience. We demonstrate the general utility of THINGsvision by relating extracted DNN activations to a number of functional MRI and behavioral datasets using representational similarity analysis, which can be performed as an integral part of the toolbox. Together, THINGSvision enables researchers across diverse fields to extract features in a streamlined manner for their custom image dataset, thereby improving the ease of relating DNNs, brain activity, and behavior, and improving the reproducibility of findings in these research fields.

sentences: Modeling tactile pleasantness across skin types at the individual level reveals a reliable and stable basic function Touch is perceived most pleasant when delivered at velocities known to optimally activate C Tactile afferents. At the group level, pleasantness ratings of touch delivered at velocities in the range 0.3-30 cm/s follows an inverted-U shape curve, with maximum pleasantness between 1 and 10 cm/s. However, the prevalence, reliability, and stability of this function at the individual level and across skin types remains unknown. Here, we tested a range of seven velocities delivered with a soft brush, on both hairy and non-hairy skin in 123 participants. We showed that the relationship between pleasantness and velocity of touch is significantly best described by a negative quadratic model at the individual level in the majority of participants both on hairy (67.1%) and non-hairy skin (62.6%). Higher interoceptive accuracy and self-reported depression were related to a better fit of the quadratic model and to the steepness of the curve, respectively. The prevalence of the quadratic model was stable across body sites (62.6%), across two experimental sessions (73-78%,), and regardless of the number of trials. Thus, the individual perception of tactile pleasantness follows a characteristic velocity-dependent function across skin types and shows trait characteristics, making it a possible biomarker for mental health disorders.

sentences: The Global Protein-RNA Interaction map of Epithelial Splicing Regulatory Protein 1 defines a post-transcriptional program that is essential for epithelial cell function The epithelial splicing regulatory proteins, ESRP1 and ESRP2 are essential for mammalian development through regulation of a global program of alternative splicing of genes involved in maintenance of epithelial cell function. To further inform our understanding of the molecular functions of ESRP1 we performed enhanced crosslinking immunoprecipitation coupled with high throughput sequencing (eCLIP) in epithelial cells of mouse epidermis. The genome-wide binding sites of ESRP1 were integrated with RNA-Seq analysis of alterations in splicing and total gene expression that result from epidermal ablation of Esrp1 and Esrp2. These studies demonstrated that ESRP1 functions in splicing regulation occur primarily through direct binding in a position-dependent manner to either promote exon inclusion or skipping. In addition, we also identified widespread binding of ESRP1 in 3 and 5 untranslated regions (UTRs) of genes involved in epithelial cell function suggesting that its post-transcriptional functions extend beyond splicing regulation.

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Revisiting the role of Toxoplasma gondii ERK7 in the maintenance and stability of the apical complex Toxoplasma gondii ERK7 is known to contribute to the integrity of the apical complex and to be involved only in the final step of the conoid biogenesis. In the absence of ERK7, mature parasites lose their conoid complex and are unable to glide, invade or egress from host cells. In contrast to a previous report, we show here that depletion of ERK7 phenocopies the depletion of the apical cap proteins AC9 or AC10. The absence of ERK7 leads to the loss of the apical polar ring, the disorganization of the basket of subpellicular microtubules and an impairment in micronemes secretion. Ultra-expansion microscopy (U-ExM) coupled to NHS-Ester staining on intracellular parasites offers an unprecedented level of resolution and highlights the disorganization of the rhoptries as well as the dilated plasma membrane at the apical pole in the absence of ERK7. Comparative proteomics analysis of wild-type and ERK7 or AC9 depleted parasites led to the disappearance of known, predicted, as well as putative novel components of the apical complex. In contrast, the absence of ERK7 led to an accumulation of microneme proteins, resulting from the defect in exocytosis of the organelles.

ImportanceThe conoid is an enigmatic, dynamic organelle positioned at the apical tip of the coccidian subgroup of the Apicomplexa, close to the apical polar ring (APR) from which the subpellicular microtubules (SPTMs) emerge and at the site of microneme and rhoptry secretory organelles exocytosis. In Toxoplasma gondii, the conoid protrudes concomitantly to microneme secretion, during egress, motility and invasion. The conditional depletion of the apical cap structural protein AC9 or AC10 leads to a disorganization of SPTMs as well as the loss of APR and conoid that result in microneme secretion defect and block in motility, invasion and egress. We show here that depletion of the kinase ERK7 phenocopies completely AC9 and AC10 mutants. Moreover, the combination of ultrastructure expansion microscopy with an NHS ester staining revealed that ERK7 depleted parasites exhibit a dilated apical plasma membrane and a mis-positioning of the rhoptry organelles.

sentences: Using Domain-Knowledge to Assist Lead Discovery in Early-Stage Drug Design We are interested in generating new small molecules which could act as inhibitors of a biological target, when there is limited prior information on target-specific inhibitors. This form of drug-design is assuming increasing importance with the advent of new disease threats for which known chemicals only provide limited information about target inhibition. In this paper, we propose the combined use of deep neural networks and Inductive Logic Programming (ILP) that allows the use of symbolic domain-knowledge (B) to explore the large space of possible molecules. Assuming molecules and their activities to be instances of random variables X and Y, the problem is to draw instances from the conditional distribution of X, given Y, B (DX|Y,B). We decompose this into the constituent parts of obtaining the distributions DX|B and DY|X,B, and describe the design and implementation of models to approximate the distributions. The design consists of generators (to approximate DX|B and DX|Y,B) and a discriminator (to approximate DY|X,B). We investigate our approach using the well-studied problem of inhibitors for the Janus kinase (JAK) class of proteins. We assume first that if no data on inhibitors are available for a target protein (JAK2), but a small numbers of inhibitors are known for homologous proteins (JAK1, JAK3 and TYK2). We show that the inclusion of relational domain-knowledge results in a potentially more effective generator of inhibitors than simple random sampling from the space of molecules or a generator without access to symbolic relations. The results suggest a way of combining symbolic domain-knowledge and deep generative models to constrain the exploration of the chemical space of molecules, when there is limited information on target-inhibitors. We also show how samples from the conditional generator can be used to identify potentially novel target inhibitors.

sentences: Role of type I interferon signaling and microglia in the abnormal long term potentiation and object place recognition deficits of male mice with a mutation of the Tuberous Sclerosis 2 gene Tuberous Sclerosis Complex (TSC) is a genetic disorder associated with high rates of intellectual disability and autism. Although previous studies focused on the role of neuronal deficits in the memory phenotypes of rodent models of TSC, the results presented here demonstrate a role for microglia in these deficits. Mice with a heterozygous null mutation of the Tsc2 gene (Tsc2+/-), show deficits in hippocampal dependent tasks, as well as abnormal long-term potentiation (LTP) in the hippocampal CA1 region. Here, we show that microglia and type I interferon signaling (IFN1) have a key role in the object place recognition (OPR; a hippocampal dependent task) deficits and abnormal LTP of Tsc2+/- male mice. Unexpectedly, we demonstrate that male, but not female, Tsc2+/- mice showed OPR deficits. Importantly, these deficits can be rescued by depletion of microglia, as well as by a genetic manipulation of a signaling pathway known to modulate microglia function (interferon-alpha/beta receptor alpha chain null mutation). In addition to rescuing the OPR deficits, depletion of microglia also reversed the abnormal LTP of the Tsc2+/- mice. Altogether, our results suggest that altered IFN1 signaling in microglia cause the abnormal LTP and OPR deficits of male Tsc2+/- mice.

sentences: Reference transcriptomes of porcine peripheral immune cells created through bulk and single-cell RNA sequencing Pigs are a valuable human biomedical model and an important protein source supporting global food security. The transcriptomes of peripheral blood immune cells in pigs were defined at the bulk cell-type and single cell levels. First, eight cell types were isolated in bulk from peripheral blood mononuclear cells (PBMCs) by cell sorting, representing Myeloid, NK cells and specific populations of T and B cells. Transcriptomes for each bulk population of cells were generated by RNA-seq with 10,974 expressed genes detected. Pairwise comparisons between cell types revealed specific expression, while enrichment analysis identified 1,885 to 3,591 significantly enriched genes across all 8 cell types. Gene Ontology analysis for the top 25% of significantly enriched genes (SEG) showed high enrichment of biological processes related to the nature of each cell type. Comparison of gene expression indicated highly significant correlations between pig cells and corresponding human PBMC bulk RNA-seq data available in Haemopedia. Second, higher resolution of distinct cell populations was obtained by single-cell RNA-sequencing (scRNA-seq) of PBMC. Seven PBMC samples were partitioned and sequenced that produced 28,810 single cell transcriptomes distributed across 36 clusters and classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, B cell, conventional CD4 and CD8 {beta} T cells, NK cells, and {gamma}{delta} T cells. Signature gene sets from the human Haemopedia data were assessed for relative enrichment in genes expressed in pig cells and integration of pig scRNA-seq with a public human scRNA-seq dataset provided further validation for similarity between human and pig data. The sorted porcine bulk RNAseq dataset informed classification of scRNA-seq PBMC populations; specifically, an integration of the datasets showed that the pig bulk RNAseq data helped define the CD4CD8 double-positive T cell populations in the scRNA-seq data. Overall, the data provides deep and well-validated transcriptomic data from sorted PBMC populations and the first single-cell transcriptomic data for porcine PBMCs. This resource will be invaluable for annotation of pig genes controlling immunogenetic traits as part of the porcine Functional Annotation of Animal Genomes (FAANG) project, as well as further study of, and development of new reagents for, porcine immunology.

sentences: Mapping the biosynthetic pathway of a hybrid polyketide-nonribosomal peptide in a metazoan Hybrid polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between megasynthases. In the nematode Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides in animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional stand-alone enzymes that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes in the biosynthetic pathway and discover a novel mechanism of trafficking intermediates between a PKS and an NRPS. Specifically, we show that the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.

sentences: Hydroxyl-radical scavenging activity of hydrogen does not significantly contribute to its biological function Since Ohsawa et al. reported a biological antioxidant function of hydrogen in 2007, researchers have now shown it to exert protective effects in a wide range of human and animal disease models. Clinical observations and scientific arguments suggest that a selective scavenging property of H2 cannot adequately explain the beneficial effects of hydrogen. However, there is no experiment challenging the original published data, which suggested that molecular hydrogen dissolved in solution reacts with hydroxyl radicals in cell-free systems. Here we report that a hydrogen-saturated solution (0.6 mM) did not significantly reduce hydroxyl radicals in the Fenton system using 1 mM H2O2. We replicated the same condition as Ohsawas study (i.e. 5 {micro}M H2O2), and observed a decrease in *OH radicals in both the H2-rich and N2-rich solutions, which may be caused by a decreased dissolved oxygen concentration. Finally, we determined the effect of hydrogen on a high-valence iron enzyme, horseradish peroxidase (HRP), and found that hydrogen could directly increase HRP activity in a dose-dependent manner. Overall, these results indicate that although H2 and *OH can react, the reaction rate is too low to have physiological function. The target of hydrogen is more complex, and its interaction with enzymes or other macro-molecules deserve more attention and in-depth study.

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Sensitisation of colonic nociceptors by TNFα is dependent on TNFR1 expression and p38 MAPK activity Visceral pain is a leading cause of morbidity in gastrointestinal diseases, which is exacerbated by the gut related side-effects of many analgesics. New treatments are needed and further understanding of the mediators and mechanisms underpinning visceral nociception in disease states is required to facilitate this. The pro-inflammatory cytokine TNF is linked to pain in patients with inflammatory bowel disease or irritable bowel syndrome and has been shown to sensitise colonic sensory neurons. Somatic, TNF-triggered thermal and mechanical hypersensitivity is mediated by TRPV1 signalling and p38 MAPK activity respectively, downstream of TNFR1 receptor activation. We therefore hypothesised that TNFR1-evoked p38 MAPK activity may also be responsible for TNF sensitisation of colonic afferent responses to the TRPV1 agonist capsaicin, and noxious distension of the bowel. Using Ca2+ imaging of dorsal root ganglia sensory neurons, we observed TNF-mediated increases in intracellular [Ca2+] and sensitisation of capsaicin responses. The sensitising effects of TNF were dependent on TNFR1 expression and attenuated by TRPV1 and p38 MAPK inhibition. Consistent with these findings, ex vivo colonic afferent fibre recordings demonstrated enhanced response to noxious ramp distention of the bowel and bath application of capsaicin following TNF pre-treatment. Responses were reversed by p38 MAPK inhibition and absent in tissue from TNFR1 knockout mice. Our findings demonstrate a contribution of TNFR1, p38 MAPK and TRPV1 to TNF-induced sensitisation of colonic afferents, highlighting the potential utility of these drug targets for the treatment of visceral pain in GI disease.

Key Points SummaryO_LITNF sensitises sensory neurons and colonic afferents to the TRPV1 agonist capsaicin.
C_LIO_LITNF mediated sensitisation of sensory neurons and colonic nociceptors is dependent on TNFR1 expression.
C_LIO_LITNF sensitisation of sensory neurons and colonic afferents to capsaicin and noxious ramp distension is abolished by inhibition of p38 MAPK.
C_LI

sentences: Heterodimers of functionally divergent ARF-GEF paralogues prevented by self-interacting dimerisation domain Functionally divergent paralogs of homomeric proteins do not form potentially deleterious heteromers, which requires distinction between self and non-self (Hochberg et al., 2018; Marchant et al, 2019; Marsh and Teichmann, 2015). In Arabidopsis, two ARF guanine-nucleotide exchange factors (ARF-GEFs) related to mammalian GBF1, named GNOM and GNL1, can mediate coatomer complex (COPI)-coated vesicle formation in retrograde Golgi-endoplasmic reticulum (ER) traffic (Geldner et al., 2003; Richter et al., 2007; Teh and Moore, 2007). Unlike GNL1, however, GNOM is also required for polar recycling of endocytosed auxin efflux regulator PIN1 from endosomes to the plasma membrane. Here we show that these paralogues form homodimers constitutively but no heterodimers. We also address why and how GNOM and GNL1 might be kept separate. These paralogues share a common domain organisation and each N-terminal dimerisation (DCB) domain can interact with the complementary fragment ({Delta}DCB) of its own and the other protein. However, unlike self-interacting DCBGNOM (Grebe et al., 2000; Anders et al., 2008), DCBGNL1 did not interact with itself nor DCBGNOM. DCBGNOM removal or replacement with DCBGNL1, but not disruption of cysteine bridges that stabilise DCB-DCB interaction, resulted in GNOM-GNL1 heterodimers which impaired developmental processes such as lateral root formation. We propose precocious self-interaction of the DCBGNOM domain as a mechanism to preclude formation of fitness-reducing GNOM-GNL1 heterodimers.

sentences: Blue and green food webs respond differently to elevation and land use While aquatic (blue) and terrestrial (green) food webs are parts of the same landscape, it remains unclear whether they respond similarly to shared environmental gradients. We use empirical community data from hundreds of sites across Switzerland, and show that blue and green food webs have different structural and ecological properties along elevation as a temperature proxy, and among various land-use types. Specifically, in green food webs, their modular structure increases with elevation and the overlap of consumers diet niche decreases, while the opposite pattern is observed in blue food webs. Such differences between blue and green food webs are particularly pronounced in farmland-dominated habitats, indicating that anthropogenic habitat modification moderates the climatic effects on food webs but differently in blue versus green systems. These findings indicate general structural differences between blue and green food webs and suggest their potential divergent future alterations through land use or climatic changes.

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Cryo-EM structure of human Nup155 reveals the biochemical basis for atrial fibrillation linked genetic mutation R391H Human nuclear pore complexes are composed of [~]32 distinct nucleoporins to facilitate bidirectional nucleo-cytoplasmic transport. Many of them have been associated with various human diseases such as an inherited mutation (R391H) in Nup155 is shown as the clinical cause of atrial fibrillation and sudden cardiac arrest. Due to the lack of structural knowledge and mechanistic insights, the roles of Nups in NPC assembly and relevance in human diseases are very restricted. Here, we show the cryo-EM structure of human Nup155 at 5.2-5.7. [A] resolution deciphered from 3 distinct particle classes: N-terminus (19-863), C-terminus (864-1337), and longer N-terminus (19-1069). It revealed intrinsic plasticity at the middle domain of Nup155 and the role of species-specific loop regions in an atypical 7-bladed {beta}-propeller domain to provide a distinct interface for Nup93 and Nup35. Due to the proximity of these Nups interacting sites near the Arginine-391 position, atrial fibrillation linked genetic mutation (R391H) causes dissociation from NPC in absence of N-terminal 112 residues.

HighlightsO_LICryo-EM structure of human Nup155 at 5.2 [A] resolution
C_LIO_LISeven bladed {beta}-propeller domain at N-terminus of Nup155 exhibited distinct features for interaction with Nup35 and Nup93
C_LIO_LIThe middle domain of Nup155 is highly dynamic in nature
C_LIO_LIStructural mapping allows mechanistic interpretation of AF linked R391H mutation
C_LI

sentences: Interaural time difference sensitivity under binaural cochlear implant stimulation even at high pulse rates up to 900 pps Spatial hearing remains one of the major challenges for bilateral cochlear implant (biCI) users, and early deaf patients in particular are often completely insensitive to interaural time differences (ITDs) delivered through biCIs. One popular theory held is that this may be due to a lack of early binaural experience. However, previously we have shown that neonatally deafened rats fitted with biCIs in adulthood quickly learn to discriminate ITDs as well as their normal hearing litter mates (Rosskothen-Kuhl et al. 2021), and perform orders of magnitude better than human biCI users. Our unique behaving biCI rat model allows us to investigate other possible limiting factors of prosthetic binaural hearing, such as the effect of stimulus pulse rate and envelope shape. Previous work has indicated that ITD sensitivity may decline substantially at the high pulse rates often used in clinical practice. We therefore measured behavioral ITD thresholds in neonatally deafened, adult implanted biCI rats to pulse trains of 50, 300, 900 and 1800 pulses per second (pps), with either rectangular or Hanning window envelopes. Our rats exhibited very high sensitivity to ITDs at pulse rates up to 900 pps for both envelope shapes, a pulse rate commonly used in clinical practice. However, ITD sensitivity declined to near zero at 1800 pps, for both Hanning and rectangular windowed pulse trains. Current clinical cochlear implant (CI) processors are often set to pulse rates [≥]900 pps, but ITD sensitivity in human CI listeners is often reported to decline sharply above [~]300 pps. Our results suggest that the relatively poor ITD sensitivity seen at >300 pps in human CI users may not reflect the hard upper limit of biCI ITD performance in the mammalian auditory pathway. Perhaps with training or better CI strategies good binaural hearing may be achievable at pulse rates high enough to allow good sampling of speech envelopes while delivering usable ITDs.

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In Utero Activation of NK Cells in Congenital CMV Infection BackgroundCongenital cytomegalovirus (CMV) infection is the most common infectious cause of birth defects and neurological damage in newborns. Despite a well-established role for NK cells in control of CMV infection in older children and adults, it remains unknown whether fetal NK cells can sense and respond to CMV infection acquired in utero.

MethodsHere, we investigate the impact of congenital CMV infection on the neonatal NK cell repertoire by assessing the frequency, phenotype, and functional profile of NK cells in cord blood samples from newborns with congenital CMV and from uninfected controls enrolled in a birth cohort of Ugandan mothers and infants.

ResultsWe find that neonatal NK cells from congenitally CMV infected newborns show increased expression of cytotoxic mediators, signs of maturation and activation, and an expansion of mature CD56-negative NK cells, an NK cell subset associated with chronic viral infections in adults. Activation was particularly prominent in NK cell subsets expressing the Fc{gamma} receptor CD16, indicating a role for antibody-mediated immunity against CMV in utero.

ConclusionThese findings demonstrate that NK cells can be activated in utero and suggest that NK cells may be an important component of the fetal and infant immune response against CMV.

sentences: A systematic approach for finding herbicide synergies Combining herbicides into a double dose is a common approach to overcome the potential for herbicide resistance by weeds. Many herbicide mixtures can be antagonistic and they are rarely synergistic. Here, 24 commercial herbicides, each representing a mode of action were used to create a matrix of all 276 unique combinations to search for new synergies in agar with Arabidopsis thaliana. Herbicides were used at an appropriate sub-lethal dose such that any synergies gave visible growth inhibition. We found five synergies including three new ones, namely mesotrione-norflurazon, mesotrione-clethodim and paraquat-clomazone. All three new synergies were reproducible in soil-grown conditions. Interestingly, all three new combinations included a bleaching herbicide, suggesting synergy might be a class specific phenomenon. We also found that mesotrione-norflurazon and mesotrione-clethodim combinations remained synergistic against lettuce (Lactuca sativa), but not tef (Eragrostis tef). Our study shows that screening herbicide mixtures against A. thaliana is an efficient approach for finding rare herbicide synergies.

sentences: Small Molecule Stabilization of PINK-1/PINK1 Improves Neurodegenerative Disease Macroautophagic recycling of dysfunctional mitochondria, known as mitophagy, is essential for mitochondrial homeostasis and cell viability. Accumulation of defective mitochondria and impaired mitophagy have been widely implicated in many neurodegenerative diseases, and loss-of-function mutations of two regulators of mitophagy, PINK1 and Parkin, are amongst the most common causes of recessive Parkinsons disease. Activation of mitophagy via pharmacological treatments may be a feasible approach for combating neurodegeneration. In this effort, we screened [~]45,000 small molecules for the ability to activate mitophagy. A high-throughput, whole-organism, phenotypic screen was conducted by monitoring stabilization of PINK-1/PINK1, a key event in mitophagy activation, in a Caenorhabditis elegans strain carrying a Ppink-1::PINK-1::GFP reporter. We obtained eight hits that induced mitophagy, as evidenced by increased mitochondrial fragmentation and autophagosome formation. Several of the compounds also reduced ATP production, oxygen consumption, mitochondrial mass, and/or mitochondrial membrane potential. Importantly, we found that treatment with two compounds, which we named PS83 and PS106 (more commonly known as sertraline) reduced neurodegenerative disease phenotypes (including delayed paralysis in a C. elegans Alzheimers model) in a PINK-1/PINK1-dependent manner. This report presents a promising step toward the identification of compounds that will stimulate mitochondrial turnover.

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KIM-1-mediated anti-inflammatory activity is preserved by MUC1 induction in the proximal tubule during ischemia-reperfusion injury. Cell-associated kidney injury molecule-1 (KIM-1) exerts an anti-inflammatory role following kidney injury by mediating efferocytosis and downregulating the NF-{kappa}B pathway. KIM-1 cleavage blunts its anti-inflammatory activities. We reported that Mucin 1 (MUC1) is protective in a mouse model of ischemia-reperfusion injury (IRI). As both KIM-1 and MUC1 are induced in the proximal tubule (PT) during IRI and are ADAM17 substrates, we tested the hypothesis that MUC1 protects KIM-1 activity. Muc1 KO mice and wild-type (WT) littermates were subjected to IRI. KIM-1, MUC1 and ADAM17 levels (and signaling pathways) were assessed by immunoblotting. PT localization was assessed by confocal microscopy and in situ proximity ligation assay. Findings were extended using human kidneys and urine, and KIM-1-mediated efferocytosis assays in mouse PT cultures. In response to tubular injury in mouse and human kidneys, we observed induction and co-expression of KIM-1 and MUC1 in the PT. Compared to WT, Muc1 KO mice had higher urinary KIM-1 and lower kidney KIM-1. KIM-1 was apical in PT of WT kidneys, but predominately with luminal debris in Muc1 KO mice. Efferocytosis was reduced in Muc1 KO PT cultures when compared to WT cells, while inflammation was increased in Muc1 KO kidneys when compared to WT mice. MUC1 was cleaved by ADAM17 in PT cultures, and blocked KIM-1 shedding in MDCK cells. We conclude that KIM-1-mediated efferocytosis and thus anti-inflammatory activity during IRI is preserved in the injured kidney by MUC1 inhibition of KIM-1 shedding.

NEW & NOTEWORTHYKIM-1 plays a key role in the recovery of the tubule epithelium during renal IRI by mediating efferocytosis and associated signaling that suppresses inflammation. Excessive cleavage of KIM-1 by ADAM17 provides decoy receptor that aggravates efferocytosis and subsequent signaling. Our data from studies in mice, patients and cultured cells show that MUC1 is also induced during IRI and competes with KIM-1 for cleavage by ADAM17. Consequently, MUC1 protects KIM-1 anti-inflammatory activity in the damaged kidney.

sentences: Neuroprotective derivatives of tacrine that target NMDA receptor and acetyl cholinesterase - Design, synthesis and biological evaluation The complex and multifactorial nature of neuropsychiatric diseases demands multi-target drugs that can intervene with various sub-pathologies underlying disease progression. Targeting the impairments in cholinergic and glutamatergic neurotransmissions with small molecules has been suggested as one of the potential disease-modifying approaches for Alzheimers disease (AD). Tacrine, apotent inhibitor of acetylcholinesterase (AChE) is the first FDA approved drug for the treatment of AD. Tacrine is also a low affinity antagonist of N-methyl-D-aspartate receptor (NMDAR). However, tacrine was withdrawn from its clinical use later due to its hepato-toxicity. With an aim to develop novel high affinity multi-target directed ligands (MTDLs) against AChE and NMDAR, with reduced hepatotoxicity, we performed in silico structure-based modifications on tacrine, chemical synthesis of the derivatives and in vitro validation of their activities. Nineteen such derivatives showed inhibition with IC50 values in the range of 18.53{+/-}2.09 to 184.09{+/-}19.23 nM against AChE and 0.27{+/-}0.05 to 38.84{+/-}9.64 M against NMDAR. Some of the selected compounds also protected rat primary cortical neurons from glutamate induced excitotoxicity. Two of the tacrine derived MTDLs, 201 and 208 exhibited in vivo efficacy in rats by protecting against behavioral impairment induced by administration of the excitotoxic agent, monosodium glutamate. Additionally, several of these synthesized compounds also exhibited promising inhibitory activities against butyrylcholinesterase and {beta}-secretase. Given the therapeutic potential of MTDLs in disease-modifying therapy, our studies revealed several promising MTDLs of which 201 appears to be a potential candidate for immediate preclinical and clinical evaluations.

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Sense of absence: Spatial perception through active sensing by insect antennal mechanosensory system Animals perceive their surroundings by using various modalities of sensory inputs to navigate their locomotion. Nocturnal insects such as crickets use mechanosensory inputs mediated by their antennae to navigate under dark conditions. Active sensing with voluntary antennal movements improves spatial information, but it remains unclear how accurately the insects can perceive the surrounding space by using their antennal system. Crickets exhibit escape behavior in response to a short air-puff, which is detected by the abdominal mechanosensory organ called cerci and is perceived as a "predator approach" signal. We placed objects of different shapes at different locations with which the cricket actively made contact using its antenna. We then examined the effects on wind-elicited escape. The crickets changed their movement trajectory depending on the shape and location of the objects so that they could avoid collision with these obstacles even when the escape behavior was triggered by another modality of stimulus. For instance, when a wall was placed in front of the crickets so that it was detected by one side of their antenna, the escape trajectory in response to a stimulus from behind was significantly biased toward the side opposite the wall. However, if the antenna on the free side without the wall was ablated, this modulation to avoid collision diminished, suggesting that the antenna on the free side provided information of "absence" of obstacles. This study demonstrated that crickets were able to perceive spatial information, including the presence or absence of objects by active sensing with their antennal system.

Summary StatementCrickets can acquire spatial information such as shape, location and orientation of objects through active sensing by antennal mechanosensory system, which also provides information about the absence of objects.

sentences: The origin and evolution of loqs2: a gene encoding an antiviral dsRNA binding protein in Aedes mosquitoes Mosquito borne viruses such as dengue, Zika, yellow fever and Chikungunya cause millions of infections every year. These viruses are mostly transmitted by two urban-adapted mosquito species, Aedes aegypti and Aedes albopictus, that appear to be more permissive to arbovirus infections compared to closely related species, although mechanistic understanding remains unknown. Aedes mosquitoes may have evolved specialized antiviral mechanisms that potentially contribute to the low impact of viral infection. Recently, we reported the identification of an Aedes specific double-stranded RNA binding protein (dsRBP), named Loqs2, that is involved in the control of infection by dengue and Zika viruses in Ae. aegypti. Loqs2 interacts with two important co-factors of the RNA interference (RNAi) pathway, Loquacious (Loqs) and R2D2, and seems to be a strong regulator of the antiviral defense. However, the origin and evolution of loqs2 remains unclear. Here, we describe that loqs2 likely originated from two independent duplications of the first dsRNA binding domain (dsRBD) of loquacious that occurred before the radiation of the Aedes Stegomyia subgenus. After its origin, our analyses suggest that loqs2 evolved by relaxed positive selection towards neofunctionalization. In fact, loqs2 is evolving at a faster pace compared to other RNAi components such as loquacious, r2d2 and Dicer-2 in Aedes mosquitoes. Unlike loquacious, transcriptomic analysis showed that loqs2 expression is tightly regulated, almost restricted to reproductive tissues in Ae. aegypti and Ae. albopictus. Transgenic mosquitoes engineered to ubiquitously express loqs2 show massive dysregulation of stress response genes and undergo developmental arrest at larval stages. Overall, our results uncover the possible origin and neofunctionalization of a novel antiviral gene, loqs2, in Aedes mosquitoes that ultimately may contribute to their effectiveness as vectors for arboviruses.

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Drosophila architectural protein CTCF is not essential for fly survival and is able to function independently of CP190 CTCF is the most likely ancestor of proteins that contain large clusters of C2H2 zinc finger domains (C2H2) and is conserved among most bilateral organisms. In mammals, CTCF functions as the main architectural protein involved in the organization of topology-associated domains (TADs). In vertebrates and Drosophila, CTCF is involved in the regulation of homeotic genes. Previously, null mutations in the dCTCF gene were found to result in death during the stage of pharate adults, which failed to eclose from their pupal case. Here, we obtained several new null dCTCF mutations and found that the complete inactivation of dCTCF appears to be limited to phenotypic manifestations of the Abd-B gene and fertility of adult flies. Many modifiers that are not associated with an independent phenotypic manifestation can significantly enhance the expressivity of the null dCTCF mutations, indicating that other architectural proteins are able to functionally compensate for dCTCF inactivation in Drosophila. We also mapped the 715-735 aa region of dCTCF as being essential for the interaction with the BTB (Broad-Complex, Tramtrack, and Bric a brac) and microtubule-targeting (M) domains of the CP190 protein, which binds to many architectural proteins. However, the mutational analysis showed that the interaction with CP190 was not essential for the functional activity of dCTCF in vivo.

HighlightsO_LIThe dCTCF 715-735 aa region interacts with the BTB and M domains of CP190
C_LIO_LIInteraction with CP190 is not essential for dCTCF function in vivo
C_LIO_LINull dCTCF mutants are viable but show severely reduced fertility
C_LI

sentences: Re-entry into mitosis and regeneration of intestinal stem cells through enteroblast dedifferentiation in Drosophila midguts Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis. In mammalian intestines, upon ablation of resident stem cells, the progenies of intestinal stem cells (ISCs) such as secretory cells and tuft cells can dedifferentiate to generate ISCs to drive epithelial regeneration, but whether and how the ISC progenies dedifferentiate to generate ISCs under physiological conditions remains unknown. Here we show that infection of pathogenic bacteria induces enteroblasts (EBs) as one type of ISC progenies to re-enter the mitotic cycle in the Drosophila intestine. The re-entry into mitosis is dependent on epithermal growth factor receptor (EGFR)-Ras signaling and ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive EBs cell-autonomously to re-enter into mitosis. In addition, we examined whether EBs gain ISC identity as a prerequisite to divide, but the immunostaining with stem cell marker Delta shows that these dividing EBs do not gain ISC identity. After employing lineage tracing experiments, we further demonstrate that EBs dedifferentiate to generate functional ISCs after symmetric divisions of EBs. Together, our study in Drosophila intestines uncovers a new role of EGFR-Ras signaling in regulating re-entry into mitosis and dedifferentiation during regeneration and reveals a novel mechanism by which ISC progenies undergo dedifferentiation through a mitotic division, which has important implication to mammalian tissue homeostasis and tumorigenesis.

sentences: Accurate model of liquid-liquid phase behaviour of intrinsically-disordered proteins from data-driven optimization of single-chain properties Many intrinsically disordered proteins (IDPs) may undergo liquidliquid phase separation (LLPS) and participate in the formation of membraneless organelles in the cell, thereby contributing to the regulation and compartmentalisation of intracellular biochemical reactions. The phase behaviour of IDPs is sequence-dependent, and its investigation through molecular simulations requires protein models that combine computational efficiency with an accurate description of intra- and intermolecular interactions. We developed a general coarse-grained model of IDPs, with residue-level detail, based on an extensive set of experimental data on single-chain properties. Ensemble-averaged experimental observables are predicted from molecular simulations, and a data-driven parameter-learning procedure is used to identify the residue-specific model parameters that minimize the discrepancy between predictions and experiments. The model accurately reproduces the experimentally observed conformational propensities of a set of IDPs. Through two-body as well as large-scale molecular simulations, we show that the optimization of the intramolecular interactions results in improved predictions of protein self-association and LLPS.

sentences: Neurotensin neurons in the central extended amygdala control energy balance Overeating and a sedentary life style are major causes of obesity and related metabolic disorders. Identification of the neurobiological processes that regulate energy balance will facilitate development of interventions for these disorders. Here we show that the Neurotensin-expressing neurons in the mouse IPAC (IPACNts), a nucleus of the central extended amygdala, bidirectionally coordinate hedonic feeding and physical activity, thereby regulating energy balance, metabolic processes and bodyweight. IPACNts are preferentially activated by consumption of highly palatable food or exposure to its taste and smell. Activating IPACNts promotes food intake in a palatability-dependent manner and decreases locomotion. Conversely, inhibiting IPACNts selectively reduces palatable food intake and dramatically enhances physical activity and energy expenditure, and in parallel stimulates physiological responses that oppose diet-induced obesity and metabolic dysfunctions. Thus, a single neuronal population, Neurotensin-expressing neurons in the IPAC, acts to control obesogenic and leptogenic processes by synergistically coordinating energy intake and expenditure with metabolism.

sentences: Sex differences in the strength of selection under facultative sex Facultative sex, the ability to reproduce both sexually and asexually, is widespread across the tree of life. In anisogamous species, the frequency of sex modulates selection on traits with sex-specific expression. Current theory on conditional gene expression posits that the strength of selection on loci only expressed by a subset of individuals, and/or in a subset of environments, is proportional to the frequency of expressers in the population. We show here that this assumption does not hold when the subsets in question are males or females (because of the Fisher condition) and is most important in facultatively sexual populations. In this case, the proportion of sexually produced offspring is not determined by male frequency (sex ratio), but on relative female investment in sexual versus asexual reproduction. This breaks the link between the frequency of expressers and selection for loci with sex-specific expression. However, certain conditions can re-establish this link, for example male traits that predict mating success better under strong male-male competition, and sex ratio affecting the relative fecundity of sexual versus asexual females. Our work highlights the importance of the Fisher condition for understanding the efficiency of selection and has implications for differences in the genetic load among sexes.

sentences: Functionality of the putative surface glycoproteins of the Wuhan spiny eel influenza virus A panel of novel influenza-like virus sequences were recently documented in jawless fish, ray-finned fish, and amphibians. Of these, the Wuhan spiny eel influenza virus (WSEIV) was found to phylogenetically cluster with influenza B viruses as a sister clade. Influenza B viruses have been historically documented to circulate only in humans, with certain virus isolates found in harbor seals. It is therefore interesting that a similar virus was potentially found in fish. Here we characterized the functionality and antigenicity of the putative hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins of the WSEIV to better understand this virus and its pandemic potential. Upon functional characterization of NA, we identified that the WSEIV NA-like protein has sialidase activity comparable to B/Malaysia/2506/2004 influenza B virus NA, making it a bona fide neuraminidase that could be inhibited by NA inhibitors. Testing of the functionality of HA was carried out including receptor specificity, stability, and preferential airway protease cleavage and showed very specific binding to monosialic ganglioside 2 (GM2). To probe the degree of conservation of target epitopes, binding of known broadly cross-reactive monoclonal antibodies targeting the influenza B HA and NA, respectively, were assessed through enzyme linked immunosorbent assays against recombinant WSEIV glycoproteins. Human serum samples of patients with antibodies to influenza B viruses were used to determine the cross-reactivity against these novel glycoproteins. Very few monoclonal antibodies - notably including pan NA antibody 1G01 - showed cross-reactivity and reactivity from human sera was basically absent. In summary, we have conducted a functional and antigenic characterization of the glycoproteins of the novel WSEIV to assess if it is indeed a bona fide influenza virus potentially circulating in ray-finned fish.

sentences: Deep learning from phylogenies to uncover the transmission dynamics of epidemics Widely applicable, accurate and fast inference methods in phylodynamics are needed to fully profit from the richness of genetic data in uncovering the dynamics of epidemics. Standard methods, including maximum-likelihood and Bayesian approaches, generally rely on complex mathematical formulae and approximations, and do not scale with dataset size. We develop a likelihood-free, simulation-based approach, which combines deep learning with (1) a large set of summary statistics measured on phylogenies or (2) a complete and compact representation of trees, which avoids potential limitations of summary statistics and applies to any phylodynamics model. Our method enables both model selection and estimation of epidemiological parameters from very large phylogenies. We demonstrate its speed and accuracy on simulated data, where it performs better than the state-of-the-art methods. To illustrate its applicability, we assess the dynamics induced by superspreading individuals in an HIV dataset of men-having-sex-with-men in Zurich. Our tool PhyloDeep is available on github.com/evolbioinfo/phylodeep.

sentences: Intranasal inhibitor blocks Omicron and other variants of SARS-CoV-2 The emergence of the SARS-CoV-2 Omicron variant capable of escaping neutralizing antibodies emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Nasal epithelium is rich in the ACE2 receptor and important for SARS-CoV-2 transmission by supporting early viral replication before seeding to the lung1. Intranasal administration of SARS-CoV-2 neutralizing antibodies or antibody fragments has shown encouraging potential as a protective measure in animal models2-5. However, there remains a need for SARS-CoV-2 blocking agents that are more economical to produce in large scale, while less vulnerable to mutational variation in the neutralization epitopes of the viral Spike glycoprotein. Here we describe TriSb92, a highly manufacturable trimeric human nephrocystin SH3 domain-derived antibody mimetic targeted against a conserved region in the receptor-binding domain of the Spike. TriSb92 potently neutralizes SARS-CoV-2 and its variants of concern, including Delta and Omicron. Intranasal administration of a modest dose of TriSb92 (5 or 50 micrograms) as early as eight hours before the challenge with SARS-CoV-2 B.1.351 efficiently protected mice from infection. The target epitope of TriSb92 was defined by cryo-EM, which revealed triggering of a conformational shift in the Spike trimer rather than competition for ACE2 binding as the molecular basis of its strong inhibitory action. Our results highlight the potential of intranasal inhibitors in protecting susceptible individuals from SARS-CoV-2 infection, and describe a novel type of inhibitor that could be of use in addressing the challenge posed by the Omicron variant.

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GenOtoScope: Towards automating ACMG classification of variants associated with congenital hearing loss Since next-generation sequencing (NGS) has become widely available, large gene panels containing up to several hundred genes can be sequenced cost-efficiently. However, the interpretation of the often large numbers of sequence variants detected when using NGS is laborious, prone to errors and often not comparable across laboratories. To overcome this challenge, the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) introduced standards and guidelines for the interpretation of sequencing variants. Further gene- and disease-specific refinements regarding hereditary hearing loss have been developed since then. With more than 200 genes associated with hearing disorders, the manual inspection of possible causative variants is especially difficult and time consuming. We developed an open-source bioinformatics tool GenOtoScope, which automates all ACMG/AMP criteria that can be assessed without further individual patient information or human curator investigation, including the refined loss of function criterion ("PVS1"). Two types of interfaces are provided: (i) a command line application to classify sequence variants in batches for a set of patients and (ii) a user-friendly website to classify single variants. We compared the performance of our tool with two other variant classification tools using two hearing loss data sets, which were manually annotated either by the ClinGen Hearing Loss Gene Curation Expert Panel or the diagnostics unit of our human genetics department. GenOtoScope achieved the best average accuracy and precision for both data sets. Compared to the second-best tool, GenOtoScope improved accuracy metric by 25.75% and 4.57% and precision metric by 52.11% and 12.13% on the two data sets respectively. The web interface is freely accessible. The command line application along with all source code, documentation and example outputs can be found via the project GitHub page.

Author summaryNew high-throughput sequencing technologies can produce massive amounts of information and are utilized by laboratories to explain the often complex genetic aetiology of hereditary diseases. The most common sensory disease, hearing loss, is often hereditary and has a high impact on a patients every-day life. To use these sequencing technologies effectively, software tools were developed that can aid researchers interpreting genetic data by semi-automatically classifying the biologic (and thus potentially medical) impact of detected variants (the alterations of the patients genome compared to the human reference genome). The available genetic variant classification tools are either not designed specifically for the interpretation of variants detected in subjects with hearing loss or they do not allow researchers to use them for batch classification of all variants detected, e. g. in a study group. To address this drawback, we developed GenOtoScope, an open-source tool that automates the pathogenicity classification of variants potentially associated with congenital hearing loss. GenOtoScope can be applied for the automatic classification of all variants detected in a set of probands.

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MVPAlab: A Machine Learning decoding toolbox for multidimensional electroencephalography data Background and ObjectiveThe study of brain function has recently expanded from classical univariate to multivariate analyses. These multivariate, machine learning-based algorithms afford neuroscientists extracting more detailed and richer information from the data. However, the implementation of these procedures is usually challenging, especially for researchers with no coding experience. To address this problem, we have developed MVPAlab, a MATLAB-based, flexible decoding toolbox for multidimensional electroencephalography and magnetoencephalography data.

MethodsThe MVPAlab Toolbox implements several machine learning algorithms to compute multivariate pattern analyses, cross-classification, temporal generalization matrices and feature and frequency contribution analyses. It also provides access to an extensive set of preprocessing routines for, among others, data normalization, data smoothing, dimensionality reduction and supertrial generation. To draw statistical inferences at the group level, MVPAlab includes a non-parametric cluster-based permutation approach.

ResultsA sample electroencephalography dataset was compiled to test all the MVPAlab main functionalities. Significant clusters (p<0.01) were found for the proposed decoding analyses and different configurations, proving the software capability for discriminating between different experimental conditions.

ConclusionsThis toolbox has been designed to include an easy-to-use and intuitive graphic user interface and data representation software, which makes MVPAlab a very convenient tool for users with few or no previous coding experience. In addition, MVPAlab is not for beginners only, as it implements several high and low-level routines allowing more experienced users to design their own projects in a highly flexible manner.

sentences: The Shu Complex Prevents Mutagenesis and Cytotoxicity of Single-Strand Specific Alkylation Lesions Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans, express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells, exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Finally, the Shu complex exhibits increased affinity for 3meC-containing DNA. Thus, our work identifies a novel mechanism for coping with alkylation adducts.

sentences: Placental and fetal characteristics of the Ohia mouse line recapitulate outcomes in human hypoplastic left heart syndrome Congenital heart defects (CHDs) are the most common birth defect worldwide. The morbidity and mortality associated with these defects is compounded by increased frequency of fetal growth abnormalities. In the Ohia mouse model of hypoplastic left heart syndrome (HLHS), the double homozygous genotype is embryonically lethal at mid-pregnancy; a time in which optimal establishment of the placenta is crucial to fetal survival. We aimed to characterize placental and fetal growth and development in the double heterozygous genotype (Sap130m/+Pcdha9m/+) to determine whether the genetic mutations associated with HLHS in the Ohia mouse also affect the placenta. The frequency of fetuses with reduced weight near term was shifted in the Sap130m/+Pcdha9m/+ fetuses compared to wildtype. This shift in fetal weight distribution in the Sap130m/+Pcdha9m/+ fetuses was associated with reduced labyrinth region area (P<0.001) and reduced fetal capillary density (P<0.001) in the placentas. Positive correlations were observed between fetal weight and placenta mRNA expression of several nutrient transporters in the Sap130m/+Pcdha9m/+ fetuses but not observed in the wildtype. Increased protein expression of Slc7a5 (P<0.05) and Slc7a8 (P<0.05) was also found in the placenta of Sap130m/+Pcdha9m/+ fetuses. This data shows, despite a potential compensatory mechanism to increase nutrient transport, abhorrent placental vascularization leads to inadequate fetal growth in the Ohia mouse model. Such differences are similar to findings in studies of human placentas and highlights the importance of this mouse model in continuing to understand the developmental links and disruptions to the heart-placenta axis.

sentences: Heterogeneity in the gene regulatory landscape of leiomyosarcoma. Soft-tissue sarcomas are group of rare but highly aggressive malignancies. It is a tremendously heterogeneous group of tumors. Characterizing inter-tumor heterogeneity is crucial for selecting suitable cancer therapy as the presence of diverse molecular subgroups of patients can be associated with disease outcome or response to treatment. However, no methods have been developed to characterize heterogeneity based on genome-wide patient-specific regulatory networks. In this work, we propose a simple but efficient approach to characterize inter-tumor regulatory network heterogeneity, which we call PORCUPINE (Principal Components Analysis to Obtain Regulatory Contributions Using Pathway-based Interpretation of Network Estimates). PORCUPINE uses as input individual patient regulatory networks, represented by estimated regulatory interactions between transcription factors and their target genes, and a list of genes assigned to biological pathways in order to identify key pathways that drive heterogeneity among individuals. We used PORCUPINE to model regulatory heterogeneity in leiomyosarcoma, one of the most common softtissue sarcomas subtypes. We applied it to 80 genome-wide leiomyosarcoma regulatory networks modeled on data from The Cancer Genome Atlas and validated the results in an independent dataset of 37 leiomyosarcoma cases from the German Cancer Research Center. PORCUPINE identified 37 pathways, including pathways that represent potential targets for treatment of subgroups of leiomyosarcoma patients, such as FGFR and CTLA4 inhibitory signaling. PORCUPINE thereby provides a robust way of analyzing and interpreting patient-specific regulatory networks and is the first step towards implementing network-informed personalized medicine in leiomyosarcoma.

sentences: bayesynergy: flexible Bayesian modelling of synergistic interaction effects in in-vitro drug combination experiments The effect of cancer therapies is often tested pre-clinically via in-vitro experiments, where the post-treatment viability of the cancer cell population is measured through assays estimating the number of viable cells. In this way, large libraries of compounds can be tested, comparing the efficacy of each treatment. Drug interaction studies focus on the quantification of the additional effect encountered when two drugs are combined, as opposed to using the treatments separately. In the bayesynergy R package, we implement a probabilistic approach for the description of the drug combination experiment, where the observed dose response curve is modelled as a sum of the expected response under a zero-interaction model and an additional interaction effect (synergistic or antagonistic). The interaction is modelled in a flexible manner, using a Gaussian process formulation. Since the proposed approach is based on a statistical model, it allows the natural inclusion of replicates, handles missing data and uneven concentration grids, and provides uncertainty quantification around the results. The model is implemented in the Stan programming language providing a computationally efficient sampler, a fast approximation of the posterior through variational inference, and features parallel processing for working with large drug combination screens.

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The Clade Displacement Index: how to detect horizontal gene transfers in unrooted gene trees While most genes of any organism are inherited vertically - i.e. from its parent organisms - sometimes they can be exchanged between unrelated species in a process known as the horizontal gene transfer (HGT). Studies of HGT contribute to our knowledge about the mechanisms of evolution, including the emergence of new pathogens, and a great deal of effort has been put into different methods of finding transferred genes. The golden standard of HGT detection is the analysis of the incongruence between the gene and the species trees. Those methods typically require rooted trees, in which the direction of evolution is known. Gene trees are typically unrooted, and rooting them is yet another step in HGT analysis, prone to errors which may lead to wrong conclusions. A natural question arises: can HGTs be detected in gene trees without rooting them at all?

It turns out that, for a particular, yet broad, class of transfers, the answer to this question is: yes. It also turns out that the same methodology can be applied to complement the bootstrap support in assessing the stability of gene tree topology. In this article, we present the Clade Displacement Index, a measure of shift of a given clades location between two trees. We derive algorithms to compute it and give several examples of its applications to HGT detection and gene tree stability analysis. We finish by pointing out directions for further studies and an example that shows that not all HGTs are detectable without knowing the location of the root of the gene tree.

A Jupyter Notebook with the implementation and applications of CDI described in this paper is available at https://github.com/mciach/CDI

sentences: Molecular mechanism of a parasite kinesin motor and implications for its inhibition Plasmodium parasites cause malaria and are responsible annually for hundreds of thousands of deaths. They have a complex life cycle in which distinct stages are transmitted between, and reproduce in, human and mosquito hosts. In the light of emerging resistance to current therapies, components of the parasite replicative machinery are potentially important targets for anti-parasite drugs. Members of the superfamily of kinesin motors play important roles in the microtubule-based replicative spindle machinery, and kinesin-5 motors are established anti-mitotic targets in other disease contexts. We therefore studied kinesin-5 from Plasmodium falciparum (PfK5) and characterised the biochemical properties and structure of the PfK5 motor domain. We found that the PfK5 motor domain is an ATPase with microtubule plus-end directed motility. We used cryo-EM to determine the motors microtubule-bound structure in no nucleotide and AMPPNP-bound states. Despite significant sequence divergence in this motor, these structures reveal that this parasite motor exhibits classical kinesin mechanochemistry. This includes ATP-induced neck-linker docking to the motor domain, which is consistent with the motors plus-ended directed motility. Crucially, we also observed that a large insertion in loop5 of the PfK5 motor domain creates a dramatically different chemical environment in the well characterised human kinesin-5 drug-binding site. Our data thereby reveal the possibility for selective inhibition of PfK5 and can be used to inform future exploration of Plasmodium kinesins as anti-parasite targets.

sentences: Delta breakthrough infections elicit potent, broad and durable neutralizing antibody responses The SARS-CoV-2 Delta variant is currently responsible for most infections worldwide, including among fully vaccinated individuals. Although these latter infections are associated with milder COVID-19 disease relative to unvaccinated subjects, the specificity and durability of antibody responses elicited by Delta breakthrough cases remain unknown. Here, we demonstrate that breakthrough infections induce serum binding and neutralizing antibody responses that are markedly more potent, durable and resilient to spike mutations observed in variants of concern than those observed in subjects who were infected only or received only two doses of COVID-19 vaccine. However, wee show that Delta breakthrough cases, subjects who were vaccinated after SARS-CoV-2 infection and individuals vaccinated three times (without infection) have serum neutralizing activity of comparable magnitude and breadth indicate that multiple types of exposure or increased number of exposures to SARS-CoV-2 antigen(s) enhance spike-specific antibody responses. Neutralization of the genetically divergent SARS-CoV, however, was moderate with all four cohorts examined, except after four exposures to the SARS-CoV-2 spike, underscoring the importance of developing vaccines eliciting broad sarbecovirus immunity for pandemic preparedness.

sentences: Quantitative cell release from plant tissues for single-cell genomics Single-cell RNA-sequencing (scRNA-seq) can provide invaluable insight into cell development, cell type identification, and plant evolution. However, the resilience of the cell wall makes it difficult to dissociate plant tissues and release individual cells. Here, we show that plant tissues can be rapidly and quantitatively dissociated if the tissues are fixed prior to enzymatic digestion. Fixation enables digestion at high temperatures at which enzymatic activity is optimal and stabilizes the plant cell cytoplasm, rendering cells resistant to mechanical shear force. This protocol was applied to maize anthers and provided suitable single-cell expression data for the identification of the tapetum, endothecium, meiocytes, and epidermis, while providing putative marker genes and gene ontology information for the identification of unknown cell types. This approach also preserves morphology of the isolated cells, permitting many cell types to be identified without staining. Our fixation-based protocol can be applied to a range of plant species and tissues with minimal optimization.

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Common variants in breast cancer risk loci predispose to distinct tumor subtypes BackgroundGenome-wide association studies (GWAS) have identified multiple common breast cancer susceptibility variants. Many of these variants have differential associations by estrogen receptor (ER), but how these variants relate with other tumor features and intrinsic molecular subtypes is unclear.

MethodsAmong 106,571 invasive breast cancer cases and 95,762 controls of European ancestry with data on 173 breast cancer variants identified in previous GWAS, we used novel two-stage polytomous logistic regression models to evaluate variants in relation to multiple tumor features (ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and grade) adjusting for each other, and to intrinsic-like subtypes.

ResultsEighty-five of 173 variants were associated with at least one tumor feature (false discovery rate <5%), most commonly ER and grade, followed by PR and HER2. Models for intrinsic-like subtypes found nearly all of these variants (83 of 85) associated at P<0.05 with risk for at least one luminal-like subtype, and approximately half (41 of 85) of the variants were associated with risk of at least one non-luminal subtype, including 32 variants associated with triple-negative (TN) disease. Ten variants were associated with risk of all subtypes in different magnitude. Five variants were associated with risk of luminal A-like and TN subtypes in opposite directions.

ConclusionThis report demonstrates a high level of complexity in the etiology heterogeneity of breast cancer susceptibility variants and can inform investigations of subtype-specific risk prediction.

sentences: Identification and Characterization of Long Non-Coding RNA in Tomato Roots under Salt Stress As one of the most important vegetable crops in the world, the production of tomatoes was restricted by salt stress. Therefore, it is of great interest to analyze the salt stress tolerance genes. As the non-coding RNAs (ncRNAs) with a length of more than 200 nucleotides, long non-coding RNAs (lncRNAs) lack the ability of protein-coding, but they can play crucial roles in plant development and response to abiotic stresses by regulating gene expression. Nevertheless, there are few studies on the roles of salt-induced lncRNAs in tomatoes. Therefore, we selected wild tomato Solanum pennellii (S. pennellii) and cultivated tomato M82 to be materials. By high-throughput sequencing, 1044 putative lncRNAs were identified here. Among them, 154 and 137 lncRNAs were differentially expressed in M82 and S. pennellii, respectively. Through functional analysis of target genes of differentially expressed lncRNAs (DE-lncRNAs), some genes were found to respond positively to salt stress by participating in Abscisic Acid (ABA) signaling pathway, brassinosteroid (BR) signaling pathway, ethylene (ETH) signaling pathway and anti-oxidation process. We also construct a salt-induced lncRNA-mRNA co-expression network to dissect the putative mechanisms of high salt tolerance in S. pennellii. We analyze the function of salt-induced lncRNAs in tomato roots at the genome-wide levels for the first time. These results will contribute to understanding the molecular mechanisms of salt tolerance in tomatoes from the perspective of lncRNAs.

sentences: Standardized nuclear markers advance metazoan taxonomy Species are the fundamental units of life and their recognition is essential for science and society. DNA barcoding, the use of a single and often mitochondrial gene, has been increasingly employed as a universal approach for the identification of animal species. However, this approach faces several challenges. Here, we demonstrate with empirical data from a number of metazoan animal lineages that multiple nuclear-encoded markers, so called universal single-copy orthologs (USCOs) performs much better than the single barcode gene to discriminate closely related species. Overcoming the general shortcomings of mitochondrial DNA barcodes, USCOs also accurately assign samples to higher taxonomic levels. These loci thus provide a powerful and unifying framework for species delimitation which considerably improves the DNA-based inference of animal species.

sentences: A new protocol for speed vernalisation of winter cereals There are many challenges facing the development of high-yielding, nutritious crops for future environments. One limiting factor is generation time, which prolongs research and plant breeding timelines. Recent advances in speed breeding protocols have dramatically reduced generation time for many short-day and long-day species by optimising light and temperature conditions during plant growth. However, winter crops with a vernalisation requirement still require up to 6-10 weeks in low-temperature conditions before transition to reproductive development. Here, we tested a suite of environmental conditions and protocols to investigate if vernalisation can be satisfied more efficiently. We identified a vernalisation method consisting of exposing seeds at the soil surface to an extended photoperiod of 22 h day:2 h night at 10{degrees}C with transfer to speed breeding conditions that dramatically reduces generation time in both winter wheat (Triticum aestivum) and winter barley (Hordeum vulgare). Implementation of this protocol achieved up to five generations per year for winter wheat or barley, instead of the two typically obtained under standard vernalisation and plant growth conditions. The protocol has great potential to enhance training and to accelerate research, pre-breeding, and breeding outcomes focussed on winter crop improvement.

sentences: A ROR2 coding variant is associated with craniofacial variation in domestic pigeons Vertebrate craniofacial morphogenesis is a highly orchestrated process that is directed by evolutionarily conserved developmental pathways 1,2. Within species, canalized developmental programs typically produce only modest morphological variation. However, as a result of millennia of artificial selection, the domestic pigeon (Columba livia) displays radical variation in craniofacial morphology within a single species. One of the most striking cases of pigeon craniofacial variation is the short beak phenotype, which has been selected in numerous breeds. Classical genetic experiments suggest that pigeon beak length is regulated by a small number of genetic factors, one of which is sex-linked (Ku2 locus) 3-5. However, the molecular genetic underpinnings of pigeon craniofacial variation remain unknown. To determine the genetic basis of the short beak phenotype, we used geometric morphometrics and quantitative trait loci (QTL) mapping on an F2 intercross between a short-beaked Old German Owl (OGO) and a medium-beaked Racing Homer (RH). We identified a single locus on the Z-chromosome that explains a majority of the variation in beak morphology in the RH x OGO F2 population. In complementary comparative genomic analyses, we found that the same locus is also strongly differentiated between breeds with short and medium beaks. Within the differentiated Ku2 locus, we identified an amino acid substitution in the non-canonical Wnt receptor ROR2 as a putative regulator of pigeon beak length. The non-canonical Wnt (planar cell polarity) pathway serves critical roles in vertebrate neural crest cell migration and craniofacial morphogenesis 6,7. In humans, homozygous ROR2 mutations cause autosomal recessive Robinow syndrome, a rare congenital disorder characterized by skeletal abnormalities, including a widened and shortened facial skeleton 8,9. Our results illustrate how the extraordinary craniofacial variation among pigeons can reveal genetic regulators of vertebrate craniofacial diversity.

sentences: Cell size asymmetries in the sea star embryo Cell size asymmetries are often linked to cell fate decisions, due to cell volumes and cell fate determinants being unequally partitioned during asymmetric cell divisions. A clear example is found in the sea urchin embryo, where a characteristic and obvious unequal 4th cleavage generates micromeres, which are necessary for mesendoderm cell fate specification. Unlike sea urchin development, sea star development is generally thought to have only equal cleavage. However, subtle cell size asymmetries can be observed in sea star embryos; whether those cell size asymmetries are consistently produced during sea star development and if they are involved in cell fate decisions remains unknown. Using confocal live imaging of early embryos we quantified cell size asymmetries in 16-cell stage embryos of two sea star species, Patiria miniata and Patiriella regularis. Using photoconversion to perform lineage tracing, we find that the position of the smallest cells of P. miniata embryos is biased toward anterior ventral tissues. However, both blastomere dissociation and mechanical removal of one small cell do not prevent dorsoventral (DV) axis formation, suggesting that embryos compensate for the loss of those cells and asymmetric partitioning of maternal determinants is not strictly necessary for DV patterning. Finally, we show that manipulating cell size to introduce artificial cell size asymmetries is not sufficient to direct the positioning of the future DV axis in P. miniata embryos. Our results show that although cell size asymmetries are consistently produced during sea star early cleavage and may be predictive of the DV axis, they are not necessary to instruct DV axis formation.

sentences: Life history traits and dispersal shape neutral genetic diversity in metapopulations Genetic diversity at population scale, depends on species life-history traits, population dynamics and local and global environmental factors. We first investigate the effect of life-history traits on the neutral genetic diversity of a single population using a deterministic mathematical model. When the population is stable, we show that semelparous species with precocious maturation and iteroparous species with delayed maturation exhibit higher diversity because their life history traits tend to balance the lifetimes of non reproductive individuals (juveniles) and adults which reproduce. Then, we extend our model to a metapopulation to investigate the additional effect of dispersal on diversity. We show that dispersal may truly modify the local effect of life history on diversity. As a result, the diversity at the global scale of the metapopulation differ from the local diversity which is only described through local life history traits of the populations. In particular, dispersal usually promotes diversity at the global metapopulation scale.

sentences: The Transcriptional Co-Activator Taz Contributes to the Differentiation of a Salivary Gland Epithelial Cell Line Towards a Myoepithelial Phenotype The salivary gland can be permanently impaired by radiation treatment for head and neck cancers. Efforts at tissue regeneration have focused on saliva-producing acinar cells. However, myoepithelial cells are also critical to gland function, but mechanisms that regulate their differentiation are poorly defined. To study myoepithelial differentiation, we employed mSG-PAC1 murine salivary gland epithelial cells. We demonstrate that mSG-PAC1 spheroids exhibit phenotypic plasticity between pro-acinar and myoepithelial cell fates. Increased expression of pro-acinar/acinar or myoepithelial RNAs was identified from spheroids cultured under different media conditions by microarray followed by gene-set enrichment analysis. Spheroids cultured with different medium components expressed proteins typical of either acinar or myoepithelial cells, as detected by immunocytochemistry. We demonstrate that the pattern of TAZ expression in the epithelial compartment of the differentiating murine salivary gland correlates with the expression of the myoepithelial marker alpha-SMA, as is the case for TAZ expression in mSG-PAC1 spheroids. Our analysis also indicates that YAP/TAZ target genes are upregulated together with myoepithelial markers. Importantly, siRNA targeting of TAZ expression in mSG-PAC1 spheroids diminished the expression of myoepithelial markers. Our results in this in vitro cell model implicate TAZ signaling in myoepithelial differentiation.

sentences: Anti-allergic and anti-inflammatory activities of black cumin extracts in in vitro and in vivo model systems Black cumin (Nigella sativa) is a widely used ingredient of traditional medicine for its broad-spectrum pharmacological actions, including anti-allergic, bronchial asthma, and anti-inflammatory properties. We sought to evaluate BC extracts efficacy for their anti-allergic and anti-inflammatory properties using a comprehensive in vitro, in vivo, and silico experimental setup. To investigate whether BC extract has anti-inflammatory, anti-allergic, and analgesic therapeutic potentials in vitro and in vivo. The activity of BC was assessed through anti-allergic activity on rat basophilic leukemia-2H3 cell line, anti-inflammatory activity on J774.1A cell line, anti-inflammatory activity by carrageenan-induced rat paw edema, analgesic activity by acetic acid-induced writhing test, and ingenuity analysis of the BC extracts in inflammation control. BC exerted potent anti-allergic activity by inhibiting antigen-induced degranulation. An anti-inflammatory effect is shown by inhibiting TNF- production. The acetic acid-induced writhing test shown a dose-dependent reduction of writhing number following BC administration. Rat paw edema test shown the dose-dependent reduction of paw edema volume following BC administration. Ingenuity Pathway Analysis (IPA) suggested BC extracts containing ferulic acid, p-coumaric acid, kaempferol, and quercetin can inhibit inflammation. This study suggests that bioactive compounds in BC extract act as an anti-allergic and anti-inflammatory agent by regulating several downstream and upstream inflammation pathways.

sentences: Dispersal between interconnected patches can reduce the total population size Human activities increasingly result in a fragmentation of natural ecosystems. However, the ecological consequences of fragmentation remain poorly understood. While some studies report that fragmentation may enhance population growth, others suggest the opposite pattern. Here we investigated how habitat connectivity affects the population size of a single species when habitat patches differ in quality. We combined dispersal experiments, in which bacterial populations of Escherichia coli were repeatedly transferred between two qualitatively different environments, with a process-based mathematical model. Both experiments and model consistently revealed that increased dispersal between patches reduced the total population size, thus demonstrating a detrimental effect of habitat connectivity on population size. This observation could be explained with a net loss of individuals upon migration from a productive to an overcrowded patch. Our findings suggest that conservation measures, which promote movement between fragmented habitats, such as dispersal corridors or stepping stones, are potentially detrimental for some species.

sentences: Generation of SIV resistant T cells and Macrophages from Nonhuman Primate Induced Pluripotent Stem Cells with Edited CCR5 locus Adoptive therapies with genetically modified somatic T cells rendered HIV resistant have shown promise for AIDS therapy. A renewable source of HIV resistant human T cells from induced pluripotent stem cells (iPSCs) would further facilitate and broaden the applicability of these therapies. Here, we report successful targeting of the CCR5 locus in iPSCs generated from peripheral blood T cells (T-iPSCs) or fibroblasts (fib-iPSCs) from Mauritian Cynomolgus macaques (MCM), using CRISPR/Cas9 technology. We found that CCR5 editing does not affect pluripotency or hematopoietic and T cell differentiation potentials of fib-iPSCs. However, deletion of CCR5 in T-iPSCs leads to selective loss of their T cell redifferentiation potential without affecting myeloid development. T cells and macrophages produced from CCR5-edited MCM- iPSCs did not support replication of the CCR5-tropic simian immunodeficiency viruses SIVmac239 (T-cell tropic) and SIVmac316 (macrophage-tropic). Overall, these studies provide a platform for further exploration of AIDS therapies based on gene-edited iPSCs in a nonhuman primate preclinical model.

sentences: Trophic specialization on unique resources in one of the most celebrated examples of sympatric speciation, Barombi Mbo crater lake cichlids Divergent ecological selection often results in trophic niche partitioning and is one of the central processes underlying sympatric speciation. However, there are still few studies of niche partitioning in putative examples of sympatric speciation in the wild. Here we conducted the first quantitative study of dietary niche partitioning in one of the most celebrated examples of sympatric speciation, Barombi Mbo cichlids, using stomach contents and stable isotope analyses. We found little evidence for trophic niche partitioning among any Barombi Mbo cichlids, even among the nine species coexisting in sympatry in the littoral zone. Stable isotope analyses supported these conclusions of minimal dietary overlap. However, we did find extraordinary dietary specialization in some species, including spongivory and feeding on terrestrial ants, both unique feeding strategies among freshwater fishes. Stomach contents of the spongivore (Pungu maclareni) were 20% freshwater sponge, notable considering that only 0.04% of all fishes consume sponges. Overall, we conclude that while there is less trophic niche partitioning than expected among Barombi Mbo cichlids, there is evidence for dietary specialization on rare resources in support of Liems paradox.

sentences: A Hierarchy of Biomolecular Proportional-Integral-Derivative Feedback Controllers for Robust Perfect Adaptation and Dynamic Performance Proportional-Integral-Derivative (PID) feedback controllers have been the most widely used controllers in industry for almost a century due to their good performance, simplicity, and ease of tuning. Motivated by their success in various engineering disciplines, PID controllers recently found their way into synthetic biology, where the design of feedback molecular control systems has been identified as an important goal. In this paper, we consider the mathematical realization of PID controllers via biomolecular interactions. We propose an array of topologies that offer a compromise between simplicity and high performance. We first demonstrate that different Proportional-Integral (PI) controllers exhibit different capabilities for enhancing the dynamics and reducing variance (cell-to-cell variability). Next, we introduce several derivative controllers that are realized based on incoherent feedforward loops acting in a feedback configuration. Alternatively, we show that differentiators can be realized by placing molecular integrators in a negative feedback loop--an arrangement that can then be augmented by PI components to yield PID feedback controllers. We demonstrate that the derivative component can be exploited for enhancing system stability, dramatically increasing the molecular control systems dynamic performance, and for reducing the noise effect on the output of interest. The PID controller features are established through various deterministic and stochastic analyses as well as numerical simulations. Finally, we provide an experimental demonstration using a recently developed hybrid setup, the cyberloop, where the controller is implemented in silico to control a biological genetic circuit in vivo. The large array of novel biomolecular PID controllers introduced here forms a basis for the design and construction of advanced high-performance biomolecular control systems that robustly regulate the dynamics of living systems.

sentences: Regulation of Chromatin Accessibility by hypoxia and HIF Reduced oxygen availability (hypoxia) can act as a signalling cue in physiological processes such as development, but also in pathological conditions such as cancer or ischaemic disease. As such, understanding how cells and organisms respond to hypoxia is of great importance. The family of transcription factors called Hypoxia Inducible Factors (HIFs) coordinate a transcriptional programme required for survival and adaptation to hypoxia. The effects of hypoxia and HIF on the chromatin accessibility landscape are still unclear. Here, using genome wide mapping of chromatin accessibility via ATAC-seq, we find hypoxia induces loci specific changes in chromatin accessibility enriched at hypoxia transcriptionally responsive genes. These changes are predominantly HIF dependent, reversible upon reoxygenation and partially mimicked by chemical HIF stabilisation independent of molecular dioxygenase inhibition. This work demonstrates that indeed, HIF stabilisation is necessary and sufficient to alter chromatin accessibility in hypoxia, with implications for our understanding of gene expression regulation by hypoxia and HIF.

sentences: In vivo CRISPR screens reveal Serpinb9 and Adam2 as regulators of immune therapy response in lung cancer How the genetic landscape of a tumor governs the tumors response to immunotherapy remains largely elusive. Here, we established a direct in vivo CRISPR/Cas9 gene editing methodology to assess the immune-modulatory capabilities of 573 putative cancer genes associated with altered cytotoxic activity in human cancers. Using KrasG12D- and BrafV600E-driven mouse lung cancer models, we identify Serpinb9 and Adam2 as our top immune suppressive and immune enhancing genes, respectively. Mechanistically, we show that Serpinb9 ablation in KrasG12D- and BrafV600E-mutant lung tumor cells greatly enhances the efficacy of cytotoxic T-cells in vitro and in vivo. ADAM2 is a cancer testis antigen broadly expressed in human cancers such as lung adenocarcinoma (13.9%), renal (74.7%), prostate (72.4%), uterine (28.6%) and invasive breast (9.5%) cancer. In our mouse models, we show that Adam2 expression is induced in KrasG12D- but not BrafV600E-driven murine lung tumors and that its expression is further enhanced by immunotherapy. We show that loss of Adam2 significantly decreases KrasG12D-lung tumor burden but blocks the efficacy of cytotoxic T-cells. Consistently, Adam2 overexpression dramatically increases tumor growth and enhances immunotherapy efficacy. Mechanistically, we find that Adam2s oncogenic function depends on modulating the tumor immune microenvironment by restraining productive type I and type II interferon responses as well as cytokine signaling, reducing the presentation of tumor-associated antigen, and modulating surface expression of several immunoregulatory receptors within Kras-driven lung tumors. Adam2 expression also leads to reduced levels of immune checkpoint inhibitors such as Pd-l1, Lag3, Tigit and Tim3. This reduced exhaustion within the tumor microenvironment may explain why ex vivo expanded and adoptively transferred cytotoxic T-cells show enhanced cytotoxic efficacy against Adam2 overexpressing lung tumors. Together, our study highlights the power of integrating cancer genomic with in vivo CRISPR/Cas9 screens to uncover how cancer-associated genetic alterations control responses to immunotherapies.

sentences: A CLEM approach to access to the ultrastructure at the graft interface in Arabidopsis thaliana Despite recent progress in our understanding of the graft union formation, we still know little about the cellular events underlying the grafting process. This is partially due to the difficulty of reliably targeting the graft interface in electron microscopy to study its ultrastructure and three-dimensional architecture. To overcome this technological bottleneck, we developed a correlative light electron microscopy approach (CLEM) to study the graft interface with high ultrastructural resolution. Grafting hypocotyls of Arabidopsis thaliana lines expressing YFP or mRFP in the endoplasmic reticulum allowed the efficient targeting of the grafting interface for under light and electron microscopy. To explore the potential of our method to study sub-cellular events at the graft interface, we focused on the formation of secondary plasmodesmata (PD) between the grafted partners. We showed that 4 classes of PD were formed at the interface and that PD introgression into the call wall was initiated equally by both partners. Moreover, the success of PD formation appeared not systematic with a third of PD not spanning the cell wall entirely. Characterizing the ultrastructural characteristics of these failed PD gives us insights into the process of secondary PD biogenesis. We showed that the thinning of the cell wall and the endoplasmic reticulum-plasma membrane tethering seem to be required for the establishment of symplastic connections between the scion and the rootstock. The resolution reached in this work shows that our CLEM method offer a new scale to the study for biological processes requiring the combination of light and electron microscopy.

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microRNAs Over-expressed in Diabetic Foot Ulcers Healing - Computational Modeling of Molecular Structure BackgroundVasculopathy associated with diabetic neuropathy are important risk factors for the diabetic foot ulcers development. Diabetic foot ulcers is severe complication that occur in about 15% of people with diabetes, being able require hospitalization and amputation in its treatment.

ObjectiveDesign in silico the molecular structure of micro-ribonucleic acid (miRNA) overexpressed in diabetic foot ulcers healing.

MethodWe performed a careful search of the nucleotide sequence of 8 miRNAs over-expressed in diabetic foot ulcers, designing in silico the molecular structure of following miRNAs: miRNA-146a, miRNA-155, miRNA-132, miRNA-191, miRNA-21, miRNA-203a, miRNA-203b, and miRNA-210. The nucleotides were taken from GenBank of National Center for Biotechnology Information genetic sequence database. The sequences acquired were aligned with the Clustal W multiple alignment algorithms. The molecular modeling of structures was built using the RNAstructure, an automated miRNAs structure modelling server.

ResultsWe showed a search for nucleotide sequence and the design of the molecular structure of following miRNA over-expressed in diabetic foot ulcers healing: miRNA-146a, miRNA-155, miRNA-132, miRNA-191, miRNA-21, miRNA-203a, miRNA-203b, and miRNA-210. We produced a tutorial on a molecular model of the 8 miRNAs overexpressed in the diabetic foot by processing in silico projection of their molecular structures.

ConclusionWe show in silico secondary structures design of selected of 8 miRNAs over-expressed in diabetic foot ulcers healing by means of computational biology.

sentences: LINE-1 activation in the cerebellum drives ataxia Previous studies have revealed that dysregulation of long interspersed nuclear element 1 (LINE-1), a dominant class of transposable elements in the human genome, correlates with neurodegeneration1-3. Yet whether LINE-1 dysregulation is causal to disease pathogenesis has not been proven directly. Here, we demonstrate that expression of evolutionarily younger LINE-1 families is elevated in the cerebella of ataxia telangiectasia (AT) patients, which was correlated with extensive downregulation of epigenetic silencers. To examine whether LINE-1 activation causes neurologic disease, we established an approach to directly target and activate the promoter of a young family of LINE-1 in mice. LINE-1 activation in the cerebellum was sufficient to lead to robust progressive ataxia. Purkinje cells in the diseased mice exhibited marked electrophysiological dysfunctions and degeneration with a significant accumulation of cytoplasmic ribonucleoprotein LINE-1Orf1p aggregates, endoplasmic reticulum (ER) stress, and DNA damage. Treatment with lamivudine, a nucleoside reverse transcriptase inhibitor, blunted the disease progression by reducing DNA damage, attenuating gliosis and interferon gene signature, and recovering the loss of key functional molecules for calcium homeostasis in Purkinje cells. This study provides direct evidence that young LINE-1 activation drives ataxia phenotype, and points to its pleiotropic effects leading to DNA damage, inflammation, and dysfunction and degeneration of neurons.

sentences: Functional analysis of the N-terminal region of Vibrio FlhG, a MinD-type ATPase in flagellar number control GTPase FlhF and ATPase FlhG are two key factors involved in regulating the flagellum number in Vibrio alginolyticus. FlhG is a paralog of the Escherichia coli cell division regulator MinD, which has a longer N-terminal region. Deleting the N-terminal region of FlhG results in cells with multiple flagella. The Q9A mutation in the DQAxxLR motif of the N-terminal region prevents it from activating the GTPase activity of FlhF in vitro and results in a multi-flagellation phenotype. The mutant FlhG protein was remarkably reduced compared to that of the wild-type protein in vivo. When the mutant FlhG was expressed at the same level as the wild-type FlhG, the number of flagella was restored to the wild-type level. Once synthesized in Vibrio cells, the N-terminal region mutation in FlhG seems not to affect the protein stability. We speculated that the flhG translation efficiency is decreased by N-terminal mutation. Our results suggest that the N-terminal region of FlhG controls the number of flagella by adjusting the FlhF activity and the amount of FlhG in vivo. We speculate that the regulation by FlhG, achieved through transcription by the master regulator FlaK, is affected by the mutations, resulting in reduced flagellar formation by FlhF.

sentences: DNA supercoiling-mediated collective behavior of co-transcribing RNA polymerases Multiple RNA polymerases (RNAPs) transcribing a gene have been known to exhibit collective group behavior, causing the transcription elongation rate to increase with the rate of transcription initiation. Such behavior has long been believed to be driven by a physical interaction or "push" between closely spaced RNAPs. However, recent studies have posited that RNAPs separated by longer distances may cooperate via the DNA segment under transcription. Here, we present a theoretical model incorporating the mechanical coupling between RNAP translocation and the torsional response of supercoiled DNA. Using stochastic simulations, we demonstrate long-range cooperation between co-transcribing RNAPs mediated by DNA supercoiling. We find that inhibiting transcription initiation can slow down the already recruited RNAPs, in agreement with recent experimental observations, and predict that the average transcription elongation rate varies non-monotonically with the rate of transcription initiation. We further show that while RNAPs transcribing neighboring genes oriented in tandem can cooperate, those transcribing genes in divergent or convergent orientations can act antagonistically, and that such behavior holds over a large range of intergenic separations. Our model makes testable predictions, revealing how the mechanical interplay between RNAPs and the DNA they transcribe can govern a key cellular process.

sentences: Predictions of the SARS-CoV-2 Omicron Variant (B.1.1.529) Spike Protein Receptor-Binding Domain Structure and Neutralizing Antibody Interactions The genome of the SARS-CoV-2 Omicron variant (B.1.1.529) was released on November 22, 2021, which has caused a flurry of media attention due the large number of mutations it contains. These raw data have spurred questions around vaccine efficacy. Given that neither the structural information nor the experimentally-derived antibody interaction of this variant are available, we have turned to predictive computational methods to model the mutated structure of the spike proteins receptor binding domain and posit potential changes to vaccine efficacy. In this study, we predict some structural changes in the receptor-binding domain that may reduce antibody interaction without completely evading existing neutralizing antibodies (and therefore current vaccines).

sentences: NDC1 is necessary for stable assembly of the nuclear pore scaffold to establish nuclear transport in early C. elegans embryos Nuclear pore complexes (NPCs) are large protein assemblies that facilitate transport of macromolecules across the nuclear envelope (NE) [1, 2]. How thousands of NPCs rapidly assemble after open mitosis to form a functional NE is not known. Recruitment of the Nup107-160 outer ring scaffold to chromatin initiates NPC assembly. The Nup53/93 complex bridges the outer ring to the central channel to form a functional pore [3-6]. Nup53 interacts with the conserved transmembrane nucleoporin Ndc1; however, how Ndc1 contributes to post-mitotic NPC assembly is unclear [7-9]. Here, we use C. elegans embryos to show that the timely formation of a functional NE after mitosis depends on Ndc1. Endogenously tagged Ndc1 is recruited early to the reforming NE and is highly mobile in the nuclear rim. 3D analysis of post-mitotic NE formation revealed a decreased NPC density in NEs of ndc1 deleted embryos - continuous nuclear membranes contained few holes where assembling NPCs are normally located. Nup160 is highly mobile in NEs depleted of Ndc1 and outer ring scaffold components are less enriched at the rim. When both ndc1 and nup53 are absent, nuclear assembly fails. Together, these data show that Ndc1 dynamically associates with the NE and promotes stable association of the outer ring scaffold in the NE to facilitate NPC assembly after open mitosis. Furthermore, Ndc1 and Nup53 function in parallel to drive nuclear assembly. We propose that Ndc1 is a dynamic membrane adaptor that helps recruit and promote the self-assembly of the nuclear pore scaffold to drive post-mitotic NPC assembly.

sentences: Neutrophils initiate the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters The loss of smell related to SARS-CoV-2 infection is one of the most prevalent symptoms of COVID-19. It is now clear that this symptom is related to the massive infection by SARS-CoV-2 of the olfactory epithelium leading to its desquamation. However, the molecular mechanism behind the destabilization of the olfactory epithelium is less clear. Using golden Syrian hamster, we show here that while apoptosis remains at a low level in damaged infected epithelium, the latter is invaded by innate immunity cells. By depleting the neutrophil population or blocking the activity of neutrophil elastase-like proteinases, we reduced the damage induced by the SARS-CoV-2 infection. Surprisingly, the impairment of neutrophil activity led to a decrease of SARS-CoV-2 infection levels in the nasal cavity. Our results indicate a counterproductive role of neutrophils leading to the release of infected cells in the lumen of the nasal cavity and thereby enhanced spreading of the virus.

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Intergenerational microbial transmission in the little skate (Leucoraja erinacea) BackgroundMicrobial transmission from parent to offspring is hypothesized to be widespread in vertebrates. However, evidence for this is limited as many evolutionarily important clades remain unexamined. There is currently no data on the microbiota associated with any Chondrichthyan species during embryonic development, despite the global distribution, ecological importance, and phylogenetic position of this clade. In this study, we take the first steps towards filling this gap by investigating the microbiota associated with embryonic development in the little skate, Leucoraja erinacea, a common North Atlantic species and popular system for chondrichthyan biology.

MethodsTo assess the potential for bacterial transmission in an oviparous chondrichthyan, we used 16S rRNA amplicon sequencing to characterize the microbial communities associated with the skin, gill, and egg capsule of the little skate, at six points during ontogeny. Community composition was analyzed using the QIIME2 pipeline and microbial continuity between stages was tracked using FEAST.

ResultsWe identify site-specific and stage-specific microbiota dominated by the bacterial phyla Proteobacteria and Bacteroidetes. This composition is similar to, but distinct from, that of previously published data on the adult microbiota of other chondrichthyan species. Our data reveal that the skate egg capsule harbors a highly diverse bacterial community-particularly on the internal surface of the capsule-and facilitates intergenerational microbial transfer to the offspring. Embryonic skin and external gill tissues host similar bacterial communities; the skin and gill communities later diverge as the internal gills and skin denticles develop.

ConclusionsOur study is the first exploration of the chondrichthyan microbiota throughout ontogeny and provides the first evidence of vertical transmission in this group.

sentences: A natural genetic variation screen identifies insulin signaling, neuronal communication, and innate immunity as modifiers of hyperglycemia in the absence of Sirt1 Variation in the onset, progression, and severity of symptoms associated with metabolic disorders such as diabetes impairs the diagnosis and treatment of at-risk patients. Diabetes symptoms, and patient variation in these symptoms, is attributed to a combination of genetic and environmental factors, but identifying the genes and pathways that modify diabetes in humans has proven difficult. A greater understanding of genetic modifiers and the ways in which they interact with metabolic pathways could improve the ability to predict a patients risk for severe symptoms, as well as enhance the development of individualized therapeutic approaches. In this study we use the Drosophila Genetic Reference Panel (DGRP) to identify genetic variation influencing hyperglycemia associated with loss of Sirt1 function. Through analysis of individual candidate functions, physical interaction networks, and Gene Set Enrichment Analysis (GSEA) we identify not only modifiers involved in canonical glucose metabolism and insulin signaling, but also genes important for neuronal signaling and the innate immune response. Furthermore, reducing the expression of several of these candidates suppressed hyperglycemia, making them ideal candidate therapeutic targets. These analyses showcase the diverse processes contributing to glucose homeostasis and open up several avenues of future investigation.

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Inability to switch from ARID1A-BAF to ARID1B-BAF impairs exit from pluripotency and commitment towards neural crest differentiation in ARID1B-related neurodevelopmental disorders The BAF complex modulates chromatin accessibility. Specific BAF configurations have functional consequences, and subunit switches are essential for cell differentiation. ARID1B and its paralog ARID1A encode for mutually exclusive BAF subunits. De novo ARID1B haploinsufficient mutations cause a neurodevelopmental disorder spectrum, including Coffin-Siris syndrome, which is characterized by neurological and craniofacial features. Here, we reprogrammed ARID1B+/- Coffin-Siris patient-derived skin fibroblasts into iPSCs and modeled cranial neural crest cell (CNCC) formation. We discovered that ARID1B is active only during the first stage of this process, coinciding with neuroectoderm specification, where it is part of a lineage-specific BAF configuration (ARID1B-BAF), which includes SMARCA4 and nine additional subunits. ARID1B-BAF acts as a gatekeeper, ensuring exit from pluripotency and lineage commitment, by attenuating NANOG, SOX2 and thousands of enhancers directly regulated by these two pluripotency factors at the iPSC stage.

In iPSCs, these enhancers are maintained active by an ARID1A-containing BAF. At the onset of differentiation, cells transition from ARID1A-BAF to ARID1B-BAF, eliciting attenuation of the NANOG/SOX2 networks, and triggering pluripotency exit. Coffin-Siris patient cells fail to perform the ARID1A/ARID1B switch and maintain ARID1A-BAF at pluripotency enhancers throughout all stages of CNCC formation. This leads to a persistent and aberrant SOX2 and NANOG activity, which impairs CNCC formation. In fact, despite showing the typical neural crest signature (TFAP2A+, SOX9+), ARID1B-haploinsufficient CNCCs are also NANOG/OCT4-positive, in stark contrast with the ARID1B-wt CNCCs, which are NANOG/OCT4-negative.

These findings suggest a connection between ARID1B mutations, neuroectoderm formation, and a pathogenic mechanism for Coffin-Siris syndrome.

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Symbiosis with rhizobia limits range expansion only in polyploid legumes O_LIBoth mutualism and polyploidy are thought to influence invasion success in plants but few studies have tested their joint effects. Mutualism can limit range expansion when plants cannot find a compatible partner in a novel habitat, or facilitate range expansion when mutualism increases a plants niche breadth. Polyploids are also expected to have greater niche breadth because of greater self-compatibility and phenotypic plasticity, increasing invasion success.
C_LIO_LIFor 839 legume species, we compiled data from published sources to estimate ploidy, symbiotic status with rhizobia, specificity on rhizobia, and the number of introduced ranges.
C_LIO_LIWe found that diploid species have had limited spread around the globe regardless of whether they are symbiotic or how many partners of rhizobia they can host. Polyploids, in contrast, have been successfully introduced to many new ranges, but interactions with rhizobia constrain their range expansion. In a hidden state model of trait evolution, we also found evidence of a high rate of re-diploidization in symbiotic legume lineages, suggesting that symbiosis and ploidy may interact at macroevolutionary scales.
C_LIO_LIOverall, our results suggest that symbiosis with rhizobia affects range expansion only in polyploid legumes.
C_LI

sentences: Precision neoantigen discovery using large-scale immunopeptidomes and composite modeling of MHC peptide presentation Major histocompatibility complex (MHC)-bound peptides that originate from tumor-specific genetic alterations, known as neoantigens, are an important class of anti-cancer therapeutic targets. Accurately predicting peptide presentation by MHC complexes is a key aspect of discovering therapeutically relevant neoantigens. Technological improvements in mass-spectrometry-based immunopeptidomics and advanced modeling techniques have vastly improved MHC presentation prediction over the past two decades. However, improvement in the sensitivity and specificity of prediction algorithms is needed for clinical applications such as the development of personalized cancer vaccines, the discovery of biomarkers for response to checkpoint blockade and the quantification of autoimmune risk in gene therapies. Toward this end, we generated allele-specific immunopeptidomics data using 25 mono-allelic cell lines and created Systematic HLA Epitope Ranking Pan Algorithm (SHERPA), a pan-allelic MHC-peptide algorithm for predicting MHC-peptide binding and presentation. In contrast to previously published large-scale mono-allelic data, we used an HLA-null K562 parental cell line and a stable transfection of HLA alleles to better emulate native presentation. Our dataset includes five previously unprofiled alleles that expand MHC binding pocket diversity in the training data and extend allelic coverage in underprofiled populations. To improve generalizability, SHERPA systematically integrates 128 mono-allelic and 384 multi-allelic samples with publicly available immunoproteomics data and binding assay data. Using this dataset, we developed two features that empirically estimate the propensities of genes and specific regions within gene bodies to engender immunopeptides to represent antigen processing. Using a composite model constructed with gradient boosting decision trees, multiallelic deconvolution and 2.15 million peptides encompassing 167 alleles, we achieved a 1.44 fold improvement of positive predictive value compared to existing tools when evaluated on independent mono-allelic datasets and a 1.15 fold improvement when evaluating on tumor samples. With a high degree of accuracy, SHERPA has the potential to enable precision neoantigen discovery for future clinical applications.

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Induction of pancreatic neoplasia in the KRAS/TP53 Oncopig: preliminary report IntroductionFive years survival of pancreatic cancer (PC) remains low. The current murine models may not adequately mimic human PC and can be inadequate for device development. To develop a more accurate large animal model of PC, we induced pancreatic tumors in Oncopigs (transgenic swine expressing KRASG12D and TP53R167H mutants).

MethodsOncopigs (n = 16) were injected via laparotomy with adenovirus expresses Cre recombinase (AdCre) with or without interleukin 8 (IL-8) by either technique 1: into the main pancreatic duct (MPD; via duodenotomy) or by technique 2: into the duct of the isolated pancreatic connecting lobe (CL). Subjects were necropsied after [≤]10 week, followed by histological analysis, cytokine expression analysis, exome sequencing and transcriptome analysis.

ResultsIn a total of 16 OCM subjects, 12 subjects received pancreatic AdCre injection by technique 2 and 9 developed pancreatic tumor (incidence outcome by technique 2: 75%). All of these subjects expired within 2-5 weeks after the tumor induction procedure. All had a similar terminal course: failure to thrive, which was secondary to gastric outlet obstruction, which was secondary to a peripancreatic phlegmon, which was secondary to pancreatitis, which appeared to be secondary to tumor. None of the OCM subjects that underwent a control injection procedure (no AdCre) nor any of the WT littermates that underwent AdCre injection with or without IL-8 developed pancreatitis. Exome and transcriptome analysis of the porcine pancreatic tumors revealed similarity in molecular signatures and pathways with human pancreatic tumors.

ConclusionOncopigs developed pancreatic cancer in a short period of time with similar molecular characteristics like human PC. Further optimization to increase the longevity of this transgenic porcine cancer model will help to investigate the stage-wise progression and therapeutic responses against pancreatic cancer.

sentences: Mechanism of dopamine traveling waves in the striatum: theory and experiment Striatal dopamine (DA) encodes reward, with recent work showing that DA release occurs in spatiotemporal waves. However, the mechanism of DA waves is unknown. Here we report that the striatal cholinergic neuropil also exhibits wave-like activity, and that the spatial scale of striatal DA release is extended by nicotinic receptors. Based on these findings we hypothesized that the local reciprocal interaction between cholinergic interneurons (CIN) and DA axons suffices to drive endogenous traveling waves. We show that the morphological and physiological properties of the CIN-DA interaction can be modeled as a reaction-diffusion system that gives rise to traveling waves. Analytically-tractable versions of the model show that the structure and the nature of propagation of CIN and DA traveling waves depend on their coupling, and that traveling waves can give rise to empirically observed correlations between these signals. Our model provides a biophysical mechanism for wave formation and predicts that the observed DA and CIN waves are strongly coupled phenomena.

sentences: The first sheep graph pan-genome reveals the spectrum of structural variations and their effects on different tail phenotypes Structural variations (SVs) are a major contributor to genetic diversity and phenotypic variations, but their prevalence and functions in domestic animals are largely unexplored. Here, we assembled 26 haplotype-resolved genome assemblies from 13 genetically diverse sheep using PacBio HiFi sequencing. We constructed a graph-based ovine pan-genome and discovered 142,422 biallelic insertions and deletions, 7,028 divergent alleles and 13,419 multiallelic variations. We then used a graph-based approach to genotype the biallelic SVs in 684 individuals from 45 domestic breeds and two wild species. Integration with RNA-seq data allows to identify candidate expression-associated SVs. We demonstrate a direct link of SVs and phenotypes by localizing the putative causative insertion in HOXB13 gene responsible for the long-tail trait and identifying multiple large SVs associated with the fat-tail. Beyond generating a benchmark resource for ovine structural variants, our study highlights that animal genetic research will greatly benefit from using a pan-genome graph rather than a single reference genome.

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Practices for Measuring 3D Organelle Morphology and Generating Surfaces with Amira Analysis of 3D structures is of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have remained staples for imaging cellular structures, they lack the ability to image in 3D. However, recent technological advances, such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM), have allowed researchers to observe cellular ultrastructure in 3D. Here, we propose a standardized protocol using the visualization software Amira to quantify organelle morphologies in 3D; this method allows researchers to produce accurate and reproducible measurements of cellular structure characteristics. We demonstrate this applicability by utilizing SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.

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sentences: GLUCOCORTICOIDS REGULATE MITOCHONDRIAL FATTY ACID OXIDATION IN FETAL CARDIOMYOCYTES The late gestational rise in glucocorticoids contributes to the structural and functional maturation of the perinatal heart. Here, we hypothesised that glucocorticoid action contributes to the metabolic switch in perinatal cardiomyocytes from carbohydrate to fatty acid oxidation. In primary mouse fetal cardiomyocytes, dexamethasone treatment induced expression of genes involved in fatty acid oxidation and increased mitochondrial oxidation of palmitate, dependent upon glucocorticoid receptor (GR). Dexamethasone did not, however, induce mitophagy or alter the morphology of the mitochondrial network. In neonatal mice, dexamethasone treatment induced cardiac expression of fatty acid oxidation genes in vivo. However, dexamethasone treatment of pregnant C57Bl/6 mice at embryonic day (E)13.5 or E16.5 failed to induce fatty acid oxidation genes in fetal hearts assessed 24 hours later. Instead, at E17.5, fatty acid oxidation genes were down-regulated by dexamethasone, as was GR itself. PGC-1, required for glucocorticoid-induced maturation of primary mouse fetal cardiomyocytes in vitro, was down-regulated in vivo in fetal hearts at E17.5, 24 hours after dexamethasone administration. Similarly, following a course of antenatal corticosteroids in a sheep model of preterm birth, both GR and PGC-1 were down-regulated in fetal heart. These data suggest endogenous glucocorticoids support the perinatal switch to fatty acid oxidation in cardiomyocytes through changes in gene expression rather than gross changes in mitochondrial volume or mitochondrial turnover. Moreover, our data suggest that treatment with exogenous glucocorticoids may interfere with normal fetal heart maturation, possibly by down-regulating GR. This has implications for clinical use of antenatal corticosteroids when preterm birth is considered a possibility.

sentences: Portrait of a genus: the genetic diversity of Zea Maize is a globally valuable commodity and one of the most extensively studied genetic model organisms. However, we know surprisingly little about the extent and potential utility of the genetic variation found in the wild relatives of maize. Here, we characterize a high-density genomic variation map from ~700 genomes encompassing maize and all wild taxa of the genus Zea, identifying over 65 million single nucleotide polymorphisms (SNPs), 8 million Insertion/Deletion (InDel) polymorphisms, and over one thousand novel inversions. The variation map reveals evidence of selection within taxa displaying novel adaptations such as perenniality and regrowth. We focus in detail on evidence of convergent adaptation in highland teosinte and temperate maize. This study not only indicates the key role of hormone related pathways in highland adaptation and flowering time related pathways in high latitude adaptation, but also identifies significant overlap in the genes underlying adaptations to both environments. To show how this data can identify useful genetic variants, we generated and characterized novel mutant alleles for two flowering time candidate genes. This work provides the most extensive sampling to date of the genetic diversity inherent in the genus Zea, resolving questions on evolution and identifying adaptive variants for direct use in modern breeding.

sentences: Automatic classification of ICA components from infant EEG using MARA Automated systems for identifying and removing non-neural ICA components are growing in popularity among adult EEG researchers. Infant EEG data differs in many ways from adult EEG data, but there exists almost no specific system for automated classification of source components from paediatric populations. Here, we adapt one of the most popular systems for adult ICA component classification for use with infant EEG data. Our adapted classifier significantly outperformed the original adult classifier on samples of naturalistic free play EEG data recorded from 10 to 12-month-old infants, achieving agreement rates with the manual classification of over 75% across two validation studies (n=44, n=25). Additionally, we examined both classifiers ability to remove stereotyped ocular artifact from a basic visual processing ERP dataset, compared to manual ICA data cleaning. Here the new classifier performed on level with expert manual cleaning and was again significantly better than the adult classifier at removing artifact whilst retaining a greater amount of genuine neural signal, operationalised through comparing ERP activations in time and space. Our new system (iMARA) offers developmental EEG researchers a flexible tool for automatic identification and removal of artifactual ICA components.

sentences: High throughput hemogram of T cells using digital holographic microscopy and deep learning T cells of the adaptive immune system provide effective protection to the human body against numerous pathogenic challenges. Current labelling methods of detecting these cells, such as flow cytometry or magnetic bead labelling, are time consuming and expensive. To overcome these limitations, the label-free method of digital holographic microscopy (DHM) combined with deep learning has recently been introduced which is both time and cost effective. In this study, we demonstrate the application of digital holographic microscopy with deep learning to classify the key CD4+ and CD8+ T cell subsets. We show that combining DHM of varying fields of view, with deep learning, can potentially achieve a classification throughput rate of 78,000 cells per second with an accuracy of 76.2% for these morphologically similar cells. This throughput rate is 100 times faster than the previous studies and proves to be an effective replacement for labelling methods.

sentences: SARS-CoV-2 triggers Golgi fragmentation via down-regulation of GRASP55 to facilitate viral trafficking The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an enveloped RNA virus. Despite the high economic and life losses caused by SARS-CoV-2, the detailed viral cycle, especially how it assembles and traffics in the secretory pathway, remains largely unknown. Here, we show that SARS-CoV-2 infection induces global alterations of the host endomembrane system, including dramatic Golgi fragmentation. Disrupting Golgi function with small molecules strongly inhibits viral infection. Furthermore, expression of several SARS-CoV-2 proteins individually is sufficient to trigger Golgi fragmentation. Significantly, SARS-CoV-2 infection down-regulates GRASP55 but up-regulates TGN46 expression, while expression of GRASP55 or knockdown of TGN46 reduces the infection rate of both USA-WA1 and Delta variants of SARS-CoV-2. Our study reveals that SARS-CoV-2 modulates Golgi structure and function via altering GRASP55 and TGN46 expression to facilitate viral trafficking, indicating the Golgi as a novel therapeutic target to block SARS-CoV-2 infection.

sentences: Sensitive and specific spectral library searching with COSS and Percolator Maintaining high sensitivity while limiting false positives is a key challenge in peptide identification from mass spectrometry data. Here, we therefore investigate the effects of integrating the machine learning-based post-processor Percolator into our spectral library searching tool COSS. To evaluate the effects of this post-processing, we have used forty data sets from two different projects and have searched these against the NIST and MassIVE spectral libraries. The searching is carried out using two spectral library search tools, COSS and MSPepSearch with and without Percolator post-processing, and using sequence database search engine MS-GF+ as a baseline comparator. The addition of the Percolator rescoring step to COSS is effective and results in a substantial improvement in sensitivity and specificity of the identifications. COSS is freely available as open source under the permissive Apache2 license, and binaries and source code are found at https://github.com/compomics/COSS

sentences: The role of oxytocin in modulating self-other distinction brain: a pharmacological fMRI study The self-other distinction is crucial in human social cognition and social interaction. Studies have found that oxytocin (OT) sharpens the self-other perceptual boundary but with mixed results. Further, little is known if the effect of OT on self-resemblance facial perception exists, especially on its neural basis. Moreover, it is unclear if OT would influence the judgment in self-other discrimination when the other is a child or an adult. In the current double-blinded, placebo-controlled study, we investigated the effect of OT on self-face perception at both behavioral and neural levels. We morphed participants faces and strangers faces to create four stimuli conditions. After being treated by either OT or placebo (PL), participants reported whether a morphed face resembles themselves, or was morphed with their own faces, while being scanned with fMRI. Behavioral results showed that people judged adult-morphed faces better than child-morphed faces. fMRI results showed that the OT group exhibited generally increased activities in the visual area and IFG for self-morphed faces. Such difference was more pronounced in the adult face compared to child face conditions. Multivariate fMRI analysis revealed that the OT group showed better classification between self-morphed versus other-morphed faces, indicating that OT increased self-other distinction, especially for adult faces and in the left hemisphere. Our study shows the significant effect of OT on self-referential brain processes, providing evidence for the potential OTs effect on a left hemisphere self network.

sentences: Stratified Test Accurately Identifies Differentially Expressed Genes Under Batch Effects in Single-Cell Data Analyzing single-cell sequencing data from large cohorts is challenging. Discrepancies across experiments and differences among participants often lead to omissions and false discoveries in differentially expressed genes. We find that the Van Elteren test, a stratified version of the widely used Wilcoxon rank-sum test, elegantly mitigates the problem. We also modified the common language effect size to supplement this test, further improving its utility. On both simulated and real patient data we show the ability of Van Elteren test to control for false positives and false negatives. A comprehensive assessment using receiver operating characteristic (ROC) curve shows that Van Elteren test achieves higher sensitivity and specificity on simulated datasets, compared with nine state-of-the-art differential expression analysis methods. The effect size also estimates the differences between cell types more accurately.

sentences: Connectomic Analysis of the Drosophila Lateral Neuron Clock Cells Reveals the Synaptic Basis of Functional Pacemaker Classes. The circadian clock orchestrates daily changes in physiology and behavior to ensure internal temporal order and optimal timing across the day. In animals, a central brain clock coordinates circadian rhythms throughout the body and is characterized by a remarkable robustness that depends on synaptic connections between constituent neurons. The clock neuron network of Drosophila, which shares network motifs with clock networks in the mammalian brain yet is built of many fewer neurons, offers a powerful model for understanding the network properties of circadian timekeeping. Here we report an assessment of synaptic connectivity within a clock network, focusing on the critical lateral neuron (LN) clock neuron classes. Our results reveal that previously identified anatomical and functional subclasses of LNs represent distinct connectomic types. Moreover, we identify a small number of clock cell subtypes representing highly synaptically coupled nodes within the clock neuron network. This suggests that neurons lacking molecular timekeeping likely play integral roles within the circadian timekeeping network. To our knowledge, this represents the first comprehensive connectomic analysis of a circadian neuronal network.

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CellSium - Versatile Cell Simulator for Microcolony Ground Truth Generation SummaryTo address the need for ground truth data for deep learning based segmentation algorithms in microfluidic live-cell imaging, we present CellSium, a cell simulator primarily aimed at synthesizing realistic image sequences of bacterial microcolonies suitable for training neural networks.

Availability and ImplementationCellSium is free and open source software under the BSD license, implemented in Python, available at https://github.com/modsim/cellsium (DOI: 10.5281/zenodo.6193033), along with documentation, usage examples and Docker images.

Contactk.noeh@fz-juelich.de

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The guidance and adhesion protein FLRT2 dimerizes in cis via dual Small-X3-Small transmembrane motifs Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these  Small-X3-Small motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules.

FieldsStructural Biology and Biophysics / Computational Biology

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Pkd1 mutation has no apparent effects on peroxisome structure or lipid metabolism BackgroundMultiple studies of tissue and cell samples from patients and pre-clinical models of autosomal dominant polycystic kidney disease report abnormal mitochondrial function and morphology and suggest metabolic reprogramming is an intrinsic feature of this disease. Peroxisomes interact with mitochondria physically and functionally, and congenital peroxisome biogenesis disorders can cause various phenotypes, including mitochondrial defects, metabolic abnormalities and renal cysts. We hypothesized that a peroxisomal defect might contribute to the metabolic and mitochondrial impairments observed in autosomal dominant polycystic kidney disease.

MethodsUsing control and Pkd1-/- kidney epithelial cells, we investigated peroxisome abundance, biogenesis and morphology by immunoblotting, immunofluorescent and live cell imaging of peroxisome-related proteins and assayed peroxisomal specific {beta}-oxidation. We further analyzed fatty acid composition by mass spectrometry in kidneys of Pkd1fl/fl; Ksp-Cre mice. We also evaluated peroxisome lipid metabolism in published metabolomics datasets of Pkd1 mutant cells and kidneys. Lastly, we investigated if the C-terminus or full-length polycystin-1 co-localize with peroxisome markers by imaging studies.

ResultsPeroxisome abundance, morphology and peroxisome-related protein expression in Pkd1-/- cells were normal, suggesting preserved peroxisome biogenesis. Peroxisomal {beta}-oxidation was not impaired in Pkd1-/- cells, and there was no obvious accumulation of very long chain fatty acids in kidneys of mutant mice. Re-analysis of published datasets provide little evidence of peroxisomal abnormalities in independent sets of Pkd1 mutant cells and cystic kidneys, while providing further evidence of mitochondrial fatty acid oxidation defects. Imaging studies with either full length polycystin-1 or its C-terminus, a fragment previously shown to go to the mitochondria, showed minimal co-localization with peroxisome markers.

ConclusionsOur studies showed that loss of Pkd1 does not disrupt peroxisome biogenesis nor peroxisomedependent fatty acid metabolism.

Key points- While mitochondrial abnormalities and fatty acid oxidation impairment have been reported in ADPKD, no studies have investigated if peroxisome dysfunction contributes to these defects.
- We investigated peroxisome morphology, biogenesis and function in cell and animal models of ADPKD and investigated whether polycystin-1 co-localized with peroxisome proteins.
- Our studies show that loss of Pkd1 does not disrupt peroxisome biogenesis nor peroxisome-dependent fatty acid metabolism.

sentences: Nef dimerization defect abrogates HIV viremia and associated immune dysregulation in the Bone Marrow-Liver-Thymus-Spleen (BLTS) humanized mouse model Loss of function mutations in the human immunodeficiency virus (HIV) negative factor (Nef) gene are associated with reduced viremia, robust T cell immune responses, and delayed acquired immunodeficiency syndrome (AIDS) progression in humans. In vitro studies have shown that mutations in the Nef dimerization interface significantly attenuate viral replication and impair host defense. However, in vivo, mechanistic studies on the role of Nef dimerization in HIV infection are lacking. Humanized rodents with human immune cells are robust platforms for investigating the interactions between HIV and the human immune system. The bone marrow-liver-thymus-spleen (BLTS) humanized mouse model carries human immune cells and lymphoid tissues that facilitate anti-viral immune responses. Here, we employed the BLTS-humanized mouse model to demonstrate that preventing Nef dimerization abrogates HIV viremia and the associated immune dysregulation. This suggests that Nef dimerization may be a therapeutic target for future HIV cure strategies.

sentences: ACE2-containing defensosomes serve as decoys to inhibit SARS-CoV-2 infection Extracellular vesicles of endosomal origin, exosomes, mediate intercellular communication by transporting substrates with a variety of functions related to tissue homeostasis and disease. Their diagnostic and therapeutic potential has been recognized for diseases such as cancer in which signaling defects are prominent. However, it is unclear to what extent exosomes and their cargo inform the progression of infectious diseases. We recently defined a subset of exosomes termed defensosomes that are mobilized during bacterial infection in a manner dependent on autophagy proteins. Through incorporating protein receptors on their surface, defensosomes mediated host defense by binding and inhibiting pore-forming toxins secreted by bacterial pathogens. Given this capacity to serve as decoys that interfere with surface protein interactions, we investigated the role of defensosomes during infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19. Consistent with a protective function, exosomes containing high levels of the viral receptor ACE2 in bronchioalveolar lavage fluid from critically ill COVID-19 patients was associated with reduced ICU and hospitalization times. We found ACE2+ exosomes were induced by SARS-CoV-2 infection and activation of viral sensors in cell culture, which required the autophagy protein ATG16L1, defining these as defensosomes. We further demonstrate that ACE2+ defensosomes directly bind and block viral entry. These findings suggest that defensosomes may contribute to the antiviral response against SARS-CoV-2 and expand our knowledge on the regulation and effects of extracellular vesicles during infection.

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Transcriptome-wide association and prediction for carotenoids and tocochromanols in fresh sweet corn kernels Sweet corn is consistently one of the most highly consumed vegetables in the U.S., providing a valuable opportunity to increase nutrient intake through biofortification. Significant variation for carotenoid (provitamin A, lutein, zeaxanthin) and tocochromanol (vitamin E, antioxidants) levels is present in temperate sweet corn germplasm, yet previous genome-wide association studies (GWAS) of these traits have been limited by low statistical power and mapping resolution. Here, we employed a high-quality transcriptomic dataset collected from fresh sweet corn kernels to conduct transcriptome-wide association studies (TWAS) and transcriptome prediction studies for 39 carotenoid and tocochromanol traits. In agreement with previous GWAS findings, TWAS detected significant associations for four causal genes, {beta}-carotene hydroxylase (crtRB1), lycopene epsilon cyclase (lcyE),{gamma} -tocopherol methyltransferase (vte4), and homogentisate geranylgeranyltransferase (hggt1) on a transcriptome-wide level. Pathway-level analysis revealed additional associations for deoxy-xylulose synthase2 (dxs2), diphosphocytidyl methyl erythritol synthase2 (dmes2), cytidine methyl kinase1 (cmk1), and geranylgeranyl hydrogenase1 (ggh1), of which, dmes2, cmk1, and ggh1 have not previously been identified through maize association studies. Evaluation of prediction models incorporating genome-wide markers and transcriptome-wide abundances revealed a trait-dependent benefit to the inclusion of both genomic and transcriptomic data over solely genomic data, but both transcriptome- and genome-wide datasets outperformed a priori candidate gene-targeted prediction models for most traits. Altogether, this study represents an important step towards understanding the role of regulatory variation in the accumulation of vitamins in fresh sweet corn kernels.

Core IdeasO_LITranscriptomic data aid the study of vitamin levels in fresh sweet corn kernels.
C_LIO_LIcrtRB1, lcyE, dxs2, dmes2, and cmk1 were associated with carotenoid traits.
C_LIO_LIvte4, hggt1, and ggh1 were associated with tocochromanol traits.
C_LIO_LITranscriptomic data boosted predictive ability over genomic data alone for some traits.
C_LIO_LIJoint transcriptome- and genome-wide models achieved the highest predictive abilities.
C_LI

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Nomograms of Human Hippocampal Volume Shifted by Polygenic Scores Nomograms are important clinical tools applied widely in both developing and aging populations. They are generally constructed as normative models identifying cases as outliers to a distribution of healthy controls. Currently used normative models do not account for genetic heterogeneity. Hippocampal Volume (HV) is a key endophenotype for many brain disorders. Here, we examine the impact of genetic adjustment on HV nomograms and the translational ability to detect dementia patients.

Using imaging data from 35,686 healthy subjects aged 44 to 82 from the UK BioBank (UKBB), we built HV nomograms using gaussian process regression (GPR), extending the application age by 20 years, including dementia critical age ranges. Using HV Polygenic Scores (HV-PGS), we built genetically adjusted nomograms from participants stratified into the top and bottom 30% of HV-PGS. This shifted the nomograms in the expected directions by [~]100 mm3 (2.3% of the average HV), which equates to 3 years of normal aging.

Clinical impact of genetically adjusted nomograms was investigated by comparing 818 subjects from the AD neuroimaging (ADNI) database diagnosed as either cognitively normal (CN), having mild cognitive impairment (MCI) or Alzheimers disease patients (AD). While no significant change in the survival analysis was found for MCI-to-AD conversion, an average of 4% decrease was found in intra-diagnostic-group variance, highlighting the importance of genetic adjustment in untangling phenotypic heterogeneity.

sentences: A parallel channel of state-dependent sensory signaling from the cholinergic basal forebrain to the auditory cortex Cholinergic basal forebrain (CBF) signaling exhibits multiple timescales of activity with classic, slow signals related to brain and behavioral states and faster, phasic signals reflecting behavioral events, including movement and reinforcement. Recent evidence suggests that the CBF may also exhibit fast, sensory-evoked responses. It remains unknown, however, whether such sensory signals target the sensory cortex and how they relate to local functional topography. Moreover, the extent to which fast and slow CBF activity interact has been largely unexplored. Here, we used simultaneous two-channel, two-photon imaging of CBF axons and auditory cortical (AC) neurons to reveal that CBF axons project a robust, non-habituating, and stimulus-specific sensory signal to the AC. Individual axon segments exhibited heterogeneous but stable tuning to auditory stimuli allowing stimulus identity to be decoded from the population. However, CBF axons displayed no tonotopy and their frequency tuning was uncoupled from that of nearby cortical neurons. Chemogenetic suppression revealed the auditory thalamus as a principal source of auditory information to the CBF. Finally, slow fluctuations in cholinergic activity modulated the fast, sensory-evoked signals in the same axons, suggesting that a multiplexed combination of fast and slow signals is projected from the CBF to the AC. Taken together, our work demonstrates a novel, non-canonical function of the CBF as a parallel channel of state-dependent sensory signaling to the sensory cortex that provides repeated representations of a broad range of sound stimuli at all points on the tonotopic map.

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scSampler: fast diversity-preserving subsampling of large-scale single-cell transcriptomic data SummaryThe number of cells measured in single-cell transcriptomic data has grown fast in recent years. For such large-scale data, subsampling is a powerful and often necessary tool for exploratory data analysis. However, the easiest random subsampling is not ideal from the perspective of preserving rare cell types. Therefore, diversity-preserving subsampling is required for fast exploration of cell types in a large-scale dataset. Here we propose scSampler, an algorithm for fast diversity-preserving subsampling of single-cell transcriptomic data. Using simulated and real data, we show that scSampler consistently outperforms existing subsam-pling methods in terms of both the computational time and the Hausdorff distance between the full and subsampled datasets.

AvailabilityscSampler is implemented in Python and is published under the MIT source license. It can be installed by pip install scsampler and used with the Scanpy pipline. The code is available on GitHub: https://github.com/SONGDONGYUAN1994/scsampler.

Contactlinwang@gwu.edu; jli@stat.ucla.edu

sentences: Multiple modes of regulation control dynamic transcription patterns during the mitosis-G1 transition Following cell division, genomes must reactivate gene expression patterns that reflect the identity of the cell. Here, we use PRO-seq to examine the mechanisms that reestablish transcription patterns after mitosis. We uncover regulation of the transcription cycle at multiple steps including initiation, promoter-proximal pause positioning and escape, poly-A site cleavage and termination during the mitotic-G1 transition. During mitosis, RNA polymerase activity is retained at initiation sites, albeit shifted in position relative to non-mitotic cells. This activity is strongly linked to maintenance of local chromatin architecture during mitosis and is more predictive of rapid gene reactivation than histone modifications previously associated with bookmarking. These molecular bookmarks, combined with sequence-specific transcription factors, direct expression of select cell growth and cell specific genes during mitosis followed by reactivation of functional gene groups with distinct kinetics after mitosis. This study details how dynamic regulation of transcription at multiple steps contributes to gene expression during the cell cycle.

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Dihydropyrimidinase-like 2 (DPYSL2) regulates breast cancer migration via a JAK/STAT3/vimentin axis The intricate neuronal wiring during development requires cytoskeletal reorganization orchestrated by signaling cues. Considering that cytoskeletal remodeling is a hallmark of cell migration, we inquired whether metastatic cancer cells exploit the axon guidance proteins to migrate. Indeed, in breast cancer patients, we found a significant correlation between the mesenchymal markers and the expression of dihydropyrimidinase-like 2 (DPYSL2), a regulator of cytoskeletal dynamics in growing axons. Strikingly, DPYSL2 knockout in mesenchymal-like cells profoundly inhibited cell migration, invasion, stemness features, tumor growth rate, and metastasis. Next, we aimed to decode the molecular mechanism underlying this phenomenon and revealed an interaction between DPYSL2 and Janus kinase 1 (JAK1). This binding is crucial for triggering signal transducer and activator of transcription 3 (STAT3) and subsequently expressing vimentin, the pro-migratory intermediate filament. Collectively, we identified DPYSL2 as a molecular link between oncogenic signaling pathways and cytoskeletal reorganization in migrating breast cancer cells.

Statement of significanceThis study shows that the axon guidance adaptor protein DPYSL2 is essential for promoting breast cancer migration. Specifically, this protein interacts with JAK1 to govern STAT3 signaling and subsequently vimentin expression.

sentences: Configurable Compartmentation Enables In vitro Reconstitution of Sustained Synthetic Biology Systems The compartmentalized and communicative nature of biological cells contributes to the complexity and endurance of living organisms. We present a general compartmentalization strategy for in vitro systems that inherits the passive transport phenomenon of biology. The strategy incorporates layered, micrometer-sized, hydrogel-based compartments featuring configurability in composition, functionality and selective permeability of biomolecules. We demonstrated the utility of the strategy by reconstituting a compartmentalized in vitro protein synthesis system which supports multiple rounds of reactions and compartmentalized living bacteria-based biosensors which sustain long-lasting functioning with markedly enhanced fitness in complex environments. The strategy should be widely applicable for constructing complex, robust and sustained in vitro synthetic molecular and cellular systems, paving the way for their practical applications.

sentences: Harmonizing Functional Connectivity Reduces Scanner Effects in Community Detection Community detection on graphs constructed from functional magnetic resonance imaging (fMRI) data has led to important insights into brain functional organization. Large studies of brain community structure often include images acquired on multiple scanners across different studies. Differences in scanner can introduce variability into the downstream results, and these differences are often referred to as scanner effects. Such effects have been previously shown to significantly impact common network metrics. In this study, we identify scanner effects in data-driven community detection results and related network metrics. We assess a commonly employed harmonization method and propose new methodology for harmonizing functional connectivity that leverage existing knowledge about network structure as well as patterns of covariance in the data. Finally, we demonstrate that our new methods reduce scanner effects in community structure and network metrics. Our results highlight scanner effects in studies of brain functional organization and provide additional tools to address these unwanted effects. These findings and methods can be incorporated into future functional connectivity studies, potentially preventing spurious findings and improving reliability of results.

sentences: Novel MOG analogues to explore the MCT2 pharmacophore, α-ketoglutarate biology and cellular effects of N-oxalylglycine -ketoglutarate (KG) is a central metabolic node with a broad influence on cellular physiology. The KG analogue N-oxalylglycine (NOG) and its membrane-permeable pro-drug derivative dimethyl-oxalylglycine (DMOG) have been extensively used as tools to study prolyl hydroxylases (PHDs) and other KG-dependent processes. In cell culture media, DMOG is rapidly converted to MOG, which enters cells through monocarboxylate transporter MCT2, leading to intracellular NOG concentrations that are sufficiently high to inhibit glutaminolysis enzymes and cause cytotoxicity. Therefore, the degree of (D)MOG instability together with MCT2 expression levels determine the intracellular targets NOG engages with and, ultimately, its effects on cell viability. Here we designed and characterised a series of MOG analogues with the aims of improving stability and exploring the functional requirements for interaction with MCT2, a relatively understudied member of the SLC16 family. We report MOG analogues that maintain ability to enter cells via MCT2, and identify compounds that do not inhibit glutaminolysis or cause cytotoxicity but can still inhibit PHDs. We use these analogues to show that glutaminolysis-induced activation of mTORC1 can be uncoupled from PHD activity. Therefore, these new compounds can help deconvolute cellular effects that result from the polypharmacological action of NOG.

sentences: Experimental increase of worker diversity benefits brood production in ants The reproductive division of labor of eusocial insects, whereby one or several queens monopolize reproduction, evolved in a context of high genetic relatedness. However, many extant eusocial species have developed strategies that decrease genetic relatedness in their colonies, suggesting some benefits of the increased diversity. Multiple studies support this hypothesis by showing positive correlations between genetic diversity and colony fitness, as well as finding effects of experimental manipulations of diversity on colony performance. However, alternative explanations could account for most of these reports, and the benefits of diversity on fitness in eusocial insects still await validation. In this study, we experimentally increased worker diversity in the ant Lasius niger while controlling for typical confounding factors. We found that experimental colonies composed of workers coming from three different source colonies produced more larvae and showed more variation in size compared to groups of workers coming from a single colony. We propose that the benefits of increased diversity stemmed from an improved division of labor. Our study confirms that worker diversity enhances colony performance, thus providing a possible explanation for the evolution of multiply mated queens and multiple-queen colonies in many species of eusocial insects.

sentences: Identification of a biosynthetic gene cluster encoding a novel lanthanide chelator in Methylorubrum extorquens AM1 While lanthanide-dependent metabolism is widespread in nature and has been proven to drive one-carbon metabolism in bacteria, details about the machinery necessary to sense, sequester, and traffic lanthanides (Ln) remain unknown. This gap in knowledge is in part because nearly all bacterial growth studies with Ln to date have used soluble chloride salts, compounds that do not reflect the insoluble Ln sources common in the natural environment. Here, we describe the changes in the metabolic machinery of Methylorubrum extorquens AM1 in response to poorly soluble Nd2O3, including 4-fold increases in orphan pqqA genes and the Ln-dependent ADHs xoxF1 and exaF compared to growth with soluble NdCl3. We report the first description of a Ln-chelator biosynthetic gene cluster, encoded by META1p4129 through META1p4138 that we named the Lanthanide Chelation Cluster (LCC). The LCC encodes a TonB dependent receptor and NRPS biosynthetic enzymes and is predicted to produce a metal-chelating molecule. As some LCC protein sequences share similarity to biosynthetic enzymes producing the Fe-chelating siderophore aerobactin, the capacity of aerobactin for binding Ln was tested. It was found that while aerobactin can bind lanthanum (La), neodymium (Nd) and lutetium (Lu) at physiological pH, providing only exogenous aerobactin did not affect growth rate or yield. The LCC was highly upregulated when M. extorquens AM1 was grown using Nd2O3 and expression in trans enabled an increase of Nd bioaccumulation by over 50%. Expression of the LCC in trans did not affect iron bioaccumulation, providing further evidence that its product is a novel Ln-chelator. Finally, expression of the LCC in trans increased Nd, dysprosium (Dy), and praseodymium (Pr) bioaccumulation from the complex Ln source NdFeB magnet swarf by over 60%, opening new strategies for sustainable recovery of these critical Rare Earth Elements.

sentences: Decellularized micro-scaffolds as stem cell carriers for regeneration of cartilage defects Cartilage regeneration remains a great challenge in orthopedic treatment owing to their avascular in nature and lack of self-healing ability. Various clinical treatment options are widely used including osteochondral graft transplantation, micro-fracture, blood clot formation and tissue debridement to recover the damaged cartilage. In this circumstance, cartilage defect recovery via tissue engineered functional micro tissue delivery is becoming an emerging trend in musculoskeletal therapeutics. In this study, functional micro-scaffolds (MS) were generated from Capra ear cartilage, and were separated into size-wise groups. The scaffolds were decellularized via NaOH treatment. The cell adhesion study indicated that Capra adipose tissue derived mesenchymal stem cells (ADMSCs) adhesion is more in ~100 m MSs in comparison to 150-300 m MSs. It may be assumed that the cells are compatible to grow on fibrous surface area (100 m) in comparison to dense surface (150-300 m). Further, 100 m MSs were transformed into functional micro tissues (FMTs) in presence of high density ADMSCs in a hanging droplet culture system. The FMTs was transferred to F127 block polymer hydrogel for 3D culture. After 21 d cultures, the FMT clusters were evaluated for quantitative gene expression. To assess the in vivo cartilage defect regeneration potential, FMTs were delivered to rabbit auricular cartilage defect for 15, 30 and 60 d studies. The H&E-stained histological analysis showed that the cartilage defect is almost healed in 60 d study in comparison to 15 and 30 d study.

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Fuscimiditide: a RiPP with {Omega}-Ester and Aspartimide Post-translational Modifications Microviridins and other {omega}-ester linked peptides (OEPs) are characterized by sidechain-sidechain linkages installed by ATP-grasp enzymes. Here we describe the discovery of a new family of OEPs, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein L-isoaspartyl methyltransferase (PIMT). We produced the first example of this new ribosomally synthesized and post-translationally modified peptide (RiPP), fuscimiditide, via heterologous expression. NMR analysis of fuscimiditide revealed that the peptide contains two ester crosslinks forming a stem-loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We have also fully reconstituted fuscimiditide biosynthesis in vitro establishing that ester formation catalyzed by the ATP-grasp enzyme is an obligate, rate-limiting first biosynthetic step. Aspartimide formation from aspartate is catalyzed by the PIMT homolog in the second step. The aspartimide moiety embedded in fuscimiditide hydrolyzes regioselectively to isoaspartate (isoAsp). Surprisingly, this isoAsp-containing protein is also a substrate for the PIMT homolog, thus driving any hydrolysis products back to the aspartimide form. Whereas aspartimide is often considered a nuisance product in protein formulations, our data here suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity.

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sentences: Object-scene conceptual regularities reveal fundamental differences between biological and artificial object vision Human vision is still largely unexplained. Computer vision made impressive progress on this front, but it is unclear to what extent artificial neural networks approximate human brain strategies. Here, we confirm this gap by testing how biological and artificial systems encode object-scene contextual regularities in natural images. Both systems represent these regularities, but the underlying information processing is markedly different. In human vision, objects and backgrounds are represented separately, with rich domain-specific representations characterizing human visual cortex. Interaction between these components occurs downstream in frontoparietal areas. Conversely, neural networks represent image components in a single entangled representation revealing reduced object-segregation abilities and impoverished domain-specific object spaces. These results show the uniqueness of human vision that allows understanding that images are not just a collection of features and points to the need for developing neural network models with a similar richness of representational content.

sentences: TREM2-H157Y Increases Soluble TREM2 Production and Reduces Amyloid Pathology The p.H157Y variant of TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) has been reported to increase Alzheimers disease (AD) risk. This mutation in the extracellular domain of TREM2 localizes at the cleavage site, leading to enhanced shedding. Here, we generated a novel Trem2 H157Y knock-in mouse model to investigate how this H157Y mutation impacts TREM2 proteolytic processing, synaptic function, and AD-related amyloid pathology. Consistent with previous in vitro findings, TREM2-H157Y increases the amount of soluble TREM2 (sTREM2) in the cortex and serum of mutant mice compared to the wild type controls. Interestingly, the Trem2 H157Y variant enhances synaptic plasticity without affecting microglial density and morphology. In the presence of amyloid pathology, TREM2-H157Y surprisingly accelerates A{beta} clearance and reduces amyloid burden and microgliosis. Taken together, our findings support a beneficial effect of the Trem2 H157Y mutation in synaptic function and in mitigating amyloid pathology. Considering the genetic association of TREM2 p.H157Y with AD, we speculate TREM2-H157Y might increase AD risk through an amyloid-independent pathway, as such its effects on tauopathy and neurodegeneration merit further investigation.

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Interpretable and tractable models of transcriptional noise for the rational design of single-molecule quantification experiments The question of how cell-to-cell differences in transcription rate affect RNA count distributions is fundamental for understanding biological processes underlying transcription. We argue that answering this question requires quantitative models that are both interpretable (describing concrete biophysical phenomena) and tractable (amenable to mathematical analysis). This enables the identification of experiments which best discriminate between competing hypotheses. As a proof of principle, we introduce a simple but flexible class of models involving a stochastic transcription rate coupled to a discrete stochastic RNA transcription and splicing process, and compare and contrast two biologically plausible hypotheses about transcription rate variation. One assumes variation is due to DNA experiencing mechanical strain, while the other assumes it is due to regulator number fluctuations. Although biophysically distinct, these models are mathematically similar, and we show they are hard to distinguish without comparing whole predicted probability distributions. Our work illustrates the importance of theory-guided data collection, and introduces a general framework for constructing and solving mathematically nontrivial continuous-discrete stochastic models.

Significance StatementThe interpretation of transcriptomic observations requires detailed models of biophysical noise that can be compared and fit to experimental data. Models of intrinsic noise, describing stochasticity in molecular reactions, and extrinsic noise, describing cell-to-cell variation, are particularly common. However, integrating and solving them is challenging, and previous results are largely limited to summary statistics. We examine two mechanistically grounded stochastic models of transcriptional variation and demonstrate that (1) well-known regimes naturally emerge in limiting cases, and (2) the choice of noise model significantly affects the RNA distributions, but not the lower moments, offering a route to model identification and inference. This approach provides a simple and biophysically interpretable means to construct and unify models of transcriptional variation.

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Hi-LASSO: High-performance Python and Apache spark packages for feature selection with high-dimensional data High-dimensional LASSO (Hi-LASSO) is a powerful feature selection tool for high-dimensional data. Our previous study showed that Hi-LASSO outperformed the other state-of-the-art LASSO methods. However, the substantial cost of bootstrapping and the lack of experiments for a parametric statistical test for feature selection have impeded to apply Hi-LASSO for practical application. In this paper, the Python package and its Spark library are efficiently implemented in a parallel manner for practice with real-world problems, as well as providing the capability of the parametric statistical tests for feature selection on high-dimensional data. We demonstrate Hi-LASSOs outperformance with various intensive experiments in a practical manner. Hi-LASSO will be efficiently and easily performed by using the packages for feature selection. Hi-LASSO packages are publicly available at https://github.com/datax-lab/Hi-LASSO under the MIT license. The packages can be easily installed by Python PIP, and additional documentation is available at https://pypi.org/project/hi-lasso and https://pypi.org/project/Hi-LASSO-spark.

Author summaryWe provide the brief presentation in the literature of Hi-LASSO comparing to Random LASSO. Then, we describe Hi-LASSOs open-source packages in Python and Apache Spark, specifying parameters. The open-source packages improves efficiency and scalability of Hi-LASSO, so that the time-consuming bootstrapping-based parametric statistical test can be practically applied for high-dimensional data. We conducted intensive experiments to assess the performance of the packages with the parametric statistical test using simulation data, semi-real datasets, and TCGA cancer dataset. The Hi-LASSO packages showed outstanding and robust performance in feature selection. The packages are available through PyPI and can be easily installed using Python PIP.

sentences: Discovery of genes that modulate flavivirus replication in an interferon-dependent manner Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent way, opening new perspectives to target weakness points in the life cycle of these viruses.

sentences: The paraventricular thalamus provides a polysynaptic brake on limbic CRF neurons to sex-dependently blunt binge alcohol drinking and avoidance behavior Bed nucleus of the stria terminalis (BNST) neurons that synthesize and release the stress neuropeptide corticotropin-releasing factor (CRF) drive binge alcohol drinking and anxiety, behaviors that are primary risk factors for alcohol use disorder (AUD) and comorbid neuropsychiatric diseases more common in women than men. Here, we show that female C57BL/6J mice binge drink more than males and have greater basal BNSTCRF neuron excitability and synaptic excitation. We identified a dense VGLUT2+ glutamatergic synaptic input from the paraventricular thalamus (PVT) that is anatomically similar in males and females. These PVTBNST neurons release glutamate directly onto BNSTCRF neurons but also engage a large BNST interneuron population to ultimately provide a net inhibition of BNSTCRF neurons, and both components of this polysynaptic PVTVGLUT2-BNSTCRF circuit are more robust in females than males. Chemogenetic inhibition of the PVTBNST projection promoted binge alcohol drinking in females without affecting males, and chemogenetic activation of the pathway was sufficient to reduce avoidance behavior in both sexes in anxiogenic contexts. Lastly, we show that withdrawal from repeated binge drinking produces a female-like phenotype in the male PVT-BNSTCRF excitatory synapse without altering the function of PVTBNST neurons per se. Our data describe a complex feedforward inhibitory PVTVGLUT2-BNSTCRF glutamatergic circuit that is more robust in females, plays sex-dependent roles in alcohol drinking and avoidance behavior, and undergoes sex-dependent alcohol-induced plasticity.

sentences: Using machine learning to design adeno-associated virus capsids with high likelihood of viral assembly We study the application of machine learning in designing adeno-associated virus (AAV) capsid sequences with high likelihood of viral assembly, i.e. capsid viability. Specifically, we design and implement Origami, a model-based optimization algorithm, to identify highly viable capsid sequences within the vast space of 2033 possibilities. Our evaluation shows that Origami performs well in terms of optimality and diversity of model-designed sequences. Moreover, these sequences are ranked according to their viability score. This helps designing experiments given budget constraint.

sentences: Heterotypic Aβ interactions facilitate amyloid assembly and modify amyloid structure It is still unclear why pathological amyloid deposition initiates in specific brain regions, nor why specific cells or tissues are more susceptible than others. Amyloid deposition is determined by the self-assembly of short protein segments called aggregation-prone regions (APRs) that favour cross-{beta} structure. Here we investigated whether A{beta} amyloid assembly can be modified by heterotypic interactions between A{beta} APRs and short homologous segments in otherwise unrelated human proteins. We identified heterotypic interactions that accelerate A{beta} assembly, modify fibril morphology and affect its pattern of deposition in vitro. Moreover, we found that co-expression of these proteins in an A{beta} reporter cell line promotes A{beta} amyloid aggregation. Importantly, reanalysis of proteomics data of A{beta} plaques from AD patients revealed an enrichment in proteins that share homologous sequences to the A{beta} APRs, suggesting heterotypic amyloid interactions may occur in patients. Strikingly, we did not find such a bias in plaques from overexpression models in mouse. Based on these data, we propose that heterotypic APR interactions may play a hitherto unrealised role in amyloid-deposition diseases.

sentences: Dietary restriction in the long-chain acyl-CoA dehydrogenase knockout mouse Patients with a disorder of mitochondrial long-chain fatty acid {beta}-oxidation (FAO) have reduced fasting tolerance and may present with hypoketotic hypoglycemia, hepatomegaly, (cardio)myopathy and rhabdomyolysis. Patients should avoid a catabolic state because it increases reliance on FAO as energy source. It is currently unclear whether weight loss through a reduction of caloric intake is safe in patients with a FAO disorder. We used the long-chain acyl-CoA dehydrogenase knockout (LCAD KO) mouse model to study the impact of dietary restriction (DR) on the plasma metabolite profile and cardiac function. For this, LCAD KO and wild type (WT) mice were subjected to DR (70% of ad libitum chow intake) for 4 weeks and compared to ad libitum chow fed mice. We found that DR had a relatively small impact on the plasma metabolite profile of WT and LCAD KO mice. Echocardiography revealed a small decrease in left ventricular systolic function of LCAD KO mice, which was most noticeable after DR, but there was no evidence of DR-induced cardiac remodeling. Our results suggest that weight loss through DR does not have acute and detrimental consequences in a mouse model for FAO disorders.

sentences: Roles of the second-shell amino acid R266 in other members of the MLE subgroup of the enolase superfamily Catalytic promiscuity is the coincidental ability to catalyze non-biological reactions in the same active site as the native biological reaction. Several lines of evidence show that catalytic promiscuity plays a role in the evolution of new enzyme functions. Thus, studying catalytic promiscuity can help identify structural features that predispose an enzyme to evolve new functions. This study identifies such a pre-adaptive residue in an N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) enzymes from the NSAR/OSBS subfamily. Previously, we identified a point mutation, R266Q, in the catalytically promiscuous Amycolatopsis sp. T-1-60 NSAR/OSBS that has a deleterious effect on NSAR activity with a lesser effect on OSBS activity (Truong et al., in preparation). We demonstrated that R266 was a pre-adaptive feature that enabled the emergence and evolution of NSAR activity in AmyNSAR/OSBS. We examined the role of the residue R266 in the evolution of NSAR activity by examining the effects of the single substitution R266Q in other members of the NSAR/OSBS subfamily including Enterococcus faecalis NSAR/OSBS, Roseiflexus castenholzii NSAR/OSBS, Lysinibacillus varians NSAR/OSBS, and Listeria innocua NSAR/OSBS, which have been previously characterized to carry out both OSBS and NSAR activities efficiently. RcNSAR/OSBS, LvNSAR/OSBS, EfNSAR/OSBS, and LiNSAR/OSBS are 49, 48, 32, and 28% identical, respectively, to AmyNSAR/OSBS. We found that while the R266Q mutation decreases NSAR activity more than OSBS activity, as expected, in most NSAR/OSBS members, the differential effects of the R266Q substitution on NSAR and OSBS activities are not as striking as observed in AmyNSAR/OSBS. In some homologs, the R266Q mutation has very deleterious effects on both OSBS and NSAR activities. Furthermore, the mutation unexpectedly decreases OSBS activity more than NSAR activity in LiNSAR/OSBS. Thus, the effects of R266Q on NSAR and OSBS activities depend on differences in sequence context between members of the NSAR/OSBS subfamily, demonstrating the complex role of epistasis in the evolution of NSAR activity in the NSAR/OSBS subfamily.

sentences: SARS-CoV-2 spike protein as a bacterial lipopolysaccharide delivery system in an overzealous inflammatory cascade Accumulating evidence indicates a potential role for bacterial lipopolysaccharide (LPS) in the overactivation of the immune response during SARS-CoV-2 infection. LPS is recognised by Toll-like receptor 4 (TLR4) in innate immunity. Here, we showed that LPS binds to multiple hydrophobic pockets spanning both the S1 and S2 subunits of the SARS-CoV-2 spike (S) protein. LPS binds to the S2 pocket with a lower affinity compared to S1, suggesting its possible role as an intermediate in the TLR4 cascade. Congruently, nuclear factor-kappa B (NF-{kappa}B) activation in vitro is strongly boosted by S2. In vivo, however, a boosting effect is observed for both S1 and S2, with the former potentially facilitated by proteolysis. Collectively, our study suggests the S protein may act as a delivery system for LPS in host innate immune pathways. The LPS binding pockets are highly conserved across different SARS-CoV-2 variants and therefore represent potential therapeutic targets.

sentences: Photoredox-catalyzed decarboxylative C-terminal differentiation for bulk and single molecule proteomics Methods for the selective labeling of biogenic functional groups on peptides are being developed and used in the workflow of both current and emerging proteomics technologies, such as single-molecule fluorosequencing. To achieve successful labeling with any one method requires that the peptide fragments contain the functional group for which the labeling chemistry is designed. In practice, only two functional groups are present on every peptide fragment regardless of the protein cleavage site, namely, an N-terminal amine and a C-terminal carboxylic acid. Developing a global-labeling technology, therefore, requires one to specifically target the N- and/or C-terminus of peptides. In this work, we showcase the first successful application of photocatalyzed C-terminal decarboxylative-alkylation for peptide mass-spectrometry and single molecule protein sequencing, that can be broadly applied to any proteome. We demonstrate that peptides in complex mixtures generated from enzymatic digests from bovine serum albumin, as well as protein mixtures from yeast and human cell extracts, can be site-specifically labeled at their C-terminal residue with a Michael acceptor. Using two distinct analytical approaches, we characterize C-terminal labeling efficiencies of greater than 50% across complete proteomes and document the proclivity of various C-terminal amino acid residues for decarboxylative-labeling, showing histidine and tryptophan to be the most disfavored. Finally, we combine C-terminal decarboxylative labeling with an orthogonal carboxylic acid labeling technology in tandem, to establish a new platform for fluorosequencing.

sentences: Loss of the extracellular matrix protein DIG-1 causes glial fragmentation, dendrite breakage, and dendrite extension defects The extracellular matrix (ECM) guides and constrains the shape of the nervous system. In C. elegans, DIG-1 is a giant ECM component that is required for fasciculation of sensory dendrites during development and for maintenance of axon positions throughout life. We identified four novel alleles of dig-1 in three independent screens for mutants affecting disparate aspects of neuronal and glial morphogenesis. First, we find that disruption of DIG-1 causes fragmentation of the amphid sheath glial cell in larvae and young adults. Second, it causes severing of the BAG sensory dendrite from its terminus at the nose tip, apparently due to breakage of the dendrite as animals reach adulthood. Third, it causes embryonic defects in dendrite fasciculation in inner labial (IL2) sensory neurons, as previously reported, as well as rare defects in IL2 dendrite extension that are enhanced by loss of the apical ECM component DYF-7, suggesting that apical and basolateral ECM contribute separately to dendrite extension. Our results highlight novel roles for DIG-1 in maintaining the cellular integrity of neurons and glia, possibly by creating a barrier between structures in the nervous system.

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Predicting Endometrial Cancer Subtypes and Molecular Features from Histopathology Images Using Multi-resolution Deep Learning Models The determination of endometrial carcinoma histological subtypes, molecular subtypes, and mutation status is a critical diagnostic process that directly affects patients prognosis and treatment options. Compared to the histopathological approach, however, the availability of molecular subtyping is limited as it can only be accurately obtained by genomic sequencing, which may be cost prohibitive. Here, we implemented a customized multi-resolution deep convolutional neural network, Panoptes, that predicts not only the histological subtypes, but also molecular subtypes and 18 common gene mutations based on digitized H&E stained pathological images. The model achieved high accuracy and generalized well on independent datasets. Our results suggest that Panoptes has potential clinical application of helping pathologists determine molecular subtypes and mutations of endometrial carcinoma without sequencing.

SignificanceRecently, molecular subtyping and mutation status are increasingly utilized in clinical practice as they offer better-informed prognosis and the possibility of individualized therapies for endometrial carcinoma patients. Taking advantage of the multi-resolution nature of the whole slide digital histopathology images, our Panoptes models integrate features of different magnification and make accurate predictions of histological subtypes, molecular subtypes, and key mutations in much faster workflows compared to conventional sequencing-based analyses. Feature extraction and visualization revealed that the model relied on human-interpretable patterns. Overall, our multi-resolution deep learning model is capable of assisting pathologists determine molecular subtypes of endometrial carcinoma, which can potentially accelerate diagnosis process.

sentences: Phosphorylation of interfacial phosphosite leads to increased binding of Rap-Raf complex The effect of phosphorylation of a serine residue in the Rap protein, residing at the complex interface of Rap-Raf complex is studied using atomistic molecular dynamics simulations. As the phosphosite of interest (SER39) is buried at the interface of the Rap-Raf complex, phosphorylation of only Rap protein was simulated and then complexed with the RBD of Raf for further analysis of complex stability. Our simulations reveal that the phosophorylation increases the binding of complex through strong electrostatic interactions and changes the charge distribution of the interface significantly. This is manifested as an increase in stable salt-bridge interactions between the Rap and Raf of the complex. Network analysis clearly shows that the phosphorylation of SER39 reorganizes the community network to include the entire region of Raf chain, including, Raf L4 loop potentially affecting downstream signalling.

sentences: Trisomy 21 induces pericentrosomal crowding disrupting early stages of primary ciliogenesis and mouse cerebellar development Primary cilia are signaling organelles essential for development and homeostasis. Loss of primary cilia is lethal, and decreased or defective cilia cause multisystemic conditions called ciliopathies. Down syndrome shares clinical overlap with ciliopathies. We previously showed that trisomy 21 diminishes primary cilia formation and function due to elevated Pericentrin, a centrosome protein encoded on chromosome 21. Pericentrin is mislocalized, creating aggregates that disrupt pericentrosomal trafficking and microtubule organization. Here, we examine the cilia-related molecules and pathways disrupted in trisomy 21 and their in vivo phenotypic relevance. Utilizing ciliogenesis time course experiments, we reveal how Pericentrin, microtubule networks, and components of ciliary vesicles are reorganized for ciliogenesis in euploid cells. Early in ciliogenesis, chromosome 21 polyploidy results in elevated Pericentrin and microtubule networks away from the centrosome that ensnare MyosinVA and EHD1, blocking mother centriole uncapping that is essential for ciliogenesis. Ciliated trisomy 21 cells have persistent trafficking defects that reduce transition zone protein localization, which is critical for Sonic hedgehog signaling. Sonic hedgehog signaling is decreased and anticorrelates with Pericentrin levels in trisomy 21 primary mouse embryonic fibroblasts. Finally, we observe decreased ciliation in vivo. A mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers. Our work reveals that elevated Pericentrin in trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding results in signaling defects consistent with the neurological deficits found in individuals with Down syndrome.

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Transcriptional organization and regulation of the Pseudomonas putida flagellar system A single region of the Pseudomonas putida genome, designated the flagellar cluster, includes 59 genes potentially involved in the biogenesis and function of the flagellar system. Here we combine bioinformatics and in vivo gene expression analyses to clarify the transcriptional organization and regulation of the flagellar genes in the cluster. We have identified eleven flagellar operons and characterized twenty-two primary and internal promoter regions. Our results indicate that synthesis of the flagellar apparatus and core chemotaxis machinery is regulated by a three-tier cascade in which fleQ is a Class I gene, standing at the top of the transcriptional hierarchy. FleQ- and{sigma} 54-dependent Class II genes encode most components of the flagellar structure, part of the chemotaxis machinery and multiple regulatory elements, including the flagellar{sigma} factor FliA. FliA activation of Class III genes enables synthesis of the filament, one stator complex and completion of the chemotaxis apparatus. Accessory regulatory proteins and an intricate operon architecture add complexity to the regulation by providing feedback and feed-forward loops to the main circuit. Because of the high conservation of the gene arrangement and promoter motifs, we believe that the regulatory circuit presented here may also apply to other environmental pseudomonads.

ORIGINALITY-SIGNIFICANCE STATEMENTThis is the first integrative study of the flagellar transcriptional cascade in Pseudomonas putida. Our results provide a new transcriptional organization featuring several operons with a nested architecture, detailed regulatory characterization of twenty-two flagellar promoters and a novel hierarchy for the regulatory circuit of the flagellar transcriptional cascade. The results presented represent a significant departure from previous models for other related bacteria. High conservation of flagellar gene organization and promoter sequences suggest that our observations may be relevant to other pseudomonads.

sentences: Evolutionary differences in the ACE2 reveals the molecular origins of COVID-19 susceptibility We explore the energetic frustration patterns associated with the binding between the SARS-CoV-2 spike protein and the ACE2 receptor protein in a broad selection of animals. Using energy landscape theory and the concept of energy frustration--theoretical tools originally developed to study protein folding--we are able to identify interactions among residues of the spike protein and ACE2 that result in COVID-19 resistance. This allows us to identify whether or not a particular animal is susceptible to COVID-19 from the protein sequence of ACE2 alone. Our analysis predicts a number of experimental observations regarding COVID-19 susceptibility, demonstrating that this feature can be explained, at least partially, on the basis of theoretical means.

sentences: Epigenetic Patterns in a Complete Human Genome The completion of the first telomere-to-telomere human genome, T2T-CHM13, enables exploration of the full epigenome, removing limitations previously imposed by the missing reference sequence. Existing epigenetic studies omit unassembled and unmappable genomic regions (e.g. centromeres, pericentromeres, acrocentric chromosome arms, subtelomeres, segmental duplications, tandem repeats). Leveraging the new assembly, we were able to measure enrichment of epigenetic marks with short reads using k-mer assisted mapping methods. This granted array-level enrichment information to characterize the epigenetic regulation of these satellite repeats. Using nanopore sequencing data, we generated base level maps of the most complete human methylome ever produced. We examined methylation patterns in satellite DNA and revealed organized patterns of methylation along individual molecules. When exploring the centromeric epigenome, we discovered a distinctive dip in centromere methylation consistent with active sites of kinetochore assembly. Through long-read chromatin accessibility measurements (nanoNOMe) paired to CUT&RUN data, we found the hypomethylated region was extremely inaccessible and paired to CENP-A/B binding. With long-reads we interrogated allele-specific, longrange epigenetic patterns in complex macro-satellite arrays such as those involved in X chromosome inactivation. Using the single molecule measurements we can clustered reads based on methylation status alone distinguishing epigenetically heterogeneous and homogeneous areas. The analysis provides a framework to investigate the most elusive regions of the human genome, applying both long and short-read technology to grant new insights into epigenetic regulation.

sentences: Preferential lipolysis of DGAT1 over DGAT2 generated triacylglycerol in Huh7 hepatocytes Hepatic steatosis is defined by accumulation of neutral lipids in lipid droplets (LDs) including triacylglycerol (TG) and steryl esters. Two distinct diacylglycerol acyltransferases (DGAT1 and DGAT2) catalyze synthesis of TG in hepatocytes. TG formed through either DGAT1 or DGAT2 appears to be preferentially directed to distinct intercellular fates, such as fatty acid production for oxidation or very-low density lipoprotein assembly, respectively. Because of the preferential use of TG generated by DGAT1 and DGAT2, we hypothesized that targeting/association of lipolytic machinery to LDs would differ depending on whether the TG stores were generated through DGAT1 or DGAT2 activities. Inhibition of DGAT1 or DGAT2 in human hepatoma cells (Huh7) incubated with oleic acid resulted in only a small change in TG accretion suggesting that the two DGATs can compensate for each other in fatty acid esterification. This compensation was not accompanied by changes in DGAT1 or DGAT2 mRNA expression. DGAT1 inhibition (TG synthesized by DGAT2) resulted in large LDs, whereas DGAT2 inhibition (TG synthesized by DGAT1) caused the accumulation of numerous small LDs. Oleic acid treatment increased mRNA and protein expression of the LD-associated protein PLIN2 but not PLIN5 or the lipase ATGL and its activator ABHD5/CGI-58. Inactivation of DGAT1 or DGAT2 did not alter expression (mRNA or protein) of ATGL, ABHD5/CGI-58, PLIN2 or PLIN5, but inactivation of both DGATs increased PLIN2 abundance despite a dramatic reduction in the number of LDs. ATGL localized preferentially to DGAT1-made LDs rather than to DGAT2-made LDs, and TG in these LDs was preferentially used for fatty acid (FA) oxidation. A combination of DGAT2 inhibitor and the pan lipase inhibitor E600 resulted in large LDs, suggesting that the small size of DGAT1-made LDs is due to a lipolytic process.

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Cataloging the potential functional diversity of Cacna1e splice variants using long-read sequencing Voltage gated calcium channels (VGCCs) regulate the influx of calcium ions in many cell types, but our lack of knowledge about the plethora of VGCC splice variants remains a gap in our understanding of calcium channel function. A recent advance in profiling gene splice variation is to use long-read RNA-sequencing technology. We sequenced Cacna1e transcripts from the rat thalamus using Oxford Nanopore sequencing, yielding the full structure of 2,110 Cacna1e splice variants. However, we observed that only 154 Cacna1e splice variants were likely to encode for a functional VGCC based on predicted amino acid sequences. We then computationally prioritized these 154 splice variants using expression and evolutionary conservation and found that four splice variants are candidate functionally distinct splice isoforms. Our work not only provides long-read sequencing of Cacna1e for the first time, but also the first computational evaluation of which Cacna1e splice variants are the best candidates for future follow-up.

SIGNIFICANCE STATEMENTVoltage gated calcium channels (Cacna1x genes) are implicated in many neurological disorders and their encoding genes are predicted to have complex patterns of alternative splicing. Previous approaches relied on short-read RNA-seq to characterize calcium channel splice variants. Here, we use long-read nanopore sequencing to establish a set of Cacna1e transcripts in the rat thalamus and use computational methods to prioritize four transcripts as functionally distinct splice isoforms. Our work to provide the field with prioritized transcripts will not only improve our understanding of Cacna1e function but its role in disease as well.

sentences: Materials in action: The look and feel of soft The softness of objects can be perceived through several senses. For instance, to judge the softness of our cats fur, we do not only look at it, we also run our fingers in idiosyncratic ways through its coat. Recently, we have shown that haptically perceived softness covaries with the compliance, viscosity, granularity, and furriness of materials (Dovencioglu et al.,2020). However, it is unknown whether vision can provide similar information about the various aspects of perceived softness. Here, we investigated this question in an experiment with three conditions: in the haptic condition, blindfolded participants explored materials with their hands, in the visual-static condition participants were presented with close-up photographs of the same materials, and in the visual-dynamic condition participants watched videos of the hand-material interactions that were recorded in the haptic condition. After haptically or visually exploring the materials participants rated them on various attributes. Our results show a high overall perceptual correspondence between the three experimental conditions. With a few exceptions, this correspondence tended to be strongest between haptic and visual-dynamic conditions. These results are discussed with respect to information potentially available through the senses, or through prior experience, when judging the softness of materials.

sentences: Developmental dynamics of the postsynaptic proteome to understand synaptic maturation and dysmaturation The postsynaptic density (PSD) is a protein condensate composed of [~]1,000 proteins beneath the postsynaptic membrane of excitatory synapses. The number, shape, and plasticity of synapses are altered during development. However, the dynamics of synaptic protein composition across development have not been fully understood. Here we show alterations of PSD protein composition in mouse and primate brains during development. Proteins involved in synapse regulation are enriched in the differentially expressed (288 decreased and 267 increased) proteins on mouse PSD after a 2-week-old. We find that the changes in PSD protein abundance in mouse brains correlate with gene expression levels in postnatal mice and perinatal primates. This alteration of PSD composition is likely to be defective in the brains of mouse models or patients with autism spectrum disorder (ASD). Finally, we demonstrate that the brain of the common marmoset (Callithrix jacchus) changes PSD composition after the juvenile period. The alteration of PSD composition after 2-month-old is distinct from that observed in mice. Our results provide a comprehensive architecture of the remodeling of PSD composition across development, which may explain the molecular basics of synapse maturation and the pathology of psychiatric disorders, such as ASD.

sentences: An environmentally ultrasensitive fluorine-19 NMR probe for monitoring protein conformational equilibria Limited chemical shift dispersion represents a major barrier to studying multi-state equilibria of large membrane proteins by 19F NMR. We describe a novel monofluoroethyl 19F probe with a dramatically larger chemical shift dispersion than existing probes. The improved conformational sensitivity and line shape enabled the detection of hidden states of a critical membrane transporter, guiding subsequent structural elucidation by single-particle cryo-electron microscopy.

sentences: Localization of muscarinic acetylcholine receptor dependent rhythm generating modules in the Drosophila larval locomotor network Mechanisms of rhythm generation have been extensively studied in motor systems that control locomotion over terrain in limbed animals; however, much less is known about rhythm generation in soft-bodied terrestrial animals. Here we explored how muscarinic acetylcholine receptor (mAChR) dependent rhythm generating networks are distributed in the central nervous system (CNS) of soft-bodied Drosophila larvae. We measured fictive motor patterns in isolated CNS preparations using a combination of Ca2+ imaging and electrophysiology while manipulating mAChR signalling pharmacologically. Bath application of the mAChR agonist oxotremorine potentiated rhythm generation in distal regions of the isolated CNS, whereas application of the mAChR antagonist scopolamine suppressed rhythm generation in these regions. Oxotremorine raised baseline Ca2+ levels and potentiated rhythmic activity in isolated posterior abdominal CNS segments as well as isolated anterior brain and thoracic regions, but did not induce rhythmic activity in isolated anterior abdominal segments. Bath application of scopolamine to reduced preparations lowered baseline Ca2+ levels and abolished rhythmic activity. These results suggest the presence of a bimodal gradient of rhythmogenicity in the larval CNS, with mAChR dependent rhythm generating networks in distal regions separated by medial segments with severely reduced rhythmogenic abilities. This work furthers our understanding of motor control in soft-bodied locomotion and provides a foundation for study of rhythm generating networks in an emerging genetically tractable locomotor system.

sentences: Using a Quadruplet Codon to Expand the Genetic Code of an Animal Genetic code expansion in multicellular organisms is currently limited to the use of repurposed amber stop codons. Here we introduce a system for the use of quadruplet codons to direct incorporation of non-canonical amino acids in vivo in an animal, the nematode worm Caenorhabditis elegans. We develop hybrid pyrrolysyl tRNA variants to incorporate non-canonical amino acids in response to the quadruplet codon UAGA. We demonstrate the efficiency of the quadruplet decoding system by incorporating photocaged amino acids into two proteins widely used as genetic tools. We use photocaged lysine to express photocaged Cre recombinase for the optical control of gene expression and photocaged cysteine to express photo-activatable caspase for light inducible cell ablation. Our approach will facilitate the routine adoption of quadruplet decoding for genetic code expansion in eukaryotic cells and multicellular organisms.

sentences: Global genetic heterogeneity in adaptive traits Understanding the genetic architecture of complex traits is a major objective in biology. The standard approach for doing so is genome-wide association studies (GWAS), which aim to identify genetic polymorphisms responsible for variation in traits of interest. In human genetics, consistency across studies is commonly used as an indicator of reliability. However, if traits are involved in adaptation to the local environment, we do not necessarily expect reproducibility. On the contrary, results may depend on where you sample, and sampling across a wide range of environments may decrease the power of GWAS because of increased genetic heterogeneity. In this study, we examine how sampling affects GWAS for a variety of phenotypes in the model plant species Arabididopsis thaliana. We show that traits like flowering time are indeed influenced by distinct genetic effects in local populations. Furthermore, using gene expression as a molecular phenotype, we show that some genes are globally affected by shared variants, while others are affected by variants specific to subpopulations. Remarkably, the former are essentially all cis-regulated, whereas the latter are predominately affected by trans-acting variants. Our result illustrate that conclusions about genetic architecture can be incredibly sensitive to sampling and population structure.

sentences: Expression and Manufacturing of Protein Therapeutics in Spirulina Arthrospira platensis (commonly known as spirulina) is a photosynthetic cyanobacterium1. It is a highly nutritious food that has been consumed for decades in the US, and even longer by indigenous cultures2. Its widespread use as a safe food source and proven scalability have driven frequent attempts to convert it into a biomanufacturing platform. But these were repeatedly frustrated by spirulinas genetic intractability. We report here efficient and versatile genetic engineering methodology for spirulina that allows stable expression of bioactive protein therapeutics at high levels. We further describe large-scale, indoor cultivation and downstream processing methods appropriate for the manufacturing of biopharmaceuticals in spirulina. The potential of the platform is illustrated by pre-clinical development and human testing of an orally delivered antibody therapeutic against campylobacter, a major cause of infant mortality in the developing world and a growing antibiotic resistance threat3,4. This integrated development and manufacturing platform blends the safety of food-based biotechnology with the ease of genetic manipulation, rapid growth rates and high productivity characteristic of microbial platforms. These features combine for exceptionally low-cost production of biopharmaceuticals to address medical needs that are unfeasible with current biotechnology platforms.

sentences: Rat ultrasonic vocalizations and novelty-induced social and non-social investigation behavior in a seminatural environment Although rats are known to emit ultrasonic vocalizations (USVs), it remains unclear whether these calls serve an auditory communication purpose. For USVs to be part of communication, the vocal signals will need to be a transfer of information between two or more conspecifics, and with the possibility to induce changes in the behavior of the recipient. Therefore, the aim of our study was to investigate the role of USVs in adult rats social and non-social investigation strategies when introduced into a large novel environment with unfamiliar conspecifics. We quantified a wide range of social and non-social behaviors in the seminatural environment, which could be affected by subtle signals, including USVs. We found that during the first hour in the seminatural environment the ability to vocalize did not affect how quickly adult rats met each other, their overall social investigation behavior, their passive social behavior nor their aggressive behavior. Furthermore, the non-social exploratory behaviors and behaviors reflecting anxiety/stress-like states were also unaffected. These results demonstrated that a disability to vocalize did not result in significant disadvantages (or changes) compared to intact conspecifics regarding social and non-social behaviors. This suggests that other (multi)sensory cues are more relevant in social interactions than USVs.

sentences: Perturbation of amygdala-cortical projections reduces ensemble coherence of palatability coding in gustatory cortex Taste palatability is centrally involved in consumption decisions--we ingest foods that taste good and reject those that dont. Gustatory cortex (GC) and basolateral amygdala (BLA) almost certainly work together to mediate palatability-driven behavior, but the precise nature of their interplay during taste decision-making is still unknown. To probe this issue, we discretely perturbed (with optogenetics) activity in BLA[->]GC axons during taste deliveries. This perturbation strongly altered GC taste responses, but while the perturbation itself was tonic (2s), the alterations were not--changes preferentially aligned with the onset times of previously-described taste response epochs, and reduced evidence of palatability-related activity in the "late-epoch" of the responses without reducing the amount of taste identity information available in the "middle epoch." Finally, BLA[->]GC perturbations changed behavior-linked taste response dynamics themselves, distinctively diminishing the abruptness of ensemble transitions into the late epoch. These results suggest that BLA "organizes" behavior-related GC taste dynamics.

sentences: Bacterial response to spatial gradients of algal-derived nutrients in a porous microplate AO_SCPLOWBSTRACTC_SCPLOWPhotosynthetic microalgae are responsible for 50% of the global atmospheric CO2 fixation into organic matter and hold potential as a renewable bioenergy source. Their metabolic interactions with the surrounding microbial community (the algal microbiome) play critical roles in carbon cycling, but due to methodological limitations, it has been challenging to examine how community is developed by spatial proximity to their algal host. Here we introduce a hydrogel-based porous microplate to co-culture algae and bacteria, where metabolites are constantly exchanged between the microorganisms while maintaining physical separation. In the microplate we found that the diatom Phaeodactylum tricornutum accumulated to cell abundances ~20 folds higher than under normal batch conditions due to constant replenishment of nutrients through the hydrogel. We also demonstrate that algal-associated bacteria, both single isolates and complex communities, responded to inorganic nutrients away from their host as well as organic nutrients originating from the algae in a spatially predictable manner. These experimental findings coupled with a mathematical model suggest that host proximity and algal culture growth phase impact bacterial community development in a taxon-specific manner through organic and inorganic nutrient availability. Our novel system presents a useful tool to investigate universal metabolic interactions between microbes in aquatic ecosystems.

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Estimating the rate of plasmid transfer with an adapted Luria-Delbrück fluctuation analysis and a case study on the evolution of plasmid transfer rate To increase our basic understanding of the ecology and evolution of conjugative plasmids, we need a reliable estimate of their rate of transfer between bacterial cells. However, accurate estimates of plasmid transfer have remained elusive due to biological and experimental complexity. Current methods to measure transfer rate can be confounded by many factors. A notable example involves plasmid transfer between different strains or species where the rate that one type of cell donates the plasmid is not equal to the rate at which the other cell type donates. Asymmetry in these rates has the potential to bias or constrain current transfer estimates, thereby limiting our capabilities for estimating transfer in microbial communities. Inspired by the classic fluctuation analysis of Luria and Delbruck, we develop a novel approach, the Luria-Delbruck method ( LDM), for estimating plasmid transfer rate. Our new approach embraces the stochasticity of conjugation departing from the current deterministic population dynamic methods. In addition, the LDM overcomes obstacles of traditional methods by not being affected by different growth and transfer rates for each population within the assay. Using stochastic simulations and experiments, we show that the LDM has high accuracy and precision for estimation of transfer rates compared to the most widely used methods, which can produce estimates that differ from the LDM estimate by orders of magnitude.

Significance StatementConjugative plasmids play significant roles in the ecology and evolution of microbial communities. Notably, antibiotic resistance genes are often encoded on conjugative plasmids. Thus, conjugation--the transfer of a plasmid copy from one cell to another--is a common way for antibiotic resistance to spread between important clinical pathogens. For both public health modeling and a basic understanding of microbial population biology, accurate estimates of this fundamental rate are of great consequence. We show that widely used methods can lead to biased estimates, deviating from true values by several orders of magnitude. Therefore, we developed a new approach, inspired by the classic fluctuation analysis of Luria and Delbruck, for accurately assessing the rate of plasmid conjugation under a variety of conditions.

sentences: Machine learning sequence prioritization for cell type-specific enhancer design Recent discoveries of extreme cellular diversity in the brain warrant rapid development of technologies to access specific cell populations, enabling characterization of their roles in behavior and in disease states. Available approaches for engineering targeted technologies for new neuron subtypes are low-yield, involving intensive transgenic strain or virus screening. Here, we introduce SNAIL (Specific Nuclear-Anchored Independent Labeling), a new virus-based strategy for cell labeling and nuclear isolation from heterogeneous tissue. SNAIL works by leveraging machine learning and other computational approaches to identify DNA sequence features that confer cell type-specific gene activation and using them to make a probe that drives an affinity purification-compatible reporter gene. As a proof of concept, we designed and validated two novel SNAIL probes that target parvalbumin-expressing (PV) neurons. Furthermore, we show that nuclear isolation using SNAIL in wild type mice is sufficient to capture characteristic open chromatin features of PV neurons in the cortex, striatum, and external globus pallidus. Expansion of this technology has broad applications in cell type-specific observation, manipulation, and therapeutics across species and disease models.

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Deep hierarchical sensory processing accounts for effects of arousal state on perceptual decision-making Arousal levels strongly affect task performance. Yet, what arousal level is optimal for a task depends on its difficulty. Easy task performance peaks at higher arousal levels, whereas performance on difficult tasks displays an inverted U-shape relationship with arousal, peaking at medium arousal levels, an observation first made by Yerkes and Dodson in 1908. It is commonly proposed that the noradrenergic locus coeruleus system regulates these effects on performance through a widespread release of noradrenaline resulting in changes of cortical gain. This account, however, does not explain why performance decays with high arousal levels only in difficult, but not in simple tasks. Here, we present a mechanistic model that revisits the Yerkes-Dodson effect from a sensory perspective: a deep convolutional neural network augmented with a global gain mechanism reproduced the same interaction between arousal state and task difficulty in its performance. Investigating this model revealed that global gain states differentially modulated sensory information encoding across the processing hierarchy, which explained their differential effects on performance on simple versus difficult tasks. These findings offer a novel hierarchical sensory processing account of how, and why, arousal state affects task performance.

Authors summaryOver a hundred years ago, it was first observed that the effect of arousal on performance depends on task difficulty: the Yerkes-Dodson effect. Difficult tasks are best solved at intermediate arousal levels, whereas easy tasks benefit from a high arousal state. Current theories on how arousal affects neural processing cannot explain this effect of task difficulty. Here, we implement a key effect of arousal on cortical processing, a change in neuronal gain, in a computational model of visual processing capable of object recognition. Across a series of experiments, we find that our model can reproduce the Yerkes-Dodson effect behaviorally and that this effect can be explained by where in the processing hierarchy different arousal states optimize sensory information encoding.

sentences: A Framework for Sensor-based Assessment of Upper-limb Functioning AO_SCPLOWBSTRACTC_SCPLOWThe ultimate goal of any upper-limb neurorehabilitation procedure is to improve upper-limb functioning in daily life. While clinic-based assessments provide an assessment of what a patient can do, they do not completely reflect what a patient does in his/her daily life. The compensatory use of the less affected upper-limb (e.g. "learned non-use") in daily life is a common behavioral pattern seen in patients with hemiparesis. To this end, there has been an increasing interest in the use of wearable sensors to objectively assess upper-limb functioning. This paper presents a framework for assessing upper-limb functioning using sensors by providing: (a) a set of definitions of important construct associated with upper-limb functioning; (b) presenting different visualization methods for evaluating upper-limb functioning, along ways to qualitatively analyze different visualization methods; and (c) two new measures for quantifying how much an upper-limb is used and the relative bias in the use of the two upper-limbs. The demonstration of some of these components is presented using data collected from inertial measurement units from a previous study. The proposed framework can help guide the future technical and clinical work in this area to realize a valid, objective, and robust tool for assessing upper-limb functioning. This will in turn drive the refinement and standardization of the assessment of upper-limb functioning.

sentences: Sub-second multi-channel magnetic control of select neural circuits in behaving flies Precisely timed activation of genetically targeted cells is a powerful tool for studying neural circuits and controlling cell-based therapies. Magnetic control of cell activity or "magnetogenetics" using magnetic nanoparticle heating of temperature-sensitive ion channels enables remote, non-invasive activation of neurons for deep-tissue applications and studies of freely behaving animals. However, the in vivo response time of thermal magnetogenetics is currently tens of seconds, which prevents the precise temporal modulation of neural activity similar to light-based optogenetics. Moreover, magnetogenetics has not provided a means to selectively activate multiple channels to drive behavior. Here we produce sub-second behavioral responses in Drosophila melanogaster by combining magnetic nanoparticles with a rate-sensitive thermoreceptor (TRPA1-A). Furthermore, by tuning the properties of magnetic nanoparticles to respond to different magnetic field strengths and frequencies, we can achieve sub-second, multichannel stimulation, analogous to multi-color optogenetic stimulation. These results bring magnetogenetics closer to the temporal resolution and multiplexed stimulation possible with optogenetics while maintaining the minimal invasiveness and deep-tissue stimulation only possible by magnetic control.

sentences: Relative DOPA Deficiency in Lightly Pigmented Skin Contributes to UV-Independent Melanoma Susceptibility Melanoma risk is 30 times higher in people with lightly pigmented skin compared to those with darkly pigmented skin. Here we show that this difference results from more than melanin pigment and its ultraviolet radiation (UVR) shielding effect. Using primary human melanocytes representing the full human skin pigment continuum and several preclinical melanoma models, we show that cell-intrinsic differences between dark and light melanocytes regulate melanocyte proliferative capacity, overall cellular differentiation state, and susceptibility to malignant transformation, independently of melanin and UV exposure. We determined that these differences result from dihydroxyphenylalanine (DOPA), a melanin precursor synthesized at higher levels in melanocytes from dark skin. Although DOPA was not previously known to have specific signaling activity, we used both high throughput pharmacologic and genetic in vivo CRISPR screens to determine that DOPA limits melanocyte and melanoma cell proliferation by directly inhibiting the muscarinic acetylcholine receptor M1 (CHRM1), a G Protein-Coupled Receptor (GPCR) not previously known to bind DOPA, nor to affect melanoma pathobiology. Pharmacologic CHRM1 antagonism in melanoma leads to depletion of c-Myc and FOXM1, both of which are proliferation drivers associated with aggressive melanoma. In preclinical mouse melanoma models using both immune deficient and syngeneic immune competent mice, pharmacologic inhibition of CHRM1 or FOXM1 inhibited tumor growth. CHRM1 and FOXM1 may be new therapeutic targets for melanoma.

sentences: The Unique Cytoarchitecture and Wiring of the Human Default Mode Network It is challenging to specify the role of the default mode network (DMN) in human behaviour. Contemporary theories, based on functional magnetic resonance imaging (MRI), suggest that the DMN is insulated from the external world, which allows it to support perceptually-decoupled states and to integrate external and internal information in the construction of abstract meanings. To date, the neuronal architecture of the DMN has received relatively little attention. Understanding the cytoarchitectural composition and connectional layout of the DMN will provide novel insights into its role in brain function. We mapped cytoarchitectural variation within the DMN using a cortical type atlas and a histological model of the entire human brain. Next, we used MRI acquired in healthy young adults to explicate structural wiring and effective connectivity. We discovered profound diversity of DMN cytoarchitecture. Connectivity is organised along the most dominant cytoarchitectural axis. One side of the axis is the prominent receiver, whereas the other side remains more insulated, especially from sensory areas. The structural heterogeneity of the DMN engenders a network-level balance in communication with external and internal sources, which is distinctive, relative to other functional communities. The neuronal architecture of the DMN suggests it is a protuberance from the core cortical processing hierarchy and holds a unique role in information integration.

sentences: From STRs to SNPs via ddRAD-seq: geographic assignment of confiscated tortoises at reduced costs Assigning individuals to their source populations is crucial for conservation research, especially for endangered species threatened by illegal trade and translocations. Genetic assignment can be achieved with different types of molecular markers, but technical advantages and cost saving are recently promoting the shift from short tandem repeats (STRs) to single nucleotide polymorphisms (SNPs). Here, we designed, developed, and tested a small panel of SNPs for cost-effective geographic assignment of individuals with unknown origin of the endangered Mediterranean tortoise Testudo hermanni. We started by performing a ddRAD-seq experiment on 70 wild individuals of T. hermanni from 38 locations. Results obtained using 3,182 SNPs are comparable to those previously obtained using STR markers in terms of genetic structure and power to identify the macro-area of origin. However, our SNPs revealed further insights into the substructure in Western populations, especially in Southern Italy. A small panel of highly informative SNPs was then selected and tested by genotyping 190 individuals using the KASP genotyping chemistry. All the samples from wild populations of known geographic origin were genetically re-assigned with high accuracy to the original population. This reduced SNPs panel represents an efficient molecular tool that enables individuals to be genotyped at low cost (less than {euro}15 per sample) for geographical assignment and identification of hybrids. This information is crucial for the management in-situ of confiscated animals and their possible re-allocation in the wild. Our methodological pipeline can easily be extended to other species.

sentences: WDR5 promotes breast cancer growth and metastasis via KMT2-independent translation regulation Metastatic breast cancer remains a major cause of cancer related deaths in women and there are few effective therapies against this advanced disease. Emerging evidence suggests that key steps of tumor progression and metastasis are controlled by reversible epigenetic mechanisms. Using an in vivo genetic screen, we identified WDR5 as an actionable epigenetic regulator that is required for metastatic progression in models of triple-negative breast cancer. We found that knockdown of WDR5 in breast cancer cells independently impaired their tumorigenic as well as metastatic capabilities. Mechanistically, WDR5 promotes cell growth by increasing ribosomal gene expression and translation efficiency in a KMT2-independent manner. Consistently, pharmacological inhibition or degradation of WDR5 impedes cellular translation rate and the clonogenic ability of breast cancer cells. Furthermore, combination of WDR5-targeting with mTOR inhibitors leads to potent suppression of translation and proliferation of breast cancer cells. These results reveal novel therapeutic strategies to treat metastatic breast cancer.

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Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of mitochondrial TCA cycle enzyme, succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect in PC tumors, triggers dysregulation of Ca2+ homeostasis, and aberrantly activates calpain and the protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a cascade of phospho- signaling where GSK3 inhibition inactivates energy sensing by AMP-kinase through dephosphorylation of the AMP-kinase {gamma} subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC tumor formation. A novel Cdk5 inhibitor, MRT3-007, reversed this phospho-cascade, invoking an anti- Warburg effect, cell cycle arrest, and senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important novel mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.

HighlightsO_LILoss of SDHB function in pheochromocytoma causes Ca2+ dysregulation, calpain activation, and aberrant activation of the protein kinase Cdk5.
C_LIO_LIHyperactive Cdk5 deregulates a GSK3/PRKAG2/AMPK signaling cascade.
C_LIO_LIp25 overexpression and consequent aberrant Cdk5 activity in chromaffin cells causes pheochromocytoma.
C_LIO_LIInhibition of Cdk5 activates the PRKAG2/AMPK/p53 signaling to rescue cell senescence and block PC tumor progression.
C_LI

Graphical Abstract

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sentences: Speech prosody supports speaker selection and auditory stream segregation in a multi-talker situation To process speech in a multi-talker environment, listeners need to segregate the mixture of incoming speech streams and focus their attention on one of them. Potentially, speech prosody could aid the segregation of different speakers, the selection of the desired speech stream, and detecting targets within the attended stream. For testing these issues, we recorded behavioral responses and extracted event-related potentials and functional brain networks from electroencephalographic signals recorded while participants listened to two concurrent speech streams, performing a lexical detection and a recognition memory task in parallel. Prosody manipulation was applied to the attended speech stream in one group of participants and to the ignored speech stream in another group. Naturally recorded speech stimuli were either intact, synthetically F0-flattened, or suppressed by the speaker. Results show that prosody - especially the parsing cues mediated by speech rate - facilitates stream selection, while playing a smaller role in auditory stream segmentation and target detection.

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Intestinal Enteroid Monolayers Model the Human Intestinal Environment for Escherichia coli Infection Enterohemorrhagic Escherichia coli O157:H7 is an enteric pathogen responsible for bloody diarrhea, hemolytic uremic syndrome, and in severe cases even death. The study of O157:H7 is difficult due to the high specificity of the bacteria for the human intestine, along with our lack of sufficiently complex human cell culture models. The recent development of human intestinal enteroids derived from intestinal crypt multipotent stem cells has allowed us to construct 2-dimensional differentiated epithelial monolayers grown in transwells that mimic the human intestine. Unlike previous studies, saline was added to the apical surface, while maintaining culture media in the basolateral well. The monolayers continued to grow and differentiate with apical saline. Apical infection with O157:H7 or commensal E. coli resulted in robust bacterial growth from 105 to over 108 over 24 hours. Despite this robust bacterial growth, commensal E. coli neither adhered to nor damaged the epithelial barrier over 30 hours. However, O157:H7 was almost fully adhered (>90%) by 18 hours with epithelial damage observed by 30 hours. O157:H7 contains the locus of enterocyte effacement (LEE) pathogenicity island responsible for attachment and damage to the intestinal epithelium. Previous studies report the ability of nutrients such as biotin, D-serine, and L-fucose to downregulate LEE gene expression. O157:H7 treated with biotin or L-fucose, but not D-serine displayed both decreased attachment and reduced epithelial damage over 36 hours. These data illustrate enteroid monolayers can serve as a suitable model for the study of O157:H7 pathogenesis, and identification of potential therapeutics.

ImportanceO157:H7 is difficult to study due to its high specificity for the human intestine and the lack of sufficiently complex human cell culture models. The recent development of human intestinal enteroids derived from intestinal crypt multipotent stem cells has allowed us to construct 2-dimensional differentiated epithelial monolayers grown in transwells that mimic the human intestine. Our data illustrates enteroid monolayers can serve as a suitable model for the study of O157:H7 pathogenesis, and allow for identification of potential therapeutics.

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Differential impact of transdiagnostic, dimensional psychopathology on multiple scales of functional connectivity IntroductionDimensional psychopathology strives to associate different domains of cognitive dysfunction with brain circuitry. Connectivity patterns as measured by functional magnetic resonance imaging (fMRI) exist at multiple scales, with global networks of connectivity composed of microscale interactions between individual nodes. It remains unclear how separate dimensions of psychopathology might differentially impact these different scales of organization.

MethodsPatients experiencing anxious misery symptomology (depression, anxiety and trauma; n = 192) were assessed for symptomology and received resting-state fMRI scans. Three modeling approaches (seed-based correlation analysis [SCA], support vector regression [SVR] and Brain Basis Set Modeling [BSS]), each relying on increasingly dense representations of functional connectivity patterns, were used to associate connectivity patterns with six different dimensions of psychopathology: anxiety sensitivity, anxious arousal, rumination, anhedonia, insomnia and negative affect. Importantly, a full 50 patients were held-out in a testing dataset, leaving 142 patients as training data.

ResultsDifferent symptom dimensions were best modeled by different scales of brain connectivity: anhedonia and anxiety sensitivity were best modeled with single connections (SCA), insomnia and anxious arousal by mesoscale patterns (SVR) and negative affect and ruminative thought by broad, cortex-spanning patterns (BBS). Dysfunction within the default mode network was implicated in all symptom dimensions that were best modeled by multivariate models.

ConclusionThese results suggest that symptom dimensions differ in the degree to which they impact different scales of brain organization. In addition to advancing our basic understanding of transdiagnostic psychopathology, this has implications for the translation of basic research paradigms to human disorders.

sentences: PARROT: a flexible recurrent neural network framework for analysis of large protein datasets The rise of high-throughput experiments has transformed how scientists approach biological questions. The ubiquity of large-scale assays that can test thousands of samples in a day has necessitated the development of new computational approaches to interpret this data. Among these tools, machine learning approaches are increasingly being utilized due to their ability to infer complex non-linear patterns from high-dimensional data. Despite their effectiveness, machine learning (and in particular deep learning) approaches are not always accessible or easy to implement for those with limited computational expertise. Here we present PARROT, a general framework for training and applying deep learning-based predictors on large protein datasets. Using an internal recurrent neural network architecture, PARROT is capable of tackling both classification and regression tasks while only requiring raw protein sequences as input. We showcase the potential uses of PARROT on three diverse machine learning tasks: predicting phosphorylation sites, predicting transcriptional activation function of peptides generated by high-throughput reporter assays, and predicting the fibrillization propensity of amyloid beta with data generated by deep mutational scanning. Through these examples, we demonstrate that PARROT is easy to use, performs comparably to state-of-the-art computational tools, and is applicable for a wide array of biological problems.

sentences: Human leukocyte antigen class II gene diversity tunes antibody repertoires to common pathogens Allelic diversity of HLA class II genes may help maintain humoral immunity against infectious diseases. We investigated the relative contribution of specific HLA class II alleles, haplotypes and genotypes on the variation of antibody responses to a variety of common pathogens in a cohort of 800 adults representing the general Arab population. We found that classical HLA class II gene heterozygosity confers a selective advantage. Moreover, we demonstrated that multiple HLA class II alleles play a synergistic role in shaping the antibody repertoire. Interestingly, associations of HLA-DRB1 genotypes with specific antigens were identified. Our findings suggest that HLA class II gene polymorphisms confer specific humoral immunity against common pathogens, which may have contributed to the genetic diversity of HLA class II loci during hominine evolution.

sentences: Synthesis, Characterization, and Nanodisc formation of Non-ionic Polymers Despite lipid-nanodiscs are increasingly used in the structural studies of membrane proteins, drug delivery and other applications, the interaction between the nanodisc-belt and the protein to be reconstituted is a major limitation. To overcome this limitation and to further broaden the scope of nanodiscs, a family of non-ionic amphiphilic polymers synthesized by hydrophobic functionalization of fructo-oligosaccharides/inulin is reported. We show the stability of lipid-nanodiscs formed by these polymers against pH and divalent metal ions, and their magnetic-alignment properties. The reported results also demonstrate that the non-ionic polymers extract membrane proteins with unprecedented efficiency.

sentences: Using herbarium samples for NGS methods - a methodological comparison Herbaria harbor a tremendous amount of plant specimens that are rarely used for plant systematic studies. The main reason is the difficulty to extract a decent quantity of good quality DNA from the preserved plant material. While the extraction of ancient DNA in animals is well established, studies including old plant material are still underrepresented. In our study we compared the standard Qiagen DNeasy Plant Mini Kit and a specific PTB-DTT protocol on two different plant genera (Xanthium L. and Salix L.). The included herbarium material covered about two centuries of plant collections. A selected subset of samples was used for a standard library preparation as well as a target enrichment approach. The results revealed that PTB-PTT resulted in higher quantity and quality regarding DNA yield. For relatively recent herbarium specimens, and despite the lower overall yield of DNA, the Qiagen Kit resulted in better sequencing results regarding the number of filtered and mapped reads. We were able to successfully sequence a sample from 1820 and conclude that it is possible to include old herbarium specimens in NGS approaches. This opens a treasure box for phylogenomic research.

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The rise and fall of the photoinhibition-related energy dissipation qI Photosynthesis converts sunlight into chemical energy, sustaining the vast majority of the biosphere. Photosystem II (PSII), the oxygen-forming enzyme that initiates photosynthesis, is however particularly prone to light-induced damage in a process known as photoinhibition, which limits the productivity of both aquatic and land photosynthesis. Photoinhibition is associated with an energy dissipation process of unknown origin, termed qI. Here, we present a detailed biophysical and biochemical in vivo study of qI in model green alga Chlamydomonas reinhardtii. Time-resolved fluorescence measurements demonstrate the origin of qI, and indicate the PSII reaction centre as the site of the quencher. Oxygen-dependence of quenching site formation, but not photoinhibition itself, is shown, suggesting that two types of PSII damage - donor and acceptor-side impairment - can be separated. We then demonstrate that the quenching loss takes place in the absence of PSII repair, and is mediated by the degradation of photoinhibited PSII cores by the FtsH protease. Finally, we integrate data ranging from picoseconds to hours in the context of structure-function excitation energy-transferring membrane patches, revealing the extent of PSII heterogeneity from the onset of photoinhibition until the breakdown of damaged PSII.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/434601v1_ufig1.gif" ALT="Figure 1">
View larger version (26K):
org.highwire.dtl.DTLVardef@d7c64dorg.highwire.dtl.DTLVardef@1c2943org.highwire.dtl.DTLVardef@d90b83org.highwire.dtl.DTLVardef@182cd12_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIUpon photoinhibition, oxygen sensitization results in an irreversible formation of quenching (qI) and inactivation of Photosystem II
C_LIO_LIqI takes place in the PSII reaction centre
C_LIO_LIPhotoinhibition-induced D1 cleavage is much slower than qI formation
C_LIO_LIFtsH metalloprotease is required to degrade quenching PSII reaction centres
C_LIO_LIA multiscale energy transfer model describes heterogeneity of PSII during photoinhibition
C_LI

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Cell surface hydrophobicity determines aggregate assembly type in Pseudomonas aeruginosa Bacteria live in spatially organized aggregates during chronic infections, where they adapt to the host environment, evade immune responses and resist therapeutic interventions. Although it is known that environmental factors such as polymers influence bacterial aggregation, it is not clear how bacterial adaptation during chronic infection impacts the formation and spatial organization of aggregates in the presence of polymers. Here we show that in an in vitro model of cystic fibrosis (CF) containing the polymers eDNA and mucin, O-specific antigen is a major factor in determining the formation of two distinct aggregate assembly types of Pseudomonas aeruginosa due to alterations in cell surface hydrophobicity. Our findings suggest that during chronic infection, interplay between cell surface properties and polymers in the environment may influence the formation and structure of bacterial aggregates, which would shed new light on the fitness costs and benefits of O-antigen production in environments such as CF lungs.

ImportanceDuring chronic infection, several factors contribute to the biogeography of microbial communities. Heterogeneous populations of Pseudomonas aeruginosa form aggregates in cystic fibrosis airways, however, the impact of this population heterogeneity on spatial organization and aggregate assembly is not well understood. In this study we found that changes in O-specific antigen determine the spatial organization of P. aeruginosa cells by altering the relative cell surface hydrophobicity. This finding suggests a role for O-antigen in regulating P. aeruginosa aggregate size and shape in cystic fibrosis airways.

sentences: HIV-1 drug resistance profiling using amino acid sequence space cartography Human immunodeficiency virus (HIV) drug resistance is a global healthcare issue. The emergence of drug resistance demands treatment adaptation. Computational methods predicting the drug resistance profile from genomic data of HIV isolates are advantageous for monitoring drug resistance in patients. Yet, the currently existing computational methods for drug resistance prediction are either not suitable for complex mutational patterns in emerging HIV strains or lack interpretability of prediction results which is of paramount importance in clinical practice. Hence, to overcome these limitations, new approaches for the HIV drug resistance prediction combining high accuracy and interpretability are required. In this work, a new methodology for the analysis of protein sequence data based on the application of generative topographic mapping was developed and applied for HIV drug resistance profiling. It allowed achieving high accuracy of resistance predictions and intuitive interpretation of prediction results. The developed approach was successfully applied for the prediction of HIV resistance towards protease, reverse-transcriptase and integrase inhibitors and in-depth analysis of HIV resistance-inducing mutation patterns. Hence, it can serve as an efficient and interpretable tool to suggest optimal treatment regimens.

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Measles virus exits human airway epithelia via infectious center sloughing Measles virus (MeV) is the most contagious human virus, but we do not fully understand why. Unlike most respiratory viruses, MeV does not infect the airway epithelium immediately. MeV traverses the epithelium within immune cells that carry it to lymphatic organs where amplification occurs. Infected immune cells then synchronously deliver large amounts of virus to the airways. However, our understanding of MeV replication in airway epithelia is limited. To model it, we use well-differentiated primary cultures of human airway epithelial cells (HAE) from lung donors. In HAE, MeV spreads directly cell-to-cell forming infectious centers that grow for [~]3-5 days, are stable for a few days, and then disappear. Transepithelial electrical resistance remains intact during the entire course of HAE infection, thus we hypothesized that MeV infectious centers may slough off while preserving epithelial function. After documenting by confocal microscopy that infectious centers progressively detach from HAE, we recovered apical washes and separated cell-associated from cell-free virus by centrifugation. Virus titers were about 10 times higher in the cell-associated fraction than in the supernatant. In sloughed infectious centers, ciliary beating persisted and apoptotic markers were not readily detected, suggesting that they retain functional metabolism. Cell-associated MeV infected primary human monocyte-derived macrophages, modeling the first stage of infection in a new host. Single-cell RNA sequencing identified wound healing, cell growth, and cell differentiation as biological processes relevant for infectious center sloughing. 5-ethynyl-2-deoxyuridine (EdU) staining located proliferating cells underneath infectious centers. Thus, cells located below infectious centers divide and differentiate to repair the extruded infected epithelial patch. As an extension of these studies, we postulate that expulsion of infectious centers through coughing and sneezing could contribute to MeVs strikingly high reproductive number by allowing the virus to survive longer in the environment and by delivering a high infectious dose to the next host.

AUTHOR SUMMARYMeasles virus (MeV) is a respiratory pathogen that infects millions worldwide each year. Although sometimes mischaracterized as an innocuous childhood disease, measles remains a leading cause of death for children under five. MeV is the most contagious human virus and requires vaccination rates above 90% to maintain herd immunity. Global decreases in vaccination rates over the past ten years contributed to recent, widespread MeV outbreaks. We uncover here a novel mechanism by which MeV exits the human airways that may explain why it is much more contagious than other viruses. We document that infected cells containing cell-associated virus slough en masse from the airway epithelial sheet. These expelled infectious centers are metabolically active and can transmit infection to primary human monocyte-derived macrophages more efficiently than cell-free virus particles. Thus, cell-associated MeV can transmit host-to-host, a new paradigm for efficient respiratory virus transmission.

sentences: The Effects of Central Nervous System Stimulants on Drosophila melanogaster Reproduction Stimulant drugs are used everyday by people around the world. The effect stimulants have on developing human fetuses is widely unknown. The fruit fly Drosophila melanogaster has become a valuable system to model the complex effects and properties of drugs in mammals. In this study, Drosophila is used to analyze the effects of stimulant exposure on reproduction to determine if stimulants cause a significant decrease in the number of offspring produced by parent generations. Caffeine, nicotine, and pseudoephedrine hydrochloride were found to significantly decrease the number of offspring in experimental populations. Further experimentation is necessary to understand the mechanisms underlying these results.

sentences: Phage Paride hijacks bacterial stress responses to kill dormant, antibiotic-tolerant cells Bacteriophages are fierce viral predators with no regard for pathogenicity or antibiotic resistance of their bacterial hosts1,2. Despite early recognition of their therapeutic potential and the current escalation of bacterial multidrug resistance, phages have so far failed to become a regular treatment option in clinical practice3-5. One reason is the occasional discrepancy between poor performance of selected phages in vivo despite high potency in vitro3,4,6,7. Similar resilience of supposedly drug-sensitive bacterial infections to antibiotic treatment has been linked to persistence of dormant cells inside patients8,9. Given the abundance of non-growing bacteria also in the environment10,11, we wondered whether some phages can infect and kill these antibiotic-tolerant cells. As shown previously12-16, most phages failed to replicate on dormant hosts and instead entered a state of hibernation or pseudolysogeny. However, we isolated a new Pseudomonas aeruginosa phage named Paride with the exciting ability to directly kill dormant, antibiotic-tolerant hosts by lytic replication, causing sterilization of deep-dormant cultures in synergy with the {beta}-lactam meropenem. Intriguingly, efficient replication of Paride on dormant hosts depends on the same bacterial stress responses that also drive antibiotic tolerance. We therefore suggest that Paride hijacks weak spots in the dormant physiology of antibiotic-tolerant bacteria that could be exploited as Achilles heels for the development of new treatments targeting resilient bacterial infections.

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Injury-Free In Vivo Delivery and Engraftment into the Cornea Endothelium Using Extracellular Matrix Shrink-Wrapped Cells Cell injection is a common clinical approach for therapeutic delivery of cells into diseased and damaged tissues in order to achieve regeneration. However, cell retention, viability, and engraftment at the injection site has generally been poor, driving the need for improved approaches. Here, we developed a technique to shrink-wrap micropatterned islands of corneal endothelial cells in a basement membrane-like layer of extracellular matrix (ECM) that enables the cells to maintain their cell-cell junctions and cytoskeletal structure while in suspension. These Monolayers exhibited the ability to rapidly engraft into intact, high-density corneal endothelial monolayers in both in vitro and in vivo model systems. Importantly, the engrafted Monolayers increased local cell density, something that the clinical-standard single cells in suspension failed to do. These results show that shrink-wrapping cells in ECM dramatically improves engraftment and provides a potential alternative to cornea transplant when low endothelial cell density is the cause of corneal blindness.

One Sentence SummaryShrink-wrapped patches of endothelial cells can rapidly attach and integrate into an intact cornea endothelium when injected into the anterior chamber, increasing cell density.

sentences: Cellular compartmentalisation and receptor promiscuity as a strategy for accurate and robust inference of position during morphogenesis Precise spatial patterning of cell fate during morphogenesis requires accurate inference of cellular position. In making such inferences from morphogen profiles, cells must contend with inherent stochasticity in morphogen production, transport, sensing and signalling. Motivated by the multitude of signalling mechanisms in various developmental contexts, we show how cells may utilise multiple tiers of processing (compartmentalisation) and parallel branches (multiple receptor types), together with feedback control, to bring about fidelity in morphogenetic decoding of their positions within a developing tissue. By simultaneously deploying specific and nonspecific receptors, cells achieve a more accurate and robust inference. We explore these ideas in the patterning of Drosophila melanogaster wing imaginal disc by Wingless morphogen signalling, where multiple endocytic pathways participate in decoding the morphogen gradient. This distributed information processing at the scale of the cell highlights how local cell autonomous control facilitates global tissue scale design.

sentences: Urgent Brain Vascular Regeneration Occurs via Lymphatic Transdifferentiation Acute ischemic stroke damages regional brain blood vessel (BV) network. Urgent recovery of basic blood flows, which represents the earliest regenerated BVs, are critical to improve clinical outcomes and minimize lethality. Although the late-regenerated BVs have been implicated to form via growing along the meninge-derived lymphatic vessels (iLVs), mechanisms underlying the early, urgent BV regeneration remain elusive. Using zebrafish cerebrovascular injury models, we show that the earliest regenerated BVs come from lymphatic transdifferentiation, a hitherto unappreciated process in vertebrates. Mechanistically, LV-to-BV transdifferentiation occurs exclusively in the stand-alone iLVs through Notch activation. In the track iLVs adhered by nascent BVs, transdifferentiation never occurs because the BV-expressing EphrineB2a paracellularly activates the iLV-expressing EphB4a to inhibit Notch activation. Suppression of LV-to-BV transdifferentiation blocks early BV regeneration and becomes lethal. These results demonstrate that urgent BV regeneration occurs via lymphatic transdifferentiation, suggesting this process and key regulatory molecules EphrinB2a/EphB4a/Notch as new post-ischemic therapeutic targets.

sentences: CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity CRISPR-Cas12a (Cpf1) is a bacterial RNA-guided nuclease that cuts double-stranded DNA (dsDNA) at sites specified by a CRISPR RNA (crRNA) guide. Additional activities have been ascribed to this enzyme in vitro: site-specific (cis) single-stranded DNA (ssDNA) cleavage and indiscriminate (trans) degradation of ssDNA, RNA, and dsDNA after activation by a complementary target. The ability of Cas12a to cleave nucleic acids indiscriminately has been harnessed for many applications, including diagnostics, but it remains unknown if it contributes to bacterial immunity. Here, we provide evidence that cleavage of ssDNA in cis or in trans by Cas12a is insufficient to impact immunity. Using LbCas12a expressed in either Pseudomonas aeruginosa or Escherichia coli, we observed that cleavage of dsDNA targets did not elicit cell death or dormancy, suggesting insignificant levels of collateral damage against host RNA or DNA. Canonical immunity against invasive dsDNA also had no impact on the replicative fitness of co-infecting ssDNA phage or plasmid in trans. Lastly, crRNAs complementary to invasive ssDNA did not provide protection, suggesting that ssDNA cleavage does not occur in vivo or is insignificant. Overall, these results suggest that CRISPR-Cas12a immunity predominantly occurs via canonical targeting of dsDNA, and that the other activities are in vitro irregularities.

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Gametogenesis in the green seaweed Ulva mutabilis coincides with massive transcriptional restructuring The molecular mechanism underlying sexual reproduction in land plants is well understood in model plants and is a target for crop improvement. However, unlike land plants, the genetic basis involved in triggering reproduction and gamete formation remains elusive in most seaweeds, which are increasingly viewed as an alternative source of functional food and feedstock for energy applications. Here, gametogenesis of Ulva mutabilis, a model organism for green seaweeds, is studied. We analyze transcriptome dynamics at different time points during gametogenesis following induction of reproduction by fragmentation and removal of sporulation inhibitors. Analyses demonstrate that 45% of the genes in the genome are differentially expressed during gametogenesis. We identified several transcription factors that potentially play a key role in the early gametogenesis of Ulva given the function of their homologs in higher plants and microalgae. In particular, the detailed expression pattern of an evolutionary conserved transcription factor containing an RWP-RK domain suggests a key role during Ulva gametogenesis. The identification of putative master regulators of gametogenesis provides a starting point for further functional characterization.

HighlightTranscriptomic analyses of gametogenesis in the green seaweed Ulva highlight the importance of a conserved RWP-RK transcription factor in induction of sexual reproduction.

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Application of machine learning methods in clinical trials for precision medicine ObjectiveA key component for precision medicine is a good prediction algorithm for patients response to treatments. We aim to implement machine learning (ML) algorithms into the response-adaptive randomization (RAR) design and improve the treatment outcomes.

Materials and MethodsWe incorporated nine ML algorithms to model the relationship of patient responses and biomarkers in clinical trial design. Such a model predicted the response rate of each treatment for each new patient and provide guidance for treatment assignment. Realizing that no single method may fit all trials well, we also built an ensemble of these nine methods. We evaluated their performance through quantifying the benefits for trial participants, such as the overall response rate and the percentage of patients who receive their optimal treatments.

ResultsSimulation studies showed that the adoption of ML methods resulted in more personalized optimal treatment assignments and higher overall response rates among trial participants. Compared with each individual ML method, the ensemble approach achieved the highest response rate and assigned the largest percentage of patients to their optimal treatments. For the real-world study, we successfully showed the potential improvements if the proposed design had been implemented in the study.

ConclusionIn summary, the ML-based RAR design is a promising approach for assigning more patients to their personalized effective treatments, which makes the clinical trial more ethical and appealing. These features are especially desirable for late-stage cancer patients who have failed all the FDA-approved treatment options and only can get new treatments through clinical trials.

sentences: Cerebello-Basal Ganglia Network Global Efficiency Predicts Cortical Network Global Efficiency The cerebellum (CB) and basal ganglia (BG) each have topographically distinct functional subregions that are functionally and anatomically interconnected with cortical regions through discrete thalamic loops and with each other via disynaptic connections, with previous work detailing high levels of functional connectivity between these phylogenetically ancient regions. It was posited that this CB-BG network provides support for cortical systems processing, spanning cognitive, emotional, and motor domains, implying subcortical network measures are strongly related to cortical network measures (Bostan & Strick, 2018); however, it is currently unknown how network measures within distinct CB-BG networks relate to cortical network measures. Here, 122 regions of interest comprising cognitive and motor CB-BG networks and 7 canonical cortical resting-state were used to investigate whether the integration (quantified using global efficiency, GE) of cognitive CB-BG network (CCBN) nodes and their segregation from motor CB-BG network (MCBN) nodes is related to cortical network GE and segregation in 233 non-related, right- handed participants (Human Connectome Project-1200). CCBN GE positively correlated with GE in the default mode, motor, and auditory networks and MCBN GE positively correlated with GE in all networks except the default mode and emotional. MCBN segregation was related to MN segregation. These findings highlight the CB-BG networks role in executive function, task switching, and verbal working memory. This work has implications for understanding cortical network organization and cortical-subcortical interactions in healthy adults and may help in deciphering subcortical differences seen in disease states.

sentences: The Ndc80-Cdt1-Ska1 complex constitute a minimal processive kinetochore-microtubule coupling unit The Ndc80 kinetochore complex is essential for robust kinetochore-microtubule (k-MT) attachments during mitosis. Ndc80 has been shown to recruit the Ska1 complex to kinetochores, where Ska1 is thought to aid in k-MT coupling by Ndc80. Our previous work has shown that Cdt1, a DNA replication licensing factor, is a novel mitotic spindle-associated protein that is also recruited to kinetochores via Ndc80 and is required for stabilizing k-MT attachments. In this study, we developed auxin-induced degron (AID)-tagging to validate the previously demonstrated mitotic role of Cdt1. We demonstrate a direct interaction between Cdt1 and Ska1 that is essential for proper recruitment of Cdt1 to kinetochores and spindle microtubules. We find that Cdk1dependent phosphorylation of Cdt1 during mitosis is critical for Ska1-binding, consequently regulating the stabilization of metaphase k-MT attachments and normal mitotic progression. Total internal reflection fluorescence microscopy (TIR-FM) experiments reveal that Cdt1 synergizes with the Ndc80 and the Ska1 complexes for microtubule-binding. Further, we show that single Cdt1 molecules form diffusive tripartite complexes with Ndc80 and Ska1 that can processively track the ends of dynamic microtubules in vitro. Taken together our data identifies a minimal molecular unit responsible for bidirectional processive tip tracking of kinetochores.

sentences: Genome size evolution in the beetle genus Diabrotica Diabrocite corn rootworms are one of the most economically significant pests of maize in the United States and Europe and an emerging model for insect-plant interactions. Genome sizes of several species in the genus Diabrotica were estimated using flow cytometry along with that of Acalymma vittatum as an outgroup. Genome sizes ranged between 1.56 - 1.64 gigabase pairs (Gb) and between 2.26-2.59 Gb, respectively, for the Diabrotica subgroups fucata and virgifera; the Acalymma vittatum genome size was around 1.65 Gb. This result indicated that a substantial increase in genome size occurred in the ancestor of the virgifera group. Further analysis of fucata group and virgifera group genome sequencing reads indicated that the genome size difference between the Diabrotica subgroups could be attributed to a higher content of transposable elements, mostly miniature inverted-transposable elements (MITEs) and gypsy-like long terminal repeat (LTR) retroelements.

sentences: PyMVPD: A toolbox for multivariate pattern dependence AO_SCPLOWBSTRACTC_SCPLOWCognitive tasks engage multiple brain regions. Studying how these regions interact is key to understand the neural bases of cognition. Standard approaches to model the interactions between brain regions rely on univariate statistical dependence. However, newly developed methods can capture multivariate dependence. Multivariate Pattern Dependence (MVPD) is a powerful and flexible approach that trains and tests multivariate models of the interactions between brain regions using independent data. In this article, we introduce PyMVPD: an open source toolbox for Multivariate Pattern Dependence. The toolbox includes pre-implemented linear regression models and artificial neural network models of the interactions between regions. It is designed to be easily customizable. We demonstrate example applications of PyMVPD using well-studied seed regions such as the fusiform face area (FFA) and the parahippocampal place area (PPA). Next, we compare the performance of different model architectures. Overall, artificial neural networks outperform linear regression. Importantly, the best performing architecture is region-dependent: MVPD subdivides cortex in distinct, contiguous regions whose interaction with FFA and PPA is best captured by different models.

sentences: Where we perceive before we look: The distribution of presaccadic attention assessed with dynamic 1/f noise Already before the onset of a saccadic eye movement, we preferentially process visual information at the upcoming eye fixation. This  presaccadic shift of attention is typically assessed via localized test items, which potentially bias the attention measurement. Here we show how presaccadic attention shapes perception from saccade origin to target when no scene-structuring items are presented. Participants made saccades into a 1/f ("pink") noise field, in which we embedded a brief orientation signal at various locations shortly before saccade onset. Local orientation discrimination performance served as a proxy for the allocation of attention. Results demonstrate that (1) saccades are preceded by shifts of attention to their goal location even if they are directed into an unstructured visual field, but the spread of attention, compared to target-directed saccades, is broad; (2) the presaccadic attention shift is accompanied by considerable attentional costs at the presaccadic eye fixation; (3) objects markedly shape the distribution of presaccadic attention - demonstrating the relevance of an item-free approach for measuring attentional dynamics across the visual field.

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Statistical and machine learning models for classification of human wear and delivery days in accelerometry data PurposeAccelerometers are increasingly utilized in healthcare research to assess human activity. Accelerometry data are often collected by mailing accelerometers to participants, who wear the accelerometers to collect data on their activity. The devices are then mailed back to the laboratory for analysis. We develop models to classify days in accelerometry data as activity from actual human wear or the delivery process. These models can be used to automate the cleaning of accelerometry datasets that are adulterated with activity from delivery.

MethodsFor the classification of delivery days in accelerometry data, we developed statistical and machine learning models in a supervised learning context using a large human activity and delivery labeled accelerometry dataset. We extracted several features, which were included to develop random forest, logistic regression, mixed effects regression, and multilayer perceptron models, while convolutional neural network, recurrent neural network, and hybrid convolutional recurrent neural network models were developed without feature extraction. Model performances were assessed using Monte Carlo cross-validation.

ResultsWe found that a hybrid convolutional recurrent neural network performed best in the classification task with an F1 score of 0.960 but simpler models such as logistic regression and random forest also had excellent performance with F1 scores of 0.951 and 0.957, respectively.

ConclusionThe models developed in this study can be used to classify days in accelerometry data as either human or delivery activity. An analyst can weigh the larger computational cost and greater performance of the convolutional recurrent neural network against the faster but slightly less powerful random forest or logistic regression. The best performing models for classification of delivery data are publicly available on the open source R package, PhysicalActivity.

sentences: Engineering transcription factor BmoR mutants for constructing multifunctional alcohol biosensors Native transcription factor-based biosensors (TFBs) have the potential for in situ detection of value-added chemicals or byproducts. However, their industrial application is limited by their ligand promiscuity, low sensitivity, and narrow detection range. Alcohols exhibit similar structures, and no reported TFB can distinguish a specific alcohol from its analogs. Here, we engineered an alcohol-regulated transcription factor, BmoR, and obtained various mutants with remarkable properties. For example, the generated signal-molecule-specific BmoRs could distinguish the constitutional isomers n-butanol and isobutanol, with insensitivity up to an ethanol concentration of 800 mM (36.9 g/L). Linear detection of 0-60 mM of a specific higher alcohol could be achieved in the presence of up to 500 mM (23.0 g/L) ethanol as background noise. Notably, two mutants with raised outputs and over 1.0 x 107-fold higher sensitivity, and one mutant with an increased upper detection limit (14.8 g/L n-butanol or isobutanol) were screened out. Using BmoR as an example, this study systematically explored the ultimate detection limit of a TFB towards its small-molecule ligands, paving the way for in situ detection in the biofuel and wine industries.

sentences: On the edge of criticality: strength-dependent perturbation unveils delicate balance between fluctuation and oscillation in brain dynamics Despite decades of research, there is still a lack of understanding of the role and generating mechanisms of the ubiquitous fluctuations and oscillations found in recordings of brain dynamics. Here, we used a strength-dependent perturbative framework to provide a causal mechanistic description of how human brain function is perched at the delicate balance between fluctuation and oscillation. Applying local strength-dependent perturbations and subsequently measuring the perturbative complexity index clearly demonstrates that the overall balance of brain dynamics is shifted towards fluctuations for providing much needed flexibility. Importantly, stimulation in the fluctuation regime modulates specific resting state network, thus providing a mechanistic explanation of experimentally reported brain dynamics. Furthermore, this framework generates specific, testable empirical predictions for human stimulation studies using strength-dependent rather than constant perturbation. Overall, the strength-dependent perturbative framework demonstrates how the human brain is poised on the edge of criticality, between fluctuations to oscillations, allowing for maximal flexibility.

sentences: Towards customized studies of G-quadruplex DNA structures in live cells G-quadruplex (G4) DNA structures are implicated in central biological processes and are considered promising therapeutic targets because of their links to human diseases such as cancer. However, functional details of how, when, and why G4 DNA structures form in vivo are largely missing leaving a knowledge gap that requires tailored chemical biology studies in relevant live-cell model systems. Towards this end, we developed a synthetic platform centered around one of the most effective and selective G4 stabilizing compounds, Phen-DC3. We used a structure-based design to equip Phen-DC3 with an amine in a position that does not interfere with G4 interactions. To evaluate the power of the approach, we next used this reactive handle to conjugate a BODIPY fluorophore and a cell-penetrating peptide to Phen-DC3. The BODIPY conjugation generated a fluorescent derivative with retained G4 selectivity, G4 stabilization, and cellular effects that revealed the localization and function of Phen-DC3 in human cells. On the other hand, the cell-penetrating peptide conjugation, while retaining G4 selectivity and stabilization, increased nuclear localization and cellular effects, showcasing the potential of this approach to modulate and direct cellular uptake e.g. as delivery vehicles. The developed platform can thus generate tailored biochemical tools for the studies of G4 biology to uncover molecular details and therapeutic approaches.

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GenomicSuperSignature: interpretation of RNA-seq experiments through robust, efficient comparison to public databases Millions of transcriptomic profiles have been deposited in public archives, yet remain underused for the interpretation of new experiments. We present a novel method for interpreting new transcriptomic datasets through near-instantaneous comparison to public archives without high-performance computing requirements. We performed Principal Component Analysis on 536 studies comprising 44,890 RNA sequencing profiles. Sufficiently similar loading vectors were aggregated to form Replicable Axes of Variation (RAV). RAVs were annotated with metadata of originating studies and samples and by gene set enrichment analysis. Functionality to associate new datasets with RAVs, extract interpretable annotations, and provide intuitive visualization are implemented as the GenomicSuperSignature R/Bioconductor package. We demonstrated the efficient and coherent database searching, robustness to batch effects and heterogeneous training data, and transfer learning capacity of our method using TCGA and rare diseases datasets. GenomicSuperSignature will aid analyzing new gene expression data in the context of existing databases using minimal computing resources.

PURPOSEMillions of transcriptomic profiles have been deposited in public archives, yet remain underused for the interpretation of new experiments. Existing methods for leveraging these public resources have focused on the reanalysis of existing data or analysis of new datasets independently. We present a novel approach to interpreting new transcriptomic datasets by near-instantaneous comparison to public archives without high-performance computing requirements.

METHODSTo identify replicable and interpretable axes of variation in any given gene expression dataset, we performed Principal Component Analysis (PCA) on 536 studies comprising 44,890 RNA sequencing profiles. Sufficiently similar loading vectors, when compared across studies, were aggregated to form Replicable Axes of Variation (RAV). RAVs were annotated with metadata of originating studies and samples and by gene set enrichment analysis. Functionality to associate new datasets with RAVs, extract interpretable annotations, and provide intuitive visualization are implemented as the GenomicSuperSignature R/Bioconductor package.

RESULTSRAVs are robust to batch effects and the presence of low-quality or irrelevant studies, and identify signals that can be lost by merging samples across the training datasets. The GenomicSuperSignature package allows instantaneous matching of PCA axes in new datasets to pre-computed RAVs, cutting down the analysis time from days to the order of seconds on an ordinary laptop. We demonstrate that RAVs associated with a phenotype can provide insight into weak or indirectly measured biological attributes in a new study by leveraging accumulated data from published datasets. Benchmarking against complementary previous works demonstrates that the RAV index 1) identifies colorectal carcinoma transcriptome subtypes that are similar to but more correlated with clinicopathological characteristics than previous disease-specific efforts and 2) can estimate neutrophil counts through transfer learning on new data comparably to the previous efforts despite major differences in training datasets and model building processes with the additional benefits of flexibility and scalability of the model application.

CONCLUSIONGenomicSuperSignature establishes an information resource and software tools to interrogate it. Prior knowledge databases are coherently linked, enabling researchers to analyze new gene expression data in the context of existing databases using minimal computing resources. The robustness of GenomicSuperSignature suggests that we can expand this approach beyond human gene expression profiles, such as single-cell RNA-seq, microbiome abundance, and different species transcriptomics datasets.

sentences: Trehalose increases tomato drought tolerance, induces defenses, and increases resistance to bacterial wilt disease Ralstonia solanacearum causes bacterial wilt disease, leading to severe crop losses. Xylem sap from R. solanacearum-infected tomato is enriched in the disaccharide trehalose. Water-stressed plants also accumulate trehalose, which increases drought tolerance via abscisic acid (ABA) signaling. Because R. solanacearum-infected plants suffer reduced water flow, we hypothesized that bacterial wilt physiologically mimics drought stress, which trehalose could mitigate. We found that R. solanacearum-infected plants differentially expressed drought-associated genes, including those involved in ABA and trehalose metabolism, and had more ABA in xylem sap. Consistent with this, treating tomato roots with ABA reduced both stomatal conductance and stem colonization by R. solanacearum. Treating roots with trehalose increased xylem sap ABA and reduced plant water use by lowering stomatal conductance and temporarily improving water use efficiency. Trehalose treatment also upregulated expression of salicylic acid (SA)-dependent tomato defense genes; increased xylem sap levels of SA and other antimicrobial compounds; and increased bacterial wilt resistance of SA-insensitive NahG tomato plants. Additionally, trehalose treatment increased xylem concentrations of jasmonic acid and related oxylipins. Finally, trehalose-treated plants were substantially more resistant to bacterial wilt disease. Together, these data show that exogenous trehalose reduced both water stress and bacterial wilt disease and triggered systemic disease resistance, possibly through a Damage Associated Molecular Pattern (DAMP) response pathway. This suite of responses revealed unexpected linkages between plant responses to biotic and abiotic stress and suggested that R. solanacearum-infected plants increase trehalose to improve water use efficiency and increase wilt disease resistance. The pathogen may degrade trehalose to counter these efforts. Together, these results suggest that treating tomatoes with exogenous trehalose could be a practical strategy for bacterial wilt management.

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Ligand Binding Prediction using Protein Structure Graphs and Residual Graph Attention Networks MotivationComputational prediction of ligand-target interactions is a crucial part of modern drug discovery as it helps to bypass high costs and labor demands of in vitro and in vivo screening. As the wealth of bioactivity data accumulates, it provides opportunities for the development of deep learning (DL) models with increasing predictive powers. Conventionally, such models were either limited to the use of very simplified representations of proteins or ineffective voxelization of their 3D structures. Herein, we present the development of the PSG-BAR (Protein Structure Graph -Binding Affinity Regression) approach that utilizes 3D structural information of the proteins along with 2D graph representations of ligands. The method also introduces attention scores to selectively weight protein regions that are most important for ligand binding.

ResultsThe developed approach demonstrates the state-of-the-art performance on several binding affinity benchmarking datasets. The attention-based pooling of protein graphs enables identification of surface residues as critical residues for protein-ligand binding. Finally, we validate our model predictions against an experimental assay on a viral main protease (Mpro)- the hallmark target of SARS-CoV-2 coronavirus.

AvailabilityThe code for PSG-BAR is made available at https://github.com/diamondspark/PSG-BAR

Contactacherkasov@prostatecentre.com

sentences: Predicting tomato field-yield using continuous monitoring of young tomato water status To address the challenge of predicting tomato yields in the field, we used whole-plant functional phenotyping to evaluate water relations under well-irrigated and drought conditions. The genotypes tested are known to exhibit variability in their yields in wet and dry fields. The examined lines included two lines with recessive mutations that affect carotenoid biosynthesis, zeta z2083 and tangerine t3406, both isogenic to the processing tomato variety M82. The two mutant lines were reciprocally grafted onto M82, and multiple physiological characteristics were measured continuously, before, during and after drought treatment in the greenhouse. A comparative analysis of greenhouse and field yields showed that the whole-canopy stomatal conductance (gsc) in the morning and cumulative transpiration (CT) were strongly correlated with field measurements of total yield (TY: r2 = 0.9 and 0.77, respectively) and plant vegetative weight (PW: r2 = 0.6 and 0.94, respectively). Furthermore, the minimum CT during drought and the rate of recovery when irrigation was resumed were both found to predict resilience.

sentences: TOR represses stress responses through global regulation of H3K27 trimethylation in plants Target of Rapamycin (TOR) functions as a central sensory hub to link a wide range of external stimuli to gene expression. However, the mechanisms underlying stimulus-specific transcriptional reprogramming by TOR remains elusive. Our in silico analysis in Arabidopsis demonstrates that TOR-repressed genes are associated with either bistable or silent chromatin states. Both states regulated by TOR signaling pathway are associated with high level of H3K27me3 deposited by CURLY LEAF (CLF) in specific context with LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Combinations of epigenetic modifications H3K4me3 and H3K27me3 implicate bistable feature which alternates between on and off state allowing rapid transcriptional changes upon external stimuli. Chromatin remodeler SWI2/SNF2 ATPase BRAHMA (BRM) activates TOR-repressed genes only at bistable chromatin domains to rapidly induce biotic stress responses. Here we demonstrated both in silico and in vivo that TOR represses transcriptional stress responses through global maintenance of H3K27me3.

sentences: A Fast Lasso-based Method for Inferring Pairwise Interactions AO_SCPLOWBSTRACTC_SCPLOWLarge-scale genotype-phenotype screens provide a wealth of data for identifying molecular alternations associated with a phenotype. Epistatic effects play an important role in such association studies. For example, siRNA perturbation screens can be used to identify pairwise gene-silencing effects. In bacteria, epistasis has practical consequences in determining antimicrobial resistance as the genetic background of a strain plays an important role in determining resistance. Existing computational tools which account for epistasis do not scale to human exome-wide screens and struggle with genetically diverse bacterial species such as Pseudomonas aeruginosa. Combining earlier work in interaction detection with recent advances in integer compression, we present a method for epistatic interaction detection on sparse (human) exome-scale data, and an R implementation in the package Pint. Our method takes advantage of sparsity in the input data and recent progress in integer compression to perform lasso-penalised linear regression on all pairwise combinations of the input, estimating up to 200 million potential effects, including epistatic interactions. Hence the human exome is within the reach of our method, assuming one parameter per gene and one parameter per epistatic effect for every pair of genes. We demonstrate Pint on both simulated and real data sets, including antibiotic resistance testing and siRNA perturbation screens.

sentences: Detection of precisely edited CRISPR/Cas9 alleles through cointroduced restriction-fragment length polymorphisms CRISPR/Cas9 is a powerful tool for producing genomic insertions and deletions (indels) to interrogate gene function. Modified CRISPR/Cas9 protocols can produce targeted genetic changes that are more precise than indels, but founder recovery is less efficient. Focusing on producing missense mutations in zebrafish using single-stranded oligo deoxynucleotide (ssODN) donor templates, we pioneered a strategy of adding synonymous changes to create novel restriction-enzyme (RE) sites, allowing detection of rare precise edits in a modified fluorescent-PCR fragment assay. We have named this process TIARS (test for incorporation of added recognition sites). Aided by TIARS, we induced two distinct amino-acid substitutions (T979I and P1387S) in the atp7a gene among somatic tissues of CRISPR-Cas9-treated F0 zebrafish. One of these F0s transmitted the allele to atp7aT979I/+ F1 progeny, and trans-heterozygosity of this allele against a null atp7a allele causes hypopigmentation, consistent with more severe pigment deficits in zebrafish or humans carrying only null mutations in atp7a/ATP7A. Design of ssODNs with novel RE recognition sites is labor-intensive, so we developed an in silico tool, TIARS Designer, and performed bioinformatic validation indicating that TIARS should be generalizable to other genes and experimental systems that employ donor template DNA.

sentences: Towards a global investigation of transcriptomic signatures through co-expression networks and pathway knowledge for the identification of disease mechanisms In this work, we attempt to address a key question in the joint analysis of transcriptomic data: can we correlate the patterns we observe in transcriptomic datasets to known molecular interactions and pathway knowledge to broaden our understanding of disease pathophysiology? We present a systematic approach that sheds light on the patterns observed in hundreds of transcriptomic datasets from over sixty indications by using pathways and molecular interactions as a template. Our analysis employs transcriptomic datasets to construct dozens of disease specific co-expression networks, alongside a human interactome network of protein-protein interactions described in the literature. Leveraging the interoperability between these two network templates, we explore patterns both common and particular to these diseases on three different levels. Firstly, at the node-level, we identify the most and least common proteins in these diseases and evaluate their consistency against the interactome as a proxy for their prevalence in the scientific literature. Secondly, we overlay both network templates to analyze common correlations and interactions across diseases at the edge-level. Thirdly, we explore the similarity between patterns observed at the disease level and pathway knowledge to identify pathway signatures associated with specific diseases and indication areas. Finally, we present a case scenario in the context of schizophrenia, where we show how our approach can be used to investigate disease pathophysiology.

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Subsurface Hydrocarbon Degradation Strategies in Low- and High-Sulfate Coal Seam Communities Identified with Activity-Based Metagenomics Environmentally relevant metagenomes and BONCAT-FACS derived translationally active metagenomes from Powder River Basin coal seams were investigated to elucidate potential genes and functional groups involved in hydrocarbon degradation to methane in coal seams with high- and low-sulfate levels. An advanced subsurface environmental sampler allowed the establishment of coal-associated microbial communities under in situ conditions for metagenomic analyses from environmental and translationally active populations. Metagenomic sequencing demonstrated that biosurfactants, aerobic dioxygenases, and anaerobic phenol degradation pathways were present in active populations across the sampled coal seams. In particular, results suggested the importance of anaerobic degradation pathways under high-sulfate conditions with an emphasis on fumarate addition. Under low-sulfate conditions, a mixture of both aerobic and anaerobic pathways were observed but with a predominance of aerobic dioxygenases. The putative low-molecular weight biosurfactant, lichysein, appeared to play a more important role compared to rhamnolipids. The methods used in this study--subsurface environmental samplers in combination with metagenomic sequencing of both total and translationally active metagenomes--offer a deeper and environmentally relevant perspective on community genetic potential from coal seams poised at different redox conditions broadening the understanding of degradation strategies for subsurface carbon.

One Sentence SummaryIdentifying hydrocarbon degradation strategies across different redox conditions via metagenomic analysis of environmental and translationally active (BONCAT-FACS) samples from subsurface coal beds.

sentences: tRNAscan-SE 2.0: Improved Detection and Functional Classification of Transfer RNA Genes tRNAscan-SE has been widely used for transfer RNA (tRNA) gene prediction for over twenty years, developed just as the first genomes were decoded. With the massive increase in quantity and phylogenetic diversity of genomes, the accurate detection and functional prediction of tRNAs has become more challenging. Utilizing a vastly larger training set, we created nearly one hundred specialized isotype-and clade-specific models, greatly improving tRNAscan-SEs ability to identify and classify both typical and atypical tRNAs. We employ a new comparative multi-model strategy where predicted tRNAs are scored against a full set of isotype-specific covariance models, allowing functional prediction based on both the anticodon and the highest-scoring isotype model. Comparative model scoring has also enhanced the programs ability to detect tRNA-derived SINEs and other likely pseudogenes. For the first time, tRNAscan-SE also includes fast and highly accurate detection of mitochondrial tRNAs using newly developed models. Overall, tRNA detection sensitivity and specificity is improved for all isotypes, particularly those utilizing specialized models for selenocysteine and the three subtypes of tRNA genes encoding a CAU anticodon. These enhancements will provide researchers with more accurate and detailed tRNA annotation for a wider variety of tRNAs, and may direct attention to tRNAs with novel traits.

sentences: Assessing the sensitivity of EEG-based frequency-tagging as a metric for statistical learning AO_SCPLOWBSTRACTC_SCPLOWStatistical Learning (SL) is hypothesized to play an important role in language development. However, the behavioral measures typically used to assess SL, particularly at the level of individual participants, are largely indirect and often have low sensitivity. Recently, a neural metric based on frequency-tagging has been proposed as an alternative and more direct measure for studying SL. Here we tested the sensitivity of frequency-tagging measures for studying SL in individual participants in an artificial language paradigm, using non-invasive EEG recordings of neural activity in humans. Importantly, we use carefully constructed controls, in order to address potential acoustic confounds of the frequency-tagging approach. We compared the sensitivity of EEG-based metrics to both explicit and implicit behavioral tests of SL, and the correspondence between these presumed converging operations. Group-level results confirm that frequency-tagging can provide a robust indication of SL for an artificial language, above and beyond potential acoustic confounds. However, this metric had very low sensitivity at the level of individual participants, with significant effects found only in 30% of participants. Conversely, the implicit behavior measures indicated that SL has occurred in 70% of participants, which is more consistent with the proposed ubiquitous nature of SL. Moreover, there was low correspondence between the different measures used to assess SL. Taken together, while some researchers may find the frequency-tagging approach suitable for their needs, our results highlight the methodological challenges of assessing SL at the individual level, and the potential confounds that should be taken into account when interpreting frequency-tagged EEG data.

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Spatially distributed infection increases viral load in a computational model of SARS-CoV-2 lung infection A key question in SARS-CoV-2 infection is why viral loads and patient outcomes vary dramatically across individuals. Because spatial-temporal dynamics of viral spread and immune response are challenging to study in vivo, we developed Spatial Immune Model of Coronavirus (SIMCoV), a scalable computational model that simulates hundreds of millions of lung cells, including respiratory epithelial cells and T cells. SIMCoV replicates viral growth dynamics observed in patients and shows how spatially dispersed infections can lead to increased viral loads. The model also shows how the timing and strength of the T cell response can affect viral persistence, oscillations, and control. By incorporating spatial interactions, SIMCoV provides a parsimonious explanation for the dramatically different viral load trajectories among patients by varying only the number of initial sites of infection, and the magnitude and timing of the T cell immune response. When the branching airway structure of the lung is explicitly represented, we find that virus spreads faster than in a 2D layer of epithelial cells, but much more slowly than in an undifferentiated 3D grid or in a well-mixed ODE model. These results illustrate how realistic spatially explicit computational models can improve understanding of within-host dynamics of SARS-CoV-2 infection.

SummaryA key question in SARS-CoV-2 infection is why viral loads and patient outcomes are so different across individuals. Because its difficult to see how the virus spreads in the lungs of infected people, we developed Spatial Immune Model of Coronavirus (SIMCoV), a computational model that simulates hundreds of millions of cells, including lung cells and immune cells. SIMCoV simulates how virus grows and then declines, and the simulations match data observed in patients. SIMCoV shows that when there are more initial infection sites, the virus grows to a higher peak. The model also shows how the timing of the immune response, particularly the T cell response, can affect how long the virus persists and whether it is ultimately cleared from the lungs. SIMCoV shows that the different viral loads in different patients can be explained by how many different places the virus is initially seeded inside their lungs. We explicitly add the branching airway structure of the lung into the model and show that virus spreads slightly faster than it would in a two-dimensional layer of lung cells, but much slower than traditional mathematical models based on differential equations. These results illustrate how realistic spatial computational models can improve understanding of how SARS-CoV-2 infection spreads in the lung.

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In Vivo Multi-Day Calcium Imaging of CA1 Hippocampus in Freely Moving Rats Reveals a High Preponderance of Place Cells and No Detectable Photobleaching Calcium imaging using GCaMP indicators and miniature microscopes has been used to image cellular populations during long timescales and in different task phases, as well as to determine neuronal circuit topology and organization. Because the hippocampus (HPC) is essential for tasks of memory, spatial navigation, and learning, calcium imaging of large populations of HPC neurons can provide new insight on cell changes over time during these tasks. All reported HPC in vivo calcium imaging experiments have been done in mouse. However, rats have many behavioral and physiological experimental advantages over mice. In this paper, we present the first (to our knowledge) in vivo calcium imaging from CA1 hippocampus in freely moving male rats. Using the UCLA Miniscope, we demonstrate that, in rat, hundreds of cells can be visualized and held across weeks. We show that calcium events in these cells are highly correlated with periods of movement, with few calcium events occurring during periods without movement. We additionally show that an extremely large percent of cells recorded during a navigational task are place cells (77.3{+/-}5.0%, surpassing the percent seen during mouse calcium imaging), and that these cells enable accurate decoding of animal position and can be held over days with consistent place fields in a consistent spatial map. A detailed protocol is included, and implications of these advancements on in vivo imaging and place field literature are discussed.

Significance statementIn vivo calcium imaging in freely moving animals allows the visualization of cellular activity across days. In this paper, we present the first in vivo Ca2+ recording from CA1 hippocampus in freely moving rats. We demonstrate that hundreds of cells can be visualized and held across weeks, and that calcium activity corresponds to periods of movement. We show that a high percentage (77.3{+/-}5.0%) of imaged cells are place cells, and that these place cells enable accurate decoding and can be held stably over days with little change in field location. Because the hippocampus is essential for many tasks involving memory, navigation, and learning, imaging of large populations of HPC neurons can shed new insight on cellular activity changes and organization.

sentences: Comparing frontal inclination angles measured from digital 3D models and 2D photographs of dry male and female crania from Croatia The frontal bone is one of the sexually dimorphic elements of the human skull that can be used for sex estimation of unidentified human remains. Numerous morphological features of the frontal bone, such as its angle of inclination, maximum anterior projection (glabella), and rounded elevations (frontal eminences) have been shown to differ between males and females. Various approaches have been developed to assess the frontal inclination in particular, and recently a method has been proposed where the angle of the frontal slope is measured from snapshots of digital three-dimensional (3D) models of human crania. However, as 3D-based investigations of skeletal material can be time-consuming and expensive, we here compare measurements of frontal angle inclination from 3D model snapshots to measurements from 2D photographs for a large sample (61 females and 61 males) of dry archaeological crania from medieval Croatia. Although angles measured from 3D snapshots and 2D photographs produced discriminant functions that classified crania by sex with similar accuracy (around 73%), the angles recorded from the 2D photographs were systematically one degree smaller than the angles recorded from the 3D images. Thus, even though both data sets were useful for sex estimation, we conclude that angles measured with the two different techniques should not be combined.

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Characterization of apicomplexan amino acid transporters (ApiATs) in the malaria parasite Plasmodium falciparum During the symptomatic human blood phase, malaria parasites replicate within red blood cells. Parasite proliferation relies on the uptake of nutrients, such as amino acids, from the host cell and the blood plasma, requiring transport across multiple membranes. Amino acids are delivered to the parasite through the parasite surrounding vacuolar compartment by specialized nutrient-permeable channels of the erythrocyte membrane and the parasitophorous vacuole membrane (PVM). However, further transport of amino acid across the parasite plasma membrane (PPM) is currently not well characterized. In this study, we focused on a family of Apicomplexan amino acid transporters (ApiATs) that comprises five members in Plasmodium falciparum. First, we localized four of the PfApiATs at the PPM using endogenous GFP-tagging. Next, we applied reverse genetic approaches to probe into their essentiality during asexual replication and gametocytogenesis. Upon inducible knockdown and targeted gene disruption a reduced asexual parasite proliferation was detected for PfApiAT2 and PfApiAT4. Functional inactivation of individual PfApiATs targeted in this study had no effect on gametocyte development. Our data suggest that individual PfApiATs are partially redundant during asexual in vitro proliferation and fully redundant during gametocytogenesis of P. falciparum parasites.

IMPORTANCEMalaria parasites live and multiply inside cells. To facilitate their extremely fast intracellular proliferation they hijack and transform their host cells. This also requires the active uptake of nutrients, such as amino acids, from the host cell and the surrounding environment through various membranes that are the consequence of the parasites intracellular lifestyle. In this manuscript we focus on a family of putative amino acid transporters termed ApiAT. We show expression and localization of four transporters in the parasite plasma membrane of Plasmodium falciparum-infected erythrocytes that represent one interface of the pathogen to its host cell. We probed into the impact of functional inactivation of individual transporters on parasite growth in asexual and sexual blood stages of P. falciparum and reveal that only two of them show a modest but significant reduction in parasite proliferation but no impact on gametocytogenesis pointing towards redundancy within this transporter family.

sentences: Assessing the utility of CASP14 models for molecular replacement The assessment of CASP models for utility in molecular replacement is a measure of their use in a valuable real-world application. In CASP7, the metric for molecular replacement assessment involved full likelihood-based molecular replacement searches; however, this restricted the assessable targets to crystal structures with only one copy of the target in the asymmetric unit, and to those where the search found the correct pose. In CASP10, full molecular replacement searches were replaced by likelihood-based rigid-body refinement of models superimposed on the target using the LGA algorithm, with the metric being the refined likelihood (LLG) score. This enabled multi-copy targets and very poor models to be evaluated, but a significant further issue remained: the requirement of diffraction data for assessment. We introduce here the relative-expected-LLG (reLLG), which is independent of diffraction data. This reLLG is also independent of any crystal form, and can be calculated regardless of the source of the target, be it X-ray, NMR or cryo-EM. We calibrate the reLLG against the LLG for targets in CASP14, showing that it is a robust measure of both model and group ranking. Like the LLG, the reLLG shows that accurate coordinate error estimates add substantial value to predicted models. We find that refinement by CASP groups can often convert an inadequate initial model into a successful MR search model. Consistent with findings from others, we show that the AlphaFold2 models are sufficiently good, and reliably so, to surpass other current model generation strategies for attempting molecular replacement phasing.

sentences: AZBA: A 3D Adult Zebrafish Brain Atlas for the Digital Age Zebrafish have made significant contributions to our understanding of the vertebrate brain and the neural basis of behavior, earning a place as one of the most widely used model organisms in neuroscience. Their appeal arises from the marriage of low cost, early life transparency, and ease of genetic manipulation with a behavioral repertoire that becomes more sophisticated as animals transition from larvae to adults. To further enhance the use of adult zebrafish, we created the first fully segmented three-dimensional digital adult zebrafish brain atlas (AZBA). AZBA was built by combining tissue clearing, light-sheet fluorescence microscopy, and three-dimensional image registration of nuclear and antibody stains. These images were used to guide segmentation of the atlas into over 200 neuroanatomical regions comprising the entirety of the adult zebrafish brain. As an open source, online azba.wayne.edu), updatable digital resource, AZBA will significantly enhance the use of adult zebrafish in furthering our understanding of vertebrate brain function in both health and disease.

sentences: A novel role of 3,5-cAMP in the regulation of actin cytoskeleton in Arabidopsis. The role of cyclic adenosine monophosphate (3,5-cAMP) in plants is not well understood, and here, we report a novel role of 3,5-cAMP in regulating the actin cytoskeleton. The 3,5-cAMP treatment affects the thermal stability of 51 proteins, including a vegetative actin isoform, ACTIN2. Consistent with the above results, the increase in 3,5-cAMP levels, obtained either by feeding or by chemical modulation of 3,5-cAMP metabolism, is sufficient to partially rescue the short hypocotyl phenotype of the actin2 actin7 mutant, severely compromised in actin level. No such complementation was measured for a positional isomer of 3,5-cAMP, 2,3-cAMP, attesting to the specificity of 3,5-cAMP treatment. Moreover, supplementation of 3,5-cAMP partly counters the activity of an actin-depolymerizing drug latrunculin B. In vitro characterization of the 3,5-cAMP - actin interaction argues against the direct binding. Instead, based on the proteomics characterization of the act2act7 mutant supplemented with 3,5-cAMP, we hypothesize that 3,5-cAMP affects cytoskeleton dynamic by modulation of calcium signaling, and actin binding proteins.

sentences: Verification of CRISPR editing and finding transgenic inserts by Xdrop Indirect sequence capture followed by short- and long- read sequencing Validation of CRISPR-Cas9 editing typically explore the immediate vicinity of the gene editing site and distal off-target sequences, which have led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kilobases-long and only affect one allele. The gold standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach allows the detection of small editing events but fails in detecting large rearrangements, in particular when only one allele is affected. Detection of large rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing. Here we implemented Xdrop, a new microfluidic technology that allows targeted enrichment of long regions (~ 100 kb) using just a single standard PCR primer set. Sequencing of the enriched CRISPR-Cas9 gene edited region in 4 cell lines on long- and short -read sequencing platforms unravelled unknown and unintended genome editing events. The analysis revealed accidental kb large insertions in 3 of the cell lines, which remained undetected using standard procedures. We also applied the targeted enrichment approach to identify the integration site of a transgene in a mouse line. The results demonstrate the potential of this technology in gene editing validation as well as in more classic transgenics.

sentences: Intestine-enriched apolipoprotein b orthologs are required for stem cell differentiation and regeneration in planarians Lipid metabolism plays an instructive role in regulating stem cell state and differentiation. However, the roles of lipid mobilization and utilization in stem cell-driven regeneration are unclear. Planarian flatworms readily restore missing tissue due to injury-induced activation of pluripotent somatic stem cells called neoblasts. Here, we identify two intestine-enriched orthologs of apolipoprotein b, apob-1 and apob-2, which mediate transport of neutral lipid stores from the intestine to target tissues including neoblasts, and are required for tissue homeostasis and regeneration. Inhibition of apob function by RNAi causes head regression and lysis in uninjured animals, and delays body axis re-establishment and regeneration of multiple organs in amputated fragments. Furthermore, apob RNAi causes expansion of the population of differentiating neoblast progeny and dysregulates expression of genes enriched in differentiating and mature cells in eight major cell type lineages. We conclude that intestine-derived lipids serve as a source of metabolites required for neoblast differentiation.

sentences: Frequent paternal mitochondrial inheritance and rapid haplotype frequency shifts in copepod hybrids Mitochondria are assumed to be maternally inherited in most animal species, and this foundational concept has fostered advances in phylogenetics, conservation, and population genetics. Like other animals, mitochondria were thought to be solely maternally inherited in the marine copepod Tigriopus californicus, which has served as a useful model for studying mitonuclear interactions, hybrid breakdown, and environmental tolerance. However, we present PCR, Sanger sequencing, and Illumina Nextera sequencing evidence that extensive paternal mitochondrial DNA (mtDNA) transmission is occurring in inter-population hybrids of T. californicus. PCR on four types of crosses between three populations (total sample size of 376 F1 individuals) with 20% genome-wide mitochondrial divergence showed 2% to 59% of F1 hybrids with both paternal and maternal mtDNA, where low and high paternal leakage values were found in different cross directions of the same population pairs. Sequencing methods further verified nucleotide similarities between F1 mtDNA and paternal mtDNA sequences. Interestingly, the paternal mtDNA in F1s from some crosses inherited haplotypes that were uncommon in the paternal population. Compared to some previous research on paternal leakage, we employed more rigorous methods to rule out contamination and false detection of paternal mtDNA due to non-functional nuclear mitochondrial DNA fragments. Our results raise the potential that other animal systems thought to only inherit maternal mitochondria may also have paternal leakage, which would then affect the interpretation of past and future population genetics or phylogenetic studies that rely on mitochondria as uniparental markers.

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CXCR6 by increasing retention of memory CD8 T cells in the ovarian tumor microenvironment promotes immunosurveillance and control of ovarian cancer PurposeResident memory CD8 T cells owing to their ability to reside and persist in peripheral tissues, impart adaptive sentinel activity and amplify local immune response, have beneficial implications for tumor surveillance and control. The current study aims to clarify the less known chemotactic mechanisms that govern the localization, retention, and residency of memory CD8 T cells in the ovarian tumor microenvironment.

Experimental DesignRNA/FACS based profiling of chemokine receptor expression in CD8+ resident memory T cells in human ovarian cancer and analyze their association with survival. Analyze chemokine receptor role in anti-tumor response and control by resident memory T cells using prophylactic mice models of ovarian cancer, treated with adoptive transfer of OT1 T cells and vaccination with maraba virus-OVA to target Ovalbumin expressing tumor.

ResultsChemokine receptor profiling of CD8+CD103+ resident memory TILs in ovarian cancer patients revealed high expression of CXCR6. Analysis of the TCGA ovarian cancer database revealed CXCR6 to be associated with CD103 and increased patient survival. Functional studies in mouse models of ovarian cancer revealed that CXCR6 is a marker of resident, but not circulatory tumor-specific memory CD8 T cells. Knockout of CXCR6 in tumor-specific CD8 T cells showed reduced retention in tumor tissues leading to diminished resident memory responses and poor control of ovarian cancer

ConclusionsCXCR6 by promoting increased retention in tumor tissues serves a critical role in resident memory T cell-mediated immunosurveillance and control of ovarian cancer. Future studies warrant exploiting CXCR6 to promote resident memory response in cancers.

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Phenotyping Polarization Dynamics Of Immune Cells Using A Lipid Droplet - Cell Pairing Microfluidic Platform The immune synapse is the tight contact zone between a lymphocyte and a cell presenting its cognate antigen. This structure serves as a signaling platform and entails a polarization of intra-cellular components, necessary to the immunological function of the cell. While the surface properties of the presenting cell are known to control the formation of the synapse, their impact on polarization has not yet been studied.

Using functional lipid droplets as tunable artificial presenting cells combined with a microfluidic pairing device, we simultaneously observe synchronized synapses and dynamically quantify polarization patterns of individual B cells. By assessing how the ligand concentration, the surface fluidity and the substrate rigidity impact this polarization, we show that its onset depends on the antigen concentration at the synapse, and that the substrate rigidity controls both its onset and its kinetics. Our experimental system enables a fine phenotyping of monoclonal cell populations based on their synaptic readout.

Significance statementTo fulfill their immune function, B cells acquire antigens presented on the surface of antigen-presenting cells and organize their cytoskeleton and lysosomal traffic towards the contact. The polarization mechanism is evolutionary conserved, making the system interesting both from the immunology and the cell biology point of view. We developed and finely characterized antigen-coated droplets as surrogate presenting cells with a physiological-flow microfluidic cell-pairing chip to explore the B cell lysosome polarization in response to changes in droplet mechanics and antigen concentration. We observed that polarization is sensitive to mechanical properties (responding more strongly but less fast on stiffer substrates) and to the local antigen concentration at the contact. Hence, surprisingly, a late cell response is sensitive to the mechanics of the contact.

sentences: Direct RNA sequencing reveals SARS-CoV-2 m6A sites and possible differential DRACH motif methylation among variants The causative agent of COVID-19 pandemic, the SARS-CoV-2 coronavirus, has a 29,903 bases positive-sense single-stranded RNA genome. RNAs exhibit about 100 modified bases that are essential for proper function. Among internal modified bases, the N6-methyladenosine, or m6A, is the most frequent, and is implicated in SARS-CoV-2 immune response evasion. Although the SARS-CoV-2 genome is RNA, almost all genomes sequenced so far are in fact, reverse transcribed complementary DNAs. This process reduces the true complexity of these viral genomes because incorporation of dNTPs hides RNA base modifications. Here, in this perspective paper, we present an initial exploration of the Nanopore direct RNA sequencing to assess the m6A residues in the SARS-CoV-2 sequences of ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10 and the 3-untranslated region. We identified 15 m6A methylated positions, of which, 6 are in ORF N. Also, because m6A is associated with the DRACH motif, we compared its distribution in major SARS-CoV-2 variants. Although DRACH is highly conserved among variants we show that variants Beta and Eta have a fourth position C>U change in DRACH at 28,884b that could affect methylation. The Nanopore technology offers a unique opportunity for the study of viral epitranscriptomics. This technique is PCR-free and is not sequencing-by-synthesis, therefore, no PCR bias and synthesis errors are introduced. The modified bases are preserved and assessed directly with no need for chemical treatments or antibodies. This is the first report of direct RNA sequencing of a Brazilian SARS-CoV-2 sample coupled with the identification of modified bases.

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Prolonged low-dose dioxin exposure impairs metabolic adaptability to high-fat diet feeding in female but not male mice ObjectiveHuman studies consistently show an association between exposure to persistent organic pollutants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, aka "dioxin"), and increased diabetes risk, but rarely consider potential sex differences. We previously showed that a single high-dose TCDD exposure (20 {micro}g/kg) decreased plasma insulin levels in both male and female mice in vivo, but effects on glucose homeostasis were sex-dependent. The purpose of the current study was to determine whether prolonged exposure to a physiologically relevant low-dose of TCDD impacts glucose homeostasis and/or the islet phenotype in a sex-dependent manner in either chow-fed or high fat diet (HFD)-fed mice.

MethodsMale and female mice were exposed to 20 ng/kg/d TCDD 2x/week for 12 weeks and simultaneously fed standard chow or a 45% HFD. Glucose homeostasis was assessed by glucose and insulin tolerance tests, and glucose-induced plasma insulin levels were measured in vivo. Histological analysis was performed on pancreas from male and female mice, and islets were isolated from females at 12 weeks for Tempo-Seq(R) analysis.

ResultsLow-dose TCDD exposure did not lead to adverse metabolic consequences in chow-fed male or female mice, or in HFD-fed males. However, TCDD accelerated the onset of HFD-induced hyperglycemia and impaired glucose-induced plasma insulin levels in female mice. TCDD caused a modest increase in islet area in males irrespective of diet, but reduced % beta cell area within islets in females. RNAseq analysis of female islets also revealed abnormal changes to endocrine and metabolic pathways in TCDD-exposed HFD-fed females compared chow-fed females.

ConclusionsOur data suggest that prolonged low-dose TCDD exposure has minimal effects on glucose homeostasis and islet morphology in chow-fed male and female mice, but promotes maladaptive metabolic responses in HFD-fed females.

sentences: Multi-modal engineering of Bst DNA polymerase for thermostability in ultra-fast LAMP reactions DNA polymerase from Geobacillus stearothermophilus, Bst DNA polymerase (Bst DNAP), is a versatile enzyme with robust strand-displacing activity that enables loop-mediated isothermal amplification (LAMP). Despite its exclusive usage in LAMP assay, its properties remain open to improvement. Here, we describe logical redesign of Bst DNAP by using multimodal application of several independent and orthogonal rational engineering methods such as domain addition, supercharging, and machine learning predictions of amino acid substitutions. The resulting Br512g3 enzyme is not only thermostable and extremely robust but it also displays improved reverse transcription activity and the ability to carry out ultrafast LAMP at 74 {degrees}C. Our study illustrates a new enzyme engineering strategy as well as contributes a novel engineered strand displacing DNA polymerase of high value to diagnostics and other fields.

sentences: Increasing phagocytosis of microglia through targeting CD33 with liposomes displaying glycan ligands CD33 is an immunomodulatory receptor expressed on microglia and genetically linked to Alzheimers disease (AD) susceptibility. While antibodies targeting CD33 have entered clinical trials to treat neurodegeneration, it is unknown whether the glycan-binding properties of CD33 can be exploited to modulate microglia. Here, we use liposomes that multivalently display glycan ligands of CD33 (CD33L liposomes) to engage CD33. We find that CD33L liposomes increase phagocytosis of cultured monocytic cells and microglia in a CD33-dependent manner. Enhanced phagocytosis strongly correlates with loss of CD33 from the cell surface and internalization of liposomes. Increased phagocytosis by treatment with CD33L liposomes is dependent on a key intracellular signaling motif on CD33 as well as the glycan-binding ability of CD33. These effects are specific to trans engagement of CD33 by CD33L liposomes, as cis engagement through insertion of lipid-linked CD33L into cells produces the opposite effect on phagocytosis. Moreover, intracerebroventricular injection of CD33L liposomes in mice enhances phagocytosis of microglia in a CD33-dependent manner. These results demonstrate that multivalent engagement of CD33 with glycan ligands can modulate microglial cell function.

sentences: Diversity and distribution of sulfur metabolism in the human gut microbiome and its association with colorectal cancer Microbial sulfidogenesis produces genotoxic hydrogen sulfide (H2S) in the human gut using inorganic (sulfate) and organic (taurine/cysteine/methionine) substrates, however the majority of studies have focused on sulfate reduction using dissimilatory sulfite reductases (Dsr). Recent evidence implicates microbial sulfidogenesis as a potential trigger of colorectal cancer (CRC), highlighting the need for comprehensive knowledge of sulfur metabolism within the human gut. Here we show that microbial sulfur metabolism is more abundant and diverse than previously studied and is statistically associated with CRC. Using ~17,000 bacterial genomes from publicly available stool metagenomes, we studied the diversity of sulfur metabolic genes in 667 participants across different health statuses: healthy, adenoma, and carcinoma. Sulfidogenic genes were harbored by 142 bacterial genera and both organic and inorganic sulfidogenic genes were associated with carcinoma. Significantly, anaerobic sulfite reductases were twice as abundant as dsr. We identified twelve potential pathways for reductive taurine metabolism including novel pathways, and prevalence of organic sulfur metabolic genes indicate these substrates may be the most abundant source of microbially derived H2S. Our findings significantly expand knowledge of microbial sulfur metabolism in the human gut, and highlight key gaps that limit understanding of its potential contributions to the pathogenesis of CRC.

sentences: ANP32A represses Wnt signaling across tissues tissues thereby protecting against joint and heart disease Wnt signaling is key to diverse homeostatic and pathological processes. This cascade is hyper-activated in osteoarthritis, the most common joint disease. Yet, fundamental aspects of Wnt signaling remain undiscovered. Here, we report that ANP32A negatively regulates Wnt signaling across tissues. In cartilage, loss of Anp32a triggered Wnt hyper-activation. Mechanistically, ANP32A directly interacted with Wnt pathway components and inhibited Wnt target genes via histone acetylation masking. Wnt antagonist treatment reduced severity of osteoarthritis in Anp32a-deficient mice preventing osteophyte formation, contrasting with cartilage-protective effects of ANP32A on oxidative stress. Hence, dual therapy targeting Wnt signaling and oxidative stress in Anp32a-deficient mice ameliorated more osteoarthritis features than individual treatments. Anp32a loss also resulted in Wnt hyper-activation in the heart with cardiac hypertrophy, and in the hippocampus, shedding light on mechanisms for reported links between ANP32A and Alzheimers disease. Collectively, this work reveals that ANP32A is a translationally relevant repressor of Wnt signaling, impacting homeostasis and disease across tissues.

sentences: Dysfunction of an energy sensor NFE2L1 triggers uncontrollable AMPK signal and glucose metabolism reprogramming NFE2L1 (also called Nrf1) acts a core regulator of redox signaling and metabolism homeostasis, and thus its dysfunction results in multiple systemic metabolic diseases. However, the molecular mechanism(s) by which NFE2L1 regulates glycose and lipid metabolism is still elusive. Here, we found that the loss of NFE2L1 in human HepG2 cells led to a lethal phenotype upon glucose deprivation. The uptake of glucose was also affected by NFE2L1 deficiency. Further experiments unveiled that although the glycosylation of NFE2L1 was monitored through the glycolysis pathway, it enabled to sense the energy state and directly interacted with AMPK. These indicate that NFE2L1 can serve as a dual sensor and regulator of glucose homeostasis. In-depth sights into transcriptome, metabolome and seahorse data further unraveled that glucose metabolism was reprogrammed by disruption of NFE2L1, so as to aggravate the Warburg effect in NFE2L1-silenced hepatoma cells, along with the mitochondrial damage observed under the electron microscope. Collectively, these demonstrate that disfunction of NFE2L1 triggers the uncontrollable signaling by AMPK towards glucose metabolism reprogramming in the liver cancer development.

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A model of Ponto-Geniculo-Occipital waves supports bidirectional control of cortical plasticity across sleep-stages During sleep, cortical network connectivity likely undergoes both synaptic potentiation and depression through system consolidation and homeostatic processes. However, how these modifications are coordinated across sleep stages remains largely unknown. Candidate mechanisms are Ponto-Geniculo-Occipital (PGO) waves, propagating across several structures during Rapid Eye Movement (REM) sleep and the transitional stage from non-REM sleep to REM sleep (pre-REM), and exhibiting sleep stage-specific dynamic patterns. To understand their impact on cortical plasticity, we built an acetylcholine-modulated neural mass model of PGO wave propagation through pons, thalamus and cortex, reproducing a broad range of electrophysiological characteristics across sleep stages. Using a population model of Spike-Time-Dependent Plasticity, we show that cortical circuits undergo different transient regimes depending on the sleep stage, with different impacts on plasticity. Specifically, PGO-induced recurrent cortical activities lead to the potentiation of cortico-cortical synapses during pre-REM, and to their depression during REM sleep. Overall, these results shed light on how the dynamical properties of sleep events propagating to cortical circuits can favor different types of local plastic changes. The variety of events occurring across sleep stages may thus be instrumental in controlling the reorganization of cortical networks from one day to the next.

Significance statementConsiderable evidence supports rescaling of cortical synaptic connec-tions during sleep, requiring both long term potentiation to consolidate newly acquired memories, and long-term depression to maintain homeostatic levels of brain activity. However, which aspects of sleep activity contribute to this bidirectional control of plasticity remains unclear. This computational modeling study suggests that widespread transient phenomena called Ponto-geniculo-occipital (PGO) waves, have a sleep-stage dependent effect on plasticity. The alternation between sleep stages can thus be exploited in combination with spontaneously occurring transients, to trigger both up- and down-regulating effects on cortical connectivity, and may explain why the basic structure of sleep-cycles is a well-preserved property across mammalian species.

sentences: Inferring the demographic history of tetraploid species from genomic data Genomic patterns of diversity and divergence are impacted by certain life history traits, reproductive systems and demographic history. The latter is characterised by fluctuations in population sizes over time, as well as by temporal patterns of introgression. For a given organism, identifying a demographic history that deviates from the standard neutral model allows a better understanding of its evolution, but also helps to reduce the risk of false positives when screening for molecular targets of natural selection. Tetraploid organisms and beyond have demographic histories that are complicated by the mode of polyploidisation, the mode of inheritance and different scenarios of gene flow between subgenomes and diploid parental species. Here we provide guidelines for experimenters wishing to address these issues through a flexible statistical framework: approximate Bayesian computation (ABC). The emphasis is on the general philosophy of the approach to encourage future users to exploit the enormous flexibility of ABC beyond the limitations imposed by generalist data analysis pipelines.

sentences: Regulation by Progestins, Corticosteroids and RU486 of Activation of Elephant Shark and Human Progesterone Receptors: An Evolutionary Perspective We investigated progestin and corticosteroid activation of the progesterone receptor (PR) from elephant shark, a cartilaginous fish belonging to the oldest group of jawed vertebrates. Comparison with human PR provides insights into the evolution of steroid activation of human PR. At 1 nM steroid, elephant shark PR is activated by progesterone, 17-hydroxy-progesterone, 20{beta}-hydroxy-progesterone, 11-deoxycorticosterone (21-hydroxyprogesterone) and 11-deoxycortisol. Human PR, in comparison, is activated at 1 nM steroid, only by progesterone and 11-deoxycorticosterone, indicating increased progestin and corticosteroid specificity during the evolution of human PR. RU486, an important clinical antagonist of human PR, did not inhibit progesterone activation of elephant shark PR. Cys-528 in elephant shark PR corresponds to Gly-722 in human PR, which is essential for RU486 inhibition of human PR. Confirming the importance of Cys-528 in elephant shark PR, RU486 inhibited progesterone activation of the Cys528Gly mutant PR. Compared to wild-type human PR, there was an increase in activation of human Gly722Cys PR by11-deoxycortisol and a decrease in activation by corticosterone, which may have been important in selection for the mutation corresponding to human glycine-722 PR that first evolved in platypus PR, a basal mammal.

sentences: Ecology dictates the value of memory for foraging bees "Ecological intelligence" hypotheses posit that animal learning and memory evolves to meet the demands posed by foraging, and together with social intelligence and cognitive buffer hypotheses, provide a key framework for understanding cognitive evolution. However, identifying the critical environments where cognitive investment reaps significant benefits has proved challenging. Here, we capitalise upon seasonal variation in forage availability for a social insect model (Bombus terrestris) to establish how the benefits of short-term memory vary with resource availability. Through analysis of over 1700 foraging trips carried out over two years, we show that short-term memory predicts foraging efficiency - a key determinant of colony fitness - in plentiful spring foraging conditions, but that this relationship is reversed during the summer floral dearth. Our results suggest that selection for enhanced cognitive abilities is unlikely to be limited to harsh environments where food is hard to find or extract, highlighting instead that the complexity of rich and plentiful environments could be a broad driver in the evolution of certain cognitive traits.

sentences: Evaluating the efficacy of Optoα1AR activation in astrocytes in modulating basal hippocampal synaptic excitation and inhibition Astrocytes play active roles at synapses and can monitor, respond, and adapt to local synaptic activity. To investigate this relationship, more tools that can selectively activate native G protein signaling pathways in astrocytes with both spatial and temporal precision are needed. Here, we tested AAV8-GFAP-Opto1AR-eYFP (Opto1AR), a viral vector to enable activation of Gq signaling in astrocytes via light-sensitive 1-adrenergic receptors. To determine if stimulating astrocytic Opto1AR modulates hippocampal synaptic transmission, recordings were made in CA1 pyramidal cells with surrounding astrocytes expressing Opto1AR, channelrhodopsin (ChR2), or GFP. Both high-frequency (20 Hz, 45-ms light pulses, 5 mW, 5 min) and low-frequency (0.5 Hz, 1-s pulses at increasing 1, 5, and 10 mW intensities, 90 s per intensity) blue light stimulation were tested. 20 Hz Opto1AR stimulation increased both inhibitory and excitatory postsynaptic current (IPSC and EPSC) frequency, and the mIPSC effect was largely reversible within 20 min. By contrast, low-frequency stimulation of Opto1AR did not modulate either IPSCs or EPSCs, whereas the same stimulation of astrocytic ChR2 was effective. These data demonstrate that Opto1AR activation in astrocytes changes synaptic excitation and inhibition in a stimulation-sensitive manner, demonstrating the efficacy and utility of GFAP-Opto1AR as a tool in studying astrocyte-neuron interactions.

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An atlas of white matter anatomy, its variability, and reproducibility based on Constrained Spherical Deconvolution of diffusion MRI Virtual dissection of white matter (WM) using diffusion MRI tractography is confounded by its poor reproducibility. Despite the increased adoption of advanced reconstruction models, early region-of-interest driven protocols based on diffusion tensor imaging (DTI) remain the dominant reference for virtual dissection protocols. Here we bridge this gap by providing a comprehensive description of typical WM anatomy reconstructed using a reproducible automated subject-specific parcellation-based approach based on probabilistic constrained-spherical deconvolution (CSD) tractography. We complement this with a WM template in MNI space comprising 68 bundles, including all associated anatomical tract selection labels and associated automated workflows. Additionally, we demonstrate bundle inter- and intra-subject variability using 40 (20 test-retest) datasets from the human connectome project (HCP) and 5 sessions with varying b-values and number of b-shells from the single-subject Multiple Acquisitions for Standardization of Structural Imaging Validation and Evaluation (MASSIVE) dataset. The most reliably reconstructed bundles were the whole pyramidal tracts, primary corticospinal tracts, whole superior longitudinal fasciculi, frontal, parietal and occipital segments of the corpus callosum and middle cerebellar peduncles. More variability was found in less dense bundles, e.g., the first segment of the superior longitudinal fasciculus, fornix, dentato-rubro-thalamic tract (DRTT), and premotor pyramidal tract. Using the DRTT as an example, we show that this variability can be reduced by using a higher number of seeding attempts. Overall inter-session similarity was high for HCP test-retest data (median weighted-dice = 0.963, stdev = 0.201 and IQR = 0.099). Compared to the HCP-template bundles there was a high level of agreement for the HCP test-retest data (median weighted-dice = 0.747, stdev = 0.220 and IQR = 0.277) and for the MASSIVE data (median weighted-dice = 0.767, stdev = 0.255 and IQR = 0.338). In summary, this WM atlas provides an overview of the capabilities and limitations of automated subject-specific probabilistic CSD tractography for mapping white matter fasciculi in healthy adults. It will be most useful in applications requiring a highly reproducible parcellation-based dissection protocol, as well as being an educational resource for applied neuroimaging and clinical professionals.

O_FIG O_LINKSMALLFIG WIDTH=182 HEIGHT=200 SRC="FIGDIR/small/464139v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@18e7c79org.highwire.dtl.DTLVardef@18e2213org.highwire.dtl.DTLVardef@5f124borg.highwire.dtl.DTLVardef@1aa242c_HPS_FORMAT_FIGEXP  M_FIG Graphical abstract(Top) shows the FWT pipeline for both CSTs, AF, and motor CC bundles. (Left to right) show the required input structural parcellation maps and a priori atlases for FWT and the resulting virtual dissection include/exclude VOIs. FWT provides two approaches to virtual dissection: (1) is a bundle-specific approach where streamlines are only seeded for the bundle of interest, (2) is a whole brain tractography followed by streamlines segmentation, (top right) shows output tractograms. (Middle) Group-averaged T1 and fODF images are generated from the HCP test-retest data, and FWT is applied to generate the HCP-atlas using the bundle-specific approach (1*). FWTs whole brain tracking and segmentation approach (2*) was applied to the HCP and MASSIVE dataset (right and left) and conducted model-based, and pair-wise similarity analyses and generated voxel-wise cumulative maps per bundle. FWT= Fun With Tracts, FS= FreeSurfer, MSBP= MultiScaleBrainParcellator, PD25= NIST Parkinsons histological, JHU= Johns Hopkins university, Juelich= Juelich university histological atlas, AC/PC= anterior commissure/posterior commissure) UKBB= UK Biobank, SUIT (spatially unbiased cerebellar atlas template), dMRI= diffusion magnetic resonance imaging, CSD= constrained spherical deconvolution, fODF= fiber orientation distribution function, CST= corticospinal tract, AF= arcuate fasciculus, CC= corpus callosum, HCP= human connectome project, MASSIVE= Multiple acquisitions for standardization of structural imaging validation and evaluation.

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A highly accurate model for screening prostate cancer using propensity index panel of ten genes Prostate-specific antigen (PSA) is a key biomarker, which is commonly used to screen patients of prostate cancer. There is a significant number of unnecessary biopsies that are performed every year, due to poor accuracy of PSA based biomarker. In this study, we identified alternate biomarkers based on gene expression that can be used to screen prostate cancer with high accuracy. All models were trained and test on gene expression profile of 500 prostate cancer and 51 normal samples. Numerous feature selection techniques have been used to identify potential biomarkers. These biomarkers have been used to develop various models using different machine learning techniques for predicting samples of prostate cancer. Our logistic regression-based model achieved highest AUROC 0.91 with accuracy 82.42% on validation dataset. We introduced a new approach called propensity index, where expression of gene is converted into propensity. Our propensitybased approach improved the performance of classification models significantly and achieved AUROC 0.99 with accuracy 96.36% on validation dataset. We also identified and ranked selected genes which can be used to discriminate prostate cancer patients from health individuals with high accuracy. It was observed that single gene-based biomarkers can only achieve accuracy around 90%. In this study, we got best performance using a panel of 10 genes; random forest model using propensity index.

HighlightsO_LIApplication of Machine learning techniques to identify Biomarkers for PRAD cancer.
C_LIO_LIHighly accurate models developed for classifying prostate cancer vs. normal sample.
C_LIO_LIIntroducing Propensity index concept for enhancing model performance.
C_LIO_LITop 10 genes identified using feature selection techniques.
C_LI

sentences: Click evoked Middle Ear Muscle Reflex: Spectral and Temporal Aspects This study describes a time series-based method of middle ear muscle reflex (MEMR) detection using bilateral clicks. Although many methods can detect changes in the OAE evoking stimulus to monitor the MEMR, they do not discriminate between true MEMR-mediated vs. artifactual changes in the stimulus. We measured MEMR in 20 young clinically normal hearing individuals using 1-second-long click trains presented at six levels (65 to 95 dB peak-to-peak SPL in 6 dB steps). Changes in the stimulus levels over the 1 second period were well-approximated by two-term exponential functions. The magnitude of ear canal pressure changes due to MEMR increased monotonically as a function of click level but non-monotonically with frequency when separated into 1/3rd-octave wide bands between 1 and 3.2 kHz. MEMR thresholds estimated using this method were lower than that obtained from a clinical tympanometer in [~]94% of the participants. A time series-based method, along with statistical tests, may provide additional confidence in detecting the MEMR. MEMR effects were smallest at 2 kHz, between 1 and 3.2 kHz, which may provide avenues for minimizing the MEMR influence while measuring other responses (e.g., the medial olivocochlear reflex).

sentences: Exploring the cytotoxic mechanisms of Pediocin PA-1 towards HeLa and HT29 cells by comparison to known bacteriocins: Microcin E492, Enterocin Heterodimer and Divercin V41. The purpose of this study was to explore potential mechanisms of cytotoxicity towards HeLa and HT29 cells displayed by Pediocin PA-1. We did this by carrying out sequence alignments and 3D modelling of related bacteriocins which have been studied in greater detail: Microcin E492, Eneterocin AB heterodimer and Divercin V41. Microcin E492 interacts with Toll-Like Receptor 4 in order to activate an apoptosis reaction, sequence alignment showed a high homology between Pediocin PA-1 and Microcin E492 and 3D modelling showed Pediocin PA-1 interacting with TLR-4 in a way reminiscent of Microcin E492. Furthermore, Pediocin PA-1 had the highest homology with the Enterocin heterodimer, particularly chain A; Enterocin has also shown to cause an apoptotic response in cancer cells. Based on this we are led to strongly believe Pediocin PA-1 interacts with TLRs in order to cause cell death. If this is the case it would explain the difference in cytotoxicity towards HeLa over HT29 cells, due to difference in expression of particular TLRs. Overall, we believe Pediocin PA-1 exhibits a dual effect which is dose dependant, like that of Microcin. Unfortunately, the COVID-19 pandemic meant that we were unable to carry out experiments in the lab, and the unavailability of important data meant we were unable to make solid conclusions but rather suggestions. However despite this we have still been able to highlight interesting findings and how these could be translated into future research and therapeutics in order to improve the quality of treatment and life of cancer patients.

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Evolutionarily stable gene clusters shed light on the common grounds of pathogenicity in the Acinetobacter calcoaceticus-baumanniicomplex Nosocomial pathogens of the Acinetobacter calcoaceticus-baumannii (ACB) complex are a cautionary example for the world-wide spread of multi- and pan-drug resistant bacteria. Aiding the urgent demand for novel therapeutic targets, comparative genomics studies between pathogens and their apathogenic relatives shed light on the genetic basis of human-pathogen interaction. Yet, existing studies are limited in taxonomic scope, sensing of the phylogenetic signal, and resolution by largely analyzing genes isolated from their functional contexts. Here, we explored more than 3,000 Acinetobacter genomes in a phylogenomic framework integrating orthology-based phylogenetic profiling and microsynteny conservation analyses. This allowed to delineate gene clusters in the type strain A. baumannii ATCC 19606 whose evolutionary conservation indicates a functional integration of the subsumed genes. These evolutionarily stable gene clusters (ESGCs) reveal metabolic pathways, transcriptional regulators residing next to their targets but also tie together sub-clusters with distinct functions to form higher-order functional modules. We shortlisted 150 ESGCs that either co-emerged with, or are found preferentially in, the pathogenic ACB clade. They unveil, at an unprecedented resolution, the genetic makeup that coincides with the manifestation of the pathogenic phenotype in the last common ancestor of the ACB clade. Key innovations are the remodeling of the regulatory-effector cascade connecting LuxR/LuxI quorum sensing via an intermediate messenger to biofilm formation, the extension of micronutrient scavenging systems, and the increase of metabolic flexibility by exploiting carbon sources that are provided by the human host. Specifically, we could show that only members of the ACB clade use kynurenine as a sole carbon and energy source, a substance produced by humans to fine-tune the antimicrobial innate immune response. In summary, this study provides a rich and unbiased set of novel testable hypotheses on how pathogenic Acinetobacter interact with and ultimately infect their human host. They disclose promising routes for future therapeutic strategies.

Author SummaryThe spread of multi- and pan-drug resistant bacterial pathogens is a world-wide threat to human health. Understanding the genetics of host colonization and infection is essential for devising novel ways of treatment. Acinetobacter baumannii, a nosocomial pathogen ranked top by the World Health Organization in the list of bacteria for which novel therapeutic approaches are needed, is a prime example. Here, we have carved out the genetic make-up that distinguishes A. baumannii and its pathogenic next relatives comprising the A. baumannii-calcoaceticus complex from other and mostly apathogenic Acinetobacter species. We found a rich spectrum of pathways and regulatory modules that reveals how the pathogens have modified biofilm formation, iron scavenging, and their carbohydrate metabolism to adapt to their human host. Among these, the capability to metabolize kynurenine is particularly intriguing. Humans produce this substance to contain bacterial invaders and to fine-tune the innate immune response. But A. baumannii and its allies have turned the table. They feed on kynurenine, and thus most likely also dysregulate human immune response. In summary, our study substantially deepens the understanding of how a highly critical pathogen interacts with its host, which substantially eases the identification of novel targets for innovative therapeutic strategies.

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SELF PRUNING 3C is a flowering repressor that modulates seed germination, root architecture and drought responses Allelic variation in the CETS (CENTRORADIALIS, TERMINAL FLOWER 1, SELF PRUNING) gene family has been shown to control agronomically important traits in many crops. CETS genes encode phosphatidylethanolamine binding proteins (PEBPs) that have a central role in flowering time control as florigenic and anti-florigenic signals. The great expansion of CETS genes in many species suggests that the functions of this family go beyond flowering. Here, we characterize the tomato SELF PRUNING 3C (SP3C) gene, and show that besides acting as a flowering repressor it also regulates seed germination and modulates root architecture. We show that loss of SP3C function in CRISPR/Cas9-generated mutant lines accelerates seed germination and increases root length with lower root side branching. Higher SP3C expression in transgenic lines promotes the opposite effects and also improves tolerance to water stress in seedlings. These discoveries provide insights into the role of SP paralogs in agronomically relevant traits and support future exploration of the involvement of CETS genes in abiotic stress responses.

HighlightThe SELF PRUNING 3C (SP3C) gene is a repressor of flowering in tomato and exhibits additional functions, acting as a repressor of seed germination and modulating root architecture.

sentences: Positive and Neutral Updating Reconsolidate Aversive Episodic Memories via Different Routes Aversive memories are long-lasting and prone to have adverse effects on our emotional wellbeing and mental health. Yet, how to remedy the maladaptive effects of aversive memories remains elusive. Using memory reactivation and emotional updating manipulations, we investigated how positive and neutral emotion updates aversive memories for reconsolidation in humans. We found that positive updating after reactivation was equivalent to neutral updating in altering true memories of the aversive story, but introduced more false memory. Moreover, an additional 12 hours of sleep reconsolidation did not further enlarge true memory differences, but attenuated the effect of reactivation and updating on false memory. Interestingly, the neutral rather than the positive updating reduced the emotional arousal of the aversive memory 24 hours later. Our findings provide novel insights into real-world therapeutic applications regarding how updating with positive and neutral emotion may reshape aversive memories, especially when taking wake- and sleep-filled reconsolidation into account.

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Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E. histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E. histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E. histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual E. histolytica RNAi pathway. These studies dramatically improve the tractability of E. histolytica and open up the possibility of applying genetics to improve understanding of this important pathogen.

Author SummaryEntamoeba histolytica is a globally important pathogen that is dramatically understudied. One of the major limitations of this organism is its challenging genome. RNAi is the state-of-the-art tool for genetic manipulation in E. histolytica, though the RNAi pathway has several noncanonical features. Here, we harnessed the RNAi pathway to enable RNAi-based forward genetics for the first time in this organism. We validated the RNAi library by performing the first E. histolytica RNAi screen and identified slow growth mutants. We showed that independently-generated mutants also exhibited slow growth phenotypes, and we characterized protein localization and domains for some of the identified slow growth genes. The RNAi library that we constructed enables modern, quantitative Illumina deep sequencing analysis to identify mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify full-length gene fragments inferred from read mapping. Our approach differs from previous approaches for analysis of RNAi screens, and it better represents the actual DNA fragments and their quantities. This study dramatically improves the tractability of this important pathogen. Moreover, the strategies behind this RNAi library, and its analysis, are novel, and can be applied to other organisms.

Author ContributionsA.B. designed and performed the experiments, except fragmentation of gDNA, immunofluorescence, and growth curves with expression of full-length or truncated proteins. R.L.S. performed SG1 and SG2 immunofluorescence experiments. M.C.R. performed growth curves with expression of full-length or truncated proteins. W.H. generated transfectants expressing full-length SG2. C.G.B. fragmented gDNA and processed the fragments to enable subsequent plasmid library construction. T.S.Y.T. and H.W.M. performed Gal/GalNAc lectin immunofluorescence experiments. S.S.H. designed the analysis strategy for Illumina sequencing data, and analyzed all Illumina sequencing data. M.L.S. oversaw the Illumina analysis approach. K.S.R. performed growth curves, conceived of the overall approach and oversaw the design and analysis of the experiments. A.B., S.S.H., and K.S.R. wrote the manuscript.

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Phyllosphere community assembly and response to drought stress on common tropical and temperate forage grasses Grasslands represent a critical ecosystem important for global food production, soil carbon storage, and water regulation. Current intensification and expansion practices add to the degradation of grasslands and dramatically increase greenhouse gas emissions and pollution. Thus, new ways to sustain and improve their productivity are needed. Research efforts focus on the plant-leaf microbiome, or phyllosphere, because its microbial members impact ecosystem function by influencing pathogen resistance, plant hormone production, and nutrient availability through processes including nitrogen fixation. However, little is known about grassland phyllospheres and their response to environmental stress. In this study, globally dominant temperate and tropical forage grass species were grown in a greenhouse under current climate conditions and drought conditions that mimic future climate predictions to understand if (i) plant host taxa influence microbial community assembly, (ii) microbial communities respond to drought stress, and (iii) phyllosphere community changes correlate to changes in plant host traits and stress-response strategies. Community analysis using high resolution sequencing revealed Gammaproteobacteria as the dominant bacterial class, which increased under severe drought stress on both temperate and tropical grasses while overall bacterial community diversity declined. Bacterial community diversity, structure, and response to drought were significantly different between grass species. This community dependence on plant host species correlated with differences in grass species traits, which became more defined under drought stress conditions, suggesting symbiotic evolutionary relationships between plant hosts and their associated microbial community. Further understanding these strategies and the functions microbes provide to plants will help us utilize microbes to promote agricultural and ecosystem productivity in the future.

IMPORTANCEGlobally important grassland ecosystems are at risk of degradation due to poor management practices compounded by predicted increases in severity and duration of drought over the next century. Finding new ways to support grassland productivity is critical to maintaining their ecological and agricultural benefits. Discerning how grassland microbial communities change in response to climate stress will help us understand how plant-microbe relationships may be useful to sustainably support grasslands in the future. In this study, phyllosphere community diversity and composition was significantly altered under drought conditions. The significance of our research is demonstrating how severe climate stress reduces bacterial community diversity, which previously was directly associated with decreased plant productivity. These findings guide future questions about functional plant-microbe interactions under stress conditions, greatly enhancing our understanding of how bacteria can increase food security by promoting grassland growth and resilience.

sentences: Deciphering 3D Human Sphygmopalpation Pulse Patterns using "X-ray" Images Acquired from Tactile Robotic Fingers Sphygmopalpation at specific locations of human wrists has been used as a medical measurement technique in China since the Han Dynasty (202 BC - 220 AD); it is now generally accepted that traditional Chinese medicine (TCM) doctors are able to decipher 28 types of basic pulse patterns using their fingertips. This TCM technique of examining individual arterial pulses by palpation has undergone an upsurge recently in popularity as a low-cost and non-invasive diagnostic technique for monitoring patient health status. We have developed a pulse sensing platform for studying and digitalizing arterial pulse patterns via a TCM approach. This platform consists of a robotic hand with three fingers for pulse measurement and an artificial neural network (ANN) together with pulse signal preprocessing for pulse pattern recognition. The platforms previously reported by other research groups or marketed commercially exhibit one or more of the following imperfections: a single channel for data acquisition, low sensitivity and rigid sensors, lack of control of the applied pressure, and in many reported works, lack of an intelligent data analysis system. The platform presented here features up to three-dimensional (3D) tactile sensing channels for recording data and uses highly sensitive capacitive MEMS (microelectromechanical systems) flexible sensing arrays, pressure-feedback-controlled robotic fingers, and machine learning algorithms. We also proposed a methodology of obtaining "X-ray" image of pulse information constructed based on the sensing data from 3 locations and 3 applied pressures (i.e., mimicking TCM doctors), which contains all arterial pulse information in both spatial and temporal spans, and which could be used as an input to a deep learning algorithm. By applying our developed platform and algorithms, 3 types of consistent pulse patterns, i.e., "Hua" [Formula], "Xi" [Formula], and "Chen" [Formula], as described by TCM doctors", could be identified in a selected group of 3 subjects who were diagnosed by TCM practitioners. We have shown the classification rates is 98.7% in training process and 84.2% in testing result for these 3 basic pulse patterns. The high classification rate of the developed platform could lead to further development of a high-level artificial intelligence system incorporating knowledge from TCM - the robotics finger system could become a standard clinical equipment for digitalizing and visualizing human arterial pulses

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The contrasting shape representations that support object recognition in humans and CNNs 1The success of Convolutional Neural Networks (CNNs) in classifying objects has led to a surge of interest in using these systems to understand human vision. Recent studies have argued that when CNNs are trained in the correct learning environment, they can emulate a key property of human vision - learning to classify objects based on their shape. While showing a shape-bias is indeed a desirable property for any model of human object recognition, it is unclear whether the resulting shape representations learned by these networks are human-like. We explored this question in the context of a well-known observation from psychology showing that humans encode the shape of objects in terms of relations between object features. To check whether this is also true for the representations of CNNs, we ran a series of simulations where we trained CNNs on datasets of novel shapes and tested them on a set of controlled deformations of these shapes. We found that CNNs do not show any enhanced sensitivity to deformations which alter relations between features, even when explicitly trained on such deformations. This behaviour contrasted with human participants in previous studies as well as in a new experiment. We argue that these results are a consequence of a fundamental difference between how humans and CNNs learn to recognise objects: while CNNs select features that allow them to optimally classify the proximal stimulus, humans select features that they infer to be properties of the distal stimulus. This makes human representations more generalisable to novel contexts and tasks.

Author summaryThe human visual system is highly adept at recognising and reasoning about objects under a wide variety of viewing conditions. Research in vision sciences has shown that humans largely rely on shape to recognise objects, but extracting object shape from a retinal image is a challenge (indeed, it is an ill-posed problem), and until recently, models of vision have been poor at classifying naturalistic images of objects. This has changed with the development of Convolutional Neural Networks (CNNs) that perform as well as humans on certain recognition tasks. Thus it makes sense to ask if we can gain insight into how humans extract shape by studying CNNs. Here, we show that there is a fundamental difference between the two systems. While humans pay particular attention to relations between an objects parts and features, we show that the shape extracted by CNNs is akin to a template, where all diagnostic features are equally important. Thus, despite their success, these results show that CNNs are currently not good models of human object recognition and machine learning models must bridge this gap in order to capture the robustness and generalisability of human vision.

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Histone acetyltransferases and deacetylases are required for virulence, conidiation, DNA damage repair, and multiple stresses resistance of Alternaria alternata Histone acetylation, which is critical for transcriptional regulation and various biological processes in eukaryotes, is a reversible dynamic process regulated by HATs and HDACs. This study determined the function of 6 histone acetyltransferases (HATs) (Gcn5, RTT109, Elp3, Sas3, Sas2, Nat3) and 6 histone deacetylases (HDACs) (Hos2, Rpd3, Hda1, Hos3, Hst2, Sir2) in the phytopathogenic fungus Alternaria alternata by analyzing targeted gene deletion mutants. Our data provide evidence that HATs and HDACs are both required for mycelium growth, cell development and pathogenicity as many gene deletion mutants ({Delta}Gcn5,{Delta} RTT109,{Delta} Elp3,{Delta} Sas3,{Delta} Nat3,{Delta} Hos2, and{Delta} Rpd3) displayed reduced growth, conidiation or virulence at varying degrees. In addition, HATs and HDACs are involved in the resistance to multiple stresses such as oxidative stress (Sas3, Gcn5, Elp3, RTT109, Hos2), osmotic stress (Sas3, Gcn5, RTT109, Hos2), cell wall-targeting agents (Sas3, Gcn5, Hos2), and fungicide (Gcn5, Hos2).{Delta} Gcn5,{Delta} Sas3 and{Delta} Hos2 displayed severe growth defects on sole carbon source medium suggesting a vital role of HATs and HDACs in carbon source utilization. More SNPs were generated in{Delta} Gcn5 in comparison to wild-type when they were exposed to ultraviolet ray. Moreover,{Delta} RTT109,{Delta} Gcn5 and{Delta} Hos2 showed severe defects in resistance to DNA-damaging agents, indicating the critical role of HATs and HDACs in DNA damage repair. These phenotypes correlated well with the differentially expressed genes in{Delta} Gcn5 and{Delta} Hos2 that are essential for carbon sources metabolism, DNA damage repair, ROS detoxification, and asexual development. Furthermore, Gcn5 is required for the acetylation of H3K4. Overall, our study provides genetic evidence to define the central role of HATs and HDACs in the pathological and biological functions of A. alternata.

ImportanceIn filamentous fungi, HATs and HDACs have been found to be involved in growth, development, virulence, synthesis of secondary metabolites and multi-stress resistance. However, the role of all HATs and HDACs in the same fungal pathogen has not been systematically explored. Our study revealed that HATs and HDACs are required for the vegetative growth, conidiation, pathogenicity, multiple stresses resistance, DNA damage repair, and carbon source utilization in A. alternata. Moreover, HATs and HDACs identified in A. alternata are highly conserved in many notorious pathogenic fungi, such as Magnaporthe grisea and Candida albicans. Therefore, this study not only systematically reveals the biological functions of twelve histone acetylation-required enzymes for the first time, but also provides a foundation for future investigations in A. alternata or other pathogenic fungi.

sentences: Y chromosomal noncoding RNAs regulate autosomal gene expression via piRNAs in mouse testis Majority of the genes expressed during spermatogenesis are autosomal. Mice with different deletions of Yq show sub-fertility, sterility and sperm abnormalities. The connection between Yq deletion and autosomal gene regulation is not well understood. We describe a novel mouse Yq-derived long noncoding RNA, Pirmy, which shows unprecedented number of splice variants in testis. Further, Pirmy transcript variants act as templates for several piRNAs. We identified ten differentially expressed autosome-encoded sperm proteins in mutant mice. Pirmy transcript variants have homology to 5/3UTRs of these deregulated autosomal genes. Thus, subfertility in Y-deleted mice appears to be a polygenic phenomenon that is partially regulated epistatically by the Y-chromosome. Our study provides novel insights into possible role of MSY-derived ncRNAs in male fertility and reproduction. Finally, sperm phenotypes from the Y-deleted mice seem to be similar to that reported in inter-specific male-sterile hybrids. Taken together, this study provides novel insights into possible role of Y-derived ncRNAs in male sterility and speciation.

sentences: Present and Future Ecological Niche Modeling of Rift Valley fever in East Africa in Response to Climate Change Rift Valley fever (RVF) has been linked with recurrent outbreaks among humans and livestock in several parts of the globe. Predicting RVFs habitat suitability under different climate scenarios offers vital information for developing informed management schemes. The present study evaluated the probable impacts of climate change on the distribution of RVF disease in East Africa (E. A.), using the maximum entropy (MaxEnt) model and the disease outbreak cases. Considering the potential of the spread of the disease in the East Africa region, we utilized two representative concentration pathways (RCP 4.5 and RCP 8.5) climate scenarios in the 2050s and 2070s (average for 2041-2060, and 2061-2080), respectively. All models had satisfactory AUC values of more than 0.809, which are considered excellent. Jackknife tests revealed that Bio4 (temperature seasonality), land use, and population density were the main factors influencing RVF distribution in the region. From the risk maps generated, we infer that, without regulations, this disease might establish itself across more extensive areas in the region, including most of Rwanda and Burundi. The ongoing trade between East African countries and changing climates could intensify RVF spread into new geographic extents with suitable habitats for the important zoonosis. The predicted suitable areas for RVF in eastern Kenya, southern Tanzania, and Somalia overlaps to a large extent where cattle keeping and pastoralism are highly practiced, thereby signifying the urgency to manage and control the disease. This work validates RVF outbreak cases effectiveness to map the diseases distribution, thus contributing to enhanced ecological modeling and improved disease tracking and control efforts in East Africa.

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Temporal dynamics of cognitive flexibility in adolescents with anorexia nervosa: a high density EEG study Cognitive rigidity is a core symptom of anorexia nervosa (AN) and is associated with treatment resistance. Nevertheless, studies on the neural basis of cognitive flexibility (CF) in adolescent AN are rare.

The aim of this study was to investigate temporal brain dynamics of CF in adolescents with AN and their tendency to experience anxiety. To address this aim, participants performed a Dimensional Change Card Sorting task during high-density EEG recording. Transient state and stable traits of anxiety were measured with the State-Trait Anxiety Inventory. Data were collected on 22 girls with AN, and 23 healthy controls (HC) (aged 12-20 years old). Evoked potentials were investigated using microstates analysis. Effects of trait and state anxiety on microstates parameters were investigated performing correlations across groups and in the AN group only.

Adolescents with AN did not differ from HC with respect to behavioral switching cost, despite a greater overall accuracy. Adolescents with AN showed altered visual orienting processes (N100 micro-state), and earlier attentional processing to task switching (P300b microstate) when compared to HC. Reduced activation in a network involving the pre-central cortex, the cerebellum, and the posterior cingulate was identified in adolescents with AN. Trait and state anxiety were correlated with atypical AN microstates across groups.

The present findings suggest CF manifests with inefficient behavioral and neural strategies in adolescents with AN, at early stages in the illness. Additionally, this study provides evidence that transient and stable tendencies to experience anxiety could represent a vulnerability to developing CF impairments in adolescent AN.

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Membrane remodeling due to mixture of multiple curvature proteins: Insights from mesoscopic continuum modeling We present an extension of the Monte Carlo based mesoscopic membrane model, where the membrane is represented as a dynamically triangulated surface and the proteins are modeled as anisotropic inclusions formulated as in-plane nematic field variables adhering to the deformable elastic sheet. In the extended model, we have augmented the Hamiltonian to study membrane deformation due to a mixture of multiple types of curvature generating proteins. This feature opens the door for understanding how multiple kinds of curvature-generating proteins may be working in a coordinated manner to induce desired membrane morphologies. For example, among other things, we study membrane deformations and tubulation due to a mixture of positive and negative curvature proteins as mimics of various proteins from BAR domain family working together for curvature formation and stabilization. We also study the effect of membrane anisotropy, which manifests as membrane localization and differential binding affinity of a given curvature protein, leading to insights into the tightly regulated cargo sorting and transport processes. Our simulation results show different morphology of deformed vesicles that depend on the curvatures and densities of the participating proteins as well as on the protein-protein and membrane-proteins interactions.

SignificanceMembrane remodeling requires highly orchestrated interactions between several types of lipids and curvature proteins. Experimentally probing the membrane deformation intermediates is non-trivial. For example, it is not known exactly how two or more different kinds of BAR domain proteins work in concert to induce and stabilize certain membrane curvatures. In this work, we use mesoscopic continuum modeling and explore the factors that induce and stabilize a range of membrane deformation due to a mixture of positive and negative curvature proteins.

sentences: CliP: subclonal architecture reconstruction of cancer cells in DNA sequencing data using a penalized likelihood model Subpopulations of tumor cells characterized by mutation profiles may confer differential fitness and consequently influence prognosis of cancers. Understanding subclonal architecture has the potential to provide biological insight in tumor evolution and advance precision cancer treatment. Recent methods comprehensively integrate single nucleotide variants (SNVs) and copy number aberrations (CNAs) to reconstruct subclonal architecture using whole-genome or whole-exome sequencing (WGS, WES) data from bulk tumor samples. However, the commonly used Bayesian methods require a large amount of computational resources, a prior knowledge of the number of subclones, and extensive post-processing. Regularized likelihood modeling approach, never explored for subclonal reconstruction, can inherently address these drawbacks. We therefore propose a model-based method, Clonal structure identification through pair-wise Penalization, or CliP, for clustering subclonal mutations without prior knowledge or post-processing. The CliP model is applicable to genomic regions with or without CNAs. CliP demonstrates high accuracy in subclonal reconstruction through extensive simulation studies. Utilizing the well-established regularized likelihood framework, CliP takes only 16 hours to process WGS data from 2,778 tumor samples in the ICGC-PCAWG study, and 38 hours to process WES data from 9,564 tumor samples in the TCGA study. In summary, a penalized likelihood framework for subclonal reconstruction will help address intrinsic drawbacks of existing methods and expand the scope of computational analysis for cancer evolution in large cancer genomic studies. The associated software tool is freely available at: https://github.com/wwylab/CliP.

sentences: PySeq2500: An open source toolkit for repurposing HiSeq 2500 sequencing systems as versatile fluidics and imaging platforms. Fluorescence microscopy is a key method in the life sciences. State of the art -omics methods combine fluorescence microscopy with complex protocols to visualize tens to thousands of features in each of millions of pixels across samples. These -omics methods require precise control of temperature, reagent application, and image acquisition parameters during iterative chemistry and imaging cycles conducted over the course of days or weeks. Automated execution of such methods enables robust and reproducible data generation. However, few commercial solutions exist for temperature controlled, fluidics coupled fluorescence imaging, and implementation of bespoke instrumentation requires specialized engineering expertise. Here we present PySeq2500, an open source Python code base and flow cell design that converts the Illumina HiSeq 2500 instrument into an open platform for programmable applications. Customizable PySeq2500 protocols enable experimental designs involving simultaneous 4-channel image acquisition, temperature control, reagent exchange, stable positioning, and sample integrity over extended experiments. To demonstrate accessible automation of complex, multi-day workflows, we use the PySeq2500 system for unattended execution of iterative indirect immunofluorescence imaging (4i). Our automated 4i method uses off-the-shelf antibodies over multiple cycles of staining, imaging, and antibody elution to build highly multiplexed maps of cell types and pathological features in mouse and postmortem human spinal cord sections. As demonstrated here, PySeq2500 enables non-specialists to develop and implement state of the art fluidics coupled imaging methods in a widely available benchtop system.

sentences: Differential sphingosine-1-phosphate receptor-1 protein expression in the dorsolateral prefrontal cortex between schizophrenia Type 1 and Type 2 Understanding the etiology and treatment approaches in schizophrenia is challenged in part by the heterogeneity of this disorder. One encouraging progress is the growing evidence that there are subtypes of schizophrenia that may relate to disease duration and premorbid severity. Recent in vitro findings of messenger ribonucleic acid (mRNA) gene expression on postmortem dorsolateral prefrontal cortex (DLPFC) showed that schizophrenia has two subtypes, those with a relatively normal DLPFC transcriptome (Type 1) and those with differentially expressed genes (Type 2). Sphingosine-1-phosphate receptor-1 (S1PR1) is one of the genes that was highly upregulated in Type 2 compared to Type 1 and controls. The impact of that finding is limited because it only can be confirmed through analysis of autopsy tissue, and the clinical characteristics such as symptoms severity or illness duration was not available from that Medical Examiner based autopsy study. However, S1PR1 has great potential because it is a target gene that can be accessed via positron emission tomography (PET) in vivo using specific radioligands (starting with [11C]CS1P1) successfully developed at our center in human brain imaging. As a preliminary study to validate this PET target in schizophrenia, S1PR1 protein expression was assessed by receptor autoradiography (ARG) using [3H]CS1P1 and immunohistochemistry (IHC) in the DLPFC from patients with schizophrenia classified as Type 1 or Type 2 based on their DLPFC transcriptomes and from controls. Our analyses demonstrate that ARG S1PR1 protein expression is significantly higher in Type 2 compared to Type 1 (p < 0.05) and controls (p < 0.05), which was consistent with previous mRNA S1PR1. These findings support the possibility that PET S1PR1 can be used as a future imaging biomarker to distinguish these subgroups of schizophrenic patients during life with obvious implications for both patient management and the design of clinical trials to validate novel pharmacologic therapies.

sentences: Aggregation pheromone 4-vinylanisole promotes the synchrony of sexual maturation in female locusts Reproductive synchrony generally occurs in various group-living animals; however, the underlying mechanisms remain largely unexplored. Here, we report that aggregation pheromone, 4-vinylanisole, plays a key role in promoting sexual maturation synchrony of female adults in the migratory locust, Locusta migratoria, a worldwide agricultural pest species that can form huge swarms with millions of individuals. Gregarious female locusts display significant synchrony of sexual maturation and oviposition whereas solitarious females and olfactory deficiency mutants do not. Only the presence of gregarious male adults can stimulate sexual maturation synchrony of female adults. Of the volatiles emitted abundantly by gregarious male adults, only 4-vinylanisole induces female sexual maturation synchrony, whereas this effect is abolished in mutants of 4- vinylanisole receptor. Interestingly, 4-vinylanisole mainly accelerates oocyte maturation of young females aged at middle developmental stages (3-4 days post adult eclosion). Juvenile hormone/vitellogenin pathway mediates female sexual maturation triggered by 4-vinylanisole. Our results highlight a "catch-up" strategy by which gregarious females synchronize their oocyte maturation and oviposition by time-dependent endocrinal response to 4-vinylanisole.

sentences: Xlnc1DCNN: An Explainable Long Non-Coding RNA Identification Tool Long non-coding RNAs (lncRNAs) play crucial roles in many biological processes and are implicated in several diseases. With the next-generation sequencing technologies, substantial un-annotated transcripts have been discovered. Classifying unannotated transcripts using biological experiments is more time-consuming and expensive than computational approaches. Several tools for identifying long non-coding RNAs are available. These tools, however, did not explain which features in their tools contributed to the prediction results. Here, we present Xlnc1DCNN, a tool for distinguishing long non-coding RNAs (lncRNAs) from protein-coding transcripts (PCTs) using a one-dimensional convolutional neural network with prediction explanations. The evaluation results of the human test set showed that Xlnc1DCNN outperformed other state-of-the-art tools in terms of accuracy and F1-score. The explanation results revealed that lncRNA transcripts were mainly identified as sequences with no conserved regions or with a region of transmembrane helix while protein-coding transcripts were mostly classified by conserved protein domains or families. The explanation results also conveyed the probably inconsistent annotations among the public databases, lncRNA transcripts which contain protein domains or families, as well as protein-coding transcripts which are nonsense-mediated decay or processed transcripts. Xlnc1DCNN is freely available at https://github.com/cucpbioinfo/Xlnc1DCNN.

sentences: Pupil-linked arousal modulates trial-by-trial motor adaptation in humans Pupil diameter, under constant illumination, is known to reflect individuals internal states, such as surprise about observation and environmental uncertainty. Despite the growing use of pupillometry in cognitive learning studies as an additional measure for examining internal states, few studies have used pupillometry in human motor learning studies. Here we provide the first detailed characterization of pupil diameter changes in a short-term reach adaptation paradigm. We measured pupil changes in 91 human participants while they adapted to abrupt, gradual, or switching force field conditions. Sudden increases in movement error caused by the introduction/reversal of the force field resulted in strong phasic pupil dilation during movement accompanied by a transient increase in tonic pre-movement baseline pupil diameter in subsequent trials. In contrast, clear changes in pupil responses were absent when the force field was gradually introduced, indicating that error drove the changes in pupil responses. Nevertheless, we found an association between baseline pupil diameter and awareness of the gradually-introduced perturbation assessed post-experimentally. In all experiments, we also found a strong co-occurrence of larger baseline pupil diameter and slower reaction and movement time after each set break. Interestingly, error-induced pupil responses gradually became insensitive after experiencing multiple reversals. Collectively, these results suggest that tonic baseline pupil diameter reflects ones belief about environmental uncertainty, whereas phasic pupil dilation during movement reflects surprise about a sensory outcome (i.e., movement error), and both effects are modulated by novelty. Our results provide a new approach for non-verbally assessing participants internal states during motor learning.

sentences: SPF45/RBM17-dependent, but not U2AF-dependent, splicing in a distinct subset of human short introns Human pre-mRNA introns vary in size from under fifty to over a million nucleotides. We searched for essential factors involved in the splicing of human short introns by screening siRNAs against 154 human nuclear proteins. The splicing activity was assayed with a model HNRNPH1 pre-mRNA containing short 56-nucleotide intron. We identified a known alternative splicing regulator SPF45 (RBM17) as a constitutive splicing factor that is required to splice out this 56-nt intron. Whole-transcriptome sequencing of SPF45-deficient cells revealed that SPF45 is essential in the efficient splicing of many short introns. To initiate the spliceosome assembly on a short intron with the truncated poly-pyrimidine tract, the U2AF-homology motif (UHM) of SPF45 competes out that of U2AF65 for binding to the UHM-ligand motif (ULM) of the U2 snRNP protein SF3b155 (SF3B1). We propose that splicing in a distinct subset of human short introns depends on SPF45 but not U2AF heterodimer.

sentences: scAEGAN: Unification of Single-Cell Genomics Data by Adversarial Learning of Latent Space Correspondences Recent progress in Single-Cell Genomics have produced different library protocols and techniques for profiling of one or more data modalities in individual cells. Machine learning methods have separately addressed specific integration challenges (libraries, samples, paired-unpaired data modalities). We formulate an unifying data-driven methodology addressing all these challenges. To this end, we design a hybrid architecture using an autoencoder (AE) network together with adversarial learning by a cycleGAN (cGAN) network, jointly referred to as scAEGAN. The AE learns a low-dimensional embedding of each condition, whereas the cGAN learns a non-linear mapping between the AE representations. The core insight is that the AE respects each samples uniqueness, whereas the cGAN exploits the distributional data similarity in the latent space. We evaluate scAEGAN using simulated data and real datasets of a single-modality (scRNA-seq), different library preparations (Fluidigm C1, CelSeq, CelSeq2, SmartSeq), and several data modalities such as paired scRNA-seq and scATAC-seq. We find that scAEGAN outperforms Seurat3 in library integration, is more robust against data sparsity, and beats Seurat 4 in integrating paired data from the same cell. Furthermore, in predicting one data modality from another, scAEGAN outperforms Babel. We conclude scAEGAN surpasses current state-of-the-art methods across several seemingly different integration challenges.

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Directional Gaussian Mixture Models of the gut microbiome elucidate microbial spatial structure The gut microbiome is spatially heterogeneous, with environmental niches contributing to the distribution and composition of microbial populations. A recently developed mapping technology, MaPS-seq, aims to characterize the spatial organization of the gut microbiome by providing data about local microbial populations. However, information about the global arrangement of these populations is lost by MaPS-seq. To address this, we propose a class of Gaussian Mixture Models (GMM) with spatial dependencies between mixture components in order to computationally recover the relative spatial arrangement of microbial communities. We demonstrate on synthetic data that our spatial models can identify global spatial dynamics, accurately cluster data, and improve parameter inference over a naive GMM. We applied our model to three MaPS-Seq datasets taken from varying regions of the mouse intestine. On cecal and distal colon datasets, we find our model accurately recapitulates known spatial behaviors of the gut microbiome, including compositional differences between mucus and lumen-associated populations. Our model also seem to capture the role of a pH gradient on microbial populations in the mouse ileum and proposes new behaviors as well.

ImportanceThe spatial arrangement of the microbes in the gut microbiome is a defining characteristic of its behavior. Various experimental studies have attempted to provide glimpses into the mechanisms that contribute to microbial arrangements. However, many of these descriptions are qualitative. We developed a computational method that takes microbial spatial data and learns many of the experimentally validated spatial factors. We can then use our model to propose previously unknown spatial behaviors. Our results demonstrate that the gut microbiome, while exceptionally large, has predictable spatial patterns that can be used to help us understand its role in health and disease.

Code availabilitygithub.com/amepas/Spatial_Mbiome

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Histone deacetylase inhibitor overrides the effect of soft hydrogel on the mechanoresponse of human mesenchymal stem cells Human Mesenchymal stem cells (hMSCs) are promising in regenerative medicine for their multi-lineage differentiation capability. It has been demonstrated that lineage specification is governed by both chemical and mechanical cues. Among all the different mechanical cues known to control hMSCs fate, substrate stiffness is the most well-studied. It has been shown that the naive mesenchymal stem cells when cultured on soft gel, they commit towards adipogenic lineage while on stiff gel they become osteogenic. Soft substrates also cause less cell spreading, less traction, less focal adhesion assembly and stress fibre formation. Furthermore, chromatin condensation increases when cells are cultured on soft substrates. As the nucleus has been postulated to be mechanosensor and mechanotransducer, in this paper we asked the question how mechanosensing and mechanoresponse process will be influenced if we reduce the chromatin condensation in hMSCs cultured on soft substrates by using an external reagent. To address this question, we treated hMSCs cultured on soft polyacrylamide (PA) gels with a histone deacetylase inhibitor (HDACi) called Valproic Acid (VA) which decondense the chromatin by hyperacetylation of histone proteins. We found that the treatment with VA overrides the effect of soft substrates on hMSCs morphology, cellular traction, nuclear localization of mechnosensory protein YAP (yes-associated protein), and differentiation. VA treated cells behaved as if they are on stiff substrates in all aspects tested here. In contrast, when we increase chromatin condensation using histone acetyl transferase inhibitor (HATi), adipogenic differentiation on soft gel increases. Furthermore, we have shown that VA controls hMSCs differentiation via activation of ERK/MAPK pathway by increasing the p-ERK expression. Collectively, these findings demonstrate for the first time that inhibiting histone deacetylase can override the mechanoresponse of hMSCs. This work will help us to fundamentally understand the mechanosignalling process and to control the hMSCs differentiation in tissue engineering and regenerative medicine.

SignificanceTo harness the clinical potential of Human mesenchymal stem cells (hMSCs), it is important to understand the working mechanism of various signals in deciding cell fate. It has been shown in the recent past that the substrate rigidity influence hMSCs differentiation. Though the mechanism is not fully understood, the mechanosignals are known to alter the cell fate via reorganizing the chromatin packing. In this work, we ask if altering chromatin packing can change the cell fate as dictated by the mechanical properties of the substrates. We have shown that almost all the known effector of mechanosignals get modified when we tweak the chromatin packing via histone deacetylase inhibitor (HDACi). This study is helpful for developing a better understanding of the mechano-signalling process. It gives us a tool to override the effect of the substrates, particularly when the mechanical properties of biomaterials are not conducive for the desired differentiation outcome. Additionally, as HDACi are being used in the therapy of cancer and different neurodegenerative diseases, our understanding of their crosstalk with tissue stiffness in controlling cell fate will help to understand any unknown and undesirable side effects of using HDACi as therapeutic agent.

sentences: A Combination Strategy of Solubility Enhancers for Effective Production of Soluble and Bioactive Human Enterokinase Enterokinase is one of the hydrolases that catalyze hydrolysis to regulate biological processes in intestinal visceral mucosa. Enterokinase plays an essential role in accelerating the process of protein digestion as it converts trypsinogen into active trypsin by accurately recognizing and cleaving a specific peptide sequence, (Asp)4-Lys. Due to its exceptional substrate specificity, enterokinase is widely used as a versatile molecular tool in various bioprocessing, especially in removing fusion tags from recombinant proteins. Despite its biotechnological importance, mass production of soluble enterokinase in bacteria still remains an unsolved challenge. Here, we present an effective production strategy of human enterokinase using tandemly linked solubility enhancers consisting of thioredoxin, phosphoglycerate kinase or maltose-binding protein. The resulting enterokinases exhibited significantly enhanced solubility and bacterial expression level while retaining enzymatic activity, which demonstrates that combinatorial design of fusion proteins has the potential to provide an efficient way to produce recombinant proteins in bacteria.

sentences: Identification of a novel osteogenetic oligodeoxynucleotide (osteoDN) that promotes osteoblast differentiation in a TLR9-independent manner Recent studies have revealed that oligodeoxynucleotides (ODNs) designed from genome sequences have the potential to regulate cell fate. Currently, ODNs that conduct cell differentiation are nanomolecular drug candidates for regenerative medicine. Herein, we demonstrate that iSN40, an 18-base ODN derived from the Lacticaseibacillus rhamnosus GG genome, promoted the differentiation and calcification of osteoblasts that play a central role during bone formation. In the murine osteoblast cell line MC3T3-E1, iSN40 enhanced alkaline phosphatase activity at the early stage of differentiation and facilitated calcium deposition at the late stage by inducing the expression of osteogenic genes such as Msx2, osterix, collagen type 1, osteopontin, and osteocalcin. Intriguingly, the CpG motif within iSN40 was not required for its osteogenetic activity, indicating that iSN40 functions in a TLR9-independent manner. These data suggest that iSN40, serving as an osteogenetic ODN (osteoDN), as a drug seed that target osteoblasts for bone regeneration.

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Temporal differentiation of resource capture and biomass accumulation as a driver of yield increase in intercropping - Intercropping, i.e. the simultaneous cultivation of different crops on the same field, has demonstrated yield advantages compared to monoculture cropping. These yield advantages have often been attributed to complementary resource use, but few studies quantified the temporal complementarity of nutrient acquisition and biomass production. Our understanding of how nutrient uptake rates of nitrogen (N) and phosphorous (P) and biomass accumulation change throughout the growing season and between different neighbors is limited.
- We conducted weekly destructive harvests to measure temporal trajectories of N and P uptake and biomass production in three crop species (oat, lupin and camelina) growing either as isolated single plants, in monocultures or as intercrops. Additionally, we quantified organic acid exudation in the rhizosphere and biological N2-fixation of lupin throughout the growing season. Logistic models were fitted to characterize nutrient acquisition and biomass accumulation trajectories.
- Nutrient uptake and biomass accumulation trajectories were curtailed by competitive interactions, resulting in earlier peak rates and lower total accumulated nutrients and biomass compared to cultivation as isolated single plants. Different pathways led to overyielding in the two mixtures. The oat-camelina mixture was characterized by a shift from belowground temporal niche partitioning of resource uptake to aboveground competition for light during the growing season. The oat-lupin mixture showed strong competitive interactions, where lupin eventually overyielded due to reliance on atmospheric N and stronger competitiveness for soil P.
- Synthesis: This study demonstrates temporal shifts to earlier peak rates of plants growing with neighbors compared to those growing alone, suggesting that the observed temporal shifts in our experiment are driven by competitive interactions rather than active plant behavior to reduce competition. The two differing pathways to overyielding in the two mixtures highlight the importance of examining temporal dynamics in intercropping systems to understand the underlying mechanisms of overyielding.

sentences: Training for object recognition with increasing spatial frequency: A comparison of deep learning with human vision. The ontogenetic development of human vision, and the real-time neural processing of visual input, both exhibit a striking similarity - a sensitivity towards spatial frequencies that progress in a coarse-to-fine manner. During early human development, sensitivity for higher spatial frequencies increases with age. In adulthood, when humans receive new visual input, low spatial frequencies are typically processed first before subsequently guiding the processing of higher spatial frequencies. We investigated to what extent this coarse-to-fine progression might impact visual representations in artificial vision and compared this to adult human representations. We simulated the coarse-to-fine progression of image processing in deep convolutional neural networks (CNNs) by gradually increasing spatial frequency information during training. We compared CNN performance, after standard and coarse-to-fine training, with a wide range of datasets from behavioural and neuroimaging experiments. In contrast to humans, CNNs that are trained using the standard protocol are very insensitive to low spatial frequency information, showing very poor performance in being able to classify such object images. By training CNNs using our coarse-to-fine method, we improved the classification accuracy of CNNs from 0% to 32% on low-pass filtered images taken from the ImageNet dataset. When comparing differently trained networks on images containing full spatial frequency information, we saw no representational differences. Overall, this integration of computational, neural, and behavioural findings shows the relevance of the exposure to and processing of input with a variation in spatial frequency content for some aspects of high-level object representations.

sentences: A closed-loop stimulation approach with real-time estimation of the instantaneous phase of neural oscillations by a Kalman filter We propose a novel method to estimate the instantaneous oscillatory phase to implement a real-time system for closed-loop sensory stimulation in electroencephalography (EEG) experiments. The method uses Kalman filter-based prediction to estimate current and future EEG signals. We tested the performance of our method in a real-time situation. We demonstrate that the performance of our method shows higher accuracy in predicting the EEG phase than the conventional autoregressive model-based method. A Kalman filter allows us to easily estimate the instantaneous phase of EEG oscillations based on the automatically estimated autoregressive model implemented in a real-time signal processing machine. The proposed method has a potential for versatile applications targeting the modulation of EEG phase dynamics and the plasticity of brain networks in relation to perceptual or cognitive functions.

sentences: Ubiquitination and degradation of NF90 by Tim-3 inhibits antiviral innate immunity Nuclear Factor 90 (NF90) is a novel virus sensor that serves to initiate antiviral innate immunity by triggering the stress granules (SGs) formation. However, the regulation of the NF90-SGs pathway remain largely unclear. We found that Tim-3, an immune checkpoint inhibitor, promotes the ubiquitination and degradation of NF90 and inhibits NF90-SGs mediated antiviral immunity. Vesicular Stomatitis Virus (VSV) infection induces the up-regulation and activation of Tim-3 in macrophages which in turn recruited the E3 ubiquitin ligase TRIM47 to the zinc finger domain of NF90 and initiated a proteasome-dependent degradation of the NF90 via K48-linked ubiquitination at Lys297. Targeted inactivation of the Tim-3 enhances the NF90 downstream SGs formation by selectively increasing the phosphorylation of PKR and eIF2a, the expression of SGs markers G3BP1 and TIA-1, and protected mice from lethal VSV challenge. These findings provide insights into the crosstalk between Tim-3 and other receptors in antiviral innate immunity and its related clinical significance.

sentences: Sharpened and mechanically durable carbon fiber electrode arrays for neural interfacing Bioelectric medicine treatments target disorders of the nervous system unresponsive to pharmacological methods. While current stimulation paradigms effectively treat many disorders, the underlying mechanisms are relatively unknown, and current neuroscience recording electrodes are often limited in their specificity to gross averages across many neurons or axons. Here, we develop a novel, durable carbon fiber electrode array adaptable to many neural structures for precise neural recording. Carbon fibers were sharpened using a reproducible blowtorch method that uses the reflection of fibers against the surface of a water bath. The arrays were developed by partially embedding carbon fibers in medical-grade silicone to improve durability. We recorded acute spontaneous electrophysiology from the rat cervical vagus nerve (CVN), feline dorsal root ganglia (DRG), and rat brain. Blowtorching resulted in fibers of 72.3 {+/-} 33.5 degree tip angle with 146.8 {+/-} 17.7 m exposed carbon. Silicone-embedded carbon fiber arrays were durable with 87.5% of fibers remaining after 50,000 passes. Observable neural clusters were recorded using sharpened carbon fiber electrodes from rat CVN (41.8 Vpp), feline DRG (101.1 Vpp), and rat brain (80.7 Vpp). Recordings from the feline DRG included physiologically-relevant signals from increased bladder pressure and cutaneous brushing. These results suggest that this carbon fiber array is a uniquely durable and adaptable neural recording device. In the future, this device may be useful as a bioelectric medicine tool for diagnosis and closed-loop neural control of therapeutic treatments and monitoring systems.

sentences: Genomic architecture controls spatial structuring in Amazonian birds Large rivers are ubiquitously invoked to explain the distributional limits and speciation of the Amazon Basins mega-diversity. However, inferences on the spatial and temporal origins of Amazonian species have narrowly focused on evolutionary neutral models, ignoring the potential role of natural selection and intrinsic genomic processes known to produce heterogeneity in differentiation across the genome. To test how genomic architecture impacts our ability to reconstruct patterns of spatial diversification across multiple taxa, we sequenced whole genomes for populations of bird species that co-occur in southeastern Amazonian. We found that phylogenetic relationships within species and demographic parameters varied across the genome in predictable ways. Genetic diversity was positively associated with recombination rate and negatively associated with the species tree topology weight. Gene flow was less pervasive in regions of low recombination, making these windows more likely to retain patterns of population structuring that matched the species tree. We further found that approximately a third of the genome showed evidence of selective sweeps and linked selection skewing genome-wide estimates of effective population sizes and gene flow between populations towards lower values. In sum, we showed that the effects of intrinsic genomic characteristics and selection can be disentangled from the neutral processes to elucidate how speciation hypotheses and biogeographic patterns are sensitive to genomic architecture.

sentences: Respiratory dysfunctions in two rodent models of chronic epilepsy and acute seizures and their link with brainstem serotonin system Patients with drug-resistant epilepsy can experience respiratory alterations notably during seizures. The mechanisms underlying this long-term alteration of respiratory function remain unclear. This study aimed at determining in rats whether epilepsy is associated with alterations of both the respiratory function and brainstem serotonin (5-HT) system. Epilepsy was triggered by pilocarpine-induced status epilepticus in rats. 30-50% of epileptic (EPI) rats exhibited sharp decrease of oxygen consumption (SDOC), low metabolic rate of oxygen and slow regular ventilation; these rats were called EPI/SDOC+ rats. These alterations were only detected in rats with chronic epilepsy, independent of behavioral seizures, persisted over the time, and were not associated with death. In these rats, 5-HT fiber density in the nucleus tractus solitarius was below that of control and EPI/SDOC-rats. Both EPI/SDOC+ rats and DBA/2 mice presenting with fatal respiratory arrest following an audiogenic-induced seizure, a model of sudden and expected death in epilepsy, had increased transcript levels of tryptophan hydroxylase 2 (p<0.001 for both strains) and 5-HT presynaptic transporter (rats: p=0.003; mice: p=0.001). Thus, our data support that 5-HT alterations are associated with chronic and acute epilepsy-related respiratory dysfunctions.

sentences: scROSHI - robust supervised hierarchical identification of single cells Identifying cell types based on expression profiles is a pillar of single cell analysis. Existing machine-learning methods identify predictive features from annotated training data, which are often not available in early-stage studies. This can lead to overfitting and inferior performance when applied to new data. To address these challenges we present scROSHI, which utilizes previously obtained cell type-specific gene sets and does not require training or the existence of annotated data. By respecting the hierarchical nature of cell type relationships and assigning cells consecutively to more specialized identities, excellent prediction performance is achieved. In a benchmark based on publicly available PBMC data sets, scROSHI outperforms competing methods when training data are limited or the diversity between experiments is large.

sentences: Generation of photocaged nanobodies for in vivo  applications using genetic code expansion and computationally guided protein engineering Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular-dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies. These variants exhibit photo-activatable binding triggered by illumination with 365nm light. We demonstrate that the photocaged nanobodies we have created are highly robust and function in a complex cellular environment. We apply them to control subcellular protein localisation in the nematode worm C. elegans. Our approach provides a rare example of computationally designed proteins being directly applied in living animals and demonstrates the importance of accounting for in vivo effects on protein-protein interactions.

sentences: Generating and testing hypotheses about the fossil record of insect herbivory with a theoretical ecospace A typical fossil flora examined for insect herbivory contains a few hundred leaves and a dozen or two insect damage types. Paleontologists employ a wide variety of metrics to assess differences in herbivory among assemblages: damage type diversity, intensity (the proportion of leaves, or of leaf surface area, with insect damage), the evenness of diversity, and comparisons of the evenness and diversity of the flora to the evenness and diversity of damage types. Although the number of metrics calculated is quite large, given the amount of data that is usually available, the study of insect herbivory in the fossil record still lacks a quantitative framework that can be used to distinguish among different causes of increased insect herbivory and to generate null hypotheses of the magnitude of changes in insect herbivory over time. Moreover, estimates of damage type diversity, the most common metric, are generated with inconsistent sampling standardization routines. Here we demonstrate that coverage-based rarefaction yields valid, reliable estimates of damage type diversity that are robust to differences among floral assemblages in the number of leaves examined, average leaf surface area, and the inclusion of plant organs other than leaves such as seeds and axes. We outline the potential of a theoretical ecospace that combines various metrics to distinguish between potential causes of increased herbivory. We close with a discussion of the most appropriate uses of a theoretical ecospace for insect herbivory, with the overlapping damage type diversities of Paleozoic gymnosperms and Cenozoic angiosperms as a brief case study.

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RosettaSurf - a surface-centric computational design approach Proteins are typically represented by discrete atomic coordinates providing an accessible framework to describe different conformations. However, in some fields proteins are more accurately represented as near-continuous surfaces, as these are imprinted with geometric (shape) and chemical (electrostatics) features of the underlying protein structure. Protein surfaces are dependent on their chemical composition and, ultimately determine protein function, acting as the interface that engages in interactions with other molecules. In the past, such representations were utilized to compare protein structures on global and local scales and have shed light on functional properties of proteins. Here we describe RosettaSurf, a surface-centric computational design protocol, that focuses on the molecular surface shape and electrostatic properties as means for protein engineering, offering a unique approach for the design of proteins and their functions. The RosettaSurf protocol combines the explicit optimization of molecular surface features with a global scoring function during the sequence design process, diverging from the typical design approaches that rely solely on an energy scoring function. With this computational approach, we attempt to address a fundamental problem in protein design related to the design of functional sites in proteins, even when structurally similar templates are absent in the characterized structural repertoire. Surface-centric design exploits the premise that molecular surfaces are, to a certain extent, independent of the underlying sequence and backbone configuration, meaning that different sequences in different proteins may present similar surfaces. We benchmarked RosettaSurf on various sequence recovery datasets and showcased its design capabilities by generating epitope mimics that were biochemically validated. Overall, our results indicate that the explicit optimization of surface features may lead to new routes for the design of functional proteins.

Author SummaryFinely orchestrated protein-protein interactions are at the heart of virtually all fundamental cellular processes. Altering these processes or encoding new functions in proteins has been a long-standing goal in computational protein design.

Protein design methods commonly rely on scoring functions that seek to identify amino acid sequences that optimize structural configurations of atoms while minimizing a variety of physics-based and statistical terms. The objectives of the large majority of computational design protocols have been focused on obtaining a predefined structural conformation. However, routinely introducing a functional aspect on designer proteins has been more challenging.

Our results suggest that the molecular surface features can be a useful optimization parameter to guide the design process towards functional surfaces that mimic known protein binding sites and interact with their intended targets. Specifically, we demonstrate that our design method can optimize experimental libraries through computational screening, creating a basis for highly specific protein binders, as well as design a potent immunogen that engages with site-specific antibodies. The ability to create proteins with novel functions will be transformative for biomedical applications, providing many opportunities for the design of novel immunogens, protein components for synthetic biology, and other protein-based biotechnologies.

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3C-seq-captured chromosome conformation of the hyperthermophilic archaeon Thermofilum adornatum Three-dimensional structure of chromosomes displays diverse patterns across the tree of life, with compartments, interaction domains and loops being quite universally observed. The archaeal kingdom remains understudied to this extent so far, despite representing an interesting area from evolutionary and other perspectives.

Here we describe the spatial chromosomal organization of a hyperthermophilic crenarchaeon Thermofilum adornatum strain 1910b based on high-throughput chromosome conformation capture (3C-seq) approach. The chromosome contact map showed a curved secondary diagonal almost orthogonal to the main one. No evidence of chromosome loops was present. We were able to identify boundaries of different strengths between chromosome interaction domains (CIDs) albeit moderate. The plaid-like patterns previously reported for Sulfolobus archaea were not observed. However, the calculation of A/B compartments divided the genome into 2 domains that were different by the density of predicted highly expressed genes and location of origins.

Further comparison of these domains with whole-genome gene expression profiles will allow to test whether these domains represent expression-associated compartments. If so, it is possible that they represent primitive compartments evolutionarily older than the plaid patterns of Sulfolobus and higher eukaryotes. Further exploration of 3D chromatin in all branches of archaeal diversity will elucidate the evolution of the links between structural and functional organization in live organisms.

sentences: A pathogenicity locus of Streptococcus gallolyticus subspecies gallolyticus Streptococcus gallolyticus subspecies gallolyticus (Sgg) is known to be strongly associated with colorectal cancer (CRC). Recent functional studies further demonstrated that Sgg actively stimulates CRC cell proliferation and promotes the development of colon tumors. However, the Sgg factors important for the pro-proliferative and pro-tumor activities of Sgg remain unclear. Here, we identified a chromosomal locus in Sgg strain TX20005. Deletion of this locus significantly reduced Sgg adherence to CRC cells and abrogated the ability of Sgg to stimulate CRC cell proliferation. Thus, we designate this locus as the Sgg pathogenicity-associated region (SPAR). More importantly, we found that SPAR is important for Sgg pathogenicity in vivo. In a gut colonization model, mice exposed to the SPAR deletion mutant showed significantly reduced Sgg load in the colonic tissues and fecal materials, suggesting that SPAR contributes to the colonization capacity of Sgg. In a mouse model of CRC, deletion of SPAR abolished the ability of Sgg to promote the development of colon tumors growth. Taken together, these results highlight SPAR as a critical pathogenicity determinant of Sgg.

sentences: Intestinal Mucin Is a Chaperone of Multivalent Copper Mucus protects the body by many mechanisms, but a role in managing toxic transition metals was not previously known. Here we report that secreted mucins, the major mucus glycoproteins coating the respiratory and intestinal epithelia, are specific copper-binding proteins. Most remarkably, the intestinal mucin, MUC2, has two juxtaposed copper binding sites, one that accommodates Cu2+ and the other Cu1+, which can be formed in situ by reduction with vitamin C. Copper is an essential trace metal because it is a cofactor for a variety of enzymes catalyzing electron transfer reactions, but copper damages macromolecules when unregulated. We observed that MUC2 protects against copper toxicity while permitting nutritional uptake into cells. These findings introduce mucins, produced in massive quantities to guard extensive mucosal surfaces, as extracellular copper chaperones and potentially important players in physiological copper homeostasis.

sentences: Re-introduction of endogenous pathways for propionyl-CoA, 1-propanol and propionate formation in Escherichia coli In a previous study, we found that 2-ketobutyrate (2-KB) was seriously degraded in Escherichia coli. In the present investigation, we tried to clarify the products of that degradation process, and intriguingly reconfirmed that 2-KB is chopped up to form propionyl-CoA, 1-propanol and propionate. This short commentary re-introduces efficient endogenous pathways for production of value-added odd-chain compounds such as propionyl-CoA-derived chemicals.

sentences: Mapping the nuclear microenvironment of genes at agenome-wide scale The nuclear folding of chromosomes relative to nuclear bodies is an integral part of gene function. Here, we demonstrate that population-based modeling--from ensemble Hi-C data--can provide a detailed description of the nuclear microenvironment of genes and its role on gene function. We define the microenvironment by the subnuclear positions of genomic regions with respect to nuclear bodies, local chromatin compaction, and preferences in chromatin compartmentalization. These structural descriptors are determined in single cell models on a genome-wide scale, thereby revealing the structural variability between cells. We demonstrate that the structural microenvironment of a genomic region is linked to its functional potential in gene transcription, replication and chromatin compartmentalization. Some chromatin regions are distinguished by their strong preferences to a single microenvironment, due to associations to specific nuclear bodies in most cells. Other chromatin shows high structural variability, which is a strong indicator of functional heterogeneity. Moreover, we identify specialized nuclear microenvironments, which distinguish chromatin in different functional states and reveal a key role of nuclear speckles in chromosome organization. We demonstrate that our method produces highly predictive 3-dimensional genome structures, which accurately reproduce data from TSA-seq, DamID, GPSeq and super-resolution imaging. Thus, our method considerably expands the range of Hi-C data analysis and is widely applicable.

sentences: Comparative Analysis of non-coding small-RNAs in P. aeruginosa Keratitis Strains with Different Antibiotic Susceptibility Pseudomonas aeruginosa, is a gram-negative bacterium causes opportunistic or nosocomial infections in immunocompromised individuals. In recent years, a steady increase in human corneal infections of P. aeruginosa has been reported with increased multi-drug resistance (MDR) or extensively drug resistance (XDR). Several non-coding sRNAs, has been identified to regulate various physiological processes in P. aeruginosa, including biofilm formation, quorum sensing. However, the regulatory mechanism of sRNAs in MDR/XDR pathways of P. aeruginosa keratitis strains is not yet studied. In this study, we identified bacterial sRNAs in publicly available P. aeruginosa keratitis genomes and investigated their regulatory role in MDR/XDR pathways using bioinformatic analysis. Totally, 46 P. aeruginosa keratitis strains from different geographical regions were included. Of 46, Eight (30%) out of Twenty-seven and Nine (52) out of Nineteen P. aeruginosa strains from India and Australia were identified as not-MDR. Whereas, 10 (38%) Indian and 9 (47%) Australian strains were identified as MDR. Eight Indian strains were identified as XDR. Out of 46 strains, 23 (50%) carried ExoU, 21(45%) carried ExoS and two (5%) strains carried both ExoU and ExoS, exotoxins for their virulence. The sRNA, SPA0021 was identified in 18 MDR/XDR and 6 not-MDR strains along with UCBPP-PA14. Interestingly, majority of the imipenem resistant P. aeruginosa keratitis strains from the present study was found to be carried SPA0023 sRNA (18 out of 30 strains). The outer membrane porin protein OprD, identified as binding target of SPA0023. Negative regulation or inactivation of OprD, reported in increased imipenem resistance in P. aeruginosa. Mutation analysis revealed that SPA0023 carrying P. aeruginosa keratitis strains contains a lesser number of amino acid changes in OprD protein than other strains. These findings indicate, imipenem resistance in SPA0023 carried strains might arose from the negative regulation or inhibition of OprD by SPA0023. However, functional studies are warranted with large number of P. aeruginosa keratitis strains to confirm the negative regulation of OprD by SPA0023 and imipenem resistance.

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Causal roles of prefrontal cortex during spontaneous perceptual switching are determined by brain state dynamics The prefrontal cortex (PFC) is thought to orchestrate cognitive dynamics. However, in tests of bistable visual perception, no direct evidence supporting such presumable causal roles of the PFC has been reported. Here, using a novel brain-state-dependent neural stimulation system, we found that three PFC regions--right frontal eye fields and anterior/posterior dorsolateral PFCs (a/pDLPFCs)--have causal effects on perceptual dynamics but the behavioural effects are detectable only when we modulated the PFC activity in brain-state-/state-history-dependent manners. Also, we revealed that the brain-dynamics-dependent behavioural causality is underpinned by transient changes in the brain state dynamics, and such neural changes are determined by structural transformations of hypothetical energy landscapes. Moreover, we identified different functions of the three PFC areas: in particular, we found that aDLPFC enhances the integration of the two PFC-active brain states, whereas pDLPFC promotes the diversity between them. This work resolves the controversy over the PFC roles in spontaneous perceptual switching and underlines brain state dynamics in fine investigations of brain-behaviour causality.

Impact statementPrefrontal causal roles are changing during bistable visual perception, which was determined by large-scale brain state dynamics and attributable to hypothetical energy landscapes that underpin the brain state dynamics.

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THE CONSTITUTIVE ACTIVITY OF THE HISTAMINE H1 RECEPTOR INTERACTION WITH THE NMDA RECEPTOR CONSEQUENCES: IN EPILEPSY ObjectivesTo explore the epileptogenic effects of several drugs specific to the antihistaminic H1 receptor on NMDA and GABA responses.

MethodsWith patch-clamp whole-cell recordings of hippocampal neurons of rats, we observe the effects of several histaminergic agonists and antagonists on the NMDA and GABA currents. During the NMDA rundown and GABA rundown, we applied the drugs and observe the effect on the responses, and compare them to control conditions.

ResultsMepyramine with nanomolar concentrations increases significantly the NMDA responses about 35 %, this effect is mimicked by another Anti H1 drug Triprolidine. Histamine alone has no effect on NMDA rundown but 100 M Histamine can reduce partially the effect of Mepyramine. An H1 receptor agonist 2,3 Bromophenyl Histamine also shows the same properties as Histamine. The H1 receptor of Histamine seems to be crucial during these experiments, blockade of its constitutive activity by Mepyramine induces a significant increase of the NMDA responses. On the GABA response, the same nanomolar dose of Mepyramine has no effect on the rundown and we also observed no effect of Histamine on the GABA rundown.

SignificanceAn interaction between Histamine H1 receptor and the NMDA is revelated by using Mepyramine in nanomolar concentration, Histamine has no effect on these responses also in these experiments Mepyramine acts as an inverse agonist blocking the constitutive activity of the H1 receptor. The constitutive activity of the H1 receptor seems to be crucial in the regulation of NMDA receptor activities.

Keys Points The use of antihistaminic H1 drugs can facilitate the apparition of epileptic seizures,
During rundown experiments, Mepyramine, a high potent H1 antagonist increased the NMDA responses, while Histamine as no effect.
On GABA rundown both Mepyramine and Histamine show no effect.
Constitutive activity of the Histamine H1 receptor seems to be crucial for the NMDA receptor activity.

sentences: Therapeutic downregulation of neuronal PAS domain 2 (Npas2) promotes surgical skin wound healing Attempts to minimize scarring remain among the most difficult challenges facing surgeons, despite the use of optimal wound closure techniques. Previously, we reported improved healing of dermal excisional wounds in circadian clock neuronal PAS domain 2 (Npas2)-null mice. In this study, we performed high-throughput drug screening to identify a compound that downregulates Npas2 activity. The hit compound (Dwn1) suppressed circadian Npas2 expression, increased murine dermal fibroblast cell migration, and decreased collagen synthesis in vitro. Based on the in vitro results, Dwn1 was topically applied to iatrogenic full-thickness dorsal cutaneous wounds in a murine model. The Dwn1-treated dermal wounds healed faster with favorable mechanical strength and developed less granulation tissue than the controls. The expression of type I collagen, Tgf{beta}1, and -smooth muscle actin was significantly decreased in Dwn1-treated wounds, suggesting that hypertrophic scarring and myofibroblast differentiation are attenuated by Dwn1 treatment. NPAS2 may represent an important target for therapeutic approaches to optimal surgical wound management.

sentences: Trends in Arabidopsis Research post genome sequencing- A Scientometric study 1.Arabidopsis thaliana, a model plant, is intensively researched because of the intrinsic advantages associated with its life cycle, genetics, and other characteristics. In the present study, we analysed the publication data of research work done on A. thaliana during 2001-2020, a period when the whole genome sequence of the plant was available to the researchers. The current meta-analysis showed that 31965 research papers were published globally on Arabidopsis during the study period. The USA topped the countries with the maximum share of publications (28.97%), followed by China, Japan, and West European countries. The analysis showed a temporal shift in the number of publications from different countries and their research focus. After 2013, the research output from China was higher than that from the USA, and there was a shift in focus from developmental biology to stress-related topics. Plant Physiology journal carried the most research work (2490 publications) on Arabidopsis, which was followed by Plant Cell (2376), the Plant Journal (2260) and the Journal of Experimental Botany (1121). However, there was a progressive decline in the publication in these journals, and a shift was evident in favour of open access journals like Frontiers in Plant Science. The publication and citation numbers also show ongoing Arabidopsis researchs relevance to plant sciences, particularly curiosity-driven and discovery-based science. This article delves into the patterns in the prominence of research areas, ideas and foci for the future Arabidopsis research roadmap.

sentences: The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50{micro}M) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0-100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3 and the 5 splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 {micro}M). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.

sentences: SpotClean adjusts for spot swapping in spatial transcriptomics data Spatial transcriptomics (ST) is a powerful and widely-used approach for profiling genome-wide gene expression across a tissue with emerging applications in molecular medicine and tumor diagnostics. Recent spatial transcriptomics experiments utilize slides containing thousands of spots with spot-specific barcodes that bind mRNA. Ideally, unique molecular identifiers at a spot measure spot-specific expression, but this is often not the case owing to bleed from nearby spots, an artifact we refer to as spot swapping. We propose SpotClean to adjust for spot swapping and, in doing so, to increase the sensitivity and precision with which downstream analyses are conducted.

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High Frequencies of PD-1+TIM3+TIGIT+CTLA4+ Functionally Exhausted SARS-CoV-2-Specific CD4+ and CD8+ T Cells Associated with Severe Disease in Critically ill COVID-19 Patients SARS-CoV-2-specific memory T cells that cross-react with common cold coronaviruses (CCCs) are present in both healthy donors and COVID-19 patients. However, whether these cross-reactive T cells play a role in COVID-19 pathogenesis versus protection remain to be fully elucidated. In this study, we characterized cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells, targeting genome-wide conserved epitopes in a cohort of 147 non-vaccinated COVID-19 patients, divided into six groups based on the degrees of disease severity. We compared the frequency, phenotype, and function of these SARS-CoV-2-specific CD4+ and CD8+ T cells between severely ill and asymptomatic COVID-19 patients and correlated this with -CCCs and {beta}-CCCs co-infection status. Compared with asymptomatic COVID-19 patients, the severely ill COVID-19 patients and patients with fatal outcomes: (i) Presented a broad leukocytosis and a broad CD4+ and CD8+ T cell lymphopenia; (ii) Developed low frequencies of functional IFN-{gamma}-producing CD134+CD138+CD4+ and CD134+CD138+CD8+ T cells directed toward conserved epitopes from structural, non-structural and regulatory SARS-CoV-2 proteins; (iii) Displayed high frequencies of SARS-CoV-2-specific functionally exhausted PD-1+TIM3+TIGIT+CTLA4+CD4+ and PD-1+TIM3+TIGIT+CTLA4+CD8+ T cells; and (iv) Displayed similar frequencies of co-infections with {beta}-CCCs strains but significantly fewer co-infections with -CCCs strains. Interestingly, the cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4+ and CD8+ T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcome seen in asymptomatic COVID-19 patients. Our results demonstrate that, the critically ill COVID-19 patients displayed fewer co-infection with -CCCs strain, presented broad T cell lymphopenia and higher frequencies of cross-reactive exhausted SARS-CoV-2-specific CD4+ and CD8+ T cells. In contrast, the asymptomatic COVID-19 patients, appeared to present more co-infections with -CCCs strains, associated with higher frequencies of functional cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells. These findings support the development of broadly protective, T-cell-based, multi-antigen universal pan-Coronavirus vaccines.

KEY POINTSO_LIA broad lymphopenia and lower frequencies of SARS-CoV-2-specific CD4+ and CD8+ T-cells were associated with severe disease onset in COVID-19 patients.
C_LIO_LIHigh frequencies of phenotypically and functionally exhausted SARS-CoV-2-specific CD4+ and CD8+ T cells, co-expressing multiple exhaustion markers, and targeting multiple structural, non-structural, and regulatory SARS-CoV-2 protein antigens, were detected in severely ill COVID-19 patients.
C_LIO_LICompared to severely ill COVID-19 patients and to patients with fatal outcomes, the (non-vaccinated) asymptomatic COVID-19 patients presented more functional cross-reactive CD4+ and CD8+ T cells targeting conserved epitopes from structural, non-structural, and regulatory SARS-CoV-2 protein antigens.
C_LIO_LIThe cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4+ and CD8+ T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcomes seen in asymptomatic COVID-19 patients.
C_LIO_LICompared to severely ill COVID-19 patients and to patients with fatal outcomes, the (non-vaccinated) asymptomatic COVID-19 patients presented higher rates of co-infection with the -CCCs strains.
C_LIO_LICompared to patients with mild or asymptomatic COVID-19, severely ill symptomatic patients and patients with fatal outcomes had more exhausted SARS-CoV-2-speccific CD4+ and CD8+ T cells that preferentially target cross-reactive epitopes that share high identity and similarity with the {beta}-CCCs strains.
C_LI

sentences: Fbxo7 promotes Cdk6 activity to inhibit PFKP and glycolysis in T cells. Deregulated Fbxo7 expression is associated with many pathologies, including anaemia, male sterility, cancer, and Parkinsons disease, demonstrating its critical role in a variety of cell types. Although Fbxo7 is an F-box protein that recruits substrates for SCF-type E3 ubiquitin ligases, it also promotes the formation of cyclin D/Cdk6/p27 complexes in an E3-ligase independent fashion. We discovered PFKP, the major gatekeeper of glycolysis, in a screen for Fbxo7 substrates. PFKP has been previously shown to be a critical substrate of Cdk6 for the viability of T-ALL cells. We investigated the molecular relationships between Fbxo7, Cdk6 and PFKP, and the functional effect Fbxo7 has on T cell metabolism, viability, and activation. Fbxo7 promotes Cdk6-independent ubiquitination and Cdk6-dependent phosphorylation of PFKP. Importantly Fbxo7-deficient cells have reduced Cdk6 activity, and haematopoietic and lymphocytic cell lines show a significant dependency on Fbxo7. Compared to WT cells, CD4+ T cells with reduced Fbxo7 expression show increased glycolysis, despite lower cell viability and activation levels. Metabolomic studies of activated CD4+ T cells confirm increased glycolytic flux in Fbxo7-deficient cells, as well as altered nucleotide biosynthesis and arginine metabolism. We show Fbxo7 expression is glucose-responsive at the mRNA and protein level, and we propose Fbxo7 inhibits PFKP and glycolysis via its activation of Cdk6.

sentences: Multi-omics approach identifies germline regulatory variants associated with hematopoietic malignancies in retriever dog breeds Histiocytic sarcoma is an aggressive hematopoietic malignancy of mature tissue histiocytes with a poorly understood etiology in humans. A histologically and clinically similar counterpart affects flat-coated retrievers (FCRs) at unusually high frequency, with 20% developing the lethal disease. The similar clinical presentation combined with the closed population structure of dogs, leading to high genetic homogeneity, makes dogs an excellent model for genetic studies of cancer susceptibility. To determine the genetic risk factors underlying histiocytic sarcoma in FCRs, we conducted multiple genome-wide association studies (GWASs), identifying two loci that confer significant risk on canine chromosomes (CFA) 5 (Pwald=4.83x10-9) and 19 (Pwald=2.25x10-7). We subsequently undertook a multi-omics approach that has been largely unexplored in the canine model to interrogate these regions, generating whole genome, transcriptome, and chromatin immunoprecipitation sequencing. These data highlight the PI3K pathway gene PIK3R6 on CFA5, and proximal candidate regulatory variants that are strongly associated with histiocytic sarcoma and predicted to impact transcription factor binding. The CFA5 association colocalizes with susceptibility loci for two hematopoietic malignancies, hemangiosarcoma and B-cell lymphoma, in the closely related golden retriever breed, revealing the risk contribution this single locus makes to multiple hematological cancers. By comparison, the CFA19 locus is unique to the FCR and harbors risk alleles associated with upregulation of TNFAIP6, which itself affects cell migration and metastasis. Together, these loci explain ~35% of disease risk, an exceptionally high value that demonstrates the advantages of domestic dogs for complex trait mapping and genetic studies of cancer susceptibility.

sentences: Maternal diet disrupts the placenta-brain axis in a sex-specific manner High maternal weight is associated with a number of detrimental outcomes in offspring, including increased susceptibility to neurological disorders such as anxiety, depression, and communicative disorders (e.g. autism spectrum disorders)1-4. Despite widespread acknowledgement of sex-biases in the prevalence, incidence, and age of onset of these disorders, few studies have investigated potential sex-biased mechanisms underlying disorder susceptibility. Here, we use a mouse model to demonstrate how maternal high-fat diet causes perinatal inflammation that influences sex-specific behavioral outcomes in offspring. In male high-fat diet offspring, increased macrophage toll like receptor 4 signaling results in excess phagocytosis of serotonin neurons in the developing dorsal raphe nucleus, decreasing serotonin bioavailability in the fetal and adult brain. Bulk sequencing from a large cohort of matched first trimester human fetal brain, placenta, and maternal decidua samples reveals sex-specific transcriptome-wide changes in placenta and brain tissue. Further, we find that fetal brain serotonin is significantly negatively correlated with maternal triglyceride accumulation (a proxy for dietary fat content) in male pregnancies only. These findings uncover a fundamental mechanism through which maternal diet may increase offspring susceptibility for neuropsychiatric disorder development.

sentences: Bio-instantiated recurrent neural networks Biological neuronal networks (BNNs) are a source of inspiration and analogy making for researchers that focus on artificial neuronal networks (ANNs). Moreover, neuroscientists increasingly use ANNs as a model for the brain. Despite certain similarities between these two types of networks, important differences can be discerned. First, biological neural networks are sculpted by evolution and the constraints that it entails, whereas artificial neural networks are engineered to solve particular tasks. Second, the network topology of these systems, apart from some analogies that can be drawn, exhibits pronounced differences. Here, we examine strategies to construct recurrent neural networks (RNNs) that instantiate the network topology of brains of different species. We refer to such RNNs as bio-instantiated. We investigate the performance of bio-instantiated RNNs in terms of: i) the prediction performance itself, that is, the capacity of the network to minimize the desired function at hand in test data, and ii) speed of training, that is, how fast during training the network reaches its optimal performance. We examine bio-instantiated RNNs in working memory tasks where task-relevant information must be tracked as a sequence of events unfolds in time. We highlight the strategies that can be used to construct RNNs with the network topology found in BNNs, without sacrificing performance. Despite that we observe no enhancement of performance when compared to randomly wired RNNs, our approach demonstrates how empirical neural network data can be used for constructing RNNs, thus, facilitating further experimentation with biologically realistic network topologies, in contexts where such aspect is desired.

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Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. Whilst the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the N-terminal FinO domain and the variable C-terminal region required for ProQ activity. Two C-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. By contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of non-functional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria.

SIGNIFICANCEProteins that interact with RNA play a vital role in controlling key functions in pathogenic bacteria. RNA-binding proteins regulate how, when and where bacteria feed, swim or interact with a host, and it is critical that we understand how RNAs associate with these proteins. ProQ is one of the three major RNA-binding proteins (RBPs) in Gram-negative bacteria. In this study, we mapped the amino acid residues of ProQ that are essential for function. We successfully identified residue substitutions that rendered the ProQ RBP both non-functional and unable to interact with RNA. Our findings raise the possibility that the Lon protease mediates a quality control mechanism of ProQ that targets this RBP in the absence of RNA. A posttranslational quality control mechanism of this type could prevent the accumulation of nonfunctional RBPs in the bacterial cytoplasm.

sentences: Optimal information loading into working memory in prefrontal cortex Working memory involves the short-term maintenance of information and is critical in many tasks. The neural circuit dynamics underlying working memory remain poorly understood, with different aspects of prefrontal cortical (PFC) responses explained by different putative mechanisms. By mathematical analysis, numerical simulations, and using recordings from monkey PFC, we investigate a critical but hitherto ignored aspect of working memory dynamics: information loading. We find that, contrary to common assumptions, optimal information loading involves inputs that are largely orthogonal, rather than similar, to the persistent activities observed during memory maintenance. Using a novel, theoretically principled metric, we show that PFC exhibits the hallmarks of optimal information loading and we find that such dynamics emerge naturally as a dynamical strategy in task-optimized recurrent neural networks. Our theory unifies previous, seemingly conflicting theories of memory maintenance based on attractor or purely sequential dynamics, and reveals a normative principle underlying the widely observed phenomenon of dynamic coding in PFC.

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Importance of cell division angle, position of cell proliferative area, and localization of AN3 in lateral organ morphology Leaf meristem is a cell proliferative zone present in the lateral organ primordia. In this study, we investigated how the proliferative zone affects the final morphology of the lateral organs. We examined how cell proliferative zones differ in the primordia of planar floral organs and polar auxin transport inhibitor (PATI)-treated leaves from normal foliage leaf primordia of Arabidopsis thaliana with a focus on the spatial accumulation pattern of ANGUSTIFOLIA3 (AN3), a key element for leaf meristem positioning. We found that organ shape changes by PATI treatment were correlated to the angle of the cell division plane relative to the leaf primordia axis in the leaf meristem (cell division angle), but not with leaf-meristem positioning, size of the leaf meristem, or the localization pattern of AN3 protein. In contrast, different shapes between sepals and petals compared with foliage leaves were associated with both altered meristem position associated with altered AN3 expression patterns and different distributions of cell division angles. These results suggest that lateral organ shapes are regulated via two aspects: position of meristem and cell division angles

Summary statementDifferent lateral organs with different morphology possess different properties of meristems; cell division angles, position of cell proliferative area and AN3 localization patterns.

sentences: Wireless endovascular nerve stimulation with a millimeter-sized magnetoelectric implant Implanted bioelectronic devices have the potential to treat disorders that are resistant to traditional pharmacological therapies; however, reaching many therapeutic nerve targets requires invasive surgeries and implantation of centimeter-sized devices. Here we show that it is possible to stimulate peripheral nerves from within blood vessels using a millimeter-sized wireless implant. By directing the stimulating leads through the blood vessels we can target specific nerves that are difficult to reach with traditional surgeries. Furthermore, we demonstrate this endovascular nerve stimulation (EVNS) with a millimeter sized wireless stimulator that can be delivered minimally invasively through a percutaneous catheter which would significantly lower the barrier to entry for neuromodulatory treatment approaches because of the reduced risk. This miniaturization is achieved by using magnetoelectric materials to efficiently deliver data and power through tissue to a digitally-programmable 0.8 mm2 CMOS system-on-a-chip. As a proof-of-principle we show wireless stimulation of peripheral nerve targets both directly and from within the blood vessels in rodent and porcine models. The wireless EVNS concept described here provides a path toward minimally invasive bioelectronics where mm-sized implants combined with endovascular stimulation enable access to a number of nerve targets without open surgery or implantation of battery-powered pulse generators.

sentences: Biochemical characterization of actin assembly mechanisms with ALS-associated profilin variants Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for many neuronal cell processes through its regulation of lipids, nuclear signals, and cytoskeletal dynamics, including actin filament assembly. Direct interactions between profilin and actin monomers inhibit actin filament polymerization. In contrast, profilin can also stimulate polymerization by simultaneously binding actin monomers and proline-rich tracts found in other proteins. Whether the ALS-associated mutations in profilin compromise these actin assembly functions is unclear. We performed a quantitative biochemical comparison of the direct and formin-mediated impact for the eight ALS-associated profilin variants on actin assembly using classic protein-binding and single-filament microscopy assays. We determined that the binding constant of each profilin for actin monomers generally correlates with the actin nucleation strength associated with each ALS-related profilin. In the presence of formin, the A20T, R136W, Q139L, and C71G variants failed to activate the elongation phase of actin assembly. This diverse range of formin-activities is not fully explained through profilin-PLP interactions, as all ALS-associated variants bind a formin-derived PLP peptide with similar affinities. However, chemical denaturation experiments suggest that the folding stability of these profilins impact some of these effects on actin assembly. Thus, changes in profilin protein stability and alterations in actin filament polymerization may both contribute to the profilin-mediated actin disruptions in ALS.

sentences: Cryo-electron tomography of native Drosophila tissues vitrified by plunge freezing Cryo-focused ion beam (cryo-FIB) milling allows thinning vitrified cells for high resolution imaging by cryo-electron tomography (cryo-ET). However, it remains challenging to apply this workflow to tissues, as they usually require high-pressure freezing for vitrification. Here we show that dissected Drosophila tissues can be directly vitrified by plunge freezing upon a short incubation in 10% glycerol. This expedites subsequent cryo-FIB/ET, enabling systematic analyses of the molecular architecture of native tissues.

sentences: The Musashi proteins are post-transcriptional activators of protein expression and alternative exon splicing in vertebrate photoreceptors The Musashi proteins, MSI1 and MSI2, are conserved RNA binding proteins with a role in the maintenance and renewal of stem cells. Contrasting with this role, retina and terminally differentiated photoreceptor cells express high levels of MSI1 and MSI2, indicating that the two proteins have a role unrelated to maintaining undifferentiated cell state. Here we show that the Musashi proteins are essential in mature photoreceptors. Combined knockout of Msi1 and Msi2 lead to loss of the retina response to light and progressive photoreceptor cell death. The two proteins are fully redundant as individual deletion of Msi1 or Msi2 did not produce a phenotype. To define the molecular functions underlying the requirement for Musashi in photoreceptors, we delineated their RNA targets and analyzed the effect of the combined Msi1/Msi2 knockout on transcript levels, pre-mRNA splicing, and protein expressions. We show distinct nuclear and cytoplasmic functions for the Musashi proteins in photoreceptor cells. In the nucleus, Musashi binding to the downstream proximal intron promotes splicing of alternative exons. Surprisingly, four conserved photoreceptor-specific alternative exons in genes critical for vision proved to be dispensable, leaving open questions about the selective pressures that lead to the conservation of these exons and the contribution of alternative splicing to the phenotype of the Musashi knockout. In the photoreceptor cell cytoplasm MSI1 and MSI2 act as activators of protein expression. The combined knockout of Msi1 and Msi2 reduced the levels of multiple proteins including proteins required for vision and photoreceptor survival.

sentences: Visual Motion Coherence Responses in Human Visual Cortex 1Random dot kinematograms (RDKs) have recently been used to train subjects with cortical scotomas to perform direction of motion discrimination, partially restoring visual motion perception. To study recovery of visual perception, it is important to understand how visual areas in normal subjects and subjects with cortical scotomas respond to RDK stimuli. Studies in normal subjects have shown that Blood Oxygen Level Dependent (BOLD) responses in human area hV5/MT+ increase monotonically with coherence, in general agreement with electrophysiology studies in primates. However, RDK responses in prior studies were obtained while the subject was performing fixation, not a motion discrimination condition. Furthermore, BOLD responses were gauged against a baseline condition of uniform illumination or static dots, potentially decreasing the specificity of responses for the spatial integration of local motion signals (motion coherence). Here we revisit this question starting from a baseline RDK condition of no coherence, thereby isolating the component of BOLD response due specifically to the spatial integration of local motion signals. In agreement with prior studies, we found that responses in area hV5/MT+ of healthy subjects were monotonically increasing when subjects fixated without performing a motion discrimination task. In contrast, when subjects were performing an RDK direction of motion discrimination task, responses in area hV5/MT+ remained flat, changing minimally, if at all, as a function of motion coherence. A similar pattern of responses was seen in area hV5/MT+ of subjects with dense cortical scotomas performing direction of motion discrimination for RDKs presented inside the scotoma. Passive RDK presentation within the scotoma elicited no significant hV5/MT+ responses. These observations shed further light on how visual cortex responses behave as a function of motion coherence, helping to prepare the ground for future studies using these methods to study visual system recovery after injury.

sentences: Avian haemosporidians (Plasmodium and Haemoproteus) status in selected bird groups (Old world Flycatchers, Warblers, Babblers, and Thrushes) of India and their phylogenetic relationships with other lineages of the world The avian haemosporidians (Plasmodium and Haemoproteus) are widely distributed and may affect the host populations from body damage at individual level to the extinction of a population. The knowledge about their status may help in future avifauna conservation plans. Hence, we examined the avian haemosporidians status, in selected bird groups (Old world Flycatchers, Warblers, Babblers, and Thrushes) of India, and their phylogenetic relationships with other known lineages of the world. We used the common genetic marker (Cytochrome b gene fragment of 479 bp) with information on the geographic distribution of parasite and host species available at MalAvi database. The prevalence of avian haemosporidians, from northern, eastern, and southern parts of India and phylogenetic genetic analysis of lineages was carried out to know the genetic relatedness among them at local and world level. The MCC tree revealed six Haemosporidian lineages in which one was common (H_MSP01) and five were unique (H_CYOPOL01, H_CHD01, H_CYORUB01, H_EUMTHA01, and P_GEOCIT01). The avian host richness Index was 2.0852. 9.9%, prevalence of Haemosporidian infection was found in 111 DNA samples belonging to 6 host species. The Haemoproteus prevalence was found to be 9.0 % across five host species (Phylloscopus trochiloides, Cyornis poliogenys, C. hainanus dialilaemus, C. rubeculoides, Eumiyas thalassinus) and Plasmodium prevalence was 0.9% in Geokichla citrina. Spatial phylogeny at global level showed H_MSP01 lineage, found in different host species in India, was genetically related to H. pallidus lineages (COLL2 and PFC1) in parts of Africa, Europe, North America, Malaysia, and Philippines. The Plasmodium lineage (P_GEOCIT01) was related to PADOM16 in Egypt with poor sequence similarity (93.89%). The statistical analysis suggested that the haemosporidians host species distribution range was directly and significantly associated with the altitude, minimum temperature, and relative humidity. H_MSP01 distribution was in accordance with H. pallidus having a wide geographic and host range.

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SiCEP3, a C-terminally encoded peptides from Setaria italica, promotes ABA import and signaling pathway C-terminally encoded peptides (CEPs) are small peptides, typically post-translationally modified, and highly conserved in many species. CEPs are known to play roles in inhibition of plant growth and regulation of development, but the mechanisms are not well understood. In this study, we searched for CEP peptides in foxtail millet (Setaria italica). The 14 peptides we identified are divided into two subfamilies. The transcripts of most SiCEPs were more abundant in roots than in other tissues. SiCEP3, SiCEP4, and SiCEP5 were also expressed at high levels in panicles. Moreover, expression of all SiCEPs was induced by biotic stress and phytohormones. SiCEP3 overexpression and application of biosynthetic SiCEP3 both inhibited the growth of seedlings. In the presence of ABA, growth inhibition and ABA content of seedlings increased with the concentration of SiCEP3. Transcripts encoding two ABA transporters and one ABA receptor were induced by SiCEP3, ABA, and the two in combination. Further analysis revealed that SiCEP3 promoted ABA transport via NRT1.2 and ABCG40. In addition, SiCEP3, ABA, or the combination inhibited the kinase activities of CEP receptors CEPR1/2. Taken together, our results indicated that the CEP-CEPR module mediates ABA signaling by regulating ABCG40, NRT1.2, and PYL4 in planta.

HighlightSiCEP3, a C-terminally encoded peptide, can promote ABA import and signaling pathway by inhibiting the kinase activity of its receptors under abiotic stress in Setaria italica.

sentences: Sex-specific regulation of metabolic health and vertebrate lifespan by AMP biosynthesis Energy homeostasis is disrupted with age, which then fuels multiple age-related pathologies. The AMP-activated protein kinase (AMPK) is the primary sensor of cellular energy in eukaryotes. However, the genetic regulation of vertebrate aging by AMPK remains poorly understood. Here, we manipulate energy levels in the turquoise killifish by mutating APRT, a key enzyme in AMP biosynthesis. These manipulations produced a male-specific lifespan extension and restored metabolic plasticity. Exploring the observed sex differences using an integrated omics approach implicated the mitochondria as an important player. Mechanistically, APRT regulated mitochondrial functions and AMPK activity, mimicking energy starvation in heterozygous cells. A fasting-like state was also detected, particularly in heterozygous males, which leads to resistance to high-fat diet. Finally, life-long intermittent fasting eliminated the male-specific longevity benefits mediated by the APRT mutation. These observations identify the AMP/AMPK axis as a sex-specific regulator of vertebrate longevity and metabolic health.

sentences: Drosophila STING protein has a role in lipid metabolism. Stimulator of interferon genes (STING) plays an important role in innate immunity by controlling type I interferon response against invaded pathogens. In this work we describe a direct but previously unknown role of STING in lipid metabolism in Drosophila. Flies with STING deletion are sensitive to starvation and oxidative stress, have reduced lipid storage and downregulated expression of lipid metabolism genes. We found that Drosophila STING interacts with lipid synthesizing enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). ACC and FAS also interact with each other, indicating that all three proteins may be components of a large multi-enzyme complex. The deletion of Drosophila STING leads to disturbed ACC localization and decreased FAS enzyme activity. Together, our results demonstrate a direct role of STING in lipid metabolism in Drosophila.

sentences: Viral origin of eukaryotic type IIA DNA topoisomerases Type II DNA topoisomerases of the family A (Topo IIA) are present in all bacteria (DNA gyrase) and eukaryotes. In eukaryotes, they play a major role in transcription, DNA replication, chromosome segregation and modulation of chromosome architecture. The origin of eukaryotic Topo IIA remains mysterious since they are very divergent from their bacterial homologues and have no orthologues in Archaea. Interestingly, eukaryotic Topo IIA have close homologues in viruses of the phylum Nucleocytoviricota, an expansive assemblage of large and giant viruses formerly known as the nucleocytoplasmic large DNA viruses (NCLDV). Topo IIA are also encoded by some bacterioviruses of the class Caudoviricetes (tailed bacteriophages). To elucidate the origin of the eukaryotic Topo IIA, we performed in-depth phylogenetic analyses combining viral and cellular Topo IIA homologs. Topo IIA encoded by bacteria and eukaryotes form two monophyletic groups nested within Topo IIA encoded by Caudoviricetes and Nucleocytoviricota, respectively. Importantly, Nucleocytoviricota remained well separated from eukaryotes after removing both bacteria and Caudoviricetes from the dataset, indicating that the separation of Nucleocytoviricota and eukaryotes is probably not due to long branch attraction artefact. The topology of our tree suggests that the eukaryotic Topo IIA was probably acquired from an ancestral member of the Nucleocytoviricota of the class Megaviricetes, before the emergence of the last eukaryotic common ancestor (LECA). This result further highlights a key role of these viruses in eukaryogenesis and suggests that early proto-eukaryotes used a Topo IIB instead of a Topo IIA for solving their DNA topological problems.

sentences: Multiview Graph Learning for single-cell RNA sequencing data Characterizing the underlying topology of gene regulatory networks is one of the fundamental problems of systems biology. Ongoing developments in high throughput sequencing technologies has made it possible to capture the expression of thousands of genes at the single cell resolution. However, inherent cellular heterogeneity and high sparsity of the single cell datasets render void the application of regular Gaussian assumptions for constructing gene regulatory networks. Additionally, most algorithms aimed at single cell gene regulatory network reconstruction, estimate a single network ignoring group-level (cell-type) information present within the datasets. To better characterize single cell gene regulatory networks under different but related conditions we propose the joint estimation of multiple networks using multiview graph learning (mvGL). The proposed method is developed based on recent works in graph signal processing (GSP) for graph learning, where graph signals are assumed to be smooth over the unknown graph structure. Graphs corresponding to the different datasets are regularized to be similar to each other through a learned consensus graph. We further kernelize mvGL with the kernel selected to suit the structure of single cell data. An efficient algorithm based on prox-linear block coordinate descent is used to optimize mvGL. We study the performance of mvGL using synthetic data generated with a diverse set of parameters. We further show that mvGL successfully identifies well-established regulators in a mouse embryonic stem cell differentiation study and a cancer clinical study of medulloblastoma.

sentences: A Biosensor for Detection of Indole Metabolites The lack of diverse health-related biosensors limits the progress towards our understanding of how the microbiome metabolism impacts health. Microbially produced indole-3-aldehyde (I3A) has been associated with reducing inflammation in diseases such as ulcerative colitis by stimulating the aryl hydrocarbon receptor (AhR) pathway. We mined the protein database for gut microbiome metabolites sensors and developed a biosensor for I3A. We engineered E. coli embedded with a single plasmid carrying a chimeric two-component system that detects I3A. Our I3A receptor characterization identified residues that contribute to the sensors high specificity in a range of 0.1-10 {micro}M. The I3A biosensor opens the door to sensing indole metabolites produced at the host-microbe interface and provides new parts for synthetic biology applications.

sentences: Molecular and functional characterization of the Drosophila melanogaster conserved smORFome Short polypeptides encoded by small open reading frames (smORFs) are ubiquitously found in eukaryotic genomes and are important regulators of physiology, development, and mitochondrial processes. Here, we focus on a subset of 194 smORFs that are evolutionarily conserved between Drosophila melanogaster and humans. Many of these smORFs are conserved broadly in the bilaterian lineage, with [~]60 conserved in plants. Within these conserved smORFs, we observed remarkably heterogenous spatial and temporal expression patterns - indicating wide-spread tissue-specific and stage-specific mitochondrial architectures. In addition, an analysis of annotated functional domains revealed a predicted enrichment of smORF polypeptides localizing to mitochondria. We conducted an embryonic ribosome profiling experiment and applied a machine learning approach, finding support for translation of 82 of these smORFs during embryogenesis. We further embarked on functional characterization using CRISPR knockout/activation, RNAi knockdown, and cDNA overexpression, revealing diverse phenotypes. This study underscores the importance of identifying smORF function in disease and phenotypic diversity.

sentences: Deep selection shapes the intragenomic diversity of rRNA genes Ribosome biogenesis in eukaryotes is supported by hundreds of ribosomal RNA (rRNA) gene copies that are encoded in the ribosomal DNA (rDNA) and are thought to undergo frequent sequence homogenization by concerted evolution. Here, we present species-wide rDNA sequence analysis in Saccharomyces cerevisiae that challenges this model of evolution. We show that rDNA copies in yeast are far from homogeneous, both among and within isolates, and that rapid sequence homogenization is unlikely because many variants avoided fixation or elimination over evolutionary time. Instead, the diversity landscape across the rDNA shows clear functional stratification, suggesting that different copy-number thresholds for selection shape rDNA diversity. Notably, variants in the most highly conserved rDNA elements are sufficiently deleterious to exhibit signatures of purifying selection even when present in only 1% of rRNA gene copies. Our results portray a complex selection landscape that shapes rDNA diversity within a single species and reveal unexpectedly deep purifying selection of multi-copy genes.

sentences: Two parallel sRNA amplification cycles contribute to RNAi inheritance in C. elegans RNA-mediated interference (RNAi) is a conserved mechanism that uses small RNAs (sRNAs) to tune gene expression. In C. elegans, exposure to dsRNA induces the production of gene-specific sRNAs that are propagated to progeny not exposed to the dsRNA trigger. We present evidence that RNAi inheritance is mediated by two parallel sRNA amplification loops. The first loop, dependent on the nuclear Argonaute HRDE-1, targets nascent transcripts, and reduces but does not eliminate productive transcription at the locus. The second loop, dependent on the conserved helicase ZNFX-1, targets mature transcripts and concentrates them in perinuclear condensates (nuage). Each amplification loop generates a distinct class of sRNAs, with the ZNFX-1 loop responsible for the bulk of sRNA production on the region targeted by the trigger. By independently targeting nascent and mature transcripts, the HRDE-1 and ZNFX-1 loops ensure maximum silencing in progeny not exposed to the trigger.

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Comparison of library preparation and sequencing depths for direct sequencing of Bordetella pertussis positive samples Whooping cough, or pertussis, is a highly transmissible respiratory infection caused by Bordetella pertussis. Due to the high burden of pertussis, vaccine programmes were introduced internationally and in Australia since the 1950s. This has resulted in a significant decrease of pertussis infections. However, since the 1990s the number of pertussis notifications has increased considerably. Currently circulating B. pertussis strains differ in vaccine antigen composition compared to strains that circulated in the pre-vaccination era. These genetic differences are thought to contribute, in part, to the re-emergence of pertussis in Australia and around the world. Whole genome sequencing (WGS) can resolve minute differences in circulating strains and provides unparalleled resolution of vaccine antigens. This high-resolution snapshot can provide clues that enable more targeted public health interventions. However, pertussis is primarily diagnosed with culture-independent diagnostic assays which offer fast turnaround result times and reduced laboratory costs, eliminating the need to culture isolates. Current WGS methods require a cultured isolate, resulting in an absence of B. pertussis genome sequences in the post vaccination era. This scarcity has, in turn, limited understanding of currently circulating strains and respective vaccine antigen compositions.

Recent advancements of WGS technologies have allowed direct sequencing of clinical specimens without the need for a cultured isolate. However, recovering reliable sequence data from clinical samples of low bacterial load infections such as B. pertussis is a pressing challenge. We sought to increase the yield of B. pertussis sequences direct from a clinical sample by evaluating widely available WGS library preparation methods.

We report that the Illumina DNA prep library preparation kit combined with deep sequencing allowed the detection of important surveillance information such as allelic variations in the B. pertussis vaccine antigens. Further, our method generates high coverage over the 23S ribosomal RNA of B. pertussis enabling macrolide resistance to be easily determined. Overall, this method can improve surveillance of B. pertussis, by monitoring changes in vaccine antigens, detecting antimicrobial resistance and guiding Public Health control interventions.

sentences: FUCCI-based live imaging platform reveals cell cycle dynamics and identifies pro-proliferative compounds in human iPSC-derived cardiomyocytes One of the major goals in cardiac regeneration research is to replace lost ventricular tissue with new cardiomyocytes. However, cardiomyocyte proliferation drops to low levels in neonatal hearts and is no longer efficient in compensating for the loss of functional myocardium in heart disease. We generated a human induced pluripotent stem cell (iPSC)-derived cardiomyocyte-specific cell cycle indicator system (TNNT2-FUCCI) to characterize regular and aberrant cardiomyocyte cycle dynamics. We visualized cell cycle progression in TNNT2-FUCCI and found G2 cycle arrest in endoreplicating cardiomyocytes. Moreover, we devised a live-cell compound screening platform to identify pro-proliferative drug candidates. We found that the alpha-adrenergic receptor agonist clonidine induced cardiomyocyte proliferation in vitro and increased cardiomyocyte cell cycle entry in neonatal mice. In conclusion, the TNNT2-FUCCI system is a valuable tool to characterize cardiomyocyte cell cycle dynamics and identify pro-proliferative candidates with regenerative potential in the mammalian heart.

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Preparation of ingestible antibodies to neutralize the binding of SarsCoV2 RBD (receptor binding domain) to human ACE2 Receptor COVID19 continues to be a serious threat to human health and mortality. There is dire need for new solutions to combat this pandemic especially for those individuals who are not vaccinated or unable to be vaccinated and continue to be exposed to the SARSCoV2. In addition, the emergence of new more transmissible variants such as delta pose additional threat from this virus.

To explore another solution for prevention and treatment of COVID 19, we have produced chicken egg derived IgY antibodies against the Receptor binding domain (RBD) of SARSCoV2 spike protein which is involved in binding to human cell ACE2 receptors. The - RBD IgY effectively neutralize the binding of RBD to ACE2 and prevent pseudovirus entry in a PRNT assay. Importantly our anti-RBD IgY also neutralize the binding of Sars CoV2 delta variant RBD to ACE2. Given that chicken egg derived IgY are safe and permissible for human consumption, we plan to develop these ingestible antibodies for prevention of viral entry in the oropharyngeal and digestive tract in humans as passive immunotherapy.

sentences: Using Obviously-Related Instrumental Variables to Increase the Predictive Power of Polygenic Scores The conventional way of boosting the predictive power of polygenic scores is to increase the GWAS sample size by meta-analyzing GWAS results of multiple cohorts. In this paper, we challenge this convention. Through simulations, we show that Instrumental Variable (IV) regression using two polygenic scores constructed from independent GWAS summary statistics outperforms the typical Ordinary Least Squares (OLS) model employing a single meta-analysis based polygenic score in terms of bias, root mean squared error, and statistical power. We verify the empirical validity of the simulations by predicting educational attainment (EA) and height in a sample of siblings from the UK Biobank. We show that IV regression between-families approaches the SNP-based heritability, and improves the predictive power of polygenic scores by 12% (height) to 22% (EA). Furthermore, IV regression within-families provides the tightest lower bound for the direct genetic effect, increasing the lower bound for EA from 2.0% to 3.4%, and for height from 28.9% to 37.7%.

sentences: Data-driven models of optimal chromatic coding in the outer retina The functional properties of the outermost retinal circuits involved in color discrimination are not well understood. Recent experimental work on zebrafish has elucidated the in-vivo activity of photoreceptors and horizontal cells as a function of the stimulus spectrum, highlighting the appearance of chromatic-opponent signals at the first synaptic connection between cones and horizontal cells. These findings, together with the observed lack of gap junctions, suggest that the mechanism yielding early color-opponency in zebrafish is dominated by inhibitory feedback. We discuss the observed neuronal activity in the context of efficient codification of chromatic information, hypothesizing that opponent chromatic signals provide optimal codification, minimizing signal redundancy. We examine whether these functional properties are general across species by studying the dynamic properties of dichromatic and trichromatic outer retinal networks. Our findings show that dominant inhibitory feedback mechanisms provide an unambiguous codification of chromatic stimuli, whereas this property is not guaranteed in networks with strong excitatory inter-cone connections, for example via gap junctions. This provides a plausible explanation for the absence of gap junctions observed in the outermost zebrafish retinal layers. In addition, our study suggests that the simplest zebrafish-like network with dominant inhibitory feedback capable of optimally codifying chromatic information requires at least two successive inhibitory feedback layers. Finally, we contrast the chromatic codification performance of zebrafish-inspired retinal networks with networks having different opsin combinations. We find that optimal combinations lead to a chromatic codification improvement of only 13% compared with zebrafish opsins, suggesting that the zebrafish retina performs nearly optimal codification of chromatic information in its habitat.

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Identification of shared bacterial strains in the vaginal microbiota of reproductive-age mothers and daughters using genome-resolved metagenomics It has been suggested that the human microbiome might be vertically transmitted from mother to offspring and that early colonizers may play a critical role in development of the immune system. Studies have shown limited support for the vertical transmission of the intestinal microbiota but the derivation of the vaginal microbiota remains largely unknown. Although the vaginal microbiota of children and reproductive age cis women differ in composition, the vaginal microbiota could be vertically transmitted. To determine whether there was any support for this hypothesis, we examined the vaginal microbiota of daughter-mother pairs from the Baltimore metropolitan area (ages 14-27, 32-51; n=39). We assessed whether the daughters microbiota was similar in composition to their mothers using metataxonomics. Permutation tests revealed that while some pairs did have similar vaginal microbiota, the degree of similarity did not exceed that expected by chance. Genome-resolved metagenomics was used to identify shared bacterial strains in a subset of the families (n=22). We found a small number of bacterial strains that were shared between mother-daughter pairs but identified more shared strains between individuals from different families, indicating that vaginal bacteria may display biogeographic patterns. Earlier-in-life studies are needed to demonstrate vertical transmission of the vaginal microbiota.

ImportanceEarly colonizers of our microbiota are theorized to play an important role in the development of our immune system, yet we know little about how these communities are established. Vertical transmission from mother to offspring at the time of birth is theorized to be a major source of early colonizers but limited evidence supporting this process has only been shown for the intestinal tract microbiota. The provenance of the vaginal microbiota is largely unknown, although some have posited it is similarly vertically transmitted. We examined the vaginal microbiota of mother-daughter pairs and found limited evidence in support of this hypothesis. However, our analysis also revealed putative biogeographic patterns in the distribution of the strains which comprise the vaginal microbiota. Our results give insight into the role of vertical transmission for the vaginal microbiota and motivate future studies on the biogeography of these bacteria.

sentences: Neuronal ROS-Induced Glial Lipid Droplet Formation is Altered by Loss of Alzheimers Disease-associated Genes A growing list of Alzheimers disease (AD) genetic risk factors is being identified, but the contribution of these genetic mutations to disease remains largely unknown. Accumulating data support a role of lipid dysregulation and excessive ROS in the etiology of AD. Here, we identified cell-specific roles for eight AD risk-associated genes in ROS-induced glial lipid droplet (LD) formation. We demonstrate that ROS-induced glial LD formation requires two ABCA transporters (ABCA1 and ABCA7) in neurons, the APOE receptor (LRP1), endocytic genes (PICALM, CD2AP, and AP2A2) in glia, and retromer genes (VPS26 and VPS35) in both neurons and glia. Moreover, ROS strongly enhances A{beta}42-toxicity in flies and A{beta}42-plaque formation in mice. Finally, an ABCA1-activating peptide restores glial LD formation in the APOE4-associated loss of LD. This study places AD risk factors in a neuron-to-glia lipid transfer pathway with a critical role in protecting neurons from ROS-induced toxicity.

sentences: Evaluation of FRET X for Single-Molecule Protein Fingerprinting Single-molecule protein identification is a novel, as of yet unrealized concept with potentially groundbreaking applications in biological research. We propose a method called FRET X (Forster Resonance Energy Transfer via DNA eXchange) fingerprinting, in which the FRET efficiency is read out between exchangeable dyes on protein-bound DNA docking strands, and accumulated FRET efficiency values constitute the fingerprint for a protein. To evaluate the feasibility of this approach, we simulated fingerprints for hundreds of proteins using a coarse-grained lattice model and experimentally demonstrated FRET X fingerprinting on a system of model peptides. Measured fingerprints are in agreement with our simulations, corroborating the validity of our modeling approach. In a simulated complex mixture of >300 human proteins of which only cysteines, lysines and arginines were labeled, a support vector machine was able to identify constituents with 95% accuracy. We anticipate that our FRET X fingerprinting approach will form the basis of an analysis tool for targeted proteomics.

sentences: Chemical Reversible Crosslinking Enables Measurement of RNA 3D Distances and Alternative Conformations in Cells Three-dimensional (3D) structures dictate the functions of RNA molecules in a wide variety of biological processes. However, direct determination of RNA 3D structures in vivo is difficult due to their large sizes, conformational heterogeneity, and dynamics. Here we present a new method, Spatial 2-Hydroxyl Acylation Reversible Crosslinking (SHARC), which uses chemical crosslinkers of defined lengths to measure distances between nucleotides in cellular RNA. Integrating crosslinking, exonuclease (exo) trimming, proximity ligation, and high throughput sequencing, SHARC enables transcriptome-wide tertiary structure contact maps at high accuracy and precision, revealing heterogeneous RNA structures and interactions. SHARC data provide constraints that improves Rosetta-based RNA 3D structure modeling at near-nanometer resolution. Integrating SHARC-exo with other crosslinking-based methods, we discover compact folding of the 7SK RNA, a critical regulator of transcriptional elongation. These results establish a new strategy for measuring RNA 3D distances and alternative conformations in their native cellular context.

sentences: Estimating the maximal growth rates of eukaryotic microbes from cultures and metagenomes via codon usage patterns Microbial eukaryotes are ubiquitous in the environment and play important roles in key ecosystem processes, including accounting for a significant portion of global primary production. Yet, our tools for assessing the functional capabilities of eukaryotic microbes in the environment are quite limited because many microbes have yet to be grown in culture. Maximum growth rate is a fundamental parameter of microbial lifestyle that reveals important information about an organisms functional role in a community. We developed and validated a genomic estimator of maximum growth rate for eukaryotic microbes, enabling the assessment of growth potential for both cultivated and yet-to-be-cultivated organisms. We produced a database of over 700 growth predictions from genomes, transcriptomes, and metagenome-assembled genomes, and found that closely related and/or functionally similar organisms tended to have similar maximal growth rates. By comparing the maximal growth rates of existing culture collections with environmentally-derived genomes we found that, unlike for prokaryotes, culture collections of microbial eukaryotes are only minimally biased in terms of growth potential. We then extended our tool to make community-wide estimates of growth potential from over 500 marine metagenomes, mapping growth potential across the global oceans. We found that prokaryotic and eukaryotic communities have highly correlated growth potentials near the ocean surface, but that this relationship disappears deeper in the water column. This suggests that fast growing eukaryotes and prokaryotes thrive under similar conditions at the ocean surface, but that there is a decoupling of these communities as resources become scarce deeper in the water column.

sentences: Increased reliability of visually-evoked activity in area V1 of the MECP2-duplication mouse model of autism Atypical sensory processing is now thought to be a core feature of the autism spectrum. Influential theories have proposed that both increased and decreased neural response reliability within sensory systems could underlie altered sensory processing in autism. Here, we report evidence for abnormally increased reliability of visual-evoked responses in layer 2/3 neurons of adult primary visual cortex in the MECP2-duplication syndrome animal model of autism. Increased response reliability was due in part to decreased response amplitude, decreased fluctuations in endogenous activity, and decreased neuronal coupling to endogenous activity. Similarly to what was observed neuronally, the optokinetic reflex occurred more reliably at low contrasts in mutant mice compared to controls. Retinal responses did not explain our observations. These data suggest that the circuit mechanisms for convolution of sensory-evoked and endogenous signal and noise may be altered in this form of syndromic autism.

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Accuracy of the pneumatic method for estimating xylem vulnerability to embolism in temperate diffuse-porous tree species O_LIThe increasing frequency of global change-type droughts has created a need for fast, accurate and widely applicable techniques for estimating xylem embolism resistance to improve forecasts of future forest changes.
C_LIO_LIWe used data from 12 diffuse-porous temperate tree species covering a wide range of xylem safety to compare the pneumatic and flow-centrifuge method for constructing xylem vulnerability curves. We evaluated the agreement between parameters estimated with both methods and the sensitivity of pneumatic measurements to the measurement duration.
C_LIO_LIThe agreement between xylem water potentials at 50% air discharged (PAD) estimated with the Pneumatron and 50% loss of hydraulic conductivity (PLC) estimated with the flow-centrifuge method was high (mean signed deviation: 0.12 MPa, Pearson correlation: 0.96 after 15 sec of gas extraction). However, the relation between the estimated slopes was more variable, resulting in lower agreement in xylem water potential at 12% and 88% PAD/PLC. All parameters were sensitive to the duration of the pneumatic measurement, with highest overall agreement between methods after 16 sec.
C_LIO_LIWe conclude that, if applied correctly, the pneumatic method enables fast and inexpensive estimations of embolism resistance for a wide range of temperate, diffuse-porous species, which makes it attractive for predicting plant performance under climate change.
C_LI

sentences: SINBAD: a flexible tool for single cell DNA methylation data DNA methylation is an epigenetic mark that has vital importance in both development and disease. Single cell bisulfite sequencing technologies enable profiling of the methylome at high resolution, providing the basis for dissecting the heterogeneity and dynamics of DNA methylation in complex tissues and over time. Despite the rapid increase in the number of experimental protocols for methylome sequencing, analytical tools designed specifically for single-cell data are lacking. We developed a computational tool, SINBAD, for efficient and standardized pre-processing, quality assessment and analysis of single cell methylation data. Starting from multiplexed sequencing reads, major analysis modules of SINBAD include preprocessing, read mapping, methylation quantification, multivariate analysis, and gene signature profiling. SINBAD provides a flexible platform to implement interoperable and robust processing of single-cell methylome data.

sentences: Dynamic integration and segregation of amygdala subregional functional circuits linking to physiological arousal The dynamical organization of brain networks is essential to support human cognition and emotion for rapid adaption to ever-changing environment. As the core nodes of emotion-related brain circuitry, the basolateral amygdala (BLA) and centromedial amygdala (CMA) as two major amygdalar nuclei, are recognized to play distinct roles in affective functions and internal states, via their unique connections with cortical and subcortical structures in rodents. However, little is known how the dynamical organization of emotion-related brain circuitry reflects internal autonomic responses in humans. Using resting-state functional magnetic resonance imaging (fMRI) with K-means clustering approach in a total of 79 young healthy individuals (cohort 1: 42; cohort 2: 37), we identified two distinct states of BLA- and CMA-based intrinsic connectivity patterns, with one state (integration) showing generally stronger BLA- and CMA-based intrinsic connectivity with multiple brain networks, while the other (segregation) exhibiting weaker yet dissociable connectivity patterns. In an independent cohort 2 of fMRI data with concurrent recording of skin conductance, we replicated two similar dynamic states and further found higher skin conductance level in the integration than segregation state. Moreover, machine learning-based Elastic-net regression analyses revealed that time-varying BLA and CMA intrinsic connectivity with distinct network configurations yield higher predictive values for spontaneous fluctuations of skin conductance level in the integration than segregation state. Our findings highlight dynamic functional organization of emotion-related amygdala nuclei circuits and networks and its links to spontaneous autonomic arousal in humans.

sentences: On the evolution of host specificity: a case study of helminths The significant variation in host specificity exhibited by parasites has been separately linked to evolutionary history and ecological factors in specific host-parasite associations. Yet, whether there are any general patterns in the factors that shape host specificity across parasites more broadly is unknown. Here we constructed a molecular phylogeny for 249 helminth species infecting free-range mammals and find that the influence of ecological factors and evolutionary history varies across different measures of host specificity. Whereas the phylogenetic range of hosts a parasite can infect shows a strong signal of evolutionary constraint, the number of hosts a parasite infects does not. Our results shed new light on the evolution of host specificity in parasites, suggesting that phylogenetic breadth may capture the evolutionary potential of a parasite to jump between hosts, whereas the number of hosts may reflect ecological opportunity. Finally, we show parasite phylogenies can also provide an alternative perspective on zoonosis by identifying which hosts are infected by a broad phylogenetic range of parasites.

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Augmentation of extracellular ATP synergizes with chemotherapy in triple negative breast cancer IntroductionBreast cancer affects two million women worldwide every year and is the most common cause of cancer-related death among women. The triple-negative breast cancer (TNBC) sub-type is associated with an especially poor prognosis because currently available therapies, fail to induce long-lasting responses. Therefore, there is an urgent need to develop novel therapies that result in durable responses. One universal characteristic of the tumor microenvironment is a markedly elevated concentration of extracellular adenosine triphosphate (eATP). Chemotherapy exposure results in further increases in eATP through its release into the extracellular space of cancer cells via P2RX channels. eATP levels are reduced by eATPases. Given that high concentrations of eATP are cytotoxic, we hypothesized that augmenting the release of eATP through P2RX channels and inhibiting extracellular ATPases would sensitize TNBC cells to chemotherapy.

MethodsTNBC cell lines MDA-MB 231, Hs 578t and MDA-MB 468 and non-tumorigenic immortalized mammary epithelial MCF-10A cells were treated with increasing concentrations the chemotherapeutic agent paclitaxel in the presence of eATPase inhibitors, specific agonists or antagonists of P2RXs with cell viability and eATP content being measured. Additionally, the mRNA, protein and cell surface expressions of the purinergic receptors P2RX4 and P2RX7 were evaluated in all examined cell lines via qRT-PCR, western blot, and flow cytometry analyses, respectively.

ResultsIn the present study, we observed dose-dependent declines in cell viability and increases in eATP in paclitaxel-treated TNBC cell lines in the presence of inhibitors of eATPases. These effects were reversed by specific antagonists of P2RXs. Similar results were observed with P2RX activators. All examined cell lines expressed both P2RX4 and P2RX7 at the mRNA, protein and cell surface levels.

ConclusionThese results reveal that eATP modulates the chemotherapeutic response in TNBC cell lines which could be exploited to enhance the efficacy of chemotherapy regimens for TNBC.

sentences: Evaluation of methods for estimating coalescence times using ancestral recombination graphs The ancestral recombination graph (ARG) is a structure that describes the joint genealogies of sampled DNA sequences along the genome. Recent computational methods have made impressive progress towards scalably estimating whole-genome genealogies. In addition to inferring the ARG, some of these methods can also provide ARGs sampled from a defined posterior distribution. Obtaining good samples of ARGs is crucial for quantifying statistical uncertainty and for estimating population genetic parameters such as effective population size, mutation rate, and allele age. Here, we use standard neutral coalescent simulations to benchmark the estimates of pairwise coalescence times from three popular ARG inference programs: ARGweaver, Relate, and tsinfer+tsdate. We compare 1) the true coalescence times to the inferred times at each locus; 2) the distribution of coalescence times across all loci to the expected exponential distribution; 3) whether the sampled coalescence times have the properties expected of a valid posterior distribution. We find that inferred coalescence times at each locus are most accurate in ARGweaver, and often more accurate in Relate than in tsinfer+tsdate. However, all three methods tend to overestimate small coalescence times and underestimate large ones. Lastly, the posterior distribution of ARGweaver is closer to the expected posterior distribution than Relates, but this higher accuracy comes at a substantial trade-off in scalability. The best choice of method will depend on the number and length of input sequences and on the goal of downstream analyses, and we provide guidelines for the best practices.

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Determining the optimal SARS-CoV-2 mRNA vaccine dosing interval for maximum immunogenicity ObjectiveEmerging evidence indicates that longer SARS-CoV-2 vaccine dosing intervals results in an enhanced immune response. However, the optimal vaccine dosing interval for achieving maximum immunogenicity is unclear.

MethodsThis study included samples from adult paramedics in Canada who received two doses of either BNT162b2 or mRNA-1273 vaccines and provided blood samples 6 months (170 to 190 days) after the first vaccine dose. The main exposure variable was vaccine dosing interval (days), categorized as "short" (first quartile), "moderate" (second quartile), "long" (third quartile), and "longest" interval (fourth quartile). The primary outcome was total spike antibody concentrations, measured using the Elecsys SARS-CoV-2 total antibody assay. Secondary outcomes included: spike and RBD IgG antibody concentrations, and inhibition of angiotensin-converting enzyme 2 (ACE-2) binding to wild-type spike protein and several different Delta variant spike proteins. We fit a multiple log-linear regression model to investigate the association between vaccine dosing intervals and the antibody concentrations.

ResultsA total of 564 adult paramedics (mean age 40 years, SD=10) were included. Compared to "short interval" ([≤]30 days), higher dosing interval quartiles (moderate: 31-38 days; long: 39-73 days and longest: [≥]74 days) were all associated with increased Elescys spike total antibody concentration. Compared to the short interval, "long" and "longest" interval quartiles were associated with higher spike and RBD IgG antibody concentrations. Similarly, increasing dosing intervals increased inhibition of ACE-2 binding to viral spike protein, regardless of the vaccine type.

ConclusionIncreased mRNA vaccine dosing intervals longer than 30 days result in higher levels of circulating antibodies and viral neutralization when assessed at 6 months.

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Phototroph-heterotroph interactions during growth and long-term starvation across Prochlorococcus and Alteromonas diversity Microbial interactions such as those between phytoplankton and bacteria been studied intensively using specific model organisms, due to their potential impact on ecosystems and biogeochemistry. Yet, to what extent interactions differ between closely related organisms, or how these interactions change over time or culture conditions, remains unclear. Here, we characterize the interactions between five strains each of two globally abundant marine microorganisms, Prochlorococcus (a phototroph) and Alteromonas (a heterotroph), from the first encounter between individual strains and over more than a year of repeated cycles of exponential growth and long-term nitrogen starvation. Prochlorococcus-Alteromonas interactions had little effect on traditional growth parameters such as Prochlorococcus growth rate, maximal fluorescence or lag phase, affecting primarily the dynamics of culture decline, which we interpret as representing cell mortality and lysis. The shape of the Prochlorococcus decline curve and the carrying capacity of the co-cultures were determined by the phototroph and not the heterotroph strains involved. Comparing various mathematical models of culture mortality suggests that Prochlorococcus death rate increases over time in mono-cultures but decreases in co-cultures, with cells potentially becoming more resistant to stress. Our results demonstrate intra-species differences in ecologically-relevant co-culture outcomes. These include the recycling efficiency of N and whether the interactions are mutually synergistic or competitive. They also highlight the information-rich growth and death curves as a useful readout of the interaction phenotype.

Significance StatementInteractions between phytoplankton and marine bacteria impact global ecosystems and biogeochemistry. Here, we explore how intra-species variability affects the interactions between Prochlorococcus, a globally abundant photosynthetic cyanobacetrium and Alteromonas, a heterotrophic bacterium that lives off and recycles organic matter. Under nitrogen starvation, Prochlorococcus growing alone increasingly accumulate damage and die, whereas in co-culture with Alteromonas they become increasingly resilient. The specific Prochlorococcus strain had a much larger effect on co-culture behavior than the Alteromonas strain, determining whether the interactions are mutually synergistic or potentially competitive. These results show how ecologically relevant outcomes of interactions may vary between closely related microorganisms, and highlight growth and death curves from laboratory (co)-cultures as information-rich views of microbial growth and death.

sentences: Genome assembly of the Australian black tiger shrimp (Penaeus monodon) reveals a fragmented IHHNV EVE sequence Shrimp are a valuable aquaculture species globally; however, disease remains a major hindrance to shrimp aquaculture sustainability and growth. Mechanisms mediated by endogenous viral elements (EVEs) have been proposed as a means by which shrimp that encounter a new virus start to accommodate rather than succumb to infection over time. However, evidence on the nature of such EVEs and how they mediate viral accommodation is limited. More extensive genomic data on Penaeid shrimp from different geographical locations should assist in exposing the diversity of EVEs. In this context, reported here is a PacBio Sequel-based draft genome assembly of an Australian black tiger shrimp (Penaeus monodon) inbred for one generation. The 1.89 Gbp draft genome is comprised of 31,922 scaffolds (N50: 496,398 bp) covering 85.9% of the projected genome size. The genome repeat content (61.8% with 30% representing simple sequence repeats) is almost the highest identified for any species. The functional annotation identified 35,517 gene models, of which 25,809 were protein-coding and 17,158 were annotated using interproscan. Scaffold scanning for specific EVEs identified an element comprised of a 9,045 bp stretch of repeated, inverted and jumbled genome fragments of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) bounded by a repeated 591/590 bp host sequence. As only near complete linear ~4 kb IHHNV genomes have been found integrated in the genome of P. monodon previously, its discovery has implications regarding the validity of PCR tests designed to specifically detect such linear EVE types. The existence of joined inverted IHHNV genome fragments also provides a means by which hairpin dsRNAs could be expressed and processed by the shrimp RNA interference (RNAi) machinery.

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AI-driven high-throughput automation of behavioral analysis and dual-channel wireless optogenetics for multiplexing brain dynamics Advances in behavioral optogenetics are limited by the absence of high-throughput pipelines for the automated analysis of behavior in freely behaving animals. Although a variety of AI algorithms has been proposed that enable automation of behavioral analysis, existing methods are generally low-throughput. In addition, optogenetic manipulation of neural circuits typically requires physical tethers to light sources, which limits the number of brain areas that can be targeted and thus constrains behavioral testing. Here, we develop a new wireless platform that combines a battery-free dual-channel optogenetic implant with an AI algorithm for high-throughput behavioral analysis. In our platform, a customized AI algorithm detected and quantified freezing behavior of rats that had undergone fear conditioning. Notably, our platform allows up to four enclosures to be monitored simultaneously. Wireless dual-channel optogenetic devices were implanted in the basolateral amygdala (BLA) to permit independent modulation of BLA principal neurons (red light, AAV-CaMKII-JAWS) or BLA interneurons (blue light, AAV-mDlx-ChR2) in freely behaving animals. In vivo validation with behaving rats demonstrates the utility of the telemetry system for large-scale optogenetic studies.

SignificanceAI algorithms can enable automation of behavioral analysis and thereby facilitate the progress on behavioral optogenetics. Successful integration of advanced wireless dual-channel optoelectronic devices with biological systems can also yield new tools and techniques for neuroscience research, particularly in the context of techniques for optogenetics. Here, we propose a new approach that combines an advanced AI algorithm with a low power wireless telemetry system, yielding powerful capabilities in the understanding of brain functions and the evaluation of the behavioral consequences of neural circuit manipulations. In vivo studies using optimized systems demonstrate high-throughput automation of behavioral manipulation and analysis via AI-powered wireless telemetry.

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Prophages in the infant gut are largely induced, and may be functionally relevant to their hosts. BackgroundBacteriophages are the most abundant biological entity on the planet, and are key components of any ecosystem they are present in. The gut virome is increasingly being implicated in disease states although these studies largely focus on lytic phages in adults. Here we identify prophages from a large infant cohort and investigate their potential functions.

ResultsWe identified 10645 vOTUs from 662 metagenomes. No core virome was found: the most prevalent vOTU was identified in 70% of the samples. The most abundant and prevalent group of phages are a novel group closely related to Bacteroides phage Hanky p00. Functional annotation of this group revealed the presence of genes in the dDTP-L-rhamnose pathway, possibly involved in the production of capsular polysaccharides. We also found an abundance of diversity generating retroelements in the phages. Additionally, paired virome data allowed us to show that the majority of prophages are induced in at least one sample and that this is not affected by the use of antibiotics in the 4 weeks prior to sampling.

ConclusionsProphages in the infant gut are largely unique to the individual and not shared. Most of them appear to be induced and so may be key drivers in shaping the bacterial microbiome. The most abundant group of phages are novel, and possess elements that may allow them to maintain differentially susceptible subpopulations of their host bacterium; whilst also containing diversity generating retroelements that could expand their host range. Prophages are important components of the infant gut that may have far reaching influences on the composition and function of the microbiome.

sentences: Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing1-3. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary4,5. Chemical labeling-based single-cell approaches introduce hundreds of cells into the MS, but direct analysis of single cells has not yet reached the necessary sensitivity, robustness and quantitative accuracy to answer biological questions6,7. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography and a novel trapped ion mobility mass spectrometer, resulting in a more than ten-fold improved sensitivity. We accurately and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators such as CDK2NA, the E2 ubiquitin ligase UBE2S, DNA topoisomerases TOP2A/B and the chromatin regulator HMGA1. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable core proteome despite perturbation, while the transcriptome appears volatile. This emphasizes substantial regulation of translation and sets the stage for its elucidation at the single cell level. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material8, posttranslational modifications and small molecule studies to gain unprecedented insights into cellular heterogeneity in health and disease.

sentences: Revealing the secrets beneath grapevine and Plasmopara viticola early communication: a picture of host and pathogen proteomes Plant apoplast is the first hub of plant-pathogen communication where pathogen effectors are recognized by plant defensive proteins and cell receptors and several signal transduction pathways are activated. As a result of this first contact, the host triggers a defence response that involves the modulation of several extra and intracellular proteins. In grapevine-pathogen interactions, little is known about the communication between cells and apoplast. Also, the role of apoplastic proteins in response to pathogens still remains a blackbox. In this study we focused on the first 6 hours after Plasmopara viticola inoculation to evaluate grapevine proteome modulation in the apoplastic fluid (APF) and whole leaf tissue. Plasmopara viticola proteome was also assessed enabling a deeper understanding of plant and pathogen communication. Our results showed that oomycete recognition, plant cell wall modifications, ROS signalling and disruption of oomycete structures are triggered in Regent after P. viticola inoculation. Our results highlight a strict relation between the apoplastic pathways modulated and the proteins identified in the whole leaf proteome. On the other hand, P. viticola proteins related to growth/morphogenesis and virulence mechanisms were the most predominant. This pioneer study highlights the early dynamics of extra and intracellular communication in grapevine defence activation that leads to the successful establishment of an incompatible interaction.

sentences: Unaffected functional recovery after spinal cord contusions at different circadian times The circadian rhythms of gene expression drive diurnal oscillations of physiological processes that determine the acute injury response including immunity, inflammation and hemostasis. While outcomes of various acute injuries are affected by the time of day at which the original insult occurred, such diurnal influences on recovery after spinal cord injury (SCI) are unknown. We report that several key regulators of circadian gene expression are differentially expressed in uninjured spinal cord tissue of naive mice at Zeitgeber time 1 (ZT1) or ZT12, where ZT0 or ZT12 are times when lights are turned on or off, respectively. However, mice that received moderate, T9 contusive SCI at ZT0 or ZT12 showed similar recovery of locomotion as determined using the ladder walking test and the Basso mouse scale (BMS) over a 6 week post-injury period. Consistent with those findings, terminal histological analysis revealed no significant differences in white matter sparing at the injury epicenter. Therefore, locomotor recovery after thoracic contusive SCI is not affected by the time of day at which the neurotrauma occurred at least when comparing the beginning to the end of the mouse active period.

sentences: VIP interneurons in mouse whisker S1 exhibit sensory and action-related signals during goal-directed behavior Vasoactive intestinal peptide-expressing (VIP) interneurons, which constitute 10-15% of the cortical inhibitory neuron population1,2, have emerged as an important cell type for regulating excitatory cell activity based on behavioral state. VIP cells in sensory cortex are potently engaged by neuromodulatory and motor inputs during active exploratory behaviors like locomotion and whisking, which in turn promote pyramidal cell firing via disinhibition3-5. Such state-dependent modulation of activity by VIP cells in sensory cortex has been studied widely in recent years. However, the function of VIP cells during goal-directed behavior is less well understood. It is not clear how task-related events like sensory stimuli, motor actions, or reward activate VIP cells in sensory cortex since there is often temporal overlap in the occurrence of these events. We developed a Go/NoGo whisker touch detection task which incorporates a post-stimulus delay period to separate sensory-driven activity from action- or reward-related activity during behavior. We used 2-photon calcium imaging to measure task-related signals of L2/3 VIP neurons in S1 of behaving mice. We report for the first time that VIP cells in mouse whisker S1 are activated by both whisker stimuli and goal-directed licking. Whisker- and lick-related signals were spatially organized in relation to anatomical columns in S1. Sensory responses of VIP cells were tuned to specific whiskers, whether or not they also displayed lick-related activity.

sentences: Quantitative MRI maps of human neocortex explored using cell type-specific gene expression analysis Quantitative MRI (qMRI) allows extraction of reproducible and robust parameter maps. However, the connection to underlying biological substrates remains murky, especially in the complex, densely packed cortex. We investigated associations in human neocortex between qMRI parameters and neocortical cell types by comparing the spatial distribution of the qMRI parameters longitudinal relaxation rate (R1), effective transverse relaxation rate (R2*), and magnetization transfer saturation (MTsat) to gene expression from the Allen Human Brain Atlas, then combining this with lists of genes enriched in specific cell types found in the human brain. As qMRI parameters are magnetic field strength-dependent, the analysis was performed on MRI data at 3T and 7T. All qMRI parameters significantly covaried with genes enriched in GABA- and glutamatergic neurons, i.e. they were associated with cytoarchitecture. The qMRI parameters also significantly covaried with the distribution of genes enriched in astrocytes (R2* at 3T, R1 at 7T), endothelial cells (R1 and MTsat at 3T), microglia (R1 and MTsat at 3T, R1 at 7T), and oligodendrocytes (R1 at 7T). These results advance the potential use of qMRI parameters as biomarkers for specific cell types.

sentences: The single-cell epigenetic regulatory landscape in mammalian perinatal testis development Spermatogenesis depends on an orchestrated series of developing events in germ cells and full maturation of the somatic microenvironment. To date, the majority of efforts to study cellular heterogeneity in testis has been focused on single-cell gene expression rather than the chromatin landscape shaping gene expression. To advance our understanding of the regulatory programs underlying testicular cell types, we analyzed single-cell chromatin accessibility profiles in more than 25,000 cells from mouse developing testis. We showed that scATAC-Seq allowed us to deconvolve distinct cell populations and identify cis-regulatory elements (CREs) underlying cell type specification. We identified sets of transcription factors associated with cell type-specific accessibility, revealing novel regulators of cell fate specification and maintenance. Pseudotime reconstruction revealed detailed regulatory dynamics coordinating the sequential developmental progressions of germ cells and somatic cells. This high-resolution data also revealed putative stem cells within the Sertoli and Leydig cell populations. Further, we defined candidate target cell types and genes of several GWAS signals, including those associated with testosterone levels and coronary artery disease. Collectively, our data provide a blueprint of the  regulon of the mouse male germline and supporting somatic cells.

sentences: A 6-CpG Validated Methylation Risk Score Model for Metabolic Syndrome: The HyperGEN and GOLDN Studies There has been great interest in genetic risk prediction using risk scores in recent years, however, the utility of scores developed in European populations and later applied to non-European populations has not been successful. In this study, we used cross-sectional data from the Hypertension Genetic Epidemiology Network (HyperGEN, N=614 African Americans (AA)) and the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN, N=995 European Americans (EA)), to create a methylation risk score (MRS) for metabolic syndrome (MetS), demonstrating the utility of MRS across race groups. To demonstrate this, we first selected cytosine-guanine dinucleotides (CpG) sites measured on Illumina Methyl450 arrays previously reported to be significantly associated with MetS and/or component conditions (CPT1A cg00574958, PHOSPHO1 cg02650017, ABCG1 cg06500161, SREBF1 cg11024682, SOCS3 cg18181703, TXNIP cg19693031). Second, we calculated the parameter estimates for the 6 CpGs in the HyperGEN data and used the beta estimates as weights to construct a MRS in HyperGEN, which was validated in GOLDN. We performed association analyses using a logistic mixed model to test the association between the MRS and MetS adjusting for covariates. Results showed the MRS was significantly associated with MetS in both populations. In summary, a MRS for MetS was a strong predictor for the condition across two ethnic groups suggesting MRS may be useful to examine metabolic disease risk or related complications across ethnic groups.

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An arsenic stress signaling code in yeast Arsenic is an environmental toxin that exists mainly as pentavalent arsenate and trivalent arsenite. Both forms activate the yeast SAPK Hog1, but with different consequences. We describe a mechanism by which cells distinguish between these arsenicals through one-step metabolism to differentially regulate the bidirectional glycerol channel Fps1, an adventitious port for arsenite. Cells exposed to arsenate reduce it to thiol-reactive arsenite, which modifies a set of cysteine residues in target proteins; whereas cells exposed to arsenite metabolize it to methylarsenite, which modifies an additional set of cysteine residues. Hog1 becomes arsenylated, which prevents it from closing Fps1. However, this block is overcome in cells exposed to arsenite through methylarsenylation of Acr3, an arsenite efflux pump that we found also regulates Fps1 directly. This adaptation allows cells to restrict arsenite entry through Fps1, but also allows its exit when produced from arsenate exposure. These results have broad implications for understanding how SAPKs activated by diverse stressors can drive stress-specific outputs.

Summary (for JCB table of contents)

Jongmin Lee, David E. Levin

Lee and Levin investigate the question of how different stressors can drive divergent outputs from an active SAPK. The work describes the mechanism by which two forms of arsenic that both activate the yeast SAPK Hog1 differentially regulate the glycerol channel Fps1.

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Sustained ability of a natural microbial community to remove nitrate from groundwater Microbial-mediated nitrate removal from groundwater is widely recognized as the predominant mechanism for nitrate attenuation in contaminated aquifers and is largely dependent on the presence of a carbon-bearing electron donor. The repeated exposure of a natural microbial community to an electron donor can result in the sustained ability of the community to remove nitrate; this phenomenon has been clearly demonstrated at the laboratory scale. However, in situ demonstrations of this ability are lacking. For this study, ethanol (electron donor) was repeatedly injected into a groundwater well (treatment) for six consecutive weeks to establish the sustained ability of a microbial community to remove nitrate. A second well (control) located up-gradient was not injected with ethanol during this time. The treatment well demonstrated strong evidence of sustained ability as evident by concomitant ethanol and nitrate removal and subsequent sulfate removal upon consecutive exposures. Both wells were then monitored for six additional weeks under natural (no injection) conditions. During the final week, ethanol was injected into both treatment and control wells. The treatment well demonstrated sustained ability as evident by concomitant ethanol and nitrate removal whereas the control did not. Surprisingly, the treatment well did not indicate a sustained and selective enrichment of a microbial community. These results suggested that the predominant mechanism(s) of sustained ability likely exist at the enzymatic- and/or genetic-levels. The results of this study demonstrated that the in situ ability of a microbial community to remove nitrate can be sustained in the prolonged absence of an electron donor. Moreover, these results implied that the electron-donor exposure history of nitrate-contaminated groundwater can play an important role nitrate attenuation.

Article Impact StatementGroundwater microbes sustain ability to remove nitrate in absence of carbon and energy source.

sentences: Differential viral RNA methylation contributes to pathogen blocking in Wolbachia-colonized arthropod Arthropod endosymbiont Wolbachia pipientis is part of a global biocontrol strategy to reduce the replication of mosquito-borne RNA viruses such as alphaviruses. We previously demonstrated the importance of a host cytosine methyltransferase, DNMT2, in Drosophila and viral RNA as a cellular target during pathogen-blocking. Here we report on the role of DNMT2 in Wolbachia-induced alphavirus inhibition in Aedes species. Expression of DNMT2 in mosquito tissues, including the salivary glands, is elevated upon virus infection. Notably, this is suppressed in Wolbachia-colonized animals, coincident with reduced virus replication and decreased infectivity of progeny virus. Ectopic expression of DNMT2 in cultured Aedes cells is proviral, increasing progeny virus infectivity, and this effect of DNMT2 on virus replication and infectivity is dependent on its methyltransferase activity. Finally, examining the effects of Wolbachia on modifications of viral RNA by LC-MS show a decrease in the amount of 5-methylcytosine modification consistent with the down-regulation of DNMT2 in Wolbachia colonized mosquito cells and animals. Collectively, our findings support the conclusion that disruption of 5-methylcytosine modification of viral RNA is a vital mechanism operative in pathogen blocking. These data also emphasize the essential role of epitranscriptomic modifications in regulating fundamental alphavirus replication and transmission processes.

sentences: A Comparison of Macaque Hair Hormone Concentration Following Enhanced Cognitive Experiences or Standard Nonhuman Primate Environmental Enrichment Experience with enriched environments positively impacts the health and wellbeing of nonhuman animals ranging from rodents to primates. Little is known, however, about the specific effects of enhanced cognitive enrichment (ECE) on nonhuman primates. The study reported here used archival samples to provide preliminary analysis of ECE on hormones associated with stress and wellbeing, as well as evaluation of persistent effects of infant social rearing. Hair samples from 24 adult male rhesus macaques were analyzed via LC-MS/MS technique for the main stress response hormones: cortisol, cortisone, and dehydroepiandrosterone. The ECE and care-as-usual (CAU) groups did not significantly differ in any of the mean analyte values. Cortisol and dehydroepiandrosterone were strongly and positively correlated in the ECE group. By contrast, for the CAU group the two analytes were weakly correlated. Within the ECE group, cortisol and dehydroepiandrosterone were significantly and positively correlated in the mother-reared group, but not in the nursery-reared group. Overall, these results provide preliminary evidence that experience with ECE could help to maintain a balanced cortisol:dehydroepiandrosterone ratio, possibly indicative of a healthy stress response. Further examination of this ratio in hair is needed to support this hypothesis. These observations may also suggest that nursery rearing could have persistent effects, including dysregulation of the hypothalamic-pituitary-adrenal axis, apparent in the unbalanced cortisol:dehydroepiandrosterone ratio. Together these findings are consistent with the growing literature that supports the use of ECE to promote nonhuman primate wellbeing and healthy development.

sentences: Overexpressing eukaryotic elongation factor 1 alpha (eEF1A) proteins to promote corticospinal axon repair after injury Although protein synthesis is hypothesized to have a pivotal role in axonal repair after central nervous system (CNS) injury, the role of core components of the protein synthesis machinery has not been examined. Notably, some elongation factors possess non-canonical functions that may further impact axonal repair. Here, we examined whether overexpressing eukaryotic elongation factor 1 alpha (eEF1A) proteins enhances the collateral sprouting of corticospinal tract (CST) neurons after unilateral pyramidotomy, along with the underlying molecular mechanisms. Compared with axonal regeneration from injured neurons, axonal sprouting from uninjured neurons occurs spontaneously after injury and may represent a more accessible form of axonal repair for clinical translation. We found that overexpressing eEF1A1, eEF1A2 or both proteins in CST neurons increased the levels of pS6, an indicator for mTOR activity, in neuronal somas. In contrast, the levels of pSTAT3 and pAKT were not increased. Strikingly, overexpressing eEF1A2 alone, but neither eEF1A1 alone nor both factors simultaneously, increased protein synthesis and actin rearrangement in CST neurons. While eEF1A1 overexpression only slightly enhanced CST sprouting across the midline into the denervated side in the cervical spinal after pyramidotomy, eEF1A2 overexpression substantially enhanced this sprouting. Surprisingly, co-overexpression of both eEF1A1 and eEF1A2 led to a sprouting phenotype similar to wild-type controls, suggesting an antagonistic effect of overexpressing both proteins. These data provide the first evidence that overexpressing a core component of the translation machinery, eEF1A2, enhances CST sprouting, likely by a combination of increased protein synthesis, mTOR signaling and actin cytoskeleton rearrangement.

sentences: The RNA m6A reader YTHDF2 controls NK cell anti-tumor and anti-viral immunity N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification on RNA. NK cells are the predominant innate lymphoid cells that mediate anti-viral and anti-tumor immunity. However, whether and how m6A modifications affect NK cell immunity remains unknown. Here, we discover that YTHDF2, a well-known m6A reader, is upregulated in NK cells upon activation by cytokines, tumors, and cytomegalovirus infection. Ythdf2 deficiency in NK cells impairs NK cell anti-tumor and anti-viral activity in vivo. YTHDF2 maintains NK cell homeostasis and terminal maturation, correlating with modulating NK cell trafficking and regulating Eomes, respectively. YTHDF2 promotes NK cell effector function and is required for IL-15-mediated NK cell survival and proliferation by forming a STAT5-YTHDF2 positive feedback loop. Transcriptome-wide screening identifies Tardbp to be involved in cell proliferation or survival as a YTHDF2-binding target in NK cells. Collectively, we elucidate the biological roles of m6A modifications in NK cells and highlight a new direction to harness NK cell anti-tumor immunity.

sentences: CYP1B1 converts procarcinogens into genotoxins in Saccharomyces cerevisiae CYP1B1 activates many chemical carcinogens into potent genotoxins, and allelic variants are risk factors in lung, breast, and prostate cancer. However, genetic instability phenotypes incurred by CYP1B1-activated metabolites have been investigated for only few compounds. In this study, we expressed human CYP1B1 in yeast strains that measure DNA damage-associated toxicity and frequencies of chromosomal translocations and mutations. DNA damage-associated toxicity was measured in a rad4 rad51 strain, defective in both DNA excision and recombinational repair. Frequencies of chromosomal translocations were measured in diploid yeast strains containing two his3 fragments, and mutation frequencies were measured by selecting for canavanine resistance (CanR) in haploid strains. These strains were exposed to benzo[a]pyrene dihydrodiol (BaP-DHD), aflatoxin B1 (AFB1), and the heterocyclic aromatic amines, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). We observed that AFB1, BaP-DHD, IQ, and MeIQx conferred toxicity in the DNA repair mutant expressing CYP1B1. Translocation frequencies increased eight-fold and three-fold after exposure to 50 M AFB1 and 33 M BaP-DHD respectively. Only a two-fold increase in mutation frequency was observed after exposure to 50 M AFB1. However, a robust DNA damage response was observed after AFB1 exposure, as measured by the induction of the small subunit of ribonucleotide reductase, Rnr3. While CYP1B1-mediated activation of BaP-DHD and heterocyclic aromatic amines was expected, strong activation of AFB1 was not. These studies demonstrate that CYP1B1-mediated activation of carcinogens does not only activate compounds to become mutagens but also can convert compounds to become potent recombinagens.

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Spatiotemporally Resolved Multivariate Pattern Analysis for M/EEG An emerging goal in neuroscience is tracking what information is represented in brain activity over time as a participant completes some task. Whilst EEG and MEG offer millisecond temporal resolution of how activity patterns emerge and evolve, standard decoding methods present significant barriers to interpretability as they obscure the underlying spatial and temporal activity patterns. We instead propose the use of a generative encoding model framework that simultaneously infers the multivariate spatial patterns of activity and the variable timing at which these patterns emerge on individual trials. An encoding model inversion allows predictions to be made about unseen test data in the same way as in standard decoding methodology. These SpatioTemporally Resolved MVPA (STRM) models can be flexibly applied to a wide variety of experimental paradigms, including classification and regression tasks. We show that these models provide insightful maps of the activity driving predictive accuracy metrics; demonstrate behaviourally meaningful variation in the timing of pattern emergence on individual trials; and achieve predictive accuracies that are either equivalent or surpass those achieved by more widely used methods. This provides a new avenue for investigating the brains representational dynamics and could ultimately support more flexible experimental designs in future.

HIGHLIGHTSO_LIWe introduce SpatioTemporally Resolved MVPA (STRM), an approach that explicitly models how successive stages of stimulus processing are distributed in both space and time in M/EEG data.
C_LIO_LIWe show that STRM is broadly applicable to diverse types of M/EEG data and outputs meaningful and interpretable maps of how neural representations evolve in space and time at millisecond resolution.
C_LIO_LIThe trial-specific deviations in activity pattern timings identified by STRM are not random, but vary systematically with inter-trial differences in behavioural, cognitive and physiological variables.
C_LIO_LIThese methods result in predictive accuracy metrics that are mostly equivalent to, or a modest improvement on, conventional methods.
C_LI

sentences: Semi-field evaluations of three botanically derived repellents against the blacklegged tick, Ixodes scapularis (Acari: Ixodidae) Three compounds derived from botanicals sources, ethyl perillyl carbonate, geranyl isovalerate, and citronellyl cyclobutane carboxylate, were tested for repellent activity against Ixodes scapularis Say in a semi-field trial. Tick drags were treated with the compounds or with N, N-diethyl-m-toluamide (DEET) at high (0.25mg/cm2) or low (0.15mg/cm2) concentrations. Negative controls included untreated drags and drags treated with acetone, the carrier for all repellents. Freshly treated drags (within 20 minutes) were used to collect I. scapularis ticks at a county park in Wisconsin. To assess effectiveness, we measured tick encounter rates, detachment rate, and time to detachment. None of the repellent treatments resulted in significantly fewer encounters compared to both control treatments. However, the percentage of ticks that detached within 3 min was significantly higher on drags treated with repellents compared to controls. DEET was the most effective, repelling 69.7 - 87% of ticks by 3 min, but the effectiveness of the three test compounds was still high, ranging from 42% to 87% of ticks detaching by 3 min. For time to detachment, there were no significant differences between DEET and the three test compounds. We conclude that these botanically-derived repellents were effective against I. scapularis in a semi-field trial and could be viable alternatives to DEET.

sentences: Neurotrophic control of size regulation during axolotl limb regeneration The mechanisms that regulate the sizing of the regenerating limb in tetrapods such as the Mexican axolotl are unknown. Upon the completion of the developmental stages of regeneration, when the regenerative organ known as the blastema completes patterning and differentiation, the limb regenerate is proportionally small in size. It then undergoes a phase of regeneration that we have called the "tiny-limb" stage, that is defined by rapid growth until the regenerate reaches the proportionally appropriate size. In the current study we have characterized this growth and have found that signaling from the limb nerves is required for its maintenance. Using the regenerative assay known as the Accessory Limb Model, we have found that the size of the limb can be positively and negatively manipulated by nerve abundance. We have additionally developed a new regenerative assay called the Neural Modified-ALM (NM-ALM), which decouples the source of the nerve from the regenerating host environment. Using the NM-ALM we discovered that non-neural extrinsic factors from differently sized host animals do not play a prominent role in determining the size of the regenerating limb. We have also discovered that the regulation of limb size is not autonomously regulated by the limb nerves. Together, these observations show that the limb nerves provide essential and instructive cues to regulate the final size of the regenerating limb.

sentences: Identifying The "Core" Transcriptome of SARS-CoV-2 Infected Cells In 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first emerged, causing the COVID-19 pandemic. Consequently, ongoing research has focused on better understanding the mechanisms underlying the symptoms of this disease. Although COVID-19 symptoms span a range of organ systems, the specific changes in gene regulation that lead to the variety of symptoms are still unclear. In our study, we used publicly available transcriptome data from previous studies on SARS-CoV-2 to identify commonly regulated genes across cardiomyocytes, human bronchial epithelial cells, alveolar type II cells, lung adenocarcinoma, human embryonic kidney cells, and patient samples. Additionally, using this common "core" transcriptome, we could identify the genes that were specifically and uniquely regulated in bronchial epithelial cells, embryonic kidney cells, or cardiomyocytes. For example, we found that genes related to cell metabolism were uniquely upregulated in kidney cells, providing us with the first mechanistic clue about specifically how kidney cells may be affected by SARS-CoV-2. Overall, our results uncover connections between the differential gene regulation in various cell types in response to the SARS-CoV-2 infection and help identify targets of potential therapeutics.

sentences: Recalled parental bonding interacts with oxytocin receptor gene polymorphism in modulating anxiety and avoidance in adult relationships Early interactions with significant individuals affect social experience throughout the course of a lifetime, as a repeated and prolonged perception of different levels of care, independence or control influences the modulation of emotional regulatory processes. As many factors play a role in shaping the expectations and features of social interaction, in this study we consider the influence of parental bonding and genetic allelic variation of oxytocin receptor gene polymorphism (rs53576) over levels of experienced anxiety and avoidance in 313 young adults belonging to two different cultural contexts, namely Italy and Singapore. Results highlight a major effect of maternal characteristics, care and overprotection, with differences between the two cultural groups. Additionally, the interaction between rs53576 and maternal overprotection suggest different environmental susceptibility in the Italian sample and the Singaporean one. Implication in clinical work and future steps are described in the conclusion.

sentences: Adsorption of Pulmonary and Exogeneous Surfactants on SARS-CoV-2 Spike Protein COVID-19 is transmitted by inhaling SARS-CoV-2 virions, which are enveloped by a lipid bilayer decorated by a "crown" of Spike protein protrusions. In the respiratory tract, virions interact with surfactant films composed of phospholipids and cholesterol that coat lung airways. Here, we explore by using coarse-grained molecular dynamics simulations the physico-chemical mechanisms of surfactant adsorption on Spike proteins. With examples of zwitterionic dipalmitoyl phosphatidyl choline, cholesterol, and anionic sodium dodecyl sulphate, we show that surfactants form micellar aggregates that selectively adhere to the specific regions of S1 domain of the Spike protein that are responsible for binding with ACE2 receptors and virus transmission into the cells. We find high cholesterol adsorption and preferential affinity of anionic surfactants to Arginine and Lysine residues within S1 receptor binding motif. These findings have important implications for informing the search for extraneous therapeutic surfactants for curing and preventing COVID-19 by SARS-CoV-2 and its variants.

sentences: Environmental drivers of body size in North American bats Bergmanns Rule--which posits that larger animals live in colder areas--is thought to influence variation in body size within species across space and time, but evidence for this claim is mixed. We tested four competing hypotheses for spatio-temporal variation in body size within bat species during the past two decades across North America. Bayesian hierarchical models revealed that spatial variation in body mass was most strongly (and negatively) correlated with mean annual temperature, supporting the heat conservation hypothesis (historically believed to underlie Bergmanns Rule). Across time, variation in body mass was most strongly (and positively) correlated with net primary productivity, supporting the resource availability hypothesis. Climate change could influence body size in animals through both changes in mean annual temperature and in resource availability. Rapid reductions in body size associated with increasing temperatures have occurred in short-lived, fecund species, but such reductions likely transpire more slowly in longer-lived species.

sentences: The archaeal division protein CdvB1 assembles into polymers that are depolymerized by CdvC The Cdv proteins constitute the cell-division system of the Crenarchaea, in a protein machinery that is closely related to the ESCRT system of eukaryotes. The CdvB paralog CdvB1 is believed to play a major role in the constricting ring that is the central actor in cell division in the crenarchaea. Here, we present an in vitro study of purified CdvB1 from the crenarchaeon M. sedula with a combination of TEM imaging and biochemical assays. We show that CdvB1 self-assembles into filamentous polymers that are depolymerized by the action of the Vps4-homolog ATPase CdvC. Using liposome flotation assays, we show that CdvB1 binds to negatively charged lipid membranes and can be detached from the membrane by the action of CdvC. Interestingly, we find that the polymerization and the membrane binding are mutually exclusive properties of the protein. Our findings provide novel insight into one of the main components of the archaeal cell division machinery.

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Fear memory in humans is consolidated over time independent of sleep BackgroundMany pathological fear memories rely on processes of fear conditioning upon an aversive event. These fear memories may continue to change after acquisition, but the factors contributing to these changes are not well understood. Sleep is known to consolidate, strengthen and change newly acquired declarative and procedural memories. However, evidence on the role of time and sleep in the consolidation of fear memories is inconclusive. Here, we used highly sensitive electrophysiological measures to examine the development of fear-conditioned responses over time and sleep in humans.

MethodsWe assessed event-related brain potentials (ERP) in 18 healthy young individuals during fear conditioning before and after a 2-hour afternoon nap or a corresponding wake interval in a counterbalanced within-subject design. The procedure involved pairing a neutral tone (CS+) with a highly unpleasant sound (US). As a control, another neutral tone (CS-) was paired with a neutral sound. Fear responses were examined before the interval during a habituation phase and an acquisition phase as well as after the interval during an extinction phase and a re-acquisition phase.

ResultsDifferential fear conditioning during acquisition was evidenced by a more negative slow ERP component preceding the CS+ compared to the CS-(stimulus-preceding negativity). This differential fear response was even stronger after the interval during re-acquisition compared to initial acquisition, but this effect was similarly pronounced after sleep and wakefulness.

ConclusionsThese findings suggest that fear memories are consolidated over time, with this effect being independent of intervening sleep.

sentences: An Omics Analysis Search and Information System (OASIS) for Enabling Biological Discovery in the Old Order Amish The "Omics Analysis Search and Information System" (OASIS), developed at the University of Maryland School of Medicine, enables discovery by allowing researchers to mine results from genome wide association studies (GWAS). When interesting signals are found, the research can immediately ask follow-up questions and get answers in real-time. OASIS provides this unique capability with a web-based, scientist-friendly search system and a variety of real-time analysis tools (linkage disequilibrium calculations, conditional analysis, and direct variant comparison) plus on-demand visualizations (boxplots, histograms, LocusZoom & Haploview plots, and pedigree charts). Because OASIS uses a web-based user interface, an understanding of programming or the UNIX operating system is not required. The OASIS application has been used to enable discovery from whole-exome, whole-genome, metabolome, transcriptome and methylome association results for Old Order Amish studies at the University of Maryland School of Medicine.

sentences: Identification and implication of tissue-enriched ligands in epithelial-endothelial crosstalk during pancreas development Development of the pancreas is driven by an intrinsic program coordinated with signals from other cell types in the epithelial environment. These intercellular communications have been so far challenging to study because of the low concentration, localized production and diversity of the signals released. Here, we combined scRNAseq data with a computational interactomic approach to identify signals involved in the reciprocal interactions between the various cell types of the developing pancreas. This in silico approach yielded 40,607 potential ligand-target interactions between the different main pancreatic cell types. Among this vast network of interactions, we focused on three ligands potentially involved in communications between epithelial and endothelial cells. Bmp7 and Wnt7b, expressed by pancreatic epithelial cells and predicted to target endothelial cells, and Sema6d, involved in the reverse interaction. In situ hybridization confirmed the localized expression of Bmp7 in the pancreatic epithelial tip cells and of Wnt7b in the trunk cells. On the contrary, Sema6d was enriched in endothelial cells. Functional experiments on ex vivo cultured pancreatic explants indicated that tip cell-produced Bmp7 restrained development of endothelial cells. This work identified ligands with a restricted tissular and cellular distribution and highlighted the role of Bmp7 in the intercellular communications shaping vessel development during pancreas organogenesis.

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Aberrant Functional Connectivity between Reward and Inhibitory Control Networks in Pre-Adolescent Binge Eating Disorder BackgroundBehavioral features of binge eating disorder (BED) suggest abnormalities in reward and inhibitory control. Studies of adult populations suggest functional abnormalities in reward and inhibitory control networks. Despite behavioral markers often developing in children, the neurobiology of pediatric BED remains unstudied.

Methods58 pre-adolescent children (aged 9-10-years) with BED and 66 age, BMI and developmentally-matched control children were extracted from the 3.0 baseline (Year 0) release of the Adolescent Brain Cognitive Development (ABCD) Study. We investigated group differences in resting-state functional MRI (rs-fMRI) functional connectivity (FC) within and between reward and inhibitory control networks. A seed-based approach was employed to assess nodes in the reward (orbitofrontal cortex, nucleus accumbens, amygdala) and inhibitory control (dorsolateral prefrontal cortex, anterior cingulate cortex) networks via hypothesis-driven seed-to- seed analyses, and secondary seed-to-voxel analyses.

ResultsOur findings revealed reduced FC between the dlPFC and amygdala, and between the anterior cingulate cortex and orbitofrontal cortex in pre-adolescent children with BED, relative to age, gender, BMI and developmentally matched controls. These findings indicating aberrant connectivity between nodes of inhibitory control and reward networks were corroborated by the whole-brain FC analyses.

ConclusionsEarly-onset BED may be characterized by diffuse abnormalities in the functional synergy between reward and cognitive control networks, without perturbations within reward and inhibitory control networks, respectively. The decreased capacity to regulate a reward-driven pursuit of hedonic foods, which is characteristic of BED, may in part, rest on this dysconnectivity between reward and inhibitory control networks.

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A single cell atlas reveals unanticipated cell type complexity in Drosophila ovaries Organ function relies on the spatial organization and functional coordination of numerous cell types. The Drosophila ovary is a widely used model system to study the cellular activities underlying organ function, including stem cell regulation, cell signaling and epithelial morphogenesis. However, the relative paucity of cell type specific reagents hinders investigation of molecular functions at the appropriate cellular resolution.

Here, we used single cell RNA sequencing to characterize all cell types of the stem cell compartment and early follicles of the Drosophila ovary. We computed transcriptional signatures and identified specific markers for nine states of germ cell differentiation, and 23 somatic cell types and subtypes. We uncovered an unanticipated diversity of escort cells, the somatic cells that directly interact with differentiating germline cysts. Three escort cell subtypes reside in discrete anatomical positions, and express distinct sets of secreted and transmembrane proteins, suggesting that diverse micro-environments support the progressive differentiation of germ cells. Finally, we identified 17 follicle cell subtypes, and characterized their transcriptional profiles. Altogether, we provide a comprehensive resource of gene expression, cell type specific markers, spatial coordinates and functional predictions for 34 ovarian cell types and subtypes.

sentences: Cellular Bases of Olfactory Circuit Assembly Revealed by Systematic Time-lapse Imaging Neural circuit assembly features simultaneous targeting of numerous neuronal processes from constituent neuron types, yet the dynamics is poorly understood. Here, we use the Drosophila olfactory circuit to investigate dynamic cellular processes by which olfactory receptor neurons (ORNs) target axons precisely to specific glomeruli in the ipsi- and contralateral antennal lobes. Time-lapse imaging of individual axons from 28 ORN types revealed a rich diversity in extension speed, innervation timing, and ipsilateral branch locations, and identified that ipsilateral targeting occurs via stabilization of transient interstitial branches. Fast imaging using adaptive optics- corrected lattice light-sheet microscopy showed that upon approaching target, many ORN types exhibit "exploring branches" consisted of parallel microtubule-based terminal branches emanating from an F-actin-rich hub. Antennal nerve ablations uncovered essential roles for bilateral interactions in contralateral target selection, and for ORN axons to facilitate dendritic refinement of postsynaptic partner neurons. Altogether, these observations provide cellular bases for wiring specificity establishment.

sentences: Fly Cell Atlas: a single-cell transcriptomic atlas of the adult fruit fly The ability to obtain single cell transcriptomes for stable cell types and dynamic cell states is ushering in a new era for biology. We created the Tabula Drosophilae, a single cell atlas of the adult fruit fly which includes 580k cells from 15 individually dissected sexed tissues as well as the entire head and body. Over 100 researchers from the fly community contributed annotations to >250 distinct cell types across all tissues. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types that are shared between tissues, such as blood and muscle cells, allowed the discovery of rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the entire Drosophila community and serves as a comprehensive reference to study genetic perturbations and disease models at single cell resolution.

sentences: debar, a sequence-by-sequence denoiser for COI-5P DNA barcode data DNA barcoding and metabarcoding are now widely used to advance species discovery and biodiversity assessments. High-throughput sequencing (HTS) has expanded the volume and scope of these analyses, but elevated error rates introduce noise into sequence records that can inflate estimates of biodiversity. Denoising --the separation of biological signal from instrument (technical) noise--of barcode and metabarcode data currently employs abundance-based methods which do not capitalize on the highly conserved structure of the cytochrome c oxidase subunit I (COI) region employed as the animal barcode. This manuscript introduces debar, an R package that utilizes a profile hidden Markov model to denoise indel errors in COI sequences introduced by instrument error. In silico studies demonstrated that debar recognized 95% of artificially introduced indels in COI sequences. When applied to real-world data, debar reduced indel errors in circular consensus sequences obtained with the Sequel platform by 75%, and those generated on the Ion Torrent S5 by 94%. The false correction rate was less than 0.1%, indicating that debar is receptive to the majority of true COI variation in the animal kingdom. In conclusion, the debar package improves DNA barcode and metabarcode workflows by aiding the generation of more accurate sequences aiding the characterization of species diversity.

sentences: CoLoRd: Compressing long reads The costs of maintaining exabytes of data produced by sequencing experiments every year has become a major issue in todays genomics. In spite of the increasing popularity of the third generation sequencing, the existing algorithms for compressing long reads exhibit minor advantage over general purpose gzip. We present CoLoRd, an algorithm able to reduce 3rd generation sequencing data by an order of magnitude without affecting the accuracy of downstream analyzes.

sentences: Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the S. cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale  crab-claw expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFCs switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.

sentences: A neuronal social trait space for first impressions in the human amygdala and hippocampus People instantaneously evaluate faces with significant agreement on evaluations of social traits. However, the neural basis for such rapid spontaneous face evaluation remains largely unknown. Here, we recorded from 490 neurons in the amygdala and hippocampus in 5 neurosurgical patients and show that amygdala and hippocampal neurons encode a social trait space. We further investigated the temporal evolution and modulation on the social trait representation, and we employed encoding and decoding models to reveal the critical social traits for the trait space. We also recorded from another 259 neurons and replicated our findings using different social traits. Lastly, the neuronal social trait space may have a behavioral consequence likely involved in the abnormal processing of social information in autism. Together, our results suggest that there exists a neuronal population code for a comprehensive social trait space in the human amygdala and hippocampus that underlie spontaneous first impressions.

sentences: Self-Supervised Deep-Learning Encodes High-Resolution Features of Protein Subcellular Localization Elucidating the diversity and complexity of protein localization is essential to fully understand cellular architecture. Here, we present cytoself, a deep-learning approach for fully self-supervised protein localization profiling and clustering. cytoself leverages a self-supervised training scheme that does not require pre-existing knowledge, categories, or annotations. Training cytoself on images of 1,311 endogenously labeled proteins from the OpenCell database reveals a highly resolved protein localization atlas that recapitulates major scales of cellular organization, from coarse classes such as nuclear, cytoplasmic and vesicular, to the subtle localization signatures of individual protein complexes. We quantitatively validate cytoselfs ability to cluster proteins into organelles and protein complex clusters using a clustering score, and show that cytoself attains higher scores than previous unsupervised or self-supervised approaches. Finally, to better understand the inner workings of our model, we dissect the emergent features from which our clustering is derived, interpret these features in the context of the fluorescence images, and analyze the performance contributions of the different components of our approach.

sentences: Dispersal syndromes affect ecosystem functioning in ciliate microcosms Dispersal is a key process mediating ecological and evolutionary dynamics. Its effects on metapopulations dynamics, population genetics or species range distribution can depend on phenotypic differences between dispersing and non-dispersing individuals (i.e., dispersal syndromes). However, scaling up to the importance of dispersal syndromes for meta-ecosystems have rarely been considered, despite intraspecific phenotypic variability is now recognised as an important factor mediating ecosystem functioning. In this study, we characterised the intraspecific variability of dispersal syndromes in twenty isolated genotypes of the ciliate Tetrahymena thermophila to test their consequences for biomass productivity in communities composed of five Tetrahymena species. To do so, dispersers and residents of each genotype were introduced, each separately, in ciliate communities composed of four other competing species of the genus Tetrahymena to investigate the effects of dispersal syndromes. We found that introducing dispersers led to a lower biomass compared to introducing residents. This effect was highly consistent across the twenty T. thermophila genotypes despite their marked differences of dispersal syndromes. Finally, we found a strong genotypic effect on biomass production, confirming that intraspecific variability in general affected ecosystem functions in our system. Our study shows that intraspecific variability and the existence of dispersal syndromes can impact the functioning of spatially structured ecosystems in a consistent and therefore predictable way.

sentences: Single-Cell Transcriptome Analysis Identifies Novel Biomarkers Involved in Major Liver Cancer Subtypes Liver cancers including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are leading cause of death worldwide. Single-cell transcriptomics studies have vast potential in advancing our understanding of cancers by defining the cellular composition of different solid tumor types. We peformed an integrated analysis using single-cell RNA sequencing (scRNA-seq) data from cancerous and healthy liver tissues in order to identify the molecular progression and intercellular heterogeneity across cell types in the liver cancer. Moreover, we performed a subtype specific analyses, separately for HCC and ICC, to identify any molecular drivers uniquely associated with these liver cancers. The scRNA-seq dataset comprising 5 healthy controls and 19 liver cancer patients were collected from Human Cell Atlas and Gene Expression Omnibus (GEO), respectively. Our analyses confirmed upregulation of four previously known malignant cell marker genes, namely, EPCAM, KRT19, KRT7 and S100P in the cancerous liver cells. Of these, KRT7 gene has been reported to be associated with ovarian cancer in the past studies. Noteworthy, four marker genes specific to the G1/S (MCM5 and PCNA) and G2/M phases (HMGB2 and CKS2) of the cell cycle were upregulated in the cancerous liver cells. This indicates that these four marker genes are actively dividing in these two phases in cancerous cells as compared to normal liver cells. Our differential expression analysis identified 2 upregulated genes (ATF3 and S100A11) and 2 downregulated genes (FCN3 and FGB) in the liver cancer. Our subtype based differential expression analysis identified 4 genes (HSPA6, LMNA, ATP1B1 and DCXR) specific to HCC and 3 genes (HSPB1, APOC3 and APOA1) specific to ICC. CD4+ T-cell, Hepatocyte, neutrophil, mesenchymal cells and liver bud hepatic cells are the predominant cell-types in liver cells. Our scRNA-seq study revealed the mesenchymal cells as potential malignant cell types in liver cancers. Our work suggests future research on developing liver cancer subtypes therapies could target these cell types and associated molecular markers.

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Replicative Aging Remodels Cell Wall and is Associated with Increased Intracellular Trafficking in Human Pathogenic Yeasts Replicative aging is an underexplored field of research in medical mycology. Cryptococcus neoformans (Cn) and Candida glabrata (Cg) are dreaded fungal pathogens that cause fatal invasive infections. The fungal cell wall is essential for yeast viability and pathogenesis. In this study, we provide data characterizing age-associated modifications to the cell wall of Cn and Cg. Here, we report that old yeast cells upregulate genes of cell wall biosynthesis, leading to cell wall reorganization, and increased levels of all major components, including glucan, chitin and its derivatives, as well as mannan. This results in a significant thickening of the cell wall in aged cells. Old generation yeast cells exhibited drastic ultrastructural changes, including the presence of abundant vesicle-like particles in the cytoplasm, and enlarged vacuoles with altered pH homeostasis. Our findings suggest that the cell wall modifications could be enabled by augmented intracellular trafficking. This work furthers our understanding of the cell phenotype that emerges during aging. It highlights differences in these two fungal pathogens and elucidates mechanisms that explain the enhanced resistance of old cells to antifungals and phagocytic attacks.

IMPORTANCECryptococcus neoformans and Candida glabrata are two opportunistic human fungal pathogens that cause life-threatening diseases. During infection, both microorganisms have the ability to persist for long periods, and treatment failure can occur even if standard testing identifies the yeasts to be sensitive to antifungals. Replicative lifespan is a trait that is measured by the number of divisions a cell undergoes before death. Aging in fungi is associated with enhanced tolerance to antifungals and resistance to phagocytosis, and characterization of old cells may help identify novel antifungal targets. The cell wall remains an attractive target for new therapies because it is essential for fungi and is not present in humans. This study shows that the organization of the fungal cell wall changes remarkably during aging and becomes thicker and is associated with increased intracellular trafficking as well as the alteration of vacuole morphology and pH homeostasis.

sentences: DOT1L activity affects cell lineage progression in the developing brain by controlling metabolic programs Cortical neurogenesis depends on the tight balance between self-renewal and differentiation of apical progenitors (APs), the key progenitor type generating all other neural cells including neocortical neurons. We here report the activity of the histone methyltransferase DOT1L as a gatekeeper for AP cell identity. Combining lineage tracing with single-cell RNA sequencing of clonally related cells, we explore consequences of DOT1L inhibition on AP lineage progression during neurogenesis in the embryonic mouse neocortex. At the cellular level, DOT1L inhibition led to increased neurogenesis driven by a shift from asymmetric self-renewing to symmetric neurogenic divisions of APs. At the molecular level, we show that DOT1L activity preserved AP identity by promoting transcription of a gene set involved in AP metabolism. On a mechanistic level, DOT1L inhibition increased expression of metabolic genes, including microcephaly-associated Asparagine synthetase (Asns) and overexpression of ASNS in APs resulted in increased neuronal differentiation. Asns expression was predicted to be controlled through EZH2 and we show that DOT1L activity allows PRC2-mediated repression of Asns expression. Importantly, inhibition of ASNS activity rescued increased AP differentiation upon DOT1L inhibition. Our data show that DOT1L activity/PRC2 crosstalk controls AP lineage progression by regulating AP metabolism, and they provide a mechanistic view on how DOT1L activity might affect neocortical neurogenesis.

sentences: Cryo-EM structure of native human thyroglobulin The thyroglobulin (Tg) protein is essential to thyroid hormone synthesis, playing a vital role in the regulation of metabolism, development and growth. Its structure is conserved among vertebrates. Tg is delivered through the secretory pathway of the thyroid follicular unit to the central colloid depository, where it is iodinated at specific tyrosine sites to form mono- or diiodotyrosine, which combine to produce triiodothyronine (T3) and thyroxine (T4), respectively. Synthesis of these hormones depends on the precise 3D structure of Tg, which has remained unknown despite decades of research. Here, we present the cryo-electron microscopy structure of human thyroglobulin (hTg) to a global resolution of 3.2 [A]. The structure provides detailed information on the location of the hTg hormonogenic sites and reveals the position as well as the role of many of its glycosylation sites. Our results offer structural insight into thyroid hormonogenesis and provide a fundamental understanding of clinically relevant hTg mutations, which can improve treatment of thyroid diseases.

sentences: Beyond resource selection: emergent spatio-temporal distributions from animal movements and stigmergent interactions A principal concern of ecological research is to unveil the causes behind observed spatio-temporal distributions of species. A key tactic is to correlate observed locations with environmental features, in the form of resource selection functions or other correlative species distribution models. In reality, however, the distribution of any population both affects and is affected by those surrounding it, creating a complex network of feedbacks causing emergent spatio-temporal features that may not correlate with any particular aspect of the underlying environment. Here, we study the way in which the movements of populations in response to one another can affect the spatio-temporal distributions of ecosystems. We construct a stochastic individual-based modelling (IBM) framework, based on stigmergent interactions (i.e. organisms leave marks which cause others to alter their movements) between and within populations. We show how to gain insight into this IBM via mathematical analysis of a partial differential equation (PDE) system given by a continuum limit. We show how the combination of stochastic simulations of the IBM and mathematical analysis of PDEs can be used to categorise emergent patterns into homogeneous vs. heterogeneous, stationary vs. perpetually-fluctuating, and aggregation vs. segregation. In doing so, we develop techniques for understanding spatial bifurcations in stochastic IBMs, grounded in mathematical analysis. Finally, we demonstrate through a simple example how the interplay between environmental features and between-population stigmergent interactions can give rise to predicted spatial distributions that are quite different to those predicted purely by accounting for environmental covariates.

sentences: Pattern separation and tuning shift in human sensory cortex underlie fear memory Animal research has recognized the role of the sensory cortex in fear memory and two key underlying mechanisms--pattern separation and tuning shift. We interrogated these mechanisms in the human sensory cortex in an olfactory differential conditioning study with a delayed (9-day) retention test. Combining affective appraisal and olfactory psychophysics with functional magnetic resonance imaging (fMRI) multivoxel pattern analysis and voxel-based tuning analysis over a linear odor-morphing continuum, we confirmed affective and perceptual learning and memory and demonstrated associative plasticity in the human olfactory (piriform) cortex. Specifically, the piriform cortex exhibited immediate and lasting enhancement in pattern separation (between the conditioned stimuli/CS and neighboring non-CS) and late-onset yet lasting tuning shift towards the CS, especially in anxious individuals. These findings highlight an evolutionarily conserved sensory cortical system of fear memory, which can underpin sensory encoding of fear/threat and confer a sensory mechanism to the neuropathophysiology of anxiety.

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How low can you go? Driving down the DNA input requirements for nanopore sequencing The requirement for large amounts of purified DNA limits many sequencing experiments, especially when seeking to avoid pre-amplification or when using third generation technology to sequence molecules directly. We wanted to test the limits of current nanopore sequencing input requirements and devised a set of experiments to evaluate extraction and library preparation approaches for low inputs.

We found an optimised bead beating approach combined with a magnetic bead protocol, rather than traditional spin columns for DNA extraction, improved both molecule length, integrity score and DNA yield. Through reducing the DNA input to as little as 6.25 % of recommended (25 ng versus 400 ng) and reaction volumes in half, library construction can be completed, and sequencing begun within 20 minutes of sample collection.

Applying these approaches, we demonstrated that our pipeline can be used as a cheap and effective method to de novo assemble a genome and identify genes from low quantities and quality of DNA. With our rapid extraction protocol using transportable equipment and low input library construction we were able to generate a de novo assembly from a single insect (Drosophila melanogaster) spanning 125 Mbp / 85 % of the reference genome, over 96.9% complete BUSCO genes, with a contig N50 over 1.2 Mbp, including chromosome arm sized contigs, for a modest consumable cost under {pound}600.

sentences: Evolution of Crassulacean Acid Metabolism (CAM) as an escape from ecological niche conservatism in Malagasy Bulbophyllum (Orchidaceae) Introductory paragraphDespite growing evidence that niche shifts are more common in flowering plants than previously thought, still little is known about the key physiological (e.g. photosynthesis) traits underlying such niche shifts. To address this question, we here combine a comprehensively sampled phylogeny for mostly epiphytic Malagasy Bulbophyllum orchids (c. 210 spp.) with climatic niche and carbon isotope-derived photosynthesis data to infer the groups spatial-temporal history and to test the role of Crassulacean Acid Metabolism (CAM), a highly water-use efficient type of photosynthesis, in facilitating niche shifts and diversification. We find that most extant species still retain niche (Central Highland) and photosynthesis (C3) states as likely present in the single mid-Miocene (c. 12.70 Ma) ancestor colonizing Madagascar. However, we also infer a major transition to CAM, linked to a late Miocene (c. 7.36 Ma) invasion of species from the sub-humid highland niche first into the islands humid eastern coastal, and then into the seasonally dry  Northwest Sambirano rainforests, yet without significant effect on diversification rates. These findings support the rarely recognized hypothesis that CAM in tropical epiphytes may be selectively advantageous even in high rainfall habitats, rather than presenting a mere adaptation to dry environments or epiphytism per se. Overall, our study qualifies CAM as an evolutionary  gateway trait that considerably widened the spatial-ecological amplitude of Madagascars most species-rich orchid genus.

sentences: In planta genome editing with CRISPR/Cas9 ribonucleoproteins In planta genome editing represents an attractive approach to engineering crops/varieties that are recalcitrant to culture-based transformation methods. Here, we report the direct delivery of CRISPR/Cas9 ribonucleoproteins into the shoot apical meristem using in planta particle bombardment and introduction of a semidwarf1 (sd1)-orthologous mutation into wheat. The triple knockout tasd1 mutant of an elite wheat variety reduced culm length by 10% without a reduction in yield.

sentences: iProMix: A decomposition model for studying the function of ACE2 based on bulk proteogenomic data for coronavirus pathogenesis Both SARS-CoV and SARS-CoV-2 use ACE2 receptors to enter epithelial cells in lung and many other tissues to cause human diseases. Genes and pathways that regulate ACE2 may facilitate/inhibit viral entry and replication, and genes and pathways that are controlled by ACE2 may be perturbed during infection, both affecting disease severity and outcomes. It is critical to understand how genes and pathways are associated with ACE2 in epithelial cells by leveraging proteomic data, but an accurate large-scale proteomic profiling at cellular resolution is not feasible at current stage. Therefore, we propose iProMix, a novel framework that decomposes bulk tissue proteomic data to identify epithelial cell component specific associations between ACE2 and other proteins. Unlike existing decomposition based association analyses, iProMix allows both predictors and outcomes to be impacted by cell type composition of the tissue and accounts for the impacts of decomposition variations and errors on hypothesis tests. It also builds in the functions to improve cell type estimation if estimates from existing literature are unsatisfactory. Simulations demonstrated that iProMix has well-controlled false discovery rate and large power in non-asymptotic settings with both correctly and mis-specified cell-type composition. We applied iProMix to the 110 adjacent normal tissue samples of patients with lung adenocarcinoma from Clinical Proteomic Tumor Analysis Consortium, and identified that interferon  and{gamma} pathways were most significantly associated with ACE2 protein abundances in epithelial cells. Interestingly, the associations were sex-specific that the positive associations were only observed in men, while in women the associations were negative.

sentences: Gut cancer increases the risk for Drosophila to be preyed upon by hunting spiders Predators are thought to prey on individuals that are in poor physical condition, although the evidence supporting this is ambiguous. We tested if sick individuals where more predated using Drosophila melanogaster flies as manipulable preys. We asked whether hunting spiders, trapped from the wild, would selectively prey upon flies with compromised health (i.e. chronically infected or cancerous) versus healthy flies under laboratory conditions. Flies chronically infected with the bacterium Providencia rettgeri, a natural Drosophila pathogen, were not selectively preyed upon by jumping spiders. We strengthened and confirmed our finding with another hunting spider species, small wolf spiders. We discuss that this result supports the hypothesis that chronic infection is associated with reduced symptoms notably to avoid the potentially deadly consequences of host predation on pathogens. We then induced colon cancer in some of the flies and asked whether the presence of cancer led to selective predation; there is little evidence for this, even in vertebrates. As the cancer developed, the incidence of predation by jumping spiders on the afflicted flies increased. We conclude that disease can have different lethal consequences through predation, even in invertebrate species, and that cancer is a factor in selective predation. Our results may explain why early tumours, but not metastasized cancers, are commonly detected in organisms in the wild, as cancer bearing individuals are rapidly eliminated due to the strong selective pressure against them.

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Temporal analysis suggests a reciprocal relationship between 3D chromatin structure and transcription To infer potential causal relationships between 3D chromatin structure, enhancers, and gene transcription, we mapped each feature in a genome-wide fashion across eight narrowly-spaced timepoints of macrophage activation. Enhancers and genes connected by loops exhibited stronger correlations between histone H3K27 acetylation and expression than can be explained by genomic distance or physical proximity alone. Changes in acetylation at looped distal enhancers preceded changes in gene expression. Changes in gene expression exhibit a directional bias at differential loop anchors; gained loops are associated with increased expression of genes oriented away from the center of the loop, while lost loops were often accompanied by high levels of transcription with the loop boundaries themselves. Taken together, these results are consistent with a reciprocal relationship in which loops can facilitate increased transcription by connecting promoters to distal enhancers while high levels of transcription can impede loop formation.

HIGHLIGHTSO_LILPS + IFN{gamma} triggers genome-wide changes in chromatin looping, enhancer acetylation, and gene expression
C_LIO_LILooped enhancer-promoter pairs exhibit ordered and correlated changes in acetylation and expression
C_LIO_LIChanges in gene expression exhibit a directional bias at differential loop anchors
C_LIO_LILost loops are associated with high levels of transcription within loop boundaries
C_LI

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The STING ligand 2'3'-cGAMP induces an NF-κB-dependent anti-bacterial innate immune response in the starlet sea anemone Nematostella vectensis In mammals, cyclic dinucleotides (CDNs) bind and activate STING to initiate an anti-viral type I interferon response. CDNs and STING originated in bacteria and are present in most animals. By contrast, interferons are believed to have emerged in vertebrates; thus, the function of CDN signaling in invertebrates is unclear. Here, we use a CDN, 23-cGAMP, to activate immune responses in a model cnidarian invertebrate, the starlet sea anemone Nematostella vectensis. Using RNA-Seq, we found that 23-cGAMP induces robust transcription of both anti-viral and anti-bacterial genes in N. vectensis. Many of the anti-viral genes induced by 23-cGAMP are homologs of vertebrate interferon stimulated genes, implying that the interferon response predates the evolution of interferons. Knockdown experiments identified a role for NF-{kappa}B in specifically inducing anti-bacterial genes downstream of 23-cGAMP. Some of these putative anti-bacterial genes were also found to be induced during Pseudomonas aeruginosa infection. We characterized the protein product of one of the putative anti-bacterial genes, the N. vectensis homolog of Dae4, and found that it has conserved anti-bacterial activity. This work suggests that a broad anti-bacterial and anti-viral transcriptional response is an evolutionarily ancestral output of 23-cGAMP signaling in animals.

Significance statementCyclic dinucleotides are signaling molecules that originated in bacteria and were subsequently acquired and co-opted by animals for immune signaling. The major cyclic dinucleotide signaling pathway in mammals results in the production of anti-viral molecules called interferons. Invertebrates such as sea anemones lack interferons, and thus it was unclear whether cyclic dinucleotide signaling would play a role in immunity in these animals. Here we report that in the anemone Nematostella vectensis, cyclic dinucleotides activate both anti-viral and anti-bacterial immune responses, and do so through a conserved pathway. These results provide insights into the evolutionary origins of innate immunity, and suggest a broader ancestral role for cyclic dinucleotide signaling that evolved toward more specialized anti-viral functions in mammals.

sentences: Prediction of local convergent shifts in evolutionary rates with phyloConverge characterizes the phenotypic associations and modularity of regulatory elements Physiological and morphological adaptations to extreme environments arise from the molecular evolution of protein-coding regions and regulatory elements (REs) that regulate gene expression. Comparative genomics methods can characterize genetic elements that underlie the organism-level adaptations, but convergence analyses of REs are often limited by their evolutionary properties. A RE can be modularly composed of multiple transcription factor binding sites (TFBS) that may each experience different evolutionary pressures. The modular composition and rapid turnover of TFBS also enables a compensatory mechanism among nearby TFBS that allows for weaker sequence conservation/divergence than intuitively expected. Here, we introduce phyloConverge, a comparative genomics method that can perform fast, fine-grained local convergence analysis of genetic elements. phyloConverge calibrates for local shifts in evolutionary rates using a combination of maximum likelihood-based estimation of nucleotide substitution rates and phylogenetic permutation tests. Using the classical convergence case of mammalian adaptation to subterranean environments, we validate that phyloConverge identifies rate-accelerated conserved non-coding elements (CNEs) that are strongly correlated with ocular tissues, with improved specificity compared to competing methods. We use phyloConverge to perform TFBS-scale and nucleotide-scale scoring to dissect each CNE into subregions with uneven convergence signals and demonstrate its utility for understanding the modularity and pleiotropy of REs. Subterranean-accelerated regions are also enriched for molecular pathways and TFBS motifs associated with neuronal phenotypes, suggesting that subterranean eye degeneration may coincide with a remodeling of the nervous system. phyloConverge offers a rapid and accurate approach for understanding the evolution and modularity of regulatory elements underlying phenotypic adaptation.

sentences: Chelator sensing and lipopeptide interplay mediates molecular interspecies interactions between soil bacilli and pseudomonads AbstractSome bacterial species are important members of the rhizosphere microbiome and confer protection to the host plant against pathogens. However, our knowledge of the multitrophic interactions determining the ecological fitness of these biocontrol bacteria in their highly competitive natural niche is still limited. In this work, we investigated the molecular mechanisms underlying interactions between B. velezensis, considered as model plant-associated and beneficial species in the Bacillus genus, and Pseudomonas as a rhizosphere-dwelling competitor. Our data show that B. velezensis boosts its arsenal of specialized antibacterials upon the perception of the secondary siderophore enantio-pyochelin produced by phylogenetically distinct pseudomonads and some other genera. We postulate that B. velezensis has developed some chelator sensing systems to learn about the identity of its surrounding competitors. Illustrating the multifaceted molecular response of Bacillus, surfactin is another crucial component of the secondary metabolome mobilized in interbacteria competition. Its accumulation not only enhances motility but, unexpectedly, the lipopeptide also acts as a chemical trap that reduces the toxicity of other lipopeptides released by Pseudomonas challengers. This in turn favors the persistence of Bacillus populations upon competitive root colonization. Our work thus highlights new ecological roles for bacterial secondary metabolites acting as key drivers of social interactions.

sentences: Niche-specific genome degradation and convergent evolution shaping Staphylococcus aureus adaptation during severe infections During severe infections, Staphylococcus aureus moves from its colonising sites to blood and tissues, and is exposed to new selective pressures, thus potentially driving adaptive evolution. Previous studies have shown the key role of the agr locus in S. aureus pathoadaptation, however a more comprehensive characterisation of genetic signatures of bacterial adaptation may enable prediction of clinical outcomes and reveal new targets for treatment and prevention of these infections. Here, we measured adaptation using within-host evolution analysis of 2,590 S. aureus genomes from 396 independent episodes of infection. By capturing a comprehensive repertoire of single-nucleotide and structural genome variations, we found evidence of a distinctive evolutionary pattern within the infecting populations compared to colonising bacteria. These invasive strains had up to 20-fold enrichments for genome degradation signatures and displayed significantly convergent mutations in a distinctive set of genes, linked to antibiotic response and pathogenesis. In addition to agr-mediated adaptation we identified non-canonical, genome-wide significant loci including sucA-sucB and stp1. The prevalence of adaptive changes increased with infection extent, emphasising the clinical significance of these signatures. These findings provide a high-resolution picture of the molecular changes when S. aureus transitions from colonisation to severe infection and may inform correlation of infection outcomes with adaptation signatures.

sentences: Ecological Stability Emerges at the Level of Strains in the Human Gut Microbiome The human gut microbiome is a complex community that harbors substantial ecological diversity at the species level, as well as at the strain level within species. In healthy hosts, species abundance fluctuations in the microbiome community are thought to be stable, and these fluctuations can be described by macroecological laws. However, it is less clear how strain abundances change over time. An open question is whether individual strains behave like species themselves, exhibiting stability and following the macroecological relationships known to hold at the species level, or whether strains have different dynamics, perhaps due to the relatively close phylogenetic relatedness of co-colonizing lineages. In this study, we sought to characterize the typical strain-level dynamics of the healthy human gut microbiome on timescales ranging from days to years. We show that genetic diversity within almost all species is stationary, tending towards a long-term typical value within hosts over time scales of several years, despite fluctuations on shorter timescales. Moreover, the abundance fluctuations of strains can be sufficiently described by a stochastic logistic model (SLM) - a model previously used to describe abundance fluctuations among species around a fixed carrying capacity - in the vast majority of cases, suggesting that strains are dynamically stable. Lastly, we find that strain abundances follow the same macroecological laws known to hold at the species level. Together, our results suggest that macroecological properties of the human gut microbiome, including its stability, emerge at the level of strains.

sentences: Simulation of pedigree vs. fully-informative marker based relationships matrices in a loblolly pine breeding population Computer simulations of breeding strategies are an essential resource for tree breeders because they allow exploratory analyses into potential long-term impacts on genetic gain and inbreeding consequences without bearing the cost, time, or resource requirements of field experiments. Previous work has modeled the potential long-term implications on inbreeding and genetic gain using random mating and phenotypic selection. Reduction in sequencing costs has enabled the use of DNA marker-based relationship matrices in addition to or in place of pedigree-based allele sharing estimates; this has been shown to provide a significant increase in the accuracy of progeny breeding value prediction. A potential pitfall of genomic selection using genetic relationship matrices is increased coancestry among selections, leading to the accumulation of deleterious alleles and inbreeding depression. We used simulation to compare the relative genetic gain and risk of inbreeding depression within a breeding program similar to loblolly pine, utilizing pedigree-based or marker-based relationships over ten generations. We saw a faster rate of purging deleterious alleles when using a genomic relationship matrix based on markers that track identity-by-descent of segments of the genome. Additionally, we observed an increase in the rate of genetic gain when using a genomic relationship matrix instead of a pedigree-based relationship matrix. While the genetic variance of populations decreased more rapidly when using genomic-based relationship matrices as opposed to pedigree-based, there appeared to be no long-term consequences on the accumulation of deleterious alleles within the simulated breeding strategy.

sentences: Who infects Whom? - Reconstructing infection chains of Mycobacterium avium ssp. paratuberculosis in an endemically infected dairy herd by use of genomic data Recent evidence of circulation of multiple strains within herds and mixed infections of cows marks the beginning of a rethink of our knowledge on Mycobacterium avium ssp. paratuberculosis (MAP) epidemiology. Strain typing opens new ways to investigate MAP transmission. This work presents a method for reconstructing infection chains in a setting of endemic Johnes disease on a well-managed dairy farm. By linking genomic data with demographic field data, strain-specific differences in spreading patterns could be quantified for a densely sampled dairy herd. Mixed infections of dairy cows with MAP are common, and some strains spread more successfully. Infected cows remain susceptible for co-infections with other MAP genotypes. The model suggested that cows acquired infection from 1-4 other cows and spread infection to 0-17 individuals. Reconstructed infection chains supported the hypothesis that high shedding animals that started to shed at an early age and showed a progressive infection pattern represented a greater risk for spreading MAP. Transmission of more than one genotype between animals was recorded. In this farm with a good MAP control management program, adult-to-adult contact was proposed as the most important transmission route to explain the reconstructed networks. For each isolate, at least one more likely ancestor could be inferred. Our study results help to capture underlying transmission processes and to understand the challenges of tracing MAP spread within a herd. Only the combination of precise longitudinal field data and bacterial strain type information made it possible to trace infection in such detail.

sentences: The Hsp90 chaperone system from the African trypanosome, Trypanosoma brucei African Trypanosomiasis is a neglected tropical disease caused by Trypanosoma brucei (T. brucei) and is spread by the tsetse fly in sub-Saharan Africa. The disease is fatal if left untreated and the currently approved drugs for treatment are toxic and difficult to administer. The trypanosome must survive in the insect vector and its mammalian host, and to adapt to these different conditions, the parasite relies on molecular chaperones called heat shock proteins. Heat shock proteins mediate the folding of newly synthesized proteins as well as prevent misfolding of proteins under normal conditions and during stressful conditions. Heat shock protein 90 (Hsp90) is one of the major molecular chaperones of the stress response at the cellular level. It functions with other chaperones and co-chaperones and inhibition of its interactions is being explored as a potential therapeutic target for numerous diseases. This study provides an in-silico overview of Hsp90 and its co-chaperones in both T. brucei brucei and T. brucei gambiense in relation to human and other kinetoplastid parasites. The evolutionary, functional, and structural analyses of Hsp90 were also shown. The updated information on Hsp90 and its co-chaperones from recently published proteomics on T. brucei was examined for the different life cycle stages and subcellular localisations. The results show a difference between T. b. brucei and T. b. gambiense with T. b. brucei encoding 12 putative Hsp90 genes, 10 of which are cytosolic and located on a single chromosome while T. gambiense encodes 5 Hsp90 genes, 3 of which are located in the cytosol. Eight putative co-chaperones were identified in this study, 6 TPR-containing and 2 non-TPR-containing co-chaperones. This study provides an updated context for studying the biology of the African trypanosome and evaluating Hsp90 and its interactions as potential drug targets.

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HOPMA: Boosting protein functional dynamics with colored contact maps In light of the recent very rapid progress in protein structure prediction, accessing the multitude of functional protein states is becoming more central than ever before. Indeed, proteins are flexible macromolecules, and they often perform their function by switching between different conformations. However, high-resolution experimental techniques such as X-ray crystallography and cryogenic electron microscopy can catch relatively few protein functional states. Many others are only accessible under physiological conditions in solution. Therefore, there is a pressing need to fill this gap with computational approaches.

We present HOPMA, a novel method to predict protein functional states and transitions using a modified elastic network model. The method exploits patterns in a protein contact map, taking its 3D structure as input, and excludes some disconnected patches from the elastic network. Combined with nonlinear normal mode analysis, this strategy boosts the protein conformational space exploration, especially when the input structure is highly constrained, as we demonstrate on a set of more than 400 transitions. Our results let us envision the discovery of new functional conformations, which were unreachable previously, starting from the experimentally known protein structures.

The method is computationally efficient and available at https://github.com/elolaine/HOPMA and https://team.inria.fr/nano-d/software/nolb-normal-modes.

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Task space exploration improves adaptation after incompatible virtual surgeries Humans have a remarkable capacity to learn new motor skills, a process that requires novel muscle activity patterns. Muscle synergies may simplify the generation of muscle patterns through the selection of a small number of synergy combinations. Learning new motor skills may then be achieved by acquiring novel muscle synergies. In a previous study, we used myoelectric control to construct virtual surgeries that altered the mapping from muscle activity to cursor movements. After compatible virtual surgeries, which could be compensated by recombining subject-specific muscle synergies, participants adapted quickly. In contrast, after incompatible virtual surgeries, which could not be compensated by recombining existing synergies, participants explored new muscle patterns, but failed to adapt. Here, we tested whether task space exploration can promote learning of novel muscle synergies, required to overcome an incompatible surgery. Participants performed the same reaching task as in our previous study, but with more time to complete each trial, thus allowing for exploration. We found an improvement in trial success after incompatible virtual surgeries. Remarkably, improvements in movement direction accuracy after incompatible surgeries occurred faster for corrective movements than for the initial movement, suggesting that learning of new synergies is more effective when used for feedback control. Moreover, reaction time was significantly higher after incompatible than after compatible virtual surgeries, suggesting an increased use of an explicit adaptive strategy to overcome incompatible surgeries. Taken together, these results indicate that exploration is important for skill learning and suggest that human participants, with sufficient time can learn new muscle synergies.

NEW & NOTEWORTHYMotor skill learning requires the acquisition of novel muscle patterns, a slow adaptive process. Here we show that learning to control a cursor after an incompatible virtual surgery, a complex skill requiring new muscle synergies, is possible when enough time for task space exploration is provided. Our results suggest that learning new synergies is related to the exceptional human capacity to acquire a wide variety of novel motor skills with practice.

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Long-term balancing selection in highly dynamic mating loci generates trans-species polymorphisms in fungi Balancing selection, an evolutionary force that retains genetic diversity, has been detected in multiple genes and organisms, such as the sexual mating loci in fungi. However, to quantify the strength of balancing selection and define the mating-related genes require a large number of specimens. In tetrapolar basidiomycete fungi, sexual type is determined by two unlinked loci, MATA and MATB. Genes in both loci defines mating type identity, control successful mating and completion of the life cycle. These loci are usually highly diverse. Previous studies have speculated, based on culture crosses, that species of the non-model genus Trichaptum (Hymenochaetales, Basidiomycota) possess a tetrapolar mating system, with multiple alleles. Here, we sequenced a hundred and eighty specimens of three Trichaptum species. We characterized the chromosomal location of MATA and MATB, the molecular structure of MAT regions and their allelic richness. Our sequencing effort was sufficient to molecularly characterize multiple MAT alleles segregating before the speciation event of Trichaptum species. Our analyses suggested that long-term balancing selection has generated trans-species polymorphisms. Mating sequences were classified in different allelic classes based on an amino acid identity (AAI) threshold supported by phylogenetics. The inferred allelic information mirrored the outcome of in vitro crosses, thus allowing us to support the degree of allelic divergence needed for successful mating. Even with the high amount of divergence, key amino acids in functional domains are conserved. The observed allelic classes could potentially generate 14,560 different mating types. We conclude that the genetic diversity of mating in Trichaptum loci is due to long-term balancing selection, with limited recombination and duplication activity. Our large number of sequenced specimens highlighted the importance of sequencing multiple individuals from different species to detect the mating-related genes, the mechanisms generating diversity and the evolutionary forces maintaining them.

Author summaryFungi have complex mating systems, and basidiomycete fungi can encode thousands of mating types. Individuals with the same mating type cannot mate. This sexual system has evolved to facilitate sexual mating, increasing the chances to recombine into advantageous allelic combination and prune deleterious alleles. We explored the genomes of hundred and eighty specimens, combined with experimental mating studies of selected specimens, from a non-model organism (Trichaptum). We characterized the genomic regions controlling sex. The mating ability of the specimens confirmed the role of the mating alleles observed in the genomic data. The detailed analyses of many specimens allowed us to observe gene duplication and rearrangements within the mating loci, increasing the diversity within these loci. We supported previous suggestions of balancing selection in this region, an evolutionary force that maintains genomic diversity. These results supports that our fungal specimens are prone to outcross, which might facilitate the adaptation to new conditions.

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Saccharomyces cerevisiae gene expression during fermentation of Pinot noir wines at industrially relevant scale During a wine fermentation, Saccharomyces cerevisiae transforms grape must through metabolic activities that generate ethanol and other compounds. Thousands of genes change expression over the course of a wine fermentation to allow S. cerevisiae to adapt to and dominate the fermentation environment. Investigations into these gene expression patterns have previously revealed genes that underlie cellular adaptation to the grape must and wine environment involving metabolic specialization and ethanol tolerance. However, the vast majority of studies detailing gene expression patterns have occurred in controlled environments that do not recapitulate the biological and chemical complexity of fermentations performed at production scale. Here, we present an analysis of the S. cerevisiae RC212 gene expression program across 40 pilot-scale fermentations (150 liters) using Pinot noir grapes from 10 California vineyards across two vintages. We observe a core gene expression program across all fermentations irrespective of vintage similar to that of laboratory fermentations, in addition to novel gene expression patterns likely related to the presence of non-Saccharomyces microorganisms and oxygen availability during fermentation. These gene expression patterns, both common and diverse, provide insight into Saccharomyces cerevisiae biology critical to fermentation outcomes at industry-relevant scales.

ImportanceThis study characterized Saccharomyces cerevisiae RC212 gene expression during Pinot noir fermentation at pilot scale (150 liters) using production-relevant conditions. The reported gene expression patterns of RC212 is generally similar to that observed in laboratory fermentation conditions, but also contains gene expression signatures related to yeast-environment interactions found in a production setting (e.g., presence of non-Saccharomyces microorganisms). Key genes and pathways highlighted by this work remain under-characterized, raising the need for further research to understand the roles of these genes and their impact on industrial wine fermentation outcomes.

sentences: Mechanistic insights into dideoxygenation in gentamicin biosynthesis Gentamicin is an important aminoglycoside antibiotic used for treatment of infections caused by Gram-negative bacteria. Although most of the biosynthetic pathway of gentamicin has been elucidated, a remaining intriguing question is how the intermediates JI-20A and JI-20B undergo a dideoxygenation to form gentamicin C complex. Here we show that the dideoxygenation process starts with GenP-catalyzed phosphorylation of JI-20A and JI-20Ba. The phosphorylated products are converted to C1a and C2a by concerted actions of two PLP (pyridoxal 5-phosphate)-dependent enzymes: elimination of water and then phosphate by GenB3 and double bond migration by GenB4. Each of these reactions liberates an imine which hydrolyses to a ketone or aldehyde and is then re-aminated by GenB3 using an amino donor. Crystal structures of GenB3 and GenB4 have guided site-directed mutagenesis to reveal crucial residues for the enzymes functions. We propose catalytic mechanisms for GenB3 and GenB4, which shed new light on the already unrivalled catalytic versatility of PLP-dependent enzymes.

sentences: Plasmid fitness costs are caused by specific genetic conflicts Plasmids play an important role in bacterial genome evolution by transferring genes between lineages. Fitness costs associated with plasmid acquisition are expected to be a barrier to gene exchange, but the causes of plasmid fitness costs are poorly understood. Single compensatory mutations are often sufficient to completely ameliorate plasmid fitness costs, suggesting that such costs are caused by specific genetic conflicts rather than generic properties of plasmids, such as their size, metabolic burden, or expression level. Here we show -- using a combination of experimental evolution, reverse genetics, and transcriptomics -- that fitness costs of two divergent large plasmids in Pseudomonas fluorescens are caused by inducing maladaptive expression of a chromosomal tailocin toxin operon. Mutations in single genes unrelated to the toxin operon, and located on either the chromosome or the plasmid, ameliorated the disruption associated with plasmid acquisition. We identify one of these compensatory loci, the chromosomal gene PFLU4242, as the key mediator of the fitness costs of both plasmids, with the other compensatory loci either reducing expression of this gene or mitigating its deleterious effects by upregulating a putative plasmid-borne ParAB operon. The chromosomal mobile genetic element Tn6291, which uses plasmids for transmission, remained upregulated even in compensated strains, suggesting that mobile genetic elements communicate through pathways independent of general physiological disruption. Plasmid fitness costs caused by specific genetic conflicts are unlikely to act as a long-term barrier to horizontal gene transfer due to their propensity for amelioration by single compensatory mutations, explaining why plasmids are so common in bacterial genomes.

sentences: A 'Variant Viewer' for investigating population-level variation in Arabidopsis thaliana High throughput sequencing has opened the doors for investigators to probe genetic variation present in large populations of organisms. In plants, the 1001 Genomes Project (1001genomes.org) is one such effort that sought to characterize the extant worldwide variation in Arabidopsis thaliana for future analyses to compare and draw upon. We developed a web application that accesses the 1001 Genomes database called The Variant Viewer, for investigators to view variants in any A. thaliana gene and within gene families. These variants may be visualized in the context of alignments of queried genes, across splice isoforms of these genes and in relation to conserved domains.

sentences: Neural entrainment is strongest to the spectral flux of slow music and depends on familiarity and beat salience Neural activity in the auditory system synchronizes to sound rhythms, and brain-environment synchronization is thought to be fundamental to successful auditory perception. Sound rhythms are often operationalized in terms of the sounds amplitude envelope. We hypothesized that - especially for music - the envelope might not best capture the complex spectro-temporal fluctuations that give rise to beat perception and synchronize neural activity. This study investigated 1) neural entrainment to different musical features, 2) tempo-dependence of neural entrainment, and 3) dependence of entrainment on familiarity, enjoyment, and ease of beat perception. In this electroencephalography study, 37 human participants listened to tempo-modulated music (1-4 Hz). Independent of whether the analysis approach was based on temporal response functions (TRFs) or reliable components analysis (RCA), the spectral flux of music - as opposed to the amplitude envelope - evoked strongest neural entrainment. Moreover, music with slower beat rates, high familiarity, and easy-to-perceive beats elicited the strongest neural response. Based on the TRFs, we could decode music stimulation tempo, but also perceived beat rate, even when the two differed. Our results demonstrate the importance of accurately characterizing musical acoustics in the context of studying neural entrainment, and demonstrate entrainments sensitivity to musical tempo, familiarity, and beat salience.

sentences: Single-embryo phosphoproteomics reveals the importance of intrinsic disorder in cell cycle dynamics Switch-like cyclin-dependent kinase (CDK)-1 activation is thought to underlie the abruptness of mitotic onset, but how CDKs can simultaneously phosphorylate many diverse substrates is unknown, and direct evidence for such phosphorylation dynamics in vivo is lacking. Here, we analysed protein phosphorylation states in single Xenopus embryos throughout synchronous cell cycles. Over a thousand phosphosites were dynamic in vivo, and assignment of cell cycle phases using egg extracts revealed hundreds of S-phase phosphorylations. Targeted phosphoproteomics in single embryos showed switch-like mitotic phosphorylation of diverse protein complexes. The majority of cell cycle-regulated phosphosites occurred in CDK consensus motifs, and 72% located to intrinsically disordered regions. Dynamically phosphorylated proteins, and documented substrates of cell cycle kinases, are significantly more disordered than phosphoproteins in general. Furthermore, 30-50% are components of membraneless organelles. Our results suggest that phosphorylation of intrinsically disordered proteins by cell cycle kinases, particularly CDKs, allows switch-like mitotic cellular reorganisation.

sentences: Mentalizing in economic games is associated with enhanced activation and connectivity in left temporoparietal junction Studies in Social Neuroeconomics have consistently reported activation in social cognition regions during interactive economic games suggesting mentalizing during economic choice. It remains important to test the involvement of neural activity associated with mentalizing in an economic games context within the same sample of participants performing the same task. We designed a novel version of the classic false-belief task in which participants observed interactions between agents in the ultimatum and trust games and were subsequently asked to infer the agents beliefs. We compared activation patterns during the economic-games false-belief task to those during the classic false-belief task using conjunction analyses. We find significant overlap in left TPJ, and dmPFC, as well as temporal pole during two task phases: belief formation and belief inference. Moreover, gPPI analyses show that during belief formation right TPJ is a target of both left TPJ and right temporal pole (TP) seed regions, while during belief inferences all seed regions show interconnectivity with each other. These results indicate that across different task types and phases, mentalizing is associated with activation and connectivity across central nodes of the social cognition network. Importantly, this is the case in the context of the novel economic-games and classic false-belief tasks.

sentences: Single-cell imaging reveals that Staphylococcus aureus is highly competitive against Pseudomonas aeruginosa on surfaces Pseudomonas aeruginosa and Staphylococcus aureus frequently occur together in polymicrobial infections, and their interactions can complicate disease progression as well as treatment options. While interactions between P. aeruginosa and S. aureus have been extensively described using planktonic batch cultures, little is known about whether and how individual cells interact with each other on solid substrates. This is important, because in infections, both species frequently colonize surfaces to form microcolony aggregates and biofilms. Here, we performed single-cell time-lapse fluorescence microscopy combined with automated image analysis to describe interactions between P. aeruginosa PAO1 with three different S. aureus strains (Cowan I, 6850, JE2) during microcolony growth on agarose surfaces. While P. aeruginosa is usually considered the dominant species, we found that the competitive balance tips in favor of S. aureus on surfaces. We observed that all S. aureus strains accelerated the onset of microcolony growth in competition with P. aeruginosa and significantly compromised P. aeruginosa growth prior to physical contact. These results suggest that S. aureus deploys mechanisms of both resource competition and interference competition via diffusible compounds. JE2 was the most competitive S. aureus strain, simply usurping P. aeruginosa microcolonies when coming into direct contact, while 6850 was the weakest competitor itself suppressed by P. aeruginosa. Moreover, P. aeruginosa reacted to the assault of S. aureus by showing increased directional growth and expedited expression of quorum sensing regulators controlling the synthesis of competitive traits. Altogether, our results reveal that quantitative single-cell live imaging has the potential to uncover microbial behaviors that cannot be predicted from batch culture studies, and thereby contribute to our understanding of interactions between pathogens that co-colonize host-associated surfaces during polymicrobial infections.

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Toxoplasma gondii subverts the host ESCRT machinery for parasite uptake of host cytosolic proteins Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole, the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of GRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PVM. These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission.

Author summaryIntracellular pathogens exploit their host to gain the resources necessary to sustain infection; however, precisely how the intracellular parasite Toxoplasma gondii acquires essential nutrients from its host remains poorly understood. Previous work showed that T. gondii is capable of internalizing host derived cytosolic proteins and delivering them to its lysosome-like compartment for degradation. However, the mechanism by which the material is trafficked across the membrane delimiting the replicative vacuole in which the parasite resides remained unclear. Here, we report a role for the parasite effector protein TgGRA14 in the recruitment of the host ESCRT machinery for the uptake of host cytosolic proteins. Important human pathogens have developed strategies for exploiting the host ESCRT machinery for intracellular subsistence. Our study sheds lights on the strategy used by a eukaryotic pathogen in subverting the host ESCRT machinery for the internalization of resources from its host cells.

sentences: Glycolytic inhibitor 2-Deoxy-D-glucose attenuates SARS-CoV-2 multiplication in host cells and weakens the infective potential of progeny virions The COVID-19 pandemic is an ongoing public health emergency of international concern. While a lot of efforts are being invested in vaccinating the population, there is also an emergent requirement to find potential therapeutics to effectively counter this fast mutating SARS-CoV-2 virus-induced pathogenicity. Virus-infected host cells switch their metabolism to a more glycolytic phenotype. This switch induced by the virus is needed for faster production of ATP and higher levels of anabolic intermediates, required for new virion synthesis and packaging. In this study, we used 2-Deoxy-D-glucose (2-DG) to target and inhibit the metabolic reprogramming induced by SARS-CoV-2 infection. Our results showed that virus infection induces glucose influx and glycolysis resulting in selective high accumulation of the fluorescent glucose/2-DG analogue, 2-NBDG in these cells. Subsequently, 2-DG inhibits glycolysis in infected cells thereby reducing the virus multiplication and alleviates the cells from virus induced cytopathic effect (CPE) and cell death. Herein, we demonstrate that the crucial Nglycosites (N331 and N343) of RBD in spike protein of progeny virions produced from 2-DG treated cells were found unglycosylated and defective with compromised infectivity potential. In line with earlier reported observations, our study also showed that 2-DG mediated metabolic inhibiton can attenuate SARS-COV-2 multiplication. In addition, mechanistic study revealed that the inhibition of SARS-COV-2 multiplication is attributed to 2-DG induced un-glycosylation of spike protein. Our findings strengthen the notion that 2-DG effectively inhibits SARS-CoV-2 multiplication. Therefore, based on its previous human trials in different types of Cancer and Herpes patients, it could be a potential molecule to study in COVID-19 patients.

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High nucleotide substitution rates associated with retrotransposon proliferation drive dynamic secretome evolution in smut pathogens Transposable elements (TEs) play a pivotal role in shaping diversity in eukaryotic genomes. The covered smut pathogen on barley, Ustilago hordei, encountered a recent genome expansion. Using long reads, we assembled genomes of 6 U. hordei strains and 3 sister species, to study this genome expansion. We found that larger genome sizes can mainly be attributed to a higher genome fraction of long terminal repeat retrotransposons (LTR-RTs). In the studied smut genomes, LTR-RTs fractions are the largest in U. hordei and are positively correlated to the mating-type locus sizes, which is up to [~]560 kb in U. hordei. Furthermore, LTR-RTs were found to be associated with higher nucleotide substitution levels, as these higher levels occur more clustered in smut species with a recent LTR-RT proliferation. Moreover, genes in genome regions with higher nucleotide substitution levels generally reside closer to LTR-RTs than other genome regions. Genome regions with many nucleotide substitutions encountered an especially high fraction of CG substitutions, which is not observed for LTR-RT sequences. The high nucleotide substitution levels particularly accelerate the evolution of secretome genes, as their more flexible nature results that substitutions often lead to amino acid alterations.

ImportanceGenomic alteration can be generated through various means, in which transposable elements (TEs) can play a pivotal role. Their mobility causes mutagenesis in itself and can disrupt the function of the sequences they insert into. Indirectly, they also impact genome evolution as their repetitive nature facilitates non-homologous recombination. Furthermore, TEs have been linked to specific epigenetic genome organizations. We report a recent TE proliferation in the genome of the barley covered smut fungus, Ustilago hordei. This proliferation is associated with a distinct nucleotide substitution regime that has a higher rate and a higher fraction of CG substitutions. This different regime shapes the evolution of genes in subjected genome regions. Our findings highlight that TEs may influence the error-rate of DNA polymerase in a hitherto unknown fashion.

sentences: Progenitor cell integration into a barrier epithelium during adult organ turnover Barrier epithelial organs face the constant challenge of sealing the interior body from the external environment while simultaneously replacing the cells that contact this environment. These replacement cells--the progeny of basal stem cells--are born without apical, barrier-forming structures such as a protective, lumen-facing membrane and occluding junctions. How stem cell progeny acquire these structures to become part of the barrier is unknown. Here we use Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM), Correlative Light-Electron Microscopy (CLEM), and volumetric imaging of live and fixed organs to investigate progenitor integration in the intestinal epithelium of adult Drosophila. We find that stem cell daughters gestate their future lumenal-apical membrane beneath a transient, basal niche formed by an umbrella-shaped occluding junction that shelters the growing cell and adheres it to mature neighbor cells. The umbrella junction both targets formation of a deep, microvilli-lined, apical invagination and closes it off from the contents of the gut lumen. When the growing cell is sufficiently mature, the umbrella junction retracts to expose this Pre-Assembled Apical Compartment (PAAC) to the gut lumen, thus incorporating the new cell into the intestinal barrier. When we block umbrella junctions, stem cell daughters grow and attempt to differentiate but fail to integrate; when we block cell growth, no umbrella junctions form and daughters arrest in early differentiation. Thus, stem cell progeny build new barrier structures in the shelter of a transient niche, where they are protected from lumenal insults until they are prepared to withstand them. By coordinating this dynamic junctional niche with progenitor cell differentiation, a physiologically active epithelial organ incorporates new cells while upholding integrity of its barrier.

sentences: Conserved α-helix-3 is crucial for structure and functions of Rad6 E2 ubiquitin-conjugating enzymes. Rad6, an E2 ubiquitin-conjugating enzyme conserved from yeast to humans, functions in transcription, genome maintenance and proteostasis. The contributions of many conserved secondary structures of Rad6 and its human homologs UBE2A and UBE2B to their biological functions are not understood. A mutant RAD6 allele with a missense substitution at alanine-126 (A126) of helix-3 that causes defects in telomeric gene silencing, DNA repair and protein degradation was reported over two decades ago. Here, using a combination of genetics, biochemical, biophysical, and computational approaches, we discovered that helix-3 A126 mutations compromise the ability of Rad6 to ubiquitinate target proteins without disrupting interactions with partner E3 ubiquitin-ligases that are required for their various biological functions in vivo. Explaining the defective in vitro or in vivo ubiquitination activities, molecular dynamics simulations and NMR showed that helix-3 A126 mutations cause local disorder of the catalytic pocket of Rad6 in addition to disorganizing the global structure of the protein to decrease its stability in vivo. We also show that helix-3 A126 mutations deform the structures of UBE2A and UBE2B, the human Rad6 homologs, and compromise the in vitro ubiquitination activity and folding of UBE2B. In summary, our studies reveal that the conserved helix-3 is a crucial structural constituent that controls the organization of catalytic pockets, enzymatic activities, and biological functions of the Rad6-family E2 ubiquitin-conjugating enzymes.

sentences: Female Bengalese finches recognize their fathers song as sexually attractive Birdsong is an important communication signal used in mate choice. In some songbirds, only males produce songs while females do not. Female birds are sensitive to inter- and intra-species song variation. Some aspects of female song preference depend on developmental experiences. For example, in Bengalese finches and zebra finches, adult females prefer the song to which they were exposed early in life, such as the fathers song. However, it is unclear whether such song preference in females is sexually motivated. The purpose of our study is to test if female Bengalese finches recognize their fathers song as sexually attractive. We measured copulation solicitation displays during playbacks of the fathers song vs. unfamiliar conspecific songs and found that across individuals, the fathers song elicited more displays than other songs. In addition, we analyzed if a birds response to a given song could be predicted by the level of similarity of that song to the fathers song. The results suggest that preference for the fathers song in this species is actually relevant to mate choice. Although more precise control is necessary in future studies to elucidate the process of preference development, our results imply the significance of early-life experience in shaping female song preference.

sentences: Hippocampal CA1 pyramidal cells do not receive monosynaptic input from thalamic nucleus reuniens Midline thalamic nuclei play a critical role in cognitive functions such as memory, decision-making and spatial navigation, by facilitating communication between the many brain regions involved in these processes. One canonical feature of thalamic interactions with the cortex or hippocampus appears to be that the thalamus receives input from, and projects to, excitatory neurons. Thalamic nucleus reuniens (NRe) is located on the midline and is viewed primarily as a relay from prefrontal cortex to hippocampal and entorhinal areas, although these connections are poorly defined at the cellular and synaptic level. Using electrophysiology and monosynaptic circuit-tracing, we found that pyramidal cells in CA1 receive no direct input from NRe. This contrasts starkly with prefrontal cortex, subiculum and entorhinal cortex, and indicates that NRe inputs to CA1 primarily drive local inhibition and not excitation they do in the other regions. The NRe to CA1 projection is thus a unique thalamic projection and as such is raising important questions about the function of NRe-mediated prefrontal control of the hippocampus.

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Differential genetic variation underlying Ammonium and Nitrate responses in Arabidopsis thaliana Nitrogen is an essential element required for plant growth and productivity. Understanding the mechanisms and natural genetic variation underlying nitrogen use in plants will facilitate engineering plant nitrogen use to maximize crop productivity while minimizing environmental costs. To understand the scope of natural variation that may influence nitrogen use, we grew 1135 Arabidopsis thaliana natural genotypes on two nitrogen sources, nitrate and ammonium, and measured both developmental and defense metabolite traits. By using different environments and focused on multiple traits, we identified a wide array of different nitrogen responses. These responses are associated with a large number of genes, most of them not previously associated with nitrogen responses. Only a small portion of these genes appear to be shared between environments or traits while most of the detected genes are predominantly specific to a developmental or defense trait under a specific nitrogen source. Finally, by using a large population we were able to identify unique nitrogen responses, like preferring ammonium or nitrate, that appear to be generated by combinations of loci rather than a few large effect loci. This suggests that it may be possible to obtain novel phenotypes in complex nitrogen responses by manipulating sets of genes with small effects rather than solely focusing on large effect single gene manipulations.

One Sentence SummaryUsing a large collection of natural genotypes, and studying both developmental and metabolic responses, we found a large number of genes that are involved in the plants nitrogen response.

sentences: partR2: Partitioning R2 in generalized linear mixed models The coefficient of determination R2 quantifies the amount of variance explained by regression coefficients in a linear model. It can be seen as the fixed-effects complement to the repeatability R (intra-class correlation) for the variance explained by random effects and thus as a tool for variance decomposition. The R2 of a model can be further partitioned into the variance explained by a particular predictor or a combination of predictors using semi-partial (part) R2 and structure coefficients, but this is rarely done due to a lack of software implementing these statistics. Here, we introduce partR2, an R package that quantifies part R2 for fixed effect predictors based on (generalized) linear mixed-effect model fits. The package iteratively removes predictors of interest from the model and monitors the change in the variance of the linear predictor. The difference to the full model gives a measure of the amount of variance explained uniquely by a particular predictor or a set of predictors. partR2 also estimates structure coefficients as the correlation between a predictor and fitted values, which provide an estimate of the total contribution of a fixed effect to the overall prediction, independent of other predictors. Structure coefficients can be converted to the total variance explained by a predictor, here called  inclusive R2, as the square of the structure coefficients times total R2. Furthermore, the package reports beta weights (standardized regression coefficients). Finally,partR2 implements parametric bootstrapping to quantify confidence intervals for each estimate. We illustrate the use of partR2 with real example datasets for Gaussian and binomial GLMMs and discuss interactions, which pose a specific challenge for partitioning the explained variance among predictors.

sentences: Quantifying the phenotypic information in mRNA abundance Quantifying the dependency between mRNA abundance and downstream cellular phenotypes is a fundamental open problem in biology. Advances in multimodal single cell measurement technologies provide an opportunity to apply new computational frameworks to dissect the contribution of individual genes and gene combinations to a given phenotype. Using an information theory approach, we analyzed multimodal data of the expression of 83 genes in the Ca2+ signaling network and the dynamic Ca2+ response in the same cell. We found that the overall expression levels of these 83 genes explain approximately 60% of Ca2+ signal entropy. The average contribution of each single gene was 16%, revealing a large degree of redundancy between genes. Using different heuristics we estimated the dependency between the size of a gene set and its information content, revealing that on average a set of 53 genes contains 90% of the information about Ca2+ signaling within the cellular transcriptional state. Our results provide the first direct quantification of information content about complex cellular phenotype that exists in mRNA abundance measurements.

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Emx1-Cre is expressed in vagal afferent nerves that regulate central cardiorespiratory functions The Emx1-IRES-Cre transgenic mouse is commonly used to direct genetic recombination in forebrain excitatory neurons. However the original study reported that Emx1-Cre is also expressed in peripheral autonomic ganglia, which could potentially affect the interpretation of targeted circuitry contributing to systemic phenotypes. Here, we report that Emx1-Cre is expressed in nodose ganglion cells whose axon travel within afferent vagal nerves and terminate in the nucleus tractus solitarius (NTS), a critical central autonomic regulatory pathway located in the dorsal medulla. Optogenetic stimulation of Emx1-Cre+ fibers reliably evoked excitatory postsynaptic currents in the NTS in vitro and evoked a parasympathetic bradycardia reflex occasionally with irregular respirations in anesthetized mice in vivo. Our study clearly demonstrates that Emx1-IRES-Cre is expressed in the vagal nerve fibers traveling within the central autonomic system involving autonomic cardiorespiratory regulation. These results raise caution when interpreting the systemic phenotypes of Emx1-IRES-Cre conditional recombinant mice, and also suggest the utility of this line to investigate the modulators of afferent vagal system.

Significance StatementEmx1-IRES-Cre mice are widely used to dissect critical circuitry underlying neurological disorders such as epilepsy. These studies often assume the Cre is expressed selectively only in forebrain excitatory neurons. However, earlier works reported that Emx1 is expressed in several peripheral tissues of the developing embryo and thus gene recombination may affect these peripheral structures. In this study, we characterized the expression and physiological functions of Emx1-Cre expressed in the afferent vagus nerve. Optogenetic stimulation of these Emx1-Cre+ vagus nerves activates the nucleus tractus solitarius neurons within the brainstem in vitro and induces brief bradycardia in vivo. This study confirmed that peripheral Emx1-Cre+ cells are involved in autonomic regulation and potentially affect transgenic mouse phenotypes.

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Efficient and stochastic mouse action switching during probabilistic decision making In probabilistic and nonstationary environments, individuals must use internal and external cues to flexibly make decisions that lead to desirable outcomes. To gain insight into the process by which animals choose between actions, we trained mice in a task with time-varying reward probabilities. In our implementation of such a "two-armed bandit" task, thirsty mice use information about recent action and action-outcome histories to choose between two ports that deliver water probabilistically. Here, we comprehensively modeled choice behavior in this task, including the trial-to-trial changes in port selection - i.e. action switching behavior. We find that mouse behavior is, at times, deterministic and, at others, apparently stochastic. The behavior deviates from that of a theoretically optimal agent performing Bayesian inference in a Hidden Markov Model (HMM). We formulate a set of models based on logistic regression, reinforcement learning, and  sticky Bayesian inference that we demonstrate are mathematically equivalent and that accurately describe mouse behavior. The switching behavior of mice in the task is captured in each model by a stochastic action policy, a history-dependent representation of action value, and a tendency to repeat actions despite incoming evidence. The models parsimoniously capture behavior across different environmental conditionals by varying the  stickiness parameter, and, like the mice, they achieve nearly maximal reward rates. These results indicate that mouse behavior reaches near-maximal performance with reduced action switching and can be described by a set of equivalent models with a small number of relatively fixed parameters.

SignificanceTo obtain rewards in changing and uncertain environments, animals must adapt their behavior. We found that mouse choice and trial-to-trial switching behavior in a dynamic and probabilistic two-choice task could be modeled by equivalent theoretical, algorithmic, and descriptive models. These models capture components of evidence accumulation, choice history bias, and stochasticity in mouse behavior. Furthermore, they reveal that mice adapt their behavior in different environmental contexts by modulating their level of  stickiness to their previous choice. Despite deviating from the behavior of a theoretically ideal observer, the empirical models achieve comparable levels of near-maximal reward. These results make predictions to guide interrogation of the neural mechanisms underlying flexible decision-making strategies.

sentences: Cytonemes coordinate asymmetric signaling and organization in the Drosophila muscle progenitor niche Asymmetric signaling and organization in the stem-cell niche determine stem-cell fates. Here, we investigate the basis of asymmetric signaling and stem-cell organization using the Drosophila wing-disc that creates an adult muscle progenitor (AMP) niche. We show that AMPs extend polarized cytonemes to contact the disc epithelial junctions and adhere themselves to the disc/niche. Niche-adhering cytonemes localize FGF-receptor to selectively adhere to the FGF-producing disc and receive FGFs in a contact-dependent manner. Activation of FGF signaling in AMPs, in turn, reinforces disc-specific cytoneme polarity/adhesion, which maintains their disc-proximal positions. Loss of cytoneme-mediated adhesion promotes AMPs to lose niche occupancy and FGF signaling, occupy a disc-distal position, and acquire morphological hallmarks of differentiation. Discrete AMP organization and diversification patterns are determined by localized expression and presentation patterns of two different FGFs in the wing-disc and their polarized target-specific distribution through niche-adhering cytonemes. Thus, cytonemes are essential for asymmetric signaling and niche-specific AMP organization.

sentences: Gotree/Goalign : Toolkit and Go API to facilitate the development of phylogenetic workflows Besides computer intensive steps, phylogenetic analysis workflows are usually composed of many small, reccuring, but important data manipulations steps. Among these, we can find file reformatting, sequence renaming, tree re-rooting, tree comparison, bootstrap support computation, etc. These are often performed by custom scripts or by several heterogeneous tools, which may be error prone, uneasy to maintain and produce results that are challenging to reproduce. For all these reasons, the development and reuse of phylogenetic workflows is often a complex task. We identified many operations that are part of most phylogenetic analyses, and implemented them in a toolkit called Gotree/Goalign. The Gotree/Goalign toolkit implements more than 120 user-friendly commands and an API dedicated to multiple sequence alignment and phylogenetic tree manipulations. It is developed in Go, which makes executables efficient, easily installable, integrable in workflow environments, and parallelizable when possible. This toolkit is freely available on most platforms (Linux, MacOS and Windows) and most architectures (amd64, i386). Sources and binaries are available on GitHub at https://github.com/evolbioinfo/gotree, Bioconda, and DockerHub.

sentences: A co-opted ISG15-USP18 binding mechanism normally reserved for deISGylation controls type I IFN signalling Type I interferon (IFN) signalling induces the expression of several hundred IFN-stimulated genes that provide an unfavourable environment for viral replication. To prevent an overexuberant response and autoinflammatory disease, IFN signalling requires tight control. One critical regulator is the ubiquitin-like protein ISG15, evidenced by autoinflammatory disease in patients with inherited ISG15 deficiencies. Current models suggest that ISG15 stabilises USP18, a well-established negative regulator of IFN signalling. USP18 also functions as an ISG15-specific peptidase, however its catalytic activity is dispensable for controlling IFN signalling. Here, we show that the ISG15-dependent stabilisation of USP18 is necessary but not sufficient for regulation of IFN signalling and that USP18 requires non-covalent interactions with ISG15 to enhance its regulatory function. Intriguingly, this trait has been acquired through co-option of a binding mechanism normally reserved for deISGylation, identifying an unexpected new function for ISG15.

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Tick hazard in the South Downs National Park (UK): species, distribution, key locations for future interventions, site density, habitats BackgroundThe South Downs National Park (SDNP) is the UKs most visited National Park, and a foci of tick-borne Lyme disease. A range of human pathogens have been detected in UK ticks and related hosts, and the first presumed autochthonous cases of tick-borne encephalitis and babesiosis were recorded in 2019-20. SDNPs key objectives include conserving wildlife and encouraging enjoyment of the countryside, so interventions are needed that reduce hazard without negatively affecting ecosystem health. To be successful these require knowledge of site hazards, and we aimed to provide this to enable action.

MethodsBritish Deer Society volunteers submitted ticks removed from deer. Key potential intervention sites were selected and ticks collected by drag-sampling six 50 m2 transects per site, in most cases twice yearly for two years. Ticks were identified in-lab (sex, life stage, species), hazard measured as tick presence, Density of Ticks (all life stages, DOT), and Density of Nymphs (DON). Sites and habitat types were analysed for association with hazard. Distribution across SDNP was mapped in a Geographic Information System (GIS), by combining and comparing our fieldwork results with records from five other data sources (recent and historic).

Results87 Ixodes ricinus (all but one adults, 82%F) were removed from 14 deer (Dama dama n=10; Capreolus capreolus n=3; 1 not recorded; tick burden, 1-35) at 12 locations (commonly woodland). Five potential key intervention sites were identified and drag-sampled 2015-16, collecting 623 ticks (238 on-transects): 53.8% nymphs, 42.5% larvae, 3.7% adults (13M, 10F). Ticks were present on-transects at all sites drag-sampled (I.ricinus at three, Haemaphysalis punctata at two). The Mens (TM, the quietist site for human visitors) had the highest DOT at 30/300 m2 (DON=30/300 m2), followed by Queen Elizabeth Country Park (QECP, the busiest) at 22/300 m2 (12/300 m2), Cowdray Estate (CE) at 8/300 m2 (6/300 m2), and Seven Sisters Country Park (SSCP) at 1/300 m2 (1/300 m2). Ditchling Beacon Nature Reserve (DBNR) was sampled 2016 only (one adult H.punctata collected). Woodland had significantly higher hazard than grazed downland, but ticks were present at all downland sites drag-sampled. GIS mapping showed I.ricinus identified in 33/37 of SDNPs 10 km2 grid squares, Ixodes hexagonus 10/37, H.punctata 7/37, Dermacentor reticulatus 1/37.

ConclusionsMapping shows tick hazard is broadly distributed across SDNP. Ixodes ricinuswas most common, though the seeming range expansion of H.punctata is concerning, particularly as it seems to thrive better on grazed downland than I.ricinus. Site specific recommendations include: management of small high hazard plots with heavy visitor numbers (QECP); signage on post-visit precautions (all sites); repellent impregnated clothing for deerstalkers (CE); flock trials to control H.punctata (SSCP, DBNR). Further research at TM, which has high tick density, may contribute to knowledge on ecological dynamics underlying infection density, and the potential use of predator re-introduction/protection as a public health intervention. Ecological research on H.punctata would aid control. The SDNP Authority is ideally placed to link and champion site-based and regional policies to reduce hazard, whilst avoiding or reducing conflict between public health and ecosystem health.

sentences: Coding and regulatory variants affect serum protein levels and common disease Circulating proteins are prognostic for human outcomes including cancer, heart failure, brain trauma and brain amyloid plaque burden. A deep serum proteome survey recently revealed close associations of serum protein networks and common diseases. The present study reveals unprecedented number of individual serum proteins that overlap genetic signatures of diseases emanating from different tissues of the body. Here, 54,469 low-frequency and common exome-array variants were compared with 4782 protein measurements in the serum of 5343 individuals of the deeply annotated AGES Reykjavik cohort. Using a study-wide significant threshold, 2019 independent exome array variants affecting levels of 2135 serum proteins were identified. These variants overlapped genetic loci for hundreds of complex disease traits, emphasizing the emerging role for serum proteins as biomarkers of and potential causative agents of multiple diseases.

sentences: Evolution of multicellularity and unicellularity in yeast S. cerevisiae to study reversibility of evolutionary trajectories. Adaptive trajectories of populations have been focus of number of studies. However, adaptive trajectories have not been studied in the context of reverse evolution. By reverse evolution, we mean a scenario where selection is reversed. In this work, we use evolution (and reversal from) of multicellularity in S. cerevisiae as a model to answer this question. When selected for fast-settling variants, multicellularity evolves rapidly in the organism. On reversing selection, unicellularity evolves from the multicellular clusters. However, the dynamic trajectories of the two processes are different. In this context, evolution is not reversed dynamically at a phenotypic level. The phenotypic reversal is not driven by reversal of the original mutations during the forward evolution. Overall, our results show that the dynamics of molecular and phenotypic trajectories of evolution are distinct, and reversal of selection leads to unique trajectories of phenotypic reversal.

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Transcriptional evaluation of the ductus arteriosus at the single-cell level uncovers a requirement for vimentin for complete closure OBJECTIVEFailure to close the ductus arteriosus immediately post-birth, patent ductus arteriosus (PDA), accounts for up to 10% of all congenital heart defects. Despite significant advances in PDA management options, including pharmacological treatment targeting the prostaglandin pathway, a proportion of patients fail to respond and must undergo surgical intervention. Thus, further refinement of the cellular and molecular mechanisms that govern vascular remodeling of this vessel is required.

APPROACH AND RESULTSAs anticipated, single-cell RNA sequencing on the ductus arteriosus in mouse embryos at E18.5, P0.5, and P5, revealed broad transcriptional alterations in the endothelial, smooth muscle, and fibroblast cell compartments. Making use of these data sets, vimentin emerged as an interesting candidate for further investigation. Subsequent studies demonstrated that, in fact, mice with genetic deletion of vimentin fail to complete vascular remodeling of the ductus arteriosus, as per presence of a functional lumen.

CONCLUSIONSThrough single-cell RNA-sequencing and by tracking closure of the ductus arteriosus postnatally in mice, we uncovered the unexpected contribution of vimentin in driving complete closure of the ductus arteriosus potentially through regulation of the Notch signaling pathway.

HIGHLIGHTSO_LISingle-cell RNA-sequencing on the ductus arteriosus at E18.5, P0.5, and P5 reveals how the ductus arteriosus undergoes drastic transcriptional changes at the single-cell level.
C_LIO_LIEndothelial cells increase levels of Vimentin, Notch1 and Jag1 transcripts soon after birth (P0.5), concurrent with ductus arteriosus closure.
C_LIO_LILoss of vimentin, the major intermediate filament protein of endothelial cells, prevents proper permanent closure of the ductus arteriosus.
C_LI

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A hemifused complex is the hub in a network of pathways to membrane fusion Membrane fusion is a critical step for many essential processes, from neurotransmission to fertilization. For over 40 years protein-free fusion driven by calcium or other cationic species has provided a simplified model of biological fusion, but the mechanisms remain poorly understood. Cation-mediated membrane fusion and permeation are essential in their own right to drug delivery strategies based on cell-penetrating peptides or cation-bearing lipid nanoparticles. Experimental studies suggest calcium drives anionic membranes to a hemifused intermediate which constitutes a hub in a network of pathways, but the pathway selection mechanism is unknown. Here we develop a mathematical model that identifies the network hub as a highly dynamical hemifusion complex. Multivalent cations drive expansion of this high tension hemifusion interface between interacting vesicles during a brief transient. The fate of this interface determines the outcome, either fusion, dead-end hemifusion or vesicle lysis. The model reproduces the unexplained finding that calcium-driven fusion of vesicles with planar membranes typically stalls at hemifusion, and we show the equilibrated hemifused state is a novel lens-shaped complex. Thus, membrane fusion kinetics follow a stochastic trajectory within a network of pathways, with outcome weightings set by a hemifused complex intermediate.

SignificanceCells use multicomponent machineries to fuse membranes for neurotransmitter and hormone release and other fundamental processes. Protein-free fusion using calcium or other multivalent cationic fusogens has long been studied as a simplifying model. Cation-mediated membrane fusion or permeation are key events for a number of current drug delivery strategies. However, the mechanisms determining outcomes are unknown. Here we develop a mathematical model that identifies a dynamic hemifusion complex as the decision hub that stochastically sets the outcome in a network of pathways. Cations transiently grow a high tension hemifusion interface between membrane-enclosed compartments, whose fate governs whether fusion, dead-end hemifusion or vesicle lysis occurs. The model provides a systematic framework to predict outcomes of cationic fusogen-mediated interactions between membrane-enclosed compartments.

sentences: Sex differences in pain-related behaviors and clinical progression of disease in mouse models of visceral pain Previous studies have reported sex differences in irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) patients, including differences in visceral pain perception. Despite this, sex differences in behavioral manifestations of visceral pain and underlying pathology of the gastrointestinal tract have been largely understudied in preclinical research. In this study, we evaluated potential sex differences in spontaneous visceral nociceptive responses, referred abdominal hypersensitivity, disease progression and bowel pathology in mouse models of acute and persistent colon inflammation. Our experiments show that females exhibit more visceral nociceptive responses and referred abdominal hypersensitivity than males in the context of acute but not persistent colon inflammation. We further demonstrate that, following acute and persistent colon inflammation, visceral pain-related behavioral responses in females and males are distinct, with increases in licking of the abdomen only observed in females and increases in abdominal contractions only seen in males. During persistent colon inflammation, males exhibit worse disease progression than females, which is manifested as worse physical appearance and higher weight loss. However, no measurable sex differences were observed in persistent inflammation-induced bowel pathology, stool consistency or fecal blood. Overall, our findings demonstrate that visceral pain-related behaviors and disease progression in the context of acute and persistent colon inflammation are sex-dependent, highlighting the importance of considering sex as a biological variable in future mechanistic studies of visceral pain as well as in the development of diagnostics and therapeutic options for chronic gastrointestinal diseases.

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Multidimensional Single-Nuclei RNA-Seq Reconstruction of Adipose Tissue Reveals Adipocyte Plasticity Underlying Thermogenic Response Adipose tissue has been classified based on its morphology and function as white, brown, or beige / brite. It plays an essential role as a regulator of systemic metabolism through paracrine and endocrine signals. Recently, multiple adipocyte subtypes have been revealed using RNA sequencing technology, going beyond simply defined morphology but by their cellular origin, adaptation to metabolic stress, and plasticity. Here, we performed an in-depth analysis of publicly available single-nuclei RNAseq from adipose tissue and utilized a workflow template to characterize adipocyte plasticity, heterogeneity, and secretome profiles. The reanalyzed dataset led to the identification of different subtypes of adipocytes including three subpopulations of thermogenic adipocytes and provided a characterization of distinct transcriptional profiles along the adipocyte trajectory under thermogenic challenges. This study provides a useful resource for further investigations regarding mechanisms related to adipocyte plasticity and trans-differentiation.

HighlightsMultidimensional transcriptome analysis at single-nucleus resolution recovers nuclei of cell types in adipose tissue

Adaptative thermogenic response results in 3 distinct mature adipose cell types

Single-nuclei transcriptomic-based secretome analysis reveals adipose cell-type-specific genes

The in vivo trajectory of adipocyte plasticity for thermogenic response reveals sets of trans-differentiation genes

Graphic Abstract

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sentences: Systematic mapping of small nucleolar RNA targets in human cells Altered expression of box C/D small nucleolar RNAs (snoRNAs) is implicated in human diseases, including cancer. Box C/D snoRNAs canonically direct site-specific, 2-O-methylation but the extent to which they participate in other functions remains unclear. To identify RNA targets of box C/D snoRNAs in human cells, we applied two techniques based on UV crosslinking, proximity ligation and sequencing of RNA hybrids (CLASH and FLASH). These identified hundreds of novel snoRNA interactions with rRNA, snoRNAs and mRNAs. We developed an informatic pipeline to rigorously call interactions predicted to direct methylation. Multiple snoRNA-rRNA interactions identified were not predicted to direct RNA methylation. These potentially modulate methylation efficiency and/or contribute to folding dynamics. snoRNA-mRNA hybrids included 1,300 interactions between 117 snoRNA families and 940 mRNAs. Human U3 is substantially more abundant than other snoRNAs and represented about 50% of snoRNA-mRNA hybrids. The distribution of U3 interactions across mRNAs also differed from other snoRNAs. Following U3 depletion, mRNAs showing altered abundance were strongly enriched for U3 CLASH targets. Most human snoRNAs are excised from pre-mRNA introns. Enrichment for snoRNA association with branch point regions of introns that contain snoRNA genes was common, suggesting widespread regulation of snoRNA maturation.

sentences: Nature-inspired Circular-economy Recycling (NaCRe) for Proteins: Proof of Concept The billion tons of synthetic polymer-based materials (i.e. plastics) produced every year are one of the greatest challenges that humanity has to face. Nature produces even more natural polymers, yet they are sustainable. For example, proteins are sequence-defined natural polymers, that are constantly recycled when living systems feed. Indeed, digestion is the protein depolymerization into amino acids (i.e. the monomers) followed by their re-assembly into new proteins of arbitrarily different sequence and function. This process breaks a common recycling paradigm where a material is recycled into itself. Organisms feed of random protein mixtures that are  recycled into new proteins whose identity depends on the cells needs at the time of protein synthesis. Currently, advanced materials are increasingly made of proteins, but the abovementioned ideal recyclability of such materials has yet to be recognized and established. In this study mixtures of several (up to >30) peptides and/or proteins were depolymerized into their amino acid constituents, and these amino acids were used to synthesize new fluorescent, and bio-active proteins extra-cellularly by using an amino acid-free cell-free transcription-translation system. Proteins with high relevance in materials engineering ({beta}-lactoglobulin films, used for water filtration, or silk fibroin solutions) were successfully recycled into biotechnologically relevant proteins (green, and red fluorescent proteins, catechol 2,3-dioxygenase). The potential long-term impact of this approach to recycling lies in its compatibility with circular-economy models where raw materials remain in use as long as possible, thus reducing the burden on the planet.

sentences: Captive-breeding and catch-and-release's effects on the reproductive success of Atlantic salmon (Salmo salar L.) Captive-breeding programs as well as and catch-and-release are among the most commonly adopted conservation practices in recreational fisheries. However, risks and benefits associated with their implementation are rarely evaluated. In the case of Atlantic Salmon, while previous studies revealed that captive-bred fish show reduced fitness compared to their wild counterparts in nature. Yet, few examined the extent and causes of their reduced reproductive success or directly compared their contribution to enhance genetic diversity to that of wild fish, including mature male parr. Furthermore, only one study specifically measured the reproductive success of caught and released Atlantic salmon in natural settings, and no study to date evaluated if released salmon are able to reproduce when released at temperature above 20{degrees}C which is known to increase post-release mortality. Here, we use high-throughput microsatellite sequencing of 38 loci to accurately assign 2500 offspring to a comprehensive set of possible parents from a supplemented Atlantic salmon population in Quebec, Canada. The resolved molecular pedigree provided informative insight on the reproductive pattern of both captive-bred salmon and caught-and-released salmon. Captive-bred salmon had fewer partners than their wild conspecifics which lead to a significant reduction of reproductive success relative to that of their wild counterparts. Supplementation of captive-bred salmon significantly contributed to increase genetic diversity but mature male parr did so to an even greater extent and significantly inflated the number of alleles found among offspring. Moreover, our results showed that that at least 83% of caught-and-released salmon did successfully reproduced although caught-and-released female salmon have a significantly reduced reproductive success, averaging 73% of the reproductive output of non-caught salmon. Reproductive success of released salmon was not influenced by water temperature over 20{degrees}C which suggests either that the studied population is locally adapted to warm waters or that they behaviorally regulated body temperature by accessing nearby thermal refugia. Our results should help refining managers ability to analyze the risks and benefits associated with captive-breeding and catch-and-release, and thus, optimize conservation practices used for the preservation of Atlantic salmon populations.

sentences: On the dynamics of spatial updating Most of our knowledge on the human neural bases of spatial updating comes from fMRI studies in which recumbent participants moved in virtual environments. As a result, little is known about the dynamic of spatial updating during real body motion. Here, we exploited the high temporal resolution of electroencephalography (EEG) to investigate the dynamics of cortical activation in a spatial updating task where participants had to remember their initial orientation while they were passively rotated about their vertical axis in the dark. After the rotations, the participants pointed towards their initial orientation. We contrasted the EEG signals with those recorded in a control condition in which participants had no cognitive task to perform during body rotations. We found that the amplitude of the P1N1 complex of the rotation-evoked potential (RotEPs) (recorded over the vertex) was significantly greater in the Updating task. The analyses of the cortical current in the source space revealed that the main significant task-related cortical activities started during the N1P2 interval (136-303 ms after rotation onset). They were essentially localised in the temporal and frontal (supplementary motor complex, dorsolateral prefrontal cortex, anterior prefrontal cortex) regions. During this time-window, the right superior posterior parietal cortex (PPC) also showed significant task-related activities. The increased activation of the PPC became bilateral over the P2N2 component (303-470 ms after rotation onset). In this late interval, the cuneus and precuneus started to show significant task-related activities. Together, the present results are consistent with the general scheme that the first task-related cortical activities during spatial updating are related to the encoding of spatial goals and to the storing of spatial information in working memory. These activities would precede those involved in higher order processes also relevant for updating body orientation during rotations linked to the egocentric and visual representations of the environment.

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Microsecond Melting and Revitrification of Cryo Samples - Protein Structure and Beam-Induced Motion We have recently introduced a novel approach to time-resolved cryo-electron microscopy (cryo-EM) that involves melting a cryo sample with a laser beam to allow protein dynamics to briefly occur in liquid, before trapping the particles in their transient configurations by rapidly revitrifying the sample. With a time-resolution of just a few microseconds, this approach is notably fast enough to study domain motions that are typically associated with the activity of proteins, but which have previously remained inaccessible. Here, we add crucial details to the characterization of our method. We show that single-particle reconstructions of apoferritin and cowpea chlorotic mottle virus (CCMV) from revitrified samples are indistinguishable from those in conventional samples, demonstrating that melting and revitrification leaves the particles intact and that they do not undergo structural changes within the spatial resolution afforded by our instrument. We also characterize how rapid revitrification affects the properties of the ice, showing that revitrified samples exhibit comparable amounts of beam-induced motion. Our results pave the way for microsecond time-resolved studies of the conformational dynamics of proteins and open up new avenues to study the vitrification process and address beam-induced specimen movement.

SynopsisMicrosecond melting and revitrification of cryo samples preserves the structure of embedded particles. The beam-induced motion of revitrified samples is comparable to that of conventional cryo samples.

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Protein undernutrition reduces the efficacy of praziquantel in a murine model of Schistosoma mansoni infection BackgroundUndernutrition and schistosomiasis are public health problems and often occur in low and middle-income countries. Protein undernutrition can alter the host-parasite environment system and aggravate the course of schistosomiasis. This study aimed to assess the impact of a protein-deficient diet on the efficacy of praziquantel.

Methodology/principal findingsThirty-day-old mice were fed with a protein-deficient diet, and 40 days later, they were individually infected with fifty Schistosoma mansoni cercariae. A 28-day-treatment with praziquantel at 100 mg/kg for five consecutive days followed by distilled water begins at the 36th day post-infection. Mice were sacrificed on the 64th day post-infection. We determined the parasitological burden, liver and intestine histomorphometry, liver injury, and immunomodulation parameters. Praziquantel treatment of infected mice fed with a standard diet (IN-PZQ) resulted in a significant reduction of worm and egg burdens and a normalization of iron and calcium levels. The therapy also improved schistosomiasis-induced hepatopathy and oxidative stress. The anti-inflammatory and immunomodulatory activities of praziquantel were also significant in these mice. When infected mice receiving the protein-deficient diet were treated with praziquantel (ILP-PZQ), the body weight loss and hepatomegaly were not alleviated, and the worm and liver egg burdens were significantly higher than those of IN-PZQ mice (P < 0.001). The treatment did not reduce the increased activities of ALT and {gamma}-GGT, the high malondialdehyde concentration, and the liver granuloma volume. The iron and calcium levels were not ameliorated and differed from those of IN-PZQ mice (P < 0.001 and P < 0.05). Moreover, in these mice, praziquantel treatment did not reverse the high level of IL-5 and the low mRNA expression of CCL3/MIP-1 and CXCL-10/IP-10 induced by S. mansoni infection.

Conclusion/significanceThese results demonstrated that a protein-deficient diet reduced the schistosomicidal, antioxidant, anti-inflammatory, and immunomodulatory activities of praziquantel.

Authors summaryAlmost 90% of people requiring schistosomiasis preventive chemotherapy in 2018 lived in sub-Saharan Africa. Besides, 205.3 million children under five years suffer and die of undernutrition in low- and middle-income countries. The physiopathology of schistosomiasis mansoni involves liver damage, oxidative stress, and perturbation of the immune response. These disturbances are intensified by undernutrition. Praziquantel is used to treat schistosomiasis, but its efficacy on the comorbidity of S. mansoni infection and undernutrition has not been investigated. We conducted this study to assess the effectiveness of praziquantel on S. mansoni infection in mice fed with a protein-deficient diet. We recorded growth retardation, hepatomegaly, and high worm and egg burdens in mice fed with a protein-deficient diet and treated with PZQ. Moreover, the treatment did not reverse the liver function injury, oxidative stress, high iron level, and low calcium level. The proinflammatory cytokine IL-5 was still high, and the gene expression of some macrophage-associated chemokines was reduced. Therefore, this study demonstrated that in a murine model of a protein-deficient diet, the efficacy of praziquantel on S. mansoni infection was reduced. It also underlines the importance of targeting protein deficiency and malnutrition in populations living in schistosomiasis endemic areas for efficient disease control.

sentences: NBI-921352, a First-in-Class, Na V 1.6 Selective, Sodium Channel Inhibitor That Prevents Seizures in Scn8a Gain-of- Function Mice, and Wild-Type Mice and Rats NBI-921352 (formerly XEN901) is a novel sodium channel inhibitor designed to specifically target NaV1.6 channels. Such a molecule provides a precision-medicine approach to target SCN8A-related epilepsy syndromes (SCN8A-RES), where gain-of-function (GoF) mutations lead to excess NaV1.6 sodium current, or other indications where NaV1.6 mediated hyper-excitability contributes to disease (Gardella and Moller, 2019; Johannesen et al., 2019; Veeramah et al., 2012). NBI-921352 is a potent inhibitor of NaV1.6 (IC50 0.051 {micro}M), with exquisite selectivity over other sodium channel isoforms (selectivity ratios of 756X for NaV1.1, 134X for NaV1.2, 276X for NaV1.7, and >583X for NaV1.3, NaV1.4, and NaV1.5). NBI-921352 is a state-dependent inhibitor, preferentially inhibiting activated (inactivated or open) channels. The state dependence leads to potent stabilization of inactivation, inhibiting NaV1.6 currents, including resurgent and persistent NaV1.6 currents, while sparing the closed/rested channels. The isoform-selective profile of NBI-921352 led to a robust inhibition of action-potential firing in glutamatergic excitatory pyramidal neurons, while sparing fast-spiking inhibitory interneurons, where NaV1.1 predominates. Oral administration of NBI-921352 prevented electrically induced seizures in a Scn8a GoF mouse, as well as in wild-type mouse and rat seizure models. NBI-921352 was effective in preventing seizures at lower brain and plasma concentrations than commonly prescribed sodium channel inhibitor antiseizure medicines (ASMs) carbamazepine, phenytoin, and lacosamide. NBI-921352 was well tolerated at higher multiples of the effective plasma and brain concentrations than those ASMs. NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A-developmental epileptic encephalopathy (SCN8A-DEE) and adult focal-onset seizures.

sentences: Multiscale modeling of tumor adaption and invasion following anti-angiogenic therapy In order to promote continued growth, a tumor must recruit new blood vessels, a process known as tumor angiogenesis. Many therapies have been tested that aim to inhibit tumor angiogenesis, thus starving the tumor of nutrients and preventing tumor growth. However, many of these therapies have been unsuccessful and can paradoxically further tumor development by leading to increased local tumor invasion and metastasis. In this study, we use agent-based modeling to examine how hypoxic and acidic conditions following anti-angiogenic therapy can influence tumor development. Under these conditions, we find that cancer cells experience a phenotypic shift to a state of higher survival and invasive capability, spreading further away from the tumor into surrounding tissue. Although anti-angiogenic therapy alone promotes tumor cell adaptation and invasiveness, we find that augmenting chemotherapy with anti-angiogenic therapy improves chemotherapeutic response and delays the time it takes for the tumor to regrow. Overall, we use computational modeling to explain the behavior of tumor cells in response to anti-angiogenic treatment in the dynamic tumor microenvironment.

sentences: Optogenetic relaxation of actomyosin contractility uncovers mechanistic roles of cortical tension during cytokinesis Actomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, it is still unknown how actomyosin contractility generates force and maintains cellular morphology. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility. The system, named OptoMYPT, combines a catalytic subunit of the type I phosphatase-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination was sufficient to induce dephosphorylation of myosin regulatory light chains and decrease in traction force at the subcellular level. The OptoMYPT system was further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We found that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system will provide new opportunities to understand cellular and tissue mechanics.

sentences: How plants minimize somatic evolution A remarkable property of plants is their ability to accumulate mutations at a very slow pace despite their potentially long lifespans, during which they continually form buds, each with the potential to become a new branch. Because replication errors in cell division represent an unavoidable source of mutations, minimizing mutation accumulation requires the minimization of cell divisions. Here we show that there exists a well defined theoretical minimum for the branching cost, defined as the number of cell divisions necessary for the creation of each branch. Most importantly, we also show that this theoretical minimum can be closely approached by a simple pattern of cell divisions in the meristematic tissue of apical buds during the generation of novel buds. Both the optimal pattern of cell divisions and the associated branching cost are consistent with recent experimental data, suggesting that plant evolution has led to the discovery of this mechanism.

sentences: Micro-region transcriptomics of fixed human tissue using Pick-Seq Spatial transcriptomics and multiplexed imaging are complementary methods for studying tissue biology and disease. Recently developed spatial transcriptomic methods use fresh-frozen specimens but most diagnostic specimens, clinical trials, and tissue archives rely on formaldehyde-fixed tissue. Here we describe the Pick-Seq method for deep spatial transcriptional profiling of fixed tissue. Pick-Seq is a form of micro-region sequencing in which small regions of tissue, containing 5-20 cells, are mechanically isolated on a microscope and then sequenced. We demonstrate the use of Pick-Seq with several different fixed and frozen human specimens. Application of Pick-Seq to a human melanoma with complex histology reveals significant differences in transcriptional programs associated with tumor invasion, proliferation, and immuno-editing. Parallel imaging confirms changes in immuno-phenotypes and cancer cell states. This work demonstrates the ability of Pick-Seq to generate deep spatial transcriptomic data from fixed and archival tissue with multiplexed imaging in parallel.

sentences: Development of methods for purification of individual biological active substances obtained from extracts of Hedysarum neglectum Hedysarum neglectum is a forage plant. Xanthone glycoside - mangiferin is extracted from this plant and used for medicine "Alpizarin". In addition to substances of xanthone nature (mangiferin and isomangiferin) Hedysarum neglectum contains sugars, vitamins and provitamins, tannins; in the underground part it contains oligomeric catechins, isoflavonoids, butylphenols, alkaloids, tannins, flavonoids, saponins, coumarins, carbohydrates, vitamin C. For selecting optimal schemes of fractionization of substances, it is necessary to resort to multi-stage schemes of group-wide (preliminary) isolation and preparative accumulation. In particular cases, it is necessary to take into account the presence of concomitant substances, as well as the effectiveness and selectivity of the sorption-chromatographic technologies used. According to the results of the studies, the use of silica gel and sefadex LH-20 for the isolation of a complex of flavonoids and gallic acid is the most effective method for the selection of the optimal variant of the preparative isolation of the total amount of BAS in mcg/ml. The results of the research allowed us to identify the target biologically active substances with a degree of extraction of at least 80%: - fractions of xanthones, flavonoids, gallic acid.

sentences: Structural insight into the individual variability architecture of the functional brain connectome Human cognition and behaviors depend upon the brains functional connectomes, which vary remarkably across individuals. However, whether and how the functional connectome individual variability architecture is structurally constrained remains largely unknown. Using tractography- and morphometry-based network models, we observed the spatial convergence of structural and functional connectome individual variability, with higher variability in heteromodal association regions and lower variability in primary regions. We demonstrated that functional variability is significantly predicted by a unifying structural variability pattern and that this prediction follows a primary-to-heteromodal hierarchical axis, with higher accuracy in primary regions and lower accuracy in heteromodal regions. We further decomposed group-level connectome variability patterns into individual unique contributions and uncovered the structural-functional correspondence that is associated with individual cognitive traits. These results advance our understanding of the structural basis of individual functional variability and suggest the importance of integrating multimodal connectome signatures for individual differences in cognition and behaviors.

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Toxicity Profile of Artesunate in Rats and Dogs AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSOBJECTIVESC_ST_ABSThe objectives of these studies were to investigate the toxicity, safety and toxicokinetics of single and multiple doses of artesunate for injection in rats and dogs.

METHODSSprague-Dawley rats and Beagle dogs were treated intravenously or intramuscularly for 28 consecutive days with doses of up to 30 mg/kg artesunate, evaluating toxicity, kinetics, genotoxicity, and cardiovascular and central nervous safety parameters after single and 4-week repeated administrations. Furthermore, respiratory parameters were evaluated after a single intravenous administration in rats.

RESULTSArtesunate was well tolerated with no mortality and only minor effects on clinical pathology parameters. Following repeated intramuscular administration, local reactions at the injection site became evident.

CONCLUSIONSThe results obtained in these studies support the safe use of artesunate in humans.

Subchronic data for repeated intravenous and intramuscular artesunate in animals are available in the literature for a maximum duration of 14-days. Published results of genotoxicity studies in vitro in bacteria or cells of animal or human origin and in vivo in animals are equivocal, with a couple of negative results following oral administration and positive results in human cells.

With the studies performed in rats and dogs for a treatment period of 28-days including measures of safety pharmacology, toxicokinetic evaluation and in vivo genotoxicity data, a comparison of toxicity and exposure of intravenous versus intramuscular artesunate for a prolonged treatment period is possible, delivering important information, when a decision between both routes has to be made for use in patients. Results of the peripheral blood micronucleus assay in rats were positive, suggesting genotoxic potential for the intravenous route of administration of artesunate, what is, beside patients, likewise relevant for production and health care personnel.

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Time series analysis of trial-to-trial variability of MEG power spectrum during rest state, unattented listening and frequency-modulated tones classification The nonstationarity of EEG/MEG signals is important for understanding the functioning of human brain. From the previous research we know that even very short, i.e. 250--500ms MEG signals are variance-nonstationary. The covariance of stochastic process is mathematically associated with its spectral density, therefore we investigate how the spectrum of such nonstationary signals varies in time.

We analyze the data from 148-channel MEG, that represent rest state, unattented listening and frequency-modulated tones classification. We transform short-time MEG signals to the frequency domain using the FFT algorithm and for the dominant frequencies 8--12 Hz we prepare the time series representing their trial-to-trial variability. Then, we test them for level- and trend-stationarity, unit root, heteroscedasticity and gaussianity and based on their properties we propose the ARMA-modelling for their description.

The analyzed time series have the weakly stationary properties independently of the functional state of brain and localization. Only their small percentage, mostly related to the cognitive task, still presents nonstationarity. The obtained mathematical models show that the spectral density of analyzed signals depends on only 2--3 previous trials.

The presented method has limitations related to FFT resolution and univariate models, but it is not computationally complicated and allows to obtain a low-complex stochastic models of the EEG/MEG spectrum variability.

Although the physiological short-time MEG signals are in principle nonstationary in time domain, its power spectrum at the dominant frequencies varies as weakly stationary stochastic process. Described technique has the possible applications in prediction of the EEG/MEG spectral properties in theoretical and clinical neuroscience.

sentences: A distinct brain expression profile for genes within large introgression deserts and under positive selection in Homo sapiens Analyses of ancient DNA from extinct hominins have provided unique insights into the complex evolutionary history of Homo sapiens, intricately related to that of the Neanderthals and the Denisovans as revealed by several instances of admixture events. These analyses have also allowed the identification of introgression deserts: genomic regions in our species that are depleted of  archaic haplotypes. The presence of genes like FOXP2 in these deserts has been taken to be suggestive of brain-related functional differences between Homo species. Here, we seek a deeper characterization of these regions, taking into account signals of positive selection in our lineage. Analyzing publicly available transcriptomic data from the human brain at different developmental stages, we found that structures outside the cerebral neocortex, in particular the cerebellum, the striatum and the mediodorsal nucleus of the thalamus show the most divergent transcriptomic profiles when considering genes within large introgression deserts and under positive selection.

sentences: Characterization Of Interspecific Gene Flows at The Genome-wide Level in a Natural Ecosystem The Massane Forest Reveals New Insights Into Horizontal Transfer In plants. Horizontal transfer (HT) refers to the exchange of genetic material between divergent species by mechanisms other than reproduction. In recent years, several studies have demonstrated HTs in eukaryotes, particularly in the context of parasitic relationships and in model species. However, very little is known about HT in natural ecosystems, especially those involving non-parasitic wild species, and the nature of the ecological relationships that promote these HTs. In this work, we conducted a pilot study investigating HTs by sequencing the genomes of 17 wild non-model species from a natural ecosystem, the Massane forest, located in southern France. To this end, we developed a new computational pipeline called INTERCHANGE that is able to characterize HTs at the whole genome level without prior annotation and directly in the raw sequencing reads. Using this pipeline, we identified 12 HT events, half of which occurred between lianas and trees. We found that only LTRs-retrotransposons and predominantly those from the Copia superfamily were transferred between these wild species. This study revealed a possible new route for HTs between non-parasitic plants and provides new insights into the genomic characteristics of horizontally transferred DNA in plant genomes.

sentences: An olfactory pattern generator for on-demand combinatorial control of receptor activities Olfactory systems employ combinatorial receptor codes for odors. Systematically generating stimuli that address the combinatorial possibilities of an olfactory code poses unique challenges. Here, we present a stimulus method to probe the combinatorial code, demonstrated using the Drosophila larva. This method leverages a set of primary odorants, each of which targets the activity of one olfactory receptor neuron (ORN) type at an optimal concentration. Our setup uses microfluidics to mix any combination of primary odorants on demand to activate any desired combination of ORNs. We use this olfactory pattern generator to demonstrate a spatially distributed olfactory representation in the dendrites of a single interneuron in the antennal lobe, the first olfactory neuropil of the larva. In the larval mushroom body, the next processing layer, we characterize diverse receptive fields of a population of Kenyon cells. The precision and flexibility of the olfactory pattern generator will facilitate systematic studies of processing and transformation of the olfactory code.

sentences: Nickel-Sulfonate Mode of Substrate Binding for Forward and Reverse Reactions of Methyl-SCoM Reductase Suggest a Radical Mechanism Involving Long Range Electron Transfer Methyl-coenzyme M reductase (MCR) catalyzes both synthesis and anaerobic oxidation of methane (AOM). Its catalytic site contains Ni at the core of Cofactor F430. The Ni ion, in its low-valent Ni(I) state lights the fuse leading to homolysis of the C-S bond of methyl-coenzyme M (methyl-SCoM) to generate a methyl radical, which abstracts a hydrogen atom from Coenzyme B (HSCoB) to generate methane and the mixed disulfide CoMSSCoB. Direct reversal of this reaction activates methane to initiate anaerobic methane oxidation. Based on crystal structures, which reveal a Ni-thiol interaction between Ni(II)-MCR and inhibitor CoMSH, a Ni(I)-thioether complex with substrate methyl-SCoM has been transposed to canonical MCR mechanisms. Similarly, a Ni(I)-disulfide with CoMSSCoB is proposed for the reverse reaction. However, this Ni(I)-sulfur interaction poses a conundrum for the proposed hydrogen atom abstraction reaction because the >6 [A] distance between the thiol group of SCoB and the thiol of SCoM observed in the structures appears too long for such a reaction. Spectroscopic, kinetic, structural and computational studies described here establish that both methyl-SCoM and CoMSSCoB bind to the active Ni(I) state of MCR through their sulfonate groups, forming a hexacoordinate Ni(I)-N/O complex, not Ni(I)-S. These studies rule out direct Ni(I)-sulfur interactions in both substrate-bound states. As a solution to the mechanistic conundrum, we propose that both forward and reverse MCR reactions emanate through long-range electron transfer from Ni(I)-sulfonate complexes with methyl-SCoM or CoMSSCoB, respectively.

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MITOCHONDRIAL GENOME REVEALS CONTRASTING PATTERN OF ADAPTIVE SELECTION IN TURTLES AND TORTOISES Testudinoidea represents an evolutionarily unique taxon comprising both turtles and tortoises. The contrasting habitats that turtles and tortoises inhabit are associated with unique physio-ecological challenges hence enable distinct adaptive evolutionary strategies. To comparatively understand the pattern and strength of Darwinian selection and physicochemical evolution in turtle and tortoise mitogenomes, we employed adaptive divergence and selection analyses. We evaluated changes in structural and biochemical properties, and codon models on the mitochondrial protein-coding genes (PCGs) among three turtles and a tortoise lineage. We used mitochondrial PCGs that constitute the crucial oxidative phosphorylation (OXPHOS) respiratory system, a critical metabolic regulator which assumes key significance in energy regulation of ectotherms.

We detected strong evidence of positive selection along the turtle lineages: Geoemydidae, Emydidae, and Platysternidae, but relatively weak signals in tortoises. The Platysternidae turtles revealed the highest gene and site-wise positive selection. In turtles, positively selected sites were prevalent in NAD2 and NAD4 genes in OXPHOS Complex I, and COB gene of Complex III, indicating convergent adaptive evolution. Besides, NAD3 was the only subunit that showed adaptive selection in both turtles and tortoises, expressing its relevance for all Testudinoidea. Structural and functional analysis revealed many sites and physiochemical changes in important conserved as well as biomedically significant regions, suggesting the influence of adaptive pressure on mitogenome functions. Hence, our study furnished novel evidence of contrasting evolutionary selective pressure acting on closely related groups such as turtles and tortoises with unique habitat preferences and associated eco-physiological challenges.

sentences: Non-toxigenic Vibrio cholerae challenge strains for evaluating vaccine efficacy and inferring mechanisms of protection Human challenge studies are instrumental for testing cholera vaccines, but these studies use outdated strains and require inpatient facilities. Here, we created next-generation isogenic Ogawa and Inaba V. cholerae challenge strains (ZChol strains) derived from a contemporary Zambian clinical isolate representative of current dominant pandemic V. cholerae. To minimize the risk of severe diarrhea these strains were rendered non-toxigenic, since antibody responses which limit V. cholerae colonization are the primary mechanism of immune protection. These strains did not cause diarrhea in infant mice and proved to accurately gauge reduction in intestinal colonization mediated by effective vaccination. They are also valuable as targets for measuring vibriocidal antibody responses. Using barcoded ZChol strains, we discovered that vaccination tightens the infection bottleneck without restricting pathogen expansion in vivo. ZChol strains have the potential to enhance the safety, relevance, and scope of future cholera vaccine challenge studies and be valuable reagents for studies of immunity to cholera.

sentences: Tracing back variations in archaeal cell division to protein domain architectures The Endosomal Sorting Complex Required for Transport (ESCRT) system is a multi-protein machinery that is involved in cell division of both Eukaryotes and Archaea. This spread across super-phyla suggests that a precursor ESCRT machinery existed already at an evolutionary early stage of life, making it a promising candidate for the (re)construction of a minimal cell division machinery. There are, however, only few experimental data about ESCRT machineries in Archaea, due to high technical challenges in cultivation and microscopy. Here, we analyse the proteins of archaeal ESCRT machineries bioinformatically on a domain level, to enable mechanistical comparison without such challenging experiments. First, we infer that there are at least three different cell division mechanisms utilizing ESCRT proteins in archaea, probably similar in their constriction mechanisms but different in membrane tethering. Second, we show that ESCRT proteins in Asgard archaea are highly similar to eukaryotic ESCRT proteins, strengthening the recently developed idea that all Eukaryotes descended from archaea. Third, we reconstruct a plausible evolutionary development of ESCRT machineries and suggest that a simple ESCRT-based constriction machinery existed in the last archaeal common ancestor. These findings not only give very interesting insights into the evolution of cell division in Archaea and Eukaryotes, but also reveal new research avenues by suggesting hypothesis-driven experiments for both, cell biology and bottom-up synthetic biology.

sentences: Understanding the emergence of contingent and deterministic exclusion in multispecies communities Competitive exclusion can be classified as deterministic or as historically contingent. While competitive exclusion is common in nature, it has remained unclear when multispecies communities should be dominated by deterministic or contingent exclusion. Here, we provide a general theoretical approach to explain both the emergence and sources of competitive exclusion in multispecies communities. We illustrate our approach on an empirical competition system between annual and perennial plant species. First, we find that the life-history of perennial species increases the probability of observing contingent exclusion by increasing their effective intrinsic growth rates. Second, we find that the probability of observing contingent exclusion increases with weaker intraspecific competition, and not with the level of hierarchical competition. Third, we find a shift from contingent exclusion to dominance with increasing numbers of competing species. Our work provides a heuristic framework to increase our understanding about the predictability of species persistence within multispecies communities.

sentences: Information transfer in mammalian glycan-based communication Glycan-binding proteins, so-called lectins, are exposed on mammalian cell surfaces and decipher the information encoded within glycans translating it into biochemical signal transduction pathways in the cell. These glycan-lectin communication pathways are complex and difficult to analyse. However, quantitative data with single cell resolution provides means to disentangle the associated signalling cascades. We chose C-Type lectin receptors (CLRs) expressed on immune cells as a model system to study their capacity to transmit information encoded in glycans of incoming particles. Lectin receptor NF{kappa}B-reporter cell lines expressing DC-SIGN, MCL, dectin-1, dectin-2, and mincle, as well as TNFR and TLR-1&2 in monocytic cell lines were characterized by comparing their efficiency to transmit glycan-encoded information. The information content was measured by following NF{kappa}B dependent GFP expression. While most receptors did transmit information to NF{kappa}B efficiently, we found dectin-2 to be an inefficient signalling receptor. Yet upon closer analysis we show that the sensitivity of the dectin-2 signal transduction pathway (EC50) can be enhanced by overexpression of its co-receptor FcR{gamma}, while its transmitted information cannot. In this context, we expanded our investigation towards the integration of multiple signal transduction pathways, which is crucial during pathogen recognition. We show how lectin receptors using a similar signal transduction pathway (dectin-1 and dectin-2) are being integrated; by striking a compromise between the lectins. By using dectin-2 and other lectins as example we demonstrate how cellular heterogeneity and the receptor itself determine the efficiency and therefore outcome of the signal transduction pathways.

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Single cell analysis of RA synovial B cells reveals a dynamic spectrum of ectopic lymphoid B cell activation and hypermutation characterized by NR4A nuclear receptor expression Ectopic lymphoid structures (ELS) can develop in rheumatoid arthritis (RA) synovial tissue, but the precise pathways of B cell activation and selection are not well understood. Here, we identified a unique B cell population in the synovium characterized by co-expression of a family of orphan nuclear receptors, NR4A1 (also known as NUR77), NR4A2 (NURR1) and NR4A3 (NOR1), that is highly enriched at both early and late stages of RA. NR4A B cells are rare in healthy peripheral blood, RA blood, and SLE kidney, but share markers with blood transcriptomic signatures that peak during RA disease flare. Using combined single cell transcriptomics and B cell receptor (BCR) sequencing, we demonstrate that NR4A synovial B cells have an activated transcriptomic profile that significantly overlaps with germinal center (GC) light zone (LZ) B cells and an accrual of somatic hypermutation that correlates with loss of naive B cell status. NR4A B cells uniquely co-express lymphotoxin {beta} and IL6, supporting important functions in ELS promotion and pro-inflammatory cytokine production. Further, the presence of shared clones in this activated B cell state and NR4A expressing synovial plasma cells (PC) and the rapid up-regulation with BCR stimulation points to in situ differentiation. Taken together, we identified a dynamic progression of B cell activation in RA synovial ELS, with NR4A transcription factors having an important role in antigen activation and local adaptive immune responses.

One sentence summaryB cells in the rheumatoid arthritis synovium undergo a spectrum of in situ activation, with the NR4A family of transcription factors having an important role in antigen stimulation, local adaptive immunity, and pathological B cell responses.

sentences: A divergent nonsense-mediated decay machinery in Plasmodium falciparum is inefficient and non-essential Nonsense-mediated decay (NMD) is a conserved mRNA quality control process that eliminates transcripts bearing a premature termination codon. In addition to its role in removing erroneous transcripts, NMD is involved in post-transcriptional regulation of gene expression via programmed intron retention in metazoans. The apicomplexan parasite Plasmodium falciparum shows relatively high levels of intron retention, but it is unclear whether these variant transcripts are functional targets of NMD. In this study, we use CRISPR-Cas9 to disrupt and epitope-tag two core NMD components: PfUPF1 (PF3D7_1005500) and PfUPF2 (PF3D7_0925800). Using RNA-seq, we find that NMD in P. falciparum is highly derived and requires UPF2, but not UPF1 for transcript degradation. Furthermore, our work suggests that the majority of intron retention in P. falciparum has no functional role and that NMD is not required for parasite growth ex vivo. We localise both PfUPF1 and PfUPF2 to puncta within the parasite cytoplasm, which may represent processing bodies - ribonucleoparticles that are sites of cytoplasmic mRNA decay. Finally, we identify a number of mRNA-binding proteins that co-immunoprecipitate with the NMD core complex and propose a model for a divergent NMD that does not require PfUPF1 and incorporates novel accessory proteins to elicit mRNA decay.

sentences: Bi-phasic effect of gelatin in myogenesis and skeletal muscle regeneration Skeletal muscle regeneration requires extracellular matrix (ECM) remodeling including an acute and transient breakdown of collagen that produces gelatin. However, the physiological function of such a remodeling process on muscle tissue repair is unclear. Here we elaborate on a bi-phasic effect of gelatin in skeletal muscle regeneration, mediated by hormetic effects of reactive oxygen species (ROS). Low-dose gelatin stimulates ROS production from NADPH oxidase 2 (NOX2) and simultaneously upregulates antioxidant system for cellular defense, reminiscent of the adaptive compensatory process during mild stress. This response triggers the release of myokine IL-6 which stimulates myogenesis and facilitates muscle regeneration. By contrast, high-dose gelatin stimulates ROS overproduction from NOX2 and mitochondrial chain complex, and ROS accumulation by suppressing antioxidant system, triggering release of TNF, which inhibits myogenesis and regeneration. Our findings reveal gelatin-ROS-IL-6/TNF signaling cascades underlying a hormetic response of myogenic cells to gelatin.

sentences: Biosynthesis of cannabinoid precursor olivetolic acid by overcoming rate-limiting steps in genetically engineered Yarrowia lipolytica Natural products acting on our central nervous systems are in utmost demand to fight against pain and mental disorders. Cannabinoids (CBDs) are proven neuroactive agents to treat anxiety, depression, chronic pain diseases, seizure, strokes and neurological disorders. The scarcity of the hemp-sourced CBD products and the prohibitive manufacturing cost limit the wide application of CBDs. Yeast metabolic engineering offers the flexibility to meet the ever-increasing market demand. In this work, we took a retrosynthetic approach and sequentially identified the rate-limiting steps to improve the biosynthesis of the CBD precursor olivetolic acid (OLA) in Yarrowia lipolytica. We debottlenecked the critical enzymatic steps to overcome the supply of hexanoyl-CoA, malonyl-CoA, acetyl-CoA, NADPH and ATPs to redirect carbon flux toward OLA. Implementation of these strategies led to an 83-fold increase in OLA titer in shaking flask experiment. This work may serve as a baseline for engineering CBD biosynthesis in oleaginous yeast species.

sentences: Hybridization and range expansion in tamarisk beetles (Diorhabda spp.) introduced to North America for classical biological control With the global rise of human-mediated translocations and invasions, it is critical to understand the genomic consequences of hybridization and mechanisms of range expansion. Conventional wisdom is that high genetic drift and loss of genetic diversity due to repeated founder effects will constrain introduced species. However, reduced genetic variation can be countered by behavioral aspects and admixture with other distinct populations. As planned invasions, classical biological control (biocontrol) agents present important opportunities to understand the mechanisms of establishment and spread in a novel environment. The ability of biocontrol agents to spread and adapt, and their effects on local ecosystems, depends on genomic variation and the consequences of admixture in novel environments. Here we use a biocontrol system to examine the genome-wide outcomes of introduction, spread, and hybridization in four cryptic species of a biocontrol agent, the tamarisk beetle (Diorhabda carinata, D. carinulata, D. elongata, and D. sublineata), introduced from six localities across Eurasia to control the invasive shrub tamarisk (Tamarix spp.) in western North America. We assembled a de novo draft reference genome and applied RADseq to over 500 individuals from laboratory cultures, the native ranges, and across the introduced range. Despite evidence of a substantial genetic bottleneck among D. carinulata in N. America, populations continue to establish and spread, possibly due to aggregation behavior. We found that D. carinata, D. elongata, and D. sublineata hybridize in the field to varying extents, with D. carinata x D. sublineata hybrids being the most abundant. Genetic diversity was greater at sites with hybrids, highlighting potential for increased ability to adapt and expand. Our results demonstrate the complex patterns of genomic variation that can result from introduction of multiple ecotypes or species for biocontrol, and the importance of understanding them to predict and manage the effects of biocontrol agents in novel ecosystems.

sentences: A short report on optimization of nucleic acid probes by DNA microarray synthesis and next generation sequencing This manuscript has been withdrawn by bioRxiv as it was submitted and made public without the full consent of all the authors. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author

sentences: Negligible impact of SARS-CoV-2 variants on CD4+ and CD8+ T cell reactivity in COVID-19 exposed donors and vaccinees. The emergence of SARS-CoV-2 variants highlighted the need to better understand adaptive immune responses to this virus. It is important to address whether also CD4+ and CD8+ T cell responses are affected, because of the role they play in disease resolution and modulation of COVID-19 disease severity. Here we performed a comprehensive analysis of SARS-CoV-2-specific CD4+ and CD8+ T cell responses from COVID-19 convalescent subjects recognizing the ancestral strain, compared to variant lineages B.1.1.7, B.1.351, P.1, and CAL.20C as well as recipients of the Moderna (mRNA-1273) or Pfizer/BioNTech (BNT162b2) COVID-19 vaccines. Similarly, we demonstrate that the sequences of the vast majority of SARS-CoV-2 T cell epitopes are not affected by the mutations found in the variants analyzed. Overall, the results demonstrate that CD4+ and CD8+ T cell responses in convalescent COVID-19 subjects or COVID-19 mRNA vaccinees are not substantially affected by mutations found in the SARS-CoV-2 variants.

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A fungal powdery mildew pathogen induces extensive local and marginal systemic changes in the Arabidopsis thaliana microbiota O_LIPowdery mildew is a foliar disease caused by epiphytically growing obligate biotrophic ascomycete fungi. How powdery mildew colonization affects host resident microbial communities locally and systemically remains poorly explored.
C_LIO_LIWe performed powdery mildew (Golovinomyces orontii) infection experiments with Arabidopsis thaliana grown in either natural soil or a gnotobiotic system and studied the influence of pathogen invasion into standing natural multi-kingdom or synthetic bacterial communities (SynComs).
C_LIO_LIWe found that after infection of soil-grown plants, G. orontii outcompetes numerous resident leaf-associated fungi. We further detected a significant shift in foliar but not root-associated bacterial communities in this setup. Pre-colonization of germ-free A. thaliana leaves with a bacterial leaf-SynCom, followed by G. orontii invasion, induced an overall similar shift in the foliar bacterial microbiota and minor changes in the root-associated bacterial assemblage. However, a standing root SynCom in root samples remained robust against foliar infection with G. orontii. Although pathogen growth was unaffected by the leaf SynCom, fungal infection caused a more than two-fold increase in leaf bacterial load.
C_LIO_LIOur findings indicate that G. orontii infection affects mainly microbial communities in local plant tissue, possibly driven by pathogen-induced changes in source-sink relationships and host immune status.
C_LI

sentences: Comparing diversification rates in lakes, rivers, and the sea The diversity of species inhabiting freshwater relative to marine habitats is striking, given that freshwater habitats encompass <1% of Earths water. The most commonly proposed explanation for this pattern is that freshwater habitats are more fragmented than marine habitats, allowing more opportunities for allopatric speciation and thus increased diversification rates in freshwater. However, speciation may be generally faster in sympatry than in allopatry, as illustrated by lacustrine radiations such as African cichlids. Differences between rivers and lakes may be important to consider when comparing diversification broadly among freshwater and marine lineages. Here I compared diversification rates of teleost fishes in marine, riverine and lacustrine habitats. I found that lakes had faster speciation and net diversification rates than other aquatic habitats. However, most freshwater diversity arose in rivers. Surprisingly, riverine and marine habitats had similar rates of net diversification. Biogeographic models suggest that lacustrine habitats are evolutionary unstable, explaining the dearth of lacustrine species in spite of their rapid diversification. Collectively, these results suggest that diversification rate differences are unlikely to explain the freshwater paradox. Instead, this pattern may be attributable to the comparable amount of time spent in riverine and marine habitats over the 200-million-year history of teleosts.

sentences: Abundant small RNAs in the reproductive tissues of the honey bee, Apis mellifera, are a plausible mechanism for epigenetic inheritance and parental manipulation of gene expression Polyandrous social insects such as the honey bee are prime candidates for parental manipulation of gene expression in offspring. Although there is good evidence for parent-of-origin effects in honey bees the epigenetic mechanisms that underlie these effects remain a mystery. Small RNA molecules such as miRNAs, piRNAs and siRNAs play important roles in transgenerational epigenetic inheritance and in the regulation of gene expression during development. Here we present the first characterisation of small RNAs present in honey bee reproductive tissues: ovaries, spermatheca, semen, fertilised and unfertilised eggs, and testes. We show that semen contains fewer piRNAs relative to eggs and ovaries, and that piRNAs and miRNAs which map antisense to genes involved in DNA regulation and developmental processes are differentially expressed between tissues. tRNA fragments are highly abundant in semen and have a similar profile to those seen in semen in other animals. Intriguingly we find abundant piRNAs that target the sex determination locus, suggesting that piRNAs may play a role in honey bee sex determination. We conclude that small RNAs play a fundamental role in honey bee gametogenesis and reproduction and provide a plausible mechanism for parent-of origin-effects on gene expression and reproductive physiology.

sentences: Single-cell transcriptional profiling reveals cellular and molecular divergence in human maternal-fetal interface Placenta play essential role in successful pregnancy, as the most important organ connecting and interplaying between mother and fetus. However, the cellular and molecular characteristics of fetal origin and maternal origin cell populations within the fetomaternal interface still is poorly understood. Here, we profiled the transcriptomes of single cells with well-defined maternal-fetal origin that consecutively localized from fetal section (FS), middle section (Mid_S) to maternal section (Mat_S) within the human full-term placenta. Then, we initially identified the cellular and molecular heterogeneity of cytotrophoblast cell (CTB) and stromal cell (STR) with the spatial location and fetal/maternal origin, also highlighted STR cells from fetal origins showed greater proliferation ability in Mat_S compared to cells from FS or Mid_S. Further, by integrating analysis with the first-trimester placental single cell transcriptome data, we revealed that a subpopulation of trophoblast progenitor-like cells (TPLCs) existed in the full-term placenta and mainly distributed in Mid_S, with high expression of pool of putative cell surface makers and unique molecular features. Moreover, through the extravillous cytotrophoblast (EVT) subsets differentiation trajectory and regulation network analysis, we proposed a putative key transcription factor PRDM6 that promoted the differentiation of endovascular extravillous trophoblast cells (enEVT). Finally, based on the integrated analyses of single cell transcriptional profiling of preeclampsia (PE) and match-trimester normal placenta, we highlighted the defective EVT subgroup composition and down-regulation of PRDM6 may lead to an abnormal enEVT differentiation process in PE. Together, our study offers important resources for better understanding of human placenta, stem cell-based therapy as well as PE, and provides new insights on the study of tissue heterogeneity, the clinical prevention and control of PE as well as the maternal-fetal interface.

sentences: Genomic organization of the autonomous regulatory domain of eyeless locus in Drosophila melanogaster In Drosophila, expression of eyeless (ey) gene is restricted to the developing eyes and central nervous system. However, the flanking genes, myoglianin (myo), and bent (bt) have different temporal and spatial expression patterns as compared to the ey. How distinct regulation of ey is maintained is mostly unknown. Earlier, we have identified a boundary element intervening myo and ey genes (ME boundary) that prevents the crosstalk between the cis-regulatory elements of myo and ey genes. In the present study, we further searched for the cis-elements that define the domain of ey and maintain its expression pattern. We identify another boundary element between ey and bt, the EB boundary. The EB boundary separates the regulatory landscapes of ey and bt genes. The two boundaries, ME and EB, show a long-range interaction as well as interact with the nuclear architecture. This suggests functional autonomy of the ey locus and its insulation from differentially regulated flanking regions. We also identify a new Polycomb Response Element, the ey-PRE, within the ey domain. The expression state of the ey gene, once established during early development is likely to be maintained with the help of ey- PRE. Our study proposes a general regulatory mechanism by which a gene can be maintained in a functionally independent chromatin domain in gene-rich euchromatin.

sentences: Evaluating sources of technical variability in the mechano-node-pore sensing pipeline and their effect on the reproducibility of single-cell mechanical phenotyping Cellular mechanical properties can reveal physiologically relevant characteristics in many cell types, and several groups have developed microfluidics-based platforms to perform single-cell mechanical testing with high throughput. However, prior work has performed only limited characterization of these platforms technical variability and reproducibility. Here, we evaluate the repeatability performance of mechano-node-pore sensing, which is a single-cell mechanical phenotyping platform developed by our research group. We measured the degree to which device-to-device variability and semi-manual data processing affected this platforms measurements of single-cell mechanical properties, and we demonstrated high repeatability across the entire technology pipeline even for novice users. We then compared results from identical mechano-node-pore sensing experiments performed by researchers in two different labs with different analytical instruments, demonstrating that the mechanical testing results from these two locations are in agreement. Our findings quantify the expectation of technical variability in mechano-node-pore sensing even in minimally experienced hands. Most importantly, we find that the repeatability performance we measured is fully sufficient for interpreting biologically relevant single-cell mechanical measurements with high confidence.

sentences: Gut bacteria regulate the pathogenesis of Huntington's disease in Drosophila Changes in the composition of gut microbiota are implicated in the pathogenesis of several neurodegenerative disorders. Here, we investigated whether gut bacteria affect the progression of Huntingtons disease (HD) in transgenic Drosophila melanogaster (fruit fly) models expressing human full-length or N-terminal fragments of mutant huntingtin (HTT) protein, here referred to as HD flies. We find that elimination of commensal gut bacteria by antibiotics reduces the aggregation of amyloidogenic N-terminal fragments of HTT and delays the development of motor defects. Conversely, colonization of HD flies with Escherichia coli (E. coli), a known pathobiont of human gut with links to neurodegeneration, accelerates HTT aggregation, aggravates immobility and shortens lifespan. Similar to antibiotics, treatment of HD flies with small compounds such as luteolin, a flavone, or crocin a beta-carotenoid, ameliorates disease phenotypes and promotes survival. Crocin prevents colonization of E. coli in the gut and alters the abundance of commensal bacteria, which may be linked to its protective effects. The opposing effects of E. coli and crocin on HTT aggregation, motor defects and survival in transgenic Drosophila models support the involvement of gut-brain networks in the pathogenesis of HD.

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Representation of illusory shapes within the topographic areas of the posterior parietal cortex The human visual system consists of multiple topographic maps that extend from the early visual cortex along the dorsal and ventral processing streams. Responses to illusory shapes within these maps have been demonstrated in the ventral stream areas, in particular the lateral occipital complex. Recently, the intraparietal sulcus of the dorsal stream has been linked to the processing of illusory shapes defined by motion. It therefore remains unclear whether the topographically organized parietal areas also respond to static illusory shapes, which would suggest their generic role in representing illusory content. Here we measured brain responses using fMRI while human participants observed flickering inducers around the fixation task. The inducers either formed an illusory diamond in the center, a triangle in the left or in the right hemifield, or were inverted such that no illusory figure was formed. We compared responses of parietal regions IPS0-IPS5 and SPL1 to each illusory figure with the non-illusory condition. To determine the role of attention in illusory shape responses we manipulated the difficulty of the fixation task. Our results show that all IPS areas responded to illusory shapes. The more posterior areas IPS0-IPS3 additionally displayed a preference towards the contralateral shapes, while the more anterior areas IPS4 and IPS5 showed response attenuation with increased task difficulty. We suggest that the IPS can represent illusory content irrespective of the perceptual mechanism that generated it. These responses may serve as a potential feedback signal that drives illusory shape responses in early and ventral visual areas.

Significance statementThe traditional view of the ventral visual pathway being solely responsible for representation of objects has recently been challenged by demonstrating illusory shape representation within the dorsal visual pathway with moving bistable stimuli. Our results provide evidence for the dorsal stream contribution to representing not only moving, but also static illusory shapes. Our results also show a functional subdivision along the topographic maps, with spatially specific shape responses in the more posterior, and attention-dependent responses in the more anterior areas. IPS areas of the dorsal stream should thus be considered in the theoretical accounts and neural models of how subjective content is generated in the brain.

sentences: Genomic and functional characterization of Pseudomonas aeruginosa-targeting bacteriophages isolated from hospital wastewater Pseudomonas aeruginosa infections can be difficult to treat and new therapeutic approaches are needed. Bacteriophage therapy is a promising alternative to traditional antibiotics, but large numbers of isolated and characterized phages are lacking. We collected 23 genetically and phenotypically diverse P. aeruginosa isolates from people with cystic fibrosis (CF) and clinical infections, and characterized their genetic, phenotypic, and prophage diversity. We then used these isolates to screen and isolate 14 new P. aeruginosa-targeting phages from hospital wastewater. Phages were characterized with genome sequencing, comparative genomics, and lytic activity screening against all 23 bacterial host isolates. For four different phages, we evolved bacterial mutants that were resistant to phage infection. We then used genome sequencing and functional analysis of the resistant mutants to study their mechanisms of phage resistance as well as changes in virulence factor production and antibiotic resistance, which differed from corresponding parent bacterial isolates. Finally, we tested two phages for their ability to kill P. aeruginosa grown in biofilms in vitro, and observed that both phages reduced viable bacteria in biofilms by least one order of magnitude. One of these phages also showed activity against P. aeruginosa biofilms grown on CF airway epithelial cells. Overall, this study demonstrates how systematic genomic and phenotypic characterization can be deployed to develop bacteriophages as precision antibiotics.

sentences: CandyCollect: At-home saliva sampling for capture of respiratory pathogens Streptococcus pyogenes is a major human-specific bacterial pathogen and a common cause of a wide range of symptoms from mild infection such as pharyngitis (commonly called strep throat) to lifethreatening invasive infection. Traditional methods for diagnosis include collecting a sample using a pharyngeal swab, which can cause discomfort and even discourage adults and children from seeking proper testing and treatment in the clinic. Saliva samples are an alternative to pharyngeal swabs. To improve the testing experience for strep throat, we developed a novel lollipop-inspired sampling platform (called CandyCollect) to capture bacteria in saliva. The device can be used in clinics or in the home and shipped back to a lab for analysis, integrating with telemedicine. CandyCollect is designed to capture bacteria on an oxygen plasma treated polystyrene surface embedded with flavoring substances to enhance the experience for children and inform the required time to complete the sampling process. In addition, the open channel structure prevents the tongue from scraping and removing the captured bacteria. Flavoring substances did not affect bacterial capture and the device has a shelf life of at least 2 months (with experiments ongoing to extend the shelf life). We performed a usability study with 17 participants who provided feedback on the device design and the dissolving time of the candy. This technology and advanced processing techniques, including polymerase chain reaction (PCR), will enable user-friendly and effective diagnosis of streptococcal pharyngitis.

sentences: Sequential and efficient neural-population coding of complex task information Recent work has highlighted that many types of variables are represented in each neocortical area. How can these many neural representations be organized together without interference, and coherently maintained/updated through time? We recorded from large neural populations in posterior cortices as mice performed a complex, dynamic task involving multiple interrelated variables. The neural encoding implied that correlated task variables were represented by uncorrelated neural-population modes, while pairs of neurons exhibited a variety of signal correlations. This finding relates to principles of efficient coding for task-specific information, with neural-population modes as the encoding unit, and applied across posterior cortical regions and layers 2/3 and 5. Remarkably, this encoding function was multiplexed with sequential neural dynamics as well as reliably followed changes in task-variable correlations through time. We suggest that neural circuits can implement time-dependent encoding in a simple way by using random sequential dynamics as a temporal scaffold.

sentences: Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype The relationship between sequence variation and phenotype is poorly understood. Here we use metabolomic analysis to elucidate the molecular mechanism underlying the filamentous phenotype of E. coli strains that carry destabilizing mutations in Dihydrofolate Reductase (DHFR). We find that partial loss of DHFR activity causes reversible filamentation despite SOS response indicative of DNA damage, in contrast to thymineless death (TLD) achieved by complete inhibition of DHFR activity by high concentrations of antibiotic Trimethoprim. This phenotype is triggered by a disproportionate drop in intracellular dTTP, which could not be explained by drop in dTMP based on the Michaelis-Menten like in vitro activity curve of Thymidylate Kinase (Tmk), a downstream enzyme that phosphorylates dTMP to dTDP. Instead, we show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Tmk is the cause of suboptimal dTTP levels. dTMP supplementation rescues filamentation and restores in vivo Tmk kinetics to Michaelis-Menten. Overall, this study highlights the important role of cellular environment in sculpting enzymatic kinetics with system level implications for bacterial phenotype.

sentences: Characterising the vegetation-rainfall relationship in the Northeast Himalaya, India Ecosystems are complex systems and are characterised by positive and negative feedbacks between the abiotic and biotic components. The response of an ecosystem to its environment can be determined by examining state diagrams, which are plots of the state variable as a function of the environmental driver. For instance, tree cover as a function of rainfall is widely used to characterise vegetation patterns. Previous studies have shown that tree cover shows bimodal distributions for intermediate rainfall regimes in Africa and South America. In this study, we construct a vegetation state diagram - by plotting vegetation cover as a function of mean annual rainfall - for Northeast India, which is part of the Eastern Himalaya and the Indo-Burma biodiversity hotspot. We use remotely sensed satellite data of Enhanced Vegetation Index (EVI) as a proxy for vegetation cover. We obtain Mean Annual Precipitation (MAP) from the CHIRPS data (Climate Hazards Group InfraRed Precipitation with Station data). We find that EVI increases monotonically as a function of MAP in the range 1000-2000 mm, after which it plateaus. The 1000 to 2000 mm MAP corresponds to the vegetation transitional zone (1200-3700 m), whereas >2000 MAP region covers the greater extent of the tropical forest (<1200 m) of NEI. In other words, we find no evidence for bimodality in tree cover or vegetation states at coarser scales in North Eastern India. Our characterisation of the state diagram for vegetation in northeast India is important to understand response to ongoing change in rainfall patterns.

sentences: eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale Even though automated functional annotation of genes represents a fundamental step in most genomic and metagenomic workflows, it remains challenging at large scales. Here, we describe a major upgrade to eggNOG-mapper, a tool for functional annotation based on precomputed orthology assignments, now optimized for vast (meta)genomic data sets. Improvements in version 2 include a full update of both the genomes and functional databases to those from eggNOG v5, as well as several efficiency enhancements and new features. Most notably, eggNOG-mapper v2 now allows: (i) de novo gene prediction from raw contigs, (ii) built-in pairwise orthology prediction, (iii) fast protein domain discovery, and (iv) automated GFF decoration. eggNOG-mapper v2 is available as a standalone tool or as an online service at http://eggnog-mapper.embl.de.

sentences: A fragment-based protein interface design algorithm for symmetric assemblies Theoretical and experimental advances in protein engineering have led to the creation of precisely defined, novel protein assemblies of great size and complexity, with diverse applications. One powerful approach involves designing a new attachment or binding interface between two simpler symmetric oligomeric protein components. The required methods of design, which present both similarities and key differences compared to problems in protein docking, remain challenging, and are not yet routine. With the aim of more fully enabling this emerging area of protein material engineering, we developed a computer program, Nanohedra, to introduce two key advances. First, we encoded in the program the construction rules (i.e. the search space parameters) that underlie all possible symmetric material constructions. Second, we developed algorithms for rapidly identifying favorable docking/interface arrangements based on tabulations of empirical patterns of known protein fragment-pair associations. As a result, the candidate poses that Nanohedra generates for subsequent amino acid interface design appear highly native-like (at the protein backbone level), while simultaneously conforming to the exacting requirements for symmetry-based assembly. A retrospective computational analysis of successful vs failed experimental studies supports the expectation that this should improve the success rate for this challenging area of protein engineering.

sentences: Isoform-specific O-glycosylation dictates Ebola virus infectivity Ebola virus glycoprotein is one of the most heavily O-glycosylated viral envelope glycoproteins. The glycoprotein possesses a large mucin-like domain responsible for the cytopathic effect on infected cells, yet its structure or potential role in early entry events is poorly defined. To understand the importance of elongated O-glycans and the individual O-glycosylation sites for viral infectivity, we performed a comprehensive characterization of site-specific glycosylation governed by the three key GalNAc-transferases, GalNAc-T1, -T2, and -T3, initiating O-glycan biosynthesis. Using TMT isobaric labelling we performed quantitative differential O-glycoproteomics on proteins produced in wild type HEK293 cells and cell lines ablated for each of the three GalNAc-Ts, as well as compared it to patterns on wild type virus-like particles. In total we found 38 and 41 O-glycosites on virus like particle-derived and recombinant GP, respectively, with well correlated sites and site-specific structures. Examination of the isoform-specific glycosylation demonstrate selective initiation of a subset of O-glycosites by each enzyme, with GalNAc-T1 having the largest contribution. We next demonstrate that O-linked glycan truncation and perturbed initiation retarded the production of viral particles and decreased infectivity of progeny virus. This work represents a comprehensive site-specific analysis of EBOV GP and sheds light on differential regulation of EBOV GP glycosylation initiated by host GalNAc-Ts. Together with the effect on viral propagation it opens prospective avenues for tailored intervention approaches and means for modulating immunogen O-glycan density.

sentences: How exposure to land use impacts and climate change may prune the tetrapod tree of life Human domination of landscapes is a key driver of biodiversity loss, with the fingerprint of climate change becoming increasingly pronounced. Frameworks and tools for identifying threats to biodiversity are required to meet Post-2020 Global Biodiversity Framework targets for 2030 that call for, among other things, reducing or halting species extinction rates (1). Hence, we compiled a phylogenetic tree for terrestrial tetrapods, mapped hotspots of geographically restricted and evolutionarily distinct lineages, and identified which hotspots may simultaneously face the highest magnitudes of land use impacts and climate change. Across a quarter of Earths surface, hotspots contain the entire ranges of 45% of tetrapods, representing 39% of terrestrial tetrapod evolutionary heritage. By 2070, we estimate 8-13% of this heritage to occur entirely within hotspots highly exposed to climate change, with 13-29% of hotspots projected to experience high exposure to both stressors simultaneously. Most hotspots at highest risk occur in countries least able to take action. Our analysis highlights the need for global ambition and coordination to avoid catastrophic loss of tetrapod evolutionary heritage.

sentences: SIRT5 is the desuccinylase of LDHA as novel cancer metastatic stimulator in aggressive prostate cancer Prostate cancer (PCa) is the most commonly diagnosed genital cancer in men worldwide. Among patients who developed advanced PCa, 80% suffered from bone metastasis, with a sharp drop in the survival rate. Despite great efforts, the details of the mechanisms underlying castration-resistant PCa (CRPC) remains unclear. SIRT5, an NAD+-dependent desuccinylase, is hypothesized to be a key regulator of various cancers. However, compared to other SIRTs, the role of SIRT5 in cancer has not been extensively studied. Here, we showed significantly decreased SIRT5 levels in aggressive PCa cells relative to the PCa stages. The correlation between the decrease in the SIRT5 level and the patients survival rate was also confirmed. Using quantitative global succinylome analysis, we characterized a significant increase of lysine 118 succinylation (K118su) of lactate dehydrogenase A (LDHA), which plays a role in increasing LDH activity. As a substrate of SIRT5, LDHA-K118su significantly increased the migration and invasion of PCa cells and LDH activity in PCa patients. This study investigated the reduction of SIRT5 and LDHA-K118su as a novel mechanism involved in PCa progression. It can also be proposed as a new target that can prevent castration-resistant PCa progression, which is key to PCa treatment.

sentences: Neuroinvasion and neurotropism by SARS-CoV-2 variants in the K18-hACE2 mouse Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) not only affects the respiratory tract but also causes neurological symptoms such as loss of smell and taste, headache, fatigue or severe cerebrovascular complications. Using transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) we investigated the spatiotemporal distribution and pathomorphological features in the CNS following intranasal infection with SARS-CoV-2 variants, also after prior influenza A virus infection. Apart from Omicron, we found all variants to frequently spread to and within the CNS. Infection was restricted to neurons and appeared to spread from the olfactory bulb mainly in basally orientated regions in the brain and into the spinal cord, independent of ACE2 expression and without evidence of neuronal cell death, axonal damage or demyelination. However, microglial activation, microgliosis and a mild macrophage and T cell dominated inflammatory response was consistently observed, accompanied by apoptotic death of endothelial, microglial and immune cells, without their apparent infection. Microgliosis and immune cell apoptosis indicate a potential role of microglia for pathogenesis and viral effect in COVID-19 and possible impairment of neurological functions, especially in long COVID. These data may also be informative for the selection of therapeutic candidates, and broadly support investigation of agents with adequate penetration into relevant regions of the CNS.

sentences: Low-cost genomics enable high-throughput isolate screening and strain-level microbiome profiling Earths environments harbor complex consortia of microbial lineages that affect processes ranging from host health to biogeochemical cycles. However, understanding the evolution and function of these microbiota has been limited by an inability to isolate individual microbial constituents and assemble their complete genomes in a high-throughput manner. Here, we present a workflow for bacterial isolation and whole-genome sequencing from complex microbiota using open-source labware and the OpenTrons automated liquid handling robotics platform. Our approach circumvents the need for isolate screening (e.g., through 16S rDNA sequencing or mass spectrometry analyses) by reducing the costs of genome-sequencing to ~$10 per bacterium. Applying the workflow, we quantified genomic diversity within 45 bacterial species in the chimpanzee gut microbiota. Results revealed hotspots of recombination in bacterial genomes and elevated transmission of plasmids between distantly related bacterial species within individual chimpanzee hosts. This study develops and applies an approach for high-throughput bacterial isolation and genome sequencing, enabling population genetic analyses of bacterial strains within complex communities not currently possible with metagenomic data alone.

sentences: Reconstructing genotypes from GWAS Summary Statistics The emergence of genomewide association studies (GWAS) has led to the creation of large repositories of human genetic variation, creating enormous opportunities for genetic research and worldwide collaboration. Methods that are based on GWAS summary statistics seek to leverage such records, overcoming barriers that often exist in individual-level data access while also offering significant computational savings. Here, we propose a novel framework that can reconstruct allelic and genotypic counts/frequencies for each SNP from case-control GWAS summary statistics. Our framework is simple and efficient without the need of any complicated underlying assumptions. Illustrating the great potential of this framework we also propose three summary-statistics-based applications implemented in a new software package (ReACt): GWAS meta-analysis (with and without sample overlap), case-case GWAS, and, for the first time, group polygenic risk score (PRS) estimation. We evaluate our methods against the current state-of-the-art on both synthetic data and real genotype data and show high performance in power and error control. Our novel group PRS method based on summary statistics could not be achieved prior to our proposed framework. We demonstrate here the potential applications and advantages of this approach. Our work further highlights the great potential of summary-statistics-based methodologies towards elucidating the genetic background of complex disease and opens up new avenues for research.

sentences: 14-3-3ζ constrains insulin secretion in pancreatic β-cells by regulating mitochondrial function While critical for neurotransmitter synthesis in the brain, members of the 14-3-3 protein family are often assumed to have redundant, over-lapping roles due to their high sequence homology and ubiquitous expression. Despite this assumption, various mammalian 14-3-3 isoforms have now been implicated in regulating cellular and organismal metabolism; however, these functions were primarily observed in cell lines or from systemic knockout mouse models. To date, we have begun to define the contributions of 14-3-3{zeta} in adipocytes, but whether 14-3-3{zeta} has additional metabolic roles in other cell types, such as the pancreatic {beta}-cell, is unclear. We previously documented a pro-survival role of 14-3-3{zeta} in MIN6 insulinoma cells, as depletion of 14-3-3{zeta} induced cell death, but paradoxically, whole-body deletion of 14-3-3{zeta} in mice resulted in significantly enlarged {beta}-cell area with no effects on insulin secretion. To better understand the role of 14-3-3{zeta} in {beta}-cells, we generated {beta}-cell-specific 14-3-3{zeta} knockout ({beta}14-3-3{zeta}KO) mice, and while no differences in {beta}-cell mass were observed, {beta}14-3-3{zeta}KO mice displayed potentiated insulin secretion due to enhanced mitochondrial function and ATP synthesis. Deletion of 14-3-3{zeta} led to profound changes to the {beta}-cell transcriptome, where pathways associated with mitochondrial respiration and oxidative phosphorylation were upregulated. Acute treatment of mouse islets and human islets with pan-14-3-3 inhibitors recapitulated the potentiation in glucose-stimulated insulin secretion (GSIS) and mitochondrial function, suggesting that 14-3-3{zeta} is a critical isoform in {beta}-cells that regulates GSIS. In dysfunctional db/db islets and islets from type 2 diabetic donors, expression of Ywhaz/YWHAZ, the gene encoding 14-3-3{zeta}, was inversely associated with insulin secretory capacity, and pan-14-3-3 protein inhibition was capable of enhancing GSIS and mitochondrial function. Taken together, this study demonstrates important regulatory functions of 14-3-3{zeta} and its related isoforms in insulin secretion and mitochondrial function in {beta}-cells. A deeper understanding of how 14-3-3{zeta} influences {beta}-cell function will further advance our knowledge of how insulin secretion from {beta}-cells is regulated.

sentences: Generation of star-shaped human induced astrocytes with a mature inflammatory phenotype for CNS disease modeling Generating astrocytes from induced pluripotent stem cells has been hampered by either prolonged differentiation -spanning over two months -or by shorter protocols that generate immature astrocytes, devoid of salient inflammation-associated astrocytic traits pivotal for CNS neuropathological modeling. We directed human neural stem cells derived from induced pluripotent stem cells to astrocytic commitment and maturity by orchestrating an astrocytic-tuned culturing environment. In under 28 days, the generated cells express canonical and mature astrocytic markers, denoted by the expression of AQP4 and, remarkably, the expression and functionality of glutamate transporter EAAT2. We also show that this protocol generates astrocytes that encompass traits critical in CNS disease modeling, such as glutathione synthesis and secretion, upregulation of ICAM-1 and a cytokine secretion profile which is on par with primary astrocytes. This protocol generates a multifaceted astrocytic model suitable for CNS in vitro disease modeling and personalized medicine through brain-on-chip technologies.

sentences: Salt-inducible kinase (SIK) inhibition is protective in a mouse model of asthma Interleukin-13 (IL-13) and other Th2 cytokines are important regulators of airway hyper-responsiveness, immune cell infiltration and inflammation in allergic asthma, and are produced when immune cells, such as type 2 innate lymphoid cells (ILC2s) and mast cells, are stimulated with IL-33. Here, we report that the IL-33-dependent secretion of IL-13 from ILC2s is prevented by inhibition of the salt-inducible kinases (SIKs), as we have shown previously in mast cells (Darling NJ et al., 2021, J. Biol. Chem. doi: 10.1016/j.jbc.2021.100428). We also report that a new SIK inhibitor with improved pharmacokinetic properties suppresses the recruitment of eosinophils to the lungs and serum immunoglobulin E (IgE) levels in the Alternaria alternata-induced model of allergic asthma. Our results suggest that drugs targeting SIK isoforms may have therapeutic potential for the treatment of asthma.

sentences: Epigenetic-structural changes in X chromosomes promote Xic pairing during early differentiation from mouse embryonic stem cells X chromosome inactivation center (Xic) pairing is robustly observed during the differentiation of embryonic stem (ES) cells from female mouse embryos, and this process is related to X chromosome inactivation, the circadian clock, intra-nucleus architecture, and metabolism. However, the mechanisms underlying the identification and approach of X chromosome pairs in the crowded nucleus are unclear. To elucidate the driving force of Xic pairing, we developed a coarse-grained molecular dynamics model of intranuclear chromosomes in ES cells and in cells 2 days after the onset of differentiation (2-days cells) by considering intrachromosome epigenetic-structural feature-dependent mechanics. The analysis of the experimental data showed X-chromosomes change to specifically softer than autosomes during the cell differentiation by the rearrangement of their distributions of open-close chromatin regions, and the simulations of these models exhibited such softening promoted the mutual approach of the Xic pair. These findings suggested that local intrachromosomal epigenetic features may contribute to the regulation of cell species-dependent differences in intranuclear architecture.

sentences: Seroprevalence of brucellosis and molecular characterization of Brucella spp. from slaughtered cattle in Rwanda Bovine brucellosis is endemic in Rwanda, although, there is paucity of documented evidence about the disease in slaughtered cattle. A cross-sectional study was conducted in slaughtered cattle (n=300) to determine the seroprevalence of anti-Brucella antibodies using the Rose Bengal Test (RBT), and indirect enzyme-linked immunosorbent assay (i-ELISA). Corresponding tissues were cultured onto a modified Centro de Investigacion y Tecnologia Agroalimentaria (CITA) selective medium and analysed for Brucella spp. using the 16S-23S ribosomal interspacer region (ITS), AMOS, and Bruce-ladder PCR assays. The RBT seroprevalence was 20.7% (62/300), and 2.9% (8/300) with i-ELISA and 2.9% (8/300) using both tests in parallel. Brucella specific 16S-23S ribosomal DNA interspace region (ITS) PCR detected Brucella DNA in 5.6% (17/300; Brucella culture prevalence). AMOS-PCR assay identified mixed B. abortus and B. melitensis (n=3), B. abortus (n=3) and B. melitensis (n=5) while Bruce-ladder PCR also identified B. abortus (n=5) and B. melitensis (n=6). The gold standard culture method combined with PCR confirmation identified 5.6% Brucella cultures which is higher than the more sensitive seroprevalence of 2.9%. This emphasizes the need to validate the serological tests in Rwanda. The mixed infection caused by B. abortus and B. melitensis in slaughtered cattle indicates cross-infection and poses a risk of exposure potential to abattoir workers. It is essential to urgently strengthen the national bovine brucellosis control program through vaccination as well as test- and-slaughter.

sentences: Making Common Fund data more Findable: Catalyzing a Data Ecosystem The Common Fund Data Ecosystem (CFDE) has created a flexible system of data federation that enables users to discover datasets from across the U.S. National Institutes of Health Common Fund without requiring that data owners move, reformat, or rehost those data. The CFDEs federation system is centered on a catalog that ingests metadata from individual Common Fund Programs Data Coordination Centers (DCCs) into a uniform metadata model that can then be indexed and searched from a centralized portal. This uniform Crosscut Metadata Model (C2M2) supports the wide variety of data types and metadata terms used by the individual DCCs and is designed to enable easy expansion to accommodate new data types. We describe its use to ingest and index data from ten DCCs.

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Pancreatic cancer ductal cell of origin drives CD73-dependent generation of immunosuppressive adenosine The microenvironment that surrounds pancreatic ductal adenocarcinoma (PDAC) is profoundly desmoplastic and immunosuppressive. Understanding initial triggers of immunosuppression during the process of pancreatic tumorigenesis would aid in establishing novel targets for effective prevention and therapy. Here, we interrogate the differential molecular mechanisms dependent on cell of origin and pathology subtype that determine immunosuppression during PDAC initiation and in established tumors. Transcriptomic analysis of cell of origin dependent-epithelial gene signatures revealed that Nt5e/CD73, a cell surface enzyme that is the pacemaker for extracellular adenosine generation, is one of the top 10% of genes over-expressed in murine tumors arising from ductal pancreatic epithelium as opposed to those rising from acinar cells. These findings were confirmed by Imaging Mass Cytometry and High-Performance Liquid Chromatography. Our data indicate that ductal activation of oncogenic mutant Kras results in loss of PTEN and elevated AKT signaling which ultimately releases CD73 suppression. Delivery of CD73 small molecule inhibitors through various delivery routes reduced tumor development and growth in genetically engineered and syngeneic mouse models. Analysis in human PDAC subtypes indicates that high Nt5e in murine ductal PDAC models overlaps with high NT5E in human PDAC Squamous and Basal Subtypes, considered to have the highest immunosuppression and worst prognosis. These findings highlight a molecular trigger of the immunosuppressive PDAC microenvironment which is dependent on ductal cell of origin, linking biology with pathological subtype classification, critical components to personalized approaches for PDAC prevention and immunotherapeutic intervention.

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sentences: HIV-1 Vif protein is stabilized by AKT-mediated phosphorylation to enhance APOBEC3G degradation HIV-1 virus has to counter anti-viral restriction factors for its successful replication after its entry in the cell. The host-pathogen dynamics operate as soon as HIV-1 interacts with the cell. HIV-1 Vif has been known for its role in degradation of APOBEC3G; a cytosine deaminase which leads to hyper mutations in the viral DNA leading to aberrant viral replication. The cellular proteins regulating the intracellular HIV-1 Vif protein levels can have profound impact on HIV-1 pathogenesis. MDM2 is known to induce degradation of Vif with subsequent effects on APOBEC3G. Here, we have identified AKT/PKB as one of the crucial regulators of HIV-1 Vif protein. The rationale for selecting Vif as a target substrate for AKT was the presence of RMRINT motif in it, which is similar to the AKT phosphorylation motif RxRxxS/T. Immunoprecipitation assay and Kinase assay revealed that AKT and Vif interact strongly with each other and Vif is phosphorylated at T20 position by AKT. This phosphorylation stabilizes HIV-1 Vif while Vif mutant T20A degrades faster. Moreover, use of dominant negative form of AKT (KD-AKT) and AKT inhibitors were found to destabilise Vif and increase its K48-ubiquitination profile. The consequences of this AKT-Vif interplay were also validated on APOBEC3G degradation, a target of Vif. AKT inhibition was found to restore APOBEC3G levels. This process can be interpreted as a strategy used by virus to prevent MDM2 mediated Vif degradation; AKT stabilises Mdm2, which then targets Vif for degradation but at the same time AKT stabilises Vif by phosphorylating it. Thus, AKT mediated stabilization of Vif might compensate for its degradation by MDM2. This study can have significant implications as HIV-1 Tat protein and growth factors like insulin activate PI3-K/AKT Kinase pathway and can potentially affect Vif and APOBEC3G protein levels and hence HIV-1 pathogenesis.

sentences: High Sensitivity and Low-Cost Flavin luciferase (FLUX)-based Reporter Gene for Mammalian Cell Expression Luciferase-based gene reporters generating bioluminescence signals are important tools for biomedical research. Amongst the luciferases, flavin-dependent enzymes use the most common, and thus most economical chemicals. However, their applications in mammalian cells are limited due to their low signals compared to other systems. Here, we constructed Flavin Luciferase for Mammalian Cell Expression (FLUX) by engineering luciferase from Vibrio campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells. We found that the FLUX reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells, significantly higher than that of firefly luciferase (Fluc). The combined use of FLUX/Fluc as target/control vectors gave the most stable signals, better than the standard set of Fluc(target)/Rluc(control). We demonstrated that FLUX can be used for testing inhibitors of the NF-{kappa}B signaling pathway, validating FLUX applications for various assays in the future.

sentences: Mitotic R-loops direct Aurora b kinase to maintain centromeric cohesion Recent work has shown that R-loops exist at mitotic centromeres, but the function of these R-loops is not well understood. Here, we report that mitotic R-loops arise in distinct locations from those formed during interphase. They accumulate on chromosome arms in prophase, where they are quickly resolved and continue to be produced at repetitive sequences including centromeres during a mitotic stall. Aurora B kinase activity is required to resolve R-loops during prophase and R-loops promote the localization of the Chromosome Passenger Complex (CPC) to the inner centromere. CPC purified from mitotic chromosomes interacts with thirty-two proteins involved with R-loop biology. One of these, the RNA regulator RBMX, controls Aurora B localization and activity in vivo. Perturbations in R-loop homeostasis or RBMX cause defects in the maintenance of centromeric cohesion due to the mislocalization of the CPC. We conclude that R-loops are generated by mitotic processes in repetitive DNA sequences, they play important roles in mitotic fidelity, and we have identified a set of mitotic R-loop regulators including the CPC and RBMX that will enable future studies of mitotic R-loops.

sentences: Unified surface and volumetric projection of physiological imaging data Projection of volumetric data onto the cortical surface is an important precursor to performing surface-based analysis. Numerous projection methods have been reported in the literature, many of which make assumptions which tie them to use with specific modalities, notably blood oxygenation level dependent (BOLD) imaging. This means that they may not be appropriate for use with modalities where subcortical tissue contributes a signal of interest. This work details a new projection that provides a number of generalisations and extensions to existing methods. Namely, it may be used to project arbitrary data without making modality-specific assumptions and can produce unified surface and volumetric representations of data (a concept also known as grayordinates space). When constrained to the same assumptions as existing methods, a comparison using simulation data shows that it produces similar outputs. When these assumptions are relaxed to project simulation data containing both cortical and subcortical signals to and from a unified surface and volume space, substantial and statistically significant differences in recovery of ground truth are observed compared to existing methods.

sentences: Ultramicrobacteria in various fermented cabbages Little is known about the ultramicrobial communities of foods. Several bacteria, including pathogenic species, can form ultramicrobial communities when exposed to harsh conditions, making their detection via conventional culture techniques difficult. This study aimed to explore ultramicrobial communities within the microbial communities of fermented cabbage products, such as kimchi, sauerkraut, and suancai, which are produced through specific manufacturing methods based on the laws and culture of respective regions. We used single-molecule real-time sequencing with tangential flow filtration for fermented cabbages after pre-filtration and transmission electron microscopy to confirm the identity of ultramicrobacteria (UMB).To the best of our knowledge, this is the first study to identify the differences between ultramicrobial communities and microbial communities of fermented cabbages. Although the size of the ultramicrobial communities was smaller than that of the latter, their diversity was not lower. In addition, some UMB underwent cell shrinkage due to unfavorable environments, while others maintained their small size regardless. Major pathogens were not detected in the ultramicrobial communities of fermented cabbages. Nevertheless, several suspicious strains were detected. Our method can be used to screen food materials for the presence of UMB undetectable via conventional methods. Ultramicrobial and microbial communities were efficiently separated using tangential flow filtration and analyzed via single-molecule real-time sequencing. The ultramicrobial communities of fermented vegetables were different from conventional microbial communities. This study provides new insights into the ecology of UMB in foods.

sentences: Cross-covariance isolate detect: a new change-point method for estimating dynamic functional connectivity Evidence of the non stationary behavior of functional connectivity (FC) networks has been observed in task based functional magnetic resonance imaging (fMRI) experiments and even prominently in resting state fMRI data. This has led to the development of several new statistical methods for estimating this time-varying connectivity, with the majority of the methods utilizing a sliding window approach. While computationally feasible, the sliding window approach has several limitations. In this paper, we circumvent the sliding window, by introducing a statistical method that finds change-points in FC networks where the number and location of change-points are unknown a priori. The new method, called cross-covariance isolate detect (CCID), detects multiple change-points in the second-order (cross-covariance or network) structure of multivariate, possibly high-dimensional time series. CCID allows for change-point detection in the presence of frequent changes of possibly small magnitudes, can assign change-points to one or multiple brain regions, and is computationally fast. In addition, CCID is particularly suited to task based data, where the subject alternates between task and rest, as it firstly attempts isolation of each of the change-points within subintervals, and secondly their detection therein. Furthermore, we also propose a new information criterion for CCID to identify the change-points. We apply CCID to several simulated data sets and to task based and resting state fMRI data and compare it to recent change-point methods. CCID may also be applicable to electroencephalography (EEG), magentoencephalography (MEG) and electrocorticography (ECoG) data. Similar to other biological networks, understanding the complex network organization and functional dynamics of the brain can lead to profound clinical implications. Finally, the R package ccid implementing the method from the paper is available from CRAN.

sentences: Mapping nationally and globally at-risk species to identify hotspots for (and gaps in) conservation Protecting habitat of species-at-risk is critical to their recovery, but can be contentious. For example, protecting species that are locally imperilled but globally common (e.g. species that only occur in a jurisdiction at the edge of their geographic range) is often thought to distract from protecting globally-imperilled species. However, such perceived trade-offs are based on the assumption that threatened groups have little spatial overlap, which is rarely quantified. Here, we compile range maps of terrestrial species-at-risk in Canada to assess the geographic overlap of nationally and globally at-risk species with each other, among taxonomic groups, and with protected areas. While many nationally-at-risk taxa only occurred in Canada at their northern range edge (median=4% of range in Canada), nationally-at-risk species were not significantly more peripheral in Canada than globally-at-risk species. Further, 56% of hotspots of nationally-at-risk taxa were also hotspots of globally-at-risk taxa in Canada, undercutting the perceived trade-off in their protection. Hotspots of nationally-at-risk taxa also strongly overlapped with hotspots of individual taxonomic groups, though less so for mammals. While strong spatial overlap across threat levels and taxa should facilitate efficient habitat protection, <7% of the area in Canadas at-risk hotspots is protected, and more than 70% of nationally and globally-at-risk species in Canada have <10% of their Canadian range protected. Our results counter the perception that protecting nationally vs. globally at-risk species are at odds, and identify critical areas to target as Canada strives to increase its protected areas and promote species-at-risk recovery.

sentences: Ecological correlates of gene family size: the draft genome of the redheaded pine sawfly Neodiprion lecontei A central goal in evolutionary biology is to determine the predictability of adaptive genetic changes. Despite many documented cases of convergent evolution at individual loci, little is known about the repeatability of gene family expansions and contractions. To address this void, we examined gene family evolution in the redheaded pine sawfly Neodiprion lecontei, a non-eusocial hymenopteran and exemplar of a pine-specialized lineage evolved from angiosperm-feeding ancestors. After assembling and annotating a draft genome, we manually annotated multiple gene families with chemosensory, detoxification, or immunity functions and characterized their genomic distributions and evolutionary history. Our results suggest that expansions of bitter gustatory receptor (GR), clan 3 cytochrome P450 (CYP3), and antimicrobial peptide (AMP) subfamilies may have contributed to pine adaptation. By contrast, there was no evidence of recent gene family contraction via pseudogenization. Next, we compared the number of genes in these same families across insect taxa that vary in diet, dietary specialization, and social behavior. In Hymenoptera, herbivory was associated with small GR and olfactory receptor (OR) families, eusociality was associated with large OR and small AMP families, and--unlike investigations in more closely related taxa--ecological specialization was not related to gene family size. Overall, our results suggest that gene families that mediate ecological interactions may expand and contract predictably in response to particular selection pressures, however, the ecological drivers and temporal pace of gene gain and loss likely varies considerably across gene families.

sentences: Genomic and epigenomic adaptation in SP-R210 (Myo18A) isoform-deficient macrophages 1Macrophages play fundamental roles in regulation of inflammatory responses to pathogens, resolution of inflammation and tissue repair, and maintenance of tissue homeostasis. The long (L) and short (S) isoforms of SP-R210/MYO18A, a macrophage receptor for surfactant protein A (SP-A) and C1q, regulate basal and inflammatory macrophage phenotype at multiple gene expression, translational, and subcellular levels in addition to their SP-A and C1q-mediated functions; disruption of L renders macrophages hyper-inflammatory, although the underlying mechanism had previously been unexplored. We asked whether disruption of the L isoform led to the hyper-inflammatory state via alteration of global genomic responses. RNA sequencing analysis of SP-R210L(DN) macrophages revealed basal and influenza induced upregulation of genes associated with inflammatory pathways, including TLR, RIG-I, NOD, and cytoplasmic DNA signaling, whereas knockdown of both SP-R210 isoforms (L and S) only resulted in increased RIG-I and NOD signaling. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis showed increased genome-wide deposition of the pioneer transcription factor PU.1 in SP-R210L(DN) compared to WT cells. ChIP-seq analysis of histone H3 methylation showed alterations in both repressive (H3K9me3 and H3K27me3) and transcriptionally active (H3K9me3) histone marks. Influenza A virus (IAV) infection, which stimulates an array of cytosolic and TLR-mediated antiviral mechanisms, resulted in differential redistribution between proximal promoter and distal sites and decoupling of PU.1 binding from Toll-like receptor regulated gene promoters in SP-R210L(DN) cells. Our findings suggest that SP-R210L-deficient macrophages are poised with an open PU.1-primed chromatin conformation to rapidly respond to inflammatory and metabolic stimuli.

sentences: RESTORING GLUTAMATE RECEPTOR SIGNALING IN PANCREATIC ALPHA CELLS RESCUES GLUCAGON RESPONSES IN TYPE 1 DIABETES Glucagon secretion from pancreatic alpha cells is crucial to prevent hypoglycemia. For reasons still unknown, people with type 1 diabetes lose this glucoregulatory mechanism and are susceptible to dangerous hypoglycemia. Here we show that alpha cells in living pancreas slices from donors with type 1 diabetes failed to secrete glucagon in response to decreases in glucose concentration, thus mirroring the in vivo unresponsiveness to hypoglycemia. Glucagon content and responses to KCl depolarization were not affected, suggesting that alpha cells retained their secretory potential. By contrast, alpha cells had severely impaired signaling via glutamate receptors of the AMPA/kainate type. Under healthy conditions, activating these receptors was required to elicit full glucagon responses to decreases in glucose levels. In type 1 diabetes, reactivating residual glutamate receptor function with the positive allosteric modulators cyclothiazide and aniracetam restored glucagon secretion in response to hypoglycemia. These positive allosteric modulators are already approved to treat other conditions and could be repurposed to prevent hypoglycemia and improve management of diabetes.

sentences: Rad52 oligomeric N-terminal domain stabilizes Rad51 nucleoprotein filaments and contributes to their protection against Srs2 Homologous recombination (HR) depends on the formation of a nucleoprotein filament of the recombinase Rad51 to scan the genome and invade the homologous sequence used as template for DNA repair synthesis. Therefore, HR is highly accurate and crucial for genome stability. Rad51 filament formation is controlled by positive and negative factors. In Saccharomyces cerevisiae, the mediator protein Rad52 catalyzes Rad51 filament formation and stabilizes them, mostly by counteracting the disruptive activity of the translocase Srs2. Srs2 activity is essential to avoid the formation of toxic Rad51 filaments, as revealed by Srs2-deficient cells. We previously reported that Rad52 SUMOylation or mutations disrupting the Rad52-Rad51 interaction suppress Rad51 filament toxicity because they disengage Rad52 from Rad51 filaments and reduce their stability. Here, we found that mutations in Rad52 N-terminal domain also suppress the DNA damage sensitivity of Srs2-deficient cells. Structural studies showed that these mutations affect the Rad52 oligomeric ring structure. Overall, in vivo and in vitro analyzes of these mutants indicate that Rad52 ring structure is important for protecting Rad51 filaments from Srs2, but can increase Rad51 filament stability and toxicity in Srs2-deficient cells. This stabilization function is distinct from Rad52 mediator and annealing activities.

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Enhanced translation of leaderless mRNAs under oxidative stress in Escherichia coli The bacterial response to oxidative stress requires the adaptation of the proteome to the hostile environment. It has been reported that oxidative stress induces a strong and global inhibition of both, transcription and translation. Nevertheless, whereas it is well known that transcription of a small group of genes is induced thanks to transcription factors such as OxyR and SoxR, an equivalent mechanism has not been described for translation. Here we report that whereas canonical translation that depends on Shine Dalgarno recognition is inhibited by oxidative stress in Escherichia coli, the translation of leaderless mRNA (lmRNA) is enhanced under such conditions. Both, inhibition of canonical translation and enhancement of lmRNA translation, depend on the production of (p)ppGpp. We propose that such a mechanism would allow bacteria to rapidly adapt their proteome to hostile conditions and is, perhaps, a general strategy to confront strong stressful conditions.

Significance statementThe regulation of translation (the production of proteins based on genetic information) is central for the adaptation to environmental changes. In Escherichia coli translation may begin through two alternative pathways. 1.- A canonical initiation that is well understood and is regulated mostly by changes in the accessibility of ribosomes to specific sequences and 2.- Initiation of leaderless mRNAs (lmRNAs) that lack these sequences and for which we do not understand the regulation process. Our results indicate that under oxidative stress, the production of (p)ppGpp in E. coli inhibits canonical translation and simultaneously enhances translation of lmRNAs, showing for the first time a natural condition where lmRNA translation is regulated and a role for (p)ppGpp in this process.

sentences: The bacterial lysate Lantigen B reduces the expression of ACE2 on primary oropharyngeal cells Background: Vercelli and coworkers recently observed that a well-established bacterial lysate (OM-85, Vifor Pharma; CH) was able to downregulate the expression of Angiotensin-Converting Enzyme 2 (ACE2) on epithelial cells. This downregulation was also associated with a reduced infectivity of cells, resulting in a reduced viral titre. We evaluated whether another bacterial lysate (Lantigen B, Bruschettini Ltd; Italy) may have similar activities. However, while OM-85 is given per os and has a systemic effect after absorption at the gut level, Lantigen B is given locoregionally. Thus, the concentration that the bacterial lysate can reach at the mucosal level seems to be promising. Methods: Oropharyngeal cells were collected from healthy donors. After 24 hours of treatment in vitro with doses of Lantigen B comparable to those that are reached in vivo, the expression of ACE2 was evaluated by direct fluorescence and flow cytometry. Results: A reduction in the number of ACE2-positive cells was observed in 80% of treated samples. Only a few donors had poor expression of ACE2, and in these donors, the downregulation was less evident or absent. Conclusions: These results suggest that Lantigen B, at pharmacological doses, could be an interesting drug to reduce ACE2 expression on oropharyngeal cells, thus contributing to the prophylaxis of COVID-19 in humans.

sentences: Three-Dimensional Loop Extrusion Loop extrusion convincingly describes how certain Structural Maintenance of Chromosome (SMC) proteins mediate the formation of large DNA loops. Yet, most of the existing computational models cannot reconcile recent in vitro observations showing that condensins can traverse each other, bypass large roadblocks and perform steps longer than its own size. To fill this gap, we propose a three-dimensional (3D) "trans-grabbing" model for loop extrusion which not only reproduces the experimental features of loop extrusion by one SMC complex, but also predicts the formation of so-called "Z-loops" via the interaction of two or more SMCs extruding along the same DNA substrate. By performing Molecular Dynamics simulations of this model we discover that the experimentally observed asymmetry in the different types of Z-loops is a natural consequence of the DNA tethering in vitro. Intriguingly, our model predicts this bias to disappear in absence of tethering and a third type of Z-loop, which has not yet been identified in experiments, to appear. Our model naturally explains road-block bypassing and the appearance of steps larger than the SMC size as a consequence of non-contiguous DNA grabbing. Finally, it is the first to our knowledge to address how Z-loops and bypassing might occur in a way that is broadly consistent with existing cis-only 1D loop extrusion models.

sentences: Xrn1 is a deNADding Enzyme Modulating Mitochondrial NAD Levels The existence of non-canonical nicotinamide adenine diphosphate (NAD) 5-end capped RNAs is now well established. Nevertheless, the biological function of this nucleotide metabolite cap remains elusive. Here, we show that the yeast Saccharomyces cerevisiae cytoplasmic 5-end exoribonuclease Xrn1 is also a NAD cap decapping (deNADding) enzyme that releases intact NAD and subsequently degrades the RNA. The significance of Xrn1 deNADding is evident in a deNADding deficient Xrn1 mutant that still retains its 5-monophosphate exonuclease activity. This mutant reveals Xrn1 deNADding is necessary for normal growth on non-fermenting sugar and is involved in modulating mitochondrial NAD-capped RNA levels and in turn intramitochondrial NAD levels. Our findings uncover a functional role for mitochondrial NAD-capped RNAs as a reservoir to maintain overall NAD homeostasis. We propose NAD-capped RNAs function as a cistern for mitochondrial NAD with Xrn1 serving as a rheostat for NAD-capped RNAs.

sentences: Latent functional connectivity underlying multiple brain states Functional connectivity (FC) studies have predominantly focused on resting state, where ongoing dynamics are thought to reflect the brains intrinsic network architecture, which is thought to be broadly relevant because it persists across brain states (i.e., is state-general). However, it is unknown whether resting state is the optimal state for measuring intrinsic FC. We propose that latent FC, reflecting shared connectivity patterns across many brain states, better captures state-general intrinsic FC relative to measures derived from resting state alone. We estimated latent FC independently for each connection using leave-one-task-out factor analysis in 7 highly distinct task states (24 conditions) and resting state using fMRI data from the Human Connectome Project. Compared to resting-state connectivity, latent FC improves generalization to held-out brain states, better explaining patterns of connectivity and task-evoked activation. We also found that latent connectivity improved prediction of behavior outside the scanner, indexed by the general intelligence factor (g). Our results suggest that FC patterns shared across many brain states, rather than just resting state, better reflects state-general connectivity. This affirms the notion of "intrinsic" brain network architecture as a set of connectivity properties persistent across brain states, providing an updated conceptual and mathematical framework of intrinsic connectivity as a latent factor.

sentences: Neuronal variability reflects probabilistic inference tuned to natural image statistics Neuronal activity in sensory cortex fluctuates over time and across repetitions of the same input. This variability is often considered detrimental to neural coding. The theory of neural sampling proposes instead that variability encodes the uncertainty of perceptual inferences. In primary visual cortex (V1), modulation of variability by sensory and non-sensory factors supports this view. However, it is unknown whether V1 variability reflects the statistical structure of visual inputs, as would be required for inferences correctly tuned to the statistics of the natural environment. Here we combine analysis of image statistics and recordings in macaque V1 to show that probabilistic inference tuned to natural image statistics explains the widely observed dependence between spike-count variance and mean, and the modulation of V1 activity and variability by spatial context in images. Our results show that the properties of a basic aspect of cortical responses -- their variability -- can be explained by a probabilistic representation tuned to naturalistic inputs.

sentences: Sequencing SARS-CoV-2 in a malaria research laboratory in Mali, West Africa: the road to sequencing the first SARS-CoV-2 genome in Mali Next generation sequencing (NGS) has become a necessary tool for genomic epidemiology. Even though the utility of genomics in human health has been proved, the genomic surveillance has never been so important until the COVID 19 pandemic. This has been evidenced with the detection of new variants of SARS-CoV-2 in the United Kingdom, South Africa and Brazil recently using genomic surveillance. Until recently, Malian scientists did not have access to any local NGS platform and samples had to be shipped abroad for sequencing. Here, we report on how we adapted a laboratory setup for Plasmodium research to generate the first complete SARS-CoV-2 genome locally. Total RNA underwent a library preparation using an Illumina TruSeq stranded RNA kit. A metagenomics sequencing was performed on an Illumina MiSeq platform following by bioinformatic analyses on a local server in Mali. We recovered a full genome of SARS-CoV-2 of 29 kb with an average depth coverage of 200x. We have demonstrated our capability of generating a high quality genome with limited resources and highlight the need to develop genomics capacity locally to solve health problems. We discuss challenges related to access to reagents during a pandemic period and propose some home-made solutions.

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Increasing Animal Cognition Research in Zoos Animal cognition covers various mental processes including perception, learning, decision-making and memory, and animal behavior is often used as a proxy for measuring cognition. Animal cognition/behavior research has multiple benefits; it provides fundamental knowledge on animal biology and evolution but can also have applied conservation and welfare applications. Zoos provide an excellent yet relatively untapped resource for animal cognition research, because they house a wide variety of species - many of which are under threat - and allow close observation and relatively high experimental control compared to the wild. Multi-zoo collaboration leads to increased sample size and species representation, which in turn leads to more robust science. However, there are salient challenges associated with zoo-based cognitive research, which are subject-based (e.g., small sample sizes at single zoos, untrained/unhabituated subjects, site effects) and human-based (e.g., time restrictions, safety concerns, and perceptions of animals interacting with unnatural technology or apparatus). We aim to increase the understanding and subsequent uptake of animal cognition research in zoos, by transparently outlining the main benefits and challenges. Importantly, we use our own research (1) a study on novelty responses in hornbills, and (2) a multi-site collaboration called the  ManyBirds project to demonstrate how challenges may be overcome. These potential options include using  drop and go apparatuses that require no training, close human contact or animal separation. This article is aimed at zoo animal care and research staff, as well as external researchers interested in zoo-based studies.

RESEARCH HIGHLIGHTSO_LIZoos are an excellent yet relatively untapped resource for animal cognition research.
C_LIO_LIZoo cognition research has historically been challenging, and traditional laboratory paradigms often do not translate well to the majority of zoos.
C_LIO_LISalient challenges of zoo-based cognitive research can be overcome by using less restrictive test apparatuses, limiting animal training and isolation, and subscribing to multi-zoo collaborative programs.
C_LI

sentences: Genomic characterisation of an mcr-1 and mcr-3-producing Escherichia coli strain isolated from pigs in France The current study is about genomic characterisation of an atypical multidrug-resistant Escherichia coli harbouring two mobilised colistin resistance (mcr) genes isolated from pigs in France. Stool samples taken from a pig farm in Avignon in the department of the Vaucluse were subjected to a molecular screening for the detection of mcr gene variants. These samples were cultured on selective LBJMR medium. Growing bacteria were identified using MALDI-TOF, followed by antibiotic susceptibility testing (AST). Whole genome sequencing (WGS) and bioinformatic genome analysis was performed. The selective culture of stools revealed the presence of an E. coli strain named Q4552 which was simultaneously positive for the mcr-1.1 and mcr-3.5 genes. This strain exhibited resistance phenotype to fourteen antibiotics, including colistin. Genome sequencing revealed a circular chromosome and eight plasmids. Genomic analysis revealed a chromosomic integration of a mobile genetic element (MGE) harbouring the mcr-1.1 gene, while the mcr-3.5 gene was plasmidic (i.e., an IncFII plasmid). Its resistome exhibited twenty-two resistance genes, explaining its multidrug resistance phenotype. The Q4552 strain is an ST-843 clone belonging to the clonal complex Cplx-568 and is the only ST type of this cplx-568 which has been isolated from animals, humans, and the environment. Here, we report the first co-occurrence of the mcr-1 and mcr-3 genes in France from a pathogenic E. coli strain isolated from a pig farm. Since this clone (ST-843) has been reported in zoonotic transmissions, programs to monitor such colistin resistant bacterium are urgently required to avoid its spread and zoonotic transmission to humans.

sentences: Metabolic control of type 2 innate lymphoid cells plasticity toward protective type 1-like cells during Mycobacterium tuberculosis infection Tissue-resident innate lymphoid cells (ILCs) regulate tissue homeostasis, protect against pathogens at mucosal surfaces and are key players at the interface of innate and adaptive immunity. How ILCs adapt their phenotype and function to environmental cues within tissues remains to be fully understood. Here, we show that Mycobacterium tuberculosis infection alters the phenotype and function of immature lung ILC2 toward a protective interferon-{gamma}-producing ILC1-like population. This differentiation is controlled by type 1 cytokines and is associated with a glycolytic program involving the transcription factor HIF1. Collectively, our data reveal how tissue-resident ILCs adapt to type 1 inflammation toward a pathogen tailored immune response.

sentences: Omnipose: a high-precision morphology-independent solution for bacterial cell segmentation Advances in microscopy hold great promise for allowing quantitative and precise readouts of morphological and molecular phenomena at the single cell level in bacteria. However, the potential of this approach is ultimately limited by the availability of methods to perform unbiased cell segmentation, defined as the ability to faithfully identify cells independent of their morphology or optical characteristics. In this study, we present a new algorithm, Omnipose, which accurately segments samples that present significant challenges to current algorithms, including mixed bacterial cultures, antibiotic-treated cells, and cells of extended or branched morphology. We show that Omnipose achieves generality and performance beyond leading algorithms and its predecessor, Cellpose, by virtue of unique neural network outputs such as the gradient of the distance field. Finally, we demonstrate the utility of Omnipose in the characterization of extreme morphological phenotypes that arise during interbacterial antagonism and on the segmentation of non-bacterial objects. Our results distinguish Omnipose as a uniquely powerful tool for answering diverse questions in bacterial cell biology.

sentences: Transcriptome profiling of barley and tomato shoot and root meristems unravels physiological variations underlying photoperiodic sensitivity The average sowing date of crops in temperate climate zones has been shifted forwards by several days, resulting in a changed photoperiod regime at the emergence stage. In the present study, we performed a global transcriptome profiling of plant development genes in the seedling stage of root and shoot apical meristems of a photoperiod-sensitive species (barley) and a photoperiod insensitive species (tomato) in short-day conditions (8h). Variant expression indicated differences in physiological development under this short day-length regime between species and tissues. The barley tissue transcriptome revealed reduced differentiation compared to tomato. In addition, decreased photosynthetic activity was observed in barley, indicating a slower physiological development of shoot meristems than in tomatoes. The photomorphogenesis controlling cryptochrome gene cry1, with an effect on physiological differentiation, showed an underexpression in barley compared to tomato shoot meristems. This might lead to a cascade of suspended sink-source activities, which ultimately delay organ development and differentiation in barley shoot meristems under short photoperiods.

sentences: Double-observer approach with camera traps: Towards an unbiased density estimation of unmarked animal populations Camera traps are a powerful tool for wildlife surveys. However, camera traps may not always detect animals passing in front. This constraint may create a substantial bias in estimating critical parameters such as the density of unmarked populations. We proposed the  double-observer approach with camera traps to counter the constraint, which involves setting up a paired camera trap at a station and correcting imperfect detection with a hierarchal capture-recapture model for stratified populations. We performed simulations to evaluate this approachs reliability and determine how to obtain desirable data for this approach. We then applied it to 12 mammals in Japan and Cameroon. The results showed that the approach could correct imperfect detection as long as paired camera traps detect animals nearly independently (Correlation coefficient < 0.2). Camera traps should be installed to monitor a predefined small focal area from different directions to satisfy this requirement. The field surveys showed that camera trap might miss animals by 3 %-40%, suggesting that current density estimation models relying on perfect detection may underestimate animal density by the same order of magnitude. We hope that our approach will be incorporated into existing density estimation models to improve their accuracy.

sentences: Automatic post-processing and merging of multiple spike-sorting analyses with Lussac The increasing site counts of multielectrodes used in extracellular recording preclude traditional manual spike-sorting and have led to the development of several new software suites with increased automation. It remains difficult, however, to find parameters enabling optimal processing of signals from all neuronal types simultaneously. Here, we describe a procedure for automatically post-processing and merging the outputs of multiple spike-sorting analyses, enabling the accumulation of results optimised for different activity features. This approach was motivated by and first applied to the analysis of cerebellar activity, in which the complex spikes challenge standard analyses. The core of our modular procedure involves the identification of cluster-fusion candidates and assurance that only fusions that increase a clearly defined cluster-quality metric are performed; important subsidiary steps that have also been automated include spike alignment and duplicate removal. The procedure was validated by recovery of injected spike waveforms. In tests combining analyses from Kilosort and MountainSort, our procedure yielded a 50 % increase in the detected number of Purkinje cell clusters with both simple and complex spikes. A further validation used the synthetic SpikeForest dataset approximating neocortical activity, in which our package also improved performance. Combining multiple analyses therefore offers a general method for improving spike-sorting, and furthermore Lussac reduces manual input and increases the objectivity of analyses.

sentences: APC-mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness Certain copy number alterations (CNAs) are strongly associated with particular cancer types. However, the mechanisms that underlie selection of specific CNAs remain unknown. Here, we identified functional relationships between recurrent CNAs in colorectal cancers (CRCs) and adenomatous polyposis coli (APC) mutations. Quantitative phenotyping of mitotic spindles highlighted APC functions at centrosomes where APC positively regulated Aurora A kinase (AURKA). Upon APC inactivation, elevated {beta}-catenin levels blocked AURKA activation, which caused chromosome instability and suppressed proliferation, resulting in the generation and selection of AURKA-activating CNAs. Arm-level amplification of chromosomes that contained AURKA and AURKA activator genes was observed in APC-mutant CRCs, early stage mouse tumours, and cells in culture, which was concomitant with an increase in growth potential. Our findings demonstrate a mechanism that restores tumour cell fitness through compensatory chromosome alterations to overcome adverse effects of prior mutations, which may affect the course of cancer type-specific CNA formation.

sentences: Partial prevention of glucocorticoid-induced osteocyte deterioration with osteocrin gene therapy Glucocorticoid (GC)-induced osteoporosis and subsequent bone fragility are preceded by death and dysfunction at the cellular level. In particular, short-term glucocorticoid excess suppresses osteocyte remodeling of the surrounding bone mineral, causes apoptosis of osteoblasts and osteocytes, and disrupts homeostatic bone remodeling. Preventing apoptosis and preserving osteocyte morphology and function could be effective means of preventing bone loss during glucocorticoid excess. We hypothesized that osteocrin, which preserves osteocyte viability and morphology in other models where osteocyte defects exist, could prevent osteocyte death and dysfunction in a GC excess model. We used a liver-targeted adeno-associated virus (AAV8) to induce osteocrin overexpression in mice one week prior to implantation with prednisolone or placebo pellets. After 28 days, tissues were collected for micro-CT and histological analysis. GC excess caused the expected reduction in cortical bone thickness and osteocyte canalicular length in control AAV8-treated mice, and these effects were blunted in mice overexpressing osteocrin. However, GC-induced changes in cortical porosity, trabecular bone mass, and gene expression were not prevented by osteocrin. While the mechanism of osteocrins effects on osteocyte morphology warrants further investigation, this study does not support a role for this model of osteocrin supplementation to combat the full skeletal effects of GC excess.

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Locally adapted oak populations along an elevation gradient display different molecular strategies to regulate bud phenology. Research conductedWith the ongoing global warming, there are serious concerns about the persistence of locally adapted populations. Indeed, with the raising of temperature, the phenological cycle of tree species may be strongly affected since higher winter temperatures may have a negative impact on endodormancy release if chilling requirements are not fulfilled during winter and late frost in spring may expose trees if buds flush too early. Thus, Environmental gradients (showing continuous variations of environmental conditions) constitute a design of choice to analyze the effect of winter dormancy in locally adapted population.

MethodsIn the present study, we used an elevation gradient in the Pyrenees to explore the gene expression network involved in dormancy regulation in natural populations of sessile oak locally adapted to temperature. Terminal buds were harvested during dormancy induction and release at different elevations. Then, gene expression was quantified using RNAseq and we used a likelihood ratio test to identify genes displaying significant dormancy, elevation or dormancy-by-elevation interaction effects.

Key resultsOur results highlight molecular processes in locally adapted populations along this elevation cline, and made it possible to identify key dormancy-by-elevation responsive genes revealing that locally adapted populations have evolved distinct molecular strategies to adapt their bud phenology in response to environmental variation (i.e. temperature).

sentences: Taxon-specific or universal? Using target capture to study the evolutionary history of rapid radiations Target capture has emerged as an important tool for phylogenetics and population genetics in non-model taxa. Whereas developing taxon-specific capture probes requires sustained efforts, available universal kits may have a lower power to reconstruct relationships at shallow phylogenetic scales and within rapidly radiating clades. We present here a newly-developed target capture set for Bromeliaceae, a large and ecologically-diverse plant family with highly variable diversification rates. The set targets 1,776 coding regions, including genes putatively involved in key innovations, with the aim to empower testing of a wide range of evolutionary hypotheses. We compare the relative power of this taxon-specific set, Bromeliad1776, to the universal Angiosperms353 kit. The taxon-specific set results in higher enrichment success across the entire family, however, the overall performance of both kits to reconstruct phylogenetic trees is relatively comparable, highlighting the vast potential of universal kits for resolving evolutionary relationships. For more detailed phylogenetic or population genetic analyses, e.g. the exploration of gene tree concordance, nucleotide diversity or population structure, the taxon-specific capture set presents clear benefits. We discuss the potential lessons that this comparative study provides for future phylogenetic and population genetic investigations, in particular for the study of evolutionary radiations.

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Quantitative Restoration of Immune Defense in Old Animals Determined by Naive Antigen-Specific CD8 T cell Numbers Older humans and animals often exhibit reduced immune responses to infection and vaccination, and this often directly correlates to the numbers and frequency of naive T (Tn) cells. We found such a correlation between reduced numbers of blood CD8+ Tn cells and severe clinical outcomes of West Nile virus (WNV) in both humans naturally exposed to, and mice experimentally infected with, WNV.

To examine possible causality, we sought to increase the number of CD8 Tn cells by treating C57BL/6 mice with IL-7 complexes (IL-7C, anti-IL-7 mAb bound to IL-7), shown previously to efficiently increase peripheral T cell numbers by homeostatic proliferation. T cells underwent robust expansion following IL-7C administration to old mice increasing the number of total T cells (>four-fold) and NS4b:H-2Db-restricted antigen-specific CD8 T cells (two-fold). This improved the numbers of NS4b-specific CD8 T cells detected at the peak of the response against WNV, but not survival of WNV challenge. IL-7C treated old animals also showed no improvement in WNV-specific effector immunity (neutralizing antibody and in vivo T cell cytotoxicity). To test quantitative limits to which CD8 Tn cell restoration could improve protective immunity, we transferred graded doses of Ag-specific precursors into old mice and showed that injection of 5,400 (but not of 1,800 or 600) adult naive WNV-specific CD8 T cells significantly increased survival after WNV. These results set quantitative limits to the level of Tn reconstitution necessary to improve immune defense in older organisms and are discussed in light of targets of immune reconstitution.

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Separate functional subnetworks of excitatory neurons show preference to periodic and random sound structures Auditory cortex (ACX) neurons are sensitive to spectro-temporal sound patterns and violations in patterns induced by rare stimuli embedded within streams of sounds. We investigate the auditory cortical representation of repeated presentations of sequences of sounds with standard stimuli (common) with an embedded deviant (rare) stimulus in two conditions - Periodic (Fixed deviant position) or Random (Random deviant position), using extracellular single-unit and 2-photon Ca+2 imaging recordings in Layer 2/3 neurons of the mouse ACX. In the population average, responses increased over repetitions in the Random-condition and were suppressed or did not change in the Periodic-condition, showing irregularity preference. A subset of neurons also showed the opposite behavior, indicating regularity preference. Pairwise noise correlations were higher in Random-condition over Periodic-condition, suggesting the role of recurrent connections. 2-photon Ca+2 imaging of excitatory (EX) and parvalbumin-positive (PV) and somatostatin-positive (SOM) inhibitory neurons, showed different categories of adaptation or change in response over repetitions (categorized by the sign of the slope of change) as observed with single units. However, the examination of functional connectivity between pairs of neurons of different categories showed that EX-PV connections behaved opposite to the EX-EX and EX-SOM pairs that show more functional connections outside category in Random-condition than Periodic-condition. Finally considering Regularity preference, Irregularity preference and no preference categories, showed that EX-EX and EX-SOM connections to be in largely separate functional subnetworks with the different preferences, while EX-PV connections were more spread. Thus separate subnetworks could underly the coding of periodic and random sound sequences.

Significance StatementStudying how the ACX neurons respond to streams of sound sequences help us understand the importance of changes in dynamic acoustic noisy scenes around us. Humans and animals are sensitive to regularity and its violations in sound sequences. Psychophysical tasks in humans show that auditory brain differentially responds to periodic and random structures, independent of the listeners attentional states. Here we show that mouse ACX L2/3 neurons detect a change and respond differentially to changing patterns over long-time scales. The differential functional connectivity profile obtained in response to two different sound contexts, suggest the stronger role of recurrent connections in the auditory cortical network. Furthermore, the excitatory-inhibitory neuronal interactions can contribute to detecting the changing sound patterns.

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Visualizing transcription sites in living cells using a genetically encoded probe specific for the elongating form of RNA polymerase II In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2). How RNAP2 transcription is regulated in the nucleus is a key to understanding the genome and cell function. The largest subunit of RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5- Pro6-Ser7) at the C-terminal domain and Ser2 is phosphorylated on an elongation form of RNAP2. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody probe exhibited numerous foci, possibly representing transcription "factories" in living HeLa cells, and foci were diminished when cells were treated with triptolide to induce RNAP2 degradation and with flavopiridol to inhibit Ser2ph. An in vitro binding assay using phospho-peptides confirmed the Ser2ph-specific binding of the mintbody. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2, such as the CDK12 and Paf1 complex component, compared to factors involved in transcription activation around the transcription start sites, such as CDK9 and BRD4. Tracking analysis revealed that RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but was more mobile compared to euchromatin domains, suggesting that the elongating RNAP2 complexes are separated from the more confined initiating clusters.

SummaryThe authors developed a genetically encoded probe to specifically detect the Ser2- phosphorylated, elongating form of RNA Polymerase II in living cells. The motion of Ser2- phosphorylated polymerase foci was more dynamic than chromatin domains, suggesting that the elongating complexes are separated from the more confined initiating clusters.

sentences: A causal test of the mechanisms by which affect state biases affective perception In this study we merged methods from machine learning and human neuroimaging to causally test the role of self-induced affect processing states in biasing the affect processing of subsequent image stimuli. To test this causal relationship we developed a novel paradigm in which (n=40) healthy adult participants observed affective neural decodings of their real-time functional magnetic resonance image (rtfMRI) responses as feedback to guide explicit regulation of their brain (and corollary affect processing) state towards a positive valence goal state. By this method individual differences in affect regulation ability were controlled. Attaining this brain-affect goal state triggered the presentation of pseudo-randomly selected affectively congruent (positive valence) or incongruent (negative valence) image stimuli drawn from the International Affective Picture Set. Separately, subjects passively viewed randomly triggered positively and negatively valent image stimuli during fMRI acquisition. Multivariate neural decodings of the affect processing induced by these stimuli were modeled using the task trial type (state- versus randomly-triggered) as the fixed-effect of a general linear mixed-effects model. Random effects were modeled subject-wise. We found that self-induction of a positive valence brain state significantly positively biased valence processing of subsequent stimuli. As a manipulation check, we validated affect processing state induction achieved by the image stimuli using independent psychophysiological response measures of hedonic valence and autonomic arousal. We also validated the predictive fidelity of the trained neural decoding models using brain states induced by an out-of-sample set of image stimuli. Beyond its contribution to our understanding of the neural mechanisms that bias affect processing this work demonstrated the viability of novel experimental paradigms triggered by pre-defined cognitive states. This line of individual differences research potentially provides neuroimaging scientists with a valuable tool for causal exploration of the roles and identities of intrinsic cognitive processing mechanisms that shape our perceptual processing of sensory stimuli.

sentences: Litter characteristics and helping context during early life shape the responsiveness of the stress axis in a wild cooperative breeder Stress responses have evolved to quickly and appropriately deal with environmental stressors in order to secure or restore homeostasis. Since the regulation of stress hormones plays a key adaptive role, the regulatory processes controlling stress hormones levels may be under high selective pressure. The social environment during early life (parents and litter characteristics) strongly affects ontogeny of the hypothalamic-pituitary-adrenal (HPA) axis. In cooperative breeders, offspring are also confronted with helpers but whether and how variation in the helping context can affect HPA axis responsiveness of offspring remains unanswered. Combining dexamethasone suppression and adrenocorticotropic hormone stimulation tests, we investigated the link between the social environment and the characteristics of the HPA axis at the early stages of life in wild Alpine marmots. We show that when raised in the presence of helpers, marmot pups exhibit a greater capacity not only to mount, but also to turn off a stress response. The capacity to mount a stress response was also higher as the pups were raised in large litters. Determining impacts of such social modulation of the HPA axis functioning on individual fitness would make an important contribution to our understanding of the evolution of cooperative breeding.

sentences: Low heritability and high phenotypic plasticity of salivary cortisol in response to environmental heterogeneity in fur seals Individuals are unique in how they interact with and respond to their environment. Correspondingly, unpredictable challenges or environmental stressors often produce an individualized response of the hypothalamic-pituitary-adrenal axis and its downstream effector cortisol. We used a fully crossed, repeated measures design to investigate the factors shaping individual variation in baseline cortisol in Antarctic fur seal pups and their mothers. Saliva samples were collected from focal individuals at two breeding colonies, one with low and the other with high density, during two consecutive years of contrasting food availability. Mothers and pups were sampled concurrently at birth and shortly before weaning, while pups were additionally sampled every 20 days. We found that heritability was low for baseline cortisol, while within-individual repeatability and among-individual variability were high. A substantial proportion of the variation in baseline cortisol could be explained in pups and mothers by a combination of intrinsic and extrinsic factors including sex, weight, day, season, and colony of birth. Our findings provide detailed insights into the individualization of endocrine phenotypes and their genetic and environmental drivers in a wild pinniped. Furthermore, the strong associations between cortisol and life history traits that we report in fur seals could have important implications for understanding the population dynamics of species impacted by environmental change.

sentences: Symbiodiniaceae conduct under natural bleaching stress during advanced gametogenesis stages of a mesophotic coral The mesophotic coral Alveopora allingi from the northern Gulf of Eilat/Aqaba, Red Sea, is affected by year-round partial coral-bleaching events. During these events, the migration of Symbiodiniaceae takes place from the coral-host mesoglea to the developed oocytes in bleached parts of colonies of A. allingi but not in the non-bleached parts. Additionally, these oocytes are abnormal, missing part of the structural material of the peripheral areas and are also significantly larger in the bleached areas of the colonies. Hence, we suggest a parasitic behavior of the symbionts or a commensalism relationship which enhance symbionts needs during bleaching periods and may boost the gametogenesis development in these corals. We propose that evolutionarily, this behavior may greatly contribute to the symbiont community survival throughout the bleaching period, and it can also be beneficial for the hosts persistence and adaptation to bleaching through the acquisition of a specific symbiont community following the bleaching event.

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Reconstructing the decline of Atlantic Cod with the help of environmental variability in the Scotia Shelf of Canada Ignoring environmental variability can lead to imprecise and inaccurate estimates of abundance and distribution of organisms. Fully embracing environmental variability can improve precision and accuracy of estimates of abundance and distribution, especially when it can be measured with lower costs. Using the example of Atlantic cod in the Scotian Shelf of the northwest Atlantic Ocean, we demonstrate the improved clarity of their historical population trends when such informative features are included. Further, the use of Bayesian spatiotemporal Conditional autoregressive models substantially improves our ability to understand the role of ecosystem variability upon cod, even when samples are incomplete or missing. Finally, by decomposing biomass into number, weight and a Hurdle process to estimate habitat conditions, we can extract much more information on what has occurred in the past and make reasoned inference on processes.

One-Sentence SummaryDeconstructing and reconstructing cod with environmental variability

sentences: Parasitic success and venom composition evolve upon specialization of parasitoid wasps to different host species AO_SCPLOWBSTRACTC_SCPLOWFemale endoparasitoid wasps usually inject venom into hosts to suppress their immune response and ensure offspring development. However, the parasitoids ability to evolve towards increased success on a given host simultaneously with the evolution of the composition of its venom has never been demonstrated. Here, we designed an experimental evolution to address this question. We crossed two parasitoid lines of Leptopilina boulardi differing both in parasitic success on different Drosophila hosts and venom composition. F2 descendants were reared on three different Drosophila species for nine generations. We tested for evolution of parasitic success over the generations and for the capacity of parasitoids selected on a given host to succeed on another host. We also tested whether the venom composition - based on a statistical analysis of the variation in intensity of the venom protein bands on SDS-PAGE 1D - evolved in response to different host species. Results showed a specialization of the parasitoids on their selection host and a rapid and differential evolution of the venom composition according to the host. Overall, data suggest a high potential for parasitoids to adapt to a new host, which may have important consequences in the field as well in the context of biological control.

sentences: Walking strides direct rapid and flexible recruitment of visual circuits for course control in <Drosophila> Flexible mapping between activity in sensory systems and movement parameters is a hallmark of successful motor control. This flexibility depends on continuous comparison of short-term postural dynamics and the longer-term goals of an animal, thereby necessitating neural mechanisms that can operate across multiple timescales. To understand how such body-brain interactions emerge to control movement across timescales, we performed whole-cell patch recordings from visual neurons involved in course control in Drosophila. We demonstrate that the activity of leg mechanosensory cells, propagating via specific ascending neurons, is critical to provide a clock signal to the visual circuit for stride-by-stride steering adjustments and, at longer timescales, information on speed-associated motor context to flexibly recruit visual circuits for course control. Thus, our data reveal a stride-based mechanism for the control of high-performance walking operating at multiple timescales. We propose that this mechanism functions as a general basis for adaptive control of locomotion.

sentences: SC2MeNetDrug: A computational tool to uncover inter-cell signaling targets and identify relevant drugs based on single cell RNA-seq data Single cell RNA sequencing (scRNA-seq) is a powerful technology to investigate the transcriptional programs in stromal, immune and tumor cells or neuron cells within the tumor or Alzheimers Disease (AD) brain microenvironment (ME) or niche. Cell-cell communications within ME play important roles in disease progression and immunotherapy response, and are novel and critical therapeutic targets. Though many tools of scRNA-seq analysis have been developed to investigate the heterogeneity and sub-populations of cells, few were designed for uncovering cell-cell communications of ME and predict the potentially effective drugs to inhibit the communications. Moreover, the data analysis processes of discovering signaling communication networks and effective drugs using scRNA-seq data are complex and involving a set of critical analysis processes and external supportive data resources, which are difficult for researchers who have no strong computational background and training in scRNA-seq data analysis. To address these challenges, in this study, we developed a novel computational tool, SC2MeNetDrug (https://fuhaililab.github.io/sc2MeNetDrug/). It was specifically designed using scRNA-seq data to identify cell types within MEs, uncover the dysfunctional signaling pathways within individual cell types, inter-cell signaling communications, and predict effective drugs that can potentially disrupt cell-cell signaling communications. SC2MeNetDrug provided a user-friendly graphical user interface to encapsulate the data analysis modules, which can facilitate the scRNA-seq data based-discovery of novel inter-cell signaling communications and novel therapeutic regimens.

sentences: A holistic insight of mycobacteriophage induced changes in mycobacterial cells Mycobacteriophages are phages that interact with mycobacteria resulting in their killing. Although lysis is the major mechanism by which mycobacteriophages cause cell death, other mechanisms may also be involved. The present study was initiated with the objective of investigating the changes that take place at the cellular level following the infection of mycobacterial cells by phage D29. To investigate this issue, we took recourse to performing immunofluorescence and electron microscopic studies. Transmission electron microscopic examination revealed the adsorption of phages on to the surface of mycobacteria, following which penetration of the tail through the thick mycoloic acid layer was seen. At later time points discrete populations of cells at different stages of lysis were observed, which comprised of completely lysed cells, in which the cells were fragmented and those at the early onset stage exhibited formation of membrane pores through which the phages and intracellular contents were released. SEM results also indicated that phages may come out through the entire surface of the cell, or alternatively through gaps in the surface. In some of the images we observed structures that apparently resembled membrane blebs which are normally encountered when cells undergo programmed cell death (PCD). In addition, we observed significant increase in DNA fragmentation as well as membrane depolarization, which are also indicative of occurrence of PCD. As several bacterial PCD pathways are mediated by the toxin-antitoxin (TA) modules, the expression profile of all the TA systems was examined before and after phage infection. Apart from specifically addressing the issue of PCD in mycobacteriophage infected cells, this investigation has led to the development of facile tools necessary for investigating mycobacteriophage-mycobacteria interactions by means of microscopic methods.

sentences: Inhibition of pyrimidine biosynthesis targets protein translation in AML. The mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes one of the rate-limiting steps in de novo pyrimidine biosynthesis, a pathway that provides essential metabolic precursors for nucleic acids, glycoproteins and phospholipids. DHODH inhibitors (DHODHi) are clinically used for autoimmune diseases and are emerging as a novel class of anti-cancer agents, especially in acute myeloid leukemia (AML) where pyrimidine starvation was recently shown to reverse the characteristic differentiation block in AML cells. Herein we show that DHODH blockade rapidly shuts down protein translation in leukemic stem cells (LSCs) by down-regulation of the multi-functional transcription factor YY1, has potent activity against AML in vivo and is well tolerated with minimal impact on normal blood development. Moreover, we find that ablation of CDK5, a gene that is recurrently deleted in AML and related disorders, increases the sensitivity of AML cells to DHODHi. Our studies provide important molecular insights and identify a potential biomarker for an emerging strategy to target AML.

sentences: GUIdEStaR (G-quadruplex, uORF, IRES, Epigenetics, Small RNA, Repeats), the integrated matadatabase: transcript-indexed binary information creation for chaining with neural network analysis GUIdEStaR integrates existing databases of various types of G-quadruplex, upstream Open Reading Frame (uORF), Internal Ribosome Entry Site (IRES), methylation to RNA and histone protein, small RNA, and repeats. GUIdEStaR consists of approx. 40,000 genes and 320,000 transcripts. An mRNA transcript is divided into 5 regions (5UTR, 3UTR, exon, intron, and biological region) where each region contains presence-absence data of 169 different types of elements. Recently, artificial intelligence (AI) based analysis of sequencing data has been gaining popularity in the area of bioinformatics. GUIdEStaR generates datasets that can be used as inputs to AI methods. At the GUIdEStaR homepage, users submit gene symbols by clicking a "Send" button, and shortly result files in CSV format are available for download at the result website. Users have an option to send the result files to their email addresses. Additionally, the entire database and the example Java codes are also freely available for download. Here, we demonstrate the database usage with three neural network classification studies-1) small RNA study for classifying transcription factor (TF) genes into either one of TF mediated by small RNA originated from SARS-CoV-2 or by human microRNA (miRNA), 2) cell membrane receptor study for classifying receptor genes as either with virus interaction or without one, and 3) nonsense mediated mRNA decay (NMD) study for classifying cell membrane and nuclear receptors as either NMD target or non-target. GUIdEStaR is available for access to the easy-to-use web-based database at www.guidestar.kr and for download at https://sourceforge.net/projects/guidestar.

sentences: Bioptim, a Python framework for Musculoskeletal Optimal Control in Biomechanics Musculoskeletal simulations are useful in biomechanics to investigate the causes of movement disorder, to estimate non-measurable physiological quantities or to study the optimality of human movement. We introduce Bioptim, an easy-to-use Python framework for biomechanical optimal control, handling musculoskeletal models. Relying on algorithmic differentiation and the multiple shooting formulation, Bioptim interfaces nonlinear solvers to quickly provide dynamically consistent optimal solutions. The software is both computationally efficient (C++ core) and easily customizable, thanks to its Python interface. It allows to quickly define a variety of biomechanical problems such as motion tracking/prediction, muscle-driven simulations, parameters optimization, multiphase problems, etc. It is also intended for real-time applications such as moving horizon estimation and model predictive control. Six contrasting examples are presented, comprising various models, dynamics, objective functions and constraints. They include data-driven simulations (i.e., a multiphase muscle driven gait cycle and an upper-limb real-time moving horizon estimation of muscle forces) and predictive simulations (i.e., a muscle-driven pointing task, a twisting somersault with a quaternion-based model, a position controller using external forces, and a multiphase torque-driven maximum-height jump motion).

sentences: Waves of sumoylation support transcription dynamics during adipocyte differentiation Tight control of gene expression networks required for adipose tissue formation and plasticity is essential for adaptation to energy needs and environmental cues. However, little is known about the mechanisms that orchestrate the dramatic transcriptional changes leading to adipocyte differentiation. We investigated the regulation of nascent transcription by the sumoylation pathway during adipocyte differentiation using SLAMseq and ChIPseq. We discovered that the sumoylation pathway has a dual function in differentiation; it supports the initial downregulation of pre-adipocyte-specific genes, while it promotes the establishment of the mature adipocyte transcriptional program. By characterizing sumoylome dynamics in differentiating adipocytes by mass spectrometry, we found that sumoylation of specific transcription factors like Ppar{gamma}/RXR and their co-factors is associated with the transcription of adipogenic genes. Our data demonstrate that the sumoylation pathway coordinates the rewiring of transcriptional networks required for formation of functional adipocytes. This study also provides an in-depth resource of gene transcription dynamics, SUMO-regulated genes and sumoylation sites during adipogenesis.

sentences: In Silico Epitope Prediction And VP1 Modelling For Foot-And-Mouth Serotype SAT 2 For Vaccine Design In East Africa Foot and Mouth Disease Virus has seven distinct, geographically localized, serotypes and a vaccination targeting one serotype does not confer immunity against another serotype. The use of inactivated vaccines is not safe and confers an immunity with a relatively shorter time. Using the VP1 sequences isolated in East Africa, we have predicted epitopes able to induce humoral and cell-mediated immunity in cattle. The Wu-Kabat variability index calculated in this study reflects the variable, including the known GH loop, and conserved regions, with the latter being good candidates for region-tailored vaccine design. Furthermore, we modelled the identified epitopes on a 3D model (PDB ID:5aca) to represent the epitopes structurally. This study can be used for in vitro and in vivo experiments.

sentences: Speech categorization reveals the role of early-stage temporal-coherence processing in auditory scene analysis Temporal coherence of sound fluctuations across spectral channels is thought to aid auditory grouping and scene segregation. Although prior studies on the neural bases of temporal-coherence processing focused mostly on cortical contributions, neurophysiological evidence suggests that temporal-coherence-based scene analysis may start as early as the cochlear nucleus (i.e., the first auditory region supporting cross-channel processing over a wide frequency range). Accordingly, we hypothesized that aspects of temporal-coherence processing that could be realized in early auditory areas may shape speech understanding in noise. We then explored whether physiologically plausible computational models could account for results from a behavioral experiment that measured consonant categorization in different masking conditions. We tested whether within-channel masking of target-speech modulations predicted consonant confusions across the different conditions, and whether predicted performance was improved by adding across-channel temporal-coherence processing mirroring the computations known to exist in the cochlear nucleus. Consonant confusions provide a rich characterization of error patterns in speech categorization, and are thus crucial for rigorously testing models of speech perception; however, to the best of our knowledge, they have not been utilized in prior studies of scene analysis. We find that within-channel modulation masking can reasonably account for category confusions, but that it fails when temporal fine structure (TFS) cues are unavailable. However, the addition of across-channel temporal-coherence processing significantly improves confusion predictions across all tested conditions. Our results suggest that temporal-coherence processing strongly shapes speech understanding in noise, and that physiological computations that exist early along the auditory pathway may contribute to this process.

sentences: Experimental sexual selection reveals rapid divergence in male and female reproductive transcriptomes and their interactions Postcopulatory interactions between the sexes in internally fertilizing species elicits both sexual conflict and sexual selection. Macroevolutionary and comparative studies have linked these processes to rapid transcriptomic evolution in sex-specific tissues and substantial transcriptomic postmating responses in females, patterns of which are altered when mating between reproductively isolated species. Here we test multiple predictions arising from sexual selection and conflict theory about the evolution of sex-specific and tissue-specific gene expression and the postmating response at the microevolutionary level. Following over 150 generations of experimental evolution under either reduced (enforced monogamy) or elevated (polyandry) sexual selection in Drosophila pseudoobscura, we found a substantial effect of sexual selection treatment on transcriptomic divergence in virgin male and female reproductive tissues (testes, male accessory glands, the female reproductive tract and ovaries). Sexual selection treatment also had a dominant effect on the postmating response, particularly in the female reproductive tract - the main arena for sexual conflict - compared to ovaries. This affect was asymmetric with monandry females typically showing more postmating responses than polyandry females, with enriched gene functions varying across treatments. The evolutionary history of the male partner had a larger effect on the postmating response of monandry females, but females from both sexual selection treatments showed unique patterns of gene expression and gene function when mating with males from the alternate treatment. Our microevolutionary results mostly confirm comparative macroevolutionary predictions on the role of sexual selection on transcriptomic divergence and altered gene regulation arising from divergent coevolutionary trajectories between sexual selection treatments.

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A Compressive Sensing Approach to Inferring Cognitive Representations with Reverse Correlation Uncovering high-level cognitive representations of categories such as faces is an elusive goal that has been frequently sought using reverse correlation, a technique employed in fields ranging from neurophysiology to cognitive psychology. In reverse correlation, subjects are asked to make perceptual judgments (e.g., "do you see a face?") about richly varying stimuli (e.g., white noise), and observed responses are then regressed against stimuli to yield reconstructions of the underlying cognitive representation. However, many thousands of stimulus-response pairs are frequently required, which severely limits the breadth of studies that are feasible using this powerful method. Techniques that are currently employed to improve efficiency, such as filtering the reconstruction, nevertheless bias the outcome. Here, we show that an advanced signal processing technique for improving sampling efficiency - compressive sensing - is directly compatible with reverse correlation. A trio of simulations are performed to demonstrate that compressive sensing can reduce the required stimulus-response pairs by up to 90% without biasing the reconstruction or can retrospectively improve the accuracy of the reconstructions on existing data. This work concludes by outlining the potential of compressive sensing to improve representation reconstruction throughout the field of neuroscience and beyond.

Significance StatementUncovering cognitive representations is an elusive goal that is increasingly pursued using the reverse correlation method, wherein human subjects make judgments about vague stimuli. Employing reverse correlation often entails collecting thousands of stimulus-response pairs, which severely limits the breadth of studies that are feasible using the method. Here we show that this methodological barrier can be overcome using compressive sensing, an advanced signal processing technique designed to improve sampling efficiency. Three sets of simulations are performed to demonstrate that compressive sensing can improve the accuracy of reconstructed cognitive representations and dramatically reduce the required number of stimulus-response pairs. This work concludes by outlining the potential of compressive sensing to improve representation reconstruction throughout the field of neuroscience and beyond.

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Multivalent designed proteins protect against SARS-CoV-2 variants of concern Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness.

One-Sentence SummaryWe designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.

sentences: Distinct Inhibitory Pathways Control Velocity and Directional Tuning in the Retina The sensory periphery is responsible for detecting ethologically relevant features of the external world, using compact, predominantly feedforward circuits. Visual motion is a particularly prevalent sensory feature, the presence of which can be a signal to enact diverse behaviors ranging from gaze stabilization reflexes, to predator avoidance or prey capture. To understand how the retina constructs the distinct neural representations required for these diverse behaviors, we investigated two circuits responsible for encoding different aspects of image motion: ON and ON-OFF direction selective ganglion cells (DSGCs). Using a combination of 2-photon targeted whole cell electrophysiology, pharmacology, and conditional knockout mice, we show that distinct inhibitory pathways independently control tuning for motion velocity and motion direction in these two cell types. We further employ dynamic clamp and numerical modeling techniques to show that asymmetric inhibition provides a velocity-invariant mechanism of directional tuning, despite the strong velocity dependence of classical models of direction selectivity. We therefore demonstrate that invariant representations of motion features by inhibitory interneurons act as computational building blocks to construct distinct, behaviorally relevant signals at the earliest stages of the visual system.

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Quantifying predator functional responses under field conditions reveals interactive effects of temperature and interference with sex and stage. O_LIPredator functional responses describe predator feeding rates and are a core component of predator-prey theory. Although originally defined as the relationship between predator feeding rates and prey densities, it is now well known that predator functional responses are shaped by a multitude of factors. Unfortunately, how these factors interact with one another remains unclear, as widely used laboratory methods for measuring functional responses are generally logistically constrained to examining a few factors simultaneously. Furthermore, it is also often unclear whether laboratory derived functional responses translate to field conditions.
C_LIO_LIOur goal was to apply an observational approach for measuring functional responses to understand how sex/stage differences, temperature, and predator interference interact to influence the functional response of zebra jumping spiders on midges under natural conditions.
C_LIO_LIWe used field feeding surveys of jumping spiders to infer spider functional responses. We applied a Bayesian model averaging approach to estimate differences among sexes and stages of jumping spiders in their feeding rates and their dependencies on midge densities, temperature, and predator interference.
C_LIO_LIWe find that females exhibit the steepest functional responses on midges, followed by juveniles, and then males, despite males being larger than juveniles. We also find that sexes and stages differ in the temperature dependence of their space clearance (aka attack) rates. We find little evidence of temperature dependence in females, whereas we find some evidence for an increase in space clearance rate at high temperatures in males and juveniles. Interference effects on feeding rates were asymmetric with little effect of interference on male feeding rates, and effects of interference on females and juveniles depending on the stage/sex from which the interference originates.
C_LIO_LIOur results illustrate the multidimensional nature of functional responses in natural settings and reveal how factors influencing functional responses can interact with one another through behavior and morphology. Further studies investigating the influence of multiple mechanisms on predator functional responses under field conditions will provide an increased understanding of the drivers of predator-prey interaction strengths and their consequences for communities and ecosystems.
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sentences: Variants identify sarcomere inter-protein contacts distinguishing inheritable cardiac muscle diseases Human ventriculum myosin ({beta}mys) powers contraction sometimes while complexed with myosin binding protein C (MYBPC3) on the myosin thick filament. The latter regulates {beta}mys activity through inter-protein contacts. Single nucleotide variants (SNVs) change protein sequence in {beta}mys or MYBPC3. They cause inheritable heart disease. When a SNV modified domain locates to an inter-protein contact it affects complex coordination. Domains involved, one in {beta}mys and the other in MYBPC3, form coordinated domains called co-domains. Co-domains are bilateral implying the potential for a shared impact from SNV modification in either domain suggesting their joint response to a common perturbation assigns location. Human population genetic divergence is the common systemic perturbation. A general contraction model with a neural/Bayes network design reveals SNV probabilities specifying correlations between domain members using 2D correlation genetics (2D-CG). It reveals co-domain locations in three common human heart diseases caused by SNVs, familial hypertrophic cardiomyopathy (FHC), dilated cardiomyopathy (DCM), and left ventricle non-compaction (LVN). Co-domain maps for DCM and LVN link MYBPC3 with two levels of myosin heads on the myosin thick filament surface implying these myosin dimers form the super-relaxed state (SRX). The FHC co-domain map involves just one myosin dimer implying the myosins do not form SRX. Comparing co-domain maps for FHC, DCM, and LVN phenotypes suggests SRX disruption involves a co-domain between MYBPC3 regulatory domain and the myosin regulatory light chain (RLC) N-terminus. The general contraction model scenarios, constructed from feed-forward neural networks, were explored with the purpose to understand how to interpret them mechanistically with basic natural language characteristics. These characteristics emerge from dependencies among inputs coded in hidden layer width and depth when they are deciphered using 2D-CG. In this application, the thick filament structural states emerge for FHC, DCM, and LVN phenotypes defining thick filament structural state joining the other standard characteristics of phenotype and pathogenicity. Emergent natural language interpretations for general network contraction models are on the horizon.

sentences: Insights into Mus musculus subspecies population structure across Eurasia revealed by whole-genome sequence analysis For more than 100 years, house mice (Mus musculus) have been used as a key animal model in biomedical research. House mice are genetically diverse, yet their genetic background at the global level has not been fully understood. Previous studies suggested that they originated in South Asia and diverged into three major subspecies almost simultaneously, approximately 350,000-500,000 years ago; however, they have spread across the world with the migration of modern humans in prehistoric and historic times ([~]10,000 years ago to present), and undergone secondary contact, which have complicated the genetic landscape of wild house mice. In this study, we sequenced the whole genomes of 98 wild house mice collected from Eurasia, particularly East Asia, Southeast Asia, and South Asia. We found that although wild house mice consist of three major genetic groups corresponding to the three major subspecies, individuals representing admixture between subspecies are much more ubiquitous than previously recognized. Furthermore, several samples showed an incongruent pattern of genealogies between mitochondrial and autosomal genomes. Using samples likely retaining the original genetic components of subspecies with least admixture, we estimated the pattern and timing of divergence among the subspecies. The results are important for understanding the genetic diversity of wild mice on a global level and the information will be particularly useful in future biomedical and evolutionary studies using laboratory mice established from these wild mice.

sentences: Evaluating brain parcellations using the distance controlled boundary coefficient One important approach to human brain mapping is to define a set of distinct regions that can be linked to unique functions. Numerous brain parcellations have been proposed, using cytoarchitectonic, structural or functional Magnetic Resonance Imaging (fMRI) data. The intrinsic smoothness of brain data, however, poses a problem for current methods seeking to compare different parcellations. For example, criteria that simply compare within-parcel to between-parcel similarity provide even random parcellations with a high value. Furthermore, the evaluation is biased by the spatial scale of the parcellation. To address this problem, we propose the Distance Controlled Boundary Coefficient (DCBC), an unbiased criterion to evaluate discrete parcellations. We employ this new criterion to evaluate existing parcellations of the human neocortex in their power to predict functional boundaries for an fMRI data set with many different tasks, as well as for resting-state data. We find that common anatomical parcellations do not perform better than chance, suggesting that task-based functional boundaries do not align well with sulcal landmarks. Parcellations based on resting-state fMRI data perform well; in some cases, as well as a parcellation defined on the evaluation data itself. Finally, multi-modal parcellations that combine functional and anatomical criteria perform substantially worse than those based on functional data alone, indicating that functionally homogeneous regions often span major anatomical landmarks. Overall, the DCBC advances the field of functional brain mapping by providing an unbiased metric that compares the predictive ability of different brain parcellations to define brain regions that are functionally maximally distinct.

sentences: Cortical tracking of unheard formant modulations derived from silently presented lip movements and its decline with age The integration of visual and auditory cues is crucial for successful processing of speech, especially under adverse conditions. Recent reports have shown that when participants watch muted videos of speakers, the phonological information about the acoustic speech envelope is tracked by the visual cortex. However, the speech signal also carries much richer acoustic details, e.g. about the fundamental frequency and the resonant frequencies, whose visuo-phonological transformation could aid speech processing. Here, we investigated the neural basis of the visuo-phonological transformation processes of these more fine-grained acoustic details and assessed how they change with ageing. We recorded whole-head magnetoencephalography (MEG) data while participants watched silent intelligible and unintelligible videos of a speaker. We found that the visual cortex is able to track the unheard intelligible modulations of resonant frequencies and the pitch linked to lip movements. Importantly, only the processing of intelligible unheard formants decreases significantly with age in the visual and also in the cingulate cortex. This is not the case for the processing of the unheard speech envelope, the fundamental frequency or the purely visual information carried by lip movements. These results show that unheard spectral fine-details (along with the unheard acoustic envelope) are transformed from a mere visual to a phonological representation. Aging affects especially the ability to derive spectral dynamics at formant frequencies. Since listening in noisy environments should capitalize on the ability to track spectral fine-details, our results provide a novel focus on compensatory processes in such challenging situations.

sentences: Genetic architecture of natural variation of cardiac performance in flies Deciphering the genetic architecture of human cardiac disorders is of fundamental importance but their underlying complexity is a major hurdle. We investigated the natural variation of cardiac performance in the sequenced inbred lines of the Drosophila Genetic Reference Panel (DGRP)1. Genome Wide Associations Studies (GWAS) identified genetic networks associated with natural variation of cardiac traits which were used to gain insights as to the molecular and cellular processes affected. Non-coding variants that we identified were used to map potential regulatory non-coding regions, which in turn were employed to predict Transcription Factors (TFs) binding sites. Cognate TFs, many of which themselves bear polymorphisms associated with variations of cardiac performance, were also validated by heart specific knockdown. Additionally, we showed that the natural variations associated with variability in cardiac performance affect a set of genes overlapping those associated with average traits but through different variants in the same genes. Furthermore, we showed that phenotypic variability was also associated with natural variation of gene regulatory networks. More importantly, we documented correlations between genes associated with cardiac phenotypes in both flies and humans, which supports a conserved genetic architecture regulating adult cardiac function from arthropods to mammals. Specifically, roles for PAX9 and EGR2 in the regulation of the cardiac rhythm were established in both models, illustrating that the characteristics of natural variations in cardiac function identified in Drosophila can accelerate discovery in humans.

sentences: Gene editing for accelerated breeding in wheat Diseases adversely affect grain yield of crop plants. Leaf rust is a major disease of wheat. As race-specific resistance breaks down, introduction of newer sources of resistance often from older accessions is necessary. Linkage drag from the donor accessions adversely impacts grain yield. As a result, CIMMYT breeding effort has shifted to using durable resistance, also referred to as race non-specific host resistance, which allows slow rusting but maintains grain yield. One of the key genes for durable resistance is Lr67 and the resistant form of this gene is absent in CIMMYT elite lines. Hence, we have initiated efforts to convert the susceptible copy of Lr67 into its resistant form directly in these elite lines using gene editing. This would eliminate backcrossing and thus save time as well as eliminate linkage drag that would accompany the resistant copy of the gene if it were to be introgressed from an older accession. As first steps, we have isolated and sequenced the genomic copies from each of the A, B, and D genome of the Lr67 gene from three elite lines and an experimental line. Identification of more than 50 single nucleotide polymorphisms (SNPs) in the open reading frames (ORF) among these lines would be useful in designing the guide RNA molecules with precision. Further, we have streamlined genetic transformation of these elite lines, a prerequisite step for gene editing.

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Apiaceae FNS I originated from F3H through tandem gene duplication BackgroundFlavonoids are specialized metabolites with numerous biological functions in stress response and reproduction of plants. Flavones are one subgroup that is produced by the flavone synthase (FNS). Two distinct enzyme families evolved that can catalyze the biosynthesis of flavones. While the membrane-bound FNS II is widely distributed in seed plants, one lineage of soluble FNS I appeared to be unique to Apiaceae species.

ResultsWe show through phylogenetic and comparative genomic analyses that Apiaceae FNS I evolved through tandem gene duplication of flavanone 3-hydroxylase (F3H) followed by neofunctionalization. Currently available datasets suggest that this event happened within the Apiaceae in a common ancestor of Daucus carota and Apium graveolens. The results also support previous findings that FNS I in the Apiaceae evolved independent of FNS I in other plant species.

ConclusionWe validated a long standing hypothesis about the evolution of Apiaceae FNS I and predicted the phylogenetic position of this event. Our results explain how an Apiaceae-specific FNS I lineage evolved and confirm independence from other FNS I lineages reported in non-Apiaceae species.

sentences: Effects of L-Dopa, SKF-38393, and Quinpirole on exploratory, anxiety- and depressive-like behaviors in pubertal female and male mice Adolescence is a phase of substantial changes in the brain, characterized by maturational remodeling of many systems. This remodeling allows functional plasticity to adapt in a changing environment but turns this period into a neurodevelopmental vulnerable window. The dopaminergic system is under morphological and physiological changes during this phase. The disruption of its balance can lead to molecular variation and abnormal behavior - representing a risk factor for neuropsychiatric disorders. In the present study, we investigated if changes in the dopaminergic tone alter mice behavior in a receptor and sex-specific manner, specifically in the beginning of puberty period. We administered L-Dopa, SKF-38393 (D1 dopamine receptor agonist) and Quinpirole (D2 dopamine receptor agonist) and tested male and female mice motor, anxiety- and depressive-like behavior. While females displayed an impaired exploratory drive, males presented an intense depressive-like response. Our results provide insights into the function of dopaminergic development in adolescent behavior and highlight the importance of studies in this time window with male and female subjects.

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Structure-function Specialisation of the Interfascicular Matrix in the Human Achilles Tendon ObjectiveTendon consists of highly aligned collagen-rich fascicles surrounded by interfascicular matrix (IFM). Some tendons act as energy stores to improve locomotion efficiency; these tendons are prone to debilitating injuries, the incidence of which increases with ageing. In equine tendons, energy storage is achieved primarily through specialisation of the IFM. However, no studies have investigated IFM structure-function specialisation in human tendons. Here, we compare the positional anterior tibialis and energy storing Achilles tendons, testing the hypothesis that the Achilles IFM has specialised composition and mechanical properties, which are lost with ageing.

MethodsWe used a multidisciplinary combination of mechanical testing, immunolocalisation and proteomics to investigate structure-function specialisations in functionally distinct human tendons and how these are altered with ageing.

ResultsThe IFM in the energy storing Achilles tendon is more elastic and fatigue resistant than the IFM in the positional anterior tibialis tendon, with a trend towards decreased fatigue resistance with age in the Achilles IFM. With ageing, alterations occur predominantly to the proteome of the Achilles IFM.

ConclusionThe Achilles tendon IFM is specialised for energy storage, and changes to its proteome with ageing are likely responsible for the observed trends towards decreased fatigue resistance. Knowledge of key energy storing specialisations and their changes with ageing offers insight towards developing effective treatments for tendinopathy.

Key messagesO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIEnergy storing tendons in animals and humans are particularly prone to tendinopathy and the incidence increases with increasing age.
C_LIO_LIPrevious work in some animal models has shown that the specialisation of tendon properties for energy storage is achieved primarily through adaptation of the interfascicular matrix, with specialisation lost in ageing. However, the structural specialisations that provide the human Achilles tendon with its energy storing ability, and how these are affected by ageing, remain to be established.
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What does this study add?O_LIWe demonstrate that the interfascicular matrix in the human Achilles tendon is specialised for energy storing, with increased elastic recoil and fatigue resistance, and that these specialisations are partially lost with ageing, likely due to alterations to the proteome of the interfascicular matrix.
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How might this impact on clinical practice or future developments?O_LIShort term, the specialist IFM mechanics we have demonstrated can be detected with new developments in ultrasound functional imaging, offering improved opportunities for contextual tendinopathy diagnostics. Personalised rehabilitation programmes can now be explored and designed specifically to target IFM mechanics.
C_LIO_LILonger term, the knowledge of key specialisations in injury prone energy storing tendons and how they are affected by ageing, offers crucial insight towards developing cell or tissue engineering treatments targeted at restoring tendon structure and function post-injury, specifically targeted at the IFM.
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sentences: Transcriptional activity and epigenetic regulation of transposable elements in the symbiotic fungus Rhizophagus irregularis Arbuscular mycorrhizal (AM) fungi form mutualistic relationships with most land plant species and have long been considered as ancient asexuals. Long-term clonal evolution would be remarkable for a eukaryotic lineage and suggests the importance of alternative mechanisms to promote genetic variability facilitating adaptation. Here, we assessed the potential of transposable elements (TEs) for generating genomic diversity. The dynamic expression of TEs during Rhizophagus irregularis spore development suggests ongoing TE activity. We find Mutator-like elements located near genes belonging to highly expanded gene families. Characterising the epigenomic status of R. irregularis provides evidence of DNA methylation and small RNA production occurring at TE loci. Our results support a potential role for TEs in shaping the genome, and roles for DNA methylation and small RNA-mediated silencing in regulating TEs. A well-controlled balance between TE activity and repression may therefore contribute to genome evolution in AM fungi.

sentences: Regeneration of humoral immunity from pluripotent stem cells by defined transcription factors Regeneration of humoral immunity from pluripotent stem cells (PSCs) is a crucial aim in translational medicine. However, reconstitution of complete, sustained, and functional B lymphopoiesis from PSCs has not yet been developed. Here, we successfully achieved regenerative B lymphopoiesis in B-cell deficient animals transplanted with PSC-derived hematopoietic progenitors (iHPCs) guided by synergistic expression of Runx1, Hoxa9, and Lhx2. Upon transplantation, the iHPCs immediately gave rise to pro/pre-B cells in recipients bone marrow, which were able to further differentiate into the entire B cell lineages, including innate B-1a, B-1b, MZ B cells, as well as adaptive FO B cells. In responding to antigen stimuli, the regenerative B cells produced adaptive humoral immune responses, sustained a prolonged antigen-specific antibody production, and formed immune-memory. Particularly, the regenerative B cells in spleen showed developing patterns of immunoglobulin chain-switch and hyper-mutation via a cross-talk with the host T follicular helper cells, which eventually formed T cell-dependent humoral responses. This study provides de novo evidence that B lymphopoiesis can be regenerated from PSCs via a HSC-independent approach, which provides insights into treating B-cell related humoral deficiencies using PSCs as unlimited cell resource.

sentences: scREMOTE: Using multimodal single cell data to predict regulatory gene relationships and to build a computational cell reprogramming model Cell reprogramming offers a potential treatment to many diseases, by regenerating specialized somatic cells. Despite decades of research, discovering the transcription factors that promote cell reprogramming has largely been accomplished through trial and error, a time-consuming and costly method. A computational model for cell reprogramming, however, could guide the hypothesis formulation and experimental validation, to efficiently utilize time and resources. Current methods often cannot account for the heterogeneity observed in cell reprogramming, or they only make short-term predictions, without modelling the entire reprogramming process. Here, we present scREMOTE, a novel computational model for cell reprogramming that leverages single cell multiomics data, enabling a more holistic view of the regulatory mechanisms at cellular resolution. This is achieved by first identifying the regulatory potential of each transcription factor and gene to uncover regulatory relationships, then a regression model is built to estimate the effect of transcription factor perturbations. We show that scREMOTE successfully predicts the long-term effect of overexpressing two key transcription factors in hair follicle development by capturing higher-order gene regulations. Together, this demonstrates that integrating the multimodal processes governing gene regulation creates a more accurate model for cell reprogramming with significant potential to accelerate research in regenerative medicine.

sentences: A multi-dimensional integrative scoring framework for predicting functional variants in the human genome Attempts to identify and prioritize functional DNA elements in coding and noncoding regions, particularly through use of in silico functional annotation data,continue to increase in popularity. However, specific functional roles may vary widely from one variant to another, making it challenging to summarize different aspects of variant function. Here we propose Multi-dimensional Annotation Class Integrative Estimation (MACIE), an unsupervised multivariate mixed model framework capable of integrating annotations of diverse origin to assess multi-dimensional functional roles for both coding and noncoding variants. Unlike existing one-dimensional scoring methods, MACIE views variant functionality as a composite attribute encompassing multiple characteristics, and estimates the joint posterior functional probability vector of each genomic position, a quantity that offers richer and more interpretable information in the presence of multiple aspects of functionality. Applied to a variety of independent coding and non-coding datasets, MACIE demonstrates powerful and robust performance in discriminating between functional and non-functional variants. We also show an application of MACIE to fine-mapping using lipids GWAS summary statistics data from the European Network for Genetic and Genomic Epidemiology Consortium.

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Artificial Intelligence Approaches to Assessing Primary Cilia Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and colocalization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.

SUMMARYThe use of Artificial Intelligence (Ai) to analyze images is emerging as a powerful, less biased, and rapid approach compared with commonly used methods. Here we trained Ai to recognize a cellular organelle, primary cilia, and analyze properties such as length and staining intensity in a rigorous and reproducible manner.

sentences: Lytic transglycosylases mitigate periplasmic crowding by degrading soluble cell wall turnover products The peptidoglycan cell wall is a predominant structure of bacteria, determining cell shape and supporting survival in diverse conditions. Peptidoglycan is dynamic and requires regulated synthesis of new material, remodeling, and turnover - or autolysis - of old material. Despite exploitation of peptidoglycan synthesis as an antibiotic target, we lack a fundamental understanding of how peptidoglycan synthesis and autolysis intersect to maintain the cell wall. Here, we uncover a critical physiological role for a widely misunderstood class of autolytic enzymes, lytic transglycosylases (LTGs). We demonstrate that LTG activity is essential to survival by contributing to periplasmic processes upstream and independent of peptidoglycan recycling. Defects accumulate in Vibrio cholerae LTG mutants due to generally inadequate LTG activity, rather than absence of specific enzymes, and essential LTG activities are likely independent of protein-protein interactions, as heterologous expression of a non-native LTG rescues growth of a conditionally LTG-null mutant. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains, also detected in the wild-type, are enriched in LTG mutants, and that LTG mutants are hypersusceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that LTGs prevent toxic crowding of the periplasm with synthesis-derived peptidoglycan polymers and contrary to prevailing models, that this autolytic function can be temporally separate from peptidoglycan synthesis.

sentences: POGZ modulates the DNA damage response in a HP1-dependent manner The heterochromatin protein HP1 plays a central role in the maintenance of genome stability, in particular by promoting homologous recombination (HR)-mediated DNA repair. However, little is still known about how HP1 is controlled during this process. Here, we describe a novel function of the POGO transposable element derived with ZNF domain protein (POGZ) in the regulation of HP1 during the DNA damage response in vitro. POGZ depletion delays the resolution of DNA double-strand breaks (DSBs) and correlates with an increased sensitivity to different DNA damaging agents, including the clinically-relevant Cisplatin and Talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair pathways by retaining the BRCA1/BARD1 complex at DSBs, in a HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonically lethal and Pogz haplo-insufficiency (Pogz+/{Delta}) results in a developmental delay, impaired intellectual abilities, a hyperactive behaviour as well as a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Importantly, Pogz+/{Delta} mice are radiosensitive and accumulate DSBs in diverse tissues, including the spleen and the brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo, with clinical implications for the WHSUS.

sentences: Parametric modulators of sex-biased conditioned fear responding Pavlovian fear conditioning is a widely used behavioral paradigm for studying associative learning in rodents. Despite early recognition that subjects may engage in a variety of both conditioned and unconditioned responses, the last several decades have seen the field narrow its focus to measure freezing as the sole indicator of conditioned fear. We previously reported that female rats were more likely than males to engage in darting, an escape-like conditioned response that is associated with heightened shock reactivity. To determine how experimental parameters contribute to the frequency of darting in both males and females, we manipulated factors such as chamber size, shock intensity, and number of trials. To better capture fear-related behavioral repertoires in our animals, we developed ScaredyRat, an open-source custom Python tool that analyzes Noldus Ethovision-generated raw data files to identify Darters and quantify both conditioned and unconditioned responses. We find that like freezing, conditioned darting occurrences scale with experimental alterations. While most darting occurs in females, we find that with an extended training protocol, darting can emerge in males as well. Collectively, our data suggest that darting reflects a behavioral switch in conditioned responding that is a product of both an individual animals sex, shock reactivity, and experimental parameters, underscoring the need for careful consideration of sex as a biological variable in classic learning paradigms.

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Challenges of γ9δ2TCR affinity maturation when using phage display BackgroundOver the past years we showed that the efficacy of {beta}T cells engineered to express a defined {gamma}{delta}TCR (TEG) depends on the functional avidity of the {gamma}9{delta}2TCR. We hypothesized that functional avidity mediated through {gamma}9{delta}2TCR in the TEG format could be further enhanced by increasing affinity of the {gamma}9{delta}2TCR.

MethodsWe attempted to overcome limited affinity of natural occurring {gamma}9{delta}2TCRs through affinity maturation by phage display using a library containing mutations in CDR1 and CDR2 of both TCR chains.

ConclusionAffinity maturation of {gamma}9{delta}2TCR by using phage display was not successful. The largest hurdle was the periplasmic expression of {gamma}9{delta}2TCR constructs in E.coli which is a prerequisite for successful phage display. The underlying reason for this lack of expression was the instability of the single chain (sc)TCR format. Expression of scTCR formats in HEK293F cells yielded only 15-20% correctly folded scTCR.

sentences: N440K variant of SARS-CoV-2 has Higher Infectious Fitness Several variants of SARS-CoV-2 have been emerging across the globe, continuing to threaten the efforts to end COVID-19 pandemic. Recent data indicate the prevalence of variants with N440K Spike substitution in several parts of India, which is under the second wave of the pandemic. Here, we first analyze the prevalence of N440K variants within the sequences submitted from India and identify a rising trend of its spread across various clusters. We then compare the replicative fitness and infectivity of a prototype of this variant with two other previously prevalent strains. The N440K variant produced ten times higher infectious viral titers than a prevalent A2a strain, and over 1000 folds higher titers than a much less prevalent A3i strain prototype in Caco2 cells. Similar results were detected in Calu-3 cells as well, confirming the increased potency of the N440K variant. Interestingly, A3i strain showed the highest viral RNA levels, but the lowest infectious titers in the culture supernatants, indicating the absence of correlation between the RNA content and the infectivity of the sample. N440K mutation has been reported in several viral sequences across India and based on our results, we predict that the higher infectious titers achieved by N440K variant could possibly lead to its higher rate of transmission. Availability of more sequencing data in the immediate future would help understand the potential spread of this variant in more detail.

sentences: Plant longevity, drought and island isolation favoured rampant evolutionary transitions towards insular woodiness Insular woodiness (IW)--the evolutionary transition from herbaceousness towards woodiness on islands--is one of the most iconic features of island floras. Since pioneering work by Darwin and Wallace, five IW drivers have been proposed: (i) favourable aseasonal climate and (ii) lack of large native herbivores promote plant longevity that (iii) results in prolonged flowering favouring outcrossing. Alternatively, (iv) competition for sunlight requires taller and stronger stems, and (v) drought favours woodiness to safeguard root-to-shoot water transport. However, information on the occurrence of IW is fragmented, hampering tests of these potential drivers. Here, we identify 1,097 insular woody species (IWS) on 375 islands, and infer at least 175 evolutionary transitions on 31 archipelagos, concentrated in six angiosperm families. Structural equation models reveal that the IWS richness on oceanic islands correlates with aseasonal favourable climate, followed by increased drought and island isolation (approximating competition). When continental islands are included, reduced herbivory pressure by large native mammals, increased drought and island isolation are most relevant. The repeated evolution of IW opens promising avenues to disentangle the variation in gene regulatory networks triggering wood formation, and emphasize individual archipelagos as laboratories of evolution, where similar environmental conditions replicate convergent evolution of similar traits.

sentences: Dominance shifts increase the likelihood of soft selective sweeps Genetic models of adaptation to a new environment have typically assumed that the alleles involved maintain a constant fitness dominance across the old and new environments. However, theories of dominance suggest that this should often not be the case. Instead, the alleles involved should frequently shift from recessive deleterious in the old environment to dominant beneficial in the new environment. Here, we study the consequences of these expected dominance shifts for the genetics of adaptation to a new environment. We find that dominance shifts increase the likelihood that adaptation occurs from the standing variation, and that multiple alleles from the standing variation are involved (a soft selective sweep). Furthermore, we find that expected dominance shifts increase the haplotypic diversity of selective sweeps, rendering soft sweeps more detectable in small genomic samples. In cases where an environmental change threatens the viability of the population, we show that expected dominance shifts of newly beneficial alleles increase the likelihood of evolutionary rescue and the number of alleles involved. Finally, we apply our results to a well-studied case of adaptation to a new environment: the evolution of pesticide resistance at the Ace locus in Drosophila melanogaster. We show that, under reasonable demographic assumptions, the expected dominance shift of resistant alleles causes soft sweeps to be the most frequent outcome in this case, with the primary source of these soft sweeps being the standing variation at the onset of pesticide use, rather than recurrent mutation thereafter.

sentences: Tracking of Anopheles stephensi in Ethiopia using mitochondrial DNA reveals pattern of spread The recent detection of the South Asian malaria vector Anopheles stephensi in the Horn of Africa (HOA) raises concerns about the impact of this mosquito on malaria transmission in the region. The mode and history of introduction is important for predicting the likelihood of continued introduction and future spread. Analysis of An. stephensi genetic diversity and population structure can provide insight into the history of the mosquito in the HOA. We investigated genetic diversity of An. stephensi in eastern Ethiopia where detection suggests a range expansion to this region to understand the history of this invasive population. We sequenced the cytochrome oxidase subunit I (COI) and cytochrome B gene (CytB) in 187 An. stephensi collected from 10 sites in Ethiopia in 2018. Phylogenetic analyses using a maximum-likelihood approach and minimum spanning network were conducted for Ethiopian sequences. Molecular identification of bloodmeal sources was also performed using universal vertebrate CytB sequencing. Six COI-CytB haplotypes were observed based on five segregating sites, with the highest number of haplotypes in the northeastern sites (Semera, Bati, and Gewana towns) relative to the southeastern sites (Kebridehar, Godey, and Degehabur) in eastern Ethiopia. In the phylogenetic and network analysis, we observed population differentiation based on the distribution of the haplotypes across the northeastern and central sites (Erer Gota, Dire Dawa, and Awash Sebat Kilo) compared to the southeastern sites and evidence of a South Asian origin of the HOA An. stephensi lineages. The presence of the putative South Asian haplotype of origin at sites closest to Ethiopias northeastern borders support route of introductions into Ethiopia from the northeast. Finally, molecular bloodmeal analysis revealed evidence of feeding on bovines, goats, dogs, and humans, as well as evidence of multiple (mixed) blood meals. In conclusion, we find support for the hypothesis for the recent expansion of An. stephensi into southeastern Ethiopia with multiple introductions. We also find evidence that supports the hypothesis that HOA An. stephensi populations originate from South Asia rather than the Arabian Peninsula. The evidence of both zoophagic and anthropophagic feeding support the potential for livestock movement to play a role in vector spread in this region.

sentences: Self-regulation of the brain's right frontal Beta rhythm using a brain-computer interface Neural oscillations, or brain rhythms, fluctuate in a manner reflecting ongoing behavior. Whether these fluctuations are instrumental or epiphenomenal to the behavior remains elusive. Attempts to experimentally manipulate neural oscillations exogenously using non-invasive brain stimulation have shown some promise, but difficulty with tailoring stimulation parameters to individuals has hindered progress in this field. We demonstrate here using electroencephalography (EEG) neurofeedback in a brain-computer interface that human participants (n=44) learned over multiple sessions across a 6-day period to self-regulate their Beta rhythm (13-20 Hz) over the right inferior frontal cortex (rIFC). The modulation was evident only during neurofeedback task performance but did not lead to offline alteration of Beta rhythm characteristics at rest, nor to changes in subsequent cognitive behavior. Likewise, a control group (n=38) who underwent training of the Alpha rhythm (8-12 Hz) did not exhibit behavioral changes. Although the right frontal Beta rhythm has been repeatedly implicated as a key component of the brains inhibitory control system, the present data suggest that its manipulation offline prior to cognitive task performance does not result in behavioral change. Thus, this form of neurofeedback training of the tonic Beta rhythm would not serve as a useful therapeutic target for disorders with dysfunctional inhibitory control as their basis.

sentences: A genome-wide CRISPR screen identifies the glycosylation enzyme DPM1 as a modifier of DPAGT1 deficiency and ER stress Partial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1-CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually cause CDGs. While both in vivo models ostensibly cause ER stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress.

sentences: Smart3-ATAC: a highly sensitive method for joint accessibility and full-length transcriptome analysis in single cells Joint single-cell measurements of gene expression and DNA regulatory element activity holds great promise as a tool to understand transcriptional regulation. Towards this goal we have developed Smart3-ATAC, a highly sensitive method which allows joint mRNA and chromatin accessibility analysis genome wide in single cells. With Smart3-ATAC, we are able to obtain the highest possible quality measurements per cell. The method combines transcriptomic profiling based on the highly sensitive Smart-seq3 protocol on cytosolic mRNA, with a novel low-loss single-cell ATAC (scATAC) protocol to measure chromatin accessibility. Compared to current droplet multiome methods, the yield of both the scATAC protocol and mRNA-seq protocol is markedly higher.

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Inhibition of BCMA signaling alters protein translation and Multiple Myeloma growth Recent T Cell therapies have been effective in the treatment of hematological cancers. However, immunotoxicity and treatment relapse pose significant clinical challenges. Here, we revealed distinctive requirement for neutralizing TNF receptor ligands APRIL and BAFF in MM and DLBCL. Furthermore, we investigated the use of BCMA decoy receptor (sBCMA-Fc) as a therapeutic inhibitor of ARPIL and BAFF. While wild-type sBCMA-Fc successfully blocked APRIL signaling with picomolar binding affinity, inhibiting tumor growth in MM models, it lacked efficacy in inhibiting DLBCL progression due to its weak binding for BAFF. To expand the therapeutic utility of sBCMA-Fc, using a protein engineering approach, we generated an affinity enhanced mutant sBCMA-Fc fusion molecule (sBCMA-Fc V3) with 4-folds and 500-folds enhancement in binding to APRIL and BAFF respectively. The sBCMA-Fc V3 clone significantly enhanced antitumor activity against both MM and DLBCL. Importantly, sBCMA-Fc V3 was proven to be a viable clinical candidate by showing adequate toxicity profile and on-target mechanism of action in nonhuman primates.

SUMMARYThis study demonstrates the dichotomous function of APRIL and BAFF in MM and DLBCL, that can be safely targeted by an engineered fusion protein designed to trap APRIL and BAFF with ultra-high binding affinity.

sentences: Recurrent chromosome reshuffling and the evolution of neo-sex chromosomes in parrots The karyotype of most birds has remained considerably stable during more than 100 million years evolution, except for some groups, such as parrots. The evolutionary processes and underlying genetic mechanism of chromosomal rearrangements in parrots, however, are poorly understood. Here, using chromosome-level assemblies of three parrot genomes (monk parakeet, blue-fronted amazon, budgerigar), we uncovered frequent chromosome fusions and fissions among parrots, with most of them being lineage-specific. In particular, at least 12 chromosomes recurrently experienced inter-chromosomal fusions in different parrot lineages. Two conserved vertebrate genes, ALC1 and PARP3, with known functions in the repair of double-strand breaks and maintenance of genome stability, were specifically lost in parrots. The loss of ALC1 was associated with multiple deletions and an accumulation of CR1-psi, a novel subfamily of transposable elements (TEs) that recently amplified in parrots, while the loss of PARP3 was associated with an inversion. Additionally, the fusion of the ZW sex chromosomes and chromosome 11 has created a pair of neo-sex chromosomes in the ancestor of parrots, and the chromosome 25 has been further added to the sex chromosomes in monk parakeet. The newly formed neo-sex chromosomes were validated by our chromosomal painting, genomic and phylogenetic analyses. Transcriptome profiling for multiple tissues of males and females did not reveal signals of female-specific selection driving the formation of neo-sex chromosomes. Finally, we identified one W-specific satellite repeat that contributed to the unusual enlargement of the W chromosome in monk parakeet. Together, the combination of our genomic and cytogenetic analyses highlight the role of TEs and genetic drift in promoting chromosome rearrangements, gene loss and the evolution of neo-sex chromosome in parrots.

sentences: Single Pulse Heating of Nanoparticle Array for Biological Applications With the ability to convert external excitation into heat, nanomaterials play an essential role in many biomedical applications. Two modes of nanoparticle (NP) array heating, nanoscale-confined heating (NCH) and macroscale-collective heating (MCH), have been found and extensively studied. Despite this, the resulting biological response at protein level remains elusive. In this study, we developed a computational model to systematically investigate the single-pulsed heating of NP array and corresponding protein denaturation/activation. We found that NCH may lead to targeted protein denaturation, however, nanoparticle heating does not lead to nanoscale selective TRPV1 channel activation. The excitation duration and NP concentration are primary factors that determine a window for targeted protein denaturation, and together with heating power, we defined quantified boundaries for targeted protein denaturation. Our results boost our understandings in the NCH and MCH under realistic physical constraints and provide a robust guidance to customize biomedical platforms with desired NP heating.

sentences: Chlamydomonas RABL4/IFT27 Mediates Phototaxis via Promoting BBSome-dependent Ciliary Export of Phospholipase D Phospholipase D (PLD) interacts with the BBSome for loading onto retrograde intraflagellar transport (IFT) trains to exit cilia in Chlamydomonas reinhardtii. PLD ciliary retention and depletion of Rab-like 4 (RABL4) GTPase IFT27 cause the same non-phototactic phenotype but not impair IFT and ciliation. Here, we show that the IFT-B1 subunit IFT27 binds its partner IFT25 to form the heterodimeric IFT25/27 in an IFT27 nucleotide state-independent manner. IFT25/27, IFT-A, and IFT-B are irrelevant for maintaining the stability of one another. GTP-loading onto IFT27 enhances the IFT25/27 affinity for IFT-B1 in cytoplasm, while GDP-loaded IFT27 does not prevent IFT25/27 from entering and cycling through cilia by integrating into IFT-B1. Upon at the ciliary tip, IFT25/27 cycles on and off IFT-B1 and this process is irrelevant with the nucleotide state of IFT27. During BBSome remodeling at the ciliary tip, IFT25/27 promotes BBSome reassembly independent of IFT27 nucleotide state, making post-remodeled BBSomes available for PLD to interact with. Therefore, IFT25/27 facilitates BBSome-dependent PLD export from cilia via controlling availability of intact BBSomes at the ciliary tip, providing a regulatory mechanism for IFT27 to mediate phototaxis in C. reinhardtii.

sentences: A potent tumour-suppressor TCF11, than its prototypic Nrf1α, is defined by similar yet different regulatory profiles of both isoforms, with a striking disparity from Nrf2 Nrf1 and Nrf2, as two principal CNC-bZIP transcription factors, regulate similar but different targets involved in a variety of biological functions for maintaining cell homeostasis and organ integrity. Of note, the unique topobiological behavior of Nrf1 makes its functions more complicated than Nrf2, because it is allowed for alternatively transcribing and selectively splicing to yield multiple isoforms (e.g., TCF11, Nrf1). In order to gain a better understanding of their similarities and differences in distinct regulatory profiles, all four distinct cell models for stably expressing TCF11, TCF11{Delta}N, Nrf1 or Nrf2 have been herein established by an Flp-In T-REx-293 system and then identified by transcriptomic sequencing. Further analysis revealed that Nrf1 and TCF11 have similar yet different regulatory profiles, although both contribute basically to positive regulation of their co-targets, which are disparate from those regulated by Nrf2. Such disparity in those gene regulation by Nrf1 and Nrf2 was further corroborated by scrutinizing comprehensive functional annotation of their specific and/or common target genes. Conversely, the mutant TCF11{Delta}N, resulting from a deletion of the N-terminal amino acids 2-156 from TCF11, resembles Nrf2 with the largely consistent structure and function. Interestingly, our further experimental evidence demonstrates that TCF11 acts as a potent tumor-repressor relative to Nrf1, albeit both isoforms possess a congruous capability to prevent malignant growth of tumor and upregulate those genes critical for improving the survival of patients with hepatocellular carcinoma.

sentences: A dissemination-prone morphotype enhances extrapulmonary organ entry by the fungus Cryptococcus neoformans Environmental pathogens, which move from ecological niches to mammalian hosts, must adapt to dramatically different environments. Microbes that disseminate farther, including the fungal meningitis pathogen Cryptococcus neoformans, require additional adaptation to diverse tissues. When C. neoformans enters the lungs, infecting cells (<10 m diameter) enlarge (>30 m diameter), then form a heterogeneous population. The brain contains uniformly small cells ([~]7 m). We demonstrate that formation of a small C. neoformans morphotype - called "seed" cells due to their disseminating ability - is critical for extrapulmonary organ entry. Seed cell formation is triggered by environmental factors, including C. neoformans environmental niche, pigeon guano. The underlying trigger, phosphate, can be released by tissue damage, potentially establishing a feed-forward loop of seed cell formation and dissemination. We demonstrate that C. neoformans size variation is not just a continuum but inducible subpopulations that change host interactions to facilitate microbe survival and spread.

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Discovery of dual inhibitors of KPC-3 and KPC-15 of Klebsiella pneumoniae :an in-silico molecular docking and dynamics study BackgroundThe development of carbapenem resistance against Klebsiella pneumoniae is a situation of grave concern and requires urgent attention. Among the KPC produced by K.pneumoniae, KPC-3, and KPC-15, play a significant role in the development of resistance to carbapenem.

Materials and methodsThe binding sites of KPC-3 and KPC-15 were predicted by the COACH server. Drug-like ligands from ZINC were then screened by ligand-based drug screening (LBVS) by keeping Relebactam as a template. The top 50,000 selected ligands were then screened by structure-based virtual screening using idock. For keeping an account of the dual inhibitors stability in complex with KPC-3 and KPC-15, MDS were carried out for each complex.

ResultsBased on consensus weighted ranks, the top 3 ligands with the dual inhibitory property are ZINC76060350 (consensus weighted rank - 1.5), ZINC05528590 (2), ZINC72290395 (3.5). All the top 3 dual inhibitors have a reasonable probability of passing through the blood-brain barrier. The RDKit and Morgan fingerprint scores between Relebactam and the top three ligands were 0.24, 0.22, 0.23, and 0.26, 0.19, 0.25, respectively (showing only 20% similarity). The MD simulation result revealed good binding stability of ligand ZINC05528590 with both KPC-3 and KPC-15, whereas ligand ZINC76060350 showed good binding stability to KPC-3.

ConclusionThe ligand ZINC05528590 could be taken forward to develop a new drug against a multi-resistant- Klebsiella pneumoniae infection. At the same time, ZINC76060350 can be considered to develop a new drug against KPC-15 resistant Klebsiella pneumoniae.

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An Engineered Nanocomposite Copper Coating with Enhanced Antibacterial Efficacy Contaminated surfaces are a major source of nosocomial infection. To reduce microbial bioburden and surface-based transmission of infectious disease, the use of antibacterial and self-sanitizing surfaces, such as copper (Cu), is being explored in clinical settings. Cu has long been known to have antimicrobial activity. However, Gram-positive microorganisms, a class that includes pathogens commonly responsible for hospital-acquired infection such as Staphylococcus aureus and Clostridioides difficile, are more resilient to its biocidal effect. Inspired by inherently bactericidal nanostructured surfaces found in nature, we have developed an improved Cu coating, engineered to contain nanoscale surface features and thus increase its antibacterial activity against a broader range of organisms. In addition, we have established a new method for facilitating the rapid and continuous release of biocidal metal ions from the coating, through incorporation of an antibacterial metal salt (ZnCl2) with a lower reduction potential than Cu. Electrophoretic deposition (EPD) was used to fabricate our coatings, which serves as a low-cost and scalable route for modifying existing conductive surfaces with complex shape. By tuning both the surface morphology and chemistry, we were able to create a nanocomposite Cu coating that decreased the microbial bioburden of Gram-positive S.aureus by 94% compared to unmodified Cu.

Table of Contents O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Antimicrobial copper (Cu) products are being deployed in clinical settings to decrease microbial bioburden and prevent surface-based transmission of infectious disease. However, Gram-positive bacteria demonstrate increased resistance to Cus biocidal effects. To improve Cus antibacterial efficacy against Gram-positive bacteria, we have developed a hydrophobic Cu coating with cytotoxic nanotopography that facilitates the rapid and continuous release of biocidal metal ions.

C_FIG_DISPLAY

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Microglia Detect Externalized Phosphatidylserine on Synapses for Elimination via TREM2 in Alzheimer's Disease Models Genetic studies implicate phagocytosis pathways in microglia to be a major Alzheimers disease (AD)-associated process. Microglia phagocytose synapses in AD mouse models, suggesting a role for microglia in region-specific synapse loss, a pathological hallmark of AD. However, whether specific synapses are targeted for elimination, and if so, how, remains to be elucidated. Here, we show that synapses externalize phosphatidylserine (PtdSer) upon challenge by {beta}-amyloid oligomers, which are then selectively engulfed by microglia. Mechanistically, we find that Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is critical for microglia to sense and preferentially engulf AD synapses. In brains of mice and humans, TREM2 dysfunction leads to exacerbation of apoptotic synapses. Our work altogether suggests a fundamental role for microglia as brain-resident macrophages to remove damaged synapses in AD. We provide mechanistic insight into how TREM2 variants associated with increased risk of developing AD may contribute to defective microglia-synapse function.

One-Sentence summaryMicroglia selectively engulf synapses in Alzheimer-like mouse brains via PtdSer-TREM2 signaling.

sentences: The Dictyostelium discoideum E3 ubiquitin ligase TrafE coordinates endolysosomal damage response and cell-autonomous immunity to Mycobacterium marinum. Cells are perpetually challenged by pathogens, protein aggregates or chemicals, that induce plasma membrane or endolysosomal compartments damage. Endolysosomal perforations are recognised as severe stress, however the mechanisms of the cellular response that ensure quality control, repair and endolysosomal homeostasis are just beginning to be unravelled. The endosomal sorting complex required for transport (ESCRT) and the autophagy machinery are recruited to damaged membranes to either repair or to remove membrane remnants. Crucial element of the endolysosomal damage response (ELDR) are factors that sense damage, paralleled by extensive tagging of the damaged organelles with signals, such as ubiquitin, required for the recruitment of ELDR components. Unattended membrane damage leads to leakage of harmful components including protons and reactive oxygen species that cause cell death. To explore ELDR key factors responsible for detection and marking of damaged compartments we use the professional phagocyte Dictyostelium discoideum. We found an evolutionary conserved E3-ligase TrafE that is robustly recruited to intracellular compartments disrupted after infection with Mycobacterium marinum or after sterile damage caused by chemical components. Importantly, we show that the absence of TrafE severely compromises the xenophagy restriction of bacteria as well as autophagy-mediated and ESCRT-mediated ELDR, resulting in early cell death.

sentences: Suppression without inhibition: How retinal computation contributes to saccadic suppression Visual perception remains stable across saccadic eye movements, despite the concurrent strongly disruptive visual flow. This stability is partially associated with a reduction in visual sensitivity, known as saccadic suppression, which already starts in the retina with reduced ganglion cell sensitivity. However, the retinal circuit mechanisms giving rise to such suppression remain unknown. Here, we describe these mechanisms using electrophysiology in mouse, pig, and macaque retina, 2-photon calcium imaging, computational modeling, and human psychophysics. We find that sequential stimuli, such as those that naturally occur during saccades, trigger three independent suppressive mechanisms in the retina. The main suppressive mechanism is triggered by contrast-reversing sequential stimuli and originates within the receptive field center of ganglion cells. It does not involve inhibition or other known suppressive mechanisms such as saturation or adaptation. Instead, it relies on temporal filtering of the inherently slow response of cone photoreceptors coupled with downstream nonlinearities. Two further mechanisms of suppression are present predominantly in ON ganglion cells and originate in the receptive field surround, highlighting a novel disparity between ON and OFF ganglion cells. The mechanisms uncovered here likely play a role in shaping the retinal output following eye movements and other natural viewing conditions where sequential stimulation is ubiquitous.

sentences: Improved correspondence of fMRI visual field localizer data after cortex-based macroanatomical alignment Studying the visual system with fMRI often requires using localizer paradigms to define regions of interest (ROIs). However, the considerable interindividual variability of the cerebral cortex represents a crucial confound for group-level analyses. Cortex-based alignment (CBA) techniques reliably reduce interindividual macroanatomical variability. Yet, their utility has not been assessed for visual field localizer paradigms, which map specific parts of the visual field within retinotopically organized visual areas. We evaluated CBA for an attention-enhanced visual field localizer mapping homologous parts of each visual quadrant in 50 participants. We compared CBA with volume-based alignment and a surface-based analysis, which did not include macroanatomical alignment. CBA led to the strongest increase in the probability of activation overlap (up to 40 percent). On the group level, CBA led to the most consistent increase in ROI size while preserving vertical ROI symmetry. Overall, our results indicate, that in addition to the increased signal-to-noise ratio of a surface-based analysis macroanatomical alignment considerably improves statistical power. These findings confirm and extend the utility of CBA for the study of the visual system in the context of group analyses. CBA should be particularly relevant when studying neuropsychiatric disorders with abnormally increased interindividual macroanatomical variability.

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Anomalous brain energy in old age by wavelet analysis of ERP during a Stroop task By event-related potentials (ERP) during a counting Stroop task it was shown that the elderly with excess in theta activity in their electroencephalogram (EEG) are at risk of cognitive decline and have a higher neuronal activity during stimulus categorization than the elderly with a normal EEG. It was suggested that this increased neuronal activity could have a compensatory function. However, the quantification of energy associated with the enhanced neuronal activity was not investigated in this group. By wavelet analysis, we measured total and relative energy in ERP during the execution of a counting Stroop task in two groups of elderly: one with excess in theta activity (Theta-EEG, n = 23) and the other with normal EEG (Normal-EEG, n = 23). In delta, theta, and alpha bands, the Theta-EEG group used a higher amount of total energy as compared to the Normal-EEG group for both types of stimuli, interference and no interference. In theta and alpha bands, the total energy was higher in the Theta-EEG group, specifically in the window of 258-516 ms, coinciding with stimulus categorization. Given that no major behavioral differences were observed between EEG groups, we suggest that a higher energy in delta, theta, and alpha bands is one of the neurobiological mechanisms that allows the Theta-EEG group to cope with the cognitive demands of the task. However, this increased energy might not be an effective mechanism in the long term as it could promote a metabolic and cellular dysregulation that would trigger the transition to cognitive impairment.

SIGNIFICANCE STATEMENTBy using wavelet transform analysis we report that the elderly with excess in theta activity show a higher energy in delta, theta, and alpha bands during the categorization of stimuli in a counting Stroop task. Our findings imply that this increase neuronal activity might be related to a dysregulated energy metabolism in the elderly with theta excess that could explain the progress to cognitive impairment in this group. The analysis of energy by wavelet transform in data obtained by ERP complements other techniques that evaluate the risk of cognitive impairment.

sentences: Computational and experimental assessment of backbone templates for computational protein design Computational protein design has taken big strides over the recent years, however, the tools available are still not at a state where a sequence can be designed to fold into a given protein structure at will and with high probability. We have here applied a recent release of Rosetta Design to redesign a set of structurally very similar proteins belonging to the Thioredoxin fold. We determined design success using a combination of a genetic screening tool to assay folding/stability in E. coli and selecting the best hits from this for further biochemical characterization. We have previously used this set of template proteins for redesign and found that success was highly dependent on template structure, a trait which was also found in this study. Nevertheless, state of the art design software is now able to predict the best template, most likely due to the introduction of the cart_bonded energy term. The template that led to the greatest fraction of successful designs was the same (a Thioredoxin from spinach) as that identified in our previous study. Our previously described redesign of Thioredoxin, which also used the spinach protein as template, however also performed well. In the present study, both these templates yielded proteins with compact folded structures, and enforces the conclusion that any design project must carefully consider different design templates. Fortunately, selecting designs using the cart_bonded energy term appears to correctly identify such templates.

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Analogous computations in working memory input, output and motor gating: Electrophysiological and computational modeling evidence Adaptive cognitive-control involves a hierarchical cortico-striatal gating system that supports selective updating, maintenance, and retrieval of useful cognitive and motor information. Here, we developed a task that independently manipulates selective gating operations into working-memory (input gating), from working-memory (output gating), and of responses (motor gating) and tested the neural dynamics and computational principles that support them. Increases in gating demands, captured by gate switches, were expressed by distinct EEG correlates at each gating level that evolved dynamically in partially overlapping time windows. Further, categorical representations of specific maintained items and of motor responses could be decoded from EEG when the corresponding gate was switching, thereby linking gating operations to prioritization. Finally, gate switching at all levels was related to increases in the motor decision threshold as quantified by the drift diffusion model. Together these results support the notion that cognitive gating operations scaffold on top of mechanisms involved in motor gating.

Author summaryHow do humans decide which information is relevant to attend to in memory, which cognitive operation to take, and when? Flexibly updating, maintenance and retrieval of relevant information from working memory (WM) are thought to be managed by gating computations in the frontostriatal network, supporting higher order learning and cognitive flexibility. Using the reference-back-2 task, we tested the key properties of gating. Namely that they are selective ("content-addressable") and that principles of cognitive "actions" (including input gating of WM, output gating from WM) are scaffold on top of the motor gating operations. Using trial-by-trial EEG indexing and quantitative computational modeling (the hierarchical drift-diffusion model) we showed that action selection at all three levels of gating have separable neural signatures but they operate partly in parallel, such that decisions about a response are processed to some degree even while the identity of the cognitive rule were uncertain. Furthermore, we showed analogous computations across levels of gating as selection of WM representation and of motor action lead to increase in the estimated decision threshold and to enhanced neural coding of the selected information thereby providing a novel link between WM gating and WM prioritization.

sentences: Protein Structure Prediction Using a Maximum Likelihood Formulation of a Recurrent Geometric Network Only [~]40% of the human proteome has structural coordinates available from experiment (i.e., X-ray crystallography, NMR spectroscopy, or cryo-EM) or homology modeling with quality templates (i.e., 30% sequence identity or greater), leaving most of the proteome structurally unsolved. Deep learning (DL) methods for predicting protein structure can help close knowledge gaps where experimental and homology models are difficult to obtain. Recent advances in these DL methods have shown promising results in expanding structural coverage to the scale of the entire human proteome, providing researchers with more complete protein structural information. Here, we improve upon an existing DL algorithm for protein structure prediction, the Recurrent Geometric Network (RGN). We first expand the training dataset to include experimental uncertainty data in the form of atomic displacement parameters, then derive a maximum likelihood loss function that incorporates this uncertainty data into model training. Compared to the original RGN, our novel maximum likelihood model improves the rate of convergence of initial model training and ultimately results in more accurate structure prediction according to the root mean square deviation (RMSD) of backbone atoms, the Global Distance Test (GDT), the Global Distance Test High Accuracy (GDT-HA), and the Template-Modeling Score (TM-Score). Our model also predicts structures with more favorable backbone torsions, which provide more accurate starting coordinates for downstream physics-based simulations. Based on these results, our maximum likelihood reformulation provides a framework for improving existing or future machine learning algorithms for protein structure prediction. The augmented dataset, data collection scripts, reformulated RGN source code, and a series of trained models are publicly available at https://github.com/SchniedersLab/likelihood-rgn.

sentences: The genome sequence of the avian vampire fly (Philornis downsi), an invasive nest parasite of Darwins finches in Galpagos The invasive avian vampire fly (Philornis downsi) is considered one of the greatest threats to the unique and endemic avifauna of the Galapagos Islands, Ecuador. The fly parasitizes nearly every passerine species, including Darwins finches, in the Galapagos. The fly is thought to have been introduced from mainland Ecuador, although the full pathway of invasion is not yet known. The majority of research to date has focused on the effects of the fly on the fitness of avian hosts and explorations of mitigation methods. A lag in research related to the genetics of this invasion demonstrates, in part, a need to develop full-scale genomic resources with which to address further questions within this system. In this study, an adult P. downsi collected from San Cristobal Island within the Galapagos archipelago was sequenced to generate a high-quality genome assembly. We examined various features of the genome (e.g., coding regions, non-coding transposable elements) and carried out comparative genomics analysis against other dipteran genomes. We identified lists of gene families that are significantly expanding/contracting in P. downsi that are related to insecticide resistance, detoxification, and potential feeding ecology and counter defense against host immune responses. The P. downsi genome assembly provides an important foundational resource for studying the molecular basis of its successful invasion in the Galapagos and the dynamics of its population across multiple islands. The findings of significantly changing gene families associated with insecticide resistance and immune responses highlight the need for further investigations into the role of different gene families in aiding the flys successful invasion. Furthermore, this genomic resource will also better help inform future research studies and mitigation strategies aimed at minimizing the flys impact on the birds of the Galapagos.

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Fluorescence exclusion: a rapid, accurate and powerful method for measuring yeast cell volume Cells exist in an astonishing range of volumes across and within species. However, our understanding of cell size control remains limited, due in large part to the challenges associated with accurate determination of cell volume. Much of our comprehension of size regulation derives from models such as budding and fission yeast, but even for these morphologically stereotypical cells, assessment of cell volume has relied on proxies and extrapolations from two-dimensional measurements. Recently, the fluorescence exclusion method (FXm) was developed to evaluate the size of mammalian cells, but whether it could be applied to smaller cells remained unknown. Using specifically designed microfluidic chips and an improved data analysis pipeline, we show here that FXm reliably detects subtle difference in the volume of fission yeast cells, even for those with altered shapes. Moreover, it allows for the monitoring of dynamic volume changes at the single-cell level with high time resolution. Collectively, our work reveals how coupling FXm with yeast genetics will bring new insights into the complex biology of cell growth.

SUMMARY STATEMENTFluorescence exclusion provides a unique method to accurately measure the volume of yeast cells at both the population and single-cell levels.

sentences: The Hair Cell Analysis Toolbox: A machine learning-based whole cochlea analysis pipeline Auditory hair cells, the whole length of the cochlea, are routinely visualized using light microscopy techniques. It is common, therefore, for one to collect more data than is practical to analyze manually. There are currently no widely accepted tools for unsupervised, unbiased, and comprehensive analysis of cells in an entire cochlea. This represents a stark gap between image-based data and other tests of cochlear function. To close this gap, we present a machine learning-based hair cell analysis toolbox, for the analysis of whole cochleae, imaged with confocal microscopy. The software presented here allows the automation of common image analysis tasks such as counting hair cells, determining their best frequency, as well as quantifying single cell immunofluorescence intensities along the entire cochlear coil. We hope these automated tools will remove a considerable barrier in cochlear image analysis, allowing for more informative and less selective data analysis practices.

sentences: Plant-insect chemical communication in ecological communities: an information theory perspective Cross-species communication, where signals are sent by one species and perceived by others, is one of the most intriguing types of communication that functionally links different species to form complex ecological networks. Yet, global changes and human activities can affect communication by increasing the fluctuations of species composition and phenology, altering signal profiles and intensity, and introducing noises. So far, most studies on cross-species communication have focused on a few specific species isolated from ecological communities. Scaling up investigations of cross-species communication to the community level is currently hampered by a lack of conceptual and practical methodologies. Here, we propose an interdisciplinary framework based on information theory to investigate mechanisms shaping cross-species communication at the community level. We use plants and insects, the cornerstones of most ecosystems, as a showcase; and focus on chemical communication as the key communication channel. We first introduce some basic concepts of information theory, then we illustrate information patterns in plant-insect chemical communication, followed by a further exploration of how to integrate information theory into ecological and evolutionary processes to form testable mechanistic hypotheses. We conclude by highlighting the importance of community-level information as a vehicle to better understand the maintenance of ecological systems, especially when facing rapid global changes.

sentences: The Euler Characteristic and Topological Phase Transitions in Complex Systems In this work, we use methods and concepts of applied algebraic topology to comprehensively explore the recent idea of topological phase transitions (TPT) in complex systems. TPTs are characterized by the emergence of nontrivial homology groups as a function of a threshold parameter. Under certain conditions, one can identify TPTs via the zeros of the Euler characteristic or by singularities of the Euler entropy. Recent works provide strong evidence that TPTs can be interpreted as a complex networks intrinsic fingerprint. This work illustrates this possibility by investigating some classic network and empirical protein interaction networks under a topological perspective. We first investigate TPT in protein-protein interaction networks (PPIN) using methods of topological data analysis for two variants of the Duplication-Divergence model, namely, the totally asymmetric model and the heterodimerization model. We compare our theoretical and computational results to experimental data freely available for gene co-expression networks (GCN) of Saccharomyces cerevisiae, also known as bakers yeast, as well as of the nematode Caenorhabditis elegans. Supporting our theoretical expectations, we can detect topological phase transitions in both networks obtained according to different similarity measures. Later, we perform numerical simulations of TPTs in four classical network models: the Erd[o]s-Renyi model, the Watts-Strogatz model, the Random Geometric model, and the Barabasi-Albert. Finally, we discuss some perspectives and insights on the topic. Given the universality and wide use of those models across disciplines, our work indicates that TPT permeates a wide range of theoretical and empirical networks.

sentences: Darting across space and time: Parametric modulators of sex-biased conditioned fear responding Pavlovian fear conditioning is a widely used behavioral paradigm for studying associative learning in rodents. Despite early recognition that subjects may engage in a variety of both conditioned and unconditioned responses, the last several decades have seen the field narrow its focus to measure freezing as the sole indicator of conditioned fear. We previously reported that female rats were more likely than males to engage in darting, an escape-like conditioned response that is associated with heightened shock reactivity. To determine how experimental parameters contribute to the frequency of darting in both males and females, we manipulated factors such as chamber size, shock intensity, and number of trials. To better capture fear-related behavioral repertoires in our animals, we developed ScaredyRat, an open-source custom Python tool that analyzes Noldus Ethovision-generated raw data files to identify Darters and quantify both conditioned and unconditioned responses. We find that like freezing, conditioned darting occurrences scale with experimental alterations. While most darting occurs in females, we find that with an extended training protocol, darting can emerge in males as well. Collectively, our data suggest that darting reflects a behavioral switch in conditioned responding that is a product of both an individual animals sex, shock reactivity, and experimental parameters, underscoring the need for careful consideration of sex as a biological variable in classic learning paradigms.

sentences: Classification of Preeclamptic Placental Extracellular Vesicles Using Femtosecond Laser-fabricated Nanoplasmonic Sensors and Machine Learning Placental extracellular vesicles (EVs) play an essential role in pregnancy by protecting and transporting diverse biomolecules that aid in fetomaternal communication. However, in preeclampsia, they have also been implicated in contributing to disease progression. Despite their potential clinical value, most current technologies cannot provide a rapid and effective means of differentiating between healthy and diseased placental EVs. To address this, we developed a fabrication process called laser-induced nanostructuring of SERS-active thin films (LINST), which produces nanoplasmonic substrates that provide exceptional Raman signal enhancement and allow the biochemical fingerprinting of EVs. After validating LINST performance with chemical standards, we used placental EVs from tissue explant cultures and demonstrated that preeclamptic and normotensive placental EVs have classifiably distinct Raman spectra following the application of both conventional and advanced machine learning algorithms. Given the abundance of placental EVs in maternal circulation, these findings will encourage immediate exploration of surface-enhanced Raman spectroscopy (SERS) as a promising method for preeclampsia liquid biopsies, while our novel fabrication process can provide a versatile and scalable substrate for many other SERS applications.

sentences: Multi-Scale Coarse Grained Model for the Stepping of Molecular Motors with application to Kinesin. Conventional kinesin, a motor protein that transports cargo within cells, walks by taking multiple steps towards the plus end of the microtubule (MT). While significant progress has been made in understanding the details of the walking mechanism of kinesin there are many unresolved issues. From a computational perspective, a central challenge is the large size of the system, which limits the scope of time scales accessible in standard computer simulations. Here, we create a general multi-scale coarse-grained model for motors that enables us to simulate the stepping process of motors on polar tracks (actin and MT) with focus on kinesin. Our approach greatly shortens the computation times without a significant loss in detail, thus allowing us to better describe the molecular basis of the stepping kinetics. The small number of parameters, which are determined by fitting to experimental data, allows us to develop an accurate method that may be adopted to simulate stepping in other molecular motors. The model enables us to simulate a large number of steps, which was not possible previously. We show in agreement with experiments that due to the docking of the neck linker (NL) of kinesin, sometimes deemed as the power stroke, the space explored diffusively by the tethered head is severely restricted allowing the step to be in a tens of microseconds. We predict that increasing the interaction strength between the NL and the motor head, achievable by mutations in the NL, decreases the stepping time but reaches a saturation value. Furthermore, the full 3-dimensional dynamics of the cargo are fully resolved in our model, contributing to the predictive power and allowing us to study the important aspects of cargo-motor interactions.

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Energetics but not development is impacted in coral embryos exposed to ocean acidification In light of the chronic stress and mass mortality reef-building corals face under climate change, it is critical to understand the processes essential to reef persistence and replenishment, including coral reproduction and development. Here we quantify gene expression and size sensitivity to ocean acidification across a set of developmental stages in the rice coral, Montipora capitata. Embryos and swimming larvae were exposed to pH treatments 7.8 (Ambient), 7.6 (Low) and 7.3 (Xlow) from fertilization to 9 days post-fertilization. Embryo and larval volume, and stage-specific gene expression were compared between treatments to determine the effects of acidified seawater on early development. While there was no measurable size differentiation between fertilized eggs and prawn chips exposed to pH 7.8, 7.6, and 7.3, early gastrula and planula raised in reduced pH treatments were significantly smaller than those raised in ambient seawater, suggesting an energetic cost to developing under low pH. However, no differentially expressed genes emerged until 9 days post-fertilization. Notably, gene expression patterns of larvae developing at pH 7.8 and pH 7.3 were more similar than those developing at pH 7.6. Larvae from pH 7.6 showed upregulation of genes involved in cell division, regulation of transcription, lipid metabolism, and oxidative stress in comparison to the other two treatments. While low pH appears to increase energetic demands and trigger oxidative stress, the developmental process is robust to this at a molecular level, with swimming larval stage reached in all pH treatments.

Summary statementThis developmental time series tracks the physiological and transcriptomic outcomes of early coral development under ambient pH (pH 7.8), and two low pH conditions (pH 7.6 and 7.3).

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Rapid detection of Staphylococcus aureus and Streptococcus pneumoniae by real-time analysis of volatile metabolites Rapid detection of pathogenic bacteria is needed for rapid diagnostics allowing adequate and timely treatment. In this study, we aimed to evaluate the technical feasibility of Secondary Electro-Spray Ionization-High Resolution Mass Spectrometry (SESI-HRMS) as a diagnostic tool for rapid detection of bacterial infections and compare its performance with the current standard of diagnostics. We compared the time required to confirm growth of the pathogenic bacteria Staphylococcus aureus and Streptococcus pneumoniae by conventional detection by culture and MAL-DI-TOF vs. detection of specific volatile organic compounds (VOCs) produced by these human pathobionts. SESI-HRMS could consistently detect VOCs produced by S. aureus or S. pneumoniae on blood agar plates within minutes, allowing to positively identify bacteria within hours. Unique S. aureus and S. pneumoniae features were detected already at bacterial densities as low as [~]103 colony forming units. Rich mass spectral fingerprints allowed for the distinction of these two bacteria on a species and even strain level. To give an incentive towards clinical application of this technology, further analyzed 17 clinical samples previously diagnosed by conventional methods. We predominantly obtained a separation of samples which showed growth (i.e. presence of living bacteria) compared to samples with no bacterial growth (i.e. presence of dead bacteria). We conclude that SESI-HRMS allows rapid identification of unique bacterial features. Further development of real-time analysis of clinical samples by SESI-HRMS will shorten the time required for microbiological diagnosis with a high level of confidence and sensitivity and should help to improve patients tailored treatment.

IMPORTANCEA timely identification of a pathogenic bacteria causing the infection is of pivotal importance for the initiation of an adequate antimicrobial therapy. In this regard, different technologies have been developed with the aim to achieve a highly reliable, specific, and overall fast identification of pathogenic bacteria. However, conventional diagnostic techniques still require long preprocessing times (hours to days) to acquire enough biological material for an accurate identification of the pathogen. Therefore, in this work, we aimed to further shorten the detection time of current gold standards for microbiological diagnostics by providing a system capable of a fast, sensitive and specific discrimination of different pathogenic bacteria. This system relies on the real-time mass spectrometric detection of volatile organic compounds (VOCs) produced by a given organism during its growth, potentially leading to a significant shortening of the time required to obtain a positive reliable diagnostic.

sentences: VMHdm/cSF-1 Neuronal Circuits Regulate Skeletal Muscle PGC1-α via the Sympathoadrenal Drive To adapt to metabolically challenging environments, the central nervous system (CNS) orchestrates metabolism of peripheral organs including skeletal muscle. The organ-communication between the CNS and skeletal muscle has been investigated, yet our understanding of the neuronal pathway from the CNS to skeletal muscle is still limited. Neurons in the dorsomedial and central parts of the ventromedial hypothalamic nucleus (VMHdm/c) expressing steroidogenic factor-1 (VMHdm/cSF-1 neurons) are key for metabolic adaptations to exercise, including increased basal metabolic rate and skeletal muscle mass in mice. However, the mechanisms by which VMHdm/cSF-1 neurons regulate skeletal muscle function remain unclear. Here, we show that VMHdm/cSF-1 neurons increase the sympathoadrenal activity and regulate skeletal muscle peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1) in mice via multiple downstream nodes. Optogenetic activation of VMHdm/cSF-1 neurons dramatically elevates mRNA levels of skeletal muscle Pgc-1, which regulates a spectrum of skeletal muscle function including protein synthesis and metabolism. Mechanistically, the sympathoadrenal drive coupled with {beta}2 adrenergic receptor ({beta}2AdR) is essential for VMHdm/cSF-1 neurons-mediated increases in skeletal muscle PGC1-. Specifically, adrenalectomy and knockout of {beta}2AdR block augmented skeletal muscle PGC1- by VMHdm/cSF-1 neuronal activation. Optogenetic functional mapping reveals that downstream nodes of VMHdm/cSF-1 neurons are functionally redundant to increase circulating epinephrine and skeletal muscle PGC1-. Collectively, we propose that VMHdm/cSF-1 neurons-skeletal muscle pathway, VMHdm/cSF-1 neurons[->]multiple downstream nodes[->]the adrenal gland[->]skeletal muscle {beta}2AdR, underlies augmented skeletal muscle function for metabolic adaptations.

sentences: Development of visual cortex in human neonates are selectively modified by postnatal experience Experience-dependent cortical plasticity is a pivotal process of human brain development and essential for the formation of most cognitive functions. Although studies found that early visual experience could influence the endogenous development of visual cortex in animals, little is known about such impact on human infants. Using the multi-modal MRI data from developing human connectome project, we revealed the early structural and functional maps in the ventral visual cortex and their development across the first month of age. Particularly, we found the postnatal experience could modulate both the cortical morphology in ventral visual cortex and the functional circuit between bilateral primary visual cortices. But the cortical myelination and overall functional circuits of ventral cortex, particularly that of the high-order visual cortex, developed without significant influence of postnatal experience in such early period. These experience-dependent cortical properties were further validated in the preterm-born infants who have longer postnatal time but immature cortical development at birth. The results confirmed that the development of cortical thickness was dominated by the postnatal experience but the functional circuit might be determined by the overall maturity of visual cortex. These data suggest in human newborns that early postnatal experience shapes the structural and functional development of the visual cortex in selective and organized pattern.

sentences: Sensorimotor feedback loops are selectively sensitive to reward While it is well established that motivational factors such as earning more money for performing well improve motor performance, how the motor system implements this improvement remains unclear. For instance, feedback-based control, which uses sensory feedback from the body to correct for errors in movement, improves with greater reward. But feedback control encompasses many feedback loops with diverse characteristics such as the brain regions involved and their response time. Which specific loops drive these performance improvements with reward is unknown, even though their diversity makes it unlikely that they are contributing uniformly. This lack of mechanistic insight leads to practical limitations in applications using reward, such as clinical rehabilitation, athletic coaching, and brain-inspired robotics. We systematically tested the effect of reward on the latency (how long for a corrective response to arise?) and gain (how large is the corrective response?) of eight distinct sensorimotor feedback loops in humans. Only the feedback responses known to rely on prefrontal associative cortices showed sensitivity to reward, while feedback responses that relied mainly on premotor and sensorimotor cortex did not show sensitivity to reward. Our results may have implications regarding feedback control performance in pathologies showing a cognitive decline, or on athletic coaching. For instance, coaching methodologies that rely on reinforcement or "reward shaping" may need to specifically target aspects of movement that rely on reward-sensitive feedback responses.

sentences: Implications of trade network structure and population dynamics for food security and equality Given trades importance to maintaining food security, it is crucial to understand the relationship between human population growth, land use, food supply, and trade. We develop a metapopulation model coupling human population dynamics to agricultural land use and food production in "patches" (regions and countries) connected via trade networks. Patches that import sparingly or fail to adjust their demand sharply in response to changes in food per capita experience food insecurity. They fall into a feedback loop between increasing population growth and decreasing food per capita, particularly if they are peripheral to the network. A displacement effect is also evident; patches that are more central and/or import more heavily preserve their natural land states. Their reliance on imports means other patches must expand their agricultural land. These results emphasize that strategies for improving food security and equality must account for the combined effects of network topology and patch-level characteristics.

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Defining the age-dependent and tissue-specific circadian transcriptome in male mice Cellular circadian clocks direct a daily transcriptional program that supports homeostasis and resilience. Emerging evidence supports age-associated changes in circadian functions. To define age-dependent changes at the systems level, we profiled the circadian transcriptome in the hypothalamus, lung, heart, kidney, skeletal muscle, and adrenal gland in 3 age groups. We found age-dependent and tissue-specific clock output changes. Aging reduced the number of rhythmically expressed genes (REGs), indicative of weakened circadian control. Many genes gained rhythmicity in old tissues, reflecting an adaptive response. REGs were enriched for the hallmarks of aging, adding a new dimension to our understanding of aging. Differential gene expression analysis found that there were temporally distinct clusters of genes in tissue-specific manner. Increased daily gene expression variability is a common feature of aged tissues. This novel analysis extends the landscape of the understanding of aging and highlights the impact of aging on circadian clock function and temporal changes in gene expression.

HIGHLIGHTS- Rhythmically expressed genes (REGs) in Young, but not Old mice, are enriched for the aging hallmarks across all tissues.
- The numbers of REGs decline across all tissues with age implicating the circadian clock in altered homeostasis.
- Age- and tissue-specific differentially expressed genes (DEGs) cluster at specific times of the day.
- Increase in gene expression variability over a day is a common feature of aging tissues.

sentences: To bin or not to bin: analyzing single-cell growth data Collection of high-throughput data has become prevalent in biology. Large datasets allow the use of statistical constructs such as binning and linear regression to quantify relationships between variables and hypothesize underlying biological mechanisms based on it. We discuss several such examples in relation to single-cell data and cellular growth. In particular, we show instances where what appears to be ordinary use of these statistical methods leads to incorrect conclusions such as growth being non-exponential as opposed to exponential and vice versa. We propose that the data analysis and its interpretation should be done in the context of a generative model, if possible. In this way, the statistical methods can be validated either analytically or against synthetic data generated via the use of the model, leading to a consistent method for inferring biological mechanisms from data. On applying the validated methods of data analysis to infer cellular growth on our experimental data, we find the growth of length in E. coli to be non-exponential. Our analysis shows that in the later stages of the cell cycle the growth rate is faster than exponential.

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TWISTED DWARF1 regulates Arabidopsis stamen development by differential activation of ABCB-mediated auxin transport Despite clear evidence that a local accumulation of auxin is likewise critical for male fertility, much less is known about the components that regulate auxin-controlled stamen development.

In this study, we analyzed physiological and morphological parameters in mutants of key players of ABCB-mediated auxin transport and spatially and temporally dissected their expression on the protein level as well as auxin fluxes in the Arabidopsis stamens. Our analyses revealed that the FKBP42, TWISTED DWARF1 (TWD1), promotes stamen elongation and, to a lesser extent, anther dehiscence, as well as pollen maturation and thus is required for seed development. Most of the described developmental defects in twd1 are shared with the abcb1 abcb19 mutant, which can be attributed to the fact that TWD1 - as a described ABCB chaperon - is a positive regulator of ABCB1 and ABCB19-mediated auxin transport. However, reduced stamen number was dependent on TWD1 but not on investigated ABCBs, suggesting additional actors down-stream of TWD1. We predict an overall housekeeping function for ABCB1 during earlier stages, while ABCB19 seems to be responsible for the key event of rapid elongation at later stages of stamen development. Our data indicate that TWD1 controls stamen development by differential activation of ABCB-mediated auxin transport in the stamen.

HighlightBy using a mix of phenotypical and imaging analyses, we here identify and functionally characterize a new master regulator of flower development.

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Detection of hematopoietic stem cell transcriptome in human fetal kidneys and kidney organoids derived from human induced pluripotent stem cells (iPSC) BackgroundIn mammalians, hematopoietic stem cells (HSC) arise in the dorsal aorta from the hemogenic endothelium, followed by their migration to fetal liver and to bone marrow. In zebrafish, kidney is the site of primary hematopoiesis. In humans, the presence of HSC in the fetal or adult kidney has not been established.

MethodsWe analyzed the presence of HSC markers in human fetal kidneys by analysis of single-cell datasets. We then analyzed in kidney organoids derived from iPSC, the presence of hematopoietic markers using transcriptome analyses.

Results12 clusters were identified of stromal, endothelial, and nephron cell type-specific markers in the two fetal stage (17 weeks) kidney datasets. Among these, expression of hematopoietic cells in Cluster 9 showed expression of primitive markers. Moreover, whole transcriptome analysis of our iPSC-derived kidney organoids revealed induction of the primitive hematopoietic transcription factor RUNX1 as found in the human fetal kidney cortex.

ConclusionsThese finding support the presence of cells expressing HSC transcriptome in human kidney. The mechanisms of the appearance of the cells with the same transcriptional features during iPSC-derived kidney organoid generation requires further investigation.

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Pyrolyzed substrates induce aromatic compound metabolism in the post-fire fungus, Pyronema domesticum Wildfires represent a fundamental and profound disturbance in many ecosystems, and their frequency and severity are increasing in many regions of the world. Fire affects soil by removing carbon in the form of CO2 and transforming remaining surface carbon into pyrolyzed organic material (PyOM). Fires also generate substantial necromass at depths where the heat kills soil organisms but does not catalyze the formation of PyOM. Pyronema species strongly dominate soil fungal communities within weeks to months after fire. However, the carbon pool (i.e. necromass or PyOM) that fuels their rise in abundance is unknown. We used a Pyronema domesticum isolate from the catastrophic 2013 Rim Fire (CA, USA) to ask if P. domesticum is capable of metabolizing PyOM. P. domesticum grew readily on agar media where the sole carbon source was PyOM (specifically, pine wood PyOM produced at 750 {degrees}C). Using RNAseq, we investigated the response of P. domesticum to PyOM and observed a comprehensive induction of genes involved in the metabolism and mineralization of aromatic compounds, typical of those found in PyOM. Lastly, we used 13C-labeled 750 {degrees}C PyOM to demonstrate that P. domesticum is capable of mineralizing PyOM to CO2. Collectively, our results indicate a robust potential for P. domesticum to liberate carbon from PyOM in post-fire ecosystems and return it to the bioavailable carbon pool.

IMPORTANCEFires are increasing in frequency and severity in many regions across the world. Thus, its critically important to understand how our ecosystems respond to inform restoration and recovery efforts. Fire transforms the soil, removing many nutrients while leaving behind both nutritious necromass and complex pyrolyzed organic matter, which is often recalcitrant. Filamentous fungi of the genus Pyronema strongly dominate soil fungal communities soon after fire. While Pyronema are key pioneer species in post-fire environments, the nutrient source that fuels their rise in abundance is unknown. In this manuscript, we used a P. domesticum isolate from the catastrophic 2013 Rim Fire (CA, USA) to demonstrate that P. domesticum metabolizes pyrolyzed organic material, effectively liberating this complex pyrolyzed carbon and returning it to the bioavailable carbon pool. The success of Pyronema in post-fire ecosystems has the potential to kick-start growth of other organisms and influence the entire trajectory of post-fire recovery.

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Auto-Immunoproteomics Analysis of COVID-19 ICU Patients Revealed Increased Levels of Autoantibodies Related to Male Reproductive System The role of autoantibodies in coronavirus disease (COVID-19) complications is not yet fully understood. The current investigation screened two independent cohorts of 97 COVID-19 patients (Discovery (Disc) cohort from Qatar (n = 49) and Replication (Rep) cohort from New York (n = 48)) utilizing high-throughput KoRectly Expressed (KREX) immunome protein-array technology. Autoantibody responses to 57 proteins were significantly altered in the COVID-19 Disc cohort compared to healthy controls (P [≤] 0.05). The Rep cohort had altered autoantibody responses against 26 proteins compared to non-COVID-19 ICU patients that served as controls. Both cohorts showed substantial similarities (r2 = 0.73) and exhibited higher autoantibodies responses to numerous transcription factors, immunomodulatory proteins, and human disease markers. Analysis of the combined cohorts revealed elevated autoantibody responses against SPANXN4, STK25, ATF4, PRKD2, and CHMP3 proteins in COVID-19 patients. KREX analysis of the specific IgG autoantibody responses indicates that the targeted host proteins are supposedly increased in COVID-19 patients. The autoantigen-autoantibody response was cross-validated for SPANXN4 and STK25 proteins using Uniprot BLASTP and sequence alignment tools. SPANXN4 is essential for spermiogenesis and male fertility, which may predict a potential role for this protein in COVID-19 associated male reproductive tract complications and warrants further research.

Significance StatementCoronavirus disease (COVID-19), caused by the SARS-CoV-2 virus, has emerged as a global pandemic with a high morbidity rate and multiorgan complications. It is observed that the host immune system contributes to the varied responses to COVID-19 pathogenesis. Autoantibodies, immune system proteins that mistakenly target the bodys own tissue, may underlie some of this variation. We screened total IgG autoantibody responses against 1,318 human proteins in two COVID-19 patient cohorts. We observed several novel markers in COVID-19 patients that are associated with male fertility, such as sperm protein SPANXN4, STK25, and the apoptotic factor ATF4. Particularly, elevated levels of autoantibodies against the testicular tissue-specific protein SPANXN4 offer significant evidence of anticipating the protein role in COVID-19 associated male reproductive complications.

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Protective efficacy of rhesus adenovirus COVID-19 vaccines against mouse-adapted SARS-CoV-2 The global COVID-19 pandemic has sparked intense interest in the rapid development of vaccines as well as animal models to evaluate vaccine candidates and to define immune correlates of protection. We recently reported a mouse-adapted SARS-CoV-2 virus strain (MA10) with the potential to infect wild-type laboratory mice, driving high levels of viral replication in respiratory tract tissues as well as severe clinical and respiratory symptoms, aspects of COVID-19 disease in humans that are important to capture in model systems. We evaluated the immunogenicity and protective efficacy of novel rhesus adenovirus serotype 52 (RhAd52) vaccines against MA10 challenge in mice. Baseline seroprevalence is lower for rhesus adenovirus vectors than for human or chimpanzee adenovirus vectors, making these vectors attractive candidates for vaccine development. We observed that RhAd52 vaccines elicited robust binding and neutralizing antibody titers, which inversely correlated with viral replication after challenge. These data support the development of RhAd52 vaccines and the use of the MA10 challenge virus to screen novel vaccine candidates and to study the immunologic mechanisms that underscore protection from SARS-CoV-2 challenge in wild-type mice.

ImportanceWe have developed a series of SARS-CoV-2 vaccines using rhesus adenovirus serotype 52 (RhAd52) vectors, which exhibits a lower seroprevalence than human and chimpanzee vectors, supporting their development as novel vaccine vectors or as an alternative Ad vector for boosting. We sought to test these vaccines using a recently reported mouse-adapted SARS-CoV-2 (MA10) virus to i) evaluate the protective efficacy of RhAd52 vaccines and ii) further characterize this mouse-adapted challenge model and probe immune correlates of protection. We demonstrate RhAd52 vaccines elicit robust SARS-CoV-2-specific antibody responses and protect against clinical disease and viral replication in the lungs. Further, binding and neutralizing antibody titers correlated with protective efficacy. These data validate the MA10 mouse model as a useful tool to screen and study novel vaccine candidates, as well as the development of RhAd52 vaccines for COVID-19.

sentences: Cryo-EM Structures of Amyloid-β 42 Filaments from Human Brain Filament assembly of amyloid-{beta} peptides ending at residue 42 (A{beta}42) is a central event in Alzheimers disease. We report the cryo-EM structures of A{beta}42 filaments from brain. Two structurally related S-shaped protofilament folds give rise to two types of filaments. Type I filaments were found mostly in the brains of individuals with sporadic Alzheimers disease and Type II filaments in individuals with familial Alzheimers disease and other conditions. The structures of A{beta}42 filaments from brain differ from those of filaments assembled in vitro. By contrast, in AppNL-F knock-in mice, A{beta}42 deposits were made of Type II filaments. Knowledge of A{beta}42 filament structures from human brain may lead to the development of inhibitors of assembly and improved imaging agents.

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Physiological changes throughout the ear due to age and noise - a longitudinal study Biological and mechanical systems, whether by their overuse or their aging, will inevitably fail. Hearing provides a poignant example of this with noise-induced and age-related hearing loss. Hearing loss is not unique to humans, however, and is experienced by all animals in the face of wild and eclectic differences in ear morphology and operation. Here we exploited the high throughput and accessible tympanal ear of the desert locust, Schistocerca gregaria (mixed sex) to rigorously quantify changes in the auditory system due to noise exposure (3 kHz pure tone at 126 dB SPL) and age. We analysed tympanal dispalcements, morphology of the auditory Mullers organ and measured activity of the auditory nerve, the transduction current and electrophysiological properties of individual auditory receptors. We found that noise mildly and transiently changes tympanal displacements, decreases both the width of the auditory nerve and the transduction current recorded from individual auditory neurons. Whereas age - but not noise - decreases the number of auditory neurons and increases their resting potential. Multiple other properties of Mullers organ were unaffected by either age or noise including: the number of supporting cells in Mullers organ or the nerve, membrane resistance and capacitance of the auditory neurons. The sound-evoked activity of the auditory nerve decreased as a function of age and this decrease was exacerbated by noise, with the largest difference during the middle of their life span. This  middle-aged deafness pattern of hearing loss mirrors that found for humans exposed to noise early in their life.

Key point summaryO_LIAge and noise lead to hearing loss.
C_LIO_LITympanal displacements have a transient and delayed reduction after noise.
C_LIO_LIThe number of auditory neurons decreases with age. The width of the auditory nerve is reduced with noise exposure.
C_LIO_LILocusts repeatedly exposed to noise lost their hearing compared to silent controls, but their hearing became similar to old silent control locusts due to age-related hearing loss dominating.
C_LIO_LIThe electrophysiological properties of the auditory neurons and the transduction current remained unchanged for aged locusts but repeated noise exposure led to a cumulative decrease in the transduction current.
C_LI

sentences: Spatial, Quantitative and Functional Deconstruction of Virus and Host Protein Interactions Inside Intact Cytomegalovirus Particles Herpesviruses assemble large enveloped particles that are difficult to characterize structurally due to their size, fragility and complex proteome with partially amorphous nature. Here we use cross-linking mass spectrometry and quantitative proteomics to derive a spatially resolved interactome map of intact human cytomegalovirus virions. This enabled the de novo allocation of 32 viral proteins into four spatially resolved virion layers, each organized by a dominant viral scaffold protein. The viral protein UL32 engages with all layers in an N-to-C-terminal radial orientation bridging nucleocapsid to viral membrane. In addition, we observed the layer-specific recruitment of 82 host proteins, a subset of which are constitutively and selectively incorporated via specific host-virus interactions. We uncover how the recruitment of PP1 phosphatase and 14-3-3 proteins by UL32 affects early and late steps during viral biogenesis. Collectively, this study provides global structural insights into the native configuration of virus and host protein interactions inside herpesvirus particles.

sentences: Measuring drift by mean deviation: unequal breeding sex ratio revisited With the growing amount and diversity of intermediate omics data complementary to genomics (e.g., DNA methylation, gene expression, and protein abundance), there is a need to develop methods to incorporate intermediate omics data into conventional genomic evaluation. The omics data helps decode the multiple layers of regulation from genotypes to phenotypes, thus forms a connected multi-layer network naturally. We developed a new method named NN-LMM to model the multiple layers of regulation from genotypes to intermediate omics features, then to phenotypes, by extending conventional linear mixed models ("LMM") to multi-layer artificial neural networks ("NN"). NN-LMM incorporates intermediate omics features by adding middle layers between genotypes and phenotypes. Linear mixed models (e.g., pedigree-based BLUP, GBLUP, Bayesian Alphabet, single-step GBLUP, or single-step Bayesian Alphabet) can be used to sample marker effects or genetic values on intermediate omics features, and activation functions in neural networks are used to capture the nonlinear relationships between intermediate omics features and phenotypes. NN-LMM had significantly better prediction performance than the recently proposed single-step approach for genomic prediction with intermediate omics data. Compared to the single-step approach, NN-LMM can handle various patterns of missing omics measures, and allows nonlinear relationships between intermediate omics features and phenotypes. NN-LMM has been implemented in an open-source package called "JWAS".

sentences: Implicitly Learned Higher Order Associations Differentiates Recent and Remote Retrieval of Temporal Order Memory Memory of an ordered sequence of distinct events requires encoding the temporal order as well as the intervals that separates these events. In this study, using order place association task where the animal learns to associate the location of the food pellet to the order of entry into the event arena, we probe the nature of temporal order memory in mice. In our task, individual trials, become distinct events, as the animal is trained to form unique association between entry order and a correct location. The inter-trial intervals (> 30 mins) are chosen deliberately to minimise the working memory contributions. We develop this paradigm initially using 4 order place associates and later extend it to 5 paired associates. Our results show that animals not only acquire these explicit (entry order to place) associations but also higher order associations that can only be inferred implicitly from the temporal order of these events. As an indicator of such higher order learning during the probe trail the mice exhibit predominantly prospective errors that declines proportionally with temporal distance. On the other hand, prior to acquiring the sequence the retrospective errors are dominant. Additionally, we also tested the nature of such acquisitions when temporal order CS is presented along with flavour as a compound stimulus comprising of order and flavour both simultaneously being paired with location. Results from these experiments indicate that the animal learns both order-place and flavour-place associations. Comparing with pure order place training, we find that the additional flavour in compound training did not interfere with the ability of the animals to acquire the order place associations. When tested remotely, pure order place associations could be retrieved only after a reminder training. Further higher order associations representing the temporal relationship between the events is markedly absent in the remote time.

sentences: Ligand-independent modulation of GIPR signaling by splice variants Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a potential drug target for metabolic disorders. It works with glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) in humans to maintain glucose homeostasis. Unlike the other two receptors, GIPR has at least 7 reported (EMBL-EBI, 2022; NCBI, 2022a, 2022b) splice variants (SVs) with previously undefined functions. To explore their roles in endogenous peptide mediated GIPR signaling, we investigated the outcome of co-expressing each of the four SVs in question with GIPR in terms of ligand binding, cAMP accumulation, Gs activation, {beta}-arrestin recruitment and cell surface localization. The effects of these SVs on intracellular cAMP responses modulated by receptor activity-modifying proteins (RAMPs) were also studied. It was found that while SVs of GIPR neither bound to the hormone nor affected its signal transduction per se, they differentially regulated GIPR-mediated cAMP and {beta}-arrestin responses. Specifically, SV1 and SV4 were preferable to Gs signaling, SV3 was biased towards {beta}-arrestin recruitment, whereas SV2 was inactive on both pathways. In the presence of RAMPs, only SV1 and SV4 synergized the repressive action of RAMP3 on GIP-elicited cAMP production. The results suggest a new form of signal bias that is constitutive and ligand-independent, thereby expanding our knowledge of biased signaling beyond pharmacological manipulation (i.e., ligand specific) as well as constitutive and ligand-dependent (e.g., SV1 of the growth hormone-releasing hormone receptor).

sentences: Colocalized, Bidirectional Optogenetic Modulations in Freely Behaving Animals with a Wireless Dual-Color Optoelectronic Probe The precise control of neural activities at both cellular and circuit levels reveals significant impacts on the fundamental neuroscience explorations and medical applications. Optogenetic methods provide efficient cell-specific modulations, and the ability of simultaneous neural activation and inhibition in the same brain region of freely moving animals is highly desirable and being actively researched. Here we report bidirectional neuronal activity manipulation accomplished by a wireless, dual-color optogenetic probe in synergy with the co-expression of two spectrally distinct opsins (ChrimsonR and stGtACR2) in a rodent model. Based on vertically assembled, thin-film microscale light-emitting diodes (micro-LEDs) with a lateral dimension of 125 x 180 {micro}m2 on flexible substrates, the dual-color probe shows colocalized red and blue emissions and allows chronic in vivo operations with desirable biocompatibilities. In addition, we discover that neurons co-expressing the two opsins can be deterministically evoked or silenced under red or blue irradiations. Implanted in behaving mice, the wirelessly controlled dual-color probe interferes with dopaminergic neurons in the ventral tegmental area (VTA), increasing or decreasing dopamine levels with colocalized red and blue stimulations. Such bidirectional regulations further generate rewarding and aversive behaviors of freely moving mice in a place preference test and interrogate social interactions among multiple mice. The technologies established here will create numerous opportunities and profound implications for brain research.

sentences: Topological morphogenesis of neuroepithelial organoids Animal organs exhibit complex topologies involving cavities and tubular networks, which underlie their form and function. However, how topology emerges during organ morphogenesis remains elusive. Here, we combine tissue reconstitution and quantitative microscopy to show that trans and cis epithelial fusion govern tissue topology and shape. These two modes of topological transitions can be regulated in neuroepithelial organoids, leading to divergent topologies. The morphological space can be captured by a single control parameter which is analogous to the reduced Gaussian rigidity of an epithelial surface. Finally, we identify a pharmacologically accessible pathway that regulates the frequency of trans and cis fusion, and demonstrate the control of organoid topology and shape. The physical principles uncovered here provide fundamental insights into the self-organization of complex tissues.

sentences: Optimization of Non-Coding Regions Improves Protective Efficacy of an mRNA SARS-CoV-2 Vaccine in Nonhuman Primates The CVnCoV (CureVac) mRNA vaccine for SARS-CoV-2 has recently been evaluated in a phase 2b/3 efficacy trial in humans. CV2CoV is a second-generation mRNA vaccine with optimized non-coding regions and enhanced antigen expression. Here we report a head-to-head study of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in nonhuman primates. We immunized 18 cynomolgus macaques with two doses of 12 ug of lipid nanoparticle formulated CVnCoV, CV2CoV, or sham (N=6/group). CV2CoV induced substantially higher binding and neutralizing antibodies, memory B cell responses, and T cell responses as compared with CVnCoV. CV2CoV also induced more potent neutralizing antibody responses against SARS-CoV-2 variants, including B.1.351 (beta), B.1.617.2 (delta), and C.37 (lambda). While CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded robust protection with markedly lower viral loads in the upper and lower respiratory tract. Antibody responses correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of an mRNA SARS-CoV-2 vaccine in nonhuman primates.

sentences: DecoPath: A web application for decoding pathway enrichment analysis The past two decades have brought a steady growth of pathway databases and pathway enrichment methods. However, the advent of pathway data has not been accompanied by an improvement with regards to interoperability across databases, thus, hampering the use of pathway knowledge from multiple databases for pathway enrichment analyses. While integrative databases have attempted to address this issue by collating pathway knowledge from multiple resources, these approaches do not account for redundant information across them. On the other hand, the majority of studies that employ pathway enrichment analyses still rely upon a single database, though the use of another resource could yield differing results, which is similarly the case when different pathway enrichment methods are employed. These shortcomings call for approaches that investigate the differences and agreements across databases and enrichment methods as their selection in the experimental design of a pathway analysis can be a crucial first step in ensuring the results of such an analysis are meaningful. Here we present DecoPath, a web application to assist in the interpretation of the results of pathway enrichment analysis. DecoPath provides an ecosystem to run pathway enrichment analysis or directly upload results and facilitate the interpretation of these results with custom visualizations that highlight the consensus and/or discrepancies at the pathway- and gene-levels. DecoPath is available at https://decopath.scai.fraunhofer.de and its source code and documentation can be found on GitHub at https://github.com/DecoPath/DecoPath.

sentences: KLF4 induces Mesenchymal-Epithelial Transition (MET) by suppressing multiple EMT-inducing transcription factors Epithelial-Mesenchymal Plasticity (EMP) refers to reversible dynamic processes where cells can transition from epithelial to mesenchymal (EMT) or from mesenchymal to epithelial (MET) phenotypes. Both these processes are modulated by multiple transcription factors acting in concert. While EMT-inducing transcription factors (TFs) - TWIST1/2, ZEB1/2, SNAIL1/2/3, GSC, FOXC2 - are well-characterized, the MET-inducing TFs are relatively poorly understood (OVOL1/2, GRHL1/2). Here, using mechanism-based mathematical modeling, we show that the transcription factor KLF4 can delay the onset of EMT by suppressing multiple EMT-TFs. Our simulations suggest that KLF4 overexpression can promote phenotypic shift toward a more epithelial state, an observation suggested by negative correlation of KLF4 with EMT-TFs and with transcriptomic based EMT scoring metrics in cancer cell lines. We also show that the influence of KLF4 in modulating EMT dynamics can be strengthened by its ability to inhibit cell-state transitions at an epigenetic level. Thus, KLF4 can inhibit EMT through multiple parallel paths and can act as a putative MET-TF. KLF4 associates with patient survival metrics across multiple cancers in a context-specific manner, highlighting the complex association of EMP with patient survival.

sentences: Double mutant of chymotrypsin inhibitor 2 stabilized through increased conformational entropy The conformational heterogeneity of a folded protein can affect both its function but also stability and folding. We recently discovered and characterized a stabilized double mutant (L49I/I57V) of the protein CI2 and showed that state-of-the-art prediction methods could not predict the increased stability relative to the wild-type protein. Here we have examined whether changed native state dynamics, and resulting entropy changes, can explain the stability changes in the double mutant protein, as well as the two single mutant forms. We have combined NMR relaxation measurements of the ps-ns dynamics of amide groups in the backbone and the methyl groups in the side-chains with molecular dynamics simulations to quantify the native state dynamics. The NMR experiments reveal that the mutations have different effects on the conformational flexibility of CI2: A reduction in conformational dynamics (and entropy) of the native state of L49I variant correlates with its decreased stability, while increased dynamics of the I57V and L49I/I57V variants correlates with their increased stability. These findings suggest that explicitly accounting for changes in native state entropy might be needed to improve the predictions of the effect of mutations on protein stability.

sentences: Response to nitrogen and salinity in Rhizophora mangle propagules varies by maternal family and population of origin Many coastal foundation plant species thrive across a range of environmental conditions, often displaying dramatic phenotypic variation in response to environmental variation. We characterized the response of propagules from six populations of the foundation species Rhizophora mangle L. to full factorial combinations of two levels of salt (15 ppt and 45 ppt) reflecting the range of salinity measured in the field populations, and two levels of nitrogen (N; no addition and amended at approximately 3 mg N per pot each week) equivalent to comparing ambient N to a rate of addition of 75 kg per hectare per year. The response to increasing salt included significant plasticity in succulence. Propagules also showed plasticity in maximum photosynthetic rate in response to N amendment, but the responses depended on the level of salt and varied by population of origin. Generally, survival was lower in high salt and high N, but the impact varied among populations. Overall, this study revealed significant phenotypic plasticity in response to salt and N level. Propagules from different populations differed in all traits measured. Variation in phenotypic plasticity and propagule survival in R. mangle may contribute to adaptation to a complex mosaic of environmental conditions and response to climate change.

sentences: SARS-CoV-2 mRNA vaccination elicits robust and persistent T follicular helper cell response in humans SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we show that frequency of TFH correlates with that of S-binding germinal center B cells. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLADPB1* 04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.

sentences: Machine learning approaches to identify sleep genes Genetics impacts sleep, yet, the molecular mechanisms underlying sleep regulation remain elusive. We built machine learning (ML) models to predict genes based on their similarity to known sleep genes. Using a manually curated list of 109 labeled sleep genes, we trained a prediction model on thousands of published datasets, representing circadian, immune, sleep deprivation, and many other processes. Our predictions fit with prior knowledge of sleep regulation and also identify several key genes/pathways to pursue in follow-up studies. We tested one of our findings, the NF-{kappa}B pathway, and showed that its genetic alteration affects sleep duration in mice. Our study highlights the power of ML to integrate prior knowledge and genome-wide data to study genetic regulation of sleep and other complex behaviors.

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Tfap2b specifies an embryonic melanocyte stem cell population that retains adult multi-fate potential Melanocytes, our pigment producing cells, are replenished from multiple stem cell niches in adult tissues. Although pigmentation traits are known risk-factors for melanoma, we know little about melanocyte stem cell (McSC) populations other than hair follicle McSCs, and lack key lineage markers with which to identify McSCs and study their function. Here, we discover that Tfap2b, and a select set of its target genes, specifies an McSC population at the dorsal root ganglia in zebrafish. Functionally, Tfap2b is required for only a few late-stage embryonic melanocytes, and instead is essential for McSC-dependent melanocyte regeneration. Fate- mapping data reveal that tfap2b-expressing McSCs have multi-fate potential, and are the cell-of- origin for large patches of adult melanocytes, and two other pigment cell types, iridophores and xanthophores. Hence, Tfap2b confers McSC identity in early development, thereby distinguishing McSCs from other neural crest and pigment cell lineages, and retains multi-fate potential in the adult zebrafish.

HighlightsO_LITfap2b and its target genes specify McSCs with mixed pigment cell identities
C_LIO_LIFunctional dependence on Tfap2b for melanocyte regeneration from the McSC
C_LIO_LItfap2b specifies ErbB-dependent McSCs at the stem cell niche
C_LIO_LIFate mapping reveals Tfap2b-McSCs have multi-fate potential for adult pigment cells
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sentences: Non-ribosomal peptide synthetase domain boundary identification and new motifs discovery based on motif-intermotifs standardized architecture Non-ribosomal peptide synthetase (NRPS) is a diverse family of biosynthetic enzymes for the assembly of bioactive peptides. Its repetitive modular structure inspired many reengineering efforts. Despite the continued expansion of microbial sequencing data, varied standards for annotating domains and modules make data-driven discoveries in NRPSs challenging. In this work, we presented a motif-and-intermotif architecture of NRPSs by partitioning the condensation (C), adenylation (A), thiolation (T) and thioesterase (TE) domains. This "standardization" enables systematic evaluation of functional couplings from a large number of NRPS sequences, resulting in the discovery of novel motifs such as glycine (G)-motif with functional implications for structural flexibility. As a proof of concept, the function of G-motif in controlling the activity of NRPS was verified by mutation experiments in an antitumor fumiquinazoline pathway. Additionally, coevolution studies clarify significant barriers associated with reengineering NRPSs, and comparison of sequences between species reveals interactions between phylogeny and substrate specificity. Our findings give an in-depth analysis and statistical insights into the sequencing characteristics of NRPSs, paving the path for future data-driven discoveries.

sentences: H3K4me3 enrichment defines neuronal age, while a youthful H3K27ac signature is recapitulated in aged neurons Neurons live for the lifespan of the individual and underlie our ability for lifelong learning and memory. However, aging alters neuron morphology and function resulting in age-related cognitive decline. It is well established that epigenetic alterations are essential for learning and memory, yet few neuron-specific genome-wide epigenetic maps exist into old age. Comprehensive mapping of H3K4me3 and H3K27ac in mouse neurons across lifespan revealed plastic H3K4me3 marking that differentiates neuronal age linked to known characteristics of cellular and neuronal aging. We determined that neurons in old age recapitulate the H3K27ac enrichment at promoters, enhancers and super enhancers from young adult neurons, likely representing a re-activation of pathways to maintain neuronal output. Finally, this study identified new characteristics of neuronal aging, including altered rDNA regulation and epigenetic regulatory mechanisms. Collectively, these findings indicate a key role for epigenetic regulation in neurons, that is inextricably linked with aging.

sentences: A CTDNEP1-lipin 1-mTOR regulatory network restricts ER membrane biogenesis to enable chromosome motions necessary for mitotic fidelity The endoplasmic reticulum (ER) dramatically restructures in open mitosis to become excluded from the mitotic spindle; however, the significance of ER reorganization to mitotic progression is not known. Here, we demonstrate that limiting ER membrane biogenesis enables mitotic chromosome movements necessary for chromosome biorientation and prevention of micronuclei formation. Aberrantly expanded ER membranes increase the effective viscosity of the mitotic cytoplasm to physically restrict chromosome dynamics - slowed chromosome motions impede correction of mitotic errors induced by transient spindle disassembly, leading to severe micronucleation. We define the mechanistic link between regulation of ER membrane biogenesis and mitotic fidelity by demonstrating that a CTDNEP1-lipin 1-mTOR regulatory network limits ER lipid synthesis to prevent chromosome missegregation. Together, this work shows that ER membranes reorganize in mitosis to enable chromosome movements necessary for mitotic error correction and reveal dysregulated lipid metabolism as a potential source of aneuploidy in cancer cells.

sentences: The AAA-ATPase Atad1 and its partners promote degradation of desmin intermediate filaments in muscle Maintenance of desmin intermediate filaments (IF) is vital for muscle plasticity and function, and their perturbed integrity due to accelerated loss or aggregation causes atrophy and myopathies. Calpain-1-mediated disassembly of ubiquitinated desmin IF is a prerequisite for desmin loss, myofibril breakdown and atrophy. Because calpain-1 does not harbor a bona fide ubiquitin binding domain, the precise mechanism for desmin IF disassembly remains unknown. Here, we demonstrate that the AAA-ATPase, ATAD1, is required to facilitate disassembly and turnover of ubiquitinated desmin IF. We identified PLAA and UBXN4 as ATAD1s interacting partners, and show they are required for desmin filaments turnover because their downregulation attenuated desmin loss upon denervation. The ATAD1-PLAA-UBXN4 complex binds desmin filaments and promotes a release of phosphorylated and ubiquitinated species into the cytosol, presenting ATAD1 as the only known AAA-ATPase that preferentially acts on phosphorylated substrates. Desmin filaments disassembly was accelerated by the coordinated functions of Atad1 and calpain-1, which interact in muscle. Thus, by extracting ubiquitinated desmin from the insoluble filament, ATAD1 may expose calpain-1 cleavage sites on desmin, consequently enhancing desmin solubilization and degradation in the cytosol.

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Understanding the mechanism of monoADP ribosylation in OsSRT1 and its linkage to the DNA repair system under stress conditions The role of sirtuins in plants are slowly unraveling. There are only reports of H3K9Ac deacetylation by OsSRT1. Here our studies shade light on its dual enzyme capability with preference for mono ADP ribosylation over deacetylation. OsSRT1 can specifically transfer the single ADP ribose group on its substrates in an enzymatic manner. This mono ADPr effect is not well known in plants, more so for deacetylases. The products of this reaction (NAM and ADP ribose) have immense negative effect on this enzyme suggesting a tighter regulation. Resveratrol, a natural plant polyphenol proves to be a strong activator of this enzyme at 150 M concentration. Under different abiotic stress conditions, we could link this ADP ribosylase activity to the DNA repair pathway by activating the enzyme PARP1. Metal stress in plants also influences these enzyme activities.

HighlightsO_LIOsSRT1 can transfer a single moiety of ADP-ribose on itself as well as other nuclear proteins like histones H3 and H2A.
C_LIO_LINAM, ADP-ribose and certain metal ions negatively regulate this ADP-ribose transfer.
C_LIO_LIADPr of OsPARP1 and OsPARP2 links OsSRT1 to DNA damage repair pathways.
C_LIO_LIOsSRT1 positively regulates the activity of OsPARP1 by ADP ribosylating it.
C_LIO_LIOn plants exposure to H2O2 (oxidative stress) and Arsenic toxicity, there is a link between the increased activity of the players of DNA damage repair system and overexpression of OsSRT1.
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sentences: Dicer-to-Argonaute switch controls biogenesis of oncogenic miRNA miRNAs are post-transcriptional regulators of gene expression, controlling biological processes from development to pathogenesis. We asked whether the reshaped functional miRNA landscape in cancers is driven by altered transcription of its precursors, or altered biogenesis and maturation of miRNAs. Integrated analysis of genomic and transcriptomic data in 9,111 samples across 10 cancer types and healthy tissues revealed a recurrent genomic switch from DICER-dependent to non-canonical Argonaute-mediated, DICER-independent, miRNA biogenesis. Experimental validation in AGO2-amplified clinical samples and cancer cell lines confirmed that canonical miRNAs can undergo maturation in a DICER-independent manner, and that elevated Argonaute levels promote selective maturation of the oncogenic miR-106b/25 cluster as shown by the altered ratio of mature miRNA to immature pri-miRNA levels. The preferential maturation of these oncogenic miRNAs, whose processing bypasses DICER1, promotes cancer progression and predicts poor prognosis. This highlights the evolution of non-canonical AGO2-dependent oncomiR processing as a novel driver pathway in cancer.

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Immune Correlates of Protection by mRNA-1273 Immunization against SARS-CoV-2 Infection in Nonhuman Primates Immune correlates of protection can be used as surrogate endpoints for vaccine efficacy. The nonhuman primate (NHP) model of SARS-CoV-2 infection replicates key features of human infection and may be used to define immune correlates of protection following vaccination. Here, NHP received either no vaccine or doses ranging from 0.3 - 100 g of mRNA-1273, a mRNA vaccine encoding the prefusion-stabilized SARS-CoV-2 spike (S-2P) protein encapsulated in a lipid nanoparticle. mRNA-1273 vaccination elicited robust circulating and mucosal antibody responses in a dose-dependent manner. Viral replication was significantly reduced in bronchoalveolar lavages and nasal swabs following SARS-CoV-2 challenge in vaccinated animals and was most strongly correlated with levels of anti-S antibody binding and neutralizing activity. Consistent with antibodies being a correlate of protection, passive transfer of vaccine-induced IgG to naive hamsters was sufficient to mediate protection. Taken together, these data show that mRNA-1273 vaccine-induced humoral immune responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP.

One-Sentence SummarymRNA-1273 vaccine-induced antibody responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP.

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Aeromonas hydrophila induces skin disturbance through mucosal microbiota dysbiosis in Pangasianodon hypophthalmus Bacterial pathogens are well-equipped to adhere to and initiate infection in teleost fish. The fish skin mucus serves as the first barrier against environmental pathogens. The mucus harbors commensal microbes that form the microbiota, impacting the host physiological and immunological regulation. However, how the skin mucosal microbiota responds to the presence of pathogens remains largely unexplored. Thus, little is known about the status of skin mucus prior to the infection with noticeable symptoms. In this study, we aim to investigate the interaction between pathogen and skin mucosal microbiota, as well as the fish skin immune responses in the presence of pathogens. By challenging striped catfish (Pangasianodon hypophthalmus) with different concentrations of bacterial pathogen Aeromonas hydrophila (AH), the skin immune response and the mucosal microbiota were examined by qPCR and 16S rRNA gene sequencing analysis. We found a pathogen concentration able to stimulate the skin immune response associated with the significant mucosal microbiota change and re-confirmed with the ex vivo fish skin model. Further analysis indicated that the change was attributed to the significant increase in opportunistic pathogens over AH. We concluded that the presence and increase of AH results in dysbiosis of mucosal microbiota that can stimulate skin immune response. We believe our work can shed some light on host-pathogen-commensal microbiota interaction and therefore contribute to aquaculture infection prevention.

IMPORTANCEThe fish skin mucosal microbiota is essentially the first barrier in response to the presence of pathogens. This study is the first study to elucidate the interaction between the AH, the skin mucosal microbiota, and the striped catfish skin at the initiation stage of infection. Our study provides a platform to study both the correlation and causation of the interaction between pathogen, the fish skin, and the skin mucosal microbiota. This work provides information that changes in the AH-induced mucosal microbiota result in skin disturbance with immune stimulation.

sentences: Improved Characterization of Balancing Selection Genome Wide and a Detailed Look at HLA Genes Balancing selection occurs when multiple alleles are kept at elevated frequencies in equilibrium due to opposing evolutionary pressures. A new statistical method was developed to test for selection using efficient Bayesian techniques. Selection signals in three different data sets, generated with variable sequencing technologies, were compared: clinical trios, HLA NGS typed samples, and whole-genome long-read samples. Genome-wide, selection was observed across multiple gene families whose biological functions favor diversification, revealing established targets as well as 45 novel genes under selection. Using high-resolution HLA typing and long-read sequencing data, for the characterization of the MHC, revealed strong selection in expected peptide-binding domains as well as previously understudied intronic and intergenic regions of the MHC. Surprisingly, SIRPA, demonstrated dramatic selection signal, second only to the MHC in most settings. In conclusion, employing novel statistical approaches and improved sequencing technologies is critical to properly analyze complex genomic regions.

sentences: Assessment of dynamics of global DNA methylation during the cryopreservation process of Pinus pinaster embryogenic lines Long-term in vitro maintenance of embryogenic lines of Pinus species has been associated with lower maturation capacity, because of this, cryopreservation protocols for the embryogenic lines are needed to maintain valuable genotypes. Since cryopreservation may induce epigenetic variations, we evaluate changes in DNA methylation levels through the course of the cryopreservation of maritime pine embryogenic lines, as compared to those lines maintained by repeated subcultures. Six maritime pine embryogenic lines were cryopreserved following a protocol that includes pre-treatments inducing osmotic stress in liquid media. The percentage of methylated cytosines (%5-mC) in total DNA was determined by using a colorimetric assay. Reactive oxygen species (ROS) accumulation in cell lines was also determined by quantifying dihydroethidium (DHE) fluorescence under a confocal laser-scanning microscope. In the first experiment, we found that global DNA methylation was significantly reduced during the cryopreservation protocol. Subsequently, we evaluated the methylation status of both cryopreserved and no cryopreserved lines (maintained by subcultures) and found differences among embryogenic lines but overall, cryopreservation did not alter %5-mC of the recovered lines while periodical subcultures increased methylation rates. In addition, maltose pretreatment did not increase significantly ROS production in embryogenic lines. Our results demonstrate that the genetic stability during cryopreservation highly depends on the embryogenic line studied, but the protocol allows maintaining methylation DNA rates in most of the recovered lines.

sentences: Sequential sampling from memory underlies action selection during abstract decision making The study of perceptual decision making in monkeys has provided insights into the process by which sensory evidence is integrated towards a decision. When monkeys make decisions with the knowledge of the motor actions the decisions bear upon, the process of evidence integration is instantiated by neurons involved in the selection of said actions. It is less clear how monkeys make decisions when unaware of the actions required to communicate their choice--what we refer to as  abstract decisions. We investigated this by training monkeys to associate the direction of motion of a noisy random-dot display with the color of two targets. Crucially, the targets were displayed at unpredictable locations after the motion stimulus was extinguished. We found that monkeys postponed decision formation until the targets were revealed. Neurons in the parietal association area LIP represented the integration of evidence leading to a choice, but as the stimulus was no longer visible, the samples of evidence must have been retrieved from short-term memory. Our results imply that when decisions are temporally unyoked from the motor actions they bear upon, decision formation is protracted until they can be framed in terms of motor actions.

sentences: Immunization with SARS-CoV-2 nucleocapsid protein triggers a pulmonary immune response in rats The SARS-CoV-2 pandemic have been affecting millions of people worldwide, since the beginning of 2020. COVID-19 can cause a wide range of clinical symptoms, which varies from asymptomatic presentation to severe respiratory insufficiency, exacerbation of immune response, disseminated microthrombosis and multiple organ failure, which may lead to dead. Due to the rapid spread of SARS-CoV-2, the development of vaccines to minimize COVID-19 severity in the world population is imperious. One of the employed techniques to produce vaccines against emerging viruses is the synthesis of recombinant proteins, which can be used as immunizing agents. Based on the exposed, the aim of the present study was to verify the systemic and immunological effects of IM administration of recombinant Nucleocapsid protein (NP), derived from SARS-CoV-2 and produced by this research group, in 2 different strains of rats (Rattus norvegicus); Wistar and Lewis. For this purpose, experimental animals received 4 injections of NP, once a week, and were submitted to biochemical and histological analysis. Our results showed that NP inoculations were safe for the animals, which presented no clinical symptoms of worrying side effects, nor laboratorial alterations in the main biochemical and histological parameters, suggesting the absence of toxicity induced by NP. Moreover, NP injections successfully triggered the production of specific anti-SARS-CoV-2 IgG antibodies by both Wistar and Lewis rats, showing the sensitization to have been well sufficient for the immunization of these strains of rats. Additionally, we observed the local lung activation of the Bronchus-Associated Lymphoid Tissue (BALT) of rats in the NP groups, suggesting that NP elicits specific lung immune response. Although pre-clinical and clinical studies are still required, our data support the recombinant NP produced by this research group as a potential immunizing agent for massive vaccination, and may represent advantages upon other recombinant proteins, since it seems to induce specific pulmonary protection.

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Integrated molecular and pharmacological characterization of patient-derived xenografts from bladder and ureteral cancers identifies new potential therapies. BackgroundMuscle-invasive bladder cancer (MIBC) and upper urinary tract urothelial carcinoma (UTUC) are molecularly heterogeneous. Despite chemotherapies, immunotherapies or anti-FGFR treatments, these tumors are still of poor outcome. Our objective was to develop a bank of patient-derived xenografts (PDXs) recapitulating molecular heterogeneity of MIBC and UTUC, to facilitate preclinical identification of therapies.

MethodsFresh tumors were obtained from patients and subcutaneously engrafted into immune-compromised mice. Patient tumors and matched PDXs were compared regarding histopathology, transcriptomic (microarrays) and genomic profiles (targeted-NGS). Several PDXs were treated with chemotherapy (cisplatin/gemcitabine) or targeted therapies (FGFR and EGFR inhibitors).

Results31 PDXs were established from 1 non-MIBC, 25 MIBC, 5 upper urinary tract tumors, including 28 urothelial (UCC) and 3 squamous-cell carcinomas (SCC). Integrated genomic and transcriptomic profiling identified PDXs of 3 different consensus molecular subtypes (Basal/Squamous, Luminal papillary and Luminal unstable), and included FGFR3-mutated PDXs. High histological and genomic concordance was found between matched patient tumor/PDX. Discordance in molecular subtypes, such as a basal/squamous patient tumor giving rise to a luminal papillary PDX, was observed (n=5) at molecular and histological levels. Ten models were treated with cisplatin-based chemotherapy and we did not observe association between subtypes and response. Of the 3 basal/squamous models treated with anti-EGFR therapy, two models were sensitive and one model, of sarcomatoid variant, was resistant. Treatment of 3 FGFR3-mutant PDXs with combined FGFR/EGFR inhibitors was more efficient than anti-FGFR3 treatment alone.

ConclusionsWe developed preclinical PDX models that recapitulate the molecular heterogeneity of MIBCs and UTUC, including actionable mutations, which will represent an essential tool in therapy development. Pharmacological characterization of the PDXs suggested that upper urinary tract and MIBCs, UCC but also SCC, with similar molecular characteristics could benefit from the same treatments including anti-FGFR for FGFR3-mutated tumors and anti-EGFR for basal ones and showed a benefit for combined FGFR/EGFR inhibition in FGFR3-mutant PDXs, compared to FGFR inhibition alone.

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Differential plasmacytoid dendritic cell phenotype and type I Interferon response in asymptomatic and severe COVID-19 infection SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure.

Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro, asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum.

This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo. These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients.

Author summarySARS-CoV-2 pandemic has resulted in millions of infections and deaths worldwide, yet the role of host innate immune responses in COVID-19 pathogenesis remains only partially characterized. Innate immunity represents the first line of host defense against viruses. Upon viral recognition, the secretion of type I and III interferons (IFN) establishes the cellular state of viral resistance, and contributes to induce the specific adaptive immune responses. Moving from in vitro evidences on the protective role played by plasmacytoid dendritic cells (pDC)-released type I IFN in the early phase of SARS-CoV-2 infection, here we characterized ex vivo the pDC phenotype and the balance between anti-viral and pro-inflammatory cytokines of COVID-19 patients stratified according to disease severity. Our study confirms in COVID-19 the crucial and protective role of pDC/type I IFN axis, whose deeper understanding may contribute to the development of novel pharmacological strategies and/or host-directed therapies aimed at boosting pDC response since the early phases of SARS-CoV-2 infection.

sentences: Meta-analytic maps of brain activity evoked by cognitive function diverge from resting state networks Spontaneous human brain activity is organized into resting state networks (RSN), complex patterns of synchronized activity that account for the majority of brain metabolism. The correspondence between these patterns and those elicited by task performance suggests that spontaneous brain activity originates from the stream of ongoing cognitive processing. Here we investigate a large number of meta-analytic activation maps obtained from Neurosynth (www.neurosynth.org) to establish that task-rest similarity can be inflated by two potential sources of bias. Applying a hierarchical module detection algorithm to a network representation of activation map similarity, we showed that the correspondence between RSN and task-evoked activity tends to hold only for the largest spatial scales. Second, we established that this correspondence is biased by the inclusion of maps related to neuroanatomical terms (e.g. "parietal", "occipital", "cingulate", etc.). Our results challenge the cognitive origin of spontaneous brain activity, suggesting that anatomically-constrained homeostatic processes could also play an important role in the inception and shaping of human resting state activity fluctuations.

sentences: Chronumental: time tree estimation from very large phylogenies Phylogenetic trees are an important tool for interpreting sequenced genomes, and their interrelationships. Estimating the date associated with each node of such a phylogeny creates a "time tree", which can be especially useful for visualising and analysing evolution of organisms such as viruses. Several tools have been developed for time-tree estimation, but the sequencing explosion in response to the SARS-CoV-2 pandemic has created phylogenies so large as to prevent the application of these previous approaches to full datasets. Here we introduce Chronumental, a tool that can rapidly infer time trees from phylogenies featuring large numbers of nodes. Chronumental uses stochastic gradient descent to identify lengths of time for tree branches which maximise the evidence lower bound under a probabilistic model, implemented in a framework which can be compiled into XLA for rapid computation. We show that Chronumental scales to phylogenies featuring millions of nodes, with chronological predictions made in minutes, and is able to accurately predict the dates of nodes for which it is not provided with metadata.

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Constitutive depletion of brain serotonin differentially affects rats' social and cognitive abilities BackgroundCentral serotonin is an essential neuromodulator for mental disorders. It appears a promising transdiagnostic marker of distinct psychiatric disorders and a common modulator of some of their key behavioral symptoms. We aimed to identify the behavioral markers of serotonergic function in rats and compare them to human deficits.

MethodsWe applied a comprehensive profiling approach in adult male Tph2-/- rats constitutively lacking central serotonin. Under classical and ethological testing conditions, we tested each individuals cognitive, social and non-social abilities and characterized the group organization (i.e. social network, hierarchy). Using unsupervised machine learning, we identified the functions most dependent on central serotonin.

ResultsIn classical procedures, Tph2-/- rats presented an unexpected normal cognitive profile. Under the complex and experimenter-free conditions of their home-cage, the same Tph2-/- rats presented drastic changes in their daily life. Brain serotonin depletion induced compulsive aggression and sexual behavior, hyperactive and hypervigilant stereotyped behavior, reduced self-care and body weight, and exacerbated corticosterone levels. Group-housed Tph2-/- rats showed strong social disorganization with disrupted social networks and hierarchical structure, which may arise from communication deficits and cognitive blunting.

ConclusionsSerotonin depletion induced a profile reminiscent of the symptomatology of impulse control and anxiety disorders. Serotonin was necessary for behavioral adaptation to dynamic social environments. In classical testing conditions, our animal model challenged the concept of an essential role of serotonin in decision-making, flexibility, and impulsivity, although developmental compensations may have occurred. These contrasting findings highlight the need to generalize the evaluation of animal models multidimensional functions within the complexity of the social living environment.

sentences: An in silico method to assess antibody fragment polyreactivity Antibodies are essential biological research tools and important therapeutic agents, but some exhibit non-specific binding to off-target proteins and other biomolecules. Such polyreactive antibodies compromise screening pipelines, lead to incorrect and irreproducible experimental results, and are generally intractable for clinical development. We designed a set of experiments using a diverse naive synthetic camelid antibody fragment ( nanobody) library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC > 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally tested our models performance on three independent nanobody scaffolds, where over 90% of predicted substitutions successfully reduced polyreactivity. Importantly, the model allowed us to diminish the polyreactivity of an angiotensin II type I receptor antagonist nanobody, without compromising its pharmacological properties. We provide a companion web-server that offers a straightforward means of predicting polyreactivity and polyreactivity-reducing mutations for any given nanobody sequence.

sentences: The quorum sensing system NprR-NprRB contributes to spreading and fitness in colony biofilms of Bacillus thuringiensis. Quorum sensing (QS) are intercellular communication mechanisms to coordinate bacterial gene expression in response to signaling molecules. In Bacillus thuringiensis the QS system NprR-NprRB (receptor protein-signaling peptide) regulates the expression of genes related to nutrient scavenging during necrotrophism and also modulates sporulation onset. However, the relevance of QS in free-living stages of B. thuringiensis is less known. In this work, we depict the contribution of this QS system to spreading in colony biofilms. Through a spreading assay in spotted colonies of B. thuringiensis Bt8741 Wt and derived mutants, we find that the spreading phenotype depends on the NprR regulator and on the extracellular signaling NprRB peptide. We also show that this phenotype is associated to an increased fitness of the bacterium in these experimental conditions. Exogenous addition of a lipopeptide surfactant was sufficient to recover spreading in the {Delta}nprR-nprRB mutant, indicating that the phenotype could be mediated by the lipopeptide kurstakin. Finally, we suggest that the spreading is relevant in nature, since it occurs in the sole presence of soil nutrients, and it is conserved in several species of Bacillus commonly found in soil. This novel function of NprR-NprRB highlights the relevance of this QS system on the evolution and on the free-lifestyle ecology of B. thuringiensis.

sentences: Heterologous Vaccination with SARS-CoV-2 Spike saRNA Prime followed by DNA Dual-Antigen Boost Induces Robust Antibody and T-Cell Immunogenicity against both Wild Type and Delta Spike as well as Nucleocapsid Antigens We assessed if immune responses are enhanced in CD-1 mice by heterologous vaccination with two different nucleic acid-based COVID-19 vaccines: a next-generation human adenovirus serotype 5 (hAd5)-vectored dual-antigen spike (S) and nucleocapsid (N) vaccine (AdS+N) and a self-amplifying and -adjuvanted S RNA vaccine (SASA S) delivered by a nano-lipid carrier. The AdS+N vaccine encodes S modified with a fusion motif to increase cell-surface expression. The N antigen is modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal compartment and increase MHC class I and II stimulation potential. The S sequence in the SASA S vaccine comprises the D614G mutation, two prolines to stabilize S in the prefusion conformation, and 3 glutamines in the furin cleavage region to increase cross-reactivity across variants. CD-1 mice received vaccination by homologous and heterologous prime > boost combinations. Humoral responses to S were the highest with any regimen including the SASA S vaccine, and IgG bound to wild type and Delta (B.1.617.2) variant S1 at similar levels. An AdS+N boost of an SASA S prime particularly enhanced both CD4+ and CD8+ T-cell responses to both wild type and Delta S peptides relative to all other vaccine regimens. Sera from mice receiving SASA S homologous or heterologous vaccination were found to be highly neutralizing of all pseudovirus strains tested: Wuhan, Beta, Delta, and Omicron strain. The findings here support the clinical testing of heterologous vaccination by an SASA S > AdS+N regimen to provide increased protection against emerging SARS-CoV-2 variants.

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Mobile microscopy and telemedicine platform assisted by deep learning for quantification of Trichuris trichiura infection. Soil-transmitted helminths (STH) are the most prevalent pathogens among the group of neglected tropical diseases (NTDs). Kato-Katz technique is the diagnosis method recommended by WHO and although is generally more sensitive than other microscopic methods in high transmission settings, it often presents a decreased sensitivity in low transmission settings and it is labour intensive. Digitizing the samples could provide a solution which allows to store the samples in a digital database and perform remote analysis. Artificial intelligence methods based on digitized samples can support diagnostics efforts by support diagnostics efforts by performing an automatic and objective quantification of disease infection.

In this work, we propose an end-to-end pipeline for microscopy image digitization and automatic analysis of digitized images of soil-transmitted helminths. Our solution includes (1) a digitalization system based on a mobile app that digitizes the microscope samples using a low-cost 3D-printed microscope adapter, (2) a telemedicine platform for remote analysis and labelling and (3) novel deep learning algorithms for automatic assessment and quantification of parasitological infection of STH.

This work has been evaluated by comparing the STH quantification using both a manual remote analysis based on the digitized images and the AI-assisted quantification against the reference method based on conventional microscopy. The deep learning algorithm has been trained and tested on 41 slides of stool samples containing 949 eggs from 6 different subjects using a cross-validation strategy obtaining a mean precision of 98,44% and mean recall of 80,94%. The results also proved the potential of generalization capability of the method at identifying different types of helminth eggs.

In conclusion, this work has presented a comprehensive pipeline using smartphone-based microscopy integrated with a telemedicine platform for automatic image analysis and quantification of STH infection using artificial intelligence models.

sentences: Enhanced envelope coding following acoustic trauma is detrimental to neural coding of speech in noise People with hearing loss struggle understanding speech in noisy backgrounds. Speech-intelligibility models highlight the importance of slow-varying envelopes of speech and noise; however, the physiological basis of impaired perception remains unclear. We provide neurophysiological evidence that, although acoustic trauma enhances both speech and noise envelopes, disruptions in cochlear tonotopicity preferentially enhance neural responses to noise in noisy speech. These results provide mechanistic insights into everyday-communication challenges created by hearing loss.

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Allele-specific knockouts reveal a role for apontic-like in the evolutionary loss of larval pigmentation in Bombyx mori The domesticated silkworm, Bombyx mori, and its wild progenitor, B. mandarina, are extensively studied as a model case of the evolutionary process of domestication. A conspicuous difference between these species is the dramatic reduction in pigmentation in both larval and adult B. mori. Here we evaluate the efficiency of CRISPR/Cas9-targeted knockouts of pigment-related genes as a tool to understand their potential contributions to domestication-associated pigmentation loss in B. mori. To demonstrate the efficacy of targeted knockouts in B. mandarina, we generated a homozygous CRISPR/Cas9-targeted knockout of yellow-y. In yellow-y knockout mutants, black body color became lighter throughout the larval, pupal and adult stages, confirming a role for this gene in pigment formation. Further, we performed allele-specific CRISPR/Cas9-targeted knockouts of the pigment-related transcription factor, apontic-like (apt-like) in B. mori x B. mandarina F1 hybrid individuals. Knockout of the B. mandarina allele of apt-like in F1 embryos results in depigmented patches on the dorsal integument of larvae, whereas corresponding knockouts of the B. mori allele consistently exhibit normal F1 larval pigmentation. These results demonstrate a contribution of apt-like to the evolution of reduced pigmentation in B. mori. Together, our results demonstrate the feasibility of CRISPR/Cas9-targeted knockouts as a tool for understanding the genetic basis of traits associated with B. mori domestication.

Brief abstractBombyx mori and its wild progenitor are an important model for the study of phenotypic evolution associated with domestication. As proof-of-principle, we used CRISPR/Cas9 to generate targeted knockouts of two pigmentation-related genes. By generating a homozygous knockout of yellow-y in B. mandarina, we confirmed this gene"s role in pigment formation. Further, by generating allele-specific knockouts of apontic-like (apt-like) in B. mori x B. mandarina F1 hybrids, we establish that evolution of apt-like contributed to reduced pigmentation during B. mori domestication.

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sentences: Subcellular proteomics of dopamine neurons in the mouse brain reveals axonal enrichment of proteins encoded by Parkinson's disease-linked genes Dopaminergic neurons modulate neural circuits and behaviors via dopamine release from expansive, long range axonal projections. The elaborate cytoarchitecture of these neurons is embedded within complex brain tissue, making it difficult to access the neuronal proteome using conventional methods. Here, we demonstrate APEX2 proximity labeling within genetically targeted neurons in the mouse brain, enabling subcellular proteomics with cell type-specificity. By combining APEX2 biotinylation with mass spectrometry, we mapped the somatodendritic and axonal proteomes of midbrain dopaminergic neurons. Our dataset reveals the proteomic architecture underlying proteostasis, axonal metabolism, and neurotransmission in these neurons. We find a significant enrichment of proteins encoded by Parkinsons disease-linked genes in striatal dopaminergic axons, including proteins with previously undescribed axonal localization. These proteomic datasets provide a resource for neuronal cell biology, and this approach can be readily adapted for study of other neural cell types.

sentences: A high throughput SEC23 functional interaction screen reveals a role for focal adhesion and extracellular matrix signalling in the regulation of COPII subunit SEC23A Proteins that enter the secretory pathway are transported from their place of synthesis in the Endoplasmic Reticulum to the Golgi complex by COPII coated carriers. The components of the COPII transport machinery have been well characterized, but the network of proteins that regulate these components in response to extracellular cues have remained largely elusive. The discovery of such regulatory proteins is crucial for understanding how cells integrate extracellular and intracellular information to fine-tune membrane traffic in the secretory pathway. A key group of proteins that plays a central role in the regulation of membrane traffic is associated with the cytoskeleton. Using high throughput microscopy of the well-established VSVG transport from the ER to the plasma membrane, we comprehensively screened 378 cytoskeleton-associated proteins for their functional interaction with the COPII components SEC23A and SEC23B using a double knockdown approach. Ranking of the transport effectors identified 20 cytoskeleton-associated proteins as the strongest functional interactors of SEC23, most of them not previously associated with the secretory pathway. Knockdown of a subgroup of these interactors (FERMT2, MACF1, MAPK8IP2, NGEF, PIK3CA, ROCK1), associated with cell adhesion, not only induced changes in focal adhesions and the expression of adhesion-related genes but led to the specific downregulation of SEC23A, a SEC23 paralogue that has been implicated in the secretion of extracellular matrix (ECM) components. Furthermore, SEC23A downregulation could also be recapitulated by plating cells on ECM and was dependent on focal adhesions function. Altogether, our results identify a network of cytoskeleton-associated proteins connecting focal adhesions and ECM-related signalling with the gene expression of the COPII secretory machinery.

sentences: Host bioenergetic parameters reveal cytotoxicity of anti-tuberculosis drugs undetected using conventional viability assays. High attrition rates in tuberculosis (TB) drug development have been largely attributed to safety, which is likely due to the use of endpoint assays measuring cell viability to detect drug cytotoxicity. In drug development of cancer, metabolic and neurological disorders, and antibiotics, cytotoxicity is increasingly being assessed using extracellular flux (XF) analysis, which measures cellular bioenergetic metabolism in real-time. Here, we adopt the XF platform to investigate the cytotoxicity of drugs currently used in TB treatment on the bioenergetic metabolism of HepG2 cells, THP-1 macrophages, and human monocyte derived macrophages (hMDM). We found that the XF analysis reveals earlier drug-induced effects on the cells bioenergetic metabolism prior to cell death, measured by conventional viability assays. Furthermore, each cell type has a distinct response to drug treatment, suggesting that more than one cell type should be considered to examine cytotoxicity in TB drug development. Interestingly, chemically unrelated drugs with different modes of action on Mycobacterium tuberculosis have similar effects on the bioenergetic parameters of the cells, thus, discouraging the prediction of potential cytotoxicity based on chemical structure and mode of action of new chemical entities. The clustering of the drug-induced effects on the hMDM bioenergetic parameters are reflected in the clustering of the effects of the drugs on cytokine production in hMDMs, demonstrating concurrence between the effects of the drugs on the metabolism and functioning of the macrophages. These findings can be used as a benchmark to establish XF analysis as a new tool to assay cytotoxicity in TB drug development.

sentences: A low sugar diet enhances Drosophila body size in males and females via sex-specific mechanisms In Drosophila, changes to dietary protein elicit different body size responses between the sexes. Whether this sex difference in nutrient-dependent body size regulation extends to other nutrients, such as dietary sugar, remains unclear. Here, we show that reducing dietary sugar enhanced body size in Drosophila male and female larvae. Indeed, the largest body size was found in larvae reared in a diet without added sugar. Despite the equivalent body size effects of a low sugar diet between males and females, we detected sex-specific changes to the insulin/insulin-like growth factor (IIS) and target of rapamycin (TOR) signaling pathways. Further, we show that the metabolic changes observed in larvae reared on a low sugar diet differ between the sexes. Thus, despite identical phenotypic responses to dietary sugar in males and females, distinct changes to cell signaling pathways and whole-body metabolism were associated with the increased body size in each sex. This highlights the importance of including both sexes in all mechanistic studies on larval growth, as males and females may use different molecular and metabolic mechanisms to achieve similar phenotypic outcomes.

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Prehension kinematics in humans and macaques AO_SCPLOWBSTRACTC_SCPLOWNon-human primates, especially rhesus macaques, have been a dominant model to study sensorimotor control of the upper limbs. Indeed, human and macaques have similar hands and homologous neural circuits to mediate manual behavior. However, few studies have systematically and quantitatively compared the manual behaviors of the two species. Such comparison is critical for assessing the validity of using the macaque sensorimotor system as a model of its human counterpart. In this study, we systematically compared the prehensile behaviors of humans and rhesus macaques using an identical experimental setup. We found human and macaque prehension kinematics to be generally similar with a few subtle differences. While the structure of the preshaping postures is similar in humans and macaques, human postures are more object-specific and human joints are less intercorrelated. Conversely, monkeys demonstrate more stereotypical grasping behaviors that are common across all grasp conditions and more variability in their postures across repeated grasps of the same object. Despite these subtle differences in manual behavior between humans and monkeys, our results bolster the use of the macaque model to understand the neural mechanisms of manual dexterity in humans.

NO_SCPLOWEWC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWANDC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWNEWSWORTHYC_SCPLOWMacaques have been a dominant animal model to study the neural mechanisms of human dexterity because they exhibit complex manual behavior. We show that the kinematics of prehension - a critical dexterous behavior - are largely similar in humans and macaques. However, human preshaping postures are more object-specific and the movement of human digits are less correlated with each other. The thumb, index, and wrist are major driver of these interspecies differences.

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Hyperglycemic O-GlcNAc transferase activity drives cancer stem cell induction in TNBC Enhanced glucose metabolism is a feature of almost all cancers, but downstream functional effects of aberrant glucose flux are difficult to mechanistically determine. The objective of this study is to characterize a mechanism by which elevated glucose level drives a tumorigenic pathway in triple negative breast cancer (TNBC). We used chemical biology methods to track how a metabolite of glucose, N-acetylglucosamine (GlcNAc), is linked to the transcriptional regulatory protein tet-methylcytosine dioxygenase 1 (TET1) as an O-linked GlcNAc post translational modification (O-GlcNAc). In this work, we revealed that intracellular protein glycosylation by O-GlcNAc is driven by high glucose levels in TNBC models, including on TET1. A single enzyme, O-GlcNAc transferase (OGT), is responsible for catalyzing protein modification of O-GlcNAc. We showed that OGT activity is higher in TNBC cell lines compared to non-tumor breast cell lines and is associated with hyperglycemia. Furthermore, enhanced OGT activity activated a pathway for cancer stem-like cell (CSC) reprogramming in TNBC cells. In our model, O-GlcNAcylated TET1 upregulated expression of splicing factor TAR-DNA binding protein (TARDBP), which drives CSC induction as well as higher OGT levels. We show that this OGT-TET1-TARDBP axis "feeds-forward" in hyperglycemic conditions both in cell lines and diet-induced obese mice, which displayed higher blood glucose levels and tumor O-GlcNAc levels than lean littermates. This data converges on a novel pathway whereby hyperglycemia drives aberrant OGT activity, activating a pathway for CSC induction in TNBC. Our findings partially explain a key aspect of how obesity is associated with TNBC risk and negative outcomes.

Statement of ImplicationThis work presents a novel mechanism to explain how obesity is a risk factor for triple-negative breast cancer via elevated sugar-transfer activity by O-GlcNAc transferase in hyperglycemia, leading to the induction of a cancer stem-like cells and revealing a targetable pathway in obesity-associated tumors.

sentences: Comparing the efficiency of monofilament and traditional nets for capturing bats Traditional mist nets used for capturing bats have several drawbacks, particularly that they are inefficient at sampling many insectivorous species. One possible alternative is to use monofilament nets, whose netting is made of single strands of yarn instead of several as regular nets, making them less detectable. To date, no study has quantified the capture efficiency of monofilament nets compared to regular mist nets for the study of bats. Here we compare capture efficiency of monofilament and regular mist nets, focusing on bat abundance and species diversity at a lowland tropical forest in southwestern Costa Rica. During our sampling period, we captured 90 individuals and 14 species in regular nets and 125 individuals and 20 species in monofilament nets. The use of monofilament nets increased overall capture rates, but most notably for insectivorous species. Species accumulation curves indicate that samples based on regular nets are significantly underestimating species diversity, most notably as these nets fail at sampling rare species. We show that incorporating monofilament nets into bat studies offers an opportunity to expand records of different guilds and rare bat species and to improve our understanding of poorly-known bat assemblages while using a popular, relatively cheap and portable sampling method.

sentences: Integrated cyto-physiological and proteomic analyses reveal new insight into CMS mechanism in a novel upland cotton CMS line LD6A Cytoplasmic male sterile (CMS) system has extensively been exploited for hybrid vigor in plant breeding programs. However, its application in many crops is limited due to poor understanding of molecular mechanism of fertility restoration. Using advanced analytical approaches, we elucidated molecular pathways regulating CMS induction and fertility restoration in cotton. Reproductive structures of a novel CMS (LD6A) and its maintainer (LD6B) line were analyzed for physiological and proteomic changes during the development process. Significant differential expression of proteins, such as Abrin, malate dehydrogenase, malic enzyme, isocitrate dehydrogenase, histone acetyltransferase was observed in CMS and its maintainer line. Transmission electron micrographs of anther tapetum showed that inner ridge of CMS mitochondria was relatively indistinct than that of LD6B with narrower membranous space at tetrad stage. Further, relatively higher reactive oxygen species were accumulated in the anther of CMS than its maintainer line at pollen mother cell and tetrad stage. We suggest that abnormal sequence of mitochondrial ribosome gene rps4 and rpl10 and high expression of ribosome-inactivating protein gene Abrin in CMS line damaged mitochondrial membrane and consequently induced pollen sterility. These data provide new insight into CMS mechanism in cotton crops and a tool to develop new CMS germplasm resources.

sentences: Identification of TAZ as the essential molecular switch in orchestrating SCLC phenotypic transition and metastasis Small cell lung cancer (SCLC) is a recalcitrant cancer featured with high metastasis. However, the exact cell type contributing to metastasis remains elusive. Using Rb1L/L/Trp53L/L mouse model, we identify the NCAMhiCD44lo/- subpopulation as SCLC metastasizing cell (SMC), which is progressively transitioned from non-metastasizing NCAMloCD44hi cell (Non-SMC). Integrative chromatin accessibility and gene expression profiling studies reveal an important role of SWI/SNF complex, and knockout of its central component, Brg1, significantly inhibits such phenotypic transition and metastasis. Mechanistically, TAZ is silenced by SWI/SNF complex during SCLC malignant progression, and its knockdown promotes SMC transition and metastasis. Importantly, ectopic TAZ expression reversely drives SMC-to-Non-SMC transition and alleviates metastasis. Single-cell RNA-sequencing analyses identify SMC as the dominant subpopulation in human SCLC metastasis, and immunostaining data show a positive correlation between TAZ and patient prognosis. These data uncover high SCLC plasticity and identify TAZ as key molecular switch in orchestrating SCLC phenotypic transition and metastasis.

sentences: Predicted binding interface between coronavirus nsp3 and nsp4 Double membrane vesicles (DMVs) in coronavirus-infected cells feature pores that span both membranes. DMV pores were observed to have six-fold symmetry and include the nsp3 protein. Co-expression of SARS-CoV nsp3 and nsp4 induces DMV formation, and elements of nsp3 and nsp4 have been identified that are essential for membrane disruption. I describe a predicted luminal binding interface between nsp3 and nsp4 that is membrane-associated, conserved in SARS-CoV-2 during the COVID-19 pandemic and in diverse coronaviruses, and stable in molecular dynamics simulation. Combined with structure predictions for the full-length nsp4 monomer and cryo-EM data, this suggests a DMV pore model in which nsp4 spans both membranes with nsp3 and nsp4 inserted into the same bilayer. This approach may be able to identify additional protein-protein interactions between coronavirus proteins.

sentences: Comparative transcriptomics reveal highly conserved regional programs between porcine and human colonic enteric nervous system The porcine gut is increasingly regarded as a useful translational model. The enteric nerve system (ENS) in the colon coordinates diverse functions. However, knowledge of the molecular profiling of porcine ENS and its similarity to that of human is limited. We identified the distinct transcriptional programs associated with functional characteristics between inner submucosal and myenteric ganglia (ISG, MG) in porcine proximal and distal colon (p-pC, p-dC) using bulk RNA sequencing (RNA-seq) and single-cell RNA-seq. Comparative transcriptomics of MG in corresponding colonic regions of porcine and human revealed highly conserved programs existing in p-pC and p-dC, which explained >90% of their transcriptomic responses to vagal nerve stimulation (VNS), suggesting that p-pC and p-dC could serve as predictors in translational studies. The conserved programs specific for inflammatory modulation were displayed in porcine with VNS. This study provides a valuable transcriptomic resource for understanding of human colonic functions and neuromodulation using porcine model.

sentences: A cocktail containing two synergetic antibodies broadly neutralizes SARS-CoV-2 and its variants including Omicron BA.1 and BA.2 Neutralizing antibodies (NAbs) can prevent and treat infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, continuously emerging variants, such as Omicron, have significantly reduced the potency of most known NAbs. The selection of NAbs with broad neutralizing activities and the identification of conserved critical epitopes are still urgently needed. Here, we identified an extremely potent antibody (55A8) by single B-cell sorting from convalescent SARS-CoV-2-infected patients that recognized the receptor-binding domain (RBD) in the SARS-CoV-2 spike (S) protein. 55A8 could bind to wild-type SARS-CoV-2, Omicron BA.1 and Omicron BA.2 simultaneously with 58G6, a NAb previously identified by our group. Importantly, an antibody cocktail containing 55A8 and 58G6 (2-cocktail) showed synergetic neutralizing activity with a half-maximal inhibitory concentration (IC50) in the picomolar range in vitro and prophylactic efficacy in hamsters challenged with Omicron (BA.1) through intranasal delivery at an extraordinarily low dosage (25 g of each antibody daily) at 3 days post-infection. Structural analysis by cryo-electron microscopy (cryo-EM) revealed that 55A8 is a Class III NAb that recognizes a highly conserved epitope. It could block angiotensin-converting enzyme 2 (ACE2) binding to the RBD in the S protein trimer via steric hindrance. The epitopes in the RBD recognized by 55A8 and 58G6 were found to be different and complementary, which could explain the synergetic mechanism of these two NAbs. Our findings not only provide a potential antibody cocktail for clinical use against infection with current SARS-CoV-2 strains and future variants but also identify critical epitope information for the development of better antiviral agents.

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Elementary Integrate-and-Fire Process Underlies Pulse Amplitudes in Electrodermal Activity Electrodermal activity (EDA) is a direct read-out of sweat-induced changes in the skins electrical conductance. Sympathetically-mediated pulsatile changes in skin sweat measured as EDA resemble an integrate-and-fire process, which yields an inverse Gaussian model as the inter-pulse interval distribution. We have previously showed that the inter-pulse intervals in EDA follow an inverse Gaussian distribution. However, the statistical structure of EDA pulse amplitudes has not yet been characterized based on the physiology. Expanding upon the integrate-and-fire nature of sweat glands, we hypothesized that the amplitude of an EDA pulse is proportional to the excess volume of sweat produced compared to what is required to just reach the surface of the skin. We modeled this as the difference of two inverse Gaussian models for each pulse, one which represents the time required to produce just enough sweat to rise to the surface of the skin and one which represents the time requires to produce the actual volume of sweat. We proposed and tested a series of four simplifications of our hypothesis, ranging from a single difference of inverse Gaussians to a single simple inverse Gaussian. We also tested four additional models for comparison, including the lognormal and gamma distributions. All models were tested on EDA data from two subject cohorts, 11 healthy volunteers during 1 hour of quiet wakefulness and a different set of 11 healthy volunteers during approximately 3 hours of controlled propofol sedation. All four models represent simplifications of our hypothesis outperformed other models across all 22 subjects, as measured by Akaikes Information Criterion (AIC), as well as mean and maximum distance from the diagonal on a quantile-quantile plot. Our broader model set of four simplifications offered a useful framework to enhance further statistical descriptions of EDA pulse amplitudes. Some of the simplifications prioritize fit near the mode of the distribution, while others prioritize fit near the tail. With this new insight, we can summarize the physiologically-relevant amplitude information in EDA with at most four parameters. Our findings establish that physiologically based probability models provide parsimonious and accurate description of temporal and amplitude characteristics in EDA.

AUTHOR SUMMARYElectrodermal activity (EDA) is an indirect read-out of the bodys sympathetic nervous system, or fight-or-flight response, measured as sweat-induced changes in the electrical conductance properties of the skin. Interest is growing in using EDA to track physiological conditions such as stress levels, sleep quality, and emotional states. Our previous worked showed that the times in between EDA pulses obeyed a specific statistical distribution, the inverse Gaussian, that arises from the physiology of EDA production. In this work, we build on that insight to analyze the amplitudes of EDA pulses. In an analysis of EDA data recorded in 11 healthy volunteers during quiet wakefulness and 11 different healthy volunteers during controlled propofol sedation, we establish that the amplitudes of EDA pulses also have specific statistical structure, as the differences of inverse Gaussians, that arises from the physiology of sweat production. We capture that structure using a series of progressively simpler models that each prioritize different parts of the pulse amplitude distribution. Our findings show a physiologically-based statistical model provides a parsimonious and accurate description EDA. This enables increased reliability and robustness in analyzing EDA data collected under any circumstance.

sentences: Pathological LSD1 mutations repress enhancer-mediated gene regulatory networks in early differentiation Lysine-specific demethylase 1 (LSD1/KDM1A) demethylates both histone and non-histone substrates, recruits repressive chromatin complexes, and is increased in cancers. De novo LSD1 mutations impairing protein function lead to a rare developmental disorder, but the molecular details of the pathology remains unclear. Using patient-derived fibroblasts, reprogrammed pluripotent stem cells, and differentiated cells, we found over 4000 differentially expressed genes and 68 transcription factors (TFs) whose motif accessibilities changed upon LSD1 mutation. An enhancer-mediated gene regulatory network approach identified transcriptional repressors with impaired activity in fibroblast and stem cells, leading to erroneous activation of their target genes. We also revealed overall decreases in TF target gene expression during early lineage differentiation of LSD1 mutant stem cells, likely caused by increased activity of repressive histone deacetylases (HDACs), co-factors of LSD1. Indeed, an HDACs inhibitor restored changes in gene expression including downregulation phenotype. Our findings characterize the molecular pathogenesis of LSD1 mutations and suggest potential therapeutic strategies for the developmental disorder and cancers caused by LSD1 dysregulations.

sentences: Exploring neural correlates of behavioral and academic resilience among children in poverty Children in poverty must contend with systems that do not meet their needs. We explored what, at a neural level, helps explain childrens resilience in these contexts. Lower coupling between lateral frontoparietal network (LFPN) and default mode network (DMN)--linked, respectively, to externally- and internally-directed thought--has previously been associated with better cognitive performance. However, we recently found the opposite pattern for children in poverty. Here, we probed ecologically-valid assessments of performance. In a pre-registered study, we investigated trajectories of network coupling over ages 9-13 and their relation to school grades and attention problems. We analyzed longitudinal data from ABCD Study (N=8366 children at baseline; 1303 below poverty). The link between cognitive performance and grades was weaker for children in poverty, highlighting the importance of ecologically-valid measures. As predicted, higher LFPN-DMN connectivity was linked to worse grades and attentional problems for children living above poverty, while children below poverty showed opposite tendencies. This interaction between LFPN-DMN connectivity and poverty related to childrens grades two years later; however, it was attenuated when controlling for baseline grades and was not related to attention longitudinally. Together, these findings suggest network connectivity is differentially related to performance in real-world settings for children above and below poverty.

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ACE2 expression in rat brain: implications for COVID-19 associated neurological manifestations We examined cell type-specific expression and distribution of rat brain angiotensin converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, in rodent brain. ACE2 is ubiquitously present in brain vasculature, with the highest density of ACE2 expressing capillaries found in the olfactory bulb, the hypothalamic paraventricular, supraoptic and mammillary nuclei, the midbrain substantia nigra and ventral tegmental area, and the hindbrain pontine nucleus, pre-Botzinger complex, and nucleus of tractus solitarius. ACE2 was expressed in astrocytes and astrocytic foot processes, pericytes and endothelial cells, key components of the blood-brain-barrier. We found discrete neuronal groups immunopositive for ACE2 in brainstem respiratory rhythm generating centers including the pontine nucleus, the parafascicular/retrotrapezoid nucleus, the parabrachial nucleus, the Botzinger and pre-Botzinger complex and the nucleus of tractus solitarius; in arousal-related pontine reticular nucleus and in gigantocellular reticular nuclei; in brainstem aminergic nuclei, including substantia nigra, ventral tegmental area, dorsal raphe, and locus coeruleus; in the epithalamic habenula, hypothalamic paraventricular and suprammamillary nuclei; and in the hippocampus. Identification of ACE2-expressing neurons in rat brain within well-established functional circuits facilitates prediction of possible neurological manifestations of brain ACE2 dysregulation during and after COVID-19 infection.

HighlightsO_LIACE2 is present in astrocytes, pericytes, and endothelia of the blood brain barrier.
C_LIO_LINeuronal ACE2 expression is shown in discrete nuclei through the brain.
C_LIO_LIBrainstem breathing, arousal-related, hypothalamic and limbic nuclei express ACE2.
C_LIO_LIACE2 is expressed in circuits potentially involved in COVID-19 pathophysiology.
C_LI

sentences: Numerical Uncertainty in Analytical Pipelines Lead to Impactful Variability in Brain Networks The analysis of brain-imaging data requires complex processing pipelines to support findings on brain function or pathologies. Recent work has shown that variability in analytical decisions, small amounts of noise, or computational environments can lead to substantial differences in the results, endangering the trust in conclusions1-7. We explored the instability of results by instrumenting a connectome estimation pipeline with Monte Carlo Arithmetic8,9 to introduce random noise throughout. We evaluated the reliability of the connectomes, their features10,11, and the impact on analysis12,13. The stability of results was found to range from perfectly stable to highly unstable. This paper highlights the potential of leveraging induced variance in estimates of brain connectivity to reduce the bias in networks alongside increasing the robustness of their applications in the classification of individual differences. We demonstrate that stability evaluations are necessary for understanding error inherent to brain imaging experiments, and how numerical analysis can be applied to typical analytical workflows both in brain imaging and other domains of computational science. Overall, while the extreme variability in results due to analytical instabilities could severely hamper our understanding of brain organization, it also leads to an increase in the reliability of datasets.

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The Features Underlying the Memorability of Objects Despite decades of study of memory, it remains unclear what makes an image memorable. There is considerable debate surrounding the underlying determinants of memory, including the roles of semantic (e.g., animacy, utility) and visual features (e.g., brightness) as well as whether the most prototypical or most atypical items are best remembered. Prior studies have relied on constrained stimulus sets, limiting any generalized view of the features that may contribute to memory. Here, we collected over one million memory ratings (N=13,946) for THINGS (Hebart et al., 2019), a naturalistic dataset of 26,107 object images designed to comprehensively sample concrete objects. First, we establish a model of object features that is predictive of image memorability, capturing over half of the explainable variance. For this model, we find that semantic features have a stronger influence than visual features on what people will remember. Second, we examined whether memorability could be accounted for by the typicality of the objects, by comparing human behavioral data, object feature dimensions, and deep neural network features. While prototypical objects tend to be the most memorable, the relationship between memorability and typicality is more complex than a simple positive or negative association and typicality alone cannot account for memorability.

SIGNIFICANCE STATEMENTWhy is it that we seem to remember and forget the same things? Our lived experiences differ, but there is remarkable consistency in what is remembered across people. Here, we collected memory performance scores for a comprehensive and diverse collection of natural object images to identify which properties determine our ability to remember. We created a model for predicting memory from object features showing that semantic properties more than visual properties contribute primarily to memorability. Further, we find that it is neither the most prototypical or atypical images that are best remembered, which suggests that typicality alone cannot account for memorability. Our findings challenge decades of prior research that suggest that the most atypical items are most memorable, informing our understanding of the features and organizational principles of memory.

sentences: BFG-PCA: tools and resources that expand the potential for binary protein interaction discovery Barcode fusion genetics (BFG) utilizes deep sequencing to improve the throughput of protein-protein interaction (PPI) screening in pools. BFG has been implemented in Yeast two-hybrid (Y2H) screens (BFG-Y2H). While Y2H requires test protein pairs to localize in the nucleus for reporter reconstruction, Dihydrofolate Reductase Protein-Fragment Complementation Assay (DHFR-PCA) allows proteins to localize in broader subcellular contexts and proves to be largely orthogonal to Y2H. Here, we implemented BFG to DHFR-PCA (BFG-PCA). This plasmid-based system can leverage ORF collections across model organisms to perform comparative analysis, unlike the original DHFR-PCA that requires yeast genomic integration. The scalability and quality of BFG-PCA were demonstrated by screening human and yeast interactions for >11,000 protein pairs. BFG-PCA showed high-sensitivity and high-specificity for capturing known interactions for both species. BFG-Y2H and BFG-PCA capture distinct sets of PPIs, which can partially be explained based on the domain orientation of the reporter tags. BFG-PCA is a high-throughput protein interaction technology to interrogate binary PPIs that exploits clone collections from any species of interest, expanding the scope of PPI assays.

sentences: Nelfinavir markedly improves lung pathology in SARS-CoV-2-infected Syrian hamsters despite lack of an antiviral effect In response to the ongoing COVID-19 pandemic, repurposing of drugs for the treatment of SARS-CoV-2 infections is being explored. The HIV protease inhibitor Nelfinavir, widely prescribed in combination with other HIV inhibitors, has been shown to inhibit in vitro SARS-CoV-2 replication. We here report on the effect of Nelfinavir in the Syrian hamster SARS-CoV-2 infection model. Although treatment of infected hamsters with either 15 or 50 mg/kg BID Nelfinavir [for four consecutive days, initiated on the day of infection] does not reduce viral RNA loads nor infectious virus titres in the lungs compared to the vehicle control, the drug reduced virus-induced lung pathology to nearly the baseline scores of healthy animals. A substantial interstitial infiltration of neutrophils is observed in the lungs of treated (both infected and uninfected) animals. The protective effect of Nelfinavir on SARS-CoV-2-induced lung pathology (at doses that are well tolerated and that result in exposures nearing those observed in HIV-infected patients) may lay the foundation for clinical studies with this widely used drug.

sentences: Thermal cycling hyperthermia sensitizes non-small cell lung cancer A549 cells to EGFR-tyrosine kinase inhibitor Erlotinib Molecular-targeted therapy has emerged as a mainstream treatment for non-small cell lung cancer (NSCLC), the most common lung cancer, which has been the leading cause of cancer death for both men and women. Erlotinib (Erl), a targeted therapy drug which triggers epidermal growth factor receptor (EGFR) pathways, has been proved to have noticeable response rate for NSCLC cells. However, it has achieved only limited treatment effect, due to intrinsic and acquired resistance among most NSCLC patients. Therefore, chemosensitizers are required to potentiate the efficacy of Erl in NSCLC treatment. The study proposed a novel thermal therapy, thermal cycling hyperthermia (TC-HT), as a supplement to amplify the effect of Erl, and proved that it can sensitize A549 NSCLC cells to Erl via the downstream of EGFR signaling cascades. In addition, we found that TC-HT not only greatly enhanced the anticancer effect of Erl but also remarkably reduced the half-maximal inhibitory concentration (IC50) to as little as 0.5 M. Besides, via regulation of thermal dosage, TC-HT alone can produce excellent antineoplastic effect without hurting the normal cells. The method is expected to be applicable to other combination therapies and may be a starter for more sophisticated, side-effect-free anticancer treatments.

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Genomic surveillance of Bacillus cereus sensu lato strains isolated from meat and poultry products in South Africa enables inter- and intra-national surveillance and source tracking Members of the Bacillus cereus sensu lato (s.l.) species complex, also known as the B. cereus group, vary in their ability to cause illness, but are frequently isolated from foods, including meat products; however, food safety surveillance efforts that employ whole-genome sequencing (WGS) often neglect these potential pathogens. Here, WGS was used to characterize B. cereus s.l. strains (n = 25) isolated during surveillance of meat products in South Africa. Strains were collected from beef, poultry, and mixed meat products obtained from (i) retail outlets, processing plants, and butcheries across six South African provinces (n = 15, 7, and 1, respectively), and (ii) imports in cold stores (n = 2). Strains were assigned to panC Groups IV, III, II, and V (n = 18, 5, 1, and 1, respectively) and spanned multiple genomospecies, regardless of the taxonomy used. All strains possessed diarrheal toxin-encoding genes, while one sequence type 26 (ST26) strain possessed cereulide (emetic toxin) synthetase-encoding genes. No strains harbored anthrax toxin- or capsule-encoding genes. The 25 strains were partitioned into 15 lineages via in silico seven-gene multi-locus sequence typing (MLST), six of which contained multiple strains sequenced in this study, which were identical or nearly identical at the whole-genome scale. Five MLST lineages contained (nearly) identical genomes collected from two or three South African provinces; one MLST lineage contained nearly identical genomes from two countries (South Africa and the Netherlands), indicating that B. cereus s.l. can spread intra- and inter-nationally via foodstuffs.

ImportanceNation-wide foodborne pathogen surveillance programs that employ high-resolution genomic methods have been shown to provide vast public health and economic benefits. However, B. cereus s.l. are often overlooked during large-scale, routine WGS efforts. Thus, to our knowledge, no studies to date have evaluated the potential utility of WGS for B. cereus s.l. surveillance and source tracking in foodstuffs. In this proof-of-concept study, we applied WGS to B. cereus s.l. strains collected via South Africas national surveillance program of domestic and imported meat products, and we provide strong evidence that B. cereus s.l. can be disseminated intra- and inter-nationally via the agro-food supply chain. Our results showcase that WGS can be used for source tracking of B. cereus s.l. in foods, although future WGS and isolate metadata collection efforts are needed to ensure that B. cereus s.l. surveillance initiatives are on par with those of other foodborne pathogens.

sentences: Diversity of Lepidoptera in urban parks at Pasir Gudang, Johor, Malaysia Urban parks are a valuable habitat for Lepidoptera, which are often neglected despite their potential role as pollinators and important food source for small animals. Lepidoptera in urban parks in Pasir Gudang are still under-studied. Yet, there is no publication on Lepidoptera in these urban parks. The objectives of this study were to determine and to compare the diversity of Lepidoptera in three urban parks (Laman Rekreasi Sungai Buluh, Tasik Dahlia and Taman Bandar) at Pasir Gudang, Johor. Visual Encounter Survey (VES) method was used to capture the Lepidoptera from February 2021 until April 2021. Four sampling sessions were conducted for each study site. A total of nine species from 117 individuals comprised of family Pieridae (43%), Nymphalidae (29%) and Lycaenidae (28%) were recorded from this study. The most abundant species were Zizula hylax and Elymnias hypermnestra, while Leptosia nina was the least abundant. The results from this study showed that the diversity of Lepidoptera have established in these three urban parks. Diversity of Lepidoptera in Tasik Dahlia was significantly lower compared to the other two study sites. The findings from this study could be useful as a baseline data for future research and conservation management of the order Lepidoptera around the urban parks and indirectly can support the sustainable development of the urban parks.

sentences: Neural circuits for dynamics-based segmentation of time series The brain must extract behaviorally relevant latent variables from the signals streamed by the sensory organs. Such latent variables are often encoded in the dynamics that generated the signal rather than in the specific realization of the waveform. Therefore, one problem faced by the brain is to segment time series based on underlying dynamics. We present two algorithms for performing this segmentation task that are biologically plausible, which we define as acting in a streaming setting and all learning rules being local. One algorithm is model-based and can be derived from an optimization problem involving a mixture of autoregressive processes. This algorithm relies on feedback in the form of a prediction error, and can also be used for forecasting future samples. In some brain regions, such as the retina, the feedback connections necessary to use the prediction error for learning are absent. For this case, we propose a second, model-free algorithm that uses a running estimate of the autocorrelation structure of the signal to perform the segmentation. We show that both algorithms do well when tasked with segmenting signals drawn from autoregressive models with piecewise-constant parameters. In particular, the segmentation accuracy is similar to that obtained from oracle-like methods in which the ground-truth parameters of the autoregressive models are known. We also test our methods on datasets generated by alternating snippets of voice recordings. We provide implementations of our algorithms at https://github.com/ttesileanu/bio-time-series.

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Validation of non-invasive body-surface gastric mapping for detecting electrophysiological biomarkers by simultaneous high-resolution serosal mapping in a porcine model Gastric disorders are increasingly prevalent, but reliable clinical tools to objectively assess gastric function are lacking. Body-surface gastric mapping (BSGM) is a non-invasive method for the detection of gastric electrophysiological biomarkers including slow wave direction, which have correlated with symptoms in patients with gastroparesis and functional dyspepsia. However, no studies have validated the relationship between gastric slow waves and body surface activation profiles. This study aimed to comprehensively evaluate the relationship between gastric slow waves and body-surface recordings. High-resolution electrode arrays were placed to simultaneously capture slow waves from the gastric serosa (32x6 electrodes at 4 mm resolution) and abdominal surface (8x8 at 20 mm inter-electrode spacing) in a porcine model. BSGM signals were extracted based on a combination of wavelet and phase information analyses. A total of 1185 individual cycles of slow waves assessed, out of which 897 (76%) were normal antegrade waves, occurring in 10/14 (71%) subjects studied. BSGM accurately detected the underlying slow wave in terms of frequency (r = 0.99, p = 0.43) as well as the direction of propagation (p = 0.41, F-measure: 0.92). In addition, the cycle-by-cycle match between BSGM and transitions of gastric slow waves in terms either or both temporal and spatial abnormalities was demonstrated. These results validate BSGM as a suitable method for non-invasively and accurately detecting gastric slow wave activation profiles from the body surface.

Single sentence summarySimultaneous recordings of the stomach using serosal and body-surface electrode arrays demonstrated reliable detection of frequency and classification of propagation.

sentences: Predicting and Visualizing STK11 Mutation in Lung Adenocarcinoma Histopathology Slides Using Deep Learning Studies have shown that STK11 mutation plays a critical role in affecting the lung adenocarcinoma (LUAD) tumor immune environment. By training an Inception-Resnet-v2 deep convolutional neural network model, we were able to classify STK11-mutated and wild type LUAD tumor histopathology images with a promising accuracy (per slide AUROC=0.795). Dimensional reduction of the activation maps before the output layer of the test set images revealed that fewer immune cells were accumulated around cancer cells in STK11-mutation cases. Our study demonstrated that deep convolutional network model can automatically identify STK11 mutations based on histopathology slides and confirmed that the immune cell density was the main feature used by the model to distinguish STK11-mutated cases.

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Momelotinib is a highly potent inhibitor of FLT3-mutant AML Kinase activating mutation in FLT3 is the most frequent genetic lesion associated with poor prognosis in acute myeloid leukemia (AML). Therapeutic response to FLT3 tyrosine kinase inhibitor (TKI) therapy is dismal, and many patients relapse even after allogenic stem cell transplantation. Despite the introduction of more selective FLT3 inhibitors, remissions are short-lived, and patients show progressive disease after an initial response. Acquisition of resistance-conferring genetic mutations and growth factor signaling are two principal mechanisms that drive relapse. FLT3 inhibitors targeting both escape mechanisms could lead to a more profound and lasting clinical responses. Here we show that the JAK2 inhibitor, momelotinib, is an equipotent type-1 FLT3 inhibitor. Momelotinib showed potent inhibitory activity on both mouse and human cells expressing FLT3-ITD, including clinically relevant resistant mutations within the activation loop at residues, D835, D839, and Y842. Additionally, momelotinib efficiently suppressed the resistance mediated by FLT3 ligand (FL) and hematopoietic cytokine activated JAK2 signaling. Interestingly, unlike gilteritinib, momelotinib inhibits the expression of MYC in leukemic cells. Consequently, concomitant inhibition of FLT3 and downregulation of MYC by momelotinib treatment showed better efficacy in suppressing the leukemia in a preclinical murine model of AML. Altogether, these data provide evidence that momelotinib is an effective type-1 dual JAK2/FLT3 inhibitor and may offer an alternative to gilteritinib. Its ability to impede the resistance conferred by growth factor signaling and activation loop mutants suggests that momelotinib treatment could provide a deeper and durable response; thus, warrants its clinical evaluation.

Key pointsO_LIMomelotinib shows high efficacy against FLT3 mutated AML cells including quizartinib-resistant activation loop variants.
C_LIO_LIMomelotinib effectively suppresses intrinsic resistance conferred by FLT3 ligand and hematopoietic cytokines (GM-CSF and IL3).
C_LI

sentences: Harnessing protein folding neural networks for peptide-protein docking Highly accurate protein structure predictions by the recently published deep neural networks such as AlphaFold2 and RoseTTAFold are truly impressive achievements, and will have a tremendous impact far beyond structural biology. If peptide-protein binding can be seen as a final complementing step in the folding of a protein monomer, we reasoned that these approaches might be applicable to the modeling of such interactions. We present a simple implementation of AlphaFold2 to model the structure of peptide-protein interactions, enabled by linking the peptide sequence to the protein c-terminus via a poly glycine linker. We show on a large non-redundant set of 162 peptide-protein complexes that peptide-protein interactions can indeed be modeled accurately. Importantly, prediction is fast and works without multiple sequence alignment information for the peptide partner. We compare performance on a smaller, representative set to the state-of-the-art peptide docking protocol PIPER-FlexPepDock, and describe in detail specific examples that highlight advantages of the two approaches, pointing to possible further improvements and insights in the modeling of peptide-protein interactions. Peptide-mediated interactions play important regulatory roles in functional cells. Thus the present advance holds much promise for significant impact, by bringing into reach a wide range of peptide-protein complexes, and providing important starting points for detailed study and manipulation of many specific interactions.

sentences: Reflective mirror-based line-scan adaptive optics OCT Line-scan OCT, incorporated with adaptive optics (AO), offers high resolution, speed and sensitivity for imaging retinal structure and function in vivo. Here, we introduce its implementation with reflective mirror-based afocal telescopes, optimized for imaging light-induced retinal activity (optoretinography) and weak retinal reflections at the cellular scale. A non-planar optical design was followed based on previous recommendations with key differences specific to a line-scan geometry. The three beam paths fundamental to an OCT system - illumination/sample, detection, and reference - were modeled in Zemax optical design software to yield theoretically diffraction-limited performance over a 2.2 deg. field-of-view and 1.5 D vergence range at the eyes pupil. The performance for imaging retinal structure was exemplified by cellular-scale visualization of retinal ganglion cells, macrophages, foveal cones, and rods in human observers. The performance for functional imaging was exemplified by resolving the light-evoked optical changes in foveal cone photoreceptors where the spatial resolution was sufficient for cone spectral classification at an eccentricity 0.3 deg. from the foveal center. This enabled the first in vivo demonstration of reduced S-cone (short-wavelength cone) density in the human foveola, thus far observed only in ex vivo histological preparations. Together, the feasibility for high resolution imaging of retinal structure and function demonstrated here holds significant potential for basic science and translational applications.

sentences: Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles Rewired and synthetic signaling networks can impart cells with new functionalities and enable efforts in engineering cell therapies and directing cell development. However, there is a need for tools to build synthetic signaling networks that are tunable, can precisely regulate target gene expression, and are robust to perturbations within the complex context of mammalian cells. Here, we use proteins derived from bacterial two-component signaling pathways to develop synthetic phosphorylation-based and feedback-controlled devices in mammalian cells with such properties. First, we isolate kinase and phosphatase proteins from the bifunctional histidine kinase EnvZ. We then use these proteins to engineer a synthetic covalent modification cycle, in which the kinase and phosphatase competitively regulate phosphorylation of the cognate response regulator OmpR, enabling analog tuning of OmpR-driven gene expression. Further, we show that the phosphorylation cycle can be extended by connecting phosphatase expression to small molecule and miRNA inputs in the cell, with the latter enabling cell-type specific signaling responses and accurate cell type classification. Finally, we implement a tunable negative feedback controller by co-expressing the kinase-driven output gene with the small molecule-tunable phosphatase. This negative feedback substantially reduces cell-to-cell noise in output expression and mitigates the effects of cell context perturbations due to off-target regulation and resource competition. Our work thus lays the foundation for establishing tunable, precise, and robust control over cell behavior with synthetic signaling networks.

sentences: Transcriptional Inactivation of TP53 and the BMP Pathway Mediates Therapy-induced Dedifferentiation and Metastasis in Prostate Cancer Unsupervised clustering and deconvolution analysis identifies a novel subtype of M-CRPC endowed with hybrid epithelial/mesenchymal (E/M) and luminal progenitor-like traits (Mesenchymal and Stem-like PC, MSPC). Analysis of patient datasets and mechanistic studies indicate that MSPC arises as a consequence of therapy-induced lineage plasticity. AR blockade instigates two separate and complementary processes: 1) transcriptional silencing of TP53 and hence acquisition of hybrid E/M and stem-like traits; and 2) inhibition of the BMP signaling, which promotes resistance to the pro-apoptotic and anti-proliferative effects of AR inhibition. The drug-tolerant prostate cancer cells generated through reprogramming are rescued by neuregulin and generate metastases in mice. Combined inhibition of HER2/3 and AR or mTORC1 exhibit efficacy in preclinical models of mixed ARPC/MSPC or MSPC, respectively. These results identify a novel subtype of M-CRPC, trace its origin to therapy-induced lineage plasticity, and reveal its dependency on HER2/3 signaling.

sentences: scAllele: a versatile tool for the detection and analysis of variants in scRNA-seq Single-cell RNA sequencing (scRNA-seq) data contain rich information at the gene, transcript, and nucleotide levels. Most analyses of scRNA-seq have focused on gene expression profiles, and it remains challenging to extract nucleotide variants and isoform-specific information. Here, we present scAllele, an integrative approach that detects single nucleotide variants, insertions, deletions, and their allelic linkage with splicing patterns in scRNA-seq. We demonstrate that scAllele achieves better performance in identifying nucleotide variants than other commonly used tools. In addition, the read-specific variant calls by scAllele enables allele-specific splicing analysis, a unique feature not afforded by other methods. Applied to a lung cancer scRNA-seq data set, scAllele identified variants with strong allelic linkage to alternative splicing, some of which being cancer-specific. scAllele represents a versatile tool to uncover multi-layer information and novel biological insights from scRNA-seq data.

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Distant water industrial fishing in developing countries: A case study of Madagascar As industrial vessels continue to expand in both extractive capacity and spatial range, concerns have grown over foreign industrial fishing occurring within the marine territories of developing countries, both legally and illegally. Madagascars status as a "least developed country", coupled with its high marine biodiversity, makes its waters particularly susceptible to fishing by distant water fishing nations (DWFNs). However, given constraints in management and research, it is unclear how foreign industrial fishing, both legal via foreign agreements and illegal, may impact local marine resources that many coastal communities depend on for food security, cultural meaning and livelihoods. We used satellite-derived fishing effort data from 2012-2020, via Global Fishing Watch, to analyze industrial fishing effort occurring within Madagascars Exclusive Economic Zone (EEZ). We documented 907,643 hours of industrial fishing within the Madagascar EEZ across 277 vessels from 17 different countries. We found that Taiwanease vessels (39.8%) using drifting longlines and Malagasy (17.2% shrimp trawlers were the most prevalent. Fishing effort was highly seasonal (68% of effort between October and February) and increased with higher global fish prices and the Indian Ocean Dipole, which is a measure of regional water temperature cycles. We also found a number of instances (17.6% of the fishing effort for 170,726 total hours) of foreign fishing vessels operating close to shore and within a number of marine protected areas. These results highlight the need for increased transparency surrounding foreign fishing agreements and unauthorized fishing within the waters of developing countries. Increases in industrial fishing effort and encroachment into near-shore areas has the potential to severely threaten current sustainable fisheries management initiatives by conservation organizations and coastal communities.

HighlightsO_LIDistant water fishing nations dominated fishing efforts within Madagascars EEZ.
C_LIO_LILonglining by foreign nations was the dominant fishing mode and increased from October-February.
C_LIO_LIMalagasy vessels focused on trawling for shrimp.
C_LIO_LIFishing effort increased during positive Indian Ocean Dipoles and with higher fish prices.
C_LIO_LIDistant water fishing nations frequently fished close to shore and sometimes within MPAs.
C_LI

sentences: Differential antibody dynamics to SARS-CoV-2 infection and vaccination Optimal immune responses furnish long-lasting (durable) antibodies protective across dynamically mutating viral variants (broad). To assess robustness of mRNA vaccine-induced immunity, we compared antibody durability and breadth after SARS-CoV-2 infection and vaccination. While vaccination delivered robust initial virus-specific antibodies with some cross-variant coverage, pre-variant SARS-CoV-2 infection-induced antibodies, while modest in magnitude, showed highly stable long-term antibody dynamics. Vaccination after infection induced maximal antibody magnitudes with enhanced longitudinal stability while infection-naive vaccinee antibodies fell with time to post-infection-alone levels. The composition of antibody neutralizing activity to variant relative to original virus also differed between groups, with infection-induced antibodies demonstrating greater relative breadth. Differential antibody durability trajectories favored COVID-19-recovered subjects with dual memory B cell features of greater early antibody somatic mutation and cross-coronavirus reactivity. By illuminating an infection-mediated antibody breadth advantage and an anti-SARS-CoV-2 antibody durability-enhancing function conferred by recalled immunity, these findings may serve as guides for ongoing vaccine strategy improvement.

sentences: scPNMF: sparse gene encoding of single cells to facilitate gene selection for targeted gene profiling Single-cell RNA sequencing (scRNA-seq) captures whole transcriptome information of individual cells. While scRNA-seq measures thousands of genes, researchers are often interested in only dozens to hundreds of genes for a closer study. Then a question is how to select those informative genes from scRNA-seq data. Moreover, single-cell targeted gene profiling technologies are gaining popularity for their low costs, high sensitivity, and extra (e.g., spatial) information; however, they typically can only measure up to a few hundred genes. Then another challenging question is how to select genes for targeted gene profiling based on existing scRNA-seq data. Here we develop the single-cell Projective Non-negative Matrix Factorization (scPNMF) method to select informative genes from scRNA-seq data in an unsupervised way. Compared with existing gene selection methods, scPNMF has two advantages. First, its selected informative genes can better distinguish cell types. Second, it enables the alignment of new targeted gene profiling data with reference data in a low-dimensional space to facilitate the prediction of cell types in the new data. Technically, scPNMF modifies the PNMF algorithm for gene selection by changing the initialization and adding a basis selection step, which selects informative bases to distinguish cell types. We demonstrate that scPNMF outperforms the state-of-the-art gene selection methods on diverse scRNA-seq datasets. Moreover, we show that scPNMF can guide the design of targeted gene profiling experiments and cell-type annotation on targeted gene profiling data.

sentences: Adrenergic Attenuation of Immune Activity is Associated with Anhedonia in Adolescents While inflammation has been implicated in depression, little is known of immune-inhibitory agents for individuals with depression. This study sought to assess whether {beta}2-agonist clenbuterol (CBL) would attenuate increased cytokine secretion in adolescents with mood and anxiety symptoms following ex vivo exposure of whole blood to lipopolysaccharide (LPS). Our focus on adolescents aimed to target a critical developmental period when psychiatric conditions often emerge and prior to chronicity effects. Participants (15.25 {+/-} 2.16 years old, 59% female) were 97 psychotropic-medication free adolescents with mood and anxiety symptoms and 33 healthy controls. All had comprehensive evaluations and dimensional assessments of psychiatric symptoms. Fasting whole-blood samples were collected and stimulated with LPS in the presence and absence of CBL for 6 hours and analyzed for 41 cytokines. Comparison analyses used Bonferroni-corrected nonparametric tests. Exploratory factor analysis reduced 41 cytokines into 5 cytokine factors in each experimental condition, and their relationships with psychiatric symptoms were examined. Levels of nine cytokines were significantly reduced by CBL treatment compared to LPS alone. A cytokine factor in the LPS+CBL condition significantly correlated with anticipatory (rho = -0.39, p = 7.4 x 10-5) and consummatory anhedonia (rho = -0.36, p = 3.3 x 10-4), which remained significant when controlling for depression. This study supports the possible inhibitory effect of CBL on immune activation. Using a data-driven method, distinctive relationships between CBL-affected cytokines and dimensional anhedonia were reported, further elucidating the role of {beta}2-agonism in adolescent affective symptomatology.

sentences: Histone deacetylase 1 maintains lineage integrity through histone acetylome refinement during early embryogenesis Histone acetylation is a pivotal epigenetic modification that controls chromatin structure and regulates gene expression. It plays an essential role in modulating zygotic transcription and cell lineage specification of developing embryos. While the outcomes of many inductive signals have been described to require enzymatic activities of histone acetyltransferases and deacetylases (HDACs), the mechanisms by which HDACs confine the utilization of the zygotic genome remain to be elucidated. Here, we show that histone deacetylase 1 (Hdac1) progressively binds to the zygotic genome from mid blastula and onward. The recruitment of Hdac1 to the genome at blastula is instructed maternally. Cis-regulatory modules (CRMs) bound by Hdac1 possess epigenetic signatures underlying distinct functions. We highlight a dual function model of Hdac1 where Hdac1 not only represses gene expression by sustaining a histone hypoacetylation state on inactive chromatin, but also maintains gene expression through participating in dynamic histone acetylation-deacetylation cycles on active chromatin. As a result, Hdac1 maintains differential histone acetylation states of bound CRMs between different germ layers and reinforces the transcriptional program underlying cell lineage identities, both in time and space. Taken together, our study reveals a comprehensive role for Hdac1 during early vertebrate embryogenesis.

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MYC drives aggressive prostate cancer by disrupting transcriptional pause release at androgen receptor targets c-MYC (MYC) is a major driver of prostate cancer tumorigenesis and progression. Although MYC is overexpressed in both early and metastatic disease and associated with poor survival, its impact on prostate transcriptional reprogramming remains elusive. We demonstrate that MYC overexpression significantly diminishes the androgen receptor (AR) transcriptional program (the set of genes directly targeted by the AR protein) in luminal prostate cells without altering AR expression. Importantly, analyses of clinical specimens revealed that concurrent low AR and high MYC transcriptional programs accelerate prostate cancer progression toward a metastatic, castration-resistant disease. Data integration of single-cell transcriptomics together with ChIP-seq revealed an increased RNA polymerase II (Pol II) promoter-proximal pausing at AR-dependent genes following MYC overexpression without an accompanying deactivation of AR-bound enhancers. Altogether, our findings suggest that MYC overexpression antagonizes the canonical AR transcriptional program and contributes to prostate tumor initiation and progression by disrupting transcriptional pause release at AR-regulated genes.

STATEMENT OF SIGNIFICANCEAR and MYC are key to prostate cancer etiology but our current understanding of their interplay is scarce. Here we show that the oncogenic transcription factor MYC can pause the transcriptional program of the master transcription factor in prostate cancer, AR, while turning on its own, even more lethal program.

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The mRNA-LNP platform's lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory Vaccines based on mRNA-containing lipid nanoparticles (LNPs) are a promising new platform used by two leading vaccines against coronavirus disease in 2019 (COVID-19). Clinical trials and ongoing vaccinations present with very high protection levels and varying degrees of side effects. However, the nature of the reported side effects remains poorly defined. Here we present evidence that LNPs used in many preclinical studies are highly inflammatory in mice. Intradermal injection of these LNPs led to rapid and robust inflammatory responses, characterized by massive neutrophil infiltration, activation of diverse inflammatory pathways, and production of various inflammatory cytokines and chemokines. The same dose of LNP delivered intranasally led to similar inflammatory responses in the lung and resulted in a high mortality rate.

In summary, here we show that the LNPs used for many preclinical studies are highly inflammatory. Thus, their potent adjuvant activity and reported superiority comparing to other adjuvants in supporting the induction of adaptive immune responses likely stem from their inflammatory nature. Furthermore, the preclinical LNPs are similar to the ones used for human vaccines, which could also explain the observed side effects in humans using this platform.

sentences: Triple Negative Breast Cancer-derived Small Extracellular Vesicles as Modulator of Biomechanics in target cells Extracellular vesicle (EV) mediated communication has recently been proposed as one of the pivotal routes in the development of cancer metastasis. EVs are nano-sized vesicles swapped between cells, carrying a biologically active content that can promote tumor-induced immune suppression, metastasis and angiogenesis. Thus, EVs constitute a potential target in cancer therapy. However, their role in triggering the premetastatic niche and in tumor spreading is still unclear. Here, we focused on the EV ability to modulate the biomechanical properties of target cells, known to play a crucial role in metastatic spreading. To this purpose, we isolated and thoroughly characterized triple-negative breast cancer (TNBC)-derived small EVs. We then evaluated variations in the mechanical properties (cell stiffness, cytoskeleton/nuclear/morphology and Yap activity rearrangements) of non-metastatic breast cancer MCF7 cells upon EV treatment. Our results suggest that TNBC-derived small EVs are able to directly modify MCF7 cells by inducing a decrease in cell stiffness, rearrangements in cytoskeleton, focal adhesions and nuclear/cellular morphology, and an increase in Yap downstream gene expression. Testing the biomechanical response of cells after EV addition might represent a new functional assay in metastatic cancer framework that can be exploited for future application both in diagnosis and in therapy.

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Structurally Constrained Effective Brain Connectivity The relationship between structure and function is of interest in many research fields involving the study of complex biological processes. In neuroscience in particular, the fusion of structural and functional data can help understanding the underlying principles of the operational networks in the brain. To address this issue, this paper proposes a constrained autoregressive model leading to a representation of "effective" connectivity that can be used to better understand how the structure modulates the function. Or simply, it can be used to find novel biomarkers characterizing groups of subjects. In practice, an initial structural connectivity representation is re-weighted to explain the functional co-activations. This is obtained by minimizing the reconstruction error of an autoregressive model constrained by the structural connectivity prior. The model has been designed to also include indirect connections, allowing to split direct and indirect components in the functional connectivity, and it can be used with raw and deconvoluted BOLD signal.

The derived representation of dependencies was compared to the well known dynamic causal model, giving results closer to known ground-truth. Further evaluation of the proposed effective network was performed on two typical tasks. In a first experiment the direct functional dependencies were tested on a community detection problem, where the brain was partitioned using the effective networks across multiple subjects. In a second experiment the model was validated in a case-control task, which aimed at differentiating healthy subjects from individuals with autism spectrum disorder. Results showed that using effective connectivity leads to clusters better describing the functional interactions in the community detection task, while maintaining the original structural organization, and obtaining a better discrimination in the case-control classification task.

HighlightsO_LIA method to combine structural and functional connectivity by using autoregressive model is proposed.
C_LIO_LIThe autoregressive model is constrained by structural connectivity defining coefficients for Granger causality.
C_LIO_LIThe usefulness of the generated effective connections is tested on simulations, ground-truth default mode network experiments, a classification and clustering task.
C_LIO_LIThe method can be used for direct and indirect connections, and with raw and deconvoluted BOLD signal.
C_LI

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A Robust and Scalable Graph Neural Network for Accurate Single Cell Classification MotivationSingle-cell RNA sequencing (scRNA-seq) techniques provide high-resolution data on cellular heterogeneity in diverse tissues, and a critical step for the data analysis is cell type identification. Traditional methods usually cluster the cells and manually identify cell clusters through marker genes, which is time-consuming and subjective. With the launch of several large-scale single-cell projects, millions of sequenced cells have been annotated and it is promising to transfer labels from the annotated datasets to newly generated datasets. One powerful way for the transferring is to learn cell relations through the graph neural network (GNN), while vanilla GNN is difficult to process millions of cells due to the expensive costs of the message-passing procedure at each training epoch.

ResultsHere, we have developed a robust and scalable GNN-based method for accurate single cell classification (GraphCS), where the graph is constructed to connect similar cells within and between labelled and unlabelled scRNA-seq datasets for propagation of shared information. To overcome the slow information propagation of GNN at each training epoch, the diffused information is pre-calculated via the approximate Generalized PageRank algorithm, enabling sublinear complexity for a high speed and scalability on millions of cells. Compared with existing methods, GraphCS demonstrates better performance on simulated, cross-platform, and cross-species scRNA-seq datasets. More importantly, our model can achieve superior performance on a large dataset with one million cells within 50 minutes.

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EGR2 promoter antisense RNA coordinates chromatin remodeling and spatial genome organization in Schwann cells. The EGR2 promoter antisense RNA (AS-RNA) recruits chromatin remodeling complexes to inhibit EGR2 transcription following peripheral nerve injury. Here we show that the EGR2-AS-RNA modulates chromatin accessibility and serves as scaffold for two distinct histone modification complexes. It binds to EZH2 and WDR5 and enables coordinate targeting of H3K27me3 and H3K4me3 to promoters of EGR2and C-JUN. Expression of the AS-RNA results in reorganization of the global chromatin landscape and quantitative changes in loop formation and in contact frequency at domain boundaries exhibiting enrichment for AP-1 genes. In addition, the EGR2-AS-RNA induces changes in hierarchical TADs and increases transcriptional activity on an inter-TAD loop between a super-enhancer regulatory hub and the promoter of mTOR. Our results show that the EGR2-AS-RNA may serve as regulator of coordinate chromatin remodeling and spatial genome organization to regulate cellular plasticity-associated transcription in Schwann cells.

Highlights- Overexpression of the EGR2-AS-RNA regulates gene expression in Schwann Cells
- The EGR2-AS-RNA binds and recruits repressive and activating chromatin modifiers on the EGR2 and C-JUN promoters, repsectively
- ATAC-seq data showed an increase in the AP-1 network accessibility/TF activity
- The EGR2-AS-RNA promotes reorganization of the global chromatin landscape, quantitative changes in loop formation and in contact frequency at domain boundaries exhibiting enrichment for AP-1 genes

sentences: Legacy effect of constant and diurnally oscillating temperatures on soil respiration and microbial community structure 1.0Laboratory incubation studies evaluating the temperature sensitivity of soil respiration often use measurements of respiration taken at a constant incubation temperature from soil that has been pre-incubated at the same constant temperature. However, such constant temperature incubations do not represent the field situation where soils undergo diurnal temperature oscillations. We investigated the effects of constant and diurnally oscillating temperatures on soil respiration and soil microbial community composition. A grassland soil from the UK was either incubated at a constant temperature of 5 {degrees}C, 10 {degrees}C, or 15 {degrees}C, or diurnally oscillated between 5 {degrees}C and 15 {degrees}C. Soil CO2 flux was measured by temporarily moving incubated soils from each of the abovementioned treatments to 5 {degrees}C, 10 {degrees}C or 15 {degrees}C, such that soils incubated at each temperature had CO2 flux measured at every temperature. We hypothesised that, irrespective of measurement temperature, CO2 emitted from the 5 {degrees}C to 15 {degrees}C oscillating incubation would be most similar to the soil incubated at 10 {degrees}C. The results showed that both incubation and measurement temperatures influence soil respiration. Incubating soil at a temperature oscillating between 5 {degrees}C and 15 {degrees}C resulted in significantly greater CO2 flux than constant incubations at 10 {degrees}C or 5 {degrees}C, but was not significantly different to the 15 {degrees}C incubation. The greater CO2 flux from soils incubated at 15 {degrees}C, or oscillating between 5 {degrees}C and 15 {degrees}C, coincided with a depletion of dissolved organic carbon and a shift in the phospholipid fatty acid profile of the soil microbial community, consistent with the thermal adaptation of microbial communities to higher temperatures. However, diurnal temperature oscillation did not significantly alter Q10. Our results suggest that daily maximum temperatures are more important than daily minimum or daily average temperatures when considering the response of soil respiration to warming.

sentences: Intracellular Oxidative Stress Levels are Significantly Associated with the Green Autofluorescence Intensity of Buthionine Sulfoximine-Treated B16-F10 Cells Since oxidative stress is a critical common pathological factor of numerous diseases, it is critical to find biomarkers for non-invasive evaluations of the levels of oxidative stress in the body. Our previous studies have indicated that epidermal green autofluorescence (AF) is a novel biomarker of this type: The oxidative stress inducer buthionine sulfoximine (BSO) can dose-dependently increase the epidermal green AF of mice, with BSO doses being significantly associated with the AF intensity. However, it is necessary to use skin cell cultures to investigate the mechanisms underlying the relationships between BSO and the green AF intensity. In our current study we found that BSO concentration-dependently increased the green AF intensity of B16-F10 cells a skin cell line, with BSO concentrations being significantly associated with the AF intensity. BSO also concentration-dependently increased the intracellular DCF signals an index of ROS levels. The green AF intensity of the cells was also significantly associated with the intracellular ROS levels. Moreover, we found that the green AF intensity was significantly associated with the cell death induced by BSO. Collectively, our study has provided first evidence indicating that the green AF intensity of skin cells is significantly associated with both intracellular ROS levels and cell death of the skin cells exposed to oxidative stress, which has indicated that green AF is a novel biomarker for both oxidative stress and cell death.

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Shade suppresses wound-induced leaf repositioning through a mechanism involving PHYTOCHROME KINASE SUBSTRATE (PKS) genes Shaded plants challenged with herbivores or pathogens prioritize growth over defense. However, most experiments have focused on the effect of shading light cues on defense responses. To investigate the potential interaction between shade-avoidance and wounding-induced Jasmonate (JA)-mediated signaling on leaf growth and movement, we used repetitive mechanical wounding of leaf blades to mimic herbivore attacks. Phenotyping experiments with combined treatments on Arabidopsis thaliana rosettes revealed that shade strongly inhibits the wound effect on leaf elevation. By contrast, petiole length is reduced by wounding both in the sun and in the shade. Thus, the relationship between the shade and wounding/JA pathways varies depending on the physiological response, implying that leaf growth and movement can be uncoupled. Using RNA-sequencing, we identified genes with expression patterns matching the hyponastic response (opposite regulation by both stimuli, interaction between treatments with shade dominating the wound signal). Among them were genes from the PKS (Phytochrome Kinase Substrate) family, which was previously studied for its role in phototropism and leaf positioning. Interestingly, we observed reduced shade suppression of the wounding effect in pks2pks4 double mutants while a PKS4 overexpressing line showed constitutively elevated leaves and was less sensitive to wounding. Our results indicate a trait-specific interrelationship between shade and wounding cues on Arabidopsis leaf growth and positioning. Moreover, we identify PKS genes as integrators of external cues in the control of leaf hyponasty further emphasizing the role of these genes in aerial organ positioning.

Author summaryPlants face different types of stressful situations without the ability to relocate to favorable environments. For example, increasing plant density reduces access to sunlight as plants start to shade each other. Foliar shading represents a stress that many plants cope with by changing their morphology. This includes elongation of stem-like structures and repositioning of leaves to favor access to unfiltered sunlight. Plants also defend themselves against various pathogens including herbivores. Defense mechanisms include the production of deterrent chemical and morphological adaptations such as stunted growth and downwards leaf repositioning. Here we studied the morphological response of plants when simultaneously facing shade and herbivore stress. When facing both stresses petiole growth was intermediate between the shade-enhanced and wound-repressed response. In contrast, the shade cue overrides the wounding cue leading to a similar upwards leaf repositioning in the combined treatments or in the response to shade alone. Using gene expression analyses and genetics we identified two members of the Phytochrome Kinase Substrate family as playing a signal integration role when plants simultaneously faced both stresses. This contributes to our understanding of the mechanisms underlying plant morphological adaptations when facing multiple stresses.

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Human photoreceptors switch from autonomous axon extension to cell-mediated process pulling during synaptic marker redistribution. Photoreceptors (PRs) are the primary visual sensory cells, and their loss leads to blindness that is currently incurable. Cell replacement therapy holds promise as a therapeutic approach to restore vision to those who have lost PRs through damage or disease. While PR transplant research is ongoing in animal models, success is hindered by our limited understanding of PR axon growth during development and regeneration. Using a human pluripotent stem cell (hPSC) reporter line that labels PRs (WA09 CRX+/tdTomato), we generated retinal organoids in order to study mechanisms of PR process extension. We found that the earliest born PRs exhibit autonomous axon extension from dynamic terminals that appear similar to projection neuron growth cones. However, as hPSC-derived PRs age from 40 to 80 days of differentiation, they lose dynamic terminals in 2D plated cultures and within 3D retinal organoids, which does not correlate with cell birth date. Using a rod-specific hPSC reporter line (WA09 NRL+/eGFP), we further determined that rod PRs never form motile growth cones. Interestingly, PRs without motile terminals are still capable of extending axons, but neurites are generated from process stretching via their attachment to motile non-PR cells, which underlies the observed differences in PR neurite lengths on different substrata. While immobile PR terminals express actin, it is less polymerized and less organized than actin present in motile terminals. However, immobile PRs do localize synaptic proteins to their terminals, suggesting a normal developmental progression. These findings help inform the development of PR transplant therapies to treat blinding diseases and provide a platform to test treatments that restore autonomous PR axon extension.

Significance StatementLoss of photoreceptors (PRs) in the retina through damage or disease causes irreversible vision loss and blindness. One treatment approach is to replace lost cells with transplanted human stem cell-derived PRs, but this requires PR axons to integrate into the host retina to restore the required neural connections. For this strategy to succeed, we need to understand how PRs extend processes to their targets during development in situ, and whether dissociated human stem cell (hPSC)-derived PRs behave in a similar fashion. In this paper, we show that hPSC-PRs have only a short window during which they are capable of autonomous axon extension, which has implications for PR transplant efforts and for our basic understanding of human retinal development.

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BWA-mem is not the best aligner for ancient DNA short reads. Xu and colleagues (Xu et al., 2021) recently suggested a new parameterisation of BWA-mem (Li, 2013) as an alternative to the current standard BWA-aln (Li and Durbin, 2009) to process ancient DNA sequencing data. The authors tested several combinations of the -k and -r parameters to optimise BWA-mems performance with degraded and contaminated ancient DNA samples. They report that using BWA-mem with -k 19 -r 2.5 parameters results in a mapping efficiency comparable to BWA-aln with -I 1024 -n 0.03 (i.e. a derivation of the standard parameters used in ancient DNA studies; (Schubert et al., 2012)), while achieving significantly faster run times.

We recently performed a systematic benchmark of four mapping software (i.e. BWA-aln, BWA-mem, NovoAlign (http://www.novocraft.com/products/novoalign), and Bowtie2 (Langmead and Salzberg, 2012) for ancient DNA sequencing data and quantified their precision, accuracy, specificity, and impact on reference bias (Oliva et al., 2021). Notably, while multiple parameterisations were tested for BWA-aln, NovoAlign, and Bowtie2, we only tested BWA-mem with default parameters.

Here, we use the alignment performance metrics from Oliva et al. to directly compare the recommended BWA-mem parameterisation reported in Xu et al. with the best performing alignment methods determined in the Oliva et al. benchmarks, and we make recommendations based on the results.

sentences: Candidatus Nitrosopolaris, a genus of putative ammonia-oxidizing archaea with a polar/alpine distribution Ammonia-oxidizing archaea (AOA) are key players in the nitrogen cycle. Here, we report four novel metagenome-assembled genomes (MAGs) assigned to the genus "UBA10452", an uncultured lineage of putative AOA in the family Nitrososphaeraceae. Analysis of other eight previously reported MAGs and publicly available amplicon sequencing data revealed that the UBA10452 lineage is predominantly found in acidic polar and alpine soils. We propose a novel Candidatus genus, Ca. Nitrosopolaris, with four species representing clear biogeographical/habitat clusters. In addition to the presence of genes involved in cold adaptation, we hypothesize that the distribution of Ca. Nitrosopolaris across the cold biosphere might also be due to the combination of a polar origin and limited dispersal capabilities throughout geological time.

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Genome instability drives epistatic adaptation in the human pathogen Leishmania How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches we provide first evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover post-transcriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in snoRNA gene dosage with changes in rRNA 2-O-methylation and pseudouridylation, suggesting translational control is an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression, and genome-independent, compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery, and may be relevant to other fast evolving, eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.

One Sentence SummaryEpistatic interactions harness genome instability for Leishmania fitness gain.

sentences: Characterisation and chemometric evaluation of 17 elements in ten seaweed species from Greenland Several Greenland seaweed species have potential as foods or food ingredients, both for local consumption and export. However, knowledge regarding their content of beneficial and deleterious elements on a species specific and geographical basis is lacking. This study investigated the content of 17 elements (As, Ca, Cd, Cr, Cu, Fe, Hg, I, K, Mg, Mn, Na, Ni, P, Pb, Se and Zn) in 77 samples of ten species (Agarum clathratum, Alaria esculenta, Ascophyllum nodosum, Fucus distichus, Fucus vesiculosus, Hedophyllum nigripes, Laminaria solidungula, Palmaria palmata, Saccharina latissima and Saccharina longicruris). Element profiles differed between species but showed similar patterns within the same family. For five species, different thallus parts were investigated separately, and showed different element profiles. A geographic origin comparison of Fucus species indicated regional differences. The seaweeds investigated were especially good sources of macrominerals (K > Na > Ca > Mg) and trace minerals, such as Fe. Iodine contents were high, especially in macroalgae of the family Laminariaceae. None of the samples exceeded the EU maximum levels for Cd, Hg or Pb, but some exceeded the stricter French regulations, especially for Cd and I. In conclusion, these ten species are promising food items.

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cageminer: an R/Bioconductor package to prioritize candidate genes by integrating GWAS and gene coexpression networks SummaryAlthough genome-wide association studies (GWAS) identify variants associated with traits of interest, they often fail in identifying causative genes underlying a given phenotype. Integrating GWAS and gene coexpression networks can help prioritize high-confidence candidate genes, as the expression profiles of trait-associated genes can be used to mine novel candidates. Here, we present cageminer, the first R package to prioritize candidate genes through the integration of GWAS and coexpression networks. Genes are considered high-confidence candidates if they pass all three filtering criteria implemented in cageminer, namely physical proximity to SNPs, coexpression with known trait-associated genes, and significant changes in expression levels in conditions of interest. Prioritized candidates can also be scored and ranked to select targets for experimental validation. By applying cageminer to a real data set, we demonstrate that it can effectively prioritize candidates, leading to >99% reductions in candidate gene lists.

Availability and implementationThe package is available at Bioconductor (http://bioconductor.org/packages/cageminer).

sentences: Circulating Low Density Neutrophils of Breast Cancer Patients are Associated with their Worse Prognosis due to the Impairment of T cell Responses Neutrophils are prominent immune components of solid tumors, which can protect against the onset of cancer (N1) or have pro-tumor activity (N2). Circulating neutrophils, divided into high density neutrophils (HDN) and low density neutrophils (LDN), functionally mirror those N1 and N2 cells, respectively. LDN, a rare subset in non-pathological conditions, have been extensively studied in cancer, due to their frequency in this disease and their pro-tumor phenotype. However, this has been mainly demonstrated in animal models and proper validation in humans is an urgent need. Here, we further enlighten the clinical impact of LDN in a cohort of breast cancer (BC) patients. We observed that LDN were practically absent in healthy donors blood, while were significantly increased in the blood of BC patients, particularly with metastatic disease. Relevant for a clinical translation, within the population of non-metastatic patients, LDN were more prevalent in patients with poor response to neoadjuvant chemotherapy than in responders. The association of a higher incidence of circulating LDN and the worse prognosis of BC patients could be explained by the pro-tumor/immunosuppressive characteristics exhibited by these cells. Namely, there are more LDN expressing the immunosuppressive marker PD-L1, than HDN. Additionally, LDN also showed increased expression of activation markers; a robust formation of neutrophil extracellular traps; an augmented phagocytic activity; and a higher capacity to release reactive oxygen species, which may contribute for tumor development and metastization. Moreover, the percentage of LDN in BC patients blood was negatively correlated with activated cytotoxic T lymphocytes and positively correlated with the immunosuppressive CCR4+ regulatory T cells, corroborating their impairment on the anti-tumor immune responses, which was further demonstrated ex vivo. Hence, this study reveals the potential of LDN as a clinical meaningful biomarker of BC response to treatment and opens new avenues for developing targeted immunotherapies.

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Characterization of SARS-CoV-2 public CD4+ αβ T cell clonotypes through reverse epitope discovery The amount of scientific data and level of public sharing produced as a consequence of the COVID-19 pandemic, as well as the speed at which these data were produced, far exceeds any previous effort against a specific disease condition. This unprecedented situation allows for development and application of new research approaches. One of the major technical hurdles in immunology is the characterization of HLA-antigen-T cell receptor (TCR) specificities. Most approaches aim to identify reactive T cells starting from known antigens using functional assays. However, the need for a reverse approach identifying the antigen specificity of orphan TCRs is increasing.

Utilizing large public single-cell gene expression and TCR datasets, we identified highly public CD4+ T cell responses to SARS-CoV-2, covering >75% of the analysed population. We performed an integrative meta-analysis to deeply characterize these clonotypes by TCR sequence, gene expression, HLA-restriction, and antigen-specificity, identifying strong and public CD4+ immunodominant responses with confirmed specificity. CD4+ COVID-enriched clonotypes show T follicular helper functional features, while clonotypes depleted in SARS-CoV-2 individuals preferentially had a central memory phenotype. In total we identify more than 1200 highly public CD4+ T cell clonotypes reactive to SARS-CoV-2. TCR similarity analysis showed six prominent TCR clusters, for which we predicted both HLA-restriction and cognate SARS-CoV-2 immunodominant epitopes. To validate our predictions we used an independent cohort of TCR repertoires before and after vaccination with ChAdOx1, a replication-deficient simian adenovirus-vectored vaccine, encoding the SARS-CoV-2 spike protein. We find statistically significant enrichment of the predicted spike-reactive TCRs after vaccination with ChAdOx1, while the frequency of TCRs specific to other SARS-CoV-2 proteins remains stable. Thus, the CD4-associated TCR repertoire differentiates vaccination from natural infection.

In conclusion, our study presents a novel reverse epitope discovery approach that can be used to infer HLA- and antigen-specificity of orphan TCRs in any context, such as viral infections, antitumor immune responses, or autoimmune disease.

HighlightsO_LIIdentification of highly public CD4+ T cell responses to SARS-CoV-2
C_LIO_LISystematic prediction of exact immunogenic HLA class II epitopes for CD4+ T cell response
C_LIO_LIMethodological framework for reverse epitope discovery, which can be applied to other disease contexts and may provide essential insights for future studies and clinical applications
C_LI

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: Computational Modeling: Human Dynamic Model Improvements in quantitative measurements of human physical activity are proving extraordinarily useful for studying the underlying musculoskeletal system. Dynamic models of human movement support clinical efforts to analyze, rehabilitate injuries. They are also used in biomechanics to understand and diagnose motor pathologies, find new motor strategies that decrease the risk of injury, and predict potential problems from a particular procedure. In addition, they provide useful constraints for underlying neural circuits. This paper describes a physics-based movement analysis method for analyzing and simulating bipedal humanoid movements. A 48 degree of freedom dynamic model of humans has been developed to report humanoid movements energetic components. It has sufficient speed and accuracy to analyze and synthesize real-time interactive applications, such as psychophysics experiments using virtual reality or human-in-the-loop teleoperation of a simulated robotic system. The dynamic model is fast and robust while still providing results sufficiently accurate to be used to believably animate a humanoid character or estimate internal joint forces used during a movement for creating effort-contingent experimental stimuli. A virtual reality environment developed as part of this research supports controlled experiments for systematically recording human behaviors.

sentences: Structural and computational insights into the SARS-CoV-2 Omicron RBD-ACE2 interaction Since SARS-CoV-2 Omicron variant (B.1.1.529) was reported in November 2021, it has quickly spread to many countries and outcompeted the globally dominant Delta variant in several countries. The Omicron variant contains the largest number of mutations to date, with 32 mutations located at spike (S) glycoprotein, which raised great concern for its enhanced viral fitness and immune escape[1-4]. In this study, we reported the crystal structure of the receptor binding domain (RBD) of Omicron variant S glycoprotein bound to human ACE2 at a resolution of 2.6 [A]. Structural comparison, molecular dynamics simulation and binding free energy calculation collectively identified four key mutations (S477N, G496S, Q498R and N501Y) for the enhanced binding of ACE2 by the Omicron RBD compared to the WT RBD. Representative states of the WT and Omicron RBD-ACE2 systems were identified by Markov State Model, which provides a dynamic explanation for the enhanced binding of Omicron RBD. The effects of the mutations in the RBD for antibody recognition were analyzed, especially for the S371L/S373P/S375F substitutions significantly changing the local conformation of the residing loop to deactivate several class IV neutralizing antibodies.

sentences: Neural signatures of compression in the retelling of past events When we retell our past experiences, we aim to reproduce some version of the original events; this reproduced version is often temporally compressed relative to the original. How does such compression of memories manifest in brain activity? One possibility is that a compressed retrieved memory manifests as a neural pattern which is more dissimilar to the original, relative to a more detailed or vivid memory. However, we argue that measuring raw dissimilarity alone is insufficient, as it confuses a variety of interesting and uninteresting changes. To address this problem, we examine brain pattern changes that are consistent across people. We show that temporal compression in individuals retelling of past events predicts systematic encoding-to-recall transformations in a number of higher associative regions. These findings elucidate how neural representations are not simply reactivated, but can also be transformed due to temporal compression during a universal form of human memory expression: verbal retelling.

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Comparison of Mucosal and Intramuscular Immunization against SARS-CoV-2 with Replication-Defective and Replicating Single-cycle Adenovirus Vaccines SARS-CoV-2 enters the body at mucosal surfaces, such as the nose and lungs. These events involve a small number of virions at these mucosal barriers and are therefore a strategic point to stop a COVID-19 infection before it starts. Despite this, most vaccines against COVID-19 are being injected into the muscle where they will not generate the highest levels of mucosal protection. The vaccines that are approved for use in humans are all replication-defective (RD) mRNA, DNA, or adenovirus (Ad) vaccines that do not amplify antigen transgenes. We developed single cycle adenovirus (SC-Ad) vectors that replicate antigen genes up to 10,000-fold in human cells, but that are disabled from producing infectious Ad particles. We show here that SC-Ad expressing the full-length SARS-CoV-2 spike protein produces 100-fold more spike protein than a matched RD-Ad-Spike vector. When Ad-permissive hamsters were immunized with these vaccines by intranasal (IN) or intramuscular (IM) routes, SC-Ad produced significantly stronger antibody responses as compared to RD-Ad against the spike protein that rose over 14 weeks after one immunization. Single IN or IM immunizations generated significant antibody responses in serum and in bronchoalveolar lavages (BALs). IN priming, but not IM priming, generated HLA-restricted CD8 T cell responses in BALs. SC-Ad-Spike generated antibodies that retain binding to spike receptor binding domains (RBDs) with mutations from new viral variants. These data suggest empowering the genomes of gene-based vaccines with the ability to amplify antigen genes can increase potency. This may be particularly advantageous when applying mucosal vaccines to combat mucosal pathogens like SARS-CoV-2.

One Sentence SummaryArming adenovirus vaccines with the ability to replicate vaccine antigen genes may increase potency for systemic, or more importantly, mucosal immunization against mucosal pathogens.

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Multidimensional Boolean Patterns in Multi-omics Data MotivationVirtually all biological systems are governed by a set of complex relations between their components. Identification of relations within biological systems involves a rigorous search for patterns among variables/parameters. Two-dimensional (involving two variables) patterns are identified using correlation, covariation, and mutual information approaches. However, these approaches are not suited to identify more complicated multidimensional relations, which simultaneously include 3, 4, and more variables.

ResultsWe present a novel pattern-specific method to quantify the strength and estimate the statistical significance of multidimensional Boolean patterns in multiomics data. In contrast with dimensionality reduction and AI solutions, patterns identified by the proposed approach may provide a better background for meaningful mechanistic interpretation of the biological processes. Our preliminary analysis suggests that multidimensional patterns may dominate the landscape of multi-omics data, which is not surprising because complex interactions between components of biological systems are unlikely to be reduced to simple pairwise interactions.

sentences: Micronized curcumin causes hyperlocomotion in zebrafish larvae Zebrafish larvae have been widely used in neuroscience and drug research and development. In the larval stage, zebrafish present a broad behavioral repertoire and physiological responses similar to adults. Curcumin (CUR), a major component of Curcuma longa L. (Zingiberaceae), has demonstrated the ability to modulate several neurobiological processes relevant to mental disorders in animal models. However, the low bioavailability of this compound can compromise its in vivo biological potential. Interestingly, it has been shown that micronization can increase the biological effects of several compounds. Thus, in this study, we compared the effects of acute exposure for 30 minutes to the following solutions: water (control), 0.1% DMSO (vehicle), 1 M CUR, or 1 M micronized curcumin (MC) in zebrafish larvae 7 days post-fertilization (dpf). We analyzed locomotor activity (open tank test), anxiety (light/dark test), and avoidance behavior (aversive stimulus test). Moreover, we evaluated parameters of oxidative status (thiobarbituric acid reactive substances and non-protein thiols levels). MC increased the total distance traveled and absolute turn angle in the open tank test. There were no significant differences in the other behavioral or neurochemical outcomes. The increase in locomotion induced by MC may be associated with a stimulant effect on the central nervous system, which was evidenced by the micronization process.

sentences: Oscillatory dynamics underlying emotion-cognition integration: differential role of theta and alpha oscillations A fundamental aspect of human mental life is the seamless ability for integration of emotion and cognition. Despite progress regarding the spatial architecture of Emotion-Cognition Integration (ECI), the time course of ECI processes remains unclear. To examine the temporal organization of brain oscillations underpinning ECI, we simultaneously manipulated emotional valence of stimuli and cognitive task demand while recording electrophysiological responses of 61 participants. They were asked to complete tasks with low (body-part judgement) and high (laterality judgement) cognitive demand while viewing other people photographs that varied on dimensions of laterality (left or right), body-part (hand or foot), and emotional valence (pain or no pain). We found increased reaction times and error rates in pain versus no pain during laterality judgement relative to body-part judgement, suggesting reciprocal inhibition between emotion and cognition. EEG results showed that 1) emotion processing (valence) occurred first in the theta band from 144 to 372 ms; 2) cognitive processing (laterality) took place in the theta band from 332 to 608 ms; 3) emotional and cognitive processes were integrated in the alpha band from 268 ms and lasted to 800 ms. These findings reveal oscillatory dynamics of the processing and integration of emotion and cognition, providing further insights into the underlying neurophysiology. This may ultimately contribute to our understanding of ECI processing in psychopathology.

sentences: ADP-ribose triggers neuronal ferroptosis via metabolic orchestrating The depletion of NAD is intimately linked to neurodegenerative diseases and aging. Excessive activation of NAD+-consuming enzymes that hydrolyze NAD to ADP-ribose and nicotinamide is closely related to neurodegenerative diseases. However, supplementation of NAD or its precursors failed to show any benefits for most patients. The molecular mechanisms underlying this are largely undefined. Here we show that ADP-ribose, as an endogenous NAD metabolite, triggers neuronal cell death by glutathione-independent ferroptosis. The neuronal cells in both the hippocampus and cerebral cortex were severely reduced under the treatment of ADP-ribose in mice. Intracellular ADP-ribose, an endogenous inhibitor of NAD-cofactor enzymes, but not NAD-consuming enzymes, is an accelerator of ferroptosis by blocking the catabolism of lipid peroxidation. Furthermore, the extracellular ADP-ribose directly binds to membrane surface nucleoside transporter ENT1 to orchestrate purine and pyrimidine metabolism. ENT1-hypoxanthine axis and glutamine-dihydroorotate-quinone pathway are independent and intersecting, which is specifically mobilized by ADP-ribose to trigger ferroptosis. Precisely because of it, we find that the neuronal death by ADP-ribose can be rescued by XO and DHODH inhibitors. Moreover, endogenous ADP-ribose levels are increased on oxidative stress and PROTAC of PARP1 maintain neuronal survival under these circumstances. We provide evidence that ADP-ribose is a key metabolite that is used for disease diagnosis and drug target. Our analyses uncover new molecular links between NAD metabolism and ferroptosis in the regulation of neuronal death, thus suggesting new strategies for the prevention and treatment of neurodegenerative diseases.

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Dicer promotes genome stability via the bromodomain transcriptional co-activator Brd4. RNA interference is essential for transcriptional silencing and genome stability, but conservation of this role in mammals has been difficult to demonstrate. Dicer1-/- mouse embryonic stem cells have microRNA-independent proliferation defects, and we conducted a CRISPR-Cas9 screen to restore viability. We identified suppressor mutations in transcriptional activators, H3K9 methyltransferases, and chromosome segregation factors, strongly resembling Dicer suppressors in fission yeast. Suppressors rescued chromosomal defects, and reversed strand-specific transcription of major satellite repeats in Dicer1-/-. The strongest suppressors were in Brd4, and in the transcriptional elongator/histone acetyltransferase Elp3. Using viable mutants and pharmaceutical inhibitors, we demonstrate that deletion of specific residues in Brd4 rescue genome instability defects of Dicer1-/- in both mammalian cells and fission yeast, implicating Dicer in coordinating transcription and replication of satellite repeats.

SummaryReplication and segregation defects in Dicer1-/- stem cells depend on centromeric transcription by Brd4, and are deeply conserved in fission yeast.

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Differential effects of soil-based agricultural diversification on arthropods and crop yield O_LIAgricultural diversification often promotes biodiversity and ecosystem services by increasing habitat diversity. However, responses to agricultural diversification are context dependent, differentially impacting functional groups of service-providing organisms and crop yields.
C_LIO_LIConservation and no tillage are promoted as agricultural diversification practices that increase soil heterogeneity and habitat diversity. Here we investigated whether soil tillage practices in canola crop fields altered arthropod biodiversity or yield, and how effects of field-scale diversification compared to landscape-scale habitat context. We focused on effects of high, medium, or no tillage on five functional groups with unique diets and reproductive strategies: (i) herbivores, (ii) kleptoparasites, (iii) parasitoids, (iv) pollinators, and (v) predators.
C_LIO_LIEffects of agricultural diversification on arthropod abundance and diversity varied across functional groups. Pollinators responded to on-farm soil diversification, benefiting from medium tillage. Predators and herbivores responded most strongly to landscape-scale habitat composition and were more abundant in landscapes with more semi-natural habitat. However, variation in arthropod communities had little effect on canola crop yield, which was lowest in fields with no tillage.
C_LIO_LIPolicy implications: Our results indicate that natural history differences among arthropod functional groups mediate how habitat availability affects biodiversity. Crop yields, however, showed no response to biodiversity of ecosystem service providers. Our research highlights the need to determine the contexts in which soil diversification practices meet a multi-faceted goal of simultaneously supporting natural biodiversity, ecosystem services, and crop yield.
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miniTurbo-based interactomics of two plasma membrane-localized SNARE proteins in Marchantia polymorpha O_LIMarchantia polymorpha is a model liverwort and its overall low genetic redundancy is advantageous for dissecting complex pathways. Proximity-dependent in vivo biotin-labelling methods have emerged as powerful interactomics tools in recent years. However, interactomics studies applying proximity labelling are currently limited to angiosperm species in plants.
C_LIO_LIHere, we established and evaluated a miniTurbo-based interactomics method in M. polymorpha using MpSYP12A and MpSYP13B, two plasma membrane- localized SNARE proteins, as baits.
C_LIO_LIWe show that our method yields a manifold of potential interactors of MpSYP12A and MpSYP13B compared to a co-immunoprecipitation approach. Our method could capture specific candidates for each SNARE.
C_LIO_LIWe conclude that a miniTurbo-based method is a feasible tool for interactomics in M. polymorpha and potentially applicable to other model bryophytes. Our interactome dataset on MpSYP12A and MpSYP13B will be a useful resource to elucidate the evolution of SNARE functions.
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sentences: MXtrodes: MXene-infused bioelectronic interfaces for multiscale electrophysiology and stimulation Soft bioelectronic interfaces for mapping and modulating excitable networks at high resolution and at large scale can enable paradigm-shifting diagnostics, monitoring, and treatment strategies. Yet, current technologies largely rely on materials and fabrication schemes that are expensive, do not scale, and critically limit the maximum attainable resolution and coverage. Solution processing is a cost-effective manufacturing alternative, but biocompatible conductive inks matching the performance of conventional metals are lacking. Here, we introduce MXtrodes, a novel class of soft, high-resolution, large-scale bioelectronic interfaces enabled by Ti3C2 MXene and scalable solution processing. We show that the electrochemical properties of MXtrodes exceed those of conventional materials, and do not require conductive gels when used in epidermal electronics. Furthermore, we validate MXtrodes in a number of applications ranging from mapping large scale neuromuscular networks in humans to delivering cortical microstimulation in small animal models. Finally, we demonstrate that MXtrodes are compatible with standard clinical neuroimaging modalities.

sentences: Host Induced Gene Silencing of the Sclerotinia sclerotiorum ABHYDROLASE-3 gene reduces disease severity in Brassica napus Sclerotinia sclerotiorum is a pathogenic fungus that infects hundreds of crop species, causing extensive yield loss every year. Chemical fungicides are used to control this phytopathogen, but with concerns about increasing resistance and impacts on non-target species, there is a need to develop alternative control measures. In the present study, we engineered Brassica napus to constitutively express a hairpin (hp)RNA molecule to silence ABHYRDOLASE-3 in S. sclerotiorum. We demonstrate the potential for Host Induced Gene Silencing (HIGS) to protect B. napus from S. sclerotiorum using leaf, stem and whole plant infection assays. The interaction between the transgenic host plant and invading pathogen was further characterized at the molecular level using dual-RNA sequencing and at the anatomical level through microscopy to understand the processes and possible mechanisms leading to increased tolerance to this damaging necrotroph. We observed significant shifts in the expression of genes relating to plant defense as well as cellular differences in the form of structural barriers around the site of infection in the HIGS-protected plants. Our results provide proof-of-concept that HIGS is an effective means of limiting damage caused by S. sclerotiorum to the plant and demonstrates the utility of this biotechnology in the development of resistance against fungal pathogens.

sentences: Principles of ecDNA random inheritance drive rapid genome change and therapy resistance in human cancers The foundational principles of Darwinian evolution are variation, selection, and identity by descent. Oncogene amplification on extrachromosomal DNA (ecDNA) is a common event, driving aggressive tumour growth, drug resistance, and shorter survival in patients1-4. Currently, the impact of non-chromosomal oncogene inheritance--random identity by descent--is not well understood. Neither is the impact of ecDNA on variation and selection. Here, integrating mathematical modeling, unbiased image analysis, CRISPR-based ecDNA tagging, and live-cell imaging, we identify a set of basic "rules" for how random ecDNA inheritance drives oncogene copy number and distribution, resulting in extensive intratumoural ecDNA copy number heterogeneity and rapid adaptation to metabolic stress and targeted cancer treatment. Observed ecDNAs obligatorily benefit host cell survival or growth and can change within a single cell cycle. In studies ranging from well-curated, patient-derived cancer cell cultures to clinical tumour samples from patients with glioblastoma and neuroblastoma treated with oncogene-targeted drugs, we show how these ecDNA inheritance "rules" can predict, a priori, some of the aggressive features of ecDNA-containing cancers. These properties are entailed by their ability to rapidly change their genomes in a way that is not possible for cancers driven by chromosomal oncogene amplification. These results shed new light on how the non-chromosomal random inheritance pattern of ecDNA underlies poor outcomes for cancer patients.

sentences: A mathematical framework for analyzing wild tomato root architecture The root architecture of wild tomato, Solanum pimpinellifolium, can be viewed as a network connecting the main root to various lateral roots. Several constraints have been proposed on the structure of such biological networks, including minimizing the total amount of wire necessary for constructing the root architecture (wiring cost), and minimizing the distances (and by extension, resource transport time) between the base of the main root and the lateral roots (conduction delay). For a given set of lateral root tip locations, these two objectives compete with each other -- optimizing one results in poorer performance on the other -- raising the question how well S. pimpinellifolium root architectures balance this network design trade-off in a distributed manner. Here, we describe how well S. pimpinellifolium roots resolve this trade-off using the theory of Pareto optimality. We describe a mathematical model for characterizing the network structure and design trade-offs governing the structure of S. pimpinellifolium root architecture. We demonstrate that S. pimpinellifolium arbors construct architectures that are more optimal than would be expected by chance. Finally, we use this framework to quantify structural differences between arbors grown in the presence of salt stress, classify arbors into four distinct architectural ideotypes, and test for heritability of variation in root architecture structure.

sentences: Large-scale voltage imaging in the brain using targeted illumination Recent improvements in genetically encoded voltage indicators enabled optical imaging of action potentials and subthreshold membrane voltage dynamics from single neurons in the mammalian brain. To perform high speed voltage imaging, widefield microscopy remains an essential tool for recording activity from many neurons simultaneously over a large anatomical area. However, the lack of optical sectioning makes widefield microscopy more prone to background signal contamination, and thus far voltage imaging using fully genetically encoded voltage indicators remains limited to simultaneous sampling of a few cells over a restricted field-of-view. We here demonstrate a strategy for large scale voltage imaging using the fully genetically encoded voltage indicator SomArchon and targeted illumination. We implemented a simple, low-cost digital micromirror device based targeted illumination strategy to restrict illumination to the cells of interest, and systematically quantified the improvement of this microscopy design theoretically and experimentally with SomArchon expressing neurons in single layer cell cultures and in the brains of awake mice. We found that targeted illumination, in comparison to widefield illumination, increased SomArchon signal contrast and reduced background cross-contamination in the brain. Such improvement permitted the reduction of illumination intensity, and thus reduced fluorescence photobleaching and prolonged imaging duration. When coupled with a high-speed, large area sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over minutes in the brain. Thus, the widefield microscopy design with an integrated targeted illumination system described here offers a simple solution for voltage imaging analysis of large neuron populations in behaving animals.

sentences: Pneumococcal within-host diversity during colonisation, transmission and treatment Characterising the genetic diversity of pathogens within the host promises to greatly improve surveillance and reconstruction of transmission chains. For bacteria, it also informs our understanding of inter-strain competition, and how this shapes the distribution of resistant and sensitive bacteria. Here we study the genetic diversity of Streptococcus pneumoniae within individual infants and their mothers by deep sequencing whole pneumococcal populations from longitudinal nasopharyngeal samples. We demonstrate deep sequencing has unsurpassed sensitivity for detecting multiple colonisation, doubling the rate at which highly invasive serotype 1 bacteria were detected in carriage compared to gold-standard methods. The greater resolution identified an elevated rate of transmission from mothers to their children in the first year of the childs life. Comprehensive treatment data demonstrated infants were at an elevated risk of both the acquisition, and persistent colonisation, of a multidrug resistant bacterium following antimicrobial treatment. Some alleles were enriched after antimicrobial treatment, suggesting they aided persistence, but generally purifying selection dominated within-host evolution. Rates of co-colonisation imply that in the absence of treatment, susceptible lineages outcompeted resistant lineages within the host. These results demonstrate the many benefits of deep sequencing for the genomic surveillance of bacterial pathogens.

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Ultrahigh-throughput directed evolution of a metal-free α/β-hydrolase with a Cys-His-Asp triad into an efficient phosphotriesterase The recent massive release of new, man-made substances into the environment requires bioremediation, but a very limited number of enzymes evolved in response are available. When environments have not encountered the potentially hazardous materials in their evolutionary history, existing enzymes have to be repurposed. The recruitment of accidental, typically low-level promiscuous activities provides a head start that, after gene duplication, can adapt and provide a selectable advantage. This evolutionary scenario raises the question whether it is possible to adaptively improve the low-level activity of enzymes recruited from non- (or only recently) contaminated environments quickly to the level of evolved bioremediators.

Here we address the evolution of phosphotriesterases (enzymes for hydrolysis of organophosphate pesticides or chemical warfare agents) in such a scenario: In a previous functional metagenomics screening we had identified a promiscuous phosphotriesterase activity of the /{beta}-hydrolase P91, with an unexpected Cys-His-Asp catalytic triad as the active site motif. We now probe evolvability of P91 using ultrahigh-throughput screening in microfluidic droplets, and test for the first time whether the unique catalytic motif of a cysteine-containing triad can adapt to achieve rates that rival existing phosphotriesterases. These mechanistically distinct enzymes achieve their high rates based on catalysis involving a metal-ion cofactor. A focussed, combinatorial library of P91 (> 105 members) was screened on-chip in microfluidic droplets by quantification of the reaction product, fluorescein. Within only two rounds of evolution P91s phosphotriesterase activity was increased {approx} 400-fold to a kcat/KM of {approx} 106 M-1s-1, matching the catalytic efficiencies of naturally evolved metal-dependent phosphotriesterases. In contrast to its homologue acetylcholinesterase that suffers suicide inhibition, P91 shows fast de-phosphorylation rates and is rate-limited by the formation of the covalent adduct rather than by its hydrolysis. Our analysis highlights how the combination of focussed, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in Nature, resulting in alternative reagents with a novel catalytic machinery.

GRAPHICAL ABSTRACT

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sentences: Advancing antimicrobial resistance monitoring in surface waters with metagenomic and quasimetagenomic methods Surface waters present a unique challenge for the monitoring of critically important antimicrobial resistance. Metagenomic approaches provide unbiased descriptions of taxonomy and antimicrobial resistance genes in many environments, but for surface water, culture independent data is insufficient to describe critically important resistance. To address this challenge and expand resistome reporting capacity of antimicrobial resistance in surface waters, we apply metagenomic and quasimetagenomic (enriched microbiome) data to examine and contrast water from two sites, a creek near a hospital, and a reservoir used for recreation and municipal water. Approximately 30% of the National Antimicrobial Resistance Monitoring Systems critically important resistance gene targets were identified in enriched data contrasted to only 1% in culture independent data. Four different analytical approaches consistently reported substantially more antimicrobial resistance genes in quasimetagenomic data compared to culture independent data across most classes of antimicrobial resistance. Statistically significant differential fold changes (p<0.05) of resistance determinants were used to infer microbiological differences in the waters. Important pathogens associated with critical antimicrobial resistance were described for each water source. While the single time-point for only two sites represents a small pilot project, the successful reporting of critically important resistance determinants is proof of concept that the quasimetagenomic approach is robust and can be expanded to multiple sites and timepoints for national and global monitoring and surveillance of antimicrobial resistance in surface waters.

sentences: USPNet: unbiased organism-agnostic signal peptidepredictor with deep protein language model Signal peptide is a short peptide located in the N-terminus of proteins. It plays an important role in targeting and transferring transmembrane proteins and secreted proteins to correct positions. Compared with traditional experimental methods to identify and discover signal peptides, the computational methods are faster and more efficient, which are more practical for the analysis of thousands or even millions of protein sequences in reality, especially for the metagenomic data. Therefore, computational tools are recently proposed to classify signal peptides and predict cleavage site positions, but most of them disregard the extreme data imbalance problem in these tasks. In addition, almost all these methods rely on additional group information of proteins to boost their performances, which, however, may not always be available. To deal with these issues, in this paper, we present Unbiased Organism-agnostic Signal Peptide Network (USPNet), a signal peptide prediction and cleavage site prediction model based on deep protein language model. We propose to use label distribution-aware margin (LDAM) loss and evolutionary scale modeling (ESM) embedding to handle data imbalance and object-dependence problems. Extensive experimental results demonstrate that the proposed method significantly outperforms all the previous methods on the classification performance. Additional study on the simulated metagenomic data further indicates that our model is a more universal and robust tool without dependency on additional group information of proteins, with the Matthews correlation coefficient improved by up to 17.5%. The proposed method will be potentially useful to discover new signal peptides from the abundant metagenomic data.

sentences: Ceylon cinnamon and its major compound Cinnamaldehyde can limit overshooting inflammatory signaling and angiogenesis in vitro: implications for COVID-19 treatment Overshooting immune reactions can occur during inflammatory responses that accompany severe infections, such as COVID-19. Cytokines, damage-associated molecular patterns (DAMPs), and reactive oxygen and nitrogen species can generate positive feedback loops of inflammation, leading to long-term complications such as vascular endothelialitis, thrombosis, endothelial dysfunction, neurological impairments, and chronic fatigue. Dexamethasone can limit inflammation by inhibiting the activation of pro-inflammatory transcription factors. High dose dexamethasone, however, has undesirable side effects. Here, we show that Ceylon cinnamon and its major compound cinnamaldehyde can mitigate inflammatory signaling in vitro. Cinnamaldehyde interferes with the dimerization of toll-like receptor 4 (TLR4), which can be activated by DAMPs like HSP60 and HMGB1. Our results suggest that supplementary treatment with Ceylon cinnamon may allow administration of lower doses of dexamethasone to avoid high dose steroid side effects. Moreover, preliminary results indicate that Ceylon cinnamon modulates angiogenesis, which is a reactive phenomenon in COVID-19.

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GTP and lipids control self-assembly and functional promiscuity of Dynamin2 molecular machinery Dynamin2 GTPase (Dyn2) is a crucial player in clathrin-mediated endocytosis. Dyn2 is tetrameric in cytoplasm and self-assembles into functional units upon membrane binding. How the curvature activities and functionality of Dyn2 emerge during self-assembly and are regulated by lipids remains unknown. Here we reconstituted the Dyn2 self-assembly process using membrane nanotubes (NT) and vesicles and characterized it using single- molecule fluorescence microscopy, optical tweezers force spectroscopy and cryo-electron microscopy. On NTs, Dyn2 first forms small subhelical oligomers, which are already curvature active and display pronounced curvature sensing properties. Conical lipids and GTP promote their further self-assembly into helical machinery mediating the NT scission. In the presence of large unilamellar vesicles (LUVs), an alternative self- assembly pathway emerges where the subhelical oligomers form membrane tethering complexes mediating LUV-NT binding. Reconstitution of tethering in the LUV system revealed that lipid mixing is controlled by conical lipid species, divalents, GTP, and SH3 binding partners of Dyn2. In membranes with a high content of lipids with negative intrinsic curvature, cryo-EM revealed putative membrane contact sites made by Dyn2 clusters. On such membranes, with GTP lowered to 0.2 mM, both membrane fission and tethering activities become possible, indicating functional promiscuity of Dyn2.

We conclude that GTP and lipids control both extent and topology of Dyn2 functional self-assembly. The function of Dyn2 oligomers evolves from curvature sensing, seen in subhelical Dyn2 oligomers, to curvature creation and fission, seen in Dyn2 helices. Under specific circumstances, such as downregulation of SH3 partners of Dyn2 and GTP depletion, membrane tethering activity can emerge in membrane systems enriched with conical lipids. Hence the Dyn2 functionality is actively adapted to lipidome, explaining its large habitat in the cells and tissues.

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Increasing the throughput of sensitive proteomics by plexDIA Current mass-spectrometry methods enable high-throughput proteomics of large sample amounts, but proteomics of low sample amounts remains limited in depth and throughput. To increase the throughput of sensitive proteomics, we developed an experimental and computational framework, plexDIA, for simultaneously multiplexing the analysis of both peptides and samples. Multiplexed analysis with plexDIA increases throughput multiplicatively with the number of labels without reducing proteome coverage or quantitative accuracy. By using 3-plex nonisobaric mass tags, plexDIA enables quantifying 3-fold more protein ratios among nanogram-level samples. Using 1 hour active gradients and first-generation Q Exactive, plexDIA quantified about 8,000 proteins in each sample of labeled 3-plex sets. plexDIA also increases data completeness, reducing missing data over 2-fold across samples. We applied plexDIA to quantify proteome dynamics during the cell division cycle in cells isolated based on their DNA content; plexDIA detected many classical cell cycle proteins and discovered new ones. When applied to single human cells, plexDIA quantified about 1,000 proteins per cell and achieved 98 % data completeness within a plexDIA set while using about 5 min of active chromatography per cell. These results establish a general framework for increasing the throughput of sensitive and quantitative protein analysis.

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sentences: Osmotic stress via calmodulin lead to the formation of stress granule in Drosophila S2 cells. Cellular stress of S2 cells leads to the formation of stress assemblies by phase separation of cytoplasmic components. We have shown that the cellular stresses of either high increase of the NaCl concentration in the extracellular medium, or a moderate one combined to amino acid starvation, leads to the formation of Sec bodies where components of the endoplasmic Reticulum exit sites (ERES) coalesce. These extracellular stresses lead to both the activation of salt inducible kinase (SIK), and to ER stress triggering the activation of the two downstream kinases IRE1 and PERK. Interestingly, the same stresses also result in the formation of a second stress assembly, the stress granules, which stores specific RNAs and RNA binding proteins. Here we asked whether stress granule formation is governed by the same pathways as Sec bodies. However, we found that the inhibition of SIK, IRE1 and PERK does not affect stress granule formation. Instead, we found that osmotic stress through the addition of either salts (including calcium chloride) or sucrose leads to the formation of stress granules. Interestingly, stress granule formation is partly modulated by calmodulin activation, suggesting the involvement of calcium signaling. Furthermore, as Sec body formation is driven by entirely different pathways, these results show that the same cells under the same stress, form two different stress assemblies by non-overlapping downstream pathway activation, perhaps explaining that they do not coalescence into a single structure.

sentences: Fate-resolved gene regulatory signatures of individual B lymphocytes in the early stages of Epstein-Barr Virus infection Epstein-Barr Virus (EBV) infection of B lymphocytes elicits diverse host responses via complex, well-adapted transcriptional control dynamics. Consequently, this host-pathogen interaction provides a powerful system to explore fundamental cellular processes that contribute to consensus fate decisions including cell cycle arrest, apoptosis, proliferation, and differentiation. Here we capture these responses and fates with matched single-cell transcriptomics and chromatin accessibility, from which we construct a genome-wide multistate model of early infection dynamics. Notably, our model captures a previously uncharacterized EBV+ analog of a multipotent activated precursor state that can yield early memory B cells. We also find that a marked global reduction in host chromatin accessibility occurs during the first stages of infection in subpopulations of EBV+ cells that display senescent and pre-apoptotic hallmarks induced by innate antiviral sensing and proliferation-linked DNA damage. However, cells in proliferative infection trajectories exhibit greater accessibility at select host sites linked to B cell activation and survival genes as well as key regions within the viral genome. To further investigate such loci, we implement a bioinformatic workflow (crisp-ATAC) to identify phenotype-resolved regulatory signatures. This customizable method applies user-specified logical criteria to produce genome-wide single-cell ATAC-and ChIP-seq range intersections that are used as inputs for cis-linkage prediction and ontology tools. The resulting tri-modal data yield exquisitely detailed hierarchical perspectives of the transforming regulatory landscape during critical stages of an oncogenic viral infection that simulates antigen-induced B cell activation and differentiation. We anticipate these resources will guide investigations of gene regulatory modules controlling EBV-host dynamics, B cell effector fates, and lymphomagenesis. To demonstrate the utility of this resource, this work concludes with the discovery of EBV infection dynamics in FCRL4+ / TBX21+ Tissue-Like Memory B cells, an unconventional subset with notable associations to numerous immune disorders.

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A multimodal AI system for out-of-distribution generalization of seizure detection Epilepsy is one of the most common severe neurological disorders worldwide. The International League Against Epilepsy (ILAE) define epilepsy as a brain disorder that generates (1) two unprovoked seizures more than 24 hrs apart, or (2) one unprovoked seizure with at least 60% risk of recurrence over the next ten years. Complete remission has been defined as ten years seizure free with the last five years medication free. This requires a cost-effective ambulatory ultra-long term out-patient monitoring solution. The common practice of self-reporting is inaccurate. Applying artificial intelligence (AI) to scalp electroencephalogram (EEG) interpretation is becoming increasingly common, but other data modalities such as electrocardiograms (ECGs) are simpler to collect and often recorded simultaneously with EEG. Both recordings contain biomarkers in the detection of seizures.

Here, we propose a state-of-the-art performing AI system that combines EEG and ECG for seizure detection, tested on clinical data with early evidence demonstrating generalization across hospitals. The model was trained and validated on the publicly available Temple University Hospital (TUH) dataset. To evaluate performance in a clinical setting, we conducted nonpatient-specific inference-only tests on three out-of-distribution datasets, including EPILEPSIAE (30 patients) and the Royal Prince Alfred Hospital (RPAH) in Sydney, Australia (31 patients shortlisted by neurologists and 30 randomly selected). Across all datasets, our multimodal approach improves the area under the receiver operating characteristic curve (AUC-ROC) by an average margin of 6.71% and 14.42% for prior state-of-the-art approaches using EEG and ECG alone, respectively. Our models state-of-the-art performance and robustness to out-ofdistribution datasets can improve the accuracy and efficiency of epilepsy diagnoses.

sentences: Insertions in SARS-CoV-2 genome caused by template switch and duplications give rise to new variants that merit monitoring The appearance of multiple new SARS-CoV-2 variants during the winter of 2020-2021 is a matter of grave concern. Some of these new variants, such as B.1.617.2, B.1.1.7, and B.1.351, manifest higher infectivity and virulence than the earlier SARS-CoV-2 variants, with potential dramatic effects on the course of the COVID-19 pandemic. So far, analysis of new SARS-CoV-2 variants focused primarily on point nucleotide substitutions and short deletions that are readily identifiable by comparison to consensus genome sequences. In contrast, insertions have largely escaped the attention of researchers although the furin site insert in the spike protein is thought to be a determinant of SARS-CoV-2 virulence and other inserts might have contributed to coronavirus pathogenicity as well. Here, we investigate insertions in SARS-CoV-2 genomes and identify 347 unique inserts of different lengths. We present evidence that these inserts reflect actual virus variance rather than sequencing errors. Two principal mechanisms appear to account for the inserts in the SARS-CoV-2 genomes, polymerase slippage and template switch that might be associated with the synthesis of subgenomic RNAs. We show that inserts in the Spike glycoprotein can affect its antigenic properties and thus merit monitoring. At least, three inserts in the N-terminal domain of the Spike (ins245IME, ins246DSWG, and ins248SSLT) that were first detected in 2021 are predicted to lead to escape from neutralizing antibodies, whereas other inserts might result in escape from T-cell immunity.

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Extended bacterial diversity of the urinary microbiome of reproductive-age healthy European women captured by culturomics and long-read amplicon sequencing The recognition of microbiome inhabiting the healthy female bladder engendered the need for comprehensive characterization of the female urinary microbiome (FUM) in health and disease. Although previous studies reported FUM composition at different taxonomic levels, progress towards reliable identification at species level is highly required. The aim of this study was to comprehensively characterize bacterial species of FUM of healthy reproductive-age European women by two complementary methodologies i.e., extended culturomics and long-read third generation sequencing of near full-length 16S rRNA gene.

A wide diversity of bacterial species was captured (297 species) with a median of 53 species/sample, including 16 putative uropathogens. Clustering FUM into community structure types revealed high inter-individual differences. Notably, there was not a single species common to all samples, although the Lactobacillus genus was detected in all samples. Lactobacillus crispatus, Lactobacillus iners and Lactobacillus mulieris were observed in high relative abundance in several samples as well as other species (e.g., Streptococcus agalactiae, Atopobium vaginae, Gardnerella vaginalis, Gardnerella swidsinskii), while more prevalent species were often low abundant members (e.g., Finegoldia magna). We captured remarkable richness within Corynebacterium spp. (25 species) and Lactobacillaceae (4 genera, 14 species). While amplicon sequencing allowed detection of more anaerobic species (e.g., 11 Peptoniphilus spp.), culturomics enabled the identification of recently recognized Gardnerella species and putative novel Corynebacterium species.

This study provided fine-grained FUM profiling at species level and revealed detailed FUM structure, which is critical to unveil the potential relationship between specific microbiome members and urinary diseases/disorders.

IMPORTANCEDespite evidence of the resident microbial community in the female lower urinary tract, bacterial species diversity and abundance in healthy women is still unclear. This study demonstrated that complementarity between optimized culture-dependent and -independent approaches is highly beneficial for comprehensive FUM species profiling by detecting higher FUM species diversity than previously reported, including identification of unreported Lactobacillaceae species and putative novel Corynebacterium species. Although some particular species were present in high relative abundance, low-abundant members were more prevalent. FUM classification into community structure types demonstrated high inter-individual differences in urinary microbiome composition among healthy women. We also report moderate correlation between culture-dependent and -independent derived data highlighting drawbacks resulting from each methodological approach. Our findings suggest that FUM bacterial diversity reported from previous studies may be underestimated. Finally, our results contribute to the fundamental knowledge of healthy FUM required for further exploration of the urinary microbiome role in urinary tract diseases.

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Ratiometric Sensing of Redox Environments Inside Individual Carboxysomes Trapped in Solution Diffusion of biological nanoparticles in solution impedes our ability to continuously monitor individuals and measure their physical and chemical properties. To overcome this, we previously developed the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap, which uses scattering to localize a particle and applies electrokinetic forces which counteract Brownian motion, thus enabling extended observation. Here, we present an improved ISABEL trap that incorporates a near-infrared scatter illumination beam and rapidly interleaves 405 and 488 nm fluorescence excitation reporter beams. With the ISABEL trap, we monitor the internal redox environment of individual carboxysomes labeled with the ratiometric redox reporter roGFP2. Carboxysomes widely vary in scattering contrast (reporting on size) and redox-dependent ratiometric fluorescence. Further, we used redox sensing to explore the chemical kinetics within intact carboxysomes, where bulk measurements may contain unwanted contributions from aggregates or interfering fluorescent proteins. Overall, we demonstrate the ISABEL traps ability to sensitively monitor nanoscale biological objects, enabling new experiments on these systems.

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sentences: Differential Expression of α,β, and γ Protocadherin Isoforms During Differentiation, Aging, and Cancer The cadherin family of cell surface glycoproteins plays a fundamental role in cell-cell recognition, thereby participating in diverse biological process such as embryonic morphogenesis and oncogenic transformation. The subset of clustered protocadherin (PCDH) genes generated from the , {beta}, and {gamma} loci, have been widely studied for their potential role in neuronal cell-cell recognition and neurogenesis, however their broader role in normal embryonic development and cancer has not been examined in detail. We utilized human embryonic stem (hES) cells to model early human development in vitro, comparing PCDH isoform transcription in diverse types of embryonic progenitors with normal adult-derived and cancer counterparts. Embryonic progenitors express genes from the  and {beta} cluster at levels comparable to that seen in the CNS, while fetal and adult-derived cells express primarily from the {gamma} cluster. Replicative senescence left fibroblasts with markedly lower expression of all isoforms. We observe that an embryonic pattern of clustered protocadherin gene expression and associated CpG island methylation is commonly associated with cancer cell lines from diverse tissue types. The differential regulation of the , {beta}, and {gamma} loci coincide with alternate regions of DNA accessibility at CTCF binding sites and lamina-associated domains and CPL expression correlated with the expression of LMNA and LMNB1. These observations support a potential role for the differential regulation of genes within the clustered protocadherin locus in selective cell-cell adhesion during embryogenesis, regeneration, cancer and aging.

sentences: HOW CONCERNING IS A SARS-COV-2 VARIANT OF CONCERN? COMPUTATIONAL PREDICTIONS AND THE VARIANTS LABELING SYSTEM We herein report a study to evaluate the use of computational prediction of SARS-CoV-2 genetic variations in improving the current variants labeling system. First, we reviewed the basis of the system developed by the World Health Organization (WHO) for the labeling of SARS-CoV-2 genetic variants and the adaptations made to it by the United States Center of Diseases Control (CDC). We observed that the labeling system is based upon the virus major attributes. However, we found that the labeling criteria of the SARS-CoV-2 variants derived from these attributes are not accurately defined and are used differently by the two health management agencies. Consequently, discrepancies exist between the labels given by WHO and CDC to same variants. Our observations suggest that giving the VOC label to a new variant is premature and might not be appropriate. Therefore, we carried out a comparative computational study to predict the effects of the mutations on the virus structure and functions of five VOCs. By linking these data to the criteria used by WHO and the CDC for variant labeling, we ascertained that comparative computational predictions of the impact of genetic variations are a better ground for rapid and more accurate labelling of SARS-CoV-2 variants. We propose to label all emergent variants VUM or VBM and to carry out computational predictive studies and thorough variants comparison, upon which more appropriate and informative labels can be attributed. Furthermore, harmonization of the variants labeling system would be globally beneficial to communicate about and fight COVID-19 pandemic.

sentences: Ongoing exposure to peritoneal dialysis fluid alters resident peritoneal macrophage phenotype and activation propensity Peritoneal dialysis (PD) is a more continuous alternative to haemodialysis, for patients with chronic kidney disease, with considerable initial benefits for survival, patient independence and healthcare costs. However, longterm PD is associated with significant pathology, negating the positive effects over haemodialysis. Importantly, peritonitis and activation of macrophages is closely associated with disease progression and treatment failure. However, recent advances in macrophage biology suggest opposite functions for macrophages of different cellular origins. While monocyte-derived macrophages promote disease progression in some models of fibrosis, tissue resident macrophages have rather been associated with protective roles. Thus, we aimed to identify the relative contribution of tissue resident macrophages to PD induced inflammation in mice. Unexpectedly, we found an incremental loss of homeostatic characteristics, anti-inflammatory and efferocytic functionality in peritoneal resident macrophages, accompanied by enhanced inflammatory responses to external stimuli. Moreover, presence of glucose degradation products within the dialysis fluid led to markedly enhanced inflammation and almost complete disappearance of tissue resident cells. Thus, alterations in tissue resident macrophages may render longterm PD patients sensitive to developing peritonitis and consequently fibrosis/sclerosis.

sentences: Multivalent peptide ligands to probe the chromocenter microenvironment in living cells Chromatin is spatially organized into functional states that are defined by both the presence of specific histone post-translational modifications (PTMs) and a defined set of chromatin-associated  reader proteins. Different models for the underlying mechanism of such compartmentalization have been proposed, including liquid-liquid phase separation (LLPS) of chromatin-associated proteins to drive spatial organization. Heterochromatin, characterized by lysine 9 methylation on histone H3 (H3K9me3) and the presence of heterochromatin protein 1 (HP1) as a multivalent reader, represents a prime example of a spatially defined chromatin state. Heterochromatin foci exhibit features of protein condensates driven by LLPS; however, the exact nature of the physicochemical environment within heterochromatin in different cell types is not completely understood. Here, we present tools to interrogate the environment of chromatin sub-compartments in the form of modular, cell-permeable, multivalent and fluorescent peptide probes. These probes can be tuned to target specific chromatin states by providing binding sites to reader proteins and can thereby integrate into the PTM-reader interaction network. As a target, here we generate probes specific to HP1, directing them to heterochromatin at chromocenters in mouse fibroblasts. Moreover, we use a polarity-sensing photoactivatable probe that photoconverts to a fluorescent state in phase-separated protein droplets and thereby reports on the local microenvironment. Equipped with this dye, our probes indeed turn fluorescent in murine chromocenters. However, image analysis and single-molecule tracking experiments reveal that the compartments are less dense and more dynamic than HP1 condensates obtained in vitro. Our results thus demonstrate that the local organization of heterochromatin in chromocenters is internally more complex than an HP1 condensate.

sentences: Comparative transcriptomic analysis of maize ear heterosis during the inflorescence meristem differentiation stage Heterosis is widely used in many crops; however, its genetic mechanisms are only partly understood. Here, we sampled inflorescence meristem (IM) ears from the single-segment substitution maize (Zea mays) line lx9801hlEW2b, containing a heterotic locus hlEW2b associated with ear width, the receptor parent lx9801, the test parent Zheng58, and their corresponding hybrids. After transcriptomic analysis, 1638 genes were identified in at least one hybrid with nonadditively expressed patterns and different expression levels between the two hybrids. In particular, 2263 (12.89%) and 2352 (14.65%) genes showed allele-specific expression (ASE) in Zheng58 x lx9801 and Zheng58 x lx9801hlEW2b, respectively. A functional analysis showed that these genes were enriched in development-related processes and biosynthesis and catabolism processes, which are potentially associated with heterosis. Additionally, nonadditive expression and ASE may fine-tune the expression levels of crucial genes (such as WUS and KNOX that control IM development) controlling auxin metabolism and ear development to optimal states, and transcriptional variation may play important roles in maize ear heterosis. The results provide new information that increases our understanding of the relationship between transcriptional variation and heterosis formation during maize ear development, which may be helpful in clarifying the genetic and molecular mechanisms of heterosis.

sentences: IRI-CCE: A Platform for Evaluating CRISPR-based Genome Editing Tools and Its Components Rapid assessment of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated Cas protein)-based genome editing (GE) tools and their components are critical aspects for successful applications in different organisms. In many bacteria, double-stranded breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient non-homologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs capable of converting C-to-T (CBE) and A-to-G (ABE) have been recently developed but yet to be tested in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising different combinations of promoters/terminators. We demonstrate the use of IRI-CEE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e), exhibiting that each independent BE has its specific editing pattern for a given target site and promoter type. Additionally, the IRI-CCE platform offers a rapid way to screen functional gRNAs. In summary, CRISPR-BE components expressed by promoters of different strengths in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and gRNAs.

sentences: COVID-eVax, an electroporated plasmid DNA vaccine candidate encoding the SARS-CoV-2 Receptor Binding Domain, elicits protective immune responses in animal models of COVID-19 The COVID-19 pandemic caused by the {beta}-coronavirus SARS-CoV-2 has made the development of safe and effective vaccines a critical global priority. To date, four vaccines have already been approved by European and American authorities for preventing COVID-19 but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle, a technology previously utilized for cancer vaccines. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 Spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax - a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein RBD - induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function and significantly lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started in Italy.

sentences: In silico analysis of the fast-growing thermophile Geobacillus sp. LC300 using a novel genome-scale metabolic model Thermophilic microorganisms show high potential for use as biorefinery cell factories. Their high growth temperatures provide fast conversion rates, lower risk of contaminations, and facilitated purification of volatile products. To date, only a few thermophilic species have been utilized for microbial production purposes, and the development of production strains is impeded by the lack of metabolic engineering tools. In this study, we constructed a genome-scale metabolic model, iGEL601, of Geobacillus sp. LC300, an important part of the metabolic engineering pipeline. The model contains 601 genes, 1240 reactions and 1305 metabolites, and the reaction reversibility is based on thermodynamics at the optimum growth temperature. Using flux sampling, the model shows high similarity to experimentally determined reaction fluxes with both glucose and xylose as sole carbon sources. Furthermore, the model predicts previously unidentified by-products, closing the gap in the carbon balance for both carbon sources. Finally, iGEL601 was used to suggest metabolic engineering strategies to maximise production of five industrially relevant compounds. The suggested strategies have previously been experimentally verified in other microorganisms, and predicted production rates are on par with or higher than those previously achieved experimentally. The results highlight the biotechnological potential of LC300 and the application of iGEL601 for use as a tool in the metabolic engineering workflow.

sentences: A family of cell wall transglutaminases is essential for appressorium development and pathogenicity in Phytophthora infestans Transglutaminases (TGases) are enzymes highly conserved among prokaryotic and eukaryotic organisms, where their role is to catalyse protein cross-linking. One of the putative TGases of Phytophthora infestans has previously been shown to be localised to the cell wall. Based on sequence similarity we were able to identify six more genes annotated as putative TGases and show that these seven genes group together in phylogenetic analysis. All of the seven proteins are predicted to contain transmembrane helices and both a TGase domain and a MANSC domain, the latter of which was previously shown to play a role in protein stability. Chemical inhibition of transglutaminase activity and silencing of the entire family of the putative cell wall TGases are both lethal to P. infestans indicating the importance of these proteins in cell wall formation and stability. The intermediate phenotype obtained with lower drug concentrations and less efficient silencing displays a number of deformations to germ tubes and appressoria. Both chemically treated and silenced lines show lower pathogenicity than the wild type in leaf infection assays. Finally, we show that appressoria of P. infestans possess the ability to build up turgor pressure and that this ability is decreased by chemical inhibition of TGases.

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Longitudinal high-resolution imaging through a flexible intravital imaging window Intravital microscopy (IVM) is a powerful technique that enables imaging of internal tissues at (sub)cellular resolutions in living animals. Here, we present a silicone-based imaging window consisting of a fully flexible, suture-less design that is ideally suited for long-term, longitudinal IVM of growing tissues and tumors. Crucially, we show that this window, without any customization, is suitable for numerous anatomical locations in mice using a rapid and standardized implantation procedure. This low-cost device represents a substantial technological and performance advance that facilitates intravital imaging in diverse contexts in higher organisms, opening new avenues for in vivo imaging of soft and fragile tissues.

One-sentence summaryThis study presents a versatile, fully flexible imaging window that acts as an implantable transparent  second skin for small laboratory animal in vivo imaging.

sentences: In vitro and in vivo co-infection and super-infection dynamics of Mayaro and Zika viruses in mosquito and vertebrate backgrounds Factors related to increasing globalization and climate change have contributed to the simultaneous increase and spread of arboviral diseases. Co-circulation of multiple arboviruses in the same geographic regions provides impetus to study the impacts of multiple arbovirus infections in a single vector. In the present study we describe co-infection and super-infection with Mayaro virus (Family Togaviridae, genus Alphavirus) and Zika virus (family Flaviviridae, genus Flavivirus) in vertebrate cells, mosquito cells, and Aedes aegypti mosquitoes to understand the interaction dynamics of these pathogens and effects on viral infection, dissemination and transmission. In Aedes aegypti mosquitoes, co-infection has a negative impact on infection and dissemination rates for Zika virus, but not Mayaro virus, when compared to single infection scenarios, and super-infection of Mayaro virus with a previous Zika virus infection resulted in increased Mayaro virus infection rates. We found that co-infection and super-infection negatively affected Zika viral replication in vertebrate cells (Vero and Huh), resulting in the complete blockage of Zika virus replication in some scenarios. At the cellular level, we demonstrate that single vertebrate and insect cells can be simultaneously infected with Zika and Mayaro viruses. This study highlights the dynamics of arboviral co- and super-infections and emphasizes the importance of considering these dynamics during risk assessment in epidemic areas.

sentences: The rapid developmental rise of somatic inhibition disengages hippocampal dynamics from self-motion Early electrophysiological brain oscillations recorded in preterm babies and newborn rodents are initially mostly ignited by bottom-up sensorimotor activity and only later can detach from external inputs. This is a hallmark of most developing brain areas including the hippocampus, which in the adult brain, functions in integrating external inputs onto internal dynamics. Such developmental disengagement from external inputs is likely a fundamental step for the proper development of cognitive internal models. Despite its importance, the developmental timeline and circuit basis for this disengagement remain unknown. To address this issue, we have investigated the daily evolution of CA1 dynamics and underlying circuits during the first two postnatal weeks of mouse development using two-photon calcium imaging in non-anesthetized pups. We show that the first postnatal week ends with an abrupt shift in the representation of self-motion in CA1. Indeed, most CA1 pyramidal cells switch from activated to inhibited by self-generated movements at the end of the first postnatal week whereas the majority of GABAergic neurons remain positively modulated throughout this period. This rapid switch occurs within two days and follows the rapid anatomical and functional surge of local somatic GABAergic innervation. The observed change in dynamics is consistent with a two-population model undergoing a strengthening of inhibition. We propose that this abrupt developmental transition inaugurates the emergence of internal cognitive models.

sentences: Oxidized thioredoxin-1 restrains the NLRP1 inflammasome At least six human proteins detect danger-associated signals, assemble into complexes called inflammasomes, and trigger pyroptotic cell death. NLRP1 was the first protein discovered to form an inflammasome, but the danger signals and molecular mechanisms that control its activation have not yet been fully established. Here, we report that the NACHT-LRR region of NLRP1 directly binds to oxidized form of thioredoxin-1 (TRX1). We found that NLRP1 requires the ATPase activity of its NACHT domain to associate with TRX1, and that this interaction represses inflammasome activation. Moreover, we discovered that several patient-derived missense mutations in the NACHT-LRR region of NLRP1 weaken TRX1 binding, leading to inflammasome hyperactivation and autoinflammatory disease. Overall, our results establish that oxidized TRX1 binds to and restrains the NLRP1 inflammasome, thereby revealing a link between the cellular redox environment and innate immunity.

sentences: Plant-associated microbiomes promote nutrient turnover in impoverished substrates of a biodiversity hotspot The substrates of the Brazilian campos rupestres have extremely low concentrations of key nutrients, mainly phosphorus, imposing severe restrictions to plant growth. Regardless, this ecosystem harbors enormous biodiversity which raises the question of how nutrients are cycled and acquired by the biosphere. To uncover the nutrient turnover potential of plant-associated microorganisms in the campos rupestres, we investigated the compositions and functions of microbiomes associated with two species of the Velloziaceae family that grow over distinct substrates (soil and rock). Amplicon, metagenomic, and metagenome-assembled genome sequence data showed that the campos rupestres harbor a novel assemblage of plant-associated prokaryotes and fungi. Compositional analysis revealed that the plant-associated soil and rock communities differed in taxonomic structure but shared a core of highly efficient colonizers that were strongly coupled with nutrient mobilization. Investigation of functional and abundance data revealed that the plant hosts actively recruit communities by exuding organic compounds and that the root-associated microbiomes possess a diverse repertoire of phosphorus turnover mechanisms. We also showed that the microbiomes of both plant species encompass novel populations capable of mobilizing nitrogen and that the substrate strongly influences the dynamics of this cycle. Our results show that the interplay between plants and their microbiomes shapes nutrient turnover in the campos rupestres. We highlight that investigation of microbial diversity is fundamental to understand plant fitness in stressful environments.

sentences: Protein-protein interaction map in pre-eclampsia through the interaction of hub genes, transcription factors and microRNAs Pre-eclampsia causes complications in pregnancy and characterized by uremia, proteinuria and hypertension in unattended cases. Blood biomarkers for pre-eclampsia are lacking. In this study, microarray gene expression data from peripheral blood of pre-eclampsia women was analyzed. In our study we developed a combined network approach for hub node prediction regulated by transcription factors and microRNAs corresponding to pre-eclampsia. Differentially expressed genes (DEGs) interaction map was constructed using STRING database. JUN, RPL35, NDUFB2, ATP5I, UQCRQ, COX7C, and FN1 were predicted as potential novel hub genes. Pathway analysis showed metabolic pathways, cytokine signaling in the immune system, Wnt, and MAPK signaling pathways involvement in pre-eclampsia. Regulatory network analysis showed that transcription factors JUN and STAT1 were connected with hub nodes, and microRNAs (miRNAs) like hsa-miR-26b-5p and hsa-miR-155-5p. In conclusion, the expression pattern of hub genes, analyzed deciphers a molecular signature for understanding the pathophysiology of pre-eclampsia and prediction of biomarkers for diagnosis.

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An Engineered Contact Lens for Passive and Sustained Release of Lifitegrast, an Anti-Dry Eye Syndrome Drug Lifitegrast is an FDA-approved drug that inhibits T-cell mediated inflammation associated with dry eye syndrome (DES). Lifitegrast is a potent inhibitor of the interaction between LFA-1 on T-cells and ICAM-1 on endothelial cells at the ocular surface. While effective in treating DES, 5% (81.2 mM) lifitegrast has low drug utilization and elicits off-target effects. Here we engineer contact lenses to release therapeutically-relevant doses of lifitegrast to every tear film for up to 10-hours. Lifitegrast is coupled to the polymer of the soft hydrogel lens via a photolabile (caged) crosslinker. Exposures of the lens to the 400-430 nm wavelengths of indoor daylight excite the caged crosslinker molecules and trigger a bond-cleavage reaction that releases authentic lifitegrast passively to the tear film. The photoproduct of the reaction remains chemically-linked to the polymer of the single-use lens. Our studies show that passive exposures of the lens to indoor light would generate an average of 990 nM lifitegrast to every tear film in a zero-order reaction for up to 10-hours. This concentration exceeds the Kd for the interaction between ICAM-1 and LFA-1 by [~]330-fold and would sustain inhibition of inflammatory responses at the ocular surface. The amount of lifitegrast released from the lens increases during exposures to outdoor sunlight. Over a 10-hour exposure to indoor light, a single lens would release 0.44% of the lifitegrast present in two drops of commercial 5% lifitegrast. Compared to tear-drop approaches, our engineered lenses would sustain the passive delivery of therapeutically-relevant doses of lifitegrast over a longer period, and exhibit improved drug utilization at a lower cost. Our technology could easily be integrated into daily-use contact lenses in order to prevent inflammation at the ocular surface, dry-eye and contact lens-mediated discomfort.

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Hypoxic Preconditioning Promotes Survivals of Human Adipocyte Mesenchymal Stem Cell via Expression of Prosurvival and Proangiogenic Biomarkers BackgroundContributing factors for improved survival of human adipocytes mesenchymal stem cells (h-AMSCs) cultured through hypoxia preconditioning, in example apoptosis inhibition involving BCL2 and HSP27 expression, trigger signal expression (VEGF), SCF expression, OCT-4 expression, and CD44+ expression.

ObjectiveTo explain the mechanism and role of hypoxic preconditioning and the optimal duration of hypoxic preconditioning exposure to improve survival of h-AMSCs so that could it could be used as a benchmark for h-AMSCs culture strategy before transplantation.

MethodsThis study was an experimental laboratory explorative study (in vitro study) with hypoxic preconditioning in human-adipose mesenchymal stem cells (h-AMSCs) cultures. This research was conducted through 4 stages ;First, Isolation and h-AMSCs culture from adipose tissue of patient (human). Second is the characterization of h-AMSCs from adipose tissue by phenotype (Flowcytometry) through CD44+, CD90+ and CD45-expression before being pre-conditioned for hypoxic treatment. Third, the hypoxic preconditioning in h-AMSCs culture (in vitro) was performed with an oxygen concentration of 1% for 24, 48 and 72 hours. Fourth, observation of survival from h-AMSCs culture was tested on the role of CD44 +, VEGF, SCF, OCT-4, BCL2, HSP27 with Flowcytometry method and apoptotic inhibition by Tunnel Assay method.

ResultsThe result of regression test showed that time difference had an effect on VEGF expression (p=0,000; {beta}=-0,482) and hypoxia condition also influenced VEGF expression (p= 0,000; {beta}=0,774). The result of path analysis showed that SCF had an effect on OCT-4 expression (p=0,000; {beta}=0,985). The regression test results showed that time effects on HSP27 expression (p=0.000; {beta}=0.398) and hypoxia precondition also affects HSP27 expression (p=0.000; {beta}=0.847). Pathway analysis showed that BCL2 expression inhibited apoptosis (p=0.030; {beta}=-0.442) and HSP27 expression also inhibited apoptosis (p=0,000; {beta}=-0.487).

ConclusionIn conclusion, hypoxic preconditioning of h-AMSC culture has proven to increase the expression of VEGF, SCF, OCT-4, and BCL2 and HSP27. This study demonstrated and explained the existence of a new mechanism of increased h-AMSC survival in cultures with hypoxic preconditioning (O2 1%) via VEGF, SCF, OCT-4, BCL2, and HSP 27. But CD 44+ did not play a role in the mechanism of survival improvement of human AMSC survival.

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A novel biosynthetic gene cluster across the Pantoea species complex is important for pathogenicity in onion Onion center rot is caused by at least four species of Pantoea (P. ananatis, P. agglomerans, P. allii, and P. stewartii subsp. indologenes). Critical onion pathogenicity determinants for P. ananatis were recently described but whether those determinants are common among other onion-pathogenic Pantoea species remains unknown. In this work, we report onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii. We identified two distinct secondary metabolite biosynthetic gene clusters in different strains of onion pathogenic P. stewartii subsp. indologenes. One cluster is similar to the previously described HiVir phosphonate biosynthetic cluster of P. ananatis and another is a novel putative phosphonate biosynthetic gene cluster, which we name "Halophos". The Halophos gene cluster was also identified in P. allii strains. Both clusters are predicted to be phosphonate biosynthetic clusters based on the presence of a characteristic phosphenolpyruvate phosphomutase (pepM) gene. The deletion of pepM gene from either the P. stewartii subsp. indologenes HiVir or Halophos clusters caused loss of necrosis on onion leaves and red onion scales, and resulted in significantly lower bacterial populations compared to the corresponding wildtype and complemented strains. Seven (halB-halH) out of eleven genes (halA-halK) in the Halophos gene cluster are required for onion necrosis phenotypes. The onion non-pathogenic strain PNA15-2 gained the capacity to cause foliar necrosis on onion via exogenous expression of a minimal seven gene Halophos cluster (halB -halH). Furthermore, cell-free culture filtrates of PNA14-12 expressing the intact Halophos-gene cluster caused necrosis on onion leaves consistent with the presence of a secreted toxin. Together, these observations indicated that pepM genes in both phosphonate biosynthetic gene clusters (HiVir and Halophos) are important for Pantoea spp. onion pathogenicity and the biosynthetic product of the Halophos cluster causes necrosis on onion leaf tissue. Overall, this is the first report of onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii.

Author summaryOnion center rot is caused by multiple Pantoea species including P. stewartii subsp. indologenes and P. allii. We identified two distinct secondary metabolite biosynthetic clusters associated with onion pathogenic strains, the validated HiVir phosphonate cluster and a putative phosphonate biosynthetic cluster that we named as Halophos based on the associated "halo" phenotype on the red onion scales. We found that pepM genes from each cluster (HiVir and Halophos) are required for onion infection by P. stewartii subsp. indologenes and P. allii but not for millet infection by P. stewartii subsp. indologenes. Conversely, the T3SS was important for millet infection by P. stewartii subsp. indologenes but not onion infection. Induction of the intact Halophos cluster was associated with the accumulation of a necrosis-inducing factor in culture, which suggests it might be a secreted phytotoxin. Seven of the eleven Halophos cluster genes are required for onion necrosis phenotypes and expression of this minimal cluster conferred a limited onion necrosis phenotype to an onion-non-pathogenic Pantoea strain. We provide evidence of a Halophos biosynthetic gene cluster to be associated with onion pathogenicity in strains of P. stewartii subsp. indologenes and P. allii.

sentences: Multi-omics profiling of Earth's biomes reveals that microbial and metabolite composition are shaped by the environment As our understanding of the structure and diversity of the microbial world grows, interpreting how it functions is of critical interest for understanding and managing the many systems microbes influence. Despite advances in sequencing, lack of standardization challenges comparisons among studies that could provide global insight. Here, we present a multi-omics, meta-analysis of a novel, diverse set of microbial community samples collected for the Earth Microbiome Project. Standardized protocols and analytical methods for characterizing microbial communities, including assessment of function using untargeted metabolomics, facilitate identification of shared microbial and metabolite features, permitting us to explore diversity at extraordinary scale. In addition to a reference database for metagenomic and metabolomic data, we provide a framework for incorporating additional studies, enabling the expansion of existing knowledge in the form of a community resource that will become more valuable with time. To provide an example of applying this database, we tested the hypothesis that metabolic potential is not limited or enriched by the environment. Our results show that the relative abundances of microbially-related metabolites vary across habitats, and highlight the power of certain chemistry - in particular alkaloids - in distinguishing Earths environments.

sentences: The impact of phosphorylated Pten at threonine 366 on cortical connectivity and behaviour The lipid phosphatase Pten (phosphatase and tensin homologue on chromosome 10) is a key tumour suppressor gene and an important regulator of neuronal signalling. Pten mutations have been identified in patients with autism spectrum disorders, characterized by macrocephaly, impaired social interactions and communication, repetitive behaviour, intellectual disability, and epilepsy. Pten enzymatic activity is regulated by a cluster of phosphorylation sites at the C-terminus of the protein. Here we specifically focussed on the role of Pten T366 phosphorylation and generated a knock-in mouse line in which Pten T366 was substituted with alanine (PtenT366A/T366A). We identify that phosphorylation of Pten at T366 controls neuron size and connectivity of brain circuits involved in sensory processing. We show in behavioural tests that PtenT366/T366A mice exhibit cognitive deficits and selective sensory impairments, with significant differences in male individuals. We identify restricted cellular overgrowth of cortical neurons in PtenT366A/T366A brains, linked to increases in both dendritic arborization and soma size. In a combinatorial approach of anterograde and retrograde monosynaptic tracing using rabies virus, we characterize differences in connectivity to the primary somatosensory cortex of PtenT366A/T366A brains, with imbalances in long-range cortico-cortical input to neurons. We conclude that phosphorylation of Pten at T366 controls neuron size and connectivity of brain circuits involved in sensory processing and propose that PTEN T366 signalling may account for a subset of autism-related functions of Pten.

sentences: An Endogenously activated antiviral state restricts SARS-CoV-2 infection in differentiated primary airway epithelial cells Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease-19 (COVID-19) pandemic, was identified in late 2019 and went on to cause over 3.3 million deaths in 15 months. To date, targeted antiviral interventions against COVID-19 are limited. The spectrum of SARS-CoV-2 infection ranges from asymptomatic to fatal disease. However, the reasons for varying outcomes to SARS-CoV-2 infection are yet to be elucidated. Here we show that an endogenously activated interferon lambda (IFN{lambda}) pathway leads to resistance against SARS-CoV-2 infection. Using a well-differentiated primary nasal epithelial cell (WD-PNEC) model from multiple adult donors, we discovered that susceptibility to SARS-CoV-2 infection, but not respiratory syncytial virus (RSV) infection, varied. One of four donors was resistant to SARS-CoV-2 infection. High baseline IFN{lambda} expression levels and associated interferon stimulated genes correlated with resistance to SARS-CoV-2 infection. Inhibition of the JAK/STAT pathway in WD-PNECs with high endogenous IFN{lambda} secretion resulted in higher SARS-CoV-2 titres. Conversely, prophylactic IFN{lambda} treatment of WD-PNECs susceptible to infection resulted in reduced viral titres. An endogenously activated IFN{lambda} response, possibly due to genetic differences, may be one explanation for the differences in susceptibility to SARS-CoV-2 infection in humans. Importantly, our work supports the continued exploration of IFN{lambda} as a potential pharmaceutical against SARS-CoV-2 infection.

sentences: Graph neural network based heterogeneous propagation scheme for classifying Alzheimer's disease Deep learning frameworks for disease classification using neuroimaging and non-imaging information require the capability of capturing individual features as well as associative information among subjects. Graphs represent the interactions among nodes, which contain the individual features, through the edges in order to incorporate the inter-relatedness among heterogeneous data. Previous graph-based approaches for disease classification have focused on the similarities among subjects by establishing customized functions or solely based on imaging features. The purpose of this paper is to propose a novel graph-based deep learning architecture for classifying Alzheimers disease (AD) by combining the resting-state functional magnetic resonance imaging and demographic measures without defining any study-specific function. We used the neuroimaging data from the ADNI and OASIS databases to test the robustness of our proposed model. We combined imaging-based and non-imaging information of individuals by categorizing them into distinctive nodes to construct a subject-demographic bipartite graph. The approximate personalized propagation of neural predictions, a recently developed graph neural network model, was used to classify the AD continuum from cognitively unimpaired individuals. The results showed that our model successfully captures the heterogeneous relations among subjects and improves the quality of classification when compared with other classical and deep learning models, thus outperforming the other models.

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Multiplatform Computational Analysis of Mast Cells in Adrenocortical Carcinoma Tumor Microenvironment IntroductionImmunotherapeutic response failure of adrenocortical carcinomas (ACC) highlights a need for novel strategies targeting immune cell populations in the tumor microenvironment (TME) to overcome tumor resistance and enhance therapeutic response. A recent study explored a new link between tumor mast cell (MC) infiltration and improved outcomes in patients with ACC. We further dissect the role of MC in TME of ACC by examining the tumor MC expression signatures and MC activity within TME to provide additional insight into potential novel immunotherapeutic targets.

MethodsUsing CIBERSORTx computational immunogenomic deconvolution algorithm to analyze ACC tumor gene mRNA expression data (TCGA, N=79), we estimated the abundance of tumor immune infiltrating MC, and assessed prognostic potential of MC signaling genes as pro- or anti- tumor signatures, as well as the impact on overall (OS) and disease-free (DFS) survival.

ResultsWe stratified MC signaling genes with survival prognostic values (OS, DFS, p<0.05) into anti-tumor (ALOX5, CCL2, CCL5, CXCL10, HDC, IL16, TNF, TPSAB1, VEGFD) and pro-tumor (CXCL1, CXCL3, CXCL8, IL4, IL13, PTGS3, TNSF4, VEGFD) groups. Anti-tumor MC signature, as the predominant phenotype, was associated with improved OS and DFS.

ConclusionThe deconvolution analysis of TCGA data identified MC infiltration in ACC microenvironment as predominantly associated with anti-tumor activity. Future studies stemming from our findings may help define the role of MC in TME and the impact on patient survival in patients with ACC. Modulation of tumor MC infiltration may serve as a potential target for novel synergistic immunotherapies for the treatment and improved survival of patients with ACC.

sentences: Resting-state network hubs are causally required in memory consolidation Resting-state networks (RSNs) detected by fMRI have been associated with cognitive function, providing insights to the relationship between brain and behavior. However, whether altered RSNs are epiphenomena or essential constructs of behavior remains unclear. Here we investigated whether post-encoding RSN hubs that are commonly engaged after similar tasks or integrate distributed areas into large networks are causally involved in memory consolidation. RSN changes following two types of spatial memory training in mice, allowing us to distinguish post-encoding hubs related to spatial memory, and verify their behavioral impact by hub inhibition. We found that functional connectivity with sensory areas was commonly strengthened in both tasks whereas frontal and striatal areas were influential in network integration. Chemogenetic suppression of each hub after learning resulted in retrograde amnesia. These results demonstrate causal and functional roles of RSN hubs in system consolidation and validate fMRI as a means to track this process.

sentences: Microbial metabolite butyrate-prodrug polymeric micelles promote gut health and treat food allergies The gut microbiome modulates the bodys response to food antigens1. Beneficial taxa, specifically butyrate-producing Clostridia, are depleted in food-allergic individuals2,3. Although butyrate is known to play important roles in regulating gut immunity and maintaining epithelial barrier function4-6, its clinical translation is challenging due to its offensive odor and quick absorption in the upper gut. Here, we developed two polymeric micelle systems, one with a neutral charge (NtL-ButM) and one with a negative charge (Neg-ButM), that release butyrate from their polymeric core in the ileum or the cecum, respectively. Treatment with NtL-ButM in germ-free (and thus butyrate-depleted) mice up-regulated genes expressing antimicrobial peptides in the ileal epithelium. We show that these butyrate-containing micelles, used in combination, restore a barrier-protective response in mice treated with either dextran sodium sulfate or antibiotics. Treatment with the micelles protects peanut-allergic mice from an anaphylactic reaction to peanut challenge and rescues their dysbiosis by increasing the abundance of Clostridium Cluster XIVa. By restoring microbial and mucosal homeostasis, these butyrate-prodrug polymeric micelles may function as a new, antigen-agnostic approach to the treatment of food allergy.

sentences: DomeVR: A setup for experimental control of an immersive dome virtual environment created with Unreal Engine 4 1Immersive virtual reality (VR) environments are a powerful tool to explore cognitive processes ranging from memory and navigation to visual processing and decision making - and to do so in a naturalistic yet controlled setting. As such, they have been employed across different species, and by a diverse range of research groups. Unfortunately, designing and implementing behavioural tasks in such environments often proves complicated. To tackle this challenge, we created DomeVR, an immersive VR environment built using Unreal Engine 4 (UE4). UE4 is a powerful game engine with photo-realistic graphics containing a visual scripting language designed for use by non-programmers. As a result, virtual environments are easily created using drag-and-drop elements. DomeVR aims to make these features accessible to neuroscience experiments. This includes a logging and synchronization system to solve timing uncertainties inherent in UE4; an interactive GUI for scientists to observe subjects during experiments and adjust task parameters on the fly, and a dome projection system for full task immersion in non-human subjects. These key features are modular and can easily be added individually into other UE4 projects. Finally, we present proof-of-principle data highlighting the functionality of DomeVR in three different species: human, macaque and mouse.

sentences: Gene editing in bacteria via SpCas9/sgRNA ribonucleoprotein complexes Gene editing has been revolutionized by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas technology in a variety of organisms. In bacteria, however, CRISPR-Cas still holds many caveats such as high toxicity of Cas9 and off-target editing effects. In this work we develop a system for the incorporation of Cas9/single guide RNA ribonucleoprotein complexes in bacteria and their successful application for gene editing via homologous recombination. Targeting of a green fluorescent protein (GFP) reporter allows for easy verification of gene-edition via conversion to blue-fluorescent protein (BFP), mediated by the well characterized 196T > C (Tyr66His) mutation.

sentences: Lactobacillus rhamnosus reduces the cytotoxic effects of Group B Streptococcus on HeLa cells Group B Streptococcus (GBS) is an opportunistic pathogen found in the vaginal tract and is a leading cause of preterm birth and neonatal illness. Aside from GBS, the vaginal tract is predominantly colonized by commensal Lactobacillus species that are thought to protect the vaginal tract from pathogens, including GBS. Studies that examined if, and how Lactobacilli modulate GBS pathogenicity remain limited. This study sought to investigate the potential protective role of Lactobacillus rhamnosus against GBS, using an in vitro model system. Immunofluorescence microscopy and Scanning Electron Microscopy (SEM) captured images of infected HeLa cells and were analyzed using the image analysis program ImageJ. Results indicate that GBS causes HeLa cell detachment unless L. rhamnosus is present. SEM images show that GBS reduces length and number of microvilli on HeLa cell surface, as well as size of secreted vesicles. L. rhamnosus partially inhibits GBS-dependent microvilli and vesicle disruption. GBS also disrupts HeLa cell F-actin fibers unless L. rhamnosus is present. These results reveal effects of GBS infection on the host cell cytoskeleton and implies a protective role of L. rhamnosus against GBS colonization.

sentences: Disruption of miR-18a alters proliferation, photoreceptor replacement kinetics, inflammatory signaling and microglia/macrophage numbers during retinal regeneration in zebrafish In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), photoreceptor loss reprograms Muller glia to function as stem cells, producing progenitors that regenerate photoreceptors. MicroRNAs (miRNAs) regulate CNS neurogenesis, but the roles of miRNAs in injury-induced neuronal regeneration are largely unknown. In the embryonic zebrafish retina, miR-18a regulates photoreceptor differentiation. The purpose of the current study was to determine, in zebrafish, the function of miR-18a during injury-induced photoreceptor regeneration. RT-qPCR, in-situ hybridization and immunohistochemistry showed that miR-18a expression increases throughout the retina between 1 and 5 days post-injury (dpi). To test miR-18a function during photoreceptor regeneration, we used homozygous miR-18a mutants (miR-18ami5012), and knocked down miR-18a with morpholino oligonucleotides. During photoreceptor regeneration, miR-18ami5012 retinas have fewer mature photoreceptors than WT at 7 and 10 dpi, but there is no difference at 14 dpi, indicating that photoreceptor regeneration is delayed. Labeling dividing cells with 5-bromo-2'-deoxyuridine (BrdU) showed that at 7 and 10 dpi, there are excess dividing progenitors in both mutants and morphants, indicating that miR-18a negatively regulates injury-induced proliferation. Tracing 5-ethynyl-2'-deoxyuridine (EdU) and BrdU-labeled cells showed that in miR-18ami5012 retinas excess progenitors migrate to other retinal layers in addition to the photoreceptor layer. Inflammation is critical for photoreceptor regeneration, and RT-qPCR showed that in miR-18ami5012 retinas, inflammatory gene expression and microglia activation are prolonged. Suppressing inflammation with dexamethasone rescues the miR-18ami5012 phenotype. Together, these data show that in the injured zebrafish retina, disruption of miR-18a alters proliferation, inflammation, the microglia/macrophage response, and the timing of photoreceptor regeneration.

sentences: IDSL.CCDB: a database for exploring inter-chemical correlations in metabolomics and exposomics datasets Inter-chemical correlations in metabolomics and exposomics datasets provide valuable information for studying relationships among reported chemicals measured in human specimens. With an increase in the size of these datasets, a network graph analysis and visualization of the correlation structure is difficult to interpret. While co-regulatory genes databases have been developed, a similar database for metabolites and chemicals have not been developed yet. We have developed the Integrated Data Science Laboratory for Metabolomics and Exposomics - Chemical Correlation Database (IDSL.CCDB), as a systematic catalogue of inter-chemical correlation in publicly available metabolomics and exposomics studies. The database has been provided via an online interface to create single compound-centric views that are clear, readable and meaningful. We have demonstrated various applications of the database to explore: 1) the chemicals from a chemical class such as Per- and Polyfluoroalkyl Substances (PFAS), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), phthalates and tobacco smoke related metabolites; 2) xenobiotic metabolites such as caffeine and acetaminophen; 3) endogenous metabolites (acyl-carnitines); and 4) unannotated peaks for PFAS. The database has a rich collection of 36 human studies, including the National Health and Nutrition Examination Survey (NHANES) and high-quality untargeted metabolomics datasets. IDSL.CCDB is supported by a simple, interactive and user-friendly web-interface to retrieve and visualize the inter-chemical correlation data. The IDSL.CCDB has the potential to be a key computational resource in metabolomics and exposomics facilitating the expansion of our understanding about biological and chemical relationships among metabolites and chemical exposures in the human body. The database is available at www.ccdb.idsl.me site.

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Proteomics profiling reveals regulation of immune response to Salmonella Typhimurium infection in mice Regulation of the immune response to Salmonella typhimurium (S. Typhimurium) infection is a complex process, influenced by genetic and environmental factors. Different inbred mouse strains show distinct levels of resistance to S. Typhimurium infection, ranging from susceptible (e.g., C57BL/6J) to resistant (e.g., DBA/2J) strains. However, the underlying molecular mechanisms contributing to the host response remain elusive. In this study, we present a comprehensive proteomics profiling of the spleen tissue from C57BL/6J and DBA/2J strains with different doses of S. Typhimurium infection by tandem tag mass coupled with two-dimensional liquid chromatography-tandem mass spectrometry. We identified and quantified 3,986 proteins, resulting in 475 differentially expressed proteins between C57BL/6J and DBA/2J strains. Functional enrichment analysis revealed that the mechanisms of innate immune responses to S. Typhimurium infection are associated with several signaling pathways, including the interferon signaling pathway. Our proteomic data also discovered a plausible gene in a genomic region that control different levels of resistance to S. Typhimurium infection. We further revealed the roles of macrophage cells and pro-inflammatory cytokines in the mechanisms under the natural resistance to S. Typhimurium. In summary, our results provide new insights into the genetic regulation of the immune response to S. Typhimurium infection in mice.

Author SummarySalmonella infection (salmonellosis) is a common zoonotic disease that mainly propagates through contaminated food and drink. Various mouse strains display prominent disparities in responses to Salmonella invasion. Elucidating the heterogeneous immune reactions between different mouse strains can shed light on the fundamental molecular mechanisms of the innate immune system. Here, we employed a combination of proteomics and systems biology approaches to provide an unprecedented panorama of the inextricably interlaced immune signaling pathways in response to Salmonella infection in mice. Our results revealed the dynamics of cell signaling molecules elicited by inflammation and established new connections among them. We also identified a new candidate gene involved in the combat with pathogens. Our proteomic data and results contribute to understanding the intricate interactions of immune responses to Salmonella infection from molecular to systemic levels.

sentences: Local regulation of extracellular vesicle traffic by the synaptic endocytic machinery Neuronal extracellular vesicles (EVs) can be locally released from presynaptic terminals, carrying cargoes that are important in intercellular signaling and disease. EVs are derived from endosomes, but it remains unclear how synaptic cargoes are directed to the EV pathway, rather than undergoing conventional retrograde endosomal transport and degradation. Here, we find that the clathrin-mediated endocytic machinery plays an unexpected role in maintaining a release-competent pool of synaptic EV cargoes. Endocytic mutants, including nervous wreck (nwk), Shibire/Dynamin, and AP-2, exhibit local depletion of multiple cargoes in EV donor terminals. Accordingly, nwk mutants phenocopy synaptic plasticity defects associated with loss of the EV cargo Synaptotagmin-4, and suppress lethality upon overexpression of the EV cargo Amyloid Precursor Protein. These EV defects are genetically separable from canonical functions of endocytic proteins in synaptic vesicle recycling and synaptic growth. This endocytic pathway opposes the endosomal retromer complex to regulate EV cargo levels, and acts upstream of synaptic cargo removal by retrograde axonal transport. Our data suggest a novel molecular mechanism that protects EV cargoes from local depletion at synapses.

sentences: Collagen has a unique SEC24 preference for efficient export from the endoplasmic reticulum SEC24 is mainly involved in cargo sorting during COPII vesicle assembly. There are four SEC24 paralogs (A to D) in mammals, which are classified into two subgroups (SEC24A/B and SEC24C/D). Pathological mutations in SEC24D cause osteogenesis imperfecta with craniofacial dysplasia in humans. sec24d mutant fish also recapitulate the phenotypes. Consistent with the skeletal phenotypes, the secretion of collagen was severely defective in mutant fish, emphasizing the importance of SEC24D in collagen secretion. However, SEC24D patient-derived fibroblasts show only a mild secretion phenotype, suggesting tissue-specificity in the secretion process. Using Sec24d KO mice and cultured cells, we show that SEC24A and SEC24B also contribute to ER export of procollagen. In contrast, fibronectin 1 requires either SEC24C or SEC24D for ER export. On the basis of our results, we propose that procollagen interacts with multiple SEC24 paralogs for efficient export from the ER, and that this is the basis for tissue-specific phenotypes resulting from SEC24 paralog deficiency.

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Crowdsourced identification of multi-target kinase inhibitors for RET- and TAU- based disease: the Multi-Targeting Drug DREAM Challenge A continuing challenge in modern medicine is the identification of safer and more efficacious drugs. Precision therapeutics, which have one molecular target, have been long promised to be safer and more effective than traditional therapies. This approach has proven to be challenging for multiple reasons including lack of efficacy, rapidly acquired drug resistance, and narrow patient eligibility criteria. An alternative approach is the development of drugs that address the overall disease network by targeting multiple biological targets ( polypharmacology). Rational development of these molecules will require improved methods for predicting single chemical structures that target multiple drug targets. To address this need, we developed the Multi-Targeting Drug DREAM Challenge, in which we challenged participants to predict single chemical entities that target pro-targets but avoid anti-targets for two unrelated diseases: RET-based tumors and a common form of inherited Tauopathy. Here, we report the results of this DREAM Challenge and the development of two neural network-based machine learning approaches that were applied to the challenge of rational polypharmacology. Together, these platforms provide a potentially useful first step towards developing lead therapeutic compounds that address disease complexity through rational polypharmacology.

Author SummaryMany modern drugs are developed with the goal of modulating a single cellular pathway or target. However, many drugs are, in fact,  dirty; they target multiple cellular pathways or targets. This phenomenon is known as multi-targeting or polypharmacology. While some strive to develop  cleaner therapeutics that eliminate secondary targets, recent work has shown that multi-targeting therapeutics have key advantages for a variety of diseases. However, while multi-targeting drugs that affect a precisely-defined profile of targets may be more effective, it is difficult to computationally predict which molecules have desirable target profiles. Here, we report the results of a competitive crowdsourcing project (the Multi-Targeting Drug DREAM Challenge), where we challenged participants to predict chemicals that have desired target profiles for cancer and neurodegenerative disease.

sentences: The Role of Glycocalyx Steric Effects on Viral Endocytosis Understanding the mechanisms of viral entry is key to aiding the development of effective anti-viral treatments. In this work, we produce a simple thermodynamic model of viral entry, which is able to predict the differences between viruses. We also show that the glycocalyx, which is often neglected in studies of cell-entry, plays a key role, and the density and structure of the glycocalyx can determine whether or not a virus can enter the cell. We further find that co-receptors play not only a signalling role, but an important thermodynamic role in viral entry. We then show that this model can be used to calculate the cell-entry probabilities of a given virus, which can shine a light on the clinical observations associated with a virus.

sentences: Perineuronal nets in HVC and plasticity in male canary song Songbirds learn their vocalizations during developmental sensitive periods of song memorization and sensorimotor learning. Some seasonal songbirds, called open-ended learners, recapitulate transitions from sensorimotor learning and song crystallization on a seasonal basis during adulthood. In adult male canaries, sensorimotor learning occurs each year in autumn and leads to modifications of the syllable repertoire during successive breeding seasons. We previously showed that perineuronal nets (PNN) expression in song control nuclei decreases during this sensorimotor learning period. Here we explored the causal link between PNN expression in adult canaries and song modification by enzymatically degrading PNN in HVC, a key song control system nucleus. Three independent experiments identified limited effects of the PNN degradation in HVC on the song structure of male canaries. They clearly establish that presence of PNN in HVC is not required to maintain general features of crystallized song. Some suggestion was collected that PNN are implicated in the stability of song repertoires but this evidence is too preliminary to draw firm conclusions and additional investigations should consider producing PNN degradations at specified time points of the seasonal cycle. It also remains possible that once song has been crystallized at the beginning of the first breeding season, PNN no longer play a key role in determining song structure which could be tested by treatments with ChABC at key step in ontogeny. It would in this context be important to develop multiple stereotaxic procedures allowing the simultaneous bilateral degradation of PNN in several song control nuclei for extended periods.

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Single-cell RNA-seq analysis of human coronary arteries using an enhanced workflow reveals SMC transitions and candidate drug targets Background and AimsThe atherosclerotic plaque microenvironment is highly complex, and selective agents that modulate plaque stability or other plaque phenotypes are not yet available. We sought to investigate the human atherosclerotic cellular environment using scRNA-seq to uncover potential therapeutic approaches. We aimed to make our workflow user-friendly, reproducible, and applicable to other disease-specific scRNA-seq datasets.

MethodsHere we incorporate automated cell labeling, pseudotemporal ordering, ligand-receptor evaluation, and drug-gene interaction analysis into an enhanced and reproducible scRNA-seq analysis workflow. Notably, we also developed an R Shiny based interactive web application to enable further exploration and analysis of the scRNA dataset.

ResultsWe applied this analysis workflow to a human coronary artery scRNA dataset and revealed distinct derivations of chondrocyte-like and fibroblast-like cells from smooth muscle cells (SMCs), and show the key changes in gene expression along their de-differentiation path. We highlighted several key ligand-receptor interactions within the atherosclerotic environment through functional expression profiling and revealed several attractive avenues for future pharmacological repurposing in precision medicine. Further, our interactive web application, PlaqView (www.plaqview.com), allows other researchers to easily explore this dataset and benchmark applicable scRNA-seq analysis tools without prior coding knowledge.

ConclusionsThese results suggest novel effects of chemotherapeutics on the atherosclerotic cellular environment and provide future avenues of studies in precision medicine. This publicly available workflow will also allow for more systematic and user-friendly analysis of scRNA datasets in other disease and developmental systems. PlaqView allows for rapid visualization and analysis of atherosclerosis scRNA-seq datasets without the need of prior coding experience. Future releases of PlaqView will feature additional larger scRNA-seq and scATAC-seq atherosclerosis-related datasets, thus providing a critical resource for the field by promoting data harmonization and biological interpretation.

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Prophylactic efficacy of riluzole against anxiety- and depressive-like behaviors in two rodent stress models BackgroundChronic stress-related illnesses, such as major depressive disorder and post-traumatic stress disorder share symptomatology, including anxiety, anhedonia, and helplessness. Across disorders, neurotoxic dysregulated glutamate (Glu) signaling may underlie symptom emergence. Current first-line antidepressant drugs, which do not directly target Glu signaling, fail to provide adequate benefit for many patients and are associated with high relapse rates. Riluzole modulates glutamatergic neurotransmission by increasing metabolic cycling and modulating signal transduction. Clinical studies exploring riluzoles efficacy in stress-related disorders have provided varied results. However, the utility of riluzole for treating specific symptom dimensions or as a prophylactic treatment has not been comprehensively assessed.

MethodsWe investigated whether chronic prophylactic riluzole ([~]12-15/kg/day p.o.) could prevent the emergence of behavioural deficits induced by unpredictable chronic mild stress (UCMS) in mice. We assessed: i) anxiety-like behaviour using the elevated-plus maze, open field test, and novelty-suppressed feeding, ii) mixed anxiety/anhedonia-like behaviour in the novelty-induced hypophagia test and, iii) anhedonia-like behaviour using the sucrose consumption test. Z-scoring summarized changes across tests measuring similar dimensions. In a separate learned helplessness (LH) cohort, we investigated whether chronic prophylactic riluzole treatment could block the development of helplessness-like behaviour.

ResultsUCMS induced an elevation in anxiety-, anhedonia-like behaviour, and overall behavioural emotionality that was blocked by prophylactic riluzole. In the LH cohort, prophylactic riluzole blocked the development of helplessness-like behaviour.

ConclusionThis study supports the utility of riluzole as a prophylactic medication for preventing anhedonia, anxiety, and helplessness symptoms associated with stress-related disorders.

sentences: Delta-range coupling between prefrontal cortex and hippocampus supported by respiratory rhythmic input from the olfactory bulb in freely behaving rats An explosion of recent findings firmly demonstrated that brain activity and cognitive function in rodents and humans are modulated synchronously with nasal respiration. Rhythmic respiratory (RR) coupling of wide-spread forebrain activity was confirmed using advanced techniques, including current source density analysis, single unit firing, and phase modulation of local gamma activity, creating solid premise for investigating how higher networks use this mechanism in their communication. Here we show essential differences in the way prefrontal cortex (PFC) and hippocampus (HC) process the RR signal from the olfactory bulb (OB) allowing dynamic PFC-HC coupling utilizing this input. We used inter-regional coherences and their correlations in rats, breathing at low rate ([~]2 Hz) at rest, outside of the short sniffing bouts. We found strong and stable OB-PFC coherence, contrasting OB-HC coherence which was low but highly variable. PFC-HC coupling, however, primarily correlated with the latter, indicating that HC access to the PFC output is dynamically regulated by the responsiveness of HC to the common rhythmic drive. This pattern was present in both theta and non-theta states of waking, whereas PFC-HC communication appeared protected from RR synchronization in sleep states. The findings help to understand the mechanism of rhythmic modulation of non-olfactory cognitive processes by the on-going regular respiration, reported in rodents as well as humans. These mechanisms may be impaired when nasal breathing is limited or in OB-pathology, including malfunctions of the OB epithelium due to infections, such as in COVID-19.

sentences: MicroRNA-202 safeguards meiotic progression by preventing premature degradation of REC8 mediated by separase MicroRNAs (miRNAs) are believed to play important roles in mammalian spermatogenesis but the in vivo functions of single miRNAs in this highly complex developmental process remain unclear. Here, we reported that miR-202, a member of the let-7 family, played an important role in mouse spermatogenesis by phenotypic evaluation of miR-202 knockout (KO) mice. In miR-202 KO mice, germ cells underwent apoptosis. Multiple processes in meiosis I including synapsis and crossover formation were disrupted, and inter-sister chromatid synapses were detected. More importantly, we found that upon miR-202 KO, meiotic-specific cohesin protein REC8 was prematurely cleaved by precociously activated separase, whose mRNA was a direct target of miR-202-3p. Our findings identify miR-202 as a novel regulator of meiosis and contribute to the list of miRNAs that play specific and important roles in developmental processes.

sentences: The Mismatch Between Neuroscience Graduate Training and Professional Skill Sets Understanding the skill sets required for career paths is a prerequisite for preparing students for those careers. Neuroscience career paths are rapidly changing as the field expands and increasingly overlaps with computational and data-heavy job sectors. With the steady growth in neuroscience trainees and the diversification of jobs for those trainees, it is important to assess whether or not our training is matching the skill sets required in the workforce. Here, we surveyed hundreds of neuroscience professionals and graduate students to assess their use and valuation of a range of skills, from bench skills to communication and management. We find that professionals with neuroscience degrees can be clustered into three main groups based on their skill sets: academic research, industry research and technical work, and non-research. Further, we find that while graduate students do not use or highly value management and communication skills, almost all neuroscience professionals report strongly needing those skills. Finally, coding and data analysis skills are widely used in academic and industry research and predict higher salaries. Our findings can help trainees assess their own skill sets as well as encourage educational leaders to offer training in management and communication-skills which may help catapult trainees into the next stages of their careers.

sentences: Estimating the overall fraction of phenotypic variance attributed to high-dimensional predictors measured with error In prospective genomic studies (e.g., DNA methylation, metagenomics, and transcriptomics), it is crucial to estimate the overall fraction of phenotypic variance (OFPV) attributed to the high-dimensional genomic variables, a concept similar to heritability analyses in genome-wide association studies (GWAS). Unlike genetic variants in GWAS, these genomic variables are typically measured with error due to technical limitation and temporal instability. While the existing methods developed for GWAS can be used, ignoring measurement error may severely underestimate OFPV and mislead the design of future studies. Assuming that measurement error variances are distributed similarly between causal and noncausal variables, we show that the asymptotic attenuation factor equals to the average intraclass correlation coefficients of all genomic variables, which can be estimated based on a pilot study with repeated measurements. We illustrate the method by estimating the contribution of microbiome taxa to body mass index and multiple allergy traits in the American Gut Project. Finally, we show that measurement error does not cause meaningful bias when estimating the correlation of effect sizes for two traits.

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Comprehensive multi-omics integration identifies differentially active enhancers during human brain development with clinical relevance BackgroundNon-coding regulatory elements (NCREs), such as enhancers, play a crucial role in gene regulation and genetic aberrations in NCREs can lead to human disease, including brain disorders. The human brain is complex and can be affected by numerous disorders; many of these are caused by genetic changes, but a multitude remain currently unexplained. Understanding NCREs acting during brain development has the potential to shed light on previously unrecognised genetic causes of human brain disease. Despite immense community-wide efforts to understand the role of the non-coding genome and NCREs, annotating functional NCREs remains challenging.

ResultsHere we performed an integrative computational analysis of virtually all currently available epigenome data sets related to human fetal brain. Our in-depth analysis unravels 39,709 differentially active enhancers (DAEs) that show dynamic epigenomic rearrangement during early stages of human brain development, indicating likely biological function. Many of these DAEs are linked to clinically relevant genes, and functional validation of selected DAEs in cell models and zebrafish confirms their role in gene regulation. Compared to enhancers without dynamic epigenomic rearrangement, these regions are subjected to higher sequence constraints in humans, have distinct sequence characteristics and are bound by a distinct transcription factor landscape. DAEs are enriched for GWAS loci for brain related traits and for genetic variation found in individuals with neurodevelopmental disorders, including autism.

ConclusionOur compendium of high-confidence enhancers will assist in deciphering the mechanism behind developmental genetics of the human brain and will be relevant to uncover missing heritability in human genetic brain disorders.

sentences: Geometric and Topological Approaches to Shape Variation in Ginkgo Leaves Leaf shape is a key plant trait that varies enormously. The diversity of leaf shape, and the range of applications for data on this trait, requires frequent methodological developments so that researchers have an up-to-date toolkit with which to quantify leaf shape. We generated a dataset of 468 leaves produced by Ginkgo biloba, and 24 fossil leaves produced by evolutionary relatives of extant Ginkgo. We quantified the shape of each leaf by developing a geometric method based on elastic curves and a topological method based on persistent homology. Our geometric method indicates that shape variation in our modern sample is dominated by leaf size, furrow depth, and the angle of the two lobes at the base of the leaf that is also related to leaf width. Our topological method indicates that shape variation in our modern sample is dominated by leaf size and furrow depth. We have applied both methods to modern and fossil material: the methods are complementary, identifying similar primary patterns of variation, but also revealing some different aspects of morphological variation. Our topological approach distinguishes long-shoot leaves from short-shoot leaves and both methods indicate that leaf shape influences or is at least related to leaf area.

sentences: An efficient method of evaluating multiple concurrent management actions on invasive populations Evaluation of the efficacy of management actions to control invasive species is crucial for maintaining funding and to provide feedback for the continual improvement of management efficiency. However, it is often difficult to assess the efficacy of control methods due to limited resources for monitoring. Many managers view effort on monitoring as effort taken away from performing management actions. We developed a method to estimate invasive species abundance, evaluate management effectiveness, and evaluate population growth overtime from a combination of removal activities (e.g., aerial gunning, trapping, ground shooting) using only data collected during removal efforts (the method of removal, the date, location, number of animals removed, and the effort). This dynamic approach allows for estimating abundance at discrete time points and the estimation of population growth between removal periods. We applied this method to removal data from Mingo National Wildlife Refuge in Missouri from December 2015 to September 2019, where the management objective is elimination. Populations of feral swine on Mingo NWR have fluctuated over time but showed more marked declines in the last 3-6 months of the time series. More dramatic declines were observed in the center of the refuge likely aided by reducing potential immigration from the rest of the refuge. To counteract population growth (from both births and immigration) the percent of the population of feral swine removed monthly must be greater than the growth rate. On average, we found that removing 10% of the population monthly had only a 50% chance of causing a population decline, whereas removing 15% of the population monthly had a 95% chance of causing a population decline. Our method provides advancement over traditional removal modeling approaches because it can be applied to evaluate management programs that use a broad range of removal techniques concurrently and whose management effort and spatial coverage vary across time.

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PASV: Automatic protein partitioning and validation using conserved residues BackgroundIncreasingly, researchers use protein-coding genes from targeted PCR amplification or direct metagenomic sequencing in community and population ecology. Analysis of protein-coding genes presents different challenges from those encountered in traditional SSU rRNA studies. Most protein-coding sequences are annotated based on homology to other computationally-annotated sequences, which can lead to inaccurate annotations. Therefore, the results of sensitive homology searches must be validated to remove false-positives and assess functionality. Multiple lines of in silico evidence can be gathered by examining conserved domains and residues identified through biochemical investigations. However, manually validating sequences in this way can be time consuming and error prone, especially in large environmental studies.

ResultsAn automated pipeline for protein active site validation (PASV) was developed to improve validation and partitioning accuracy for protein-coding sequences, combining multiple sequence alignment with expert domain knowledge. PASV was tested using commonly misannotated proteins: ribonucleotide reductase (RNR), alternative oxidase (AOX), and plastid terminal oxidase (PTOX). PASV partitioned 9,906 putative Class I alpha and Class II RNR sequences from bycatch in a global viral metagenomic investigation with >99% true positive and true negative rates. PASV predicted the class of 2,579 RNR sequences in >98% agreement with manual annotations. PASV correctly partitioned all 336 tested AOX and PTOX sequences.

ConclusionsPASV provides an automated and accurate way to address post-homology search validation and partitioning of protein-coding marker genes. Source code is released under the MIT license and is found with documentation and usage examples on GitHub at https://github.com/mooreryan/pasv.

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High contrast, moving targets in an emerging target paradigm promote fast visuomotor responses during visually guided reaching Humans have a remarkable capacity to rapidly interact with the surrounding environment, often by transforming visual input into motor output on a moment-to-moment basis. But what visual features promote rapid reaching? High contrast, fast-moving targets elicit strong responses in the superior colliculus (SC), a structure associated with express saccades and implicated in rapid electromyographic (EMG) responses on upper limb muscles. To test the influence of stimulus properties on rapid reaches, we had human subjects perform visually guided reaches to moving targets varied by speed (experiment 1) or speed and contrast (experiment 2), in an emerging target paradigm which has recently been shown to robustly elicit fast visuomotor responses. Our analysis focused on stimulus-locked responses (SLRs) on upper limb muscles. SLRs appear within <100 ms of target presentation, and as the first wave of muscle recruitment, they have been hypothesized to arise from the SC. Across 32 subjects studied in both experiments, 97% expressed SLRs in the emerging target paradigm, whereas only 69% expressed SLRs in an immediate response paradigm towards static targets. Faster moving targets (experiment 1) evoked large magnitude SLRs, while high contrast fast moving targets (experiment 2) evoked short latency, large magnitude SLRs. In some instances, SLR magnitude exceeded the magnitude of movement aligned activity. Both large magnitude and short latency SLRs were correlated with short latency reach reaction times. Our results support the hypothesis that, in scenarios requiring expedited responses, a subcortical pathway originating in the SC elicits the earliest wave of muscle recruitment, expediting reaction times.

New & NoteworthyHow does the brain rapidly transform vision into action? Here, by recording upper limb muscle activity, we find that high contrast and fast moving targets are highly effective at evoking rapid visually guided reaches. We surmise that a brainstem circuit originating in the superior colliculus contributes to the most rapid reaching responses. When time is of the essence, cortical areas may serve to prime this circuit, and elaborate subsequent phases of recruitment.

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Estimates of persistent inward currents are reduced in upper limb motor units of older adults Aging is a natural process that causes alterations in the neuromuscular system, which contribute to weakness and reduced quality of life. Reduced firing rates of individual motor units (MUs) likely contribute to weakness, but the mechanisms underlying reduced firing rates are not clear. Persistent inward currents (PICs) are crucial for the initiation, gain control, and maintenance of motoneuron firing, and are directly proportional to the level of monoaminergic input. Since the concentration of monoamines (i.e. serotonin and norepinephrine) are reduced with age, we sought to determine if estimates of PICs are reduced in older (>60 years old) compared to younger adults (<35 years old). We decomposed MU spike trains from high-density surface electromyography over the biceps brachii and triceps brachii during isometric ramp contractions to 20% of maximum. Estimates of PICs (i.e. {Delta}F) were computed using the paired MU analysis technique. Regardless of the muscle, peak firing rates of older adults were reduced by ~1.6 pulses per second (pps) (P = 0.0292), and {Delta}F was reduced by ~1.9 pps (P < 0.0001), compared to younger adults. We further found that age predicted {Delta}F in older adults (P = 0.0261), resulting in a reduction of ~1pps per decade, but there was no relationship in younger adults (P = 0.9637). These findings suggest that PICs are reduced in older adults, and, further, age is a significant predictor of estimates of PICs in older adults. Reduced PIC magnitude represents one plausible mechanism for reduced firing rates and weakness in older individuals.

KEY POINTSO_LIPersistent inward currents play an important role in the neural control of human movement and are influenced by neuromodulation via monoamines originating in the brainstem.
C_LIO_LIDuring aging, motor unit firing rates are reduced, and there is deterioration of brainstem nuclei, which may reduce persistent inward currents in alpha motoneurons.
C_LIO_LIHere we show that estimates of persistent inward currents ({Delta}F) of both elbow flexor and extensor motor units are reduced in older adults.
C_LIO_LIEstimates of persistent inward currents have a negative relationship with age in the older adults, but not young.
C_LIO_LIThis novel mechanism may play a role in alteration motor firing rates that occurs with aging, which may have consequences for motor control.
C_LI

sentences: A hetero-oligomeric remorin-receptor complex regulates plant development Plant growth and development are modulated by both biotic and abiotic stress. Increasing evidence suggests that cellular integration of the corresponding signals occurs within preformed hubs at the plasma membrane called nanodomains. These membrane sub-compartments are organized by multivalent molecular scaffold proteins, such as remorins. Here, we demonstrate that Group 1 remorins form a hetero-oligomeric complex at the plasma membrane. While these remorins are functionally redundant for some pathways their multivalency also allows the recruitment of specific interaction partners. One of them, the receptor-like kinase REMORIN-INTERACTING RECEPTOR 1 (RIR1), that acts redundantly with the closely related receptor NILR2, is specifically recruited by REM1.2 in a phosphorylation-dependent manner. Overlapping developmental phenotypes suggest that the REM/RIR complex regulates key developmental pathways.

sentences: Quantitative analysis of ZFY and CTCF reveals dependent recognition of tandem zinc finger proteins. The human genome contains around 800 C2H2 Zinc Finger Proteins (ZFPs), and many of them are composed of long tandem arrays of zinc fingers. Current motif prediction models assume longer finger arrays correspond to longer DNA-binding motifs and higher specificity. However, recent experimental efforts to identify ZFP binding sites in vivo contradict this assumption, with many having short motifs. Here, we systematically test how multiple zinc fingers contribute to binding for three model ZFPs: Zinc Finger Y (ZFY), CTCF, and ZNF343. Using ZFY, which contains 13 fingers, we quantitatively characterize its binding specificity with several methods, including Affinity-seq, HT-SELEX, Spec-seq and fluorescence anisotropy, and find evidence for  dependent recognition where downstream fingers can recognize some extended motifs only in the presence of an intact core site. For the genomic insulator CTCF, additional high-throughput affinity measurements reveal that its upstream specificity profile depends on the strength of the core, violating presumed additivity and positionindependence. Moreover, the effect of different epigenetic modifications within the core site depends on the strength of flanking upstream site, providing new insight into how the previously identified intellectual disability-causing and cancer-related mutant R567W disrupts upstream recognition and deregulates CTCFs methylation sensitivity. Lastly, we used ZNF343 as example to show that a simple iterative motif analysis strategy based on a small set of prefixed cores can reveal the dependent relationship between cores and upstream motifs. These results establish that the current underestimation of ZFPs motif lengths is due to our lack of understanding of intrinsic properties of tandem zinc finger recognition, including irregular motif structure, variable spacing, and dependent recognition between sub-motifs. These results also motivate a need for better recognition models beyond additive, position-weight matrix to predict ZFP specificities, occupancies, and the molecular mechanisms of disease mutations.

sentences: Improving the RNA velocity approach using long-read single cell sequencing The concept of RNA velocity has been recently developed that allowed to look at the otherwise static single-cell RNA sequencing data in a dynamic way, which permitted inferences about cell fates. However, the more precise parameters, such as the number of exons/introns, can also be determined using long-read methods. Comparing the numbers of exons and introns allows including more genes for downstream velocity analysis and resolves the precise cell fate. The recently developed concept of "RNA velocity" concerns with dynamic changes in mRNA expression and complements single-cell RNA sequencing (scRNA-seq) data, which are static snapshots of a certain cell state taken at a given time point1. RNA velocity measures the change in mRNA abundance by differentiating the newly transcribed unspliced pre-mRNAs from mature spliced mRNAs. The rapidly developing long-read sequencing technology lends itself for RNA velocity analysis of scRNA-seq data, which was previously performed primarily using second-generation sequencing.

sentences: The essential transcriptional regulator HDP1 drives expansion of the inner membrane complex during early sexual differentiation of malaria parasites. Transmission of Plasmodium falciparum and other malaria parasites requires their differentiation from asexual blood stages into gametocytes, the non-replicative sexual stage necessary for transmission to the mosquito vector. This transition involves changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. However, the genomes of malaria parasites have been noted for their dearth of transcriptional and chromatin regulators and the molecular mediators of these changes remain largely unknown. We recently identified HomeoDomain Protein 1 (HDP1) as a DNA-binding protein that enhances the expression of key genes that are critical for early sexual differentiation. The discovery of a homeodomain-like DNA-binding protein marks a new class of transcriptional regulator in malaria parasites outside of the better-characterized ApiAP2 family. In this study, we show that HDP1 facilitates the necessary upregulation of inner membrane complex components during early gametocytogenesis that gives P. falciparum gametocytes they characteristic shape and is required for gametocyte maturation and parasite transmission.

sentences: Neutralization of variant under investigation B.1.617 with sera of BBV152 vaccinees The drastic rise in the number of cases in Maharashtra, India has created a matter of concern for public health experts. Twelve isolates of VUI lineage B.1.617 were propagated in VeroCCL81 cells and characterized. Convalescent sera of the COVID-19 cases and recipients of BBV152 (Covaxin) were able to neutralize VUI B.1.617.

sentences: Transient inhibition to light explains stronger V1 responses to dark stimuli Neurons in the primary visual cortex (V1) receive excitation and inhibition from two different pathways processing lightness (ON) and darkness (OFF). V1 neurons overall respond more strongly to dark than light stimuli (Yeh, Xing and Shapley, 2010; Kremkow et al., 2014), consistent with a preponderance of darker regions in natural images (Ratliff et al., 2010), as well as human psychophysics (Buchner & Baumgartner, 2007). However, it has been unclear whether this "dark-dominance" is due to more excitation from the OFF pathway (Jin et al., 2008) or more inhibition from the ON pathway (Taylor et al., 2018). To understand the mechanisms behind dark-dominance, we record electrophysiological responses of individual simple-type V1 neurons to natural image stimuli and then train biologically inspired convolutional neural networks to predict the neurons responses. Analyzing a sample of 74 neurons (in anesthetized, paralyzed cats) has revealed their responses to be more driven by dark than light stimuli, consistent with previous investigations (Yeh et al., 2010; Kremkow et al., 2013). We show this asymmetry to be predominantly due to slower inhibition to dark stimuli rather than by stronger excitation from the thalamocortical OFF pathway. Consistent with dark-dominant neurons having faster responses than light-dominant neurons (Komban et al., 2014), we find dark-dominance to solely occur in the early latencies of neurons responses. Neurons that are strongly dark-dominated also tend to be less orientation selective. This novel approach gives us new insight into the dark-dominance phenomenon and provides an avenue to address new questions about excitatory and inhibitory integration in cortical neurons.

sentences: A feature-based network analysis and fMRI meta-analysis reveal three distinct types of prosocial decisions Tasks that measure correlates of prosocial decision-making share one common feature: agents can make choices that increase the welfare of a beneficiary. However, prosocial decisions vary widely as a function of other task features. The diverse ways that prosociality is defined and the heterogeneity of prosocial decisions have created challenges for interpreting findings across studies and identifying their neural correlates. To overcome these challenges, we aimed to organize the prosocial decision-making task-space of neuroimaging studies. We conducted a systematic search for studies in which participants made decisions to increase the welfare of others during fMRI. We identified shared and distinct features of these tasks and employed an unsupervised graph-based approach to assess how various forms of prosocial decision-making are related in terms of their low-level components (e.g., task features like potential cost to the agent or potential for reciprocity). Analyses uncovered three clusters of prosocial decisions, which we labeled cooperation, equity, and altruism. This feature-based representation of the task structure was supported by results of a neuroimaging meta-analysis that each type of prosocial decisions recruited diverging neural systems. Results clarify some of the existing heterogeneity in how prosociality is conceptualized and generate insight for future research and task paradigm development.

sentences: The use of disinfectant in barn cleaning alters microbial composition and increases carriage of Campylobacter jejuni in broiler chickens To maintain food safety and flock health in broiler chicken production, biosecurity approaches to keep chicken barns free of pathogens are important. Canadian broiler chicken producers must deep clean their barns with chemical disinfectants at least once annually (full disinfection; FD) and may wash with water (water-wash; WW) throughout the year. However, many producers use FD after each flock, assuming a greater efficacy of more stringent cleaning protocols, although little information is known regarding how these two cleaning practices affect pathogen population and gut microbiota. In the current study, a cross-over experiment over four production cycles was conducted in seven commercial chicken barns to compare WW and FD. We evaluated the effects of barn cleaning method on the commercial broiler performance, cecal microbiota composition, pathogen occurrence and abundance, as well as short-chain fatty acid concentrations in the month-old broiler gut. The 30-day body weight and mortality rate were not affected by the barn cleaning methods. The WW resulted in a modest but significant effect on the structure of broiler cecal microbiota (weighted-UniFrac; adonis p = 0.05, and unweighted-UniFrac; adonis p = 0.01), with notable reductions in Campylobacter jejuni occurrence and abundance. In addition, the WW group had increased cecal acetate, butyrate and total short-chain fatty acid concentrations, which were negatively correlated with C. jejuni abundance. Our results support the use of WW over FD to enhance the activity of the gut microbiota and potentially reduce zoonotic transmission of C. jejuni in broiler production without previous disease challenges.

sentences: Markov state models of proton- and gate-dependent activation in a pentameric ligand-gated ion channel Ligand-gated ion channels conduct currents in response to chemical stimuli, mediating electrochemical signaling in neurons and other excitable cells. For many channels the mechanistic details of gating remain unclear, partly due to limited structural data and simulation timescales. Here, we used enhanced sampling to simulate the pH-gated channel GLIC, and construct Markov state models (MSMs) of gating transitions. Consistent with new functional recordings reported here in oocytes, our analysis revealed differential effects of protonation and mutation on free-energy wells. Clustering of closed-versus open-like states enabled estimation of open probabilities and transition rates in each condition, while higher-order clustering affirmed conformational trends in gating. Furthermore, our models uncovered state- and protonation-dependent symmetrization among subunits. This demonstrates the applicability of MSMs to map energetic and conformational transitions between ion-channel functional states, and how they correctly reproduce shifts upon activation or mutation, with implications for modeling neuronal function and developing state-selective drugs.

sentences: A reduction methodology for fluctuation driven population dynamics Lorentzian distributions have been largely employed in statistical mechanics to obtain exact results for heterogeneous systems. Analytic continuation of these results is impossible even for slightly deformed Lorentzian distributions, due to the divergence of all the moments (cumulants). We have solved this problem by introducing a pseudo-cumulants expansion. This allows us to develop a reduction methodology for heterogeneous spiking neural networks subject to extrinsinc and endogenous fluctuations, thus obtaining an unified mean-field formulation encompassing quenched and dynamical disorder sources.

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Multimodal image fusion via deep generative models Recently, it has become progressively more evident that classic diagnostic labels are unable to accurately and reliably describe the complexity and variability of several clinical phenotypes. This is particularly true for a broad range of neuropsychiatric illnesses such as depression and anxiety disorders or behavioural phenotypes such as aggression and antisocial personality. Patient heterogeneity can be better described and conceptualized by grouping individuals into novel categories, which are based on empirically-derived sections of intersecting continua that span both across and beyond traditional categorical borders. In this context, neuroimaging data carry a wealth of spatiotemporally resolved information about each patients brain. However, they are usually heavily collapsed a priori through procedures which are not learned as part of model training, and consequently not optimized for the downstream prediction task. This is due to the fact that every individual participant usually comes with multiple whole-brain 3D imaging modalities often accompanied by a deep genotypic and phenotypic characterization, hence posing formidable computational challenges.

In this paper we design and validate a deep learning architecture based on generative models rooted in a modular approach and separable convolutional blocks (which result in a 20-fold decrease in parameter utilization) in order to a) fuse multiple 3D neuroimaging modalities on a voxel-wise level, b) efficiently convert them into informative latent embeddings through heavy dimensionality reduction, c) maintain excellent generalizability and minimal information loss. As proof of concept, we test our architecture on the well characterized Human Connectome Project database (n=974 healthy subjects), demonstrating that our latent embeddings can be clustered into easily separable subject strata which, in turn, map to extremely different phenotypical information (including organic, neuropsychological, personality variables) which was not included in the embedding creation process.

The ability to extract meaningful and separable phenotypic information from brain images alone can aid in creating multi-dimensional biomarkers able to chart spatio-temporal trajectories which may correspond to different pathophysiological mechanisms unidentifiable to traditional data analysis approaches. In turn, this may be of aid in predicting disease evolution as well as drug response, hence supporting mechanistic disease understanding and also empowering clinical trials.

sentences: Collective regulation of metabolism, development and longevity through a socially exchanged fluid Socially exchanged fluids, like seminal fluid and milk, allow individuals to molecularly influence conspecifics. Many social insects have a social circulatory system, where food and endogenously produced molecules are transferred mouth-to-mouth (stomodeal trophallaxis), connecting all the individuals in the society. To understand how these endogenous molecules relate to colony life, we used quantitative proteomics to investigate the trophallactic fluid within colonies of the carpenter ant Camponotus floridanus. We show that different stages of the colony life cycle circulate different types of proteins: young colonies prioritize direct carbohydrate processing; mature colonies prioritize accumulation and transmission of stored resources. Further, colonies circulate proteins implicated in oxidative stress, ageing, and social insect caste determination, potentially acting as superorganismal hormones. Brood-caring individuals that are also closer to the queen in the social network (nurses) showed higher abundance of oxidative stress-related proteins. Thus, trophallaxis behavior provides a mechanism for distributed metabolism in social insect societies.

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Aquaglyceroporin AQP7's affinity for its substrate glycerol AQP7 is one of the four human aquaglyceroporins that facilitate glycerol transport across the cell membrane, a biophysical process that is essential in human physiology. Therefore, it is interesting to compute AQP7s affinity for its substrate (glycerol) with reasonable certainty to compare with the experimental data suggesting high affinity in contrast with most computational studies predicting low affinity. In this study aimed at computing the AQP7-glycerol affinity with high confidence, we implemented a direct computation of the affinity from unbiased equilibrium molecular dynamics (MD) simulations of three all-atom systems constituted with 0.16M, 4.32M, and 10.23M atoms, respectively. These three sets of simulations manifested a fundamental physics law that the intrinsic fluctuations of pressure in a system are inversely proportional to the system size (the number of atoms in it). These simulations showed that the computed values of glycerol-AQP7 affinity are dependent upon the system size (the inverse affinity estimations were, respectively, 47.3 mM, 1.6 mM, and 0.92 mM for the three model systems). In this, we obtained a lower bound for the AQP7-glycerol affinity (an upper bound for the dissociation constant). Namely, the AQP7-glycerol affinity is stronger than 1087/M (the dissociation constant is less than 0.92 mM). Additionally, we conducted hyper steered MD (hSMD) simulations to map out the Gibbs free-energy profile. From the free-energy profile, we produced an independent computation of the AQP7-glycerol dissociation constant being approximately 0.18 mM.

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sentences: Comparing transmission reconstruction models with Mycobacterium tuberculosis whole genome sequence data Pathogen genomic epidemiology is now routinely used worldwide to interrogate infectious disease dynamics. Multiple computational tools that reconstruct transmission networks by coupling genomic data with epidemiological modelling have been developed. The resulting inferences are often used to inform outbreak investigations, yet to date, the performance of these transmission reconstruction tools has not been compared specifically for tuberculosis, a disease process with complex epidemiology that includes variable latency periods and within-host heterogeneity. Here, we carried out a systematic comparison of seven publicly available transmission reconstruction tools, evaluating their accuracy in predicting transmission events in both simulated and real-world Mycobacterium tuberculosis outbreaks. No tool was able to fully resolve transmission networks, though both the single-tree and multi-tree input implementations of TransPhylo identified the most epidemiologically supported transmission events and the fewest false positive links. We observed a high degree of variability in the transmission networks inferred by each approach. Our findings may inform an end-users choice of tools in future tuberculosis transmission analyses and underscore the need for caution when interpreting transmission networks produced using probabilistic approaches.

sentences: Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in non-structural protein 14 (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-[A] resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.

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Reconstitution and Expression of mcy Gene Cluster in The Model Cyanobacterium Synechococcus 7942 Reveals a Roll of MC-LR in Cell Division Cyanobacterial blooms pose a serious threat to public health due to the presence of cyanotoxins. The most common cyanotoxins, microcystins (MCs), can cause acute poisoning at high concentrations and hepatocellular carcinoma following chronic exposure. Among all MC variants, MC-LR produced by Microcystis aeruginosa PCC 7806 is the most common toxic MC. Although the biosynthetic pathway for MC-LR has been proposed, experimental support of this pathway is lacking. In an effort to experimentally validate this pathway, we expressed the 55 kb microcystin biosynthetic gene cluster (mcy cluster) (mcyA-J) and produced MC-LR in the model cyanobacterium Synechococcus 7942. We designed and constructed the strong bidirectional promoter biPpsbA2 between mcyA and mcyD, reassembled the mcy cluster in yeast by transformation-associated recombination (TAR cloning), transformed the gene cluster into the NSII site of Synechococcus 7942, and successfully expressed MC-LR at a level of 0.006-0.018 fg cell-1 day-1. The expression of MC-LR led to abnormal cell division and the filamentation of Synechococcus 7942 cells, further analysis proved a role of MC-LR in functional assembly of the cell division protein FtsZ, by competing its GTP binding site. These results represent the first synthetic biological expression of the mcy cluster and the autotrophic production of MC-LR in a photosynthetic model organism, which lays the foundation for resolving the MC biosynthesis pathway. The suggested role of MC-LR in cell division reveals a mechanism of how blooming cyanobacteria gain a competitive edge over their non-blooming counterparts.

HighlightsO_LIWe expressed the 55 kb mcy cluster and produced MC-LR in the model cyanobacterium Synechococcus 7942.
C_LIO_LIThis is the first realized production of MC in the model non-toxin-production cyanobacteria from CO2 by photosynthesis.
C_LIO_LICompared with the self-replicating plasmid, the recombination of the mcy cluster into the genome of Synechococcus 7942 is more suitable for the heterologous production of microcystin.
C_LIO_LIMC-LR inhibits cell division by irreversibly competing the GTP binding domain of the cell division protein FtsZ.
C_LIO_LIThe newly discovered effect of MC-LR on cell division reveals a mechanism of how blooming cyanobacteria gain competitive edge over their non-blooming counterparts.
C_LI

sentences: An instance-specific causal framework for learning intercellular communication networks that define microenvironments of individual tumors Cells within a tumor microenvironment (TME) dynamically communicate and influence each others cellular states through an intercellular communication network (ICN). In cancers, intercellular communications underlie immune evasion mechanisms of individual tumors. We developed an individualized causal analysis framework for discovering tumor-specific ICNs. Using head and neck squamous cell carcinoma (HNSCC) tumors as a testbed, we first mined single-cell RNA-sequencing data to discover gene expression modules (GEMs) that reflect the states of transcriptomic processes within tumor and stromal single cells. By deconvoluting bulk transcriptomes of HNSCC tumors profiled by The Cancer Genome Atlas (TCGA), we estimated the activation states of these transcriptomic processes in individual tumors. Finally, we applied individualized causal network learning to discover an ICN within each tumor. Our results show that cellular states of cells in TMEs are coordinated through ICNs that enable multi-way communications among epithelial, fibroblast, endothelial, and immune cells. Further analyses of individual ICNs revealed structural patterns that were shared across subsets of tumors, leading to the discovery of 4 different subtypes of networks that underlie disparate TMEs of HNSCC. Patients with distinct TMEs exhibited significantly different clinical outcomes. Our results show that the capability of estimating individual ICNs reveals heterogeneity of ICNs and sheds light on the importance of intercellular communication in impacting disease development and progression.

sentences: A phenotypic rescue approach identifies lineage regionalization defects in a mouse model of DiGeorge syndrome. TBX1 is a key regulator of pharyngeal apparatus (PhAp) development. Vitamin B12 treatment partially rescues aortic arch patterning defects of Tbx1+/- embryos. Here we show that it also improves cardiac outflow tract septation and branchiomeric muscle anomalies of Tbx1 hypomorphic mutants. At molecular level, the in vivo vB12 treatment let us to identify genes that were dysregulated by Tbx1 haploinsufficiency and rescued by treatment. We found that SLUG, encoded by the rescued gene Snai2, identified a population of mesodermal cells that was partially overlapping with but distinct from ISL1+ and TBX1+ populations. In addition, SLUG+ cells were mislocalized and had a greater tendency to aggregate in Tbx1+/- and Tbx1-/- embryos and vB12 treatment restore cellular distribution. Adjacent neural crest-derived mesenchymal cells, which do not express TBX1, were also affected, showing enhanced segregation from cardiopharyngeal mesodermal cells. We propose that TBX1 regulates cell distribution in core mesoderm and the arrangement of multiple lineages within the PhAp.

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Ionic remodelling following myocardial infarction explains phenotypic variability in ECG and arrhythmic substrate but not in ejection fraction AimsSudden death after myocardial infarction (MI) is associated with electrophysiological heterogeneities and ionic remodelling, which are reflected as variable phenotypes. Low ejection fraction (EF) is used in risk stratification, but its mechanistic links with the post-MI electrophysiological heterogeneities are unknown. We aim to unravel how phenotypic ECG and EF variability in post-MI may be explained by the impact of ionic remodelling on spatio-temporal dispersion of repolarization using human biventricular electromechanical modelling and simulation.

Methods and ResultsMultiple post-MI ECG phenotypes observed clinically were investigated using multi-scale modelling and simulation, calibrated and evaluated using experimental and clinical data. Acute stage T-wave inversion was caused by delayed repolarization in the epicardial border zone (BZ), and Brugada phenocopy was generated by repolarization delay and activation failure in the BZ. Upright tall T-waves in chronic MI represented large repolarisation dispersion between BZ and surrounding tissue, which promoted ectopic propagation at fast pacing. T-wave morphology alternans were present at fast-pacing due to prolonged refractoriness in the mid-myocardium. Post-MI ionic remodelling reduced EF through inhibition of calcium transient amplitude, but the EF at resting heart rate was not sensitive to the extent of repolarisation heterogeneity and the risk of repolarisation abnormalities at fast pacing.

ConclusionsIn acute post-MI, ionic remodelling and its effect on refractoriness and propagation failure in the BZ have a strong impact on phenotypic ECG variability, whereas in chronic post-MI, the repolarisation dispersion across the BZ is crucial. T-wave and QT abnormalities are better indicators of repolarisation heterogeneities than EF in post-MI.

sentences: Novel antibodies for the simple and efficient enrichment of native O-GlcNAc modified peptides Antibodies against post-translational modifications (PTMs) such as lysine acetylation, ubiquitin remnants, or phosphotyrosine have resulted in significant advances in our understanding of the fundamental roles of PTMs in biology. However, the roles of a number of PTMs remain largely unexplored due to the lack of robust enrichment reagents. The addition of N-acetylglucosamine to serine and threonine residues (O-GlcNAc) by the O-GlcNAc transferase (OGT) is a PTM implicated in numerous biological processes and disease states but with limited techniques for its study. Here, we evaluate a new mixture of anti-O-GlcNAc monoclonal antibodies for the immunoprecipitation of native O-GlcNAcylated peptides from cells and tissues. The anti-O-GlcNAc antibodies display good sensitivity and high specificity toward O-GlcNAc-modified peptides, and do not recognize O-GalNAc or GlcNAc in extended glycans. Applying this antibody-based enrichment strategy to synaptosomes from mouse brain tissue samples, we identified over 1,300 unique O-GlcNAc-modified peptides and over 1,000 sites using just a fraction of sample preparation and instrument time required in other landmark investigations of O-GlcNAcylation. Our rapid and robust method greatly simplifies the analysis of O-GlcNAc signaling and will help to elucidate the role of this challenging PTM in health and disease.

sentences: Stability of neocortical synapses across sleep and wake Sleep is important for brain plasticity, but its exact function remains mysterious. An influential but controversial idea is that a crucial function of sleep is to drive widespread downscaling of excitatory synaptic strengths. Here we used real-time sleep classification, ex vivo measurements of postsynaptic strength, and in vivo optogenetic monitoring of thalamocortical synaptic efficacy to ask whether sleep and wake states can constitutively drive changes in synaptic strength within the neocortex of juvenile rats. We found that miniature EPSC amplitudes onto L4 and L2/3 pyramidal neurons were stable across sleep and wake dense epochs in both primary visual (V1) and prefrontal cortex (PFC). Further, chronic monitoring of thalamocortical synaptic efficacy in V1 of freely behaving animals revealed stable responses across even prolonged periods of natural sleep and wake. Together these data demonstrate that neocortical synaptic strengths are remarkably stable across sleep and wake states, and provide strong evidence against the view that sleep drives widespread synaptic downscaling at neocortical synapses.

sentences: An insight into SARS-CoV-2 Membrane protein interaction with Spike, Envelope, and Nucleocapsid proteins Intraviral protein-protein interactions are crucial for replication, pathogenicity, and viral assembly. Among these, virus assembly is a critical step as it regulates the arrangements of viral structural proteins and helps in the encapsulation of genomic material. SARS-CoV-2 structural proteins play an essential role in the self-rearrangement, RNA encapsulation, and mature virus particle formation. In SARS-CoV, the membrane protein interacts with the envelope and spike protein in Endoplasmic Reticulum Golgi Intermediate Complex (ERGIC) to form an assembly in the lipid bilayer, followed by membrane-ribonucleoprotein (nucleocapsid) interaction. In this study, we tried to understand the interaction of membrane proteins interaction with envelope, spike, and nucleocapsid proteins using protein-protein docking. Further, simulation studies performed up to 100 ns to examine the stability of protein-protein complexes of Membrane-Envelope, Membrane-Spike, and Membrane-Nucleocapsid. Prime MM-GBSA showed high binding energy calculations than the docked complex. The interactions identified in our study will be of great importance, as it provides valuable insight into the protein-protein complex, which could be the potential drug targets for future studies.

sentences: The chromatin-remodeling enzyme Smarca5 regulates erythrocyte aggregation via Keap1-Nrf2 signaling Although thrombosis has been extensively studied using various animal models, however, our understanding of the underlying mechanism remains elusive. Here, using zebrafish model, we demonstrated that smarca5-deficient red blood cells (RBCs) formed blood clots in the caudal vein plexus that mimics venous thrombosis. We further used the anti-thrombosis drugs to treat smarca5zko1049a embryos and found that a thrombin inhibitor, argatroban, partially prevented blood clot formation in smarca5zko1049a. To explore the regulatory mechanism of smarca5 in RBC homeostasis, we profiled the chromatin accessibility landscape and transcriptome features in RBCs from smarca5zko1049a and their siblings and found that both the chromatin accessibility at the keap1a promoter and expression of keap1a were decreased. Keap1 is a suppressor protein of Nrf2, which is a major regulator of oxidative responses. We further identified that the expression of hmox1a, a downstream target of Keap1-Nrf2 signaling pathway, was markedly increased upon smarca5 deletion. Importantly, overexpression of keap1a or knockdown of hmox1a partially rescued the blood clot formation, suggesting that the disrupted Keap1-Nrf2 signaling is responsible for the venous thrombosis-like phenotypes in smarca5 mutants. Together, our study using zebrafish smarca5 mutants not only characterizes a novel role for smarca5 in blood clot formation, but also provides a new venous thrombosis animal model to support drug screening and pre-clinical therapeutic assessments to treat thrombosis.

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Preclinical studies for plant-based oral enzyme replacement therapy (Oral-ERT) in Pompe disease knockout mice with transgenic tobacco seeds expressing human GAA (tobrhGAA) Genetic deficiency of acid -glucosidase (GAA) results in glycogen storage disease type II (GSDII) or Pompe disease (PD) encompassing at least four clinical subtypes of varying severity (infantile; childhood, juvenile and late onset). Our objective is to develop an innovative and affordable approach for enzyme replacement therapy (ERT) via oral administration (Oral-ERT) to maintain a sustained, therapeutic level of enzyme on a daily basis to improve efficacy of treatment and quality of life for people living with Pompe disease. A consensus at a 2019 US Acid Maltase Deficiency (AMDA) conference suggested that a multi-pronged approach including gene therapy, diet, exercise, etc. must be evaluated for a successful treatment of Pompe disease. Tobacco seeds contain the metabolic machinery that is more compatible with mammalian glycosylation-phosphorylation and processing. Previously, we have shown that a lysate from transgenic tobacco seeds expressing human GAA (tobrhGAA) was enzymatically active and can correct enzyme deficiency in cultured PD cells and in adult lymphocytes of Pompe patients and in vivo in disease-relevant tissues in GAA knockout (KO) mice when administered IP.

We have extended these pre-clinical studies in PD knockout (KO) mice with ground tobrhGAA seeds that supports proof-of-concept for Oral-ERT for future clinical trials. Briefly in GAA KO mice, Oral-ERT with ground tobrhGAA seeds showed significant reversal of fore-limb and hind-limb muscle weakness, increased motor coordination/balance/strength and mobility, improved spontaneous learning, increased GAA baseline activity in tissues, reduced glycogen in tissues and negible serum titers to GAA. Pharmacokinetics showed maximum serum GAA concentration (Cs) at 8-10 hr and peak urine excretion at 10-12 hr. The tobrhGAA was taken up in PD fibroblast, lymphoid and myoblast cells. Enzyme kinetics compared favorably or superior to placental hGAA, plus alglucosidase alfa or other rhGAAs for Km, Vmax, pH optima, thermal heat stability and IC50 for inhibitors. The tobrhGAA in seeds was extremely stable stored for 15 years at room temperature. NGS-genome sequencing of the tobrhGAA and wild-type plants and RNA expression profiles was performed and will be posted on our website. Thus, Oral-ERT with ground tobrhGAA seeds is an innovative approach that overcomes some of the challenges of alglucosidase alfa-ERT and provides a more effective, safe and significantly less expensive treatment.

sentences: Fabrication and Characterization Methods for Investigating Cell-Matrix Interactions in Environments Possessing Spatial Orientation Heterogeneity Fibrillar collagen is a ubiquitous structural protein that plays a significant role in determining the mechanical properties of various tissues. The constituent collagen architecture can give direct insight into the respective functional role of the tissue due to the strong structure-function relationship that is exhibited. In such tissues, matrix structure can vary across local subregions contributing to mechanical heterogeneity which can be implicated in tissue function or failure. The post-myocardial infarction scar environment is an example of note where mechanically insufficient collagen can result in impaired cardiac function and possibly tissue rupture due to post-MI cellular response and matrix interactions. In order to further develop the understanding of cell-matrix and cell-cell interactions within heterogeneous environments, we developed a method of heterogeneous collagen gel fabrication which produces a region of randomly oriented fibers directly adjacent to an interconnected region of anisotropic alignment. To fully capture and evaluate the degree of alignment and spatial orientation heterogeneity, several image processing and automated analysis methods were employed. Our analysis revealed the successful fabrication of an interconnected spatially heterogeneous collagen gel possessing distinct regions of random or preferential alignment. Additionally, embedded cell populations were observed to recognize and reorient with their underlying and surrounding architectures through our cell-centric analysis techniques.

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InterARTIC: an interactive web application for whole-genome nanopore sequencing analysis of SARS-CoV-2 and other viruses MotivationInterARTIC is an interactive web application for the analysis of viral whole-genome sequencing (WGS) data generated on Oxford Nanopore Technologies (ONT) devices. A graphical interface enables users with no bioinformatics expertise to analyse WGS experiments and reconstruct consensus genome sequences from individual isolates of viruses, such as SARS-CoV-2. InterARTIC is intended to facilitate widespread adoption and standardisation of ONT sequencing for viral surveillance and molecular epidemiology.

Worked exampleWe demonstrate the use of InterARTIC for the analysis of ONT viral WGS data from SARS-CoV-2 and Ebola virus, using a laptop computer or the internal computer on an ONT GridION sequencing device. We showcase the intuitive graphical interface, workflow customisation capabilities and job-scheduling system that facilitate execution of small- and large-scale WGS projects on any common virus.

ImplementationInterARTIC is a free, open-source web application implemented in Python. The application can be downloaded as a set of pre-compiled binaries that are compatible with all common Ubuntu distributions, or built from source. For further details please visit: https://github.com/Psy-Fer/interARTIC/.

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A Deep Learning Framework for Prediction of Clinical Drug Response of Cancer Patients and Identification of Drug Sensitivity Biomarkers using Preclinical Samples BackgroundPrediction of the response of cancer patients to different treatments and identification of biomarkers of drug sensitivity are two major goals of individualized medicine. In this study, we developed a deep learning framework called TINDL, completely trained on preclinical cancer cell lines, to predict the response of cancer patients to different treatments. TINDL utilizes a tissue-informed normalization to account for the tissue and cancer type of the tumours and to reduce the statistical discrepancies between cell lines and patient tumours. In addition, this model identifies a small set of genes whose mRNA expression are predictive of drug response in the trained model, enabling identification of biomarkers of drug sensitivity.

ResultsUsing data from two large databases of cancer cell lines and cancer tumours, we showed that this model can distinguish between sensitive and resistant tumours for 10 (out of 14) drugs, outperforming various other machine learning models. In addition, our siRNA knockdown experiments on 10 genes identified by this model for one of the drugs (tamoxifen) confirmed that all of these genes significantly influence the drug sensitivity of the MCF7 cell line to this drug. In addition, genes implicated for multiple drugs pointed to shared mechanism of action among drugs and suggested several important signaling pathways.

ConclusionsIn summary, this study provides a powerful deep learning framework for prediction of drug response and for identification of biomarkers of drug sensitivity in cancer.

sentences: An Image Reconstruction Framework for Characterizing Early Vision We developed an image-computable observer model of the initial visual encoding that operates on natural image input, based on the framework of Bayesian image reconstruction from the excitations of the retinal cone mosaic. Our model extends previous work on ideal observer analysis and evaluation of performance beyond psychophysical discrimination, takes into account the statistical regularities of the visual environment, and provides a unifying framework for answering a wide range of questions regarding the visual front end. Using the error in the reconstructions as a metric, we analyzed variations of the number of different photoreceptor types on human retina as an optimal design problem. In addition, the reconstructions allow both visualization and quantification of information loss due to physiological optics and cone mosaic sampling, and how these vary with eccentricity. Furthermore, in simulations of color deficiencies and interferometric experiments, we found that the reconstructed images provide a reasonable proxy for modeling subjects percepts. Lastly, we used the reconstruction-based observer for the analysis of psychophysical threshold, and found notable interactions between spatial frequency and chromatic direction in the resulting spatial contrast sensitivity function. Our method is widely applicable to experiments and applications in which the initial visual encoding plays an important role.

sentences: Nonstoichiometric balanced complexes: Implications on the effective deficiency of the underlying metabolic network The deficiency of a (bio)chemical reaction network can be conceptually interpreted as a measure of its ability to support exotic dynamical behavior and/or multistationarity. The classical definition of deficiency relates to the capacity of a network to permit variations of the complex formation rate vector at steady state, irrespective of the network kinetics. However, the deficiency is by definition completely insensitive to the fine details of the directionality of reactions as well as bounds on reaction fluxes. While the classical definition of deficiency can be readily applied in the analysis of unconstrained, weakly reversible networks, it only provides an upper bound in the cases where relevant constraints on reaction fluxes are imposed. Here we propose the concept of effective deficiency, which provides a more accurate assessment of the networks capacity to permit steady state variations at the complex level for constrained networks of any reversibility patterns. The effective deficiency relies on the concept of nonstoichiometric balanced complexes, which we have already shown to be present in real-world biochemical networks operating under flux constraints. Our results demonstrate that the effective deficiency of real-world biochemical networks is smaller than the classical deficiency, indicating the effects of reaction directionality and flux bounds on the variation of the complex formation rate vector at steady state.

sentences: Single-cell sequencing of the Drosophila embryonic heart and muscle cells during differentiation and maturation The developing Drosophila heart consists of cardioblasts that differentiate into different types of cardiomyocytes and pericardial cells. A large body of work has identified numerous genes and pathways involved in heart specification and differentiation, downstream of cardiac transcription factors, such as Tinman (NKX2-5) and Dorsocross1/2/3 (TBX5). The advent of single-cell RNA sequencing (scRNAseq) technology allowed us for the first time to describe the transcriptome of different cardiac cell types in the Drosophila model at high resolution. Here, we applied scRNAseq on sorted cells of late-stage Drosophila embryos expressing a cardiac GFP reporter. We find distinct expression profiles of cardioblasts as they mature to cardiomyocytes, as well as discretely clustering pericardial cells, including a set expressing Tinman that potentially assist in heart morphogenesis. In addition, we describe other cell types that were sequenced as by-catch due to low but distinct extracardiac expression of the GFP reporter. Our studies on wildtype cardioblasts will be the foundation for investigating developmental profiles in mutant backgrounds and for generating gene regulatory networks at single-cell resolution during cardiogenesis.

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Soil collected in the Great Smoky Mountains National Park yielded a novel Listeria species, L. swaminathanii, effectively expanding the sensu stricto clade to ten species Soil samples collected in the Great Smoky Mountains National Park yielded a Listeria isolate that could not be classified to the species level. Whole-genome sequence-based average nucleotide identity BLAST and in silico DNA-DNA Hybridization analyses confirmed this isolate to be a novel Listeria sensu stricto species with the highest similarity to L. marthii (ANI=93.9%, isDDH=55.9%). Additional whole-genome-based analysis using the Genome Taxonomy Database Toolkit, an automated program for classifying bacterial genomes, further supported delineation as a novel Listeria sensu stricto species, as this tool failed to assign a species identification but identified L. marthii as the closest match. Phenotypic and genotypic characterization results indicate that this species is nonpathogenic. Specifically, the novel Listeria species described here is phenotypically (i) non-hemolytic and (ii) negative for phosphatidylinositol-specific phospholipase C activity; the draft genome lacks all virulence genes found in the Listeria pathogenicity island 1 (LIPI-1), as well as the internalin genes inlA and inlB. While the type strain for the new species is phenotypically catalase-negative (an unusual characteristic for Listeria sensu stricto species), its genome contained an apparently intact catalase gene (kat); hence assessment of this phenotype with future isolates will be important. Rapid species identification systems (Listeria API, VITEK 2, VITEK MS) misidentified this novel species as either L. monocytogenes, L. innocua, or L. marthii. We propose the name L. swaminathanii, and the type strain is FSL L7-0020T (=ATCC TSD-239T).

IMPORTANCEL. swaminathanii is a novel sensu stricto species that originated from a US National Park, and its place of origin is ultimately preventing this species from achieving valid status. The US National Park Service restricts strain accessibility and open access is currently a prerequisite for species validation. Essentially the only criteria that was not met for L. swaminathanii validation is accessibility of the type strain, therefore nomenclature status should not negate the significance of this discovery. As a novel sensu stricto species, L. swaminathanii expands the group of species whose presence is associated with an increased risk of an L. monocytogenes contamination, and therefore could play an important role in public health. While developers of Listeria spp. detection methods historically only included validly publish species in their validation studies, L. swaminathanii is unequivocally a sensu stricto species and should be included as well.

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Ultraviolet radiation modulates DNA methylation in melanocytes Ultraviolet radiation (UVR) is the principal causal factor for melanoma; albeit the underlying mechanisms remain unclear. While the mutagenic properties of UVR are irrefutable, the role of UVR-induced mutations in the initiation of melanoma is controversial which highlights the gap in our knowledge of the initial critical molecular mechanisms of UVR-induced melanomagenesis. To investigate the potential non-mutational mechanisms of UVR-induced melanomagenesis, we studied the role of UVR in modulating DNA methylation changes in melanocytes via next-generation sequencing-based methodologies. Here we show that UVR directly causes stable changes in the DNA methylome and transcriptome, one month after exposure. Genomic features associated with transcription were protected from 5mC alterations whereas CpG sites found in intergenic regions were more likely to be affected. Additionally, the long-term effects of UVR seem to perturb signaling pathways important for melanocyte biology. Interestingly, UVR-sensitive CpG sites were found to be prognostic of overall patient survival and highlighted a subset of CpG sites that may be relevant in melanomagenesis.

SignificanceWe report a novel finding that ultraviolet radiation (UVR) induces DNA methylation changes along with stable alterations in gene expression in cultured melanocytes. Our results provide experimental evidence of UVR-induced epigenetic rewiring, which may be implicated in the susceptibility to melanomagenesis, independently of its mutational effects. These findings offer novel insight into the role of UVR in the initiation and pathogenesis of melanoma via a currently underappreciated mechanism.

sentences: MIA3/TANGO1 enables efficient secretion by constraining COPII vesicle budding Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum, an essential process in eukaryotic cells. In vertebrates, the Mia3 gene encodes two major forms of Transport ANd Golgi Organization Protein 1 (TANGO1S and TANGO1L). Here, using genome engineering of human cells, light microscopy, secretion assays, genomics, and proteomics we show that disruption of the longer form, TANGO1L, results in relatively minor defects in secretory pathway organization and function including limited impacts on procollagen secretion. In contrast, loss of both long and short forms results in major defects in cell organization and secretion. These include a failure to maintain the localization of ERGIC53 and SURF4 to the ER-Golgi Intermediate Compartment and dramatic changes to the ultrastructure of the ER-Golgi interface. Disruption of TANGO1 expression also causes significant changes in early secretory pathway gene and protein expression. Disruption of both TANGO1L and TANGO1S expression impairs secretion not only of large proteins, including procollagens, but of all types of secretory cargo including small soluble proteins. Our data support a general role for Mia3/TANGO1 in maintaining both secretory pathway structure and function in vertebrate cells.

sentences: Engineering efficient termination of bacteriophage T7 RNA polymerase transcription The bacteriophage T7 expression system is one of the most prominent transcription systems used in biotechnology and molecular-level research. However, T7 RNA polymerase is prone to read-through transcription due to its high processivity. As a consequence, enforcing efficient transcriptional termination is difficult. The termination hairpin found natively in the T7 genome is adapted to be inefficient, exhibiting 62% termination efficiency in vivo and even lower efficiency in vitro. In this study, we engineered a series of sequences that outperform the efficiency of the native terminator hairpin. By embedding a previously discovered 8-nucleotide T7 polymerase pause sequence within a synthetic hairpin sequence, we observed in vivo termination efficiency of 91%; by joining two short sequences into a tandem 2-hairpin structure, termination efficiency was increased to 98% in vivo and 91% in vitro. This study also tests the ability of these engineered sequences to terminate transcription of the Escherichia coli RNA polymerase. Two out of three of the most successful T7 polymerase terminators also facilitated termination of the bacterial polymerase with around 99% efficiency.

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RedOak: a reference-free and alignment-freestructure for indexing a collection of similargenomes BackgroundAs the cost of DNA sequencing decreases, high-throughput sequencing technologies become increasingly accessible to many laboratories. Consequently, new issues emerge that require new algorithms, including tools for indexing and compressing hundred to thousands of complete genomes.

ResultsThis paper presents RedOak, a reference-free and alignment-free software package that allows for the indexing of a large collection of similar genomes. RedOak can also be applied to reads from unassembled genomes, and it provides a nucleotide sequence query function. This software is based on a k-mer approach and has been developed to be heavily parallelized and distributed on several nodes of a cluster. The source code of our RedOak algorithm is available at https://gite.lirmm.fr/doccy/RedOak.

ConclusionsRedOak may be really useful for biologists and bioinformaticians expecting to extract information from large sequence datasets.

sentences: Travelling wave and asymptotic analysis of a multiphase moving boundary model for engineered tissue growth We derive a multiphase, moving boundary model to represent the development of tissue in vitro in a porous tissue engineering scaffold. We consider a cell, extra-cellular liquid and a rigid scaffold phase, and adopt Darcys law to relate the velocity of the cell and liquid phases to their respective pressures. Cell-cell and cell-scaffold interactions which can drive cellular motion are accounted for by utilising relevant constitutive assumptions for the pressure in the cell phase. We reduce the model to a nonlinear reaction-diffusion equation for the cell phase, coupled to a moving boundary condition for the tissue edge, the diffusivity being dependent on the cell and scaffold volume fractions, cell and liquid viscosities, and parameters that relate to cellular motion. Numerical simulations reveal that the reduced model admits three regimes for the evolution of the tissue edge at large-time: linear, logarithmic and stationary. Employing travelling wave and asymptotic analysis, we characterise these regimes in terms of parameters related to cellular production and motion. The results of our investigation allow us to suggest optimal values for the governing parameters, so as to stimulate tissue growth in an engineering scaffold.

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Fine-grained topographic organization within somatosensory cortex during resting-state and emotional face-matching task and its association with ASD traits BACKGROUNDSensory atypicalities are particularly common in autism spectrum disorders (ASD). Nevertheless, our knowledge about the divergence of the underlying somatosensory region and its association with ASD phenotype features is limited.

METHODSWe applied a data-driven approach to map the fine-grained variations in functional connectivity of the primary somatosensory cortex (S1) to the rest of the brain in 240 autistic and 164 neurotypical individuals from the EU-AIMS LEAP dataset, aged between 7 and 30. We estimated the S1 connection topography ( connectopy) during rest and during the emotional face-matching (Hariri) task, an established measure of emotion reactivity, and accessed its association with a set of clinical and behavioral variables.

RESULTSWe demonstrated that the S1 connectopy is organized along a dorsoventral axis, mapping onto the somatotopic organization of S1. We found that its spatial characteristics were linked to the individuals adaptive functioning skills, as measured by the Vineland Adaptive Behavior Scales, across the whole sample. Higher functional differentiation characterized the S1 connectopies of individuals with higher daily life adaptive skills. Notably, we detected significant differences between rest and the Hariri task in the S1 connectopies, as well as their projection maps onto the rest of the brain suggesting a task-modulating effect on S1 due to emotion processing.

CONCLUSIONSVariation of daily life adaptive skills appears to be reflected in the brains mesoscale neural circuitry, as shown by the S1 connectivity profile, which is also differentially modulated during rest and emotional processing.

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Landscape composition mediates suppression of major pests by natural enemies in conventional cruciferous vegetables BackgroundConservation biological control provides an environment-friendly approach to improve the efficacy of natural enemies. Although numerous studies have demonstrated the potential of semi-natural habitats in promoting biological control in organic or unsprayed agroecosystems, few studies were conducted in conventional agricultural fields. In this study, we investigated the effects of landscape composition on the major pests of cruciferous vegetables and on the assemblages of their natural enemies in southeastern China.

ResultsHabitat diversity, particularly increasing grassland proportion in the landscape, had a positive impact in controlling both small-sized pests (aphids, leaf miners, thrips and flea beetles) and Plutella xylostella. This increasing proportion also promoted greater abundance and diversity of canopy-dwelling predators, more forests supported a higher diversity of airborne enemies (parasitoids and canopy-dwelling predators) as well as a higher abundance of ground-dwelling predators. A general increase in habitat diversity was beneficial to parasitoids and ground-dwelling predators. Additionally, the proportion of forest, grassland, and non-cruciferous vegetable area, as well as habitat diversity, affected the compositions of natural enemy communities. Moreover, inconsistent effects of non-cruciferous and grassland habitats were found between sampling regions for small-sized pests and canopy-dwelling predators. Moreover, the scale at which pests and natural enemies abundance and richness responded most to landscape composition varied with their feeding range and dispersal ability.

ConclusionOur study provides evidence that increasing the amount of semi-natural habitats and habitat diversity can result in lower pest and higher natural enemy abundance in conventional cruciferous agroecosystems. Regional conditions and spatial scales also should be considered in designing the agricultural landscape mosaic.

sentences: Subthalamic nucleus stabilizes movements by reducing neural spike variability in monkey basal ganglia: chemogenetic study AbstractsThe subthalamic nucleus (STN) projects to the external pallidum (GPe) and internal pallidum (GPi), the relay and output nuclei of the basal ganglia (BG), respectively, and plays an indispensable role in controlling voluntary movements. To elucidate the neural mechanism by which the STN controls GPe/GPi activity and movements, we utilized a chemogenetic method to reversibly suppress the motor subregion of the STN in three macaque monkeys (Macaca fuscata, both sexes) engaged in reaching tasks. Systemic administration of chemogenetic ligands prolonged movement time and increased spike train variability in the GPe/GPi, but only slightly affected firing rate modulations. Across-trial analyses revealed that the irregular discharge activity in the GPe/GPi coincided with prolonged movement time. STN suppression also induced excessive abnormal movements in the contralateral forelimbs, which was preceded by STN and GPe/GPi phasic activity changes. Our results suggest that the STN stabilizes spike trains in the BG and achieves stable movements.

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Close-kin mark-recapture methods to estimate demographic parameters of mosquitoes Close-kin mark-recapture (CKMR) methods have recently been used to infer demographic parameters such as census population size and survival for fish of interest to fisheries and conservation. These methods have advantages over traditional mark-recapture methods as the mark is genetic, removing the need for physical marking and recapturing that may interfere with parameter estimation. For mosquitoes, the spatial distribution of close-kin pairs has been used to estimate mean dispersal distance, of relevance to vector-borne disease transmission and novel biocontrol strategies. Here, we extend CKMR methods to the life history of mosquitoes and comparable insects. We derive kinship probabilities for mother-offspring, father-offspring, full-sibling and half-sibling pairs, where an individual in each pair may be a larva, pupa or adult. A pseudo-likelihood approach is used to combine the marginal probabilities of all kinship pairs. To test the effectiveness of this approach at estimating mosquito demographic parameters, we develop an individual-based model of mosquito life history incorporating egg, larva, pupa and adult life stages. The simulation labels each individual with a unique identification number, enabling close-kin relationships to be inferred for sampled individuals. Using the dengue vector Aedes aegypti as a case study, we find the CKMR approach provides unbiased estimates of adult census population size, adult and larval mortality rates, and larval life stage duration for logistically feasible sampling schemes. Considering a simulated population of 3,000 adult mosquitoes, estimation of adult parameters is accurate when a total of 1,000 adult females are sampled biweekly-to-fortnightly over a three month period. Estimation of larval parameters is accurate when adult sampling is supplemented with a total of 4,000 larvae sampled biweekly over the same period. As the cost of genome sequencing declines, these methods hold great promise for characterizing the demography of mosquitoes and comparable insects of epidemiological and agricultural significance.

Author summaryClose-kin mark-recapture (CKMR) methods are a genetic analogue of traditional mark-recapture methods in which the frequency of marked individuals in a sample is used to infer demographic parameters such as census population size and mean dispersal distance. In CKMR, the mark is a close-kin relationship between individuals (parents and offspring, siblings, etc.). While CKMR methods have mostly been applied to aquatic species to date, opportunities exist to apply them to insects and other terrestrial species. Here, we explore the application of CKMR to mosquitoes, with Aedes aegypti, a primary vector of dengue, chikungunya and yellow fever, as a case study. By analyzing simulated Ae. aegypti populations, we find the CKMR approach provides unbiased estimates of adult census population size, adult and larval mortality rates, and larval life stage duration. Optimal sampling schemes are consistent with Ae. aegypti ecology and field studies, requiring only minor adjustments to current mosquito surveillance programs. This study represents the first theoretical exploration of the application of CKMR to an insect species, and demonstrates its potential for characterizing the demography of insects of epidemiological and agricultural importance.

sentences: Longitudinal single-cell epitope and RNA-sequencing reveals the immunological impact of type 1 interferon autoantibodies in critical COVID-19 Type I interferon (IFN-I) neutralizing autoantibodies have been found in some critical COVID-19 patients; however, their prevalence and longitudinal dynamics across the disease severity scale, and functional effects on circulating leukocytes remain unknown. Here, in 284 COVID-19 patients, we found IFN-I autoantibodies in 19% of critical, 6% of severe and none of the moderate cases. Longitudinal profiling of over 600,000 peripheral blood mononuclear cells using multiplexed single-cell epitope and transcriptome sequencing from 54 COVID-19 patients, 15 non-COVID-19 patients and 11 non-hospitalized healthy controls, revealed a lack of IFN-I stimulated gene (ISG-I) response in myeloid cells from critical cases, including those producing anti-IFN-I autoantibodies. Moreover, surface protein analysis showed an inverse correlation of the inhibitory receptor LAIR-1 with ISG-I expression response early in the disease course. This aberrant ISG-I response in critical patients with and without IFN-I autoantibodies, supports a unifying model for disease pathogenesis involving ISG-I suppression via convergent mechanisms.

sentences: PyPlr: A versatile, integrated system of hardware and software for researching the human pupillary light reflex We introduce PyPlr--a versatile, integrated system of hardware and software to support a broad spectrum of research applications concerning the human pupillary light reflex (PLR). PyPlr is a custom Python library for integrating a research-grade video-based eye-tracker system with a light source and streamlining stimulus design, optimisation and delivery, device synchronisation, and extraction, cleaning, and analysis of pupil data. We additionally describe how full-field, homogenous stimulation of the retina can be realised with a low-cost integrating sphere that serves as an alternative to a more complex Maxwellian view setup. Users can integrate their own light source, but we provide full native software support for a high-end, commercial research-grade 10-primary light engine that offers advanced control over the temporal and spectral properties of light stimuli as well as spectral calibration utilities. Here, we describe the hardware and software in detail and demonstrate its capabilities with two example applications: 1) pupillometer-style measurement and parametrisation of the PLR to flashes of white light, and 2) comparing the post-illumination pupil response (PIPR) to flashes of long and short-wavelength light. The system holds promise for researchers who would favour a flexible approach to studying the PLR and the ability to employ a wide range of temporally and spectrally varying stimuli, including simple narrowband stimuli.

sentences: Identification of gene products involved in plant colonization by Pantoea sp. YR343 using a diguanylate cyclase expressed in the presence of plants Microbial colonization of plant roots is a highly complex process that requires the coordination and regulation of many gene networks, yet the functions of many of these gene products remain poorly understood. Pantoea sp. YR343, a gamma-proteobacterium isolated from the rhizosphere of Populus deltoides, forms robust biofilms along the root surfaces of Populus and possesses plant growth-promoting characteristics. The mechanisms governing biofilm formation along plant roots by bacteria, including Pantoea sp. YR343, are not fully understood and many genes involved in this process have yet to be discovered. In this work, we identified three diguanylate cyclases in the plant-associated microbe Pantoea sp. YR343 that are expressed in the presence of plant roots, One of these diguanylate cyclases, DGC2884 localizes to discrete sites in the cells and its overexpression results in reduced motility and increased EPS production and biofilm formation. We then performed a genetic screen by expressing this diguanylate cyclase from an inducible promoter in order to identify candidate downstream effectors of c-di-GMP signaling which may be involved in root colonization by Pantoea sp. YR343. Further, we demonstrate the importance of other domains in DGC2884 to its activity, which in combination with the genes identified by transposon mutagenesis, may yield insights into activity and regulation of homologous enzymes in medically and agriculturally relevant microbes.

sentences: Diminished miRNA activity is associated with aberrant cytoplasmic intron retention in ALS pathogenesis Intron retention (IR) is now recognized as a dominant splicing event during motor neuron (MN) development, however the role and regulation of intron-retaining transcripts (IRTs) localized to the cytoplasm remain particularly understudied. By resolving the spatiotemporal dynamics of IR underlying distinct stages of MN lineage restriction, we identify a cytoplasmic group of IRTs that is not associated with reduced expression of their own genes but instead with an upregulation of predicted target genes of specific miRNAs, the motifs of which are enriched within the intronic sequences of this group. Next, we show that ALS-causing VCP mutations lead to a selective increase in IR of this particular class of introns. This in turn temporally coincides with an increase in the expression level of predicted target genes of these miRNAs, providing a potential mechanistic insight into ALS pathogenesis. Altogether, we propose a novel role for the cytoplasmic intronic sequences in regulating miRNA activity through miRNA sequestration, which potentially contributes to ALS pathogenesis.

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Successional stages in infant gut microbiota maturation BackgroundDisturbances in the primary colonization of the infant gut can result in life-long consequences and have been associated with a range of host conditions. Although early life factors have been shown to affect the infant gut microbiota development, our current understanding of the human gut colonization in early life remains limited.

To gain more insights in the unique dynamics of this rapidly evolving ecosystem, we investigated the microbiota over the first year of life in eight densely sampled infants (total number of samples, n=303). To evaluate gut microbiota maturation transition towards an adult configuration, we compared the microbiome composition of the infants to the Flemish Gut Flora Project population (n=1,106).

ResultsWe observed the infant gut microbiota to mature through three distinct, conserved stages of ecosystem development. Across these successional gut microbiota maturation stages, genus predominance was observed to shift from Escherichia over Bifidobacterium to Bacteroides. Both disease and antibiotic treatment were observed to be associated occasionally with gut microbiota maturation stage regression, a transient setback in microbiota maturation dynamics. Although the studied microbiota trajectories evolved to more adult-like constellations, microbiome community typing against the background of the Flemish Gut Flora Project (FGFP) cohort clustered all infant samples within the (in adults) potentially dysbiotic Bact2 enterotype.

ConclusionWe confirmed similarities between infant gut microbial colonization and adult dysbiosis. A profound knowledge about the primary gut colonization process in infants might provide crucial insights into how the secondary colonization of a dysbiotic adult gut can be redirected.

sentences: Modeling gene x environment interactions in PTSD using glucocorticoid-induced transcriptomics in human neurons Post-traumatic stress disorder (PTSD) results from severe trauma exposure, but the extent to which genetic and epigenetic risk factors impact individual clinical outcomes is unknown. We assessed the impact of genomic differences following glucocorticoid administration by examining the transcriptional profile of human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons and live cultured peripheral blood mononuclear cells from combat veterans with PTSD (n=5) and without PTSD (n=5). This parallel examination in baseline and glucocorticoid-treated conditions resolves cell-type specific and diagnosis-dependent elements of stress response, and permits discrimination of gene expression signals associated with PTSD risk from those induced by stress. Computational analyses revealed neuron-specific glucocorticoid-response expression patterns that were enriched for transcriptomic patterns observed in clinical PTSD samples. PTSD-specific signatures, albeit underpowered, accurately stratify veterans with PTSD relative to combat-exposed controls. Overall, in vitro PTSD and glucocorticoid response signatures in blood and brain cells represent exciting new platforms with which to test the genetic and epigenetic mechanisms underlying PTSD, identify biomarkers of PTSD risk and onset, and conduct drug-screening to identify novel therapeutics to prevent or ameliorate clinical phenotypes.

sentences: How effective are frogs in regulating crop pest population in a natural multi-trophic system?? Potential of frogs as important natural pest control agents has been highlighted earlier. But the effectiveness of frogs in regulating the pest load in intensive agricultural landscape in a multi-trophic system is not clear. We performed controlled field experiment in paddy field with a varying density (observed in high and low agricultural intensity (AI) areas) of a commonly found frog species and compared the pest and pest predator build-up. The consumption rate of the model amphibian was studied using enclosure experiment. The consequent trophic cascade effect of frogs on both crop pest and other arthropod pest predator was analyzed using mathematical population growth models. Although frogs consumed pests, they could not reduce crop pest abundance. although a lesser frog density found in high AI areas significantly affected the pest predator abundance. Based on the functional response result, mathematical growth models demonstrated that with a constant harvesting factor (Hollings Type II) frogs will always have a negative impact on the beneficial natural enemy population due to intraguild predation thereby limiting its potential as a pest regulator. Our study challenges the notion of frogs as an effective pest control agent and argues that increasing habitat diversity might improve overall biological pest suppression.

sentences: Selective Ablation of 3' RNA ends and Processive RTs Facilitate Direct cDNA Sequencing of Full-length Host Cell and Viral Transcripts Alternative splicing (AS) is necessary for viral proliferation in host cells and a critical regulatory component of viral gene expression. Conventional RNA-seq approaches provide incomplete coverage of AS due to their short read-lengths and are susceptible to biases and artifacts introduced in prevailing library preparation methodologies. Moreover, viral splicing studies are often conducted separately from host cell transcriptome analysis, precluding an assessment of the viral manipulation of host splicing machinery. To address current limitations, we developed a quantitative full-length direct cDNA sequencing strategy to simultaneously profile viral and host cell transcripts. This nanopore-based approach couples processive reverse transcriptases with a novel one-step chemical ablation of 3 RNA ends (termed CASPR) which decreases ribosomal RNA reads and enriches for poly-adenylated coding sequences. We extensively validate our approach using synthetic reference transcripts and show CASPR doubles the breadth of coverage per transcript and increases detection of long transcripts (>4kb), while being functionally equivalent to PolyA+ selection for transcript quantification. We used our approach to interrogate host cell and HIV-1 transcript dynamics during viral reactivation and identified novel putative HIV-1 host factors containing exon skipping or novel intron retentions and delineated the HIV-1 transcriptional state associated with these differentially regulated host factors.

sentences: Genome-wide CRISPR screens identify novel regulators of wild-type and mutant p53 stability Tumour suppressor p53 (TP53) is the most frequently mutated gene in cancer. Several hotspot p53 mutants not only lose tumour suppressive capabilities, but also function in a dominant-negative manner, suppressing canonical wild-type p53 function. Furthermore, some hotspot p53 mutants promote oncogenesis by gain-of-function mechanisms. Levels of p53 are regulated predominantly through regulation of protein stability and while wild-type p53 is normally kept at very low levels at steady-state, p53 mutants are often stabilized in tumours, which may be vital for their oncogenic properties. Here, we systematically profiled the factors that regulate protein stability of wild-type and mutant p53 using marker-based genome-wide CRISPR screens. We found that most proteins that regulate wild-type p53 also regulate a subset of p53 mutants with the exception of p53 R337H regulators, which are largely private to this mutant. Mechanistically, we identified FBXO42 as novel positive regulator of a subset of mutant p53 comprising R273H, R248Q and R248W. We show that, FBXO42 acts together with CCDC6 to regulate USP28-mediated p53 stabilization. Our work also identifies C16orf72 as negative regulator of wild-type p53 stability as well as stability of all p53 mutants tested. C16orf72 is amplified in breast cancer, and we show that C16orf72 regulates p53 levels in mammary epithelium of mice and its overexpression can drive breast cancer formation. Together, this work provides a network view of the process that regulates p53 stability, which might provide clues for targeting of wild-type or mutant p53 in cancer.

sentences: Unique Microglial Transcriptomic Signature within the Hippocampal Neurogenic Niche Microglia, the resident immune cells of the brain, are crucial in the development of the nervous system. Recent evidence demonstrates that microglia modulate adult hippocampal neurogenesis by inhibiting cell proliferation of neural precursors and survival both in vitro and in vivo, thus maintaining a balance between cell division and cell death in the neural stem cell pool. There are increasing reports suggesting these microglia found in neurogenic niches differ from their counterparts in non-neurogenic areas. Here, we present evidence that microglia in the hippocampal neurogenic niche are a specialized population that express genes known to regulate neurogenesis. By comprehensively profiling myeloid lineage cells in the hippocampus using single cell RNA-sequencing, we resolve transcriptomic differences in microglia originating from the subgranular zone. These cells have lower expression of genes associated with homeostatic microglia and increased expression of genes associated with phagocytosis. Intriguingly, this small yet distinct population expresses a gene signature with substantial overlap with previously characterized phenotypes, including disease associated microglia (DAM), a particularly unique and compelling microglial state.

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Gene expression and DNA methylation consistently predict cancer mutation states In studies of cellular function in cancer, researchers are increasingly able to choose from many -omics assays as functional readouts. Choosing the correct readout for a given study can be difficult, and which layer of cellular function is most suitable to capture the relevant signal may be unclear. In this study, we consider prediction of cancer mutation status (presence or absence) from functional -omics data as a representative problem. Since functional signatures of cancer mutation have been identified across many data types, this problem presents an opportunity to quantify and compare the ability of different -omics readouts to capture signals of dysregulation in cancer. The TCGA Pan-Cancer Atlas contains genetic alteration data including somatic mutations and copy number variants (CNVs), as well as several -omics data types. From TCGA, we focus on RNA sequencing, DNA methylation arrays, reverse phase protein arrays (RPPA), microRNA, and somatic mutational signatures as -omics readouts.

Across a collection of genes recurrently mutated in cancer, RNA sequencing tends to be the most effective predictor of mutation state. Surprisingly, we found that for many of the genes we considered, one or more other data types are approximately equally effective predictors. Performance was more variable between mutations than it was between data types for the same mutation, and there was often little difference between the top data types. We also found that combining data types into a single multi-omics model provided little or no improvement in predictive ability over the best individual data type. Based on our results, for the design of studies focused on the functional outcomes of cancer mutations, there are often multiple -omics types that can serve as effective readouts, although gene expression seems to be a reasonable default option.

sentences: The Conserved Colletotrichum spp. Effector CEC3 Induces Nuclear Expansion and Cell Death in Plants Plant pathogens secrete small proteins, known as effectors, that promote infection by manipulating host cells. Members of the phytopathogenic fungal genus Colletotrichum collectively have a broad host range and generally adopt a hemibiotrophic lifestyle that includes an initial biotrophic phase and a later necrotrophic phase. We hypothesized that Colletotrichum fungi use a set of conserved effectors during infection to support the two phases of their hemibiotrophic lifestyle. This study aimed to examine this hypothesis by identifying and characterizing conserved effectors among Colletotrichum fungi. Comparative genomic analyses using genomes of ascomycete fungi with different lifestyles identified seven effector candidates that are conserved across the genus Colletotrichum. Transient expression assays showed that one of these conserved effectors, CEC3, induces nuclear expansion and cell death in Nicotiana benthamiana, suggesting that CEC3 is involved in promoting host cell death during infection. Nuclear expansion and cell death induction were commonly observed in CEC3 homologs from four different Colletotrichum species that vary in host specificity. Thus, CEC3 proteins could represent a novel class of core effectors with functional conservation in the genus Colletotrichum.

sentences: Combined landscape of single-nucleotide variants and copy-number alterations in clonal hematopoiesis Implicated in the development of hematological malignancies (HM) and cardiovascular mortality, clonal hematopoiesis (CH) in apparently healthy individuals has been investigated by detecting either single-nucleotide variants and indels (SNVs/indels) or copy number alterations (CNAs), but not both. Here by combining targeted sequencing of 23 CH-related genes and array-based CNA detection of blood-derived DNA, we have delineated the landscape of CH-related SNVs/indels and CNAs in a general population of 11,234 individuals, including 672 with subsequent HM development. Both CH-related lesions significantly co-occurred, which combined, affected blood count, hypertension, and the mortality from HM and cardiovascular diseases depending on the total number of both lesions, highlighting the importance of detecting both lesions in the evaluation of CH.

sentences: Ultra-deep sequencing reveals no evidence of oncogenic mutations or enrichment by ex vivo CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells As CRISPR-based therapies enter the clinic, evaluation of the safety remains a critical and still active area of study. While whole genome sequencing is an unbiased method for identifying somatic mutations introduced by ex vivo culture and genome editing, this methodology is unable to attain sufficient read depth to detect extremely low frequency events that could result in clonal expansion. As a solution, we utilized an exon capture panel to facilitate ultra-deep sequencing of >500 tumor suppressors and oncogenes most frequently altered in human cancer. We used this panel to investigate whether transient delivery of high-fidelity Cas9 protein targeted to three different loci (using guide RNAs (gRNAs) corresponding to sites at AAVS1, HBB, and ZFPM2) at day 4 and day 10 timepoints post-editing resulted in the introduction or enrichment of oncogenic mutations. In three separate primary human HSPC donors, we identified a mean of 1,488 variants per Cas9 treatment (at <0.07% limit of detection). After filtering to remove germline and/or synonymous changes, a mean of 3.3 variants remained per condition, which were further reduced to six total mutations after removing variants in unedited treatments. Of these, four variants resided at the predicted off-target site in the myelodysplasia-associated EZH2 gene that were subject to ZFPM2 gRNA targeting in Donors 2 and 3 at day 4 and day 10 timepoints. While Donor 1 displayed on-target cleavage at ZFPM2, we found no off-target activity at EZH2. Sanger sequencing revealed a homozygous single nucleotide polymorphism (SNP) at position 14bp distal from the Cas9 protospacer adjacent motif in EZH2 that eliminated any detectable off-target activity. We found no evidence of exonic off-target INDELs with either of the AAVS1 or HBB gRNAs. These findings indicate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP outside the seed region of the gRNA protospacer is sufficient to eliminate Cas9 off-target activity with this method of delivery into primary, repair competent human HSPCs.

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Measuring mitochondrial electron transfer complexes in previously frozen cardiac tissue from the offspring of sow: A model to assess exercise induced mitochondrial bioenergetics changes Measuring the mitochondrial electron transfer complex (ETC) profile from previously frozen heart tissue samples from offspring born to an exercised sow provided descriptive data about exercise-induced mitochondrial biochemical changes in heart tissue from the offspring born to the exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

SUMMARYPreparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

sentences: Weak representation of awake/sleep states by local field potentials in aged mice Senescence affects various aspects of sleep, and it remains unclear how sleep-related neuronal network activity is altered by senescence. Here, we recorded local field potential signals from multiple brain regions covering the forebrain in young (10-week-old) and aged (2-year-old) mice. Interregional LFP correlations across these brain regions showed smaller differences between awake and sleep states in aged mice. Multivariate analyses with machine learning algorithms with uniform manifold approximation and projection (UMAP) and robust continuous clustering (RCC) demonstrated that these LFP correlational patterns in aged mice less represented awake/sleep states than those in young mice. By housing aged mice in an enriched environment, the LFP patterns were restored to those observed in young mice. Our results demonstrate senescence-induced changes in neuronal activity at the network level and provide insight into the prevention of pathological symptoms associated with sleep disturbance in senescence.

sentences: Purification of immunostimulatory glucose homopolymer from broccoli (Brassica oleracea var. italica) We prepared broccoli (Brassica oleracea var. italica) neutral polysaccharides (flow-through fractions of anion exchange column chromatography from hot water extracts) from different broccoli cultivars and compared their immunostimulatory effects in the silkworm muscle contraction assay. The cultivars showed a wide range of activity, with the cultivar  Winter dome showing the highest specific activity (more than 100 times higher than curdlan). Furthermore, the active substance was purified by gel filtration column chromatography. The active substance showed heterogeneous molecular weights of more than 270 kDa. Sugar composition analysis of the purified fraction revealed that more than 95% of its sugar component was glucose, suggesting that the immunostimulatory neutral polysaccharide from broccoli cultivar  Winter dome was a homopolymer of glucose. The purified fraction also induced TNF production in cultured mouse macrophage cells. These results suggest that the glucose homopolymer in broccoli has an immunostimulatory effect on both arthropod and mammalian immune system.

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Exploring Proteomes of Robust Yarrowia lipolytica Isolates Cultivated in Biomass Hydrolysate Reveal Key Processes Impacting Mixed Sugar Utilization, Lipid Accumulation, and Degradation Yarrowia lipolytica is an oleaginous yeast exhibiting robust phenotypes beneficial for industrial biotechnology. The phenotypic diversity found within the undomesticated Y. lipolytica clade from various origins illuminates desirable phenotypic traits not found in the conventional laboratory strain CBS7504, which include xylose utilization, lipid accumulation, and growth on undetoxified biomass hydrolysates. Currently, the related phenotypes of lipid accumulation and degradation when metabolizing non-preferred sugars (e.g., xylose) associated with biomass hydrolysates are poorly understood, making it difficult to control and engineer in Y. lipolytica. To fill this knowledge gap, we analyzed the genetic diversity of five undomesticated Y. lipolytica strains and identified singleton genes and genes exclusively shared by strains exhibiting desirable phenotypes. Strain characterizations from controlled bioreactor cultures revealed that the undomesticated strain YB420 used xylose to support cell growth and maintained high lipid levels while the conventional strain CBS7504 degraded cell biomass and lipids when xylose was the sole remaining carbon source. From proteomic analysis, we identified carbohydrate transporters, xylose metabolic enzymes and pentose phosphate pathway proteins stimulated during the xylose uptake stage for both strains. Furthermore, we distinguished proteins in lipid metabolism (e.g., lipase, NADPH generation, lipid regulators, {beta}-oxidation) activated by YB420 (lipid maintenance phenotype) or CBS7504 (lipid degradation phenotype) when xylose was the sole remaining carbon source. Overall, the results relate genetic diversity of undomesticated Y. lipolytica strains to complex phenotypes of superior growth, sugar utilization, lipid accumulation and degradation in biomass hydrolysates.

IMPORTANCEYarrowia lipolytica is an important industrial oleaginous yeast due to its robust phenotypes for effective conversion of inhibitory lignocellulosic biomass hydrolysates into neutral lipids. While lipid accumulation has been well characterized in this organism, its interconnected lipid degradation phenotype is poorly understood during fermentation of biomass hydrolysates. Our investigation into the genetic diversity of undomesticated Y. lipolytica strains, coupled with detailed strain characterization and proteomic analysis, revealed metabolic processes and regulatory elements conferring desirable phenotypes for growth, sugar utilization, and lipid accumulation in undetoxified biomass hydrolysates by these natural variants. This study provides a better understanding of the robust metabolism of Y. lipolytica and suggests potential metabolic engineering strategies to enhance its performance.

sentences: Isoform-specific mutation in Dystonin-b gene causes late-onset protein aggregate myopathy and cardiomyopathy Dystonin (DST), which encodes cytoskeletal linker proteins, express three tissue-selective isoforms: neural DST-a, muscular DST-b, and epithelial DST-e. DST mutations cause different disorders including hereditary sensory and autonomic neuropathy 6 (HSAN-VI) and epidermolysis bullosa simplex; however, etiology of the muscle phenotype in DST-related diseases has been unclear. Because DST-b contains all of the DST-a-encoding exons, known HSAN-VI mutations could affect both DST-a and DST-b isoforms. To investigate the specific function of DST-b in striated muscles, we generated a Dst-b-specific mutant mouse model harboring a nonsense mutation. Dst-b mutant mice exhibited late-onset protein aggregate myopathy and cardiomyopathy without neuropathy. We observed desmin aggregation, focal myofibrillar dissolution, and mitochondrial accumulation in striated muscles, which are common characteristics of myofibrillar myopathy. We also found nuclear inclusions containing p62, ubiquitin, and SUMO proteins with nuclear envelope invaginations as a unique pathological hallmark in Dst-b mutation-induced cardiomyopathy. RNA sequencing analysis revealed changes in expression of genes responsible for cardiovascular functions. In silico analysis identified DST-b alleles with nonsense mutations in populations worldwide, suggesting that some unidentified hereditary myopathy and cardiomyopathy are caused by DST-b mutations. Here, we demonstrate that the Dst-b isoform is essential for long-term maintenance of striated muscles.

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Prostaglandin PGE2 receptor EP4 regulates microglial phagocytosis and increases susceptibility to diet-induced obesity BackgroundIn rodents, susceptibility to diet-induced obesity requires microglial activation, but the molecular components of this pathway remain incompletely defined. Prostaglandin E2 (PGE2) levels increase in the mediobasal hypothalamus during high fat diet (HFD) feeding, and the PGE2 receptor EP4 regulates microglial activation state and phagocytic activity, suggesting a potential role for microglial EP4 signaling in obesity pathogenesis.

MethodMetabolic phenotyping, as assessed by body weight, energy expenditure, glucose, and insulin tolerance, was performed in microglia-specific EP4 knockout (MG-EP4 KO) mice and littermate controls on HFD. Morphological and gene expression analysis of microglia, and a histological survey of microglia-neuron interactions in the arcuate nucleus was performed. Phagocytosis was assessed using in vivo and in vitro pharmacological techniques.

ResultsMicroglial EP4 deletion markedly reduced weight gain and food intake in response to HFD feeding. In correspondence with this lean phenotype, insulin sensitivity was also improved in the HFD-fed MG-EP4 KO mice though glucose tolerance remained surprisingly unaffected. Mechanistically, EP4-deficient microglia showed an attenuated phagocytic state marked by reduced CD68 expression and fewer contacts with POMC neuron soma and processes. These cellular changes observed in the microglial EP4 knockout mice corresponded with an increased density of POMC neurites extending into the paraventricular nucleus.

ConclusionThese findings reveal that microglial EP4 signaling promotes body weight gain and insulin resistance during HFD feeding. Furthermore, the data suggest that curbing microglial phagocytic function may preserve POMC cytoarchitecture and PVN input to limit overconsumption during diet-induced obesity.

sentences: A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell-matrix adhesions. While biochemical mechanisms that regulate activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM II heavy chains and myofilament assembly. We found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein levels of UNC-45A were decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at epithelial junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.

sentences: Mechanical torque promotes bipolarity of the mitotic spindle through multi-centrosomal clustering Intracellular forces shape cellular organization and function. One example is the mi-totic spindle, a cellular machine consisting of multiple chromosomes and centrosomes which interact via dynamic microtubule filaments and motor proteins, resulting in complicated spatially dependent forces. For a cell to divide properly, is important for the spindle to be bipolar, with chromosomes at the center and multiple centrosomes clustered into two  poles at opposite sides of the chromosomes. Experimental observations show that in unhealthy cells, the spindle can take on a variety of patterns. What forces drive each of these patterns? It is known that attraction between centrosomes is key to bipolarity, but what the prevents the centrosomes from collapsing into a monopolar configuration? Here, we explore the hypothesis that torque rotating chromosome arms into orientations perpendicular to the centrosome-centromere vector promotes spindle bipolarity. To test this hypothesis, we construct a pairwise-interaction model of the spindle. On a continuum version of the model, an integro-PDE system, we perform linear stability analysis and construct numerical solutions which display a variety of spatial patterns. We also simulate a discrete particle model resulting in a phase diagram that confirms that the spindle bipolarity emerges most robustly with torque. Altogether, our results suggest that rotational forces may play an important role in dictating spindle patterning.

sentences: Revealing Free Energy Landscape from MD Data via Conditional Angle Partition Tree Deciphering the free energy landscape of biomolecular structure space is crucial for understanding many complex molecular processes, such as protein-protein interaction, RNA folding, and protein folding. A major source of current dynamic structure data is Molecular Dynamics (MD) simulations. Several methods have been proposed to investigate the free energy landscape from MD data, but all of them rely on the assumption that kinetic similarity is associated with global geometric similarity, which may lead to unsatisfactory results. In this paper, we proposed a new method called Conditional Angle Partition Tree to reveal the hierarchical free energy landscape by correlating local geometric similarity with kinetic similarity. Its application on the benchmark alanine dipeptide MD data showed a much better performance than existing methods in exploring and understanding the free energy landscape. We also applied it to the MD data of Villin HP35. Our results are more reasonable on various aspects than those from other methods and very informative on the hierarchical structure of its energy landscape.

sentences: Synaptic and intrinsic potentiation in O-LM interneurons is induced by theta patterns of stimulation Oriens lacunosum-moleculare (O-LM) interneurons display a non-conventional form of long-term synaptic potentiation (LTP) conferred by calcium-permeable AMPA receptors (CP-AMPAR). So far, this form of LTP has been induced in O-LM cells by physiologically unrealistic protocols. We report here the induction of both synaptic and intrinsic potentiation in O-LM interneurons following stimulation of afferent glutamatergic inputs in the theta ({theta}) frequency range. LTP is induced by synaptic activation of CP-AMPAR whereas long-term potentiation of intrinsic excitability (LTP-IE) results from the mGluR1-dependent down-regulation of Kv7 voltage-dependent potassium channel and hyperpolarization activated and cyclic nucleotide-gated (HCN) channel through the depletion of phosphatidylinositol-4,5-bi-phosphate (PIP2). LTP and LTP-IE are reversible, demonstrating that both synaptic and intrinsic changes are bidirectional in O-LM cells. We conclude that physiological stimuli such as {theta} patterns induce synaptic and intrinsic potentiation in O-LM interneurons.

sentences: A rigorous framework for detecting SARS-CoV-2 spike protein mutational ensemble from genomic and structural features The recent release of SARS-CoV-2 genomic data from several countries has provided clues into the potential antigenic drift of the coronavirus population. In particular, the genomic instability observed in the spike protein necessitates immediate action and further exploration in the context of viralhost interactions. By temporally tracking 527,988 SARS-CoV-2 genomes, we identified invariant and hypervariable regions within the spike protein. We evaluated combination of mutations from SARS-CoV-2 lineages and found that maximum number of lineage-defining mutations were present in the N-terminal domain (NTD). Based on mutant 3D-structural models of known Variants of Concern (VOCs), we found that structural properties such as accessibility, secondary structural type, and intra-protein interactions at local mutation sites are greatly altered. Further, we observed significant differences between intra-protein networks of wild-type and Delta mutant, with the latter showing dense intra-protein contacts. Extensive molecular dynamics simulations of D614G mutant spike structure with hACE2 further revealed dynamic features with 47.7% of mutations mapping on flexible regions of spike protein. Thus, we propose that significant changes within spike protein structure have occurred that may impact SARS-CoV-2 pathogenesis, and repositioning of vaccine candidates is required to contain the spread of COVID-19 pathogen.

sentences: Glucose-1,6-bisphosphate, a key metabolic regulator, is synthesized by a distinct family of α-D-phosphohexomutases widely distributed in prokaryotes The reactions of -D-phosphohexomutases (PHM) are ubiquitous, key to primary metabolism and essential for several processes in all domains of life. The functionality of these enzymes relies on an initial auto-phosphorylation step which requires the presence of -D-glucose-1,6-bisphosphate (Glc-1,6-BP). While well investigated in vertebrates, the origin of this activator compound in bacteria is unknown. Here we show that the Slr1334 protein from the unicellular cyanobacterium Synechocysitis sp. PCC 6803 is a Glc-1,6-BP-synthase. Biochemical analysis revealed that Slr1334 efficiently converts fructose-1,6-bisphosphate (Frc-1,6-BP) and -D-glucose-1-phosphate/-D-glucose-6-phosphate into Glc-1,6-BP and also catalyzes the reverse reaction. As inferred from phylogenetic analysis, the slr1334 product belongs to a primordial subfamily of PHMs that is present especially in deeply branching bacteria and also includes human commensals and pathogens. Remarkably, the homologue of Slr1334 in the human gut bacterium Bacteroides salyersiae catalyzes the same reaction, suggesting a conserved and essential role for the members of this PHM subfamily.

sentences: Sister chromatid exchanges induced by perturbed replication are formed independently of homologous recombination factors Sister chromatid exchanges (SCEs) are products of joint DNA molecule resolution, and are considered to form through homologous recombination (HR). Indeed, upon generation of irradiation-induced DNA breaks, SCE induction was compromised in cells deficient for canonical HR factors BRCA1, BRCA2 and RAD51. Contrarily, replication-blocking agents, including PARP inhibitors, induced SCEs independently of BRCA1, BRCA2 and RAD51. PARP inhibitor-induced SCEs were enriched at common fragile sites (CFSs), and were accompanied by post-replicative single-stranded DNA (ssDNA) gaps. Moreover, PARP inhibitor-induced replication lesions were transmitted into mitosis, suggesting that SCEs originate from mitotic processing of under-replicated DNA. We found that DNA polymerase theta (POLQ) was recruited to mitotic DNA lesions, and loss of POLQ resulted in reduced SCE numbers and severe chromosome fragmentation upon PARP inhibition in HR-deficient cells. Combined, our data show that PARP inhibition generates under-replicated DNA, which is transferred into mitosis and processed into SCEs, independently of canonical HR factors.

sentences: The Fragile X Mental Retardation Protein protects the lung from xenobiotic stress by facilitating the Integrated Stress Response Stress response pathways protect the lung from the damaging effects of environmental toxicants. Here we investigate the role of the Fragile X Mental Retardation Protein (FMRP), a multifunctional protein implicated in stress responses, in the lung. We report that FMRP is expressed in murine and human lungs, in the airways and more broadly. Analysis of airway stress responses in mice and in a murine cell line ex vivo, using the well-established Naphthalene (Nap) injury model, reveals that FMRP-deficient cells exhibit increased expression of markers of oxidative and genotoxic stress and increased cell death. We find that FMRP-deficient cells fail to actuate the Integrated Stress Response Pathway (ISR) and upregulate the transcription factor ATF4. Knockdown of ATF4 expression phenocopies the loss of FMRP. We extend our analysis of the role of FMRP to human bronchial BEAS-2B cells, using a 9, 10-Phenanthrenequinone air pollutant model, to find that FMRP-deficient BEAS-2B also fail to actuate the ISR and exhibit greater susceptibility. Taken together, our data suggest that FMRP has a conserved role in protecting the airways by facilitating the ISR.

sentences: Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitro transport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD> 50 {micro}M), and this monomer binds reversibly to spTorA (KD{approx} 1 {micro}M). While TorD binds to membranes (KD{approx} 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.

sentences: A functional topography within the cholinergic basal forebrain for processing sensory cues associated with reward and punishment Basal forebrain cholinergic neurons (BFCNs) project throughout the cortex to regulate arousal, stimulus salience, plasticity, and learning. Although often treated as a monolithic structure, the basal forebrain features distinct connectivity along its anteroposterior axis that could impart regional differences in BFCN processing. Here, we performed simultaneous bulk calcium imaging from anterior and posterior BFCNs over a one-month period of variable reinforcement learning in mice. BFCNs in both regions showed equivalently weak responses to unconditioned visual stimuli and anticipated rewards. Anterior BFCNs in the horizontal limb of the diagonal band were more responsive to reward omission, more accurately classified behavioral outcomes, and more closely tracked fluctuations in pupil-indexed global brain state. Posterior BFCNs in globus pallidus and substantia innominata were more responsive to unconditioned auditory stimuli, orofacial movements, aversive reinforcement, and showed robust associative plasticity for punishment-predicting cues. These results identify a functional topography that diversifies cholinergic modulatory signals broadcast to downstream brain regions.

sentences: Age-related increase in matrix stiffness downregulates α-Klotho in chondrocytes and induces cartilage degeneration Enhanced mechanistic insight into age-related knee osteoarthritis (KOA) is an essential step to promote successful translation of animal research to bedside interventions. To this end, the goal of these studies was to interrogate molecular mechanisms driving age-related KOA in a mouse model and correspond findings to human knee cartilage. Unbiased mass spectrometry proteomics of cartilage tissue revealed PI3K/Akt signaling was the predominant pathway disrupted over time in male, but not female, mice. This finding was consistent with a significantly accelerated KOA progression in males when compared to female counterparts. In probing for upstream regulators of these age-dependent alterations, we found that -Klotho, a suppressor of PI3K/Akt signaling and potent longevity protein, significantly decreased with aging in both mouse and human knee cartilage. Upstream of these alterations, we found that age-related increases in matrix stiffness initiated a cascade of altered nuclear morphology and downregulated -Klotho expression, ultimately impairing chondrocyte health. Conversely, reducing matrix stiffness increased -Klotho expression in chondrocytes, thus enhancing their chondrogencity and cartilage integrity. Collectively, our findings establish a novel mechanistic link between age-related alterations in ECM biophysical properties and regulation of cartilage health by -Klotho.

sentences: Dynlt3, a novel fundamental regulator of melanosome movement, distribution, maturation and transfer Skin pigmentation is dependent on cellular processes including melanosome biogenesis, transport, maturation and transfer to keratinocytes. However, how the cells finely control these processes in space and time to ensure proper pigmentation remains unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for efficient melanosome transport, maturation and transfer. In melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional (convective) motion leading to their peripheral location in the cell, and are not fully matured. Stage IV melanosomes are more acidic, but still heavily pigmented, resulting in a less efficient melanosome transfer. Finally, the level of Dynlt3 is dependent on {beta}-catenin activity, revealing a novel function of the Wnt/{beta}-catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, position and maturation of melanosomes required for their transfer.

sentences: ERPs responses to dominance features from human faces Social dominance is an important feature of social life. Dominance has been proposed to be one of two trait dimensions underpinning social judgments of human faces. Yet, the neural bases of the ability to identify different dominance levels in others based on facial features remains poorly understood. Here, we used event-related potentials (ERPs) to determine the temporal dynamics of facial dominance evaluation based on facial features signaling physical strength/weakness in humans. Twenty-seven participants performed a dominance perception task where they passively viewed faces with different dominance levels. Dominance levels did not modulate an early component of face processing, known as the N170 component, but did modulate the Late Positive Potential (LPP) component. These findings indicate that participants inferred dominance levels at a late stage of face evaluation. Furthermore, the highest level of dominant faces and the lowest level of submissive faces both elicited higher LPP amplitudes than faces with a neutral dominance level. Taken together, the present study provides new insights regarding the dynamics of the neurocognitive processes underlying facial dominance evaluation.

sentences: Inhibitors of L-type calcium channels show therapeutic potential for treating SARS-CoV-2 infections by preventing virus entry and spread COVID-19 is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus (CoV)-2 (SARS-CoV-2). The virus is responsible for an ongoing pandemic and concomitant public health crisis around the world. While vaccine development is proving to be highly successful, parallel drug development approaches are also critical in the response to SARS-CoV-2 and other emerging viruses. Coronaviruses require Ca2+ ions for host cell entry and we have previously shown that Ca2+ modulates the interaction of the viral fusion peptide with host cell membranes. In an attempt to accelerate drug development, we tested a panel of L-type calcium channel blocker (CCB) drugs currently developed for other conditions, to determine whether they would inhibit SARS-CoV-2 infection in cell culture. All the CCBs tested showed varying degrees of inhibition, with felodipine and nifedipine strongly limiting SARS-CoV-2 entry and infection in epithelial lung cells at concentrations where cell toxicity was minimal. Further studies with pseudo-typed particles displaying the SARS-CoV-2 spike protein suggested that inhibition occurs at the level of virus entry. Overall, our data suggest that certain CCBs have potential to treat SARS-CoV-2 infections and are worthy of further examination for possible treatment of COVID-19.

sentences: Using a combination of CRISPR/Cas9, behavioural experiments and functional analysis to characterise taste receptors in honeybees Honeybees (Apis mellifera) need their fine sense of taste to evaluate nectar and pollen sources. Gustatory receptors (Grs) translate taste signals into electrical responses. In vivo experiments primarily demonstrate collective responses of the whole Gr-set, but little is known about the individual impact of receptors. Here, we disentangle for the first time the contributions of three gustatory receptors (AmGr1-3) in sugar sensing of honeybees by combining CRISPR/Cas9 mediated genetic knock-out, electrophysiology and behaviour. AmGr1 responds to multiple sugars. Bees lacking this receptor have a reduced response to sucrose and glucose but not to fructose. AmGr2 acts as co-receptor of AmGr1 in a heterologous expression system, but honeybee knock-out mutants perform normally. Eliminating AmGr3 while preserving AmGr1 and AmGr2 abolishes the perception of fructose but not of sucrose. We thus dissociate the roles of AmGr1, AmGr2 and AmGr3 in honeybee taste perception.

sentences: The Anna Karenina model of beta cell maturation in development and their dedifferentiation in type 1 and type 2 diabetes Loss of mature {beta} cell function and identity, or {beta} cell dedifferentiation, is seen in all types of diabetes mellitus. Two competing models explain {beta} cell dedifferentiation in diabetes. In the first model, {beta} cells dedifferentiate in the reverse order of their developmental ontogeny. This model predicts that dedifferentiated {beta} cells resemble {beta} cell progenitors. In the second model, {beta} cell dedifferentiation depends on the type of diabetogenic stress. This model, which we call the "Anna Karenina" model, predicts that in each type of diabetes, {beta} cells dedifferentiate in their own way, depending on how their mature identity is disrupted by any particular diabetogenic stress. We directly tested the two models using a {beta} cell-specific lineage-tracing system coupled with RNA-sequencing in mice. We constructed a multidimensional map of {beta} cell transcriptional trajectories during the normal course of {beta} cell postnatal development and during their dedifferentiation in models of both type 1 diabetes (NOD) and type 2 diabetes (BTBR-Lepob/ob). Using this unbiased approach, we show here that despite some similarities between immature and dedifferentiated {beta} cells, {beta} cells dedifferentiation in the two mouse models is not a reversal of developmental ontogeny and is different between different types of diabetes.

sentences: Localization of TRP channels in healthy oral mucosa from human donors The oral cavity is exposed to a remarkable range of noxious and innocuous conditions, including temperature fluctuations, mechanical forces, inflammation and environmental and endogenous chemicals. How such changes in the oral environment are sensed by oral cells and tissues is not completely understood. Transient receptor potential (TRP) ion channels are a diverse family of molecular receptors that are activated by chemicals, temperature changes, and tissue damage. In non-neuronal cells, TRP channels play roles in inflammation, as well as tissue development and maintenance. In somatosensory neurons, TRP channels mediate nociception, thermosensation and chemosensation. To assess whether TRP channels might be involved in environmental sensing in the human oral cavity, we investigated the distribution of TRP channels in human tongue and hard palate. Oral biopsies were collected from volunteers and underwent fluorescent immunohistochemistry followed by confocal imaging. We analyzed immunoreactivity of TRP channels in human oral epithelia including TRPV3, TRPV4, TRPV1, TRPM8, and TRPA1. TRPV3 and TRPV4 were expressed in epithelial cells with inverse expression patterns where they are likely to contribute to epithelial development and integrity. TRPA1 immunoreactivity was found in fibroblasts, subsets immune cells, and neurons, consistent with known roles of TRPA1 in sensory transduction, as well as in response to damage and inflammation. TRPM8 immunoreactivity was found in lamina propria cells and some neuronal subpopulations including some neurons within the end bulbs of Krause, consistent with a role in thermal sensation. TRPV1 immunoreactivity was identified in intraepithelial nerve fibers, in some end bulbs of Krause, and in epithelial cells, consistent with roles in nociception and thermosensation. Immunoreactivity of TRPM8 and TRPV1 in end bulbs of Krause suggest that these structures contain a variety of neuronal afferents, including those that mediate nociception, thermosensation and mechanotransduction. Collectively, these studies support the role of TRP channels in oral environmental surveillance and response.

sentences: STOPPING A CONTINUOUS MOVEMENT: A NOVEL APPROACH TO INVESTIGATING MOTOR CONTROL Flexible, adaptive behavior is critically dependent on inhibitory control. For example, if you suddenly notice you are about to step on a tack and would prefer not to, the ability to halt your ongoing movement is critical. To address limitations in existing approaches for studying your ability to rapidly terminate your movement ("stopping"), we developed a novel stop task. This task requires termination of ongoing motor programs, provides a direct measure of SSRT, and allows for comparison of the same behavior (stopping) in conditions that elicit either prepared or reactive inhibitory control. Here, we present and evaluate our novel Continuous Movement Stop Task (CMST). We examined several versions of the task in a total of 49 participants. Our data reveal that the CMST is effectively able to dissociate stopping behavior between the planned and unplanned conditions. Additionally, within the subset of participants for which we measured speed, we found that participants initiated stopping (with respect to the stop signal) significantly earlier on planned stop compared to unplanned stop trials. Finally, participants took longer to arrive at full motor arrest (i.e. SSRT) following stop initiation on planned than on unplanned stop trials. This novel task design will enable a more precise quantification of stopping behavior and, in conjunction with neuroscientific methods, could provide more rigorous characterization of brain networks underlying stopping.

sentences: Arctic evergreening - encroaching allelopathy in a management blind spot Management blind spots, i.e., influential processes not included in management models, may undermine management objectives and reduce system resilience. Here we address one such blind spot in the management of Rangifer in Norway. The current model is based on regulation of pastures and population vital rates through reindeer density, but lacks validation of the underlying assumption of forage stability, which is a critical blind spot. We report pasture changes over 18 growing seasons, under variable reindeer densities and a changing climate. We show a strong increase of the native evergreen, allelopathic crowberry, contrasted by smaller increases of deciduous dwarf-shrubs and stagnation in herbaceous species. We found no evidence of reindeer density influencing these vegetation changes. Our results unveil a trajectory of Arctic evergreening and increasing allelopathy, and a shift towards lower biodiversity and ecosystem functioning. It exemplifies an ongoing change in a management blind spot that greatly impacts social-ecological systems.

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Robust Prediction of Patient-Specific Clinical Response to Unseen Drugs From in vitro Screens Using Context-aware Deconfounding Autoencoder Accurate and robust prediction of patient-specific responses to drug treatments is critical for drug development and personalized medicine. However, patient data are often too scarce to train a generalized machine learning model. Although many methods have been developed to utilize cell line data, few of them can reliably predict individual patient clinical responses to new drugs due to data distribution shift and confounding factors. We develop a novel Context-aware Deconfounding Autoencoder (CODE-AE) that can extract common biological signals masked by context-specific patterns and confounding factors. Extensive studies demonstrate that CODE-AE effectively alleviates the out-of-distribution problem for the model generalization, significantly improves accuracy and robustness over state-of-the-art methods in both predicting patient-specific ex vivo and in vivo drug responses purely from in vitro screens and disentangling intrinsic biological signals from confounding factors. Using CODE-AE, we screened 50 drugs for 9,808 cancer patients and discovered novel personalized anti-cancer therapies and drug-response biomarkers.

Contact: lxie@iscb.org

sentences: Generating and testing the efficacy of transgenic Cas9 in Tribolium castaneum CRISPR/Cas9 genome editing has now expanded to many insect species, including Tribolium castaneum. However, compared to Drosophila melanogaster, the CRISPR toolkit of T. castaneum is limited. A particularly apparent gap is the lack of Cas9 transgenic animals, which generally offer higher editing efficiency. We address this by creating and testing transgenic beetles expressing Cas9. We generated two different constructs bearing basal heat shock promoter-driven Cas9, two distinct 3 UTRs, and one containing Cas9 fused to EGFP by a T2A peptide. For each construct, we were able to generate a line that is homozygous viable, though variable reductions in reproductive success with each construct were noted. Analyses of Cas9 activity in each transgenic line demonstrated that both designs are capable of inducing CRISPR-mediated changes in the genome in the absence of heat induction. Overall, these resources enhance the accessibility of CRISPR/Cas9 genome editing for the Tribolium research community and provide a benchmark against which to compare future transgenic Cas9 lines.

sentences: E. coli can eat DNA as excellent nitrogen source to grow quickly Is DNA or RNA a good nutrient? Although scientists have raised this question for dozens of years, few textbooks mention the nutritional role of nucleic acids. Paradoxically, mononucleotides are widely added to infant formula milk and animal feed for improving Intestinal health. Here, found that E. coli can grow well in the medium with DNA as carbon and nitrogen sources. More interestingly, in the presence of glucose and DNA, bacteria grew more rapidly, showing that bacteria can use DNA as an excellent nitrogen source. Surprisingly, the amount of DNA in culture media decreased but its length remained unchanged, demonstrating that E. coli ingested long DNA directly. The gene expression study shows that E. coli mainly ingests DNA before digestion and digests it in the periplasm. Bifidobacterium bifidum can also use DNA as the nitrogen source for growth but not efficiently as E. coli. This study is of great significance to study DNA metabolism and utilization in organisms. It also lays a foundation to understand the nutritional function of DNA for intestinal flora and human health.

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Wavelength-dependent effects of artificial light at night on phytoplankton growth and community structure Artificial light at night (ALAN) is increasingly recognised as a disruptive form of environmental pollution, impacting many physiological and behavioural processes that may scale up to population and community-level effects. Mounting evidence from animal studies show that the severity and type of the impact depends on the wavelength and intensity of ALAN. This knowledge has been instrumental for informing policy-making and planning for wildlife-friendly illumination. However, most of this evidence comes from terrestrial habitats, while research testing alternative wavelength illumination in marine environments is lagging behind. In this study we investigated the effect of such alternative ALAN colours on marine primary producers. Specifically, we tested the effect of green, red, and natural white LED illumination at night, compared to a dark control, on the growth of a green microalgae as well as the biomass, diversity and composition of a phytoplankton assemblage. Our findings show that green ALAN boosted chlorophyll production at the exponential growth stage, resulting in higher biomass production in the green algae Tetraselmis suesica. All ALAN wavelengths affected the biomass and diversity of the assemblage with the red and green ALAN having the stronger effects, leading to higher overall abundance and selective dominance of specific diatom species compared to white ALAN and the dark control.

SynthesisOur work indicates that the wavelength of artificial light sources in marine areas should be carefully considered in management and conservation plans. In particular, green and red light should be used with caution in coastal areas, where there might be a need to strike a balance between the strong effects of green and red light on marine primary producers with the benefit they bring to other organisms.

sentences: Dorsal color variation among subspecies of the "Oregon" Dark-eyed Junco complex Initial descriptions of avian subspecies were based on expert opinions of geographic variation in phenotypes and are inherently subjective. Although best practices for subspecies delimitation continue to be debated, reassessing subspecies limits with current, quantitative methods is important toward refining and improving taxonomic treatments. Plumage coloration is the basis of many subspecies diagnoses, but is potentially problematic because of the historical lack of quantitative methods to quantify color. Recently developed methods, such as colorimetry, provide repeatable measurements of color variation that can be used to reassess subspecies limits. In this study, we reassess color variation among subspecies of the Oregon Dark-eyed Junco (Junco hyemalis [oreganus Group]) complex, in which differences in back and hood color were established as diagnostic characters. We measured back and hood brightness and hue in 206 museum specimens among five Oregon Dark-eyed Junco subspecies using a colorimeter. We then compared mean measurements among subspecies and conducted a discriminant function analysis to assess how well dorsal color predicted subspecies. Our data correctly classified only 67.9% of males and 82.5% of females to their presumed subspecies. Furthermore, no adjacent subspecies pairs passed the "75% rule" due to extensive overlap in plumage characters. Thus, back color alone is not as effective in diagnosing Oregon Dark-eyed Junco subspecies as originally described, suggesting a possible taxonomic revision. Specifically, similarity in phenotypic and genetic data suggests that some combination of thurberi, montanus, and shufeldti may be lumped to recognize broad, clinal variation in dorsal color alongside clinal variation in other phenotypes and extensive gene flow.

sentences: Pathogenic variants of sphingomyelin synthase SMS2 disrupt lipid landscapes in the secretory pathway Sphingomyelin is a dominant sphingolipid in mammalian cells. Its production in the trans-Golgi traps cholesterol synthesized in the ER to promote formation of a sphingomyelin/sterol gradient along the secretory pathway. This gradient marks a fundamental transition in physical membrane properties that help specify organelle identify and function. We previously identified mutations in sphingomyelin synthase SMS2 that cause osteoporosis and skeletal dysplasia. Here we show that SMS2 variants linked to the most severe bone phenotypes retain full enzymatic activity but fail to leave the ER owing to a defective autonomous ER export signal. Cells harboring pathogenic SMS2 variants accumulate sphingomyelin in the ER and display a disrupted transbilayer sphingomyelin asymmetry. These aberrant sphingomyelin distributions also occur in patient-derived fibroblasts and are accompanied by imbalances in cholesterol organization, glycerophospholipid profiles and lipid order in the secretory pathway. We postulate that pathogenic SMS2 variants undermine the capacity of osteogenic cells to uphold nonrandom lipid distributions that are critical for their bone forming activity.

sentences: Rapid tissue-specific screening of Wolbachia, Cardinium and Rickettsia in Flies (Diptera: Sepsidae; Drosophilidae) Maternally transmitted endosymbionts can negatively influence the reproduction of their arthropod hosts (e.g. male-killing, cytoplasmic incompatibility). However, such infections are rarely assessed in insect models such as Sepsidae or Drosophilidae that are routinely used in sexual selection studies. To detect infection and bacterial localisation in the host, we developed and optimised a tissue-specific multiplex screening protocol for Wolbachia, Cardinium and Rickettsia that can be completed in a day. The robustness of the protocol was tested with the screening of multiple species and populations of flies commonly used in reproductive studies (N=147 flies; n=426 tissues). With triplex PCRs and more effective duplex PCRs, we detected both single and co-infections in most individuals from both families (Drosophilidae | Sepsidae; Single infection: 51.4% | 62.7%; Dual infection: 29.2% | 9.3%; Triple infection: 4.2% | 0%). Surprisingly, we documented the presence of all three reproductive bacteria in 32 wild-caught drosophilids from Singapore. Also, we note that most sepsid populations (19 out of 22) tested positive for Cardinium. We found that the Rickettsia infection was overall low, but it was predominantly detected in the gastrointestinal tract instead of the reproductive tract, suggesting a potential horizontal transmission. Finally, we found that amplicon sequences of equivalent sizes between the three tissues from the same individuals share at least 98.8% identity, which suggests that the same endosymbiont strain inhabits within the whole arthropod. Overall, we believe this protocol is effective in detecting co-infections and understanding the transmission of various reproductive endosymbionts. It can also be used to assess endosymbiont infections in other insects.

sentences: Convergent Usage of Amino Acids in Human Cancers as a Reversed Process of Tissue Development Genome and transcriptome-wide amino acid usage preference across different species is a well-studied phenomenon in molecular evolution, but its characteristics and implication in cancer evolution and therapy remain largely unexplored. Here, we analyzed large-scale transcriptome/proteome profiles such as TCGA, GTEx, and CPTAC and found that compared to normal tissues, different cancer types showed a convergent pattern towards using biosynthetically low-cost amino acids. Such a pattern can be accurately captured by a single index based on the average biosynthetic energy cost of amino acids, termed Energy Cost Per Amino Acid (ECPA). With this index, we further compared the trends of amino acid usage and the contributing genes in cancer and tissue development and revealed their reversed patterns. Finally, focusing on the liver, a tissue with a dramatic increase in ECPA during development, we found that EPCA represented a powerful biomarker that could distinguish liver tumors from normal liver samples consistently across 11 independent patient cohorts (AUROC = ~0.99) and outperformed any index based on single genes. Our study reveals an important principle underlying cancer evolution and suggests the global amino acid usage as a system-level biomarker for cancer diagnosis.

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Resolution of glycogen and glycogen-degrading activities reveals correlates of Lactobacillus crispatus dominance in a cohort of young African women BackgroundA healthy vaginal microbiome is dominated by Lactobacillus species that produce lactic acid, lowering vaginal pH and limiting colonization by pathogens. Lactobacillus dominance (LD) is established during puberty, but many women, especially those of Black race, lose LD during their reproductive years. Glycogen is thought to be a key host nutrient that supports vaginal lactobacilli and their fermentative lactic acid production, but mechanisms of glycogen utilization by Lactobacillus species are incompletely understood. By partitioning glycogen and glycogen-derived maltodextrin, as well as the activity of glycogen-degrading pullulanase enzymes, this work refines understanding of vaginal glycogen catabolism and identifies correlates of LD.

ResultsVaginal swab samples were collected from a cohort of young women with limited sexual experience in Thika, Kenya (N=17, ages 17-20). Metagenomic profiling of the vaginal microbiome revealed that most samples exhibited LD, particularly dominant Lactobacillus crispatus. Amylopullulanase activity, cleavage of glycogen -1,4 and -1,6 linkages by individual/multifunctional enzymes, showed a significant positive correlation with glycogen-derived maltodextrin, but no relationship with L. crispatus dominance. Pullulanase activity, which specifically targets glycogen -1,6 linkages, was 3-fold higher in L. crispatus-dominated samples and significantly correlated with D-lactic acid levels. Metagenomics and targeted PCR revealed that 36% of L. crispatus-dominated metagenomes from our African cohort lacked a functional L. crispatus pullulanase (pulA) gene, a 3-fold higher frequency of gene loss than that seen in metagenomes from European and North American women. Our findings suggest pulA gene loss or inactivation may correspond with reductions in L. crispatus abundance, pullulanase activity and lactic acid levels compared to samples dominated by pulA-competent L. crispatus.

ConclusionsOur results indicate that although amylase activity drives the accumulation of glycogen catabolites in vaginal fluid, pullulanase appears to specifically contribute to maximal D-lactic acid production by L. crispatus. However, this is only possible when a functional pulA gene is present, which was not the case in a substantial proportion of young African women with dominant L. crispatus. Scaling this analysis to a larger cohort will address whether genomic and enzymatic indicators of L. crispatus pullulanase activity are predictive of sustained LD and vaginal health.

sentences: Combining Multi-Dimensional Molecular Fingerprints to Predict hERG Cardiotoxicity of Compounds At present, drug toxicity has become a critical problem with heavy medical and economic burdens. acLQTS (acquired Long QT Syndrome) is acquired cardiac ion channel disease caused by drugs blocking the hERG channel. Therefore, it is necessary to avoid cardiotoxicity in the drug design and computer models have been widely used to fix this plight. In this study, we present a molecular fingerprint based on the molecular dynamic simulation and uses it combined with other molecular fingerprints (multi-dimensional molecular fingerprints) to predict hERG cardiotoxicity of compounds. 203 compounds with hERG inhibitory activity (pIC50) were retrieved from a previous study and predicting models were established using four machine learning algorithms based on the single and multi-dimensional molecular fingerprints. Results showed that MDFP has the potential to be an alternative to traditional molecular fingerprints and the combination of MDFP and traditional molecular fingerprints can achieve higher prediction accuracy. Meanwhile, the accuracy of the best model, which was generated by consensus of four algorithms with multi-dimensional molecular fingerprints, was 0.694 (RMSE) in the test dataset. Besides, the number of hydrogen bonds from MDFP has been determined as a critical factor in the predicting models, followed by rgyr and sasa. Our findings provide a new sight of MDFP and multi-dimensional molecular fingerprints in building models of hERG cardiotoxicity prediction.

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Partitioning of ribonucleoprotein complexes from the cellular actin cortex The cell cortex plays a crucial role in cell mechanics, signaling, and development. However, little is known about the influence of the cortical meshwork on the spatial distribution of cytoplasmic biomolecules. Here, we describe a new fluorescence microscopy method to infer the intracellular distribution of labeled biomolecules with sub-resolution accuracy. Unexpectedly, we find that RNA-binding proteins are partially excluded from the cytoplasmic volume adjacent to the plasma membrane that corresponds to the actin cortex. Complementary diffusion measurements of RNA-protein complexes suggest that a rudimentary model based on excluded volume interactions can explain this partitioning effect. Our results suggest the actin cortex meshwork may play a role in regulating the biomolecular content of the volume immediately adjacent to the plasma membrane.

TeaserA novel microscopy technique reveals partial exclusion of RNA-protein complexes from the actin cortex due to their large hydrodynamic size.

sentences: Joint language production: an electrophysiological investigation of simulated lexical access on behalf of task partner This study investigates in a joint action setting a well-established effect in speech production, cumulative semantic interference, an increase in naming latencies when naming a series of semantically related pictures. In a joint action setting, two task partners take turns naming pictures. Previous work in this setting demonstrated that naming latencies increase not only with each semantically related picture speakers named themselves, but also with each picture named by the partner (Hoedemaker, Ernst, Meyer, & Belke, 2017; Kuhlen & Abdel Rahman, 2017). This suggests that speakers pursue lexical access on behalf of their partner. In two electrophysiological experiments (N=30 each) we investigated the neuro-cognitive signatures of such simulated lexical access. As expected, in both experiments speakers naming latency increased with successive naming instances within a given semantic category. Correspondingly, speakers EEG showed an increasing posterior positivity between 250-400ms, an ERP modulation typically associated with lexical access. However, unlike previous experiments, speakers were not influenced by their partners picture naming. Accordingly, we found no electrophysiological evidence of lexical access. To reconcile these findings we pooled behavioral data from five experiments (N=144). Within this large sample we find empirical evidence for partner-elicited interference. Furthermore, our data suggests that speakers may be less affected by their partners naming response in settings with remotely located task partners (as in present experiments). We conclude that speakers do not always represent their partners naming response and that our experimental setting may have limited the participants evaluation of the task as a joint action.

sentences: CHD4 conceals aberrant CTCF-binding sites at TAD interiors by regulating chromatin accessibility in mESCs CTCF plays a critical role in the 3D chromatin organization by determining the TAD borders. Although CTCF primarily binds at the TAD borders, there also exist putative CTCF-binding sites within TADs, which are spread throughout the genome by retrotransposition. However, the detailed mechanism responsible for masking these putative CTCF-binding sites remains elusive. Here, we show that the ATP-dependent chromatin remodeler, CHD4, regulates chromatin accessibility to conceal aberrant CTCF-binding sites embedded in H3K9me3-enriched heterochromatic B2 SINEs in mouse embryonic stem cells (mESCs). Upon CHD4 depletion, these aberrant CTCF-binding sites become accessible, and aberrant CTCF recruitment occurs at the TAD interiors, resulting in disorganization of the local TADs. Furthermore, RNA-binding intrinsically disordered domains of CHD4 is required to prevent the aberrant CTCF bindings. Lastly, CHD4 is required for the repression of B2 SINE transcripts. These results highlight the CHD4-mediated mechanism that safeguards the appropriate CTCF bindings and associated TAD organizations in mESCs.

sentences: Distinct information-seeking modes in healthy and addicted individuals As massive amounts of information are becoming available to people understanding the mechanisms underlying information-seeking is more pertinent today than ever. In this study, we investigate the underlying motivations to seek out information in healthy and addicted individuals. We developed a novel decision-making task and a novel computational model which allow to dissociate the relative contribution of two motivating factors to seek out information: a desire for novelty and a desire to reduce uncertainty. To investigate whether/how the motivations to seek out information vary between healthy and addicted individuals, in addition to healthy controls we included a sample of individuals with gambling disorder- a form of addiction without the confound of substance consumption and characterized by compulsive gambling. Our results indicate that healthy subjects and problem gamblers adopt distinct information-seeking "modes". Healthy information-seeking behavior was mostly motivated by a desire for novelty. Problem gamblers, on the contrary, displayed reduced novelty-seeking and an increased desire to reduce uncertainty (general information-seeking) compared to healthy controls. Our findings not only shed new light on the motivations driving healthy and addicted individuals to seek out information, but they also have important implications for treatment and diagnosis of behavioral addiction.

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Facilitation of TRKB activation by the angiotensin II receptor type-2 (AT2R) agonist C21 Blockers of angiotensin II type-1 receptor (AT1R) exert antidepressant-like effects by indirectly facilitating the activation of the angiotensin II type-2 receptor (AT2R), which leads to increased surface expression and transactivation of tropomyosin-related kinase B receptors (TRKB). Compound 21 (C21) is a non-peptide AT2R agonist that produces neuroprotective effects. However, the behavioral effects of C21 and its involvement with the brain-derived neurotrophic factor (BDNF)-TRKB system still need further investigation.

The aim of the present study was to assess the effect of C21 on the activation of TRKB and its consequences on conditioned fear. The administration of C21 (0.1-10M/15min) increased the surface levels of TRKB but was not sufficient to increase the levels of phosphorylated TRKB (pTKB) in cultured cortical neurons from rat embryos. Consistent with increased TRKB surface expression, C21 (10M/15min or 3 days) facilitated the effect of BDNF (0.1ng/ml/15min) on pTRKB in these cells. In contextual fear conditioning, the freezing time of C21-treated (administered intranasally) wild-type mice was decreased compared to the vehicle-treated group, but no effect of C21 was observed in BDNF.het animals. We observed no effect of C21 in the elevated plus-maze test for anxiety.

Taken together, our results indicate that C21 facilitated BDNF effect by increasing the levels of TRKB on the cell surface and reduced the freezing time of mice in a BDNF-dependent manner, but not through a general anxiolytic-like effect.

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Structural mapping of antibody landscapes to human betacoronavirus spike proteins Preexisting immunity against seasonal coronaviruses (CoV) represents an important variable in predicting antibody responses and disease severity to Severe Acute Respiratory Syndrome CoV-2 (SARS-2) infections. We used electron microscopy based polyclonal epitope mapping (EMPEM) to characterize the antibody specificities against {beta}-CoV spike proteins in sera from healthy donors (HDs) or SARS-2 convalescent donors (CDs). We observed that most HDs possessed antibodies specific to seasonal human CoVs (HCoVs) OC43 and HKU1 spike proteins while the CDs showed reactivity across all human {beta}-CoVs. Detailed molecular mapping of spike-antibody complexes revealed epitopes that were differentially targeted by antibodies in preexisting and convalescent serum. Our studies provide an antigenic landscape to {beta}-HCoV spikes in the general population serving as a basis for cross-reactive epitope analyses in SARS-2 -infected individuals.

One-Sentence summaryWe present the epitope mapping of polyclonal antibodies against beta-coronavirus spike proteins in human sera.

sentences: Necrosis-induced apoptosis promotes regeneration in Drosophila wing imaginal discs Regeneration is a complex process that requires a coordinated genetic response to tissue loss. Signals from dying cells are crucial to this process and are best understood in the context of regeneration following programmed cell death, like apoptosis. Conversely, regeneration following unregulated forms of death such as necrosis have yet to be fully explored. Here we have developed a method to investigate regeneration following necrosis using the Drosophila wing imaginal disc. We show that necrosis stimulates regeneration at an equivalent level to that of apoptosis-mediated cell death and activates a similar response at the wound edge involving localized JNK signaling. Unexpectedly however, necrosis also results in significant apoptosis far from the site of ablation, which we have termed necrosis-induced apoptosis (NiA). This apoptosis occurs independent of changes at the wound edge and importantly does not rely on JNK signaling. Furthermore, we find that blocking NiA limits proliferation and subsequently inhibits regeneration, suggesting that tissues damaged by necrosis can activate programmed cell death at a distance from the injury to promote regeneration.

sentences: Transcription-coupled DNA repair underlies variation in persister awakening and the emergence of resistance Persisters constitute a population of temporarily antibiotic-tolerant variants in an isogenic bacterial population and are considered an important cause of relapsing infections. It is currently unclear how cellular damage inflicted by antibiotic action is reversed upon persister state exit and how this relates to antibiotic resistance development. We demonstrate that persisters, upon fluoroquinolone treatment, accumulate oxidative damage which is repaired through nucleotide excision repair. Detection of the damage occurs via transcription-coupled repair using UvrD-mediated backtracking or Mfd-mediated displacement of the RNA polymerase. This competition results in heterogeneity in persister awakening lags. Most persisters repair the oxidative DNA damage, displaying a mutation rate equal to the untreated population. However, the promutagenic factor Mfd increases the mutation rate in a persister subpopulation. Our data provide in-depth insight in the molecular mechanisms underlying persister survival and pinpoints Mfd as an important molecular factor linking persistence to resistance development.

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COVID-19 lung disease shares driver AT2 cytopathic features with Idiopathic pulmonary fibrosis BackgroundIn the aftermath of Covid-19, some patients develop a fibrotic lung disease, i.e., post-COVID-19 lung disease (PCLD), for which we currently lack insights into pathogenesis, disease models, or treatment options.

MethodUsing an AI-guided approach, we analyzed > 1000 human lung transcriptomic datasets associated with various lung conditions using two viral pandemic signatures (ViP and sViP) and one covid lung-derived signature. Upon identifying similarities between COVID-19 and idiopathic pulmonary fibrosis (IPF), we subsequently dissected the basis for such similarity from molecular, cytopathic, and immunologic perspectives using a panel of IPF-specific gene signatures, alongside signatures of alveolar type II (AT2) cytopathies and of prognostic monocyte-driven processes that are known drivers of IPF. Transcriptome-derived findings were used to construct protein-protein interaction (PPI) network to identify the major triggers of AT2 dysfunction. Key findings were validated in hamster and human adult lung organoid (ALO) pre-clinical models of COVID-19 using immunohistochemistry and qPCR.

FindingsCOVID-19 resembles IPF at a fundamental level; it recapitulates the gene expression patterns (ViP and IPF signatures), cytokine storm (IL15-centric) and the AT2 cytopathic changes, e.g., injury, DNA damage, arrest in a transient, damage-induced progenitor state, and senescence-associated secretory phenotype (SASP). These immunocytopathic features were induced in pre-clinical COVID models (ALO and hamster) and reversed with effective anti-CoV-2 therapeutics in hamsters. PPI-network analyses pinpointed ER stress as one of the shared early triggers of both diseases, and IHC studies validated the same in the lungs of deceased subjects with COVID-19 and SARS-CoV-2-challenged hamster lungs. Lungs from tg-mice, in which ER stress is induced specifically in the AT2 cells, faithfully recapitulate the host immune response and alveolar cytopathic changes that are induced by SARS-CoV-2.

InterpretationLike IPF, COVID-19 may be driven by injury-induced ER stress that culminates into progenitor state arrest and SASP in AT2 cells. The ViP signatures in monocytes may be key determinants of prognosis. The insights, signatures, disease models identified here are likely to spur the development of therapies for patients with IPF and other fibrotic interstitial lung disease.

FundingThis work was supported by the National Institutes for Health grants R01-GM138385 and AI155696 and funding from the Tobacco-Related disease Research Program (R01RG3780).

One Sentence SummarySevere COVID-19 triggers cellular processes seen in fibrosing Interstitial Lung Disease

sentences: Small molecules enhance the potency of natural antimicrobial peptides The skin-associated microbiome plays an important role in general well-being and in a variety of treatable conditions. In this regard, endogenous antimicrobial peptides have a role in controlling the microbial population. We demonstrate here that certain small molecular species can amplify the potency of naturally-occurring antimicrobial peptides. For example, Niacinamide is a vitamin B3 analogue naturally found in foods and widely used in topical skin care products, and here we have investigated its cooperativity with the human antimicrobial peptide LL37 on the bacterium Staphylococcus aureus. We have also studied two other structurally related B3 analogs. We observed a clear synergistic effect of niacinamide and, to some extent, methyl niacinamide, whereas isonicotinamide showed no significant cooperativity with LL37. Adaptively-biased molecular dynamics simulations revealed that the analogs partition into the head group region of an anionic bilayer used to mimic the bacterial membrane. The observed effects on the physical properties of the membrane are well correlated with experimental activity. In contrast, the analogs have little effect on zwitterionic bilayers which mimic a mammalian membrane. We conclude that these vitamin B3 analogues can potentiate the activity of host peptides by modulating the physical properties of the bacterial membrane, and to a lesser extent through direct interactions with the peptide. The level of cooperativity is strongly dependent on the detailed chemistry of the additive, suggesting an opportunity to fine-tune the behaviour of host peptides.

sentences: Modelling global mRNA dynamics during Drosophila embryogenesis reveals a relationship between mRNA degradation and P-bodies Regulation of mRNA degradation is critical for a diverse array of cellular processes and developmental cell fate decisions. Although many methods for determining mRNA half-lives rely on transcriptional inhibition or metabolic labelling, these manipulations can influence half-lives or introduce errors in the measurements. Here we use a non-invasive method for estimating mRNA half-lives globally in the early Drosophila embryo using a total RNA-seq time series and Gaussian process regression to model the dynamics of premature and mature mRNAs. We show how regulation of mRNA stability is used to establish a range of mature mRNA dynamics during embryogenesis, despite shared transcription profiles. Our data suggest that mRNA half-life is not dependent on translation efficiency, but instead we provide evidence that short half-life mRNAs are more strongly associated with P-bodies. Moreover, we detect an enrichment of mRNA 3 ends in P-bodies in the early embryo, consistent with 5 to 3 degradation occurring in P-bodies for at least a subset of mRNAs. We discuss our findings in relation to recently published data suggesting that the primary function of P-bodies in other biological contexts is mRNA storage.

sentences: Unsupervised Models of Mouse Visual Cortex Task-optimized deep convolutional neural networks are the most quantitatively accurate models of the primate ventral visual stream. However, such networks are implausible as models of the mouse visual system because mouse visual cortex has both lower retinal resolution and a shallower hierarchy than the primate. Moreover, the category supervision deep networks typically receive is neither ethologically relevant to the mouse in semantic content, nor realistic in quantity. As a result, standard supervised deep neural networks have proven quantitatively ineffective at modeling mouse visual data. Here, we develop and evaluate models that remedy these structural and functional gaps. We first demonstrate that shallow hierarchical architectures applied to lower resolution images improve match to neural responses, both in electro-physiological and calcium imaging data. We then show that networks trained using contrastive embedding methods, a recent unsupervised learning objective that requires no semantic labeling, achieve neural prediction performance that substantially exceed that of the same architectures trained in a supervised manner, across a wide variety of architecture types. Combining these better structural and functional priors yields models that are the most quantitatively accurate match to mouse visual responses to natural scenes, significantly surpassing that of prior attempts using primate-specific models, and approaching the inter-animal consistency level of the data itself. We further find that these shallow unsupervised models transfer to a wide variety of non-categorical visual tasks better than categorization-trained models. Taken together, our results suggest that mouse visual cortex is a low-resolution, shallow network that makes best use of the mouses limited resources to create a light-weight, general-purpose visual system - in contrast to the deep, high-resolution, and more task-specific visual system of primates.

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Sciviewer enables interactive visual interrogation of single-cell RNA-Seq data from the Python programming environment SummaryVisualizing two-dimensional (2D) embeddings (e.g. UMAP or tSNE) is a key step in interrogating single-cell RNA sequencing (scRNA-Seq) data. Subsequently, users typically iterate between programmatic analyses (e.g. clustering and differential expression) and visual exploration (e.g. coloring cells by interesting features) to uncover biological signals in the data. Interactive tools exist to facilitate visual exploration of embeddings such as performing differential expression on user-selected cells. However, the practical utility of these tools is limited because they dont support rapid movement of data and results to and from the programming environments where the bulk of data analysis takes place, interrupting the iterative process. Here, we present the Single-cell Interactive Viewer (Sciviewer), a tool that overcomes this limitation by allowing interactive visual interrogation of embeddings from within Python. Beyond differential expression analysis of user-selected cells, Sciviewer implements a novel method to identify genes varying locally along any user-specified direction on the embedding. Sciviewer enables rapid and flexible iteration between interactive and programmatic modes of scRNA-Seq exploration, illustrating a useful approach for analyzing high-dimensional data.

Availability and implementationCode and examples are provided at https://github.com/colabobio/sciviewer

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MycoPhylo experiment reveals how mycorrhiza types and phylogenetic relationships affect soil biodiversity and functioning O_LINatural forests and abandoned agricultural lands are increasingly replaced by monospecific forest plantations that have poor capacity to support biodiversity and ecosystem services. Natural forests harbour plants belonging to different mycorrhiza types that differ in their microbiome and carbon and nutrient cycling properties.
C_LIO_LIHere we describe the MycoPhylo field experiment that encompasses 116 woody plant species from three mycorrhiza types and 237 plots, with plant diversity and mycorrhiza type diversity ranging from one to four and one to three per plot, respectively.
C_LIO_LIThe MycoPhylo experiment enables us to test hypotheses about the plant species, species diversity, mycorrhiza type, and mycorrhiza type diversity effects and their phylogenetic context on soil microbial diversity and functioning and soil processes.
C_LIO_LIAlongside with other experiments in the TreeDivNet consortium, MycoPhylo will contribute to our understanding of the tree diversity effects on soil biodiversity and ecosystem functioning across biomes, especially from the mycorrhiza type and phylogenetic conservatism perspectives.
C_LI

sentences: Analysis of the PcrA-RNA polymerase complex reveals a helicase interaction motif and a role for PcrA/UvrD helicase in the suppression of R-loops. The PcrA/UvrD helicase binds directly to RNA polymerase (RNAP) but the structural basis for this interaction and its functional significance have remained unclear. In this work we used biochemical assays and hydrogen-deuterium exchange coupled to mass spectrometry to study the PcrA-RNAP complex. We find that PcrA binds tightly to a transcription elongation complex in a manner dependent on protein:protein interaction with the conserved PcrA C-terminal Tudor domain. The helicase binds predominantly to two positions on the surface of RNAP. The PcrA C-terminal domain engages a conserved region in a lineage-specific insert within the {beta} subunit which we identify as a helicase interaction motif present in many other PcrA partner proteins, including the nucleotide excision repair factor UvrB. The catalytic core of the helicase binds near the RNA and DNA exit channels and blocking PcrA activity in vivo leads to the accumulation of R-loops. We propose a role for PcrA as an R-loop suppression factor that helps to minimise conflicts between transcription and other processes on DNA including replication.

sentences: Proteomic analysis of human milk reveals nutritional and immune benefits in the colostrum from mothers with COVID-19 The range of benefits breastfeeding provides neonates and infants include nutrition, improved neonatal survival, and reduced morbidity from certain diseases. It also aids maternal health by speeding postpartum recovery. However, due to concern about the risk of SARS-CoV-2 transmission and the lack of evidence of breastmilks protective effects against the virus, whether mothers with COVID-19 should be encouraged to breastfeed is under debate. Here, we present the results of proteomic and glycoproteomic studies of breast milk (colostrum and mature milk) from mothers with confirmed COVID-19. All colostrum samples exhibited significantly upregulated immune-related proteins, especially whey proteins with antiviral properties against SARS-CoV-2, and increased glycosylation levels and heterogeneity at those proteins. Such adaptive differences in milk from COVID-19 mothers tend to fade in mature milk from the same mothers one month postpartum. These results suggest the immune benefits of colostrum from mothers with COVID-19 and provide molecular-level insights that aid breastmilk feeding decisions in cases of active infection.

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Piezo1 and BKCa channels in human atrial fibroblasts: interplay and remodelling in atrial fibrillation AimsAtrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging population of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC-presence and -activity associated with AF.

Methods and ResultsUsing primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC-signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline and iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent  big potassium channels, BKCa. In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of BKCa current during stretch. No co-immunoprecipitation of Piezo1 and BKCa was detected.

ConclusionsHuman atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BKCa. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data support the presence and interplay of Piezo1 and BKCa in human atrial fibroblasts in the absence of physical interactions between the two channel proteins.

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Turnover Regulation of the Rho GTPase Cdc42 by Heat Shock Protein Chaperones and the MAPK Pathway Scaffold Bem4 All cells maintain an axis of polarity that directs the orientation of growth. Cell polarity can be reorganized during development and in response to extrinsic cues to produce new cell types. Rho GTPases are central regulators of cell polarity and signal-dependent cell differentiation. We show here that one of the best understood Rho GTPases, the highly conserved yeast Cdc42p, is turned over by members of the Heat Shock family of Proteins (HSPs). The Hsp40p chaperone, Ydj1p, was required for turnover of Cdc42p by the NEDD4 E3 ubiquitin ligase, Rsp5p, in the proteosome. Cdc42p turnover was regulated by HSPs at high temperatures, and in aging cells where the protein formed aggregates, implicating HSPs in Rho GTPase quality control. We also show that Cdc42pQ61L, which mimics the active (GTP-bound) conformation of the protein, was turned over at elevated levels by Ydj1p and Rsp5p. A turnover-defective version of Cdc42pQ61L led to multibudding phenotypes, implicating Cdc42 turnover in singularity in cell polarization. Cdc42p turnover also impacted MAP kinase pathway specificity. A pathway-specific scaffold, Bem4p, stabilized Cdc42p levels, which biased Cdc42p function in one MAPK pathway over another. Turnover regulation of Rho GTPases by HSPs and scaffolds provides new dimensions to the regulation of cell polarity and signal-dependent morphogenesis.

Significance StatementRho GTPases are switch-like proteins that govern major decisions in cell polarity and signaling in eukaryotes. We elucidate here a pathway that turns over the yeast Rho GTPase Cdc42p, which is mediated by the heat-shock family of proteins (HSPs) and the NEDD4-type E3 ubiquitin ligase Rsp5p. This finding provides a way for HSPs to exert their widespread effects on morphogenetic responses, phenotypic plasticity, and signaling pathways. We also found that turnover of an active version of Cdc42p is critical for modulating cell polarity. Cdc42p turnover also impacted its function in a pathway specific setting, as stabilization of Cdc42p by Bem4p (SmgGDS-type scaffold) influenced the activity of a specific MAPK pathway. HSPs may regulate Rho GTPase turnover in many systems.

sentences: No impact of functional connectivity of the motor system on the resting motor threshold: A replication study. The physiological mechanisms of corticospinal excitability and factors influencing its measurement with transcranial magnetic stimulation are still poorly understood. A recent study reported an impact of functional connectivity between the primary motor cortex and dorsal premotor cortex on the resting motor threshold of the dominant hemisphere. We aimed to replicate these findings in a larger sample of 38 healthy right-handed subjects with data from both hemispheres. Resting-state functional connectivity was assessed between the primary motor cortex and five a-priori defined motor-relevant regions on each hemisphere as well as interhemispherically between both primary motor cortices. Following the procedure by the original authors, we included age, the cortical grey matter volume and coil to cortex distance as further predictors in the analysis. We report replication models for the dominant hemisphere as well as an extension to data from both hemispheres and support the results with Bayes factors. Functional connectivity between the primary motor cortex and dorsal premotor cortex did not explain variability in the resting motor threshold and we obtained moderate evidence for the absence of this effect. In contrast, coil to cortex distance could be confirmed as an important predictor with strong evidence. These findings contradict the previously proposed effect, thus questioning the notion of the dorsal premotor cortex playing a major role in modifying corticospinal excitability.

sentences: Made together, replayed together: Context reinstatement during sleep guides memory consolidation Newly formed memories arent quarantined one from another when first encoded; rather, they are interlinked with other memories that were encoded in temporal proximity or share semantic features. In this study, we selectively biased memory processing during sleep in humans to test the hypothesis that memories contexts influence their consolidation. Participants formed 18 idiosyncratic narratives, each linking four objects together. They then memorized random on-screen spatial positions assigned to each of the 72 objects. During sleep, 12 object-specific sounds were unobtrusively presented, thereby cuing the associated memories and improving spatial recall for those objects. As predicted, memory for uncued objects that were contextually linked with cued objects benefited as well. The correlational structure of spectral power for electrophysiological responses revealed that sleep spindles supported context reinstatement and predicted context-related memory benefits. Moreover, context-specific electrophysiological activity patterns emerged during sleep. We conclude that when an individual memory is reactivated during sleep, the corresponding encoding context is also reinstated, impacting consolidation and enhancing subsequent retrieval of associated knowledge.

sentences: Desolvation Energy Explains Partitioning of Client Proteins into Condensates Membraneless organelles are cellular compartments that form by liquid-liquid phase separation of one or more components. Other molecules, such as other proteins and nucleic acids, will distribute between the cytoplasm and the liquid compartment in accordance with the thermodynamic drive to lower the free energy of the system. The resulting distribution colocalizes molecular species, to carry out a diversity of functions. Two factors could drive this partitioning: the difference in solvation between the dilute versus dense phase, and intermolecular interactions between the client and scaffold proteins. Here, we develop a set of knowledge-based potentials that allow for the direct comparison between desolvation energy and pairwise interaction energy terms, and use these to examine experimental data from two systems: protein cargo dissolving within phase-separated droplets made from FG repeat proteins of the nuclear pore complex, and client proteins dissolving within phase-separated FUS droplets. We find close agreement between desolvation energies of the client proteins and the experimentally determined values of the partition coefficients, while pairwise interaction energies between client and scaffold show weaker correlations. These results show that client stickiness is sufficient to explain differential partitioning of clients within these two phase-separated systems without taking into account the composition of the condensate. This suggests that selective trafficking of client proteins to distinct membraneless organelles requires recognition elements beyond the client sequence composition.

sentences: A Strategy to Quantify Myofibroblast Activation on a Continuous Spectrum Myofibroblasts are a highly secretory and contractile phenotype most commonly identified by the de novo expression and assembly of alpha-smooth muscle actin stress fibers. Traditionally, this activation process has been thought of as a binary process, with cells being labeled as "activated" or "quiescent (non-activated)". More recently, this view has been expanded to consider activation on a continuous spectrum. However, there is no established method to quantify a cells position on this spectrum, and as a result, the binary labeling system is still widely used. While transcriptomic analyses provide a continuous measure of myofibroblast markers, a faster and more facile screening method is needed. To this end, we utilized optical microscopy and machine learning methods to quantify myofibroblast activation on a spectrum. We first measured size and shape features of over 1,000 individual cardiac fibroblasts and found that these features provide enough information to predict activation state, on the binary scale, with 94% accuracy as compared to manual classification. We next performed dimensionality reduction techniques on these features to create a continuous scale of activation. Importantly, this new classification system captures a range of fibroblast activation states, but still possesses inherent bias due to choice of morphological features. Thus, we next used self-supervised machine learning to create a second continuous labeling system free from biases associated with the manually measured features. Lastly, we compared our findings for mechanically activated cardiac fibroblasts to a distribution of cell phenotypes generated from transcriptomic data using single-cell RNA sequencing. Altogether, these results demonstrate a continuous spectrum of activation from fibroblast to myofibroblast and provide a strategy to quantify a cells position on that spectrum.

sentences: An analytical solution simulating growth of Lewy bodies This paper reports a minimal model simulating the growth of a Lewy body (LB). To the best of our knowledge, this is the first model simulating Lewy body growth. The LB is assumed to consist of a central spherical core, which is composed of membrane fragments and various dysfunctional intracellular organelles, and a halo, which is composed of alpha-synuclein fibrils. Membrane fragments and alpha-synuclein monomers are assumed to be produced in the soma at constant rates. The growth of the core and the halo are simulated by the Finke-Watzky model. Analytical (closed-form) solutions describing the growth of the core and the halo are obtained. A sensitivity analysis in terms of model parameters is performed.

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Disbalance of the intestinal epithelial cell turnover and apoptosis in a rat model of sporadic Alzheimer's disease BackgroundDyshomeostasis of the gastrointestinal (GI) system is investigated as a potential contributor to metabolic dysfunction, systemic and neuro-inflammation recognized as important pathophysiological drivers of neurodegeneration. Gastrointestinal redox dyshomeostasis and dysfunctional brain-gut incretin axis have been reported in the rat model of insulin-resistant brain state (IRBS)-driven neurodegeneration induced by intracerebroventricular administration of streptozotocin (STZ-icv). The aim was to assess i) whether GI oxidative stress is accompanied by structural and functional changes of the epithelial barrier; ii) whether the brain glucose-dependent insulinotropic polypeptide receptor (GIP-R) is also involved in redox regulation of the gut; and iii) whether the STZ-icv brain-gut axis is resistant to pharmacological inhibition of the brain GIP-R.

MethodsForty three-month-old male Wistar rats were treated with 3mg/kg STZ-icv or vehicle. One month later the animals were randomized to receive either saline or 85 g/kg GIP-R inhibitor [Pro3]-GIP intracerebroventricularly and sacrificed 30 minutes later. Thiobarbituric acid reactive substances (TBARS) were measured in plasma and duodenum. Duodenal sections were subjected to morphometric analysis. Caspase-3 expression and activation were analyzed by western blot and spatial signal analysis was done by multiplex fluorescent signal amplification (MFSA). Data were analyzed by linear and linear mixed modeling, and exploration was done by principal component analysis.

ResultsInhibition of the brain GIP-R decreased plasma TBARS in the controls and the STZ-icv animals and increased duodenal TBARS only in the controls. Acute inhibition of brain GIP-R affects duodenal epithelial cell, but not villus structure, while all morphometric parameters were altered in the STZ-icv-treated animals. Morphometric changes in the STZ-icv animals were accompanied by reduced levels of activated and total regulator of apoptosis - caspase-3. Acute inhibition of brain GIP-R inactivated duodenal apoptosis at the level of caspase-3 activation.

ConclusionsBrain GIP-R is involved in the regulation of the systemic and duodenal redox homeostasis and epithelial function. Duodenal oxidative stress in the STZ-icv rats is accompanied by the resistance of the brain-gut GIP axis and morphological changes indicative of abnormal epithelial cell turnover and dysfunctional GI barrier. Dysfunction of the brain-gut incretin axis might be an important etiopathogenetic factor in neurodegeneration and a potential pharmacological target.

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Vibrio tarriae sp. nov., a novel member of the Cholerae clade isolated from across the United States A number of bacteria with close resemblance to Vibrio cholerae has been isolated over the years by the Centers for Disease Control and Prevention (CDC), which could not be assigned a proper taxonomic designation based on preliminary identification methods. Nine such isolates have been found to share 16S rRNA gene identity exceeding 99% with V. cholerae, yet DNA-DNA hybridization (60.4-62.1%) and average nucleotide identity values (94.4-95.1%) were below the species cut-off, indicating a potentially novel species. Phylogenetic analysis of core genomes places this group of isolates in a monophyletic clade, within the "Cholerae clade," but distinct from any other species. Extensive phenotypic characterization reveals unique biochemical properties that distinguish this novel species from V. cholerae. Comparative genomic analysis reveals a unique set of siderophore genes, suggesting that iron acquisition strategies could be vital for the divergence of the novel species from a common ancestor with V. cholerae. Based on genetic, phylogenetic, and phenotypic differences observed, we propose these isolates represent a novel species of the genus Vibrio, for which the name Vibrio tarriae sp. nov. is proposed. Strain 2521-89 (= DSM 112461 = CCUG 75318), isolated from lake water, is the type strain.

Author NotesThe GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 2521-89 is MW773202.1. The genome sequences (genome assemblies) of strains 2521-89, 2523-88, 2015V-1076, 2016V-1018, 2016V-1062, 2017V-1038, 2017V-1070 and 2017V-1124 are deposited under the accession number NZ_CP022353.1, QKKG01000001.1, QKKH00000000.1, QKKI00000000.1, QKKJ00000000.1, QKKK00000000.1, QKKM00000000.1, and QKKN00000000.1, respectively. All the whole genome sequences are deposited under bioproject ID: PRJNA391152.

sentences: AltumAge: A Pan-Tissue DNA-Methylation Epigenetic Clock Based on Deep Learning Several age predictors based on DNA methylation, dubbed epigenetic clocks, have been created in recent years, with the vast majority based on regularized linear regression. This study explores the improvement in the performance and interpretation of epigenetic clocks using deep learning. First, we gathered 143 publicly available data sets from several human tissues to develop AltumAge, a neural network framework that is a highly accurate and precise age predictor. Compared to ElasticNet, AltumAge performs better for within-data set and cross-data set age prediction, being particularly more generalizable in older ages and new tissue types. We then used deep learning interpretation methods to learn which methylation sites contributed to the final model predictions. We observe that while most important CpG sites are linearly related to age, some highly-interacting CpG sites can influence the relevance of such relationships. Using chromatin annotations, we show that the CpG sites with the highest contribution to the model predictions were related to gene regulatory regions in the genome, including proximity to CTCF binding sites. We also found age-related KEGG pathways for genes containing these CpG sites. Lastly, we perform downstream analyses of AltumAge to explore its applicability and compare its age acceleration with Horvaths model. We show that our neural network approach predicts higher age acceleration for tumors and for cells that exhibit age-related changes in vitro, such as replicative senescence and mitochondrial dysfunction. Altogether, our neural network approach provides significant improvement and flexibility to current epigenetic clocks for both performance and model interpretability.

sentences: Genome-wide association study suggests an independent genetic basis of zinc and cadmium concentrations in fresh sweet corn kernels Despite being one of the most consumed vegetables in the United States, the elemental profile of sweet corn (Zea mays L.) is limited in its dietary contributions. To address this through genetic improvement, a genome-wide association study was conducted for the concentrations of 15 elements in fresh kernels of a sweet corn association panel. In concordance with mapping results from mature maize kernels, we detected a probable pleiotropic association of zinc and iron concentrations with nicotianamine synthase5 (nas5), which purportedly encodes an enzyme involved in synthesis of the metal chelator nicotianamine. Additionally, a pervasive association signal was identified for cadmium concentration within a recombination suppressed region on chromosome 2. The likely causal gene underlying this signal was heavy metal ATPase3 (hma3), whose counterpart in rice, OsHMA3, mediates vacuolar sequestration of cadmium and zinc in roots, whereby regulating zinc homeostasis and cadmium accumulation in grains. In our association panel, hma3 associated with cadmium but not zinc accumulation in fresh kernels. This finding implies that selection for low cadmium will not affect zinc levels in fresh kernels. Although less resolved association signals were detected for boron, nickel, and calcium, all 15 elements were shown to have moderate predictive abilities via whole-genome prediction. Collectively, these results help enhance our genomics-assisted breeding efforts centered on improving the elemental profile of fresh sweet corn kernels.

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Computational Structure Prediction Provides a Plausible Mechanism for Electron Transfer by the Outer Membrane Protein Cyc2 from Acidithiobacillus ferrooxidans Cyc2 is the key protein in the outer membrane of Acidithiobacillus ferrooxidans that mediates electron transfer between extracellular inorganic iron and the intracellular central metabolism. This cytochrome c is specific for iron and interacts with periplasmic proteins to complete a reversible electron transport chain. A structure of Cyc2 has not yet been characterized experimentally. Here we describe a structural model of Cyc2, and associated proteins, to highlight a plausible mechanism for the ferrous iron electron transfer chain. A comparative modeling protocol specific for trans membrane beta barrel (TMBB) proteins in acidophilic conditions (pH ~2) was applied to the primary sequence of Cyc2. The proposed structure has three main regimes: extracellular loops exposed to low-pH conditions, a TMBB, and a N-terminal cytochrome-like region within the periplasmic space. The Cyc2 model was further refined by identifying likely iron and heme docking sites. This represents the first computational model of Cyc2 that accounts for the membrane microenvironment and the acidity in the extracellular matrix. This approach can be used to model other TMBBs which can be critical for chemolithotrophic microbial growth.

Importance of workAcidithiobacillus ferrooxidans can oxidize both iron and reduced sulfur compounds and plays a key role in metal sulfide ore bioleaching used for the industrial recovery of metals. A. ferrooxidans has also been explored as a potential organism for emerging technologies such as e-waste recycling and biofuel production. Synthetic biology efforts are hampered by lack of knowledge about the mechanisms of iron oxidation and reduction, which is mediated by the Cyc2 transmembrane beta barrel (TMBB) protein.

sentences: Compensatory CSF2-driven macrophage activation promotes adaptive resistance to CSF1R inhibition in breast-to-brain metastasis Tumor microenvironment-targeted therapies are emerging as promising treatment options for different cancer types. Tumor-associated macrophages and microglia (TAMs) represent an abundant non-malignant cell type in brain metastases and have been proposed to modulate metastatic colonization and outgrowth. We used an inhibitor of colony stimulating factor 1 receptor (CSF1R) to target TAMs at distinct stages of the metastatic cascade in preclinical breast-to-brain metastasis models and found that CSF1R inhibition leads to anti-tumor responses in prevention and intervention trials. However, in established brain metastases, compensatory CSF2Rb-STAT5-mediated pro-inflammatory TAM activation blunted the ultimate efficacy of CSF1R inhibition by inducing neuro-inflammation gene signatures in association with wound repair responses that fostered tumor recurrence. Consequently, combined blockade of CSF1R and STAT5 signaling led to sustained tumor control, a normalization of microglial activation states and amelioration of neuronal damage.

sentences: Deciphering Brassica plant defence responses to cabbage white butterfly egg-associated molecular patterns Brassica plants activate a strong hypersensitive response (HR)-like necrosis underneath eggs of cabbage white butterflies, but their molecular response to eggs is poorly understood. Here, we developed a method to generate egg wash to identify potential insect egg-associated molecular patterns (EAMPs) inducing HR-like necrosis. We found that egg wash, containing compounds from Pieris eggs, induced a similar response as eggs. We show that wash of hatched eggs, of egg glue, and of accessory reproductive glands (ARG) that produce this glue, also induced HR-like necrosis, whereas removal of the glue from eggs resulted in a reduced response. Eggs of Pieris butterflies induced callose deposition, production of reactive oxygen species and cell death in B. nigra and B. rapa leaf tissue, also in plants that did not express HR-like necrosis. Finally, only washes from Pieris eggs induced defence genes and ethylene production, whereas egg wash of a generalist moth did not. Our results indicate that EAMPs are in the egg glue and that the response in B. nigra is specific to Pieris species. Our study expands knowledge on the Brassica-Pieris-egg interaction, and paves the way for identification of EAMPs in Pieris egg glue and corresponding receptor in Brassica spp.

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Drastic mutations can be hidden in short-read mapping: thousands of mutation clusters in human genome are explained by short-range template switching Variation within human genomes is distributed unevenly and variants show spatial clustering. DNA-replication related template switching is a poorly known mutational mechanism capable of causing major chromosomal rearrangements as well as creating short inverted sequence copies that appear as local mutation clusters in sequence comparisons. We reanalyzed haplotype-resolved genome assemblies representing 25 human populations and multinucleotide variants aggregated from 140,000 human sequencing experiments. We found local template switching to explain thousands of complex mutation clusters across the human genome, the loci segregating within and between populations with a small number appearing as de novo mutations. We developed computational tools for genotyping candidate template switch loci using short-read sequencing data and for identification of template switch events using both short-read data and genotype data. These tools will enable building a catalogue of affected loci and studying the cellular mechanisms behind template switching both in healthy organisms and in disease. Strikingly, we noticed that widely-used analysis pipelines for short-read sequencing data - capable of identifying single nucleotide changes - may miss TSM-origin inversions of tens of base pairs, potentially invalidating medical genetic studies searching for causative alleles behind genetic diseases.

Author summaryMutations are not randomly distributed in genomes and they often appear as clusters of nearby changes. We earlier showed that a poorly known mechanism in DNA replication can create short inverted copies of nearby sequence and that these events then show as mutation clusters in sequence comparison. Using the latest DNA sequencing and variation data we show that the human genome contains thousands of mutation clusters consistent with this mechanism and that novel mutations are created at a significant rate. Strikingly we observed that widely used methods for processing DNA sequencing data may completely miss these mutations. This has significance e.g. in medical genetic studies aiming to identify mutations causing genetic diseases.

sentences: Fluorescence lifetime imaging of pH along the secretory pathway Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein (GFP), pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution is unsuitable to follow the rapid transit of cargo between organelles. We therefore applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway and compared the pH values obtained by the FLIM-based measurements with those obtained by conventional ratiometric imaging. Then, we analyzed the dynamic pH changes within cells treated with Bafilomycin A1, to block the vesicular ATPase, and Brefeldin A, to block ER-Golgi trafficking. Finally, we followed the pH changes of newly-synthesized molecules of the inflammatory cytokine tumor necrosis factor (TNF)- while they were in transit from the endoplasmic reticulum via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution, and can be used to assess organellar pH in disease models.

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Unbiased pangenome graphs MotivationPangenome variation graphs model the mutual alignment of collections of DNA sequences. A set of pairwise alignments implies a variation graph, but there are no scalable methods to generate such a graph from these alignments. Existing related approaches depend on a single reference, a specific ordering of genomes, or a de Bruijn model based on a fixed k-mer length. A scalable, self-contained method to build pangenome graphs without such limitations would be a key step in pangenome construction and manipulation pipelines.

ResultsWe design the seqwish algorithm, which builds a variation graph from a set of sequences and alignments between them. We first transform the alignment set into an implicit interval tree. To build up the variation graph, we query this tree-based representation of the alignments to reduce transitive matches into single DNA segments in a sequence graph. By recording the mapping from input sequence to output graph, we can trace the original paths through this graph, yielding a pangenome variation graph. We present an implementation that operates in external memory, using disk-backed data structures and lock-free parallel methods to drive the core graph induction step. We demonstrate that our method scales to very large graph induction problems by applying it to build pangenome graphs for several species.

Availabilityseqwish is published as free software under the MIT open source license. Source code and documentation are available at https://github.com/ekg/seqwish. seqwish can be installed via Bioconda https://bioconda.github.io/recipes/seqwish/README.html or GNU Guix https://github.com/ekg/guix-genomics/blob/master/seqwish.scm.

Contactegarris5@uthsc.edu

sentences: Patrolling human SLE haematopoietic progenitors demonstrate enhanced extramedullary colonisation; implications for peripheral tissue injury Systemic Lupus erythematosus (SLE) is an autoimmune disease where bone-marrow-derived haematopoietic cells have a key role in its pathogenesis with accumulating evidence suggesting an aberrant function of haematopoietic stem/progenitor cells (HSPCs). By employing next-generation sequencing, we compared the gene transcription signatures of CD34+ HSPCs deriving from either the bone marrow or HSPCs patrolling the bloodstream of healthy and individuals with SLE, seeking common transcriptional pathways that may have been modified between steady and disease states. Our findings indicate that circulating and bone marrow-derived HSPCs are distinct in steady and diseased states. Non-mobilised, SLE-derived circulating HSPCs demonstrated enhanced engrafting and altered differentiation capacities. Importantly, xenotransplantation of circulating HSPCs in humanised mice showed that human peripheral blood HSPCs possess the ability for extramedullary organ colonisation to the kidneys. SLE CD34+ HSPCs homing and engraftment at extramedullary sites such as the spleen and kidneys may participate in peripheral tissue injury.

sentences: Lower promoter activity of the ST8SIA2 gene has been favored in evolving human collective brains ST8SIA2 is the main factor regulating expression of the phenotype involved in schizophrenia. Lowered promoter activity of the ST8SIA2 gene is considered to be protective against schizophrenia by conferring tolerance to psychosocial stress. Here, we examined the promoter type composition of anatomically modern humans (AMHs) and archaic humans (AHs; Neanderthals and Denisovans), and compared the promoter activity at the population level (population promoter activity; PPA) between them. In AMHs, the TCT-type, showing the second lowest promoter activity, was most prevalent in the ancestral population of non-Africans. However, the detection of only the CGT-type from AH samples and recombination tracts in AH sequences showed that the CGT- and TGT-types, exhibiting the two highest promoter activities, were common in AH populations. Furthermore, interspecies gene flow occurred into AMHs from AHs and into Denisovans from Neanderthals, influencing promoter-type compositions independently in both AMHs and AHs. The difference of promoter-type composition makes PPA unique in each population. East and Southeast Asian populations show the lowest PPA. This results from the selective increase of the CGC-type, showing the lowest promoter activity, in these populations. Every non-African population shows significantly lower PPA than African populations, resulting from the TCT-type having the highest prevalence in the ancestral population of non-Africans. In addition, PPA reduction is also found among subpopulations within Africa via a slight increase of the TCT-type. These findings indicate a trend toward lower PPA in the spread of AMHs, interpreted as a continuous adaptation to psychosocial stress arising in migration. This trend is considered as genetic tuning for the evolution of collective brains. The inferred promoter-type composition of AHs differed markedly from that of AMHs, resulting in higher PPA in AHs than in AMHs. This suggests that the trend toward lower PPA is a unique feature in AMH spread.

sentences: Widespread premature transcription termination of Arabidopsis thaliana NLR genes by the spen protein FPA Genes involved in disease resistance are some of the fastest evolving and most diverse components of genomes. Large numbers of nucleotide-binding, leucine-rich repeat receptor (NLR) genes are found in plant genomes and are required for disease resistance. However, NLRs can trigger autoimmunity, disrupt beneficial microbiota or reduce fitness. It is therefore crucial to understand how NLRs are controlled. Here we show that the RNA-binding protein FPA mediates widespread premature cleavage and polyadenylation of NLR transcripts, thereby controlling their functional expression and impacting immunity. Using long-read Nanopore direct RNA sequencing, we resolved the complexity of NLR transcript processing and gene annotation. Our results uncover a co-transcriptional layer of NLR control with implications for understanding the regulatory and evolutionary dynamics of NLRs in the immune responses of plants.

sentences: Network architecture producing swing to stance transitions in an insect walking system The walking system of the stick insect is one of the most thoroughly described invertebrate systems. We know a lot about the role of sensory input in the control of stepping of a single leg. However, the neuronal organization and connectivity of the central neural networks underlying the rhythmic activation and coordination of leg muscles still remain elusive. It is assumed that these networks can couple in the absence of phasic sensory input due to the observation of spontaneous recurrent patterns (SRPs) of coordinated motor activity equivalent to fictive stepping-phase transitions. Here we sought to quantify the phase of motor activity within SRPs in the isolated and interconnected meso- and metathoracic ganglia. We show that SRPs occur not only in the meso-, but also in the metathoracic ganglia of the stick insect, discovering a qualitative difference between them. We construct a network based on neurophysiological data capable of reproducing the measured SRP phases to investigate this difference. By comparing network output to the biological measurements we confirm the plausibility of the architecture and provide a hypothesis to account for these qualitative differences. The neural architecture we present couples individual central pattern generators to reproduce the fictive stepping-phase transitions observed in deafferented stick insect preparations after pharmacological activation, providing insights into the neural architecture underlying coordinated locomotion.

sentences: CDCA-seq resolve the different chromatin structure on integral circular DNAs Although extrachromosomal DNA (ecDNA) has been intensively studied for several decades, the mechanisms underlying its tumorigenic effects have been revealed only recently. In the majority of conventional sequencing studies, the high-throughput short-read sequencing largely ignores the epigenetic status of most ecDNA regions except for the junctional areas. Here, we developed the sequencing of enzyme-accessible chromatin in circular DNA (CCDA-seq) method, which uses methylase to label open chromatin without fragmentation and exonuclease to enrich the ecDNA sequencing depth, followed by long-read nanopore sequencing. Using CCDA-seq, we observed significantly different patterns in nucleosome/regulator binding in ecDNA at a single-molecule resolution. These results deepen the understanding of ecDNA regulatory mechanisms.

sentences: Crystal structure of chloroplast fructose-1,6-bisphosphate aldolase from the green alga Chlamydomonas reinhardtii The Calvin-Benson cycle fixes carbon dioxide into organic triosephosphates through the collective action of eleven conserved enzymes. Regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco-mediated carboxylation, requires two lyase reactions catalyzed by fructose-1,6-bisphosphate aldolase (FBA). While cytoplasmic FBA has been extensively studied in non-photosynthetic organisms, functional and structural details are limited for chloroplast FBA encoded by oxygenic phototrophs. Here we determined the crystal structure of plastidial FBA from the unicellular green alga Chlamydomonas reinhardtii (Cr). We confirm that CrFBA folds as a TIM barrel, describe its catalytic pocket and homo-tetrameric state. Multiple sequence profiling classified the photosynthetic paralogs of FBA in a distinct group from non-photosynthetic paralogs. We mapped the sites of thiol- and phospho-based post-translational modifications known from photosynthetic organisms and predict their effects on enzyme catalysis.

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ONTdeCIPHER: An amplicon-based nanopore sequencing pipeline for tracking pathogen variants MotivationAmplicon-based nanopore sequencing is increasingly used for molecular surveillance during epidemics (e.g. ZIKA, EBOLA) or pandemics (e.g. SARS-CoV-2). However, there is still a lack of versatile and easy-to-use tools that allow users with minimal bioinformatics skills to perform the main steps of downstream analysis, from quality testing to SNPs effect to phylogenetic analysis.

ResultsHere, we present ONTdeCIPHER, an amplicon-based Oxford Nanopore Technology (ONT) sequencing pipeline to analyze the genetic diversity of SARS-CoV-2 and other pathogenes. Our pipeline integrates 13 bioinformatics tools. With a single command line and a simple configuration file, users can pre-process their data and obtain the sequencing statistics, reconstruct the consensus genome, identify variants and their effects for each viral isolate, infer lineage and, finally perform multi-sequence alignments and phylogenetic analyses.

Availability and implementationONTdeCIPHER is available at https://github.com/emiracherif/ONTdeCIPHER

Contactemira.cherif@ird.fr

Supplementary informationSupplementary data are available at ...

sentences: An amino residue that guides the correct photoassembly the water-oxidation complex but not required for high affinity Mn2+ binding The assembly of the Mn4O5Ca cluster of the photosystem II (PSII) starts from the initial binding and photooxidation of the first Mn2+ at a high affinity site (HAS). Recent cryo-EM apo-PSII structures reveal an altered geometry of amino ligands in this region and suggest the involvement of D1-Glu189 ligand in the formation of the HAS. We now find that Gln and Lys substitution mutants photoactivate with reduced quantum efficiency compared to the wild-type. However, the affinity of Mn2+ at the HAS in D1-E189K was very similar to the wild-type (~2.2 M). Thus, we conclude that D1-E189 does not form the HAS (~2.9 M) and that the reduced quantum efficiency of photoactivation in D1-E189K cannot be ascribed to the initial photooxidation of Mn2+ at the HAS. Besides reduced quantum efficiency, the D1-E189K mutant exhibits a large fraction of centers that fail to recover activity during photoactivation starting early in the assembly phase, becoming recalcitrant to further assembly. Fluorescence relaxation kinetics indicate on the presence of an alternative route for the charge recombination in Mn-depleted samples in all studied mutants and exclude damage to the photochemical reaction center as the cause for the recalcitrant centers failing to assemble and show that dark incubation of cells reverses some of the inactivation. This reversibility would explain the ability of these mutants to accumulate a significant fraction of active PSII during extended periods of cell growth. The failed recovery in the fraction of inactive centers appears to a reversible mis-assembly involving the accumulation of photooxidized, but non-catalytic high valence Mn at the donor side of photosystem II, and that a reductive mechanism exists for restoration of assembly capacity at sites incurring mis-assembly. Given the established role of Ca2+ in preventing misassembled Mn, we conclude that D1-E189K mutant impairs the ligation of Ca2+ at its effector site in all PSII centers that consequently leads to the mis-assembly resulting in accumulation of non-catalytic Mn at the donor side of PSII. Our data indicate that D1-E189 is not functionally involved in Mn2+ oxidation\binding at the HAS but rather involved in Ca2+ ligation and steps following the initial Mn2+ photooxidation.

sentences: The timing of natural killer cell response in coronavirus infection: a concise model perspective Coronaviruses, including SARS-CoV, MERS-CoV, and SARS-CoV-2 cause respiratory diseases with remarkably heterogeneous progression. This in part reflects the viral ability to influence the cytokine secretion and thereby the innate immune system. Especially the viral interference of IFN-I signaling and the subsequent deficiency of innate immune response in the early phase have been associated with rapid virus replication and later excessive immune responses. We propose a mathematical framework to analyze IFN-I signaling and its impact on the interaction motif between virus, NK cells and macrophages. The model recapture divergent dynamics of coronavirus infections including the possibility for elevated secretion of IL-6 and IFN-{gamma} as a consequence of exacerbated macrophage activation. Dysfunction of NK cells recruitment increase disease severity by leading to a higher viral load peak, the possibility for excessive macrophage activation, and an elevated risk of the cytokine storm. Thus the model predicts that delayed IFN-I signaling could lead to pathogenicity in the latter stage of an infection. Reversely, in case of strong NK recruitment from infected cells we predict a possible chronic disease state with moderate and potentially oscillating virus/cytokine levels.

sentences: Neural activity in human brainstem adapts to sound-level statistics Optimal perception requires adaptation to sounds in the environment. Adaptation involves representing the acoustic stimulation history in neural response patterns, for example, by altering response magnitude or latency as sound-level context change. Neurons in the auditory brainstem of rodents are sensitive to acoustic stimulation history and sound-level context (often referred to as sensitivity to stimulus statistics), but the degree to which the human brainstem exhibits such neural adaptation is unclear. In six electroencephalography experiments with over 125 participants, we demonstrate that the response latency of the human brainstem is sensitive to the history of acoustic stimulation over a few tens of milliseconds. We further show that human brainstem responses adapt to sound-level context in, at least, the last 44 ms, but that neural sensitivity to sound-level context decreases when the time window over which acoustic stimuli need to be integrated becomes wider. Our study thus provides evidence of adaptation to sound-level context in the human brainstem and of the timescale over which sound-level information affects neural responses to sound. The research delivers an important link to studies on neural adaptation in non-human animals.

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RF4Del: A Random Forest approach for accurate deletion detection Efficiently detecting genomic structural variants (SVs) is a key step to grasp the "missing heritability" underlying complex traits involved in major evolutionary processes such as speciation, phenotypic plasticity, and adaptive responses. Yet, the SV-based genotype/trait association studies are still largely overlooked mainly due to the lack of reliable detection methods. Here, we present a random forest (RF) method for accurate deletion identification: RF4Del. By relying on the analysis of the mapping profiles, data already available in most sequencing projects, RF4Del can easily and quickly call deletions.

Several classic and ensemble learning strategies were carefully evaluated using proper benchmark data. RF4Del was trained and tested on simulated data from the model species Drosophila melanogaster to detect deletions. The model consists of 13 features extracted from a mapping file. We show that RF4Del outperforms established SV callers (DELLY, Pindel) with higher overall performance (F1-score > 0.75; 6x-12x sequence coverage) and is less affected by low sequencing coverage and deletion size variations. RF4Del could learn from a compilation of sequence patterns linked to a given SV. Such models can then be combined to form a learning system able to detect all types of SVs in a given genome, beyond the one used in our study. https://github.com/alvesrcoo/eletric-scheep

sentences: Beneficial effects of prolonged 2-phenylethyl alcohol inhalation on chronic distress-induced anxio-depressive-like phenotype in female mice Chronic distress-induced hypothalamic-pituitary-adrenal axis deregulations have been associated with the development of neuropsychiatric disorders such as anxiety and depression. Currently available drugs treating such pathological conditions have limited efficacy and diverse side effects, revealing the need of new safer strategies. Aromatic plant-based compounds are largely used in herbal medicine due to their therapeutic properties on mood, physiology, and general well-being. The purpose of this study was to investigate the effects of 2-phenylethyl alcohol (PEA), one of the pharmacologically active constituents of rose essential oil, on chronic corticosterone (CORT)-induced behavioral and neurobiological changes in female mice. Animals followed a prolonged PEA inhalation exposure (30 min per day) for 15 consecutive days prior to behavioral evaluation with open-field, forced swim and novelty-suppressed feeding tests. CORT treatment induced an anxio-depressive-like phenotype, evidenced by a reduced locomotor activity in the open-field, and an increased latency to feed in the novelty-suppressed feeding paradigms. To elucidate the neural correlates of our behavioral results, cerebral cFos expression analysis was further performed to provide a global map of neuronal activity. The altered feeding behavior was accompanied by a significant decrease in the number of cFos-positive cells in the olfactory bulb, and altered brain connectivity as shown by cross-correlation-based network analysis. CORT-induced behavioral and neurobiological alterations were reversed by prolonged PEA inhalation, suggesting a therapeutic action that allows regulating the activity of neural circuits involved in sensory, emotional and feeding behaviors. These findings might contribute to better understand the therapeutic potential of PEA on anxio-depressive symptoms.

sentences: Programmable Microbial Ink for 3D Printing of Living Materials Produced from Genetically Engineered Protein Nanofibers Living cells have the capability to synthesize molecular components and precisely assemble them from the nanoscale to build macroscopic living functional architectures under ambient conditions.1-3 The emerging field of living materials has leveraged microbial engineering to produce materials for various applications, but building 3D structures in arbitrary patterns and shapes has been a major challenge.1-14 We set out to develop a new bioink, termed as "microbial ink" that is produced entirely from genetically engineered microbial cells, programmed to perform a bottom-up, hierarchical self-assembly of protein monomers into nanofibers, and further into nanofiber networks that comprise extrudable hydrogels. We further demonstrate the 3D printing of functional living materials by embedding programmed Escherichia coli (E. coli) cells and nanofibers into microbial ink, which can sequester toxic moieties, release biologics and regulate its own cell growth through the chemical induction of rationally designed genetic circuits. This report showcases the advanced capabilities of nanobiotechnology and living materials technology to 3D-print functional living architectures.

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Heterotrimeric G protein α subunit (GPA1) regulates the response to low-nitrogen stress in Arabidopsis by interacting with AtNRT1.4 and AtATG8a Efficient nitrogen absorption and utilization are important factors for higher plants to increase yield and reduce eutrophication (caused by excessive use of nitrogen fertilizers). Heterotrimeric G proteins, including three subunits of , {beta}, and {gamma}, participate in the pathway regulating nitrogen absorption and utilization in plants. However, the regulatory mechanism remains largely obscured. In this study, our results revealed that the G protein  subunit (AtGPA1) mutant gpa1-4 was tolerant to low-nitrogen stress in Arabidopsis. AtGPA1 was shown to directly interact with a nitrate transporter (AtNRT1.4) and a key autophagy-related protein (AtATG8a) on the plasma membrane using the yeast hybrid system and pull-down assay (in vitro) and BiFC assay (in vivo). GUS staining and subcellular localization showed that AtGPA1 and AtNRT1.4 were co-expressed in roots and leaf veins and on the plasma membrane. Under low-nitrate conditions, the single mutant gpa1-4 and NRT1.4RNAi plants (AtNRT1.4RNA interference plants) and the double mutant NRT1.4RNAi/gpa1-4 plants (AtNRT1.4RNA interference plants on a gpa1-4 background) were healthier than the wild type plants. Moreover, the phenotype of the double mutant NRT1.4RNAi/gpa1-4 plants was closer to that of the NRT1.4RNAi plants compared to that of the gpa1-4 mutants. The results of the nitrate efflux rate assay in roots were consistent with the phenotypic changes under low-nitrogen conditions. These results indicated that AtGPA1 is an upstream factor that regulated the response to low-nitrogen stress through interaction with AtNRT1.4. In addition, we found that transgenic plants overexpressing AtATG8a were more tolerant to low-nitrogen stress, and their phenotype was similar to that of gpa1-4 mutants and double mutant ATG8aOX/gpa1-4 plants (AtATG8a overexpressing plants on a gpa1-4 mutant background). Further, autophagosome observations were consistent with the phenotypes in mutant plants, indicating that AtGPA1 regulated the response to low-nitrogen stress in Arabidopsis plants by affecting the autophagosome assembly. Our findings may provide a new model for improving nitrogen-use efficiency through genetical modification to boost crop yields.

One sentence summaryAtGPA1 negative regulates low nitrogen stress response by interaction with a nitrate transporter, AtNRT1.4 and an autophagy-related protein, AtATG8a in Arabidopsis.

sentences: Protist and Fungal Metaxin-Like Proteins: Relationship to Vertebrate Metaxins that Function in Protein Import into Mitochondria Metaxin-like proteins are shown to be encoded in the genomes of a wide range of protists and fungi. The metaxin proteins were originally described in humans and mice, and were experimentally demonstrated to have a role in the import of nascent proteins into mitochondria. In this study, metaxin-like proteins of protists and fungi predicted from genome sequences were identified by criteria including their sequence homology with vertebrate metaxin proteins and the existence of distinctive metaxin protein domains. Protists of diverse taxa, including amoebae, protozoa, phytoplankton, downy mildews, water molds, and algae, were found to possess genes for metaxin-like proteins. With fungi, the important taxonomic divisions (phyla) of Ascomycota, Basidiomycota, Chytridiomycota, Mucoromycota, and Zoopagomycota had species with metaxin-like protein genes. The presence of distinctive GST_N_Metaxin, GST_C_Metaxin, and Tom37 domains in the predicted proteins indicates that the protist and fungal proteins are related to the vertebrate metaxins. However, the metaxin-like proteins are not direct homologs of vertebrate metaxins 1, 2, or 3, but have similarity to each of the three. The alignment of the metaxin-like proteins of a variety of protists with vertebrate metaxins 1 and 2 showed about 26% and 19% amino acid identities, respectively, while for fungal metaxin-like proteins, the identities were about 29% and 23%. The different percentages with the two vertebrate metaxins indicates that the metaxin-like proteins are both metaxin 1-like and, to a lesser degree, metaxin 2-like. The secondary structures of protist and fungal metaxin-like proteins both consist of nine -helical segments, the same as for the vertebrate metaxins, with a negligible contribution from {beta}-strand. Phylogenetic analysis demonstrated that the protist and fungal metaxin-like proteins and the vertebrate metaxins form distinct and separate groups, but that the groups are derived from a common ancestral protein sequence.

sentences: Ultrashort treatment with telacebec alone and with companion drugs in immunocompetent and immunosuppressed mouse footpad models of Buruli ulcer The antimicrobial treatment of Mycobacterium ulcerans infection, or Buruli ulcer (BU), has a long duration and is therefore burdensome and linked to indirect costs for affected patients. The new antimycobacterial drug telacebec (Q203) has previously shown promising treatment-shortening potential in mouse models of BU. In the present study, we investigated the potential of Q203 to reduce the treatment duration further. The first experiment investigated the possibility of cure by one, three or five doses of Q203 (2 mg/kg) with or without a companion drug (bedaquiline, BDQ, clofazimine, CFZ, or clarithromycin, CLR) in immunocompetent BALB/c mice. The second experiment assessed the effect of five doses of Q203 with or without BDQ or CFZ on Mycobacterium ulcerans infection of immunocompromised SCID-beige mice with the aim to evaluate the contribution of host immunity to treatment efficacy. In BALB/c mice, a treatment duration as short as 3 days was sufficient to prevent relapse in nearly all footpads and a single dose of Q203 with or without BDQ or CFZ prevented relapse in approximately 50% of footpads. Unlike in BALB/c mice, a small percentage of SCID-beige mouse footpads were culture-positive after a treatment duration of five days, highlighting an important role of host immunity for M. ulcerans clearance. Our results confirm the marked potency and prolonged bactericidal and sterilizing effects of Q203, even in immunocompromised SCID-beige mice.

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HYBRID REACTION-DIFFUSION AND CLOCK-AND-WAVEFRONT MODEL FOR THE ARREST OF OSCILLATIONS IN THE SOMITOGENESIS SEGMENTATION CLOCK AO_SCPLOWBSTRACTC_SCPLOWThe clock and wavefront paradigm is arguably the most widely accepted model for explaining the embryonic process of somitogenesis. According to this model, somitogenesis is based upon the interaction between a genetic oscillator, known as segmentation clock, and a differentiation wavefront, which provides the positional information indicating where each pair of somites is formed. Shortly after the clock and wavefront paradigm was introduced, Meinhardt presented a conceptually different mathematical model for morphogenesis in general, and somitogenesis in particular. Recently, Cotterell et al. rediscovered an equivalent model by systematically enumerating and studying small networks performing segmentation. Cotterell et al. called it a progressive oscillatory reaction-diffusion (PORD) model. In the Meinhardt-PORD model, somitogenesis is driven by short-range interactions and the posterior movement of the front is a local, emergent phenomenon, which is not controlled by global positional information. With this model, it is possible to explain some experimental observations that are incompatible with the clock and wavefront model. However the Meinhardt-PORD model has some important disadvantages of its own. Namely, it is quite sensitive to fluctuations and depends on very specific initial conditions (which are not biologically realistic). In this work, we propose an equivalent Meinhardt-PORD model, and then amend it to couple it with a wavefront consisting of a receding morphogen gradient. By doing so, we get a hybrid model between the Meinhardt-PORD and the clock-and-wavefront ones, which overcomes most of the deficiencies of the two originating models.

Somitogenesis, the process by which somites are formed, is an essential developmental stage in many vertebrates. This process occurs with a strikingly regular periodicity, that is preserved among embryos of a single species. The clock and wavefront paradigm is arguably the most widely accepted model for explaining somitogenesis. However, it is incapable of explaining some experimental facts, like the appearance of somites in the absence of an external wavefront (i.e. a receding morphogen gradient). Shortly after the clock and wavefront paradigm was introduced, Meinhardt presented a conceptually different mathematical model for morphogenesis in general, and somitogenesis in particular. Recently, Cotterell et al. rediscovered an equivalent model by systematically enumerating and studying small networks performing segmentation, and called it a progressive oscillatory reaction-diffusion (PORD) model. The Meinhardt-PORD model tackles some of the deficiencies of the clock and wave-front models, but it has some serious issues of its own. In the present work, we introduce an equivalent Meinhardt-PORD model, and then amend it to couple it with a receding morphogen gradient. By doing so, we get a hybrid model that incorporates characteristics of the Meinhardt-PORD and clock-and-wavefront models. We show that this hybrid model under-goes a bifurcation, from a stable to an unstable limit cycle, as the value of the parameter accounting for a background regulatory input (associated to the receding morphogen gradient) decreases. This bifurcation allows the model to explain why somites can form in the absence of an external wavefront, reassesses the role of the receding morphogen gradient as a conductor for somitogenesis, and makes the model behavior robust to random fluctuations, as well as independent from specific initial conditions (the latter, are two of the weak points of the Meinhardt-PORD model). We argue that this findings provide convincing evidence that reaction-diffusion and positional information (receding morphogen gradient) mechanisms could work together in somitogenesis.

sentences: Self-consistent migration puts tight constraintson the spatio-temporal organization ofspecies-rich metacommunities Biodiversity is often attributed to a dynamic equilibrium between immigration of species and competition-driven extinction. This equilibrium forms a common basis for studying ecosystem assembly from a static reservoir of migrants-the mainland. Yet, natural ecosystems often consist of many coupled communities (i.e. metacommunities) and migration occurs between these communities. The pool of migrants then depends on what is sustained in the ecosystem, which in turn depends on the dynamic migrant pool. This chicken-and-egg problem of survival and migration is poorly understood in communities of many competing species, except for the neutral case - the "unified neutral theory of biodiversity". Employing spatio-temporal simulations and mean-field analyses, we show that self-consistent migration puts rather tight constraints on the dynamic migration-extinction equilibrium. When the number of species is large, even weak competitions push species to the edge of their global extinction, such that the overall diversity is highly sensitive to perturbations in demographic parameters, including growth and dispersal rates. When migration is short-range, the resulting spatiotemporal abundance patterns follow broad scale-free distributions that correspond to a directed percolation phase transition. The qualitative agreement of our results for short-range and long-range migration suggests that this self-organization process is a general property of species-rich metacommunities. Our study shows that self-sustaining metacommunities are highly sensitive to environmental change and provides insights into how biodiversity can be rescued and maintained.

sentences: A simple method to separate birds and insects in single-pol weather radar data Recent and archived data from weather radar networks are extensively used for quantification of continent-wide bird migration pattern. While discriminating birds from weather signals is well established, insect contamination is still a problem. We present a simple method combining two doppler radar products within a single Gaussian-mixture model to estimate the proportions of birds and insects within a single measurement, as well as the density and speed of birds and insects. The method can be applied to any existing archives of vertical bird profiles, such as the ENRAM repository (enram.eu) with no need to recalculate the huge amount of original polar volume data, which often are not available.

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Hyperbolic Geometry-Based Deep Learning Methods to Produce Population Trees from Genotype Data The production of population-level trees using the genomic data of individuals is a fundamental task in the field of population genetics. Typically, these trees are produced using methods like hierarchical clustering, neighbor joining, or maximum likelihood. However, such methods are non-parametric: they require all data to be present at the time of tree formation, and the addition of new data points necessitates the regeneration of the entire tree, a potentially expensive process. They also do not easily integrate with larger workflows. In this study, we aim to address these problems by introducing parametric deep learning methods for tree formation from genotype data. Our models specifically create continuous representations of population trees in hyperbolic space, which has previously proven highly effective in embedding hierarchically structured data. We present two different architectures - a multi-layer perceptron (MLP) and a variational autoencoder (VAE) - and we analyze their performance using a variety of metrics along with comparisons to established tree-building methods. Both models tested produce embedding spaces that reflect human evolutionary history. In addition, we demonstrate the generalizability of these models by verifying that addition of new samples to an existing tree occurs in a semantically meaningful manner. Finally, we use Dasguptas Cost to compare the quality of trees generated by our models to those produced by established methods. Despite the fact that the benchmark methods are directly fit on the evaluation data, our models are able to outperform some of these and achieve highly comparable performance overall.

Author summaryTree production is a vital task in population genetics, but current approaches fall prey to several common shortfalls. Most notably, they lack the ability to add new data points after tree generation, and they are often difficult to use in larger pipelines. By leveraging cutting-edge advances pairing deep learning with hyperbolic geometry, we develop multiple models designed to rectify these issues. Through experiments on a dataset of humans from globally widespread ancestries, we demonstrate the generalizability of our models to new data, and we also show strong empirical performance with respect to currently used methods. In addition, we show that the data representations produced by our models are semantically meaningful and reflect known facts about human evolutionary history. Finally, we discuss the additional benefits our models could provide, including improved visualization, greater privacy preservation, and improved integration with downstream machine learning tasks. In conclusion, we present models that are accurate, flexible, and generalizable, with the potential to facilitate a variety of further applications.

sentences: A salvaging strategy enables stable metabolite provisioning among free-living bacteria All organisms rely on complex metabolites such as amino acids, nucleotides, and cofactors for essential metabolic processes. Some microbes synthesize these fundamental ingredients of life de novo, while others rely on uptake to fulfill their metabolic needs. Although certain metabolic processes are inherently  leaky, the mechanisms enabling stable metabolite provisioning among microbes in the absence of a host remain largely unclear. In particular, how can metabolite provisioning among free-living bacteria be maintained under the evolutionary pressure to economize resources? Salvaging, the process of  recycling and reusing, can be a metabolically efficient route to obtain access to required resources. Here, we show experimentally how precursor salvaging in engineered Escherichia coli populations can lead to stable, long-term metabolite provisioning. We find that salvaged cobamides (vitamin B12 and related enzyme cofactors) are readily made available to non-productive population members, yet salvagers are strongly protected from overexploitation due to partial metabolite privatization. We also describe a previously unnoted benefit of precursor salvaging, namely the removal of the non-functional, proliferation-inhibiting precursor. As long as compatible precursors are present, any microbe possessing the terminal steps of a biosynthetic process can, in principle, forgo de novo biosynthesis in favor of salvaging. Consequently, precursor salvaging likely represents a potent, yet overlooked, alternative to de novo biosynthesis for the acquisition and provisioning of metabolites in free-living bacterial populations.

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IHRW: An Improved Hypergraph Random Walk Model for Predicting Three-Drug Therapy Drug combination therapy is a well-established concept in the treatment of complex diseases due to its fewer side effects, lower toxicity, and better efficacy. However, it is challenging to identify efficacious drug combinations from many drug candidates. Computational models could greatly reduce the cost, but most models did not use data for more than two-drug combinations and could not predict three-drug therapy. However, three-drug combinations account for about 21% of the known combinations, which is a very important type of treatment. Here, we utilized higher-order information and developed an improved hypergraph random walk model (IHRW) for three-drug therapy prediction. This is the first method to explore the combination of three drugs.

As a result, the case studies of breast cancer, lung cancer, and colon cancer showed that IHRW had a powerful ability to predict potential efficacious three-drug combinations, which provides new prospects for complex disease treatment. The code of IHRW is freely available at https://github.com/wangqi27/IHRW.

sentences: Mmp14-controlled extracellular matrix homeostasis is required for circadian rhythm The circadian clock regulates extracellular matrix (ECM) homeostasis but whether altered ECM homeostasis regulates the clock was unknown. Thus, we generated mice with a Col1a2-Cre-ERT2 conditional deletion of matrix metalloproteinase-14 (Mmp14) to disrupt matrix homeostasis in postnatal mice. We show here that tamoxifen-treated Col1a2-Cre-ERT2::Mmp14F/F mice developed a severe tendon matrix phenotype with dorsiflexion of hind limbs, tissue thickening, and accumulation of hydroxylysine aldehyde (pyridinoline) crosslinked narrow-diameter collagen fibrils typically seen accumulating during embryonic development. Proteomic analysis of tamoxifen-treated postnatal mice showed [~]16% of detected proteins lost (176 proteins) or reversed (19 proteins) time-dependent rhythmicity. Ontological analysis identified  cytoskeleton as an affected system. Subsequent studies in CRISPR-Cas9 Mmp14 null cells showed decreased F-/G-actin ratio, decreased cell spreading, elevated levels of pyridinoline crosslink factor Plod2, nuclear translocation of transcription factor Pitx2, and loss of circadian rhythm. Thus, our study shows a bidirectional dependence of collagen-ECM homeostasis and the circadian clock.

sentences: Oligomeric amyloid beta prevents myelination in a clusterin-dependent manner White matter loss has been described as a common occurrence in Alzheimers disease (AD) patients for multiple decades. However, it remains unclear why oligodendrocyte progenitor cells (OPCs) fail to repair myelin deficits in these patients. Here, we show that clusterin, a risk factor for late-onset AD, is produced by OPCs and inhibits their differentiation into oligodendrocytes. Specifically, we demonstrate that a unique subset of OPCs produces clusterin. We show that phagocytosis of debris, including amyloid beta (A{beta}) and myelin, drives the upregulation of clusterin in OPCs. We confirm, in vivo, that A{beta} oligomers drive clusterin upregulation and that OPCs phagocytose A{beta}. Furthermore, we show that clusterin is a potent inhibitor of OPC differentiation and prevents the production of myelin proteins. Finally, we demonstrate that clusterin inhibits OPC differentiation by significantly reducing the production of IL-9 by OPCs. Our data reveals that clusterin may be responsible for the lack of myelin repair observed in AD and is a promising therapeutic target for AD-associated cognitive decline.

sentences: Marked enhancement of neutralizing antibody and IFN-γ T-cell responses by GX-19N DNA booster in mice primed with inactivated vaccine In response to the COVID-19 pandemic, an unprecedented level of vaccine development has occurred. As a result, various COVID-19 vaccines have been approved for use. Among these, inactivated virus particle (VP) vaccines have been widely used worldwide, but additional vaccination strategies are needed because of the short duration of immune responses elicited by these vaccines. Here, we evaluated homologous and heterologous prime-boost regimens using a VP vaccine and GX-19N DNA vaccine for their ability to enhance the protective immune response against SARS-CoV-2. We demonstrated that a heterologous prime-boost regimen with the VP vaccine and GX-19N DNA vaccine resulted in enhanced SRBD- & N-specific antibody responses, compared to the homologous VP vaccine prime-boost vaccination. In addition, the neutralizing antibody response was significantly improved with the heterologous VP prime-DNA boost regimen, and the neutralizing antibody induced with the heterologous prime-boost regimen did not decrease against the SARS-CoV-2 variant of concern (VOC). The heterologous VP prime-DNA boost regimen not only significantly increased S- and N-specific IFN-{gamma} T-cell responses, but also induced an equivalent level of T-cell response against SARS-CoV-2 VOCs. Our results provide new insights into prophylactic vaccination strategies for COVID-19 vaccination.

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When inefficient speech-motor control affects speech comprehension: atypical electrophysiological correlates of language prediction in stuttering It is well attested that people predict forthcoming information during language comprehension. The literature presents different proposals on how this ability could be implemented. Here, we tested the hypothesis according to which language production mechanisms have a role in such predictive processing. To this aim, we studied two electroencephalographic correlates of predictability during speech comprehension - pretarget alpha-beta (8-30 Hz) power decrease and the post-target N400 event-related potential (ERP) effect, - in a population with impaired speech-motor control, i.e., adults who stutter (AWS), compared to typically fluent adults (TFA). Participants listened to sentences that could either constrain towards a target word or not, allowing or not to make predictions. We analyzed time-frequency modulations in a silent interval preceding the target and ERPs at the presentation of the target. Results showed that, compared to TFA, AWS display: i) a widespread and bilateral reduced power decrease in posterior temporal and parietal regions, and a power increase in anterior regions, especially in the left hemisphere (high vs. low constraining) and ii) a reduced N400 effect (non-predictable vs. predictable). The results suggest a reduced efficiency in generating predictions in AWS with respect to TFA. Additionally, the magnitude of the N400 effect in AWS is correlated with alpha power change in the right pre-motor and supplementary motor cortex, a key node in the dysfunctional network in stuttering. Overall, the results support the idea that processes and neural structures prominently devoted to speech planning and execution support prediction during language comprehension.

Significance StatementThe study contributes to the developing enterprise of investigating language production and comprehension not as separate systems, but as sets of processes which may be partly shared. We showed that a population with impaired speech-motor control, i.e., adults who stutter, are characterized by atypical electrophysiological patterns associated with prediction in speech comprehension. The results highlight that an underlying atypical function of neural structures supporting speech production also affects processes deployed during auditory comprehension. The implications are twofold: on the theoretical side, the study supports the need for a more integrated view of language comprehension and production as human capabilities, while on the applied and clinical side, these results might open new venues for efficient treatments of developmental stuttering.

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OmicsView: omics data analysis through interactive visual analytics With advances in NGS technologies, transcriptional profiling of human tissue across many diseases is becoming more routine, leading to the generation of petabytes of data deposited in public repositories. There is a need for bench scientists with little computational expertise to be able to access and mine this data to understand disease pathology, identify robust biomarkers of disease and the effect of interventions (in vivo or in vitro). To this end we release an open source analytics and visualization platform for expression data called OmicsView, http://omicsview.org.

This platform comes preloaded with 1000s of samples across many disease areas and normal tissue, including the GTEx database, all processed with a harmonized pipeline. We demonstrate the power and ease-of-use of the platform by means of a Crohns disease data mining exercise where we can quickly uncover disease pathology and identify strong biomarkers of disease and response to treatment.

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Fungal gasdermin-like proteins are controlled by proteolytic cleavage Gasdermins are a family of pore-forming proteins controlling an inflammatory cell death reaction in the mammalian immune system. The pore-forming ability of the gasdermin proteins is released by proteolytic cleavage with the removal of their inhibitory C-terminal domain. Recently, gasdermin-like proteins have been discovered in fungi and characterized as cell death-inducing toxins in the context of conspecific non-self discrimination (allorecognition). Although functional analogies have been established between mammalian and fungal gasdermins, the molecular pathways regulating gasdermin activity in fungi remain largely unknown. Here, we characterize a gasdermin-based cell death reaction, controlled by the het-Q allorecognition genes in the filamentous fungus Podospora anserina. We show that the cytotoxic activity of the HET-Q1 gasdermin is controlled by proteolysis. HET-Q1 loses a [~]5 kDa C-terminal fragment during the cell death reaction in presence of a subtilisin-like serine protease, termed HET-Q2. Mutational analyses and successful reconstitution of the cell death reaction in a heterologous host (Saccharomyces cerevisiae) suggest that HET-Q2 directly cleaves HET-Q1 to induce cell death. By analysing the genomic landscape of het-Q1 homologs in fungi, we uncovered that the vast majority of the gasdermin genes are clustered with protease-encoding genes. These HET-Q2-like proteins carry either subtilisin-like or caspase-related proteases, which in some cases correspond to the N-terminal effector domain of NOD-like receptor proteins (NLRs). This study thus reveals the proteolytic regulation of gasdermins in fungi and establishes evolutionary parallels between fungal and mammalian gasdermin-dependent cell death pathways.

SignificanceThe recent discovery of gasdermin-like proteins in fungi have brought to light that this family of pore-forming proteins controls cell death in two of the major eukaryotic kingdoms, fungi and mammals. Yet, the regulation of cytotoxicity of the fungal gasdermins and their molecular pathways remain uncharacterized. Here, we describe the regulation through proteolytic cleavage of the fungal gasdermin HET-Q1 and uncover that majority of fungal gasdermins are genomically clustered with protease-encoding genes. Some of these genes encode proteins with caspase-related domains and/or are members of a family of immune receptors in mammals and plants. Overall, this work contributes towards our understanding of the evolution of gasdermin-dependent cell death, enlightening multiple evolutionary parallels between signaling pathways in mammals and fungi.

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LMO2 is critical for early metastatic events in breast cancer Metastasis is responsible for the majority of breast cancer-related deaths, however identifying the cellular determinants of metastasis has remained challenging. Here, we identified a minority population of immature THY1+/VEGFA+ tumor epithelial cells in human breast tumor biopsies that display angiogenic features and are marked by the expression of the oncogene, LMO2. Higher abundance of LMO2+ basal cells correlated with tumor endothelial content and predicted poor distant recurrence-free survival in patients. Using MMTV-PyMT/Lmo2CreERT2 mice, we demonstrated that Lmo2 lineage- traced cells have a higher propensity to metastasize. LMO2 knockdown in human breast tumors reduced lung metastasis by impairing intravasation, leading to a reduced frequency of circulating tumor cells. Mechanistically, we find that LMO2 binds to STAT3 and is required for STAT3 activation by TNF and IL6. Collectively, our study identifies a population of metastasis-initiating cells with angiogenic features and establishes the LMO2-STAT3 signaling axis as a therapeutic target in breast cancer metastasis.

One sentence summaryLMO2 modulates STAT3 signaling in breast cancer metastasis.

sentences: The push-pull intercrop Desmodium does not repel, but intercepts and kills pests Over two decades ago, scientists developed a push-pull intercropping strategy that received critical acclaim for synergizing food security with ecosystem resilience in smallholder farming. The strategy suppresses Lepidopteran pests in maize through a combination of a repellent intercrop (push), commonly Desmodium spp., and an attractive, dead-end border crop (pull). Key is the intercrops constitutive release of volatiles that repel herbivores. Surprisingly, however, we found that Desmodium does not constitutively release volatiles, and only minimally upon herbivory. Further, in oviposition choice settings, Spodoptera frugiperda, a devastating invasive pest, was not repelled by Desmodium volatiles. In search of an alternative mechanism, we found that neonate larvae strongly preferred Desmodium over maize. However, their development stagnated and none survived. In addition, larvae were frequently seen impaled and immobilized by the dense network of silica-fortified, non-glandular trichomes. Thus, entirely different from repelling adult moths, Desmodium intercepts and decimates dispersing offspring. As a hallmark of sustainable pest control, maize-Desmodium intercropping has inspired countless efforts trying to emulate a stimulo-deterrent diversion in other cropping systems. However, detailed knowledge of the actual mechanisms is required to rationally improve the strategy, and translate the concept into other cropping systems.

sentences: SYMBIOSIS: Synthetic Manipulable Biobricks via Orthogonal Serine Integrase Systems Serine integrases are emerging as one of the most powerful biological tools for synthetic biology. They have been widely used across genome engineering and genetic circuit design. However, developing serine integrase-based tools for directly/precisely manipulating synthetic biobricks is still missing. Here, we report SYMBIOSIS, a versatile method that can robustly manipulate DNA parts in vivo and in vitro. First, we proposed a "Keys match Locks" model to demonstrate that three orthogonal serine integrases are able to irreversibly and stably switch on seven synthetic biobricks with high accuracy in vivo. Then, we demonstrated that purified integrases can facilitate the assembly of "Donor" and "Acceptor" plasmids in vitro to construct composite plasmids. Finally, we used SYMBIOSIS to assemble different chromoprotein genes and create novel colored Escherichia coli. We anticipate that our SYMBIOSIS strategy will accelerate synthetic biobricks manipulation, genetic circuit design, and multiple plasmids assembly for synthetic biology with broad potential applications.

sentences: Type 1 piliated uropathogenic Escherichia coli hijack the host immune response by binding to CD14 A key attribute of persistent or recurring bacterial infections is the ability of the pathogen to evade the hosts immune response. Many Enterobacteriaceae express type 1 pili, a pre-adapted virulence trait, to invade host epithelial cells and establish persistent infections. However, the molecular mechanisms and strategies by which bacteria actively circumvent the immune response of the host remain poorly understood. Here, we identified CD14, the major co-receptor for lipopolysaccharide detection, on dendritic cells as a previously undescribed binding partner of FimH, the protein located at the tip of the type 1 pilus of Escherichia coli. The FimH amino acids involved in CD14 binding are highly conserved across pathogenic and non-pathogenic strains. Binding of pathogenic bacteria to CD14 lead to reduced dendritic cell migration and blunted expression of co-stimulatory molecules, both rate-limiting factors of T cell activation. While defining an active molecular mechanism of immune evasion by pathogens, the interaction between FimH and CD14 represents a potential target to interfere with persistent and recurrent infections, such as urinary tract infections or Crohns disease.

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Standard Intein Gene Expression Ramps (SIGER) for protein-independent expression control Coordination of multi-gene expression is one of the key challenges of metabolic engineering for the development of cell factories. Constraints on translation initiation and early ribosome kinetics of mRNA are imposed by features at the start of the gene, referred to as the "gene ramp", such as rare codons and mRNA secondary structures forming in relation with the 5UTR. These features strongly influence translation yield and protein quality by regulating ribosome distribution on mRNA strands. The utilization of genetic expression sequences, such as promoters and 5UTRs in combination with different target genes leads to a wide variety of gene ramp compositions with irregular translation rates leading to unpredictable levels of protein yield and quality. Here, we present the Standard Intein Gene Expression Ramps (SIGER) system for controlling protein expression. The SIGER system uses inteins to uncouple a characterized gene ramp from a target protein. We generated sequence-specific gene expression sequences for two inteins (DnaB and DnaX) that display defined levels of protein expression. Additionally, we used inteins that possess the ability to release the C-terminal fusion protein in vivo to avoid impairment of protein functionality by the fused intein. Overall, our results show that SIGER systems are unique tools to mitigate the undesirable effects of gene ramp variation and to control the relative ratios of enzymes involved in molecular pathways.

Graphical abstract

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sentences: Genomic stock structure of the marine teleost tarakihi (Nemadactylus macropterus) provides evidence of fine-scale adaptation and a temperature-associated cline amid panmixia Tarakihi (Nemadactylus macropterus) is an important fishery species with widespread distribution around New Zealand and off the southern coasts of Australia. However, little is known about whether the populations are locally adapted or genetically structured. To address this, we conducted whole-genome resequencing of 175 tarakihi from around New Zealand and Tasmania (Australia) to obtain a dataset of 7.5 million genome-wide and high-quality single nucleotide polymorphisms (SNPs). Variant filtering, FST-outlier analysis, and redundancy analysis (RDA) were used to evaluate population structure, adaptive structure, and locus-environment associations. A weak but significant level of neutral genetic differentiation was found between tarakihi from New Zealand and Tasmania (FST = 0.0054-0.0073, P [≤] 0.05), supporting the existence of at least two separate reproductive stocks. No clustering was detected among the New Zealand populations ([FE]ST < 0.001, P = 0.77). Outlier-based, presumably adaptive variation suggests fine-scale adaptive structure between locations around central New Zealand off the east (Wairarapa, Cape Campbell, and Hawkes Bay) and the west coast (Tasman Bay/Golden Bay and Upper West Coast of South Island). Allele frequencies from 55 loci were associated with at least one of six environmental variables, of which 47 correlated strongly with yearly mean water temperature. Although genes associated with these loci are linked to various functions, the most common functions were integral components of membrane and cilium assembly. Projection of the RDA indicates the existence of a latitudinal temperature cline. Our work provides the first genomic insights supporting panmixia of tarakihi in New Zealand and evidence of a genomic cline that appears to be driven by the temperature gradients, together providing crucial information to inform the stock assessment of this species, and to widen the insights of the ecological drivers of adaptive variation in a marine species.

sentences: The 4.4 A structure of the giant Melbournevirus virion belonging to the Marseilleviridae family Members of Marseilleviridae, one family of icosahedral giant viruses classified in 2012 have been identified worldwide in all types of environments. The virion shows a characteristic internal membrane extrusion at the five-fold vertices of the capsid, but its structural details need to be elucidated. We now report the 4.4 [A] cryo-electron microscopy structure of the Melbournevirus capsid. An atomic model of the major capsid protein (MCP) shows a unique cup structure on the trimer that accommodates additional proteins. A polyalanine model of the penton base protein shows internally extended N- and C-terminals, which indirectly connect to the internal membrane extrusion. The Marseilleviruses share the same orientational organisation of the MCPs as PBCV-1 and CroV, but do not appear to possess a protein akin to the "tape measure" of these viruses. Minor capsid proteins named PC-{beta}, zipper, and scaffold are proposed to control the dimensions of the capsid during assembly.

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Cell-free Expression of Proton-Coupled Folate Transporter in the Presence of Nanodiscs The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

HighlightsO_LICell free expression of PCFT without any lipids or detergents resulted in quantitative precipitation of in-situ synthesized PCFT.
C_LIO_LIAdditives for expression of PCFT in the soluble fraction were identified.
C_LI

sentences: Patient-Derived Organoids Recapitulate Intrinsic Immune Landscapes and Progenitor Populations of Glioblastoma. Glioblastoma stem cells (GSCs) are highly self-renewing, resistant to therapy, and are able to form lethal tumors1, 2. Tumor organoids have been developed to study tumor evolution1-4, and while GSCs can form organoids for glioblastoma multiforme, our understanding of their intrinsic immune, metabolic, genetic, and molecular programs is limited. To address this, we deeply characterized GSC-derived GBM organoids using a modified protocol (GBMOsm) from several patient-derived GSCs and found they develop into complex 3D tissues with unique self-organization, cancerous metabolic states, and burdensome genetic landscapes. We discovered that GBMOsc recapitulate the presence of two important cell populations thought to drive GBM progression, SATB2+ and HOPX+ progenitors. Despite being devoid of immune cells, transcriptomic analysis across GBMOsc revealed an immune-like molecular program, enriched in cytokine, antigen presentation and processing, T-cell receptor inhibitors, and interferon genes. We determined that SATB2+ and HOPX+ populations contribute to this immune and interferon landscape in GBM in vivo and GBMOsm. Our work deepens our understanding of the intrinsic molecular and cellular architecture of GSC-derived GBMO and defines a novel GBMOsc intrinsic immune-like program.

sentences: Neuronal modulation in the mouse superior colliculus during covert visual selective attention Covert visual attention is accomplished by a cascade of mechanisms distributed across multiple brain regions. Recent studies in primates suggest a parcellation in which visual cortex is associated with enhanced representations of relevant stimuli, whereas subcortical circuits are associated with selection of visual targets and suppression of distractors. Here we identified how neuronal activity in the superior colliculus (SC) of head-fixed mice is modulated during covert visual attention. We found that spatial cues modulated both firing rate and spike-count correlations, and that the cue-related modulation in firing rate was due to enhancement of activity at the cued spatial location rather than suppression at the uncued location. This modulation improved the neuronal discriminability of visual-change-evoked activity between contralateral and ipsilateral SC neurons. Together, our findings indicate that neurons in the mouse SC contribute to covert visual selective attention by biasing processing in favor of locations expected to contain relevant information.

sentences: Three-dimensional structure of the single domain cupredoxin AcoP Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper center coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in term of copper-binding ligands, copper center geometry, redox potential, as well as biological function. AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features: highly resistant to acidic pH, it possesses a constrained green-type copper center of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structure of AcoP at 1.65 [A] resolution unveils a typical cupredoxin fold with extended loops, never observed in previously characterized cupredoxins, that might be involved in the interaction of AcoP with its physiological partners. Moreover, the structure shows that the green color of AcoP cannot be attributed to nonclassical copper ligands, its green-colored copper center raising from a long Cu-S (Cys) bond, determined by both X-ray diffraction and EXAFS. The crystal structures of two AcoP mutants confirm that the active center of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active center rigidity due to the presence of an extensive hydrogen bond network.

sentences: Orb-dependent polyadenylation contributes to PLP expression and centrosome scaffold assembly As the microtubule-organizing centers (MTOCs) of most cells, centrosomes engineer the bipolar mitotic spindle required for error-free mitosis. Drosophila Pericentrin (PCNT)-like protein (PLP) is a key centrosome component that directs formation of a pericentriolar material (PCM) scaffold required for PCM organization and MTOC function. Here, we investigate the post-transcriptional regulation of plp mRNA. We identify conserved binding sites for cytoplasmic polyadenylation element binding (CPEB) proteins within the plp 3-untranslated region and examine the role of the CPEB ortholog, oo18 RNA-binding protein (Orb), in plp mRNA regulation. Our data show Orb biochemically interacts with plp mRNA and promotes PLP protein expression. Loss of orb, but not orb2, diminishes PLP levels in embryonic extracts. Consequently, PLP localization to centrosomes and function in PCM scaffolding is compromised in orb mutant embryos, resulting in genome instability and embryonic lethality. Moreover, we find PLP over-expression can restore centrosome scaffolding and rescue the cell division defects caused by orb depletion. Our data suggest Orb modulates PLP expression at the level of plp mRNA polyadenylation and showcases the post-transcriptional regulation of core, conserved centrosomal mRNAs as critical for centrosome function.

sentences: Actin turnover required for adhesion-independent bleb migration Cell migration is critical for many vital processes, such as wound healing, as well as harmful processes, like cancer metastasis. Experiments have highlighted the diversity in migration strategies employed by cells in physiologically relevant environments. In 3D fibrous matrices and confinement between two surfaces, some cells migrate using round membrane protrusions, called blebs. In bleb-based migration, the role of substrate adhesion is thought to be minimal, and it remains unclear if a cell can migrate without any adhesion complexes. We present a 2D computational fluid-structure model of a cell using cycles of bleb expansion and retraction in a channel with several geometries. The cell model consists of a plasma membrane, an underlying actin cortex, and viscous cytoplasm. Cellular structures are immersed in viscous fluid which permeates them, and the fluid equations are solved using the method of regularized Stokeslets. Simulations show that the cell cannot effectively migrate when the actin cortex is modeled as a purely elastic material. We find that cells do migrate in rigid channels if actin turnover is included with a viscoelastic description for the cortex. Our study highlights the non-trivial relationship between cell rheology and its external environment during migration with cytoplasmic streaming.

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IGF1 Signaling in Temporomandibular Joint Fibrocartilage Stem Cells Regulates Cartilage Growth and Homeostasis in Mice ObjectiveInvestigate functional roles of Igf1 in fibrocartilage stem cell (FCSC) for temporomandibular joint (TMJ) cartilage growth and homeostasis.

MethodsGli1-CreER+; RosaTdTomato mice were used for validating FCSCs lineage labeling efficiency. In Gli1-/Col2-CreER+; Igf1fl/fl mice, TMJ cartilage morphological and functional changes were characterized at 4 weeks and 5 months after Igf1 deletion. H&E, Safranine O and immuno-histochemistry staining were performed. FCSCs specificity were characterized using EdU and TUNEL staining. A unilateral anterior crossbite (UAC) mouse model was generated for mimicking TMJ osteoarthritis status.

ResultsIn Gli1-CreER+; RosaTdTomato mice, RFP labeled FCSCs showed favorable proliferative capacity. 4 weeks after Igf1 deletion, Gli1+ and Col2+ cell lineages led to distinct pathological changes of TMJ cartilage morphology. A more serious reduction of cartilage thickness and cell density were found in the superficial layers in Gli1-CreER+; Igf1fl/fl mice. 5 months after Igf1 deletion, more severe disordered cell arrangement in TMJ cartilage were found in both groups with Gli1+ and Col2+ specific deletion of Igf1. Immunostaining showed that PI3K/Akt signaling pathway was blocked in the superficial layers of TMJ in Gli1-CreER+; RosaTdTomato mice. Finally, deletion of Igf1 in FCSCs significantly aggravated osteoarthritis (OA) phenotypic changes in TMJ in UAC mice model, characterized in decreased cartilage thickness, cell numbers and loss of extracellular matrix secretion.

ConclusionIgf1 deletion disrupted stem cell functions of FCSCs, leading to disordered cell distribution during TMJ growth, as well as exaggerated the OA process in TMJ under pathological condition. In TMJ cartilage, Igf1 expression in FCSCs is critical for PI3K/Akt activation, which may be involved in regulating FCSCs self-renewal and differentiation.

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How big should this object be? Perceptual influences on viewing-size preferences When viewing objects depicted in a frame, observers prefer to view large objects like cars in larger sizes and smaller objects like cups in smaller sizes. That is, the visual size of an object that "looks best" is linked to its typical physical size in the world. Why is this the case? One intuitive possibility is that these preferences are driven by semantic knowledge: For example, when we recognize a sofa, we access our knowledge about its real-world size, and this influences what size we prefer to view the sofa within a frame. However, might visual processing play a role in this phenomenon--that is, do visual features that are related to big and small objects look better at big and small visual sizes, respectively, even when observers do not have explicit access to semantic knowledge about the objects? To test this possibility, we used "texform" images, which are synthesized versions of recognizable objects, which critically retain local perceptual texture and coarse contour information, but are no longer explicitly recognizable. To test for visual size preferences, we first used a size adjustment task, and the results were equivocal. However, clear results were obtained using a two-interval forced choice task, in which each texform was presented at the preferred visual size of its corresponding original image, and a visual size slightly bigger or smaller. Observers consistently selected the texform presented at the canonical visual size as the more aesthetically pleasing one. An additional control experiment ruled out alternative explanations related to size priming effects. These results suggest that the preferred visual size of an object depends not only on explicit knowledge of its real-world size, but also can be evoked by mid-level visual features that systematically covary with an objects real-world size.

HighlightsO_LIWe prefer to view large objects like cars large, and small objects like cups small
C_LIO_LIIntuitively, such preferences may be driven by our knowledge of object sizes
C_LIO_LIWe used unrecognizable texforms of objects that preserved mid-level visual features
C_LIO_LISimilar viewing size preferences can be revealed with these texforms
C_LIO_LISuch preferences thus arise not only from knowledge but also from visual processing
C_LI

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Cancer cell-specific MHCII expression as a determinant of the immune infiltrate organization and function in the non-small cell lung cancer tumor microenvironment IntroductionIn patients with non-small cell lung cancer (NSCLC), the prognostic significance of the tumor microenvironment (TME) immune composition has been demonstrated using single or dual-marker staining on sequential tissue sections. While these studies show that relative abundance and localization of immune cells are important parameters, deeper analyses of the NSCLC TME are necessary to refine the potential application of these findings to clinical care. Currently, the complex spatial relationships between cells of the NSCLC TME and potential drivers contributing to its immunologic composition remain unknown.

MethodsWe employed multispectral quantitative imaging on the lung adenocarcinoma TME in 153 patients with resected tumors. On a single slide per patient, we evaluated the TME with markers for CD3, CD8, CD14, CD19, major histocompatibility complex II (MHCII), cytokeratin, and DAPI. Image analysis including tissue segmentation, phenotyping, and spatial localization were performed.

ResultsSpecimens where [≥]5% of lung cancer cells expressed MHCII (MHCIIhi TME) demonstrated increased levels of CD4+ and CD8+ T cell and CD14+ cell infiltration. In the MHCIIhi TME, the immune infiltrate was closer to cancer cells and expressed an activated phenotype. Morphologic image analysis revealed cancer cells in the MHCIIhi TME more frequently interfaced with CD4+ and CD8+ T cells. Patients with an MHCIIhi TME experienced improved overall survival (p=0.046).

ConclusionsLung cancer cell-specific expression of MHCII associates with levels of immune cell infiltration, spatial localization, and activation status within the TME. This suggest cancer cell-specific expression of MHCII may represent a biomarker for the immune systems recognition and activation against the tumor.

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Interactions between Diameter, Myelination and the Na/K pump Affect Axonal Resilience to High Frequency Spiking Axons reliably conduct action potentials between neurons and/or other targets. Axons have widely variable diameters and can be myelinated or unmyelinated. Although the effect of these factors on propagation speed is well studied, how they constrain axonal resilience to high frequency spiking is incompletely understood. Maximal firing frequencies range from ~ 1 Hz to > 300 Hz across neurons, but the process by which Na/K pumps counteract Na+ influx is slow, and it is unclear the extent to which slow Na+ removal is compatible with high frequency spiking. Modeling the process of Na+ removal shows that large diameter axons are more resilient to high frequency spikes than small diameter axons, because of their slow Na+ accumulation. In myelinated axons, the myelinated compartments between nodes of Ranvier act as a  reservoir to slow Na+ accumulation and increase the reliability of axonal propagation. We now find that slowing the activation of K+ current can increase the Na+ influx rate, and the effect of minimizing the overlap between Na+- and K+-currents on spike propagation resilience depends on complex interactions among diameter, myelination and the Na/K pump density. Our results suggest that, in neurons with different channel gating kinetic parameters, different strategies may be required to improve the reliability of axonal propagation.

Significance StatementThe reliability of spike propagation in axons is determined by complex interactions among ionic currents, ion pumps and morphological properties. We use compartment-based modeling to reveal that interactions of diameter, myelination and the Na/K pump determine the reliability of high frequency spike propagation. By acting as a  reservoir of nodal Na+ influx, myelinated compartments efficiently increase propagation reliability. Although spike broadening was thought to oppose fast spiking, its effect on spike propagation is complicated, depending on the balance of Na+ channel inactivation gate recovery, Na+ influx and axial charge. Our findings suggest that slow Na+ removal influences axonal resilience to high frequency spike propagation, and that different strategies may be required to overcome this constraint in different neurons.

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Meta-analysis of tree diversity effects on the abundance, diversity and activity of herbivores' enemies O_LIThe natural enemies hypothesis predicts that the abundance and diversity of antagonists such as predators and parasitoids of herbivores increases with the diversity of plants, which can lead to more effective top-down control of insect herbivores. However, although the hypothesis has received large support in agricultural systems, fewer studies have been conducted in forest ecosystems and a comprehensive synthesis of previous research is still lacking.
C_LIO_LIWe conducted a meta-analysis of 65 publications comparing the diversity, abundance or activity of various groups of natural enemies (including birds, bats, spiders and insect parasitoids) in pure vs. mixed forest stands. We tested the effects of forest biome, natural enemy taxon and type of study (managed vs experimental forest).
C_LIO_LIWe found a significant positive effect of forest tree diversity on natural enemy abundance and diversity but not on their activity. The effect of tree diversity on natural enemies was stronger towards lower latitudes but was not contingent on the natural enemy taxon level.
C_LIO_LIOverall, our study contributes toward a better understanding of the "natural enemies hypothesis" in forest systems and provides new insights about the mechanisms involved. Furthermore, we outline potential avenues for strengthening forest resistance to the growing threat of herbivorous insects.
C_LI

sentences: The mechanism of MinD stability modulation by MinE in Min protein dynamics The patterns formed both in vivo and in vitro by the Min protein system have attracted much interest because of the complexity of their dynamic interactions given the apparent simplicity of the component parts. Despite both the experimental and theoretical attention paid to this system, the details of the biochemical interactions of MinD and MinE, the proteins responsible for the patterning, are still unclear. For example, no model consistent with the known biochemistry has yet accounted for the observed dual role of MinE in the membrane stability of MinD. Until now, a statistical comparison of models to the time course of Min protein concentrations on the membrane has not been carried out. Such an approach is a powerful way to test existing and novel models that are difficult to test using a purely experimental approach. Here, we extract time series from previously published fluorescence microscopy time lapse images of in vitro experiments and fit two previously described and one novel mathematical model to the data. We find that the novel model, which we call the Asymmetric Activation with Bridged Stability Model, fits the time-course data best. It is also consistent with known biochemistry and explains the dual MinE role via MinE-dependent membrane stability that transitions under the influence of rising MinE to membrane instability with positive feedback. Our results reveal a more complex network of interactions between MinD and MinE underlying Min-system dynamics than previously considered.

sentences: Human gaze is precisely aligned with the foveolar cone topography of both eyes The small physical depression of the human retina, the fovea, is the retinal locus of prime visual resolution, achieved by a peaking topography of the light sensitive cone photoreceptor outer segments 1-3 and a post-receptor wiring scheme preserving high-density sampling 4,5. Humans dynamically direct their gaze such that the retinal images of objects of interest fall onto the foveola, the central one-degree diameter of the fovea 6-8, but it is yet unclear if a relationship between the individual photoreceptor topography at this location and visual fixation behavior exists 9,10. By employing adaptive optics in vivo imaging and micro-stimulation 11-13, we created topographical maps of the complete foveolar cone mosaics in both eyes of 20 healthy participants while simultaneously recording the retinal location of a fixated visual object in a psychophysical experiment with cellular resolution. We found that the locus of fixation was systematically shifted away from the topographical centers towards a naso-superior quadrant on the retina, about 5 minutes of arc of visual angle on average, with a mirror symmetrical trend between fellow eyes. In cyclopean view, the topographical centers were superior to the fixated target, corresponding to areas in the visual field usually more distant 14,15 and thus containing higher spatial frequencies. Given the large variability in foveal topography between individuals, and the surprising precision with which fixation is repeatedly directed to just a small bouquet of cones in the foveola, these findings demonstrate a finely tuned, functionally relevant link between the development of the cellular mosaic of photoreceptors and visual behavior.

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Blocking antibodies against integrin-a3, integrin-aM, and integrin-aMb2 de-differentiate myofibroblasts and reverse lung and kidney fibroses in a mouse model Fibrosis is involved in 45% of deaths in the United States, and no treatment exists to reverse the progression of the disease. Myofibroblasts are key to the progression and maintenance of fibrosis. We investigated features of cell adhesion necessary for monocytes to differentiate into myofibroblasts, seeking to identify pathways key to myofibroblast differentiation. Blocking antibodies against integrins 3, M, and M{beta}2 de-differentiate myofibroblasts in vitro, lower the pro-fibrotic secretome of myofibroblasts, and reverse lung and kidney fibrosis in vivo. Decorins collagen-binding peptide directs blocking antibodies (against integrins-3, -M, -M{beta}2) to both fibrotic lungs and fibrotic kidneys, reducing the dose of antibody necessary to reverse fibrosis. This targeted immunotherapy blocking key integrins may be an effective therapeutic for the treatment and reversal of fibrosis.

SummaryBlocking antibodies against integrins-3, -M, and -M{beta}2 can be targeted to sites of fibrosis, reverse lung and kidney fibroses, and offer the potential to bring immunotherapy to fibrosis

sentences: Supergene formation is associated with a major shift in genome-wide patterns of diversity in a butterfly Selection shapes genetic diversity around target mutations, yet little is known about how selection on specific loci affects the genetic trajectories of populations, including their genome-wide patterns of diversity and demographic responses. Adaptive introgression provides a way to assess how adaptive evolution at one locus impacts whole-genome biology. Here we study the patterns of genetic variation and geographic structure in a neotropical butterfly, Heliconius numata, and its closely related allies in the so-called melpomene-silvaniform subclade. H. numata is known to have evolved a supergene via the introgression of an adaptive inversion about 2.2 million years ago, triggering a polymorphism maintained by balancing selection. This locus controls variation in wing patterns involved in mimicry associations with distinct groups of co-mimics, and butterflies show disassortative mate preferences and heterozygote advantage at this locus. We contrasted patterns of genetic diversity and structure 1) among extant polymorphic and monomorphic populations of H. numata, 2) between H. numata and its close relatives, and 3) between ancestral lineages in a phylogenetic framework. We show that H. numata populations which carry the introgressed inversions in a balanced polymorphism show markedly distinct patterns of diversity compared to all other taxa. They show the highest diversity and demographic estimates in the entire clade, as well as a remarkably low level of geographic structure and isolation by distance across the entire Amazon basin. By contrast, monomorphic populations of H. numata as well as its sister species and their ancestral lineages all show the lowest effective population sizes and genetic diversity in the clade, and higher levels of geographical structure across the continent. This suggests that the large effective population size of polymorphic populations could be a property associated with harbouring the supergene. Our results are consistent with the hypothesis that the adaptive introgression of the inversion triggered a shift from directional to balancing selection and a change in gene flow due to disassortative mating, causing a general increase in genetic diversity and the homogenisation of genomes at the continental scale.

sentences: Genetic and genomic analysis of oxygen consumption in mice We estimated genetic parameters of oxygen consumption (OC), OC per metabolic body weight (OCMBW), and body weight at three through eight weeks of age in divergently selected mice populations, with an animal model considering maternal genetic, common litter environmental, and cytoplasmic inheritance effects. Cytoplasmic inheritance was considered based on maternal lineage information. For OC, estimated direct heritability was moderate (0.32) and estimated maternal heritability and proportion of the variance of cytoplasmic inheritance effects to the phenotypic variance were very low (both <0.03), implying that causal genes for OC could be located on autosomes. To assess this hypothesis, we attempted to identify possible candidate causal genes by performing pool-seq using pooled DNA samples from mice in high and low OC lines and selective signature detection. We made a list of possible candidate causal genes for OC, including those relating to electron transport chain and ATP-binging proteins (Ndufa12, Sdhc, Atp10b, etc.), Prr16 encoding Largen protein, Cry1 encoding a key component of the circadian core oscillator, and so on. The results could contribute to elucidate the genetic mechanism of OC, an indicator for maintenance energy requirement and therefore feed efficiency.

sentences: In silico logical modelling to uncover cooperative interactions in cancer The multistep development of cancer involves the cooperation between multiple molecular lesions, as well as complex interactions between cancer cells and the surrounding tumour microenvironment. The search for these synergistic interactions using experimental models made tremendous contributions to our understanding of oncogenesis. Yet, these approaches remain labour intensive and challenging. To tackle such a hurdle, an integrative, multidisciplinary effort is required. In this article, we highlight the use of logical computational models combined to experimental validations as an effective approach to identify cooperative mechanisms and therapeutic strategies in the context of cancer biology. In silico models overcome limitations of reductionist approaches by capturing tumour complexity, and by generating powerful testable hypotheses. We review representative examples of logical models reported in the literature and their validation. We then provide further analyses of our logical model of Epithelium to Mesenchymal Transition (EMT), searching for additional cooperative interactions involving inputs from the tumour microenvironment and gain of function mutations in NOTCH.

sentences: Diurnal activity budget of Hippopotamus amphibius at Bui National Park, Ghana Animals apportion time for their daily behavioral activities. We studied the activity budget of Hippopotamus amphibius at the Black Volta River in the Bui National Park, Ghana. We performed instantaneous scan sample using ground count survey between August 2020 to July 2021. We observed that H. amphibius spent most of their day time resting (54.75 %), followed by feeding (22.93 %), walking (19.2 %) and touching (3.12 %). We found significant difference in the time spent between the daily activities (H = 41.67; p < 0.0001). Except activities involving touching, we found no significant seasonal differences in feeding, resting and walking activities by H. amphibius. Understanding the behavioral activities of H. amphibius will enhance management and conservation of the animals.

sentences: SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a novel mechanism by which the SARS-CoV-2 virus co-opts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-{beta}. We reveal that the SARS-CoV-2 encoded Non-Structural Protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein. This interaction enhances the binding of GIGYF2 to the mRNA cap-binding protein 4EHP, thereby repressing the translation of the Ifnb1 mRNA. Depletion of GIGYF2 or 4EHP significantly enhances IFN-{beta} production, leading to reduced viral infection. Our findings reveal a new target for rescuing the antiviral innate immune response to SARS-CoV-2 and other RNA viruses.

sentences: HOTARU: Automatic sorting system for large scale calcium imaging data Currently, calcium imaging allows for the long-term recording of large-scale neuronal activity in diverse states. However, it remains difficult to extract neuronal dynamics from recorded imaging data. In this study, we propose an improved CNMF-based algorithm and an effective method for extracting cell shapes with fewer false positives and false negatives caused by image processing. We also showed that the values obtained during image processing can be combined and used for false positive determination of cells. For the CNMF algorithm, we combined cell-by-cell regularization and baseline shrinkage estimation, which greatly improved its stability and robustness. We applied these methods to artificial and real data and confirmed their effectiveness. Our method is simpler and faster, detects more cells with lower firing rates and signal-to-noise ratios, and enhance the quality of the extracted cell signals. These advances can improve the standard of downstream analysis and contribute to progress in neuroscience.

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Narcolepsy and the Dissociation of REM Sleep and Cataplexy through Ambient Temperature Manipulation Narcolepsy is characterized by increased REM sleep propensity and cataplexy. Although narcolepsy is caused by the selective loss or dysfunction of hypocretin (Hcrt) neurons within the lateral hypothalamus (LH), mechanisms underlying REM sleep propensity and cataplexy remain to be elucidated. We have recently shown that wild type (WT) mice increase REM sleep expression when exposed to thermoneutral ambient temperature (Ta) warming during the light (inactive) phase. We hypothesized that the loss of Hcrt may lead to exaggerated responses with respect to increased REM sleep and cataplexy during Ta warming. To test this hypothesis, Hcrt-KO mice were implanted for chronic sleep recordings and housed in a temperature-controlled cabinet. Sleep-wake expression and both spontaneous cataplexy and food-elicited cataplexy were evaluated at constant Ta and during a Ta manipulation protocol. Here we show several unexpected findings. First, Hcrt-KO mice show opposite circadian patterns with respect to REM sleep responsiveness to thermoneutral Ta warming compared to WT mice. As previous demonstrated, WT mice increased REM sleep when Ta warming is presented during the inactive (light) phase, whereas Hcrt-KO showed a significant decrease in REM sleep expression. In contrast, Hcrt-KO mice increased REM sleep expression upon exposure to Ta warming when presented during the active (dark) phase, a circadian time when WT mice showed no significant changes in REM sleep as a function of Ta. Second, we found that REM sleep and cataplexy can be dissociated through Ta manipulation. Specifically, although Ta warming significantly increased REM sleep expression in Hcrt-KO mice during the active phase, cataplexy bout number and total cataplexy duration significantly decreased. In contrast, cataplexy expression was favoured during Ta cooling when REM sleep expression significantly decreased. Finally, video actigraphy and sleep-wake recordings in Hcrt-KO mice demonstrated that Ta manipulation did not significantly alter waking motor activity patterns or waking or NREM sleep durations. These data suggest that neural circuits gating REM sleep and cataplexy expression can be dissociated with Ta manipulation.

Statement of SignificanceCataplexy and the loss of muscle tone has historically been viewed as a component of REM sleep that inappropriately intrudes into wakefulness. In addition to fragmented sleep, the intrusion of REM-like events into wakefulness has led to the hypothesis that narcolepsy represents a dysregulation of boundary state control. We show that REM sleep and cataplexy can be dissociated during the dark (active) phase through Ta manipulation. Such dissociation may provide clues regarding the cause of boundary state instability in Hcrt-KO mice, as well as provide a new method to interrogate mechanisms of REM sleep and cataplexy.

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A unicellular walker embodies a finite state machine Cells are complex biochemical systems whose behavior emerges from interactions among myriad molecular components. Computation is often invoked as a general framework for navigating this cellular complexity. However, the manner in which cells might embody computational processes such that theories of computation, including finite state machine models, could be productively applied, remains to be seen. Here we demonstrate finite state machine-like processing embodied in cells using the walking behavior of Euplotes eurystomus, a ciliate that walks across surfaces using fourteen motile appendages (cirri). We found that cellular walking entails highly regulated transitions between a discrete set of gait states. The set of observed transitions decomposes into a small group of high-probability, temporally irreversible transitions forming a cycle and a large group of low-probability time-symmetric transitions, thus revealing stereotypy in sequential patterns of state transitions. Taken together, these findings implicate a finite state machine-like process. Cirri are connected by microtubule bundles, and we found that the dynamics of cirri involved in different state transitions are associated with the structure of the microtubule bundle system. Perturbative experiments revealed that the fibers mediate gait coordination, suggesting a mechanical basis of gait control.

One sentence summaryThe ciliate Euplotes walks across surfaces with an unusual gait, which involves modular subcellular structure and combines elements of stereotypy and variability according to a computational process actively coordinated by a microtubule fiber system.

sentences: Flexibility of Functional Neuronal Assemblies Supports Human Memory Summary ParagraphEpisodic memories, or consciously accessible memories of unique events, represent a key aspect of human cognition. Evidence from rodent models suggests that the neural representation of these complex memories requires cooperative firing of groups of neurons on short time scales1, organized by gamma oscillations2, 3. These co-firing groups, termed "neuronal assemblies,"4 represent a fundamental neurophysiological unit supporting memory. Using microelectrode data from neurosurgical patients5, we identify neuronal assemblies in the human MTL and show that they exhibit consistent organization in their firing pattern based on gamma phase information. We connect these properties to memory performance across recording sessions. Finally, we describe how human neuronal assemblies flexibly adjust6-9 over longer time scales. Our findings provide key evidence linking assemblies to human episodic memory for the first time.

sentences: A PKA Inhibitor Motif within Smoothened Controls Hedgehog Signal Transduction The Hedgehog (Hh) cascade is central to development, tissue homeostasis, and cancer. A pivotal step in Hh signal transduction is the activation of GLI transcription factors by the atypical G protein-coupled receptor (GPCR) Smoothened (SMO). How SMO activates GLI has remained unclear for decades. Here we show that SMO employs a decoy substrate sequence to physically block the active site of the PKA catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular, and organismal models, we demonstrate that interfering with SMO / PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism we uncovered echoes one utilized by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a new mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

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Modulating the selective utilization of carbon sources by engineering the 3rd and 4th helices of the DNA-binding domain of catabolite control protein A (CcpA) in Bacillus licheniformis The gram-positive bacterium Bacillus licheniformis exhibits obvious selective utilization on carbon sources. This process is mainly governed by the global regulator catabolite control protein A (CcpA), which can recognize and bind to multiple target genes widely distributed in metabolic pathways. Although the DNA-binding domain of CcpA has been predicted, the influence of key amino acids on target gene recognition and binding remains elusive. In this study, the impact of Lys31, Ile42 and Leu56 on in vitro protein-DNA interactions and in vivo carbon source selective utilization was investigated. The results showed that alanine substitution of Lys31 and Ile42, located within the 3rd helices of the DNA-binding domain, significantly weakened the binding strength between CcpA and target genes. These mutations also lead to alleviated repression of xylose utilization in the presence of glucose. On the other hand, the Leu56Arg mutant in the 4th helices exhibited enhanced binding affinity compared with that of the wild-type one. When this mutant was used to replace the native one in B. licheniformis cells, the selective utilization of glucose over xylose increased. The above research results are helpful for a deep understanding of how microorganisms can flexibly sense and adapt to changes in the external environment. Additionally, they can provide important theoretical basis for the rational design of biomass utilization and environmental adaptability of B. licheniformis cell factories.

ImportanceBacillus licheniformis is widely used in producing various valuable products, such as  enzymes, industrial chemicals and biocides. The carbon catabolite regulation process in the utilization of raw materials is crucial to maximizing the efficiency of this microbial cell factory. CcpA plays an important role in this process. This study represents a new paradigm to investigate the structure-function relationship in CcpA by fluorescence polarization experiments in vitro. The results also uncover key amino acids in the DNA-binding domain that affect the selective utilization of carbon sources. These results provide a theoretical basis for the rational design of industrial microorganisms.

sentences: Engineering Proprioceptive Implants via Surgical and Regenerative Approaches: Preliminary Interpretations The periodontal ligaments are a group of specialized connective tissue fibres with vascular and neural elements that essentially attach a tooth to the alveolar bone. Endosseous dental implant replacing a lost tooth, gets ankylosed to the alveolar bone without intervening periodontal fibres (osseointegration). Hence, proprioception, one of the most important function of periodontal ligament is not elicited by commercial dental implants currently in use. To salvage the flaw, in our proof-of-principle trial in rabbits, biodegradable nanofibres were coiled around the additive manufactured (AM) customized titanium implants. Further, human dental pulp stem cells (DPSCs), adult mesenchymal stem cells of neuro-ectodermal origin, were seeded on the nanofibrous coated, orthotopically placed 3D-printed titanium implants and were induced to differentiate into neural cell lineages. The invivo anchoring mechanism of these biodegradable neuro-supportive scaffold coated implants could probably be "proprioceptive osseointegration" instead of defaults events leading to normal "osseointegration" and could exhibit features similar to periodontium, having possible anastomosis between the severed nerve terminals present in the wall of the extraction socket relaying to/from brain and newly differentiated neural cells present in the regenerated neo-tissue complex, gradually replacing the biodegradable scaffold and may eventually results in the development of proprioceptive osseointegrated root-form endosseous dental implants in near future.

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Discovering differential genome sequence activity with interpretable and efficient deep learning Discovering sequence features that differentially direct cells to alternate fates is key to understanding both cellular development and the consequences of disease related mutations. We introduce Expected Pattern Effect and Differential Expected Pattern Effect, two black-box methods that can interpret genome regulatory sequences for cell type-specific or condition specific patterns. We show that these methods identify relevant transcription factor motifs and spacings that are predictive of cell state-specific chromatin accessibility. Finally, we integrate these methods into framework that is readily accessible to non-experts and available for download as a binary or installed via PyPI or bioconda at https://cgs.csail.mit.edu/deepaccess-package/.

Author SummaryWithin the genome are the instructions to build all the cell types that make up the human body. However, understanding these instructions and how and when these instructions go wrong in cancer or genetically inherited disease is an open problem. Deep neural networks provide powerful models to learn the relationship between DNA sequence and functional consequence across many different cell types, such as whether a particular stretch of DNA is accessible and genes in that region can be expressed or is inaccessible and therefore genes are inactive. Despite these advances, a major setback in deep learning is that it is challenging to understand what patterns of DNA sequences a deep learning model has learned to associate with a particular genomic function, whether these patterns are significant, and how to determine whether these patterns are specific to a particular cell type or are general "housekeeping" patterns that function across many cell types. We introduce Expected Pattern Effect and Differential Expected Pattern Effect, two methods which allow us to evaluate the significance of particular patterns of DNA sequence features on models trained to predict function across multiple cell types, and apply this to problems of transcription factor binding and DNA accessibility across multiple cell types.

sentences: Reduced infectivity but increased immune escape of the new SARS-CoV-2 variant of concern Omicron A new detected SARS-CoV-2 variant Omicron (B.1.1.529) had reported from more than 80 countries. In the past few weeks, a new wave of infection driven by Omicron is in progress. Omicron Spike (S) protein pseudotyped virus was used to determine the effect of S mutations on its capacity of infectivity and immune evasion. Our results showed the lower entry efficiency and less cleavage ability of Omicron than D614G variant. Pseudotype-based neutralizing assay was performed to analyze neutralizing antibodies elicited by previously infection or the RBD-based protein subunit vaccine ZF2001 against the Omicron variant. Sera sampled at around one month after symptom onset from 12 convalescents who were previously infected by SARS-CoV-2 original strain shows a more than 20-fold decrease of neutralizing activity against Omicron variant, when compared to D614G variant. Among 12 individuals vaccinated by RBD subunit vaccine, 58.3% (7/12) sera sampled at 15-60 days after 3rd-dose vaccination did not neutralize Omicron. Geometric mean titers (GMTs, 50% inhibitory dose [ID50]) of these sera against Omicron were 9.4-fold lower than against D614G. These results suggested a higher risk of Omicron breakthrough infections and reduced efficiency of the protective immunity elicited by existing vaccines. There are important implications about the modification and optimization of the current epidemic prevention and control including vaccine strategies and therapeutic antibodies against Omicron variant.

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Integrin signaling via actin cytoskeleton activates MRTF/SRF to entrain circadian clock The circadian clock is entrained to daily environmental cues. Integrin-linked intracellular signaling via actin cytoskeleton dynamics transduces cellular niche signals to induce Myocardin-related Transcription Factor (MRTF)/Serum Response Factor (SRF)-mediated transcription. So far, how the integrin-associated signaling cascade may transmit cellular physical cues to entrain circadian clock remains to be defined. Using combined pharmacological and genetic approaches, here we show that the transcription factors mediating integrin to actin cytoskeleton signaling, MRTF-A and SRF, exert direct transcriptional control of core clock components, and that this signaling cascade modulates key properties of clock circadian activity. Pharmacological inhibition of MRTF/SRF activity by disrupting actin polymerization significantly augmented clock amplitude with period shortening, whereas an actin polymerizing compound attenuated oscillation amplitude with period lengthening. Genetic loss-of-function of Srf or Mrtf mimics that of actin-depolymerizing agents, validating the role of actin dynamics in driving clock function. Furthermore, integrin-mediated focal adhesion with extracellular matrix and its downstream signaling modulates the circadian clock, as blockade of integrin, focal adhesion kinase or Rho-associated kinase (ROCK) increased clock amplitude and shortened period length. Mechanistically, we identify specific core clock transcription regulators, Per1, Per2 and Nr1d1, as direct target genes of MRTF-A/SRF. Collectively, our findings uncovered an integrin-actin cytoskeleton-MRTF/SRF signaling cascade in linking clock entrainment to its extracellular microenvironment, which may mediate cellular adaptation to its physical niche.

Author SummaryThe circadian clock anticipates and adapts to environmental changes. Interestingly, serum, as a universal clock synchronizing signal, drives intracellular actin cytoskeleton reorganization through modulation of MRTF/SRF activity. However, mechanisms that may transduce extracellular niche signals to circadian clock remains to be defined. We hypothesize that integrin-mediated intracellular signaling to actin cytoskeleton links extracellular microenvironment with MRTF/SRF transcriptional regulation to control clock function. Using small molecules and genetic approaches targeting distinct steps of integrin-actin cytoskeleton-MRTF/SRF signaling cascade, we uncover the effects of this pathway in controlling circadian clock oscillation. We also identify specific core clock regulators as direct gene target genes of MRTF and SRF-mediated transcriptional control. Our study revealed how integrin-mediated cellular interaction with its physical environment influences its intrinsic clock properties through signaling transduction via actin cytoskeleton remodeling, and that this mechanism may facilitate circadian clock adaptation to cellular physical niche.

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Distinct and common features of numerical and structural chromosomal instability across different cancer types A large proportion of tumours is characterised by numerical or structural chromosomal instability (CIN), defined as an increased rate of gaining or losing whole chromosomes (W-CIN) or of accumulating structural aberrations (S-CIN). Both W-CIN and S-CIN are associated with tumourigenesis, cancer progression, treatment resistance and clinical outcome. Although W-CIN and S-CIN can co-occur, they are initiated by different molecular events. By analysing tumour genomic data from 33 cancer types, we show that the majority of tumours with high levels of W-CIN underwent whole genome doubling, whereas S-CIN levels are strongly associated with homologous recombination deficiency. Both CIN phenotypes are prognostic in several cancer types. Most drugs are less efficient in high-CIN cell lines, but we also report compounds and drugs which should be investigated as targets for W-CIN or S-CIN. By analysing associations between CIN and bio-molecular entities with pathway and gene expression levels, we complement gene signatures of CIN and report that the drug resistance gene CKS1B is strongly associated with S-CIN. Finally, we propose a potential copy number-dependent mechanism to activate the PI3K pathway in high-S-CIN tumours.

1. Simple summaryMany cancer cells are chromosomally unstable, a phenotype describing a tendency for accumulating chromosomal aberrations. Entire chromosomes tend to be gained or lost, which is called whole chromosome instability (W-CIN). Structural chromosomal instability (S-CIN) describes an increased rate of gaining, losing or translocating smaller parts of chromosomes. Here, we analyse data from 33 cancer types to find differences and commonalities between W-CIN and S-CIN. We find that W-CIN is strongly linked to whole genome doubling (WGD), whereas S-CIN is associated with a specific DNA damage repair pathway. Both W-CIN and S-CIN are difficult to target using currently available compounds and have distinct prognostic values. The activity of the drug resistance gene CKS1B is associated with S-CIN, which merits further investigation. In addition, we identify a potential copy number-based mechanism promoting signalling of the important PI3K cancer pathway in high-S-CIN tumours.

sentences: Dynamical interactions reconfigure the gradient of cortical timescales A hierarchy of local timescales with a back (sensory)-to-front (prefrontal) gradient reflects brain region specialization. However, cognitive processes emerge from the coordinated activity across regions, and the corresponding timescales should refer to the interactions rather than to regional activity. Using edgewise connectivity on magnetoencephalography signals, we demonstrate a reverse front-to-back gradient when non-local interactions are prominent. Thus, the timescales are dynamic and reconfigure between back-to-front and front-to-back patterns.

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A new drug candidate for glycogen storage disorders enhances glycogen catabolism: Lessons from Adult Polyglucosan Body Disease models This work employs Adult Polyglucosan Body Disease (APBD) models to explore the efficacy and mechanism of action of 144DG11, a new polyglucosan-reducing lead compound discovered by a high-throughput screen (HTS). APBD is an adult onset glycogen storage disorder (GSD) manifesting as a debilitating progressive axonopathic leukodystrophy. APBD is caused by glycogen branching enzyme (GBE) deficiency leading to poorly branched and insoluble glycogen inclusions, which precipitate as neuropathogenic polyglucosans (PG). 144DG11 led to prolonged survival and improved motor parameters in a GBE knockin (Gbeys/ys) APBD mouse model. Histopathologically, 144DG11 reduced PG and glycogen levels in brain, liver, heart, and peripheral nerve. Indirect calorimetry experiments revealed that 144DG11 increases carbohydrate burn at the expense of fat burn, suggesting metabolic mobilization of pathogenic PG. These results were also reflected at the cellular level by increased glycolytic, mitochondrial and total ATP production. Mechanistically, we show that the molecular target of 144DG11 is the lysosomal membrane protein LAMP1, whose interaction with the compound, similar to LAMP1 knockdown, enhanced autolysosomal degradation of glycogen and lysosomal acidification. Enhanced mitochondrial activity and lysosomal modifications were also the most pronounced effects of 144DG11 in APBD patient fibroblasts as discovered by image-based multiparametric phenotyping analysis and corroborated by proteomics. In summary, this work presents a broad mechanistic and target-based characterization of 144DG11 in in vivo and cell models of the prototypical GSD APBD. This investigation warrants development of 144DG11 into a safe and efficacious GSD therapy.

One Sentence SummaryA new compound, demonstrated to ameliorate APBD in vivo and ex vivo by autophagic catabolism of glycogen, may potentially become a universal drug for glycogen storage disorders.

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Control of Ca2+ signals by astrocyte nanoscale morphology at tripartite synapses Much of the Ca2+ activity in astrocytes is spatially restricted to microdomains and occurs in fine processes that form a complex anatomical meshwork, the so-called spongiform domain. A growing body of literature indicates that those astrocytic Ca2+ signals can influence the activity of neuronal synapses and thus tune the flow of information through neuronal circuits. Because of technical difficulties in accessing the small spatial scale involved, the role of astrocyte morphology on Ca2+ microdomain activity remains poorly understood. Here, we use computational tools and idealized 3D geometries of fine processes based on recent super-resolution microscopy data to investigate the mechanistic link between astrocytic nanoscale morphology and local Ca2+ activity. Simulations demonstrate that the nano-morphology of astrocytic processes powerfully shapes the spatio-temporal properties of Ca2+ signals and promotes local Ca2+ activity. The model predicts that this effect is attenuated upon astrocytic swelling, hallmark of brain diseases, which we confirm experimentally in hypo-osmotic conditions. Upon repeated neurotransmitter release events, the model predicts that swelling hinders astrocytic signal propagation. Overall, this study highlights the influence of the complex morphology of astrocytes at the nanoscale and its remodeling in pathological conditions on neuron-astrocyte communication at so-called tripartite synapses, where astrocytic processes come into close contact with pre- and postsynaptic structures.

Table of ContentsO_ST_ABSMain PointsC_ST_ABSO_LIAstrocyte nano-morphology favors the compartmentalization of biochemical signals
C_LIO_LIThis compartmentalization promotes local Ca2+ activity and signal propagation robustness
C_LIO_LIIn contrast, its pathological remodeling upon swelling attenuates Ca2+ activity
C_LI

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Our simulation results demonstrate that the nano-morphology of astrocytic processes, consisting in the alternation of nodes and shafts, favors the compartmentalization of biochemical signals. This compartmentalization promotes local Ca2+ activity and signal propagation robustness. Astrocyte swelling, observed in pathological conditions such as brain injury, stroke and epilepsy, results in an increased shaft width without altering node size. Our results suggest that such pathological alterations of the nanoscale morphology of astrocytes result in a decreased local Ca2+ activity, which we confirm experimentally in hypo-osmotic conditions. Upon repeated neuronal stimuli, we predict that swelling hinders astrocytic signal propagation. Overall, this study highlights the impact of astrocyte nano-morphology on astrocyte activity at tripartite synapses, in health and disease.

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Feasibility of an Inhalable Ultrasound Contrast Agent to Enhance Airway Imaging IntroductionUltrasound is a relatively inexpensive and non-ionizing imaging modality, but is under-utilized in large airway assessments due to poor image quality. No commercially available contrast agents currently exist for sonographic evaluation of the respiratory system, nor has a respiratory route of microbubble contrast agent (MCA) administration been previously described for the enhancement of airway imaging.

MethodsWe conducted a feasibility study to assess proof-of-concept for an inhalable ultrasound MCA composed of lipid-encapsulated decaflourobutane gas. The MCA was nebulized and administered as an aerosol through the lumen of an ex vivo porcine trachea, with image enhancement evaluated by comparing images pre- and post-exposure. Additionally, primary human bronchial epithelial (hBE) cells from three donors were differentiated at an air-liquid interface and exposed apically to 25 L of undiluted MCA or vehicle control to assess contrast agent-induced cytotoxicity and inflammation. Basolateral medium was collected 24-hours post-exposure and lactate dehydrogenase (LDH) and interleukin-8 (IL-8) concentrations were measured as biomarkers of cytotoxicity and inflammation, respectively.

ResultsContrast microbubbles remained intact following nebulization and enhanced sonographic delineation of ex vivo porcine tracheal walls, indicating adherence of the nebulized MCA to the lumenal mucosa. No significant cytotoxic or inflammatory effects were observed in cultured hBE cells following exposure to MCA.

ConclusionsWe present proof-of-concept for an inhaled MCA for the enhancement of sonographic evaluations of the large airways. Pending further evaluations for safety and effectiveness, inhaled MCA may be feasible for clinical ultrasound applications, such as enhancing ultrasound-guided tracheal intubation, detecting airway bleeds, or monitoring large airway diseases in pediatric populations.

sentences: Development of the myzozoan aquatic parasite Perkinsus marinus as a versatile experimental genetic model organism The phylum Perkinsozoa is an aquatic parasite lineage that has devastating effects on commercial and natural mollusc populations, and also comprises parasites of algae, fish and amphibians. They are related to, and share much of their biology with, dinoflagellates and apicomplexans and thus offer excellent genetic models for both parasitological and evolutionary studies. Genetic transformation has been previously achieved for select Perkinsus spp. but with few tools for transgene expression and only limited selection efficacy. We thus sought to expand the power of experimental genetic tools for Perkinsus marinus -- the principal perkinsozoan model to date. We constructed a modular plasmid assembly system that enables expression of multiple genes simultaneously. We developed an efficient selection system for three drugs, puromycin, bleomycin and blasticidin, that achieves transformed cell populations in as little as three weeks. We developed and quantified eleven new promoters of variable expression strength. Furthermore, we identified that genomic integration of transgenes is predominantly via non-homologous recombination and often involves transgene fragmentation including deletion of some introduced elements. To counter these dynamic processes, we show that bi-cistronic transcripts using the viral 2A peptides can couple selection systems to the maintenance of the expression of a transgene of interest. Collectively, these new tools and insights provide new capacity to efficiently genetically modify and study Perkinsus as an aquatic parasite and evolutionary model.

sentences: Zebrafish cornea formation and homeostasis reflect robustness in camera-type eye biology, regardless of its environment The cornea is the transparent tissue covering the camera-type eyes. This position makes this tissue prone to environmental aggressions, and physical alteration can have a drastic negative impact on sight. The current knowledge on zebrafish corneal physiology is too scarce to get an understanding on its strategy to maintain a clear sight in aquatic environment. In this study, we focus on corneal formation and maturation in zebrafish. After describing the morphological changes taking place from 3 days post fertilization to adulthood, we analyzed cell proliferation. We show that label retaining cells appear just before 1 month of age. Our cell proliferation study, combined to the study of Pax6a and krtt1c19e expression, demonstrate a long maturation process. This process ends with a solid patterning of corneal innervation. Finally, corneal abrasion wound healing process recapitulates the maturation process, via a plasticity period. Altogether, our study decipher the maturation steps of an aquatic cornea. These findings will facilitate the use of zebrafish as model of corneal physiology studies.

sentences: Micro-Meta App: an interactive software tool to facilitate the collection of microscopy metadata based on community-driven specifications For the information content of microscopy images to be appropriately interpreted, reproduced, and meet FAIR (Findable Accessible Interoperable and Reusable) principles, they should be accompanied by detailed descriptions of microscope hardware, image acquisition settings, image pixel and dimensional structure, and instrument performance. Nonetheless, the thorough documentation of imaging experiments is significantly impaired by the lack of community-sanctioned easy-to-use software tools to facilitate the extraction and collection of relevant microscopy metadata. Here we present Micro-Meta App, an intuitive open-source software designed to tackle these issues that was developed in the context of nascent global bioimaging community organizations, including BioImaging North America (BINA) and QUAlity Assessment and REProducibility in Light Microscopy (QUAREP-LiMi), whose goal is to improve reproducibility, data quality and sharing value for imaging experiments. The App provides a user-friendly interface for building comprehensive descriptions of the conditions utilized to produce individual microscopy datasets as specified by the recently proposed 4DN-BINA-OME tiered-system of Microscopy Metadata model. To achieve this goal the App provides a visual guide for a microscope-user to: 1) interactively build diagrammatic representations of hardware configurations of given microscopes that can be easily reused and shared with colleagues needing to document similar instruments. 2) Automatically extracts relevant metadata from image files and facilitates the collection of missing image acquisition settings and calibration metrics associated with a given experiment. 3) Output all collected Microscopy Metadata to interoperable files that can be used for documenting imaging experiments and shared with the community. In addition to significantly lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training users that have limited knowledge of the intricacies of light microscopy experiments. To ensure wide-adoption by microscope-users with different needs Micro-Meta App closely interoperates with MethodsJ2 and OMERO.mde, two complementary tools described in parallel manuscripts.

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Inhibitors of the Small Membrane (M) Protein Viroporin Prevent Zika Virus Infection Flaviviruses, including Zika virus (ZIKV), are a significant global health concern, yet no licensed antivirals exist to treat disease. The small M (Membrane) protein plays well-defined roles during viral egress, yet remains within virion membranes following release and maturation. However, it is unclear whether M plays a functional role in this setting. Here, we show that M forms oligomeric membrane-permeabilising channels in vitro, with increased activity at acidic pH and sensitivity to the prototypic channel-blocker, rimantadine. In turn, rimantadine blocked an early stage of ZIKV cell culture infection.

Molecular dynamics (MD) generated rationalised structure-based channel models, comprising hexameric arrangements of dual trans-membrane protomers. Interestingly, His28 protonation increased channel opening, consistent with activation within acidifying endosomes. Models contained a predicted lumenal binding site for rimantadine, as well as a second target region on the membrane-exposed periphery. In silico screening enriched for repurposed drugs/compounds predicted to bind to one or other site. Multiple hits displayed potency in vitro and in cell culture, supporting the relevance of channel models. Finally, rimantadine effectively blocked ZIKV viraemia in a preclinical model, supporting that M constitutes a physiologically relevant antiviral target, for either repurposing rimantadine, or the development of new ZIKV therapies.

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Analysis of single-cell RNA-sequencing data to identify quiescent and proliferating neural cell populations in Glioblastoma BackgroundDiffuse Glioblastoma (GBM) has high mortality and remains one of the most challenging type of cancer to treat. Identifying and characterizing the cells populations driving tumor growth and therapy resistance has been particularly difficult owing to marked inter and intra tumoral heterogeneity observed in these tumors. These tumorigenic populations contain long lived cells associated with latency, immune evasion and metastasis.

MethodsHere, we analyzed the single-cell RNA-sequencing data of high grade glioblastomas from four different studies using integrated analysis of gene expression patterns, cell cycle stages and copy number variation to identify gene expression signatures associated with quiescent and cycling neuronal tumorigenic cells.

ResultsThe results show that while cycling and quiescent cells are present in GBM of all age groups, they exist in a much larger proportion in pediatric glioblastomas. These cells show similarities in their expression patterns of a number of pluripotency and proliferation related genes. Upon unbiased clustering, these cells explicitly clustered on their cell cycle stage. Quiescent cells in both the groups specifically overexpressed a number of genes for ribosomal protein, while the cycling cells were enriched in the expression of high-mobility group and heterogeneous nuclear ribonucleoprotein group genes. A number of well-known markers of quiescence and proliferation in neurogenesis showed preferential expression in the quiescent and cycling populations identified in our analysis. Through our analysis, we identify ribosomal proteins as key constituents of quiescence in glioblastoma stem cells.

ConclusionsThis study identifies gene signatures common to adult and pediatric glioblastoma quiescent and cycling stem cell niches. Further research elucidating their role in controlling quiescence and proliferation in tumorigenic cells in high grade glioblastoma will open avenues in more effective treatment strategies for glioblastoma patients.

sentences: Computational analysis of vertebrate myoglobins reveals aggregation resistance in aquatic birds and higher surface hydrophobicity in fish Myoglobin is the major oxygen carrying protein of vertebrate muscle, and high myoglobin net charge is known to hold evolutionary significance as a molecular signature of secondarily aquatic diving capacity in mammals. However, the evolution of myoglobins electrostatic properties in non-mammalian vertebrates, such as birds, has not been investigated. Here, we used a new deep learning-based protein folding algorithm to model the tertiary structures of myoglobin from 302 vertebrate species and performed a comparative analysis of their net charge, positively charged solvent-accessible surface area, and negatively charged solvent-accessible surface area. For avian myoglobins, we also calculated selection pressure ({omega}). The results suggest that the myoglobins of diving avians, specifically those of the penguins (Sphenisciformes) and diving ducks (Aythyini), have highly positively charged electrostatic surfaces, which evolved via positive selection to reduce aggregation propensity and allow greater storage of oxygen for extended underwater foraging. In contrast, galliform myoglobins are under high purifying selection. Distribution of charged atoms on myoglobin surface was more indicative of high myoglobin content than net charge. We also found inter-class differences in net charge; bird myoglobins are the most positively charged and reptile and amphibian myoglobins are the most negatively charged, and net charge seems to be negatively associated with herbivory within mammals. Finally, we propose an equation that describes the relationship between myoglobin net charge and concentration better than the previously suggested logarithmic function. Our findings offer novel insights into the diversification of myoglobin in vertebrate clades and highlight the power of computational structural approaches for zoological and evolutionary research.

sentences: Non-invasive imaging of gene expression and protein secretion dynamics in living mice uncovers ectopic prothrombin expression as driver of thrombosis in cancer The topology of gene expression and protein localization is a crucial characteristic of life, where the spatiotemporal dynamic of secretory proteins instruct higher order organization, including the orchestration of developmental and adaptive programs. However tools to non-invasively interrogate the fate of secretory proteins in vivo are scarce. Here we introduce a genetic tagging strategy for in vivo imaging of the secretion and expression dynamics of secretory proteins in living animals. Applying this to a prototypical liver-derived secretory protein, we demonstrate that this approach, combined with optical in-vivo imaging, uncovers extrahepatic prothrombin expression in multiple novel anatomical sites (including testes, placenta, brain, kidney, heart and lymphatic system) and in emerging tumors, resulting in significant amounts of tumor-derived prothrombin in the blood with procoagulant properties. Syngeneic cell lines from this mouse model enable unravelling regulatory mechanisms in high resolution, and in a scalable format ex vivo. Beyond discovering new functions of proteins in a targeted manner, this model allows identifying rheostats in the cross-talk between gene expression and availability of a secretory protein. It is also a valuable resource for uncovering novel (tissue-specific) therapeutic vulnerabilities.

sentences: Myocardial BDNF regulates cardiac bioenergetics through the transcription factor Yin Yang 1 Circulating Brain-derived Neurotrophic Factor (BDNF) is markedly decreased in heart failure patients. Both BDNF and its receptor, Tropomyosin Related Kinase Receptor (TrkB), are expressed in cardiomyocytes, however the role of myocardial BDNF signaling in cardiac pathophysiology is poorly understood. We found that cardiac-specific TrkB knockout (cTrkB KO) mice displayed a blunted adaptive cardiac response to exercise, with attenuated upregulation of transcription factor networks controlling mitochondrial biogenesis/metabolism, including Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1). The cTrkB KO mice developed an exacerbated heart failure progression with transaortic constriction. The downregulation of PGC-1 in cTrkB KO mice exposed to exercise or TAC resulted in decreased cardiac energetics. We further unraveled that BDNF induces PGC-1 upregulation and bioenergetics through a novel signaling pathway, the pleiotropic transcription factor Yin Yang 1 (YY1). Taken together, our findings suggest that myocardial BDNF plays a critical role in regulating cellular energetics in the cardiac stress response.

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Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19 Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS-CoV-2 infection is critical for developing treatments for severe COVID-19. Here we show that in COVID-19 patients, circulating plasmacytoid dendritic cells (pDCs) decline early after symptom onset and this correlated with COVID-19 disease severity. This transient depletion coincides with decreased expression of antiviral type I IFN and the systemic inflammatory cytokines CXCL10 and IL-6. Importantly, COVID-19 disease severity correlated with decreased pDC frequency in peripheral blood. Using an in vitro stem cell-based human pDC model, we demonstrate that pDCs directly sense SARS-CoV-2 and in response produce multiple antiviral (IFN and IFN{lambda}1) and inflammatory (IL-6, IL-8, CXCL10) cytokines. This immune response is sufficient to protect epithelial cells from de novo SARS-CoV-2 infection. Targeted deletion of specific sensing pathways identified TLR7-MyD88 signaling as being crucial for production of the antiviral IFNs, whereas TLR2 is responsible for the inflammatory IL-6 response. Surprisingly, we found that SARS-CoV-2 engages the neuropilin-1 receptor on pDCs to mitigate the antiviral IFNs but not the IL-6 response. These results demonstrate distinct sensing pathways used by pDCs to elicit antiviral vs. immunopathological responses to SARS-CoV-2 and suggest that targeting neuropilin-1 on pDCs may be clinically relevant for mounting TLR7-mediated antiviral protection.

One Sentence SummarypDCs sense SARS-CoV-2 and elicit antiviral protection of lung epithelial cells through TLR7, while recognition of TLR2 elicits an IL-6 inflammatory response associated with immunopathology.

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SARS-CoV-2 is internalized by pDCs via a yet unknown endocytic mechanism. The intracellular TLR7 sensor detects viral RNA and induces a signaling cascade involving MyD88-IRAK4-TRAF6 (1) to induce CXCL10 and, via IRF7 phosphorylation and translocation, inducing type I and III Interferons (2). Once secreted, type I and III IFNs initiate autocrine and paracrine signals that induce the expression of IFN stimulated genes (ISGs), thereby facilitating an antiviral response that can protect the cell against infection. However, SARS-CoV-2, has the intrinsic property to facilitate CD304 signaling, potentially by interfering with IRF7 nuclear translocation, thereby inhibiting type I IFN production and thus reducing the antiviral response generated by the pDC (4). Furthermore, the SARS-CoV-2 envelope (E) glycoprotein is sensed by the extracellular TLR2/6 heterodimer and this facilitates production of the inflammatory IL-6 cytokine (5). Illustration was created with BioRender.com

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sentences: A novel approach to the functional classification of retinal ganglion cells Retinal neurons are remarkedly diverse based on structure, function and genetic identity. Classifying these cells is a challenging task, requiring multimodal methodology. Here, we introduce a novel approach for retinal ganglion cell (RGC) classification, based on pharmacogenetics combined with immunohistochemistry and large-scale retinal electrophysiology. Our novel strategy allows grouping of cells sharing gene expression and understanding how these cell classes respond to basic and complex visual scenes. Our approach consists of several consecutive steps. First, the spike firing frequency is increased in RGCs co-expressing a certain gene (Scnn1a or Grik4) using excitatory DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) in order to single out activity originating specifically from these cells. Their spike location is then combined with post hoc immunostaining, to unequivocally characterize their anatomical and functional features. We grouped these isolated RGCs into multiple clusters based on spike train similarities. Using this novel approach. we were able to extend the pre-existing list of Grik4 expressing RGC types to a total of 8 and, for the first time, we provide a phenotypical description of 13 Scnn1a-expressing RGCs. The insights and methods gained here can guide not only RGC classification but neuronal classification challenges in other brain regions as well.

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Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAM, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals.

We have characterized and optimized the activity of SpG and SpRY in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to efficient mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm can predict SpG and SpRY activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.

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TMT-based proteomic analysis of the antibiotic effects of ShangKeHuangShui against Straphylococcus aureus. Post-traumatic infection is a serious orthopedic trauma complication commonly caused by Staphylococcus aureus (SAU). ShangKeHuangShui, also known as Yellow Aqueous Concentrate of Traumatology Herbs (YACTH), is prepared from six Chinese herbal medicines, which has been used for decades in our hospital to prevent post-traumatic infection.

In the present study, we investigated the in vitro antibacterial effects and underlying mechanism of YACTH against SAU. YACTH exhibited significant antibacterial activity against SAU with the minimum inhibitory concentration (MIC) of 8.625 mg/mL. Proteomic analysis based on Tandem mass tag (TMT) showed different protein expression levels in SAU under the YACTH and control conditions. Compared to the control group, the expression level of 490 proteins of YACTH treated group significantly changed (>1.2 fold, P <0.05). Biological informatics analysis showed that these differential proteins were widely involved in a variety of biological processes and multiple metabolic pathways. We then selected 26 target proteins to further conduct Parallel reaction monitoring (PRM). The results revealed that the 9 down-regulated proteins were mainly involved in RNA polymerase, oxidative phosphorylation, ABC transporter and glycolysis; whereas, the 17 up-regulated proteins are related to RNA degradation, mismatch repair and ribosome.

In conclusion, YACTH, which contains the water-soluble components of six Chinese herbs, is first reported to exhibit antibacterial activity against SAU in vitro. The results provide novel insights into the antibacterial mechanisms of YACTH on protein networks, which may help us find new potential antibiotic targets.

sentences: Canonical Wnt Signaling and the Regulation of Divergent Mesenchymal Fgf8 expression in Axolotl Limb Development and Regeneration The expression of Fibroblast growth factors (Fgf) ligands in a specialized epithelial compartment, the Apical Ectodermal Ridge (AER), is a conserved feature of limb development across vertebrate species. In vertebrates, Fgf 4, 8, 9, and 17 are all expressed in the AER. An exception to this paradigm is the salamander (axolotl) developing and regenerating limb, where key Fgf ligands are expressed in the mesenchyme. The mesenchymal expression of Amex.Fgf8 in axolotl has been suggested to be critical for regeneration. To date, there is little knowledge regarding what controls Amex.Fgf8 expression in the axolotl limb mesenchyme. A large body of mouse and chick studies have defined a set of transcription factors and canonical Wnt signaling as the main regulators of epidermal Fgf8 expression in these organisms. In this study, we address the hypothesis that alterations to one or more of these components during evolution has resulted in mesenchymal Amex.Fgf8 expression in the axolotl. To sensitively quantify gene expression with spatial precision, we combined optical clearing of whole-mount axolotl limb tissue with single molecule fluorescent in situ hybridization and a semi-automated quantification pipeline. Several candidate upstream components were found expressed in the axolotl ectoderm, indicating that they are not direct regulators of Amex.Fgf8 expression. We found that Amex.Wnt3a is expressed in axolotl limb epidermis, similarly to chicken and mouse. However, unlike in amniotes, Wnt target genes are activated preferentially in limb mesenchyme rather than in epidermis. Inhibition and activation of Wnt signaling results in downregulation and upregulation of mesenchymal Amex.Fgf8 expression respectively. These results implicate a shift in tissue responsiveness to canonical Wnt signaling from epidermis to mesenchyme as one step contributing to the unique mesenchymal Amex.Fgf8 expression seen in the axolotl.

sentences: The role of vimentin-nuclear interactions in persistent cell motility through confined spaces The ability of cells to move through small spaces depends on the mechanical properties of the cellular cytoskeleton and on nuclear deformability. In mammalian cells, the cytoskeleton is comprised of three interacting, semi-flexible polymer networks: actin, microtubules, and intermediate filaments (IF). Recent experiments of mouse embryonic fibroblasts with and without vimentin have shown that the IF vimentin plays a role in confined cell motility. We, therefore, develop a minimal model of cells moving through confined geometries that effectively includes all three types of cytoskeletal filaments with a cell consisting of an actomyosin cortex and a deformable cell nucleus and mechanical connections between the two cortices--the outer actomyosin one and the inner nuclear one. By decreasing the amount of vimentin, we find that the cell speed is typically faster for vimentin-null cells as compared to cells with vimentin. Vimentin-null cells also contain more deformed nuclei in confinement. Finally, vimentin affects nucleus positioning within the cell. By positing that as the nucleus position deviates further from the center of mass of the cell, microtubules become more oriented in a particular direction to enhance cell persistence or polarity, we show that vimentin-nulls are more persistent than vimentin-full cells. The enhanced persistence indicates that the vimentin-null cells are more subjugated by the confinement since their internal polarization mechanism that depends on cross-talk of the centrosome with the nucleus and other cytoskeletal connections is diminished. In other words, the vimentin-null cells rely more heavily on external cues. Our modeling results present a quantitative interpretation for recent experiments and have implications for understanding the role of vimentin in the epithelial-mesenchymal transition.

sentences: A fijivirus major viroplasm protein shows RNA-stimulated ATPase activity by adopting pentameric and hexameric assemblies of dimers The fijivirus Mal de Rio Cuarto virus (MRCV) causes a devastating maize disease. Its non-structural protein P9-1, which shows ATPase and RNA binding activities, is the major component of the intracellular viroplasm where virus replication takes place. Here, we established that the 24 C-terminal residues (C-arm) of P9-1 are required for the formation of viroplasm-like structures (VLS) in vivo and for the protein multimerization in vitro. Employing an integrative structural approach, we found that the C-arm is dispensable for P9-1 dimer assembly, but essential for the formation of doughnut-shaped pentamers and hexamers of dimers (decamers and dodecamers). Both assemblies, larger than those reported for other reoviruses, contain disordered loops oriented towards the inner pore of the structures, where RNA binding sites and conditional proteasome-mediated degradation signals (PEST) were predicted. In vitro assays demonstrated that ssRNA binding is favored towards P9-1 (do)decamers over the dimeric {Delta}C-arm version. In addition, although both P9-1 and P9-1{Delta}C-arm catalyze the hydrolysis of ATP with similar activity values, an RNA-stimulated ATPase activity was only observed in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. Computational studies revealed a stronger preference of phosphate moieties to the decamer in the pore and the C-arm regions, suggesting that the allosteric communication between the ATP and RNA binding sites is favored with this protein arrangement. Overall, our work reveals the structural versatility of a major viroplasm protein providing unprecedented insights into fijivirus viroplasm assembly and function and establishes the structural basis for the development of antiviral strategies against the Mal de Rio Cuarto crop disease.

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Pluripotency-Independent Induction of Human Trophoblast Stem Cells from Fibroblasts Recent studies demonstrated that human trophoblast stem-like cells (hTS-like cells) can be derived from naive embryonic stem cells or be induced from somatic cells by the pluripotency factors, OSKM. This raises two main questions; (i) whether human induced TSCs (hiTSCs) can be generated independently to pluripotent state or factors and (ii) what are the mechanisms by which hTSC state is established during reprogramming. Here, we identify GATA3, OCT4, KLF4 and MYC (GOKM) as a pluripotency-independent combination of factors that can generate stable and functional hiTSCs, from both male and female fibroblasts. By using single and double knockout (KO) fibroblasts for major pluripotency genes (i.e. SOX2 or NANOG/PRDM14) we show that GOKM not only is capable of generating hiTSCs from the KO cells, but rather that the efficiency of the process is increased. Through H3K4me2 and chromatin accessibility profiling we demonstrate that GOKM target different loci and genes than OSKM, and that a significant fraction of them is related to placenta and trophoblast function. Moreover, we show that GOKM exert a greater pioneer activity compared to OSKM. While GOKM target many specific hTSC loci, OSKM mainly target hTSC loci that are shared with hESCs. Finally, we reveal a gene signature of trophoblast-related genes, consisting of 172 genes which are highly expressed in blastocyst-derived TSCs and GOKM-hiTSCs but absent or mildly expressed in OSKM-hiTSCs.

Taken together, these results imply that not only is the pluripotent state, and SOX2 specifically, not required to produce functional hiTSCs, but that pluripotency-specific factors actually interfere with the acquisition of the hTSC state during reprogramming.

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Single-cell RNA profiling of Plasmodium vivax liver stages reveals parasite- and host- specific transcriptomic signatures and drug targets The resilience of Plasmodium vivax, the most widely distributed malaria-causing parasite in humans worldwide, is attributed to its ability to produce dormant liver forms known as hypnozoites, which can activate weeks, months, or even years after an initial mosquito bite. The factors underlying hypnozoite formation and activation are poorly understood, as is the parasites influence on the host hepatocyte. Here, we shed light on transcriptome-wide signatures of both the parasite and the infected host cell by sequencing over 1,000 P. vivax liver forms at single-cell resolution. We distinguish between replicating schizonts and hypnozoites at the transcriptional level, identifying key differences in transcripts encoding for RNA-binding proteins associated with cell fate. In infected hepatocytes, we show that genes associated with energy metabolism and antioxidant stress response were upregulated, and those involved in the host immune response downregulated, suggesting both schizonts and hypnozoites alter the host intracellular environment. The transcriptional markers in schizonts, hypnozoites, and infected hepatocytes revealed here pinpoint potential factors underlying dormancy and can inform drug targets against P. vivax liver-stage infection.

IMPORTANCEIn humans, malaria-causing Plasmodium parasites invade and replicate in the liver before mounting an infection in the blood. A few Plasmodium species, including P. vivax, can cause relapsing infections due to forms that persist in the liver known as hypnozoites. While interventions targeting the hypnozoite can prevent clinical malaria, little is known about their biology at the molecular level. Transcriptome-wide assessment of individual hypnozoites has not been accessible due to the technical challenges associated with liver stage infections. To overcome technical limitations, we performed single-cell transcriptional profiling of liver stages generated using an in vitro infection platform. In elucidating the transcriptional signatures of P. vivax liver forms, as well as hepatocytes in response to infection, our data provide new insights into these host-parasite interactions. An improved understanding of these interactions will allow for novel drug or vaccine interventions targeting either parasite- or hepatocyte-specific metabolic pathways essential for successful liver stage development and disease progression.

sentences: Extreme value analysis of intracellular cargo transport by motor proteins Extreme-value analysis (EVA) deals with deviations of data from the median of probability distributions. It has been used for various purposes, such as predicting disasters and analyzing sports records. Herein, we extended the use of EVA to investigate nanoscale runners within cells. Motor proteins such as kinesin and dynein run along microtubules, which are protein-containing rails, to deliver cargo (material needed for cells). While the velocity of these runners is markedly affected by large cargos in viscous intracellular environments, the return-level EVA plots showed that the velocity near no-load conditions disclosed the physical difference between the two runners, resulting from their load (force)-velocity relationships. The concave-up force-velocity relationship of dynein hampered the independent and identically distributed conditions for the retrograde velocity dataset and the abnormality of the return-level plot of EVA. Our findings underscore the importance of EVA for assessing the physical properties of proteins in vivo.

sentences: Interfacial tension driven open droplet microfluidics We developed an open channel droplet microfluidic system that autonomously generates droplets at low Ca (~10-4-10-3) by leveraging competing hydrostatic and capillary pressure. With only our open channel polytetrafluoroethylene (PTFE) device, pipettes, and commercially available carrier fluid, we produce hundreds of microliter droplets; tubing, electronics, or pumps are not required, making droplet technology feasible for research labs without external flow generators. Furthermore, we demonstrated conceptual applications that showcase the process of droplet generation, splitting, transport, incubation, mixing, and sorting in our system. Unlike conventional droplet microfluidics, the open nature of the device enables the use of physical tools such as tweezers and styli to directly access the system; with this, we developed a new method of droplet sorting and transfer that capitalizes on the Cheerios effect, the aggregation of buoyant objects along a liquid interface. Our platform offers enhanced usability, direct access to the droplet contents, easy manufacturability, compact footprint, and high customizability. This design is a first step in exploring the space of power-free open droplet microfluidic systems and provide design rules for similar channel designs.

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Weight perturbation learning outperforms node perturbation on broad classes of temporally extended tasks Biological constraints often impose restrictions for plausible plasticity rules such as locality and reward-based rather than supervised learning. Two learning rules that comply with these restrictions are weight (WP) and node (NP) perturbation. NP is often used in learning studies, in particular as a benchmark; it is considered to be superior to WP and more likely neurobiologically realized, as the number of weights and therefore their perturbation dimension typically massively exceed the number of nodes. Here we show that this conclusion no longer holds when we take two biologically relevant properties into account: First, tasks extend in time. This increases the perturbation dimension of NP but not WP. Second, tasks are low dimensional, with many weight configurations providing solutions. We analytically delineate regimes where these properties let WP perform as well as or better than NP. Further we find qualitative features of the weight and error dynamics that allow to distinguish which of the rules underlie a learning process: in WP, but not NP, weights mediating zero input diffuse and gathering batches of subtasks in a trial decreases the number of trials required. The insights suggest new learning rules, which combine for specific task types the advantages of WP and NP. Using numerical simulations, we generalize the results to networks with various architectures solving biologically relevant and standard network learning tasks. Our findings suggest WP and NP as similarly plausible candidates for learning in the brain and as similarly important benchmarks.

Statement of significanceNeural networks can learn by first perturbing the network weights or the activity of neurons and thereafter consolidating perturbations that improve the network performance. Weight perturbation learning is considered less efficient, useful and biologically plausible, because there are many more connection weights than neurons, such that generating beneficial perturbations seems less likely. We show that the argument does no longer hold when accounting for two features common in biology: tasks extend in time and the neuronal dynamics are low dimensional. In particular, we find that perturbing the weights is comparably good or better in various biologically relevant and standard network learning applications. This indicates that weight perturbation learning is similarly useful and a plausible candidate for learning in the brain.

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Light/dark and temperature cycling modulate metabolic electron flow in Pseudomonas aeruginosa biofilms Sunlight drives phototrophic metabolism, which affects redox conditions and produces substrates for non-phototrophs. These environmental parameters fluctuate daily due to Earths rotation, and non-phototrophic organisms can therefore benefit from the ability to respond to, or even anticipate, such changes. Circadian rhythms, such as diurnal changes in body temperature, in host organisms can also affect local conditions for colonizing bacteria. Here, we investigated the effects of light/dark and temperature cycling on biofilms of the opportunistic pathogen Pseudomonas aeruginosa PA14. We grew biofilms in the presence of a respiratory indicator dye and found that greater dye reduction occurred in biofilm zones that formed during dark intervals and at lower temperatures. This pattern formation occurred with cycling of blue, red, or far-red light, and a screen of mutants representing potential sensory proteins identified two with defects in pattern formation specifically under red light cycling. We also found that the physiological states of biofilm subzones formed under specific light and temperature conditions were retained during subsequent condition cycling. Light/dark and temperature cycling affected expression of genes involved in primary metabolic pathways and redox homeostasis, including those encoding electron transport chain components. Consistent with this, we found that cbb3-type oxidases contribute to dye reduction under light/dark cycling conditions. Together, our results indicate that cyclic changes in light exposure and temperature have lasting effects on redox metabolism in biofilms formed by a non-phototrophic, pathogenic bacterium.

IMPORTANCEOrganisms that do not obtain energy from light can nevertheless be affected by diurnal changes in light exposure. Many aspects of animal and fungal physiology fluctuate in response to these changes, including body temperature and the activities of antioxidant and other redox enzymes that play roles in metabolism. Whether redox metabolism is affected by light/dark and temperature cycling in bacteria that colonize such circadian organisms has not been studied in detail. Here we show that growth under light/dark and temperature cycling leads to rhythmic changes in redox metabolism in Pseudomonas aeruginosa and identify proteins involved in this response. P. aeruginosa is a major cause of healthcare-associated infections and designated as a serious threat by the CDC due to its recalcitrance during treatments. Our findings have the potential to inform therapeutic strategies that incorporate controlled light exposure or consider P. aeruginosas responses to conditions in the host.

sentences: Regulation of lateral root development by shoot-sensed far-red light via HY5 is nitrate-dependent and involves the NRT2.1 nitrate transporter. Plants are very effective in responding to environmental changes during competition for light and nutrients. Low Red:Far-Red (low R:FR)-mediated neighbor detection allows plants to compete successfully with other plants for available light. This above-ground signal can also reduce lateral root growth by inhibiting lateral root emergence, a process that might help the plant invest resources in shoot growth. Nitrate is an essential nutrient for plant growth and Arabidopsis thaliana responds to low nitrate conditions by enhancing nutrient uptake and reducing lateral and main root growth. There are indications that low R:FR signaling and low nitrate signaling can affect each other. It is unknown which response is prioritized when low R:FR light- and low nitrate signaling co-occur. We investigated the effect of low nitrate conditions on the low R:FR response of the A. thaliana root system in agar plate media, combined with the application of supplemental Far-Red (FR) light to the shoot. We observed that under low nitrate conditions main and lateral root growth was reduced, but more importantly, that the response of the root system to low R:FR was suppressed. Consistently, a loss-of-function mutant of a nitrate transporter gene NRT2.1 lacked low R:FR-induced lateral root reduction and its root growth was hypersensitive to low nitrate. ELONGATED HYPOCOTYL5 (HY5) plays an important role in the root response to low R:FR and we found that it was less sensitive to low nitrate conditions with regards to lateral root growth. In addition, we found that low R:FR increases NRT2.1 expression and that low nitrate enhances HY5 expression. HY5 also affects NRT2.1 expression, however, it depended on the presence of ammonium in which direction this effect was. Replacing part of the nitrogen source with ammonium also removed the effect of low R:FR on the root system, showing that changes in nitrogen sources can be crucial for root plasticity. Together our results show that nitrate signaling can repress low R:FR responses and that this involves signaling via HY5 and NRT2.1.

sentences: A Chitin-Binding Protein Ultra-highly expressed in the Outer Fold of Mantle is Related to Shell Colour in Pacific Oyster Crassostrea gigas Molluscs constitute the second largest phylum of animals in the world, and shell colour and stripes are one of their most important phenotypic characteristics. Studies on the mechanism of shell pigmentation help understand the evolutionary and ecological significance of shell colour and serve as the basis for shell colour breeding. In this study, a matched-pair design was used in comparing the black- and white-striped mantles of the same oyster. The result showed that the stripes of shell surface are corresponding to the stripes of the mantle edge. Transcriptomic analysis identified an uncharacterized protein gene (we named it as Crassostrea gigas chitin-binding protein, CgCBP) highly expressed in the black mantles. Three folds (inner fold, middle fold and outer fold) were found on the mantle edge of oyster, but only the outer fold has the same colour as the shell. Transcriptomic comparison indicated that the three folds of the mantle edge are functionally differentiated, and the CgCBP gene is ultra-highly expressed only in the outer fold. We obtained new black and white shell periostraca from the shell notching experiment and found their structural differences by scanning electron microscope. Chitin was successfully extracted and identified from the shell periostraca. Proteomic analysis revealed differences in protein composition between the two shell periostraca. Particularly, the black shell periostraca have more proteins related to melanin biosynthesis and chitin binding compared with the white ones, and melanin particles were observed in the black mantle edge using transmission electron microscope. Magnetic bead binding and Western blot experiments showed that the CgCBP protein can specifically bind to chitin and in vivo RNAi screening indicated that CgCBP knockdown can change the structure of the shell periostracum and reduce its pigmentation. All these results suggest that the outer fold of mantle may have more important roles in shell pigmentation, shell periostracum structure is functionally correlated with shell pigmentation, and the CgCBP gene ultra-highly expressed in the outer fold may influence shell pigmentation by affecting its periostracum structure.

sentences: Cytometric Analysis of Diverse Glaucophyte Species Reveals Distinctive Signals Useful for Fluorescence-Based Detection and Sorting Glaucophytes, red algae and viridiplants (green algae and land plants) are formally united in the supergroup Archaeplastida. Although diverse molecular and genomic evidence suggest the common origin of the three Archaeplastida lineages, the lack of a robust glaucophyte knowledgebase has limited comprehensive evaluations of competing hypotheses. Glaucophytes are rare and apparently confined to freshwater habitats. However, the distribution and diversity of these algae have not been thoroughly explored owing to challenges with detecting and isolating novel specimens. Here we examined the cytometric signatures of representative species of the genera Cyanophora, Cyanoptyche, Glaucocystis and Gloeochaete for a distinctive signal that would aid identification. Most glaucophytes analyzed presented a relatively high red fluorescence signal due to the presence of the blue phycobiliproteins C-phycocyanin and allophycocyanin. Cell-size differences and the concurrent presence of the red phycobiliprotein phycoerythrin in other algal lineages, such as red algae and cryptophytes, allowed us to distinguish glaucophytes from other photosynthetic cells containing blue phycobiliproteins. Our results indicate that the peculiar autofluorescence signal of glaucophytes will facilitate further identification and isolation on novel specimens of this scarce but important algal group.

sentences: Revealing cell-fate bifurcations from transcriptomic trajectories of hematopoiesis Inspired by Waddingtons illustration of an epigenetic landscape, cell-fate transitions have been envisioned as bifurcating dynamical systems, wherein the dynamics of an exogenous signal couples to a cells enormously complex signaling and transcriptional machinery, eliciting a qualitative transition in the collective state of a cell - its fate. It remains unclear, however, whether the dynamical systems framework can go beyond a word-based caricature of the system and provide sharp quantitative insights that further our understanding of differentiation. Single-cell RNA sequencing (scRNA-seq), which measures the distributions of possible transcriptional states in large populations of differentiating cells, provides an alternate view, in which development is marked by the individual concentration variations of a myriad of genes. Here, starting from formal mathematical derivations, we challenge these transcriptomic trajectories to a rigorous statistical evaluation of whether they display signatures consistent with bifurcations. After pinpointing bifurcations along transcriptomic trajectories of the neutrophil branch of hematopoeitic differentiation we are able to further leverage the primitive features of a linear instability to identify the single-direction in gene expression space along which the bifurcation unfolds and identify possible gene contributors. This scheme identifies transcription factors long viewed to play a crucial role in the process of neutrophil differentiation in addition to identifying a host of other novel genetic players. Most broadly speaking, we provide evidence that, though very high-dimensional, a bifurcating dynamical systems formalism might be appropriate for the process of cellular differentiation and that it can be leveraged to provide insights. Ambitiously, our work attempts to take a step beyond data-analysis and towards the construction of falsifiable mathematical models that describe the dynamics of the entire transcriptome.

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Functional alterations in cortical processing of speech in glioma-infiltrated cortex Recent developments in the biology of malignant gliomas have demonstrated that glioma cells interact with neurons through both paracrine signaling and electrochemical synapses. Glioma-neuron interactions consequently modulate the excitability of local neuronal circuits, and it is unclear the extent to which glioma-infiltrated cortex can meaningfully participate in neural computations. For example, gliomas may result in a local disorganization of activity that impedes the transient synchronization of neural oscillations. Alternatively, glioma-infiltrated cortex may retain the ability to engage in synchronized activity, in a manner similar to normal-appearing cortex, but exhibit other altered spatiotemporal patterns of activity with subsequent impact on cognitive processing. Here, we use subdural electrocorticography to sample both normal-appearing and glioma-infiltrated cortex during speech. We find that glioma-infiltrated cortex engages in synchronous activity during task performance in a manner similar to normal-appearing cortex, but recruits a diffuse spatial network. On a temporal scale, we show that glioma-infiltrated cortex has lower capacity for information encoding when performing nuanced tasks such as speech production of monosyllabic versus polysyllabic words. As a result, temporal decoding strategies for distinguishing monosyllabic from polysyllabic words were feasible for signals arising from normal-appearing cortex, but not from glioma-infiltrated cortex. These findings inform our understanding of cognitive processing in chronic disease states and have implications for neuromodulation and prosthetics in patients with malignant gliomas.

Significance StatementAs gliomas proliferate, they infiltrate healthy brain tissue. Often, patients with such tumors in the language areas of the brain develop aphasia. Understanding how gliomas interact with normal neural circuits is critical for developing neuroprostheses that restore speech. Recent evidence demonstrates that glioma cells interact synaptically with neurons, and thus can modulate neural circuits. However, it is unclear the extent to which glioma-infiltrated cortex participates in cognitive processing. Using electrocorticography to record both glioma-infiltrated and normal-appearing cortex during speech, we found that glioma-infiltrated cortex is capable of coordinated neural responses, but has reduced capacity for information encoding. Instead, glioma-infiltrated cortex recruits a broader network of cortical regions during speech, which may represent a compensatory mechanism with implications for future neuroprostheses.

sentences: Combined MEK and STAT3 inhibition reprograms stromal inflammation to overcome immunotherapy resistance in pancreatic cancer Pancreatic ductal adenocarcinoma (PDAC) is characterized by immune exclusion, pro-inflammatory polarization of cancer-associated fibroblasts (CAF), and resistance to immune checkpoint inhibition (ICI). We have previously demonstrated that reciprocally activated RAS/MEK/ERK and JAK/STAT3 pathways mediate therapeutic resistance, while combined MEK and STAT3 inhibition (MEKi+STAT3i) overcomes such resistance in preclinical models. We now show that combined MEKi+STAT3i not only alters stromal architecture but also uncovers stromal plasticity by revealing a substantial attenuation of Il6/Cxcl1-expressing secretory and Lrrc15-expressing myofibroblastic CAF phenotypes with a concomitant enrichment of Ly6a/Cd34-expressing CAF phenotypes exhibiting mesenchymal progenitor-like properties via single-cell RNA sequencing in Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox(PKT) mice. This remodeling of CAF heterogeneity is associated with reprogramming of immunosuppressive myeloid populations and enhanced trafficking of CD8+ T-cells which exhibit a distinct effector transcriptional program. These MEKi+STAT3i-mediated repercussions are in part CAF-dependent, since CRISPR/Cas9 genetic silencing of CAF-restricted Mek1/Stat3 mitigates inflammatory CAF polarization and myeloid infiltration in vivo. Addition of MEKi+STAT3i to PD-1 blockade overcomes ICI resistance by significantly augmenting anti-tumor responses and dramatically improving survival in PKT mice compared with anti-PD-1 monotherapy. The addition of MEKi+STAT3i to PD-1 blockade not only augments the recruitment of activated and memory T-cell populations, but also improves their degranulating capacity and functional cytotoxicity compared to PD-1 blockade alone. Importantly, treatment of a patient with chemotherapy-refractory metastatic PDAC with MEKi (Trametinib), STAT3i (Ruxolitinib), and PD-1 inhibitor (Nivolumab) was well-tolerated and yielded clinical benefit. These data uncover a novel paradigm in which combined MEKi+STAT3i reprograms stromal inflammation and immune tolerance to overcome immunotherapy resistance in PDAC.

sentences: Neurogrid simulates cortical cell-types, active dendrites, and top-down attention A central challenge for systems neuroscience and artificial intelligence is to understand how cognitive behaviors arise from large, highly interconnected networks of neurons. Digital simulation is linking cognitive behavior to neural activity to bridge this gap in our understanding at great expense in time and electricity. A hybrid analog-digital approach, whereby slow analog circuits, operating in parallel, emulate graded integration of synaptic currents by dendrites while a fast digital bus, operating serially, emulates all-or-none transmission of action potentials by axons, may improve simulation efficacy. Due to the latters serial operation, this approach has not scaled beyond millions of synaptic connections (per bus). This limit was broken by following design principles the neocortex uses to minimize its wiring. The resulting hybrid analog-digital platform, Neurogrid, scales to billions of synaptic connections, between up to a million neurons, and simulates cortical models in real-time using a few watts of electricity. Here, we demonstrate that Neurogrid simulates cortical models spanning five levels of experimental investigation: biophysical, dendritic, neuronal, columnar, and area. Bridging these five levels with Neurogrid revealed a novel way active dendrites could mediate top-down attention.

sentences: Plant immune system activation is necessary for efficient interaction with auxin secreting beneficial bacteria Plants continuously monitor the presence of microorganisms through their immune system to establish an adaptive response. Unlike immune recognition of pathogenic bacteria, how beneficial bacteria interact with the plant immune system is not well understood. Analysis of colonization of Arabidopsis thaliana by auxin producing beneficial bacteria revealed that activation of the plant immune system is necessary for efficient bacterial colonization and auxin secretion. A feedback loop is established in which bacterial colonization triggers an immune reaction and production of reactive oxygen species, which, in turn, stimulate auxin production by the bacteria. Auxin promotes bacterial survival and efficient root colonization, allowing the bacteria to inhibit fungal infection and promote plant health.

sentences: Structural connectivity-based segmentation of the human entorhinal cortex The medial (MEC) and lateral entorhinal cortex (LEC), widely studied in rodents, are well defined and characterized. In humans, however, the exact locations of their homologues remain uncertain. Previous functional magnetic resonance imaging (fMRI) studies have subdivided the human EC into posterior-medial (pmEC) and anterior-lateral (alEC) parts, but uncertainty remains about the choice of imaging modality and seed regions, in particular in light of a substantial revision of the classical model of EC connectivity based on novel insights from rodent anatomy. Here, we used structural, not functional imaging, namely diffusion tensor imaging (DTI) and probabilistic tractography to segment the human EC based on differential connectivity to other brain regions known to project selectively to MEC or LEC. We defined MEC as more strongly connected to presubiculum and retrosplenial cortex (RSC), and LEC as more strongly connected to distal CA1 and proximal subiculum (dCA1pSub) and orbitofrontal cortex (OFC). Although our DTI segmentation had a larger medial-lateral component than in previous fMRI studies, our results show that the human MEC and LEC homologues have a border oriented both towards the posterior-anterior and medial-lateral axes, supporting the differentiation between pmEC and alEC.

sentences: Reinforcement learning modeling reveals a reward-history-dependent strategy underlying reversal learning in squirrel monkeys Insight into psychiatric disease and development of therapeutics relies on behavioral tasks that study similar cognitive constructs in multiple species. The reversal learning task is one popular paradigm that probes flexible behavior, aberrations of which are thought to be important in a number of disease states. Despite widespread use, there is a need for a high-throughput primate model that can bridge the genetic, anatomic, and behavioral gap between rodents and humans. Here, we trained squirrel monkeys, a promising preclinical model, on an image-guided deterministic reversal learning task. We found that squirrel monkeys exhibited two key hallmarks of behavior found in other species: integration of reward history over many trials and a side-specific bias. We adapted a reinforcement learning model and demonstrated that it could simulate monkey-like behavior, capture training-related trajectories, and provide insight into the strategies animals employed. These results validate squirrel monkeys as a model in which to study behavioral flexibility.

sentences: Detectability of biotin tags by LC-MS/MS The high affinity of biotin to streptavidin has made it one of the most widely used affinity tags in proteomics. Early methods used biotin for enrichment alone and mostly ignored the biotin labeled peptide. Recent advances in labeling led to an increase in biotinylation efficiency and shifted the interest to detection of the site of biotinylation. This increased confidence in identification and provides additional structural information yet it requires efficient release of the biotinylated protein/peptide and sensitive separation and detection of biotinylated peptides by LC-MS/MS. Despite its long use in affinity proteomics the effect of biotinylation on the chromatographic, ionization, and fragmentation behaviour and ultimate detection of peptides is not well understood. To address this we compare two commercially-available biotin labels EZ-Link Sulfo-NHS-Biotin and Sulfo-NHS-SS-Biotin, the latter one containing a labile linker to efficiently release biotin to determine the effects of peptide modification on peptide detection. We describe an increase of hydrophobicity and charge reduction with increasing number of biotin labels attached. Based on our data we recommend gradient optimization to account for more hydrophobic biotinylated peptides and include singly charged precursors to account for charge reduction by biotin.

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Brain monoaminergic activity during predator inspection in the Trinidadian guppy (Poecilia reticulata) To understand the processes underpinning social decision-making, we need to determine how internal states respond to information gathered from the social environment. Brain monoamine neurotransmitters are key in the appraisal of the social environment and can reflect the internal state underlying behavioural responses to social stimuli. Here we determined the effects of conspecific partner cooperativeness during predator inspection on brain monoamine metabolic activity in Trinidadian guppies (Poecilia reticulata). We quantified the concentration of dopamine, serotonin and their metabolites across brain sections sampled immediately after ostensibly experiencing cooperation or defection from social partners whilst inspecting a predator model. Our results indicate dopaminergic and serotonergic activity differs with the cooperativeness experienced; these different neurotransmission profiles are likely to affect the expression and regulation of downstream behaviours that ultimately contribute to the patterning of cooperative interactions among individuals in the population.

List of abbreviations used

sentences: Protein embeddings and deep learning predict binding residues for various ligand classes One important aspect of protein function is the binding of proteins to ligands, including small molecules, metal ions, and macromolecules such as DNA or RNA. Despite decades of experimental progress many binding sites remain obscure. Here, we proposed bindEmbed21, a method predicting whether a protein residue binds to metal ions, nucleic acids, or small molecules. The Artificial Intelligence (AI)-based method exclusively uses embeddings from the Transformer-based protein Language Model (pLM) ProtT5 as input. Using only single sequences without creating multiple sequence alignments (MSAs), bindEmbed21DL outperformed MSA-based predictions. Combination with homology-based inference increased performance to F1=48{+/-}3% (95% CI) and MCC=0.46{+/-}0.04 when merging all three ligand classes into one. All results were confirmed by three independent data sets. Focusing on very reliably predicted residues could complement experimental evidence: For the 25% most strongly predicted binding residues, at least 73% were correctly predicted even when ignoring the problem of missing experimental annotations. The new method bindEmbed21 is fast, simple, and broadly applicable - neither using structure nor MSAs. Thereby, it found binding residues in over 42% of all human proteins not otherwise implied in binding and predicted about 6% of all residues as binding to metal ions, nucleic acids, or small molecules.

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Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids BackgroundAccurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that allows flexibility for high and mid-throughput analysis for three blood-based proteomes: naive plasma, plasma depleted of the 14 most abundant proteins, and dried blood.

MethodsOptimal conditions for sample preparation and DIA-MS analysis were established in plasma then automated and adapted for depleted plasma and whole blood. The MS workflow was modified to facilitate sensitive high-throughput or deep profile analysis with mid-throughput analysis. Analytical performance was evaluated from 5 complete workflows repeated over 3 days as well as a linearity analysis of a 5-6-point dilution curve.

ResultUsing our high-throughput workflow, 74%, 93%, 87% of peptides displayed an inter-day CV<30% in plasma, depleted plasma and whole blood. While the mid-throughput workflow had 67%, 90%, 78% of peptides in plasma, depleted plasma and whole blood meeting the CV<30% standard. Lower limits of detection and quantitation were determined for proteins and peptides observed in each biofluid and workflow. Combining the analysis of both high-throughput plasma fractions exceeded the number of reliably identified proteins for individual biofluids in the mid-throughput workflows.

ConclusionThe workflow established here allowed for reliable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow on a large scale will facilitate the translation of candidate markers into clinical use.

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Nonlinear super-resolution signal processing for subcellular analysis of calcium dynamics Optical (fluorescence) imaging of ionic dynamics has revolutionized neuroscience as it allows the study of neural activity across spatially identified populations. Quantification of fluorescence signals is commonly performed using ratiometric measures, like the {Delta}F/F. Although these measures are robust and easy to implement, they do not take advantage of the temporal information available in time-varying signals. Moreover, since a baseline (reference) period needs to be selected, their application is often limited to the quantification of stimulus-evoked activity. Here, we present a new approach, called ARES, based on the quantification of residuals after linear autoregression. We demonstrate the utility of ARES to quantify the functional dynamics of stimulus representation in cortical networks and show that it improves the spatial and temporal resolution with respect to {Delta}F/F. We further show that ARES can be used to study subcellular calcium dynamics and exemplify its utility to describe the spatiotemporal dynamics of calcium signal localization in compartmental network recordings. ARES offers a novel method for quantitative analysis of optical imaging data

Author summaryTwo-photon calcium imaging is a powerful and non-invasive method to measure and visualize the activity of neural populations in single cell and subcellular resolution. However, the slow dynamics of both the calcium signal itself and its indicators make it difficult to relate the timing and origin of the neural activity to the calcium signal. The most commonly used analysis method, {Delta}F/F, uses a reference baseline to quantify signal ratiometrically. This results in several limitations, especially in accounting for the time varying fluctuations in fluorescence values. Here we present a new method for the analysis of calcium signals, called ARES, in which a sliding window is used to predict the future values of the signal and deviations from this prediction are recorded as the signal. This has the advantage that shifts in baseline do not affect the processing, and that the method has an improved spatial and temporal resolution with respect to {Delta}F/F.

sentences: Gut regulates brain synaptic assembly through neuroendocrine signaling pathway The gut-brain axis plays an essential role in regulating neural development in response to environmental stimuli, such as microbes or nutrients. Defects in gut-brain communication can lead to various neurological disorders. However, it remains unknown whether gut plays any intrinsic role in regulating neuronal development. Through a genetic screen in C. elegans, we uncovered that an intrinsic Wnt-endocrine pathway in gut regulates synaptic development and neuronal activity in brain. Specifically, the Wnt signaling upregulates the expression of the neuropeptide NLP-40 in the gut, which then facilitates presynaptic assembly through the neuronal expressed GPCR AEX-2 receptor during development. The NLP-40 acts most likely through modulating neuronal activity and promoting synaptic protein trafficking. Therefore, this study reveals a novel role of gut in synaptic development in the brain.

sentences: Ex-vivo anticoagulants affect the mechanical properties of human blood platelets with implications for in vitro functional mechanophenotyping. Inherited platelet disorders affecting the human platelet cytoskeleton result in increased bleeding risk. However, deciphering their impact on cytoskeleton-dependent intrinsic biomechanics of platelets remains challenging and represents an unmet need from a diagnostic and prognostic perspective. It is currently unclear whether ex vivo anticoagulants used for the collection of peripheral blood impact the mechanophenotype of cellular components of blood. Using unbiased, high-throughput functional mechanophenotyping of single human platelets by deformability cytometry, we found that ex vivo anticoagulants are a critical pre-analytical variable that differentially influences platelet deformation, their size and functional response to agonists by altering the cytoskeleton. We applied our findings to characterize the functional mechanophenotype of platelets from a patient with Myosin Heavy Chain 9 (MYH9) related macrothrombocytopenia. Our data suggest that platelets from MYH9 p.E1841K mutation in humans affecting platelet non-muscle myosin heavy chain IIa (NMMHC-IIA) are biomechanically less deformable in comparison to platelets from healthy individuals.

sentences: Dive performance and aquatic thermoregulation of the world's smallest mammalian diver, the American water shrew (Sorex palustris) Allometry predicts that the 12-17 g American water shrew (Sorex palustris)--the worlds smallest mammalian diver--will have the highest diving metabolic rate coupled with the lowest total body oxygen storage capacity, skeletal muscle buffering capacity, and glycolytic potential of any endothermic diver. Consistent with expectations, and potentially owing to their low thermal inertia, water shrews had a significantly higher diving metabolic rate in 10{degrees}C (8.77 mL O2 g-1 hr-1) compared to 30{degrees}C water (6.57 mL O2 g-1 hr-1). Unlike larger-bodied divers, muscle myoglobin contributed minimally (7.7-12.4%) to total onboard O2 stores of juvenile and adult water shrews, respectively, but was offset by high blood O2 carrying capacities (26.4-26.9 vol. %). Diving was predominantly aerobic, as only 1.2-2.3% of dives in 10 and 30{degrees}C water, respectively, exceeded the calculated aerobic dive limits at these temperatures (10.8-14.4 sec). The mean voluntary dive time of water shrews during 20-min trials in 3-30{degrees}C water was 5.0{+/-}0.1 sec (N=25, n=1628), with a mean maximum dive time of 10.1{+/-}0.4 sec. However, the average dive duration (6.9{+/-}0.2 sec, n=257) of radio-telemetered shrews exclusively foraging in a simulated riparian environment (3{degrees}C water) for 12- to 28-hr suggest that mean (but not maximum) dive times of water shrews in the wild may be longer. Mean dive duration, duration of the longest dive, and total time in water all decreased significantly as water temperature declined, suggesting that shrews employed behavioral thermoregulation to defend against immersion hypothermia. Additionally, free-diving shrews in the 24-hr trials consistently elevated core body temperature by [~]1{degrees}C immediately prior to initiating aquatic foraging bouts, and ended these bouts when body temperature was still at or above normal resting levels ([~]37.8{degrees}C). We suggest this observed pre-dive hyperthermia aids to heighten the impressive somatosensory physiology, and hence foraging efficiency, of this diminutive predator while submerged.

sentences: Robust T cell activation requires an eIF3-driven burst in T cell receptor translation Activation of T cells requires a rapid surge in cellular protein synthesis. However, the role of translation initiation in the early induction of specific genes remains unclear. Here we show human translation initiation factor eIF3 interacts with select immune system related mRNAs including those encoding the T cell receptor (TCR) subunits TCRA and TCRB. Binding of eIF3 to the TCRA and TCRB mRNA 3-untranslated regions (3-UTRs) depends on CD28 coreceptor signaling and regulates a burst in TCR translation required for robust T cell activation. Use of the TCRA or TCRB 3-UTRs to control expression of an anti-CD19 chimeric antigen receptor (CAR) improves the ability of CAR-T cells to kill tumor cells in vitro. These results identify a new mechanism of eIF3-mediated translation control that can aid T cell engineering for immunotherapy applications.

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Unifying genetic association tests via regression: Prospective and retrospective, parametric and non-parametric, and genotype- and allele-based tests Genetic association analysis, evaluating the relationship between genetic markers and complex and heritable traits, is the basis of genome-wide association studies. In response, many association tests have been developed, and they are generally classified as prospective vs. retrospective, parametric vs. non-parametric, and genotype- vs. allele-based association tests. While method classification is useful, it is confusing and challenging for practitioners to decide on the  optimal test to use for their data. Although there are known differences between some of the popular association tests, we provide new results that show the analytical connections between the different tests for both population- and family-based study designs.

ResumeInserer votre resume ici. We will supply a French abstract for those authors who cant prepare it themselves.

sentences: uniPort: a unified computational framework for single-cell data integration with optimal transport Single-cell data integration can provide a comprehensive molecular view of cells. Here we introduce uniPort, a unified single-cell data integration framework which combines a coupled Variational Autoencoder (coupled-VAE) and Minibatch Unbalanced Optimal Transport (Minibatch-UOT). It leverages both highly variable common and dataset-specific genes for integration and is scalable to large-scale and partially overlapping datasets. uniPort jointly embeds heterogeneous single-cell multi-omics datasets into a shared latent space. It can further construct a reference atlas for online prediction across datasets. Meanwhile, uniPort provides a flexible label transfer framework to deconvolute spatial heterogeneous data using optimal transport space, instead of embedding latent space. We demonstrate the capability of uniPort by integrating a variety of datasets, including single-cell transcriptomics, chromatin accessibility and spatially resolved transcriptome data. uniPort software is available at https://github.com/caokai1073/uniPort.

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CRAFT: a bioinformatics software for custom prediction of circular RNA functions Circular RNAs (circRNAs), transcripts generated by backsplicing, are particularly stable and pleiotropic molecules, whose dysregulation drives human diseases and cancer by modulating gene expression and signaling pathways. CircRNAs can regulate cellular processes by different mechanisms, including interaction with microRNAs (miRNAs) and RNA-binding proteins (RBP), and encoding specific peptides. The prediction of circRNA functions is instrumental to interpret their impact in diseases, and to prioritize circRNAs for functional investigation. Currently, circRNA functional predictions are provided by web databases that do not allow custom analyses, while self-standing circRNA prediction tools are mostly limited to predict only one type of function, mainly focusing on the miRNA sponge activity of circRNAs. To solve these issues, we developed CRAFT (CircRNA Function prediction Tool), a freely available computational pipeline that predicts circRNA sequence and molecular interactions with miRNAs and RBP, along with their coding potential. Analysis of a set of circRNAs with known functions has been used to appraise CRAFT predictions and to optimize its setting. CRAFT provides a comprehensive graphical visualization of the results, links to several knowledge databases, and extensive functional enrichment analysis. Moreover, it originally combines the predictions for different circRNAs. CRAFT is a useful tool to help the user explore the potential regulatory networks involving the circRNAs of interest and generate hypotheses about the cooperation of circRNAs into the modulation of biological processes.

Key pointsO_LICRAFT is a self standing tool for comprehensive circRNA function prediction.
C_LIO_LICRAFT functions include circRNA sequence reconstruction, microRNA and RNA-binding protein response elements and coding potential prediction.
C_LIO_LIPredictions for multiple circRNAs are connected to infer possible cooperation networks and illustrate the potential impact of circRNAs on biological and disease processes.
C_LI

sentences: A long non-coding RNA in the let-7 complex acting as a potent and specific death effector of cancer cells The let-7 complex in Drosophila encodes three evolutionarily conserved microRNAs: miR-100, let-7, and miR-125. These act as heterochronic genes in regulating developmental timing in response to the steroid hormone ecdysone and play important roles in cell differentiation. Here we identify two additional long non-coding RNAs in the let-7 complex, we named let-A and let-B. Both are transcribed in the large first intron of the primary RNA encoding the microRNAs. We show these RNAs to be sequentially expressed in early pupal stages in response to ecdysone signaling, albeit exhibiting a different expression pattern compared to the microRNA let-7. Surprisingly, ectopic expression of let-A in Drosophila cancer cells induces rapid cell death. Dead cells further release RNA molecules in the medium that is becoming toxic to other cancer cells. In vivo grown tumors lose their tumorigenicity after being incubated in the let-A induced medium. Moreover, feeding flies carrying transplanted tumor cells with such induced medium leads to reduced growth of tumors in a subset of hosts. Our results uncover a new lncRNA which can act as a potent and specific cell death effector for Drosophila tumor cells.

sentences: Safety and Immunogenicity Evaluation of Inactivated whole-virus-SARS-COV-2 In Mice As Emerging Vaccine Development In Egypt The current worldwide pandemic COVID-19 is causing severe human health problems, with high numbers of mortality rates and huge economic burdens that require an urgent demand for safe, and effective and vaccine development. Our study was the first trail to development and evaluation of safety and immune response to inactivated whole SARS-COV-2 virus vaccine adjuvanted with aluminium hydroxide. We used characterized SARS-COV-2 strain, severe acute respiratory syndrome coronavirus 2 isolates (SARS-CoV-2/human/EGY/Egy-SERVAC/2020) with accession numbers; MT981440; MT981439; MT981441; MT974071; MT974069 and MW250352 at GenBank that isolated from Egyptian patients SARS-CoV-2-positive. Development of the vaccine was carried out in a BSL - 3 facilities and the immunogenicity was determined in mice at two doses (55{micro}g and 100{micro}g per dose). All vaccinated mice were received a booster dose 14 days post first immunization. Our results demonstrated distinct cytopathic effect on the vero cell monolayers induced through SARS-COV-2 propagation and the viral particles were identified as Coronaviridae by transmission electron microscopy. SARS-CoV-2 was identified by RT-PCR performed on the cell culture. Immunogenicity of the developed vaccine indicated the high antigen-binding and neutralizing antibody titers, regardless the dose concentration, with excellent safety profiles.However, no deaths or clinical symptoms in mice groups. The efficacy of the inactivated vaccine formulation was tested by wild virus challenge the vaccinated mice and detection of viral replication in lung tissues. Vaccinated mice recorded complete protection from challenge infection three weeks post second dose. SARS-COV-2 replication was not observed in the lungs of mice following SARS-CoV-2 challenge, regardless of the level of serum neutralizing antibodies. This finding will support the future trials for evaluation an applicable SARS-CoV-2 vaccine candidate.

sentences: Interpretable, scalable, and transferrable functional projection of large-scale transcriptome data using constrained matrix decomposition Large-scale transcriptome data, such as single-cell RNA-sequencing data, have provided unprecedented resources for studying biological processes at the systems level. Numerous dimensionality reduction methods have been developed to visualize and analyze these transcriptome data. In addition, several existing methods allow inference of functional variations among samples using gene sets with known biological functions. However, it remains challenging to analyze transcriptomes with reduced dimensions that are interpretable in terms of dimensions directionalities, transferrable to new data, and directly expose the contribution of individual genes. In this study, we used gene set non-negative principal component analysis (gsPCA) and non-negative matrix factorization (gsNMF) to analyze large-scale transcriptome datasets. We found that these methods provide low-dimensional information about the progression of biological processes in a quantitative manner, and their performances are comparable to existing functional variation analysis methods in terms of distinguishing multiple cell states and samples from multiple conditions. Remarkably, upon training with a subset of data, these methods allow predictions of locations in the functional space using data from experimental conditions that are not exposed to the models. Specifically, our models predicted the extent of progression and reversion for cells in the epithelial-mesenchymal transition (EMT) continuum. These methods revealed conserved EMT program among multiple types of single cells and tumor samples. Finally, we demonstrate this approach is broadly applicable to data and gene sets beyond EMT and provide several recommendations on the choice between the two linear methods and the optimal algorithmic parameters. Our methods show that simple constrained matrix decomposition can produce to low-dimensional information in functionally interpretable and transferrable space, and can be widely useful for analyzing large-scale transcriptome data.

sentences: Transiently increased intercommunity regulation characterizes concerted cell phenotypic transition Phenotype transition takes place in many biological processes such as differentiation and reprogramming. A fundamental question is how cells coordinate switching of expressions of clusters of genes. Through analyzing single cell RNA sequencing data in the framework of transition path theory, we studied how such a genome-wide expression program switching proceeds in three different cell transition processes. For each process we reconstructed a reaction coordinate describing the transition progression, and inferred the gene regulation network (GRN) along the reaction coordinate. In all three processes we observed common pattern that the effective number and strength of regulation between different communities increase first and then decrease. The change accompanies with similar change of the GRN frustration, defined as overall confliction between the regulation received by genes and their expression states, and GRN heterogeneity. While studies suggest that biological networks are modularized to contain perturbation effects locally, our analyses reveal a general principle that during a cell phenotypic transition intercommunity interactions increase to concertedly coordinate global gene expression reprogramming, and canalize to specific cell phenotype as Waddington visioned.

sentences: Functional study of Leishmania braziliensis protein arginine methyltransferases (PRMTs) reveals that PRMT1 and PRMT5 are required for macrophage infection In trypanosomatids, regulation of gene expression occurs mainly at the posttranscriptional level, and RNA-binding proteins (RBPs) are key players in determining the fates of transcripts. RBPs are major targets of protein arginine methyltransferases (PRMTs), which posttranslationally regulate the RNA-binding capacity and other macromolecular interactions of RBPs by transferring methyl groups to protein arginine residues. Herein, we present the results of a study that functionally characterized the five predicted PRMTs in Leishmania braziliensis by gene knockout and endogenous protein HA tagging using CRISPR/Cas9 gene editing. We report that arginine methylation profiles vary among Leishmania species and that target protein methylation changes across different L. braziliensis life cycle stages, with higher PRMT expression in the promastigote stages than in the axenic amastigote stage. Knockout of some of the L. braziliensis PRMTs led to significant changes in global arginine methylation patterns without affecting promastigote axenic growth. Deletion of either PRMT1 or PRMT3 disrupted most type I PRMT activity, resulting in a global increase in monomethyl arginine (MMA) levels, which is mainly catalyzed by PRMT7. Putative targets and/or PRMT-interacting proteins were identified by coimmunoprecipitation using HA-tagged PRMTs, revealing a network of target RBPs and suggesting functional interactions between them and a relevant participation in epigenetic control of gene expression. Finally, we demonstrate that L. braziliensis PRMT1 and PRMT5 are required for efficient macrophage infection in vitro, and that in the absence of PRMT1 and PRMT5, axenic amastigote proliferation is impaired. The results indicate that arginine methylation is modulated across life cycle stages in L. braziliensis and show possible functional overlap and cooperation among the different PRMTs in targeting proteins. Overall, our data suggest important regulatory roles of these proteins throughout the L. braziliensis life cycle, showing that arginine methylation is important for parasite-host cell interactions.

sentences: Structures of active melanocortin-4 receptor--Gs-protein complexes with NDP-α-MSH and setmelanotide The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a G-protein coupled receptor and a prime target for the treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R- Gs-protein complexes with two recently FDA-approved drugs, the peptide agonists NDP--MSH and setmelanotide, with 2.9 [A] and 2.6 [A] resolution. Together with signaling data, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6 regulated receptor activation. In both structures, different ligand binding modes of NDP--MSH, a high-affinity variant of the endogenous agonist, and setmelanotide, an anti-obesity drug with biased signaling, underline the key role of TM3 for ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-TM3 interplay subsequently impacts the receptor- Gs-protein interfaces, mainly at intracellular loop 2. These structures reveal mechanistic details of MC4R activation or inhibition and provide important insights into receptor selectivity that will facilitate the development of tailored anti-obesity drugs.

sentences: Sequentially-reduced representation of artificial neural network to determine cardiovascular intrinsic frequencies Analysis of cardiovascular waveforms provides valuable clinical information about the state of health and disease. The intrinsic frequency (IF) method is a recently introduced framework that uses a single arterial pressure waveform to extract physiologically relevant information about the cardiovascular system. The clinical usefulness and physiological accuracy of the IF method have been well-established via several preclinical and clinical studies. However, the computational complexity of the current L2 optimization solver for IF calculations remains a bottleneck for practical deployment of the IF method in real-time settings. In this paper, we propose a machine learning (ML)-based methodology for determination of IF parameters from a single carotid waveform. We use a sequentially-reduced Feedforward Neural Network (FNN) model for mapping carotid waveforms to the output parameters of the IF method, thereby avoiding the non-convex L2 minimization problem arising from the conventional IF approach. Our methodology also includes procedures for data pre-processing, model training, and model evaluation. In our model development, we used both clinical and synthetic waveforms. Our clinical database is composed of carotid waveforms from two different sources: the Huntington Medical Research Institutes (HMRI) iPhone Heart Study and the Framingham Heart Study (FHS). In the HMRI and FHS clinical studies, various device platforms such as piezoelectric tonometry, optical tonometry (Vivio), and an iPhone camera were used to measure arterial waveforms. Our blind clinical test shows very strong correlations between IF parameters computed from the FNN-based method and those computed from the standard L2 optimization-based method (i.e., R[≥]0.93 and P-value [≤]0.005 for each IF parameter). Our results also demonstrate that the performance of the FNN-based IF model introduced in this work is independent of measurement apparatus and of device sampling rate.

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Glucose decoration on wall-teichoic acid is required for phage adsorption and InlB-mediated virulence in Listeria ivanovii Listeria ivanovii (Liv) is an intracellular Gram-positive pathogen that primarily infects ruminants, but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes (Lmo). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall-teichoic acid, or WTA) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor, Internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene, liv1070 that encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by UPLC-MS analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention to the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting the trade-off between phage resistance and virulence attenuation may be a general feature in the Listeria genus.

ImportanceListeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants, and occasionally in immunocompromised humans. Recent investigations revealed that, in its better-known cousin Listeria monocytogenes, strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently resulting in disconnection of one of the bacteriums major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria and the host may be a feature common to the Listeria genus.

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Mutations of SARS-CoV-2 variants of concern escaping Spike-specific T cells The amino acid (AA) mutations that characterise the different variants of concern (VOCs), which replaced the ancestral SARS-CoV-2 Wuhan-Hu-1 isolate worldwide, provide biological advantages such as increased infectivity and partial escape from humoral immunity. Here we analysed the impact of these mutations on vaccination- and infection-induced Spike-specific T cells. We confirmed that, in the majority of infected or vaccinated individuals, different mutations present in a single VOC (Delta) or a combined mosaic of more than 30 AA substitutions and deletions found in Alpha, Beta, Gamma, Delta and Omicron VOCs cause modest alteration in the global Spike-specific T cell response. However, distinct numerically dominant Spike-specific CD4 and CD8 T cells preferentially targeted regions affected by AA mutations and do not recognise the mutated peptides. Importantly, some of these mutations, such as N501Y (present in Alpha, Beta, Gamma, and Omicron) and L452R (present in Delta), known to provide biological advantage to SARS-CoV-2 in terms of infectivity also abolished CD8 T cell recognition.

Taken together, our data show that while global mRNA vaccine- and infection-induced Spike-specific T cells largely tolerate the diverse mutations present in VOCs, single Spike-specific T cells might contribute to the natural selection of SARS-CoV-2 variants.

sentences: HiCHub: A Network-Based Approach to Identify Domains of Differential Interactions from 3D Genome Data Chromatin architecture is important for gene regulation. Existing algorithms for the identification of interactions changes focus on loops between focal loci. Here we develop a network-based algorithm HiCHub to detect chromatin interaction changes at larger scales. It identifies clusters of genomic elements in physical proximity in one state that exhibit concurrent decreases in interaction among them in the opposite state. The hubs exhibit concordant changes in chromatin state and expression changes, supporting their biological significance. HiCHub works well with data of limited sequencing coverage and facilitates the integration of the one-dimensional epigenetic landscape onto the chromatin architecture. HiCHub provides an approach for finding extended architectural changes and contributes to the connection with transcriptional output. HiCHub is freely available at https://github.com/WeiqunPengLab/HiCHub.

sentences: SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome Viral infection involves complex set of events orchestrated by multiple viral proteins. To identify functions of SARS-CoV-2 proteins, we performed transcriptomic analyses of cells expressing individual viral proteins. Expression of Nsp14, a protein involved in viral RNA replication, provoked a dramatic remodeling of the transcriptome that strongly resembled that observed following SARS-CoV-2 infection. Moreover, Nsp14 expression altered the splicing of more than 1,000 genes and resulted in a dramatic increase in the number of circRNAs, which are linked to innate immunity. These effects were independent of the Nsp14 exonuclease activity and required the N7-guanine-methyltransferase domain of the protein. Activation of the NFkB pathway and increased expression of CXCL8 occurred early upon Nsp14 expression. We identified IMPDH2, which catalyzes the rate-limiting step of guanine nucleotides biosynthesis, as a key mediator of these effects. Nsp14 expression caused an increase in GTP cellular levels, and the effect of Nsp14 was strongly decreased in presence of IMPDH2 inhibitors. Together, our data demonstrate an unknown role for Nsp14 with implications for therapy.

sentences: Mutational meltdown of microbial altruists in Streptomyces coelicolor colonies In colonies of the filamentous multicellular bacterium Streptomyces coelicolor, a sub-population of cells arise that hyper-produce metabolically costly antibiotics, resulting in division of labor that maximizes colony fitness. Because these cells contain large genomic deletions that cause massive reductions to individual fitness, their behavior is altruistic, much like worker castes in social insects or somatic cells in multicellular organisms. To understand the reproductive and genomic fate of these mutant cells after their emergence, we use experimental evolution by serially transferring populations via spore-to-spore transfer for 25 cycles, reflective of the natural mode of bottlenecked transmission for these spore-forming bacteria. We show that, in contrast to wild-type cells, altruistic mutant cells continue to significantly decline in fitness during transfer while they delete larger and larger fragments from their chromosome ends. In addition, altruistic mutants acquire a roughly 10-fold increase in their base-substitution rates possibly due to mutations in genes for DNA replication and repair. Ecological damage, caused by reduced sporulation, coupled with irreversible DNA damage due to point mutations and deletions, leads to an inevitable and irreversible type of mutational meltdown in these cells. Taken together, these results suggest that the altruistic cells arising in this division of labor are analogous to reproductively sterile castes of social insects.

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VExD: A curated resource for human gene expression following viral infection MotivationHuman viruses cause significant mortality, morbidity, and economic disruption worldwide. The human gene expression response to viral infection can yield important insights into the detrimental effects to the host. To date, hundreds of studies have performed genome-scale profiling of the effect of viral infection on human gene expression. However, no resource exists that aggregates human expression results across multiple studies, viruses, and tissue types.

ResultsWe developed the Virus Expression Database (VExD), a comprehensive curated resource of transcriptomic studies of viral infection in human cells. We have processed all studies within VExD in a uniform manner, allowing users to easily compare human gene expression changes across conditions.

Availability and ImplementationVExD is freely accessible at https://vexd.cchmc.org for all modern web browsers. An Application Programming Interface (API) for VExD is also available. The source code is available at https://github.com/pdexheimer/vexd.

Contactphillip.dexheimer@cchmc.org, matthew.weirauch@cchmc.org

sentences: Single-molecule Dynamic In-Solution Inhibition Assay: A Method for Full Kinetic Profiling of Drug Candidate Binding to GPCRs in Native Membranes Kinetic profiling of drug-target interactions using surface-based label-free technologies is well established for water-soluble pharmaceutical targets but is difficult to execute for membrane proteins in general and G-protein-coupled receptors (GPCRs) in particular. That is because surface immobilization of GPCRs tends to alter their configuration and function, leading to low target coverage and non-specific binding. We here describe a novel assay for kinetic profiling of drug binding to the GPCR human beta 2 adrenergic receptor ({beta}2AR). The assay involves temporally-resolved imaging of the binding of individual {beta}2AR-containing cell membrane-derived liposomes to a surface-immobilized ligand in the presence of screened drugs. This approach allowed to determine association and dissociation constants of {beta}2AR and suspended alprenolol (antagonist) and fenoterol (agonist). The set-up combines a 384 well-plate sensor chip with automated liquid handling and the assay takes minutes to complete, making it well adapted for drug screening campaigns.

sentences: Learning interpretable cellular and gene signature embeddings from single-cell transcriptomic data The advent of single-cell RNA sequencing (scRNA-seq) technologies has revolutionized transcriptomic studies. However, large-scale integrative analysis of scRNA-seq data remains a challenge largely due to unwanted batch effects and the limited transferabilty, interpretability, and scalability of the existing computational methods. We present single-cell Embedded Topic Model (scETM). Our key contribution is the utilization of a transferable neural-network-based encoder while having an interpretable linear decoder via a matrix tri-factorization. In particular, scETM simultaneously learns an encoder network to infer cell type mixture and a set of highly interpretable gene embeddings, topic embeddings, and batch effect linear intercepts from multiple scRNA-seq datasets. scETM is scalable to over 106 cells and confers remarkable cross-tissue and cross-species zero-shot transfer-learning performance. Using gene set enrichment analysis, we find that scETM-learned topics are enriched in biologically meaningful and disease-related pathways. Lastly, scETM enables the incorporation of known gene sets into the gene embeddings, thereby directly learning the associations between pathways and topics via the topic embeddings.

sentences: ABA represses TOR and root meristem activity through nuclear exit of the SnRK1 kinase The phytohormone abscisic acid (ABA) promotes plant tolerance to major stresses like drought, partly by modulating plant growth. However, the underlying mechanisms are poorly understood. Here, we show that cell proliferation in the Arabidopsis thaliana root meristem is controlled by the interplay between three kinases, SNF1-RELATED KINASE 2 (SnRK2), the main driver of ABA signaling, the SnRK1 energy sensor, and the growth-promoting TARGET OF RAPAMYCIN (TOR) kinase. Under favorable conditions, the SnRK11 catalytic subunit is enriched in the nuclei of root cells and this is accompanied by normal cell proliferation and meristem size. Depletion of SnRK2s in a snrk2.2 snrk2.3 double mutant causes constitutive cytoplasmic localization of SnRK11 and a reduction in meristem size, suggesting that, under non-stress conditions, SnRK2s enable growth by retaining SnRK11 in the nucleus. In response to elevated ABA levels, SnRK11 translocates to the cytoplasm and this is accompanied by inhibition of TOR, decreased cell proliferation and meristem size. Blocking nuclear export with leptomycin B abrogates ABA-driven SnRK11 relocalization to the cytoplasm and the inhibition of TOR. Fusion of SnRK11 to an SV40 nuclear localization signal leads to defective TOR repression in response to ABA, demonstrating that SnRK11 nuclear exit is a premise for this repression. Altogether, we demonstrate that SnRK2-dependent changes in SnRK11 subcellular localization are crucial for the regulation of TOR activity and root growth in response to ABA. Such swift relocalization of key regulators may represent a more general strategy of sessile organisms like plants to rapidly respond to environmental changes.

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SweepCluster: A SNP clustering tool for detecting gene-specific sweeps in prokaryotes BackgroundThe gene-specific sweep is a selection process where an advantageous mutation along with the nearby neutral sites in a gene region increases the frequency in the population. It has been demonstrated to play important roles in ecological differentiation or phenotypic divergence in microbial populations. Therefore, identifying gene-specific sweeps in microorganisms will not only provide insights into the evolutionary mechanisms, but also unravel potential genetic markers associated with biological phenotypes. However, current methods were mainly developed for detecting selective sweeps in eukaryotic data of sparse genotypes and are not readily applicable to prokaryotic data. Furthermore, some challenges have not been sufficiently addressed by the methods, such as the low spatial resolution of sweep regions and lack of consideration of the spatial distribution of mutations.

ResultsWe proposed a novel gene-centric and spatial-aware approach for identifying gene-specific sweeps in prokaryotes and implemented it in a python tool SweepCluster. Our method searches for gene regions with a high level of spatial clustering of pre-selected polymorphisms in genotype datasets assuming a null distribution model of neutral selection. The pre-selection of polymorphisms is based on their genetic signatures, such as elevated population subdivision, excessive linkage disequilibrium, or significant phenotype association. Performance evaluation using simulation data showed that the accuracy and sensitivity of the clustering algorithm in SweepCluster is above 90%. The application of SweepCluster in two real datasets from the bacteria Streptococcus pyogenes and Streptococcus suis showed that the impact of pre-selection was dramatic and significantly reduced the uninformative signals. We validated our method using the genotype data from Vibrio cyclitrophicus, the only available dataset of gene-specific sweeps in bacteria, and obtained a concordance rate of 78%. We noted that the concordance rate could be underestimated due to distinct reference genomes and clustering strategies. The application to the human genotype datasets showed that SweepCluster is also applicable to eukaryotic data and recovered the known sweep regions in a wide dynamic range of pre-selection parameters.

ConclusionsSweepCluster is applicable to a broad category of datasets. It will be valuable for detecting gene-specific sweeps in diverse genotypic data and provide novel insights on adaptive evolution.

sentences: The genetic basis of interspecies cooperation and conflict in naturally-occurring isolates of a model plant symbiont Given the need to predict the outcomes of evolution in host-associated microbiomes, whether microbial and host fitnesses tend to trade off, generating conflict, remains a pressing question. Examining the relationships between host and microbe fitness proxies at both the phenotypic and genomic levels can illuminate the mechanisms underlying interspecies cooperation and conflict. We conducted genome-wide association studies (GWAS) to examined naturally-occurring genetic variation in 191 strains of the model microbial symbiont, Ensifer meliloti, paired with each of two host Medicago truncatula genotypes in single- or multi-strain experiments. Using this data, we determine how multiple proxies of microbial and host fitness were related to one another and test key predictions about mutualism evolution at the genomic scale, while also addressing the challenge of measuring microbial fitness. We found little evidence for interspecies fitness conflict; loci tended to have concordant effects on both microbe and host fitnesses, even in environments with multiple co-occurring microbes. Our results emphasize the importance of quantifying microbial relative fitness for understanding microbiome evolution and thus harnessing microbiomes to improve host fitness. Additionally, we find that mutualistic coevolution between hosts and microbes potentially acts to maintain, rather than erode, genetic diversity, thus, explaining why variation in mutualism traits persists in nature.

sentences: Tyr is Responsible for the Cctq1a QTL and Links Developmental Environment to Central Corneal Thickness Determination Central corneal thickness is a quantitative trait with important associations to human health. In a phenotype-driven approach studying corneal thickness of congenic derivatives of C57BLKS/J and SJL/J mice, the critical region for a quantitative trait locus influencing corneal thickness, Cctq1a, was delimited to a 10-gene interval. Exome sequencing, RNAseq, and studying independent mutations eliminated multiple candidate genes and confirmed one. Though the causative gene, Tyr, has no obvious direct function in the transparent cornea, studies with multiple alleles on matched genetic backgrounds, both in isolation and genetic complementation crosses, confirmed allelism of Tyr-Cctq1a; albino mice lacking Tyr function had thin corneas. Albino mice also had increased axial length. Because albinism exposes eyes to increased light, the effect of dark-rearing was tested and found to rescue central corneal thickness. In sum, the results point to an epiphenomenon; developmental light exposure interacts with genotype as an important determinate of adult corneal thickness.

sentences: Disruption of the surfactant protein A receptor SP-R210L (CD245α/MYO18Aα) alters respiratory function and iron sequestration in alveolar macrophages of aged mice Previous studies demonstrated that the host defense collectins, surfactant protein A and complement component 1q, modulate tissue-dependent macrophage activation, pathogen clearance, and regulatory macrophage functions through the receptor SP-R210, which consists of two isoforms SP-R210L and SP-R210S. These isoforms are encoded by alternatively spliced mRNAs of the Myo18a MYO18A gene in mice and humans. The present study in conditional transgenic mice revealed novel age-related functions of the SP-R210L isoform in modulating pulmonary mechanics, iron sequestration in alveolar macrophages (AMs), and life-long maintenance of the alveolar macrophage population. Our findings support the novel idea that SP-R210L-deficient AMs undergo bi-directional epigenetic adaptation that results in chronic dysregulation of broncho-alveolar function, immune homeostasis, and maintenance of oncotic balance at the airway-capillary interface. Disruption of SP-R210L increases the risk for development of severe interstitial lung disease during development and aging.

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Single gene targeted nanopore sequencing enables simultaneous identification and antimicrobial resistance detection of sexually transmitted infections ObjectivesTo develop a simple DNA sequencing test for simultaneous identification and antimicrobial resistance (AMR) detection of multiple sexually transmitted infections (STIs).

MethodsReal-time PCR (qPCR) was initially performed to identify Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) infections among a total of 200 vulvo-vaginal swab samples from female sex workers in Ecuador. qPCR positive samples plus qPCR negative controls for these STIs were subjected to single gene targeted PCR MinION-nanopore sequencing using the smartphone operated MinIT.

ResultsAmong 200 vulvo-vaginal swab samples 43 were qPCR positive for at least one of the STIs. Single gene targeted nanopore sequencing generally yielded higher pathogen specific read counts in qPCR positive samples than qPCR negative controls. Of the 26 CT, NG or MG infections identified by qPCR, 25 were clearly distinguishable from qPCR negative controls by read count. Discrimination of TV qPCR positives from qPCR negative controls was poorer as many had low pathogen loads (qPCR cycle threshold >35) which produced few specific reads. Real-time AMR profiling revealed that 3/3 NG samples identified had gyrA mutations associated with fluoroquinolone resistance, while none of the MG samples possessed 23S rRNA gene mutations contributing to macrolide resistance.

ConclusionsSingle gene targeted nanopore sequencing for diagnosing and simultaneously identifying key antimicrobial resistance markers for four common genital STIs shows promise. Further work to optimise accuracy, reduce costs and improve speed may allow sustainable approaches for managing STIs and emerging AMR in resource poor and laboratory limited settings.

sentences: dSeqSb: A systems biology approach to decipher dynamics of 1 host-pathogen interactions using temporal dual RNA-seq data Infection triggers a dynamic cascade of reciprocal events between host and pathogen wherein the host activates complex mechanisms to recognise and kill pathogens while the pathogen adjusts its virulence and fitness to avoid eradication by the host. The interaction between the pathogen and the host results in large-scale changes in gene expression in both organisms. Dual RNA-seq, the simultaneous detection of host and pathogen transcripts, has become a leading approach to unravel complex molecular interactions between the host and the pathogen and is particularly informative for intracellular organisms. The amount of in vitro and in vivo dual RNA-seq data is rapidly growing which demands computational pipelines to effectively analyse such data. In particular, holistic, systems-level, and temporal analyses of dual RNA-seq data are essential to enable further insights into the host-pathogen transcriptional dynamics and potential interactions. Here, we developed an integrative network-driven bioinformatics pipeline, dRNASb, a systems biology-based computational pipeline to analyse temporal transcriptional clusters, incorporate molecular interaction networks (e.g., protein-protein interactions), identify topologically and functionally key transcripts in host and pathogen, and associate host and pathogen temporal transcriptome to decipher potential between-species interactions. The pipeline is applicable to various dual RNA-seq data from different species and experimental conditions. As a case study, we applied dRNASb to analyse temporal dual RNA-seq data of Salmonella-infected human cells, which enabled us to uncover genes contributing to the infection process and their potential functions and to identify potential host-pathogen interactions between host and pathogen genes. Overall, dRNASb has the potential to identify key genes involved in bacterial growth or host defence mechanisms for future uses as therapeutic targets.

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Benchmarking topological accuracy of bacterial phylogenomic workflows using in silico evolution Phylogenetic analyses are widely used in microbiological research, for example to trace the progression of bacterial outbreaks based on whole-genome sequencing data. In practice, multiple analysis steps such as de novo assembly, alignment and phylogenetic inference are combined to form phylogenetic workflows. Comprehensive benchmarking of the accuracy of complete phylogenetic workflows is lacking.

To benchmark different phylogenetic workflows, we simulated bacterial evolution under a wide range of evolutionary models, varying the relative rates of substitution, insertion, deletion, gene duplication, gene loss and lateral gene transfer events. The generated datasets corresponded to a genetic diversity usually observed within bacterial species ([≥]95% average nucleotide identity). We replicated each simulation three times to assess replicability. In total, we benchmarked seventeen distinct phylogenetic workflows using 8 different simulated datasets.

We found that recently developed k-mer alignment methods such as kSNP and SKA achieve similar accuracy as reference mapping. The high accuracy of k-mer alignment methods can be explained by the large fractions of genomes these methods can align, relative to other approaches. We also found that the choice of de novo assembly algorithm influences the accuracy of phylogenetic reconstruction, with workflows employing SPAdes or SKESA outperforming those employing Velvet. Finally, we found that the results of phylogenetic benchmarking are highly variable between replicates.

We conclude that for phylogenomic reconstruction k-mer alignment methods are relevant alternatives to reference mapping at species level, especially in the absence of suitable reference genomes. We show de novo genome assembly accuracy to be an underappreciated parameter required for accurate phylogenomic reconstruction.

Impact statementPhylogenetic analyses are crucial to understand the evolution and spread of microbes. Among their many applications is the reconstruction of transmission events which can provide information on the progression of pathogen outbreaks. For example, to investigate foodborne outbreaks such as the 2011 outbreak of Escherichia coli O104:H4 across Europe. As different microbes evolve differently, it is important to know which phylogenetic workflows are most accurate when working with diverse bacterial data. However, benchmarks usually consider only a limited dataset. We therefore employed a range of simulated evolutionary scenarios and benchmarked seventeen phylogenetic workflows on these simulated datasets. An advantage of our simulation approach is that we know a priori what the outcome of the analyses should be, allowing us to benchmark accuracy. We found significant differences between phylogenetic workflows and were able to dissect which factors contribute to phylogenetic analysis accuracy. Taken together, this new information will hopefully enable more accurate phylogenetic analysis of bacterial outbreaks.

Data summaryA Zenodo repository is available at https://doi.org/10.5281/zenodo.5036179 containing all simulated genomes, all alignments produced by phylogenetic workflows and .csv files summarising the topological accuracies of phylogenies produced based on these alignments. Code is available at https://github.com/niekh-13/phylogenetic_workflows.

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Brain transcriptomic profiling reveals common alterations across neurodegenerative and psychiatric disorders Neurodegenerative and neuropsychiatric disorders (ND-NPs) are multifactorial, polygenic and complex behavioral phenotypes caused by brain abnormalities. Large-scale collaborative efforts have tried to identify the genetic architecture of these conditions. However, specific and shared underlying molecular pathobiology of brain illnesses is not clear. Here, we examine transcriptome-wide characterization of eight conditions, using a total of 2,633 post-mortem brain samples from patients with Alzheimers disease (AD), Parkinsons disease (PD), Progressive Supranuclear Palsy (PSP), Pathological Aging (PA), Autism Spectrum Disorder (ASD), Schizophrenia (Scz), Major Depressive Disorder (MDD), and Bipolar Disorder (BP)-in comparison with 2,078 brain samples from matched control subjects.

Similar transcriptome alterations were observed between NDs and NPs with the top correlations obtained between Scz-BP, ASD-PD, AD-PD, and Scz-ASD. Region-specific comparisons also revealed shared transcriptome alterations in frontal and temporal lobes across NPs and NDs. Co-expression network analysis identified coordinated dysregulations of cell-type-specific modules across NDs and NPs. This study provides a transcriptomic framework to understand the molecular alterations of NPs and NDs through their shared- and specific gene expression in the brain.

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CryoGrid-PIXUL-RNA: High throughput RNA isolation platform for tissue transcript analysis Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in life sciences analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such specimens remain, in comparison, inefficient. To mitigate this bottleneck, we have developed a low-cost user-friendly system, the CryoGrid, that consists of CryoBlock, thermometer/thermocouple, and QR coded CryoTrays to freeze and store frozen tissue fragments, and hand-held CryoCore tool for tissue sampling supported by iPad and Google apps to display tissues, direct coring and share metadata.

RNA is one of the most studied analytes. There is a decades-long history of developing methods to isolate and analyze RNA. Still, the throughput of sampling and RNA extraction from tissues has not matched that of the high throughput transcriptome analytical platforms. To address this need, we have integrated the CryoGrid system with PIXUL-based methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. TRIzol is commonly used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. We developed a PIXUL-based TRIzol-free RNA isolation fast protocol that uses a buffer containing proteinase K (PK). Virtually every disease (and often therapeutic agents toxicity) is a systemic syndrome but often only one organ is examined. CryoGrid-PIXUL, integrated with either TRIzol or PK buffer RNA isolation protocols, yielded similar RNA profiles in a multiorgan (brain, heart, kidney and liver) mouse model of sepsis. Thus, RNA isolation using the CryoGrid-PIXUL system combined with the PK buffer offers an inexpensive user-friendly workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.

sentences: The Host Cell's Endoplasmic Reticulum Proteostasis Network Profoundly Shapes the Protein Sequence Space Accessible to HIV Envelope The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, however, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody and drug escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s activation. This phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is activated, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.

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Sexual dimorphism in epilepsy and comorbidities in Dravet syndrome mice carrying a targeted deletion of exon 1 of the Scn1a gene ObjectiveDravet Syndrome (DS) is a catastrophic form of paediatric epilepsy associated with multiple comorbidities mainly caused by mutations in the SCN1A gene. DS progresses in three different phases termed febrile, worsening and stabilization stage. Mice that are haploinsufficient for Scn1a faithfully model each stage of DS, although various aspects have not been fully described, including the temporal appearance and sex differences of the epilepsy and comorbidities. The aim of the present study was to investigate the epilepsy landscape according to the progression of DS and the long-term co-morbidities in the Scn1a(+/-)tm1Kea DS mouse line that are not fully understood yet.

MethodsMale and female F1.Scn1a(+/+) and F1.Scn1a(+/-)tm1Kea mice were assessed in the hyperthermia model or monitored by video electroencephalogram (vEEG) and wireless video-EEG according to the respective stage of DS. Long-term comorbidities were investigated through a battery of behaviour assessments in [~]6 month-old mice.

ResultsAt P18, F1.Scn1a(+/-)tm1Kea mice showed the expected sensitivity to hyperthermia-induced seizures. Between P21 and P28, EEG recordings in F1.Scn1a(+/-)tm1Kea mice combined with video monitoring revealed a high frequency of SRS and SUDEP. Power spectral analyses of background EEG activity also revealed that low EEG power in multiple frequency bands was associated with SUDEP risk in F1.Scn1a(+/-)tm1Kea mice during the worsening stage of DS. Later, SRS and SUDEP rates stabilized and then declined in F1.Scn1a(+/-)tm1kea mice. SRS and SUDEP in F1.Scn1a(+/-)tm1kea mice displayed variations with the time of day and sex, with female mice displaying higher numbers of seizures and greater SUDEP risk. F1.Scn1a(+/-)tm1kea mice [~]6 month- old displayed fewer behavioural impairments than expected including hyperactivity, impaired exploratory behaviour and poor nest building performance.

SignificanceThese results reveal new features of this model that will optimize use and selection of phenotype assays for future studies on the mechanisms, diagnosis, and treatment of DS.

Key point boxO_LIScn1a(+/-)tm1kea DS mouse model faithfully reproduces the three stages of DS
C_LIO_LISex of F1.Scn1a(+/-)tm1kea mice influences the epilepsy phenotype
C_LIO_LIF1.Scn1a(+/-)tm1kea develop some of the long-term comorbidities of DS
C_LI

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Activity-mediated accumulation of potassium induces a switch in firing pattern and neuronal excitability type During normal neuronal activity, ionic concentration gradients across a neurons membrane are often assumed to be stable. Prolonged spiking activity, however, can reduce transmembrane gradients and affect voltage dynamics. Based on mathematical modeling, we investigated the impact of neuronal activity on ionic concentrations and, consequently, the dynamics of action potential generation. We find that intense spiking activity on the order of a second suffices to induce changes in ionic reversal potentials and to consistently induce a switch from a regular to an intermittent firing mode. This transition is caused by a qualitative alteration in the systems voltage dynamics, mathematically corresponding to a co-dimension-two bifurcation from a saddle-node on invariant cycle (SNIC) to a homoclinic orbit bifurcation (HOM). Our electrophysiological recordings in mouse cortical pyramidal neurons confirm the changes in action potential dynamics predicted by the models: (i) activity-dependent increases in intracellular sodium concentration directly reduce action potential amplitudes, an effect typically attributed solely to sodium channel inactivation; (ii) extracellular potassium accumulation switches action potential generation from tonic firing to intermittently interrupted output. Thus, individual neurons may respond very differently to the same input stimuli, depending on their recent patterns of activity and/or the current brain-state.

Author summaryIonic concentrations in the brain are not constant. We show that during intense neuronal activity, they can change on the order of seconds and even switch neuronal spiking patterns under identical stimulation from a regular firing mode to an intermittently interrupted one. Triggered by an accumulation of extracellular potassium, such a transition is caused by a specific, qualitative change in of the neuronal voltage dynamics - a so-called bifurcation - which affects crucial features of action-potential generation and bears consequences for how information is encoded and how neurons behave together in the network. Also, changes in intracellular sodium can induce measurable effects, like a shrinkage of spike amplitude that occurs independently of the fast amplitude-effects attributed to sodium channel inactivation. Taken together, our results demonstrate that a neuron can respond very differently to the same stimulus, depending on its previous activity or the current brain state. This finding may be particularly relevant when other regulatory mechanisms of ionic homeostasis are challenged, for example, during pathological states of glial impairment or oxygen deprivation. Finally, Categorization of cortical neurons as intrinsically bursting or regular spiking may be biased by the ionic concentrations at the time of the observation, highlighting the non-static nature of neuronal dynamics.

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Age-related change in task-evoked amygdala-prefrontal circuitry: a multiverse approach with an accelerated longitudinal cohort aged 4-22 years The amygdala and its connections with medial prefrontal cortex (mPFC) play central roles in the development of emotional processes. While several studies have suggested that this circuitry exhibits functional changes across the first two decades of life, findings have been mixed - perhaps resulting from differences in analytic choices across studies. Here we used multiverse analyses to examine the robustness of task-based amygdala-mPFC function findings to analytic choices within the context of an accelerated longitudinal design (4-22 years- old; N=98; 183 scans; 1-3 scans/participant). Participants, recruited from the greater Los Angeles area, completed an event-related emotional face (fear, neutral) task. Parallel analyses varying in preprocessing and modeling choices found that age-related change estimates for amygdala reactivity were more robust than task-evoked amygdala-mPFC functional connectivity to varied analytical choices. Specification curves indicated evidence for age-related decreases in amygdala reactivity to faces, though within-participant changes in amygdala reactivity could not be differentiated from between-participant differences. In contrast, amygdala--mPFC functional connectivity results varied across methods much more, and evidence for age-related change in amygdala-mPFC connectivity was not consistent. Generalized psychophysiological interaction (gPPI) measurements of connectivity were especially sensitive to whether a deconvolution step was applied. Our findings demonstrate the importance of assessing the robustness of findings to analysis choices, although the age-related changes in our current work cannot be overinterpreted given low test-retest reliability. Together, these findings highlight both the challenges in estimating developmental change in longitudinal cohorts and the value of multiverse approaches in developmental neuroimaging for assessing robustness of results. (Preprint: https://www.biorxiv.org/content/10.1101/2021.10.08.463601v1).

Key PointsO_LIMultiverse analyses applied to fMRI data are valuable for determining the robustness of findings to varied analytical choices
C_LIO_LIIn the current study, age-related change estimates for amygdala reactivity were relatively robust to analytical decisions, though gPPI functional connectivity analyses were much more sensitive, leading some estimates to flip sign
C_LIO_LIBoth test-retest reliability and robustness to analytical choices are important considerations for developmental research
C_LI

sentences: Comprehensive evaluation of machine learning models and gene expression signatures for prostate cancer prognosis using large population cohorts Overtreatment remains the pervasive problem in prostate cancer (PCa) management due to the highly variable and often indolent course. Molecular signatures derived from gene expression profiling have played critical roles in PCa treatment decision-making. Many gene expression signatures have been developed to improve the risk stratification of PCa and some of them have already been translationally applied to clinical practice, however, no comprehensive evaluation was performed to compare the performances of the signatures. In this study, we conducted a systematic and unbiased evaluation of 15 machine learning (ML) algorithms and 30 published PCa gene expression-based prognostic signatures leveraging 10 transcriptomics datasets with 1,558 primary PCa patients from public data repositories. The results revealed that survival analysis models outperformed binary classification models for risk assessment, and the performances of the survival analysis methods - Cox model regularized with ridge penalty (Cox-Ridge) and partial least squares regression for Cox model (Cox-PLS) - were generally more robust than the other methods. Based on the Cox-Ridge algorithm, a few top prognostic signatures that have comparable or even better performances than the commercial panels have been identified. The findings from the study will greatly facilitate the identification of existing prognostic signatures that are promising for further validations in prospective studies and promote the development of robust prognostic models to guide clinical decision-making. Moreover, the study provided a valuable data resource from large primary PCa cohorts, which can be used to develop, validate, and evaluate novel statistical methodologies and molecular signatures to improve PCa management.

sentences: Structure of a Plant-Specific Partitivirus Capsid Reveals a Novel Coat Protein Architecture with a Hypervariable Protrusion Persistent plant viruses may be the most common viruses in wild plants. A growing body of evidence for mutualism between such viruses and their hosts, suggests that they play an important role in ecology and agriculture. Here we present the structure of a plant-specific partitivirus capsid at 2.9 [A] resolution by Cryo-EM. Structural features, including the T=1 arrangement of 60 coat protein dimers, are shared with fungal partitiviruses and the picobirnavirus lineage of dsRNA viruses. However, the topology of the capsid is markedly different with protrusions emanating from, and partly comprising, the binding interface of coat protein dimers. We show that a disordered region at the apex of the protrusion is not required for capsid assembly and represents a hypervariable site characteristic of the plant-specific partitiviruses. These results suggest a structural basis for the acquisition of additional functions by partitivirus coat proteins that enables mutualistic relationships with diverse plant hosts.

sentences: The hormone Fibroblast Growth Factor 19 stimulates water intake Fibroblast growth factor 19 (FGF19) is a hormone with pleiotropic metabolic functions, leading to ongoing development of analogues for the treatment of metabolic disorders. On the other hand, FGF19 is overexpressed in a sub-group of hepatocellular carcinoma (HCC) patients and has oncogenic properties. It is therefore crucial to precisely define FGF19 effects, notably chronic exposure to elevated concentrations of the hormone. We used hydrodynamic gene transfer approach to generate a transgenic mouse model with long-term FGF19 hepatic overexpression. Here we describe a novel effect of FGF19, namely stimulation of water intake. This phenotype, lasting at least over a 6-month period, depends on signaling in the central nervous system and is independent of FGF21, although it mimics some of its features. We further show that HCC patients with high levels of circulating FGF19 have a reduced natremia, indicating dispogenic features. The present study provides evidence of a new activity for FGF19, which could be clinically relevant in the context of FGF19 overexpressing cancers and treatment of metabolic disorders by FGF19 analogues.

sentences: A divergent protein kinase A in the human pathogen Leishmania is associated with developmental morphogenesis Parasitic protozoa of the genus Leishmania cause human leishmaniasis. They cycle between the phagolysosome of mammalian macrophages, where they reside as round intracellular amastigotes, and the mid-gut of female sand flies, which they colonize as elongated extracellular promastigotes. Shifting promastigotes to a lysosome-like environment (pH 5.5 and 37C, 5% CO) initiates their development into amastigotes. Previous studies suggested a role for protein kinase A (PKA) in this differentiation process. Here, we describe a new, divergent, regulatory subunit (PKAR3) found only in a limited member of the family Kinetoplastidae. In L. donovani, phosphorylation of PKAR3 was regulated by the differentiation signal and coincided with parasite morphogenesis during stage development. LdPKAR3 was bound to the subpellicular microtubules cell cortex via a formin homology (FH2)-like domain at the tip of a large and divergent N-terminal domain. Immunoprecipitation, fluorescence resonance energy transfer (FRET), proteomics analyses, and structural modeling showed that PKAR3 selectively binds the C3 isoform of the PKA catalytic subunit in a holoenzyme complex. In promastigotes, PKAR3 recruited PKAC3 to the subpellicular microtubules at the central cortex. After exposure to a differentiation signal, PKAR3 homogenously distributed across the entire cortex, in concert with cell rounding. Deleting the genes encoding either the R3 or C3 subunit resulted in premature rounding of the promastigote population, indicating that PKA determines the normal elongated shape. Regulation of Leishmania developmental morphogenesis by interaction with the subpellicular microtubule corset is a novel function for a unique PKA complex not present in the host cell.

sentences: Neighbor-specific gene expression revealed from physically interacting cells during mouse embryonic development Cells continuously communicate with the neighboring cells during development. Direct interaction of different cell types can induce molecular signals dictating lineage specification and cell fate decisions. The current single cell RNAseq (scRNAseq) technology cannot study cell contact dependent gene expression due to the loss of spatial information. To overcome this issue and determine cell contact specific gene expression during embryogenesis, we performed RNA sequencing of physically interacting cells (PICseq) and assessed alongside our single cell transcriptomes (scRNAseq) derived from developing mouse embryos between embryonic day (E) 7.5 and E9.5. Analysis of PICseq data identifies an interesting suite of gene expression signatures depending on neighboring cell types. For instance, neural progenitor (NP) cells expressed Nkx2-1 when interacting with definitive endoderm (DE) and DE cells expressed Gsc when interacting with NP. Based on the identified cell contact specific genes, we devised a means to predict the neighboring cell types from individual cell transcriptome. We further developed spatial-tSNE to show the pseudo-spatial distribution of cells in a 2-dimensional space. In sum, we suggest an approach to study contact specific gene regulation during embryogenesis.

sentences: Role of BMP signaling during early development of the annelid Capitella teleta The mechanisms regulating nervous system development are still unknown for a wide variety of taxa. In insects and vertebrates, bone morphogenetic protein (BMP) signaling plays a key role in establishing the dorsal-ventral (D-V) axis and limiting the neuroectoderm to one side of that axis, leading to speculation about the conserved evolution of centralized nervous systems. Studies outside of insects and vertebrates show a more diverse picture of what, if any role, BMP signaling plays in neural development across Bilateria. This is especially true in the morphologically diverse Spiralia (~Lophotrochozoa). Despite several studies of D-V axis formation and neural induction in spiralians, there is no consensus for how these two processes are related, or whether BMP signaling may have played an ancestral role in either process. To determine the function of BMP signaling during early development of the spiralian annelid Capitella teleta, we incubated embryos and larvae in BMP4 protein for different amounts of time. Adding exogenous BMP protein to early-cleaving C. teleta embryos had a striking effect on formation of the brain, eyes, foregut, and ventral midline in a time-dependent manner. However, adding BMP did not block brain or VNC formation or majorly disrupt the D-V axis. We identified three key time windows of BMP activity. 1) BMP treatment around birth of the 3rd-quartet micromeres caused the loss of the eyes, radialization of the brain, and a reduction of the foregut, which we interpret as a loss of A- and C-quadrant identities with a possible trans-fate switch to a D-quadrant identity. 2) Treatment after birth of micromere 4d induced formation of a third ectopic brain lobe, eye, and foregut lobe, which we interpret as a trans-fate switch of B-quadrant micromeres to a C-quadrant identity. 3) Continuous BMP treatment from late cleavage (4d + 12h) through mid-larval stages resulted in a modest expansion of Ct-chrdl expression in the dorsal ectoderm and a concomitant loss of the ventral midline (neurotroch ciliary band). Loss of the ventral midline was accompanied by a collapse of the bilaterally-symmetric ventral nerve cord, although the total amount of neural tissue did not appear to be greatly affected. Our results compared to those from other annelids and molluscs suggest that BMP signaling was not ancestrally involved in delimiting neural tissue to one region of the D-V axis. However, the effects of ectopic BMP on quadrant-identity during cleavage stages may represent a non-axial organizing signal that was present in the last common ancestor of annelids and mollusks. Furthermore, in the last common ancestor of annelids, BMP signaling may have functioned in patterning ectodermal fates along the D-V axis in the trunk. Ultimately, studies on a wider range of spiralian taxa are needed to determine the role of BMP signaling during neural induction and neural patterning in the last common ancestor of this group. Ultimately, these comparisons will give us insight into the evolutionary origins of centralized nervous systems and body plans.

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Three-dimensional label-free histological imaging of whole organs by microtomy-assisted autofluorescence tomography Three-dimensional (3D) histology is vitally important to characterize disease-induced tissue heterogeneity at the individual cell level. However, it remains a scientific challenge for both high-quality 3D imaging and volumetric reconstruction. Here we propose a label-free, automated, and ready-to-use 3D histological imaging technique, termed microtomy-assisted autofluorescence tomography with ultraviolet excitation (MATE). With the combination of block-face imaging and serial microtome sectioning, MATE can achieve rapid and label-free imaging of paraffin-embedded whole organs at an acquisition speed of 1 cm3 per 4 hours with a voxel resolution of 1.2 x 1.2 x 10 m3. We demonstrate that MATE enables simultaneous visualization of cell nuclei, fiber tracts, and blood vessels in mouse/human brains without tissue staining or clearing. Moreover, diagnostic features, such as nuclear size and packing density, can be quantitatively extracted with high accuracy. MATE is augmented to the current slide-based 2D histology, holding great promise for facilitating histopathological interpretation at the cell level to analyze complex tissue heterogeneity in 3D.

Significance StatementConventional 3D histology based on spatial registration of serial histochemically-stained thin tissue slices is fundamentally labor-intensive and inaccurate. Here, we propose a rapid and label-free 3D histological imaging technique (i.e., MATE) that enables high-resolution imaging of complex whole organs without tissue staining or clearing. MATE is fully automated to provide a series of distortion- and registration-free images with intrinsic absorption-based contrast, demonstrating great potential as a routine tissue analysis tool that can seamlessly fit into the current clinical practice to facilitate the applications of histopathological interpretation at the subcellular level.

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GFAP-directed Inactivation of Men1 Exploits Glial Cell Plasticity in Favor of Neuroendocrine Reprogramming BACKGROUND & AIMSEfforts to characterize the signaling mechanisms that underlie gastroenteropancreatic neoplasms (GEP-NENs) are precluded by a lack of comprehensive model systems that recapitulate pathogenesis. Investigation into a potential cell-of-origin for gastrin-secreting NENs revealed a role for enteric glia in neuroendocrine cell specification. Here we investigated the hypothesis that loss of menin in glial cells stimulated neuroendocrine differentiation and tumorigenesis.

METHODSUsing Cre-lox technology, we generated a conditional glial fibrillary acidic protein-directed Men1 knockout (GFAP{Delta}Men1) mouse model. Cre specificity was confirmed using a tdTomato reporter. GFAP{Delta}Men1 mice were evaluated for GEP-NEN development and neuroendocrine cell hyperplasia. siRNA-mediated Men1 silencing in a rat enteric glial cell line was performed in parallel.

RESULTSGFAP{Delta}Men1 mice developed pancreatic NENs, in addition to pituitary prolactinomas that phenocopied the human MEN1 syndrome. GFAP{Delta}Men1 mice exhibited gastric neuroendocrine hyperplasia that coincided with a significant loss of GFAP expression. Mechanistically, Men1 deletion induced reprogramming from a mature glial phenotype toward a neuroendocrine lineage. Furthermore, blockade of Hedgehog signaling in enteric glia attenuated neuroendocrine hyperplasia by restricting the neuroendocrine cell fate.

CONCLUSIONSGFAP-directed Men1 inactivation exploits glial cell plasticity in favor of neuroendocrine differentiation.

GRAPHICAL ABSTRACT

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sentences: Whole-genome sequencing analysis reveals the population history of Mus musculus in Madagascar In Madagascar, the house mouse (Mus musculus) is thought to have colonized with human activities and is now one of the most abundant rodents on the island. In this study, we determined the whole-genome sequences of five Madagascar house mice captured from the wild. We examined the evolutionary history of the population by analyzing the mitochondrial and autosomal genomes. We confirmed that the mitochondrial genome lineages of Madagascar house mice formed a monophyletic clade placed at one of the most basal positions in the species. An analysis of autosomal genomic sequences indicates that the Madagascar house mice are genetically members of M. m. castaneus (CAS), but also contain genetic elements of M. m. domesticus (DOM) resulting from hybridization between subspecies. The signature of a strong population bottleneck 1000-3000 years ago was observed in both mitochondrial and autosomal genomic data. All samples showed strong genetic affinity to many CAS samples across a wide range of Indian Ocean coastal and island regions, with divergence time estimated around 4000 years ago. These findings support that the Madagascar house mice started to colonize the island with human agricultural activity, and experienced complex history for the establishment.

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Simple, sensitive and cost-effective detection of wAlbB Wolbachia in Aedes mosquitoes, using loop mediated isothermal amplification combined with the electrochemical biosensing method BackgroundWolbachia is an endosymbiont bacterium generally found in about 40% of insects, including mosquitoes, but it is absent in Aedes aegypti which is an important vector of several arboviral diseases. The evidence that Wolbachia trans-infected Ae. aegypti mosquitoes lost their vectorial competence and became less capable of transmitting arboviruses to human hosts highlights the potential of using Wolbachia- based approaches for prevention and control of arboviral diseases. Recently, release of Wolbachia trans-infected Ae. aegypti has been deployed widely in many countries for the control of mosquito-borne viral diseases. Field surveillance and monitoring of Wolbachia presence in released mosquitoes is important for the success of these control programs. So far, a number of studies have reported the development of loop mediated isothermal amplification (LAMP) assays to detect Wolbachia in mosquitoes, but the methods still have some specificity issues.

Methodology/Principal FindingsWe describe here the development of a LAMP combined with the DNA strand displacement-based electrochemical sensor (BIOSENSOR) method to detect wAlbB Wolbachia in trans-infected Ae. aegypti. Our developed LAMP primers were more specific to wAlbB detection than those of the previous published ones if the assays were conducted with low-cost and non-specific detecting dyes. The detection capacity of our LAMP technique was 3.8 nM and the detection limit reduced to 2.16 fM when combined with the BIOSENSOR. Our study demonstrates that the BIOSENSOR can also be applied as a stand-alone method for detecting Wolbachia; and it showed high sensitivity when used with the crude DNA extracts of macerated mosquito samples without DNA purification.

Conclusions/SignificanceOur results suggest that both LAMP and BIOSENSOR, either used in combination or stand-alone, are robust and sensitive. The methods have good potential for routine detection of Wolbachia in mosquitoes during field surveillance and monitoring of Wolbachia-based release programs, especially in countries with limited resources.

Author SummaryMosquito-borne diseases such as dengue, chikungunya, Zika, and yellow fever are transmitted to humans mainly by the bites of Aedes aegypti mosquitoes. Controlling these diseases relies mostly on the use of insecticides, in which the efficiency has been reduced through development of insecticide resistance in mosquitoes. Wolbachia is the endosymbiotic bacteria that are naturally found in 40% of insects, including mosquitoes. The bacteria could protect their hosts from viral infections and could also cause sterility in host populations, therefore, providing an opportunity to use them for disease control. Application of a Wolbachia-based strategy needs simple, rapid and sensitive methods for detecting the bacteria in released mosquitoes. In this paper, we develop the combined methods of LAMP and BIOSENSORS for detecting wAlbB Wolbachia in mosquitoes. Our positive LAMP reaction can be visualized by color change from violet to blue at a sensitivity of [≥] 60 pg of genomic DNA. When used in combination with the BIOSENSOR method, the sensitivity increased a million fold without losing specificity. Our study indicates that both developed methods, either used in combination or stand-alone, are efficient and cost-effective, hence, it could be applied for routine surveys of Wolbachia in mosquito control programs that use Wolbachia-based approaches.

sentences: Further perceptions of probability: in defence of trial-by-trial estimation models Many events we experience are binary and probabilistic, such as the weather (rain or no rain) and the outcome of medical tests (negative or positive). Extensive research in the behavioural sciences has addressed peoples ability to learn stationary probabilities (i.e., probabilities that stay constant over time) of such events, but only recently have there been attempts to model the cognitive processes whereby people learn - and track - non-stationary probabilities. The old debate on whether learning occurs trial-by-trial or by occasional shifts between discrete hypotheses has been revived in this context. Trial-by-trial estimation models - such as the delta-rule model - have been successful in describing human learning in various contexts. It has been argued, however, that behaviour on non-stationary probability learning tasks is incompatible with trial-by-trial learning and can only be explained by models in which learning proceeds through hypothesis testing. Here, we show that this conclusion was premature. By combining two well-supported concepts from cognitive modelling - delta-rule learning and drift diffusion evidence accumulation - we reproduce all behavioural phenomena that were previously used to reject trial-by-trial learning models. Moreover, a quantitative model comparison shows that this model accounts for the data better than a model based on hypothesis testing. In the spirit of cumulative science, our results demonstrate that a combination of two well-established theories of trial-by-trial learning and evidence accumulation is sufficient to explain human learning of non-stationary probabilities.

sentences: Binding specificity of ASHH2 CW-domain towards H3K4me1 ligand is coupled to its structural stability through its α1-helix The CW-domain binds to histone-tail modifications found in different protein families involved in epigenetic regulation and chromatin remodelling. CW-domains recognize the methylation state of the fourth lysine on histone 3, and could therefore be viewed as a reader of epigentic information. The specificity towards different methylation states such as me1, me2 or me3 depends on the particular CW subtype. For example, the CW domain of ASHH2-methyltransferase binds preferentially to H3K4me1, MORC3 binds to both H3K4me2 and me3 modifications, while ZCWPW1 is more specific to H3K4me3. The structural basis for these preferential bindings are not well understood, and recent research suggests that a more complete picture will emerge if dynamical and energetic assessments are included in the analysis of interactions. This study uses fold assessment by NMR in combination with mutagenesis, ITC affinity measurements and thermal denaturation studies to investigate possible couplings between ASHH2 CW selectivity towards H3K4me1 and the stabilization of the domain and loops implicated in binding. Key elements of the binding site - the two tryptophans and the 1-helix form and maintain the binding pocket were perturbed by mutagenesis and investigated. Results show that 1-helix maintains the overall stability of the fold via the I915 and L919 residues, and that correct binding consolidates the loops designated {eta}1, {eta}3, as well as the C-terminal. This consolidation is incomplete for H3K4me3 binding to CW, which experiences a decrease in overall thermal stability upon binding. Moreover, loop-mutations not directly involved in the binding site nonetheless affect the equillibrium positions of key residues.

sentences: On the reproductive mechanism of Gram-negative protocells One of the major challenges of understanding early life or a bottom-up approach of building a synthetic cell is to drive the process of reproduction. To achieve reproduction completely independent of molecular biological processes, here we worked with bacterial protoplasts under environmental conditions of early Earth. Rather than reproducing in a chaotic manner due to the loss of molecular biological co-ordination, cells in our study reproduced in a defined sequence of steps. Lifecycle of cells in our study was either similar to lipid vesicles undergoing osmotic shrinkage or to lipid vesicles undergoing expansion when placed in an elongational flow path. Fission of parent cell into multiple daughter cells was achieved via forces intrinsic to cell like the Brownian motion of intracellular constituents, asymmetric conditions on either side of the membrane or induced by membrane phase-separation. In short, our study demonstrates how cells reproduced prior to the evolution of molecular biological processes.

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HEPG2 EXPRESSION OF MIR-202 & TRIB-1 UNDER METABOLIC AND INFLAMMATORY STRESS. BackgroundThe burden of cardiovascular disease (CVD) is such that affects both developed and developing countries with high rates of mortality and morbidity. Cardiovascular diseases are highly polymorphic across its various risk factors. Human polymorphisms of trib-1 gene have been implicated to be associated with risk factors for CVD. Trib-1 gene is a known target for microRNA-202 which consequently could have an effect on its stability. The objective of this study was to evaluate the expression of miR-202 in an hepatic cell line under in vitro conditions of metabolic and inflammatory stress and the effect on trib-1 level.

Materials and MethodsHepG2 cells cultured under in vitro conditions of high glucose and cytokine stimulation of concentrations of varying time intervals were harvested and mRNA/microRNA extracted using the spin column-based centrifugation, reversed transcribed and analysed for endogenous expressions of trib-1 and miR-202 using qPCR. One-ANOVA followed by Dunnetts multiple comparison test was used to test for significance (P<0.05) across samples.

ResultsIt was observed that there was a significant decrease in trib-1 levels under these conditions of high glucose and cytokine stimulation and also with the combination of both whilst there was a consistent pattern of upregulation of MIR-202 under this conditions.

ConclusionTaken together this study reveals that miR-202 is expressed in HepG2 cells, and a possible interaction between trib-1 and MIR-202 which could affect trib-1 stability and also the potentials for MIR-202 to be involved in some cellular activities in HepG2 cells relating to these conditions.

sentences: ERK3-MK5 signaling regulates myogenic differentiation and muscle regeneration by promoting FoxO3 degradation The physiological functions and downstream effectors of the atypical mitogen-activated protein kinase ERK3 remain to be characterized. We recently reported that mice expressing catalytically-inactive ERK3 (Mapk6KD/KD) exhibit a reduced post-natal growth rate as compared to control mice. Here, we show that genetic inactivation of ERK3 impairs post-natal skeletal muscle growth and adult muscle regeneration after injury. Loss of MK5 phenocopies the muscle phenotypes of Mapk6KD/KD mice. At the cellular level, genetic or pharmacological inactivation of ERK3 or MK5 induces precocious differentiation of C2C12 or primary myoblasts, concomitant with MyoD activation. Reciprocally, ectopic expression of activated MK5 inhibits myogenic differentiation. Mechanistically, we show that MK5 directly phosphorylates FoxO3, promoting its degradation and reducing its association with MyoD. Depletion of FoxO3 rescues in part the premature differentiation of C2C12 myoblasts observed upon inactivation of ERK3 or MK5. Our findings reveal that ERK3 and its substrate MK5 act in a linear signaling pathway to control post-natal myogenic differentiation.

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Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-Translational Modification Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with protein L-isoaspartyl methyltransferase (PIMT) homologs. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an L-aspartate sidechain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates.

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sentences: The Anti-Burkholderia Lasso Peptide Ubonodin Co-Opts the Siderophore Receptor PupB for Cellular Entry New antibiotics are needed as bacterial infections continue to be a leading cause of death. Notorious among antibiotic-resistant bacteria is the Burkholderia cepacia complex (Bcc), which infects cystic fibrosis patients, causing lung function decline. We recently discovered a novel ribosomally synthesized and post-translationally modified peptide (RiPP), ubonodin, with potent activity against several Burkholderia pathogens. Ubonodin inhibits RNA polymerase, but only select Bcc strains were susceptible, indicating that having a conserved cellular target does not guarantee activity. Given the cytoplasmic target, we speculate that cellular uptake of ubonodin determines susceptibility. Here, we report a new outer membrane siderophore receptor, PupB, that is required for ubonodin uptake in B. cepacia. Loss of PupB renders B. cepacia resistant to ubonodin, whereas expressing PupB sensitizes a resistant strain. Thus, outer membrane transport is the major determinant of ubonodins spectrum of activity. We also show that PupB is activated by a TonB protein and examine a transcriptional pathway that further regulates PupB. Finally, we elucidate the complete cellular uptake pathway for ubonodin by also identifying its inner membrane transporter in B. cepacia. Our work unravels central steps in the mechanism of action of ubonodin and establishes a general framework for dissecting RiPP function.

sentences: Age-related differences in encoding-retrieval similarity and their relationship to false memory Typical aging is associated with increases in false memory rates among older adults. Such errors are frequently associated with differential neural activity during encoding and retrieval in older compared to younger adults within visual cortices, hippocampus, and front-parietal regions. It remains unknown how pattern similarity reductions relate to false memories in healthy aging. Using encoding-retrieval similarity (ERS) analyses in a sample of younger and older adults, we examined how the similarity of neural patterns between memory phases associated with target and lure objects was impacted by age and contributed to false memory rates. Single-item ERS for targets and lures was reduced by age throughout much of the ventral visual stream and the posterior hippocampus. Furthermore, ERS associated with perceptual lures within the visual stream maintained differential relationships with false memory. Finally, a global ERS metric accounted for age deficits in single-item ERS, but did not contribute to false memory rates. These findings highlight the contribution of age-related reductions in ERS across multiple representational levels to false memories in healthy aging.

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Evolution of Phenotypic Plasticity under Host-Parasite Interactions Robustness and plasticity are essential features that allow biological systems to cope with complex and variable environments. Through the evolution of a given environment, the former, the insensitivity of phenotypes, is expected to increase, whereas the latter, the changeability of phenotypes, tends to diminish. However, in nature, plasticity is preserved to a certain degree. One possible cause for this is environmental variation, with one of the most important "environmental " factors being inter-species interactions. As a first step toward investigating phenotypic plasticity in response to an ecological interaction, we present the study of a simple two-species system consisting of hosts and parasites. Hosts are expected to evolve to achieve a phenotype that optimizes fitness and increases the robustness of the corresponding phenotype by reducing phenotypic fluctuations. Conversely, plasticity evolves in order to avoid certain phenotypes being attacked by parasites. By simulating evolution using the host gene-expression dynamics model, we analyze the evolution of genotype-phenotype mapping. If the interaction is weak, the fittest phenotype of the host evolves to reduce phenotypic variances. In contrast, if a sufficient degree of interaction occurs, the phenotypic variances of hosts increase to escape parasite attacks. For the latter case, we found two strategies: if the noise in the stochastic gene expression is below a certain threshold, the phenotypic variance increases via genetic diversification, whereas above the threshold, it is increased due to noise-induced phenotypic plasticity. We examine how the increase in the phenotypic variances due to parasite interactions influences the growth rate of a single host, and observed a trade-off between the two. Our results help elucidate the roles played by noise and genetic mutations in the evolution of phenotypic plasticity and robustness in response to host-parasite interactions.

Author summaryPlasticity and phenotypic variability induced by internal or external perturbations are common features of biological systems. However, certain environmental conditions initiate evolution to increase fitness and, in such cases, phenotypic variability is not advantageous, as has been demonstrated by previous laboratory and computer experiments. As a possible origin for such plasticity, we investigated the role of host-parasite interactions, such as those between bacteria and phages. Different parasite types attack hosts of certain phenotypes. Through numerical simulations of the evolution of host genotype-phenotype mapping, we found that, if the interaction is sufficiently strong, hosts increase phenotypic plasticity by increasing phenotypic fluctuations. Depending on the degree of noise in gene expression dynamics, there are two distinct strategies for increasing the phenotypic variances: via stochasticity in gene expression or via genetic variances. The former strategy, which can work over a faster time scale, leads to a decline in fitness, whereas the latter reduces the robustness of the fitted state. Our results provide insights into how phenotypic variances are preserved and how hosts can escape being attacked by parasites whose genes mutate to adapt to changes in parasites. These two host strategies, which depend on internal and external conditions, can be verified experimentally, for example, via the transcriptome analysis of microorganisms.

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Single Cell Atlas of Human Putamen Reveals Disease Specific Changes in Synucleinopathies: Parkinson's Disease and Multiple System Atrophy Understanding disease biology at a cellular level from disease specific tissues is imperative for effective drug development for complex neurodegenerative diseases. We profiled 87,086 nuclei from putamen tissue of healthy controls, Parkinsons Disease (PD), and Multiple System Atrophy (MSA) subjects to construct a comprehensive single cell atlas. Although both PD and MSA are manifestations of -synuclein protein aggregation, we observed that both the diseases have distinct cell-type specific changes. We see a possible expansion and activation of microglia and astrocytes in PD compared to MSA and controls. Contrary to PD microglia, we found absence of upregulated unfolded protein response in MSA microglia compared to controls. Differentially expressed genes in major cell types are enriched for genes associated with PD-GWAS loci. We found altered expression of major neurodegeneration associated genes -- SNCA, MAPT, LRRK2, and APP -- at cell-type resolution. We also identified disease associated gene modules using a network biology approach. Overall, this study creates an interactive atlas from synucleinopathies and provides major cell-type specific disease insights.

Link to interactive atlas will be made available at the time of publication.

sentences: Cryo-EM single particle structure refinement and map calculation using Servalcat In 2020, cryo-EM single particle analysis achieved true atomic resolution, thanks to technological developments in hardware and software. The number of high resolution reconstructions continues to grow, increasing the importance of accurate determination of atomic coordinates. Here, a new Python package and program called Servalcat is presented that is designed to facilitate atomic model refinement. Servalcat implements a refinement pipeline, using the program REFMAC5 from the CCP4 package. After the refinement, Servalcat calculates a weighted Fo - Fc difference map, which was derived from Bayesian statistics. This map helps manual and automatic model building in real space, as is common practice in crystallography. The Fo - Fc map helps visualisation of weak features including hydrogen densities. Although hydrogen densities are weak, they are stronger than in electron density maps produced by X-ray crystallography, and some hydrogen atoms are even visible at [~] 1.8 [A] resolution. Servalcat also facilitates atomic model refinement under symmetry constraints. If a point group symmetry has been applied to the map during reconstruction, the asymmetric unit model is refined with appropriate symmetry constraints.

sentences: Joint modeling of choices and reaction times based on Bayesian contextual behavioral control In cognitive neuroscience and psychology, reaction times are an important behavioral measure. However, in instrumental learning and goal-directed decision making experiments, findings often rely only on choice probabilities from a value-based model, instead of reaction times. Recent advancements have shown that it is possible to connect value-based decision models with reaction time models, for example in a joint reinforcement learning and diffusion decision model. We propose a novel joint model of both choices and reaction times by combining a mechanistic account of Bayesian sequential decision making with a sampling procedure. Specifically, we use a recent context-specific Bayesian forward planning model which we extend by an MCMC sampler to obtain both choices and reaction times. We show that we can explain and reproduce a rather wide range of well-known experimental findings in value based-decision making as well as classical inhibition and switching tasks. First, we use the proposed model to explain how instrumental learning and automatized behavior result in decreased reaction times and improved accuracy. Second, we reproduce classical results in the Eriksen flanker task. Third, we reproduce established findings in task switching. These findings show that the proposed joint behavioral model may describe common underlying processes in all these types of decision making paradigms.

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Rossmann-toolbox: a deep learning-based protocol for the prediction and design of cofactor specificity in Rossmann-fold proteins The Rossmann fold enzymes are involved in essential biochemical pathways such as nucleotide and amino acid metabolism. Their functioning relies on interaction with cofactors, small nucleoside-based compounds specifically recognized by a conserved {beta}{beta} motif shared by all Rossmann fold proteins. While Rossmann methyltransferases recognize only a single cofactor type, the S-Adenosylmethionine (SAM), the oxidoreductases, depending on the family, bind nicotinamide (NAD, NADP) or flavin-based (FAD) cofactors. In this study, we show that despite its short length, the {beta}{beta} motif unambiguously defines the specificity towards the cofactor. Following this observation, we trained two complementary deep learning models for the prediction of the cofactor specificity based on the sequence and structural features of the {beta}{beta} motif. A benchmark on two independent test sets, one containing {beta}{beta} motifs bearing no resemblance to those of the training set, and the other comprising 38 experimentally confirmed cases of rational design of the cofactor specificity, revealed the nearly perfect performance of the two methods. The Rossmann-toolbox protocols can be accessed via the webserver at https://lbs.cent.uw.edu.pl/rossmann-toolbox and are available as a Python package at https://github.com/labstructbioinf/rossmann-toolbox.

Key pointsO_LIThe Rossmann fold encompasses a multitude of diverse enzymes involved in most of the essential cellular pathways
C_LIO_LIProteins belonging to the Rossmann fold co-evolved with their nucleoside-based cofactors and require them for the functioning
C_LIO_LIManipulating the cofactor specificity is an important step in the process of enzyme engineering
C_LIO_LIWe developed an end-to-end pipeline for the prediction and design of the cofactor specificity of the Rossmann fold proteins
C_LIO_LIOwing to the utilization of deep learning approaches the pipeline achieved nearly perfect accuracy
C_LI

sentences: Sexual Conflict Over Survival In Trinidadian Guppies Differences in allele frequencies at autosomal genes between males and females in a population can result from two scenarios. Unresolved sexual conflict over survival produces allelic differentiation between the sexes. However, given the substantial mortality costs required to produce allelic differences between males and females at each generation, it remains unclear how many loci within the genome experience significant sexual conflict over survival. Alternatively, recent studies have shown that similarity between autosomal and Y sequence, arising from duplication onto the Y, can create perceived allelic differences, and this represents potentially resolved sexual conflict. However, Y duplications are most likely in species with large non-recombining regions, in part because they simply represent larger targets for duplications. We assessed the genomes of 120 wild-caught guppies, which experience extensive predation- and pathogen-induced mortality and have a relatively small ancestral Y chromosome. We identified seven autosomal genes that show allelic differences between male and female adults. Five of these genes show clear evidence of whole or partial gene duplication to the Y chromosome, suggesting that the male-specific region of the guppy Y chromosome, although relatively small, may nonetheless act as a hotspot for the resolution of sexual conflict. The remaining two genes show evidence of partial homology to the Y. Overall, our findings suggest that the guppy genome experiences a very low level of unresolved sexual conflict over survival, and instead the Y chromosome, despite its small ancestral size and recent origin, acts as a major mechanism of conflict resolution.

sentences: MCIBox: A Toolkit for Single-molecule Multi-way Chromatin Interaction Visualization and Micro-Domains Identification The emerging ligation-free three-dimensional (3D) genome mapping technologies can identify multiplex chromatin interactions with single-molecule precision. These technologies offer new insight into high-dimensional chromatin organization and gene regulation, but also introduce new challenges in data visualization and analysis. To overcome these challenges, we developed MCIBox, a toolkit for Multi-way Chromatin Interaction (MCI) analysis, including a visualization tool and a platform for identifying micro-domains with clustered single-molecule chromatin complexes. MCIBox is based on various clustering algorithms integrated with dimensionality reduction methods that can display multiplex chromatin interactions at single-molecule level, allowing users to explore chromatin extrusion patterns and super-enhancers regulation modes in transcription, and to identify single-molecule chromatin complexes that are clustered into micro-domains. Furthermore, MCIBox incorporates a two-dimensional kernel density estimation algorithm to identify micro-domains boundaries automatically. These micro-domains were stratified with distinctive signatures of transcription activity and contained different cell cycle associated genes. MCIBox could potentially distinguish the specificity of single-molecule chromatin interaction patterns in various phases of a cell cycle or cell types.

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A hierarchical GBP network promotes cytosolic LPS recognition and sepsis Bacterial lipopolysaccharide (LPS) is one of the most bioactive substances known. Trace amounts trigger robust immunity to infection but also life-threatening sepsis causing millions of deaths each year. LPS contamination of the cytosol elicits a caspase-dependent inflammasome pathway promoting cytokine release and host cell death. Here, we report an immune GTPase network controls multiple steps in this pathway by genome-engineering mice to lack 7 different guanylate-binding proteins (GBPs). Gbp2-/- and Gbp3-/- mice had severe caspase-11-driven defects that protected them from septic shock. Gbp2 recruited caspase-11 for LPS recognition whereas Gbp3 assembled and trafficked the pyroptotic pore-forming protein, gasdermin D, after caspase-11 cleavage. Together, our results identify a new functional hierarchy wherein different GBPs choreograph sequential steps in the non-canonical inflammasome pathway to control Gram-negative sepsis.

One-Sentence SummaryImmune GTPase network orchestrates hierarchical immunity to bacterial products in vivo

sentences: The geometry of domain-general performance monitoring representations in the human medial frontal cortex Controlling behavior to flexibly achieve desired goals depends on the ability to monitor ones own performance. It is unknown how performance monitoring can be both flexible to support different tasks and specialized to perform well on each. We recorded single neurons in the human medial frontal cortex while subjects performed two tasks that involve three types of cognitive conflict. Neurons encoding predicted conflict, conflict, and error in one or both tasks were intermixed, forming a representational geometry that simultaneously allowed task specialization and generalization. Neurons encoding conflict retrospectively served to update internal estimates of control demand. Population representations of conflict were compositional. These findings reveal how representations of evaluative signals can be both abstract and task-specific and suggest a neuronal mechanism for estimating control demand.

sentences: Improved assessments of milk microbiota composition via sample preparation and DNA extraction methods Although bacterial detection by 16S rRNA gene amplicon DNA sequencing is now a widely-applied technique, standardized methods for sample preparation and DNA extraction are needed to ensure accuracy, reproducibility, and scalability for automation. To develop these methods for bovine milk, we assembled a bacterial cell mock community (BCMC) containing bacterial species commonly found in milk. Variations to the following methods were examined: BCMC enumeration method (colony enumeration or microscopy), sample volume (200 l to 30 ml), sample storage condition (frozen in PBS or 25% glycerol or exposure to freeze-thaw cycles), cell lysis method (bead-beating, vortex, enzymatic), and DNA extraction procedure (MagMAX Total, MagMAX CORE, and MagMAX Ultra 2.0, with and without either Proteinase K or RNase A). Cell enumeration by microscopy was shown to be more accurate for quantification of the BCMC contents. We found that least 10 mL ([≥] 104 cells in high quality milk) is needed for reproducible bacterial detection by 16S rRNA gene amplicon DNA sequencing, whereas variations in storage conditions caused minor differences in the BCMC. For DNA extraction and purification, a mild lysis step (bead-beating for 10 s at 4 m/s or vortexing at 1800 rpm for 10 s) paired with the MagMAX Total kit and Proteinase K digestion provided the most accurate representation of the BCMC. These approaches were confirmed to provide similar results in commercial milk samples. Overall, variations in cell lysis methods conferred the greatest changes to milk microbiota composition and therefore should be optimized for sample type.

sentences: Identification of bacteria by poly-aromatic hydrocarbons biosensors Human health is consistently threatened by different species of pathogenic bacteria. To fight the spread of diseases, it is important to develop rapid methods for bacterial identification. Over the years, different kinds of biosensors were developed for this cause. Another environmental risk are poly-aromatic hydrocarbons (PAHs) that may be emitted from industrial facilities and pollute environmental water and soil. One of the methods for their purification is conducted by the addition of bacteria that can degrade the PAHs, while the bacteria itself can be filtrated at the end of the process. Although many studies reported monitoring of the PAHs degradation by fluorescence, not much attention was dedicated to studying the influence of the PAHs on the intrinsic fluorescence of the degrading bacteria. In this work, we apply synchronous fluorescence (SF) measurements to study the ability of the 5 PAHs: 9-Antracene carboxylic acid (9ACA), Pyrene, Perylene, Pentacene, and Chrysene to interact with bacteria and change its fluorescence spectra. We show that upon incubation of each PAH with the bacterium E.coli only the 2 PAHs 9ACA and Perylene cause an intensity decrease in the emission at {lambda} = 300 - 375 nm, which derives from the emission of Tyrosine and Tryptophane (TT). Also, we show that upon incubation of 9ACA and Perylene with 5 different pathogenic bacteria, the intensity increase or decrease in the TT emission is unique to each bacterial species. Based on this observation, we suggest that the PAHs 9ACA and Perylene can be utilized as biosensors for bacterial identification.

sentences: Evidence for Normalization as a Fundamental Operation Across the Human Visual Cortex Divisive normalization of the neural responses by the activity of the neighboring neurons has been proposed as a fundamental operation in the nervous system based on its success in predicting neural responses recorded in primate electrophysiology studies. Nevertheless, experimental evidence for the existence of this operation in the human brain is still scant. Here, using functional MRI, we explored the role of normalization across the visual hierarchy in the human visual cortex. Using stimuli form the two categories of human bodies and houses, we presented objects in isolation or in clutter and asked participants to attend or ignore the stimuli.Focusing on the primary visual area V1, the object-selective regions LO and pFs, and the category-selective regions EBA and PPA, we first modeled single-voxel responses using a weighted sum, a weighted average, and a normalization model and demonstrated that the response to multiple stimuli could best be described by a model that takes normalization into account. We then explored the observed effects of attention on cortical responses and demonstrated that these effects were predicted by the normalization model, but not by the weighted sum or the weighted average models. Our results thus provide compelling evidence for normalization as a canonical computation operating in the human brain.

sentences: Topography-induced large-scale anti-parallel collective migration in vascular endothelium Collective migration of vascular endothelial cells is central for embryonic development, angiogenesis, and wound closure. Although physical confinement of cell assemblies has been shown to elicit specific patterns of collective movement in various cell types, endothelial migration in vivo often occurs without confinement. Here we show that unconfined endothelial cell monolayers on microgrooved substrates that mimic the anisotropic organization of the extracellular matrix exhibit a new type of collective movement that takes the form of a periodic pattern of anti-parallel cell streams. We further establish that the development of these streams requires intact cell-cell junctions and that stream sizes are particularly sensitive to groove depth. Finally, we show that modeling the endothelial cell sheet as an active fluid with the microgrooves acting as constraints on cell orientation predicts the occurrence of the periodic anti-parallel cell streams as well as their lengths and widths. We posit that in unconfined cell assemblies, physical factors that constrain or bias cellular orientation such as anisotropic extracellular matrix cues or directed flow-derived shear forces dictate the pattern of collective cell movement.

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Distinct signatures of loss of consciousness during Focal Impaired Awareness (FIA) versus Focal to Bilateral Tonic-Clonic (FBTC) seizures Loss of consciousness (LOC) is a hallmark of many epileptic seizures and carries risks of serious injury and sudden death. While cortical sleep-like activities accompany LOC during focal impaired awareness (FIA) seizures, the mechanisms of LOC during focal to bilateral tonic-clonic (FBTC) seizures remain unclear. Quantifying differences in markers of cortical activation and ictal recruitment between FIA and FBTC seizures may also help to understand their different consequences for clinical outcomes and to optimize neuromodulation therapies.

We quantified clinical signs of LOC and intracranial EEG (iEEG) activity during 129 FIA and 50 FBTC from 41 patients. We characterized iEEG changes both in the seizure onset zone (SOZ) and in areas remote from SOZ with a total of 3386 electrodes distributed across brain areas. First, we compared the dynamics of iEEG sleep-like activities: slow-wave activity (SWA; 1-4 Hz) and beta/delta ratio (B/D; a validated marker of cortical activation) during FIA vs. FBTC. Second, we quantified differences between FBTC and FIA for a marker validated to detect ictal cross-frequency coupling: phase-locked high-gamma (PLHG; high gamma phased locked to low frequencies) and a marker of ictal recruitment: the epileptogenicity index (i.e. the number of channels crossing an energy ratio threshold for high vs. low frequency power). Third, we assessed changes in iEEG activity preceding and accompanying behavioral generalization onset and their correlation with electromyogram (EMG) channels. In addition, we analyzed human cortical multi-unit activity recorded with Utah arrays during three FBTC.

Compared to FIA, FBTC seizures were characterized by deeper LOC and by stronger increases in SWA in parieto-occipital cortex. FBTC also displayed more widespread increases in cortical activation (B/D), ictal cross-frequency coupling (PLHG) and ictal recruitment (epileptogenicity index). Even before generalization, FBTC displayed deeper LOC; this early LOC was accompanied by a paradoxical increase in B/D in fronto-parietal cortex. Behavioral generalization coincided with complete loss of responsiveness and a subsequent increase in high-gamma in the whole brain, which was especially synchronous in deep sources and could not be explained by EMG. Similarly, multi-unit activity analysis of FBTC revealed sustained increases in cortical firing rates during and after generalization onset in areas remote from the SOZ.

Unlike during FIA, LOC during FBTC is characterized by a paradoxical increase in cortical activation and neuronal firing. These findings suggest differences in the mechanisms of ictal LOC between FIA and FBTC and may account for the more negative prognostic consequences of FBTC.

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Classification of plant growth-promoting bacteria inoculation status and prediction of growth-related traits in tropical maize using hyperspectral image and genomic data Recent technological advances in high-throughput phenotyping have created new opportunities for the prediction of complex traits. In particular, phenomic prediction using hyper-spectral reflectance could capture various signals that affect phenotypes genomic prediction might not explain. A total of 360 inbred maize lines with or without plant growth-promoting bacterial inoculation management under nitrogen stress were evaluated using 150 spectral wavelengths ranging from 386 to 1021 nm and 13,826 single-nucleotide polymorphisms. Six prediction models were explored to assess the predictive ability of hyperspectral and genomic data for inoculation status and plant growth-related traits. The best models for hyperspectral prediction were partial least squares and automated machine learning. The Bayesian ridge regression and BayesB were the best performers for genomic prediction. Overall, hyper-spectral prediction showed greater predictive ability for shoot dry mass and stalk diameter, whereas genomic prediction was better for plant height. The prediction models that simultaneously accommodated both hyperspectral and genomic data resulted in a predictive ability as high as that of phenomics or genomics alone. Our results highlight the usefulness of hyperspectral-based phenotyping for management and phenomic prediction studies.

Core ideasO_LIHyperspectral reflectance data can classify plant growth-promoting bacteria inoculation status
C_LIO_LIPhenomic prediction performs better than genomic prediction depending on the target phenotype
C_LIO_LIAutoML is a promising approach for automating hyperparameter tuning for classification and prediction
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sentences: Exploring the structure and mechanics of thin supported inverse bicontinuous cubic phase lipid films Inverse bicontinuous cubic phase membranes are ubiquitous in nature but their properties and functions are still not fully understood. To shed light on this topic, we herein realize thin supported cubic phase lipid films, characterize their structure and provide the first study of the mechanical properties of these non-lamellar architectures.

sentences: Evaluation role of miR-124 in neurodegenerative diseases: literature review and in silico analysis Neurodegenerative diseases (ND) are characterized by loss of function and structure of neurons. NDs like Alzheimers disease (AD) and Parkinsons disease (PD) have high burden on the society and patients. Currently microRNAs (miRNAs) approach is growing. miRNAs express in different tissues, especially in the central neuron systems (CNS). miRNAs have a dynamic role in the CNS among this miRNAs, miR-124 significantly express in the CNS. Studies on miR-124 have shown that miR-124 improves ND. In this study, we evaluated the role of miR-124 in the ND by literature review and in silico analysis. We used Pubmed database to find miR-124 function in the Alzheimers disease, Parkinsons disease, Multiple sclerosis, Huntingtons disease and amyotrophic lateral sclerosis. To better understand the role of miR-124 in the neurons, RNA-seq data form miR-124-deleted neuronal cells extracted from GEO database and analyzed in Galaxy platform. According literature review miR-124 attenuates inflammation and apoptosis in the ND by target NF-kb signaling pathway and regulation of BAX/BCL-2. miR-124 targets BACE1 and decreases level of A{beta}. RNA-seq data showed miR-124 downregulation, an increase in chemokine gene like CCL1 and cytokine-cytokine receptor-interaction, as well as MAPK-signaling pathway. Our study shows that miR-124 can be promising therapeutic approaches to ND.

sentences: Single immunization with recombinant ACAM2000 vaccinia viruses expressing the spike and the nucleocapsid proteins protect hamsters against SARS-CoV-2 caused clinical disease Increasing cases of SARS-CoV-2 breakthrough infections from immunization with predominantly spike protein based COVID-19 vaccines highlight the need for alternative vaccines using different platforms and/or antigens. In this study, we expressed SARS-CoV-2 spike and nucleocapsid proteins in a novel vaccinia virus ACAM2000 platform (rACAM2000). Following a single intramuscular immunization, the rACAM2000 co-expressing the spike and nucleocapsid proteins induced significantly improved protection against SARS-CoV-2 challenge in comparison to rACAM2000 expressing the individual proteins in a hamster model, as shown by reduced weight loss and quicker recovery time. The protection was associated with reduced viral loads, increased neutralizing antibody titre and reduced neutrophil-to-lymphocyte ratio. Thus, our study demonstrates that the rACAM2000 expressing a combination of the spike and nucleocapsid antigens is a promising COVID-19 vaccine candidate and further studies will investigate if the rACAM2000 vaccine candidate can induce a long lasting immunity against infection of SARS-CoV-2 variants of concern.

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Is the Success of Adaptive Therapy in Metastatic Castrate Resistant Prostate Cancer Influenced by Cell-Type-Dependent Production of Prostate Specific Antigen? Prostate-specific antigen (PSA) is the most common serum marker for prostate cancer. It is used to detect prostate cancer, to assess responses to treatment and recently even to determine when to switch treatment on and off in adaptive therapy protocols. However, the correlation between PSA and tumor volume is poorly understood. There is empirical evidence that some cancer cell types produce more PSA than others. Still, recent mathematical cancer models assume either that all cell types contribute equally to PSA levels, or that only specific subpopulations produce PSA at a fixed rate.

Here, we compare time to competitive release of the PSA-based adaptive therapy protocol by Zhang et al. with that of the standard of care based on continuous maximum tolerable dose under different assumptions on PSA production. In particular, we assume that androgen dependent, androgen producing, and androgen independent cells may contribute to the PSA production to different extents.

Our results show that, regardless the assumption on how much each type contributes to PSA production, the time to competitive release is always longer under adaptive therapy than under the standard of care. However, in some cases, e.g., if the androgen-independent cells are the only PSA producers, adaptive therapy protocol by Zhang et al. cannot be applied, because the PSA value never reaches half of its initial size and therefore therapy is never discontinued.

Furthermore, we observe that in the adaptive therapy protocol, the number of treatment cycles and their length strongly depend on the assumptions about the PSA contribution of the three types. Our results support the belief that a better understanding of patient-specific PSA dynamics will lead to more successful adaptive therapies.

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Single-cell generalized trend model (scGTM): a flexible and interpretable model of gene expression trend along cell pseudotime MotivationModeling single-cell gene expression trends along cell pseudotime is a crucial analysis for exploring biological processes. Most existing methods rely on nonparametric regression models for their flexibility; however, nonparametric models often provide trends too complex to interpret. Other existing methods use interpretable but restrictive models. Since model interpretability and flexibility are both indispensable for understanding biological processes, the single-cell field needs a model that improves the interpretability and largely maintains the flexibility of nonparametric regression models.

ResultsHere we propose the single-cell generalized trend model (scGTM) for capturing a genes expression trend, which may be monotone, hill-shaped, or valley-shaped, along cell pseudotime. The scGTM has three advantages: (1) it can capture non-monotonic trends that are still easy to interpret, (2) its parameters are biologically interpretable and trend informative, and (3) it can flexibly accommodate common distributions for modeling gene expression counts. To tackle the complex optimization problems, we use the particle swarm optimization algorithm to find the constrained maximum likelihood estimates for the scGTM parameters. As an application, we analyze several single-cell gene expression data sets using the scGTM and show that it can capture interpretable gene expression trends along cell pseudotime and reveal molecular insights underlying the biological processes.

Availability and implementationThe Python package scGTM is open-access and available at https://github.com/ElvisCuiHan/scGTM.

Contactjli@stat.ucla.edu

sentences: Facilitation between ecosystem engineers, salt marsh grass and mussels, produces pattern formation on salt marsh shorelines Interspecific facilitation between ecosystem engineers, such as salt marsh grass and mussel aggregations, is a key process that structures communities and enhances biodiversity. Scale-dependent pattern formation via self-organization is ubiquitous in terrestrial, aquatic and marine ecosystems. Despite their prevalence and ecological importance, these two phenomena have rarely been linked. We provide empirical evidence that the facilitative interaction in salt marshes between smooth cordgrass Spartina alterniflora and the ribbed mussel Geukensia demissa produces distinct spatial patterns along marsh shorelines. These findings advance our understanding of linkages between facilitation and pattern formation in nature, and are particularly relevant to conservation and restoration of salt marshes threatened by climate change and sea-level rise.

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BioISO: an objective-oriented application for assisting the curation of genome-scale metabolic models As the reconstruction of Genome-Scale Metabolic Models becomes standard practice in systems biology, the number of organisms having at least one metabolic model at the genome-scale is peaking at an unprecedented scale. The automation of several laborious tasks, such as gap-finding and gap-filling, allowed to develop GSMMs for poorly described organisms. However, such models quality can be compromised by the automation of several steps, which may lead to erroneous phenotype simulations.

The Biological networks constraint-based In Silico Optimization (BioISO) is a computational tool aimed at accelerating the reconstruction of Genome-Scale Metabolic Models. This tool facilitates the manual curation steps by reducing the large search spaces often met when debugging in silico biological models. BioISO uses a recursive relation-like algorithm and Flux Balance Analysis to evaluate and guide debugging of in silico phenotype simulations. The potential of BioISO to guide the debugging of model reconstructions was showcased using GSMMs available in literature and compared with the results of two other state-of-the-art gap-filling tools (Meneco and fastGapFill). Furthermore, BioISO was used as Menecos gap-finding algorithm to reduce the number of proposed solutions (reaction sets) for filling the gaps.

BioISO was implemented as a webserver available at https://bioiso.bio.di.uminho.pt; and integrated into merlin as a plugin. BioISOs implementation as a Python package can also be retrieved from https://github.com/BioSystemsUM/BioISO.

sentences: Comparative single-cell transcriptional atlases of Babesia species reveal conserved and species-specific expression profiles Babesia is a genus of Apicomplexan parasites that infect red blood cells in vertebrate hosts. Pathology occurs during rapid replication cycles in the asexual blood-stage of infection. Current knowledge of Babesia replication cycle progression and regulation is limited and relies mostly on comparative studies with related parasites. Due to limitations in synchronizing Babesia parasites, fine-scale time-course transcriptomic resources are not readily available. Single-cell transcriptomics provides a powerful unbiased alternative for profiling asynchronous cell populations. Here, we applied single-cell RNA sequencing to three Babesia species (B. divergens, B. bovis, and B. bigemina). We used analytical approaches and algorithms to map the replication cycle and construct pseudo-synchronized time-course gene expression profiles. We identify clusters of co-expressed genes showing just-in-time expression profiles, with gradually cascading peaks throughout asexual development. Moreover, clustering analysis of reconstructed gene curves reveals coordinated timing of peak expression in epigenetic markers and transcription factors. Using a regularized Gaussian Graphical Model, we reconstructed co-expression networks and identified conserved and species-specific nodes. Motif analysis of a co-expression interactome of AP2 transcription factors identified specific motifs previously reported to play a role in DNA replication in Plasmodium species. Finally, we present an interactive web-application to visualize and interactively explore the datasets.

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Kinome state is predictive of cell viability in pancreatic cancer tumor and stroma cell lines Numerous aspects of cellular signaling are regulated by the kinome - the network of over 500 protein kinases that guides and modulates information transfer throughout the cell. The key role played by both individual kinases and assemblies of kinases organized into functional subnetworks leads to kinome dysregulation being a key driver of many diseases, particularly cancer. In the case of pancreatic ductal adenocarcinoma (PDAC), a variety of kinases and associated signaling pathways have been identified for their key role in the establishment of disease as well as its progression. However, the identification of additional relevant therapeutic targets has been slow and is further confounded by interactions between the tumor and the surrounding tumor microenvironment. Fundamentally, it is an open question as to the degree to which knowledge of the state of the kinome at the protein level is able to provide insight into the downstream phenotype of the cell.

In this work, we attempt to link the state of the kinome, or kinotype, with cell viability in representative PDAC tumor and stroma cell lines. Through the application of both regression and classification models to independent kinome perturbation and kinase inhibitor cell screen data, we find that the inferred kinotype of a cell has a significant and predictive relationship with cell viability. While regression models perform poorly, we find that classification approaches are able to predict drug viability effects. We further find that models are able to identify a set of kinases whose behavior in response to perturbation drive the majority of viability responses in these cell lines. Using the models to predict new compounds with cell viability effects and not in the initial data set, we conducted a validation screen that confirmed the accuracy of the models. These results suggest that characterizing the state of the protein kinome provides significant opportunity for better understanding signaling behavior and downstream cell phenotypes, as well as providing insight into the broader design of potential therapy design for PDAC.

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Untargeted metabolomic analyses reveal the diversity and plasticity of the specialized metabolome in seeds of different Camelina sativa varieties Despite the essential role of Specialized Metabolites (SMs) in the interaction of plants with the environment, studying the ability of crop seeds to produce these protective compounds has been neglected. Furthermore, seeds produce a myriad of SMs providing an interesting model to investigate their diversity and plasticity. Camelina sativa gained a lot of interest in the past few years as rustic oil seed crop. A characterization of seed SM landscapes in six camelina genotypes grown in the field and harvested during five growing seasons has been undertaken in this work. This allowed a comprehensive annotation of seed SMs combining analyses that cluster SMs based on their chemical structures and co-accumulation patterns. These data showed broad effects of the environment on the stimulation of the seed-specialized metabolome. Among well annotated compounds, flavonols were identified as the metabolic class characterised by high plasticity, revealing significant variable accumulation according to the year and/or the genotype. Lastly, a deeper characterisation of primary metabolites and lipids in two selected genotypes has been performed. We showed that, in addition to flavonols, alkaloids and glucosinolates displayed a higher phenotypic plasticity with respect to most of the primary metabolites, including some sugars and major storage compounds such as fatty acids, proteins and most lipid classes (e.g. DAG, TAG), but similar plasticity compared to free aminoacids and carboxylic acids. This work highlighted major and unexplored effects of the environment on the seed specialized metabolome demonstrating that seeds exhibit a dynamic and plastic metabolism, with an impact on seed quality.

Significance statementSeeds produce a myriad of Specialized Metabolites (SMs) with an essential role in the interaction of plants with the environment. We characterized SM landscapes, primary metabolites and lipid composition in the seeds of camelina genotypes grown in the open field in five consecutive growing seasons. Our results showed the predominant effect of the environment on the regulation of the seed - specialized metabolome, with a potential impact on seed quality of camelina that may also occur in other oilseed crops.

sentences: The angular gyrus is engaged by autobiographical recall not object-semantics, or event-semantics: Evidence from contrastive propositional speech production A long history of neuropsychology and neuroimaging has implicated the angular gyrus (AG) with a myriad of cognitive functions. We investigated the functional engagement of AG by three forms of memory: 1) episodic/autobiographical memory 2) object semantic-memory, and 3) event-semantic processing using the previously under-examined naturalistic task of propositional speech production. We conducted an ALE meta-analysis of imaging studies of propositional speech, followed by an fMRI study in which we conducted direct cross-domain comparisons. The results from the meta-analysis suggest that the AG is only engaged as part of the propositional speech network when the task carries an autobiographical component. This conclusion was strongly supported by the results from the fMRI study. The key fMRI findings were: 1) AG was positively engaged during autobiographical memory retrieval. 2) The AG was strongly deactivated for definitions of object semantics and non-propositional speech. 3) The level of AG activation increased with the degree to which the event descriptions relied on input from the episodic memory system. 4) Critically, the AG showed a very different pattern to that of known semantic representation regions (the anterior temporal lobe; ATL) - whilst AG activation increased with the autobiographical nature of the task, the ATL was equally responsive to all conditions. These results provide clear evidence that the AG is not acting as a semantic hub. In contrast, the AG activation profile directly mirrored that found in the wider autobiographical retrieval network. We propose that information stored elsewhere in the episodic system is temporally buffered online in the AG during autobiographical retrieval/memory construction.

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Strain-Programmable Patch for Diabetic Wound Healing Chronic wounds with impaired healing capability such as diabetic foot ulcers (DFU) are devastating complications in diabetic patients, inflicting rapidly growing clinical and economic burdens in aging societies. Despite recent advances in therapeutic approaches, limited benefits of the existing solutions highlight the critical need for novel therapeutic solutions for diabetic wound healing. Here we propose a strain-programmable patch capable of rapid robust adhesion on and programmable mechanical contraction of wet wounded tissues over days to offer a new therapeutic platform for diabetic wounds. The strain-programmable patch, consisting of a dried bioadhesive layer and a pre-stretched elastomer backing, implements a hydration-based shape-memory mechanism to achieve both uniaxial and biaxial contractions and stress remodeling of wet wounds in a programmable manner. We develop theoretical and numerical models to rationally guide the strain-programming and mechanical modulation of wounds. In vivo rodent and ex vivo human skin culture models validate the programmability and efficacy of the proposed platform and identify mechanisms of action for accelerated diabetic wound healing.

One Sentence SummaryA strain-programmable bioadhesive patch is developed for accelerated closure and healing of wounds in diabetic mice and human skin.

sentences: Comprehensive Structure and Functional Adaptations of the Yeast Nuclear Pore Complex Nuclear Pore Complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the yeast NPC in which the inner ring is resolved by cryo-EM at - helical resolution to show how flexible connectors tie together different structural and functional layers in the spoke. These connectors are targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and karyopherins have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We also provide evidence for three major NPC variants that foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies to provide a comprehensive model of the in situ NPC with a radially-expanded inner ring. Our model reveals novel features of the central transporter and nuclear basket, suggests a role for the lumenal ring in restricting dilation and highlights the structural plasticity required for transport by the NPC.

sentences: Reward enhances memory via age-varying online and offline neural mechanisms across development Reward motivation enhances memory through interactions between mesolimbic, hippocampal, and cortical systems -- both during and after encoding. Developmental changes in these distributed neural circuits may lead to age-related differences in reward-motivated memory and the underlying neural mechanisms. Converging evidence from cross-species studies suggests that subcortical dopamine signaling is increased during adolescence, which may lead to stronger memory representations of rewarding, relative to mundane, events and changes in the contributions of underlying subcortical and cortical brain mechanisms across age. Here, we used fMRI to examine how reward motivation influences the "online" encoding and "offline" post-encoding brain mechanisms that support long-term associative memory from childhood to adulthood. We found that reward motivation led to both age-invariant as well as adolescent-specific enhancements in associative memory after 24 hours. Furthermore, reward-related memory benefits were linked to age-varying neural mechanisms. During encoding, interactions between the prefrontal cortex and ventral tegmental area (VTA) were associated with better high-reward memory to a greater degree with increasing age. Pre-to post-encoding changes in functional connectivity between the anterior hippocampus and VTA were also associated with better high-reward memory, but more so at younger ages. Our findings suggest that there may be developmental shifts -- from offline subcortical to online cortical processes -- in the brain mechanisms supporting reward-motivated memory.

sentences: Isolation of Cell cycle delay, Pro-metaphase arrest, and c-metaphase inducing agents from leaf aqueous extract of Clerodendrum viscosum Vent. Clerodendrum viscosum is a traditionally used medicinal plant. The present study aimed to analyze cell cycle delay, pro-metaphase arrest, and c-metaphase inducing effects of the petroleum ether fraction (AQPEF) of leaf aqueous extract of C. Viscosum (LAECV). The LAECV was fractionated with petroleum ether and its metaphase arrest, cell cycle delay, and c-metaphase inducing activities were tested on A. cepa root tip cells. The AQPEF induced cell cycle delay, and colchicine like metaphase, c-metaphase, in A. cepa root tip cells. Thus, the present study explores AQPEF as an active fraction of LAECV having metaphase arresting, cell cycle delay, and c-metaphase inducing potentials.

sentences: The methyl phosphate capping enzyme Bin3 is a stable component of the fission yeast telomerase holoenzyme The telomerase holoenzyme is critical for maintaining eukaryotic genome integrity. In addition to a reverse transcriptase and an RNA template, telomerase contains additional proteins that protect the telomerase RNA and promote holoenzyme assembly. Here we report that the methyl phosphate capping enzyme (MePCE) Bin3 is a stable component of the S. pombe telomerase holoenzyme. Bin3 associates with the telomerase holoenzyme and U6 snRNA through an interaction with the recently described LARP7 family member Pof8, and we demonstrate that these two factors are evolutionarily linked in fungi. Our data suggest that the association of Bin3 with telomerase is independent of its methyltransferase activity, but rather that Bin3 functions in telomerase holoenzyme assembly by promoting TER1 accumulation and Pof8 recruitment to TER1. Taken together, this work yields new insight into the composition, assembly, and regulation of the telomerase holoenzyme in fission yeast as well as the breadth of its evolutionary conservation.

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Structure and dynamics of human disease-complication network A complication is an unanticipated disease arisen following, induced by a disease, a treatment or a procedure. We compile the Human Disease-Complication Network from the medical data and investigate the characteristics of the network. It is observed that the modules of the network are dominated by the classes of diseases. The relations between modules are unveiled in detail. Three nontrivial motifs are identified from the network. We further simulate the dynamics of motifs with the Boolean dynamic model. Each motif represents a specific dynamic behavior, which is potentially functional in the disease system, such as generating temporal progressions and governing the responses to fluctuating external stimuli.

Author summaryAdvances in molecular biology lead to a new discipline of network medicine, investigating human diseases in a networked structure perspective. Recently, clinical records have been introduced to the research of complex networks of diseases. An important available medical dataset that has been overlooked so far is the complications of diseases, which are vital for human beings. We compile the Human Disease-Complication Network, representing the causality between the upstream diseases and their downstream complications. This work not only helps us to comprehend why certain groups of diseases appear collectively, but also provides a new paradigm to investigate the dynamics of disease progression. For clinical applications, the investigation of complications may yield new approaches to disease prevention, diagnosis and treatment.

sentences: Unsupervised deep learning method for cell segmentation Advances in the artificial neural network have made machine learning techniques increasingly more important in image analysis tasks. Recently, convolutional neural networks (CNN) have been applied to the problem of cell segmentation from microscopy images. However, previous methods used a supervised training paradigm in order to create an accurate segmentation model. This strategy requires a large amount of manually labeled cellular images, in which accurate segmentations at pixel level were produced by human operators. Generating training data is expensive and a major hindrance in the wider adoption of machine learning based methods for cell segmentation. Here we present an alternative strategy that uses unsupervised learning to train CNNs without any human-labeled data. We show that our method is able to produce accurate segmentation models. More importantly, the algorithm is applicable to both fluorescence and bright-field images, requiring no prior knowledge of signal characteristics and requires no tuning of parameters.

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Integrative analysis of the hydroxypyruvate reductases revealing their distinct roles in photorespiration of Chlamydomonas Photorespiration plays an important role in maintaining normal physiological metabolism in higher plants and other oxygenic organisms such as algae. The unicellular eukaryotic organism Chlamydomonas is reported to have a different photorespiration system from that in higher plants, and only two out of nine genes encoding photorespiratory enzymes have been experimentally characterized. Hydroxypyruvate reductase (HPR), which is responsible for the conversion of hydroxypyruvate into glycerate, is poorly understood and not yet explored in Chlamydomonas. To identify the candidate genes encoding hydroxypyruvate reductase in Chlamydomonas (CrHPR) and uncover their elusive functions, we performed sequence comparison, enzyme activity measurement, subcellular localization, and analysis of knockout/knockdown strains. Together we identify five proteins to be good candidates as CrHPRs, all of which are detected with the activity of hydroxypyruvate reductase. CrHPR1, a NADH-dependent enzyme in mitochondria, may function as the major component of photorespiration, and deletion of CrHPR1 causes severe photorespiratory defects. CrHPR2 takes parts in the cytosolic bypass of photorespiration as the compensatory pathway of CrHPR1 for the reduction of hydroxypyruvate. CrHPR4, with NADH as the cofactor, may participate in photorespiration by acting as the chloroplastidial glyoxylate reductase in glycolate-quinone oxidoreductase system. Therefore, our results reveal that the CrHPRs are far more complex than previously recognized, and provide a greatly expanded knowledge base for studies to understand how CrHPRs perform their functions in photorespiration. These will facilitate the genetic engineering for crop improvement by synthetic biology.

Brief summaryIdentification and characterization of genes encoding hydroxypyruvate reductases in Chlamydomonas, demonstrating difference in the enzymatic activity, subcellular location, as well as function in photorespiration.

sentences: Nutrient and salt depletion synergistically boosts glucose metabolism in individual bacteria The interaction between a cell and its environment shapes fundamental intracellular processes such as cellular metabolism. In most cases growth rate is treated as a proximal metric for understanding the cellular metabolic status. However, changes in growth rate might not reflect metabolic variations in individuals responding to environmental fluctuations. Here we use single-cell microfluidics-microscopy combined with transcriptomics, proteomics and mathematical modelling to quantify the accumulation of glucose within Escherichia coli cells. In contrast to the current consensus, we reveal that environmental conditions which are comparatively unfavourable for growth, where both nutrients and salinity are depleted, increase glucose accumulation rates in individual bacteria and population subsets. We find that these changes in metabolic function are underpinned by variations at the translational and posttranslational level but not at the transcriptional level and are not dictated by changes in cell size. The metabolic response-characteristics identified greatly advance our fundamental understanding of the interactions between bacteria and their environment and have important ramifications when investigating cellular processes where salinity plays an important role.

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yHydra: Deep Learning enables an Ultra Fast Open Search by Jointly Embedding MS/MS Spectra and Peptides of Mass Spectrometry-based Proteomics Mass spectrometry-based proteomics allows to study all proteins of a sample on a molecular level. The ever increasing complexity and amount of proteomics MS-data requires powerful and yet efficient computational and statistical analysis. In particular, most recent bottom-up MS-based proteomics studies consider either a diverse pool of post-translational modifications, employ large databases - as in metaproteomics or proteogenomics, contain multiple isoforms of proteins, include unspecific cleavage sites or even combinations thereof and thus face a computationally challenging situation regarding protein identification. In order to cope with resulting large search spaces, we present a deep learning approach that jointly embeds MS/MS spectra and peptides into the same vector space such that embeddings can be compared easily and interchangeable by using euclidean distances. In contrast to existing spectrum embedding techniques, ours are learned jointly with their respective peptides and thus remain meaningful. By visualizing the learned manifold of both spectrum and peptide embeddings in correspondence to their physicochemical properties our approach becomes easily interpretable. At the same time, our joint embeddings blur the lines between spectra and protein sequences, providing a powerful framework for peptide identification. In particular, we build an open search, which allows to search multiple ten-thousands of spectra against millions of peptides within seconds. yHydra achieves identification rates that are compatible with MSFragger. Due to the open search, delta masses are assigned to each identification which allows to unrestrictedly characterize post-translational modifications. Meaningful joint embeddings allow for faster open searches and generally make downstream analysis efficient and convenient for example for integration with other omics types.

Availability(under MIT license) https://github.com/tzom/yHydra

Contactbernhard.renard@hpi.de

sentences: Genetic Control of Pluripotency Epigenome Determines Differentiation Bias in Mouse Embryonic Stem Cells Genetically diverse pluripotent stem cells (PSCs) display varied, heritable responses to differentiation cues in the culture environment. By harnessing these disparities through derivation of embryonic stem cells (ESCs) from the BXD mouse genetic reference panel, along with C57BL/6J (B6) and DBA/2J (D2) parental strains, we demonstrate genetically determined biases in lineage commitment and identify major regulators of the pluripotency epigenome. Upon transition to formative pluripotency using epiblast-like cells (EpiLCs), B6 quickly dissolves naive networks adopting gene expression modules indicative of neuroectoderm lineages; whereas D2 retains aspects of naive pluripotency with little bias in differentiation. Genetic mapping identifies 6 major trans-acting loci co-regulating chromatin accessibility and gene expression in ESCs and EpiLCs, indicating a common regulatory system impacting cell state transition. These loci distally modulate occupancy of pluripotency factors, including TRIM28, P300, and POU5F1, at hundreds of regulatory elements. One trans-acting locus on Chr 12 primarily impacts chromatin accessibility in ESCs; while in EpiLCs the same locus subsequently influences gene expression, suggesting early chromatin priming. Consequently, the distal gene targets of this locus are enriched for neurogenesis genes and were more highly expressed when cells carried B6 haplotypes at this Chr 12 locus, supporting genetic regulation of biases in cell fate. Spontaneous formation of embryoid bodies validated this with B6 showing a propensity towards neuroectoderm differentiation and D2 towards definitive endoderm, confirming the fundamental importance of genetic variation influencing cell fate decisions.

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Proteogenomics Reveals how Metastatic Melanoma Modulates the Immune System to Allow Immune Evasion Malignant melanoma (MM) develops from the melanocytes and in its advanced stage is the most aggressive type of skin cancer. Here we report a comprehensive analysis on a prospective cohort study, including non-tumor, primary and metastasis tissues (n=77) with the corresponding plasma samples (n=56) from patients with malignant melanoma. The tumors and surrounding tissues were characterized with a combination of high-throughput analyses including quantitative proteomics, phosphoproteomics, acetylomics, and whole exome sequencing (WES) combined with in-depth histopathology analysis. Melanoma cell proliferation highly correlates with dysregulation at the proteome, at the posttranslational- and at the transcriptome level. Some of the changes were also verified in the plasma proteome. The metabolic reprogramming in melanoma includes upregulation of the glycolysis and the oxidative phosphorylation, and an increase in glutamine consumption, while downregulated proteins involved in the degradation of amino acids, fatty acids, and the extracellular matrix (ECM) receptor interaction. The pathways most dysregulated in MM including the MAP kinases-, the PI3K-AKT signaling, and the calcium homeostasis, are among the most affected by mutations, thus, dysregulation in these pathways can be manifested as drivers in melanoma development and progression.

The phosphoproteome analysis combined with target-based prediction mapped 75% of the human kinome. Melanoma cell proliferation was driven by two key factors: i) metabolic reprogramming leading to upregulation of the glycolysis and oxidative phosphorylation, supported by HIF-1 signaling pathway and mitochondrial translation; and ii) a dysregulation of the immune system response, which was mirrored by immune system processes in the plasma proteome. Regulation of the melanoma acetylome and expression of deacetylase enzymes discriminated between groups based on tissue origin and proliferation, indicating a way to guide the successful use of HDAC inhibitors in melanoma. The disease progression toward metastasis is driven by the downregulation of the immune system response, including MHC class I and II, which allows tumors to evade immune surveillance. Altogether, new evidence is provided at different molecular levels to allow improved understanding of the melanoma progression, ultimately contributing to better treatment strategies.

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sentences: RNA surveillance controls 3D genome structure via stable cohesin-chromosome interaction The 3D architecture that the genome is folded into is regulated by CTCF, which determines domain borders, and cohesin, which generates interactions within domains. However, organisms lacking CTCF have been reported to still have cohesin-mediated 3D structures with strong borders. How this can be achieved and precisely regulated are yet unknown. Using in situ Hi-C, we found that 3-end RNA processing factors coupled with proper transcription termination are a cis-acting determinant that regulates the localization and dynamics of cohesin on the chromosome arms. Loss of RNA processing factors, including nuclear exosome and Pfs2, destabilizes cohesin from the 3-ends of convergent genes and results in decreased cohesin-mediated domain boundaries. We observed the co-localization between Rad21 and a wide range of 3end RNA processing/termination factors. Further, deletion of Rrp6 leads to cohesin redistribution to facultative heterochromatin, resulting in improper domain boundaries. Importantly, we observed that knockdown of Rrp6Exosc10 caused a defect in cohesin binding and loss of local topologically associating domains (TADs) interactions in mouse embryonic stem cells. Based on these findings, we propose a novel function of the RNA surveillance pathway in 3D genome organization via cohesin complex, which provides the molecular basis underlying the dynamics of cohesin function.

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Avoiding Data Loss: Synthetic MRIs Generated from Diffusion Imaging Can Replace Corrupted Structural Acquisitions For Freesurfer-Seeded Tractography 1Magnetic Resonance Imaging (MRI) motion artefacts frequently complicate structural and diffusion MRI analyses. While diffusion imaging is easily  scrubbed of motion affected volumes, the same is not true for structural images. Structural images are critical to most diffusion-imaging pipelines thus their corruption can lead to disproportionate data loss. To enable diffusion-image processing when structural images have been corrupted, we propose a means by which synthetic structural images can be generated from diffusion MRI. This technique combines multi-tissue constrained spherical deconvolution, which is central to many existing diffusion analyses, with the Bloch equations which allow simulation of MRI intensities given scanner parameters and magnetic resonance (MR) tissue properties. We applied this technique to 32 scans, including those acquired on different scanners, with different protocols and with pathology present. The resulting synthetic T1w and T2w images were visually convincing and exhibited similar tissue contrast to acquired structural images. These were also of sufficient quality to drive a Freesurfer-based tractographic analysis. In this analysis, probabilistic tractography connecting the thalamus to the primary sensorimotor cortex was delineated with Freesurfer, using either real or synthetic structural images. Tractography for real and synthetic conditions was largely identical in terms of both voxels encountered (Dice 0.88 - 0.95) and mean fractional anisotropy (intrasubject absolute difference 0.00 - 0.02). We provide executables for the proposed technique in the hope that these may aid the community in analysing datasets where structural image corruption is common, such as studies of children or cognitively impaired persons.

HighlightsO_LIWe propose a simple means of synthesizing T1w and T2w images from diffusion data
C_LIO_LIThe proposed method worked well for a variety of acquisitions
C_LIO_LISynthetic images showed tissue contrast akin to acquired images
C_LIO_LISynthetic images were high enough quality to be used for Freesurfer seeded diffusion tractography
C_LIO_LIThis method enables analysis of datasets where motion has corrupted acquired structural MRIs
C_LI

sentences: DeLUCS: Deep Learning for Unsupervised Classification of DNA Sequences We present a novel Deep Learning method for the Unsupervised Clustering of DNA Sequences (DeLUCS) that does not require sequence alignment, sequence homology, or (taxonomic) identifiers. DeLUCS uses Frequency Chaos Game Representations (FCGR) of primary DNA sequences, and generates "mimic" sequence FCGRs to self-learn data patterns (genomic signatures) through the optimization of multiple neural networks. A majority voting scheme is then used to determine the final cluster assignment for each sequence. The clusters learned by DeLUCS match true taxonomic groups for large and diverse datasets, with accuracies ranging from 77% to 100%: 2,500 complete vertebrate mitochondrial genomes, at taxonomic levels from sub-phylum to genera; 3,200 randomly selected 400 kbp-long bacterial genome segments, into clusters corresponding to bacterial families; three viral genome and gene datasets, averaging 1,300 sequences each, into clusters corresponding to virus subtypes. DeLUCS significantly outperforms two classic clustering methods (K-means++ and Gaussian Mixture Models) for unlabelled data, by as much as 47%. DeLUCS is highly effective, it is able to cluster datasets of unlabelled primary DNA sequences totalling over 1 billion bp of data, and it bypasses common limitations to classification resulting from the lack of sequence homology, variation in sequence length, and the absence or instability of sequence annotations and taxonomic identifiers. Thus, DeLUCS offers fast and accurate DNA sequence clustering for previously intractable datasets.

sentences: Pupil size dynamics predict dLGN firing mode over a wide range of timescales The way in which sensory systems encode information, even at early processing stages, is modulated according to the internal state of the animal. Internal states, such as arousal, are often characterized by relating neural variables to a single "level" of arousal, defined using a behavioral variable such as locomotion or pupil size. Here, we extend the notion of arousal-related modulation by showing that multifaceted aspects of pupil size dynamics can predict changes in neural activity. Specifically, we show that the phases of pupil size fluctuations occurring over multiple timescales modulate firing mode in the dorsal lateral geniculate nucleus (dLGN) of mice. Increased firing rates, driven by a preponderance of tonic spiking, occurred during pupil dilations, while burst spikes preferentially occurred during contractions. We further find that these activity modulations could not be explained solely by pupil size per se, or by transitions between overt locomotion and quiescence, and extend to periods of patterned stimulus viewing. We conclude that dLGN spiking activity is modulated by pupil-indexed arousal processes on various timescales, with implications for the determination of arousal-related cortical rhythms, and for the transfer of sensory information to the cortex.

sentences: Class II LBD genes ZmLBD5 and ZmLBD33 regulate gibberellin and abscisic acid biosynthesis Lateral organ boundaries domain (LBD) proteins are plant-specific transcription factors. Class I LBD members are widely reported to be pivotal for organ development, however, the role of class II members is unknown in cereal crops. Class II LBD proteins are distinguished from class I by the lack of a Gly-Ala-Ser (GAS) peptide and leucine-zipper-like coiled-coil domain, which is thought to be essential for protein dimerization. In this study, ZmLBD5 and ZmLBD33 form homo- and hetero-dimers, like class I members. At seedling stage, ZmLBD5 promoted biomass accumulation (shoot dry weight and root dry weight), root development (root length, root number, and root volume), and organ expansion (leaf area), while ZmLBD33 repressed these processes and display a dwarf phenotype. Both ZmLBD5 and ZmLBD33 displayed negative roles in drought tolerance mainly by increasing stomatal density and stomatal aperture. RNA sequencing, gene ontology enrichment analysis, and transient luciferase expression assays indicated that ZmLBD5 and ZmLBD33 are mainly involved in the regulation of the TPS-KS-GA2ox gene module, which comprises key enzymatic genes upstream of GA and ABA biosynthesis. GA1 content increased in ZmLBD5-overexpressing seedlings, while GA3 and abscisic acid content decreased in both transgenic seedlings. Consequently, exogenous GA1 or GA3 undoubtedly rescued the dwarf phenotype of ZmLBD33-overexpressing plants, with GA1 performing better. The study of ZmLBD5 and ZmLBD33 sheds light on the function of the class II LBD gene family in maize.

sentences: Identification of the receptor-binding protein of Clostridium difficile phage CDHS-1 reveals a new class of receptor-binding domains. As natural bacterial predators, bacteriophages have the potential to be developed to tackle antimicrobial resistance, but our exploitation of them is limited by understanding their vast uncharacterised genetic diversity1,2. Fascinatingly, this genetic diversity reflects many ways that phages can make proteins, performing similar functions that together form the familiar phage particle. Critical to infection are phage receptor-binding proteins (RBPs) that bind bacterial  receptors and initiate bacterial entry3. Here we identified and characterised Gp22, a novel RBP for phage CDHS-1 that infects pathogenic C. difficile, but that had no recognisable RBPs. We showed that Gp22 antibodies neutralised CDHS-1 infection and used immunogold-labelling and transmission electron microscopy to identify their location on the capsid. The Gp22 three-dimensional structure was resolved by X-ray crystallography revealing a new RBP class with an N-terminal L-shaped -helical superhelix domain and a C-terminal Mg2+-binding domain. The findings provide novel insights into C. difficile phage biology and phage-host interactions. This will facilitate optimal phage development and future engineering strategies4,5. Furthermore, the AlphaFold2-predicted Gp22 structure, which was strikingly accurate, paves the way for a structurome based transformation and guidance of future phage studies where many proteins lack sequence homology but have recognisable protein structures.

sentences: No leeway to enhance carbon sequestration and stock capacity via changes to forest management Forest management interventions can act as value-based agents to remove CO2 from the atmosphere and slow anthropogenic climate change and thus might play a strategic role in the framework of the EU forestry-based mitigation strategy. To what extent diversified management actions could lead to quantitatively important changes in carbon sequestration potential and stocking capacity at the tree level remains to be thoroughly assessed. To that end, we used a state-of-the-science bio-geochemically based forest growth model to assess effects of multiple alternative forest management scenarios on plant net primary productivity (NPP) and potential carbon woody stocks (pCWS) under differing scenarios of climate change. The experiments indicated that the capacity of trees to assimilate and store atmospheric CO2 in recalcitrant standing woody tissue is already being attained as its optimum under business-as-usual forest management conditions regardless of the different climate change scenarios investigated. Nevertheless, on the long-term and under increasing atmospheric CO2 concentration and warming, managed forests show both higher productivity and a larger pool of stored carbon than unmanaged ones as long as forest thinning and tree harvesting are of moderate intensity.

sentences: Reproductive skew, fitness costs, and winner-loser effects in social-dominance evolution Social hierarchies can increase reproductive skew in group-living animals. Using game theory we investigate how the opportunity for differently ranked individuals to acquire resources influences reproductive skew, costs of hierarchy formation, and winner and loser effects. Individuals adjust their aggressive and submissive behaviour through reinforcement learning. The learning is based on perceived rewards and penalties, which depend on relative fighting ability. From individualbased simulations we determine evolutionary equilibria of traits that control an individuals learning. We examine situations that differ in the extent of monopolisation of contested resources by dominants and in the amounts of uncontested resources that are distributed independently of rank. With costly fighting, we find that stable dominance hierarchies form, such that reproductive skew mirrors the distribution of resources over ranks. Individuals pay substantial costs of interacting, in particular in high-skew situations, with the highest costs paid by intermediately ranked individuals. For cases where dominants monopolise contested resources there are notable winner and loser effects, with winner effects for high ranks and very pronounced loser effects for lower ranks. The effects are instead weak when acquired resources increase linearly with rank. We compare our results on contest costs and winner-loser effects with field and experimental observations.

sentences: A pilot study to define and identify future priorities into Allocasuarina robusta recovery as part of a community program The Allocasuarina robusta pilot study investigated the process involved to facilitate seed recruitment as part of a threatened species project. Several experiments occurred, each examining a specific attribute in the seed recruitment process. A. robusta is a threatened species of national and local significance. The research design would help land managers and communities to conserve A. robusta. The investigation aimed to improve seed recruitment in A. robusta occurring under natural conditions. The experiment results highlighted several experimental design flaws and identified opportunities to increase community participation as part of the recovery program.

sentences: Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has outcompeted previously prevalent variants and become a dominant strain worldwide. We report here structure, function and antigenicity of its full-length spike (S) trimer in comparison with those of other variants, including Gamma, Kappa, and previously characterized Alpha and Beta. Delta S can fuse membranes more efficiently at low levels of cellular receptor ACE2 and its pseudotyped viruses infect target cells substantially faster than all other variants tested, possibly accounting for its heightened transmissibility. Mutations of each variant rearrange the antigenic surface of the N-terminal domain of the S protein in a unique way, but only cause local changes in the receptor-binding domain, consistent with greater resistance particular to neutralizing antibodies. These results advance our molecular understanding of distinct properties of these viruses and may guide intervention strategies.

sentences: MultiMAP: Dimensionality Reduction and Integration of Multimodal Data Multimodal data is rapidly growing in many fields of science and engineering, including single-cell biology. We introduce MultiMAP, an approach for dimensionality reduction and integration of multiple datasets. MultiMAP recovers a single manifold on which all of the data resides and then projects the data into a single low-dimensional space so as to preserve the structure of the manifold. It is based on a framework of Riemannian geometry and algebraic topology, and generalizes the popular UMAP algorithm1 to the multimodal setting. MultiMAP can be used for visualization of multimodal data, and as an integration approach that enables joint analyses. MultiMAP has several advantages over existing integration strategies for single-cell data, including that MultiMAP can integrate any number of datasets, leverages features that are not present in all datasets (i.e. datasets can be of different dimensionalities), is not restricted to a linear mapping, can control the influence of each dataset on the embedding, and is extremely scalable to large datasets. We apply MultiMAP to the integration of a variety of single-cell transcriptomics, chromatin accessibility, methylation, and spatial data, and show that it outperforms current approaches in preservation of high-dimensional structure, alignment of datasets, visual separation of clusters, transfer learning, and runtime. On a newly generated single-cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq) and single-cell RNA-seq (scRNA-seq) dataset of the human thymus, we use MultiMAP to integrate cells along a temporal trajectory. This enables the quantitative comparison of transcription factor expression and binding site accessibility over the course of T cell differentiation, revealing patterns of transcription factor kinetics.

sentences: A Novel Membrane Protein in the Rhodobacter sphaeroides LH1-RC Photocomplex We present a cryo-EM structure of the monomeric light-harvesting-reaction center (LH1-RC) core complex from photosynthetic purple bacterium Rhodobacter (Rba.) sphaeroides at 2.9 [A] resolution. The LH1 complex forms a C-shaped structure composed of 14 {beta}-polypeptides around the RC with a large ring opening. From the cryo-EM density map, a previously unrecognized integral membrane protein, referred to as protein-U, was identified. Protein-U has a U-shaped conformation near the LH1-ring opening and was annotated as a hypothetical protein in the Rba. sphaeroides genome. Deletion of protein-U resulted in a mutant strain that expressed a much-reduced amount of the dimeric LH1-RC, indicating an important role for protein-U in dimerization of the LH1-RC complex. PufX was located opposite protein-U on the LH1-ring opening, and both its position and conformation differed from that of previous reports of dimeric LH1-RC structures obtained at low-resolution. Twenty-six molecules of the carotenoid spheroidene arranged in two distinct configurations were resolved in the Rba. sphaeroides LH1 and were positioned within the complex to block its pores. Our findings offer a new view of the core photocomplex of Rba. sphaeroides and the connections between structure and function in bacterial photocomplexes in general.

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Rules for the self-assembly of ESCRT-III on endosomes We, the authors, have withdrawn this manuscript. During our work on the revision of this manuscript for eLife, it became clear that several key results of the manuscript were not reproducible by my lab. The rules for ESCRT-III assembly and the model that we have proposed in this manuscript are no longer supported by the data. Currently we are performing additional experiments to test some of our conclusions further. Therefore, we do not wish this work to be cited.

We sincerely apologize to the scientific community for any inconvenience resulting from this manuscript.

If you have any questions, please contact the corresponding author.

sentences: Multi-Task Brain Network Reconfiguration is Inversely Associated with Human Intelligence Intelligence describes the general cognitive ability level of a person. It is one of the most fundamental concepts in psychological science and is crucial for effective adaption of behavior to varying environmental demands. Changing external task demands have been shown to induce reconfiguration of functional brain networks. However, whether neural reconfiguration between different tasks is associated with intelligence has not yet been investigated. We used fMRI data from 812 subjects to show that higher scores of general intelligence are related to less brain network reconfiguration between resting state and seven different task states as well as to network reconfiguration between tasks. This association holds for all functional brain networks except the motor system, and replicates in two independent samples (N = 138, N = 184). Our findings suggest that the intrinsic network architecture of individuals with higher intelligence scores is closer to the network architecture as required by various cognitive demands. Multi-task brain network reconfiguration may, therefore, represent a neural reflection of the behavioral positive manifold - the essence of the concept of general intelligence. Finally, our results support neural efficiency theories of cognitive ability and reveal insights into human intelligence as an emergent property from a distributed multi-task brain network.

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Cohesin is essential for the timely activation of condensin and anaphase segregation Chromosome segregation requires the separation of sister chromatids and the sustained condensation of chromatids during anaphase. Yeast cohesin has a dual function on metaphase chromosomes, holding sister chromatids together and organising individual chromatids into loops. Cleavage by separase is thought to remove all cohesin from chromosomes at the anaphase onset. This fulfils the requirement for chromatid separation, but it is unclear how the structure of segregating chromatids is maintained following cleavage. Here we demonstrate that auxin-mediated degradation of cohesins kleisin in anaphase, causes catastrophic chromosome mis-segregation demonstrating that cohesin affects post-metaphase processes. We identify a pool of cohesin bound to anaphase/telophase chromosomes and demonstrate that its inactivation causes defects in the organisation of centromeric regions and condensin function. Our data thus uncovers an unsuspected role for cohesin on anaphase/telophase chromosomes that is essential for the fidelity of segregation.

One sentence summaryCohesin complexes on segregating chromosomes provide centromere structure and regulate condensin activity, demonstrating that cohesin has active roles after metaphase.

sentences: The Daphnia carapace and the origin of novel structures Understanding how novel structures arise is a central question in evolution. Novel structures are often defined as structures that are not derived from (homologous to) any structure in the ancestor1. The carapace of the water flea Daphnia magna is a bivalved "cape" of exoskeleton that has been proposed to be one of many novel arthropod structures that arose through repeated co-option of genes that also pattern insect wings2-4. To determine whether the Daphnia carapace is a novel structure, we compare the expression of pannier, araucan, and vestigial between Daphnia, Parhyale, and Tribolium. Our results suggest that the Daphnia carapace did not arise by co-option, but instead derives from an exite (lateral lobe) that emerges from an ancestral proximal leg segment that was incorporated into the Daphnia body wall. The Daphnia carapace therefore appears to be homologous to the Parhyale tergal plate and the insect wing5. Remarkably, the vestigial-positive region that gives rise to the Daphnia carapace appears to be present in Parhyale6 and Tribolium as a small, inconspicuous protrusion. Similarly, the vestigial-positive regions that form thoracic tergal plates in Parhyale appear to be present in Daphnia, even though Daphnia does not form thoracic tergal plates. Thus, rather than a novel structure resulting from gene co-option, the Daphnia carapace appears to have arisen from a shared, ancestral tissue (morphogenetic field) that persists in a cryptic state in other arthropod lineages. Cryptic persistence of unrecognized serial homologs may thus be a general solution for the origin of novel structures.

sentences: Utilizing proteomics and phosphoproteomics to predict ex vivo drug sensitivity across genetically diverse AML patients Acute Myeloid Leukemia (AML) affects 20,000 patients in the US annually with a five-year survival rate of approximately 25%. One reason for the low survival rate is the high prevalence of clonal evolution that gives rise to heterogeneous sub-populations of leukemia. This genetic heterogeneity is difficult to treat using conventional therapies that are generally based on the detection of a single driving mutation. Thus, the use of molecular signatures, consisting of multiple functionally related transcripts or proteins, in making treatment decisions may overcome this hurdle and provide a more effective way to inform drug treatment protocols. Toward this end, the Beat AML research program prospectively collected genomic and transcriptomic data from over 1000 AML patients and carried out ex vivo drug sensitivity assays to identify signatures that could predict patient-specific drug responses. The Clinical Proteomic Tumor Analysis Consortium is in the process of extending this cohort to collect proteomic and phosphoproteomic measurements from a subset of these patient samples to evaluate the hypothesis that proteomic signatures can robustly predict drug response in AML patients. We sought to examine this hypothesis on a sub-cohort of 38 patient samples from Beat AML with proteomic and drug response data and evaluate our ability to identify proteomic signatures that predict drug response with high accuracy. For this initial analysis we built predictive models of patient drug responses across 26 drugs of interest using the proteomics and phosphproteomics data. We found that proteomics-derived signatures provide an accurate and robust signature of drug response in the AML ex vivo samples, as well as related cell lines, with better performance than those signatures derived from mutations or mRNA expression. Furthermore, we found that in specific drug-resistant cell lines, the proteins in our prognostic signatures represented dysregulated signaling pathways compared to parental cell lines, confirming the role of the proteins in the signatures in drug resistance. In conclusion, this pilot study demonstrates strong promise for proteomics-based patient stratification to predict drug sensitivity in AML.

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Across functional boundaries: making non-neutralizing antibodies to neutralize HIV-1 and mediate Fc-mediated effector killing of infected cells In HIV-1 infection, many antibodies (Abs) are elicited to Envelope (Env) epitopes that are conformationally masked in the native trimer and are only available for antibody recognition after the trimer binds host cell CD4. Among these are epitopes within the Co-Receptor Binding Site (CoRBS) and the constant region 1 and 2 (C1-C2 or Cluster A region). In particular, C1-C2 epitopes map to the gp120 face interacting with gp41 in the native,  closed Env trimer present on HIV-1 virions or expressed on HIV-1 infected cells. Antibodies targeting this region are therefore non-neutralizing and their potential as mediators of antibody depended cellular cytoxicity (ADCC) of HIV-1 infected cells diminished by a lack of available binding targets. Here we present the design of Ab-CD4 chimeric proteins that consist of the Ab-IgG1 of a CoRBS or Cluster A specificity to the extracellular domain 1 and 2 of human CD4. Our Ab-CD4 hybrids induce potent ADCC against infected primary CD4+ T cells and neutralize tier 1 and 2 HIV-1 viruses. Furthermore, competition binding experiments reveal that the observed biological activities rely on both the antibody and CD4 moieties confirming their cooperativity in triggering conformational rearrangements of Env. Our data indicate the utility of these Ab-CD4 hybrids as antibody therapeutics effective in eliminating HIV-1 through the combined mechanisms of neutralization and ADCC. This is also the first report of single-chain-Ab-based molecules capable of opening  closed Env trimers on HIV-1 particles/infected cells to expose the Cluster A region and activate ADCC and neutralization against these non-neutralizing targets.

ImportanceHighly conserved epitopes within the co-receptor binding site (CoRBS) and constant region 1 and 2 (C1C2 or Cluster A) are only available for antibody recognition after the HIV-1 Env trimer binds host cell CD4, therefore they are not accessible on virions and infected cells where the expression of CD4 is downregulated. Here we have developed new antibody fusion molecules in which domains 1 and 2 of soluble human CD4 are linked with monoclonal antibodies of either the CoRBS or Cluster A specificity. We optimized the conjugation sites and linker lengths to allow each of these novel bispecific fusion molecules to recognize native "closed" Env trimers and induce the structural rearrangements required for exposure of the epitopes for antibody binding. Our in vitro functional testing shows that our Ab-CD4 molecules can efficiently target and eliminate HIV-1 infected cells through antibody depended cellular cytoxicity (ADCC) and inactivate HIV-1 virus through neutralization.

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Morphometric relationships and their contribution to biomass and cannabinoid yield in hybrids of hemp (Cannabis sativa) The breeding of hybrid cultivars of hemp (Cannabis sativa L.) is not well described, especially the segregation and inheritance of traits that are important for yield. A total of 23 families were produced from genetically diverse parents to investigate the inheritance of morphological traits and their association with biomass accumulation and cannabinoid yield. In addition, a novel classification method for canopy architecture was developed. The strong linear relationship between wet and dry biomass provided an accurate estimate of final dry stripped floral biomass. Of all field and aerial measurements, basal stem diameter was determined to be the single best selection criterion for final dry stripped floral biomass yield. Along with stem diameter, canopy architecture and stem growth predictors described the majority of the explainable variation of biomass yield. Within-family variance for morphological and cannabinoid measurements reflected the heterozygosity of the parents. While selfed populations suffered from inbreeding depression, hybrid development in hemp will require at least one inbred parent to achieve uniform growth and biomass yield. Nevertheless, floral phenology remains a confounding factor in selection because of its underlying influence on biomass production highlighting the need to understand the genetic basis for flowering time in the breeding of uniform cultivars.

HighlightStem and canopy architecture traits are superior predictors of floral biomass yield and offer a good indication of hybrid uniformity in field plantings of genetically diverse cannabinoid hemp populations.

sentences: Chloroplasts in plant cells show active glassy behavior under low light conditions Plants have developed intricate mechanisms to adapt to changing light conditions. Besides photo- and heliotropism - the differential growth towards light and the diurnal motion with respect to sunlight - chloroplast motion acts as a fast mechanism to change the intracellular structure of leaf cells. While chloroplasts move towards the sides of the plant cell to avoid strong light, they accumulate and spread out into a layer on the bottom of the cell at low light to increase the light absorption efficiency. Although the motion of chloroplasts has been studied for over a century, the collective organelle-motion leading to light adapting self-organized structures remains elusive. Here we study the active motion of chloroplasts under dim light conditions, leading to an accumulation in a densely packed quasi-2D layer. We observe burst-like re-arrangements and show that these dynamics resemble colloidal systems close to the glass transition by tracking individual chloroplasts. Furthermore, we provide a minimal mathematical model to uncover relevant system parameters controlling the stability of the dense configuration of chloroplasts. Our study suggests that the meta-stable caging close to the glass-transition in the chloroplast mono-layer serves a physiological relevance. Chloroplasts remain in a spread-out configuration to increase the light uptake, but can easily fluidize when the activity is increased to efficiently re-arrange the structure towards an avoidance state. Our research opens new questions about the role that dynamical phase transitions could play in self-organized intracellular responses of plant cells towards environmental cues.

sentences: High Cellular Uptake Gene Delivery Platform with Chitosan and L-arginine Complex for Cancer Treatment Gene therapy outperforms chemotherapy in limiting the harm to human body. A major impediment to the progress of gene therapy is a lack of proper carriers. Chitosan, derived from shellfish, is a desirable candidate for nucleic acid delivery because of its outstanding biodegradability and biocompatibility. However, previous works show that chitosan has poor cell membrane permeability. In this study, we synthesize a complex delivery system for gene therapy by compounding chitosan with L-arginine, and test the encapsulation, uptake, and cancer cell treatment power. Encapsulation efficiency of the chitosan/L-arginine complex (CA) can reach more than 90% in weight. Flow cytometry reveal that siRNA delivered by the complex can be taken in by cells 10 times better than free siRNA and 2 times better than chitosan alone. Confocal imaging confirmed the high cellular uptake, and siRNA tests in HeLa cells indicate the successful silencing of the target gene (RRM2). The complex serves as a gene delivery system that is capable of loading and delivering a variety of genetic materials in which the specific nucleic acid molecule to be delivered could be altered at will.

sentences: TGF-β1 drives Th9 but not Treg cells upon allergen exposure TGF-{beta}1 is known to have a pro-inflammatory impact by inducing Th9 cells, while it also induces anti-inflammatory Treg cells (Tregs). In the context of allergic airway inflammation (AAI) its dual role can be of critical importance in influencing the outcome of the disease. Here we demonstrate that TGF-{beta} acts in AAI by driving effector T cells into Th9 cells, while Tregs differentiate independently. Induction of experimental AAI and airway hyperreactivity in a mouse model with inducible genetic ablation of the TGF{beta}-receptor 2 (TGFBR2) on CD4+T cells significantly reduced the disease phenotype. Further, it blocked the induction of Th9 cell frequencies, but increased Treg cells. To translate these findings into a human clinically relevant context, Th9 and Treg cells were quantified both locally in induced sputum and systemically in blood of allergic rhinitis and asthma patients with or without allergen-specific immunotherapy (AIT). Natural allergen exposure induced local and systemic Th2, Th9 cell and reduced Tregs, while therapeutic allergen exposure by AIT suppressed Th2 and Th9 cell frequencies along with TGF-{beta} and IL-9 secretion. Altogether, these findings support that neutralization of TGF-{beta} represents a viable therapeutic option in allergy and asthma, not posing the risk of immune dysregulation by impacting Tregs.

sentences: Reuse of a wing venation gene-regulatory network in patterning the eyespot rings of butterflies Novel organismal traits might reuse ancestral gene-regulatory networks (GRNs) in their development, but data supporting this mechanism are still sparse. Here we propose that a conserved venation gene network, used in patterning insect wings, has been recruited to pattern the sharp and distinct rings of color in butterfly eyespot development. This network involves Decapentaplegic (Dpp), acting as a central morphogen, activating optix and spalt at different concentration thresholds. It also involves Spalt cross-regulating optix resulting in the formation of a sharp boundary between the two genes. First, using in situ hybridizations, we show that dpp is expressed in the eyespot centers during the early pupal stage and that CRISPR-Cas9 embryonic knockouts of dpp, optix, and spalt result in the loss of eyespots, loss of the orange ring, and loss of the black scales, respectively. Furthermore, using RNAi during the pupal stage, we show that dpp down-regulation results in the complete or partial loss of eyespots. Using CRISPR disruptions of optix or spalt, followed by immunostainings, we show that Spalt represses optix in the central region of the eyespot, limiting optix expression to a more peripheral ring. We further illustrate that optix disruptions alter scale pigmentation and scale ultrastructure as they transform orange to brown scales. Altogether, the regulatory interactions between dpp, spalt and optix mimic those found in earlier larval developmental stages, in the anterior compartment of an insects wing, where these genes play a role in venation patterning. The network similarities suggest that the venation GRN was co-opted to aid in the differentiation of the eyespot rings.

sentences: A Class of Simple Biomolecular Antithetic Proportional-Integral-Derivative Controllers Proportional-Integral-Derivative (PID) feedback controllers have been the most widely used controllers in industry for almost a century due to their good performance, simplicity, and ease of tuning. Motivated by their success in various engineering disciplines, PID controllers recently found their way into synthetic biology, where the design of feedback molecular control systems has been identified as an important goal. In this paper, we consider the mathematical realization of PID controllers via biomolecular interactions. We propose an array of topologies that offer a compromise between simplicity and high performance. We first demonstrate that different Proportional-Integral (PI) controllers exhibit different capabilities for enhancing the dynamics and reducing variance (cell-to-cell variability). Next, we introduce several derivative controllers that are realized based on incoherent feedforward loops acting in a feedback configuration. Alternatively, we show that differentiators can be realized by placing molecular integrators in a negative feedback loop--an arrangement that can then be augmented by PI components to yield PID feedback controllers. We demonstrate that the derivative component can be exploited for enhancing system stability, dramatically increasing the molecular control systems dynamic performance, and for reducing the noise effect on the output of interest. The PID controller features are established through various deterministic and stochastic analyses as well as numerical simulations. Finally, we provide an experimental demonstration using a recently developed hybrid setup, the cyberloop, where the controller is implemented in silico to control a biological genetic circuit in vivo. The large array of novel biomolecular PID controllers introduced here forms a basis for the design and construction of advanced high-performance biomolecular control systems that robustly regulate the dynamics of living systems.

sentences: Discovery of a Novel Inhibitor for Chikungunya Virus Chikungunya virus (CHIKV) is the etiological agent of chikungunya fever, a (re)emerging arbovirus infection, that causes severe and often persistent arthritis, representing a serious health problem worldwide for which no antivirals are currently available. Despite the efforts over the last decade to identify and optimize new inhibitors or to reposition existing drugs, no compound has progressed to clinical trials and prophylaxis is based on vector control, which has shown limited success in containing the virus. Herein, we screened 36 compounds using a replicon system and ultimately identified 3-methyltoxoflavin with activity against CHIKV using cell assays (EC50 200 nM). We have additionally screened 3-methyltoxoflavin against a panel of viruses and showed that it also inhibits yellow fever virus (IC50 370 nM, SI=3.2 in Huh-7 cells). 3-methyltoxoflavin is a known protein disulfide isomerase (PDI) inhibitor, and inhibitor of alphaviruses which likely depends on this host protein to aid in facilitating disulfide bond formation and isomerization, since alphaviruses require conserved cysteine residues for proper folding and assembly of the E1 and E2 envelope glycoproteins. In summary we demonstrate that 3-methyltoxoflavin has activity against CHIKV and may represent a starting point for optimization to develop inhibitors to this and other viruses.

sentences: The Neural Determinants of Beauty What are the conditions that determine whether the medial orbito-frontal cortex (mOFC), in which activity correlates with the experience of beauty derived from different sources, becomes co-active with sensory areas of the brain during the experience of sensory beauty? We addressed this question by studying the neural determinants of facial beauty. The perception of faces correlates with activity in a number of brain areas, but only when a face is perceived as beautiful is the mOFC also engaged. The enquiry thus revolved around the question of whether a particular pattern of activity, within or between areas implicated in face perception, emerges when a face is perceived as beautiful, and which determines that there is, as a correlate, activity in mOFC. 17 subjects of both genders viewed and rated facial stimuli according to how beautiful they perceived them to be while the activity in their brains was imaged with functional magnetic resonance imaging. A univariate analysis revealed parametrically scaled activity within several areas in which the strength of activity correlated with the declared intensity of the aesthetic experience of faces; the list included the mOFC and two core areas strongly implicated in the perception of faces - the occipital face area (OFA), fusiform face area (FFA)- and, additionally, the cuneus. Multivariate analyses showed strong patterns of activation in the FFA and the cuneus and weaker patterns in the OFA and the pSTS. It is only when specific patterns emerged in these areas that there was co-activation of the mOFC, in which a strong pattern also emerged during the experience of facial beauty. A psychophysiological interaction analysis with mOFC as the seed area revealed the involvement of the right FFA and the right OFA. We conjecture that these collective patterns of activity constitute the neural basis for the experience of facial beauty, bringing us a step closer to understanding the neural determinants of aesthetic experience.

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Unsupervised cell functional annotation for single-cell RNA-Seq One of the first steps in the analysis of single cell RNA-Sequencing data (scRNA-Seq) is the assignment of cell types. While a number of supervised methods have been developed for this, in most cases such assignment is performed by first clustering cells in low-dimensional space and then assigning cell types to different clusters. To overcome noise and to improve cell type assignments we developed UNIFAN, a neural network method that simultaneously clusters and annotates cells using known gene sets. UNIFAN combines both, low-dimensional representation for all genes and cell specific gene set activity scores to determine the clustering. We applied UNIFAN to human and mouse scRNA-Seq datasets from several different organs. As we show, by using knowledge on gene sets, UNIFAN greatly outperforms prior methods developed for clustering scRNA-Seq data. The gene sets assigned by UNIFAN to different clusters provide strong evidence for the cell type that is represented by this cluster making annotations easier.

Softwarehttps://github.com/doraadong/UNIFAN

sentences: Balancing selection on genomic deletion polymorphisms in humans A key question in biology is why genomic variation persists in a population for extended periods. Recent studies have identified examples of genomic deletions that have remained polymorphic in the human lineage for hundreds of millennia, ostensibly owing to balancing selection. Nevertheless, genome-wide investigations of ancient and possibly adaptive deletions remain an imperative exercise. Here, we used simulations to show an excess of ancient allele sharing between modern and archaic human genomes that cannot be explained solely by introgression or ancient structure under neutrality. We identified 63 deletion polymorphisms that emerged before the divergence of humans and Neanderthals and are associated with GWAS traits. We used empirical and simulation-based analyses to show that the haplotypes that harbor these functional ancient deletions have likely been evolving under time- and geography-dependent balancing selection. Collectively, our results suggest that balancing selection may have maintained at least 27% of the functional deletion polymorphisms in humans for hundreds of thousands of years.

sentences: Exploring the miRNA-mediated response to combined stresses in melon plants Climate change has been associated with a higher incidence of combined adverse environmental conditions that can promote a significant decrease in crop productivity. However, knowledge on how a combination of stresses might affect plant development is still scarce. MicroRNAs (miRNAs) have been proposed as potential targets for improving crop-productivity. Here, we have combined deep-sequencing, computational characterization of responsive miRNAs and validation of their regulatory role in a comprehensive analysis of melons response to several combinations of four stresses (cold, salinity, short day, and infection with a fungus). Twenty-two miRNA families responding to double and/or triple stresses were identified. The regulatory role of the differentially expressed miRNAs was validated by quantitative measurements of the expression of the corresponding target genes. A high proportion (ca. 60%) of these families (mainly highly conserved miRNAs targeting transcription factors) showed a non-additive response to multiple stresses in comparison with that observed under each one of the stresses individually. Among those miRNAs showing non-additive response to stress-combinations, most interactions were negative suggesting the existence of functional convergence in the miRNA-mediated response to combined stresses. Taken together, our results provide compelling evidences that the response to combined stresses cannot be easily predicted from the study individual stresses.

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Biomass, microbial composition and functions are responsible for the differential removal of trace organic chemicals in biofiltration systems Biofiltration processes help to remove trace organic chemicals (TOrCs) both in wastewater and drinking water treatment systems. However, the detailed TOrCs biotransformation mechanisms as well as the underlying drivers behind the variability of site specific transformation processes remain elusive. In this study, we used laboratory batch incubations to investigate the biotransformation of 51 TOrCs in eight bioactive filter materials of different origins treating a range of waters, from wastewater effluents to drinking water. Microscopy, 16S rRNA amplicon and whole metagenome sequencing for assessing associations between the biotransformation rate constants, microbial composition and genetic potential complemented chemical analysis. We observed strong differences in the mean global removal of TOrCs between the individual sand filters (-1.4% to 58%), which were mirrored in overall biomass, microbial community composition, and enzyme encoding genes. From the six investigated biomass markers, ATP turned out to be a major predictor of the mean global biotransformation rate, while compound specific biotransformations were correlated with the microbial community composition. High biomass ecosystems were indicated in our systems by a dominance of Nitrospirae, but individual TOrC biotransformation was statistically connected to rare taxa (< 2%) such as Hydrogenophaga, or indiviudal functions such as the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase encoding genes. In general, this study provides new insights into so far rarely addressed variability of TOrCs biotransformation. We propose novel biological indicators for the removal performance of TOrCs in biofiltration systems, highlighting the role of living biomass in predicting and normalizing the global transformation, and the role of the microbial community for the individual transformation of TOrCs in engineered and natural systems.

Contribution to the Field StatementTrace organic chemicals (TOrCs) are an emerging problem in the aquatic environment that has attracted global attention over the last decade. Recent research efforts on this topic have increased our knowledge on the transformation of TOrCs and various technologies have been developed to improve their removal. In this study, we investigated a wide range of biotransformation of TOrCs by eight sand filter materials from wastewater and water treatment plants. Biotransformation rate constants were calculated using first-order kinetics to evaluate TOrC removal performance. We reevaluated the role of biomass and could thus explain a greater part of the global TOrC removal performance. The remaining variation in removal rates of individual compounds correlated with the microbiome of the biofilter. Rare biosphere lineages and specific enzyme categories genes were correlated with the removal of certain compounds. In summary, our research identified future indicators for successful biotransformation of TOrCs in biofilter systems.

sentences: Relocation of rDNA repeats for repair is dependent on SUMO-mediated nucleolar release by the Cdc48/p97 segregase Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to active transcription and their repetitive nature. Compartmentalization of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be released to the nucleoplasm to allow repair by homologous recombination. The process of rDNA relocation is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation targets CLIP-cohibin for disassembly mediated by the Cdc48/p97 chaperone, which recognizes SUMOylated CLIP-cohibin through its cofactor, Ufd1. Consistent with a conserved mechanism, UFD1L depletion impairs rDNA release in human cells. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity.

sentences: Near-infrared imaging in fission yeast by genetically encoded biosynthesis of phycocyanobilin Near-infrared fluorescent protein (iRFP) is a bright and stable fluorescent protein with excitation and emission maxima at 690 nm and 713 nm, respectively. Unlike the other conventional fluorescent proteins such as GFP, iRFP requires biliverdin (BV) as a chromophore because iRFP originates from bacteriophytochrome. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and biosynthesis of PCB allows live-cell imaging with iRFP in the fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV, and therefore did not show any iRFP fluorescence. The brightness of iRFP attached to PCB was higher than that of iRFP attached to BV in vitro and in fission yeast. We introduced SynPCB, a previously reported PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids, which contain genes required for the SynPCB system and the iRFP-fused marker proteins. These tools not only enable the easy use of iRFP in fission yeast and the multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the door to new opportunities for near-infrared fluorescence imaging in a wider range of living organisms.

sentences: Thriving of hyperthermophilic microbial communities from a deep-sea sulfidic hydrothermal chimney under electrolithoautotrophic conditions with nitrate as electron acceptor. Recent studies have shown the presence of an abiotic electrical current across the walls of deep-sea hydrothermal chimneys, allowing the growth of electroautotrophic microbial communities. To understand the role of the different phylogenetic groups and metabolisms involved, this study focused on an electrotrophic enrichment, with nitrate as electron acceptor. The biofilm density, the community composition, the organic products released, and the electrical consumption were monitored by FISH confocal microscopy, qPCR, Metabarcoding, MNR and potentiostat measurements. A statistic analysis by PCA showed the correlation between the different parameters in 3 distinct temporal phases. The Archaeoglobales have been shown to play a key role in the development of the community, as first colonizers and producing pyruvate, therefor used as organic source for heterotrophs. Some Thermococcales showed the ability to perform electrofermentation of this pyruvate into acetate and H2. Finally, through subcultures of the community, we showed the development of a larger biodiversity over time. This observed phenomenon could explain the biodiversity development in hydrothermal context where energy sources are transient and unstable.

sentences: Pathology dynamics in healthy-toxic protein interaction and the multiscale analysis of neurodegenerative diseases Neurodegenerative diseases are frequently associated with aggregation and propagation of toxic proteins. In particular, it is well known that along with amyloid-beta, the tau protein is also driving Alzheimers disease. Multiscale reaction-diffusion models can assist in our better understanding of the evolution of the disease. We have modified the heterodimer model in such a way that it can now capture some of critical characteristics of this evolution such as the conversion time from healthy to toxic proteins. We have analyzed the modified model theoretically and validated the theoretical findings with numerical simulations.

sentences: The assessment of similarity vectors of fingerprint and UMLS in adverse drug reaction prediction Identifying and controlling adverse drug reactions is a complex problem in the pharmacological field. Despite the studies done in different laboratory stages, some adverse drug reactions are recognized after being released, such as Rosiglitazone. Due to such experiences, pharmacists are now more interested in using computational methods to predict adverse drug reactions. In computational methods, finding and representing appropriate drug and adverse reaction features are one of the most critical challenges. Here, we assess fingerprint and target as drug features; and phenotype and unified medical language system as adverse reaction features to predict adverse drug reaction. Meanwhile, we show that drug and adverse reaction features represented by similarity vectors can improve adverse drug prediction. In this regard, we propose four frameworks. Two frameworks are based on random forest classification and neural networks as machine learning methods called F_RF and F_NN, respectively. The rest of them improve two state-of-art matrix factorization models, CS and TMF, by considering target as a drug feature and phenotype as an adverse reaction feature. However, machine learning frameworks with fewer drug and adverse reaction features are more accurate than matrix factorization frameworks. In addition, the F_RF framework performs significantly better than F_NN with ACC = %89.15, AUC = %96.14 and AUPRC = %92.9. Next, we contrast F_RF with some well-known models designed based on similarity vectors of drug and adverse reaction features. Unlike other methods, we do not remove rare reactions from the data set in our frameworks. The data and implementation of proposed frameworks are available at http://bioinformatics.aut.ac.ir/ADRP-ML-NMF/.

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A discovery-based proteomics approach identifies protein disulfide isomerase (PDIA1) as a biomarker of β cell stress in type 1 diabetes BackgroundActivation of stress pathways intrinsic to the {beta} cell are thought to both accelerate {beta} cell death and increase {beta} cell immunogenicity in type 1 diabetes (T1D). However, information on the timing and scope of these responses is lacking.

MethodsTo identify temporal and disease-related changes in islet {beta} cell protein expression, data independent acquisition-mass spectrometry was performed on islets collected longitudinally from NOD mice and NOD-SCID mice rendered diabetic through T cell adoptive transfer.

FindingsIn islets collected from female NOD mice at 10, 12, and 14 weeks of age, we found a time-restricted upregulation of proteins involved in the maintenance of {beta} cell function and stress mitigation, followed by loss of expression of protective proteins that heralded diabetes onset. Pathway analysis identified EIF2 signaling and the unfolded protein response, mTOR signaling, mitochondrial function, and oxidative phosphorylation as commonly modulated pathways in both diabetic NOD mice and NOD-SCID mice rendered acutely diabetic by adoptive transfer, highlighting this core set of pathways in T1D pathogenesis. In immunofluorescence validation studies, {beta} cell expression of protein disulfide isomerase A1 (PDIA1) and 14-3-3b were found to be increased during disease progression in NOD islets, while PDIA1 plasma levels were increased in pre-diabetic NOD mice and in the serum of children with recent-onset T1D compared to age and sex-matched non-diabetic controls.

InterpretationWe identified a common and core set of modulated pathways across distinct mouse models of T1D and identified PDIA1 as a potential human biomarker of {beta} cell stress in T1D.

sentences: Therapeutic efficacy of an oral nucleoside analog of remdesivir against SARS-CoV-2 pathogenesis in mice. The COVID-19 pandemic remains uncontrolled despite the rapid rollout of safe and effective SARS-CoV-2 vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. Additionally, the emergence of SARS-CoV-2 variants of concern with their potential to escape therapeutic monoclonal antibodies emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parental nucleoside of remdesivir, which targets the highly conserved RNA-dependent RNA polymerase. GS-621763 exhibited significant antiviral activity in lung cell lines and two different human primary lung cell culture systems. The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 significantly reduced viral load, lung pathology, and improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral currently in human clinical trial, proved both drugs to be similarly efficacious. These data demonstrate that therapy with oral prodrugs of remdesivir can significantly improve outcomes in SARS-CoV-2 infected mice. Thus, GS-621763 supports the exploration of GS-441524 oral prodrugs for the treatment of COVID-19 in humans.

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Detection of velogenic avian paramyxoviruses in rock doves in New York City, New York Avian paramyxovirus 1 (APMV-1), also known as Newcastle disease virus (NDV), causes severe and economically important disease in poultry around the globe. Although a limited amount of APMV-1 strains in urban areas have been characterized, the role of the urban wild bird population as an APMV-1 reservoir is unclear. Since urban birds may have an important role for long-term circulation of the virus, fecal and swab samples were collected by community scientists from wild birds in New York City (NYC), New York, United States. These samples were screened for APMV-1 and genotypically characterized by sequencing of the complete genome. A total of 885 samples were collected from NYC parks and from a local wildlife rehabilitation clinic from October 2020 through June 2021. Eight birds (1.1 %) screened positive for the APMV-1 nucleoprotein gene by conventional reverse transcription polymerase chain reaction (RT-PCR), and two live viruses were isolated via egg culture. The F protein cleavage site, an indicator of pathogenicity, was present in the two samples fully sequenced by next generation sequencing, and positioned 112R R K K R F117. Phylogenetic analysis of the F gene coding sequence classified both isolates into genotype VI, a diverse and predominant genotype responsible for NDV outbreaks in pigeon and dove species worldwide.

ImportanceHere we describe the first large-scale effort to screen for APMV-1 in New York Citys wild bird population as part of the New York City Virus Hunters program, a community science initiative. We have characterized two isolates of NDV, with phylogenetic analyses suggesting diversity in established and circulating strains of pigeon paramyxoviruses. Our isolates are also domestic reference strains for future APMV-1 vaccine developments. Future surveillance in this region may contribute to our understanding of NDVs evolution and genetic diversity, as well as inform poultry husbandry and vaccination practices in New York State.

sentences: Dynamic subcortical modulators of human default mode network function The brains  default mode network (DMN) enables flexible switching between internally and externally focused cognition. Precisely how this modulation occurs is not well understood, although may involve key subcortical mechanisms, including hypothesized influences from the basal forebrain (BF) and mediodorsal thalamus (MD). Here, we used ultra-high field (7T) functional magnetic resonance imaging to examine the involvement of the BF and MD across states of task-induced DMN activity modulation. Specifically, we mapped DMN activity suppression ( deactivation) when participants transitioned between rest and externally focused task performance, as well as DMN activity engagement ( activation) when task performance was internally (i.e., self) focused. Consistent with recent rodent studies, the BF showed overall activity suppression with DMN cortical regions when comparing the rest to external task conditions. Further analyses, including dynamic causal modelling, confirmed that the BF drove changes in DMN cortical activity during these rest-to-task transitions. The MD, by comparison, was specifically engaged during internally focused cognition and demonstrated a broad excitatory influence on DMN cortical activation. These results provide the first direct evidence in humans of distinct basal forebrain and thalamic circuit influences on the control of DMN function and suggest novel mechanistic avenues for ongoing translational research.

sentences: The B1.351 and P.1 variants extend SARS-CoV-2 host range to mice Receptor recognition is a major determinant of viral host range, infectivity and pathogenesis. Emergences have been associated with serendipitous events of adaptation upon encounters with novel hosts, and the high mutation rate of RNA viruses may explain their frequent host shifts. SARS-CoV-2 extensive circulation in humans results in the emergence of variants, including variants of concern (VOCs) with diverse mutations notably in the spike, and increased transmissibility or immune escape. Here we show that, unlike the initial and Delta variants, the three VOCs bearing the N501Y mutation can infect common laboratory mice. Contact transmission occurred from infected to naive mice through two passages. This host range expansion likely results from an increased binding of the spike to the mouse ACE2. Together with the observed contact transmission, it raises the possibility of wild rodent secondary reservoirs enabling the emergence of new variants.

sentences: Hypertrophic Chondrocytes Serve as a Reservoir for Unique Marrow Associated Skeletal Stem and Progenitor Cells, Osteoblasts, and Adipocytes During Skeletal Development Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; R26-tdTomatof/+ and Col10a1Cre; R26-tdTomatof/+) in combination with a PDGFRa-H2B-GFP transgenic line, single cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage.

sentences: Sulfide toxicity as key control on anaerobic oxidation of methane in eutrophic coastal sediments Coastal zones account for significant global marine methane emissions to the atmosphere. In coastal ecosystems, the tight balance between microbial methane production and oxidation in sediments prevents most methane from escaping to the water column. Anthropogenic activities, causing eutrophication and bottom water deoxygenation, could disrupt this balance in the microbial methane cycle and lead to increased methane release from coastal sediments. Here, we combined microbiological and biogeochemical analyses of sediments from three sites along a bottom water redox gradient (oxic-hypoxic-euxinic) in the eutrophic Stockholm Archipelago to investigate the impact of anthropogenically-induced redox shifts on microbial methane cycling. At both the hypoxic and euxinic site, sediments displayed a stronger depletion of terminal electron acceptors at depth and a shoaling of the sulfate-methane transition zone in comparison to the oxic site. Porewater methane and sulfide concentrations and potential methane production rates were also higher at the hypoxic and euxinic site. Analyses of metagenome-assembled genomes and 16S rRNA gene profiling indicated that methanogens became more abundant at the hypoxic and euxinic site, while anaerobic methane-oxidizing archaea (ANME), present in low coverage at the oxic site, increased at the hypoxic site but virtually disappeared at the euxinic site. A 98% complete genome of an ANME-2b Ca. Methanomarinus archaeon had genes encoding a complete reverse methanogenesis pathway, several multiheme cytochromes, and a sulfite reductase predicted to detoxify sulfite. Based on these results, we infer that sulfide exposure at the euxinic site led to toxicity in ANME, which, despite the abundance of substrates at this site, could no longer thrive. These mechanistic insights imply that the development of euxinia, driven by eutrophication, could disrupt the coastal methane biofilter, leading to increased benthic methane release and potential increased methane emissions from coastal zones to the atmosphere.

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Common breast cancer risk loci predispose to distinct tumor subtypes BackgroundGenome-wide association studies (GWAS) have identified multiple common breast cancer susceptibility variants. Many of these variants have differential associations by estrogen receptor (ER), but how these variants relate with other tumor features and intrinsic molecular subtypes is unclear.

MethodsAmong 106,571 invasive breast cancer cases and 95,762 controls of European ancestry with data on 173 breast cancer variants identified in previous GWAS, we used novel two-stage polytomous logistic regression models to evaluate variants in relation to multiple tumor features (ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and grade) adjusting for each other, and to intrinsic-like subtypes.

ResultsEighty-five of 173 variants were associated with at least one tumor feature (false discovery rate <5%), most commonly ER and grade, followed by PR and HER2. Models for intrinsic-like subtypes found nearly all of these variants (83 of 85) associated at P<0.05 with risk for at least one luminal-like subtype, and approximately half (41 of 85) of the variants were associated with risk of at least one non-luminal subtype, including 32 variants associated with triple-negative (TN) disease. Ten variants were associated with risk of all subtypes in different magnitude. Five variants were associated with risk of luminal A-like and TN subtypes in opposite directions.

ConclusionThis report demonstrates a high level of complexity in the etiology heterogeneity of breast cancer susceptibility variants and can inform investigations of subtype-specific risk prediction.

sentences: A new fold in TANGO1 evolved from SH3 domains for the export of bulky cargos Bulky cargos like procollagens, apolipoproteins, and mucins exceed the size of conventional COPII vesicles. During evolution a process emerged in metazoans, predominantly governed by the TANGO1 protein family, that organizes cargo at the exit sites of the endoplasmic reticulum and facilitates export by the formation of tunnel-like connections between the ER and Golgi. Cargorecognition appears to be mediated by an SH3-like domain, however, just how the vastly different cargos are recognized remains elusive. Based on structural and dynamic data as well as interaction studies from NMR spectroscopy presented here, we show that the luminal cargo-recognition domain of TANGO1 adopts a new functional fold for which we suggest the term MOTH (MIA, Otoraplin, TALI/TANGO1 homology) domain. Also, differences in the structural properties within the domain family suggest fundamentally different mechanisms of cargo-recognition in vertebrates and invertebrates. Similarly, in vertebrates, it is proposed that cargo-specificity is mediated by structural differences within the vertebrate TANGO1 and TALI MOTH domains themselves.

sentences: Conserved residues in the extracellular loop 2 regulate Stachel-mediated activation of ADGRG2 Cleavage and dissociation of a large N-terminal fragment and the consequent unmasking of a short sequence (Stachel) remaining on the N-terminus have been proposed as mechanisms of activation of some members of the adhesion G protein-coupled receptor (aGPCR) family. However, the identity of residues that play a role in the activation of aGPCRs by the cognate Stachel remains largely unknown. Protein sequence alignments revealed a conserved stretch of residues in the extracellular loop 2 (ECL2) of all 33 members of the aGPCR family. ADGRG2, an orphan aGPCR, plays a major role in male fertility, Ewing sarcoma cell proliferation, and parathyroid cell function. We used ADGRG2 as a model aGPCR and generated mutants of the conserved residues in the ECL2 via site-directed mutagenesis. We show that tryptophan and isoleucine in the ECL2 are required for receptor stability and surface expression in the HEK293 cells. By adjusting the receptor surface expression levels, we show that the ECL2 mutation ablates the Stachel-mediated activation of multiple signaling pathways of ADGRG2. This study provides a novel understanding of the role of the ECL2 in Stachel-mediated signaling and degradation of ADGRG2, which may lay the foundation for the rational design of therapeutics to target aGPCRs.

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The Contribution of Environmental Enrichment to Phenotypic Variation in Mice and Rats The reproducibility and translation of neuroscience research is assumed to be undermined by introducing environmental complexity and heterogeneity. Rearing laboratory animals with minimal (if any) environmental stimulation is thought to control for biological variability but may not adequately test the robustness of our animal models. Standard laboratory housing is associated with reduced demonstrations of species typical behaviors and changes in neurophysiology that may impact the translation of research results. Modest increases in environmental enrichment (EE), mitigate against insults used to induce animal models of disease, directly calling into question the translatability of our work. This may in part underlie the disconnect between preclinical and clinical research findings. Enhancing environmental stimulation for our model organisms promotes ethological natural behaviors but may simultaneously increase phenotypic trait variability. To test this assumption, we conducted a systematic review and evaluated coefficients of variation between EE and standard housed mice and rats. Given findings of suboptimal reporting of animal laboratory housing conditions, we also developed a methodological reporting table for enrichment use in neuroscience research. Our data show that animals housed in environmental enrichment were not more variable than those in standard housing. Therefore, environmental heterogeneity introduced into the laboratory, in the form of enrichment, does not compromise data integrity. Overall, human life is complicated and by embracing such nuanced complexity into our laboratories we may paradoxically improve upon the rigor and reproducibility of our research.

Significance StatementEnvironmental complexity is thought to increase phenotypic variability, undermining research translation. We conducted a systematic review and compared coefficients of variation between environmentally enriched and standard housed laboratory animals. Despite there being no differences in variability across several phenotypic traits, there are stark contrasts in the display of ethological natural behaviors between these housing conditions. Environmental enrichment is recognized as being beneficial for animal welfare and mitigates against insults used to induce animal models of disease. In contrast, standard laboratory cages are recognized as being impoverished and  unnatural. From these observations, it is apparent that our current "gold standard" caging system is not a true control condition as it does not adequately test the robustness of our animal models.

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Context, competition, and symbiont-induced bet-hedging between Dictyostelium discoideum and Paraburkholderia Symbiotic interactions change with environmental context. Measuring these context-dependent effects in hosts and symbionts is critical to determining the nature of symbiotic interactions. We investigated context-dependence in the symbiosis between social amoeba hosts and their inedible Paraburkholderia bacterial symbionts, where the context is the abundance of host food bacteria. Paraburkholderia have been shown to harm hosts dispersed to food-rich environments, but aid hosts dispersed to food-poor environments by allowing hosts to carry food bacteria. Through measuring symbiont density and host spore production, we show that this food context matters in three other ways. First, it matters for symbionts, who suffer a greater cost from competition with food bacteria in the food-rich context. Second, it matters for host-symbiont conflict, changing how symbiont density negatively impacts host spore production. Third, data-based simulations show that symbiosis often provides a long-term fitness advantage for hosts after rounds of growth and dispersal in variable food-contexts, especially when conditions are harsh with little food. These results show how food context can have many consequences for the Dictyostelium-Paraburkholderia symbiosis and that both sides can frequently benefit.

Impact StatementMany organisms form symbiotic relationships with other species. These symbioses often exhibit context-dependence, where the sign or magnitude of one partners effect on the other will change in different environments. Context-dependent effects make it difficult to assign interactions to categories like mutualisms or antagonisms because they involve both benefits and costs depending on the environment. However, in some cases, accounting for context-dependence can clarify an interaction so that it more easily fits a mutualism or antagonism. We investigated context-dependence using the symbiosis between Dictyostelium discoideum and two symbiotic Paraburkholderia species. In this symbiosis, Paraburkholderia bacteria allow hosts to carry food bacteria to food-poor contexts, where hosts rarely survive without food, but reduce host fitness in the more hospitable food-rich contexts. The effect of food context on Paraburkholderia symbionts is unknown. We show that Paraburkholderia symbionts are also affected by this context, through facing reduced competition after being dispersed by hosts to food-poor contexts. We also identify a new way that symbionts affect hosts, where symbiont density reduces host fitness, but less so in food-poor contexts. Finally, we use simulations to show that infected hosts benefit in the long-term across variable food contexts, especially in the harshest environments with little food. These results show that context-dependence in symbiosis can have many consequences for hosts and symbionts, though in general for D. discoideum and Paraburkholderia, both are likely to benefit.

sentences: Topographically distinct adaptive landscapes for skulls, skeletons, and size explain the dietary adaptive radiation of Carnivora (Mammalia) AO_SCPLOWBSTRACTC_SCPLOWModels of adaptive radiation were originally developed to explain the early, rapid appearance of distinct modes of life within diversifying clades. Phylogenetic tests of this hypothesis have yielded limited support for temporally declining rates of phenotypic evolution across diverse clades, but the concept of an adaptive landscape that links form to fitness, while also crucial to these models, has received more limited attention. Using methods that assess the temporal accumulation of morphological variation and estimate the topography of the underlying adaptive landscape, I found evidence of an early partitioning of craniodental morphological variation in Carnivora (Mammalia) that occurs on an adaptive landscape with multiple peaks, consistent with classic ideas about adaptive radiation. Although strong support for this mode of adaptive radiation is present in traits related to diet, its signal is not present in body mass data or for traits related to locomotor behavior and substrate use. These findings suggest that adaptive radiations may occur along some axes of ecomorphological variation without leaving a signal in others and that their dynamics are more complex than simple univariate tests might suggest.

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Pancreatic ductal adenocarcinoma cells employ integrin α6β4 to form hemidesmosomes and regulate cell proliferation Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin 6{beta}4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin {beta}4 using siRNA reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, indicating a requirement for 6{beta}4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC.

Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827.

sentences: Fauxcurrence: simulating multi-species occurrences for null models in species distribution modelling and biogeography Defining appropriate null expectations for species distribution hypotheses is important because sampling bias and spatial autocorrelation can produce realistic, but ecologically meaningless, geographic patterns. Generating null species occurrences with similar spatial structure to observed data can help overcome these problems, but existing methods focus on single or pairs of species and do not incorporate between-species spatial structure that may occlude comparative biogeographic analyses. Here, we describe an algorithm for generating randomised species occurrence points that mimic the within- and between-species spatial structure of real datasets and implement it in a new R package - fauxcurrence. The algorithm can be implemented on any geographic domain for any number of species, limited only by computing power. To demonstrate its utility, we apply the algorithm to two common analysis-types: testing the fit of species distribution models (SDMs) and evaluating niche-overlap. The method works well on all tested datasets within reasonable timescales. We found that many SDMs, despite a good fit to the data, were not significantly better than null expectations and identified only two cases (out of a possible 32) of significantly higher niche divergence than expected by chance. The package is user-friendly, flexible and has many potential applications beyond those tested here, such as joint SDM evaluation and species co-occurrence analysis, spanning the areas of ecology, evolutionary biology and biogeography.

sentences: Changes in movement characteristics in response to private and social information acquisition of socially foraging fish To overcome the cost of competition resulting from foraging in a group, individuals may balance their use of private (i.e., acquired from personal sampling) and social (i.e., acquired by watching other individuals) information to adjust their foraging strategy accordingly. Reliability of private information about environmental characteristics, such as the spatial distribution of prey, is thus likely to affect individual movement and social interactions during foraging. Our aim was to investigate how movement characteristics of foraging individuals changed as they acquired reliable private information about the spatial occurrence of prey in a foraging context. We allowed guppies (Poecilia reticulata) to develop the reliability of their private knowledge about prey spatial occurrence by repeatedly testing shoals in a foraging task under three experimental distributions of prey: 1) aggregated prey forming three patches located in fixed locations, 2) scattered distribution of prey with random locations, or 3) no prey (used as control). We then applied tracking methods to obtain individual time series of spatial coordinates from which we computed a suite of movement variables reflecting search effort, social proximity and locomotion characteristics during foraging, to examine changes occurring over repeated trials and to investigate which best explained foraging success. We show that foraging shoals became more efficient at finding and consuming food over the first three days by increasing their time spent active. Over time, individuals foraging on either scattered or aggregated prey travelled greater distances, showed an increasing distance to their closest neighbour and became slightly more stochastic in their acceleration profile, compared to control individuals. We found that behaviour changed as private information increased over time, with a clear behavioural shift occurring on the third testing day. Social proximity was the major predictor of foraging success in the absence of prior foraging information, while search effort became the most important predictors of foraging success as information increased. In conclusion, we show that individual movement patterns changed as they acquired private information. Contrary to our predictions, the spatial distribution of prey did not affect any of the movement variables of interest. Our results emphasise the importance of information in shaping movement behaviour in animals.

sentences: Fast bacterial growth reduces antibiotic accumulation and efficacy Phenotypic variations between individual microbial cells play a key role in the resistance of microbial pathogens to pharmacotherapies. Nevertheless, little is known about cell individuality in antibiotic accumulation. Here we hypothesize that phenotypic diversification can be driven by fundamental cell-to-cell differences in drug transport rates. To test this hypothesis, we employed microfluidics-based single-cell microscopy, libraries of fluorescent antibiotic probes and mathematical modelling. This approach allowed us to rapidly identify phenotypic variants that avoid antibiotic accumulation within populations of Escherichia coli, Pseudomonas aeruginosa, Burkholderia cenocepacia and Staphylococcus aureus. Crucially, we found that fast growing phenotypic variants avoid macrolide accumulation and survive treatment without genetic mutations. These findings are in contrast with the current consensus that cellular dormancy and slow metabolism underlie bacterial survival to antibiotics. Our results also show that fast growing variants display significantly higher expression of ribosomal promoters before drug treatment compared to slow growing variants. Drug-free active ribosomes facilitate essential cellular processes in these fast growing variants, including efflux that can reduce macrolide accumulation. Using this new knowledge, we phenotypically engineered bacterial populations by eradicating variants that displayed low antibiotic accumulation through the chemical manipulation of their outer membrane inspiring new avenues to overcome current antibiotic treatment failures.

sentences: TP53 promotes lineage commitment of human embryonic stem cells through ciliogenesis and sonic hedgehog signaling Aneuploidy, defective differentiation, and inactivation of the tumor suppressor TP53 all occur frequently during tumorigenesis. Here, we probe the potential links among these cancer traits by inactivating TP53 in human embryonic stem cells (hESCs). TP53-/- hESCs exhibit increased proliferation rates, mitotic errors, and low-grade structural aneuploidy; produce poorly differentiated immature teratomas in mice; and fail to differentiate into neural progenitor cells (NPC) in vitro. Genome-wide CRISPR screen reveals requirements of ciliogenesis and sonic hedgehog (Shh) pathways for hESC differentiation into NPCs. TP53 deletion causes abnormal ciliogenesis in neural rosettes. In addition to restraining cell proliferation through CDKN1A, TP53 activates the transcription of BBS9, which encodes a ciliogenesis regulator required for proper Shh signaling and NPC formation. This developmentally regulated transcriptional program of TP53 promotes ciliogenesis, restrains Shh signaling, and commits hESCs to neural lineages.

sentences: Analysis of visual processing capabilities and neural coding strategies of a detailed model for laminar cortical microcircuits in mouse V1 Neural networks of the brain that process visual information have structural properties that differ significantly from those of neural networks which are commonly used for visual processing in AI, such as Convolutional Neural Networks (CNNs). But it has remained unknown how these structural differences are related to network function. We analyze visual processing capabilities of a large-scale model for area V1 that arguably provides the most comprehensive accumulation of anatomical and neurophysiological data that is currently available. Its network structure turns out to induce a number of characteristic visual processing capabilities of the brain, in particular the capability to multiplex different visual processing tasks, also on temporally dispersed visual information, with remarkable robustness to noise. This V1 model also exhibits a number of characteristic neural coding properties of the brain, which provide explanations for its superior noise robustness. Since visual processing in the brain is substantially more energy-efficient than implementations of CNNs in common computer hardware, such brain-like neural network models are likely to have also an impact on technology: As blueprints for visual processing in more energy-efficient neuromorphic hardware.

sentences: Mutations in Auxilin cause parkinsonism via impaired clathrin-mediated trafficking at the Golgi apparatus and synapse Parkinsons disease (PD) is a common neurodegenerative motor disorder characterized in part by neuropathological lesions in the nigrostriatal pathway. Loss of function mutations in Auxilin, the major neuronal clathrin uncoating protein, cause an aggressive form of juvenile onset PD. How mutations in Auxilin cause PD, is currently not understood. Here, we generated a novel mouse model carrying an endogenous pathogenic Auxilin mutation that phenocopies neurological features observed in patients, including motor impairments and seizures. Unbiased mapping of the Auxilin interactome identified synaptic and Golgi-resident clathrin adaptor proteins as novel interactors. Impaired clathrin-mediated trafficking in mutant Auxilin mice, both at the Golgi and the synapse, results in neuropathological lesions in the nigrostriatal pathway. Collectively, these results provide molecular mechanisms of PD pathogenesis in Auxilin mutation carriers, reinforcing a key role for clathrin-mediated trafficking in PD, and expand our understanding of the cellular function of Auxilin.

sentences: Quantification of macromolecular biomass composition for constraint-based metabolic modeling Genome-scale metabolic models (GEMs) are mathematical representations of metabolism that allow for in silico simulation of metabolic phenotypes and capabilities. A prerequisite for these predictions is an accurate representation of the biomolecular composition of the cell necessary for replication and growth, implemented in GEMs as the so-called biomass objective function (BOF). The BOF contains the metabolic precursors required for synthesis of the cellular macro- and micromolecular constituents (e.g. protein, RNA, DNA), and its composition is highly dependent on the particular organism, strain, and growth condition. Despite its critical role, the BOF is rarely constructed using specific measurements of the modeled organism, drawing the validity of this approach into question. Thus, there is a need to establish robust and reliable protocols for experimental condition-specific biomass determination. Here, we address this challenge by presenting a general pipeline for biomass quantification, evaluating its performance on Escherichia coli K-12 MG1655 sampled during balanced exponential growth under controlled conditions in a batch-fermentor set-up. We significantly improve both the coverage and molecular resolution compared to previously published workflows, quantifying 91.6% of the biomass. Our measurements display great correspondence with previously reported measurements, and we were also able to detect subtle characteristics specific to the particular E. coli strain. Using the modified E. coli GEM iML1515a, we compare the feasible flux ranges of our experimentally determined BOF with the original BOF, finding that the changes in BOF coefficients considerably affect the attainable fluxes at the genome-scale.

sentences: The Ventrolateral Anterior Temporal Lobe is Commonly Engaged by Both Mental State Inference and Semantic Association Tasks A key challenge for neurobiological models of social cognition is to elucidate whether brain regions are specialised for that domain. In recent years, discussion surrounding the role of anterior temporal regions epitomises such debates; some argue the anterior temporal lobe (ATL) is part of a domain-specific network for social processing, while others claim it comprises a domain-general hub for semantic representation. In the present study, we used ATL-optimised fMRI to map the contribution of different ATL structures to a variety of paradigms frequently used to probe a crucial social ability, namely  theory of mind (ToM). Using multiple tasks enables a clearer attribution of activation to ToM as opposed to idiosyncratic features of stimuli. Further, we directly explored whether these same structures are also activated by a non-social task probing semantic representations. We revealed that common to all of the tasks was activation of a key ventrolateral ATL region that is often invisible to standard fMRI. This constitutes novel evidence in support of the view that the ventrolateral ATL contributes to social cognition via a domain-general role in semantic processing, and against claims of a specialised social function.

sentences: ResistoXplorer: a web-based tool for visual, statistical and exploratory data analysis of resistome data The study of resistomes using whole metagenomic sequencing enables high throughput identification of resistance genes in complex microbial communities, such as the human microbiome. Over recent years, sophisticated and diverse pipelines have been established to facilitate raw data processing and annotation. Despite the progress, there are no easy-to-use tools for comprehensive visual, statistical, and functional analysis of resistome data. Thus, exploration of the resulting large complex datasets remains a key bottleneck requiring robust computational resources and technical expertise, which creates a significant hurdle for advancements in the field. Here, we introduce ResistoXplorer, a user-friendly tool that integrates recent advancements in statistics and visualization, coupled with extensive functional annotations and phenotype collection, to enable high-throughput analysis of common outputs generated from metagenomic resistome studies. ResistoXplorer contains three modules- the  Antimicrobial Resistance Gene Table module offers various options for composition profiling, functional profiling and comparative analysis of resistome data; the  Integration module supports integrative exploratory analysis of resistome and microbiome abundance profiles derived from metagenomic samples; finally, the  Antimicrobial Resistance Gene List module enables users to intuitively explore the associations between antimicrobial resistance genes and the microbial hosts using network visual analytics to gain biological insights. ResistoXplorer is publicly available at http://www.resistoxplorer.no.

sentences: Differential serotonin uptake mechanisms at the human maternal-fetal interface Mechanisms regulating serotonin (5-HT) homeostasis at the maternal-fetal interface are important for proper placental functioning and fetal (neuro)development. Here we studied 5-HT uptake mechanisms in human primary trophoblasts, feto-placental endothelial cells and cord blood platelets, all isolated directly after birth. Trophoblasts and cord blood platelets demonstrated high-affinity 5-HT uptake with similar Michaelis constant (Km) values (0.60{+/-}0.27 and 0.65{+/-}0.18 M, respectively). In contrast, feto-placental endothelial cells displayed saturation kinetics only over the low-affinity range of 5-HT concentrations (Km=782{+/-}218 M). 5-HT uptake into trophoblasts was inhibited by various psychotropic drugs targeting high-affinity serotonin transporter (SERT), and into feto-placental endothelial cells by an inhibitor of low-affinity transporters. SERT mRNAs were abundant in trophoblasts, but sparse in feto-placental endothelial cells; the opposite was found for transcripts of the low-affinity plasma membrane monoamine transporter (PMAT). These results show for the first time the presence of functional 5-HT uptake systems in feto-placental endothelial cells and fetal platelets, cells in direct contact with the fetal blood plasma. Data also emphasize sensitivity of 5-HT transport into trophoblasts, cells facing maternal blood, to various psychotropic drugs. The multiple, high- and low-affinity, systems present for cellular 5-HT uptake highlight the importance of maintaining 5-HT homeostasis at the maternal-fetal interface.

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Animal lifestyle changes acceptable mass limits for attached tags O_LIAnimal-attached devices have transformed our understanding of vertebrate ecology. To minimize tag-related harm for these studies, researchers have long advocated that tag masses should not exceed 3% of the animals body mass. However, this proposition ignores tag forces generated as a result of animal movement.
C_LIO_LIUsing data from collar-attached accelerometers on diverse free-ranging terrestrial animals, we detail a tag-based acceleration method (TbAM) in which we quantify animal athleticism in terms of fractions of animal movement time devoted to different collar-recorded accelerations. The varying accelerations are converted to forces imposed on the animals based on the acceleration and tag mass and allow derivation of defined force limits, including those amounting to 3% of the animals mass, for specified fractions of any animals active time.
C_LIO_LIWe demonstrate how species athleticism is the principal determinant of tag forces, whereas body mass is of little importance. Forces exerted by  3% tags were mostly equivalent to 4-19% of the animals masses during moving, with a maximum of 54% in a hunting cheetah. Cumulative frequency curves of tag acceleration for periods when animals were active, all showed a characteristic sigmoid pattern, which was displaced further to the right as higher acceleration activities accounted for an increasing proportion of any animals time. Specifying that tags should exert forces that are less than 3% of the animals body mass for 95% of the time led to corrected tag masses constituting between 1.6% and 2.98% of our study animals masses, with values depending on animal athleticism.
C_LIO_LIRecognition that animal athleticism affects tag forces of their carriers fundamentally changes how acceptable tag mass limits should be determined by ethics bodies. In order to have a scientifically robust acceptable threshold to limit the forces experienced by an animal carrier, we suggest practitioners derive a similar cumulative acceleration profile for their study species and use a minimum of the 95% limits on the plot (although higher limits may be more appropriate).
C_LI

sentences: ATR kinase inhibition induces thymineless death in proliferating CD8+ T cells ATR kinase is a central regulator of the DNA damage response (DDR) and cell cycle checkpoints. However, little is known about the role of ATR in the cell cycle and the impact of DDR inhibitors in immune cells in the absence of DNA damage. We previously showed that the ATR inhibitor AZD6738 (ATRi) combines with radiation to generate delayed, CD8+ T cell-dependent antitumor responses in mouse models of cancer. Here, we show that ATRi induces untimely CDK1 activity during S phase in CD8+ T cells and this induces origin firing and simultaneous degradation of dNTP synthesis and salvage enzymes. These pleiotropic effects of ATRi in proliferating CD8+ T cells induce deoxyuridine contamination in genomic DNA, R loops, RNA-DNA polymerase collisions, and death. Remarkably, thymidine significantly rescues ATRi-induced CD8+ T cell death. Our data identifies critical considerations for the design of clinical ATRi regimens with genotoxic chemo- and radiation and immunotherapies.

sentences: A systematic examination of learning in the invasive ant Linepithema humile reveals very rapid development of short and long-term memories The Argentine ant (Linepithema humile) is one of the most damaging and widespread invasive ant species worldwide. However, control attempts often fail due to insufficient bait uptake, or bait abandonment. Increasing preference for, and consumption of, is thus an important requirement for successful control. Associative learning and within-nest information transfer might be a potential tool for achieving this goal. Here, we conducted a detailed and systematic investigation of olfactory learning and side learning in Argentine ants. The ants showed very strong and rapid side learning, choosing the correct arm in a Y-maze 65% of time after just one visit, and 84% correct after two. Odour learning was even more rapid, with just one visit to a flavoured food source, reached by a scented runway, leading to 85% choices for the corresponding scent on a Y-maze. Further experiments demonstrated that having two cues (runway odour and food flavour) does not improve learning significantly over just one cue. This rapid learning is long-lasting, with one exposure to a runway odour associated with a reward resulting in a strong preference (73%) for this odour even after 48 hours. Food flavour information is transferred efficiently between nestmates in the nest, driving preference: naive ants housed with ants fed on flavoured food show a strong preference (77%) for that odour after 24 hours. Our results demonstrate the impressive learning abilities of Linepithema humile, which coupled with efficient intranidal information transfer and strong use of pheromonal recruitment may help explain their ability to discover and then dominate resources. However, these strengths could potentially be used against them, by exploiting learning and information transfer to increase toxic bait uptake during control efforts. Steering ant preference by leveraging learning might be an underappreciated tool in invasive alien species control.

sentences: Structural basis for ultrapotent neutralization of human metapneumovirus Human metapneumovirus (hMPV) is a leading cause of morbidity and hospitalization among children worldwide, however, no vaccines or therapeutics are currently available for hMPV disease prevention and treatment. The hMPV fusion (F) protein is the sole target of neutralizing antibodies. To map the immunodominant epitopes on the hMPV F protein, we isolated a panel of human monoclonal antibodies (mAbs), and the mAbs were assessed for binding avidity, neutralization potency, and epitope specificity. We found the majority of the mAbs target diverse epitopes on the hMPV F protein, and we discovered multiple mAb binding approaches for antigenic site III. The most potent mAb, MPV467, which had picomolar potency, was examined in prophylactic and therapeutic mouse challenge studies, and MPV467 limited virus replication in mouse lungs when administered 24 hrs before or 72 hrs after viral infection. We determined the structure of MPV467 in complex with the hMPV F protein using cryo-electron microscopy to a resolution of 3.3 [A], which revealed a complex novel prefusion-specific epitope overlapping antigenic sites II and V on a single protomer. Overall, our data reveal new insights into the immunodominant antigenic epitopes on the hMPV F protein, identify a new mAb therapy for hMPV F disease prevention and treatment, and provide the discovery of a unique pre-fusion-specific epitope on the hMPV F protein.

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SARS-CoV-2 spike glycoprotein-reactive T cells can be readily expanded from COVID-19 vaccinated donors IntroductionThe COVID-19 vaccine was designed to provide protection against infection by the severe respiratory coronavirus 2 (SARS-CoV-2) and coronavirus disease 2019 (COVID-19). However, the vaccines efficacy can be compromised in patients with immunodeficiencies or the vaccine-induced immunoprotection suppressed by other comorbidity treatments, such as chemotherapy or immunotherapy. To enhance the protective role of the COVID-19 vaccine, we have investigated a combination of the COVID-19 vaccination with ex vivo enrichment and large-scale expansion of SARS-CoV-2 spike glycoprotein-reactive CD4+ and CD8+ T cells.

MethodsSARS-CoV-2-unexposed donors were vaccinated with two doses of the BNT162b2 SARS-CoV-2 vaccine. The peripheral blood mononuclear cells of the vaccinated donors were cell culture-enriched with T cells reactive to peptides derived from SARS-CoV-2 spike glycoprotein. The enriched cell cultures were large-scale expanded using the rapid expansion protocol (REP) and the peptide-reactive T cells evaluated.

ResultsWe show that vaccination with the SARS-CoV-2 spike glycoprotein-based mRNA COVID-19 vaccine induced humoral response against SARS-CoV-2 spike glycoprotein in all tested healthy SARS-CoV-2-unexposed donors. This humoral response was found to correlate with the ability of the donors PBMCs to become enriched with SARS-CoV-2 spike glycoprotein-reactive CD4+ and CD8+ T cells. Using an 11-day rapid expansion protocol, the enriched cell cultures were expanded nearly a thousand fold, and the proportions of the SARS-CoV-2 spike glycoprotein-reactive T cells increased.

ConclusionsThese findings show for the first time that the combination of the COVID-19 vaccination and ex vivo T cell large-scale expansion of SARS-CoV-2-reactive T cells could be a powerful tool for developing T cell-based adoptive cellular immunotherapy of COVID-19.

sentences: Computational design of peptide therapeutics: how useful are sequence-based protein-protein interaction predictors? Engineering peptides to achieve a desired therapeutic effect through the inhibition of a specific target activity or protein interaction is a non-trivial task. Few of the existing in silico peptide design algorithms generate target-specific peptides. Instead, many methods produce peptides that achieve a desired effect through an unknown mechanism. In contrast with resource-intensive high-throughput experiments, in silico screening is a cost-effective alternative that can prune the space of candidates when engineering target-specific peptides. Using a set of FDA-approved peptides we curated specifically for this task, we assess the applicability of several sequence-based protein-protein interaction predictors as a screening tool within the context of peptide therapeutic engineering. We show that similarity-based protein-protein interaction predictors are more suitable for this purpose than the state-of-the-art deep learning methods publicly available at the time of writing. We also show that this approach is mostly useful when designing new peptides against targets for which naturally-occurring interactors are already known, and that deploying it for de novo peptide engineering tasks may require gathering additional target-specific training data. Taken together, this work offers evidence that supports the use of similarity-based protein-protein interaction predictors for peptide therapeutic engineering, especially peptide analogs.

sentences: Myasthenia gravis-specific aberrant neuromuscular gene expression by medullary thymic epithelial cells in thymoma Myasthenia gravis (MG) is a neurological disease caused by autoantibodies against neuromuscular-associated proteins. While MG is frequently developed in thymoma patients, the etiologic factors for MG are not well understood. Here, by constructing a comprehensive atlas of thymoma using bulk and single-cell RNA-seq, we identified ectopic expression of neuromuscular molecules in MG-associated thymoma (MG-thymoma). These molecules were originated from a distinct subpopulation of medullary thymic epithelial cells (mTECs), which we named neuromuscular mTECs (nmTECs). MG-thymoma also exhibited microenvironments dedicated to autoantibody production, including ectopic germinal center formation, T follicular helper cell accumulation, and type 2 conventional dendritic cell migration. Cell-cell interaction analysis also predicted the interaction between nmTECs and T/B cells via CXCL12-CXCR4. The enrichment of nmTECs presenting neuromuscular molecules within MG-thymoma was further confirmed by immunohistochemically and by cellular composition estimation from MG-thymoma transcriptome. Altogether, this study suggests that nmTECs play a significant role in MG pathogenesis via ectopic expression of neuromuscular molecules.

sentences: Reverse engineering of a mechanistic model of gene expression using metastability and temporal dynamics Differentiation can be modeled at the single cell level as a stochastic process resulting from the dynamical functioning of an underlying Gene Regulatory Network (GRN), driving stem or progenitor cells to one or many differentiated cell types. Metastability seems inherent to differentiation process as a consequence of the limited number of cell types. Moreover, mRNA is known to be generally produced by bursts, which can give rise to highly variable non-Gaussian behavior, making the estimation of a GRN from transcriptional profiles challenging. In this article, we present CARDAMOM (Cell type Analysis from scRna-seq Data achieved from a Mixture MOdel), a new algorithm for inferring a GRN from timestamped scRNA-seq data, which crucially exploits these notions of metastability and transcriptional bursting. We show that such inference can be seen as the successive resolution of as many regression problem as timepoints, after a preliminary clustering of the whole set of cells with regards to their associated bursts frequency. We demonstrate the ability of CARDAMOM to infer a reliable GRN from in silico expression datasets, with good computational speed. To the best of our knowledge, this is the first description of a method which uses the concept of metastability for performing GRN inference.

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Rcompadre and Rage - two R packages to facilitate the use of the COMPADRE and COMADRE databases and calculation of life history traits from matrix population models O_LIMatrix population models (MPMs) are an important tool for biologists seeking to understand the causes and consequences of variation in vital rates (e.g., survival, reproduction) across life cycles. Empirical MPMs describe the age- or stage-structured demography of organisms and usually represent the life history of a population during a particular time frame at a specific geographic location.
C_LIO_LIThe COMPADRE Plant Matrix Database and COMADRE Animal Matrix Database are the most extensive resources for MPM data, collectively containing >12,000 individual projection matrices for >1,100 species globally. Although these databases represent an unparalleled resource for researchers, land managers, and educators, the current computational tools available to answer questions with MPMs impose significant barriers to potential COM(P)ADRE database users by requiring advanced knowledge to handle diverse data structures and program custom analysis functions.
C_LIO_LITo close this knowledge gap, we present two interrelated R packages designed to (i) facilitate the use of these databases by providing functions to acquire, quality control, and manage both the MPM data contained in COMPADRE and COMADRE, and a users own MPM data (Rcompadre), and (ii) present a range of functions to calculate life history traits from MPMs in support of ecological and evolutionary analyses (Rage). We provide examples to illustrate the use of both.
C_LIO_LIRcompadre and Rage will facilitate demographic analyses using MPM data and contribute to the improved replicability of studies using these data. We hope that this new functionality will allow researchers, land managers, and educators to unlock the potential behind the thousands of MPMs and ancillary metadata stored in the COMPADRE and COMADRE matrix databases, and in their own MPM data.
C_LI

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Germline ERBB3 mutation in familial non-small cell lung carcinoma: expanding the role of the ErbB family in oncogenesis. BackgroundLung cancer is the commonest cause of cancer deaths worldwide. Although strongly associated with smoking, predisposition to lung cancer is also heritable with multiple common risk variants identified. Rarely, dominantly inherited non-small-cell lung cancer (NSCLC) has been reported due to somatic mutations in EGFR/ErbB1 and ERBB2.

MethodsGermline exome sequencing was performed in a multi-generation family with autosomal dominant NSCLC, including an affected child. Tumour samples were also sequenced. Full-length wild-type (wtErbB3) and mutant ERBB3 (mutErbB3) constructs were transfected into HeLa cells. Protein expression, stability, and sub-cellular localisation were assessed; and cellular proliferation, pAkt/Akt, and pERK levels were determined.

ResultsA novel germline variant in ERBB3 (c.1946T>G: p.Iso649Arg), coding for receptor tyrosineprotein kinase erbB-3 (ErbB3), was identified, with appropriate segregation. There was no loss-of-heterozygosity in tumour samples. Both wtErbB3 and mutErbB3 were stably expressed. MutErbB3-transfected cells demonstrated an increased ratio of the 80kD form (which enhances proliferation) compared to the full-length (180kD) form. MutErbB3 and wtErbB3 had similar punctate cytoplasmic localisation pre- and post-EGF stimulation; however, EGFR levels decreased faster post-stimulation in mutErbB3-transfected cells, suggesting more rapid processing of the mutErbB3/EGFR heterodimer. Cellular proliferation was increased in mutErbB3-transfected cells compared to wtErbB3 transfection. MutErbB3-transfected cells also showed decreased pAkt/tAkt ratios and increased pERK/tERK 30 minutes post-stimulation compared to wtErbB3 transfection, demonstrating altered signalling pathway activation by mutErbB3. Cumulatively, these results support this mutation as tumorogenic.

ConclusionsThis is the first reported family with a germline ERBB3 mutation causing heritable NSCLC, furthering understanding of the ErbB family pathway in oncogenesis.

sentences: Evolution of butterfly-plant networks over time, as revealed by Bayesian inference of host repertoire The study of herbivorous insects underpins much of the theory that concerns the evolution of species interactions. In particular, Pieridae butterflies and their host plants have served as a model system for studying evolutionary arms-races. To learn more about the coevolution of these two clades, we reconstructed ancestral ecological networks using stochastic mappings that were generated by a phylogenetic model of host-repertoire evolution. We then measured if, when, and how two ecologically important structural features of the ancestral networks (modularity and nestedness) evolved over time. Our study shows that as pierids gained new hosts and formed new modules, a subset of them retained or recolonized the ancestral host(s), preserving connectivity to the original modules. Together, host-range expansions and recolonizations promoted a phase transition in network structure. Our results demonstrate the power of combining network analysis with Bayesian inference of host-repertoire evolution to understand changes in complex species interactions over time.

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Droplet sample preparation for single-cell proteomics applied to the cell cycle Many biological processes, such as the cell division cycle, are reflected in protein covariation across single cells. This covariation can be quantified and interpreted by single-cell proteomics with sufficiently high throughput and accuracy. Toward this goal, we developed the nano-ProteOmic sample Preparation (nPOP) method for single-cell proteomics. nPOP uses piezo acoustic dispensing to isolate individual cells in 300 picoliter volumes and performs all subsequent preparation steps in small droplets on a fluorocarbon-coated slide. This design enables simultaneous sample preparation of thousands of single cells, including lysing, digesting, and labeling individual cells in volumes below 20 nl. We used nPOP to prepare 1,888 single cells and 128 negative controls in a single batch. Their analysis enabled quantifying the covariation between thousands of proteins and cell-cycle protein markers. Many protein sets covaried with the cell cycle similarly across all cell types and states, reflecting cell-type independent cell cycle functions. However, the cell cycle covariation of other protein sets differed markedly between cell types, even within subpopulation of melanoma cells expressing markers for drug-resistance priming. The cells expressing these markers accumulated in the G1 phase of the cell cycle and exhibited different covariation of enzymes catabolizing glucose. These results demonstrate that protein covariation across single cells may reveal functionally concerted biological differences between closely related cell states.

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sentences: VEGF-A, PDGF-BB and HB-EGF engineered for promiscuous super affinity to the extracellular matrix improve wound healing in a model of type 1 diabetes Chronic non-healing wounds, frequently caused by diabetes, lead to lower quality of life, infection, and amputation. These wounds have limited treatment options. We have previously engineered growth factors to bind to exposed extracellular matrix (ECM) in the wound environment using the heparin-binding domain of placental growth factor-2 (PlGF-2123-144), which binds promiscuously to ECM proteins. Here, in the type 1 diabetic (T1D) NOD mouse model, engineered growth factors improved both re-epithelialization and granulation tissue formation. Engineered growth factors were even more potent in combination, and the "triple therapy" of vascular endothelial growth factor-A (VEGF-PlGF-2123-144), platelet-derived growth factor-BB (PDGF-BB-PlGF-2123-144), and heparin-binding epidermal-growth factor (EGF-PlGF-2123-144) both improved wound healing and remained at the site of administration for significantly longer than wild-type growth factors. In addition, we also found that changes in the cellular milieu of a wound, including changing amounts of M1 macrophages, M2 macrophages and effector T cells, are most predictive of wound healing success in the NOD mouse model. These results suggest that the triple therapy of VEGF-PlGF-2123-144, PDGF-BB-PlGF-2123-144, and EGF-PlGF-2123-144 may be an effective therapy for chronic non-healing wounds in that occur as a complication of diabetes.

sentences: Nuclear lamins promote protrusion dynamics and collective, confined migration in vivo Cells migrate collectively through confined environments during development and cancer metastasis. While the nucleus, a large and stiff organelle, impedes cell migration between non-deformable pillars in vitro, its function in vivo may vary depending on the microenvironment. Further, it is unknown how nuclei contribute to collective migration in vivo and whether nuclei in different positions within cell collectives experience different forces. Here, we use border cell migration in the fly ovary as an in vivo model to investigate the effects of confined, collective migration on nuclei and the contribution of nuclear lamins to migration. We found severe yet transient nuclear deformations occur, particularly in the leading cell, as border cells squeeze through tiny crevices between germline cells, termed nurse cells. Leading cells extend protrusions between nurse cells, which may pry open space to allow the cluster to advance. Here we report that the leading cell nuclei deformed as they moved into leading protrusions. Then as protrusions widened, the nucleus recovered a more circular shape. These data suggest that lead cell nuclei may help protrusions expand and thereby enlarge the migration path. To test how nuclei might promote or impede border cell migration, we investigated nuclear lamins, proteins that assemble into intermediate filaments and structurally support the nuclear envelope. Depletion of the Drosophila B-type lamin, Lam, from the outer, motile border cells, but not the inner, nonmotile polar cells, impeded border cell migration, whereas perturbations of the A-type lamin, LamC, did not. While wild type border cell clusters typically have one large leading protrusion as they delaminate from the anterior follicular epithelium, clusters depleted of B-type lamin had multiple, short-lived protrusions, resulting in unproductive cluster movement and failure to progress along the migration path. Further, border cell nuclei depleted of B-type lamins were small, formed blebs, and ruptured. Together, these data indicate that B-type lamin is requied for nuclear integrity, which in turn stabilizes the leading protrusion and promotes overall cluster polarization and collective movement through confined spaces.

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TRPM2 deficiency protects against atherosclerosis by inhibiting TRPM2-CD36 inflammatory axis in macrophages Atherosclerosis is the major cause of ischemic heart diseases and ischemic brain stroke, which are the leading causes of mortality worldwide. The central pathological features of atherosclerosis include macrophage infiltration and foam cell formation. However, the detailed mechanisms regulating these two processes remain unclear. Here we show that oxidative stress-activated Ca2+-permeable TRPM2 plays a key role in the pathogenesis of atherosclerosis. Trpm2 deletion produces a potent protective effect against atherosclerosis in ApoE-/- mice fed with a high-fat diet (HFD), as evidenced by reduced atherosclerotic plaque burden, decreased macrophage load and suppressed inflammasome activation in the vessel wall. Moreover, we show that Trpm2 deletion or inhibition reduces oxidized low-density lipoprotein (oxLDL) uptake by macrophages, suppresses macrophage infiltration induced by monocyte chemoattractant protein-1 (MCP1), and prevents the impairment of macrophage emigration caused by oxLDL. Intriguingly, we uncover that activation of CD36, an oxLDL receptor, can promote the activation of TRPM2, and vice versa, the CD36-mediated inflammatory cascade in atherosclerosis is dependent on TRPM2. In transfected HEK293T cells, CD36 ligands oxLDL and TSP1 induce TRPM2 activation in a CD36-dependent manner. Deleting Trpm2 or inhibiting TRPM2 activity in cultured macrophages suppresses the CD36 signaling cascade induced by oxLDL and TSP1. Our studies establish TRPM2-CD36 axis as a new mechanism underlying atherogenesis, and suggest TRPM2 as an effective therapeutic target for atherosclerosis.

HIGHLIGHTSO_LITrpm2 deletion protects against atherosclerosis in ApoE-/- mice fed with a high-fat diet (HFD)
C_LIO_LITrpm2 deficiency reduces atherosclerotic lesions by minimizing foam cell formation, inhibiting macrophage infiltration and preserving macrophage emigration
C_LIO_LITRPM2 activation is required for CD36-induced oxLDL uptake and subsequent inflammatory responses
C_LIO_LIThe ligands of CD36, oxLDL and TSP1, activate TRPM2, thereby perpetuating TRPM2-CD36 inflammatory cycle in atherogenesis cascade
C_LIO_LIOur data establish TRPM2-CD36 axis as a new atherogenesis mechanism and TRPM2 as a novel therapeutic target for atherosclerosis
C_LI

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY TRPM2-mediated Ca2+ signal is essential for CD36 induced oxLDL uptake and atherosclerosis in ApoE-/- mice fed with a high-fat diet (HFD). The activation of CD36 and TRPM2 form a positive feedback loop in atherogenesis.

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ionbot: a novel, innovative and sensitive machine learning approach to LC-MS/MS peptide identification Mass spectrometry-based proteomics generates vast amounts of signal data that require computational interpretation to obtain peptide identifications. Dozens of algorithms for this task exist, but all exploit only part of the acquired data to judge a peptide-to-spectrum match (PSM), ignoring important information such as the observed retention time and fragment ion peak intensity pattern. Moreover, only few identification algorithms allow open modification searches that can substantially increase peptide identifications.

We here therefore introduce ionbot, a novel open modification search engine that is the first to fully merge machine learning with peptide identification. This core innovation brings the ability to include a much larger range of experimental data into PSM scoring, and even to adapt this scoring to the specifics of the data itself. As a result, ionbot substantially increases PSM confidence for open searches, and even enables a further increase in peptide identification rate of up to 12% by also considering lower-ranked, co-eluting matches for a fragmentation spectrum. Moreover, the exclusive use of machine learning for scoring also means that any future improvements to predictive models for peptide behavior will also result in more sensitive and accurate peptide identification.

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Comparative analysis of RNA enrichment methods for preparation of Cryptococcus neoformans RNA sequencing libraries Ribosomal RNA (rRNA) is the major RNA constituent of cells, therefore most RNA sequencing (RNA-Seq) experiments involve removal of rRNA. This process, called RNA enrichment, is done primarily to reduce cost: without rRNA removal, deeper sequencing would need to be performed to balance the sequencing reads wasted on rRNA. The ideal RNA enrichment method would remove all rRNA without affecting other RNA in the sample. We have tested the performance of three RNA enrichment methods on RNA isolated from Cryptococcus neoformans, a fungal pathogen of humans. We show that the RNase H depletion method unambiguously outperforms the commonly used Poly(A) isolation method: the RNase H method more efficiently depletes rRNA while more accurately recapitulating the expression levels of other RNA observed in an unenriched "gold standard". The RNase H depletion method is also superior to the Ribo-Zero depletion method as measured by rRNA depletion efficiency and recapitulation of protein-coding gene expression levels, while the Ribo-Zero depletion method performs moderately better in preserving non-coding RNA (ncRNA). Finally, we have leveraged this dataset to identify novel long non-coding RNA (lncRNA) genes and to accurately map the C. neoformans mitochondrial rRNA genes.

ARTICLE SUMMARYWe compare the efficacy of three different RNA enrichment methods for RNA-Seq in Cryptococcus neoformans: RNase H depletion, Ribo-Zero depletion, and Poly(A) isolation. We show that the RNase H depletion method, which is evaluated in C. neoformans samples for the first time here, is highly efficient and specific in removing rRNA. Additionally, using data generated through these analyses, we identify novel long non-coding RNA genes in C. neoformans. We conclude that RNase H depletion is an effective and reliable method for preparation of C. neoformans RNA-Seq libraries.

sentences: Temporal Window based Feature Extraction Technique for Motor-Imagery EEG Signal Classification Electroencephalogram (EEG) based motor-imagery classification is one of the most popular Brain Computer Interface (BCI) research areas due to its portability and low cost. In this paper, we have compared Wavelet Energy-entropy based different prediction models and empirically proven that temporal window based approach in motor-imagery classification provides more consistent and better results than popular filter-bank approach. In order to examine the robustness and stability of the proposed method, we have also employed multiple types of classifiers at the end and found that mix-bagging (bagging ensemble learning with multiple types of learners) technique out-smarts other frequently used classifiers. In our study, BCI Competition II Data-set III has been used with four experimental setup: (a) The whole signal (for each trial) as one segment, (b) The whole signal (for each trial) is divided into non-overlapping segments, (c) The whole signal (for each trial) is divided into overlapping segments, and (d) The filter-bank approach where the whole signal (each trial) is segmented based on different frequency bands. The result obtained from the experiment (c) i.e. 91.43% classification accuracy which outperforms the other approaches not only in this paper but to best of our knowledge it is the highest performance for this dataset so far.

sentences: Ribosome collisions in bacteria promote ribosome rescue by triggering mRNA cleavage by SmrB Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation. In bacteria, it remains unclear how rescue pathways distinguish ribosomes stalled in the middle of a transcript from actively translating ribosomes. In a genetic screen in E. coli, we discovered a novel rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmRNA (which acts on truncated mRNAs) to rescue upstream ribosomes. SmrB is recruited by ribosome collisions; cryo-EM structures of collided disomes from E. coli and B. subtilis reveal a distinct and conserved arrangement of the individual ribosomes and the composite SmrB binding site. These findings reveal the underlying mechanism by which ribosome collisions trigger ribosome rescue in bacteria.

sentences: Omicron-specific mRNA vaccine induced potent neutralizing antibody against Omicron but not other SARS-CoV-2 variants The emerging SARS-CoV-2 variants of concern (VOC) harbor mutations associated with increasing transmission and immune escape, hence undermine the effectiveness of current COVID-19 vaccines. In late November of 2021, the Omicron (B.1.1.529) variant was identified in South Africa and rapidly spread across the globe. It was shown to exhibit significant resistance to neutralization by serum not only from convalescent patients, but also from individuals receiving currently used COVID-19 vaccines with multiple booster shots. Therefore, there is an urgent need to develop next generation vaccines against VOCs like Omicron. In this study, we develop a panel of mRNA-LNP-based vaccines using the receptor binding domain (RBD) of Omicron and Delta variants, which are dominant in the current wave of COVID-19. In addition to the Omicron- and Delta-specific vaccines, the panel also includes a "Hybrid" vaccine that uses the RBD containing all 16 point-mutations shown in Omicron and Delta RBD, as well as a bivalent vaccine composed of both Omicron and Delta RBD-LNP in half dose. Interestingly, both Omicron-specific and Hybrid RBD-LNP elicited extremely high titer of neutralizing antibody against Omicron itself, but few to none neutralizing antibody against other SARS-CoV-2 variants. The bivalent RBD-LNP, on the other hand, generated antibody with broadly neutralizing activity against the wild-type virus and all variants. Surprisingly, similar cross-protection was also shown by the Delta-specific RBD-LNP. Taken together, our data demonstrated that Omicron-specific mRNA vaccine can induce potent neutralizing antibody response against Omicron, but the inclusion of epitopes from other variants may be required for eliciting cross-protection. This study would lay a foundation for rational development of the next generation vaccines against SARS-CoV-2 VOCs.

sentences: Cooperative binding of TCR and CD4 to pMHC enhances TCR sensitivity Antigen recognition of CD4+ T cells by the T cell receptor (TCR) can be greatly enhanced by the coreceptor CD41-7. Yet, understanding of the molecular mechanism is hindered by the ultra-low affinity of CD4 binding to class-II peptide-major histocompatibility complexes (pMHC)1,7-10. Using two-dimensional (2D) mechanical-based assays, we determined a CD4-pMHC interaction to have 3-4 logs lower affinity than cognate TCR-pMHC interactions8, and to be susceptible to increased dissociation by forces (slip bond)5,8,11. In contrast, CD4 binds TCR-prebound pMHC at 5-6 logs higher affinity, forming TCR-pMHC-CD4 trimolecular bonds that are prolonged by force (catch bond)5,8,11 and modulated by protein mobility on the cell membrane, indicating profound TCR-CD4 cooperativity. Consistent with a tri-crystal structure12, using DNA origami as a molecular ruler to titrate spacing between TCR and CD4 indicates 7-nm proximity enables optimal trimolecular bond formation with pMHC. Our results reveal how CD4 augments TCR antigen recognition.

sentences: A brain-based universal measure of attention: predicting task-general and task-specific attention performance and their underlying neural mechanisms from task and resting state fMRI Attention is central for many aspects of cognitive performance, but there is no singular measure of a persons overall attentional functioning across tasks. To develop a universal measure that integrates multiple components of attention, we collected data from more than 90 participants performing three different attention-demanding tasks during fMRI. We constructed a suite of whole-brain models that can predict a profile of multiple attentional components - sustained attention, divided attention and tracking, and working memory capacity - from a single fMRI scan type within novel individuals. Multiple brain regions across the frontoparietal, salience, and subcortical networks drive accurate predictions, supporting a universal (general) attention factor across tasks, which can be distinguished from task-specific attention factors and their neural mechanisms. Furthermore, connectome-to-connectome transformation modeling enhanced predictions of an individuals attention-task connectomes and behavioral performance from their rest connectomes. These models were integrated to produce a new universal attention measure that generalizes best across multiple, independent datasets, and which should have broad utility for both research and clinical applications.

sentences: Unlocking the microblogging potential for science and medicine Twitter is one of the most popular microblogging and social networking services, where users can post, retweet, comment, and engage in collaborative discussions. However, improper usage of Twitter can be detrimental to science and even have a negative impact on mental health. Thus, analyzing tweets and Twitter data of various researchers will help us to deduce appropriate ways of using Twitter to advance in our research careers. Existing literature has analyzed the activity of scientists on Twitter, such as studying the relationship between Twitter mentions and article citations, determining the benefits of Twitter in the development and distribution of scientific knowledge, relevant metrics for prediction of highly cited articles, and type of content that researchers tweet etc. For example, Eysenbach et al performed an analysis of tweets and citations and how one can predict citations using tweets. Most of the existing literature analyzed a limited number of researchers, compromising the generalizability of derived results. In our study, we have taken a comprehensive and systematic approach to analyze 167,000 scientists who published research papers on PubMed using data-driven methods. We observed various parameters like number of followers, number of friends, citation count and K-index, in the light of gender, ancestry and profession of the researchers.

sentences: Microfluidic system-based time-course tracking of physical proximity between cells and its effect on gene expression for elucidating live single cancer-immune cell interactions Cell-cell communication and physical interactions play a vital role in cancer initiation, homeostasis, progression, and immune response. Here, we report a system that combines live capture of different cell types, co-incubation, time-lapse imaging, and gene expression profiling of doublets using a microfluidic integrated fluidic circuit (IFC) that enables measurement of physical distances between cells and the associated transcriptional profiles due to cell-cell interactions. The temporal variations in natural killer (NK) - triple-negative breast cancer (TNBC) cell distances were tracked and compared with terminally profiled cellular transcriptomes. The results showed the time-bound activities of regulatory modules and alluded to the existence of transcriptional memory. Our experimental and bioinformatic approaches serve as a proof of concept for interrogating live cell interactions at doublet resolution, which can be applied across different cancers and cell types.

sentences: Stress induced TDP-43 mobility loss independent of stress granules TAR DNA binding protein 43 (TDP-43) is closely related to the pathogenesis of amyotrophic lateral sclerosis (ALS) and translocates to stress granules (SGs). The role of SGs as aggregation-promoting "bioreactors" for TDP-43, however, is still under debate. We analyzed TDP-43 mobility and localization under different stress and recovery conditions using live cell single-molecule tracking and super-resolution microscopy. Besides reduced mobility within SGs, a stress induced decrease of TDP-43 mobility in the cytoplasm and the nucleus was observed. Stress removal led to a recovery of TDP-43 mobility, which strongly depended on the stress duration.  Stimulated-emission depletion microscopy (STED) and  tracking and localization microscopy (TALM) revealed not only TDP-43 substructures within stress granules but also numerous patches of slow TDP-43 species throughout the cytoplasm. The data provide new insights into the aggregation of TDP-43 in living cells and provide evidence suggesting that TDP-43 oligomerization takes place in the cytoplasm separate from SGs.

sentences: γδ intraepithelial lymphocytes facilitate pathological epithelial cell shedding via CD103-mediated granzyme release. Excessive shedding of enterocytes into the intestinal lumen is observed in inflammatory bowel disease and is correlated with disease relapse. However, the mechanisms underlying this phenomenon remain unclear. Intraepithelial lymphocytes (IEL) expressing the {gamma}{delta} T-cell receptor (TCR) provide surveillance of the intestinal mucosa at steady-state, which is regulated, in part, by CD103. Intravital microscopy of lipopolysaccharide (LPS)-treated mice revealed that {gamma}{delta} IELs make extended contact with shedding enterocytes. These prolonged interactions require CD103 engagement by E-cadherin, as CD103 blockade significantly reduces LPS-induced shedding. Furthermore, we find that granzymes A and B, but not perforin, are required for cell shedding, and that these granzymes are released by {gamma}{delta} IELs both constitutively and following CD103/E-cadherin ligation. These findings indicate that extracellular granzyme facilitates shedding, likely through cleavage of extracellular matrix proteins. Our results uncover a previously unrecognized role for {gamma}{delta} IELs in facilitating pathological cell shedding in a CD103- and granzyme-dependent manner.

sentences: A connectome-based model of conscious access in monkey cortex A growing body of evidence suggests that conscious perception of a sensory stimulus triggers an all-or-none activity across multiple cortical areas, a phenomenon called  ignition. In contrast, the same stimulus, when undetected, induces only transient activity. In this work, we report a large-scale model of the macaque cortex based on recently quantified structural connectome data. We use this model to simulate a detection task, and demonstrate how a dynamical bifurcation mechanism produces ignition-like events in the model network. Within this framework, the model predicts that feedforward excitatory transmission is primarily mediated by the fast AMPA receptors to ensure rapid signal propagation from sensory to associative areas. In contrast, a large fraction of the inter-areal feedback projections and local recurrent excitation depend on the slow NMDA receptors, to ensure ignition of distributed frontoparietal activity. Our model predicts, counterintuitively, that fast-responding sensory areas contain a higher ratio of NMDA to AMPA receptors compared to association cortical areas that show slow, sustained activity. We validate this prediction using in-vitro receptor autoradiography data. Finally, we show how this model can account for various behavioral and physiological effects linked to consciousness. Together, these findings clarify the neurophysiological mechanisms of conscious access in the primate cortex and support the concept that gradients of receptor densities along the cortical hierarchy contribute to distributed cognitive functions.

sentences: Design of the SARS-CoV-2 RBD vaccine antigen improves neutralizing antibody response The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is the primary target of neutralizing antibodies and is a component of almost all vaccine candidates. Here, RBD immunogens were created with stabilizing amino acid changes that improve the neutralizing antibody response, as well as characteristics for production, storage, and distribution. A computational design and in vitro screening platform identified three improved immunogens, each with approximately nine amino acid changes relative to the native RBD sequence and four key changes conserved between immunogens. The changes are adaptable to all vaccine platforms, are compatible with established changes in SARS-CoV-2 vaccines, and are compatible with mutations in emerging variants of concern. The immunogens elicit higher levels of neutralizing antibodies than native RBD, focus the immune response to structured neutralizing epitopes, and have increased production yields and thermostability. Incorporating these variant-independent amino acid changes in next-generation vaccines may enhance the neutralizing antibody response and lead to pan-SARS-CoV-2 protection.

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Common signatures of differential microRNA expression in Parkinson's and Alzheimer's disease brains BackgroundDysregulation of microRNA (miRNA)-mediated gene expression has been implicated in the pathogenesis and course of many neurodegenerative diseases including Parkinsons disease (PD). However, the functionally relevant miRNAs remain largely unknown. Previous meta-analyses on differential miRNA expression data in post-mortem PD brains have highlighted several miRNAs showing consistent and statistically significant effects. However, these meta-analyses were based on exceedingly small sample sizes.

MethodsIn this study, we quantified the expression of the four most compelling PD candidate miRNAs from these meta-analyses in the superior temporal gyrus (STG) of one of the largest case-control post-mortem brain datasets available (261 samples), thereby quadruplicating previously investigated sample sizes. Furthermore, we probed for common differential miRNA expression signatures with Alzheimers disease (AD) by also analyzing these miRNAs in post-mortem STG of 190 AD patients and controls and by testing six top AD miRNAs in the PD brains.

ResultsOf all ten analyzed miRNAs, PD candidate miRNA homo sapiens (hsa-) miR-132-3p showed evidence for differential expression in both PD (p=4.89E-06) and AD (p=3.20E-24), and AD miRNAs hsa-miR-132-5p (p=4.52E-06) and hsa-miR-129-5p (p=0.0379) showed evidence for differential expression in PD. Combining these novel data with previously published data substantially improved the statistical support (=3.85E-03 using Bonferroni correction) of the corresponding meta-analyses clearly and compellingly implicating these miRNAs in both PD and AD. Furthermore, hsa-miR-132-3p/-5p (but not hsa-miR-129-5p) showed association with neuropathological Braak PD staging (p=3.51E-03/p=0.0117), suggesting that these miRNAs may play a role in -synuclein aggregation beyond the early disease phase.

ConclusionsOur study represents the largest independent assessment of recently highlighted candidate brain miRNAs in PD and AD post-mortem brain samples, to date. Our results implicate hsa-miR-132-3p/-5p and hsa-miR-129-5p to be differentially expressed in both PD and AD brains, potentially pinpointing shared pathogenic mechanisms across these neurodegenerative diseases.

sentences: CCPE: Cell Cycle Pseudotime Estimation for Single Cell RNA-seq Data The rapid development of single-cell RNA-sequencing (scRNA-seq) technologies makes it possible to characterize cellular heterogeneity by detecting and quantifying transcriptional changes at the single-cell level. Pseudotime analysis enables to characterize the continuous progression of various biological processes, such as cell cycle. Cell cycle plays an important regulatory role in cell fate decisions and differentiation and is also often regarded as a confounder in scRNA-seq data analysis when analyzing the role of other factors on transcriptional regulation. Therefore, accurate prediction of cell cycle pseudotime and identify cell stages are important steps for characterizing the development-related biological processes, identifying important regulatory molecules and promoting the analysis of transcriptional heterogeneity. Here, we develop CCPE, a novel cell cycle pseudotime estimation method to characterize cell cycle timing and determine cell cycle phases from single-cell RNA-seq data. CCPE uses a discriminative helix to characterize the circular process and estimates pseudotime in the cell cycle. We evaluated the model performance based on a variety of simulated and real scRNA-seq datasets. Our results indicate that CCPE is an effective method for cell cycle estimation and competitive in various downstream analyses compared with other existing methods. CCPE successfully identified cell cycle marker genes and is robust to dropout events in scRNA-seq data. CCPE also has excellent performance on small datasets with fewer genes or cells. Accurate prediction of the cell cycle in CCPE effectively contributes to cell cycle effect removal across cell types or conditions.

sentences: Engineering the AEG1 promoter from cotton to develop male sterile lines This work reports on modifying the Upstream Regulatory Module (URM, 1.5 Kb region upstream of the open reading frame) of Anther Expressing Gene 1 (AEG1) from cotton to achieve anther specific activity. AEG1 was identified in a previous study aimed to isolate a promoter with tapetum specific activity. Such a promoter could then be used to express barnase and barstar genes for developing male sterile and restorer lines for hybrid seed production in cotton. The AEG1 URM was observed to be active in tapetum as well as in roots making it unusable to drive the expression of barnase gene. Analysis of the URM showed the presence of several root specific motifs. Two modified AEG1 URMs were developed, by removing or mutating these motifs and its activity checked in tobacco. The activity of one of the modified URMs, AEG1({Delta}Bmut) was restricted to the anther tissue as observed using the reporter gene {beta}-glucuronidase. The study also demonstrates that male sterile lines could be developed in tobacco using the AEG1({Delta}Bmut) URM to express the barnase gene. This work thus shows the possibility of engineering promoters to achieve tissue specificity and to develop male sterile lines in cotton.

sentences: Cannabinoids accumulate in mouse breast milk and differentially regulate lipid signaling molecules involved in infant development Maternal cannabis use during lactation may expose developing infants to cannabinoids (CBs) such as tetrahydrocannabinol (THC) and cannabidiol (CBD). CBs modulate lipid signaling molecules in the central nervous system in age- and cell-dependent ways, but their influence on the lipid composition of breastmilk has yet to be established. This study investigates the effects of THC, CBD, or their combination on milk lipids by analyzing the stomach contents of CD1 mouse pups that have been nursed by dams injected with CBs on postnatal days (PND) 1-10 collected 2 hours after the last injection on PND10. HPLC/MS/MS was used to identify and quantify over 80 endogenous lipid species and cannabinoids in pup stomach contents. We show that CBs differentially accumulate in milk, lead to widespread decreases in free fatty acids, decreases in N-acyl methionine species, increases N-linoleoyl species, as well as modulate levels of endogenous CBs (eCBs) AEA, 2-AG, and their structural congeners. Our data indicate the passage of CBs to pups through breast milk and that maternal CB exposure alters breast milk lipid compositions.

sentences: Rab11 endosomes coordinate centrosome number and movement following mitotic exit The last stage of cell division involves two daughter cells remaining interconnected by a cytokinetic bridge that is cleaved in a process called abscission. During pre-abscission, we identified that the centrosome moves in a Rab11-dependent manner towards the cytokinetic bridge in human cells grown in culture and in an in vivo vertebrate model, Danio rerio (zebrafish). Rab11-endosomes are dynamically organized in a Rab11-GTP dependent manner at the centrosome during pre-abscission and this organization is required for the centrosome protein, pericentrin, to be enriched at the centrosome. Using zebrafish embryos, we found that reduction in pericentrin expression or optogenetically disrupting Rab11-endosome function inhibited centrosome movement towards the cytokinetic bridge and abscission resulting in daughter cells prone to being binucleated and/or having supernumerary centrosomes. These studies suggest that Rab11-endosomes contribute to centrosome function during pre-abscission by regulating pericentrin organization resulting in appropriate centrosome movement towards the cytokinetic bridge and subsequent abscission.

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Synthetic nanobody-SARS-CoV-2 receptor-binding domain structures identify distinct epitopes The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands unprecedented attention. We report four X-ray crystal structures of three synthetic nanobodies (sybodies) (Sb16, Sb45 and Sb68) bind to the receptor-binding domain (RBD) of SARS-CoV-2: binary complexes of Sb16-RBD and Sb45-RBD; a ternary complex of Sb45-RBD-Sb68; and Sb16 unliganded. Sb16 and Sb45 bind the RBD at the ACE2 interface, positioning their CDR2 and CDR3 loops diametrically. Sb16 reveals a large CDR2 shift when binding the RBD. Sb68 interacts peripherally at the ACE2 interface; steric clashes with glycans explain its mechanism of viral neutralization. Superposing these structures onto trimeric spike (S) protein models indicates these sybodies bind conformations of the mature S protein differently, which may aid therapeutic design.

One Sentence SummaryX-ray structures of synthetic nanobodies complexed with the receptor-binding domain of the spike protein of SARS-CoV-2 reveal details of CDR loop interactions in recognition of distinct epitopic sites.

sentences: Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: role of a conserved aromatic cage Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the  clade and the {beta} clade. Most  clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in {beta} clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_{beta}IB1). A structural comparison of available PLDs structures reveals a conserved aromatic cage in the  clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular-dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the  clade, Loxosceles intermedia IA1 (Li_IA) and Loxosceles laeta III1 (Ll_III1), and one from the {beta} clade, St_{beta}IB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_{beta}IB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the  clade PLDs. Here, we confirmed the membrane binding site of  and {beta} clade PLDs on choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the  clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.

sentences: Mouse CHD4-NURD is required for spermatogonia survival and normal meiotic progression Testis development and sustained germ cell production in adults rely on the establishment and maintenance of spermatogonia stem cells and their proper differentiation into spermatocytes. Chromatin remodeling complexes regulate critical processes during gamete development by restricting or promoting accessibility of DNA repair and gene expression machineries to the chromatin. Here, we investigated the role of CHD4 and CHD3 catalytic subunits of the NURD complex during spermatogenesis. Germ cell-specific deletion of Chd4 early in gametogenesis, but not Chd3, resulted in arrested early gamete development due to failed cell survival of neonate undifferentiated spermatogonia stem cell population. Candidate assessment revealed that CHD4 controls expression of Dmrt1 and its downstream target Plzf, both described as prominent regulators of spermatogonia stem cell maintenance. Our results show the requirement of CHD4 in mammalian gametogenesis pointing to functions in gene expression early in the process.

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IMPAIRED OLFACTORY NETWORK FUNCTIONAL CONNECTIVITY IN PARKINSON'S DISEASE: A NOVEL MARKER FOR DISEASE PROGRESSION ObjectiveDetermine the neural basis of olfactory impairment in akinetic-rigid (PDAR) and tremor predominant (PDT) Parkinsons disease subtypes.

MethodsWe combined resting-state fMRI (rs-fMRI) with seed based functional connectivity (FC) in order to delineate the olfactory networks functional connectivity (ON FC) between PDAR and PDT patients. We then contrasted their ON FC patterns with cognitively normal (CN) subjects. All three groups were closely matched in age, demographic variables, and adjusted for relative cognitive performance. Olfactory function was measured using the University of Pennsylvania Smell Identification Test (UPSIT).

ResultsUPSIT scores were lower in akinetic-rigid vs tremor subtypes; ON FC values were lower in PDAR compared to PDT and CN, and followed the trend observed in UPSIT scores. UPSIT scores and ON FC values were significantly correlated, reflecting the effects of PD pathologies.

ConclusionsThe results show that olfactory function differs between PDAR and PDT suggesting a correlation between PD-related motor symptoms and olfactory deficits. ON FC differences accounts for the impaired olfactory functions observed between PDAR and PDT. PDAR is known to have worse clinical outcomes and faster cognitive decline compared to PDT; therefore, PD-related olfactory dysfunction may serve as a novel metric for enhancing PD prognosis.

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An Analysis of gRNA Sequence Dependent Cleavage Highlights the Importance of Genomic Context on CRISPR-Cas Activity CRISPR-Cas9 is a powerful DNA editing tool. A gRNA directs Cas9 to cleave any DNA sequence with a PAM. However, some gRNA sequences mediate cleavage at higher efficiencies than others. To understand this, numerous studies have screened large gRNA libraries and developed algorithms to predict gRNA sequence dependent activity. These algorithms do not predict other datasets as well as their training dataset and do not predict well between species. To better understand these discrepancies, we retrospectively examine sequence features that impact gRNA activity in 39 published data sets. We find strong evidence that the genomic context, which can be defined as the DNA content outside of the gRNA/target sequence itself, greatly contributes to differences in gRNA dependent activity. Context underlies variation in activity often attributed to differences in gRNA sequence. This understanding will help guide future work to understand Cas9 activity as well as efforts to identify optimal gRNAs and improve Cas9 variants.

HighlightsO_LISpecies-specific genomic context drives variability in gRNA activity in a PAM proximal sequence-dependent manner
C_LIO_LIIncreased PAM specificity of Cas9 and/or increased Cas9/gRNA expression reduces the impact of species-specific context
C_LIO_LICurrent gRNA prediction algorithms trained on species are not expected to predict activity in another species
C_LI

sentences: An 18S V4 rDNA metabarcoding dataset of protist diversity in the Atlantic inflow to the Arctic Ocean, through the year and down to 1000 m depth. Arctic marine protist communities have been understudied due to challenging sampling conditions, in particular during winter and in deep waters. The aim of this study was to improve our knowledge on Arctic protist diversity through the year, both in the epipelagic (< 200 m depth) and mesopelagic zones (200-1000 m depth). Sampling campaigns were performed in 2014, during five different months, to capture the various phases of the Arctic primary production: January (winter), March (pre-bloom), May (spring bloom), August (post-bloom) and November (early winter). The cruises were undertaken west and north of the Svalbard archipelago, where warmer Atlantic waters from the West Spitsbergen Current meets cold Arctic waters from the Arctic Ocean. From each cruise, station, and depth, 50 L of sea water were collected and the plankton was size-fractionated by serial filtration into four size fractions between 0.45-200 {micro}m, representing the picoplankton, nanoplankton and microplankton. In addition vertical net hauls were taken from 50 m depth to the surface at selected stations. From the plankton samples DNA was extracted, the V4 region of the 18S rRNA-gene was amplified by PCR with universal eukaryote primers and the amplicons were sequenced by Illumina high-throughput sequencing. Sequences were clustered into Amplicon Sequence Variants (ASVs), representing protist genotypes, with the dada2 pipeline. Taxonomic classification was made against the curated Protist Ribosomal Reference database (PR2). Altogether 6,536 protist ASVs were obtained (including 54 fungal ASVs). Both ASV richness and taxonomic composition were strongly dependent on size-fraction, season, and depth. ASV richness was generally higher in the smaller fractions, and higher in winter and the mesopelagic samples than in samples from the well-lit epipelagic zone during summer. During spring and summer, the phytoplankton groups diatoms, chlorophytes and haptophytes dominated in the epipelagic zone. Parasitic and heterotrophic groups such as Syndiniales and certain dinoflagel-lates dominated in the mesopelagic zone all year, as well as in the epipelagic zone during the winter. The dataset is available at https://doi.org/10.17882/79823, (Egge et al., 2014).

sentences: A Novel Local Ablation Approach for Explaining Multimodal Classifiers With the growing use of multimodal data for deep learning classification in healthcare research, more studies have begun to present explainability methods for insight into multimodal classifiers. Among these studies, few have utilized local explainability methods, which could provide (1) insight into the classification of each sample and (2) an opportunity to better understand the effects of demographic and clinical variables within datasets (e.g., medication of subjects in electrophysiology data). To the best of our knowledge, this opportunity has not yet been explored within multimodal classification. We present a novel local ablation approach that shows the importance of each modality to the correct classification of each class and explore the effects of demographic and clinical variables upon the classifier. As a use-case, we train a convolutional neural network for automated sleep staging with electroencephalogram (EEG), electrooculogram (EOG), and electromyogram (EMG) data. We find that EEG is the most important modality across most stages, though EOG is particular important for non-rapid eye movement stage 1. Further, we identify significant relationships between the local explanations and subject age, sex, and state of medication which suggest that the classifier learned specific features associated with these variables across multiple modalities and correctly classified samples. Our novel explainability approach has implications for many fields involving multimodal classification. Moreover, our examination of the degree to which demographic and clinical variables may affect classifiers could provide direction for future studies in automated biomarker discovery.

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Pre- and postsynaptically expressed spike-timing-dependent plasticity contribute differentially to neuronal learning A plethora of experimental studies have shown that long-term synaptic plasticity can be expressed pre- or postsynaptically depending on a range of factors such as developmental stage, synapse type, and activity patterns. The functional consequences of this diversity are not clear, although it is understood that whereas postsynaptic expression of plasticity predominantly affects synaptic response amplitude, presynaptic expression alters both synaptic response amplitude and short-term dynamics. In most models of neuronal learning, long-term synaptic plasticity is implemented as changes in connective weights. The consideration of long-term plasticity as a fixed change in amplitude corresponds more closely to post-than to presynaptic expression, which means theoretical outcomes based on this choice of implementation may have a postsynaptic bias. To explore the functional implications of the diversity of expression of long-term synaptic plasticity, we adapted a model of long-term plasticity, more specifically spike-timing-dependent plasticity (STDP), such that it was expressed either independently pre- or postsynaptically, or in a mixture of both ways. We compared pair-based standard STDP models and a biologically tuned triplet STDP model, and investigated the outcomes in a minimal setting, using two different learning schemes: in the first, inputs were triggered at different latencies, and in the second a subset of inputs were temporally correlated. We found that presynaptic changes adjusted the speed of learning, while postsynaptic expression was more efficient at regulating spike timing and frequency. When combining both expression loci, postsynaptic changes amplified the response range, while presynaptic plasticity allowed control over postsynaptic firing rates, potentially providing a form of activity homeostasis. Our findings highlight how the seemingly innocuous choice of implementing synaptic plasticity by single weight modification may unwittingly introduce a postsynaptic bias in modelling outcomes. We conclude that pre- and postsynaptically expressed plasticity are not interchangeable, but enable complimentary functions.

Author summaryDifferences between functional properties of pre- or postsynaptically expressed long-term plasticity have not yet been explored in much detail. In this paper, we used minimalist models of STDP with different expression loci, in search of fundamental functional consequences. Biologically, presynaptic expression acts mostly on neurotransmitter release, thereby altering short-term synaptic dynamics, whereas postsynaptic expression affects mainly synaptic gain. We compared models where plasticity was expressed only presynaptically or postsynaptically, or in both ways. We found that postsynaptic plasticity had a bigger impact over response times, while both pre- and postsynaptic plasticity were similarly capable of detecting correlated inputs. A model with biologically tuned expression of plasticity also completed these tasks over a range of frequencies. Also, postsynaptic spiking frequency was not directly affected by presynaptic plasticity of short-term plasticity alone, however in combination with a postsynaptic component, it helped restrain positive feedback, contributing to activity homeostasis. In conclusion, expression locus may determine affinity for distinct coding schemes while also contributing to keep activity within bounds. Our findings highlight the importance of carefully implementing expression of plasticity in biological modelling, since the locus of expression may affect functional outcomes in simulations.

sentences: Differential expression of glycosyltransferases identified through comprehensive pan-cancer analysis Despite accumulating evidence supporting a role for glycosylation in cancer progression and prognosis, the complexity of the human glycome and glycoproteome poses many challenges to understanding glycosylation-related events in cancer. In this study, a multifaceted genomics approach was applied to analyze the impact of differential expression of glycosyltransferases (GTs) in 16 cancers. An enzyme list was compiled and curated from numerous resources to create a consensus set of GTs. Resulting enzymes were analyzed for differential expression in cancer, and findings were integrated with experimental evidence from other analyses, including: similarity of healthy expression patterns across orthologous genes, miRNA expression, automatically-mined literature, curation of known cancer biomarkers, N-glycosylation impact, and survival analysis. The resulting list of GTs comprises 222 human enzymes based on annotations from five databases, 84 of which were differentially expressed in more than five cancers, and 14 of which were observed with the same direction of expression change across all implicated cancers. 25 high-value GT candidates were identified by cross-referencing multimodal analysis results, including PYGM, FUT6 and additional fucosyltransferases, several UDP-glucuronosyltransferases, and others, and are suggested for prioritization in future cancer biomarker studies. Relevant findings are available through OncoMX at https://data.oncomx.org, and the overarching pipeline can be used as a framework for similarly analysis across diverse evidence types in cancer. This work is expected to improve the understanding of glycosylation in cancer by transparently defining the space of glycosyltransferase enzymes and harmonizing variable experimental data to enable improved generation of data-driven cancer biomarker hypotheses.

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Increased heterogeneity and task-related reconfiguration of functional connectivity within a lexicosemantic network in autism Autism spectrum disorder (ASD) is highly heterogeneous in etiology and clinical presentation. Findings on intrinsic functional connectivity (FC) or task-induced FC in ASD have been inconsistent including both over- and underconnectivity and diverse regional patterns. As FC patterns change across different cognitive demands, a novel and more comprehensive approach to network architecture in ASD is to examine the change in FC patterns between rest and task states, referred to as reconfiguration. This approach is suitable for investigating inefficient network connectivity that may underlie impaired behavioral functioning in clinical disorders. We used functional magnetic resonance imaging (fMRI) to examine FC reconfiguration during lexical processing, which is often affected in ASD, with additional focus on interindividual variability. Thirty adolescents with ASD and a matched group of 23 typically developing (TD) participants completed a lexicosemantic decision task during fMRI, using multiecho-multiband pulse sequences with advanced BOLD signal sensitivity and artifact removal. Regions of interest (ROIs) were selected based on task-related activation across both groups, and FC and reconfiguration were compared between groups. The ASD group showed increased interindividual variability and overall greater reconfiguration than the TD group. An ASD subgroup with typical performance accuracy (at the level of TD participants) showed reduced similarity and typicality of FC during the task. In this ASD subgroup, greater FC reconfiguration was associated with increased language skills. Findings suggest that intrinsic functional networks in ASD may be inefficiently organized for lexicosemantic decisions and may require greater reconfiguration during task processing, with high performance levels in some individuals being achieved through idiosyncratic mechanisms.

HighlightsFC reconfiguration is a comprehensive approach to examining network architecture
Functional networks are inefficiently organized for lexicosemantic decisions in ASD
ASD may require greater reconfiguration during task processing
Some ASD individuals achieve high performance through idiosyncratic mechanisms

sentences: Factors that Influence Career Choice Among Different Populations of Neuroscience Trainees Enhancing the diversity of the scientific workforce is critical to achieving the mission of NIH: "To seek fundamental knowledge about the nature and behavior of living systems and the application of that knowledge to enhance health, lengthen life, and reduce the burdens of illness and disability." However, specific groups have historically been, and continue to be, underrepresented in the biomedical research workforce, especially academia. Career choice is a multi-factorial process that evolves over time; among all trainees, expressed interest in faculty research careers decreases over time in graduate school, but that trend is amplified in women and members of historically underrepresented racial and ethnic groups (Fuhrmann, Halme, OSullivan, & Lindstaedt, 2011; Gibbs, McGready, Bennett, & Griffin, 2014; C. Golde & Dore, 2004; Roach & Sauermann, 2017; Sauermann & Roach, 2012). Neuroscience as a discipline has characteristics that may exacerbate the overall trends seen in the life sciences, such as a greater growth in the number of awarded neuroscience PhDs than in other life sciences fields (US National Science Foundation, 2016b). This work was designed to investigate how career interest changes over time among recent neuroscience PhD graduates, and whether differences in career interests are associated with social identity (i.e. gender and race/ethnicity), experiences in graduate school and postdoctoral training (e.g. relationship with advisor; feelings of belonging), and personal characteristics (e.g. confidence in ones potential to be an independent researcher). We report results from a survey of 1,479 PhD neuroscientists (including 16% underrepresented (UR) and 54% female scientists). We saw repeated evidence that individual preferences about careers in general, and academic careers specifically, predict current career interest. These statistically significant preferences mostly had medium to low effect size that varied by career type. These findings were mediated by social identity and experiences in graduate school and postdoctoral training. Our findings highlight the important influence of the advisor in shaping a trainees career path, and the ways in which academic culture is perceived as unwelcoming or incongruent with the values or priorities of certain groups. For women, issues of work/life balance and structural issues of academia, and for UR women in particular, lower confidence in their ability to be an independent researcher, affected their interest in academia. Both women and UR men in our study report a lower importance of autonomy in their careers. UR respondents report feeling less like they were a part of the social and intellectual community. However, they have formed beneficial relationships with faculty outside their PhD institutions that, particularly for UR women, are associated with increased interest in academia. Our findings suggest several areas for positive growth, ways to change how we think about the impact of mentorship, and policy and programmatic interventions that extend beyond trying to change or "fix" the individual and instead recognize the systemic structures that influence career choices.

sentences: BSImp: imputing partially observed methylation patterns for evaluating methylation heterogeneity DNA methylation is one of the most studied epigenetic modifications that has applications ranging from transcriptional regulation to aging, and can be assessed by bisulfite sequencing (BS-seq) at single base-pair resolution. The permutations of methylation statuses at bisulfite converted reads reflect the methylation patterns of individual cells. These patterns at specific genomic locations are sought to be indicative of cellular heterogeneity within a cellular population, which are predictive of developments and diseases; therefore, methylation heterogeneity has potentials in early detection of these changes. Computational methods have been developed to assess methylation heterogeneity using methylation patterns formed by four CpGs, but the nature of shotgun sequencing often give partially observed patterns, which makes very limited data available for downstream analysis. While many programs are developed to impute methylation levels genomewide, currently there is only one method developed for recovering partially observed methylation patterns; however, the program needs lots of data to train and cannot be used directly; therefore, we developed a probabilistic-based imputation method that uses information from neighbouring sites to recover partially observed methylation patterns speedily. It is demonstrated to allow for the evaluation of methylation heterogeneity at three times more regions genome-wide with high accuracy for data with moderate depth. To make it more user-friendly we also provide a computational pipeline for genome-screening, which can be used in both evaluating methylation levels and profiling methylation patterns genomewide for all cytosine contexts, which is the first of its kind. Our method allows for accurate estimation of methylation levels and makes evaluating methylation heterogeneity available for much more data with reasonable coverage, which has important implications in using methylation heterogeneity for monitoring changes within the cellular populations that were impossible to detect for the assessment of development and diseases.

sentences: Functional cooperativity mediated by rationally selected combinations of human monoclonal antibodies targeting the henipavirus receptor binding protein Hendra virus (HeV) and Nipah virus (NiV), the prototypic members of the Henipavirus (HNV) genus, are emerging, zoonotic paramyxoviruses known to cause severe disease across six mammalian orders, including humans (Eaton et al., 2006). While several research groups have made strides in developing candidate vaccines and therapeutics against henipaviruses, such countermeasures have not been licensed for human use, and significant gaps in knowledge about the human immune response to these viruses exist. To address these gaps, we isolated a large panel of human monoclonal antibodies (mAbs) from the B cells of an individual with prior occupation-related exposure to the equine HeV vaccine (Equivac(R) HeV). Competition-binding and hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies identified at least six distinct antigenic sites on the HeV/NiV receptor binding protein (RBP) that are recognized by human mAbs. Antibodies recognizing multiple antigenic sites potently neutralized NiV and/or HeV isolates in vitro. The most potent class of cross-reactive antibodies achieved neutralization by blocking viral attachment to the host cell receptors ephrin-B2 and ephrin-B3. Antibodies from this class mimic receptor binding by inducing a receptor-bound conformation to the HeV-RBP protein tetramer, exposing an epitope that appears to lie hidden in the interface between protomers within the HeV-RBP tetramer. Antibodies that recognize this cryptic epitope potently neutralized HeV and NiV. Flow cytometric studies using cell-surface-displayed HeV-RBP protein showed that cross-reactive, neutralizing mAbs from each of these classes cooperate for binding. In a highly stringent hamster model of NiVB infection, antibodies from both classes reduced morbidity and mortality and achieved synergistic protection in combination and provided therapeutic benefit when combined into two bispecific platforms. These studies identified multiple candidate mAbs that might be suitable for use in a cocktail therapeutic approach to achieve synergistic antiviral potency and reduce the risk of virus escape during treatment.

sentences: Molecular atlas of the human brain vasculature at the single-cell level A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains poorly understood. Here, we performed single-cell RNA sequencing of 599,215 freshly isolated endothelial, perivascular and other tissue-derived cells from 47 fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We uncover extensive molecular heterogeneity of healthy fetal and adult human brains and across eight vascular-dependent CNS pathologies including brain tumors and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict numerous endothelial-to-perivascular cell ligand-receptor crosstalk including immune-related and angiogenic pathways, thereby unraveling a central role for the endothelium within brain neurovascular unit signaling networks. Our single-cell brain atlas provides insight into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

sentences: Hemagglutinin stability determines influenza A virus susceptibility to a broad-spectrum fusion inhibitor Arbidol Understanding mechanisms of resistance to antiviral inhibitors can reveal nuanced features of targeted viral mechanisms and, in turn, lead to improved strategies for inhibitor design. Arbidol is a broad-spectrum antiviral which binds to and prevents the fusion-associated conformational changes in the trimeric influenza hemagglutinin (HA). The rate-limiting step during HA-mediated membrane fusion is the release of the hydrophobic fusion peptides from a conserved pocket on HA. Here, we investigated how destabilizing or stabilizing mutations in or near the fusion peptide affect viral sensitivity to Arbidol. The degree of sensitivity was proportional to the extent of fusion peptide stability on the pre-fusion HA: stabilized mutants were more sensitive, and destabilized ones resistant to Arbidol. Single-virion membrane fusion experiments for representative Wild Type and mutant viruses demonstrated that resistance is a direct consequence of fusion-peptide destabilization not dependent on reduced Arbidol binding to HA at neutral pH. Our results support the model whereby the probability of individual HAs extending to engage the target membrane is determined by the composite of two critical forces: a  tug on the fusion peptide by the extension of the central coiled-coil on HA, and the key interactions stabilizing fusion peptide in the pre-fusion pocket. Arbidol increases the free-energy penalty for coiled-coil extension, but destabilizing mutations decrease the free-energy cost for fusion peptide release, accounting for the observed resistance. Our findings have broad implications for fusion-inhibitor design, viral mechanisms of resistance, and our basic understanding of HA-mediated membrane fusion.

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Increased gut microbial mucin foraging promotes clearance of a parasitic worm BACKGROUND & AIMSHost-secreted gastrointestinal mucus plays a key role in the expulsion of intestinal nematode parasites. A balance between mucin secretion by the host and the gut microbial mucin foraging is essential to maintain the intestinal homeostasis, yet little is known about how changes in the mucin-microbiome interactions affect worm infection. Here, we aimed to examine how increased mucin foraging activity by the microbiome changes the course of the parasitic infection by modulating the host immune responses.

METHODSWe employed a gnotobiotic mouse model containing a 14-member synthetic human gut microbiota that facilitates functional interpretations, including diet-driven manipulation of the microbiota toward mucin foraging. We infected the mice with a robust murine nematode, Trichuris muris, that closely resembles human infection with Trichuris trichiura. We investigated the temporal dynamics of worm infection including worm burden and the host immune responses, and connected these readouts to the microbial changes and metabolic activity toward mucin foraging.

RESULTSThe microbial mucin foraging was further increased during worm infection only in mice with pre-enhanced mucin degrading capacity. The elevated mucin foraging coincides with a shift in host immune responses from susceptible (chronic, Th1 type) to resistant (acute, Th2 type), which promotes worm clearance. The relative abundances of mucin-generalist bacteria drammatically increased during worm clearance, but not during worm retention.

CONCLUSIONSThese results point to a mechanism whereby skewing the metabolic activity of the microbiome toward mucin glycoproteins promotes resistance to worm infection. Our study documents a clinically-relevant, novel link in the microbiome-parasite-host immune axis, which is important prerequisite knowledge in treating parasitic infections.

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Litter commensal bacteria can limit the horizontal gene transfer of antimicrobial resistance to Salmonella in chickens Fostering a balanced gut microbiome through the administration of beneficial microbes that can competitively exclude pathogens has gained a lot of attention and use in human and animal medicine. However, little is known about how microbes affect the horizontal gene transfer of antimicrobial resistance (AMR). To shed more light on this question, we challenged neonatal broiler chicks raised on reused broiler chicken litter - a complex environment made up of decomposing pine shavings, feces, uric acid, feathers, and feed, with Salmonella Heidelberg (S. Heidelberg), a model pathogen. Neonatal chicks challenged with S. Heidelberg and raised on reused litter were more resistant to S. Heidelberg cecal colonization than chicks grown on fresh litter. Furthermore, chicks grown on reused litter were at a lower risk of colonization with S. Heidelberg strains that encoded AMR on IncI1 plasmids. We used 16S rRNA gene sequencing and shotgun metagenomics to show that the major difference between chicks grown on fresh litter and reused litter was the microbiome harbored in the litter and ceca. The microbiome of reused litter samples was more uniform and enriched in functional pathways related to the biosynthesis of organic and antimicrobial molecules than fresh litter samples. We found that E. coli was the main reservoir of plasmids encoding AMR and that the IncI1 plasmid was maintained at a significantly lower copy per cell in reused litter compared to fresh litter. These findings support the notion that commensal bacteria play an integral role in the horizontal transfer of plasmids encoding AMR to pathogens like Salmonella.

Importance/SignificanceAntimicrobial resistance spread is a worldwide health challenge, stemming in large part, from the ability of microorganisms to share their genetic material through horizontal gene transfer. To address this issue, many countries and international organization have adopted a One health approach to curtail the proliferation of antimicrobial resistant bacteria. This includes the removal and reduction of antibiotics used in food animal production and the development of alternatives to antibiotics. However, there is still a significant knowledge gap in our understanding of how resistance spreads in the absence of antibiotic selection and the role commensal bacteria play in reducing antibiotic resistance transfer. In this study, we show that commensal bacteria play a key role in reducing the horizontal gene transfer of antibiotic resistance to Salmonella and provide the identity of the bacterial species that potentially perform this function in broiler chickens and also postulate the mechanism involved.

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Strategies of tolerance reflected in two North American maple genomes Maples (the genus Acer) represent important and beloved forest, urban, and ornamental trees distributed throughout the Northern hemisphere. They exist in a diverse array of native ranges and distributions, across spectrums of tolerance or decline, and have varying levels of susceptibility to biotic and abiotic stress. Among Acer species, several stand out in their importance to economic interest. Here we report the first two chromosome-scale genomes for North American species, Acer negundo and Acer saccharum. Both assembled genomes contain scaffolds corresponding to 13 chromosomes, with A. negundo at a length of 442 Mb, N50 of 32 Mb and 30,491 genes, and A. saccharum at 626 Mb, N50 of 46 Mb, and 40,074 genes. No recent whole genome duplications were detected, though A. saccharum has local gene duplication and more recent bursts of transposable elements, as well as a large-scale translocation between two chromosomes. Genomic comparison revealed that A. negundo has a smaller genome with recent gene family evolution that is predominantly contracted and expansions that are potentially related to invasive tendencies and tolerance to abiotic stress. Examination of expression from RNA-Seq obtained from A. saccharum grown in long-term aluminum and calcium soil treatments at the Hubbard Brook Experimental Forest, provided insights into genes involved in aluminum stress response at the systemic level, as well as signs of compromised processes upon calcium deficiency, a condition contributing to maple decline.

Significance statementThe first chromosome-scale assemblies for North American members of the Acer genus, sugar maple (Acer saccharum) and boxelder (Acer negundo), as well as transcriptomic evaluation of abiotic stress response in A. saccharum. This integrated study describes in-depth aspects contributing to each species approach to tolerance and applies current knowledge in many areas of plant genome biology with Acer physiology to help convey the genomic complexities underlying tolerance in broadleaf tree species.

sentences: Functional architecture of cerebral cortex during naturalistic movie-watching Characterizing the functional organization of cerebral cortex is a fundamental step in understanding how different kinds of information are processed in the brain. Neuroimaging studies over the past decades have uncovered the function of many cortical areas in response to selected stimulus categories. However, it is still unclear how cortical areas are organized during naturalistic visual and auditory stimulation. Here we used high-resolution functional MRI data from 176 human subjects to map the macro-architecture of the entire cerebral cortex based on responses to a 60-minute audiovisual movie stimulus. A data-driven clustering approach revealed a map of 24 functional areas/networks, each related to a specific aspect of sensory or cognitive processing. The map included three distinct executive control (domain-general) networks which showed a strong push-pull interaction with domain-specific regions in visual, auditory, and language cortex. The cortical parcellation scheme presented here provides a comprehensive and unified map of functionally defined areas, which could replace a large set of functional localizer maps.

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Integration of Diverse Transcriptomics Datasets using Random Forest to Predict Universal Functional Pathways in Tfr Cells MotivationT follicular regulatory (Tfr) cells are a specialized cell subset that controls humoral immunity. Despite a number of individual transcriptomic studies on these cells, core functional pathways have been difficult to uncover due to the substantial transcriptional overlap of these cells with other effector cell types, as well as transcriptional changes occurring due to disease settings. Developing a core transcriptional module for Tfr cells that integrates multiple cell type comparisons as well as diverse disease settings will allow a more accurate prediction of functional pathways. Researchers studying allergic reactions, immune responses to vaccines, autoimmunity and cancer could use this gene set to better understand the roles of Tfr cells in controlling disease progression.

Additional cell types beyond Tfr cells that have similar features of transcriptomic complexity within diverse disease settings may also be studied using similar approaches. High-throughput sequencing technologies allow the generation of large datasets that require specific tools to best interpret the data. The development of a core transcriptional module for Tfr cells will allow investigators to determine if Tfr cells may have functional roles within their biological systems with little knowledge of Tfr biology. With this work, we have addressed the need of core gene modules to define specific subsets of immune cells.

ResultsWe introduce an integrated "core Tfr cell gene module" that can be incorporated into GSEA analysis using various input sizes. The integrated core Tfr gene module was built using transcriptomic studies in Tfr cells from several different tissues, disease settings, and cell type comparisons. Random forest was used to integrate the transcriptomic studies to generate the core gene module. A GSEA gene set was formulated from the integrated core Tfr gene module for incorporation into end-user friendly GSEA. The gene sets are presented along with random genes taken from the GTEX data set and are presented as GMT files. The user can upload the gene set to the GSEA website or any gene set tool which takes GMT files. We also present the full results of the model including p-values calculated by random forest. This provides users with more flexibility in choosing a p-value cutoff that is most appropriate for the experimental setting.

AvailabilityThe core Tfr gene sets are freely available at: https://github.com/alosdiallo/TFR_Model. We have also included all of the code and data used in developing these gene sets. The code and results are released under an MIT license.

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: Environmental responsiveness of flowering time in cassava genotypes and associated transcriptome changes Cassava is an important food security crop in tropical regions of the world. Cassava improvement by breeding is limited by its delayed and poor production of flowers, such that cassava flowering under field conditions indirectly lengthens the breeding cycle. By studying genotype and environment interaction under two Nigerian field conditions (Ubiaja and Ibadan) and three controlled temperature conditions (22{degrees}C/18{degrees}C, 28/24{degrees}C and 34/30{degrees}C (day/night)), we found that while early flowering genotypes flowered at similar times and rates under all growing conditions (unfavorable and favorable field and controlled-temperature environments), late flowering genotypes were environmentally sensitive such that they were substantially delayed in unfavorable environments. Flowering times of late genotypes approached the flowering time of early flowering genotypes under relatively cool Ubiaja field conditions and in growth chambers at 22{degrees}C, whereas warmer temperatures elicited a delaying effect. Analysis of field and controlled temperature transcriptomes in leaves revealed that conditions that promote early flowering in cassava have low expression of the flowering repressor gene TEMPRANILLO 1 (TEM1), before and after flowering, among others. Field transcriptomes showed that the balance between flower promoting and inhibitory signaling, appeared to correlate with flowering time across the environments and genotypes.

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Cell atlas of the human ocular anterior segment: Tissue-specific and shared cell types The anterior segment of the eye consists of the cornea, iris, ciliary body, crystalline lens and aqueous humor outflow pathways. Together, these tissues are essential for the proper functioning of the eye. Disorders of vision have been ascribed to defects in all of them; some, including glaucoma and cataract, are among the most prevalent causes of blindness in the world. To characterize the cell types that comprise these tissues, we generated an anterior segment cell atlas of the human eye using high throughput single-nucleus RNA sequencing (snRNAseq). We profiled 191,992 nuclei from non-diseased anterior segment tissues from 6 human donors, identifying >60 cell types. Many of these cell types were discrete, whereas others, especially in lens and cornea, formed continua corresponding to known developmental transitions that persist in adulthood. Having profiled each tissue separately, we performed an integrated analysis of the entire anterior segment revealing that some cell types are unique to single structure whereas others are shared across tissues. The integrated cell atlas was then used to investigate cell type-specific expression patterns of more than 900 human ocular disease genes identified either through Mendelian inheritance patterns or genome-wide association studies (GWAS).

SIGNIFICANCE STATEMENTSeveral of the most prevalent blinding ocular conditions worldwide, including glaucoma, cataract and uncorrected refractive error, involve structures of the anterior segment of the human eye, which consists of the cornea, iris, ciliary body, crystalline lens and aqueous humor outflow pathways. In addition to providing transcriptomic profiles of the cell types within individual tissues, this work contributes to our understanding of the relatedness and diversity of these cell types across contiguous tissues by generating an integrated anterior segment cell atlas and documenting the expression of over 900 disease-associated genes in each cell type. By allowing simultaneous interrogation of cell-type specific expression of genes across multiple tissues, the atlas may yield broad insight into normal and disease-associated anterior segment functions.

sentences: A Single Cell Atlas of Lung Development Lung organogenesis requires precisely timed shifts in the spatial organization and function of parenchymal cells, especially during the later stages of lung development. To investigate the mechanisms governing lung parenchymal dynamics during development, we performed a single cell RNA sequencing (scRNA-seq) time-series yielding 92,238 epithelial, endothelial, and mesenchymal cells across 8 time points from embryonic day 12 (E12) to postnatal day 14 (P14) in mice. We combined new computational analyses with RNA in situ hybridization to explore transcriptional velocity, fate likelihood prediction, and spatiotemporal localization of cell populations during the transition between the saccular and alveolar stages. We interrogated this atlas to illustrate the complexity of type 1 pneumocyte function during the saccular and alveolar stages, and we demonstrate an integrated view of the cellular dynamics during lung development.

sentences: Profiling Genetic Diversity Reveals the Molecular Basis for Balancing Function with Misfolding in Alpha-1 Antitrypsin Genetic variation of alpha-1 antitrypsin (AAT) is responsible for alpha-1-antitrypsin deficiency (AATD) leading to gain-of-toxic aggregation in the liver and loss-of-function on neutrophil elastase (NE) inhibitory activity in the lung contributing to chronic obstructive pulmonary disease (COPD) during aging. To probe the molecular basis for how biology designs the protein fold to achieve balance between sequence, function and structure contributing to AATD in the population, we measured the intracellular monomer and polymer, secreted monomer and polymer and NE inhibitory activity of 75 alpha-1-antitrypsin (AAT) variants. To address the complex folding dynamics affecting the form and function of the protein fold that is differentially impacted by variants in the population, we applied a Gaussian process regression (GPR) based machine learning approach termed variation spatial profiling (VSP). By using a sparse collection of extant variants to link genotype to phenotype, VSP maps spatial covariance (SCV) relationships that quantitate the functional value of every residue in the wild-type (WT) AAT sequence with defined uncertainty in the context of its protein fold design. The SCV-based uncertainty allows us to pinpoint critical short- and long-range residue interactions involving 3 regions-the N-terminal (N1), middle (M2) and carboxyl-terminal (C3) of AAT polypeptide sequence that differentially contribute to the balance between function and misfolding of AAT, thus providing an unanticipated platform for precision therapeutic development for liver and lung disease. By understanding mechanistically the complex fold design of the metastable WT AAT fold, we posit that GPR-based SCV provides a foundation for understanding the evolutionary design of the fold from the ensemble of structures found in the population driving biology for precision management of AATD in the individual.

sentences: Deliberation gated by opportunity cost adapts to context with urgency Finding the right amount of deliberation, between insufficient and excessive, is a hard decision making problem that depends on the value we place on our time. Average-reward, putatively encoded by tonic dopamine, serves in existing reinforcement learning theory as the stationary opportunity cost of time, and of deliberation in particular. However, this cost often varies with environmental context that can change over time. Here, we introduce an opportunity cost of deliberation estimated adaptively on multiple timescales to account for non-stationary contextual factors. We use it in a simple decision-making heuristic based on average-reward reinforcement learning (AR-RL) that we call Performance-Gated Deliberation (PGD). We propose PGD as a strategy used by animals wherein deliberation cost is implemented directly as urgency, a previously characterized neural signal effectively controlling the speed of the decision-making process. We show PGD outperforms AR-RL solutions in explaining behaviour and urgency of non-human primates in a context-varying random walk prediction task and is consistent with relative performance and urgency in a context-varying random dot motion task. We make readily testable predictions for both neural activity and behaviour and call for an integrated research program in cognitive and systems neuroscience around the value of time.

sentences: PRMT5 regulates ovarian follicle development by facilitating Wt1 translation Protein arginine methyltransferase 5 (Prmt5) is the major type II enzyme responsible for symmetric dimethylation of arginine. Here, we found PRMT5 was expressed at high level in ovarian granulosa cells of growing follicles. Inactivation of Prmt5 in granulosa cells resulted in aberrant follicle development and female infertility. In Prmt5-knockout mice, follicle development was arrested with disorganized granulosa cells in which WT1 expression was dramatically reduced and the expression of steroidogenesis-related genes was significantly increased. The premature differentiated granulosa cells were detached from oocytes and follicle structure was disrupted. Mechanism studies revealed that Wt1 expression was regulated by PRMT5 at the protein level. PRMT5 facilitated IRES-dependent translation of Wt1 mRNA by methylating HnRNPA1. Moreover, the upregulation of steroidogenic genes in Prmt5-deficient granulosa cells was repressed by Wt1 overexpression. These results demonstrate PRMT5 participates in granulosa cell lineage maintenance by inducing Wt1 expression. Our study uncovers a new role of post-translational arginine methylation in granulosa cell differentiation and follicle development.

sentences: Age-related neural dedifferentiation for individual stimuli: An across-participant pattern similarity analysis Age-related neural dedifferentiation - reductions in the regional specificity and precision of neural representations - is proposed to compromise the ability of older adults to form sufficiently distinct neural representations to support episodic memory encoding. The computational model that spurred investigations of age-related neural dedifferentiation initially characterized this phenomenon as a reduction in the specificity of neural patterns for individual items or stimuli. Most investigations have focused on reductions in neural differentiation for patterns of neural activity associated with category level information, such as reduced neural selectivity between categories of visual stimuli (e.g., scenes, objects, and faces). Here, I report a novel across-participant pattern similarity analysis method to measure neural distinctiveness for individual stimuli that were presented to participants on a single occasion. Measures of item level pattern similarity during encoding showed a graded positive subsequent memory effect in younger, with no significant subsequent memory effect in older adults. These results suggest that age-related reductions in the distinctiveness of neural patterns for individual stimuli during age differences in memory encoding. Moreover, a measure of category level similarity demonstrated a significant subsequent memory effect associated with item recognition (regardless of an object source memory detail), whereas the effect in older was associated with source memory. These results converge with predictions of computational models of dedifferentiation showing age-related reductions in the distinctiveness of neural patterns across multiple levels of representation.

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Robust Characterization of Non-Invasive Post-Traumatic Osteoarthritis Mouse Model ObjectiveBiochemical and molecular changes involved in the pathophysiology of post-traumatic arthritis (PTOA) have not been fully understood. This study used non-invasive mouse models to study biochemical, biomechanical and pain-related behavior changes induced in mice following repetitive mechanical knee loading. Mouse models were used to reflect the effects of the early stages of PTOA in humans.

MethodsForty-eight twelve week old male mice were obtained for three groups: normal control without mechanical loading, trauma (24 hours after loading), and PTOA (early OA) groups. For the non-invasive PTOA mouse model, cyclic comprehensive loading (9 N) was applied on the left knee joint of each mouse. Biochemical and molecular changes induced by mechanical loading were analyzed after loading was completed. Blood and cartilage were collected and further examined using gene expression analysis. Grading of the tissue sections was completed using the osteoarthritis research society international (OARSI) scale. Biomechanical features of mechanically loaded knee joints were determined after 24 hours (Trauma) and three weeks (PTOA) post-mechanical loading sessions to examine the development of PTOA, respectively.

ResultsThe loaded left knee joint showed a greater ROS/RNS signal than the right knee that was not loaded. There was an increase in cartilage damage and MMP activity in the affected knee as the intensity of MabCII680 and MMP750 signal increased in the mechanical loaded joints as compared to unloaded control knee joints. There was also an increase in the difference of viscoelastic energy dissipation ability (tan {delta}) in PTOA. The OA score increased significantly in mechanically loaded knee joints.

ConclusionThis study showed that biomechanical, biochemical, and behavioral characteristics of the murine PTOA groups are significantly different from the control group. These results validate that the current mouse model can be used for translational studies to examine PTOA.

sentences: Mechanochemical tuning of a kinesin motor essential for malaria parasite transmission Kinesins are a superfamily of molecular motors that undertake ATP-dependent microtubule-based movement or regulate microtubule dynamics. Plasmodium species, which cause malaria and kill hundreds of thousands annually, encode 8-9 kinesins in their genomes. Of these, two - kinesin-8B and kinesin-8X -are canonically classified as kinesin-8s, which in other eukaryotes typically modulate microtubule dynamics. Unexpectedly, Plasmodium kinesin-8B is required for development of the flagellated male gamete in the mosquito host, and its absence completely blocks parasite transmission. To understand the molecular basis of kinesin-8Bs essential role, we characterised the in vitro properties of the kinesin-8B motor domains from P. berghei and P. falciparum. Both motors drive plus-end directed ATP-dependent microtubule gliding, but also catalyse ATP-dependent microtubule depolymerisation. We determined the microtubule-bound structures of these motors using cryo-electron microscopy. P. berghei and P. falciparum kinesin-8B exhibit a very similar mode of microtubule interaction, in which Plasmodium-distinct sequences at the microtubule-kinesin interface influence motor function. Intriguingly, however, P. berghei kinesin-8B exhibits a non-canonical structural response to ATP analogue binding such that neck linker docking is not induced. Nevertheless, the neck linker region is absolutely required for motility and depolymerisation activities of these motors. Taken together, these data suggest that the mechanochemistry of Plasmodium kinesin-8Bs has been functionally tuned to efficiently contribute to flagella formation.

sentences: Risk-utility tradeoff shapes memory strategies for evolving patterns Keeping a memory of evolving stimuli is ubiquitous in biology, an example of which is immune memory for evolving pathogens. However, learning and memory storage for dynamic patterns still pose challenges in machine learning. Here, we introduce an analytical energy-based framework to address this problem. By accounting for the tradeoff between utility in keeping a high-affinity memory and the risk in forgetting some of the diverse stimuli, we show that a moderate tolerance for risk enables a repertoire to robustly classify evolving patterns, without much fine-tuning. Our approach offers a general guideline for learning and memory storage in systems interacting with diverse and evolving stimuli.

sentences: Evolutionary ecology of natural comammox Nitrospira populations Microbes commonly exists in diverse and complex communities where species interact, and their genomic repertoires evolve over time. Our understanding of species interactions and evolution has increased in the last decades, but most studies of evolutionary dynamics are based on single species in isolation or in experimental systems composed of few interacting species. Here, we use the microbial ecosystem found in groundwater-fed sand filters as a model to avoid this limitation. In these open systems, diverse microbial communities experience relatively stable conditions, and the coupling between chemical and biological processes is generally well defined. Metagenomic analysis of 12 sand filters revealed systematic co-occurrence of at least five comammox Nitrospira species, likely promoted by low ammonium concentrations. These Nitrospira species showed intra-population sequence diversity, although possible clonal expansion was detected in few abundant local comammox populations. They showed low homologous recombination and strong purifying selection, the latter process being especially strong in genes essential in energy metabolism. Positive selection was detected on genes related to resistance to foreign DNA and phages. We found that, compared to other habitats, groundwater-fed sand filters impose strong purifying selection and low recombination on comammox Nitrospira populations. These results suggest that evolutionary processes are more affected by habitat type than by species identity. Together, this study improves our understanding of species interactions and evolution in complex microbial communities, and sheds light on the environmental dependency of evolutionary processes.

sentences: A guide to examining intramuscular fat formation and its cellular origin in skeletal muscle Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build and maintain the extracellular matrix that acts as a molecular myofiber scaffold. In addition, FAPs are indispensable for myofiber regeneration as they secrete a multitude of beneficial factors sensed by the muscle stem cells (MuSCs). In diseased states, however, FAPs are the cellular origin of intramuscular fat and fibrotic scar tissue. This fatty fibrosis is a hallmark of sarcopenia and neuromuscular diseases, such as Duchenne Muscular Dystrophy. One significant barrier in determining why and how FAPs differentiate into intramuscular fat is effective preservation of adipocytes, especially in frozen tissue sections. Conventional methods of skeletal muscle tissue processing, such as snap-freezing, do not properly preserve the morphology of individual adipocytes, thereby preventing accurate visualization and quantification. Here, we describe a protocol that provides robust preservation of adipocyte morphology in skeletal muscle sections allowing visualization, imaging, and quantification of intramuscular fat. We also outline how to process a portion of muscle tissue for RT-qPCR, enabling users to confirm observed changes in fat formation by viewing differences in expression of adipogenic genes. Additionally, we will describe how our protocol can be adopted to visualize adipocytes by whole mount immunofluorescence of muscle samples. Finally, we will outline how to combine this protocol with genetic lineage tracing of Pdgfr-expressing FAPs to study the adipogenic conversion of FAPs. Our protocol consistently yields high-resolution and morphologically accurate immunofluorescent images of adipocytes that, along with confirmation by RT-qPCR, allows for robust, rigorous, and reproducible visualization and quantification of intramuscular fat. Together, our analysis pipeline is the first step to improve our understanding of how FAPs differentiate into intramuscular fat and provides a framework to validate novel interventions to prevent fat formation.

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Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1 Relaxin/insulin-like family peptide receptor 1 (RXFP1) mediates relaxins antifibrotic effects and has reduced expression in the lung and skin of patients with fibrotic interstitial lung disease (fILD) including idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). This may explain the failure of relaxin-based anti-fibrotic treatments in SSc, but the regulatory mechanisms controlling RXFP1 expression remain largely unknown. This study aimed to identify regulatory elements of RXFP1 that may function differentially in fibrotic fibroblasts.

We identified and evaluated a distal regulatory region of RXFP1 in lung fibroblasts using a luciferase reporter system. Using serial deletions, an enhancer upregulating pGL3-promoter activity was localized to the distal region between -584 to -242bp from the distal transcription start site (TSS). This enhancer exhibited reduced activity in IPF and SSc lung fibroblasts. Bioinformatic analysis identified two clusters of activator protein 1 (AP-1) transcription factor binding sites within the enhancer. Site-directed mutagenesis of the binding sites confirmed that only one cluster reduced activity (-358 to -353 relative to distal TSS). Co-expression of FOS in lung fibroblasts further increased enhancer activity. In vitro complex formation with a labeled probe spanning the functional AP-1 site using nuclear proteins isolated from lung fibroblasts confirmed a specific DNA/protein complex formation. Application of antibodies against JUN and FOS resulted in the complex alteration, while antibodies to JUNB and FOSL1 did not. Analysis of AP-1 binding in 5 pairs of control and IPF lung fibroblasts detected positive binding more frequently in control fibroblasts. Expression of JUN and FOS was reduced and correlated positively with RXFP1 expression in IPF lungs.

In conclusion, we identified a distal enhancer of RXFP1 with differential activity in fibrotic lung fibroblasts involving AP-1 transcription factors. Our study provides insight into RXFP1 downregulation in fILD and may support efforts to reevaluate relaxin-based therapeutics alongside upregulation of RXFP1 transcription.

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Bridging functional annotation gaps in non-model plant genes with AlphaFold, DeepFRI and small molecule docking BackgroundFunctional annotation assigns descriptive biological meaning to genetic sequences. Limited availability of manually curated or experimentally validated plant genes from a diverse range of taxa poses a significant challenge for functional annotation in non-model organisms. Accurate computational approaches are required. We argue that recent breakthroughs in deep learning have the potential to not only narrow the functional annotation gap between non-model and model plant organisms, but also annotate and reveal novel functions even for genes with no homologs in public databases.

ResultsDeep learning models were applied to functionally annotate a set of previously published differentially expressed genes. Predicted protein structures and functional annotations were generated using the AlphaFold protein structure and DeepFRI protein language inference models respectively. The resulting structures and functional annotations were validated using small molecule docking experiments. DeepFRI and AlphaFold models not only correctly annotated differentially expressed genes, but also revealed detailed mechanisms involving protein-protein interactions.

ConclusionsDeep learning models are capable of inferring novel functions and achieving high accuracy in functional annotation. Their increased use in plant research will result in major improvements in annotations for non-model plants that are underrepresented in genome databases. We illustrate how integrating protein structure prediction, functional residue prediction, and small molecule docking can infer plausible protein-protein interactions and yield additional mechanistic insights. This approach will aid in the selection of candidate genes for further study from differential expression studies that generate large gene lists.

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Epistasis at the SARS-CoV-2 RBD Interface and the Propitiously Boring Implications for Vaccine Escape At the time of this writing, December 2021, potential emergence of vaccine escape variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a grave global concern. The interface between the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein and the host receptor (ACE2) overlap with the binding site of principal neutralizing antibodies (NAb), limiting the repertoire of viable mutations. Nonetheless, variants with multiple mutations in the RBD have rose to dominance. Non-additive, epistatic relationships among RBD mutations are apparent, and assessing the impact of such epistasis on the mutational landscape is crucial. Epistasis can substantially increase the risk of vaccine escape and cannot be completely characterized through the study of the wild type (WT) alone. We employed protein structure modeling using Rosetta to compare the effects of all single mutants at the RBD-NAb and RBD-ACE2 interfaces for the WT, Delta, Gamma, and Omicron variants. Overall, epistasis at the RBD interface appears to be limited and the effects of most multiple mutations are additive. Epistasis at the Delta variant interface weakly stabilizes NAb interaction relative to ACE2 interaction, whereas in the Gamma variant, epistasis more substantially destabilizes NAb interaction. Although a small, systematic trend towards NAb destabilization not observed for Delta or Gamma was detected for Omicron, and despite bearing significantly more RBD mutations, the epistatic landscape of the Omicron variant closely resembles that of Gamma. These results suggest that, although Omicron poses new risks not observed with Delta, structural constraints on the RBD hamper continued evolution towards more complete vaccine escape. The modest ensemble of mutations relative to the WT that are currently known to reduce vaccine efficacy is likely to comprise the majority of all possible escape mutations for future variants, predicting continued efficacy of the existing vaccines.

SignificanceEmergence of vaccine escape variants of SARS-CoV-2 is arguably the most pressing problem during the COVID-19 pandemic as vaccines are distributed worldwide. We employed a computational approach to assess the risk of antibody escape resulting from mutations in the receptor-binding domain of the spike protein of the wild type SARS-CoV-2 virus as well as the Delta, Gamma, and Omicron variants. At the time of writing, December, 2021, Omicron is poised to replace Delta as the dominant variant worldwide. The efficacy of the existing vaccines against Omicron could be substantially reduced relative to the WT and the potential for vaccine escape is of grave concern. Our results suggest that although Omicron poses new evolutionary risks not observed for the Delta variant, structural constraints on the RBD make continued evolution towards more complete vaccine escape unlikely. The modest set of escape-enhancing mutations already identified for the wild type likely include the majority of all possible mutations with this effect.

sentences: In vitro pectate lyase activity and carbon uptake assays and whole genome sequencing of Bap strains for a pectin defective pathway The pectin lyase activity of 59 Bacillus amyloliquefaciens subsp. plantarum (Bap) strains was tested in vitro on Pectate Agar (PA) and Tris-Spizizen Salts (TSS) medium. Bap strains were cultured on TSA medium and washed three times with sterile water before the inoculation on PA media. Higher and lower pectate lyase activity were observed in six (AP193, AP203, AP299, AP80, AP102, and AP52) and four (AP 194, AP214, AP215, and AP305) Bap strains compared to other Bap strains. A total of 12 Bap strains (AP67, AP71, AP77, AP78, AP85, AP102, AP108, AP135, AP143, AP189, AP192, and AP193) grew vigorously on TSS medium. A total of six Bap strains (AP194, AP204, AP214, AP216, AP219, and HD73) had lower growth compared to other Bap strains. Pectin (1%) were used for in vitro PA and TSS medium. Pectate lyase and utilization activity were not found in Bacillus thuringiensis subsp. kurstaki strain HD73 compared to Bap strains. A draft genome sequence for strains AP194 and AP214 that were negative for pectin utilization were generated using an Illumina MiSeq. RAST analysis revealed that the pectin-associated gene altronate hydrolase (uxaA) absent in AP 214 strain. Multiple amino acid alignments of exuT and uxuB gene sequence showed dissimilarities among AP194, AP214, and reference Bap strains.

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The Effect of Blood Viscosity on Shear-Induced Hemolysis using a Magnetically Levitated Shearing Device IntroductionBlood contacting medical devices, including rotary blood pumps, can cause shear-induced blood damage that may lead to adverse effects in patients. Due in part to an inadequate understanding of how cell-scale fluid mechanics impact red blood cell membrane deformation and damage, there is currently not a uniformly accepted engineering model for predicting blood damage caused by complex flow fields within ventricular assist devices (VADs).

MethodsWe empirically investigated hemolysis in an axial Couette flow device typical of a rotary VAD to expand our current understanding of shear-induced blood damage in two ways. First, we used a magnetically levitated device to accurately control the shear rate and exposure time experienced by blood and to minimize the effects of other uncharacterized stresses. Second, we explored the effects of both hematocrit and plasma viscosity on shear-induced hemolysis to characterize blood damage based on the viscosity-independent shear rate, rather than on shear stress.

ResultsOver a shear rate range of 20,000-80,000 1/s, the Index of Hemolysis was found to be largely independent of hematocrit, bulk viscosity, or the suspension media viscosity.

ConclusionIt is recommended that future investigations of shear-induced blood damage report findings with respect to the viscosity-neutral term of shear rate, in addition to the bulk whole blood viscosity measured at an appropriate shear rate relevant to the flow conditions of the device.

sentences: Integrated data-driven reannotation of the Kluyveromyces marxianus genome reveals an expanded protein coding repertoire Many new biotechnology applications make use of non-traditional yeast species that possess characteristics and traits that are advantageous in specific settings. To a large extent, this is possible because of advances in molecular and genomic technologies that provide opportunities to study and manipulate the genomes of diverse yeasts. There remains, however, a substantial gap between what we know about well-studied models versus emerging industrial yeast species. In this study, we applied a multi-faceted omics analysis to better understand organisation of protein coding gene expression in Kluyveromyces marxianus, a yeast widely used in food and industrial biotechnology. We combined advanced transcriptomic techniques for mapping 5 and 3 ends of RNA transcripts with ribosome profiling to explore the transcriptional and translational landscapes of this yeast. This allowed us to improve the genome annotation and identify over 300 un-annotated or mis-annotated genes. We discovered numerous examples of novel proteoforms due to use of alternative transcription or translation start sites, many genes with translated upstream open reading frames (uORFs) and a novel case of programmed ribosomal frameshifting. Only some of newly discovered features are shared with distant species, for example, being present in Saccharomyces cerevisiae orthologs. The exploration of shared and unique features may provide an insight into the evolution of gene regulation in pre- and post-WGD yeasts. The processed data has been made available on the GWIPS-viz and Trips-Viz browsers, thus providing an accurate data-driven annotation of transcripts and their protein coding regions along with quantitative information on their transcription and translation.

sentences: Northward Expansion of Amblyomma americanum (Acari: Ixodidae) into Southern Michigan Amblyomma americanum (Linnaeus) (Acari: Ixodidae) (lone star tick) is an aggressive, generalist parasite that vectors numerous important human and animal pathogens. In recent decades its geographic range has expanded northwards from endemic regions in the southeastern and southcentral US. In 2019 five questing A. americanum comprising two life stages were detected at one site in Berrien County, in southwestern Michigan, satisfying one CDC criterium for an established population for the first time in the state. To better characterize the northern extent of emerging A. americanum, we conducted active surveillance (i.e., drag sampling) in summer 2020 throughout Michigans southern counties and detected one adult A. americanum from each of six widespread sites, including where they had been detected in 2019. A larger established population was identified at another site in Berrien County, which yielded 691 A. americanum comprising three life stages. Questing tick phenologies at this site were similar to that reported for other regions. Statewide surveillance in 2021 revealed no A. americanum outside of Berrien County, but establishment criteria were met again at the two sites where established populations were first detected respectively in 2019 and 2020. These observations may represent the initial successful invasion of A. americanum into Michigan. Data from passive (1999-2020) and active surveillance (2004-2021) efforts, including a domestic animal sentinel program (2015-2018), are reported to provide context for this nascent invasion. Continued active surveillance is needed to help inform the public, medical professionals, and public health officials of the health risks associated with this vector.

sentences: Self-assembling short immunostimulatory duplex RNAs with broad spectrum antiviral activity The current COVID-19 pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III), in a wide range of human cell types. These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique conserved sequence motif (sense strand: 5-C, antisense strand: 3-GGG) that mediates end-to-end dimer self-assembly of these RNAs by Hoogsteen G-G base-pairing. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory siRNAs, their activity is independent of TLR7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad spectrum inhibition of infections by many respiratory viruses with pandemic potential, including SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza A, as well as the common cold virus HCoV-NL63 in both cell lines and human Lung Chips that mimic organ-level lung pathophysiology. These short dsRNAs can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics at low cost.

sentences: Antarctic biodiversity predictions through substrate qualities and environmental DNA Antarctic conservation science is important to enhance Antarctic policy and to understand alterations of terrestrial Antarctic biodiversity. Antarctic conservation will have limited long term effect in the absence of large-scale biodiversity data, but if such data were available, it is likely to improve environmental protection regimes. To enable Antarctic biodiversity prediction across continental spatial scales through proxy variables, in the absence of baseline surveys, we link Antarctic substrate-derived environmental DNA (eDNA) sequence data from the remote Antarctic Prince Charles Mountains to a selected range of concomitantly collected measurements of substrate properties. We achieve this using a statistical method commonly used in machine learning. We find neutral substrate pH, low conductivity, and some substrate minerals to be important predictors of presence for basidiomycetes, chlorophytes, ciliophorans, nematodes, or tardigrades. Our bootstrapped regression reveals how variations of the identified substrate parameters influence probabilities of detecting eukaryote phyla across vast and remote areas of Antarctica. We believe that our work may improve future taxon distribution modelling and aid targeting logistically challenging biodiversity surveys.

sentences: Structural Mechanics of the Alpha-2-Macroglobulin Transformation Alpha-2-Macroglobulin (A2M) is the critical pan-protease inhibitor of the innate immune system. When proteases cleave the A2M bait region, global structural transformation of the A2M tetramer is triggered to entrap the protease. The structural basis behind the cleavage-induced transformation and the protease entrapment remains unclear. Here, we report cryo-EM structures of native- and intermediate-forms of the Xenopus laevis egg A2M homolog (A2Moo or ovomacroglobulin) tetramer at 3.7-4.1 [A] and 6.4 [A] resolution, respectively. In the native A2Moo tetramer, two pairs of dimers arrange into a cross-like configuration with four 60 [A]-wide bait-exposing grooves. Each bait in the native form threads into an aperture formed by three macroglobulin domains (MG2, MG3, MG6). The bait is released from the narrowed aperture in the induced protomer of the intermediate form. We propose that the intact bait region works as a "latch-lock" to block futile A2M transformation until its protease-mediated cleavage.

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Hemagglutination Inhibition (HAI) Antibody Landscapes after Vaccination with diverse H7 hemagglutinin (HA) proteins BackgroundA systemic evaluation of the antigenic differences of the H7 influenza hemagglutinin (HA) proteins, especially for the viruses isolated after 2016, are limited. The purpose of this study was to investigate the antigenic differences of major H7 strains with an ultimate aim to discover H7 HA proteins that can elicit protective receptor-blocking antibodies against co-circulating H7 influenza strains.

MethodA panel of nine H7 influenza strains were selected from 3,633 H7 HA amino acid sequences identified over the past two decades (2000-2018). The sequences were expressed on the surface of virus like particles (VLPs) and used to vaccinate C57BL/6 mice. Serum samples were collected and tested for hemagglutination-inhibition (HAI) activity. The vaccinated mice were challenged with lethal dose of H7N9 virus, A/Anhui/1/2013.

ResultsVLPs expressing the H7 HA antigens elicited broadly reactive antibodies each of the selected H7 HAs, except the A/Turkey/Italy/589/2000 (Italy/00) H7 HA. A putative glycosylation due to an A169T substitution in antigenic site B was identified as a unique antigenic profile of Italy/00. Introduction of the putative glycosylation site (H7 HA-A169T) significantly altered the antigenic profile of HA of the A/Anhui/1/2013 (H7N9) strain.

ConclusionThis study identified key amino acid mutations that result in severe vaccine mismatches for future H7 epidemics. Future universal influenza vaccine candidates will need to focus on viral variants with these key mutations.

sentences: Acorde: unraveling functionally-interpretable networks of isoform co-usage from single cell data Alternative splicing (AS) is a highly-regulated post-transcriptional mechanism known to modulate isoform expression within genes and contribute to cell-type identity. However, the extent to which alternative isoforms establish co-expression networks that may relevant in cellular function has not been explored yet. Here, we present acorde, a pipeline that successfully leverages bulk long reads and single-cell data to confidently detect alternative isoform co-expression relationships. To achieve this, we developed and validated percentile correlations, a novel approach that overcomes data sparsity and yields accurate co-expression estimates from single-cell data. Next, acorde uses correlations to cluster co-expressed isoforms into a network, unraveling cell type-specific alternative isoform usage patterns. By selecting same-gene isoforms between these clusters, we subsequently detect and characterize genes with co-differential isoform usage (coDIU) across neural cell types. Finally, we predict functional elements from long read-defined isoforms and provide insight into biological processes, motifs and domains potentially controlled by the coordination of post-transcriptional regulation.

sentences: Hingepoint emergence in mammalian spinal neurulation Neurulation is the process in early vertebrate embryonic development during which the neural plate folds to form the neural tube. Spinal neural tube folding in the posterior neuropore changes over time, first showing a median hingepoint, then both the median hingepoint and dorsolateral hingepoints, followed by dorsolateral hingepoints only. The biomechanical mechanism of hingepoint formation in the mammalian neural tube is poorly understood. Here, we employ a mechanical finite element model to study neural tube formation. The computational model mimics the mammalian neural tube using microscopy data from mouse and human embryos. While intrinsic curvature at the neural plate midline has been hypothesized to drive neural tube folding, intrinsic curvature was not sufficient for tube closure in our simulations. We achieved neural tube closure with an alternative model combining mesoderm expansion, non-neural ectoderm expansion and neural plate adhesion to the notochord. Dorsolateral hingepoints emerged in simulations with low mesoderm expansion and zippering. We propose that zippering provides the biomechanical force for dorsolateral hingepoint formation in settings where the neural plate lateral sides extend above the mesoderm. Together, these results provide a new perspective on the biomechanical and molecular mechanism of mammalian spinal neurulation.

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Topological Data Analysis of Human Brain Networks Through Order Statistics Understanding the topological characteristics of the brain network across a population is central to understanding brain functions. The abstraction of human connectome as a graph has been pivotal in gaining insights on the topological features of the brain network. The development of group-level statistical inference procedures in brain graphs while accounting for the heterogeneity and randomness still remains a difficult task. In this study, we develop a robust statistical framework based on persistent homology using the order statistics for analyzing brain networks. The use of order statistics greatly simplifies the computation of the persistent barcodes. We validate the proposed methods using comprehensive simulation studies and subsequently apply to the resting-state functional magnetic resonance images. We conclude a statistically significant topological difference between the male and female brain networks.

Author summaryWe fit a random graph model to the brain network and compute the expected persistent barcodes using order statistics. This novel approach significantly simplifies the computation of expected persistent barcodes, which otherwise requires complex theoretical constructs. Subsequently, the proposed statistical framework is used to discriminate if two groups of brain networks are topologically different. The method is applied in determining the sexual dimorphism in the shape of resting-state functional magnetic resonance images.

sentences: Gradual evolution of allopolyploidy in Arabidopsis suecica The majority of diploid organisms have polyploid ancestors. The evolutionary process of polyploidization (and subsequent re-diploidization) is poorly understood, but has frequently been conjectured to involve some form of "genome shock" -- partly inspired by studies in crops, where polyploidy has been linked to major genomic changes such as genome reorganization and subgenome expression dominance. It is unclear, however, whether such dramatic changes would be characteristic of natural polyploidization, or whether they are a product of domestication. Here, we study polyploidization in Arabidopsis suecica (n = 13), a post-glacial allopolyploid species formed via hybridization of A. thaliana (n = 5) and A. arenosa (n = 8). We generated a chromosome-level genome assembly of A. suecica and complemented it with polymorphism and transcriptome data from multiple individuals of all species. Despite a divergence of [~]6 Mya between the two ancestral species and appreciable differences in their genome composition, we see no evidence of a genome shock: the A. suecica genome is highly colinear with the ancestral genomes, there is no subgenome dominance in expression, and transposable element dynamics appear to be stable. We do, however, find strong evidence for changes suggesting gradual adaptation to polyploidy. In particular, the A. thaliana subgenome shows upregulation of meiosis-related genes, possibly in order to prevent aneuploidy and undesirable homeologous exchanges that are frequently observed in experimentally generated A. suecica, and the A. arenosa subgenome shows upregulation of cyto-nuclear related processes, possibly in response to the new cytoplasmic environment of A. suecica, with plastids maternally inherited from A. thaliana.

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The neuroprotective effect of meloxicam in a transient ischemia model involves an increase in axonal sprouting but a decrease in new neuron formation after 7 days ofreperfusion The inflammatory response plays an important role in neuroprotection and regeneration after ischemic insult. The use of non-steroidal anti-inflammatory drugs has been a matter of debate as to whether they have beneficial or detrimental effects. In this context, the effects of the anti-inflammatory agent meloxicam have been scarcely documented after stroke, but its ability to inhibit both cyclooxygenase isoforms (1 and 2) could be a promising strategy to modulate post-ischemic inflammation. This study analyzed the effect of the anti-inflammatory agent meloxicam in a transient focal ischemia model in rats, measuring its neuroprotective effect after 48 hours and 7 days of reperfusion and the effects of the treatment on the glial scar and regenerative events such as the generation of new progenitors in the subventricular zone and axonal sprouting at the edge of the damaged area. We show that meloxicams neuroprotective effects remained after 7 days of reperfusion even if its administration was restricted to the two first days after ischemia. Moreover, meloxicam treatment modulated glial scar reactivity, which matched with an increase in axonal sprouting. However, this treatment decreased the formation of neuronal progenitor cells. This study discusses the dual role of anti-inflammatory treatments after stroke and encourages the careful analysis of both the neuroprotective and the regenerative effects in preclinical studies.

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Istradefylline, an adenosine A2a receptor antagonist, ameliorates neutrophilic airway inflammation and psoriasis in mice ObjectiveExtracellular adenosine is produced from secreted ATP by cluster of differentiation (CD)39 and CD73. Both are critical nucleotide metabolizing enzymes of the adenosine generating pathway and are secreted by neuronal or immune cells. Adenosine plays a role in energy processes, neurotransmission, and endogenous regulation of inflammatory responses. Istradefylline is a selective adenosine A2a receptor (A2aR) antagonist used for the treatment of Parkinsons disease. We have reported that adenosine primes hypersecretion of interleukin (IL)-17A via A2aR. Istradefylline, as well as an inhibitor of CD39 (ARL67156) and an inhibitor of CD73 (AMP-CP), suppressed IL-17A production, and the administration of istradefylline to mice with experimental autoimmune encephalomyelitis (EAE) led to the marked amelioration of the disease. These previous results suggest that adenosine is an endogenous modulator of neutrophilic inflammation. We investigated the effect of istradefylline, ARL67156 and AMP-CP on other mouse models of neutrophilic inflammation.

MethodsWe tested the effect of istradefylline, ARL67156 and AMP-CP on OVA-induced neutrophilic airway inflammation or imiquimod (IMQ)-induced psoriasis in mice. These two model mice received these drugs orally or percutaneously, respectively. The production of IL-17A in the lung and ear thickness were used as an index of the effects.

ResultsWe show that istradefylline, ARL67156 and AMP-CP suppressed the OVA-induced IL-17A production in the lung and IMQ-induced psoriasis.

ConclusionThese results indicate that adenosine-mediated IL-17A production plays a role in neutrophilic inflammation models, and moreover, istradefylline, ARL67156, and AMP-CP are effective in animal models of neutrophilic inflammation. Some clinical relevancies in COVID-19 are discussed.

sentences: Disinfection of SARS-CoV-2 contaminated surfaces of personal items with UVC-LED disinfection boxes The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted from person-to-person by close contact, small aerosol respiratory droplets and potentially via contact with contaminated surfaces. Here, we investigated the effectiveness of commercial UVC-LED disinfection boxes in inactivating SARS-CoV-2 contaminated surfaces of personal items. We contaminated glass, metal and plastic samples representing the surfaces of personal items such as smartphones, coins or credit cards with SARS-CoV-2 formulated in an organic matrix mimicking human respiratory secretions. For disinfection, the samples were placed at different distances from UVC emitting LEDs inside commercial UVC-LED disinfection boxes and irradiated for different time periods (up to 10 minutes). High viral loads of SARS-CoV-2 were effectively inactivated on all surfaces after 3 minutes of irradiation. Even 10 seconds of UVC-exposure strongly reduced viral loads. Thus, UVC-LED boxes proved to be an effective method for disinfecting SARS-CoV-2 contaminated surfaces that are typically found on personal items.

sentences: ROPIUS0: A deep learning-based protocol for protein structure prediction and model selection and its performance in CASP14 Protein structure prediction has recently been revolutionized when AlphaFold2 [1] predicted protein structures with near-experimental accuracy in the latest CASP14 season of critical assessment of methods of protein structure prediction (CASP). Among numerous implications, this breakthrough has led to a rapidly growing number of high-quality structural models [2]. We present a protocol ROPIUS0 for protein structure prediction and model selection and discuss its benefits in the new era of structure prediction. At the core of the ROPIUS0 protocol is the deep learning module developed for the selection of protein structural models. It is shown that the direct use of predicted inter-residue distances may be sufficient to discriminate between correct and incorrect protein folds, considering only a small fraction of predicted distances. We extensively tested the protocol: In the latest CASP14 prediction season, a ROPIUS0 variant based on model selection ranked 13th in the category of tertiary structure prediction. Its performance is on par with top-performing automated prediction servers when tested on the CASP13 dataset, and it performs similarly on a CAMEO dataset. The results suggest ways to improve searching for structurally similar and homologous proteins without considerably increasing speed. Our new open-source threading tool based on comparing a subset of inter-residue distances demonstrates the effectiveness and application of the deep learning module of the ROPIUS0 protocol.

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CUB domain containing protein 1 (CDPC1) is a target for radioligand therapy in castration resistant prostate cancer PurposeRadioligand therapy (RLT) is relatively unexplored in metastatic castration resistant prostate cancer (mCRPC), with much of the focus having been on bone seeking radionuclides and PSMA-directed RLT. Herein, we evaluated if CUB domain containing protein 1 (CDCP1) can be exploited to treat mCRPC with RLT, particularly for subsets like small cell neuroendocrine prostate cancer (SCNC) that would not be expected to respond to current options.

Experimental DesignCDCP1 mRNA levels were evaluated in the RNA-seq data from 119 recent mCRPC biopsies. Protein expression was assessed in twelve SCNC and adenocarcinoma patient derived xenografts. Saturation binding assays were performed with 4A06, a recombinant human antibody that targets the CDCP1 ectodomain. The feasibility of imaging and treating mCRPC in vivo was tested with 89Zr-4A06 and 177Lu-4A06.

ResultsCDCP1 mRNA expression was observed in over 90% of mCRPC biopsies, including SCNC and in adenocarcinoma with low FOLH1 (PSMA) levels. A modest anticorrelation was observed between CDCP1 and PTEN. Overall survival was not significantly different based on CDCP1 mRNA levels, regardless of PTEN status. Full length and/or cleaved CDCP1 was expressed in ten of twelve PDX samples. Bmax values of ~22,000 and ~6,200 fmol/mg were calculated for two human prostate cancer cell lines. Five prostate cancer models were readily detected in vivo with 89Zr-4A06. 177Lu-4A06 significantly suppressed the growth of DU145 tumors compared to control.

ConclusionsThe antitumor data and the overexpression of CDCP1 reported herein provide the first evidence promoting CDCP1 directed RLT as a treatment strategy for mCRPC.

Statement of Translational RelevanceNew targets for RLT are needed to address the subset of mCRPC that cannot be treated with bone seeking radionuclides or PSMA directed RLT. We report herein the first data credentialing CDCP1 as a target for mCRPC, in both adenocarcinoma and neuroendocrine subtypes. Combined with low expression in normal human tissues, these data provide a compelling scientific rationale for testing CDCP1 directed RLT clinically in mCRPC patients alone or in combination with other systemic therapies.

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Metabolic-imaging of human glioblastoma explants: a new precision-medicine model to predict tumor treatment response early BackgroundGlioblastoma (GB) is the most severe form of brain cancer, with a 12-15 month median survival. Surgical resection, temozolomide (TMZ) and radiotherapy (RT) remain the primary therapeutic options for GB, and no new therapies have been introduced in recent years. This therapeutic standstill is primarily due to preclinical models that do not reflect the complexity of GB cell biology and fail to test anti-cancer treatments valuably. Better preclinical models for compound screening are therefore needed.

MethodsIn-vitro 3D glioblastoma live explants (GB-EXPs) were derived from patients resected tumors. We then applied metabolic imaging by fluorescence lifetime imaging microscopy (FLIM) on to GB-EXPs to assess drug response, using TMZ as our benchmark drug. Whole-transcriptome and whole-exome analyses were performed.

ResultsUsing FLIM we were able to stratify samples in Responder and Non-Responder tumors. Our functional precision medicine approach was successfully completed within 1 week of surgery. FLIM-based tumor samples stratification was well reflected at the molecular level, highlighting new targets associated with TMZ treatment and identifying a molecular signature associated with survival.

ConclusionsTo the best of our knowledge, this is the first time that FLIM-based metabolic imaging is used on live glioblastoma 3D explants to test anti-neoplastic drugs rapidly. FLIM-based readouts of drug response in GB explants could improve precision treatment of patients with GB and the identification of new anti-GB drugs.

O_LIPatients tumors can be tested for several individual anti-cancer treatments early after surgery
C_LIO_LIFLIM metabolic imaging is an accurate and rapid in vitro tumor treatment response assessment tool
C_LIO_LIOur model can be exploited to identify new drug molecular targets and applied to other cancers
C_LI

Importance of the studyIn this article, we propose an accurate glioblastoma anti-cancer treatment response 3D-approach consisting of FLIM-based metabolic imaging applied directly to in-vitro vital patient-derived glioblastoma explants. Unlike other models, drug response of live explants is assessed within 1 week from patients surgery, therefore with minimal tampering of natural tumor cyto-architecture, preventing the conventional structural and molecular divergence that the tumor undergoes after long time of in vitro culturing and with no animal involvement. This novel in vitro approach for glioblastoma treatment assessment could be exploited as a tool, in a range of clinical and laboratory settings to improve patients clinical management. Selection of the optimal drug regimen for glioblastoma patients remains a clinical challenge and a method to assess the tumor specific response to several therapies at an early stage could be transformative for glioblastoma patients management. Furthermore, this model can be easily used for large-scale screening of new pharmaceutical compounds.

sentences: Tree diversity effects on productivity depend on mycorrhizae and life strategies in a temperate forest experiment Tree species are known to predominantly interact either with arbuscular mycorrhizal (AM) or ectomycorrhizal (EM) fungi. However, there is a knowledge gap whether these mycorrhizae differently influence biodiversity-ecosystem functioning (BEF) relationships and whether a combination of both can increase community productivity. In 2015, we established a tree-diversity experiment by growing tree communities with varying species-richness levels (1, 2, or 4 species), and either with AM or EM tree species, or a combination of both. We investigated basal area and annual basal area increment from 2015 to 2020 as proxy for community productivity. We found significant positive relationships between tree species richness and community productivity, which strengthened over time. Further, AM and EM tree species differently influenced productivity; however, there was no overyielding when AM and EM trees grew together. EM tree communities were characterized by low productivity in the beginning, but an increase of increment over time, and showed overall strong biodiversity effects. For AM tree communities the opposite was true. While young trees did not benefit from the presence of the other mycorrhizal type, dissimilar mechanisms underlying BEF relationships in AM and EM trees indicate that maximizing tree and mycorrhizal diversity may increase ecosystem functioning in the long run.

sentences: Stromal inflammation is a targetable driver of hematopoietic aging Hematopoietic aging is marked by a loss of regenerative capacity and skewed differentiation from hematopoietic stem cells (HSC) leading to dysfunctional blood production. Signals from the bone marrow (BM) niche dynamically tailor hematopoiesis, but the effect of aging on the niche microenvironment and the contribution of the aging niche to blood aging still remains unclear. Here, we characterize the inflammatory milieu in the aged marrow cavity that drives both stromal and hematopoietic remodeling. We find decreased numbers and functionality of osteogenic mesenchymal stromal cells (MSC) at the endosteum and expansion of pro-inflammatory perisinusoidal MSCs with deterioration of sinusoidal endothelium in the central marrow, which together create a degraded and inflamed old niche. Molecular mapping at single cell resolution confirms disruption of cell identities and enrichment of inflammatory response genes in niche populations. Niche inflammation, in turn, drives chronic activation of emergency myelopoiesis pathways in old HSCs and multipotent progenitors (MPP), which promotes myeloid differentiation at the expense of lymphoid and erythroid commitment and hinders hematopoietic regeneration. Remarkably, niche deterioration, HSC dysfunction and defective hematopoietic regeneration, can be improved by blocking inflammatory IL-1 signaling. Our results demonstrate that targeting niche inflammation is a tractable strategy to restore blood production during aging.

sentences: A water-reward task assay for evaluating mouse mutualistic cooperative behavior Social cooperation is fundamentally important for group animals but rarely studied with mice because of their natural aggressiveness. In the present work, we induced pairs of mice to develop a mutualistic cooperative behavior in a non-divided chamber. Each mouse was first trained to learn to use a water dispenser by occupying a particular zone served as a switch to the dispenser. Two trained mice were then put into a chamber containing two separate zones jointly controlling two dispensers. We recorded the latency before each co-drinking, the number and cumulated time of co-drinking each day during the test. These parameters served as quantitative measurements of cooperative behavior in mice. The whole procedure includes preparation, training and testing phases, which take 15 days in total. This assay provides detailed procedures and analytical methods for investigators to characterize and quantify the mutualistic cooperative behavior. The use of mice as subjects allows convenient coupling to other behavior assays and is amiable to genetic manipulations for mechanistic study.

sentences: Generative and interpretable machine learning for aptamer design and analysis of in vitro sequence selection Selection protocols such as SELEX, where molecules are selected over multiple rounds for their ability to bind to a target molecule of interest, are popular methods for obtaining binders for diagnostic and therapeutic purposes. With the increasing amount of such high-throughput experimental data available, machine learning techniques have become increasingly popular for molecular datasets analysis. Here, we show that Restricted Boltzmann Machines (RBMs), a two-layer neural network architecture, can successfully be trained on sequence ensembles from SELEX experiments for thrombin aptamers, and used to estimate the fitness of the sequences obtained through the experimental protocol. As a direct consequence, we show that trained RBMs can be exploited to classify as well as generate novel molecules. To confirm our findings, we experimentally verify the generated sequences from RBM.

sentences: Optical measurement of voltage sensing by endogenous ion channel A primary goal of molecular physiology is to understand how conformational changes of proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging method for measuring the conformational changes of the voltage sensors of endogenous ion channel proteins within live tissue, without genetic modification. We synthesized GxTX-594, a variant of the peptidyl tarantula toxin guangxitoxin-1E, conjugated to a fluorophore optimal for two-photon excitation imaging through light-scattering tissue. GxTX-594 targets the voltage sensors of Kv2 proteins, which form potassium channels and plasma membrane-endoplasmic reticulum junctions. GxTX-594 dynamically labels Kv2 proteins on cell surfaces in response to voltage stimulation. To interpret dynamic changes in labeling intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to labeling intensity. We used two-photon excitation imaging of rat brain slices to image Kv2 proteins in neurons. This imaging method enabled identification of conformational changes of endogenous Kv2 voltage sensors in tissue.

sentences: Mutualists construct the ecological conditions that trigger the transition from parasitism The evolution of mutualism between hosts and initially parasitic symbionts represents a major transition in evolution. Although vertical transmission of symbionts during host reproduction and partner control both favour the stability of mutualism, these mechanisms require specifically evolved features that may be absent in the first place. Therefore, the first steps of the transition from parasitism to mutualism may suffer from the cost of mutualism at the organismic level. We hypothesize that spatial structure can lead to the formation of higher selection levels favouring mutualism. This resembles the evolution of altruism, with the additional requirement that the offspring of mutualistic hosts and symbionts must co-occur often enough. Using a spatially explicit agentbased model we demonstrate that, starting from a parasitic system with global dispersal, the joint evolution of mutualistic effort and local dispersal of hosts and symbionts leads to a stable coexistence between parasites and mutualists. The evolution of local dispersal mimics vertical transmission and triggers the formation of mutualistic clusters, counteracting the organismic selection level of parasites that maintain global dispersal. The transition occurs when mutualistic symbionts increase the density of hosts, which strengthens competition between hosts and disfavours hosts inhabiting areas dominated by parasitic symbionts: mutualists construct the ecological conditions that allow their own spread. Therefore, the transition to mutualism may come from an eco-evolutionary feedback loop involving spatially structured population dynamics.

sentences: The cell growth cycle in budding yeast: testing hypotheses. Metabolic oscillation percolate throughout cellular physiology. The transcriptome oscillates between gene cohorts encoding for biosynthesis and growth, and for catabolism and stress response. However, long protein half-lives dampen effects on protein abundance, and the function of periodic transcription remains elusive. To test three prevailing hypotheses, we performed RNA-seq analysis during a dynamic state of the system, just preceding a bifurcation of system dynamics to a longer period. A few enzymes with short protein half-live and high transcript abundance amplitude could play a direct role in metabolic dynamics (hypothesis H1), e.g., carbonic anhydrase, sulfate uptake and the glyoxylate cycle. Substantial changes in the relative duration of expression of the antagonistic cohorts precede the bifurcation, supporting the idea of a function in cellular resource allocation (H2). The pulse-width modulation model, a mathematical formulation of this idea, explains the dependence of the cycle period on the culture growth rate. This pulse-like model of cell growth provides a first theoretical framework, where the phenomenon is understood as a mechanism of cellular resource allocation and protein homeostasis. The universal temporal program of transcription encodes the spatial structure of the cell, and we further suggest a function in pattern formation of the growing cell (H3).

sentences: Progression and Resolution of SARS-1 CoV-2 Infection in Golden Syrian Hamsters To catalyze SARS-CoV-2 research including development of novel interventive and preventive strategies, we characterized progression of disease in depth in a robust COVID-19 animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at day 2, 4, 7, 14, and 28 days post-inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal timepoints, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 post-inoculation, corresponding with widespread necrosis and inflammation. At day 7, when infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 post-inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

sentences: A model for the intrinsic limit of cancer therapy: duality of treatment-induced cell death and treatment-induced stemness AO_SCPLOWBSTRACTC_SCPLOWIntratumor cellular heterogeneity and non-genetic cell plasticity in tumors pose a recently recognized challenge to cancer treatment. Because of the dispersion of initial cell states within a clonal tumor cell population, a perturbation imparted by a cytocidal drug only kills a fraction of cells. Due to dynamic instability of cellular states the cells not killed are pushed by the treatment into a variety of functional states, including a "stem-like state" that confers resistance to treatment and regenerative capacity. This immanent stress-induced stemness competes against cell death in response to the same perturbation and may explain the near-inevitable recurrence after any treatment. This double-edged-sword mechanism of treatment complements the selection of preexisting resistant cells in explaining post-treatment progression. Unlike selection, the induction of a resistant state has not been systematically analyzed as an immanent cause of relapse. Here, we present a generic elementary model and analytical examination of this intrinsic limitation to therapy. We show how the relative proclivity towards cell death versus transition into a stem-like state, as a function of drug dose, establishes either a window of opportunity for containing tumors or the inevitability of progression following therapy. The model considers measurable cell behaviors independent of specific molecular pathways and provides a new theoretical framework for optimizing therapy dosing and scheduling as cancer treatment paradigms move from "maximal tolerated dose," which may promote therapy induced-stemness, to repeated "minimally effective doses" (as in adaptive therapies), which contain the tumor and avoid therapy-induced progression.

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Antagonistic relationship of NuA4 with the Non-Homologous End-Joining machinery at DNA damage sites. The NuA4 histone acetyltransferase complex, apart from its known role in gene regulation, has also been directly implicated in the repair of DNA double-strand breaks (DSBs), favoring homologous recombination (HR) in S/G2 during the cell cycle. Here, we investigate the antagonistic relationship of NuA4 with non-homologous end joining (NHEJ) factors. We show that budding yeast Rad9, the 53BP1 ortholog, can inhibit NuA4 acetyltransferase activity when bound to chromatin in vitro. While we previously reported that NuA4 is recruited at DSBs during the S/G2 phase, we can also detect its recruitment in G1 when genes for NHEJ factors Rad9, Yku80 and Nej1 are mutated. This is accompanied with the binding of single-strand DNA binding protein RPA and Rad52, indicating DNA end resection in G1 as well as recruitment of the HR machinery. This NuA4 recruitment to DSBs in G1 depends on both Xrs2 and Lcd1/Ddc2 and is linked to the hyper-resection phenotype of NHEJ mutants. It also implicates NuA4 in the resection-based single-strand annealing (SSA) repair pathway along Rad52. Interestingly, we identified two novel non-histone acetylation targets of NuA4, Nej1 and Yku80. Acetyl-mimicking mutant of Nej1 inhibits repair of DNA breaks by NHEJ, decreases its interaction with other core NHEJ factors such as Yku80 and Lif1 and favors end resection. Altogether, these results establish a strong reciprocal antagonistic regulatory function of NuA4 and NHEJ factors in repair pathway choice and suggests a role of NuA4 in alternative repair mechanism that involves DNA-end resection in G1.

Author SummaryDNA double-strand breaks (DSBs) are one of the most harmful form of DNA damage. Cells employ two major repair pathways to resolve DSBs: Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ). Here we wanted to dissect further the role played by the NuA4 (Nucleosome acetyltransferase of histone H4) complex in the repair of DSBs. Budding yeast NuA4 complex, like its mammalian homolog TIP60 complex, has been shown to favor repair by HR. Here, we show that indeed budding yeast NuA4 and components of the NHEJ repair pathway share an antagonistic relationship. Deletion of NHEJ components enables increased recruitment of NuA4 in the vicinity of DSBs, possible through two independent mechanisms, where NuA4 favors the end resection process which implicates it in repair by single-strand annealing (SSA), an alternate homology-based repair pathway. Additionally, we also present two NHEJ core components as new targets of NuA4 acetyltransferase activity and suggest that these acetylation events can disassemble the NHEJ repair complex from DSBs, favoring repair by HR. Our study demonstrates the importance of NuA4 in the modulation of DSB repair pathway choice.

sentences: Linking Parasitism to Network Centrality and the Impact of Sampling Bias in its Interpretation Group living is beneficial for individuals, but also comes with costs. One such cost is the increased possibility of pathogen transmission because increased numbers or frequencies of social contacts are often associated with increased parasite abundance or diversity. The social structure of a group or population is paramount to patterns of infection and transmission. Yet, for various reasons, studies investigating the links between sociality and parasitism in animals, especially in primates, have only accounted for parts of the group (e.g., only adults), which is likely to impact the interpretation of results. Here, we investigated the relationship between social network centrality and an estimate of gastrointestinal helminth infection intensity in a whole group of Japanese macaques (Macaca fuscata). We then tested the impact of omitting parts of the group on this relationship. We aimed to test: (1) whether social network centrality - in terms of the number of partners (degree), frequency of interactions (strength), and level of social integration (eigenvector) - was linked to parasite infection intensity (estimated by eggs per gram of faeces, EPG); and, (2) to what extent excluding portions of individuals within the group might influence the observed relationship. We conducted social network analysis on data collected from one group of Japanese macaques over three months on Koshima Island, Japan. We then ran a series of knock-out simulations. General linear mixed models showed that, at the whole-group level, network centrality was positively associated with geohelminth infection intensity. However, in partial networks with only adult females, only juveniles, or random subsets of the group, the strength of this relationship - albeit still generally positive - lost statistical significance. Furthermore, knock-out simulations where individuals were removed but network metrics were retained from the original whole-group network, showed that this is partly a power issue and partly a real effect of incomplete network. Our study indicates that sampling bias can thus hamper our ability to detect real network effects involving social interaction and parasitism. In addition to supporting earlier results linking geohelminth infection to Japanese macaque social networks, this work introduces important methodological considerations for research into the dynamics of social transmission, with implications for infectious disease epidemiology, population management, and health interventions.

sentences: Group II Intron-Like Reverse TranscriptasesFunction in Double-Strand Break Repair by Microhomology-Mediated End Joining Bacteria encode free-standing reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. Here, we found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when transferred into Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase{theta} and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ) in vitro and in vivo. We also found that a group II intron RT can function similarly to G2L4 RT in DNA repair, with reciprocal substitutions at the active site showing an I residue favors MMEJ and an A residue favors primer extension in both enzymes. The DNA repair functions of these enzymes utilize conserved structural features of non-LTR-retroelement RTs, including human LINE-1 and other eukaryotic non-LTR-retrotransposon RTs, suggesting such enzymes may have an inherent ability to function in DSBR in a wide range of organisms.

sentences: Long term analysis of social structure: evidence of age-based consistent associations in male Alpine ibex Despite its recognized importance for understanding the evolution of animal sociality as well as for conservation, long term analysis of social networks of animal populations is still relatively uncommon. We investigated social network dynamics in males of a gregarious mountain ungulate (Alpine ibex, Capra ibex) over ten years focusing on groups, sub-groups and individuals, exploring the dynamics of sociality over different scales. Despite the social structure changing between seasons, the Alpine ibex population was highly cohesive: fission-fusion dynamics lead almost every male in the population to associate with each other male at least once. Nevertheless, we found that male Alpine ibex showed preferential associations that were maintained across seasons and years. Age seemed to be the most important factor driving preferential associations while other characteristics, such as social status, appeared less crucial. We also found that centrality measures were influenced by age and were also related to individual physical condition. The multi-scale and long-term frame of our study helped us show that ecological constrains, such as resource availability, may play a role in shaping associations in a gregarious species, but they cannot solely explain sociality and preferential association that are likely also to be driven by life-history linked physiological and social needs. Our results highlight the importance of long-term studies based on individually recognizable subjects to help us build on our understanding of the evolution of animal sociality.

sentences: Towards a causal role of Broca's area in language: A TMS-EEG study on syntactic prediction Categorical predictions have been proposed as the key mechanism supporting the fast pace of syntactic composition in human language. Accordingly, grammar-based expectations facilitate the analysis of incoming syntactic information--e.g., hearing the determiner "the" enhances the prediction of a noun--which is then checked against a single or few other word categories. Previous functional neuroimaging studies point towards Brocas area in the left inferior frontal gyrus (IFG) as one fundamental cortical region involved in categorical prediction during on-line language processing. Causal evidence for this hypothesis is however still missing. In this study, we combined Electroencephalography (EEG) and Transcranial Magnetic Stimulation (TMS) to test whether Brocas area is functionally relevant in predictive mechanisms for language. Specifically, we transiently perturbed Brocas area during the categorical prediction phase in two-word constructions, while simultaneously measuring the Event-Related Potential (ERP) correlates of syntactic composition. We reasoned that if Brocas area is involved in predictive mechanisms for syntax, disruptive TMS during the processing of the first word (determiner/pronoun) would mitigate the difference in ERP responses for predicted and unpredicted categories when composing basic phrases and sentences. Contrary to our hypothesis, perturbation of Brocas area at the predictive stage did not affect the ERP correlates of basic composition. The correlation strength between the electrical field induced by TMS and the magnitude of the EEG response on the scalp further confirmed this pattern. We discuss the present results in light of an alternative account of the role of Brocas area in syntactic composition, namely the bottom-up integration of words into constituents.

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Hierachical Resampling for Bagging in Multi-Study Prediction with Applications to Human Neurochemical Sensing We propose the "study strap ensemble," which combines advantages of two common approaches to fitting prediction models when multiple training datasets ("studies") are available: pooling studies and fitting one model versus averaging predictions from multiple models each fit to individual studies. The study strap ensemble fits models to bootstrapped datasets, or "pseudo-studies." These are generated by resampling from multiple studies with a hierarchical resampling scheme that generalizes the randomized cluster bootstrap. The study strap is controlled by a tuning parameter that determines the proportion of observations to draw from each study. When the parameter is set to its lowest value, each pseudo-study is resampled from only a single study. When it is high, the study strap ignores the multi-study structure and generates pseudo-studies by merging the datasets and drawing observations like a standard bootstrap. We empirically show the optimal tuning value often lies in between, and prove that special cases of the study strap draw the merged dataset and the set of original studies as pseudo-studies. We extend the study strap approach with an ensemble weighting scheme that utilizes information in the distribution of the covariates of the test dataset.

Our work is motivated by neuroscience experiments using real-time neurochemical sensing during awake behavior in humans. Current techniques to perform this kind of research require measurements from an electrode placed in the brain during awake neurosurgery and rely on prediction models to estimate neurotransmitter concentrations from the electrical measurements recorded by the electrode. These models are trained by combining multiple datasets that are collected in vitro under heterogeneous conditions in order to promote accuracy of the models when applied to data collected in the brain. A prevailing challenge is deciding how to combine studies or ensemble models trained on different studies to enhance model generalizability.

Our methods produce marked improvements in simulations and in this application. All methods are available in the studyStrap CRAN package.

sentences: Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 M, 1 hour) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 {micro}M) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.

sentences: Determination of the Optical Properties of Thylakoid Membranes from Whole Leaf Reflectance Measurements Using a Capacitor and Unbound Electron Model This paper demonstrates that a capacitor equivalent along with unbound electrons can be used to model thylakoid membranes in grana stacks. From whole leaf reflectance measurements at normal incidences at 660nm wavelength and taken from the literature (Hu et al. 2020), refractive indices are obtained from the Fresnels equation for transverse electric (TE) and transverse magnetic (TM) polarization. The TE and TM polarizations for external reflectance depict the Brewster angle at which the magnitude of the reflected electric vector is zero; the internal reflections show that theres a narrow angle window of about 10 degrees before the internally refracted light goes into critical angle. The clustering and separation of reflection measurements with angle of incidence is explained using Fresnels equation; the cross-over angle beyond the Brewsters angle for internal reflection at which reflectance measurements separate with incidence angle is given by [Formula]. The predicted relaxation times from a capacitor and unbound electron model gave favorable comparisons against commonly reported fluorescence times in the 0.1 to 1 ns range (our results gave 0.5-0.8 ns). The di-electric constant for the membrane is estimated to be 5. The stacking number (number of grana layers) is consistent with the light penetration depth (skin depth). The magnetic permeability was shown to be close to that of vacuum and therefore the thylakoid lacks any magnetic properties as would be expected for such a transparent media. An in-vivo estimate based on thermal equilibrium of molecules for the permanent dipole moment of the chlorophyll molecule gave 2,025D (Debye).

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Evolutionary divergence of the Wsp signal transduction system in β- and γ-proteobacteria Bacteria rapidly adapt to their environment by integrating external stimuli through diverse signal transduction systems. Pseudomonas aeruginosa, for example, senses surface-contact through the Wsp signal transduction system to trigger the production of cyclic di-GMP. Diverse mutations in wsp genes that manifest enhanced biofilm formation are frequently reported in clinical isolates of P. aeruginosa, and in biofilm studies of Pseudomonas spp. and Burkholderia cenocepacia. In contrast to the convergent phenotypes associated with comparable wsp mutations, we demonstrate that the Wsp system in B. cenocepacia does not impact intracellular cyclic di-GMP levels unlike that in Pseudomonas spp. Our current mechanistic understanding of the Wsp system is entirely based on the study of four Pseudomonas spp. and its phylogenetic distribution remains unknown. Here, we present the first broad phylogenetic analysis to date to show that the Wsp system originated in the {beta}-proteobacteria then horizontally transferred to Pseudomonas spp., the sole member of the {gamma}-proteobacteria. Alignment of 794 independent Wsp systems with reported mutations from the literature identified key amino acid residues that fall within and outside annotated functional domains. Specific residues that are highly conserved but uniquely modified in B. cenocepacia likely define mechanistic differences among Wsp systems. We also find the greatest sequence variation in the extracellular sensory domain of WspA, indicating potential adaptations to diverse external stimuli beyond surface-contact sensing. This study emphasizes the need to better understand the breadth of functional diversity of the Wsp system as a major regulator of bacterial adaptation beyond B. cenocepacia and select Pseudomonas spp.

ImportanceThe Wsp signal transduction system serves as an important model system for studying how bacteria adapt to living in densely structured communities known as biofilms. Biofilms frequently cause chronic infections and environmental fouling, and they are very difficult to eradicate. In Pseudomonas aeruginosa, the Wsp system senses contact with a surface, which in turn activates specific genes that promote biofilm formation. We demonstrate that the Wsp system in Burkholderia cenocepacia regulates biofilm formation uniquely from that in Pseudomonas species. Furthermore, a broad phylogenetic analysis reveals the presence of the Wsp system in diverse bacterial species, and sequence analyses of 794 independent systems suggest that the core signaling components function similarly but with key differences that may alter what or how they sense. This study shows that Wsp systems are highly conserved and more broadly distributed than previously thought, and their unique differences likely reflect adaptations to distinct environments.

sentences: A Deep Learning Proteomic Scale Approach for Drug Design Computational approaches have accelerated novel therapeutic discovery in recent decades. The Computational Analysis of Novel Drug Opportunities (CANDO) platform for shotgun multi-target therapeutic discovery, repurposing, and design aims to improve their efficacy and safety by employing a holistic approach by computing interaction signatures between every drug/compound and a large library of non-redundant protein structures corresponding to the human proteome fold space. These signatures are compared and analyzed to determine if a given drug/compound is efficacious and safe for a given indication/disease. In this study, we used a deep learning based autoencoder to first reduce the dimensionality of CANDO computed drug-proteome interaction signatures. We then employed a reduced conditional variational autoencoder to generate novel drug-like compounds when given a target encoded "objective" signature. Using this model, we designed compounds to recreate the interaction signatures for twenty approved and experimental drugs and showed that 16/20 designed compounds are predicted to be significantly (p-value[≤] .05) more behaviorally similar relative to all corresponding controls, and 20/20 are predicted to be more behaviorally similar relative to a random control. We further observed that redesigns of objectives developed via rational drug design perform significantly better than those derived from natural sources (p-value[≤] .05), suggesting that the model has learned an abstraction of rational drug design. We also show that designed compounds are structurally diverse and synthetically feasible when compared to their respective objective drugs despite consistently high predicted behavioral similarity. Finally, we generated new designs that enhance thirteen drugs/compounds associated with non-small cell lung cancer and anti-aging properties using their predicted proteomic interaction signatures. This work represents a significant step forward in automating holistic therapeutic design with machine learning, and subsequently offers a reduction in the time needed to generate novel, effective, and safe drug leads for any indication.

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Theoretical model of efficient phagocytosis driven by curved membrane proteins and active cytoskeleton forces Phagocytosis is the process of engulfment and internalization of comparatively large particles by the cell, that plays a central role in the functioning of our immune system. We study the process of phagocytosis by considering a simplified coarse grained model of a three-dimensional vesicle, having uniform adhesion interaction with a rigid particle, in the presence of curved membrane proteins and active cytoskeletal forces. Complete engulfment is achieved when the bending energy cost of the vesicle is balanced by the gain in the adhesion energy. The presence of curved (convex) proteins reduces the bending energy cost by self-organizing with higher density at the highly curved leading edge of the engulfing membrane, which forms the circular rim of the phagocytic cup that wraps around the particle. This allows the engulfment to occur at much smaller adhesion strength. When the curved proteins exert outwards protrusive forces, representing actin polymerization, at the leading edge, we find that engulfment is achieved more quickly and at lower protein density. We consider spherical as well as non-spherical particles, and find that non-spherical particles are more difficult to engulf in comparison to the spherical particles of the same surface area. For non-spherical particles, the engulfment time crucially depends upon the initial orientation of the particles with respect to the vesicle. Our model offers a mechanism for the spontaneous self-organization of the actin cytoskeleton at the phagocytic cup, in good agreement with recent high-resolution experimental observations.

Significance StatementPhagocytosis is an important process that plays a central role in our immune system, and also during nutrient uptake, drug delivery and pathogen invasion. Despite its biological importance, it is still not understood how the actin cytoskeleton is coordinated spatio-temporally during the phagocytosis process. We present a simple model that explains the dynamics and self-organization of the membrane and the actin cytoskeleton during the engulfment process, based on curved membrane activators of actin polymerization.

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Determining salmon provenance with automated otolith reading Synthetic otolith marks are used at hundreds of hatcheries throughout the Pacific Rim to record the release location of salmon. Each year, human readers examine tens of thousands of otolith samples to identify the marks in salmon that are caught. The data inform dynamic management practices that maximize allowable catch while preserving populations, and guide hatchery investments. However, the method is limited by the time required to process otoliths, the inability to distinguish between wild and un-marked hatchery fish, and in some cases classification processes are limited by the subjective decisions of human readers. Automated otolith reading using computer vision has the potential to improve on all three of these limitations. Our work advances the field of automated otolith reading through a novel otolith classification algorithm that uses two neural networks trained with an adversarial algorithm to achieve 93% classification accuracy between four hatchery marks and unmarked otoliths. The algorithm relies on hemisection images of the otolith exclusively: no additional biological data are needed. Our work demonstrates a novel technique with modest training requirements that achieves unprecedented accuracy. The method can be easily adopted in existing otolith labs, scaled to accommodate additional marks, and does not require tracking additional information about the fish that the otolith was retrieved from.

Author summaryMany fish harvested in commercial fisheries have a bone-like structure in their inner ear called an otolith. Otoliths are useful because as they grow changes in the fishs environment create unique patterns that can be interpreted years later--similar to the way tree rings can be used to determine a trees age and rate of growth in different seasons. Hatcheries use this phenomenon to create unique patterns in the otoliths of fish they release that identify their origin. Trained otolith "readers" examine tens of thousands of otolith samples as part of commercial fishery management every year. The data help resource managers protect returns to wild streams and help hatcheries track the survival rates of their releases. However, human reading is time consuming, limited in the types of patterns that can be identified and can be subjective. Applying modern machine learning techniques to otolith reading has the potential to improve on all of these challenges. We developed a computational method that identifies hatchery marks in digital images of salmon otoliths without human input. In a test with otoliths containing one of four hatchery marks and unmarked otoliths, the method accurately classified 93% of otoliths. The method can be adopted in otolith labs and can be further improved by expanding the sample size used to train and test the method.

sentences: Evolutionary selection on synonymous codons in RNA G-quadruplex structural region In addition to the amino acid sequence information, synonymous codons can encode multiple regulatory and structural signals in protein coding region. In this study, we investigated how synonymous codons have been adapted to the formation of RNA G-quadruplex (rG4) structure. We found a universal selective pressure acting on synonymous codons to facilitate rG4 formation in five eukaryotic organisms. While G-rich codons are preferred in rG4 structural region, C-rich codons are selectively unpreferred for rG4 structures. Genes codon usage bias, nucleotide composition and evolutionary rate can account for the selective variations on synonymous codons among rG4 structures within a species. Moreover, rG4 structures in translational initiation region showed significantly higher selective pressures than those in translational elongation region. These results bring us another dimension of evolutionary selection on synonymous codons for proper RNA structure and function.

sentences: helixCAM: A Platform for Programmable Cellular Assembly in Bacteria and Human Cells Interactions between cells are indispensable for creating structure and signaling. The ability to direct precise cell-cell interactions would be powerful for engineering tissues, understanding signaling pathways, and directing immune cell targeting. In humans, intercellular interactions are mediated by cell adhesion molecules (CAMs). However, CAMs are natively expressed by many cells and have cross-reactivity, making them unsuitable for programming specific interactions. Here, we showcase "helixCAM," a platform for engineering novel CAMs by presenting coiled-coil peptides on the cell surface. helixCAMs were able to create specific cell-cell interactions and direct patterned aggregate formation in bacteria and human cells. We built rationally designed helixCAM libraries and discovered novel high-performance helixCAM pairs. High-affinity helixCAMs were then used for multicellular engineering applications, such as spherical layering, adherent cell targeting, and surface patterning. The helixCAMs are an expandable platform for directing complex multicellular assemblies, and we foresee its utility in tissue engineering, immunology, and developmental biology.

sentences: Large positive ecological changes of small urban greening actions The detrimental effects of human-induced environmental change on people and other species are acutely manifested in urban environments. While urban greenspaces are known to mitigate these effects and support functionally diverse ecological communities, evidence of the ecological outcomes of urban greening remains scarce. We use a longitudinal observational design to provide empirical evidence of the putative ecological benefits of greening actions. We show how a small greening action quickly led to large positive changes in the richness, demographic dynamics, and network structure of a depauperate insect community. An increase in the diversity and complexity of the plant community led to, after only three years, a large increase in insect species richness, a greater probability of occurrence of insects within the greenspace, and a higher number and diversity of interactions between insects and plant species. We demonstrate how large ecological benefits may be derived from investing in small greening actions and how these contribute to bring indigenous species back to greenspaces where they have become rare or locally extinct. Our findings provide crucial evidence that support best practice in greenspace design and contribute to re-invigorate policies aimed at mitigating the negative impacts of urbanisation on people and other species.

sentences: Implications for food safety of the size and location of fragments of lead shotgun pellets embedded in hunted carcasses of small game animals intended for human consumption Carcasses of common pheasants (Phasianus colchicus) killed by hunters using shotguns are widely used or sold in the United Kingdom and elsewhere for human consumption. Almost all of the birds are shot using shotgun pellets composed principally of lead (Pb). Lead shotgun pellets often fragment on impact within the bodies of gamebirds, leaving small lead particles in the meat that are difficult for consumers to detect and remove and from which a greater proportion of lead is likely to be absorbed. Chronic exposure to even low levels of lead is associated with negative health effects in humans and especially in groups particularly vulnerable to the effects of lead, which include young children and pregnant women. Our study used a high-resolution computerised tomography X-ray scanner to locate, in three dimensions, metal fragments embedded within carcasses of eight pheasants sold for human consumption in the UK. Small radio-dense fragments (<2 mm diameter), assumed to be metallic lead, were present in all of the pheasant carcasses examined (mean number: 39 per carcass) and many were too small (<0.1 mm diameter) and too distant from the nearest large shotgun pellet for it to be practical for consumers to detect and remove them without discarding a large proportion of otherwise usable meat. Consumers of carcasses of pheasants killed using lead shotgun ammunition are likely to be exposed to elevated levels of dietary lead, even if careful food preparation is practiced to remove shotgun pellets and the most damaged tissue.

sentences: SURFMAP: a software for mapping in two dimensions protein surface features Molecular cartography using two-dimensional (2D) representation of protein surfaces has been shown to be very promising for protein surface analysis. Here, we present SURFMAP, a free standalone and easy-to-use software that enables the fast and automated 2D projection of either predefined features of protein surface (i.e., electrostatic potential, Kyte-Doolittle hydrophobicity, stickiness, and surface relief) or any descriptor encoded in the temperature factor column of a PDB file. SURFMAP uses a pseudo-cylindrical sinusoidal "equal-area" projection that has the advantage of preserving the area measures. It provides the user with (i) 2D maps that enable the easy and visual analysis of protein surface features of interest and (ii) maps in a text file format allowing the fast and straightforward quantitative comparison of 2D maps of homologous proteins.

sentences: Genome Size Estimation and Full-length Transcriptome of Sphingonotus tsinlingensis: Genetic Background for the Drought-Adapted Grasshopper Sphingonotus Fieber, 1852 (Orthoptera: Acrididae) is a species-rich grasshopper genus with ~146 species. All species of this genus prefer dry environments, such as: desert, steppe, sand, and stony benchland. This genomic study aimed to understand the evolution and ecology of these grasshopper species. Here, the genome size of Sphingonotus tsinlingensis was estimated using flow cytometry and the first high-quality full-length transcriptome of this species is presented, which may serve as a reference genetic resource for the drought-adapted grasshopper species of Sphingonotus Fieber. The genome size of Sphingonotus tsinlingensis was ~12.8 Gb. Based on the 146.98 Gb Pacbio isoform sequencing data, 221.47 Mb full-length transcripts were assembled. Among these transcripts, 88,693 non-redundant isoforms were identified with an average length of 2,497 bp and an N50 value of 2,726 bp, which was much longer than the formal grasshopper transcriptome assemblies. A total of 48,502 protein coding sequences were determined, and 37,569 were annotated in public gene function databases. A total of 36,488 simple tandem repeats, 12,765 long non-coding RNAs, and 414 transcription factors were also identified. According to gene functions, 70 heat shock proteins and 61 P450 genes that may correspond to drought adaptation of S. tsinlingensis were identified. The genome of Sphingonotus tsinlingensis is an ultra-large and complex genome. Full-length transcriptome sequencing is an ideal strategy for genomic research. This is the first full-length transcriptome of the genus Sphingonotus. The assembly parameters were better than all known grasshopper transcriptomes. This full-length transcriptome may be used to understand its genetic background and the evolution and ecology of grasshoppers.

sentences: Bifurcating Neurons In The Anterior Thalamic Nuclei The anterior thalamic nuclei (ATN) form a nodal point within a distributed memory network. The conventional view of ATN function describes segregated efferents to different terminal regions. By contrast, we found that bifurcating neurons are common within the anteromedial nucleus (AM) of the ATN. A substantial proportion of AM neurons ([~]36% within a region of interest) showed collateral projections when one of two retrograde neurotracers, Cholera Toxin Subunit B (CTB) conjugated to either Alexa Fluor(R) 488 or 594, was placed in the medial prefrontal cortex (mPFC) or dorsal subiculum (dSub). A marked degree of collateralization ([~]20% AM neurons) was also found when neurotracers were placed in mPFC and caudal retrosplenial cortex (cRSC); about 10% showed collaterals when the cRSC was paired with either dSub or vHF; the fewest (6%) was found for mPFC paired with the ventral hippocampal formation (vHF). A generally similar range of percentages of bifurcating neurons was found in the adjacent nucleus reuniens (Re). Evidence that AM neurons project simultaneously to many distantly-located structures provides a new perspective on ATN function. These neurons would facilitate direct coordination among key neural structures to support memory and may explain the strong association between the ATN and diencephalic amnesia.

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Chronic Aryl Hydrocarbon Receptor Activity Phenocopies Smoking-induced Skeletal Muscle Impairment BackgroundCOPD patients exhibit skeletal muscle atrophy, denervation, and reduced mitochondrial oxidative capacity. Whilst chronic tobacco smoke exposure is implicated in COPD muscle impairment, the mechanisms involved are ambiguous. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that activates detoxifying pathways with numerous exogenous ligands, including tobacco smoke. Whereas transient AHR activation is adaptive, chronic activation can be toxic. On this basis, we tested the hypothesis that chronic smoke-induced AHR activation causes adverse muscle impact.

MethodsWe used clinical patient muscle samples, and in vitro (C2C12 myotubes) and in vivo models (mouse), to perform gene expression, mitochondrial function, muscle and neuromuscular junction morphology, and genetic manipulations (adeno-associated virus-mediated gene transfer).

Results16 weeks tobacco smoke exposure in mice caused: muscle atrophy, neuromuscular junction degeneration, and reduced oxidative capacity. Similarly, smoke exposure reprogrammed the muscle transcriptome, with down-regulation of mitochondrial and neuromuscular junction genes. In mouse and human patient specimens, smoke exposure increased muscle AHR signaling. Mechanistically, experiments in cultured myotubes demonstrated that smoke condensate activated the AHR, caused mitochondrial impairments, and induced an AHR-dependent myotube atrophy. Finally, to isolate the role of AHR activity, expression of a constitutively active AHR mutant without smoke exposure caused atrophy and mitochondrial impairments in cultured myotubes, and muscle atrophy and neuromuscular junction degeneration in mice.

ConclusionsThese results establish that chronic AHR activity, as occurs in smokers, phenocopies the atrophy, mitochondrial impairment and neuromuscular junction degeneration caused by chronic tobacco smoke exposure.

sentences: Identification of a Golgi-localized altORF peptide reveals a minimal Golgi targeting motif Prior work has identified signal sequences and motifs that are necessary and sufficient to target proteins to specific subcellular regions and organelles such as the plasma membrane, nucleus, Endoplasmic Reticulum, and mitochondria. In contrast, minimal sequence motifs that are sufficient for Golgi localization remain largely elusive. In this work, we identified a 37 amino acid alternative open reading frame (altORF) within the mRNA of the centromere protein, CENP-R. This altORF peptide localizes specifically to the cytoplasmic surface of the Golgi apparatus. Through mutational analysis, we identify a minimal 10 amino acid sequence and a critical cysteine residue that are necessary and sufficient for Golgi localization. Pharmacological perturbations suggest that this peptide is modified through palmitoylation to promote its localization. Together, our work defines a minimal sequence that is sufficient for Golgi targeting and provide a valuable Golgi marker for live cell imaging.

sentences: Mobile antimicrobial resistance genes in probiotics Even though people around the world tend to consume probiotic products for their beneficial health effects on a daily basis, recently, concerns were outlined regarding the uptake and potential intestinal colonisation of the bacteria that they transfer. These bacteria are capable of executing horizontal gene transfer (HGT) which facilitates the movement of various genes, including antimicrobial resistance genes (ARGs), among the donor and recipient bacterial populations. Within our study, 47 shotgun sequencing datasets deriving from various probiotic samples (isolated strains and metagenomes) were bioinformatically analysed. We detected more than 70 ARGs, out of which rpoB mutants conferring resistance to rifampicin, tet(W/N/W) and potentially extended-spectrum beta-lactamase (ESBL) coding TEM-116 were the most common. Numerous ARGs were associated with integrated mobile genetic elements, plasmids or phages promoting the HGT. Our findings raise clinical and public health concerns as the consumption of probiotic products may lead to the transfer of ARGs to human gut bacteria.

sentences: Privacy Preserving RNA-Model Validation Across Laboratories Reproducibility of results obtained using RNA data across labs remains a major hurdle in cancer research. Often, molecular predictors trained on one dataset cannot be applied to another due to differences in RNA library preparation and quantification. While current RNA correction algorithms may overcome these differences, they require access to all patient-level data, which necessitates the sharing of training data for predictors when sharing predictors. Here, we describe SpinAdapt, an unsupervised RNA correction algorithm that enables the transfer of molecular models without requiring access to patient-level data. It computes data corrections only via aggregate statistics of each dataset, thereby maintaining patient data privacy. Furthermore, SpinAdapt can correct new samples, thereby enabling evaluation of validation cohorts. Despite an inherent tradeoff between privacy and performance, SpinAdapt outperforms current correction methods that require patient-level data access. We expect this novel correction paradigm to enhance research reproducibility and patient privacy. Finally, SpinAdapt lays a mathematical framework that can be extended to other -omics modalities.

sentences: Bacterial hopanoids: a newly identified potent inducer of monocyte to macrophage differentiation Monocyte to macrophage differentiation is an extremely essential cellular and immunological process aimed to combat the assault of foreign pathogens as well as this process has immense importance in the maintenance of physiological homeostasis. Differentiated macrophage, serving as specialized phagocytes is an indispensable component of innate immunity. Besides this, being a well-documented antigen-presenting cell (APC) macrophages also function as a key regulator of both humoral and cell-mediated immunity: the two integral components of adaptive immunity. Bacterial hopanoids are a primitive analogous of eukaryotic sterols that are established as a prominent immunomodulator. In this study, we have demonstrated the isolation protocol of neutral lipid fractions from different Gram-negative bacteria, and adopting several analytical approaches we proposed that the extracted lipid fractions may contain hopanoid as an active component. We have delved deeper into the biological effect of these probable hopanoids like compounds. Here, by considering the several structural and functional attributes like altered phenotype, expression of macrophage-specific markers, increased intracellular organelles, acquirement of enhanced phagocytic and inflammatory property, induction of autophagy, etc we have established hopanoids as a potential inducer of monocyte to macrophage differentiation. Thus, our study has unraveled a new immunomodulator present in Gram-negative bacteria and would undoubtedly help to understand the intricacies of host-pathogen interaction in a better and conclusive manner.

sentences: TOR complex 2 promotes proteasome dependent Pub1 (NEDD4 family E3 ligase) turnover in fission yeast. Cells respond to changing nutrient environments by adjusting the abundance of surface nutrient transporters and receptors. This can be achieved by modulating ubiquitin-dependent endocytosis, which in part is regulated by the NEDD4 family of E3 ligases. Here we report novel regulation of Pub1, a fission yeast Schizosaccharomyces pombe member of the NEDD4-family of E3 ligases. We show that nitrogen stress inhibits Pub1 function, thereby increasing the abundance of the amino acid transporter Aat1 at the plasma membrane and enhancing sensitivity to the toxic arginine analogue canavanine. We show that TOR complex 2 (TORC2) signalling negatively regulates Pub1, thus TORC2 mutants under nutrient stress have decreased Aat1 at the plasma membrane and are resistant to canavanine. Inhibition of TORC2 signalling increases Pub1 phosphorylation, and this is dependent on Gsk3 activity. Addition of the Tor inhibitor Torin1 increases phosphorylation of Pub1 at serine 199 (S199) by 2.5-fold, and Pub1 protein levels in S199A phospho-ablated mutants are reduced. S199 is conserved in NEDD4 and is located immediately upstream of a WW domain required for protein interaction. Together, we describe how the major TORC2 nutrient-sensing signalling network regulates environmental control of Pub1 to modulate the abundance of nutrient transporters.

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Development of a Ribosome Profiling Protocol to Study Translation in the yeast Kluyveromyces marxianus Kluyveromyces marxianus is an interesting and important yeast because of particular traits like thermotolerance and rapid growth, and applications in food and industrial biotechnology. Knowing how K. marxianus responds and adapts to changing environments is important to achieve a full understanding of the its biology and to develop bioprocesses. For this, a full suite of omics tools to measure and compare global patterns of gene expression and protein synthesis is needed. Whereas transcriptome analysis by RNA-Seq quantifies mRNA abundance, ribosome profiling allows codon-resolution of translation on a genome-wide scale by deep sequencing of ribosome locations on mRNAs and is emerging as a valuable tool to study translation control of gene expression. We report here the development of a ribosome profiling method for K. marxianus and we make the procedure available as a step by step protocol. To aid in the analysis and sharing of ribosome profiling data, we also added the K. marxianus genome as well as transcriptome and ribosome profiling data to the publicly accessible GWIPS-viz and Trips-Viz browsers. Users are able to upload custom ribosome profiling and RNA-Seq data to both browsers, therefore allowing easy analysis and sharing of data. As many studies only focus on the use of RNA-Seq to study K. marxianus in different environments, the availability of ribosome profiling is a powerful addition to the K. marxianus toolbox.

Graphical abstractDevelopment of a Ribosome Profiling protocol to study gene expression in the thermotolerant yeast Kluyveromyces marxianus.

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sentences: The EGFR/ErbB inhibitor neratinib modifies the neutrophil phosphoproteome and promotes apoptosis and clearance by airway macrophages Dysregulated neutrophilic inflammation can be highly destructive in chronic inflammatory diseases due to prolonged neutrophil lifespan and continual release of histotoxic mediators in inflamed tissues. Therapeutic induction of neutrophil apoptosis, an immunologically silent form of cell death, may be beneficial in these diseases, provided that the apoptotic neutrophils are efficiently cleared from the tissue. Our previous research identified ErbB inhibitors as able to induce neutrophil apoptosis and reduce neutrophilic inflammation both in vitro and in vivo (Rahman et al., 2019). Here we extend that work using a clinical ErbB inhibitor, neratinib, which has the potential to be repurposed in inflammatory diseases. We show that neratinib reduces neutrophilic migration to an inflammatory site in zebrafish larvae. Neratinib upregulates efferocytosis and reduces the number of persisting neutrophil corpses in mouse models of acute, but not chronic, lung injury, suggesting the drug may have therapeutic benefits in acute inflammatory settings. Phosphoproteomics analysis of human neutrophils shows that neratinib modifies the phosphorylation of proteins regulating apoptosis, migration and efferocytosis. This work identifies a potential mechanism for neratinib in treating acute lung inflammation by upregulating the clearance of dead neutrophils and, through examination of the neutrophil phosphoproteome, provides important insights into the mechanisms by which this may be occurring.

sentences: HMMploidy: inference of ploidy levels from short-read sequencing data The inference of ploidy levels from genomic data is important to understand molecular mechanisms underpinning genome evolution. However, current methods based on allele frequency and sequencing depth variation do not have power to infer ploidy levels at low- and mid-depth sequencing data, as they do not account for data uncertainty. Here we introduce HMMploidy, a novel tool that leverages the information from multiple samples and combines the information from sequencing depth and genotype likelihoods. We demonstrate that HMMploidy outperforms existing methods in most tested scenarios, especially at low-depth with large sample size. We apply HMMploidy to sequencing data from the pathogenic fungus Cryptococcus neoformans and retrieve pervasive patterns of aneuploidy, even when artificially downsampling the sequencing data. We envisage that HMMploidy will have wide applicability to low-depth sequencing data from polyploid and aneuploid species.

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Fast and accurate joint inference of coancestry parameters for populations and/or individuals We introduce a fast, new algorithm for inferring jointly the FST parameters describing genetic distances among a set of populations and/or unrelated diploid individuals, and a tree representing their genetic structure, from allele count data. While the inferred tree typically reflects historical processes of splitting and divergence, its aim is to represent the actual genetic variance, with FST values specified by branch lengths. We generalise two major approaches to defining FST, via correlations and mismatch probabilities of sampled allele pairs, which measure shared and non-shared components of genetic variance. A diploid individual can be treated as a population of two gametes, which allows inference of coancestry coefficients for individuals as well as for populations, or a combination of the two. A simulation study illustrates that our fast method-of-moments estimation of FST values, simultaneously for multiple populations/individuals, gains statistical efficiency over pairwise approaches by pooling information about ancestral allele frequencies. We apply our approach to genome-wide genotypes from the 26 worldwide human populations of the 1000 Genomes Project. We first analyse at the population level, then a subset of individuals and in a final analysis we pool individuals from the more homogeneous populations. This flexible analysis approach gives many advantages over traditional approaches to population structure/coancestry, including visual and quantitative assessments of long-standing questions about the relative magnitudes of within- and between-population genetic differences.

Author summaryWe propose new ways to measure, and visualise in a tree, the genetic distances among a set of populations using allele frequency data. The two genomes within a diploid individual can be treated as a small population, which allows a flexible framework for investigating genetic variation within and between populations. Genetic structure can be accurately and efficiently represented in a tree with nodes representing either homogeneous populations or genetically diverse individuals, for example due to admixture. We first generalise the long-established measure of genetic distance, FST, to tree-structured populations and individuals, finding that two measures are required for each pair of populations, corresponding to their shared and and non-shared genetic variation. We show using a simulation study that our novel tree-based estimators are more efficient than current pairwise estimators, and we illustrate the potential for novel ways to explore and visualise genetic variation within and between populations using a worldwide human genetic dataset.

sentences: Impact of sterilization on a conjugated polymer based bioelectronic patch Translation into the clinic of organic bioelectronic devices having conjugated polymers as the active material will hinge on their long-term operation in vivo. This will require the device to be subject to clinically approved sterilization techniques without a deterioration in its physical and electronic properties. To date, there remains a gap in the literature addressing the impact of this critical pre-operative procedure on the properties of conjugated polymers. This study aims to address this gap by assessing the physical and electronic properties of a sterilized porous bioelectronic patch having polyaniline as the conjugated polymer. The patch was sterilized by autoclave, ethylene oxide and gamma ({gamma}-) irradiation at 15, 25, and 50 kGy doses. Autoclaving resulted in cracking and macroscopic degradation of the patch, while patches sterilized by {gamma}-irradiation at 50 kGy exhibited reduced mechanical and electronic properties, attributed to chain scission and non-uniform crosslinking caused by the high dose irradiation. Ethylene oxide and {gamma}-irradiation at 15 and 25 kGy sterilization appeared to be the most effective at maintaining the mechanical and electronic properties of the patch, as well as inducing a minimal immune response as revealed by a receding fibrotic capsule after 4 weeks implantation. Our findings pave the way towards closing the gap for the translation of organic bioelectronic devices from acute to long-term in vivo models.

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The quantitative genetics of the endemic prevalence of infectious diseases: Indirect Genetic Effects dominate heritable variation and response to selection Infectious diseases have profound effects on life, both in nature and agriculture. Despite the availability of well-established epidemiological theory, however, a quantitative genetic theory of the host population for the endemic prevalence of infectious diseases is almost entirely lacking. While several studies have demonstrated the relevance of the transmission dynamics of infectious diseases for heritable variation and response to selection of the host population, our current theoretical framework of quantitative genetics excludes these dynamics. As a consequence, we do not know which genetic effects of the host population determine the prevalence of an infection, and have no concepts of breeding value and heritable variation for endemic prevalence.

Here we integrate quantitative genetics and epidemiology, and propose a quantitative genetic theory for R0 and for the endemic prevalence of an infectious disease. We first identify the genetic factors that determine the prevalence of an infection, using an approach founded in epidemiological theory. Subsequently we investigate the population level consequences of individual genetic variation, both for R0 and for the endemic prevalence. Next, we present expressions for the breeding value and heritable variation, for both prevalence and individual binary disease status, and show that these parameters depend strongly on the level of the prevalence. Results show that heritable variation for endemic prevalence is substantially greater than currently believed, and increases when prevalence approaches zero, while heritability of individual disease status goes to zero. As a consequence, response of prevalence to selection accelerates considerably when prevalence goes down, in contrast to predictions from classical theory. Finally, we show that most of the heritable variation for the endemic prevalence of an infection is hidden due to indirect genetic effects, suggesting a key role for kin-group selection both in the evolutionary history of current populations and for genetic improvement strategies in animals and plants.

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Toxoplasma gondii phosphatidylserine flippase complex ATP2B-CDC50.4 critically participates in microneme exocytosis Regulated microneme secretion governs motility, host cell invasion and egress in the obligate intracellular apicomplexans. Intracellular calcium oscillations and phospholipid dynamics critically regulate micronemes exocytosis. Despite its importance for the lytic cycle of these parasites, molecular mechanistic details about exocytosis are still missing. Some members of the P4-ATPases act as flippases, changing the phospholipid distribution by translocation from the outer to the inner leaflet of the membrane. Here, the localization and function of the repertoire of P4-ATPases was investigated across the lytic cycle of Toxoplasma gondii. Of relevance, ATP2B and the non-catalytic subunit cell division control protein 50.4 (CDC50.4) form a stable heterocomplex at the parasite plasma membrane, essential for microneme exocytosis. This complex is responsible for flipping phosphatidylserine (PS), which presumably acts as a lipid mediator for the organelle fusion with the plasma membrane. DOC2.1, a previously described key egress and invasion factor, is shown here to be affected in its function in egress upon mutation on residues putatively involved in calcium binding. This study points toward the importance of PS in microneme exocytosis and unveils subtle differences in the signaling cascades leading to organelle secretion between intracellular and extracellular parasites to ensure egress and invasion, respectively.

Author SummaryBiological membranes display diverse functions, including membrane fusion, which are conferred by a defined composition and organization of proteins and lipids. Apicomplexan parasites possess specialized secretory organelles (micronemes), implicated in motility, invasion and egress from host cells. Microneme exocytosis is already known to depends on phosphatidic acid for its fusion with the plasma membrane. Here we identify a type P4-ATPase and its CDC50 chaperone (ATP2B-CDC50.4) that act as flippase and contribute to the enrichment of phosphatidylserine (PS) in the inner leaflet of the parasite plasma membrane. PS and the previously described C2-containing protein DOC2.1 differentially participate in microneme exocytosis in the context of environmental changes. Overall, our results shed light on the importance membrane homeostasis and lipid composition in controlling microneme secretion.

sentences: Loss-of-function of OTUD7A in the schizophrenia-associated 15q13.3 deletion impairs synapse development and function in human neurons Identifying causative gene(s) within disease-associated large genomic regions of copy number variants (CNVs) is challenging. Here, by targeted sequencing of genes within schizophrenia (SZ)-associated CNVs in 1,779 SZ cases and 1,418 controls, we identified three rare putative loss-of-function (LoF) mutations in OTU deubiquitinase 7A (OTUD7A) within the 15q13.3 deletion in cases, but none in controls. To tie OTUD7A LoF with any SZ-relevant cellular phenotypes, we modeled the OTUD7A LoF mutation, rs757148409, in human induced pluripotent stem cell (hiPSC)-derived induced excitatory neurons (iNs) by CRISPR/Cas9 engineering. The mutant iNs showed a [~]50% decrease in OTUD7A expression without undergoing nonsense-mediated mRNA decay. The mutant iNs also exhibited marked reduction of dendritic complexity, density of synaptic proteins GluA1 and PSD-95, and neuronal network activity. Congruent with the neuronal phenotypes in mutant iNs, our transcriptomic analysis showed that the set of OTUD7A LoF-downregulated genes was enriched for those relating to synapse development and function, and was associated with SZ and other neuropsychiatric disorders. These results suggest that OTUD7A LoF impairs synapse development and neuronal function in human neurons, providing mechanistic insight into the possible role of OTUD7A in driving neuropsychiatric phenotypes associated with the 15q13.3 deletion.

sentences: Screening thousands of transcribed coding and non-coding regions reveals sequence determinants of RNA polymerase II elongation potential Organismal growth and development rely on RNA Polymerase II (RNAPII) synthesizing the appropriate repertoire of messenger RNAs (mRNAs) from protein-coding genes. Productive elongation of full-length transcripts is essential for mRNA function, however what determines whether an engaged RNAPII molecule will terminate prematurely or transcribe processively remains poorly understood. Notably, despite a common process for transcription initiation across RNAPII-synthesized RNAs1, RNAPII is highly susceptible to termination when transcribing non-coding RNAs such as upstream antisense RNAs (uaRNAs) and enhancers RNAs (eRNAs)2, suggesting that differences arise during RNAPII elongation. To investigate the impact of transcribed sequence on elongation potential, we developed a method to screen the effects of thousands of INtegrated Sequences on Expression of RNA and Translation using high-throughput sequencing (INSERT-seq). We found that higher AT content in uaRNAs and eRNAs, rather than specific sequence motifs, underlies the propensity for RNAPII termination on these transcripts. Further, we demonstrate that 5 splice sites exert both splicing-dependent and autonomous, splicing-independent stimulation of transcription, even in the absence of polyadenylation signals. Together, our results reveal a potent role for transcribed sequence in dictating gene output at mRNA and non-coding RNA loci, and demonstrate the power of INSERT-seq towards illuminating these contributions.

sentences: Genomic comparison of eight closed genomes of multidrug resistant Salmonella enterica strains isolated from broiler farms and processing plants in Trinidad and Tobago Salmonella enterica is an important foodborne pathogen worldwide. We used long and short read sequencing to close genomes of eight multidrug resistant (MDR) Salmonella enterica strains, belonging to serovars Infantis (2), Albany, Oranienburg, I 4,[5],12:i:-, Javiana, Schwarzengrund, and Kentucky from broiler chicken farms and processing plants in Trinidad and Tobago. They also belonged to seven different sequence types (STs- 32, 292, 1510, 19, 24, and 96). Seven of the strains demonstrated multi-drug resistance with the presence of at least three AMR genes. Three isolates contained the quinolone resistance gene qnrB19 in plasmids (CFSAN103840, CFSAN103854, and CFSAN103872). The extended spectrum {beta}-lactamase genes blaCTX-M-65 (CFSAN103796) and blaTEM-1 (CFSAN103852) were detected in this study. The genomes closed in this study will be useful for future source tracking and outbreak investigations in Trinidad and Tobago and worldwide.

sentences: Reconstruction of distinct vertebrate gastrulation modes via modulation of key cell behaviours in the chick embryo The morphology of gastrulation driving the internalisation of the mesoderm and endoderm differs dramatically among vertebrate species. It ranges from involution of epithelial sheets of cells through a circular blastopore in amphibians to ingression of mesenchymal cells through a primitive streak in amniotes. By targeting signalling pathways controlling critical cell behaviours in the chick embryo, we generated crescent- and ring-shaped mesendoderm territories in which cells can or cannot ingress. These alterations subvert the formation of the chick primitive streak into the gastrulation modes seen in amphibians, reptiles and teleost fish. Our experimental manipulations are supported by a theoretical framework linking cellular behaviors to self-organized multi-cellular flows in the accompanying paper. All together, this suggests that the evolution of gastrulation movements are largely determined by the shape of and cell behaviours in the mesendoderm territory across different species, and controlled by a relatively small number of signalling pathways.

sentences: Hackflex: low cost Illumina Nextera Flex sequencing library construction We developed a low-cost method for the production of Illumina-compatible sequencing libraries that allows up to 14 times more libraries for high-throughput Illumina sequencing to be generated for the same cost. We call this new method Hackflex. Quality of library preparation was tested by constructing libraries from E. coli MG1655 genomic DNA using either Hackflex, standard Nextera Flex or a variation of standard Nextera Flex in which the bead-linked transposase is diluted prior to use. In order to test the library quality for genomes with a higher and a lower GC content, library construction methods were also tested on P. aeruginosa PAO1 and S. aureus ATCC25923, respectively. We demonstrated that Hackflex can produce high quality libraries and yields a highly uniform coverage, equivalent to the standard Nextera Flex kit. We show that strongly size selected libraries produce sufficient yield and complexity to support de novo microbial genome assembly, and that assemblies of the large insert libraries can be much more contiguous than standard libraries without strong size selection. We introduce a new set of sample barcodes that are distinct from standard Illumina barcodes, enabling Hackflex samples to be multiplexed with samples barcoded using standard Illumina kits. Using Hackflex, we were able to achieve a per sample reagent cost for library prep of A$7.22 (USD$5.60), which is 9.87 times lower than the Standard Nextera Flex protocol at advertised retail price. An additional simple modification and further simplification of the protocol by omitting the wash step enables a further price reduction to reach an overall 14-fold cost saving. This method will allow researchers to construct more libraries within a given budget, thereby yielding more data and facilitating research programs where sequencing large numbers of libraries is beneficial.

sentences: Large-scale geographic survey provides insights into the colonization history of a major aphid pest on its cultivated apple host in Europe, North America and North Africa AO_SCPLOWBSTRACTC_SCPLOWWith frequent host shifts involving the colonization of new hosts across large geographical ranges, crop pests are good models for examining the mechanisms of rapid colonization. The microbial partners of pest insects may also be involved in or affected by colonization processes, which has been little studied so far. We investigated the demographic history of the rosy apple aphid, Dysaphis plantaginea, a major pest of the cultivated apple (Malus domestica) in Europe, North Africa and North America, as well as the diversity of its microbiota. We genotyped a comprehensive sample of 714 colonies from Europe, Morocco and the US using mitochondrial (CytB and CO1), bacterial (16s rRNA and TrnpB), and 30 microsatellite markers. We detected five populations spread across the US, Morocco, Western and Eastern Europe and Spain. Populations showed weak genetic differentiation and high genetic diversity, except the ones from Morocco and North America that are likely the result of recent colonization events. Coalescent-based inferences revealed high levels of gene flow among populations during the colonization but did not allow determining the sequence of colonization of Europe, North America and Morroco by D. plantaginea, likely because of the weak genetic differentiation and the occurrence of gene flow among populations. We found that D. plantaginea rarely hosts other endosymbiotic bacteria than its obligate nutritional symbiont Buchnera aphidicola. This suggests that secondary endosymbionts did not play an important role in the rapid spread of the rosy apple aphid. These findings have fundamental importance for understanding pest colonization processes and implications for sustainable pest control programs.

sentences: Assessing the global impact of targeted conservation actions on species abundance In recent years, vertebrate population abundance has declined at unprecedented rates (WWF 2020). In response, targeted conservation measures - such as breeding programs or species-specific habitat management - have been applied to halt population declines, aid population recovery, and reduce and reverse the loss of biodiversity (Salafsky et al. 2008; Bolam et al. 2020). Until now, assessments of conservation actions have focused on the extent to which they reduce extinction risk, impact populations within protected areas, or increase the global area of land under protection (Hoffmann et al. 2010, 2015; Barnes et al. 2016; Maxwell et al. 2020; Bolam et al. 2020; Grace et al. 2021a). Here, we record and analyze conservation actions for 26,904 vertebrate populations from 4,629 species, to measure the impact of targeted conservation on vertebrate abundance. Using a counterfactual approach to represent population trends in the absence of conservation, we demonstrate that targeted actions have delivered substantial positive effects on the abundance of recipient vertebrate populations worldwide. We show that, in the absence of conservation, a global indicator of vertebrate abundance would have declined even more. Positive population trends were associated with vertebrate populations subject to species or habitat management. We demonstrate that targeted conservation actions can help to reverse global biodiversity loss and show the value of counterfactual analysis for impact evaluation - an important step towards reversing biodiversity declines.

sentences: The conserved histone chaperone Spt6 facilitates DNA replication and mediates genome instability Histone chaperones are an important class of proteins that regulate chromatin accessibility for DNA-templated processes. Spt6 is a conserved histone chaperone and key regulator of transcription and chromatin structure. However, its functions outside of these roles have been little explored. In this work, we demonstrate a role for S. cerevisiae Spt6 in DNA replication and more broadly as a regulator of genome stability. Spt6 binds the replication machinery and depletion or mutation of Spt6 impairs DNA replication in vivo. Additionally, spt6 mutants are sensitive to DNA replication stress inducing agents, with increased sensitivity when combined with loss of DNA replication associated factors. Furthermore, spt6 mutants have elevated levels of DNA double strand breaks and recombination. These effects appear to be independent of R-loops, which are not elevated in spt6 mutants. Our results identify Spt6 as a regulator of genome stability, at least in part through a role in DNA replication.

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Cyclic uniaxial mechanical load enhances chondrogenesis through entraining the molecular circadian clock ObjectiveThe biomechanical environment plays a key role in regulating cartilage formation, but current understanding of mechanotransduction pathways in chondrogenic cells is still incomplete. Amongst the combination of external factors that control chondrogenesis are temporal cues that are governed by the cell-autonomous circadian clock. However, mechanical stimulation has not yet directly been proven to modulate chondrogenesis via entraining the circadian clock in chondroprogenitor cells.

DesignThe purpose of this study was to establish whether mechanical stimuli entrain the core clock in chondrogenic cells, and whether augmented chondrogenesis caused by mechanical loading was at least partially mediated by the synchronised, rhythmic expression of the core circadian clock genes, chondrogenic transcription factors, and cartilage matrix constituents.

ResultsWe report here, for the first time, that cyclic uniaxial mechanical load applied for 1 hour for a period of 6 days entrains the molecular clockwork in chondroprogenitor cells during chondrogenesis in limb bud-derived micromass cultures. In addition to the several core clock genes, the chondrogenic markers SOX9, ACAN, and COL2A1 also followed a robust sinusoidal rhythmic expression pattern. These rhythmic conditions significantly enhanced cartilage matrix production and upregulated marker gene expression. The observed chondrogenesis-promoting effect of the mechanical environment was at least partially attributable to its entraining effect on the molecular clockwork, as co-application of the small molecule clock modulator longdaysin attenuated the stimulatory effects of mechanical load.

ConclusionsResults from this study suggest that an optimal biomechanical environment enhances tissue homeostasis and histogenesis during early chondrogenesis through entraining the molecular clockwork.

sentences: Visual search for upright bigrams predicts reading fluency in children Fluent reading is an important milestone in education, but we lack a clear understanding of why children vary so widely in attaining this milestone. Language-related factors such as rapid automatized naming (RAN) and phonological awareness have been identified as important factors that influence reading fluency. Of theoretical interest is also, however, whether aspects of visual processing influence reading fluency. To investigate this issue, we tested primary school children (n = 68) on four tasks: two reading fluency tasks (word reading and passage reading), a RAN task to measure naming speed, and a visual search task using letters and bigrams to measure visual processing. As expected, the RAN score was strongly correlated with reading fluency. In addition, visual processing of bigrams was correlated with reading fluency. Importantly, this association was specific to upright but not inverted bigrams, and to bigrams with normal but not large letter spacing. Thus, reading fluency in children is accompanied by specialized changes in upright bigram processing. We propose that bigram processing during visual search could complement existing measures of language processing to understand individual differences in reading fluency.

sentences: Relative abundance data can misrepresent heritability of the microbiome Host genetics can shape microbiome composition, but to what extent it does, remains unclear. Like any other complex trait, this question can be addressed by estimating the heritability (h2) of the microbiome - the proportion of variance in the abundance of each taxon that is attributable to host genetic variation. However, unlike most complex traits, microbiome heritability is typically based on relative abundance data, where taxon-specific abundances are expressed as the proportion of the total microbial abundance in a sample. We derived an analytical approximation for the heritability that one obtains when using such relative abundances and we uncovered three problems: 1) The interdependency between taxa leads to imprecise heritability estimates. 2) Large sample size leads to high false discovery rates, overestimating the number of heritable taxa. 3) Microbial co-abundances lead to biased heritability estimates. We conclude that caution must be taken when interpreting heritability estimates and comparing values across studies.

sentences: Predictive systems biomarkers of response to immune checkpoint inhibitors Cancer cells can leverage several cell-intrinsic and -extrinsic mechanisms to escape immune system recognition. The inherent complexity of the tumor microenvironment, with its multicellular and dynamic nature, poses great challenges for the extraction of biomarkers of immune response and immunotherapy efficacy. Here, we use RNA-seq data combined with different sources of prior-knowledge to derive system-based signatures of the tumor microenvironment, quantifying immune-cell composition and intra- and inter-cellular communications. We applied multi-task learning to these signatures to predict different hallmarks of immune responses and derive cancer-type-specific models based on interpretable systems biomarkers. By applying our models to independent RNA-seq data from cancer patients treated with PD-1 inhibitors, we demonstrated that our method to Estimate Systems Immune Response (EaSIeR) accurately predicts therapeutic outcome. We anticipate that EaSIeR will be a valuable tool to provide a holistic description of immune responses in complex and dynamic systems such as tumors using available RNA-seq data.

sentences: Sustained plumage divergence despite weak genomic differentiation and broad sympatry in sister species of Australian woodswallows (Artamus spp.) Plumage divergence can function as a strong premating barrier when species come into secondary contact. When it fails to do so, the results are often genome homogenization and phenotypic hybrids at the zone of contact. This is not the case in the largely sympatric masked- and white-browed woodswallow species (Passeriformes: Artamidae: Artamus spp) complex in Australia where phenotypic integrity is sustained despite no discernible mitochondrial structure. This lack of structure may suggest recent divergence, ongoing gene flow or both, and phenotypic hybrids are reported albeit rarely. Here, we further assessed the population structure and differentiation across the species nuclear genomes using ddRAD-seq. As found in the mitochondrial genome, no structure or divergence within or between the two species is detected in the nuclear genome. This coarse sampling of the genome nonetheless revealed peaks of differentiation around the genes SOX5 and Axin1. Both are involved in the Wnt/{beta}-catenin signaling pathway, which regulates feather development. Reconstruction of demographic history supports a scenario with ongoing gene flow during divergence but involving shifts in direction and magnitude. Our study informs how divergent plumage morphs may arise and be sustained despite whole-genome homogenization and reveals new candidate genes potentially involved in plumage divergence.

sentences: Trans-channel fluorescence learning improves high-content screening for Alzheimer's disease therapeutics In microscopy-based drug screens, fluorescent markers carry critical information on how compounds affect different biological processes. However, practical considerations may hinder the use of certain fluorescent markers. Here, we present a deep learning method for overcoming this limitation. We accurately generated predicted fluorescent signals from other related markers and validated this new machine learning (ML) method on two biologically distinct datasets. We used the ML method to improve the selection of biologically active compounds for Alzheimers disease (AD) from high-content high-throughput screening (HCS). The ML method identified novel compounds that effectively blocked tau aggregation, which would have been missed by traditional screening approaches unguided by ML. The method improved triaging efficiency of compound rankings over conventional rankings by raw image channels. We reproduced this ML pipeline on a biologically independent cancer-based dataset, demonstrating its generalizability. The approach is disease-agnostic and applicable across diverse fluorescence microscopy datasets.

sentences: A Biodegradable, Tacrolimus-releasing Nerve Wrap Promotes Peripheral Nerve Regeneration Axonal regeneration following nerve repair is slow and often incomplete, resulting in poor functional recovery and sometimes lifelong disability. Yet, there are no FDA-approved therapies available to promote nerve regeneration. Tacrolimus accelerates axonal regeneration, but systemic side-effects presently outweigh its potential benefits for peripheral nerve surgery. We have developed a biodegradable drug delivery system for the sustained local release of tacrolimus at the nerve repair site, with suitable properties for large-scale manufacturing and clinical application, aiming to promote axonal regeneration and functional recovery with minimal systemic drug exposure. Tacrolimus is encapsulated in polycarbonate-urethane nanofibers and electrospun to generate an implantable nerve wrap that releases therapeutic doses of bioactive tacrolimus over 31 days. Size and drug loading are adjustable for applications in small and large caliber nerves, and the wrap degrades within 120 days into biocompatible byproducts. Tacrolimus released from the nerve wrap promotes axon elongation in vitro and accelerates nerve regeneration and functional recovery in preclinical nerve repair models while systemic drug exposure is reduced by 80% compared to systemic delivery. Given its surgical suitability and preclinical efficacy and safety, this system may provide a readily translatable approach to support axonal regeneration and recovery in patients undergoing nerve surgery.

sentences: Genomic databanks and targeted assays help characterise mosquito incursions within Australia Biosecurity strategies aiming to restrict the spread of invasive pests can benefit from knowing where new incursions have come from. This knowledge can be acquired using genomic databanks, where genetic variation in incursion samples is compared against variation in reference samples. Here, we use genomic databanks to investigate domestic incursions of two mosquito species in Australia. We used a deep learning method to trace a 2021 invasion of Aedes aegypti in Tennant Creek, Northern Territory, to Townsville, Queensland, and to trace Ae. albopictus incursions between several invaded and uninvaded islands in the Torres Strait. We observed high precision of tracing despite 30-70 generations separating incursions and source samples. Targeted assays also improved precision, in this case by comparing Wolbachia infection data to mitochondrial DNA variation in Ae. aegypti. For Ae. albopictus, incursions detected in 2021 were traced to different islands than incursions detected in 2019. Patterns of relatedness and inbreeding indicated that Tennant Creek was likely invaded by Ae. aegypti from one family, whereas Torres Strait incursions involved distinct kinship groups. Our results highlight the value of repeated tracing with genomic databanks that remain informative over years, and demonstrate how additional targeted assays (e.g. Wolbachia) can improve inferences.

sentences: The Selenoprotein P 3' untranslated region is an RNA binding protein platform that fine tunes selenocysteine incorporation Selenoproteins contain the 21st amino acid, selenocysteine (Sec), which is incorporated at select UGA codons when a specialized hairpin sequence, the Sec insertion sequence (SECIS) element, is present in the 3 UTR. Aside from the SECIS, selenoprotein mRNA 3 UTRs are not conserved between different selenoproteins within a species. In contrast, the 3-UTR of a given selenoprotein is often conserved across species, which supports the hypothesis that cis-acting elements in the 3-UTR other than the SECIS exert post-transcriptional control on selenoprotein expression. In order to determine the function of one such SECIS context, we chose to focus on the plasma selenoprotein, SELENOP, which is required to maintain selenium homeostasis as a selenium transport protein that contains 10 Sec residues. It is unique in that its mRNA contains two SECIS elements in the context of a highly conserved 843-nucleotide 3 UTR. Here we have used RNA affinity chromatography and identified PTBP1 as the major RNA binding protein that specifically interacts with the sequence between the two SECIS elements. We then used CRISPR/Cas9 genome editing to delete two regions surrounding the first SECIS element. We found that these sequences are involved in regulating SELENOP mRNA and protein levels, which are inversely altered as a function of selenium concentrations.

sentences: Increased parasite load is associated with reduced metabolic rates and escape responsiveness in pumpkinseed sunfish host Wild animals have parasites that can compromise their physiological and/or behavioural performance. Yet, the extent to which parasite load is related to intraspecific variation in performance traits within wild populations remains relatively unexplored. We used pumpkinseed sunfish (Lepomis gibbosus) and their endoparasites as a model system to explore the effects of infection load on host aerobic metabolism and escape performance. Metabolic traits (standard and maximum metabolic rates, aerobic scope) and fast-start escape responses following a simulated aerial attack by a predator (responsiveness, response latency, and escape distance) were measured in fish from across a gradient of visible (i.e. trematodes causing black spot disease counted on fish surfaces) and non-visible (i.e. cestodes in fish abdominal cavity counted post-mortem) endoparasite infection. We found that a higher infection load of non-visible endoparasites was related to lower standard and maximum metabolic rates, but not aerobic scope in fish. Non-visible endoparasite infection load was also related to decreased responsiveness of the host to a simulated aerial attack. Visible endoparasites were not related to changes in metabolic traits nor fast-start escape responses. Our results suggest that infection with parasites that are inconspicuous to researchers can result in intraspecific variation in physiological and behavioral performance in wild populations, highlighting the need to more explicitly acknowledge and account for the role played by natural infections in studies of wild animal performance.

sentences: DynaTMT: A user-friendly tool to process combined SILAC/TMT data The measurement of protein dynamics by proteomics to study cell remodeling has seen increased attention over the last years. This development is largely driven by a number of technological advances in proteomics methods. Pulsed stable isotope labeling in cell culture (SILAC) combined with tandem mass tag (TMT) labeling has evolved as a gold standard for profiling protein synthesis and degradation. While the experimental setup is similar to typical proteomics experiments, the data analysis proves more difficult: After peptide identification through search engines, data extraction requires either custom scripted pipelines or tedious manual table manipulations to extract the TMT-labeled heavy and light peaks of interest. To overcome this limitation, which deters researchers from using protein dynamic proteomics, we developed a user-friendly, browser-based application that allows easy and reproducible data analysis without the need for scripting experience. In addition, we provide a python package that can be implemented in established data analysis pipelines. We anticipate that this tool will ease data analysis and spark further research aimed at monitoring protein translation and degradation by proteomics.

sentences: Identification of key molecules and biological processes in TCF21 treated tumor pericytes Colorectal cancer has become a major public health problem in the US. Transcription factor 21 (TCF21) is reported to be silenced in colorectal cancer tissues. However, the mechanism of TCF21 in tumor pericytes is still unclear. In our study, we aim to identify the key biological processes and signaling pathways by analyzing the RNA-seq data. The GSE200064 was produced by the Illumina NovaSeq 6000 (Homo sapiens). The KEGG and GO analyses showed that MAPK signaling pathway and complement/coagulation cascades are the major changed signaling pathways in the progression of tumor pericytes with overexpression of TCF21. Moreover, we identified several interactive molecules including VEGFA, MMP2, CCL2, COL3A1, COL1A2, CXCL12, ELN, PDGFRB, VWF, and APOE. These findings may benefit the study of colorectal cancer treatment.

sentences: Longitudinal analysis of humoral immunity against SARS-CoV-2 Spike in convalescent individuals up to 8 months post-symptom onset Functional and lasting immune responses to the novel coronavirus (SARS-CoV-2) are currently under intense investigation as antibody titers in plasma have been shown to decline during convalescence. Since the absence of antibodies does not equate to absence of immune memory, we sought to determine the presence of SARS-CoV-2-specific memory B cells in COVID-19 convalescent patients. In this study, we report on the evolution of the overall humoral immune responses on 101 blood samples obtained from 32 COVID-19 convalescent patients between 16 and 233 days post-symptom onset. Our observations indicate that anti-Spike and anti-RBD IgM in plasma decay rapidly, whereas the reduction of IgG is less prominent. Neutralizing activity in convalescent plasma declines rapidly compared to Fc-effector functions. Concomitantly, the frequencies of RBD-specific IgM+ B cells wane significantly when compared to RBD-specific IgG+ B cells, which increase over time, and the number of IgG+ memory B cells which remain stable thereafter for up to 8 months after symptoms onset. With the recent approval of highly effective vaccines for COVID-19, data on the persistence of immune responses are of central importance. Even though overall circulating SARS-CoV-2 Spike-specific antibodies contract over time during convalescence, we demonstrate that RBD-specific B cells increase and persist up to 8 months post symptom onset. We also observe modest increases in RBD-specific IgG+ memory B cells and importantly, detectable IgG and sustained Fc-effector activity in plasma over the 8-month period. Our results add to the current understanding of immune memory following SARS-CoV-2 infection, which is critical for the prevention of secondary infections, vaccine efficacy and herd immunity against COVID-19.

sentences: Reconstructing human early embryogenesis in vitro with pluripotent stem cells Understanding human development is of fundamental biological and clinical importance. Yet despite its significance, insights into early developmental events in humans still remain largely unknown. While recent advances show that stem cells can mimic embryogenesis1-9 to unravel hidden developmental mechanisms, a stem cell-based model of early human embryogenesis is lacking. Here, we use human extended pluripotent stem cells10to reconstitute early human development in 3-dimensions and recapitulate early embryo-like events. We first perform a systematic characterisation to reveal unique signalling requirements for building the human pre-implantation blastocyst. Further, we show that these in vitro stem cell-derived blastocyst-like structures are able to undertake spatiotemporal self-organisation to mimic peri-implantation remodelling in which a polarised rosette opens up the amniotic cavity within a developing disc. The hallmarks of human early development displayed by this stem cell-based in vitro model mimics features of embryonic day 3 to day 9/10 of natural development. Thus, this platform represents a tractable model system to contribute to the basic understanding of cellular and molecular mechanisms governing early embryonic events in humans and to provide valuable insights into the design of differentiation protocols for human stem cells in clinical applications.

sentences: Comparative analysis of Alzheimer's disease knock-in model brain transcriptomes implies changes to energy metabolism as a causative pathogenic stress Energy production is the most fundamentally important cellular activity supporting all other functions, particularly in highly active organs such as brains. Here, we summarise transcriptome analyses of young adult (pre-disease) brains from a collection of eleven early-onset familial Alzheimers disease (EOfAD)-like and non-EOfAD-like mutations in three zebrafish genes. The one cellular activity consistently predicted as affected by only the EOfAD-like mutations is oxidative phosphorylation that produces most of the brains energy. All the mutations were predicted to affect protein synthesis. We extended our analysis to knock-in mouse models of APOE alleles and found the same effect for the late onset Alzheimers disease risk allele {varepsilon}4. Our results support a common molecular basis for initiation of the pathological processes leading to both early and late onset forms of Alzheimers disease and illustrate the utility of both zebrafish and knock-in, single EOfAD mutation models for understanding the causes of this disease.

sentences: Organization of the catecholaminergic system in the short-lived fish Nothobranchius furzeri The catecholaminergic system has received much attention based on its regulatory role in a wide range of brain functions and its relevance in aging and neurodegenerative diseases. In the present study, we analyzed the neuroanatomical distribution of catecholaminergic neurons based on tyrosine hydroxylase (TH) immunoreactivity in the brain of adult Nothobranchius furzeri. In the telencephalon, numerous TH+ neurons were observed in the olfactory bulbs and the ventral telencephalic area, arranged as strips extending through the rostrocaudal axis. We found the largest TH+ groups in the diencephalon at the preoptic region level, the ventral thalamus, the pretectal region, the posterior tuberculum, and the caudal hypothalamus. In the dorsal mesencephalic tegmentum, we identified a particular catecholaminergic group. The rostral rhombencephalon housed TH+ cells in the locus coeruleus and the medulla oblongata, distributing in a region dorsal to the inferior reticular formation, the vagal lobe, and the area postrema. Finally, scattered TH+ neurons were present in the ventral spinal cord and the retina. From a comparative perspective, the overall organization of catecholaminergic neurons is consistent with the general pattern reported for other teleosts. However, Nothobranchius furzeri shows some particular features, including the presence of catecholaminergic cells in the midbrain. This work provides a detailed neuroanatomical map of the catecholaminergic system of Nothobranchius furzeri, a powerful aging model, also contributing to the phylogenetic understanding of one of the most ancient neurochemical systems.

sentences: TAK1: a potential therapeutic target for retinal neovascularization Retinal neovascularization, or pathological angiogenesis in the retina, is a leading cause of blindness in developed countries. Transforming growth factor-{beta}-activated kinase 1 (TAK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) activated by TGF-{beta}1 and other pro-inflammatory cytokines. TAK1 is also a key mediator of inflammation, innate immune responses, apoptosis and tissue homeostasis and plays an important role in physiological angiogenesis. Its role in pathological angiogenesis, particularly in retinal neovascularization, remains unclear. We investigated the regulatory role of TAK1 in pathological angiogenesis in the retina. Transcriptome analysis of human retina featuring retinal neovascularization revealed enrichment of known TAK1-mediated signaling pathways. Selective inhibition of TAK1 activation by 5Z-7-oxozeaenol attenuated aberrant retinal angiogenesis in rats following oxygen-induced retinopathy. Transcriptome profiling revealed that TAK1 activation in human microvascular endothelial cells under TNF stimulation led to increase the gene expression related to cytokines and leukocyte-endothelial interaction, mainly through nuclear factor kappa B (NF{kappa}B) signaling pathways. These results reveal that inhibition of TAK1 signaling may have therapeutic value for the treatment of pathological angiogenesis in the retina.

sentences: Disruption of the microphysiological niche alters matrix deposition and causes loss of mitochondrial Ca2+ control in skeletal muscle fibers Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. Here, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca2+ handling in adult mouse skeletal muscle fibers. Adult skeletal muscle fibers were isolated from mouse toe muscle either by collagenase-induced dissociation of the ECM or by mechanical dissection that leaves the contiguous ECM intact. Experiments were generally performed four hours after cell isolation. At this time, there were striking differences in the gene expression patterns between fibers isolated with the two methods; 24h after cell isolation, enzymatically dissociated fibers had transcriptomic signatures resembling dystrophic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria in enzymatically dissociated than in mechanically dissected fibers. Similar increases in free cytosolic [Ca2+] during repeated tetanic stimulation were accompanied by marked mitochondrial Ca2+ uptake only in enzymatically dissociated muscle fibers. The aberrant mitochondrial Ca2+ uptake was partially prevented by the mitochondrial Ca2+ uniporter inhibitor Ru360 and by cyclosporine A and NV556, which inhibit the mitochondrial protein Ppif (also called cyclophilin D). Importantly, inhibition of Ppif with NV556 significantly improved survival of mice with mitochondrial myopathy in which muscle mitochondria take up excessive amounts of Ca2+ even with an intact ECM. In conclusion, skeletal muscle fibers isolated by collagenase-induced dissociation of the ECM display aberrant mitochondrial Ca2+ uptake, which involves a Ppif-dependent mitochondrial Ca2+ influx resembling that observed in mitochondrial myopathies.

sentences: Structural mechanism underpinning Thermus oshimai Pif1-mediated G-quadruplex unfolding G-quadruplexes (G4s) are unusual DNA structures and can stall DNA replication, causing genomic instability for the cell. Although the solved crystal structure of the DHX36 helicase demonstrated that G4 was specifically targeted by a DHX36-specific motif (DSM), lack of complete structural details for general G4-resolving helicases without specific target motifs remains a barrier to the complete understanding of the molecular basis underlying the recognition and unfolding of G4s. Herein, we present the first X-ray crystal structure of the Thermus oshimai Pif1 (ToPif) complexed with a G4, thereby mimicking the physiological G4 formed during DNA replication. Strictly different from the previous determined G4-helicase structure of DHX36, our structure revealed that ToPif1 recognizes the entire native G4 via a cluster of amino acids at domains 1B/2B constituting a G4-Recognizing Surface (GRS). The overall topology of the G4 structure solved in this work maintains its three-layered propeller-type G4 topology, with no significant reorganization of G-tetrads upon protein binding. The three G-tetrads in G4 were differentially recognized by GRS residues mainly through electrostatic, ionic interactions and hydrogen bonds formed between the GRS residues and the ribose-phosphate backbone. Our structure explains how helicases from distinct superfamilies adopt different strategies for recognizing and unfolding G4s.

sentences: Hormonal contraceptives alter amphetamine place preference and responsivity in the intact female rat. Hormonal contraceptives (HCs) are commonly used among reproductive aged women and alter the physiological state of the user by interfering with endogenous hormone concentrations and their actions on the reproductive tract. As hormones such as estradiol and progesterone modulate the incidence of substance abuse disorders in women, it is important to consider the influence HCs have on the female brain and behavior. This experiment explores how female sex steroid hormonal states associated with the rat estrous cycle, and modulating those states with HCs, influences measures of drug preference and responsivity. First, rats underwent food-light Pavlovian conditioning to measure conditioned orienting, a known predictor of amphetamine (AMP) place preference. Then, rats were conditioned and tested for AMP place preference with either an HC-implant or during estrous cycle stages associated with different ovarian hormone levels (i.e., proestrus (P) or metestrus/diestrus (M/D) while recording ultrasonic vocalizations (USVs) as an index of hedonic responsivity. Because of dopamines (DA) role in modulation of AMP actions, DA cell activity and availability were examined using tyrosine hydroxylase and FOS immunohistochemistry after final AMP challenge. Conditioned orienting did not differ between cycling and HC-implanted. P rats emitted more USVs during conditioning, showed higher AMP place preference throughout testing, and had higher DA cell activity in the substantia nigra compared to M/D and HC-implanted rats. Sex steroid hormone serum concentration and uterine horn thickness predicted some but not all of these measures. This experiment suggests ovarian hormones affect drug preference and responsivity, while providing novel insight into how hormone-altering contraceptives may reduce these measures.

sentences: Inter-microscope comparability of dental microwear texture data obtained from obtained from different optical profilometers. Dental microwear texture analysis has become a well-established approach for dietary inference and reconstruction of mammals and other tetrapods, both extant and extinct. As the amount of available data grows immensely, researchers could benefit from combining data gathered by others to perform meta-analyses. However, different devices used to capture three-dimensional surface scans for DMTA are known to produce variation even when measuring the same surface. Here we compare DMTA data of 36 guinea pigs that received different diets in a controlled feeding experiment, measured on a confocal disc-scanning and a confocal laser-scanning microscope. We are testing different pre-analysis filtering protocols to mitigate differences. We find inter-microscopes and filter-related differences for the majority of analysed 41 DMTA parameters. Certain microscope-specific filter routines resulted in less differences than other pre-analysis protocols. We further identify DMTA parameters which were stable regardless of microscope or pre-analysis treatment. Overall, the results obtained on both microscopes show the same dietary differentiation between guinea pig feeding groups, which supports that DMTA is a suitable method to obtain repeatable, objective dietary inferences. We finally propose a roadmap to enhance data exchange and inter-lab comparability and collaboration in the future.

sentences: Population-level variation of enhancer expression identifies novel disease mechanisms in the human brain Identification of risk variants for neuropsychiatric diseases within enhancers underscores the importance of understanding the population-level variation of enhancers in the human brain. Besides regulating tissue- and cell-type-specific transcription of target genes, enhancers themselves can be transcribed. We expanded the catalog of known human brain transcribed enhancers by an order of magnitude by generating and jointly analyzing large-scale cell-type-specific transcriptome and regulome data. Examination of the transcriptome in 1,382 brain samples in two independent cohorts identified robust expression of transcribed enhancers. We explored gene-enhancer coordination and found that enhancer-linked genes are strongly implicated in neuropsychiatric disease. We identified significant expression quantitative trait loci (eQTL) for 25,958 enhancers which mediate 6.8% of schizophrenia heritability, mostly independent from standard gene eQTL. Inclusion of enhancer eQTL in transcriptome-wide association studies enhanced functional interpretation of disease loci. Overall, our study characterizes the enhancer-gene regulome and genetic mechanisms in the human cortex in both healthy and disease states.

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Cerebrovascular response to exercise interacts with individual genotype and amyloid-beta deposition to influence response inhibition with aging The etiology of cognitive dysfunction associated with Alzheimers disease (AD) and dementia is multifactorial. Yet, mechanistic interactions among key neurobiological factors linked to AD pathology are unclear. This study tested the effect of interactions between cerebrovascular function, individual genotype, and structural brain pathology on response inhibition performance, an early and sensitive indicator of cognitive executive dysfunction with aging.

We quantified cerebrovascular response (CVR) to moderate-intensity aerobic exercise using transcranial doppler ultrasound and global amyloid-beta (A{beta}) deposition using positron emission tomography in a group of cognitively normal older adults genotyped as APOE4 carriers and noncarriers. We quantified response inhibition during a cognitive Stroop test.

Individuals with blunted CVR possessed greater A{beta} deposition. There was CVR-by-carrier status-by-A{beta} interaction on response inhibition. Blunted CVR was associated with impaired response inhibition specifically in carriers. Despite having greater A{beta} deposition, carriers with higher CVR demonstrated better response inhibition.

Cerebrovascular interactions with individual genotype and structural brain pathology may provide a physiologically-informed target for precision-medicine approaches for early treatment and prevention of cognitive dysfunction with aging.

HighlightsO_LINeurobiological interactions between CVR, APOE genotype, and A{beta} are behaviorally significant.
C_LIO_LIBlunted CVR to exercise is associated with impaired response inhibition specifically in APOE4 carriers.
C_LIO_LIAPOE4 carriers with more robust CVR have higher response inhibition performance, despite having greater A{beta} deposition.
C_LIO_LIAssessment of multifactorial neurobiological variables offers an early and sensitive biomarker of cognitive behavioral dysfunction with aging.
C_LI

sentences: Histone deacetylase inhibitor induces acetyl-CoA depletion leading to lethal metabolic stress in RAS-pathway activated cells RAS-mutant cancers are among the most refractory to treatment. Apart from new G12C genotype targeted therapies, strategies to kill RAS-mutant cells by directly targeting RAS or its downstream effectors have been mostly unsuccessful, mainly due to pathway redundancy and heterogeneities in RAS-induced phenotypes. Here we identified a RAS-phenotype that can be targeted by the histone deacetylase inhibitor (HDACi) romidepsin. We showed that the hyperacetylation induced by romidepsin depleted acetyl-CoA, the cell donor substrate for acetylation, and led to metabolic stress and death in KRAS-activated cells. Elastic net analysis on transcriptomics from a 608-cell panel confirmed that HDACi sensitivity was linked to a difference in profiles in two pathways involved in acetyl-CoA metabolism. The analysis of a clinical dataset confirmed that perturbation of the two acetyl-CoA pathways were correlated with HDACi sensitivity in patients treated with belinostat. Our analysis suggests the potential utility of a RAS-associated acetyl-CoA phenotype to sharpen treatment choices for RAS-activated tumors.

sentences: The iron-sulfur cluster is critical for DNA binding by human DNA polymerase ϵ DNA polymerase {varepsilon} (Pol{varepsilon}) is a key enzyme for DNA replication in eukaryotes. It is attached to a helicase and performs DNA synthesis on the leading strand. Recently it was shown that the catalytic domain of yeast Pol{varepsilon} (Pol{varepsilon}CD) contains a [4Fe-4S] cluster located at the base of the processivity domain (P-domain) and coordinated by four conserved cysteines. In this work, we have shown that human Pol{varepsilon}CD (hPol{varepsilon}CD) expressed in bacterial cells also contains an iron-sulfur cluster. In comparison, recombinant hPol{varepsilon}CD produced in insect cells contains an eight-fold-lower level of iron. Interestingly, the iron content correlates with the level of DNA-binding molecules, which suggests an important role of the iron-sulfur cluster in hPol{varepsilon} interaction with DNA. Indeed, mutation of two conserved cysteines that coordinate the cluster abolished template:primer binding and, therefore, DNA polymerase and proofreading exonuclease activities. We propose that the cluster regulates the conformation of the P-domain, which, like a gatekeeper, controls access to a DNA-binding cleft for a template:primer. In addition, we performed kinetic and binding studies of hPol{varepsilon}CD. The binding studies demonstrated low affinity of hPol{varepsilon}CD to DNA and a strong effect of salt concentration on stability of the hPol{varepsilon}CD/DNA complex. Pre-steady-state kinetic studies have shown a maximal polymerization rate constant of 51.5 s-1 and a relatively low affinity to incoming dNTP with an apparent KD of 105 M. This work provides notable insight into the role of a [4Fe-4S] cluster in Pol{varepsilon} function.

sentences: Reducing the efforts to create reproducible analysis code with FieldTrip The analysis of EEG and MEG data typically requires a lengthy and complicated sequence of analysis steps, often requiring large amounts of computations, which are ideally represented in analysis scripts. These scripts are often written by researchers without formal training in computer science, resulting in the quality and readability of these analysis scripts to be highly dependent on individual coding expertise and style. Even though the computational outcomes and interpretation of the results can be correct, the inconsistent style and quality of analysis scripts make reviewing the details of the analysis difficult for other researchers that are either involved in the study or not, and the quality of the scripts might compromise the reproducibility of obtained results. This paper describes the design and implementation of a strategy that allows complete reproduction of MATLAB-based scripts with little extra efforts on behalf of the user, which we have implemented as part of the FieldTrip toolbox. Starting from the researchers idiosyncratic pipeline scripts, this new functionality allows researchers to automatically create and publish analysis pipeline scripts in a standardized format, along with all relevant intermediate data. We demonstrate the functionality and validate its effectiveness by applying it to the analysis of a recently published MEG study.

sentences: Interrogation of genome-wide, experimentally dissected gene regulatory networks reveals mechanisms underlying dynamic cellular state control Pooled CRISPRi-mediated silencing of >1,000 transcriptional regulators expressed in single colorectal adenocarcinoma cells, followed by single-cell RNA-seq profiling at two timepoints, 1 day and 4 days, allowed reverse engineering the underlying tumor context-specific, causal regulatory network. Furthermore, the availability of experimentally derived, highly multiplexed gene reporter assays for each regulator, as identified by this analysis, allowed accurate assessment of differential protein activity following silencing of each regulator, thus providing proof-of-concept for generating comprehensive, tissue-specific networks of transcriptional and post-translational interactions. Analysis of this causal network allowed elucidation of complex autoregulatory mechanisms that have eluded previous computational approaches and supported systematic elucidation of cooperative mechanisms, where one regulatory protein can modulate the activity of another regulatory protein, as well as transcriptional mimicry, where one regulatory protein can phenocopy others.

sentences: Delta-range coupling between prefrontal cortex and hippocampus supported by respiratory rhythmic input from the olfactory bulb An explosion of recent findings firmly demonstrated that brain activity and cognitive function in rodents and humans are modulated synchronously with nasal respiration. Rhythmic respiratory (RR) coupling of wide-spread forebrain activity was confirmed using advanced techniques, including current source density analysis, single unit firing, and phase modulation of local gamma activity, creating solid premise for investigating how higher networks use this mechanism in their communication. Here we show essential differences in the way prefrontal cortex (PFC) and hippocampus (HC) process the RR signal from the olfactory bulb (OB) allowing dynamic PFC-HC coupling utilizing this input. We used inter-regional coherences and their correlations in rats, breathing at low rate ([~]2 Hz) at rest, outside of the short sniffing bouts. We found strong and stable OB-PFC coherence, contrasting OB-HC coherence which was low but highly variable. PFC-HC coupling, however, primarily correlated with the latter, indicating that HC access to the PFC output is dynamically regulated by the responsiveness of HC to the common rhythmic drive. This pattern was present in both theta and non-theta states of waking, whereas PFC-HC communication appeared protected from RR synchronization in sleep states. The findings help to understand the mechanism of rhythmic modulation of non-olfactory cognitive processes by the on-going regular respiration, reported in rodents as well as humans. These mechanisms may be impaired when nasal breathing is limited or in OB-pathology, including malfunctions of the OB epithelium due to infections, such as in COVID-19.

sentences:
FINE-SCALE ADAPTATIONS TO ENVIRONMENTAL VARIATION AND GROWTH STRATEGIES DRIVE PHYLLOSPHERE METHYLOBACTERIUM DIVERSITY. Methylobacterium is a prevalent bacterial genus of the phyllosphere. Despite its ubiquity, little is known about the extent to which its diversity reflects neutral processes like migration and drift, versus environmental filtering of life history strategies and adaptations. In two temperate forests, we investigated how phylogenetic diversity within Methylobacterium was structured by biogeography, seasonality, and growth strategies. Using deep, culture-independent barcoded marker gene sequencing coupled with culture-based approaches, we uncovered a considerable diversity of Methylobacterium in the phyllosphere. We cultured different subsets of Methylobacterium lineages depending upon the temperature of isolation and growth (20 {degrees}C or 30 {degrees}C), suggesting long-term adaptation to temperature. To a lesser extent than temperature adaptation, Methylobacterium diversity was also structured across large (>100km; between forests) and small geographical scales (<1.2km within forests), among host tree species, and was dynamic over seasons. By measuring growth of 79 isolates at different temperature treatments, we observed contrasting growth performances, with strong lineage- and season-dependent variations in growth strategies. Finally, we documented a progressive replacement of lineages with a high-yield growth strategy typical of cooperative, structured communities, in favor of those characterized by rapid growth, resulting in convergence and homogenization of community structure at the end of the growing season. Together our results show how Methylobacterium is phylogenetically structured into lineages with distinct growth strategies, which helps explain their differential abundance across regions, host tree species, and time. This works paves the way for further investigation of adaptive strategies and traits within a ubiquitous phyllosphere genus.

ImportanceMethylobacterium is a bacterial group tied to plants. Despite its ubiquity and importance to their hosts, little is known about the processes driving Methylobacterium community dynamics. By combining traditional culture-dependent and independent (metabarcoding) approaches, we monitored Methylobacterium diversity in two temperate forests over a growing season. On the surface of tree leaves, we discovered remarkably diverse and dynamic Methylobacterium communities over short temporal (from June to October) and spatial scales (within 1.2 km). Because we cultured different subsets of Methylobacterium diversity depending on the temperature of incubation, we suspected that these dynamics partly reflected climatic adaptation. By culturing strains in lab conditions mimicking seasonal variations, we found that diversity and environmental variations were indeed good predictors of Methylobacterium growth performances. Our findings suggest that Methylobacterium community dynamics at the surface of tree leaves results from the succession of strains with contrasted growth strategies in response to environmental variations.

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Dynamic Cognitive States Predict Individual Variability in Behavior and Modulate with EEG Functional Connectivity during Working Memory Fluctuations in strategy, attention, or motivation can cause large variability in performance across task trials. Typically, this variability is treated as noise, and assumed to cancel out, leaving supposedly stable relationships among behavior, neural activity, and experimental task conditions. Those relationships, however, could change with a participants internal cognitive states, and variability in performance may carry important information regarding those states, which cannot be directly measured. Therefore, we used a mathematical, state-space modeling framework to estimate internal states from measured behavioral data, quantifying each participants sensitivity to factors such as past errors or distractions, to predict their reaction time fluctuations. We show how modeling these states greatly improves trial-by-trial prediction of behavior. Further, we identify EEG functional connectivity features that modulate with each state. These results illustrate the potential of this approach and how it could enable quantification of intra- and inter-individual differences and provide insight into their neural bases.

Statement of RelevanceCognitive behavioral performance and its neural bases vary both across individuals and within individuals over time. Understanding this variability may be key to the success of clinical or educational interventions. Internal cognitive states reflecting differences in strategy, attention, and motivation may drive much of these inter- and intra-individual differences, but often cannot be reliably controlled or measured in cognitive neuroscience research. The mathematical modeling framework developed here uses measured data to estimate a participants dynamic, internal cognitive states, with each state derived from specific factors hypothesized to affect attention, motivation or strategy. The results highlight potential sources of behavioral variability and reveal EEG features that modulate with each state. Our method quantifies and characterizes individual behavioral differences and highlights their underlying neural mechanisms, which could be used for future targeted training or neuromodulation therapies to improve cognitive performance.

sentences: Squidpy: a scalable framework for spatial single cell analysis Spatial omics data are advancing the study of tissue organization and cellular communication at an unprecedented scale. Here, we present Squidpy, a Python framework that brings together tools from omics and image analysis to enable scalable description of spatial molecular data, such as transcriptome or multivariate proteins. Squidpy provides both infrastructure and numerous analysis methods that allow to efficiently store, manipulate and interactively visualize spatial omics data.

sentences: Nuclear and plastid phylogenomic analyses provide insights into the reticulate evolution, species delimitation and biogeography of the Sino-Japanese disjunctive Diabelia (Caprifoliaceae) Understanding biological diversity and the mechanisms of the Sino-Japanese disjunctions are major challenges in eastern Asia biogeography. The Sino-Japanese flora has been broadly studied as an ideal model for plant phylogeography. Diabelia (Caprifoliaceae) is an East Asian genus, with a disjunctive distribution across the Sino-Japanese region. However, relationships within Diabelia remain elusive. In this study, we reconstructed the phylogeny of Diabelia and inferred historical biogeography and evolutionary patterns based on nuclear and plastid sequences from target enrichment and genome skimming approaches, respectively. We found that the main clades within Diabelia were discordant between nuclear and plastid trees. Both nuclear and plastid phylogenetic analyses supported five main clades: D. serrata, D. tetrasepala, D. sanguinea, D. spathulata var. stenophylla and D. spathulata var. spathulata. Species network analyses revealed that Diabelia tetrasepala is likely the result of a hybridization event. Divergence time estimation and ancestral area reconstructions showed that Diabelia originated in Japan during the early Miocene, with subsequent vicariance and dispersal events between Japan and Korea, and between Japan and China. Overall, our results support the division of Diabelia into five main clades and the recognition of five species in the genus. This research provides new insights in the species delimitation and speciation processes of taxonomically complex lineages such as Diabelia.

sentences: The Age-Dependent Decline in Neuron Growth Potential in the CNS is Associated with an Age-Related Dysfunction of Neuronal Mitochondria The age of incidence of Spinal Cord Injury (SCI) and the average age of people living with SCI is continuously increasing. In contrast, SCI is extensively modelled in young adult animals, hampering translation of research to clinical application. While there has been significant progress in manipulating axon growth after injury, how it is impacted by aging impacts this is still unknown. Aging is associated with a decline in mitochondrial functions, whereas mitochondria are essential to successful neurite and axon growth. Using isolation and culture of adult cortical neurons, we have analyzed mitochondrial changes in 2-, 6-, 12- and 18-month mice. We observed reduced neurite growth in older neurons. Older neurons also showed dysfunctional respiration, reduced membrane potential, and altered mitochondrial membrane transport proteins; however mitochondrial DNA (mtDNA) abundance and cellular ATP were increased. Taken together, these data suggest dysfunctional mitochondria in older neurons are involved in the age-dependent reduction in neuron growth. Both normal aging and traumatic injury are associated with mitochondrial dysfunction, posing a challenge for an aging SCI population as the two elements can compound one another to worsen injury outcomes. The results of this study highlight this as an area of great interest in CNS trauma.

sentences: Cold survival and its molecular mechanisms in a locally adapted nematode population Since Darwin, evolutionary biologists have sought to understand the drivers and mechanisms of natural trait diversity. The field advances toward this goal with the discovery of phenotypes that vary in the wild, their relationship to ecology, and their underlying genes. Here, we established resistance to extreme low temperature in the free-living nematode Caenorhabditis briggsae as an ecological and evolutionary model system. We found that C. briggsae strains of temperate origin were strikingly more cold-resistant than those isolated from tropical localities. Transcriptional profiling revealed expression patterns unique to the resistant temperate ecotype, including dozens of genes expressed at high levels even after multiple days of cold-induced physiological slowdown. Mutational analysis validated a role in cold resistance for seven such genes. As the temperate C. briggsae population likely diverged only ~700 years ago from tropical ancestors, our findings highlight a candidate case of very rapid, robust, and genetically complex adaptation, and shed light on the mechanisms at play.

sentences: Genetic Encoding of a Highly Photostable, Long Lifetime Fluorescent Amino Acid for Imaging in Mammalian Cells Acridonylalanine (Acd) is a fluorescent amino acid that is highly photostable, with a high quantum yield and long fluorescence lifetime in water. These properties make it superior to existing genetically encodable fluorescent amino acids for monitoring protein interactions and conformational changes through fluorescence polarization or lifetime experiments, including fluorescence lifetime imaging microscopy (FLIM). Here, we report the genetic incorporation of Acd using engineered pyrrolysine tRNA synthetase (RS) mutants that allow for efficient Acd incorporation in both E. coli and mammalian cells. We compare protein yields and amino acid specificity for these Acd RSs to identify an optimal construct. We also demonstrate the use of Acd in FLIM, where its long lifetime provides strong contrast compared to endogenous fluorophores and engineered fluorescent proteins, which have lifetimes less than 5 ns.

sentences: Thrombocytopenia and splenic platelet directed immune responses after intravenous ChAdOx1 nCov-19 administration Summary paragraphVaccines against SARS-CoV-2 are based on a range of novel vaccine platforms, with adenovirus-based approaches (like ChAdOx1 nCov-19) being one of them. Recently a rare and novel complication of SARS-CoV-2 targeted adenovirus vaccines has emerged: thrombosis with thrombocytopenia syndrome (TTS). TTS is characterized by low platelet counts, clot formation at unusual anatomic sites and platelet-activating PF4-polyanion antibodies reminiscent of heparin-induced thrombocytopenia. Here, we employ in vitro and in vivo models to characterize the possible mechanisms of this platelet-targeted autoimmunity. We show that intravenous but not intramuscular injection of ChAdOx1 nCov-19 triggers platelet-adenovirus aggregate formation and platelet activation. After intravenous injection, these aggregates are phagocytosed by macrophages in the spleen and platelet remnants are found in the marginal zone and follicles. This is followed by a pronounced B-cell response with the emergence of circulating antibodies binding to platelets. Our work contributes to the understanding of TTS and highlights accidental intravenous injection as potential mechanism for post-vaccination TTS. Hence, safe intramuscular injection, with aspiration prior to injection, could be a potential preventive measure when administering adenovirus-based vaccines.

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Alkaline phosphatase activity of serum affects osteogenic differentiation cultures Fetal bovine serum (FBS) is a widely used supplement in cell culture medium, despite its known variability in composition which greatly affects cellular function and consequently the outcome of studies. In bone tissue engineering, the deposited mineralized matrix is one of the main outcome parameters, but using different brands of FBS can result in large variations. Alkaline phosphatase (ALP) is present in FBS. Not only is ALP used to judge the osteogenic differentiation of bone cells, it may affect deposition of mineralized matrix. The present study focused on the enzymatic activity of ALP in FBS of different suppliers and its contribution to mineralization in osteogenic differentiation cultures. It was hypothesized that culturing cells in a medium with high intrinsic ALP activity of FBS will lead to higher mineral deposition compared to media with lower ALP activity. The used FBS types were shown to have significant differences in enzymatic ALP activity. Our results indicate that the ALP activity of the medium not only affected the deposited mineralized matrix but also the osteogenic differentiation of cells as measured by a changed cellular ALP activity of human bone marrow derived mesenchymal stromal cells (hBMSC). In media with low inherent ALP activity, the cellular ALP activity was increased and played the major role in the mineralization process; while, in media with high intrinsic ALP activity contribution from the serum, less cellular ALP activity was measured and the ALP activity of the medium also contributed to mineral formation substantially. Our results highlight the diverse effects of ALP activity intrinsic to FBS on osteogenic differentiation and matrix mineralization and how FBS can determine the experimental outcomes, in particular for studies investigating matrix mineralization. Once again, the need to replace FBS with more controlled and known additives is highlighted.

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sentences: Privacy-Preserving Federated Neural Network Learning for Disease-Associated Cell Classification Training accurate and robust machine learning models requires a large amount of data that is usually scattered across data-silos. Sharing or centralizing the data of different healthcare institutions is, however, unfeasible or prohibitively difficult due to privacy regulations. In this work, we address this problem by using a novel privacy-preserving federated learning-based approach, PriCell, for complex machine learning models such as convolutional neural networks. PriCell relies on multiparty homomorphic encryption and enables the collaborative training of encrypted neural networks with multiple healthcare institutions. We preserve the confidentiality of each institutions input data, of any intermediate values, and of the trained model parameters. We efficiently replicate the training of a published state-of-the-art convolutional neural network architecture in a decentralized and privacy-preserving manner. Our solution achieves an accuracy comparable to the one obtained with the centralized solution, with an improvement of at least one-order-of-magnitude in execution time with respect to prior secure solutions. Our work guarantees patient privacy and ensures data utility for efficient multi-center studies involving complex healthcare data.

sentences: Solution structure of the phosphatidylinositol 3-phosphate binding domain from the Legionella effector SetA Legionella pneumophila is a facultative intracellular pathogen that causes Legionnaires disease or Pontiac fever in humans upon accidental inhalation of Legionella-contaminated aerosols. During infection, L. pneumophila secretes more than 300 effectors into the host for the biogenesis of a replication-permissive niche, known as the Legionella containing vacuole (LCV). Among these, a large number of effectors harbor protein domains that recognize specific phosphoinositide (PI) lipids and mediate the anchoring of these effectors to the surface of LCV or other host membrane-bound organelles. The Legionella effector SetA contains a unique C-terminal domain (SetA-CTD) that has been shown to specifically bind with phosphatidylinositol-3-phosphate (PI(3)P) and target SetA to endosomes and LCVs. Here, we report the NMR solution structure of SetA-CTD, which mainly comprises a four -helix bundle. The structure reveals a basic pocket at one end of the -helix bundle for PI(3)P binding and two hydrophobic loops for membrane insertion. Mutations of key residues involved in lipid binding result in the loss of SetA in membrane association and endosomal localization. Structural comparison with other PI(3)P-binding domains highlights a general theme applied by multiple families of phosphoinositide-binding domains across species.

sentences: A transcriptomic atlas of Aedes aegypti reveals detailed functional organization of major body parts and gut regional specializations in sugar-fed and blood-fed adult females Mosquito vectors transmit numerous pathogens, but large gaps remain in our understanding of their physiology. To facilitate future explorations of mosquito biology, with specific attention to the major vector Aedes aegypti, we have created Aegypti-Atlas (http://aegyptiatlas.buchonlab.com/), an online resource hosting RNAseq profiles of Ae. Aegypti body parts (head, thorax, abdomen, gut, Malpighian tubules, and ovaries), gut regions (crop, proventriculus, anterior and posterior midgut, and hindgut), and a time course of blood meal digestion in the gut. Using Aegypti-Atlas, we provide new insights into the regionalization of gut function, blood feeding response, and immune defenses. We find that the anterior and posterior regions of the mosquito midgut possess clearly delineated digestive specializations which are preserved in the blood-fed state. Blood feeding initiates the sequential transcriptional induction and repression/depletion of multiple cohorts of peptidases throughout blood meal digestion. With respect to defense, immune signaling components, but not recognition or effector molecules, show enrichment in ovaries. Basal expression of antimicrobial peptides is dominated by two genes, holotricin and gambicin, that are expressed in the carcass and the digestive tissues, respectively, in a near mutually exclusive manner. In the midgut, gambicin and other immune effector genes are almost exclusively expressed in the anterior regions, while the posterior midgut exhibits the hallmarks of immune tolerance. Finally, in a cross-species comparison between the midguts of Ae. aegypti and Anopheles gambiae, we observe that regional digestive and immune specializations are closely conserved, indicating that our data may yield inferences that are broadly relevant to multiple mosquito vector species. We further demonstrate that the expression of orthologous genes is highly correlated, with the exception of a  species signature comprising a small number of highly/disparately expressed genes. With this work, we show the potential of Aegypti-Atlas to unlock a more complete understanding of mosquito biology.

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Effectiveness of the protected areas on the Mornington Peninsula for the common resident shorebird species using citizen science data In the absence of comprehensive survey data this study used citizen science bird counts, extracted from the Atlas of Living Australia, to assess which species benefit most from protected areas. This was done by fitting temporal models using the Integrated Laplace Approximation (INLA) method.

The trends for five resident shorebird species were compared to the Australian Pied Oystercatcher, with significantly steeper upward trends identified for the Black-fronted Dotterel, Red-capped Dotterel and Red-kneed Dotterel. Steeper upward trends were observed in protected than unprotected areas for the Black-fronted Dotterel, Masked Lapwing and Red-kneed Dotterel.

This work suggests that, with some limitations, statistical models can be used with citizen science data for monitoring the persistence of resident shorebirds and for investigating factors that are impacting these data. The results for the Dotterel species in protected areas are particularly encouraging.

sentences: Floral scents of a deceptive plant are hyperdiverse and under population-specific phenotypic selection Floral scent is a key mediator in plant-pollinator interactions; however, little is known to what extent intraspecific scent variation is shaped by phenotypic selection, with no information yet in deceptive plants. We recorded 291 scent compounds in deceptive moth fly-pollinated Arum maculatum from various populations north vs. south of the Alps, the highest number so far reported in a single plant species. Scent and fruit set differed between regions, and some, but not all differences in scent could be explained by differential phenotypic selection in northern vs. southern populations. Our study is the first to provide evidence that phenotypic selection is involved in shaping geographic patterns of floral scent in deceptive plants. The hyperdiverse scent of A. maculatum might result from the plants imitation of various brood substrates of its pollinators.

sentences: Striatal dopamine encodes the relationship between actions and reward Despite the well-described role of dopamine in the striatum for skill learning and movement, its function in encoding actions and their outcomes for goal-directed action is less clear. Here, rats acquired two actions for distinct outcomes while we simultaneously recorded dopamine release in the dorsomedial striatum as these action-outcome associations were incremented and selectively degraded. Goal-directed actions generated a lateralized dopamine signal that reflected the strength of the action-outcome association and tracked increments and decrements in the specific action-outcome relationship, which was sensitive to changes in both contingency and outcome identity. This lateralized signal was updated by a dopamine action value signal that was broadcast bilaterally during the action and updated by a reward prediction error following exposure to the outcome. Our results establish, therefore, a dual role for striatal dopamine signaling during goal-directed actions: bilateral trial-by-trial burst signaling encoding action values, which modifies the long-term action-outcome association, reflected in emergent lateralization of dopamine release during actions.

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The nuclear ubiquitin ligase adaptor SPOP is a conserved regulator of C9orf72 dipeptide toxicity A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the C9orf72 repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs (PR)50 and (GR)50 are highly toxic when expressed in C. elegans and this toxicity depends on nuclear localization of the DPR. In an unbiased genome-wide RNAi screen for suppressors of (PR)50 toxicity, we identified 12 genes that consistently suppressed either the developmental arrest and/or paralysis phenotype evoked by (PR)50 expression. All of these genes have vertebrate homologs and 7/12 contain predicted nuclear localization signals. One of these genes was spop-1, the C. elegans homolog of SPOP, a nuclear localized E3 ubiquitin ligase adaptor only found in metazoans. SPOP is also required for (GR)50 toxicity and functions in a genetic pathway that includes cul-3, which is the canonical E3 ligase partner for SPOP. Genetic or pharmacological inhibition of SPOP in mammalian primary spinal cord motor neurons suppressed DPR toxicity without affecting DPR expression levels. Finally, we find that genetic inhibition of bet-1, the C. elegans homolog of the known SPOP ubiquitination targets BRD2/3/4, suppresses the protective effect of SPOP mutations. Together, these data suggest a model in which SPOP promotes the DPR-dependent ubiquitination and degradation of BRD proteins. We speculate the pharmacological manipulation of this pathway, which is currently underway for multiple cancer subtypes, could also represent a novel entry point for therapeutic intervention to treat C9 FTD/ALS.

Significance statementThe G4C2 repeat expansion in the C9orf72 gene is a major cause of Fronto-Temporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS). Unusual translation of the repeat sequence produces two highly toxic dipeptide repeat proteins, PRX and GRX, which accumulate in the brain tissue of individuals with these diseases. Here, we show that PR and GR toxicity in both C. elegans and mammalian neurons depends on the E3 ubiquitin ligase adaptor SPOP. SPOP acts through the bromodomain protein BET-1 to mediate dipeptide toxicity. SPOP inhibitors, which are currently being developed to treat SPOP-dependent renal cancer, also protect neurons against DPR toxicity. Our findings identify a highly conserved and  druggable pathway that may represent a new strategy for treating these currently incurable diseases.

sentences: Sensorimotor transformation underlying odor-modulated locomotion in walking Drosophila Most real-world behaviors are performed with incomplete information. Odor-guided locomotion, an ecologically important behavior essential to an animals survival, is an example of such a behavior. Different odors activate different patterns of olfactory receptor neuron (ORN) classes providing information about which odor is present but does not provide any navigational information. In this study, we investigate the sensorimotor transformation that relates ORN activation to locomotion changes in Drosophila by optogenetically activating different combinations of ORN classes and measuring the resulting changes in locomotion. Three features describe this sensorimotor transformation: First, locomotion depends on both the instantaneous firing frequency (f) and its change (df); the two together serve as short-term memory that allows the fly to automatically adapt its motor program to sensory context. Second, the mapping between f-df and locomotor parameters such as speed or curvature is distinct for each pattern of activated ORNs. Finally, the sensorimotor mapping changes with time after odor exposure allowing integration of information over a longer timescale.

sentences: Combining genotypes and T cell receptor distributions to infer genetic loci determining V(D)J recombination probabilities Every T cell receptor (TCR) repertoire is shaped by a complex probabilistic tangle of genetically determined biases and immune exposures. T cells combine a random V(D)J recombination process with a selection process to generate highly diverse and functional TCRs. The extent to which an individuals genetic background is associated with their resulting TCR repertoire diversity has yet to be fully explored. Using a previously published repertoire sequencing dataset paired with high-resolution genome-wide genotyping from a large human cohort, we infer specific genetic loci associated with V(D)J recombination probabilities using genome-wide association inference. We show that V(D)J gene usage profiles are associated with variation in the TCRB locus and, specifically for the functional TCR repertoire, variation in the major histocompatibility complex locus. Further, we identify specific variations in the genes encoding the Artemis protein and the TdT protein to be associated with biasing junctional nucleotide deletion and N-insertion, respectively. These results refine our understanding of genetically-determined TCR repertoire biases by confirming and extending previous studies on the genetic determinants of V(D)J gene usage and providing the first examples of trans genetic variants which are associated with modifying junctional diversity. Together, these insights lay the groundwork for further explorations into how immune responses vary between individuals.

sentences: Lag-optimized BOLD cerebrovascular reactivity derived from breathing task data has a stronger relationship with baseline cerebral blood flow Cerebrovascular reactivity (CVR) is an important indicator of cerebrovascular health and is commonly studied with the Blood Oxygenation Level Dependent functional MRI (BOLD-fMRI) response to a vasoactive stimulus. There is theoretical and empirical evidence to suggest that baseline cerebral blood flow (CBF) modulates the BOLD signal amplitude, and that baseline CBF may influence BOLD-CVR estimates. We address how some pertinent data acquisition and modelling choices affect the relationship between baseline CBF and BOLD-CVR: whether BOLD-CVR is modelled with breathing task data or just resting-state data, and whether BOLD-CVR amplitudes are optimized for hemodynamic lag effects. For the relationship between baseline CBF and BOLD-CVR, we assessed both between-subject correlations of average GM values and within-subject spatial correlations across cortical regions. Our results suggest that a simple breathing task addition to a resting-state scan, alongside lag-optimization within BOLD-CVR modelling, can improve BOLD-CVR correlations with baseline CBF, both between- and within-subjects, likely because these CVR estimates are more physiologically accurate. We report positive coupling between baseline CBF and BOLD-CVR, both between and within subjects; the physiological explanation of this positive coupling is unclear, and future research with larger sample sizes and more tightly controlled vasoactive stimuli is needed. Understanding how baseline vascular physiology relates to dynamic cerebrovascular processes will bring further insights into what drives between and within subject participant variability in BOLD-CVR measurements and related measurements of cerebrovascular function. These insights are particularly relevant when interpreting results in populations with altered vascular and/or metabolic baselines or impaired cerebrovascular reserve.

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Metacommunities from bacteria to birds: stronger environmental effects in mediterranean than in tropical ponds. The metacommunity concept provides a theoretical framework that aims at explaining organism distributions by a combination of environmental filtering, dispersal and drift. With the development of statistical tools to quantify and partially isolate the role of each of these processes, empirical metacommunity studies have multiplied worldwide. However, few works attempt a multi-taxon approach and even fewer compare two distant biogeographical regions using the same methodology. Under this framework, we tested the expectation that temperate (mediterranean-climate) pond metacommunities would be more influenced by environmental and spatial processes than tropical ones, because of stronger environmental gradients and greater isolation of waterbodies.

We surveyed 30 tropical and 32 mediterranean temporary ponds from Costa Rica and Spain, respectively, and obtained data on 49 environmental variables (including limnological, hydrogeomorphological, biotic, climatic, and landscape variables). We characterized the biological communities of Bacteria and Archaea (from both the water column and the sediments), phytoplankton, zooplankton, benthic invertebrates, amphibians and birds, and estimated the relative role of space and environment on metacommunity organization for each group and region, by means of variation partitioning using Generalized Additive Models (GAMs).

Environmental selection was important in both tropical and mediterranean ponds, but markedly stronger in the latter, probably due to their larger limnological heterogeneity. Spatialized environment and pure spatial effects were greater in the tropics, related to higher climatic heterogeneity and dispersal processes (e.g. restriction, surplus) acting at different scales. The variability between taxonomic groups in spatial and environmental contributions was very wide. Effects on passive and active dispersers were similar within regions but different across regions, with higher environmental effects in mediterranean active dispersers. The residual (unexplained) variation was larger in tropical pond metacommunities, suggesting a higher role for stochastic processes and/or effects of biotic interactions in the tropics. Overall, these results provide support, for a wide variety of organisms related to aquatic habitats, for the classical view of stronger abiotic niche constraints in temperate areas compared to the tropics.

sentences: On the Contribution of Reproductive and Offspring Investment on Fertility: Human and Animal Societies Differences in investment into reproduction or offspring rearing are plentiful throughout the world, from the cells inside our bodies to complex sociological interactions among humans. Such differences can lead to profound impacts on species fitness, fertility, and reproductive rates, sometimes in startling ways. In this paper, we create a simple game-theoretical model to qualitatively investigate the effects of such differential investment. We focus on fertility in human societies and show that more wealthy individuals produce more offspring within a a mating group. However, when assortative mating mechanisms are introduced, this effectively leads to a speciation event, and a higher reproduction rate for poorer individuals is noticed, capturing what we call the "wealthy-to-poor switch". We discuss extensions and implications of this work to nupital gifts in ecology and to clonal competition in cancer cell lines under the influence of treatment.

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LiPyphilic: A Python toolkit for the analysis of lipid membrane simulations Molecular dynamics simulations are now widely used to study emergent phenomena in lipid membranes with complex compositions. Here, we present LiPyphilic - a fast, fully tested, and easy to install Python package for analysing such simulations. Analysis tools in LiPyphilic include the identification of cholesterol flip-flop events, the classification of local lipid environments, and the degree of interleaflet registration. LiPyphilic is both force field and resolution agnostic, and thanks to the powerful atom selection language of MDAnalysis it can handle membranes with highly complex compositions. LiPyphilic also offers two on-the-fly trajectory transformations to i) fix membranes split across periodic boundaries and ii) perform nojump coordinate unwrapping. Our implementation of nojump unwrapping accounts for fluctuations in box volume under the NPT ensemble -- an issue that most current implementations have overlooked. The full documentation of LiPyphilic, including installation instructions, is available at https://lipyphilic.readthedocs.io/en/latest.

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Neuroanatomical organization and functional roles of PVN MC4R pathways in physiological and behavioral regulations ObjectiveThe paraventricular nucleus of hypothalamus (PVN) is an integrative center in the brain orchestrating a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is undoubtedly involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ pathway and its role in diverse physiological and behavioral regulations have not been fully understood. Here we aimed to better characterize the input-output organization of PVNMC4R+ neurons and further test their potential functional roles beyond feeding.

MethodsUsing a combination of viral tools, we performed a comprehensive mapping of PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermogenesis, cardiovascular control and other behavioral regulations beyond feeding.

ResultsWe found that PVNMC4R+ neurons broadly innervate many different brain regions known to be important not only for feeding but also for neuroendocrine and autonomic control of thermogenesis and cardiovascular function, including but not limited to preoptic area, median eminence, parabrachial nucleus, locus coeruleus, nucleus of solitary tract, ventrolateral medulla and thoracic spinal cord. Contrary to broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs from limited brain regions, including medial preoptic nucleus, arcuate and dorsomedial hypothalamic nuclei, and supraoptic nucleus. Consistent with broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure and brown adipose tissue thermogenesis. Strikingly, these physiological changes accompanied an unexpected repetitive bedding-removing behavior followed by hypoactivity and resting-like behavior.

ConclusionsOur results clarify the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermogenesis, cardiovascular function and other behavioral regulations.

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The mating system affects the temperature sensitivity of male and female fertility O_LITo mitigate effects of climate change it is important to understand species responses to increasing temperatures. This has often been done by studying survival or activity at temperature extremes. Before such extremes are reached, however, effects on fertility may already be apparent.
C_LIO_LISex differences in the thermal sensitivity of fertility (TSF) could impact species persistence under climate warming because female fertility is typically more limiting to population growth than male fertility. However, little is known about sex differences in TSF.
C_LIO_LIHere we first demonstrate that the mating system can strongly influence TSF using the seed beetle Callosobruchus maculatus. We exposed populations carrying artificially induced mutations to two generations of short-term experimental evolution under alternative mating systems, manipulating the opportunity for natural and sexual selection on the mutations. We then measured TSF in males and females subjected to juvenile or adult heat stress.
C_LIO_LIPopulations kept under natural and sexual selection had higher fitness, but similar TSF, compared to control populations kept under relaxed selection. However, females had higher TSF than males, and strikingly, this sex difference had increased over only two generations in populations evolving under sexual selection.
C_LIO_LIWe hypothesized that an increase in male-induced harm to females during mating had played a central role in driving this evolved sex difference, and indeed, remating under conditions limiting male harassment of females reduced both male and female TSF. Moreover, we show that manipulation of mating system parameters in C. maculatus generates intraspecific variation in the sex difference in TSF equal to that found among a diverse set of studies on insects.
C_LIO_LIOur study provides a causal link between the mating system and TSF. Sexual conflict, (re)mating rates, and genetic responses to sexual selection differ among ecological settings, mating systems and species. Our study therefore also provides mechanistic understanding for the variability in previously reported TSFs which can inform future experimental assays and predictions of species responses to climate warming.
C_LI

sentences: Population ecology of seabirds in Mexican Islands at the California Current System The Baja California Pacific Islands (BCPI) is a seabird hotspot in the southern California Current System supporting 129 seabird breeding populations of 23 species and over one million birds annually. These islands had a history of environmental degradation because of invasive alien species, human disturbance, and contaminants that caused the extirpation of 27 seabird populations. Most of the invasive mammals have been eradicated and breeding colonies have been restored with social attraction techniques. We have systematic information for most of the breeding populations since 2008. To assess population trends, we analyzed data and present results for 19 seabird species on ten island groups. The maximum number of breeding pairs for each nesting season was used to estimate the population growth rate ({lambda}) for each species at every island colony. We performed a nonparametric bootstrapping to assess whether seabird breeding populations are increasing or decreasing. San Benito, Natividad, and San Jeronimo are the top three islands in terms of abundance of breeding pairs. The most widespread species is Cassins Auklet with 14 colonies. Twenty-three populations of 13 species are significantly increasing while eight populations of six species are decreasing. We did not find statistical significance for 30 populations, however, 20 have {lambda}>1 which suggest they are growing. Seven of the 18 species for which we estimated a regional population trend are significantly increasing, including three surface-nesting species: Brown Pelican, Elegant Tern and Laysan Albatross, and four burrow-nesting species: Ainleys and Ashy Storm-Petrels, and Craveris and Guadalupe Murrelet. Our results suggest that the BCPI support healthy and growing populations of seabirds that have shown to be resilient to extreme environmental conditions such as the "Blob", and that such resilience has been strengthen from conservation and restoration actions such as the eradication of invasive mammals and social attraction techniques.

sentences: γ-TuRCs are required for asymmetric microtubule nucleation from the somatic Golgi of Drosophila neurons Microtubules are polarised polymers nucleated by multi-protein {gamma}-tubulin ring complexes ({gamma}-TuRCs). Within neurons, microtubule polarity is plus-end-out in axons and mixed or minus-end-out in dendrites. Previously we showed that within the soma of Drosophila sensory neurons {gamma}-tubulin localises asymmetrically to Golgi stacks, Golgi-derived microtubules grow asymmetrically towards the axon, and growing microtubule plus-ends are guided towards the axon and restricted from entering dendrite in a Kinesin-2-dependent manner (Mukerjee et al., 2020). Here we show that depleting {gamma}-TuRCs perturbs the direction of microtubule growth from the Golgi stacks, consistent with a model for asymmetric microtubule nucleation involving {gamma}-TuRCs and other nucleation-promoting factors. We also directly observe microtubule turning along microtubule bundles and show that depleting APC, proposed to link Kinesin-2 to plus ends, reduces microtubule turning and increases plus end growth into dendrites. These results support a model of asymmetric nucleation and guidance within the neuronal soma that helps establish and maintain overall microtubule polarity.

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Amyloidogenic proteins in the SARS-CoV and SARS-CoV-2 proteomes The phenomenon of protein aggregation is associated with a wide range of human diseases. Our knowledge on the aggregation behaviour of viral proteins, however, is still rather limited. Here, we investigated this behaviour in the the SARS-CoV and SARS-CoV-2 proteomes. An initial analysis using a panel of sequence-based predictors suggested the presence of multiple aggregation-prone regions in these proteomes, and revealed an enhanced aggregation propensity in some SARS-CoV-2 proteins. We then studied the in vitro aggregation of predicted aggregation-prone SARS-CoV-2 proteins, including the signal sequence peptide and fusion peptide 1 of the spike protein, a peptide from the NSP6 protein (NSP6-p), the ORF10 protein, and the NSP11 protein. Our results show that these peptides and proteins form aggregates via a nucleation-dependent mechanism. Moreover, we demonstrated that the aggregates of NSP11 are toxic to mammalian cell cultures. These findings provide evidence about the aggregation of proteins in the SARS-CoV-2 proteome.

SignificanceThe aggregation of proteins is linked with human disease in a variety of ways. In the case of viral infections, one could expect that the aberrant aggregation of viral proteins may damage the host cells, and also that viral particles may trigger the misfolding and aggregation of host proteins, resulting in damage to the host organism. Here we investigate the aggregation propensity of SARS-CoV-2 proteins and show that many of them can form aggregates that are potentially cytotoxic. In perspective, these results suggest that a better understanding of the effects of viruses on the human protein homeostasis system could help future therapeutic efforts.

sentences: In silico analysis and in planta production of recombinant ccl21/IL1β protein and characterization of its in vitro anti-tumor and immunogenic activity CCL21 has an essential role in anti-tumor immune activity. Epitopes of IL1{beta} have adjuvant activity without causing inflammatory responses. CCR7 and its ligands play a vital role in the immune balance; specifically, in transport of T lymphocytes and antigen-presenting cells such as dendritic cells to the lymph nodes. This study aimed to produce epitopes of CCL21 and IL1{beta} as a recombinant protein and characterize its in vitro anti-tumor and immunogenic activity. A codon-optimized ccl21/IL1{beta} gene was designed and synthesized from human genes. Stability and binding affinity of CCL21/IL1{beta} protein and CCR7 receptor were examined through in silico analyses. The construct was introduced into N. tabacum to produce this recombinant protein and the structure and function of CCL21/IL1{beta} were examined. Purified protein from transgenic leaves generated a strong signal in SDS PAGE and western blotting assays. FTIR measurement and MALDI-TOF/TOF mass spectrography showed that ccl21/IL-1{beta} was correctly expressed in tobacco plants. Potential activity of purified CCL21/IL1{beta} in stimulating the proliferation and migration of MCF7CCR7+ cancer cell line was investigated using the wound healing method. The results demonstrated a decrease in survival rate and metastasization of cancer cells in the presence of CCL21/IL1{beta}, and IC50 of CCL21 on MCF7 cells was less than that of non-recombinant protein. Agarose assay on PBMCsCCR7+ showed that CCL21/IL1{beta} has biological activity and there is a distinguishable difference between chemokinetic (CCL21) and chemotactic (FBS) movements. Overall, the results suggest that CCL21/IL1{beta} could be considered an effective adjuvant in future in vivo and clinical tests.

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Muscle fatigue revisited - Insights from optically pumped magnetometers Muscle fatigue is well characterized electromyographically, nevertheless only information about summed potential differences is detectable. In contrast, recently developed quantum sensors optically pumped magnetometers (OPMs) offer the advantage of recording both the electrical current propagation in the muscle as well as its geometry, by measuring the magnetic field generated by the muscular action potentials. Magnetomyographic investigation of muscle fatigue is still lacking and it is an open question whether fatigue is characterized similarly in magnetomyography (MMG) compared to electromyography (EMG). Herein, we investigated the muscle fatigue during a 3x1-min strong isometric contraction of the rectus femoris muscle of 12 healthy subjects using simultaneous EMG-MMG (4-channel surface EMG and 4 OPM along the rectus femoris muscle).

Both EMG and MMG showed the characteristic frequency decrease in the signal magnitude during isometric contraction, which is typical for muscle fatigue. In addition, it was shown that the main part of this frequency decrease seems to occur in the circular component of the magnetic field around the muscle fibers and less longitudinally along the muscle fibers. Overall, these results show not only that magnetomyography is capable of reproducing the electromyographic standards in identifying muscular fatigue, but it also adds relevant information about the spatial characterization of the signal. Therefore, OPM-MMG offers new insights for the study of muscular activity and might serve as a new, supplementary neurophysiological method.

sentences: A New Protocol for Targeted insertion using CRISPR-Cas9, Oligo Single-Stranded DNA and Protoplast Regeneration Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50.0% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60-nt donor containing 27 nt in each homologous arm, 6 of 22 regenerated N. benthamiana plants showed targeted insertions, and one contained a precise insertion of a 6-bp HindIII site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants.

sentences: Selective activation of subthalamic nucleus output quantitatively scales movements The subthalamic nucleus (STN), a diencephalic nucleus strongly connected with the basal ganglia, is a common target for deep brain stimulation (DBS) treatments of Parkinsonian motor symptoms. The STN is thought to suppress movement by enhancing inhibitory basal ganglia output via the indirect pathway, and disrupting STN output using DBS can restore movement in Parkinsons patients. However, previous studies on STN DBS usually relied on electrical stimulation, which cannot selectively target STN output neurons. Moreover, most studies did not measure behavior precisely during STN manipulations. Here we selectively stimulated STN projection neurons, by expressing channelrhodopsin in vesicular glutamate transporter 2 (VGlut2)-positive projection neurons in the STN. We also quantified behavior in freely moving male mice with high spatial and temporal resolution using 3D motion capture at 200 frames per second. We found STN stimulation resulted in movements with very short latencies (10-15 ms). Unilateral stimulation quantitatively determines orientation, head yaw, and head roll consistently in the ipsiversive direction, toward thee side of stimulation. On the other hand, head raising (increased head pitch) was observed after stimulation on either side. Strikingly, a single pulse of light was sufficient to generate movement, and there was a highly linear relationship between stimulation frequency and kinematics. These results question the common assumption that STN suppresses movement; instead they suggest that STN output can precisely specific action parameters via direct projections to brainstem regions.

sentences: Delayed effects of climate on vital rates lead to demographic divergence in Amazonian forest fragments Deforestation often results in landscapes where remaining forest habitat is highly fragmented, with remnants of different sizes embedded in an often highly contrasting matrix. Local extinction of species from individual fragments is common, but the demographic mechanisms underlying these extinctions are poorly understood. It is often hypothesized that altered environmental conditions in fragments drive declines in reproduction, recruitment, or survivorship. The Amazon basin, in addition to experiencing continuing fragmentation, is experiencing climate change related increases in the frequency and intensity of droughts and unusually wet periods. Whether plant populations in tropical forest fragments are particularly susceptible to extremes in precipitation remains unclear. Most studies of plants in fragments are relatively short (1-6 years), focus on a single life-history stage, and often do not compare to populations in continuous forest. Even fewer studies consider delayed effects of climate on demographic vital rates despite the importance of delayed effects in studies that consider them. Using a decade of demographic and climate data from an experimentally fragmented landscape in the Central Amazon, we assess the effects of climate on populations of an understory herb (Heliconia acuminata, Heliconiaceae). We used distributed lag non-linear models to understand the delayed effects of climate (measured as standardized precipitation evapotranspiration index, SPEI) on survival, growth, and flowering. We detected delayed effects of climate up to 36 months. Extremes in SPEI in the previous year reduced survival, drought in the wet season 8-11 months prior to the February census increased growth, and drought two dry seasons prior increased flowering probability. Effects of extremes in precipitation on survival and growth were more pronounced in forest fragments compared to continuous forest. The complex delayed effects of climate and habitat fragmentation in our study point to the importance of long-term demography experiments in understanding the effects of anthropogenic change on plant populations.

sentences: Neural harmonics of syntactic structure Can neural rhythms reflect purely internal syntactic processes in multi-word constructions? To test this controversial conjecture - relevant to language in particular and cognition more broadly - we recorded electroencephalographic and behavioural data as participants listened to isochronously presented sentences of varying in syntactic complexity. Each trial comprised ten concatenated sentences and was either fully grammatical (regular) or rendered ungrammatical via randomly distributed word order violations. We found that attending the regular repetition of abstract syntactic categories (phrases and sentences) generates neural rhythms whose harmonics are mathematically independent of word rate. This permits to clearly separate endogenous syntactic rhythms from exogenous speech rhythms. We demonstrate that endogenous but not exogenous rhythms predict participants grammaticality judgements, and allow for the neural decoding of regular vs. irregular trials. Neural harmonic series constitute a new form of behaviourally relevant evidence for syntactic competence.

sentences: Heterologous expression of human pro-inflammatory Caspase-1 in Saccharomyces cerevisiae and comparison to pro-apoptotic Caspase-8 Caspases are a family of cysteine proteases that play an essential role in inflammation, apoptosis, cell death, and development. Here we delve into the effects caused by heterologous expression of human Caspase-1 in the yeast Saccharomyces cerevisiae and compare them to those of Caspase-8. Overexpression of both caspases in the heterologous model led to their activation, and caused mitochondrial depolarization, ROS production, damage to different organelles, and cell death. All these effects were dependent on their protease activity, and Caspase-8 was more aggressive than Caspase-1. Growth arrest could be at least partially explained by dysfunction of the actin cytoskeleton as a consequence of the processing of the yeast Bni1 formin, which we identify here as a likely direct substrate of both caspases. Through the modulation of the GAL1 promoter by using different galactose:glucose ratios in the culture medium, we have established a scenario in which Caspase-1 is sufficiently expressed to become activated while yeast growth is not impaired. Finally, we used the yeast model to explore the role of death-fold domains (DD) of both caspases in their activity. Peculiarly, the DDs of either caspase showed an opposite involvement in its intrinsic activity, as the deletion of the caspase activation and recruitment domain (CARD) of Caspase-1 enhanced its activity, while the deletion of the death effector domain (DED) of Caspase-8 diminished it. We propose the yeast model as a useful and manageable tool to explore Caspase-1 structure-function relationships, the impact of mutations or the activity of putative inhibitors or regulators.

sentences: Improving antibody thermostability based on statistical analysis of sequence and structural consensus data The use of Monoclonal Antibodies (MAbs) as therapeutics has been increasing over the past 30 years due to their high specificity and strong affinity towards the target. One of the major challenges towards their use as drugs is their low thermostability, which impacts both efficacy as well as manufacturing and delivery. To aid the design of thermally more stable mutants, consensus sequence-based method has been widely used. These methods typically have a success rate of about 50% with maximum melting temperature increment ranging from 10 to 32{degrees}C. In order to improve the prediction performance, we have developed a new and fast MAbs specific method by adding a 3D structural layer to the consensus sequence method. This is done by analyzing the close-by residue pairs which are conserved in more than eight hundred MAbs 3D structures. Major advantage of this structural level assessment is in significantly reducing the false positives by almost half from the consensus sequence method alone. In summary, combining consensus sequence and structural residue pair covariance methods, we developed an inhouse application for predicting human MAb thermostability to guide protein engineers to design more stable molecules. This application has shown success in designing MAb engineering panels in multiple biologics programs.

sentences: Instant three color multi-plane fluorescence microscopy One of the most widely used microscopy techniques in biology and medicine is fluorescence microscopy, offering high specificity in labeling as well as maximum sensitivity. For live cell imaging, the ideal fluorescence microscope should offer high spatial resolution, fast image acquisition, three-dimensional sectioning, and multi-color detection. However, most existing fluorescence microscopes have to compromise between these different requirements. Here, we present a multi-plane multi-color wide-field microscope that uses a dedicated beam-splitter for recording volumetric data in eight focal planes and for three emission colors with frame rates of hundreds of volumes per second. We demonstrate the efficiency and performance of our system by three-dimensional imaging of multiply labeled fixed and living cells.

sentences: Highly replicated evolution of parapatric ecotypes Parallel evolution of ecotypes occurs when selection independently drives the evolution of similar traits across similar environments. The multiple origin of ecotypes is often inferred on the basis of a phylogeny which clusters populations according to geographic location and not by the environment they occupy. However, the use of phylogenies to infer parallel evolution in closely related populations is problematic because gene flow and incomplete lineage sorting can uncouple the genetic structure at neutral markers from the colonization history of populations. Here, we demonstrate multiple origins within ecotypes of an Australian wildflower, Senecio lautus. We observed strong genetic structure as well as phylogenetic clustering by geography and show that this is unlikely due to gene flow between parapatric ecotypes, which is surprisingly low. We further confirm this analytically by demonstrating that phylogenetic distortion due to gene flow often requires higher levels of migration than those observed in S. lautus. Our results imply that selection can repeatedly create similar phenotypes despite the perceived homogenizing effects of gene flow.

sentences: Nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants We are in the midst of the historic coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Although countless efforts to control the pandemic have been attempted--most successfully, vaccination1-3--imbalances in accessibility to vaccines, medicines, and diagnostics among countries, regions, and populations have been problematic. Camelid variable regions of heavy chain-only antibodies (VHHs or nanobodies)4 have unique modalities: they are smaller, more stable, easier to customize, and, importantly, less expensive to produce than conventional antibodies5, 6. We present the sequences of nine alpaca nanobodies that detect the spike proteins of four SARS-CoV-2 variants of concern (VOCs)--namely, the alpha, beta, gamma, and delta variants. We show that they can quantify or detect spike variants via ELISA and lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays7. The panel of nanobodies broadly neutralized viral infection by pseudotyped SARS-CoV-2 VOCs. Structural analyses showed that a P86 clone targeted epitopes that were conserved yet unclassified on the receptor-binding domain (RBD) and located inside the N-terminal domain (NTD). Human antibodies have hardly accessed both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins undetected by conventional antibodies and maintains activity against spike proteins carrying escape mutations.

sentences: Using embedded alginate microparticles to tune the properties of in situ forming poly(N-isopropylacrylamide)-graft-chondroitin sulfate bioadhesive hydrogels for replacement and repair of the nucleus pulposus of the intervertebral disc Low back pain (LBP) is a major public health issue associated with degeneration of the intervertebral disc (IVD). The early stages of degeneration are characterized by the dehydration of the central, gelatinous portion of the IVD, the nucleus pulposus (NP). One possible treatment approach is to replace the NP in the early stages of IVD degeneration with a hydrogel that restores healthy biomechanics while supporting tissue regeneration. The present study evaluates a novel thermosensitive hydrogel based on poly(N-isopropylacrylamide-graft-chondroitin sulfate) (PNIPAAM-g-CS) for NP replacement. The hypothesis was tested that the addition of freeze-dried, calcium crosslinked alginate microparticles (MPs) to aqueous solutions of PNIPAAm-g-CS would enable tuning of the rheological properties of the injectable solution, as well as the bioadhesive and mechanical properties of the thermally precipitated composite gel. Further, we hypothesized that the composite would support encapsulated cell viability and differentiation. Structure-material property relationships were evaluated by varying MP concentration and diameter. The addition of high concentrations (50 mg/mL) of small MPs (20 {+/-} 6 {micro}m) resulted in the greatest improvement in injectability, compressive mechanical properties, and bioadhesive strength of PNIPAAm-g-CS. This combination of PNIPAAM-g-CS and alginate MPs supported the survival, proliferation, and differentiation of adipose derived mesenchymal stem cells (ADMSCs) towards an NP-like phenotype in the presence of soluble GDF-6. When implanted ex vivo into the intradiscal cavity of degenerated porcine IVDs, the formulation restored the compressive and neutral zone (NZ) stiffnesses to intact values and resisted expulsion under lateral bending. Overall, results indicate the potential of the hydrogel composite to serve as a scaffold for supporting NP regeneration. This work uniquely demonstrates that encapsulation of re-hydrating polysaccharide-based MPs may be an effective method for improving key functional properties of in situ forming hydrogels for orthopaedic tissue engineering applications.

sentences: Tracing the genetics of neurological disease to the mutation-directed addition of single hydrogen bonds Mutations causative of neurological and neurodegenerative disease can occur in coding regions that specify protein domains of low sequence complexity. These autosomal dominant mutations can be idiosyncratic in their recurrent appearance at the same amino acid. Here we report studies of recurrent mutations in proline residues located within low complexity (LC) domains associated with the neurofilament light chain protein, the microtubule-associated tau protein, and the heterogeneous nuclear RNPA2 protein. All such mutations manifest their effects by directing formation of variant proteins endowed with the addition of a single, main chain hydrogen bond specified by the variant amino acid replacing proline. Here we show that methylation of the peptide backbone nitrogen atom associated with these variant amino acids eliminates the aberrant hydrogen bond and restores normal protein function.

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leADS: improved metabolic pathway inference based on active dataset subsampling Metabolic pathways are composed of reaction sequences catalyzed by enzymes. The set of reactions within and between cells comprises a reactome. Pathways and reactomes can be predicted from organismal or multi-organismal genomes using rule-based or machine learning methods. While machine learning methods overcome issues of probability and scale associated with rule-based methods, several complications remain that can degrade performance including inadequately labeled training data, missing feature information, and inherent imbalances in the distribution of pathways within a dataset. Here, we present leADS (multi-label learning based on active dataset subsampling), a machine learning method, that uses subsampling to reduce the negative impact of training loss due to class imbalance. We demonstrate leADs performance using organismal and multi-organismal datasets in relation to other machine learning pathway prediction methods.

Availability and implementationleADS is available under the GNU license at github.com/hallamlab/leADS. A wiki, including a tutorial, is available at github.com//hallamlab/leADS/wiki

Contactshallam@mail.ubc.ca

sentences: CADHERIN mediated AMIS localisation Individual cells within de novo polarising tubes and cavities must integrate their forming apical domains into a centralised apical membrane initiation site (AMIS). This is necessary to enable organised lumen formation within multi-cellular tissue. Despite the well documented importance of cell division in localising the AMIS, we have found a division-independent mechanism of AMIS localisation that relies instead on Cadherin-mediated cell-cell adhesion. Our study of de novo polarising mouse embryonic stem cells (mESCs) cultured in 3D suggest that cell-cell adhesion directs the localisation of apical proteins such as PAR-6 to a centralised AMIS. Unexpectedly, we also found that mESC cell clusters lacking functional E-Cadherin were still able to form a lumen-like cavity in the absence of AMIS localisation and did so at a later stage of development via a  closure mechanism, instead of via hollowing. This work suggests that there are two, interrelated mechanisms of apical polarity localisation: cell adhesion and cell division. Alignment of these mechanisms in space allows for redundancy in the system and ensures the development of a coherent epithelial structure within a growing organ.

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Maximizing the Anti-tumor Potential of Immune Checkpoint Blockade through Modulation of Myeloid-specific CXCL16 and STAT1 Signaling Sensitivity to immune checkpoint blockades (ICB) depends on the overall balance of immunogenic and immunosuppressive signals in the tumor immune microenvironment (TIME). Chemotherapy as an immunostimulatory strategy showed potential in improving ICBs clinical efficacy. Yet, evolution of highly plastic tumor-associated myeloid cells hinders ICBs potential to reach its full therapeutic potential. In this study, we leveraged single-cell transcriptomic and trajectory analyses to delineate TIME dynamics after chemotherapy priming. We found that metronomic chemotherapy (MCT) treatment led to an accelerated T cell exhaustion through CXCL16-mediated recruitment of peripheral immature myeloid cells and expansion of STAT1-driven PD-L1 expressing myeloid cells. Inhibiting STAT1 signaling in MCT-primed breast cancer relieved T cell exhaustion and significantly enhanced the efficacy of anti-PD-1 ICB treatment. Our study leveraged single-cell analyses to dissect the dynamics of breast cancer TIME and provides a pre-clinical rationale to translate the anti-STAT1 plus anti-PD-1 combinatorial immunotherapy regimen to maximize ICBs efficacy.

Manuscript SummarySingle-cell analyses on low dose chemotherapy primed breast tumor-associated immune cells demonstrates a parallel coexistence of immunogenic and immunosuppressive myeloid cell subsets. Modulating STAT1 signaling in the tumor microenvironment fine-tunes immunogenic and immunosuppressive balance and maximizes the anti-PD-1 immunotherapy efficacy in chemotherapy-primed breast cancer.

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Interdisciplinary analyses of Bronze Age communities from Western Hungary reveal complex population histories In this study we report 20 ancient shotgun genomes from present-day Western Hungary (3530 - 1620 cal BCE), mainly from previously understudied Baden, Somogyvar-Vinkovci, Kisapostag, and Encrusted Pottery archaeological cultures. Besides analysing archaeological, anthropological and genetic data, 14C and strontium isotope measurements complemented reconstructing the dynamics of the communities discovered at the site Balatonkeresztur. Our results indicate the appearance of an outstandingly high Mesolithic hunter-gatherer ancestry in the largest proportion (up to ~46%) among Kisapostag associated individuals, despite this component being thought to be highly diluted by the Early Bronze Age. We show that hunter-gatherer ancestry was likely derived from a previously unrecognised source in Eastern Europe that contributed mostly to prehistoric populations in Central Europe and the Baltic region. We revealed a patrilocal residence system and local female exogamy for this Kisapostag population that was also the genetic basis of the succeeding community of the Encrusted Pottery culture, represented by a mass grave that likely resulted from an epidemic. We also created a bioinformatic pipeline dedicated for archaeogenetic data processing. By developing and applying analytical methods for analysing genetic variants we found carriers of aneuploidy and inheritable genetic diseases. Furthermore, based on genetic and anthropological data, we present here the first female facial reconstruction from the Bronze Age Carpathian Basin.

SignificanceHere we present a genomic time transect study from the Carpathian Basin (3530 - 1620 cal BCE), that sheds light on local and interregional population processes. We not only discovered long-distance mobility to provide detailed analysis of yet understudied Bronze Age communities, but we also recovered a previously hidden remnant hunter-gatherer genetic ancestry and its contribution to various populations in Eastern and Central Europe. We integrated 14C and strontium isotope measurements to the interdisciplinary interpretation of a site with 19 individuals analysed, where patrilocal social organisation and several health-related genetic traits were detected. Furthermore, we developed new methods and method standards for computational analyses of archaic DNA, implemented to our newly developed and freely available bioinformatic pipeline.

sentences: Multimodal fusion analysis of functional, cerebrovascular and structural neuroimaging in healthy ageing subjects Cognitive ageing is a complex process which requires multimodal approach. Neuroimaging can provide insights into brain morphology, functional organization and vascular dynamics. However, most neuroimaging studies of ageing have focused on each imaging modality separately, limiting the understanding of interrelations between processes identified by different modalities and the interpretation of neural correlates of cognitive decline in ageing. Here, we used linked independent component analysis as a data-driven multimodal approach to jointly analyze magnetic resonance imaging of grey matter density, cerebrovascular, and functional network topographies. Neuroimaging and behavioural data (n = 215) from the Cambridge Centre for Ageing and Neuroscience study were used, containing healthy subjects aged 18 to 88. In the output components, fusion was found between structural and cerebrovascular topographies in multiple components with cognitive-relevance across the lifespan. A component reflecting global atrophy with regional cerebrovascular changes and a component reflecting right frontoparietal network activity were correlated with fluid intelligence over and above age and gender. No meaningful fusion between functional network topography and structural or cerebrovascular signals was observed. We propose that integrating multiple neuroimaging modalities allows to better characterize brain pattern variability and to differentiate brain changes in healthy ageing.

sentences: Interacting synapses stabilise both learning and neuronal dynamics in biological networks Distinct synapses influence one another when they undergo changes, with unclear consequences for neuronal dynamics and function. Here we show that synapses can interact such that excitatory currents are naturally normalised and balanced by inhibitory inputs. This happens when classical spike-timing dependent synaptic plasticity rules are extended by additional mechanisms that incorporate the influence of neighbouring synaptic currents and regulate the amplitude of their efficacy changes accordingly. The resulting control of excitatory plasticity by inhibitory activation, and vice versa, gives rise to quick and long-lasting memories as seen experimentally in receptive field plasticity paradigms. In models with additional dendritic structure, we observe experimentally reported clustering of co-active synapses that depends on initial connectivity and morphology. Finally, in recurrent neuronal networks, rich and stable dynamics with high input sensitivity emerge, providing transient activity that resembles recordings from motor cortex. Our model provides a general framework for codependent plasticity that frames individual synaptic modifications in the context of population-wide changes, allowing us to connect micro-level physiology with network-wide phenomena.

sentences: Trade-offs in biosensor optimization for dynamic pathway engineering Recent progress in synthetic biology allows the construction of dynamic control circuits for metabolic engineering. This technology promises to overcome many challenges encountered in traditional pathway engineering, thanks to their ability to self-regulate gene expression in response to bioreactor perturbations. The central components in these control circuits are metabolite biosensors that read out pathway signals and actuate enzyme expression. However, the construction of metabolite biosensors is a major bottleneck for strain design, and a key challenge is to understand the relation between biosensor dose-response curves and pathway performance. Here we employ multiobjective optimization to quantify performance trade-offs that arise in the design and calibration of metabolite biosensors. Our approach reveals strategies for tuning dose-response curves along an optimal trade-off between production flux and the cost of an increased expression burden on the host. We explore properties of control architectures built in the literature, and identify their advantages and caveats in terms of performance and robustness to growth conditions and leaky promoters. We demonstrate the optimality of a control circuit for glucaric acid production in Escherichia coli, which has been shown to increase titer by 2.5-fold as compared to static designs. Our results lay the groundwork for the automated design of control circuits for pathway engineering, with applications in the food, energy and pharmaceutical sectors.

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Non-random sister chromatid segregation mediates rDNA copy number maintenance in Drosophila Although considered to be exact copies of each other, sister chromatids can segregate non-randomly in some cases. For example, sister chromatids of the X and Y chromosomes segregate non-randomly during asymmetric division of male germline stem cells (GSCs) in D. melanogaster. Here we identify that the ribosomal DNA (rDNA) loci, which are located on the X and Y chromosomes, and an rDNA-binding protein, Indra, are required for non-random sister chromatid segregation (NRSS). We provide the evidence that NRSS is a mechanism by which GSCs recover rDNA copy number, which occurs through unequal sister chromatid exchange, counteracting the spontaneous copy number loss that occurs during aging. Our study reveals an unexpected role for NRSS in maintaining germline immortality through maintenance of a vulnerable genomic element, rDNA.

One Sentence SummaryrDNA copy number maintenance by non-random sister chromatid segregation contributes to germline immortality in Drosophila

sentences: Raman imaging and statistical methods for analysis various type of human brain tumor Spectroscopic methods provide information on the spatial localization of biochemical components based on the analysis of vibrational spectra. Raman spectroscopy and Raman imaging can be used to analyze various types of human brain tissue. The objective of this study is employment of Raman spectroscopy and Raman imaging to evaluate the Raman biomarkers to distinguish tumor types. We have demonstrated that bands characteristic for carotenoids (1156 cm-1, 1520 cm-1), proteins (1004 cm-1), fatty acids (1444 cm-1, 1655 cm-1) and cytochrome (1585 cm-1) can be used as universal biomarkers to distinguish aggressiveness in human brain tumor. The sensitivity and specificity obtained from PLS-DA have been over 85%. Only for pituitary adenoma the specificity is lower and takes equal 46%. The presented results confirm the potential applications of vibrational spectroscopy methods in oncological diagnostics.

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GuideMaker: Software to design CRISPR-Cas guide RNA pools in non-model genomes BackgroundCRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing the CRISPR-Cas guide RNAs for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for atypical projects with 1) non-standard Cas enzymes, 2) non-model organisms, or 3) projects that need to design a panel of guide RNAs (gRNA) for genome-wide screens.

FindingsGuideMaker can rapidly design gRNAs for gene targets across the genome from a degenerate protospacer adjacent motif (PAM) and a GenBank file. The tool applies Hierarchical Navigable Small World (HNSW) graphs to speed up the comparison of guide RNAs. This allows the user to design gRNAs targeting all genes in a typical bacterial genome in about 1-2 minutes.

ConclusionsGuidemaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind, it can efficiently process smaller eukaryotic genomes as well. GuideMaker is available as command-line software, a stand-alone web application, and a tool in the CyCverse Discovery Environment. All versions are available under a Creative Commons CC0 1.0 Universal Public Domain Dedication.

sentences: Genesis of bursting activity: Role of inhibitory constraints on persistent Na+ currents Some neurons express a plateau of spike activity greatly outlasting a stimulus, whereas others do not. This difference could be due to differential expression of ion channels (e.g., persistent Na+, and Ca2+) or similar expression of channels that differ in their properties. We compared three Aplysia neurons with varying capacity to generate plateau potentials: B51 with self-terminating plateaus, B64 with plateau potentials that do not self-terminate, and the regular spiking neuron B8. Our results indicate the three neurons expressed outward currents IA and ID, voltage-gated Ca2+ currents ICaL and ICaR, and persistent inward INaP. The most notable difference observed was a larger IA, ID, and IKCa currents in B8, and the plateau generating B64 did not express IKCa. Computational models suggest outward currents suppress and temporally constrain the plateau potential and that inward Ca2+ currents suppress plateau potentials when coupled with IKCa.

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Five years of citizen science and standardized field surveys reveal a threatened urban Eden for wild bees in Brussels, Belgium O_LIUrbanisation is often put forward as an important driver of biodiversity loss, including for pollinators such as wild bees. However, recent evidence shows that the mosaics of urban green spaces, and in particular certain categories of informal urban green spaces (IGS), can play an important role to help native wild bees thrive in cities.
C_LIO_LIHere, we describe the results of five years of citizen science and standardised field surveys of wild bees conducted at the Friche Josaphat, a 24-ha urban wasteland in the Brussels-Capital Region (Belgium). These field surveys were initiated following the planned restructuring and partial destruction of this site by the regional authorities.
C_LIO_LIWe recorded a total of 2,507 specimens belonging to 127 species of wild bees, i.e. 60.5% of the 210 species recorded regionally, including nine that are threatened with extinction at national or European scales. The Friche Josaphat encompasses a significant share of the functional and phylogenetic diversity of wild bees known from the Brussels-Capital Region and is one of the most species-rich localities known to date for wild bees in Belgium.
C_LIO_LICollectively, our results highlight the strong complementarity of citizen science and academic approaches in biodiversity surveys, and they reaffirm that wastelands are essential components of urban biodiversity. Our study stresses the need to provide biodiverse IGS with a formal status within the mosaic of urban green spaces, but also to acknowledge and safeguard their natural capital and the multiple ecosystem services they provide.
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sentences: Flagellum and toxin phase variation impacts intestinal colonization and disease development in a mouse model of Clostridioides difficile infection Clostridioides difficile is a major nosocomial pathogen that can cause severe, toxin-mediated diarrhea and pseudomembranous colitis. Recent work has shown that C. difficile exhibits heterogeneity in swimming motility and toxin production in vitro through phase variation by site-specific DNA recombination. The recombinase RecV reversibly inverts the flagellar switch sequence upstream of the flgB operon, leading to the ON/OFF expression of flagellum and toxin genes. How this phenomenon impacts C. difficile virulence in vivo remains unknown. We identified mutations in the right inverted repeat that reduced or prevented flagellar switch inversion by RecV. We introduced these mutations into C. difficile R20291 to create strains with the flagellar switch "locked" in either the ON or OFF orientation. These mutants exhibited a loss of flagellum and toxin phase variation during growth in vitro, yielding precisely modified mutants suitable for assessing virulence in vivo. In a hamster model of acute C. difficile infection, the phase-locked ON mutant caused greater toxin accumulation than the phase locked OFF mutant but did not differ significantly in the ability to cause acute disease symptoms. In contrast, in a mouse model, preventing flagellum and toxin phase variation affected the ability of C. difficile to colonize the intestinal tract and to elicit weight loss, which is attributable to differences in toxin production during infection. These results show that the ability of C. difficile to phase vary flagella and toxins influences colonization and disease development and suggest that the phenotypic variants generated by flagellar switch inversion have distinct capacities for causing disease.

sentences: '4-Aminoquinazoline-6,7-diol' derivatives for enhanced EGFR binding (as inhibitor) against Lung cancer The Epidermal growth factor receptor (EGFR) have been considered as potential targets for cancer therapy due to their role in regulating proliferation and survival of cancer cells. In the present study, the four  4-aminoquinazoline-6, 7-diol derivative molecules were selected to the theoretically investigate their inhibitory activity against EGFR protein. Molecules were evaluated on the basis of: (i) in-silico cytotoxic assay on human cancer cell lines expressing EGFR (A549 and A431), (ii) in-silico kinase assay, (iii) impact of molecular interaction on EGFR, (iv) molecular interaction stability, (v) inhibitory impact of molecules on combined expression of EGF-EGFR and (v) ADME observations. Studies were observed on the scale of two known EGFR inhibitors. In-silico cytotoxicity screening results demonstrated that the A431 lung cancer cell line was vulnerable to compound abc1-4. However, A549 cells were less sensitive to our designed molecules. From kinase inhibition assay results, compound abc1&2 were found to be an inhibitor against EGFR. According to the molecular interaction analysis, compounds abc1-4 performed in similar fashion of positive controls, acting at the catalytic site of EGFR. Altogether, these potent compounds, with compromised bioavailability, could likely be developed as a promising EGFR targeted drug(s) for cancer therapy.

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Neural Representations of Food-Related Attributes in the Human Orbitofrontal Cortex During Choice Deliberation in Anorexia Nervosa Decisions about what to eat recruit the orbitofrontal cortex (OFC) and involve the evaluation of food-related attributes, such as taste and health. These attributes are utilized differently by healthy individuals and patients with disordered eating behavior, but it is unclear whether these attributes are decodable from activity in the OFC in both groups and whether neural representations of these attributes are differentially related to decisions about food. We used fMRI combined with behavioral tasks to investigate the representation of taste and health attributes in the human OFC and the role of these representations in food choices in healthy individuals and patients with anorexia nervosa (AN). We found that subjective ratings of tastiness and healthiness could be decoded from patterns of activity in the OFC in both groups. However, health-related patterns of activity in the OFC were more related to the magnitude of choice preferences among patients with AN than healthy individuals. These findings suggest that maladaptive decision-making in AN is associated with more consideration of health information represented by the OFC during deliberation about what to eat.

Significance StatementAn open question about the orbitofrontal cortex (OFC) is whether it supports the evaluation of food-related attributes during deliberation about what to eat. We found that healthiness and tastiness information were decodable from patterns of neural activity in the OFC in both patients with anorexia nervosa (AN) and healthy controls. Critically, neural representations of health were more strongly related to choices in patients with AN, suggesting that maladaptive overconsideration of healthiness during deliberation about what to eat is related to activity in the OFC. More broadly, these results show that activity in the human OFC is associated with the evaluation of relevant attributes during value-based decision-making. These findings may also guide future research into the development of treatments for AN.

sentences: Quantification of salt stress in wheat leaves by Raman spectroscopy and machine learning The salinity level of the growing medium has diverse effects on the development of plants, including both physical and biochemical changes. To determine the salt stress level of a plant endures, one can measure these structural and chemical changes. Raman spectroscopy and biochemical analysis are some of the most common techniques in the literature. Here, we present a combination of machine learning and Raman spectroscopy with which we can both find out the biochemical change that occurs while the medium salt concentration changes and predict the level of salt stress a wheat sample experiences accurately using our trained regression models. In addition, by applying different machine learning algorithms, we compare the level of success for different algorithms and determine the best method to use in this application. Production units can take actions based on the quantitative information they get from the trained machine learning models related to salt stress, which can potentially increase efficiency and avoid the loss of crops.

sentences: Peripheral NOD-like receptor deficient inflammatory macrophages trigger neutrophil infiltration disrupting daytime locomotion Inflammation is known to disrupt normal behavior, yet the underlying neuroimmune interactions remain elusive. Here, we investigated whether inappropriate macrophage-evoked inflammation alters CNS control of daily-life animal locomotion using a set of zebrafish mutants selected for specific macrophage dysfunction and microglia deficiency. Large-scale genetic and computational analyses revealed that NOD-like receptor nlrc3l mutants are capable of normal motility and visuomotor response, but preferentially swim less in the daytime, suggesting low motivation rather than physical impairment. Examining their brain activities and structures implicate impaired dopaminergic descending circuits, where neutrophils abnormally infiltrate. Furthermore, neutrophil depletion recovered daytime locomotion. Restoring wild-type macrophages reversed behavioral and neutrophil aberrations, while three other microglia-lacking mutants failed to phenocopy nlrc3l mutants. Overall, we reveal how peripheral inflammatory macrophages with elevated pro-inflammatory cues (including il1b, tnfa, cxcl8a) in the absence of microglia co-opt neutrophils to infiltrate the brain, thereby enabling local modulation of neural circuits affecting spontaneous locomotion.

sentences: Gene duplications and phylogenomic conflict underlie major pulses of phenotypic evolution in gymnosperms Inferring the intrinsic and extrinsic drivers of species diversification and phenotypic disparity across the Tree of Life is a major challenge in evolutionary biology. In green plants, polyploidy (or whole-genome duplication, WGD) is known to play a major role in microevolution and speciation1, but the extent to which WGD has shaped macroevolutionary patterns of diversification and phenotypic innovation across plant phylogeny remains an open question. Here we examine the relationship of various facets of genomic evolution--including gene and genome duplication, genome size, and chromosome number--with macroevolutionary patterns of phenotypic innovation, species diversification, and climatic occupancy in gymnosperms. We show that genomic changes, such as WGD and genome-size shifts, underlie the origins of most major extant gymnosperm clades, and notably our results support an ancestral WGD in the gymnosperm lineage. Spikes of gene duplication typically coincide with major spikes of phenotypic innovation, while increased rates of phenotypic evolution are typically found at nodes with high gene-tree conflict, representing historic population-level dynamics during speciation. Most shifts in gymnosperm diversification since the rise of angiosperms are decoupled from putative WGDs and instead are associated with increased rates of climatic occupancy evolution, particularly in cooler and/or more arid climatic conditions, suggesting that ecological opportunity, especially in the later Cenozoic, and environmental heterogeneity have driven a resurgence of gymnosperm diversification. Our study provides critical insight on the processes underlying diversification and phenotypic evolution in gymnosperms, with important broader implications for the major drivers of both micro- and macroevolution in plants.

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Lifestyle and Genetic Factors Modify Parent-of-Origin Effects on the Human Methylome Backgroundparent-of-origin effects (POE) play important roles in development and complex disease and thus understanding their regulation and associated molecular and phenotypic variation are warranted. Previous studies have mainly focused on the detection of genomic regions or phenotypes regulated by POE. Understanding whether POE may be modified by environmental or genetic exposures is important for understanding of the source of POE-associated variation, but only a few case studies addressing these modifiable POE exist.

Methodsin order to understand this high order of POE regulation, we screened 101 genetic and environmental factors such as "predicted mRNA expression levels" of DNA methylation/imprinting machinery genes and early/late lifestyle/environmental exposures. POE-mQTL-modifier interaction models were proposed to test the potential of these factors to modify POE at DNA methylation using data from Generation Scotland: The Scottish Family Health Study(N=2315).

Resultsa set of vulnerable/modifiable POE-CpGs were identified (modifiable-POE-regulated CpGs, N=3). Four factors, "lifetime smoking status" and "predicted mRNA expression levels" of TET2, SIRT1 and KDM1A, were found to significantly modify the POE on the three CpGs in both discovery and replication datasets. Importantly, the POE on one of the CpGs were modified by both genetic and environmental factors. We further identified plasma protein and health-related phenotypes associated with the methylation level of one of the identified CpGs.

Conclusionsthe modifiable POE identified here revealed an important yet indirect path through which genetic background and environmental exposures introduce their effect on DNA methylation, motivating future comprehensive evaluation of the role of these modifiers in complex diseases.

Key MessagesO_LIPrevious population studies showed that parent-origin-effects(POE) on human methylome can be widespread and affect health-related traits and diseases.
C_LIO_LIWhether the POE remained stable throughout the life or can be modified by genetic or environmental factors were largely unknown.
C_LIO_LIBy systematically screening 101 genetic and environmental factors in a large cohort(GS:SFHS) we provided the first population-level replicated evidence that those measuring lifestyle (smoking) and predicted expression of DNA methylation- or imprinting-machinery genes are amongst the factors that can modulate the POE of mQTLs for a set of CpG sites.
C_LIO_LIWe found those modifiable-POE-regulated CpGs are also phenotypically relevant - one is associated with the plasma levels of CLEC4C and health-related phenotypes such as HDL levels.
C_LIO_LIThe modifiable POE identified here revealed an important yet indirect path through which genetic background and environmental exposures introduce their effect on DNA methylation and their potential phenotypic consequences. This also provided a paradigm for further studies to explore how environmental and genetic effects can be integrated at methylation level.
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sentences: The Impact of Neuron Morphology on Cortical Network Architecture It has become increasingly clear that the neurons in the cerebral cortex are not randomly interconnected. This wiring specificity can result from synapse formation mechanisms that interconnect neurons depending on their activity or genetically defined identity. Here we report that in addition to these synapse formation mechanisms, the structural composition of the neuropil provides a third prominent source by which wiring specificity emerges in cortical networks. This structurally determined wiring specificity reflects the packing density, morphological diversity and similarity of the dendritic and axonal processes. The higher these three factors are, the more recurrent the topology of the networks. Conversely, low density, diversity and similarity yield feedforward networks. These principles predict connectivity patterns from subcellular to network scales that are remarkably consistent with empirical observations from a rich body of literature. Thus, cortical network architectures reflect the specific morphological properties of their constituents to much larger degrees than previously thought.

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A potent myeloid response is rapidly activated in the lungs of premature Rhesus macaques exposed to intra-uterine inflammation Intrauterine inflammation/infection (IUI), which is present in up to 40% of premature births, leads to elevated levels of pro-inflammatory mediators and microbial products within the amniotic fluid, which come in close contact to fetal mucosae. Yet, knowledge on the fetal mucosal responses to IUI exposure remains limited. To address these questions, we used a non-human primate model of IUI, in which pregnant Rhesus macaques received intra-amniotic (IA) LPS, compared with IA saline.

We found that IA LPS exposure induced a robust and rapid inflammation of the fetal lung, but not the intestine. This inflammatory response was characterized by high levels of pro-inflammatory cytokines in the lung and the alveolar wash, and a potent myeloid cell response, dominated by neutrophils and monocytes/macrophages. scRNAseq analyses of fetal lungs showed that the infiltrating (neutrophils and inflammatory monocytes) and the resident (alveolar and interstitial macrophages) myeloid cells exhibited transcriptional profiles consistent with exposure to TLR ligands, as well as to cytokines, notably IL-1 and TNF. However, blocking IL-1 signaling or TNF, alone or simultaneously by administering inhibitors intra-amniotically and subcutaneously to the dam only partially blunted fetal lung inflammation.

Together, our novel data indicate that the fetal innate immune system can mount a rapid multi-factorial mucosal innate response to IUI, responding both to direct signaling by bacterial products and to indirect cytokine-mediated pathways of activation. These data thus provide more mechanistic insights into the association between IUI exposure and the post-natal lung morbidities of the premature infant.

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Long-Term Inactivation of Sodium Channels as a Mechanism of Adaptation in CA1 Pyramidal Neurons Many hippocampal CA1 pyramidal cells function as place cells, increasing their firing rate when a specific place field is traversed. The dependence of CA1 place cell firing on position within the place field is asymmetric. We investigated the source of this asymmetry by injecting triangular depolarizing current ramps to approximate the spatially-tuned, temporally-diffuse depolarizing synaptic input received by these neurons while traversing a place field. Ramps were applied to CA1 pyramidal neurons from male rats in vitro (slice electrophysiology) and in silico (multi-compartmental NEURON model). Under control conditions, CA1 neurons fired more action potentials at higher frequencies on the up-ramp versus the down-ramp. This effect was more pronounced for dendritic compared to somatic ramps. We incorporated a four-state Markov scheme for NaV1.6 channels into our model and calibrated the spatial dependence of long-term inactivation according to the literature; this spatial dependence was sufficient to explain the difference in dendritic versus somatic ramps. Long-term inactivation reduced the firing frequency by decreasing open-state occupancy, and reduced spike amplitude during trains by decreasing occupancy in closed states, which comprise the available pool. PKC activator phorbol-dibutyrate, known to reduce NaV long-term inactivation, removed spike amplitude attenuation in vitro more visibly in dendrites and greatly reduced adaptation, consistent with our hypothesized mechanism. Intracellular application of a peptide inducing long-term NaV inactivation elicited spike amplitude attenuation during spike trains in the soma and greatly enhanced adaptation. Our synergistic experimental/computational approach shows that long-term inactivation of NaV1.6 is a key mechanism of adaptation in CA1 pyramidal cells.

Significance statementThe hippocampus plays an important role in certain types of memory, in part through context-specific firing of "place cells"; these cells were first identified in rodents as being particularly active when an animal is in a specific location in an environment, called the place field of that neuron. In this in vitro/in silico study, we found that long-term inactivation of sodium channels causes adaptation in the firing rate that could potentially skew the firing of CA1 hippocampal pyramidal neurons earlier within a place field. A computational model of the sodium channel revealed differential regulation of spike frequency and amplitude by long-term inactivation, which may be a general mechanism for spike frequency adaptation in the central nervous system.

sentences: Loss of SUMO-specific protease 2 causes isolated glucocorticoid deficiency by blocking adrenal cortex zonal transdifferentiation SUMOylation is a dynamic posttranslational modification, that provides fine-tuning of protein function involved in the cellular response to stress, differentiation, and tissue development. In the adrenal cortex, an emblematic endocrine organ that mediates adaptation to physiological demands, the SUMOylation gradient is inversely correlated with the gradient of cellular differentiation raising important questions about its role in functional zonation and the response to stress. Considering that SUMO-specific protease 2 (SENP2), a deSUMOylating enzyme, is upregulated by ACTH/PKA signalling within the zona Fasciculata (zF), we generated mice with adrenal-specific Senp2 loss to address these questions. Disruption of SENP2 activity in steroidogenic cells leads to specific hypoplasia of the zF, a blunted reponses to ACTH and isolated glucocorticoid deficiency. Mechanistically, overSUMOylation resulting from SENP2 loss shifts the balance between ACTH/PKA and WNT/{beta}-catenin signalling leading to repression of PKA activity and ectopic activation of {beta}-catenin. At the cellular level, this blocks transdifferentiation of {beta}-catenin-positive zona Glomerulosa cells into zF cells and sensitises them to premature apoptosis. Our findings indicate that the SUMO pathway is critical for adrenal homeostasis and stress responsiveness.

sentences: piRNAs of Caenorhabditis elegans broadly silence nonself sequences through functionally random targeting Small non-coding RNAs such as piRNAs serve as guides for Argonaute proteins, enabling sequence-specific, post-transcriptional regulation of gene expression. The piRNAs of Caenorhabditis elegans have been observed to bind targets with high mismatch tolerance, and appear to lack specific transposon targets, unlike piRNAs in Drosophila melanogaster and other organisms. These observations support a model in which C. elegans piRNAs provide a broad, indiscriminate net of silencing, acting in competition with siRNAs associated with the CSR-1 Argonaute that specifically protect self genes from silencing. However, the breadth of piRNA targeting has not been subject to in-depth quantitative analysis, nor has it been explained how piRNAs are distributed across sequence space to achieve complete coverage. Through a bioinformatic analysis of piRNA sequences, incorporating an original metric of piRNA-target distance, we demonstrate that C. elegans piRNAs are functionally random, in that their coverage of sequence space is comparable to that of random sequences. By possessing a sufficient number of distinct, essentially random piRNAs, C. elegans is able to target arbitrary nonself sequences with high probability. This result elucidates the mechanism by which newly transcribed mRNAs in C. elegans are classified as self or nonself, and has implications for piRNA evolution and biogenesis.

sentences: Modularity and connectivity of nest structure scale with colony size Large body sizes have evolved structures to facilitate resource transport. Like unitary organisms, social insect colonies must transport information and resources. Colonies with more individuals may experience transport challenges similar to large-bodied organisms. In ant colonies, transport occurs in the nest, which may consist of structures that facilitate movement. We examine three attributes of nests that might have evolved to mitigate transport challenges related to colony size: (1) subdivision - nests of species with large colonies are more subdivided to reduce crowd viscosity; (2) branching - nest tunnels increase branching in species with large colonies to reduce travel distances; and (3) short-cuts - nests of species with large colonies have cross-linking tunnels to connect distant parts of the nest and create alternative routes. We test these hypotheses by comparing nest structures of species with different colony sizes in phylogenetically controlled meta-analyses. Our findings support the hypothesis that nest subdivision and branching evolved to mitigate transport challenges related to colony size. Nests of species with large colonies contain more chambers and branching tunnels. The similarity in how ant nests and bodies of unitary organisms have evolved in response to increasing size suggests common solutions across taxa and levels of biological organization.

sentences: The megabase-scale crossover landscape is independent of sequence divergence Meiotic recombination frequency varies along chromosomes and strongly correlates with sequence divergence. However, the causality underlying this correlation is unclear. To untangle the relationship between recombination landscapes and polymorphisms, we characterized the genome-wide recombination landscape in the absence of polymorphisms, using Arabidopsis thaliana homozygous inbred lines in which a few hundred genetic markers were introduced through mutagenesis. We found that megabase-scale recombination landscapes in inbred lines are strikingly similar to the recombination landscapes in hybrids, with the sole exception of heterozygous large rearrangements where recombination is prevented locally. In addition, we found that the megabase-scale recombination landscape can be accurately predicted by chromatin features. Our results show that polymorphisms are not causal for the shape of the megabase-scale recombination landscape, rather, favor alternative models in which recombination and chromatin shape sequence divergence across the genome.

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Automatic grading of cervical biopsies by combining full and self-supervision In computational pathology, the application of Deep Learning to the analysis of Whole Slide Images (WSI) has provided results of unprecedented quality. Due to their enormous size, WSIs have to be split into small images (tiles) which are first encoded and whose representations are then agglomerated in order to solve prediction tasks, such as prognosis or treatment response. The choice of the encoding strategy plays a key role in such algorithms. Current approaches include the use of encodings trained on unrelated data sources, full supervision or self-supervision. In particular, self-supervised learning (SSL) offers a great opportunity to exploit all the unlabelled data available. However, it often requires large computational resources and can be challenging to train. On the other end of the spectrum, fully-supervised methods make use of valuable prior knowledge about the data but involve a costly amount of expert time.

This paper proposes a framework to reconcile SSL and full supervision and measures the trade-off between long SSL training and annotation effort, showing that a combination of both has the potential to substantially increase performance. On a recently organized challenge on grading Cervical Biopsies, we show that our mixed supervision scheme reaches high performance (weighted accuracy (WA): 0.945), outperforming both SSL (WA: 0.927) and transfer learning from ImageNet (WA: 0.877). We further provide insights and guidelines to train a clinically impactful classifier with a limited expert and/or computational workload budget. We expect that the combination of full and self-supervision is an interesting strategy for many tasks in computational pathology and will be widely adopted by the field.

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Accurate self-assessment in 2D: protein contact map quality estimation by deep evolutionary reconciliation MotivationProtein contact maps have proven to be a valuable tool in the deep learning revolution of protein structure prediction, ushering in the recent breakthrough by AlphaFold2. However, self-assessment of the quality of predicted structures is typically performed at the granularity of 3D coordinates as opposed to directly exploiting the rotation- and translation-invariant 2D contact maps.

ResultsHere we present rrQNet, a deep learning method for self-assessment in 2D by contact map quality estimation. Our approach is based on the intuition that for a contact map to be of high quality, the residue pairs predicted to be in contact should be mutually consistent with the evolutionary context of the protein. The deep neural network architecture of rrQNet implements this intuition by cascading two deep modules--one encoding the evolutionary context and the other performing evolutionary reconciliation. The penultimate stage of rrQNet estimates the quality scores at the interacting residue-pair level, which are then aggregated for estimating the quality of a contact map. This design choice offers versatility at varied resolutions from individual residue pairs to full-fledged contact maps. Trained on multiple complementary sources of contact predictors, rrQNet facilitates generalizability across various contact maps. By rigorously testing using publicly available datasets and comparing against several in-house baseline approaches, we show that rrQNet accurately reproduces the true quality score of a predicted contact map and successfully distinguishes between accurate and inaccurate contact maps predicted by a wide variety of contact predictors.

AvailabilityThe open-source rrQNet software package is freely available at https://github.com/Bhattacharya-Lab/rrQNet.

Contactdbhattacharya@vt.edu

sentences: Evolutionary history of mammalian UDP-glucuronosyltransferase (UGT)1 and UGT2 families: the emergence of UGT2B subfamily in eutherians after the diversification of flowering plants The UDP-glucuronosyltransferase (UGT) gene family is responsible for the transfer of glucuronic acid to exogenous and endogenous chemicals. Based on the highly diversified number of genes, the mammalian UGT1A and UGT2B subfamily genes are believed to be involved in the conjugation reactions of xenobiotic metabolism. However, it is speculated that the UGT2 family genes are not involved in the xenobiotic metabolism of avian species due to the less diverse number of genes. In this study, we aimed to investigate the evolutionary history of mammalian UGT1 and UGT2 family genes and determine when the diversification of UGT2B genes occurred. We also attempted to identify the main factors responsible for the diversification of UGT genes. By examining the genomic information and feeding habits of 67 species representing each mammalian family, we discovered that the UGT2B genes emerged in the Eutheria on or after Cretaceous period and that their number were higher in plant-eating mammals (herbivore or omnivore) than in carnivorous mammals. We also found that the UGT2B genes in some herbivorous mammals underwent positive selection. In contrast, the diversity of the UGT1 family genes was inherited from the common ancestor of birds and mammals. Thus, our findings suggest that the emergence of angiosperms (flowering plants) and the occurrence of "animal-plant warfare" influenced the evolution of this gene family involved in the xenobiotic metabolism of eutherians. Furthermore, future research investigating the marsupials and birds that do not possess UGT2B genes is required to elucidate the mechanisms underlying the metabolism of chemical substances in these species.

sentences: Analyzing and Reconciling Colocalization and Transcriptome-wide Association Studies from the Perspective of Inferential Reproducibility Transcriptome-wide association studies and colocalization analysis are popular computational approaches for integrating genetic association data from molecular and complex traits. They show the unique ability to go beyond variant-level genetic association evidence and implicate critical functional units, e.g., genes, in disease etiology. However, in practice, when the two approaches are applied to the same molecular and complex trait data, the inference results can be markedly different. This paper systematically investigates the inferential reproducibility between the two approaches through theoretical derivation, numerical experiments, and analyses of 4 complex trait GWAS and GTEx eQTL data. We identify two classes of inconsistent inference results. We find that the first class of inconsistent results may suggest an interesting biological phenomenon, i.e., horizontal pleiotropy; thus, the two approaches are truly complementary. The inconsistency in the second class can be understood and effectively reconciled. To this end, we propose a novel approach for locus-level colocalization analysis. We demonstrate that the joint TWAS and locus-level colocalization analysis improves specificity and sensitivity for implicating biological-relevant genes.

sentences: Nonlinear averaging in 2D: intraspecific variation in two interacting species and consequences for population dynamics Recent empirical and theoretical work shows that intraspecific trait variation (ITV) is prevalent and ecologically important, and should thus be taken into account in ecological models. What is lacking, however, is a comprehensive understanding of the joint effects of ITV in two interacting species (two-dimensional ITV). Here we address this gap for the cases where interspecific individual-by-individual interactions are affected by the trait values of both participants. Using nonlinear averaging in two dimensions, we show for several interaction functions how ITV affects average predation rates or competition coefficients. We develop an intuition for the direction and magnitude of this effect by using a Taylor approximation based on the local curvatures of the interaction function, the trait means, and the trait variances and covariances. We then incorporate the estimated average interaction parameters into simple competition and predator-prey models to derive the expected population-dynamic consequences. We show that two-dimensional ITV can have quantitative effects on abundances, as well as qualitative effects, such as stabilizing or destabilizing coexistence. Our approach can straightforwardly be applied to other interaction functions and dynamical systems and thus provides a valuable tool for understanding the joint effect of trait variation in two interacting species.

sentences: Miniaturizing wet scrubbers for aerosolized droplet capture Aerosols dispersed and transmitted through the air (e.g., particulate matter pollution, bioaerosols) are ubiquitous and one of the leading causes of adverse health effects and disease transmission. A variety of sampling methods (e.g., filters, cyclones, impactors) have been developed to assess personal exposures. However, a gap still remains in the accessibility and ease-of-use of these technologies for people without experience or training in collecting airborne samples. Additionally, wet scrubbers (large non-portable industrial systems) utilize liquid sprays to remove aerosols from the air; the goal is to "scrub" (i.e., clean) the exhaust of industrial smokestacks, not collect the aerosols for analysis. Inspired by wet scrubbers, we developed a device fundamentally different from existing portable air samplers by using aerosolized microdroplets to capture aerosols in personal spaces (e.g., homes, offices, schools). Our aerosol-sampling device is the size of a small teapot, can be operated without specialized training, and features a winding flow path in a supersaturated relative humidity environment enabling droplet growth. The integrated open mesofluidic channels shuttle coalesced droplets to a collection chamber for subsequent sample analysis. Here, we present the experimental demonstration of aerosol capture into water droplets. Iterative study optimized the non-linear flow manipulating baffles and enabled an 83% retention of the aerosolized microdroplets in the confined volume of our device. As a proof-of-concept for aerosol capture into a liquid medium, 0.5-3 {micro}m model particles were used to evaluate aerosol capture efficiency. Finally, we demonstrate the device can capture and keep a bioaerosol (bacteriophage MS2) viable for downstream analysis.

sentences: Tightly-coupled inhibitory and excitatory functional networks in the early visual cortex Intracortical inhibition plays a critical role in shaping activity patterns in the mature cortex. However, little is known about the structure of inhibition in early development prior to the onset of sensory experience, a time when spontaneous activity exhibits long-range correlations predictive of mature functional networks. Here, using calcium imaging of GABAergic neurons in the early ferret visual cortex, we show that spontaneous activity in inhibitory neurons is already highly organized into distributed modular networks before visual experience. Inhibitory neurons exhibit spatially modular activity with long-range correlations and precise local organization that is in quantitative agreement with excitatory networks. Furthermore, excitatory and inhibitory networks are strongly co-aligned at both millimeter and cellular scales. These results demonstrate a remarkable degree of organization in inhibitory networks early in the developing cortex, providing support for computational models of self-organizing networks and suggesting a mechanism for the emergence of distributed functional networks during development.

sentences: Decoding assembly of alpha-helical transmembrane pores through intermediate states Membrane-active pore-forming alpha-helical peptides and proteins are well known for their dynamic assembly mechanism and it has been critical to delineate the pore-forming structures in the membrane. Previously, attempts have been made to elucidate their assembly mechanism and there is a large gap due to complex pathways by which these membrane-active pores impart their effect. Here we demonstrate the multi-step structural assembly pathway of alpha-helical peptide pores formed by a 37 amino-acid synthetic peptide, pPorU based on the natural porin from Corynebacterium urealyticum using single-channel electrical recordings. More specifically, we report detectable intermediates states during membrane insertion and pore formation of pPorU. The fully assembled pore is functional and exhibited unusually large stable conductance and voltage-dependent gating, generally applicable to a range of pore-forming proteins. Furthermore, we used rationally designed mutants to understand the role of specific amino acids in the assembly of these peptide pores. Mutant peptides that differ from wild-type peptides produced noisy, unstable intermediate states and low conductance pores, demonstrating sequence specificity in the pore-formation process supported by molecular dynamics simulations. We suggest that our study contributes to understanding the mechanism of action of alpha-helical pores and antimicrobial peptides and should be of broad interest to bioengineers to build peptide-based nanopore sensors.

sentences: A burst of regulatory and protein innovation at the origin of placental mammals drove the emergence of placenta and underpins divergent early pregnancy strategies in modern mammals. Summary paragraphThe origin of live birth in mammals [~]148 million years ago was a dramatic shift in reproductive strategy, yet the molecular changes that established mammal viviparity are largely unknown. Although progesterone receptor signalling predates the origin of mammals andis highly conserved in, and critical for, successful mammal pregnancy, it alone cannot explain the origin and subsequent diversity of implantation strategies throughout the placental mammal radiation. MiRNAs are known to be flexible and dynamic regulators with a well established role in the pathophysiology of mammal placenta. We propose that a dynamic core microRNA (miRNA) network originated early in placental mammal evolution, responds to conserved mammal pregnancy cues (e.g. progesterone), and facilitates species-specific responses. Here we identify 13 miRNAs that arose at the origin of placental mammals and were subsequently retained in all descendent lineages. The expression of these 13 miRNAs in response to early pregnancy molecules is regulated in a species-specific manner in endometrial epithelia of species with extremes of implantation strategies. Furthermore, these 13 miRNAs preferentially target 84 proteins under positive selective pressure on ancestral eutherian. Discovery of this core "live-birth" toolkit and specifically adapted proteins helps explain the origin and evolution of the placenta in mammals.

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Sulfonylureas target the neurovascular response to decrease Alzheimer's pathology Hyperexcitability is a defining feature of Alzheimers disease (AD), where aberrant neuronal activity is both a cause and consequence of AD. Therefore, identifying novel targets that modulate cellular excitability is an important strategy for treating AD. ATP-sensitive potassium (KATP) channels are metabolic sensors that modulate cellular excitability. Sulfonylureas are KATP channel antagonists traditionally used to combat hyperglycemia in diabetic patients by inhibiting pancreatic KATP channels, thereby stimulating insulin release. However, KATP channels are not limited to the pancreas and systemic modulation of KATP channels has pleotropic physiological effects, including profound effects on vascular function. Here, we demonstrate that human AD patients have higher cortical expression of vascular KATP channels, important modulators of vasoreactivity. We demonstrate that peripheral treatment with the sulfonylurea and KATP channel inhibitor, glyburide, reduced the aggregation and activity-dependent production of amyloid-beta (A{beta}), a hallmark of AD, in mice. Since glyburide does not readily cross the blood brain barrier, our data suggests that glyburide targets vascular KATP channel activity to reduce arterial stiffness, improve vasoreactivity, and normalize pericyte-endothelial cell morphology, offering a novel therapeutic target for AD.

Graphical abstractTargeting vascular KATP channel activity for the treatment of Alzheimers disease pathology.

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: Limited overlap of eQTLs and GWAS hits due to systematic differences in discovery Most signals in genome-wide association studies (GWAS) of complex traits point to noncoding genetic variants with putative gene regulatory effects. However, currently identified expression quantitative trait loci (eQTLs) explain only a small fraction of GWAS signals. By analyzing GWAS hits for complex traits in the UK Biobank, and cis-eQTLs from the GTEx consortium, we show that these assays systematically discover different types of genes and variants: eQTLs cluster strongly near transcription start sites, while GWAS hits do not. Genes near GWAS hits are enriched in numerous functional annotations, are under strong selective constraint and have a complex regulatory landscape across different tissue/cell types, while genes near eQTLs are depleted of most functional annotations, show relaxed constraint, and have simpler regulatory landscapes. We describe a model to understand these observations, including how natural selection on complex traits hinders discovery of functionally-relevant eQTLs. Our results imply that GWAS and eQTL studies are systematically biased toward different types of variants, and support the use of complementary functional approaches alongside the next generation of eQTL studies.

sentences: Lumican regulates cervical corticospinal axon collateralization via non-autonomous crosstalk between distinct corticospinal neuron subpopulations Corticospinal neurons (CSN) are the cortical projection neurons that innervate the spinal cord and some brainstem targets with segmental precision to control voluntary movement of specific functional motor groups, limb sections, or individual digits. CSN subpopulations exhibit striking axon targeting specificity from development into maturity: Evolutionarily newer rostrolateral CSN exclusively innervate bulbar-cervical targets (CSNBC-lat), while evolutionarily older caudomedial CSN (CSNmedial) are more heterogeneous, with distinct subpopulations extending axons to either bulbar-cervical or thoraco-lumbar segments. However, molecular regulation over specificity of CSN segmental target innervation is essentially unknown. The cervical cord, with its evolutionarily enhanced precision of forelimb movement, is innervated by multiple CSN subpopulations, suggesting inter-neuronal interactions in establishing cervical corticospinal circuitry. Here, we identify that Lumican, previously unrecognized in axon development, controls the balance of innervation between CSNBC-lat and CSNmedial within the cervical spinal cord. Remarkably, Lumican, an extracellular matrix protein expressed by CSNBC-lat, non-cell-autonomously suppresses axon collateralization in the cervical cord by CSNmedial. Intersectional viral labeling and mouse genetics further identify that Lumican controls axon collateralization by multiple CSN subpopulations in caudomedial sensorimotor cortex. These results identify inter-axonal molecular crosstalk between CSN subpopulations as a novel mechanism controlling corticospinal circuitry, target density, and competitive specificity. Further, this mechanism has potential implications for evolutionary diversification of corticospinal circuitry with finer scale precision.

sentences: Mental health explains individual deviations from normative range in cognition-associated brain states Links between cognitive deficits and psychiatric disorders have been studied predominantly at the group level, leaving unique individual characteristics largely elusive. Here, we applied normative modeling to UK Biobank data (N=18,634) and estimated the individual-level interplay of large-scale brain networks over time (i.e., dynamic brain state) as a function of general cognitive ability. Abnormality in such brain states was linked to individual variation in mental health. Specifically, brain state measures including fractional occupancy that indicates the brain state probability over time were estimated using a Hidden Markov Model, followed by a Gaussian process regression to estimate the normative range of these brain state measures from general cognitive ability. Abnormality scores per participant were quantified to represent the degree of deviations relative to the estimated population norm. We found significant associations between the abnormality scores of several brain states and individuals overall mental health. Our findings suggest potential impact of mental health on dynamic brain states that subserve cognitive functions and shed light on the relevant brain mechanisms underlying cognitive deficits in mental illness.

sentences: Effects of hyperinsulinemia on pancreatic cancer development and the immune microenvironment revealed through single-cell transcriptomics Hyperinsulinemia is independently associated with increased risk and mortality of pancreatic cancer. We recently reported that a [~]50% reduction in pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in mice could be achieved with reduced insulin production. However, only female mice remained normoglycemic and only the gene dosage of rodent-specific Ins1 alleles was tested in our previous model. Moreover, we did not delve into the molecular and cellular mechanisms associated with modulating hyperinsulinemia. Here, we studied PanIN lesion development in both male and female Ptf1aCreER;KrasLSL-G12D mice lacking the rodent specific Ins1 gene, and possessing one or two alleles of the wild-type Ins2 gene to modulate insulin production. High-fat diet induced hyperinsulinemia was transiently and modestly reduced, without affecting glucose tolerance, in male and female mice with only one allele of Ins2. Genetic reduction of insulin production resulted in mice with a tendency for less PanIN and acinar-to-ductal metaplasia (ADM) lesions. Using single-cell transcriptomics, we found hyperinsulinemia affected multiple cell types in the pancreas, with the most statistically significant effects on local immune cell populations, which were highly represented in our analysis. Specifically, hyperinsulinemia modulated pathways associated with protein translation, MAPK-ERK signaling, and PI3K-AKT signaling, which were changed in epithelial cells and subsets of immune cells. These data suggest a role for the immune microenvironment in hyperinsulinemia-driven PanIN development. Together with our previous work, we propose that mild suppression of insulin levels may be useful in preventing pancreatic cancer by acting on multiple cell types.

sentences: Identification of common key genes and pathways between Covid-19 and lung cancer by using protein-protein interaction network analysis COVID-19 is indeed an infection that is caused by a recently found coronavirus group, a type of virus proven to cause human respiratory diseases. The high mortality rate was observed in patients who had pre-existing health conditions like cancer. However, the molecular mechanism of SARS-CoV-2 infection in lung cancer patients was not discovered yet at the pathway level. This study was about determining the common key genes of COVID-19 and lung cancer through network analysis. The hub genes associated with COVID-19 and lung cancer were identified through Protein-Protein interaction analysis. The hub genes are ALB, CXCL8, FGF2, IL6, INS, MMP2, MMP9, PTGS2, STAT3 and VEGFA. Through gene enrichment, it is identified both COVID-19 and lung cancer have a common pathway in EGFR tyrosine kinase inhibitor resistance, IL-17 signalling pathway, AGE-RAGE signalling pathway in diabetic complications, HIF-1 signalling pathway and pathways in cancer.

sentences: Linking Diabetes mellitus to SARS-CoV-2 infection through differential targeting of the microRNAs in the Pancreas tissue Coronavirus Disease 2019 (COVID-19) severity and Diabetes mellitus affect each other bidirectionally. The plus-sense single-stranded RNA (+ssRNA) genome of the SARS-CoV-2 virus can be targeted and suppressed by the host cells microRNAs (miRNAs). Using the differential gene expression analysis between the mock-infected and the SARS-CoV-2-infected pancreatic tissue, we report five Diabetes-associated genes that are upregulated due to SARS-CoV-2 infection in the hESC pancreas tissues. Ten miRNAs regulating these five genes can potentially target the SARS-CoV-2 genome. We hypothesize that the SARS-CoV-2 genome copies in the infected human pancreas cell compete with the host cells native genes in being regulated by the native miRNAs. It leads to the reduced miRNA-regulation and, thus, the upregulation of the Diabetes-associated native genes. Thus, the resultant new-onset or elevated Diabetic symptoms may worsen the condition of COVID-19 patients.

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1,25(OH)2D3 attenuates   hepatic insulin resistance  in rats with streptozotocin -induced diabetes via antioxidation AimThis study was conducted to explore the mechanism by which 1,25(OH)2D3 ameliorates hepatic insulin resistance in rats with streptozotocin (STZ)-induced type 2 diabetes.

MethodsSprague Dawley (SD) rats were randomly divided into five groups: normal, control, and 1,25(OH)2D3 administered at 0.075, 0.15, and 0.3 g/kg/d. Tissue and blood samples were obtained from all five groups after 4 and 12 weeks of treatment. Morphological changes in the livers were observed with HE staining. The levels of fasting blood glucose (FBG), insulin, and lipids were determined using an automatic biochemistry analyzer. The levels of hepatic glycogen, superoxide dismutase (SOD), malondialdehyde (MDA), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were analyzed by spectrophotometry. Protein expression of AKT and GLUT2 was examined by western blotting.

Results1,25(OH)2D3 treatment reduced the FBG content as well as hepatic MDA, AST, and ALT levels, improved the activity of SOD, and enhanced the expression of GLUT2 and AKT in diabetic rats. These findings suggest that 1,25(OH)2D3, exerts antioxidant effects and ameliorates insulin resistance in the liver.

Conclusion1,25(OH)2D3 is a potential agent that could be used in the treatment of insulin resistance in the liver.

sentences: Tissue-wide coordination of calcium signaling regulates the epithelial stem cell pool during homeostasis Skin epidermal homeostasis is maintained via constant regeneration by stem cells, which must communicate to balance their self-renewal and differentiation. A key molecular pathway, Ca2+ signaling has been implicated as a signal integrator in developing and wounded epithelial tissues[1, 2, 3, 4]. Yet how stem cells carry out this signaling across a regenerative tissue remains unknown due to significant challenges in studying signaling dynamics in live mice, limiting our understanding of the mechanisms of stem cell communication during homeostasis. To interpret high dimensional signals that have complex spatial and temporal patterns, we combined optimized imaging of Ca2+ signaling in thousands of epidermal stem cells in living mice with a new machine learning tool, Geometric Scattering Trajectory Homology (GSTH). Using a combination of signal processing, data geometry, and topology, GSTH captures patterns of signaling at multiple scales, either between direct or distant stem cell neighbors. Here we show that epidermal stem cells display dynamic intercellular Ca2+ signaling among neighborhoods of up to 10 cells that is surprisingly coordinated and directed through time across a pool of thousands of stem cells. We find that this collective coordination is an emergent property of the stem cell compartment, distinct from excitatory quiescent neuronal tissues. We demonstrate that cycling stem cells, specifically G2 cells, govern homeostatic patterns of Ca2+ signaling. Stem cells in different cell cycle stages dynamically regulate localization of the gap junction component Connexin43 (Cx43). Lastly, we uncouple global from local communication and identify Cx43 as the molecular mediator necessary for connectivity between local signaling neighborhoods. This work provides resolution in how stem cells at different stages of the cell cycle communicate and how that diversity of phases is essential for tissue wide communication and signaling flow during epidermal regeneration. Our approach provides a framework to investigate stem cell populations and their signaling dynamics, previously not possible.

sentences: Uncertainty quantification in subject-specific estimation of local vessel mechanical properties Quantitative estimation of local mechanical properties remains critically important in the ongoing effort to elucidate how blood vessels establish, maintain, or lose mechanical homeostasis. Recent advances based on panoramic digital image correlation (pDIC) have made high-fidelity 3D reconstructions of small-animal (e.g., murine) vessels possible when imaged in a variety of quasi-statically loaded configurations. While we have previously developed and validated inverse modeling approaches to translate pDIC-measured surface deformations into biomechanical metrics of interest, our workflow did not heretofore include a methodology to quantify uncertainties associated with local point estimates of mechanical properties. This limitation has compromised our ability to infer biomechanical properties on a subject-specific basis, such as whether stiffness differs significantly between multiple material locations on the same vessel or whether stiffness differs significantly between multiple vessels at a corresponding material location. In the present study, we have integrated a novel uncertainty quantification and propagation pipeline within our inverse modeling approach, relying on empirical and analytic Bayesian techniques. To demonstrate the approach, we present illustrative results for the ascending thoracic aorta from three mouse models, quantifying uncertainties in constitutive model parameters as well as circumferential and axial tangent stiffness. Our extended workflow not only allows parameter uncertainties to be systematically reported, but also facilitates both subject-specific and group-level statistical analyses of the mechanics of the vessel wall.

sentences: Segmentation of Pancreatic Ductal Adenocarcinoma (PDAC) and surrounding vessels in CT images using deep convolutional neural networks and Texture Descriptors Fully automated and volumetric segmentation of critical tumors may play a crucial role in diagnosis and surgical planning. One of the most challenging tumor segmentation tasks is localization of Pancreatic Ductal Adenocarcinoma (PDAC). Exclusive application of conventional methods does not appear promising. Deep learning approaches has achieved great success in the computer aided diagnosis, especially in biomedical image segmentation. This paper introduces a framework based on convolutional neural network (CNN) for segmentation of PDAC mass and surrounding vessels in CT images by incorporating powerful classic features, as well. First, a 3D-CNN architecture is used to localize the pancreas region from the whole CT volume using 3D Local Binary Pattern (LBP) map of the original image. Segmentation of PDAC mass is subsequently performed using 2D attention U-Net and Texture Attention U-Net (TAU-Net). TAU-Net is introduced by fusion of dense Scale-Invariant Feature Transform (SIFT) and LBP descriptors into the attention U-Net. An ensemble model is then used to cumulate the advantages of both networks using a 3D-CNN. In addition, to reduce the effects of imbalanced data, a new loss function is proposed as a weighted combination of three classic losses including Generalized Dice Loss (GDL), Weighted Pixel-Wise Cross Entropy loss (WPCE) and boundary loss. Due to insufficient sample size for vessel segmentation, we used the above-mentioned pre-trained networks and fin-tuned them. Experimental results show that the proposed method improves the Dice score for PDAC mass segmentation in portal-venous phase by 7.52% compared to state-of-the-art methods (from 53.08% to 60.6%) in term of DSC. Besides, three dimensional visualization of the tumor and surrounding vessels can facilitate the evaluation of PDAC treatment response.

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Clustering single-cell RNA-seq data by rank constrained similarity learning MotivationRecent breakthroughs of single-cell RNA sequencing (scRNA-seq) technologies offer an exciting opportunity to identify heterogeneous cell types in complex tissues. However, the unavoidable biological noise and technical artifacts in scRNA-seq data as well as the high dimensionality of expression vectors make the problem highly challenging. Consequently, although numerous tools have been developed, their accuracy remains to be improved.

ResultsHere, we introduce a novel clustering algorithm and tool RCSL (Rank Constrained Similarity Learning) to accurately identify various cell types using scRNA-seq data from a complex tissue. RCSL considers both local similarity and global similarity among the cells to discern the subtle differences among cells of the same type as well as larger differences among cells of different types. RCSL uses Spearmans rank correlations of a cells expression vector with those of other cells to measure its global similarity, and adaptively learns neighbour representation of a cell as its local similarity. The overall similarity of a cell to other cells is a linear combination of its global similarity and local similarity. RCSL automatically estimates the number of cell types defined in the similarity matrix, and identifies them by constructing a block-diagonal matrix, such that its distance to the similarity matrix is minimized. Each block-diagonal submatrix is a cell cluster/type, corresponding to a connected component in the cognate similarity graph. When tested on 16 benchmark scRNA-seq datasets in which the cell types are well-annotated, RCSL substantially outperformed six state-of-the-art methods in accuracy and robustness as measured by three metrics.

AvailabilityThe RCSL algorithm is implemented in R and can be freely downloaded at https://github.com/QinglinMei/RCSL.

Contactguojunsdu@gmail.com, zcsu@uncc.edu

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: A Snapshot of the Global Drinking Water Virome: Diversity and Metabolic Potential Vary with Residual Disinfectant Use Viruses are important drivers of microbial community ecology and evolution, influencing microbial mortality, metabolism, and horizontal gene transfer. However, the effects of viruses remain largely unknown in many environments, including in drinking water systems. Drinking water metagenomic studies have offered a whole community perspective of bacterial impacts on water quality, but have not yet considered the influences of viruses. In this study, we address this gap by mining viral DNA sequences from publicly available drinking water metagenomes from distribution systems in six countries around the world. These datasets provide a snapshot of the taxonomic diversity and metabolic potential of the global drinking water virome; and provide an opportunity to investigate the effects of geography, climate, and drinking water treatment practices on viral diversity. Both environmental conditions and differences in sample processing were found to influence the viral composition. Using free chlorine as the residual disinfectant was associated with clear differences in viral taxonomic diversity and metabolic potential, with significantly fewer viral populations and less even viral community structures than observed in distribution systems without residual disinfectant. Additionally, drinking water viruses carry antibiotic resistance genes (ARGs), as well as genes to survive oxidative stress and nitrogen limitation. Through this study, we have demonstrated that viral communities are diverse across drinking water systems and vary with the use of residual disinfectant. Our findings offer directions for future research to develop a more robust understanding of how virus-bacteria interactions in drinking water distribution systems affect water quality.

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The wheat multi-pathogen resistance gene, Lr67res, confers a novel gain-of-function phenotype involving anion fluxes Partial resistance to multiple biotrophic fungal pathogens in wheat (Triticum aestivum L.) is conferred by the Lr67 gene, which encodes a Sugar Transport Protein 13 (STP13) family hexose-proton symporter variant. Two mutations (G144R, V387L) differentiate the resistant and susceptible protein variants (Lr67res and Lr67sus). The molecular function of the Lr67res protein is not well understood. We functionally characterized the wheat Lr67res protein variant using two heterologous expression systems - Xenopus laevis oocytes and Saccharomyces cerevisiae yeast. Wheat and barley (Hordeum vulgare) were used to verify disease resistance capability of Lr67/STP13 variants. The Lr67res allele, but not Lr67sus, induced large sugar-independent, anion-dominated currents in oocytes and an increased sensitivity to ions in yeast, implicating a novel gain-of-function. We demonstrate that the single mutant variant, Lr67susG144R, confers disease resistance in wheat and that transgenic barley (Hordeum vulgare L.) plants expressing the orthologous HvSTP13 gene carrying the equivalent mutations present in Lr67res exhibited increased resistance to Puccinia hordei. NaCl treatment was found to induce leaf tip necrosis in Lr67res wheat. An Lr67res-like gain-of-function can be introduced into orthologous plant hexose transporters via single amino acid mutation, highlighting the possibility of generating disease resistance in other crop species, especially with gene editing.

One sentence summaryThe wheat Lr67res protein responsible for multipathogen resistance has a gain-of-function over the Lr67sus protein, that is associated with anion fluxes and likely contributes to disease resistance.

sentences: Smell-induced gamma oscillations in human olfactory cortex are required for accurate perception of odor identity Studies of neuronal oscillations have contributed substantial insight into the mechanisms of visual, auditory and somatosensory perception. However, progress in such research in the human olfactory system has lagged behind. As a result, the electrophysiological properties of the human olfactory system are poorly understood, and in particular, whether stimulus-driven high frequency oscillations play a role in odor processing is unknown. Here, we used direct intracranial recordings from human piriform cortex during an odor identification task to show that three key oscillatory rhythms are an integral part of the human olfactory cortical response to smell: odor induces theta, beta and gamma rhythms in human piriform cortex. We further show that these rhythms have distinct relationships with perceptual behavior. Odor-elicited gamma oscillations occur only during trials in which the odor is accurately perceived, and features of gamma oscillations predict odor identification accuracy, suggesting they are critical for odor identity perception in humans. We also found that the amplitude of high-frequency oscillations is organized by the phase of low frequency signals shortly following sniff onset, only when odor is present. Our findings reinforce previous work on theta oscillations, suggest that gamma oscillations in human piriform cortex are important for perception of odor identity, and constitute a robust identification of the characteristic electrophysiological response to smell in the human brain. Future work will determine whether the distinct oscillations we identified reflect distinct perceptual features of odor stimuli.

sentences: Improved two-photon imaging of GPCR-based optogenetic neurotransmitter sensors using orthogonally polarized excitation Fluorescent proteins such as GFP are best excited by light that is polarized parallel to the dipole axis of the fluorophore. In most cases, fluorescent proteins are randomly oriented, resulting in unbiased images even when polarized light is used for excitation, e.g. in two-photon microcopy. Here we reveal a surprisingly strong polarization sensitivity in a class of GPCR-based neurotransmitter sensors where the fluorophore is anchored on both ends. In tubular structures such as dendrites, this effect led to a complete loss of membrane signal in dendrites running parallel to the polarization direction of the excitation beam. Our data reveal a major problem for two-photon measurements of neurotransmitter concentration that has not been recognized by the neuroscience community. To remedy the sensitivity to dendritic orientation, we designed an optical device that generates interleaved pulse trains of orthogonal polarization, removing the orientation bias from images. The passive device, which we inserted in the beam path of an existing two-photon microscope, also removed the strong direction bias in second harmonic generation (SHG) images. We conclude that for optical measurements of transmitter concentration with GPCR-based sensors, orthogonally polarized excitation is essential.

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MicroRNAs are deeply linked to the emergence of the complex octopus brain Soft-bodied cephalopods such as the octopus are exceptionally intelligent invertebrates with a highly complex nervous system that evolved independently from vertebrates. Because of elevated RNA editing in their nervous tissues, we hypothesized that RNA regulation may play a major role in the cognitive success of this group. We thus profiled mRNAs and small RNAs in 18 tissues of the common octopus. We show that the major RNA innovation of soft-bodied cephalopods is a massive expansion of the miRNA gene repertoire. These novel miRNAs were primarily expressed in neuronal tissues, during development, and had conserved and thus likely functional target sites. The only comparable miRNA expansions happened, strikingly, in vertebrates. Thus, we propose that miRNAs are intimately linked to the evolution of complex animal brains.

One-Sentence SummarymiRNAs are deeply linked to the emergence of complex brains.

sentences: Selecting an effective amplitude threshold for neural spike detection This paper assesses and challenges whether commonly used methods for defining amplitude thresholds for spike detection are optimal. This is achieved through empirical testing of single amplitude thresholds across multiple recordings of varying SNR levels. Our results suggest that the most widely used noise-statistics-driven threshold can suffer from parameter deviation in different noise levels. The spike-noise-driven threshold can be an ideal approach to set the threshold for spike detection, which suffers less from the parameter deviation and is robust to sub-optimal settings.

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Relaxation of purifying selection suggests low effective population size in eusocial Hymenoptera and solitary pollinating bees With one of the highest number of parasitic, eusocial and pollinator species among all insect orders, Hymenoptera features a great diversity of lifestyles. At the population genetic level, such life-history strategies are expected to decrease effective population size and the efficiency of purifying selection. In this study, we tested this hypothesis by estimating the relative rate of non-synonymous substitution in 169 species to investigate the variation in natural selection efficiency throughout the hymenopteran tree of life. We found no effect of parasitism, but we show that relaxed selection is associated with eusociality, suggesting that the division of reproductive labour decreases effective population size in ants, bees and wasps. Unexpectedly, the effect of eusociality is marginal compared to a striking and widespread relaxation of selection in both social and non social bees, which indicates that these keystone pollinator species generally feature low effective population sizes. This widespread pattern suggests specific constraints in pollinating bees potentially linked to limited resource availability and high parental investment. The particularly high load of deleterious mutations we report in the genome of these crucial ecosystem engineer species also raises new concerns about their ongoing population decline.

This article has been peer-reviewed and recommended by Peer Community in Evolutionary Biology https://doi.org/10.24072/pci.evolbiol.100120

sentences: Maize (Zea mays L.) nucleoskeletal proteins regulate nuclear envelope remodeling and function in stomatal complex development and pollen viability. In eukaryotes, the nuclear envelope (NE) encloses chromatin and separates it from the rest of the cell. The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex physically bridges across the NE, linking nuclear and cytoplasmic components. In plants, these LINC complexes are beginning to be ascribed roles in cellular and nuclear functions, including chromatin organization, regulation of nuclei shape and movement, and cell division. Homologs of core LINC components, KASH and SUN proteins, have previously been identified in maize. Here, we characterized the presumed LINC-associated maize nucleoskeletal proteins NCH1 and NCH2, homologs of members of the plant NMCP/CRWN family, and MKAKU41, homologous to AtKAKU4. All three proteins localized to the nuclear periphery when transiently and heterologously expressed as fluorescent protein fusions in Nicotiana benthamiana. Overexpression of MKAKU41 caused dramatic changes in the organization of the nuclear periphery, including nuclear invaginations that stained positive for non-nucleoplasmic markers of the inner and outer NE, and the ER. The severity of these invaginations was altered by changes in LINC connections and the actin cytoskeleton. In maize, MKAKU41 appeared to share genetic functions with other LINC components, including control of nuclei shape, stomatal complex development, and pollen viability. Overall, our data show that NCH1, NCH2, and MKAKU41 have characteristic properties of LINC-associated plant nucleoskeletal proteins, including interactions with NE components suggestive of functions at the nuclear periphery that impact the overall nuclear architecture.

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Efficacy of a Broadly Neutralizing SARS-CoV-2 Ferritin Nanoparticle Vaccine in Nonhuman Primates The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine in nonhuman primates (NHPs). High-dose (50 {micro}g) SpFN vaccine, given twice within a 28 day interval, induced a Th1-biased CD4 T cell helper response and a peak neutralizing antibody geometric mean titer of 52,773 against wild-type virus, with activity against SARS-CoV-1 and minimal decrement against variants of concern. Vaccinated animals mounted an anamnestic response upon high-dose SARS-CoV-2 respiratory challenge that translated into rapid elimination of replicating virus in their upper and lower airways and lung parenchyma. SpFNs potent and broad immunogenicity profile and resulting efficacy in NHPs supports its utility as a candidate platform for SARS-like betacoronaviruses.

One-Sentence SummaryA SARS-CoV-2 Spike protein ferritin nanoparticle vaccine, co-formulated with a liposomal adjuvant, elicits broad neutralizing antibody responses that exceed those observed for other major vaccines and rapidly protects against respiratory infection and disease in the upper and lower airways and lung tissue of nonhuman primates.

sentences: β-Carotene oxidation products - function and safety {beta}-Carotene oxidation products have newly discovered bioactivity in plants and animals. Synthetic fully oxidized {beta}-carotene (OxBC) has application in supporting livestock health, with potential human applications. The safety of synthetic OxBC has been evaluated. An Ames test showed weak-to-moderate mutagenicity in only one cell line at high concentrations. A mouse micronucleus assay established a non-toxic dose of 1800 mg/kg body weight, and no bone marrow micronuclei were induced. Plant sources of {beta}-carotene inevitably contain varying levels of natural OxBC. Vegetable powders and dried forages can be especially rich. Intakes of natural OxBC for humans and livestock alike have been estimated. The exposure range for humans (1-22 mg/serving) is comparable to the safe intake of {beta}-carotene (<15 mg/d). In livestock, OxBC in alfalfa can contribute ~550-850 mg/head/d for dairy cattle but in forage-deficient poultry feed much less (~1 ppm). Livestock intake of supplemental synthetic OxBC is comparable to OxBC potentially available from traditional plant sources. Human intake of synthetic OxBC in meat from livestock fed OxBC is similar to a single serving of food made with carrot powder. It is concluded that consumption of synthetic OxBC at levels comparable to natural OxBC is safe for humans and animals.

sentences: Humanization of antibodies using a machine learning approach on large-scale repertoire data Monoclonal antibody therapeutics are often produced from non-human sources (typically murine), and can therefore generate immunogenic responses in humans. Humanization procedures aim to produce antibody therapeutics that do not elicit an immune response and are safe for human use, without impacting efficacy. Humanization is normally carried out in a largely trial-and-error experimental process. We have built machine learning classifiers that can discriminate between human and non-human antibody variable domain sequences using the large amount of repertoire data now available. Our classifiers consistently outperform existing best-in-class models, and our output scores exhibit a negative relationship with the experimental immunogenicity of existing antibody therapeutics. We used our classifiers to develop a novel, computational humanization tool, Hu-mAb, that suggests mutations to an input sequence to reduce its immunogenicity. For a set of existing therapeutics with known precursor sequences, the mutations suggested by Hu-mAb show significant overlap with those deduced experimentally. Hu-mAb is therefore an effective replacement for trial- and-error humanization experiments, producing similar results in a fraction of the time. Hu-mAb is freely available to use at opig.stats.ox.ac.uk/webapps/humab.

sentences: Peptidome profiling for the immunological stratification in sepsis: a proof of concept study Sepsis has been called "the graveyard of pharmaceutical companies" due to the numerous failed clinical trials. The lack of tools to monitor the immunological status in sepsis constrains the development of immunomodulatory therapies. Here, we evaluated a test based on whole plasma peptidome acquired in a MALDI-TOF-mass spectrometer used for bacterial biotyping and machine-learning algorithms to discriminate the different immunological phases of sepsis. In this proof of concept study, two discrete lipopolysaccharide-(LPS) induced murine models emulating the pro- and anti-inflammatory phases that occur during sepsis were evaluated. The LPS group was inoculated with a single high dose of LPS, recalling the proinflammatory phase, and the IS groups was subjected to increasing doses of LPS to induce the anti-inflammatory/immunosuppression phase. Unstimulated mice served as controls. Both experimental groups showed the hallmarks of pro- and anti- inflammatory phases respectively; the LPS group showed leukopenia and higher levels of cytokines and tissue damage markers, and the IS group showed neutrophilia, lymphopenia and significantly lower antibody titers upon immunization. Principal component analysis of the plasma peptidomes formed three discrete clusters that mostly coincided with the experimental groups. In addition, machine-learning algorithms discriminated the different experimental groups with a sensitivity and specificity of up to 95.7% and 90.9% respectively. Our data reveal the potential of plasma peptidome analysis by MALDI-TOF-mass spectrometry as a simple, speedy and readily transferrable method for sepsis patient stratification that would contribute to therapeutic decision-making based on their immunological status.

sentences: Diamond Raman Laser and Yb Fiber Amplifier for In Vivo Multiphoton Fluorescence Microscopy Here we introduce a fiber amplifier and a diamond Raman laser that output high powers (6.5 W, 1.3 W) at valuable wavelengths (1060 nm, 1250 nm) for multiphoton excitation of red-shifted fluorophores. These custom excitation sources are both simple to construct and cost-efficient in comparison to similar custom and commercial alternatives. Furthermore, they operate at a repetition rate (80 MHz) that allows fast image acquisition using resonant scanners. We demonstrate our systems compatibility with fast resonant scanning, the ability to acquire neuronal images, and the capability to image vasculature at deep locations (>1 mm) within the mouse cerebral cortex.

sentences: Knockout of E-cadherin in adult mouse epithelium results in emphysema and airway disease Chronic obstructive pulmonary disease (COPD) is a devastating lung disease, characterized by a progressive decline in lung function, alveolar loss (emphysema), and airflow limitation due to excessive mucus secretion (chronic bronchitis), that can occur even after the injurious agent is removed. It is slated to rise to the 3rd leading cause of death due to chronic disease by 2030 globally, and the 4th leading cause of death due to chronic disease in the USA. While there is substantial evidence indicating loss of E-cadherin in the lung epithelium of patients with COPD, it is not known if this is causal to the disease. We investigated if loss of E-cadherin can result in lung disease using in both in vitro models of primary, differentiated human cells and in mouse models. Using a cell type-specific promoter using Cre/LoxP mice system to knock-out E- cadherin in ciliated and alveolar epithelial cell (Type 1 and Type 2) populations in adult mouse models, we determined that loss of E-cadherin caused airspace enlargement, as well as increased airway hyperresponsiveness indicating that it does have a causative role in causing COPD. Strategies to upregulate CDH1 (encodes for E-cadherin) in CHBEs and cigarette-smoke injured NHBEs can rescue the dysfunctional epithelium.

sentences: Antimicrobial Agent Triclosan Inhibits Acetylcholinesterase Activity in Vitro in a Dose-Dependent Manner The antimicrobial agent, Triclosan, is widely used in many consumer products. It has been designated as a "contaminant of emerging concern (CEC)" because its exposure is known to cause adverse ecological and human health effects. Triclosan is not labelled as GRAS/GRAE (generally recognized as safe and effective), but its use is still prevailing. In vivo studies have revealed that exposure to triclosan results in a decreased acetylcholinesterase (AChE) activity. However mechanistic insights into AChE inhibition by triclosan are missing. Using in vitro AChE activity assay with purified AChE, we show that triclosan acts as a direct inhibitor of AChE and inhibits AChE activity in a dose-dependent manner. Given the function of AChE, any alteration in its activity can be neurotoxic. Our results provide important mechanistic insights into triclosan induced neurotoxicity with AChE as a target.

sentences: A reference-quality, fully annotated genome from a Puerto Rican individual Until 2019, the human genome was available in only one fully-annotated version, GRCh38, which was the result of 18 years of continuous improvement and revision. Despite dramatic improvements in sequencing technology, no other genome was available as an annotated reference until 2019, when the genome of an Ashkenazi individual, Ash1, was released. In this study, we describe the assembly and annotation of a second individual genome, from a Puerto Rican individual whose DNA was collected as part of the Human Pangenome project. The new genome, called PR1, is the first true reference genome created from an individual of African descent. Due to recent improvements in both sequencing and assembly technology, and particularly to the use of the recently completed CHM13 human genome as a guide to assembly, PR1 is more complete and more contiguous than either GRCh38 or Ash1. Annotation revealed 37,755 genes (of which 19,999 are protein-coding), including 12 additional gene copies that are present in PR1 and missing from CHM13. 57 genes have fewer copies in PR1 than in CHM13, 9 map only partially, and 3 genes (all non-coding) from CHM13 are entirely missing from PR1.

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Expression of Unfolded Protein Response Genes in Post-transplantation Liver Biopsies Cholestatic liver diseases are a major source of morbidity and mortality that can progress to end-stage liver disease. There are few effective medical therapies for primary biliary cholangitis, primary sclerosing cholangitis and other cholestatic liver diseases, in part, due to our incomplete understanding of the pathogenesis of cholestatic liver injury. The hepatic unfolded protein response (UPR) is an adaptive cellular response to endoplasmic reticulum stress that is important in the pathogenesis of many liver diseases and recent animal studies have demonstrated the importance of the UPR in the pathogenesis of cholestatic liver injury. However, the role of the UPR in human cholestatic liver diseases is largely unknown. In this study, we utilized liver biopsies from patients after liver transplantation as a disease model to determine the transcriptional profile and hepatic UPR gene expression that is associated with liver injury and cholestasis. RNA-seq analysis revealed that patients with hyperbilirubinemia had enhanced expression of hepatic UPR pathways. Alternatively, liver biopsy samples from patients with acute rejection had enhanced gene expression of LAG3 and CDK1. Pearson correlation analysis of serum alanine aminotransferase, aspartate aminotransferase and total bilirubin levels demonstrated significant correlations with the hepatic expression of several UPR genes, as well as genes involved in hepatic bile acid metabolism and inflammation. In contrast, serum alkaline phosphatase levels were correlated with the level of hepatic bile acid metabolism gene expression but not liver UPR gene expression.

ConclusionOverall, these data indicate that hepatic UPR pathways are increased in cholestatic human liver biopsy samples and supports an important role of the UPR in the mechanism of human cholestatic liver injury.

sentences: Molecular origin of somatostatin-positive neuron vulnerability Reduced somatostatin (SST) and SST-positive (SST+) neurons are hallmarks of neurological disorders and associated with mood disturbances, but their origin are unknown. Chronic psychosocial stress induces behavioral emotionality deficits and deregulates unfolded protein response (UPR) of the endoplasmic reticulum (ER) preferentially in SST+ neurons. Here we confirm that chronic stress increases ER stress levels in SST+ neurons of mouse prefrontal cortex, and show that genetically suppressing ER stress in SST+ neurons, but not in pyramidal neurons, normalized psychosocial stress-induced behavioral emotionality. Forced expression of SST precursor protein (preproSST), mimicking psychosocial stress-induced early proteomic changes, induces ER stress, whereas mature SST or processing-incompetent preproSST does not. Biochemical analyses further show that psychosocial stress induces SST protein aggregation under elevated ER stress conditions. These results demonstrate that SST processing is a SST+ neuron-intrinsic vulnerability factor under conditions of sustained or over-activated UPR in the ER, hence negatively impacting SST+ neuron functions.

sentences: Dysregulated coordination of MAPT exon 2 and exon 10 splicing underlies different tau pathologies in PSP and AD Understanding regulation of MAPT splicing is important to the etiology of many nerurodegenerative diseases, including Alzheimer disease (AD) and progressive supranuclear palsy (PSP), in which different tau isoforms accumulate in pathologic inclusions. MAPT, the gene encoding the tau protein, undergoes complex alternative pre-mRNA splicing to generate six isoforms. Tauopathies can be categorized by the presence of tau aggregates containing either 3 (3R) or 4 (4R) microtubule binding domain repeats (determined by inclusion/exclusion of exon 10), but the role of the N terminal domain of the protein, determined by inclusion/exclusion of exons 2 and 3 has been less well studied. Using an unbiased correlational screen in human brain tissue, we observed coordination of MAPT exons 2 and 10 splicing. Expression of exon 2 splicing regulators and subsequently exon 2 inclusion are differentially disrupted in PSP and AD brain, resulting in the accumulation of 1N4R isoforms in PSP and 0N isoforms in AD temporal cortex. Furthermore, we identified different N-terminal isoforms of tau present in neurofibrillary tangles, dystrophic neurites and tufted astrocytes, indicating a role for differential N-terminal splicing in the development of disparate tau neuropathologies. We conclude that N-terminal splicing and combinatorial regulation with exon 10 inclusion/exclusion is likely to be important to our understanding of tauopathies.

sentences: Maturation of human intestinal epithelium from pluripotency in vitro Methods to generate human intestinal tissue from pluripotent stem cells (PSCs) open new inroads into modeling intestine development and disease. However, current protocols require organoid transplantation into an immunocompromised mouse to achieve matured and differentiated epithelial cell states. Inspired by developmental reconstructions from primary tissues, we establish a regimen of inductive cues that enable stem cell maturation and epithelial differentiation entirely in vitro. We show that the niche factor Neuregulin1 (NRG1) promotes morphological change from proliferative epithelial cysts to matured epithelial tissue in three-dimensional cultures. Single-cell transcriptome analyses reveal differentiated epithelial cell populations, including diverse secretory and absorptive lineages. Comparison to multi-organ developmental and adult intestinal cell atlases confirm the specificity and maturation state of cell populations. Altogether, this work opens a new direction to use in vitro matured epithelium from human PSCs to study human intestinal epithelium development, disease, and evolution in controlled culture environments.

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Integration of visual motion and pursuit signals in areas V3A and V6+ across cortical depth using 9.4T fMRI Neural mechanisms underlying a stable perception of the world during pursuit eye movements are not fully understood. Both, perceptual stability as well as perception of real (i.e. objective) motion are the product of integration between motion signals on the retina and efference copies of eye movements. Human areas V3A and V6 have previously been shown to have strong objective ( real) motion responses. Here we used high-resolution laminar fMRI at ultra-high magnetic field (9.4T) in human subjects to examine motion integration across cortical depths in these areas. We found an increased preference for objective motion in areas V3A and V6+ i.e. V6 and possibly V6A towards the upper layers. When laminar responses were detrended to remove the upper-layer bias present in all responses, we found a unique, condition-specific laminar profile in V6+, showing reduced mid-layer responses for retinal motion only. The results provide evidence for differential, motion-type dependent laminar processing in area V6+. Mechanistically, the mid-layer dip suggests a special contribution of retinal motion to integration, either in the form of a subtractive (inhibitory) mid-layer input, or in the form of feedback into extragranular or infragranular layers. The results show that differential laminar signals can be measured in high-level motion areas in the human occipitoparietal cortex, opening the prospect of new mechanistic insights using non-invasive brain imaging.

Significance StatementVisual stability and our ability to differentiate between self-induced and real motion are central to our visual sense. Both require the integration of two signals - retinal motion and copies of muscle commands used for eye movements (efference copies). A reasonable assumption is that either the efference copy or the result of integration will be conveyed to high-level visual regions along with visual retinal input, possibly differentially across cortical depth as the input sources differ. Our ultra-high field recordings present the first laminar evidence of differential signal processing of retinal and objective motion signals in area V6+, and present a first window into a mechanistic understanding of visual high-level motion processing.

sentences: Photothermal Dye-based Subcellular-sized Heat Spot Enabling the Modulation of Local Cellular Activities Thermal engineering at microscale such as the control and measurement of temperature is a key technology in basic biological research and biomaterials development, which remains challenge yet. Here, we engineered the polymeric nanoparticle, in which a fluorescent temperature sensory dye and a photothermal dye were embedded in its polymer matrices, termed nanoHT. When a near infrared laser at 808 nm is illuminated to the particle, it enables to create the subcellular-sized heat spot in a live cell, where fluorescence thermometry allows the read out of the temperature increment concurrently at individual heat spots. Owing to the controlled local heating, we found that the cell death of HeLa cells was induced at the certain temperature at rate of a few seconds. It should be also noted that the cell death was triggered from the very local heat spot at subcellular level. Furthermore, nanoHT was applied for the induction of muscle contraction of the C2C12 myotube by heat. We successfully showed that the heat-induced contraction took place at the limited area of a single myotube according to the alteration of protein-protein interactions related to the contraction event. These studies demonstrated that even a single heat spot provided by a photothermal material could be very effective in altering cellular functions, paving the way for novel photothermal therapies.

sentences: Lis1-dynein drives corona compaction and error-correction at kinetochores Mitotic cell division requires that kinetochores form microtubule attachments that can segregate chromosomes and control mitotic progression via the spindle assembly checkpoint. During prometaphase, kinetochores shed a distal domain called the fibrous corona as microtubule attachments form and mature. This shedding is mediated, in part, by the minus-end directed motor dynein, which  strips kinetochore cargoes along K-fibre microtubules towards the pole. While the main molecular players are well understood, relatively little is known about how dynein stripping is regulated and how it responds to increasing microtubule occupancy. Lis1 is a conserved dynein regulator that associates with kinetochores and is mutated in the severe neurodevelopmental disease lissencephaly. Here, we have combined loss-of-function studies, high-resolution imaging and engineered separation-of-function mutants to define how Lis1 contributes to dynein-mediated corona stripping. We show that cells depleted of Lis1 fail to fully dissemble the corona and delay in metaphase as a result of persistent checkpoint activation. Furthermore, we find that while kinetochore-tethered Lis1-dynein is required for attachment error-correction, the contribution of Lis1 to corona disassembly can be mediated by a rapidly cycling cytosolic pool. These findings support the idea that Lis1 contextualises dynein function at kinetochores to maintain corona disassembly into metaphase and prevent chromosome mis-segregation.

sentences: Skill and value in recruitment forecasting Forecasting variation in the recruitment to fish stocks is one of the most challenging and long-running problems in fisheries science and essentially remains unsolved today. Traditionally recruitment forecasts are developed and evaluated based on explanatory and goodness-of-fit approaches that do not reflect their ability to predict beyond the data on which they were developed. Here we propose a new generic framework that allows the skill and value of recruitment forecasts to be assessed in a manner that is relevant to their potential use in an operational setting. We assess forecast skill based on predictive power using a retrospective forecasting approach inspired by meterology, and emphasise the importance of assessing these forecasts relative to a baseline. We quantify the value of these forecasts using an economic cost-loss decision model that is directly relevant to many forecast users. We demonstrate this framework using four stocks of lesser sandeel (Ammodytes marinus) in the North Sea, showing for the first time in an operationally realistic setting that skilful and valuable forecasts are feasible in two of these areas. This result shows the ability to produce valuable short-term recruitment forecasts, and highlights the need to revisit our approach to and understanding of recruitment forecasting.

sentences: Double knockout CRISPR screen in cancer resistance to T cell cytotoxicity Immunotherapy has transformed cancer treatments; however, a large fraction of patients encounter resistance. Such resistance is mediated by complex factors, often involving interactions between multiple genes. Thus, it is crucially important to identify genetic interactions between genes that are significantly mutated in cancer patients and those involved in immune responses, ideally the ones that currently have chemical compounds for direct targeting. To systematically interrogate such genetic interactions that mediate cancer cells response to T cell killing, we designed an asymmetric CRISPR/Cas9 dual perturbation library targeting the matched combinations between significantly mutated tumor suppressors and immune resistance genes. We performed a combinatorial double knockout screen on 1,159 gene pairs and identified those where joint loss-of-function renders altered cellular response to T cell cytotoxicity. With individual double knockout constructs we validated these genetic interactions including Jak1-Trp53, Jak1-Kmt2d, and Ifngr1-Kmt2d. Interactions between significantly mutated tumor suppressors and potentially druggable immune resistance genes may offer insights on potential new concepts of how to target clinically relevant cancer mutations with currently available agents. This study also provides a technology platform and an asymmetric CRISPR double knockout library for interrogating genetic interactions between cancer mutations and immune resistance pathways under various settings.

sentences: A protein microarray-based in vitro transglutaminase assay platform for epitope mapping and peptide discovery Transglutaminases (TGs) are a family of crosslinking enzymes catalyzing the formation of intra- and inter-molecular glutamine-lysine isopeptide bonds in a calcium dependent manner. Activation of transglutaminases is pathogenically associated with severe human diseases including neurodegenerations, cardiovascular diseases, and autoimmune diseases. Although continuous efforts determining the enzymes substrate preference have been witnessed, a high-throughput assay platform with the "omic" efficiency is still missing for the global identification of substrate-specific TG modification sites. In this study we report a protein microarray-based in vitro TG assay platform for rapid and large-scale (up to 30000 reactions per chip) determination of the glutamine (Q)-bearing TG modification motifs. With this platform we identified the Q16 in superoxide dismutase 1 and Q109 in alpha-synuclein as the primary modification sites for tissue transglutaminase (TG2), the most ubiquitous member of the enzyme family. Of particular interest, we found a close match between the modification motifs and published vaccine epitope sequences in alpha-synuclein, implying an essential and intrinsic role transglutaminase might play in the determination of immunodominant epitopes. Our data collectively suggest the glutamine and its follow-up five residues on the C terminal of a protein compose a minimal determinant motif for TG2 modification and the TG2 modification motifs determined by our platform could finally correspond to the substrates epitope sequences in antigen processing. To screen for site-specific interfering peptides and assist gene editing-based protein engineering, we optimized through onchip amino-acid scanning the TG2 modification motif QQIV in the extracellular matrix protein fibronectin and obtained 14 variants with significantly higher TG2 reactivity that might serve as the competitive inhibitor peptides and 1 with lower reactivity. We further confirmed this optimization approach with 12-mer peptides, the longest ones that could be synthesized on the chip. Taken together, our synthetic transglutaminase assay platform might be able to deliver a precise epitope blueprint for immunotherapeutic targeting and provide proof-of-concept and directional studies for TG-based peptide discovery and protein design.

sentences: Tbx2 is essential for cochlear inner hair cell development and regeneration Atoh1 is essential for the development of outer hair cells (OHCs) and inner hair cells (IHCs) in the mammalian cochlea. Whereas Ikzf2 is necessary for OHC development, the key gene for IHC development remains unknown. We found that deleting Tbx2 in neonatal IHCs led to their trans-differentiation into OHCs, by repressing 26.7% of IHC and inducing 56.3% of OHC genes, including Ikzf2. More importantly, persistent expression of Tbx2 together with transient Atoh1 effectively reprogramed non-sensory supporting cells into new IHCs expressing functional IHC marker vGlut3. The differentiation status of these new IHCs is much more advanced than those previously reported. Thus, Tbx2 is essential for IHC development, and its co-upregulation with Atoh1 in supporting cells represents a new approach for treating IHC degeneration-related deafness.

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DNA supercoiling restricts the transcriptional bursting of neighboring eukaryotic genes DNA supercoiling has emerged as a major contributor to gene regulation in bacteria. The impact of DNA supercoiling on transcription dynamics in eukaryotes is less clear. Here, using single-molecule dual-color RNA imaging in budding yeast, we show that transcriptional bursting of the divergent and tandem GAL genes is coupled. Upon topoisomerase degradation, supercoils that buildup from transcription inhibit subsequent transcription at neighboring genes, thereby reducing their simultaneous bursting. GAL gene transcription is inhibited more by negative than by positive supercoiling accumulation. Unlike bacteria, wildtype yeast has sufficient topoisomerase levels to minimize inhibition from supercoils at adjacent genes. Overall, we discover fundamental differences in supercoiling-mediated gene regulation between bacteria and yeast and show that rapid supercoiling release in eukaryotes ensures proper gene expression of neighboring genes.

One sentence summaryTranscription causes twisting of the DNA double helix, which can inhibit transcription of adjacent genes.

sentences: TGFβ2 regulates human trabecular meshwork cell contractility via ERK and ROCK pathways with distinct signaling crosstalk dependent on the culture substrate Transforming growth factor beta 2 (TGF{beta}2) is a major contributor to the pathologic changes occurring in human trabecular meshwork (HTM) cells in primary open-angle glaucoma. Receptor binding of TGF{beta}2 activates non-canonical extracellular-signal-regulated kinase (ERK) and Rho-associated kinase (ROCK) signaling pathways, both broadly affecting HTM cell behavior. However, exactly how these signaling pathways converge to regulate pathologic HTM cell contractility associated with glaucomatous dysfunction is unclear. Here, we investigated the molecular mechanism underlying TGF{beta}2-induced pathologic HTM cell contractility, and the crosstalk between ERK and ROCK signaling pathways. We compared soft biomimetic hydrogels composed of collagen type I, elastin-like polypeptide, and hyaluronic acid with conventional stiff glass coverslips. Results show that HTM cell morphology and filamentous (F)-actin organization was affected by the underlying culture substrate: TGF{beta}2 increased HTM cell contractility via ERK and ROCK signaling pathways by differentially regulating F-actin, -smooth muscle actin, fibronectin, and phospho-myosin light chain in cells grown on hydrogels compared to glass. Importantly, we showed that ERK inhibition further increased TGF{beta}2-induced phospho-myosin light chain levels in HTM cells on hydrogels, but not on glass, which translated into hypercontractility of three-dimensional (3D) HTM cell-laden hydrogels. ROCK inhibition had precisely the opposite effects and potently relaxed the TGF{beta}2-induced hydrogels. This suggests that ERK signaling negatively regulates ROCK-mediated HTM cell contractility, and that impairment of this crosstalk balance contributes to the pathologic contraction associated with the glaucomatous stressor TGF{beta}2. These findings emphasize the critical importance of using 3D tissue-mimetic extracellular matrix substrates for investigating HTM cell physiology and glaucomatous pathophysiology in vitro.

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Microtubule-associated IQD9 guides cellulose synthase velocity to shape seed mucilage O_LIArabidopsis seeds release large capsules of mucilaginous polysaccharides, which are shaped by an intricate network of cellulosic microfibrils. Cellulose synthase complexes is guided by the microtubule cytoskeleton, but it is unclear which proteins mediate this process in the seed coat epidermis (SCE).
C_LIO_LIUsing reverse genetics, we identified IQ67 DOMAIN 9 (IQD9) and KINESIN LIGHT CHAIN-RELATED 1 (KLCR1) as two highly expressed genes during seed development and comprehensively characterized their roles for cell wall polysaccharide biosynthesis and cortical microtubule (MT) organization.
C_LIO_LIMutations in IQD9 as well as in KLCR1 lead to compact mucilage capsules with aberrant cellulose distribution, which can be rescued by transgene complementation. Double mutant analyses revealed that their closest paralogs (IQD10 and KLCR2, respectively) are not required for mucilage biosynthesis. IQD9 physically interacts with KLCR1 and localizes to cortical MTs to maintain their organization in SCE cells. Similar to the previously identified TONNEAU1 (TON1) RECRUITING MOTIF 4 (TRM4) protein, IQD9 is required to maintain the velocity of cellulose synthases.
C_LIO_LIOur results demonstrate that IQD9, KLCR1 and TRM4 are MT-associated proteins that are required for seed mucilage architecture. This study provides the first direct evidence that members of the IQD, KLCR and TRM families have overlapping roles in guiding the distribution of cell wall polysaccharides. Therefore, SCE cells provide an attractive system to further decipher the complex genetic regulation of polarized cellulose deposition.
C_LI

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Aggression and multimodal signaling in noise in the European robin The effect on anthropogenic noise on acoustic signals is widely studied although the extent to which this is due to short-term flexibility or long-term adaptation is unclear. Anthropogenic noise may disrupt signals used to mediate aggressive interactions, leading to more physical aggression between opponents. One solution to this problem is to switch signaling effort to a less noisy modality (e.g. the visual modality). In the present study we ask whether urban and rural European robins (Erithacus rubecula) use multi-modal signals flexibly in response to anthropogenic noise during territorial defense. We predicted that during simulated intrusions with experimental noise, European robins would increase their physical aggression, respond with more visual threat displays and sing fewer songs, i.e. show a multi-modal shift. We also predicted that urban birds, living in noisier habitats, would be generally more aggressive than rural birds. The results showed that urban birds were more aggressive than rural robins, but an increase in aggression with experimental noise was seen only in the rural birds. Urban birds also used visual signals more often than rural birds. However, birds did not use visual signals more in experimental noise. Instead, both urban and rural robins sang at higher rates under noise conditions, thus increasing signaling effort in the noisy modality. These results point to a complex role of immediate plasticity and longer-term processes in affecting communication during aggressive interactions under anthropogenic noise.

Significance StatementHuman activity has an enormous effect on wildlife, including on their social behavior. Animals living in urban areas often tend to be more aggressive than those living in rural areas, which may be due to urban acoustic noise making communication between individuals more difficult. In a study with a common songbird, the European robin, we investigated the role of urban acoustic noise in aggression and territorial communication. Urban robins were more aggressive than rural robins, and additional noise in the territory increased aggression in rural but not urban robins. Robins increased their singing effort but did not increase visual signals in acoustic noise. These results suggest that noise can indeed make animals behave more aggressively although the effect may depend on how noisy it is already. These results further our understanding of how human-made noise changes animal communication and social behavior.

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A system for multiplexed selection of aptamers with exquisite specificity without counter-selection Aptamers have proven to be valuable tools for the detection of small molecules due to their remarkable ability to specifically discriminate between structurally similar molecules. Most aptamer selection efforts have relied on counter-selection to eliminate aptamers that exhibit unwanted cross-reactivity to interferents or structurally similar relatives to the target of interest. However, because the affinity and specificity characteristics of an aptamer library are fundamentally unknowable a priori, it is not possible to determine the optimal counter-selection parameters. As a result, counter-selection experiments require trial-and-error approaches that are inherently inefficient and may not result in aptamers with the best combination of affinity and specificity. In this work, we describe a high-throughput screening process for generating high-specificity aptamers to multiple targets in parallel, while also eliminating the need for counter-selection. We employ a platform based on a modified benchtop sequencer to conduct a massively-parallel aptamer screening process that enables the selection of highly-specific aptamers against multiple structurally similar molecules in a single experiment, without any counter-selection. As a demonstration, we have selected aptamers with high affinity and exquisite specificity for three structurally similar kynurenine metabolites that differ by a single hydroxyl group in a single selection experiment. This process can easily be adapted to other small-molecule analytes, and should greatly accelerate the development of aptamer reagents that achieve exquisite specificity for their target analytes.

Significance statementAptamers offer the exciting potential to discriminate between structurally similar small molecules. However, generating such highly specific aptamers has been proven challenging using the conventional process of counter-selection. In this work, we describe a high-throughput screening platform that can characterize the specificity of millions of aptamers towards a group of structurally related molecules in a single experiment and generate exquisitely specific aptamers without any counter-selection. As exemplars, we generated aptamers with high affinity and specificity towards three structurally related kynurenine metabolites using our platform. Our platform can be readily adapted to other small molecule targets and should therefore accelerate the development of aptamer reagents with exquisite specificity.

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Lithium orotate is more potent, effective, and less toxic than lithium carbonate in a mouse model of mania BackgroundLithium carbonate (LiCO) is a mainstay treatment option for the prevention of mood-episode recurrences in bipolar disorder (BD). Unfortunately, the narrow therapeutic index of LiCO is associated with common thyroid and kidney complications that lead to high rates of non-compliance. Lithium orotate (LiOr) is an alternative compound that is suggested to possess uptake properties that would allow for reduced dosing to mitigate toxicity and compliance concerns.

MethodsDose responses were established for LiOr and LiCO in male and female mice using an amphetamine-induced hyperlocomotion (AIH) murine (C57BL/6) model (n = 5-11); AIH captures manic elements of BD and is sensitive to a dose-dependent lithium blockade. These studies were followed by an examination of the relative toxicities of LiOr and LiCO over 14 consecutive daily administrations (n = 5-7).

ResultsA partial block of AIH was maintained by LiCO at doses of 15 mg/kg or greater in males and 20 mg/kg or greater in females. In contrast, a near-complete blockade was observed for LiOr concentrations equal to or exceeding 1.5 mg/kg in both males and females, indicating improved efficacy and potency. Prior application of polyethylene glycol-400 completely blocked the effects of LiOr on AIH while sparing LiCO, suggesting differences in transport between the two compounds. LiCO, but not LiOr, elicited polydipsia in both sexes, elevated serum creatinine levels in males, and increased serum TSH expression in females.

ConclusionsLiOr demonstrates superior efficacy, potency, and tolerability to LiCO in both male and female mice as a result of select transport-mediated uptake.

sentences: Dynamical processing of orientation precision in the primary visual cortex In our daily visual environment, the primary visual cortex (V1) detects oriented features from a large variety of orientation distributions. The precision of these distributions is fluctuating from one part of the visual scenery to another, and it remains unclear how V1 can account for this range of input configurations in order to achieve robust orientation recognition performances. To answer this question, we used natural-like stimuli and characterised the responses of V1 neurons to quantified variations of orientation precision. Generally, decrements of orientation precision correlated with decreased single neuron tuning, but thirty percent of the recorded neurons remained robustly tuned to imprecise inputs. The existence of these resilient neurons, which could be explained by a model of recurrent cortical inhibition, allows to encode both the orientation and its precision, improving the robustness of the overall population response in V1. Altogether, our results support the notion that the processing of orientation involves precision-specific neurons and slow recurrent dynamics, which possibly constitutes an experimental signature of predictive processes occurring in V1.

sentences: Destabilization of the holo-DNA Polδ by loss of Pol32 confers conditional lethality that can be suppressed by stabilizing Pol31-Pol3 interaction DNA Polymerase {delta} plays an essential role in genome replication and in the preservation of genome integrity. In S. cerevisiae, Pol{delta} consists of three subunits: Pol3 (the catalytic subunit), Pol31 and Pol32. We have constructed pol31 mutants by alanine substitution at conserved amino acids, and identified three alleles that do not confer any disadvantage on their own, but which suppress the cold-, HU- and MMS-hypersensitivity of yeast strains lacking Pol32. We have shown that Pol31 and Pol32 are both involved in translesion synthesis, error-free bypass synthesis, and in preservation of replication fork stability under conditions of HU arrest. We identified a solvent exposed loop in Pol31 defined by two alanine substitutions at T415 and W417. Whereas pol31-T4l5A compromises polymerase stability at stalled forks, pol31-W417A is able to suppress many, but not all, of the phenotypes arising from pol32{Delta}. ChIP analyses showed that the absence of Pol32 destabilizes Pole and Pol at stalled replication forks, but does not interfere with checkpoint kinase activation. We show that the Pol31-W417A-mediated suppression of replicationstress sensitivity in pol32{Delta} stems from enhanced interaction between Pol3 and Pol31, which stabilizes a functional Pol{delta}.

sentences: Accurate estimation of cell-type resolution transcriptome in bulk tissue through matrix completion Single cell RNA-seq (scRNA-seq) has been widely used to uncover cellular heterogeneity, however, the constraints of cost make it impractical as a routine on large patient cohorts. Here we present ENIGMA, a method that accurately deconvolute bulk tissue RNA-seq into single cell-type resolution given the knowledge gained from scRNA-seq. ENIGMA applies a matrix completion strategy to minimize the distance between mixture transcriptome and weighted combination of cell type-specific expression, allowing quantification of cell type proportions and reconstruction of cell type-specific transcriptome. The superior performance of ENIGMA was validated in simulated and realistic datasets, including disease-related tissues, demonstrating its ability in novel biological findings.

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Barley guanine nucleotide exchange factor HvGEF14 is an activator of the susceptibility factor HvRACB and supports host cell entry by Blumeria graminis f.sp. hordei In barley (Hordeum vulgare), the function of ROPs appears central to polar cell development and the interaction outcome with parasitic fungi but little is known about ROP activation. Guanine nucleotide exchange factors (GEFs) facilitate the exchange of GDP with GTP and thereby turn ROPs into a signalling-activated ROP-GTP state. Plants possess a unique class of GEFs harbouring a plant specific ROP nucleotide exchanger domain (PRONE). We performed phylogenetic analyses and annotated barley PRONE GEFs. HvGEF14 is expressed in leaf epidermal tissue and downregulated after inoculation with Blumeria graminis f.sp. hordei. Protein-protein interaction assays indicate that HvGEF14 and the type I barley ROP protein HvRACB can interact in yeast and in planta. Overexpression of HvGEF14 further recruited the ROP-GTP downstream interactor HvRIC171 to the cell periphery and let to interaction of HvRACB with an extended CRIB (Cdc42/Rac Interactive Binding motif) peptide of HvRIC171 in a similar manner as constitutively activated HvRACB. Finally, the over expression of HvGEF14 led to enhanced susceptibility to fungal entry while HvGEF14 RNAi provoked a trend to more penetration resistance. HvGEF14 might therefore play a role in the activation of HvRACB in barley epidermal cells, which is crucial for fungal penetration success.

HighlightThe activated GTPase HvRACB is a susceptibility factor of barley in response to Blumeria graminis f.sp. hordei. The newly discovered guanine nucleotide exchange factor HvGEF14 is a HvRACB activator.

sentences: When the ventral visual stream is not enough: A deep learning account of medial temporal lobe involvement in perception The medial temporal lobe (MTL) supports a constellation of memory-related behaviors. Its involvement in perceptual processing, however, has been subject to enduring debate. This debate centers on perirhinal cortex (PRC), an MTL structure at the apex of the ventral visual stream (VVS). Here we leverage a deep learning framework that approximates visual behaviors supported by the VVS--i.e. lacking PRC. We first apply this approach retroactively, modeling 30 published visual discrimination experiments: Excluding non-diagnostic stimulus sets, there is a striking correspondence between VVS-modeled and PRC-lesioned behavior, while each are outperformed by PRC-intact participants. We corroborate and extend these results with a novel experiment, directly comparing PRC-intact human performance to electrophysiological recordings from the macaque VVS: PRC-intact participants outperform a linear readout of high-level visual cortex. By situating lesion, electrophysiological, and behavioral results within a shared computational framework, this work resolves decades of seemingly inconsistent findings surrounding PRC involvement in perception.

sentences: Response outcome gates the effect of spontaneous cortical state fluctuations on perceptual decisions Sensory responses of cortical neurons are more discriminable when evoked on a base-line of desynchronized spontaneous activity, but cortical desynchronization has not generally been associated with more accurate perceptual decisions. Here we show that mice perform more accurate auditory judgements when activity in the auditory cortex is elevated and desynchronized before stimulus onset, but only if the previous trial was an error, and that this relationship is occluded if previous outcome is ignored. We confirmed that the outcome-dependent effect of brain state on performance is neither due to idiosyncratic associations between the slow components of either signal, nor to the existence of specific cortical states evident only after errors. Instead, errors appear to gate the effect of cortical state fluctuations on discrimination accuracy. Neither facial movements nor pupil size during the baseline were associated with accuracy, but they were predictive of measures of responsivity, such as the probability of not responding to the stimulus or of responding prematurely. These results suggest that the functional role of cortical state on behavior is dynamic and constantly regulated by performance monitoring systems.

sentences: Kiawah and Seabrook islands are a critical site for the rufa red knot (Calidris canutus rufa) The rufa Red Knot (Calidris canutus rufa) is a migratory shorebird that performs one of the longest known migrations of any bird species -- from their breeding grounds in the Canadian Arctic to their nonbreeding grounds as far south as Tierra del Fuego -- and has experienced a population decline of over 85% in recent decades. During migration, knots rest and refuel at stopover sites along the Atlantic Coast, including Kiawah and Seabrook islands in South Carolina. Here, we document the importance of Kiawah and Seabrook islands for knots by providing population and stopover estimates during their spring migration. We conducted on-the-ground surveys between 19 February - 20 May 2021 to record the occurrence of individually marked knots. In addition, we quantified the ratio of marked to unmarked knots and deployed geolocators on knots captured in the area. Using a superpopulation model, we estimated a minimum passage population of 17,247 knots (~41% of the total rufa knot population) and an average stopover duration of 47 days. Our geolocator results also showed that knots using Kiawah and Seabrook islands can bypass Delaware Bay and fly directly to the Canadian Arctic. Finally, our geolocators, combined with resighting data from across the Atlantic Flyway, indicate that a large network of more than 70 coastal sites mostly concentrated along the coasts of Florida, Georgia, South Carolina, and North Carolina provide stopover and overwintering habitat for the knots we observed on Kiawah and Seabrook islands. These findings corroborate that Kiawah and Seabrook islands should be recognized as critical sites in the knot network and, therefore, a conservation priority. As a result, the threats facing the sites -- such as prey management issues, anthropogenic disturbance, and sea level rise -- require immediate attention.

sentences: Tethering designer short double-stranded RNA to mRNA for co-delivery of molecularly-targeted adjuvants and antigens towards cancer vaccination Immunostimulatory adjuvants play pivotal roles in mRNA cancer vaccines. Herein, mRNA architectonics-based approach was employed to enhance immunostimulatory properties of mRNA, by tethering immunostimulatory short 24-base-pair double-stranded RNA (dsRNA) to mRNA via hybridization, for specific stimulation of retinoic acid-inducible gene-I (RIG-I), an innate immune receptor triggering effective cellular immunity. This approach ensures the co-delivery of mRNA and adjuvants to obtain antigen expression and immune activation in the same antigen presenting cells for effective vaccination, and allows the tuning of immunostimulatory effects by controlling dsRNA number to avoid safety concerns associated with excessive immunostimulation. Thorough in vitro screening provided optimal mRNA structure that induces the secretion of a broad spectrum of proinflammatory molecules and effectively activated mouse and human dendritic cells, with minimal effect on antigen translation efficiency of mRNA. The optimal mRNA formulation improved the potency of mRNA vaccine to induce cytotoxic immune responses after encapsulation into mRNA lipoplex, and exhibited high therapeutic efficiency in lung metastatic model of melanoma using mRNA encoding tumor associated antigen. Furthermore, this system is versatile to potentiate vaccination effect with the use of mRNA polyplex micelle.

sentences: Suppression of aminoglycoside-induced premature termination codon readthrough by the TRP channel inhibitor AC1903 Nonsense mutations, which occur in ~11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as gentamicin and G418 permit PTC readthrough and so may address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we addressed the hypothesis that TRP non-selective cation channels contribute to the variable effect of aminoglycosides by controlling their cellular uptake. To attempt to identify the channel type involved, we tested AC1903, a 2-aminobenzimidazole derivative recently reported to selectively inhibit TRPC5 cation channels. AC1903 consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cell line and patient-derived JEB01 keratinocytes. In an effort to validate the suggested role of TRPC5, we tested an independent and more potent inhibitor called Pico145, which affects channels containing TRPC1, TRPC4 and TRPC5 but not other TRPCs or other channels. Unexpectedly, Pico145 was completely without effect, suggesting that AC1903 may work through other or additional targets. Consistent with this suggestion, AC1903 inhibited multiple TRPC channels including homomeric TRPC3, TRPC4, TRPC5, TRPC6 as well as concatemeric TRPC4-C1 and TRPC5-C1 channels, all with low micromolar IC50 values. It also inhibited TRPV4 channels but had weak or no effects on TRPV1 and no effect on another non-selective cation channel, PIEZO1. Overall, our study reveals a suppressor of aminoglycoside-mediated PTC readthrough (i.e., AC1903) but suggests that this compound has previously unrecognised effects. These effects require further investigation to determine the molecular mechanism by which AC1903 suppresses aminoglycoside uptake and PTC readthrough.

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System genetics in the rat HXB/BXH family identifies Tti2 as a pleiotropic quantitative trait gene for adult hippocampal neurogenesis and serum glucose Neurogenesis in the adult hippocampus contributes to learning and memory in the healthy brain but is dysregulated in metabolic and neurodegenerative diseases. The molecular relationships between neural stem cell activity, adult neurogenesis, and global metabolism are largely unknown. Here we applied unbiased systems genetic methods to quantify genetic covariation among adult neurogenesis and metabolic phenotypes in peripheral tissues of a genetically diverse family of rat strains, derived from a cross between the spontaneously hypertensive (SHR/OlaIpcv) strain and Brown Norway (BN-Lx/Cub). The HXB/BXH family is a very well established model to dissect genetic variants that modulate metabolic and cardiovascular disease and we have accumulated deep phenome and transcriptome data in a FAIR-compliant resource for systematic and integrative analyses. Here we measured rates of precursor cell proliferation, survival of new neurons, and gene expression in the hippocampus of the entire HXB/BXH family, including both parents. These data were combined with published metabolic phenotypes to detect a neurometabolic quantitative trait locus (QTL) for serum glucose and neuronal survival. We subsequently fine-mapped a key phenotype to a locus that includes the telo2-interacting protein 2 gene (Tti2)--a chaperone that modulates the activity and stability of PIKK kinases. To validate variants in or near Tti2 as a cause for differences in neurogenesis and glucose levels, we generated a targeted frameshift mutation on the SHR/OlaIpcv background. Heterozygous SHR-Tti2+/- mutants had lower rates of hippocampal neurogenesis and hallmarks of dysglycemia compared to wild-type littermates. Our findings highlight Tti2 as a causal genetic and molecular link between glucose metabolism and structural brain plasticity. In humans, more than 800 genomic variants are linked to TTI2 expression, seven of which have associations to protein and blood stem cell factor concentrations, blood pressure and frontotemporal dementia.

Author summaryMetabolic and neurological disorders are often comorbid, suggesting that biological pathways which orchestrate peripheral homeostasis and the integrity of the nervous system intersect. The genetic architecture behind these relationships is still poorly described, in part because molecular processes in the human brain are very difficult to study. We thus used a rodent genetic reference population to investigate links between adult hippocampal neurogenesis-- a cellular plasticity mechanism important for learning flexibility--and metabolism. We measured adult neurogenesis in the family of 30 HXB/BXH rat recombinant inbred strains, who are characterised by stable differences in metabolism, behaviour, and gene expression levels. Because gene variants affecting distinct traits segregated into different members of the family, we discovered that previously published phenotypes correlated to adult neurogenesis due to shared genomic sequence. We found that expression levels of Tti2--a part of a specialised protein chaperone complex regulating stability of PIKK kinases--were concomitantly influencing adult neurogenesis and serum glucose levels. In human populations hundreds of genomic variants regulate TTI2 expression, potentially affecting brain function and glucose homeostasis.

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Staphylococci planktonic and biofilm environments differentially affect macrophage immune activation and osteoclastogenic differentiation. Implant-related bone infections are a major complication in orthopedic surgery that lead to inflammation and bone destruction. Bacterial biofilm formation on the implant is discussed to polarize the immune response towards tolerance and to facilitate bacterial persistence. In addition to their role in the early immune response, macrophages are osteoclast precursor cells. Therefore, macrophages can link inflammation and RANKL-mediated osteoclastogenic bone destruction.

We investigated the influence of Staphylococcus aureus (SA) and epidermidis (SE) biofilm formation on immune function and osteoclastogenesis using RAW264.7 cells and conditioned media (CM) of planktonic and biofilm cultures in the presence and absence of the osteoclastogenic transcription factor RANKL. Analysis of immune cell activation, metabolic activity and osteoclast formation revealed that a planktonic environment causes a pro-inflammatory response. This was also partially induced by biofilm CM. Simultaneous stimulation with CM and RANKL suppressed osteoclast formation in favor of a long-term immune activation. While the early macrophage response towards CM was dominated by glycolysis, the CM and RANKL approach shifted metabolism towards increased mitochondrial biomass and activity. This was most evident in biofilm CM. We further showed that planktonic CM effects are mediated through activation of TLR signaling and induction of IFN-{beta} production. In biofilm CM, high lactate levels seem to significantly contribute to the modulation of macrophages.

Our results can contribute to find targets for therapeutic intervention that restore an effective pro-inflammatory immune response, which could help to control implant-related bone infections.

ImportanceThe number of endoprosthetic and bone reconstructive surgeries is rising and accordingly incidences of implant-related bone infections increase. Biofilm formation on the implant surface protects bacteria against most antibiotics and the hosts immune response. Thus, novel therapeutic strategies for prevention or treatment of biofilm formation are needed. Furthermore, bone infections are associated with osteoclast-mediated bone destruction, which promotes infection progression and ultimately leads to implant loosening. We investigated the effect of staphylococci-mediated biofilm formation on macrophage immune activation and osteoclastogenic differentiation, respectively. In planktonic and biofilm environments, immune cell activation dominated over osteoclastogenesis. The effects of planktonic environments were mainly mediated through activation of TLR signaling and resulted in a pronounced pro-inflammatory and IFN-{beta} macrophage immune response. In biofilm environments, high lactate levels impaired osteoclast formation but also prevented an effective long-term immune activation. Therefore, targeting biofilm-derived metabolites but also IFN-{beta} signaling might be promising novel treatment strategies.

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RiboReport - Benchmarking tools for ribosome profiling-based identification of open reading frames in bacteria Small proteins, those encoded by open reading frames, with less than or equal to 50 codons, are emerging as an important class of cellular macromolecules in all kingdoms of life. However, they are recalcitrant to detection by proteomics or in silico methods. Ribosome profiling (Ribo-seq) has revealed widespread translation of sORFs in diverse species, and this has driven the development of ORF detection tools using Ribo-seq read signals. However, only a handful of tools have been designed for bacterial data, and have not yet been systematically compared. Here, we have performed a comprehensive benchmark of ORF prediction tools which handle bacterial Ribo-seq data. For this, we created a novel Ribo-seq dataset for E. coli, and based on this plus three publicly available datasets for different bacteria, we created a benchmark set by manual labeling of translated ORFs using their Ribo-seq expression profile. This was then used to investigate the predictive performance of four Ribo-seq-based ORF detection tools we found are compatible with bacterial data (Reparation_blast, DeepRibo, Ribo-TISH and SPECtre). The tool IRSOM was also included as a comparison for tools using coding potential and RNA-seq coverage only. DeepRibo and Reparation_blast robustly predicted translated ORFs, including sORFs, with no significant difference for those inside or outside of operons. However, none of the tools was able to predict a set of recently identified, novel, experimentally-verified sORFs with high sensitivity. Overall, we find there is potential for improving the performance, applicability, usability, and reproducibility of prokaryotic ORF prediction tools that use Ribo-Seq as input.

Key pointsO_LICreated a benchmark set for Ribo-seq based ORF prediction in bacteria
C_LIO_LIDeepRibo the first choice for bacterial ORF prediction tasks
C_LIO_LITool performance is comparable between operon vs single gene regions
C_LIO_LIIdentification of novel sORF with DeepRibo is, with restrictions, possible, by using the top 100 novel sORFs sorted by rank.
C_LIO_LIExperimental results show that considering translation initiation site data could boost the detection of novel small ORFs
C_LIO_LIDetermination of novel sORFs in E. coli using a new experimental protocol to enrich for translation initiation site. These data-set shows that still a significant part (here 8 out 24, so 1/3) are not detected dispute sufficient Ribo-seq signal. An additional 7 could be recovered using translation initiation site protocols.
C_LIO_LITools should embrace the use of replicate data and improve packaging, usability and documentation.
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sentences: Substrate topology and cognate immunity modulate antibacterial toxicity of Tae cell wall hydrolases Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance (NMR) and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.

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Intravenous haloperidol and cocaine alter the distribution of T CD4+ and B lymphocytes and NKT cells in rats Modulation of dopamine transmission evokes strong behavioral effects that can be achieved by psychoactive drugs such as haloperidol or cocaine. Cocaine non-specifically increases dopamine transmission by blocking dopamine active transporter (DAT) and evokes behavioral arousal, while haloperidol is a non-specific dopamine D2 receptor antagonist with sedative effects. Interestingly, dopamine has been found to affect immune cells in addition to its action in the central nervous system. Here we address the possible interactions between haloperidol and cocaine and their effects on both immune cells and behavior in freely moving rats. We use an intravenous model of haloperidol and binge cocaine administration to evaluate the drugs impact on the distribution of lymphocyte subsets in both the peripheral blood and the spleen. We assess the drugs behavioral effects by measuring locomotor activity. Cocaine evoked a pronounced locomotor response and stereotypic behaviors, both of which were completely blocked after pretreatment with haloperidol. The results suggest that blood lymphopenia which was induced by haloperidol and cocaine (except for NKT cells), is independent of dopaminergic activity and most likely results from the massive secretion of corticosterone. Haloperidol pretreatment prevented the cocaine-induced decrease in NKT cell numbers. On the other hand, the increased systemic dopaminergic activity after cocaine administration is a significant factor in retaining T CD4+ and B lymphocytes in the spleen.

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sentences: Highly efficient healing of critical sized articular cartilage defect in situ using a chemically nucleoside-modified mRNA-enhanced cell therapy Critical sized cartilage defects heal poorly and MSC-based therapies holds promise functional cartilage regeneration either used alone or in combination with growth factors. However, Recombinant protein growth factors were proven to have minimal benefits while to have adverse side effects and high cost. Nonviral mRNA delivery provides a promising, alternative approach to delivering therapeutic proteins within defect lesion for an extended period of time. Despite successful therapeutic outcome in bone and other vascularized tissues, the therapeutic application of mRNA in poorly vascularized tissues such as cartilage is still facing many challenges and rarely studied. We report here using chemically modified messenger RNA encoding TGF-{beta}3(TGF-{beta}3 cmRNA) to enhance the therapeutic efficacy of BMSCs to efficient repair of cartilage defect. Local administration of TGF-{beta}3 cmRNA enhanced BMSCs therapy restored critical-sized cartilage defects in situ in a rat model within 6 weeks with structural and molecular markers similar to its nature counterparts. In addition, the development of osteoarthritis caused by cartilage damage was prevented by this mRNA-enhanced BMSCs therapy evidenced by minimal late-stage OA pharmacological presentations. This novel mRNA enhanced-MSC technology extend the development of new therapeutic approaches for treating functional cartilage repair.

sentences: Non-target effects of ten essential oils on the egg-parasitoid Trichogramma evanescens Essential oils (EOs) are increasingly used as biopesticides due to their insecticidal potential. This study addresses their non-target effects on a biological control agent: the egg parasitoid Trichogramma evanescens. In particular, we tested whether EOs affected parasitoid fitness either directly, by decreasing pre-imaginal survival, or indirectly, by disrupting parasitoids orientation abilities. The effect of Anise, Fennel, Sweet orange, Basil, Coriander, Oregano, Peppermint, Mugwort, Rosemary and Thyme EOs were studied on five strains of T. evanescens. Specific experimental setups were developed, and data obtained from image analysis were interpreted with phenomenological models fitted with Bayesian inference. Results highlight the fumigant toxicity of EOs on parasitoid development. Anise, Fennel, Basil, Coriander, Oregano, Peppermint and Thyme EOs are particularly toxic and drastically reduce the emergence rate of T. evanescens. Most EOs also affect parasitoid behavior: (i) Basil, Coriander, Oregano, Peppermint, Mugwort and Thyme EOs are highly repellent for naive female parasitoids; (ii) Anise and Fennel EOs can have repellent to attractive effects depending on strains; and (iii) Sweet orange, Oregano and Rosemary EOs have no detectable impact on orientation behavior. This study shows that EOs fumigation have non-target effects on egg parasitoids. This highlights the need to cautiously precise the deployment framework of biopesticides in an agroecological perspective.

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Predicting the Functional Impact of KCNQ1 Variants with Artificial Neural Networks Recent advances in experimental and computational protein structure determination have provided access to high-quality structures for most human proteins and mutants thereof. However, linking changes in structure in protein mutants to functional impact remains an active area of method development. If successful, such methods can ultimately assist physicians in taking appropriate treatment decisions. This work presents three artificial neural network (ANN)-based predictive models that classify four key functional parameters of KCNQ1 variants as normal or dysfunctional using PSSM-based evolutionary and/or biophysical descriptors. Recent advances in predicting protein structure and variant properties with artificial intelligence (AI) rely heavily on the availability of evolutionary features and thus fail to directly assess the biophysical underpinnings of a change in structure and/or function. The central goal of this work was to develop an ANN model based on structure and physiochemical properties of KCNQ1 potassium channels that performs comparably or better than algorithms using only on PSSM-based evolutionary features. These biophysical features highlight the structure-function relationships that govern protein stability, function, and regulation. The input sensitivity algorithm incorporates the roles of hydrophobicity, polarizability, and functional densities on key functional parameters of the KCNQ1 channel. Inclusion of the biophysical features outperforms exclusive use of PSSM-based evolutionary features in predicting activation voltage dependence and deactivation time. As AI is increasing applied to problems in biology, biophysical understanding will be critical with respect to  explainable AI, i.e., understanding the relation of sequence, structure, and function of proteins. Our model is available at www.kcnq1predict.org.

Author summaryHeartbeat is maintained by electrical impulses generated by ion-conducting channel proteins in the heart such as the KCNQ1 potassium channel. Pathogenic variants in KCNQ1 can lead to channel loss-of-function and predisposition to fatal life-threatening irregularities of heart rhythm (arrhythmia). Machine learning methods that can predict the outcome of a mutation on KCNQ1 structure and function would be of great value in helping to assess the risk of a heart rhythm disorder. Recently, machine learning has made great progress in predicting the structures of proteins from their sequences. However, there are limited studies that link the effect of a mutation and change in protein structure with its function. This work presents the development of neural network models designed to predict mutation-induced changes in KCNQ1 functional parameters such as peak current density and voltage dependence of activation. We compare the predictive ability of features extracted from sequence, structure, and physicochemical properties of KCNQ1. Moreover, input sensitivity analysis connects biophysical features with specific functional parameters that provides insight into underlying molecular mechanisms for KCNQ1 channels. The best performing neural network model is publicly available as a webserver, called Q1VarPredBio, that delivers predictions about the functional phenotype of KCNQ1 variants.

sentences: Efficiency of the synthetic self-splicing RiboJ ribozyme is robust to cis- and trans-changes in genetic background The expanding knowledge of the variety of synthetic genetic elements has enabled the construction of new and more efficient genetic circuits and yielded novel insights into molecular mechanisms. However, context dependence, in which interactions between proximal (cis) or distal (trans) elements affect the behaviour of these elements, can reduce their general applicability or predictability. Genetic insulators, which mitigate unintended context-dependent cis-interactions, have been used to address this issue. One of the most commonly used genetic insulators is a self-splicing ribozyme called RiboJ, which can be used to decouple upstream 5 UTR in mRNA from downstream sequences (e.g., open reading frames). Despite its general use as an insulator, there has been no systematic study quantifying the efficiency of RiboJ splicing or whether this autocatalytic activity is robust to trans- and cis-genetic context. Here, we determine the robustness of RiboJ splicing in the genetic context of six widely divergent E. coli strains. We also check for possible cis-effects by assessing two SNP versions close to the catalytic site of RiboJ. We show that mRNA molecules containing RiboJ are rapidly spliced even during rapid exponential growth and high levels of gene expression, with a mean efficiency of 98%. We also show that neither the cis- nor trans-genetic context has a significant impact on RiboJ activity, suggesting this element is robust to both cis- and trans-genetic changes.

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Non-invasive sex genotyping of paiche Arapaima gigas by qPCR: An applied bioinformatic approach to identify sex differences Arapaima gigas, one of the largest freshwater fish in the world, is suffering from high fishing pressure and habitat loss, which threaten the conservation status of its natural populations. Of great cultural importance to Amazonian people, the paiche or pirarucu A. gigas is in high demand for its meat, ornamental uses and other byproducts such as scales. Aquaculture is a feasible solution to this dilemma. However, the fact that A. gigas presents no sexual dimorphism until it is 5 years old and its long period to sexual maturity are major obstacles for brood-stock management and fingerling production. Thus, the aim of this study was to develop a molecular tool for non-invasive genotypic sexing of paiche throughout its life cycle. We collected samples from gonads, fins and gill mucus of juvenile specimens from local facilities for histological and molecular analysis. Based on the recently available genome sequence of the paiche and making use of current NGS method, we implemented a novel approach, called Genome Differences by Unmapped Reads, to identify DNA sex markers. We found a Male-Specific Region (MSR), identified as MSR_3728, to be present only in males. Next, we designed two specific sets of primers on this region to identify genotypic sex by qPCR assays. Both primer sets, MSR_107 and MSR_129, detected males with 100% accuracy. Then we developed a duplex qPCR reaction for each primer set along a reference gene, analyzed the melting curves and detected males by observing two distinct peaks, one for MSR_107 or MSR_129 and one for the reference, while females only presented the reference peak. The same results were obtained for gonads, fins and interestingly, a non-invasive source from gill mucus samples. Finally, the gonads were evaluated histologically in a double-blind test, showing 100% accuracy with qPCR assay for identifying males and females. Data clearly demonstrated a novel pipeline approach for identifying DNA sex markers, followed by a quick, non-invasive, cost-effective duplex qPCR method for sexing A. gigas. These results may be valuable to efficient paiche aquaculture and conservation studies, helping to reduce the fishing pressure on its natural populations.

Highlights of the manuscript- Implementation of a novel approach, called Genome Differences by Unmapped Reads, to identify DNA sex markers.
- Finding of a Male-Specific Region (MSR), present only in males.
- Development of a duplex qPCR to identify genotypic sex through non-invasive sampling.

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Efficient high-resolution TMS mapping of the human motor cortex by nonlinear regression Transcranial magnetic stimulation (TMS) is a powerful tool to investigate causal structure-function relationships in the human brain. However, a precise delineation of the effectively stimulated neuronal populations is notoriously impeded by the widespread and complex distribution of the induced electric field.

Here, we propose a method that allows rapid and feasible cortical localization at the individual subject level. The functional relationship between electric field and behavioral effect is quantified by combining experimental data with numerically modelled fields to identify the cortical origin of the modulated effect. Motor evoked potentials (MEPs) from three finger muscles were recorded for a set of random stimulations around the primary motor area. All induced electric fields were nonlinearly regressed against the elicited MEPs to identify their cortical origin.

We could distinguish cortical muscle representation with high spatial resolution and localized them primarily on the crowns and rims of the precentral gyrus. A post-hoc analysis revealed exponential convergence of the method with the number of stimulations, yielding a minimum of about 180 random stimulations to obtain stable results.

Establishing a functional link between the modulated effect and the underlying mode of action, the induced electric field, is a fundamental step to fully exploit the potential of TMS. In contrast to previous approaches, the presented protocol is particularly easy to implement, fast to apply, and very robust due to the random coil positioning and therefore is suitable for practical and clinical applications.

sentences: Photon-free (s)CMOS camera characterization for artifact reduction in high- and super-resolution microscopy Modern implementations of widefield fluorescence microscopy often rely on sCMOS cameras, but this camera architecture inherently features pixel-to-pixel variations. Such variations lead to image artifacts and render quantitative image interpretation difficult. Although a variety of algorithmic corrections exists, they require a thorough characterization of the camera, which typically is not easy to access or perform. Here, we developed a fully automated pipeline for camera characterization based solely on thermally generated signal, and implemented it in the popular open-source software Micro-Manager and ImageJ/Fiji. Besides supplying the conventional camera maps of noise, offset and gain, our pipeline also gives access to dark current and thermal noise as functions of the exposure time. This allowed us to avoid structural bias in single-molecule localization microscopy (SMLM), which without correction is substantial even for scientific-grade, cooled cameras. In addition, our approach enables high-quality 3D super-resolution as well as live-cell time-lapse microscopy with cheap, industry-grade cameras. As our approach for camera characterization does not require any user interventions or additional hardware implementations, numerous correction algorithms demanding camera characterization become easily applicable.

sentences: The Landscape-Based Protein Stability Analysis and Network Modeling of Multiple Conformational States of the SARS-CoV-2 Spike D614 Mutant: Conformational Plasticity and Frustration-Driven Allostery as Energetic Drivers of Highly Transmissible Spike Variant The structural and functional studies of the SARS-CoV-2 spike protein variants revealed an important role of the D614G mutation that is shared across many variants of concern(VOCs), suggesting the effect of this mutation on the enhanced virus infectivity and transmissibility. The recent structural and biophysical studies provided important evidence about multiple conformational substates of the D614G spike protein. The development of a plausible mechanistic model which can explain the experimental observations from a more unified thermodynamic perspective is an important objective of the current work. In this study, we employed efficient and accurate coarse-grained simulations of multiple structural substates of the D614G spike trimers together with the ensemble-based mutational frustration analysis to characterize the dynamics signatures of the conformational landscapes. By combining the local frustration profiling of the conformational states with residue-based mutational scanning of protein stability and network analysis of allosteric interactions and communications, we determine the patterns of mutational sensitivity in the functional regions and sites of variants. We found that the D614G mutation may induce a considerable conformational adaptability of the open states in the SARS-CoV-2 spike protein without compromising folding stability and integrity of the spike protein. The results suggest that the D614G mutant may employ a hinge-shift mechanism in which the dynamic couplings between the site of mutation and the inter-protomer hinge modulate the inter-domain interactions, global mobility change and the increased stability of the open form. This study proposes that mutation-induced modulation of the conformational flexibility and energetic frustration at the inter-protomer interfaces may serve as an efficient mechanism for allosteric regulation of the SARS-CoV-2 spike proteins.

sentences: Optimal BR signalling is required for adequate cell wall orientation in the Arabidopsis root meristem The plant steroid hormones brassinosteroids (BRs) regulate growth in part through altering the properties of the cell wall, the extracellular matrix of plant cells. Conversely, cell wall signalling connects the state of cell wall homeostasis to the BR receptor complex and modulates BR activity. Here we report that both pectin-triggered cell wall signalling and impaired BR signalling result in altered cell wall orientation in the Arabidopsis root meristem. BR-induced defects in the orientation of newly placed walls are associated with aberrant localization of the cortical division zone but with normal specification of its positioning. Tissue- specific perturbations of BR signalling revealed that the cellular malfunction is unrelated to previously described whole organ growth defects. Thus, tissue type separates the pleiotropic effects of cell wall/BR signals and highlights their importance during cell wall placement.

sentences: Mechanisms underlying microglial colonization of developing neural retina in zebrafish Microglia are brain-resident macrophages that function as the first line of defense in brain. Embryonic microglial precursors originate in peripheral mesoderm and migrate into the brain during development. However, the mechanism by which they colonize the brain is incompletely understood. The retina is one of the first brain regions to accommodate microglia. In zebrafish, embryonic microglial precursors use intraocular hyaloid blood vessels as a pathway to migrate into the optic cup via the choroid fissure. Once retinal progenitor cells exit the cell cycle, microglial precursors associated with hyaloid blood vessels start to infiltrate the retina preferentially through neurogenic regions, suggesting that colonization of retinal tissue depends upon the neurogenic state. Along with blood vessels and retinal neurogenesis, IL34 also participates in microglial precursor colonization of the retina. Altogether, CSF receptor signaling, blood vessels, and neuronal differentiation function as cues to create an essential path for microglial migration into developing retina.

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Spatially confined sub-tumor microenvironments orchestrate pancreatic cancer pathobiology Pancreatic ductal adenocarcinoma (PDAC) remains resistant to most treatments and demonstrates a complex pathobiology. Here, we deconvolute regional heterogeneity in the human PDAC tumor microenvironment (TME), a long-standing obstacle, to define precise stromal contributions to PDAC progression. Large scale integration of histology-guided multiOMICs with clinical data sets and functional in vitro models uncovers two microenvironmental programs in PDAC that were anchored in fibroblast differentiation states. These sub-tumor microenvironments (subTMEs) co-occurred intratumorally and were spatially confined, producing patient-specific cellular and molecular heterogeneity associated with shortened patient survival. Each subTME was uniquely structured to support discrete aspects of tumor biology: reactive regions rich in activated fibroblast communities were immune-hot and promoted aggressive tumor progression while deserted regions enriched in extracellular matrix supported tumor differentiation yet were markedly chemoprotective. In conclusion, PDAC regional heterogeneity derives from biologically distinct reactive and protective TME elements with a defined, active role in PDAC progression.

Graphical Abstract & Key findings

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C_LIO_LISubTMEs exhibit distinct immune phenotypes and CAF differentiation states
C_LIO_LIDifferent subTMEs are either tumor-promoting or chemoprotective
C_LIO_LIIntratumoral subTME co-occurrence links stromal heterogeneity to patient outcome
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sentences: Genetic Analysis Suggests a Surface of PAT-4 (ILK) that Interacts with UNC-112 (kindlin) The transmembrane protein integrin plays a crucial role in the attachment of cells to the extracellular matrix. Integrin recruits many proteins intracellularly, including a four protein complex (Kindlin, Integrin linked kinase (ILK), PINCH, and parvin). C. elegans muscle provides an excellent model to study integrin adhesion complexes. In C. elegans, UNC-112 (Kindlin) binds to the cytoplasmic tail of PAT-3 ({beta}-integrin) and to PAT-4 (ILK). We previously reported that PAT-4 binding to UNC-112 is essential for the binding of UNC-112 to PAT-3. Although there are crystal structures for ILK and a kindlin, there is no co-crystal structure available. To understand the molecular interaction between PAT-4 (ILK) and UNC-112 (Kindlin), we took a genetic approach. First, we isolated mutant PAT-4 proteins that cannot bind to UNC-112. Then, we isolated suppressor mutant UNC-112 proteins that restore interaction with mutant PAT-4 proteins. Second, these mutant PAT-4 proteins cannot localize to attachment structures in nematode muscle, but upon co-expression of an UNC-112 suppressor mutant protein, the mutant PAT-4 protein could localize to attachment structures. Third, overexpression of a mutant PAT-4 protein results in disorganization of the adhesion plaques at muscle cell boundaries, and co-expression of the UNC-112 supressor mutant protein alleviates this defect. Thus, we demonstrate that UNC-112 binding to PAT-4 is required for the localization and function of PAT-4 in integrin adhesion complexes in vivo. The missense mutations were mapped onto homology models of PAT-4 and UNC-112, and taking into account previously isolated mutations, we suggest a surface of PAT-4 that binds to UNC-112.

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Human Cytomegalovirus uses a Host Stress Response to Balance the Elongation of Saturated/Monounsaturated and Polyunsaturated Very Long Chain Fatty Acids Stress and virus infection are known to regulate lipid metabolism in cells. Human cytomegalovirus infection induces fatty acid (FA) elongation and increases the cellular abundance of lipids with very long-chain FA tails (VLCFAs). While reprogramming of metabolism can be stress-related, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood. In this study, we engineered cells to knockout PKR-like ER kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMV infection. We found that in HCMV-infected cells, PERK promotes the increase in the levels of phospholipids with saturated FA (SFA) and monounsaturated FA (MUFA) VLCFAs tails. Consistent with the SFA/MUFA lipidome changes, PERK enhances the protein levels of FA elongase 7 (ELOVL7), which elongates SFA and MUFA VLCFAs. Additionally, we found that increases in the elongation of polyunsaturated fatty acids (PUFAs) associated with HCMV infection was independent of PERK and that lipids with PUFA tails accumulated in HCMV-infected PERK knockout cells. Consistent with the PUFA lipidome changes, the protein levels of ELOVL5, which elongates PUFAs, are increased by HCMV infection through a PERK-independent mechanism. These observations show that PERK differentially regulates ELOVL7 and ELOVL5, creating a balance between the synthesis of lipids with SFA/MUFA tails and PUFA tails. Additionally, we found that PERK was necessary for virus replication and the infectivity of released viral progeny. Overall, our findings indicate that PERK--and more broadly, ER stress--may be necessary for membrane biogenesis needed to generate infectious HCMV virions.

IMPORTANCEHCMV is a common herpesvirus that establishes lifelong persistent infections. While infection is asymptomatic in most people, HCMV causes life-threatening illnesses in immunocompromised people, including transplant recipients and cancer patients. Additionally, HCMV infection is a leading cause of congenital disabilities. HCMV replication relies on lipid synthesis. Here, we demonstrated that the ER stress mediator, PERK, controls fatty acid (FA) elongation and cellular abundance of several types of lipids following HCMV infection. Specifically, we found that PERK promotes FA elongase 7 synthesis of lipids with saturated/monounsaturated very long-chain FA tails which are important for building the viral membrane of infectious HCMV virions. Overall, our study shows that PERK is an essential host factor that supports HCMV replication and promotes lipidome changes caused by HCMV infection.

sentences: Mapping RNA-binding proteins in human B cells and Tcells upon differentiation B cells and T cells are key players in the defence against infections and malignancies. To exert their function, B cells and T cells differentiate into effector and memory cells. Tight regulation of these differentiation processes is key to prevent their malfunction, which can result in life-threatening disease. Lymphocyte differentiation relies on the appropriate timing and dosage of regulatory molecules, and post-transcriptional gene regulation (PTR) is a key player herein. PTR includes the regulation through RNA-binding proteins (RBPs), which control the fate of RNA and its translation into proteins. To date, a comprehensive RBP expression map throughout lymphocyte differentiation is lacking. Using transcriptome and proteome analyses, we here provide an RBP expression map for human B cells and T cells. We observed that even though the overall RBP expression is conserved, the relative RBP expression is distinct between B cells and T cells. Differentiation into effector and memory cells alters the RBP expression, resulting into preferential expression of different classes of RBPs. For instance, whereas naive T cells express high levels of translation-regulating RBPs, effector T cells preferentially express RBPs that modulate mRNA stability. Lastly, we found that cytotoxic CD8+ and CD4+ T cells express a common RBP repertoire. Combined, our study reveals a cell type-specific and differentiation-dependent RBP expression landscape in human lymphocytes, which will help unravel the role of RBPs in lymphocyte function.

sentences: Optineurin mediates delivery of membrane from transferrin receptor-positive endosomes to autophagosomes Autophagy is a conserved quality control mechanism that removes damaged proteins, organelles and invading bacteria through lysosome-mediated degradation. During autophagy several organelles including endoplasmic reticulum, mitochondria, plasma membrane and endosomes contribute membrane for autophagosome formation. However, the mechanisms and proteins involved in membrane delivery to autophagosomes are not clear. Optineurin (OPTN), a cytoplasmic adaptor protein, is involved in promoting maturation of phagophores into autophagosomes; it is also involved in regulating endocytic trafficking and recycling of transferrin receptor (TFRC). Here, we have examined the role of optineurin in the delivery of membrane from TFRC-positive endosomes to autophagosomes. Only a small fraction of autophagosomes was positive for TFRC, indicating that TFRC-positive endosomes could contribute membrane to a subset of autophagosomes. The percentage of TFRC-positive autophagosomes was reduced in Optineurin knockout mouse embryonic fibroblasts (Optn-/- MEFs) in comparison with normal MEFs. Upon over-expression of optineurin, the percentage of TFRC-positive autophagosomes was increased in Optn-/- MEFs. Unlike wild-type optineurin, a disease-associated mutant, E478G, defective in ubiquitin binding, was not able to enhance formation of TFRC-positive autophagosomes in Optn-/- MEFs. TFRC degradation mediated by autophagy was decreased in optineurin deficient cells. Our results suggest that optineurin mediates delivery of TFRC and perhaps associated membrane from TFRC-positive endosomes to autophagosomes, and this may contribute to autophagosome formation.

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Genetic diversity of wild and cultivated Coffea canephora in northeastern DR Congo and the implications for conservation. PremiseMany cultivated coffee varieties descend from Coffea canephora, commonly known as Robusta coffee. The Congo Basin has a century long history of Robusta coffee cultivation and breeding, and is hypothesized to be the region of origin of many of the cultivated Robusta varieties. Since little is known about the genetic composition of C. canephora in this region, we assessed the genetic diversity of wild and cultivated C. canephora shrubs in the Democratic Republic of the Congo.

MethodsUsing 18 microsatellite markers, we studied the genetic composition of wild and backyard-grown C. canephora shrubs in the Tshopo and Ituri provinces, and from the INERA Yangambi Coffee Collection. We assessed genetic clustering patterns, genetic diversity, and genetic differentiation between populations.

Key resultsGenetic differentiation was relatively strong between wild and cultivated C. canephora shrubs, and both gene pools harbored multiple unique alleles. Strong genetic differentiation was also observed between wild populations. The level of genetic diversity in wild populations was similar to that of the INERA Yangambi Coffee Collection, but local wild genotypes were mostly missing from that collection. Shrubs grown in the backyards were genetically similar to the breeding material from INERA Yangambi.

ConclusionsMost C. canephora that is grown in local backyards originated from INERA breeding programs, while a few shrubs were obtained directly from surrounding forests. The INERA Yangambi Coffee Collection could benefit from an enrichment with local wild genotypes, to increase the genetic resources available for breeding purposes, as well as to support ex situ conservation.

sentences: Analysis of motor unit spike trains estimated from high-density surface electromyography is highly reliable across operators There is a growing interest in decomposing high-density surface electromyography (HDsEMG) into motor unit spike trains to improve knowledge on the neural control of muscle contraction. However, the reliability of decomposition approaches is sometimes questioned, especially because they require manual editing of the outputs. We aimed to assess the inter-operator reliability of the identification of motor unit spike trains. Eight operators with varying experience in HDsEMG decomposition were provided with the same data extracted using the convolutive kernel compensation method. They were asked to manually edit them following established procedures. Data included signals from three lower leg muscles and different submaximal intensities. After manual analysis, 126 {+/-} 5 motor units were retained (range across operators: 119-134). A total of 3380 rate of agreement values were calculated (28 pairwise comparisons x 11 contractions/muscles x 4-28 motor units). The median rate of agreement value was 99.6%. Inter-operator reliability was excellent for both mean discharge rate and time at recruitment (intraclass correlation coefficient > 0.99). These results show that when provided with the same decomposed data and the same basic instructions, operators converge toward almost identical results. Our data have been made available so that they can be used for training new operators.

sentences: Protection of Hamsters Challenged with SARS-CoV-2 after Two Doses of MVC-COV1901 Vaccine Followed by a Single Intranasal Booster with Nanoemulsion Adjuvanted S-2P Vaccine Intramuscular vaccines have greatly reduced hospitalization and death due to severe COVID-19. However, most countries are experiencing a resurgence of infection driven predominantly by the Delta and Omicron variants of SARS-CoV-2. In response, booster dosing of COVID-19 vaccines has been implemented in many countries to address waning immunity and reduced protection against the variants. However, intramuscular boosting fails to elicit mucosal immunity and therefore does not solve the problem of persistent viral carriage and transmission, even in patients protected from severe disease. In this study, two doses of stabilized prefusion SARS-CoV-2 spike (S-2P)-based intramuscular vaccine adjuvanted with Alum/CpG1018, MVC-COV1901, were used as a primary vaccination series, followed by an intranasal booster vaccination with nanoemulsion (NE01)-adjuvanted S-2P vaccine in a hamster model to demonstrate immunogenicity and protection from viral challenge. Here we report that this vaccination regimen resulted not only in the induction of robust immunity and protection against weight loss and lung pathology following challenge with SARS-CoV-2, but also led to increased viral clearance from both upper and lower respiratory tracts. Our findings showed that intramuscular MVC-COV1901 vaccine followed by a booster with intranasal NE01-adjuvanted vaccine promotes protective immunity against both viral infection and disease, suggesting that this immunization protocol may offer a solution in addressing a significant, unmet medical need for both the COVID-19 and future pandemics.

sentences: The Disease-Associated Proteins Drosophila Nab2 and Ataxin-2 Interact with Shared RNAs and Coregulate Neuronal Morphology Nab2 encodes a conserved polyadenosine RNA-binding protein (RBP) with broad roles in post-transcriptional regulation, including in poly(A) RNA export, poly(A) tail length control, transcription termination, and mRNA splicing. Mutation of the Nab2 human ortholog ZC3H14 gives rise to an autosomal recessive intellectual disability, but understanding of Nab2/ZC3H14 function in metazoan nervous systems is limited, in part because no comprehensive identification of metazoan Nab2/ZC3H14-associated RNA transcripts has yet been conducted. Moreover, many Nab2/ZC3H14 functional protein partnerships likely remain unidentified. Here we present evidence that Drosophila melanogaster Nab2 interacts with the RBP Ataxin-2 (Atx2), a neuronal translational regulator, and implicate these proteins in coordinate regulation of neuronal morphology and adult viability. We then present the first high-throughput identifications of Nab2- and Atx2-associated RNAs in Drosophila brain neurons using an RNA immunoprecipitation-sequencing (RIP-Seq) approach. Critically, the RNA interactomes of each RBP overlap, and Nab2 exhibits high specificity in its RNA associations in neurons in vivo, associating with a small fraction of all polyadenylated RNAs. The identities of shared associated transcripts (e.g. drk, me31B, stai) and of transcripts specific to Nab2 or Atx2 (e.g. Arpc2, tea, respectively) promise insight into neuronal functions of and interactions between each RBP. Significantly, Nab2-associated RNAs are overrepresented for internal A-rich motifs, suggesting these sequences may partially mediate Nab2 target selection. Taken together, these data demonstrate that Nab2 opposingly regulates neuronal morphology and shares associated neuronal RNAs with Atx2, and that Drosophila Nab2 associates with a more specific subset of polyadenylated mRNAs than its polyadenosine affinity alone may suggest.

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Cell-free chromatin particles released from dying cells inflict mitochondrial damage and ROS production in living cells Several hundred billion to a trillion cells die in the body every day and release cell free chromatin particles (cfChPs) which enter into the circulation, or are released locally into extracellular compartments of the body. We have reported that cfChPs from the dying cells can readily enter into living cells and damage their DNA. To test the hypothesis that internalised cfChPs might also inflict mitochondrial damage, we treated NIH3T3 mouse fibroblast cells with cfChPs isolated from sera of healthy individuals (10ng), or co-cultured the cells with hypoxia induced dying NIH3T3 cells. Abundant cfChPs could be detected in the cytoplasm of the treated cells by 4h. The latter was associated with evidence of mitochondrial damage in the form of ultra-structural changes, increased mitochondrial mass, alterations in mitochondrial shape, upregulation of the mitochondrial outer membrane protein TOM20, and changes in mitochondrial membrane potential. We also detected increased fluorescence signals of {gamma}-H2AX and p-ATM signifying double-strand breaks in mitochondrial DNA. There was marked increase in production of mitochondrial superoxide (ROS) as detected by MitoSOX Red, and activation of the intracellular antioxidant enzyme superoxide dismutase-1. Mitochondrial damage and ROS production could be inhibited by a cfChPs deactivating agent viz. anti-histone antibody complexed nanoparticles. Given that 1x109-1x1012 cells die in the body every day, we propose that cfChPs are major physiological triggers for mitochondrial damage and ROS production with an important bearing on human health and disease. Deactivation of cfChPs may provide a novel therapeutic approach to retard ageing and associated degenerative conditions that have been linked to oxidative stress.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/474529v1_ufig1.gif" ALT="Figure 1">
View larger version (28K):
org.highwire.dtl.DTLVardef@1111c19org.highwire.dtl.DTLVardef@4bd7eaorg.highwire.dtl.DTLVardef@14c47e7org.highwire.dtl.DTLVardef@104a4e6_HPS_FORMAT_FIGEXP  M_FIG C_FIG Legend: Cell-free chromatin particles (cfChPs) that circulate in blood, and those that are released locally from dying cells, can readily enter into healthy cells to damage their mitochondria leading to ROS production.

HighlightsO_LICell-free chromatin particles (cfChPs) are continuously released from dying cells
C_LIO_LIcfChPs can readily enter into living cells
C_LIO_LIThe internalised cfChPs inflict physical damage to mitochondria
C_LIO_LIcfChPs induced mitochondrial damage results in ROS production and oxidative stress
C_LIO_LIcfChPs are novel triggers for ROS production with implications for health and disease
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sentences: Identifying nearshore nursery habitats for sharks in the Eastern Tropical Pacific from fisheries landings and interviews The Eastern Tropical Pacific (ETP) comprising the coasts of Costa Rica, Panama, Colombia and Ecuador, represents an area of high marine biodiversity that supports productive fisheries and acts as an important migratory corridor for many marine species. Despite its biological importance, the ETP is understudied and lacks sufficient data for science-based fisheries management and conservation decision-making. This study aims to consolidate understanding of the current and historical distribution of sharks and mobulid rays in the ETP. We used interviews of coastal community stakeholders to document traditional knowledge of shark and mobulid ray species and distributions. We also analysed small-scale fisheries landings data, where available, to quantify local exploitation patterns and the importance of sharks and rays in small-scale fisheries catches. All shark species landed in the dominant nearshore gillnet fishery show very low mean individuals weights (<5 kg), indicating that the fisheries are dominated by juveniles, captured. Aside from smooth-hounds (Mustelus spp.), the scalloped hammerhead shark, Sphyrna lewini, is the most frequently landed shark species in the region by weight and number, with peaks in abundance between April - July. From 132 interviews in 51 communities across the three countries, and landings data from two small-scale fisheries sites, we identified 41 sites in 12 broad geographical zones as important shark nursery habitats. Of these sites, 68% were associated closely with large mangrove systems of the ETP, highlighting the importance of this habitat for shark life history. No patterns were seen in the occurrence or distribution of mobulid rays in coastal areas. Marine protected areas and responsible fishing zones cover 37% of identified nursery habitats in the ETP, 30% in Costa Rica, 48% in Panama and 30% in Colombia. These findings provide an important benchmark of the conservation status of sharks in the ETP and allow for the prioritisation of research and policy-making.

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Covalent Docking and Molecular Dynamics Simulations Reveal the Specificity-Shifting Mutations Ala237Arg and Ala237Lys in TEM Beta-Lactamase The rate of modern drug discovery using experimental screening methods still lags behind the rate at which pathogens mutate, underscoring the need for fast and accurate predictive simulations of protein evolution. Multidrug-resistant bacteria evade our defenses by expressing a series of proteins, the most famous of which is the 29-kilodalton enzyme, TEM {beta}-lactamase. Considering these challenges, we applied a covalent docking heuristic to measure the effects of all possible alanine 237 substitutions in TEM due to this codons importance for catalysis and effects on the binding affinities of commercially-available {beta}-lactam compounds. In addition to the usual mutations that reduce substrate binding due to steric hindrance, we identified two distinctive specificity-shifting TEM mutations, Ala237Arg and Ala237Lys, and their respective modes of action. Notably, we discovered and verified through minimum inhibitory concentration assays that, while these mutations and their bulkier side chains lead to steric clashes that curtail ampicillin binding, these same groups foster salt bridges with the negatively-charged side-chain of the cephalosporin cefixime, widely used in the clinic to treat multi-resistant bacterial infections. To measure the stability of these unexpected interactions, we used molecular dynamics simulations and found the binding modes to be stable despite the application of biasing forces. Finally, we found that both TEM mutants also bind strongly to other drugs containing negatively-charged R-groups, such as carumonam and ceftibuten. As with cefixime, this increased binding affinity stems from a salt bridge between the compounds negative moieties and the positively-charged side chain of the arginine or lysine, suggesting a shared mechanism. In addition to reaffirming the power of using simulations as molecular microscopes, our results can guide the rational design of next-generation {beta}-lactam antibiotics and bring the community closer to retaking the lead against the recurrent threat of multidrugresistant pathogens.

Author SummaryResistance to antibiotics is a major public health threat. Microorganisms are able to resist commonly used drugs by evolving and expressing enzymes capable of neutralizing antibiotics. Understanding the relationships between structural elements in these enzymes and their drug-clearing functions can lead to crucial insights for the discovery of next-generation antibiotics. In this study, we have used cutting-edge computational modeling methods to predict the effects of all naturally-occurring variations of an important region of the binding site of TEM {beta}-lactamase, one of the major resistance-granting enzymes in bacteria. In an effort to identify patterns that could be useful for drug discovery, our simulations sought to understand how chemical changes in the tested region can affect resistance against a collection of over 90 widely used antibiotics. Crucially, through our simulations, we have identified a pathway for bacterial resistance against {beta}-lactam antibiotics containing a negatively-charged moiety. We have also elucidated the mechanism behind the gain of resistance, which involves strong interactions between the drugs negative moieties and the positively-charged chemical shifts resulting from the mutation. Finally, we validated our predictions against fitness experiments for two commonly used antibiotics, which qualitatively corroborated our most important findings.

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Importance of Mobile Genetic Element Immunity in Numerically Abundant Trichodesmium Clades The colony-forming cyanobacteria Trichodesmium spp. are considered one of the most important nitrogen-fixing genera in the warm, low nutrient, open ocean. Despite this central biogeochemical role, many questions about their evolution, physiology, and trophic interactions remain unanswered. To address these questions, we describe the genetic potential of the genus via significantly improved genomic assemblies of strains Trichodesmium thiebautii H94, Trichodesmium erythraeum 2175, and 17 new Trichodesmium metagenome-assembled genomes (MAGs, >50% complete) from hand-picked, Trichodesmium colonies spanning the Atlantic Ocean. Phylogenomics identified [~]four N2 fixing clades of Trichodesmium across the transect, with T. thiebautii dominating the colony-specific reads. Pangenomic analyses showed that all T. thiebautii MAGs are enriched in defense mechanisms and encode a vertically inherited Type III-B Clustered Regularly Interspaced Short Palindromic Repeats and associated protein-based immunity system (CRISPR-Cas hereafter). Surprisingly, this CRISPR-Cas system was absent in all T. erythraeum genomes and MAGs, vertically inherited by T. thiebautii, and correlated with increased signatures of horizontal gene transfer. Multiple lines of evidence indicate that the CRISPR-Cas system is functional in situ: 1. Trichodesmium CRISPR spacer sequences with 100% identical hits to field-assembled, putative phage genome fragments were identified, 2. High Trichodesmium spacer sequence variability indicating rapid adaptation, and 3. metaproteomic and transcriptomic expression analyses detecting the CRISPR-Cas system components in Trichodesmium colonies from the Atlantic and Pacific Oceans. These data suggest that phage or mobile genetic element immunity in T. thiebautii could contribute to their success, gene diversity, and numerical dominance over T. erythraeum in the oceans, thus warranting further Trichodesmium virome investigations.

Significance statementOur work identifies CRISPR-Cas immunity as a phylogenetically distinct, environmentally expressed factor in the speciation of closely related N2-fixing Trichodesmium clades. These findings suggest that differential phage predation and resistance could be a previously overlooked selective pressure in the genus, potentially leading to the current numerical dominance of T. thiebautii over T. erythraeum in the oceans. Furthermore, while the currently CO2-limited T. erythraeum is expected to be a  winner of anthropogenic climate change, their predicted higher phage sensitivity than T. thiebautii could challenge this outcome.

sentences: Designing BH3-mimetic Peptide Inhibitors for the Viral Bcl-2 Homologs A179L and BHRF1: Importance of long-range electrostatic interactions Viruses have evolved strategies to prevent apoptosis of infected cells at early stages of infection. The viral proteins (vBcl-2s) from specific viral genes adopt a helical fold that is structurally similar to that of mammalian anti-apoptotic Bcl-2 proteins and exhibit little sequence similarity. Hence vBcl-2 homologs are attractive targets to prevent viral infection. However, very few studies have focused on developing inhibitors for vBcl-2 homologs. In this study, we have considered two vBcl-2 homologs, A179L from African swine fever virus and BHRF1 from Epstein-Barr virus. We generated two sets of 8000 randomized BH3-like sequences from eight wild-type pro-apoptotic BH3 peptides. During this process, the four conserved hydrophobic residues and an Asp residue were retained at their respective positions and all other positions were substituted randomly without any bias. We constructed 8000 structures each for A179L and BHRF1 in complex with BH3-like sequences. Histograms of interaction energies calculated between the peptide and the protein resulted in negatively skewed distributions. The BH3-like peptides with high helical propensities selected from the negative tail of respective interaction energy distributions exhibited more favorable interactions with A179L and BHRF1 and they are rich in basic residues. Molecular dynamics studies and electrostatic potential maps further revealed that both acidic and basic residues favorably interact with A179L while only basic residues have the most favorable interactions with BHRF1. As in mammalian homologs, the role of long range interactions and non-hotspot residues have to be taken into account while designing specific BH3-mimetic inhibitors for vBcl-2 homologs.

sentences: Detecting patterns of accessory genome coevolution in bacterial species using data from thousands of bacterial genomes Bacterial genomes exhibit widespread horizontal gene transfer, resulting in highly variable genome content that complicates the inference of genetic interactions. In this study, we develop a method for detecting coevolving genes from large datasets of bacterial genomes that we call a "coevolution score". The method is based on pairwise comparisons of closely related individuals, analogous to a pedigree study in eukaryotic populations. This approach avoids the need for an accurate phylogenetic tree and allows very large datasets to be analyzed for signatures of recent coevolution. We apply our method to all of the more than 3 million pairs of genes from the entire annotated Staphylococcus aureus accessory genome of 2,756 annotated genes using a database of over 40,000 whole genomes. We find many pairs of genes that that appear to be gained or lost in a coordinated manner, as well as pairs where the gain of one gene is associated with the loss of the other. These pairs form networks of dozens of rapidly coevolving genes, primarily consisting of genes involved in metal resistance, virulence, mechanisms of horizontal gene transfer, and antibiotic resistance, particularly the SCCmec complex. Our results reflect the fact that the evolution of many bacterial pathogens since the middle of the twentieth century has largely been driven by antibiotic resistance gene gain, and in the case of S. aureus the SCCmec complex is the most prominent of these elements driving the evolution of resistance. The frequent coincidence of these gene gain or loss events suggests that S. aureus switch between antibiotic-resistant niches and antibiotic-susceptible ones. While we focus on gene gain and loss, our method can also detect genes which tend to acquire substitutions in tandem or, in datasets that include phenotypic information, genotype-phenotype or phenotype-phenotype coevolution.

sentences: Neural Network Organization for Courtship Song Feature Detection in Drosophila Animals communicate using sounds in a wide range of contexts, and auditory systems must encode behaviorally relevant acoustic features to drive appropriate reactions. How feature detection emerges along auditory pathways has been difficult to solve due to challenges in mapping the underlying circuits and characterizing responses to behaviorally relevant features. Here, we study auditory activity in the Drosophila melanogaster brain and investigate feature selectivity for the two main modes of fly courtship song, sinusoids and pulse trains. We identify 24 new cell types of the intermediate layers of the auditory pathway, and using a new connectomic resource, FlyWire, we map all synaptic connections between these cell types, in addition to connections to known early and higher-order auditory neurons - this represents the first map of the auditory pathway. We additionally determine the sign (excitatory or inhibitory) of most synapses in this auditory connectome. We find that auditory neurons display a continuum of preferences for courtship song modes, and that neurons with different song mode preferences are highly interconnected in a network that lacks hierarchical structure. Among this network, frequency tuning is centered on the range of frequencies present in song, whereas pulse rate tuning extends to rates outside of song, suggesting that these neurons form a basis set for downstream processing. Our study provides new insights into the organization of auditory coding within the Drosophila brain.

sentences: SOX15 regulates stem cell pluripotency and promotes neural fate during differentiation by activating Hes5 SOX2 and SOX15 are Sox family transcription factors enriched in embryonic stem cells (ESCs). The role of SOX2 in activating gene expression programs essential for stem cell self-renewal and acquisition of pluripotency during somatic cell reprogramming is well-documented. However, the contribution of SOX15 to these processes is unclear and often presumed redundant with SOX2 largely because overexpression of SOX15 can partially restore self-renewal in SOX2-deficient ESCs. Here, we show that SOX15 contributes to stem cell maintenance by cooperating with ESC-enriched transcriptional coactivators to ensure optimal expression of pluripotency-associated genes. We demonstrate that SOX15 depletion compromises reprogramming of fibroblasts to pluripotency which cannot be compensated by SOX2. Ectopic expression of SOX15 promotes the reversion of a post-implantation, epiblast stem cell state back to a pre-implantation, ESC-like identity even though SOX2 is expressed in both cell states. We also uncover a role of SOX15 in lineage specification, by showing that loss of SOX15 leads to defects in commitment of ESCs to neural fates. SOX15 promotes neural differentiation by binding to and activating a previously uncharacterized distal enhancer of a key neurogenic regulator, Hes5. Together, these findings identify a multifaceted role of SOX15 in induction and maintenance of pluripotency and neural differentiation.

sentences: Juvenile Huntington's Disease and Other PolyQ diseases, Update on Neurodevelopmental Character and Comparative Bioinformatic Review of Transcriptomic Data Polyglutamine (PolyQ) diseases are neurodegenerative disorders caused by the CAG repeat expansion mutation in affected genes resulting in toxic proteins containing a long chain of glutamines. There are nine PolyQ diseases: Huntingtons disease (HD), spinocerebellar ataxias (types 1, 2, 3, 6, 7, and 17), dentatorubral-pallidoluysian atrophy (DRPLA), and spinal bulbar muscular atrophy (SBMA). In general, longer CAG expansions and longer glutamine tracts lead to earlier disease presentations in PolyQ patients. Rarely, cases of extremely long expansions are identified for PolyQ diseases, and they consistently lead to juvenile or sometimes very severe infantile-onset polyQ syndromes. In apparent contrast to the very long CAG tracts, shorter CAGs and PolyQs in proteins seems to be the evolutionary factor enhancing human cognition. Therefore, polyQ tracts in proteins can be modifiers of brain development and disease drivers, which contribute neurodevelopmental phenotypes in juvenile- and adult-onset PolyQ diseases. Therefore we performed a bioinformatics review of published RNAseq polyQ expression data resulting from the presence of polyQ genes in search of neurodevelopmental expression patterns and comparison between diseases. The expression data were collected from cell types reflecting stages of development such as iPSC, neuronal stem cell, neurons, but also the adult patients and models for PolyQ disease. Our comparative bioinformatic review highlighted several (neuro)developmental pathways and genes identified within PolyQ diseases and mouse models responsible for neural growth, synaptogenesis, and synaptic plasticity.

sentences: A Unifying Statistical Framework to Discover Disease Genes from GWAS Genome-wide association studies (GWAS) identify genomic loci associated with complex traits, but it remains an open challenge to identify the genes underlying the association signals. Here, we extend the equations of statistical fine-mapping, to compute the probability that each gene in the human genome is targeted by a causal variant, given a particular trait. Our computations are enabled by several key innovations. First, we partition the genome into optimal linkage disequilibrium blocks, enabling genome-wide detection of trait-associated genes. Second, we unveil a comprehensive mapping that associates genetic variants to the target genes they affect. The combined performance of the map on high-throughput functional genomics and eQTL datasets supersedes the state of the art. Lastly, we describe an algorithm which learns, directly from GWAS data, how to incorporate prior knowledge into the statistical computations, significantly improving their accuracy. We validate each component of the statistical framework individually and in combination. Among methods to identify genes targeted by causal variants, this paradigm rediscovers an unprecedented proportion of known disease genes. Moreover, it establishes human genetics support for many genes previously implicated only by clinical or preclinical evidence, and it discovers an abundance of novel disease genes with compelling biological rationale.

sentences: Rational Drug Design of Targeted and Enzyme Cleavable Vitamin E Analogs as Neoadjuvant to Chemotherapy: In Vitro and In Vivo Evaluation on Reduction of Cardiotoxicity of Doxorubicin. Traditional drug design focus on specific target (s) expressed by cancer cells. However, cancer cells outsmart the interventions by activating survival pathways and/or downregulating cell death pathways. As the research in molecular biology of cancer grows exponentially, new methods of drug designs are needed to target multiple pathways/targets which are involved in survival of cancer cells. Vitamin E analogues including a-tocopheryl succinate (-TOS) is a well-known anti-tumoregenic agent which is well studied both in vitro and in vivo tumor models. However, lack of targeting cancer cells and unexpected toxicity along with the poor water solubility of -TOS compelled a rational drug design using both targeting and cleavable technologies incorporated in the new drug design. A plethora of Vitamin E derivatives (AMP-001, 002 and 003) were synthesized, characterized and studied for the improved efficacy and lowered toxicity in various cancer cells in vitro. Preliminary studies revealed AAAPT leading candidates reduced the invasive potential of brain tumor stem cells, synergized with different drugs and different treatments. AAAPT leading drug AMP-001 enhanced the therapeutic index of front-line drug Doxorubicin in triple negative breast cancer (TNBC) tumor rat model preserving the ventricular function when used as a neoadjuvant to Doxorubicin. These results may pave the way for reducing the cardiotoxicity of chemotherapy in clinical settings.

sentences: Functional connectome of brainstem nuclei involved in autonomic, limbic, pain and sensory processing in living humans from 7 Tesla resting state fMRI Despite remarkable advances in mapping the functional connectivity of the cortex, the functional connectivity of subcortical regions is understudied in living humans. This is the case for brainstem nuclei that control vital processes, such as autonomic, limbic, nociceptive and sensory functions. This is because of the lack of precise brainstem nuclei localization, of adequate sensitivity and resolution in the deepest brain regions, as well as of optimized processing for the brainstem. To close the gap between the cortex and the brainstem, on 20 healthy subjects, we computed a correlation-based functional connectome of 15 brainstem nuclei involved in autonomic, limbic, nociceptive, and sensory function (superior and inferior colliculi, ventral tegmental area-parabrachial pigmented nucleus complex, microcellular tegmental nucleus-prabigeminal nucleus complex, lateral and medial parabrachial nuclei, vestibular and superior olivary complex, superior and inferior medullary reticular formation, viscerosensory motor nucleus, raphe magnus, pallidus, and obscurus, and parvicellular reticular nucleus - alpha part) with the rest of the brain. Specifically, we exploited 1.1mm isotropic resolution 7 Tesla resting-state fMRI, ad-hoc coregistration and physiological noise correction strategies, and a recently developed probabilistic template of brainstem nuclei. Further, we used 2.5mm isotropic resolution resting-state fMRI data acquired on a 3 Tesla scanner to assess the translatability of our results to conventional datasets. We report highly consistent correlation coefficients across subjects, confirming available literature on autonomic, limbic, nociceptive and sensory pathways, as well as high interconnectivity within the central autonomic network and the vestibular network. Interestingly, our results showed evidence of vestibulo-autonomic interactions in line with previous work. Comparison of 7 Tesla and 3 Tesla findings showed high translatability of results to conventional settings for brainstem-cortical connectivity and good yet weaker translatability for brainstem-brainstem connectivity. The brainstem functional connectome might bring new insight in the understanding of autonomic, limbic, nociceptive and sensory function in health and disease.

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A dual sequence and culture-based survey of maize rhizosphere protists reveals dominant,plant-enriched, and culturable community members Protists play important roles in shaping the microbial community of the rhizosphere. However, there is still a limited understanding of how plants shape the protist community, and how well protist isolate collections might represent rhizosphere protist composition and function in downstream studies. We sought to determine whether maize roots select for a distinct protist community in the field, and whether the common or dominant members of that community are readily culturable using standard protist isolation methods. We sequenced 18S and 16S rRNA genes from the rhizospheres of maize grown in two sites, and isolated 103 protists into culture from the same roots. While field site had the greatest effect, rhizospheres in both sites had distinct protist composition from the bulk soils, and certain taxa were enriched in both sites. Enriched taxa were correlated to bacterial abundance patterns. The isolated protists represented six supergroups, and the majority corresponded to taxa found in the sequencing survey. Twenty-six isolates matched eight of the 89 core rhizosphere taxa. This study demonstrates that maize roots select for a distinct protist community, but also illustrate the potential challenges in understanding the function of the dominant protist groups in the rhizosphere.

Originality-Significance StatementThis is the first study comparing cultivation-dependent and independent methods for studying the protist community of plant roots, and the first untargeted analysis of the maize rhizospheres effect on protist communities. We show that maize in different sites select for distinct communities and overlapping enriched taxa, but that isolating the most important plant-associated protists may be a challenge for researchers.

sentences: Pattern dynamics and stochasticity of the brain rhythms Our current understanding of brain rhythms is based on quantifying their instantaneous or time-averaged characteristics. What remains unexplored, is the actual structure of the waves--their shapes and patterns over finite timescales. To address this, we used two independent approaches to link wave forms to their physiological functions: the first is based on quantifying their consistency with the underlying mean behavior, and the second assesses "orderliness" of the waves features. The corresponding measures capture the waves characteristic and abnormal behaviors, such as atypical periodicity or excessive clustering, and demonstrate coupling between the patterns dynamics and the animals location, speed and acceleration. Specifically, we studied patterns of{theta} and{gamma} waves, and Sharp Wave Ripples, and observed speed-modulated changes of the waves cadence, an antiphase relationship between orderliness and acceleration, as well as spatial selectiveness of patterns. Further-more, we found an interdependence between orderliness and regularity: larger deviations from steady oscillatory behavior tend to accompany disarrayed temporal cluttering of peaks and troughs. Taken together, our results offer a complementary--mesoscale--perspective on brain wave structure, dynamics, and functionality.

sentences: Fibrin is a critical regulator of neutrophil effector function at mucosal barrier sites Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we document that the clotting factor fibrin is a critical regulator of neutrophil function at mucosal barriers. We demonstrate that fibrin engages neutrophils through the M{beta}2 integrin receptor and activates effector functions, including the production of reactive oxygen species and NET formation. These immune-protective neutrophil functions become tissue damaging in the context of impaired plasmin-mediated fibrinolysis. Indeed, the accumulation of fibrin due to Mendelian genetic defects in plasmin leads to severe oral mucosal immunopathology in mice and humans. Concordantly, genetic polymorphisms in the human PLG gene, encoding plasminogen, are associated with common forms of the oral mucosal disease periodontitis. Our work uncovers fibrin as a critical regulator of neutrophil effector function within the mucosal tissue microenvironment and suggests fibrin-neutrophil engagement as a pathogenic instigator and therapeutic target in common mucosal disease.

sentences: Chromosomal instability mediates immune exclusion and response to cytotoxic chemotherapy in colorectal liver metastases The genomic drivers of immune exclusion in colorectal cancer liver metastases (CRCLM) remain poorly understood. Chromosomal instability (CIN), resulting in aneuploidy and genomic rearrangements, is the central pathway of mismatch repair-proficient colorectal cancer pathogenesis; however, it is unknown whether CIN impacts the outcomes of patients with limited spread of CRCLM treated with curative intent cytotoxic chemotherapy and surgery. Herein, we examined the relationship between CIN and the molecular subtypes of CRCLM, immune signaling, treatment sensitivity, and patient outcomes in three independent CRCLM patient cohorts. We established that a previously developed 70-gene CIN signature (CIN70) is a reliable measure of CIN, encompassing features of both aneuploidy and cellular proliferation. We demonstrated that tumors with the canonical subtype of CRCLM exhibit elevated levels of CIN and aneuploidy. Genomically unstable tumors were associated with an immune-depleted tumor microenvironment, and patients with genomically unstable tumors were at increased risk for disease progression in adverse metastatic sites, resulting in poor progression-free and overall survival. However, high-CIN tumors were particularly susceptible to DNA-damaging chemotherapies, including topoisomerase inhibitors, as well as radiation therapy. Treatment with genotoxic agents depleted CIN-rich cell populations, which resulted in a concomitant increase in intratumoral CD8+ T-cells in patients with primary rectal, breast, and bladder cancer. Taken together, we propose a mechanistic explanation for why cytotoxic chemotherapy can augment anti-tumor immunity and improve outcomes in patients with genomically unstable cancers.

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Use of object detection in camera trap image identification: assessing a method to rapidly and accurately classify human and animal detections for research and application in recreation ecology Camera traps are increasingly used to answer complex ecological questions. However, the rapidly growing number of images collected presents technical challenges. Each image must be classified to extract data, requiring significant labour, and potentially creating an information bottleneck. We applied an object-detection model (MegaDetector) to camera trap data from a study of recreation ecology in British Columbia, Canada. We tested its performance in detecting humans and animals relative to manual image classifications, and assessed efficiency by comparing the time required for manual classification versus a modified workflow integrating object-detection with manual classification. We also evaluated the reliability of using MegaDetector to create an index of human activity for application to the study of recreation impacts to wildlife. In our application, MegaDetector detected human and animal images with 99% and 82% precision, and 95% and 92% recall respectively, at a confidence threshold of 90%. The overall time required to process the dataset was reduced by over 500%, and the manual processing component was reduced by 840%. The index of human detection events from MegaDetector matched the output from manual classification, with a mean 0.45% difference in estimated human detections across site-weeks. Our test of an open-source object-detection model showed it performed well in partially classifying a camera trap dataset, significantly increasing processing efficiency. We suggest that this tool could be integrated into existing camera trap workflows to accelerate research and application by alleviating data bottlenecks, particularly for surveys processing large volumes of human images. We also show how the model and workflow can be used to anonymize human images prior to classification, protecting individual privacy.

Impact StatementWe developed and tested a workflow for classifying camera trap images that integrated an existing object-detection model with manual image classification. Our workflow demonstrates an increase in efficiency of 500% over manual labelling, and additionally includes a method to anonymize human images prior to archiving and classification. We provide an example of the application of these tools to ease data processing, particularly for studies focused on recreation ecology which record high volumes of human images. Data lags due to processing delays have the potential to result in sub-optimal conservation decisions, which may be alleviated by accelerated processing. To our knowledge, this is the first in-depth assessment of the practical application of such technology to real world workflows focused on human detections.

sentences: LYRUS: A Machine Learning Model for Predicting the Pathogenicity of Missense Variants Single amino acid variations (SAVs) are a primary contributor to variations in the human genome. Identifying pathogenic SAVs can aid in the diagnosis and understanding of the genetic architecture of complex diseases, such as cancer. Most approaches for predicting the functional effects or pathogenicity of SAVs rely on either sequence or structural information. Nevertheless, previous analyses have shown that methods that depend on only sequence or structural information may have limited accuracy. Recently, researchers have attempted to increase the accuracy of their predictions by incorporating protein dynamics into pathogenicity predictions. This study presents < Lai Yang Rubenstein Uzun Sarkar > (LYRUS), a machine learning method that uses an XGBoost classifier selected by TPOT to predict the pathogenicity of SAVs. LYRUS incorporates five sequence-based features, six structure-based features, and four dynamics-based features. Uniquely, LYRUS includes a newly-proposed sequence co-evolution feature called variation number. LYRUSs performance was evaluated using a dataset that contains 4,363 protein structures corresponding to 20,307 SAVs based on human genetic variant data from the ClinVar database. Based on our dataset, the LYRUS classifier has a higher accuracy, specificity, F-measure, and Matthews correlation coefficient (MCC) than alternative methods including PolyPhen2, PROVEAN, SIFT, Rhapsody, EVMutation, MutationAssessor, SuSPect, FATHMM, and MVP. Variation numbers used within LYRUS differ greatly between pathogenic and neutral SAVs, and have a high feature weight in the XGBoost classifier employed by this method. Applications of the method to PTEN and TP53 further corroborate LYRUSs strong performance. LYRUS is freely available and the source code can be found at https://github.com/jiaying2508/LYRUS.

sentences: Transient beta modulates decision thresholds during human action-stopping Action-stopping in humans involves bursts of beta oscillations in prefrontal-basal ganglia regions. To determine the functional role of these beta bursts we took advantage of the Race Model framework describing action-stopping. We incorporated beta bursts in three race model variants, each implementing a different functional contribution of beta to action-stopping. In these variants, we hypothesized that a transient increase in beta could 1) modulate decision thresholds, 2) change stop accumulation rates, or 3) promote the interaction between the Stop and the Go process. We then tested the model predictions using EEG recordings in humans performing a Stop-signal task. We found that the model variant in which beta increased decision thresholds for a brief period of time best explained the empirical data. The model parameters fitted to the empirical data indicated that beta bursts involve a stronger decision threshold modulation for the Go process than for the Stop process. This suggests that prefrontal beta influences stopping by temporarily holding the response from execution. Our study further suggests that human action-stopping could be multi-staged with the beta acting as a pause, increasing the response threshold for the Stop process to modulate behavior successfully. Our novel approach of introducing transient oscillations into the race model and testing against human neurophysiological data allowed us to discover potential mechanisms of prefrontal beta, possibly generalizing its role in situations requiring executive control over actions.

sentences: Src42A is required for E-cadherin dynamics at cell junctions during Drosophila axis elongation Src kinases are important regulators of cell adhesion. Here we explored the function of Src42A in junction remodelling during Drosophila gastrulation. Src42A is required for tyrosine phosphorylation at bicellular and tricellular junctions in germband cells and localizes to hotspots of mechanical tension. The role of Src42A was investigated using maternal RNAi and by CRISPR-Cas9-induced germline mosaics. We find that during cell intercalations, Src42A is required for the contraction of junctions at anterior-posterior cell interfaces. The planar polarity of E-cadherin is compromised and E-cadherin accumulates at tricellular junctions after Src42A knock down. Furthermore, we provide evidence that Src42A acts in parallel to Abl, which has also been implicated in cell intercalations. Our data suggest that Src42A is involved in two related processes, one affecting tension generated by the planar polarity of MyoII and secondly, it can act as a signalling factor at tAJs in controlling E-cadherin residence time.

sentences: Modeling lung cell development using human pluripotent stem cells Human pluripotent stem cells (hPSC) differentiations can capture developmental phenotypes and processes. They are useful for studying fundamental biological mechanisms driving tissue morphogenesis and cell lineage development. Here, we show temporal development of lung cell lineages using hPSC that recapitulate developmental milestones observed in primary tissue, the generation of renewable fetal lung epithelial spheroids, and the functional utility of the lung models at different differentiation stages for cystic fibrosis disease modeling. We first show the presence of hPSC-derived lung progenitor cells reminiscent of early trimester lung development and containing basal stem cells that generate renewable airway spheroids. Maturation and polarization in air liquid interface (ALI) generates additional epithelial cell lineages found in adult airways, including pulmonary neuroendocrine, brush, mature basal, ciliated and secretory cell types. Finally, pseudotime and RNA velocity analyses of the integrated datasets from fetal and ALI stages reveal both previously identified and new cell lineage relationships. Overall, hPSC differentiation can capture aspects of human lung development and potentially provide important insight into congenital causes of diseases.

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A deep dilated convolutional residual network for predicting interchain contacts of protein homodimers MotivationDeep learning has revolutionized protein tertiary structure prediction recently. The cutting-edge deep learning methods such as AlphaFold can predict high-accuracy tertiary structures for most individual protein chains. However, the accuracy of predicting quaternary structures of protein complexes consisting of multiple chains is still relatively low due to lack of advanced deep learning methods in the field. Because interchain residue-residue contacts can be used as distance restraints to guide quaternary structure modeling, here we develop a deep dilated convolutional residual network method (DRCon) to predict interchain residue-residue contacts in homodimers from residue-residue co-evolutionary signals derived from multiple sequence alignments of monomers, intrachain residue-residue contacts of monomers extracted from true/predicted tertiary structures or predicted by deep learning, and other sequence and structural features.

ResultsTested on three homodimer test datasets (Homo_std dataset, DeepHomo dataset, and CASP14-CAPRI dataset), the precision of DRCon for top L/5 interchain contact predictions (L: length of monomer in a homodimer) is 43.46%, 47.15%, and 24.81% respectively, which is substantially better than two existing deep learning interchain contact prediction methods. Moreover, our experiments demonstrate that using predicted tertiary structure or intrachain contacts of monomers in the unbound state as input, DRCon still performs reasonably well, even though its accuracy is lower than when true tertiary structures in the bound state are used as input. Finally, our case study shows that good interchain contact predictions can be used to build high-accuracy quaternary structure models of homodimers.

AvailabilityThe source code of DRCon is available at https://github.com/jianlin-cheng/DRCon.

Contactchengji@missouri.edu

sentences: Marker-free characterization of single live circulating tumor cell full-length transcriptomes The identification and characterization of circulating tumor cells (CTCs) are important for gaining insights into the biology of metastatic cancers, monitoring disease progression, and medical management of the disease. The limiting factor that hinders enrichment of purified CTC populations is their sparse availability, heterogeneity, and altered phenotypic traits relative to the tumor of origin. Intensive research both at the technical and molecular fronts led to the development of assays that ease CTC detection and identification from the peripheral blood. Most CTC detection methods use a mix of size selection, immune marker based white blood cells (WBC) depletion, and positive enrichment antibodies targeting tumor-associated antigens. However, the majority of these methods either miss out on atypical CTCs or suffer from WBC contamination. Single-cell RNA sequencing (scRNA-Seq) of CTCs provides a wealth of information about their tumors of origin as well as their fate and is a potent method of enabling unbiased identification of CTCs. We present unCTC, an R package for unbiased identification and characterization of CTCs from single-cell transcriptomic data. unCTC features many standard and novel computational and statistical modules for various analysis tasks. These include a novel method of scRNA-Seq clustering, named Deep Dictionary Learning using K-means clustering cost (DDLK), expression based copy number variation (CNV) inference, and combinatorial, marker-based verification of the malignant phenotypes. DDLK enables robust segregation of CTCs and WBCs in the pathway space, as opposed to the gene expression space. We validated the utility of unCTC on scRNA-Seq profiles of breast CTCs from six patients, captured and profiled using an integrated ClearCell(R) FX and PolarisTM workflow that works by the principles of size-based separation of CTCs and marker based WBC depletion.

sentences: An Issue of Concern: Unique Truncated ORF8 Protein Variants of SARS-CoV-2 Open reading frame 8 (ORF8) protein is one of the most evolving accessory proteins in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). It was previously reported that the ORF8 protein inhibits presentation of viral antigens by the major histocompatibility complex class I (MHC-I) and interacts with host factors involved in pulmonary inflammation. The ORF8 protein assists SARS-CoV-2 to evade immunity and replication. Among many contributing mutations, Q27STOP, a mutation in the ORF8 protein defines the B.1.1.7 lineage of SARS-CoV-2, which is engendering the second wave of COVID-19. In the present study, 47 unique truncated ORF8 proteins (T-ORF8) due to the Q27STOP mutations were identified among 49055 available B.1.1.7 SARS-CoV-2 sequences. The results show that only one of the 47 T-ORF8 variants spread to over 57 geo-locations in North America, and other continents which includes Africa, Asia, Europe and South America. Based on various quantitative features such as amino acid homology, polar/non-polar sequence homology, Shannon entropy conservation, and other physicochemical properties of all specific 47 T-ORF8 protein variants, a collection of nine possible T-ORF8 unique variants were defined. The question of whether T-ORF8 variants work similarly to ORF8 has yet to be investigated. A positive response to the question could exacerbate future COVID-19 waves, necessitating severe containment measures.

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Transmission of Candidatus Anaplasma camelii to laboratory animals by camel-specific keds, Hippobosca camelina Anaplasmosis, caused by infection with bacteria of the genus Anaplasma is an important veterinary and zoonotic disease. The characterization of transmission has concentrated on ticks and little is known about non-tick vectors of livestock anaplasmosis. This study investigated the presence of Anaplasma spp. in camels in northern Kenya and whether the hematophagous camel ked, Hippobosca camelina, acts as a vector. Camels (n = 976) and > 10,000 keds were sampled over a three-year study period and the presence of Anaplasma species was determined by PCR-based assays targeting the Anaplasmataceae 16S rRNA gene. Camels were infected by  Candidatus Anaplasma camelii occurring from 63 - 78% during the dry (September 2017), wet (June-July 2018), and late wet seasons (July-August 2019). 10 - 29% of camel keds harbored  Ca. Anaplasma camelii acquired from infected camels during blood feeding. We determined whether Anaplasma positive camel keds could transmit  Ca. Anaplasma camelii to small laboratory animals via blood-feeding. We show competence in pathogen transmission and subsequent infection in mice and rabbits by both direct detection in blood smears and subsequent molecular identification by PCR. Transmission of  Ca. Anaplasma camelii to mice (8 - 47%) and rabbits (25%) occurred readily after ked bites. Hence, we demonstrate, for the first time, the potential of H. camelina as a vector of anaplasmosis. This key finding provides the basis for establishing ked control programmes for improvement of livestock and human health.

Author summaryHematophagous flies such as Tabanids and Stomoxys, among other biting flies, are mechanical transmitters of various pathogens such as African trypanosomes and Anaplasma species. However, little is known about the role of common camel-specific biting keds (also known as camel flies or louse flies, genus Hippobosca) in pathogen transmission. Keds inflict painful bites to access host blood, and in the process may transmit bacterial hemopathogens frequently detected in both camels and their keds. We confirmed by experimental blood-feeding, gene amplification, and amplicon sequencing that camel keds can transmit "Candidatus Anaplasma camelii" from naturally-infected camels to healthy mice and rabbits. The high prevalence of camel anaplasmosis throughout the year in northern Kenya could be explained by the infestation camel-specific H. camelina, whose capacity as efficient fliers, unlike ticks, promotes disease transmission and maintenance within and among camel herds. Although this study focused on the transmission of Anaplasma sp. by camel keds, it is possible that other hemopathogens could also be transmitted by these flies through a similar mechanism. Notably, in the absence of their preferred hosts, keds occasionally bite humans and other vertebrates they come across in order to acquire bloodmeals, and in the process could transmit zoonotic pathogens.

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Striatin 3 and MAP4K4 cooperate towards oncogenic growth and tissue invasion in medulloblastoma MAP4K4 has been correlated with increased cell motility and reduced proliferation in mammalian cells. Consequences and regulation of this dichotomous functionality of MAP4K4 in tumor cells remained elusive. We find that MAP4K4 interacts with the STRN3 protein in medulloblastoma and that STRN3 and MAP4K4 exert opposing functions in Hippo tumor suppressor signaling and clonal growth. However, depletion of either STRN3 or MAP4K4 reduces migration and tissue invasion, and concomitant loss of both proteins halts tumor cell growth in the cerebellar tissue. Mechanistically, STRN3 couples MAP4K4 to the protein phosphatase 2A, which inactivates growth repressing activities of MAP4K4. In parallel, STRN3 enables growth factor-induced PKC{theta} activation and the direct phosphorylation of VASPS157 by MAP4K4, which are necessary for efficient migration and tissue invasion. Thus, STRN3 controls MAP4K4 functionality in growth factor-activated tumor cells, and the cooperation of the two proteins promotes invasiveness through nPKCs and VASP and increases proliferation via inactivation of Hippo signaling. Targeting of the STRN3-MAP4K4 cooperation could reactivate Hippo signaling and repress invasiveness in growth factor-driven tumors.

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sentences: Synthesis and characterization of peptide conjugated human serum albumin nanoparticles for targeted cardiac uptake and drug delivery. Congestive heart failure, a prominent cardiovascular disease results primarily from myocardial infarction or ischemia. Milrinone (MRN), a widely used clinical drug for heart failure, improves myocardial contractility and cardiac function through its inotropic and vasodilatory effects. However, lacking target specificty, it exhibits low bioavailability and lower body retention time. Therefore, in this study, angiotensin II (AT1) peptide conjugated human serum albumin nanoparticles (AT1-HSA-MRN-NPs) have been synthesized for targeted delivery of MRN to the myocardium, overexpressing AT1 receptors under heart failure. The NPs were surface functionalized through a covalent conjugation reaction between HSA and AT1. Nanoparticle size was 215.2{+/-}4.7 nm and zeta potential -28.8{+/-}2.7 mV and cumulative release of MRN was [~]72% over 24 hrs. The intracellular uptake of nanoparticles and cell viability was studied in H9c2 cells treated with AT1-MRN-HSA-NPs vs the control non-targeted drug, MRN Lactate under normal, hypoxic and hypertrophic conditions. The uptake of AT1-HSA-MRN-NPs in H9c2 cells was significantly higher as compared to non-targeted nanoparticles, and the viability of H9c2 cells treated with AT1-MRN-HSA-NPs vs MRN Lactate was 73.4{+/-}1.4% vs 44.9{+/-}1.4%, respectively. Therefore, AT1-HSA-MRN-NPs are safe for in vivo use and exhibit superior targeting and drug delivery characteristics for treatment of heart failure.

sentences: Supracellular organization confers directionality and mechanical potency to migrating pairs of cardiopharyngeal progenitor cells Physiological and pathological morphogenetic events involve a wide array of collective movements, suggesting that these multicellular arrangements confer biochemical and biomechanical properties that contribute to tissue scale organization. The cardiopharyngeal progenitors of the tunicate Ciona provide the simplest possible model of collective cell migration. They form cohesive bilateral cell pairs, leader-trailer polarized along the migration path as they migrate between the ventral epidermis and trunk endoderm. Here, circumventing difficulties in quantifying cellular mechanics in live embryos, we use the Cellular Potts Model to computationally probe the distributions of forces consistent with the shapes and collective polarity of migrating cell pairs. Combining computational modeling, confocal microscopy, and molecular perturbations, we first determine that cardiopharyngeal progenitors display hallmarks of supracellular organization, with differential distributions of protrusive forces, cell-matrix adhesion, and myosin-based retraction forces along the leader-trailer axis. Combined 4D simulations and experimental observations suggest that cell-cell communication helps establish a hierarchy that contributes to aligning collective polarity with the direction of migration, as observed with three or more cells both in silico and in vivo. Our approach reveals emerging properties of the migrating collective. Specifically, cell pairs are more persistent, thus migrating over longer distances, and presumably with higher accuracy. Finally, simulations suggest that polarized cell pairs literally join forces to deform the trunk endoderm, as they migrate through the extracellular space. We thus propose that the polarized supracellular organization of cardiopharyngeal progenitors confers emergent physical properties that determine mechanical interactions with their environment during morphogenesis.

sentences: The adapted Activity-By-Contact model for enhancer-gene assignment and its application to single-cell data Identifying regulatory regions in the genome is of great interest for understanding the epigenomic landscape in cells. One fundamental challenge in this context is to find the target genes whose expression the regulatory regions affect. A recent successful method is the Activity-By-Contact (ABC) model [10] which scores enhancer-gene interactions based on enhancer activity and the contact frequency of an enhancer to its target gene. It requires two types of assays to measure enhancer activity, which limits the applicability. The ABC score describes regulatory interactions entirely from a genes perspective, and does not account for all the candidate target genes of an enhancer. Moreover, there is no implementation available which would allow for an integration with transcription factor (TF) binding information or an efficient analysis of single-cell data. We show that the ABC-model is comparably accurate with only one assay to measure enhancer activity. Further, we demonstrate that it can yield a higher accuracy by adapting the enhancer activity according to the number of contacts the enhancer has to its candidate target genes. We combined our adapted ABC-model with TF binding information and illustrate an analysis of a single-cell ATAC-seq data set of the human heart. We were able to characterise cell type-specific regulatory interactions as well as to prioritise candidate TFs that drive cell type-specific expression. All executed processing steps are incorporated into our new computational pipeline STARE. The software is available at https://github.com/schulzlab/STARE.

sentences: Seronegative MSM at high-risk of HIV-1 acquisition show Immune quiescent profile with normal immune response against common antigens HIV infection still represents a major public health problem worldwide, and a vaccine remains elusive. The study of HIV-exposed seronegative individuals (HESN) brings important information about the natural resistance to HIV, allows a better understanding of the infection and opens doors for new preventive and therapeutic strategies. Among HESN groups there are some men who have sex with men (MSM) with high-risk sexual behaviors, who represent an adequate cohort for the study of HESN because of their major exposure to HIV in the absence of infection. This study aimed to compare the immunological profile of Colombian seronegative MSM with different risk sexual behaviors. Sixty MSM at high-risk (n=16) and low-risk (n=44) of HIV-1 acquisition were included. No sex worker nor homozygous delta 32 mutation subjects were included. All the participants were negative for anti-HIV-1/2 antibodies and HIV-1 proviral DNA. The high-risk MSM presented a higher frequency of sexual partners in the last 3 months previous to the studys enrollment (Median 30 vs. 2), lifetime sexual partners (Median 1708 vs. 26), and unprotected anal intercourse (Median 12.5 vs. 2) than low-risk MSM. This group also showed a quiescent profile of T cells and NK cells, with a significantly lower percentage of CD4+CD38+, CD4+HLADR-CD38+, CD4+Ki67+ T cells, NKG2D+ NK cells (CD3-CD16+CD56+), a significantly higher percentage of CD4+HLADR-CD38- and a tendency to show a higher percentage of CD8+HLADR+CD38-T cells, than the low-risk group. Likewise, they showed higher mRNA levels of Serpin A1 from PBMCs. The results suggest that this cohort of MSM could be HESN individuals and their resistance would be explained by a quiescent profile of T cells and NK cells, and increased expression of Serpin A1. It is necessary to continue the study of MSM at high-risk of exposure to HIV-1 to better understand the natural resistance to HIV.

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The Prediction of Active Ingredients and Polypharmacological Mechanisms of Orthosiphon stamineus Benth. (Lamiaceae) of Dai medicine based on Network Pharmacology BackgroundOrthosiphon stamineus Benth. was a dietary supplement or a traditional folk herb with widespread clinical application, but it was lack of comprehensive understanding of its active ingredients and polypharmacological mechanisms. The aim of this work was to systematically study its natural compounds and the molecular mechanisms of the O. stamineus via network pharmacology.

MethodsCompounds from O. stamineus were collected by literature retrieval and evaluated by SwissADME with the physicochemical property (ADMET model) and the likelihood for a natural medicine. The connection of active ingredients and target genes was built and confirmed by Cytoscape and AutoDock vina. Then, Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were separately performed to obtain a more in-depth understanding of O. stamineus. Finally, the relationship among active ingredients, targets, and diseases was built to clarify the polypharmacological mechanisms and found relevant active substances for further drug discovery.

ResultsA total of 159 compounds of O. stamineus were collected, and 22 potentially active ingredients were screened out. The Ingredient-Target Interaction Network was built with 12 flavonoids, 3 diterpenes, 3 phenols, and 4 volatile oils, and 65 targets. The Docking analysis indicated that the ingredient-target interaction network was reliable; most ligand-receptor had a strong binding affinity (lowest binding energy: -6.9 kcal/mol). After pathway analysis, 185 significant biological processes and 36 signal pathways were found, and the ingredient-target-disease network of O. stamineus was constructed for polypharmacological mechanisms.

ConclusionOur study clarified the polypharmacological mechanisms via the relationship among active ingredients, targets, and diseases and provided better guidance for subsequent experiments and potential active ingredients for drug discovery or health promotion.

sentences: The crystal structure of TRPM2 MHR1/2 domain reveals a conserved Zn2+-binding domain essential for ligand binding and activity Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable, non-selective cation channel involved in diverse physiological processes such as immune response, apoptosis and body temperature sensing. TRPM2 is activated by ADP-ribose (ADPR) and 2'-deoxy-ADPR in a Ca2+-dependent manner. While two species-specific binding sites exist for ADPR, a binding site for 2'-deoxy-ADPR is not known yet. Here, we report the crystal structure of the MHR1/2 domain of TRPM2 from zebrafish (Danio rerio) and show binding of both ligands to this domain. We identified a so-far unrecognized Zn2+-binding domain that was not resolved in previous cryo-EM structures and that is conserved in most TRPM channels. In combination with patch clamp experiments, we comprehensively characterize the effect of the Zn2+-binding domain on TRPM2 activation. Our results provide insight into a conserved structural element essential for channel activity.

sentences: Deep Mutagenesis of a Transporter for Uptake of a Non-Native Substrate Identifies Conformationally Dynamic Regions The serotonin transporter, SERT, catalyzes serotonin reuptake at the synapse to terminate neurotransmission via an alternating access mechanism, and SERT inhibitors are the most widely prescribed antidepressants. Here, deep mutagenesis is used to determine the effects of nearly all amino acid substitutions on human SERT surface expression and transport of the fluorescent substrate analogue APP+, identifying many mutations that enhance APP+ import. Comprehensive simulations of the entire ion-coupled import process reveal that while binding of the native substrate, serotonin, reduces free energy barriers between conformational states to promote SERT dynamics, the conformational free energy landscape in the presence of APP+ instead resembles Na+ bound-SERT, with a higher free energy barrier for transitioning to an inward-facing state. The deep mutational scan for SERT-catalyzed import of APP+ finds mutations that promote the necessary conformational changes that would otherwise be facilitated by the native substrate. Indeed, hundreds of gain-of-function mutations for APP+ import are found along the permeation pathway, most notably mutations that favor opening of a solvent-exposed intracellular vestibule. The mutagenesis data support the simulated mechanism in which the neurotransmitter and a symported sodium share a common cytosolic exit pathway to achieve coupling. Furthermore, the mutational landscape for SERT surface trafficking, which likely filters out misfolded sequences, reveals that residues along the permeation pathway are mutationally tolerant, providing plausible evolutionary pathways for changes in transporter properties while maintaining folded structure.

sentences: Methylene blue, Mycophenolic acid, Posaconazole, and Niclosamide inhibit SARS-CoV-2Omicron variant BA.1 infection of human airway epithelial explant cultures Sublineages of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Omicron variants continue to amass mutations in the spike (S) glycoprotein, which leads to immune evasion and rapid spread of the virus across the human population. Here we demonstrate the susceptibility of the Omicron variant BA.1 (B.1.1.529.1) to four repurposable drugs, Methylene blue (MB), Mycophenolic acid (MPA), Posaconazole (POS), and Niclosamide (Niclo) in post-exposure treatments of primary human airway cell cultures. MB, MPA, POS, and Niclo are known to block infection of human nasal and bronchial airway epithelial explant cultures (HAEEC) with the Wuhan strain, and four variants of concern (VoC), Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28), Delta (B.1.617.2) (1, 2). Our results not only show broad anti-coronavirus effects of MB, MPA, POS and Niclo, but also demonstrate that the Omicron variant BA.1 (B.1.1.529.1) sheds infectious virus from HAEEC over at least 15 days, and maintains both intracellular and extracellular viral genomic RNA without overt toxicity, suggesting viral persistence. The data underscore the broad effects of MB, MPA, POS, and Niclo against SARS-CoV-2 and the currently circulating VoC, and reinforce the concept of repurposing drugs in clinical trials against COVID-19.

sentences: FtsZ is essential until the late stage of constriction 1There has been recent debate as to the source of constriction force during cell division. FtsZ can generate a constriction force on tubular membranes in vitro, suggesting it may generate the constriction force in vivo. However, another study showed that mutants of FtsZ did not affect the rate of constriction, whereas mutants of the PG assembly did, suggesting that PG assembly may push the constriction from the outside. Supporting this model, two groups found that cells that have initiated constriction can complete septation while the Z ring is poisoned with PC190723. PC19 arrests treadmilling but leaves FtsZ in place. We sought to determine if a fully assembled Z ring is necessary during constriction. To do this, we used a temperature sensitive FtsZ mutant, FtsZ84. FtsZ84 behaves as wild type at 30{degrees} C, but it disassembles from the Z ring within one min upon a temperature jump to 42{degrees} C. Following the temperature jump we found that cells in early constriction stop constricting. Cells that had progressed to the later stage of division finished constriction without a Z ring. These results show that in Escherichia coli, an assembled Z ring is essential for constriction except in the final stage, contradicting the simplest interpretation of previous studies using PC19.

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Structural modeling of the hERG potassium channel and associated drug interactions The voltage-gated potassium channel, KV11.1, encoded by the human Ether-a-go-go- Related Gene (hERG) is expressed in cardiac myocytes, where it is crucial for the membrane repolarization of the action potential. Gating of hERG channel is characterized by rapid, voltage-dependent, C-type inactivation, which blocks ion conduction and is suggested to involve constriction of the selectivity filter. Mutations S620T and S641A/T within the selectivity filter region of hERG have been shown to alter the voltage- dependence of channel inactivation. Because hERG channel blockade is implicated in drug-induced arrhythmias associated with both the open and inactivated states, we used Rosetta to simulate effects of hERG S620T and S641A/T mutations to elucidate conformational changes associated with hERG channel inactivation and differences in drug binding between the two states. Rosetta modeling of the S641A fast-inactivating mutation revealed a lateral shift of F627 side chain in the selectivity filter into the central channel axis along the ion conduction pathway and formation of four lateral fenestrations in the pore. Rosetta modeling of the non-inactivating mutations S620T and S641T suggested a potential molecular mechanism preventing F627 side chain from shifting into the ion conduction pathway during the proposed inactivation process. Furthermore, we used Rosetta docking to explore the binding mechanism of highly selective and potent hERG blockers - dofetilide, terfenadine, and E4031. Our structural modeling correlate well with existing experimental evidence involving interactions of these drugs with key hERG residues Y652 and F656 inside the pore and reveal potential ligand binding interactions within fenestration region in an inactivated state.

Significance StatementComputational models of hERG potassium channel provide structural insights into an inactivated state and associated drug interactions. Our computational approach will be useful to study ion channel modulation by small molecules.

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Extracellular vesicles Transfer Polarized Mitochondria and Increase Cellular Energetics in Ischemic Endothelial Cells We have demonstrated, for the first time that microvesicles, a sub-type of extracellular vesicles (EVs) derived from hCMEC/D3: a human brain endothelial cell (BEC) line transfer polarized mitochondria to recipient BECs in culture and to neurons in mice acute brain cortical and hippocampal slices. This mitochondrial transfer increased ATP levels by 100 to 200-fold (relative to untreated cells) in the recipient BECs exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. We have also demonstrated that transfer of microvesicles, the larger EV fraction, but not exosomes resulted in increased mitochondrial function in hypoxic endothelial cultures. Gene ontology and pathway enrichment analysis of EVs revealed a very high association to glycolysis-related processes. In comparison to heterotypic macrophage- derived EVs, BEC-derived EVs demonstrated a greater selectivity to transfer mitochondria and increase endothelial cell survival under ischemic conditions.

HighlightsO_LIMicrovesicles transfer mitochondria to endothelial cells and brain slice neurons
C_LIO_LIMitochondrial transfer increased ATP in ischemic brain endothelial cells (BECs)
C_LIO_LITransfer of microvesicles increased mitochondrial function in hypoxic BECs
C_LIO_LITransfer of exosomes did not affect mitochondrial function in hypoxic BECs
C_LIO_LIHomotypic BEC-derived EVs result in greater ATP levels in the recipient BECs
C_LI

sentences: Simulated attack reveals how lesions affect network properties in post-stroke aphasia Aphasia is one of the most prevalent cognitive syndromes caused by stroke. The rarity of premorbid imaging and heterogeneity of lesion size and extent obfuscates the links between the local effects of the lesion, global anatomical network organization, and aphasia symptoms. We applied a simulated attack approach to examine the effects of 39 stroke lesions on network topology by simulating their effects in a control sample of 36 healthy brain networks. We focused on measures of global network organization thought to support overall brain function and resilience in the whole brain and within the left hemisphere. After removing lesion volume from the network topology measures and behavioral scores (the Western Aphasia Battery Aphasia Quotient; WAB-AQ), four behavioral factor scores obtained from a neuropsychological battery, and a factor sum), we compared the behavioral variance accounted for by simulated post-stroke connectomes to that observed in the randomly permuted data. Overall, global measures of network topology in the whole brain and left hemisphere accounted for 10% variance or more of the WAB-AQ and the lexical factor score beyond lesion volume and null permutations. Streamline networks provided more reliable point estimates than FA networks. Edge weights and network efficiency were weighted most highly in predicting the WAB-AQ for FA networks. Overall, our results suggest that global network measures can provide modest statistical value predicting overall aphasia severity, but less value in predicting specific behaviors. Variability in estimates could be induced by premorbid ability, deafferentation and diaschisis, and neuroplasticity following stroke.

sentences: Guanosine-specific single-stranded ribonuclease effectors of a phytopathogenic fungus potentiate host immune responses Plants activate immunity upon recognition of pathogen-associated molecular patterns. Although phytopathogens have evolved a set of effector proteins to counteract plant immunity, some effectors are perceived by hosts and induce immune responses. Here, we show that two secreted ribonuclease effectors, SRN1 and SRN2, encoded in a phytopathogenic fungus, Colletotrichum orbiculare, induce cell death in a signal peptide- and catalytic residue-dependent manner, when transiently expressed in Nicotiana benthamiana. The pervasive presence of SRN genes across Colletotrichum species suggested the conserved roles. Using a transient gene expression system in cucumber (Cucumis sativus), an original host of C. orbiculare, we show that SRN1 and SRN2 potentiate host pattern-triggered immunity. Consistent with this, C. orbiculare SRN1 and SRN2 deletion mutants exhibited increased virulence on the host. In vitro analysis revealed that SRN1 specifically cleaves single-stranded RNAs at guanosine, leaving a 3'-end phosphate. This activity has not been reported in plants. Importantly, the potentiation of C. sativus responses by SRN1 and SRN2 depends on the signal peptide and ribonuclease catalytic residues, suggesting that secreted SRNs cleave RNAs in apoplast and are detected by the host. We propose that the pathogen-derived apoplastic guanosine-specific single-stranded endoribonucleases lead to immunity potentiation in plants.

sentences: Modelling complex population structure using F-statistics and Principal Component Analysis Human genetic diversity is shaped by our complex history. Data-driven methods such as Principal Component Analysis (PCA) are an important population genetic tool to understand this method. Here, I contrast PCA with a set of statistics motivated by trees (F-statistics). Here, I show that these two methods are closely related, and I derive explicit connections between the two approaches. I show that F-statistics have a simple geometrical interpretation in the context of PCA, and that orthogonal projections are the key concept to establish this link. I illustrate my results on two examples, one of local, and one of global human diversity. In both examples, I find that just using the first few PCs provides good population structure is sparse, and only a few components contribute to most statistics. Based on these results, I develop novel visualizations that allow for investigating specific hypotheses, checking the assumptions of more sophisticated models. My results extend F-statistics to non-discrete populations, moving towards more complete and less biased descriptions of human genetic variation.

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CO2 supply modulates lipid remodelling, photosynthetic and respiratory activities in Chlorella species Microalgae represent potential solutions to reduce the atmospheric CO2 level through photosynthesis. To boost CO2 fixation by microalgae it is essential to understand physiologic and metabolic responses at the base of CO2 assimilation and carbon flow. In this work two Trebouxiophyceae species, Chlorella sorokiniana and Chlorella vulgaris, were investigated for their metabolic responses to high and low CO2 (air level) availability. High CO2 availability resulted in an increase in biomass accumulation in both species but with a different chloroplast and mitochondrial responses. In C. sorokiniana we observed increased polar lipids and protein amount and a balanced NADPH redox state and a similar total respiration in the two conditions analysed. In contrast, in C. vulgaris high CO2 level caused an increase in TAG accumulation and a higher NADPH consumption suggesting a CO2 dependent increase of reducing power consumption in the chloroplast, which in turn influences the redox state of the mitochondria by lowering total dark respiration. Several rearrangements of the photosynthetic machinery were observed in both species, which differ from those described for the model organism Chlamydomonas reinhardtii. In the case of C. reinhardtii, adaptation of the photosynthetic apparatus to different CO2 availability relies on the translational repressor NAB1. NAB1 homologous protein could be identified only in C. vulgaris but lacked the regulation mechanisms previously described in C. reinhardtii. These findings highlight that the acclimation strategies to cope with a fluctuating inorganic carbon supply are diverse among green microalgae and point to new biotechnological strategies to boost CO2 fixation.

One sentence summaryHigh/low CO2 availability induces cell responses as lipids remodelling, adaptations of the photosynthetic apparatus and modulation of mitochondrial respiration not conserved among green algae

sentences: A subcellular cookie cutter for spatial genomics in human tissue Intracellular heterogeneity contributes significantly to cellular physiology and, in a number of debilitating diseases, cellular pathophysiology. This is greatly influenced by distinct organelle populations and to understand the aetiology of disease it is important to have tools able to isolate and differentially analyse organelles from precise location within tissues. Here we report the development of a subcellular biopsy technology that facilitates the isolation of organelles, such as mitochondria, from human tissue. We compared the subcellular biopsy technology to laser capture microdissection (LCM) that is the state of art technique for the isolation of cells from their surrounding tissues. We demonstrate an operational limit of (>20m) for LCM and then, for the first time in human tissue, show that subcellular biopsy can be used to isolate mitochondria beyond this limit.

sentences: Restoration of high-sensitivity patterned vision in motion with an engineered light-gated G protein-coupled receptor Inherited retinal degenerations (IRDs) result in blindness due to apoptotic cell death of rods and cones, but spare other retinal neurons, providing a potential that delivery of a light-activated signaling protein to surviving neurons may restore vision. We previously demonstrated that aspects of vision could be restored by introduction into surviving cells of a G protein-coupled receptor for glutamate (mGluR) bearing a tethered photoswitchable agonist. However, this system, containing one photoswitchable agonist per glutamate binding site, yielded low sensitivity, responding only to visual stimuli at the intensity of bright outdoor light, similar to channelrhodopsins. To increase sensitivity, we designed a multi-branched photoswitch, bearing four light-activatable glutamates for each glutamate binding site. When tethered to a modified mGluR2 expressed in retinal ganglion cells via intravitreal AAV gene delivery, this photoswitch boosted sensitivity by ~100-fold compared to the unbranched (single photo-ligand) photoswitch. This improvement in sensitivity enabled an IRD mouse model (rd1) to perform visually-guided object recognition under incidental room light and pattern recognition using standard LCD computer displays. The restored line pattern differentiation approached the acuity reported for normal mouse vision. Pattern recognition functioned as well as wildtype vision with line patterns moving at speeds of up to 36{degrees}/s. In summary, this two-component chemical-optogenetic approach combines high sensitivity and high acuity with superior motion vision, and, unlike optogenetic gene therapy, can be adjusted for dose, upgraded, as new photoswitches are developed, and discontinued at will.

sentences: Bovine neutrophils release extracellular traps and cooperate with macrophages in Mycobacterium avium subsp. paratuberculosis clearance in vitro Mycobacterium avium subsp. paratuberculosis (Map) is the underlying pathogen causing bovine paratuberculosis (PTB), an enteric granulomatous disease that mainly affects ruminants and for which an effective treatment is needed. Macrophages are the primary target cells for Map, which survives and replicates intracellularly by inhibiting phagosome maturation. Neutrophils are present at disease sites during the early stages of the infection, but seem to be absent in the late stage, in contrast to healthy tissue. Although neutrophil activity has been reported to be impaired following Map infection, their role in PTB pathogenesis has not been fully defined. Neutrophils are capable of releasing extracellular traps consisting of extruded DNA and proteins that immobilize and kill microorganisms, but this mechanism has not been evaluated against Map. Our main objective was to study the interaction of neutrophils with macrophages during an in vitro mycobacterial infection. For this purpose, neutrophils and macrophages from the same animal were cultured alone or together in the presence of Map or Mycobacterium bovis Bacillus-Calmette-Guerin (BCG). Extracellular trap release, mycobacteria killing as well as IL-1{beta} and IL-8 release were assessed. Neutrophils released extracellular traps against mycobacteria when cultured alone and in the presence of macrophages without direct cell contact, but resulted inhibited in direct contact. Macrophages were extremely efficient at killing BCG, but ineffective at killing Map. In contrast, neutrophils showed similar killing rates for both mycobacteria. Co-cultures infected with Map showed the expected killing effect of combining both cell types, whereas co-cultures infected with BCG showed a potentiated killing effect beyond the expected one, indicating a potential synergistic cooperation. In both cases, IL-1{beta} and IL-8 levels were lower in co-cultures, suggestive of a reduced inflammatory reaction. These data indicate that cooperation of both cell types can be beneficial in terms of decreasing the inflammatory reaction while the effective elimination of Map can be compromised. These results suggest that neutrophils are effective at Map killing and can exert protective mechanisms against Map that seem to fail during PTB disease after the arrival of macrophages at the infection site.

sentences: A single vesicle fluorescence-bleaching assay for multi-parameter analysis of proteoliposomes by total internal reflection fluorescence microscopy Reconstitution of membrane proteins into model membranes is an essential approach for their functional analysis under chemically defined conditions. Established model-membrane systems used in ensemble average measurements are limited by sample heterogeneity and insufficient knowledge of lipid and protein content at the single vesicle level, which limits quantitative analysis of vesicle properties and prevents their correlation with protein activity. Here, we describe a versatile total internal reflection fluorescence microscopy-based bleaching protocol that permits parallel analyses of multiple parameters (physical size, tightness, unilamellarity, membrane protein content and orientation) of individual proteoliposomes prepared with fluorescently tagged membrane proteins and lipid markers. The approach makes use of commercially available fluorophores including the commonly used nitrobenzoxadiazole (NBD) dye and may be applied to deduce functional molecular characteristics of many types of reconstituted fluorescently tagged membrane proteins.

sentences: STED super-resolution imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes Understanding the plasma membrane nano-scale organisation and dynamics in living cells requires microscopy techniques with high spatial and temporal resolution, permitting for long acquisition times, and that allow for the quantification of membrane biophysical properties such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolutions, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile Red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dye is showcased by live-cell real-time 3D-STED recordings of bleb formation and lipid exchange during membrane fusion, as well as by STED-fluorescence correlation spectroscopy (STED-FCS) experiments for the simultaneous quantification of membrane dynamics and lipid packing, which correlate in model and live-cell membranes.

sentences: High-resolution single-molecule long-fragment rRNA gene amplicon sequencing for uncultured bacterial and fungal communities Although several large-scale environmental microbial projects have been initiated in the past two decades, understanding of the role of complex microbiotas is still constrained by problems of detecting and identifying unknown microorganisms1-6.Currently, hypervariable regions of rRNA genes as well as internal transcribed spacer regions are broadly used to identify bacteria and fungi within complex communities7,8, but taxonomic and phylogenetic resolution is hampered by insufficient sequencing length9-11. Direct sequencing of full length rRNA genes is currently limited by read length using second generation sequencing or sacrificed quality and throughput by using single molecule sequencing. We developed a novel method to sequence and assemble nearly full length rRNA genes using second generation sequencing.Benchmarking was performed on mock bacterial and fungal communities as well as two forest soil samples. The majority of rRNA gene sequences of all species in the mock community samples were successfully recovered with identities above 99.5% compared to the reference sequences. For soil samples we obtained exquisite coverage with identification of a large number of putative new species, as well as high abundance correlation between replicates. This approach provides a cost-effective method for obtaining extensive and accurate information on complex environmental microbial communities.

sentences: Leishmania major formins are cytosolic actin bundler play an important role in cell physiology Formins are a highly conserved eukaryotic family of proteins that regulate actin dynamics. They play important physiological roles in cell adhesion, motility, vesicular trafficking and cytokinesis. Although sequence analysis of Trypanosomatida genomes predicted multiple formin-encoding genes, none of them are functionally characterized yet. We report here experimental identification and functional characterization of two constitutively expressed formins from the trypanosomatid protozoa Leishmania major viz. LmForminA and LmForminB. These formins exhibited irregular cytosolic distribution that co-localized with actin patches. Co-sedimentation assay and surface plasmon resonance confirmed that purified LmForminA and B FH2 domains can bind actin, albeit with differential affinity. Interestingly, both LmForminA and B FH2 domains were found to be actin bundlers as revealed by low-speed co-sedimentation assay and TIRF microscopy. LmForminA and B also had actin-nucleating activities, which was abolished by mutating their conserved Ile residue crucial for actin assembly. The Ile-mutant formins, however, retained their actin binding and bundling properties. Treatment of Leishmania cells with formin inhibitor SMIFH2 severely perturbed parasite growth and morphology indicating that Lmformins are physiologically important and may be considered as novel drug targets.

sentences: Marchantia polymorpha gene expression atlas reveals the hierarchy of abiotic stress responses and conservation of diurnal gene expression Abiotic stress has a dramatic impact on ecosystems and the yield of crops, and global warming will likely result in more frequent and intense stresses. However, due to the complexity of the underlying signalling pathways and the fact that stresses often occur in combinations in nature, our knowledge of how plants acclimatize to abiotic stress is still lacking. Here, we used a plant with minimal regulatory network redundancy, Marchantia polymorpha, to study how seven abiotic stresses alone and in combination affect the phenotype, gene expression, and activity of biological pathways. Worryingly, we see no evidence of conservation of gene expression to abiotic stress between model organisms, suggesting that each species develops unique strategies to cope with abiotic stress. Interestingly, we observed that certain stresses are able to suppress others, while specific combinations can elucidate novel transcriptomic responses. Finally, we provide two online databases to study abiotic stress responses and diurnal gene expression data in Marchantia.

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Xanthomonas infection transforms the apoplast into an accessible and habitable niche for Salmonella enterica The physiology of plant hosts can be dramatically altered by phytopathogens. Xanthomonas hortorum pv. gardneri is one such pathogen that creates an aqueous niche within the leaf apoplast by manipulating the plant via the transcription activator-like effector AvrHah1. Simultaneous immigration of X. gardneri and Salmonella enterica to healthy tomato leaves results in increased survival of S. enterica as Xanthomonas infection progresses. However, the fate of S. enterica following arrival on actively infected leaves has not been examined. We hypothesized that the water-soaking caused by X. gardneri could facilitate the ingression of S. enterica into the apoplast, and that this environment would be conducive for growth. We found that a water-soaked apoplast, abiotically or Xanthomonas-infected, enabled surface S. enterica to passively localize to the protective apoplast and facilitated migration of S. enterica to distal sites within the aqueous apoplast. AvrHah1 contributed to the protection and migration of S. enterica early in X. gardneri infection. Xanthomonas-infected apoplasts facilitated prolonged survival and promoted S. enterica replication compared to healthy apoplasts. Access to an aqueous apoplast in general protects S. enterica from immediate exposure to irradiation whereas, the altered environment created by Xanthomonas infection provides growth-conducive conditions for S. enterica. Overall, we have characterized an ecological relationship in which host infection converts an unreachable niche to a habitable environment.

ImportanceBacterial spot disease caused by Xanthomonas species devastates tomato production worldwide. Salmonellosis outbreaks from consumption of raw produce have been linked to the arrival of Salmonella enterica on crop plants in the field via contaminated irrigation water. Considering that Xanthomonas is difficult to eradicate, it is highly likely that S. enterica arrives on leaves pre-colonized by Xanthomonas with infection underway. Our study demonstrates that infection and disease fundamentally alter the leaf, resulting in redistribution and change in abundance of a phyllosphere bacterial member. These findings contribute to our understanding of how S. enterica manages to persist on leaf tissue despite lacking the ability to liberate nutrients from plant cells. More broadly, this study reveals a mechanism by which physiochemical changes to a host environment imposed by a plant pathogen can convert an uninhabitable leaf environment into a hospitable niche for select epiphytic microbes.

sentences: Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing The mRNA 5 cap structure serves both to protect transcripts from degradation and promote their translation. Cap removal is thus an integral component of mRNA turnover that is carried out by cellular decapping enzymes, whose activity is tightly regulated and coupled to other stages of the mRNA decay pathway. The poxvirus vaccinia virus (VACV) encodes its own decapping enzymes, D9 and D10, that act on cellular and viral mRNA, but may be regulated differently than their cellular counterparts. Here, we evaluated the targeting potential of these viral enzymes using RNA sequencing from cells infected with wild-type and decapping mutant versions of VACV as well as in uninfected cells expressing D10. We found that D9 and D10 target an overlapping subset of viral transcripts but that D10 plays a dominant role in depleting the vast majority of human transcripts, although not in an indiscriminate manner. Unexpectedly, the splicing architecture of a gene influences how robustly its corresponding transcript is targeted by D10, as transcripts derived from intronless genes are less susceptible to enzymatic decapping by D10. As all VACV genes are intronless, preferential decapping of transcripts from intron-encoding genes provides an unanticipated mechanism for the virus to disproportionately deplete host transcripts and remodel the infected cell transcriptome.

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Global evolutionary dynamics and resistome analysis of Clostridioides difficile ribotype 017 Clostridioides difficile PCR ribotype (RT) 017 ranks among the most successful strains of C. difficile in the world. In the past three decades, it has caused outbreaks on four continents, more than other "epidemic" strains, however, our understanding of the genomic epidemiology underpinning the spread of C. difficile RT 017 is limited. Here, we performed high-resolution phylogenomic and Bayesian evolutionary analyses on an updated and more representative dataset of 282 non-clonal C. difficile RT 017 isolates collected worldwide between 1981 and 2019. These analyses place an estimated time of global dissemination between 1953 and 1983 and identified the acquisition of the ermB-positive transposon Tn6194 as a key factor behind global emergence. This coincided with the introduction of clindamycin, a key inciter of C. difficile infection, into clinical practice in the 1960s. Based on the genomic data alone, the origin of C. difficile RT 017 could not be determined, however, geographical data and records of population movement suggest that C. difficile RT 017 had been moving between Asia and Europe since the Middle Ages and was later transported to North America around 1860 (95% CI: 1622 - 1954). A focused epidemiological study of 45 clinical C. difficile RT 017 genomes from a cluster in a tertiary hospital in Thailand revealed that the population consisted of two groups of multidrug-resistant (MDR) C. difficile RT 017 and a group of early, non-MDR C. difficile RT 017. The significant genomic diversity within each MDR group suggests that although they were all isolated from hospitalised patients, there was likely a reservoir of C. difficile RT 017 in the community that contributed to the spread of this pathogen.

Impact statementThis study utilises genomic sequence data from 282 non-clonal C. difficile ribotype (RT) 017 isolates collected from around the world to delineate the origin and spread of this epidemic lineage, as well as explore possible factors that have driven its success. It also reports a focused epidemiological investigation of a cluster of C. difficile RT 017 in a tertiary hospital in Thailand to identify possible sources of transmission in this specific setting.

Data summaryAll new WGS data generated in this study has been submitted to the European Nucleotide Archive under the BioProject PRJEB44406 (sample accession ERS6268756 - ERS6268798). The complete genome of C. difficile MAR286 was submitted to GenBank under BioProject PRJNA679085 (accession CP072118). Details of all genomes included in the final analyses are available in the Supplementary Document, available at 10.6084/m9.figshare.14544792.

sentences: Combinatorial approach of binary colloidal crystals (BCCs) and CRISPR activation to improve induced pluripotent stem cell differentiation into neurons Conventional methods of neuronal differentiation for human induced pluripotent stem cells (iPSCs) are tedious and complicated, involving multi-stage protocols with complex cocktails of growth factors and small molecules. Artificial extracellular matrix with defined surface topography and chemistry represents a promising venue to improve the neuronal differentiation in vitro. In the present study, we test the impact of a type of colloidal self-assembled patterns called binary colloidal crystals (BCCs) in neuronal differentiation. We developed a CRISPR activation (CRISPRa) iPSC platform that constitutively expresses the dCas9-VPR system, which allows robust activation of the proneural transcription factor NEUROD1 to rapidly induce neuronal differentiation within seven days. We showed that the combinatorial use of BCCs can further improve this neuronal differentiation system. In particular, our results indicate that fine tuning of silica and polystyrene size is critical to generate specific topographies to improve neuronal differentiation and branching. BCCs with 5 m silica and 100 nm carboxylated polystyrene has the most prominent effect on increasing neurite outgrowth and more complex ramification, while BCCs with 2m silica and 65nm carboxylated polystyrene is better in promoting neuronal enrichment. These results indicate that biophysical cues can support rapid differentiation and improve neuronal maturation. In summary, our combinatorial approach of CRISPRa and BCCs provides a robust and rapid pipeline for in vitro production of human neurons. Specific BCCs can be adapted to late stages of neuronal differentiation protocols to improve neuronal maturation, which have important implications in tissue engineering, in vitro biological studies and disease modeling.

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The Case for Retaining Natural Language Descriptions of Phenotypes in Plant Databases and a Web Application as Proof of Concept MotivationFinding similarity across phenotypic descriptions is not straightforward, with previous successes in computation requiring significant expert data curation. Natural language processing of free text phenotype descriptions is often easier to apply than intensive curation. It is therefore critical to understand the extent to which these techniques can be used to organize and analyze biological datasets and enable biological discoveries.

ResultsA wide variety of approaches from the natural language processing domain perform as well as similarity metrics over curated annotations for predicting shared phenotypes. These approaches also show promise both for helping curators organize and work through large datasets as well as for enabling researchers to explore relationships among available phenotype descriptions. Here we generate networks of phenotype similarity and share a web application for querying a dataset of associated plant genes using these text mining approaches. Example situations and species for which application of these techniques is most useful are discussed.

AvailabilityThe dataset used in this work is available at https://git.io/JTutQ. The code for the analysis performed here is available at https://git.io/JTutN and https://git.io/JTuqv. The code for the web application discussed here is available at https://git.io/Jtv9J, and the application itself is available at https://quoats.dill-picl.org/.

sentences: Structure Insight into Photosystem I Octamer from Cyanobacteria Diversity of photosystem oligomers is essential to understand how photosynthetic organisms adopted to light conditions. Given by the structural and physiological significance, the assemblies of PSI supercomplex is of great interest in both chloroplast and cyanobacteria recently. In this study, two novel photosystem I supercomplexes were isolated for the first time from the low light incubated culture of filamentous cyanobacterium Anabaena sp. PCC 7120. These complexes were defined as PSI hexamers and octamers through biochemical and biophysical characterization. Their 77K emission spectra indicated that the red forms of chlorophylls seemed not to be affected during oligomerization. By cryo-EM single particle analysis, a near-atomic (7.0 [A]) resolution structure of PSI octamer was resolved, and the molecular assemblies of stable PSI octamer was revealed.

sentences: Tropical tree growth sensitivity to climate is driven by species intrinsic growth rate and leaf traits A better understanding of how climate affects growth in tree species is essential for improved predictions of forest dynamics under climate change. Long-term climate averages (mean climate) and short-term deviations from these averages (anomalies) both influence tree growth, but the rarity of long-term data integrating climatic gradients with tree censuses has so far limited our understanding of their respective role, especially in tropical systems. Here, we combined 49 years of growth data for 509 tree species across 23 tropical rainforest plots along a climatic gradient to examine how tree growth responds to both climate means and anomalies, and how species functional traits mediate these tree growth responses to climate. We showed that short-term, anomalous increases in atmospheric evaporative demand and solar radiation consistently reduced tree growth. Drier forests and fast-growing species were more sensitive to water stress anomalies. In addition, species traits related to water use and photosynthesis partly explained differences in growth sensitivity to both long-term and short-term climate variations. Our study demonstrates that both climate means and anomalies shape tree growth in tropical forests, and that species traits can be leveraged to understand these demographic responses to climate change, offering a promising way forward to forecast tropical forest dynamics under different climate trajectories.

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Exploration of Antibacterial Activities of Berberis royleana Fractions Extracts ObjectivesPrepare various solvent extracts of Berberis royleana (areal part of plant) to determine the in vitro antibacterial potential of methanolic, ethyl acetate, chloroform, n-hexane and water extracts of B. royleana against various bacterial isolates and Compare the efficacy of outstanding antimicrobial extracts of B. royleana with commonly used antibiotics. Berberis species are medicinally important plants, produce various metabolites and used as treatment for multiple complications. Berberis royleana is a rare specie belongs to genus Berberis.

MethodsIn the current study the areal parts of the plant were isolated to explore antibacterial activities. Antibacterial activities were done using standard procedures. The antibacterial activities of different fractions were tested by 100 g methanolic, ethyl acetate, chloroform, n-hexane and water fractions of B. royleana against bacterial isolates Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella Typhi and Proteus spp. The ciprofloxacin (5g) was used as a positive control and DMSO as a negative control.

ResultsAll fractions showed zone of inhibition against the growth of tested bacterial isolates. Methanolic fractions have maximum ZI against S. aureus and K. pneumoniae (25.7{+/-}1.5 mm), S. aureus (23{+/-}2.7), Salmonella Typhi (25{+/-}1), water fraction have Klebsiella pneumoniae (24.4 {+/-}2.5), Salmonella Typhi (23 {+/-}1 mm), S. aureus (21{+/-}2.8 mm) and the n-hexane fraction exhibits ZI against K. pneumoniae (24.7{+/-}1.5), Salmonella Typhi (24{+/-}2) S. aureus, ethyl acetate maximum zone against E. coli (16.6{+/-}3.1) and chloroform fraction showed maximum ZI against S. Typhi (21.6{+/-}3.3).

ConclusionFrom current report it may be concluded that B. royleana extracts have medicinally effective potentials against drugs resistant bacteria.

sentences: Differential Roles of Positive and Negative Valence Systems in the Development of Post-Traumatic Stress Psychopathology Negative and positive valence systems (NVS and PVS) pertain to processing of aversive and rewarding stimuli, respectively. Post-Traumatic Stress Disorder (PTSD) has been typically associated with hyper-responsivity of the NVS, and more recently, with deficient PVS functionality. The respective roles of these systems in early PTSD development have yet to be resolved. Here, we assessed neurobehavioral indicators of PVS and NVS longitudinally among 171 adult civilians at 1-, 6-, and 14-months following trauma exposure (TP1, TP2, and TP3). Using the  Safe or Risky Domino Choice (SRDC) game during fMRI, NVS and PVS functionality (i.e., activity and connectivity) were indicated by the amygdala and ventral striatum (VS) responses to punishments and rewards, respectively. The complementary functionality of these systems was behaviorally assessed as the percentage of risky choices taken during the game. Results revealed that increased amygdala functionality at TP1 was associated with greater PTSD severity at TP1 and TP3, specifically with hyperarousal and intrusion. Decreased VS functionality at TP1 was associated with greater PTSD severity at TP3, specifically with avoidance. Explainable machine learning revealed the primacy of PVS over NVS functionality at TP1 in predicting PTSD severity at TP3. Behaviorally, fewer risky choices were associated with more severe symptoms at TP1, especially with intrusion and avoidance. Overall, these results suggest a differential and potentially complementary involvement of NVS and PVS in PTSD development following trauma. Early therapeutics for PTSD in the immediate aftermath of trauma may thus target both negative and positive valence processing.

sentences: CDK11 Promotes Centromeric Transcription to Maintain Centromeric Cohesion during Mitosis Actively-transcribing RNA polymerase (RNAP)II is remained on centromeres to maintain centromeric cohesion during mitosis although it is largely released from chromosome arms. This pool of RNAPII plays an important role in centromere functions. However, the mechanism of RNAPII retention on mitotic centromeres is poorly understood. We here demonstrate that Cdk11 depletion-induced centromeric cohesion defects are largely independent of Bub1. We further show that Cdk11 depletion and expression of its kinase-dead version significantly reduce both RNAPII and elongating RNAPII (pSer2) levels on centromeres, and also decrease centromeric transcription without altering the protein expression of cohesin and cohesion-regulators. Interestingly, enhanced centromeric transcription by THZ1 treatment or overexpression of CENP-B DNA-binding domain completely rescues Cdk11-depletion defects. These results suggest that Cdk11 promotes centromeric cohesion through facilitating centromeric transcription. Mechanistically, Cdk11 binds and phosphorylates RNAPII to promote transcription. Furthermore, mitosis-specific degradation of G2/M Cdk11-p58 recapitulates Cdk11-depletion defects. Altogether, our findings establish Cdk11 as an important regulator of centromeric transcription and as part of the mechanism for retaining RNAPII on centromeres during mitosis.

sentences: Microbial retention and resistances in stormwater quality improvement devices treating road runoff Current knowledge about the microbial communities that occur in in urban road runoff is scarce. Road runoff of trafficked roads can be heavily polluted and is treated by stormwater quality improvement devices (SQIDs). However, microbes may influence the treatment process of these devices or could lead to stress resistant opportunistic microbial strains. In this study, the microbial community in the influent, effluent and the filter materials for the removal of dissolved heavy metals of two different SQIDs were analyzed to determine the microbial load, retention, composition, and mobile resistance genes. Although the microbes were replaced by new taxa in the effluent, there was no major retention of microbial genera. Further, the bacterial abundance of the SQIDs effluent was relatively stable over time. The heavy metal content correlated with intl1 and with microbial genera. The filter media itself was enriched with Intl1 gene cassettes, carrying several heavy metal and multidrug resistance genes (e.g. czrA, czcA, silP, mexW and mexI), indicating that this is a hot spot for horizontal gene transfer. Overall, the results shed light on road runoff microbial communities, and pointed to distinct bacterial communities within the SQIDs, which subsequently influence the microbial community and the genes released with the treated water.

sentences: Structural dynamics determine voltage and pH gating in human voltage-gated proton channel Voltage-gated ion channels are key players of electrical signaling in cells. As a unique subfamily, voltage-gated proton (Hv) channels are standalone voltage sensors without separate ion conductive pores. They are gated by both voltage and transmembrane proton gradient (i.e {Delta}pH), serving as acid extruders in most cells. Amongst their many functions, Hv channels are known to regulate the intracellular pH of human spermatozoa and compensate for the charge and pH imbalances caused by NADPH oxidases in phagocytes. Like the canonical voltage sensors, the Hv channel is a bundle of 4 helices (named S1 through S4), with the S4 segment carrying 3 positively charged Arg residues. Extensive structural and electrophysiological studies on voltage-gated ion channels generally agree on an outwards movement of the S4 segment upon activating voltage, but the real time conformational transitions are still unattainable. With purified human voltage-gated proton (hHv1) channel reconstituted in liposomes, we have examined its conformational dynamics at different voltage and pHs using the single molecule fluorescence resonance energy transfer (smFRET). Here we provided the first glimpse of real time conformational trajectories of the hHv1 voltage sensor and showed that both voltage and pH gradient shift the conformational dynamics of the S4 segment to control channel gating. Our results suggested the biological gating is determined by the conformational distributions of the hHv1 voltage sensor, rather than the conformational transitions between the presumptive  resting and  activated conformations. We further identified H140 as the key residue sensing extracellular pH and showed that both the intracellular and extracellular pH sensors act on the voltage sensing S4 segment to enrich the resting conformations. Taken together, we proposed a model that explains the mechanisms underlying voltage and pH gating in Hv channels, which may also serve as a general framework to understand the voltage sensing and gating in other voltage-gated ion channels.

sentences: Effect of ambient fluid rheology on oscillatory instabilities in filament-motor systems Filaments and filament bundles such as microtubules or actin interacting with molecular motors such as dynein or myosin constitute a common motif in biology. Synthetic mimics, examples being artificial muscles and reconstituted active networks, also feature active filaments. A common feature of these filament-motor systems is the emergence of stable oscillations as a collective dynamic response. Here, using a combination of classical linear stability analysis and non-linear numerical solutions, we study the dynamics of a minimal filament-motor system immersed in model viscoelastic fluids. We identify steady states, test the linear stability of these states, derive analytical stability boundaries, and investigate emergent oscillatory solutions and their properties. We show that the interplay between motor activity, aggregate elasticity and fluid viscoelasticity allows for stable oscillations or limit cycles to bifurcate from steady states. For highly viscous Newtonian media, frequencies at onset decay with viscosity  as [Formula]. In viscoelastic fluids that have the same viscosity as the Newtonian fluid but additionally allow for temporary energy storage, emergent limit cycles are associated with higher frequencies. The magnitude of the increase in the frequency depends on motor mechanochemistry and the interplay between fluid relaxation time-scales and time-scales associated with motor binding and unbinding. Our results suggest that stability and dynamical response in filamentous active systems may be controlled by tailoring the rheology of the ambient environment.

sentences: Protrusion growth driven by myosin-generated force Actin-based protrusions are found on the surface of all eukaryotic cells, where they support diverse biological activities essential for life. Models of protrusion growth hypothesize that actin filament assembly provides the mechanical force for bending the plasma membrane outward. However, membrane-associated myosin motors are also abundant in protrusions, though their potential for contributing growth-promoting force remains unexplored. Using a novel inducible system that docks myosin motor domains to membrane binding modules with temporal control, we found that the application of myosin-generated force to the plasma membrane is sufficient for driving robust elongation of protrusions. Protrusion growth scaled with motor accumulation, required active, barbed end-directed force, and was independent of cargo delivery or the recruitment of canonical barbed end elongation factors. Application of growth-promoting force was also supported by structurally distinct myosin motor domains and membrane binding modules. We conclude that myosin-generated force can drive protrusion growth and this mechanism is likely active in diverse biological contexts.

sentences: Loss of full-length dystrophin expression results in major cell-autonomous abnormalities in proliferating myoblasts Duchenne muscular dystrophy (DMD) affects myofibers and muscle stem cells (SC), causing progressive muscle degeneration and repair defects. It is not known whether dystrophic myoblasts--the effector cells of muscle growth and regeneration--are affected. Using a combination of transcriptomic, molecular and functional analyses we demonstrate, to our knowledge for the first time, convergent cell-autonomous abnormalities in primary mouse and human dystrophic myoblasts. In Dmdmdx mouse myoblasts lacking full-length dystrophin transcripts, expression of 170 other genes was significantly altered. Myod1 (p=2.9e-21) and key muscle genes controlled by MyoD (Myog, Mymk, Mymx, epigenetic regulators, ECM interactors, calcium signalling and fibrosis genes) were significantly downregulated. Gene ontology enrichment analysis indicated significant alterations in genes involved in muscle development and function. These transcriptomic abnormalities translated into increased proliferation (p=3.0e-3), reduced chemotaxis towards both sera-rich (p=3.8e-2) and cytokine-containing medium (p=1.0e-2), and significantly accelerated differentiation in 3D organotypic cultures. These altered myoblast functions are essential for muscle regeneration. The defects were caused by the loss of expression of full-length dystrophin as strikingly similar and not exacerbated alterations were also observed in dystrophin-null Dmdmdx-{beta}geo myoblasts. Corresponding abnormalities were identified in human DMD primary myoblasts and in an established dystrophic mouse muscle (SC5) cell line, confirming universal, cross-species and cell-autonomous nature of this defect. These results demonstrate the disease continuum: DMD defects in satellite cells cause myoblast dysfunctions diminishing muscle regeneration, which is essential to counteract myofiber degeneration. Contrary to the established belief, our data identify myoblasts as a novel and important therapeutic target for treatment of this lethal disease.

sentences: Accounting for dispersal improves the understanding of species abundance patterns A long-standing question in ecology is how are species population distributed across space. The highest abundance has been hypothesized to be in the spatial or niche center, though mixed patterns from empirical studies has triggered a recent debate. Here we propose a conceptual framework based on environmental suitability and dispersal to interpret the mixed evidence. We demonstrate that the highest abundance could occur in the spatial center, in the niche center, or somewhere in-between the two centers, depending on the environmental setup and dispersal ability. We found that spatial and niche centers rarely overlap, suggesting the counteracting effect between the two factors, rather than reinforcement, is the norm in determining abundance patterns. The varied locations of highest abundance mirror the mixed evidence in literature, suggesting the "abundant-centre" and "abundant-niche centre" hypotheses are not mutually exclusive. This highlights the importance in understanding the biogeographic patterns through the lens of underlying mechanisms.

sentences: Population dynamics and ranging behaviors of provisioned silvered langur (Trachypithecus cristatus) in Peninsular Malaysia Tourists are attracted to the Bukit Melawati Kuala Selangor (BMKS) of Peninsular Malaysia, a small hill park, both for its status as a historical site and the free-ranging silvered langurs (Trachypithecus cristatus) that come for provisioning. We assessed the population trends and group sizes of T. cristatus over 10 years in the BMKS and examined their ranging patterns. Comparisons of observed populations between 2005 (190 individuals) and 2017 (193 individuals) revealed the stable demography and group sizes of the six T. cristatus groups in the BMKS. Based on a total of 185 location points of the six groups in 2017, their mean ranging area was 3.6 ha with a range of 0.86 to 6.93 ha with extensive spatial overlap. We also found a significant positive relationship between the six groups ranges and group sizes in 2017. Additionally, qualitative ecological comparisons with a previous study on T. cristatus in 1965 (before provisioning) suggest that the artificial food supply in the study area could modify the population dynamics and socioecology of T. cristatus. The modifications might alter their range size and territoriality in the BMKS. Overall, we found that provisioning had negative effects on the ecology of T. cristatus in the BMKS. Therefore, modifying management policies, such as banning feeding and implementing educational programs, may contribute to their proper conservation.

sentences: Net-shaped DNA nanostructure designed for rapid/sensitive detection and potential inhibition of SARS-CoV-2 virus We present a net-shaped DNA nanostructure (called "DNA Net" herein) design strategy for selective recognition and high-affinity capture of the intact SARS-CoV-2 virions through spatial pattern-matching and multivalent interactions between the aptamers (targeting wild type spike-RBD) positioned on the DNA Net and the trimeric spike glycoproteins displayed on the viral outer surface. Carrying a designer nanoswitch, the DNA Net-aptamers releases fluorescent signal upon virus binding that is easily read by a hand-held fluorimeter for a rapid (in 10 mins), simple (mix- and-read), sensitive (PCR equivalent), room temperature compatible, and inexpensive ([~] $1.26/test) COVID-19 test assay. The DNA Net-aptamers also impede authentic wild-type SARS-CoV-2 infection in cell culture with a near 1x103-fold enhancement of the monomeric aptamer. Furthermore, our DNA Net design principle and strategy can be customized to tackle other life-threatening and economically influential viruses like influenza and HIV, whose surfaces carry class-I viral envelope glycoproteins like the SARS-CoV-2 spikes in trimeric forms.

sentences: Synthetic lethal screening identifies existing drugs with selective viability effects on Neurofibromatosis type-1 model systems Neurofibromatosis type 1 (NF1) is a genetic multi-system disorder. Symptoms include near universal benign neurofibromas, as well as malignant tumours, including generally fatal malignant peripheral nerve sheath tumours. There are limited therapies for any NF1-associated tumours; therefore, there is a clear clinical need to discover new drugs that specifically target NF1-deficient tumour cells. Using a Drosophila NF1-KO cell model, we used synthetic lethal screening to identify candidate drug targets for NF1-deficient tumours and performed statistical enrichment analysis to identify further targets. We then assessed the top 72 candidate synthetic lethal partner genes to NF1 using Variable Dose Analysis, resulting in 15 candidate genes that decreased NF1-KO viability by >10% and were novel druggable targets for NF1. Autophagy inhibitors Chloroquine (CQ) and Bafilomycin A1 resulted in a significant reduction in NF1-KO cell viability, which was conserved in a panel of human NF1 mutant cell lines. AZT and Enzalutamide also selectively reduced NF1 mutant cell viability in human cell lines. Furthermore, the effect of CQ was conserved in a Drosophila NF1-mutant in vivo model. This study highlights two key points: 1) The use of Drosophila cells as a model to screen for drugs specifically targeting NF1 mutant cells was highly successful as candidate interactions were conserved across a panel of human NF1 mutant cells and an in vivo fly NF1 mutant model, and 2) NF1-deficient cells have vulnerability to disruption of the autophagy pathway, telomerase activity, and AR activity. These pathways/drugs represent promising targets for the potential treatment of NF1-associated tumours.

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Experimental evolution in TP53 deficient human gastric organoids recapitulates tumorigenesis The earliest events during human tumor initiation are poorly characterized but may hold clues as to how to detect and prevent malignancy. Here we model occult pre-neoplasia by engineering TP53 deficiency, a common and early event in gastric cancer, into human gastric organoids. By performing experimental evolution in multiple clonally derived cultures over two years we define causal relationships between this common initiating genetic lesion and resulting phenotypes. TP53 loss elicited progressive aneuploidy, including copy number alterations and complex structural variants that are common in gastric cancers and which follow preferred temporal orders. Longitudinal single cell sequencing of TP53 deficient gastric organoids similarly indicates progression towards malignant transcriptional programs. Moreover, lineage tracing with expressed cellular barcodes demonstrates reproducible dynamics whereby initially rare subclones with shared transcriptional programs repeatedly attain clonal dominance. This powerful platform for experimental evolution exposes stringent selection, clonal interference and a striking degree of phenotypic convergence in pre-malignant epithelial organoids, implying that the earliest stages of tumorigenesis may be predictable while illuminating evolutionary constraints and barriers to malignant transformation.

Graphical abstract

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sentences: Characterization of an α-glucosidase enzyme conserved in Gardnerella spp. isolated from the human vaginal microbiome Gardnerella spp. in the vaginal microbiome are associated with bacterial vaginosis, a dysbiosis in which a lactobacilli dominant microbial community is replaced with mixed aerobic and anaerobic bacteria including Gardnerella species. The co-occurrence of multiple Gardnerella species in the vaginal environment is common, but different species are dominant in different women. Competition for nutrients, particularly glycogen present in the vaginal environment, could play an important role in determining the microbial community structure. Digestion of glycogen into products that can be taken up and further processed by bacteria requires the combined activities of several enzymes collectively known as amylases, which belong to glycoside hydrolase family 13 (GH13) within the CAZy classification system. GH13 is a large and diverse family of proteins, making prediction of their activities challenging. SACCHARIS annotation of the GH13 family in Gardnerella resulted in identification of protein domains belonging to eight subfamilies. Phylogenetic analysis of predicted amylase sequences from 26 Gardnerella genomes demonstrated that a putative -glucosidase-encoding sequence, CG400_06090, was conserved in all species in the genus. The predicted -glucosidase enzyme was expressed, purified and functionally characterized. The enzyme was active on a variety of maltooligosaccharides over a broad pH range (4.0 - 8) with maximum activity at pH 7. The Km, kcat and kcat/Km values for the substrate 4-nitrophenyl -D-glucopyranoside were 8.3 M, 0.96 min-1 and 0.11 M-1min-1 respectively. Glucose was released from maltose, maltotriose, maltotetraose and maltopentaose, but no products were detected on thin layer chromatography when the enzyme was incubated with glycogen. Our findings show that Gardnerella spp. produce an -glucosidase enzyme that may contribute to the complex and multistep process of glycogen metabolism by releasing glucose from maltooligosaccharides.

sentences: PP1 inhibitor-2 promotes PP1γ positive regulation of synaptic transmission Inhibitor-2 (I-2) is a prototypic inhibitor of protein phosphatase-1 (PP1), a major serine-threonine phosphatase that regulates synaptic plasticity and learning and memory. Although I-2 is a potent inhibitor of PP1 in vitro, our previous work has elucidated that, in vivo, I-2 may act as a positive regulator of PP1. Here we show that I-2 and PP1{gamma}, but not PP1, positively regulate synaptic transmission in hippocampal neurons. Moreover, we demonstrated that I-2 enhances PP1{gamma} interaction with its major synaptic scaffold, neurabin, by Forster resonance energy transfer (FRET)/Fluorescence lifetime imaging microscopy (FLIM) studies, while having a limited effect on PP1 autoinhibitory phosphorylation. Furthermore, our study indicates that the effect of I-2 on PP1 activity in vivo is dictated by I-2 threonine-72 phosphorylation. Our work thus demonstrates a molecular mechanism by which I-2 positively regulates PP1 function in synaptic transmission.

sentences: Introspection confidence predicts EEG decoding of self-generated thoughts and meta-awareness The neurophysiological bases of mind wandering (MW) - an experiential state wherein attention is disengaged from the external environment in favour of internal thoughts, and state meta-awareness are poorly understood. In parallel, the relationship between introspection confidence in experiential state judgements and neural representations remains unclear. Here, we recorded EEG whilst participants completed a listening task within which they made experiential state judgments and rated their confidence. Alpha power was reliably greater during MW episodes, with unaware MW further associated with greater delta and theta power. Multivariate pattern classification analysis revealed that MW, and meta-awareness can be decoded from the distribution of power in these three frequency bands. Critically, we show that individual decoding accuracies positively correlate with introspection confidence. Our results reaffirm the role of alpha oscillations in MW, implicate lower frequencies in meta-awareness, and are consistent with the proposal that introspection confidence indexes neurophysiological discriminability of representational states.

sentences: Evaluation of new polymer-iodine complexes for the fabrication of medical devices. Povidone-iodine has been a true success story in the fight against infections by harnessing the antimicrobial and antiviral properties of elemental iodine for water-based applications. However, to date there has been little success in implementing iodine attributes in water-insoluble engineering plastics. Here, we describe the first development of biocidal active polyamide- and polyurethane-iodine complexes at laboratory and commercially relevant scales. These polymer-iodine materials are active against a broad range of microorganisms, including bacteria, yeast and fungi, and can be used as base materials for medical devices. The use of new polymer-iodine complexes for infection prevention in medical devices, such as sutures, catheters and drains, or wound care is expected to have significant positive effects at reducing healthcare-acquired infections. In addition, the materials are expected to find significant applications in other fields, such as air handling with the production of biocidal face masks and air filters to control spread of pathogens.

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Selective whole genome amplification as a tool to enrich specimens with low Treponema pallidum genomic DNA copies for whole genome sequencing Downstream next generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome, and the capture and removal of CpG-methylated host DNA using the NEBNext(R) Microbiome DNA Enrichment Kit followed by MDA with the REPLI-g Single Cell Kit as enrichment methods to improve the yields of T. pallidum DNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93-98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit propagated isolates, containing >14 T. pallidum genomic copies/l of sample for SWGA and >129 genomic copies/l for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens, showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which have been challenging until now.

ImportanceSyphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogen Treponema pallidum subspecies pallidum. Primary syphilis typically presents as single or multiple mucocutaneous lesions, and if left untreated, can progress through multiple stages with varied clinical manifestations. Molecular studies often rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past two decades, WGS data is needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of a SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers of T. pallidum.

sentences: Allele specific PCR for a major marker of levamisole resistance in Haemonchus contortus Haemonchus contortus is a haematophagous parasitic nematode that infects small ruminants and causes significant animal health concerns and economic losses within the livestock industry on a global scale. Treatment primarily depends on broad-spectrum anthelmintics, however, resistance is established or rapidly emerging against all major drug classes. Levamisole (LEV) remains an important treatment option for parasite control, as resistance to LEV is less prevalent than to members of other major classes of anthelmintics. LEV is an acetylcholine receptor (AChR) agonist that, when bound, results in paralysis of the worm. Numerous studies implicated the AChR sub-unit, ACR-8, in LEV sensitivity and in particular, the presence of a truncated acr-8 transcript or a deletion in the acr-8 locus in some resistant isolates. Recently, a single non-synonymous SNP in acr-8 conferring a serine-to-threonine substitution (S168T) was identified that was strongly associated with LEV resistance. Here, we investigate the role of genetic variation at the acr-8 locus in a controlled genetic cross between the LEV susceptible MHco3(ISE) and LEV resistant MHco18(UGA 2004) isolates of H. contortus. Using single worm PCR assays, we found that the presence of S168T was strongly associated with LEV resistance in the parental isolates and F3 progeny of the genetic cross surviving LEV treatment. We developed and optimised an allele-specific PCR assay for the detection of S168T and validated the assay using laboratory isolates and field samples that were phenotyped for LEV resistance. In the LEV-resistant field population, a high proportion (>75%) of L3 encoded the S168T variant, whereas the variant was absent in the susceptible isolates studied. These data further support the potential role of acr-8 S168T in LEV resistance, with the allele-specific PCR providing an important step towards establishing a sensitive molecular diagnostic test for LEV resistance.

sentences: Spatiotemporal control of cell cycle acceleration during axolotl spinal cord regeneration Axolotls are uniquely able to resolve spinal cord injuries, but little is known about the mechanisms underlying spinal cord regeneration. We previously found that tail amputation leads to reactivation of a developmental-like program in spinal cord ependymal cells (Rodrigo Albors et al., 2015), characterized by a high-proliferation zone emerging 4 days post-amputation (Rost et al., 2016). What underlies this spatiotemporal pattern of cell proliferation, however, remained unknown. Here, we use modelling, tightly linked to experimental data, to demonstrate that this regenerative response is consistent with a signal that recruits ependymal cells during 85 hours after amputation within ~830{square}m of the injury. We adapted FUCCI technology to axolotls (AxFUCCI) to visualize cell cycles in vivo. AxFUCCI axolotls confirmed the predicted appearance time and size of the injury-induced recruitment zone and revealed cell cycle synchrony between ependymal cells. Our modeling and imaging move us closer to understanding bona fide spinal cord regeneration.

sentences: Innate and adaptive immunity to transfused allogenic red blood cells in mice requires MyD88 Red blood cell (RBC) transfusion therapy is essential for the survival of patients with hematological disorders such as sickle cell anemia. A potentially fatal complication of transfusion is development of non-ABO alloantibodies to polymorphic RBC antigens, yet mechanisms of alloantibody formation remain unclear. Human and mouse RBCs acquire a "storage lesion" prior to transfusion, which in mice contributes to immunogenicity. We previously reported that mouse splenic dendritic cells (DCs) are required for RBC alloimmunization and are activated by sterile and leukoreduced mouse RBCs after storage. Yet how syngeneic RBCs activate innate immune pathways to induce DC activation is unknown. We now show that DC activation to transfused RBCs occurs regardless of alloantigen presence, suggesting that RBC damage induced during storage triggers innate immune receptors. We discovered an unexpected dependence of RBC alloimmunization on the Toll-like receptor (TLR) signaling adaptor molecule MyD88. TLRs are a class of pattern recognition receptors (PRRs) that regulate DC activation and signal through two adaptor molecules, MyD88 and TRIF. We show that the inflammatory cytokine response, DC activation, and the subsequent alloantibody response to transfused syngeneic RBCs require MyD88 but not TRIF, suggesting a restricted set of PRRs are responsible for sensing RBCs and triggering alloimmunization.

sentences: SYNAPTIC DYSFUNCTION UNDERLIES ALTERATIONS IN THE INITIATION OF GOAL-DIRECTED BEHAVIORS: IMPLICATIONS FOR HIV-1 ASSOCIATED APATHY Individuals living with human immunodeficiency virus type 1 (HIV-1) exhibit an increased prevalence of neuropsychiatric comorbities (e.g., apathy) relative to their seronegative counterparts. Given the profound functional consequences associated with apathy, conceptualizing the multidimensional neuropsychiatric syndrome, and associated neural mechanisms, following chronic HIV-1 viral protein exposure remains a critical need. HIV-1 associated apathy was examined by quantifying goal-directed behaviors, indexed using voluntary wheel running, during the diurnal and nocturnal cycle. Apathetic behaviors in the HIV-1 Tg rat were characterized by a profound decrease in the number of running bouts during both the diurnal and nocturnal cycle, supporting a prominent deficit in the self-initiation of spontaneous behaviors. Additionally, HIV-1 Tg animals exhibited a decreased reinforcing efficacy of voluntary wheel running during the nocturnal cycle. Following the completion of voluntary wheel running, synaptic dysfunction in medium spiny neurons (MSNs) of the nucleus accumbens (NAc) was examined as a potential neural mechanism underlying HIV-1 associated apathy. HIV-1 Tg animals displayed prominent synaptic dysfunction in MSNs of the NAc, characterized by decreased synaptic connectivity and a population shift towards an immature dendritic spine phenotype relative to control animals. Synaptic dysfunction accounted for 42.0% to 68.5% of the variance in the number of running bouts affording a key neural mechanism underlying the self-initiation of spontaneous behaviors. The establishment of a fundamental neural mechanism underlying apathy affords a key target for the development of novel therapeutics and cure strategies for affective alterations associated with HIV-1.

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Small Directional Treadmill Perturbations Induce Differential Gait Stability Adaptation Introducing unexpected perturbations to challenge gait stability is an effective approach to investigate balance control strategies. Little is known about the extent to which people can respond to small perturbations during walking. This study aimed to determine how subjects adapted gait stability to multidirectional perturbations with small magnitudes applied on a stride-by-stride basis. Ten healthy young subjects walked on a treadmill that either briefly decelerated belt speed ("stick"), accelerated belt speed ("slip"), or shifted the platform medial-laterally at right leg mid-stance. We quantified gait stability adaptation in both anterior-posterior and medial-lateral directions using margin of stability and its components, base of support and extrapolated center of mass. Gait stability was disrupted upon initially experiencing the small perturbations as margin of stability decreased in the stick, slip, and medial shift perturbations and increased in the lateral shift perturbation. Gait stability metrics were generally disrupted more for perturbations in the coincident direction. Subjects employed both feedback and feedforward strategies in response to the small perturbations, but mostly use feedback strategies during adaptation. Subjects primarily used base of support (foot placement) control in the lateral shift perturbation and extrapolated center of mass control in the slip and medial shift perturbations. These findings provide new knowledge about the extent of gait stability adaptation to small magnitude perturbations applied on a stride-by-stride basis and reveal potential new approaches for balance training interventions to target foot placement and center of mass control.

NEW & NOTEWORTHYLittle is known about if and how humans can adapt to small magnitude perturbations experienced on a stride-by-stride basis during walking. Here, we show that even small perturbations disrupted gait stability and that subjects could still adapt their reactive balance control. Depending on the perturbation direction, subjects might prefer adjusting their foot placement over their center of mass and vice versa. These findings could help potentially tune balance training to target specific aspects of balance.

sentences: Medial prefrontal activity at encoding determines enhanced recognition of threatening faces after 1.5 years Studies demonstrated that faces with threatening emotional expressions are better remembered than non-threatening faces. However, whether this memory advantage persists over years and which neural systems underlie such an effect remains unknown. Here, we investigated recognition of incidentally encoded faces with angry, fearful, happy, sad and neutral expressions over >1.5 years (N= 89). Univariate analyses showed that threatening faces (angry, fearful) were better recognized than happy and neutral faces after >1.5 years, and that the threat-related memory enhancement was driven by forgetting of non-threatening faces. Multivariate principal component analysis (PCA) confirmed the discriminative performance between threatening and non-threatening faces. With an innovative Behavioral Pattern Similarity Analysis (BPSA) approach and functional magnetic resonance imaging (fMRI) acquisition during encoding, we further found that the long-term memory advantage for threatening faces were underpinned by differential neural encoding in the left inferior occipital gyrus (IOG) and right ventromedial prefrontal/orbitofrontal cortex (vmPFC/OFC). Our study provides the first evidence that threatening facial expressions lead to persistent face recognition over periods of >1.5 years and differential encoding-related activity in the visual cortex and medial prefrontal cortex may underlie this effect.

sentences: KSTAR: An algorithm to predict patient-specific kinase activities from phosphoproteomic data Kinase inhibitors are one of the largest classes of FDA-approved drugs and are major targets in oncology. Although kinase inhibitors have played an important role in improving cancer outcomes, major challenges still exist, including the development of resistance and failure to respond to treatments. Improvements for tumor profiling of kinase activity would be an important step in improving treatment outcomes and identifying effective kinase targets. Here, we present a graph- and statistics-based algorithm, called KSTAR, which harnesses the phosphoproteomic profiling of human cells and tissues by predicting kinase activity profiles from the observed phosphorylation of kinase substrates. The algorithm is based on the hypothesis that the more active a kinase is, the more of its substrates will be observed in a phosphoproteomic experiment. This method is error- and bias-aware in its approach, overcoming challenges presented by the variability of phosphoproteomic pipelines, limited information about kinase-substrate relationships, and limitations of global kinase-substrate predictions, such as training set bias and high overlap between predicted kinase networks. We demonstrate that the predicted kinase activities: 1) reproduce physiologically-relevant expectations and generates novel hypotheses within cell-specific experiments, 2) improve the ability to compare phosphoproteomic samples on the same tissues from different labs, and 3) identify tissue-specific kinase profiles. Global benchmarking and comparison to other algorithms demonstrates that KSTAR is particularly superior for predicting tyrosine kinase activities and, given its focus on utilizing more of the available phosphoproteomic data, significantly less sensitive to study bias. Finally, we apply the approach to complex human tissue biopsies in breast cancer, where we find that KSTAR activity predictions complement current clinical standards for identifying HER2-status - KSTAR can identify clinical false positives, patients who will fail to respond to inhibitor therapy, and clinically defined HER2-negative patients that might benefit from HER2-targeted therapy. KSTAR will be useful for both basic biological understanding of signaling networks and for improving clinical outcomes through improved clinical trial design, identification of new and/or combination therapies, and for identifying the failure to respond to targeted kinase therapies.

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Mentorship as a strategy to improve research ability of students and young researchers in Africa: an exploratory study and initial findings on the CORE Africa Research Mentorship Scheme Mentorship provides an opportunity to support skill development, improve research ability, promote interest in research and offer career advice. The need for research mentorship in Africa is well-recognised. However, there is scarce literature on the development of such programmes and their potential impacts on students and young researchers in Africa (SYRIA). This study documents the development of the CORE Africa Research Mentorship Scheme (CARMS), and reports the outcomes and challenges experienced over a two-year period, from 2018 to 2020.

41 mentors and mentees from Cameroon, Uganda and Nigeria participated in the programme. Mentors were based in Africa and the UK, while mentees were all based in Africa. Mentees gained knowledge and skills in various research areas including proposal writing, research methods, data analysis, report writing and research publication. Nine mentees gained their first research publication through the CARMS and three successfully completed their first grant proposal. Three mentees were supported by their mentor to develop PhD research proposals and five others gained skills that helped them secure paid research jobs. Eleven mentees have  graduated from the programme, seven of which are currently enrolled as trainee mentors. Mentees appreciated the opportunity to improve on their research skills and felt that the programme gave them a  safe environment to freely express their concerns.

Main challenges encountered were: difficulty finding suitable mentors, communication barriers, poorly defined mentorship goals, dealing with mentees lack of knowledge/experience of  basic research concepts and funding limitations. This programme had several positive impacts on the knowledge and skills of mentees and demonstrates the importance of mentorship in research capacity strengthening. Sustaining such programmes requires investments in training and development, to ensure that mentees are continuously and adequately supported.

sentences: On the Use of Z-Scores for Fine-Mapping with Related Individuals Using summary statistics from genome-wide association studies (GWAS) has been widely used for fine-mapping complex traits in humans. The statistical framework was largely developed for unrelated samples. Though it is possible to apply the framework to fine-mapping with related individuals, extensive modifications are needed. Unfortunately, this has often been ignored in summary-statistics-based fine-mapping with related individuals. In this paper, we show in theory and simulation what modifications are necessary to extend the use of summary statistics to related individuals. The analysis also demonstrates that though existing summary-statistics-based fine-mapping methods can be adapted for related individuals, they appear to have no computational advantage over individual-data-based methods.

sentences: MG132 induces progerin clearance and improves disease phenotypes in fibroblasts of patients affected with Hutchinson-Gilford Progeria-like syndromes Progeroid Syndromes (PS), including Hutchinson-Gilford Progeria Syndrome (HGPS, OMIM #176670), are premature and accelerated aging that clinically resemble some aspects of advancing physiological aging. Most classical HGPS patients carry a de novo point mutation within exon 11 of the LMNA gene encoding A-type Lamins. This mutation activates a cryptic splice site leading to the deletion of 50 amino acids at its carboxy-terminal domain, resulting in a truncated and permanently farnesylated Prelamin A called Prelamin A {Delta}50 or Progerin that accumulates in HGPS cell nuclei and is a hallmark of the disease. Some patients with PS carry other LMNA mutations affecting exon 11 splicing, leading to defects in nuclear A-type Lamins and are named "HGPS-like" patients. They also produce Progerin and/or other truncated Prelamin A isoforms ({Delta}35 and {Delta}90). We recently found that MG132, a proteasome inhibitor, induced progerin clearance in classical HGPS through autophagy activation and splicing regulation. Here, we show that MG132 induces aberrant prelamin A clearance and improves cellular phenotypes in HGPS-like patient cells. These results provide preclinical proof of principle for the use of a promising class of molecules toward a potential therapy for children with HGPS-like, who may therefore be eligible for inclusion in a therapeutic trial based on this approach, together with classical HGPS patients.

sentences: Allele mining, evolutionary genetic analysis of TaHKT1;5 gene and evaluation of salinity stress in selected lines of wheat. This study reports the novel allelic diversity in HKT 1;5 gene (High Affinity Potassium Transporter) in bread wheat and its phylogenetic relationship among the paralogs/orthologs of in Triticum aestivum and its wild relatives. HKT 1;5 gene is a known and pivotal gene associated with the salinity tolerance in plants upon the discrimination of K+ over Na+ in leaves without change in Na+ concentration in root. This gene was sequenced in a diverse collection of bread wheat, durum wheat, wild relatives, and ditelosomic lines. Sequence analysis in bread wheat led to the identification of four alleles, which could be distinguished by number of SNPs. Sequence comparison between monocot bread wheat and dicot Arabidopsis thaliana revealed that the HKT1;5 gene is conserved at level of exonic regions; however, the presence of transposable elements especially in intronic regions is further intriguing towards evolutionary relatedness. Two paralogous or major alleles observed in Triticum monoccum and Aegilops tauschii were further categorized as sub-alleles based on their SNPs comparison. This gene was absent in T. urartu in accordance with existing evidence, while it was found in A. speltoides (an allelic variant) with a few base pairs insertion in the exon1 region causing a frameshift mutation with an altered amino acids and genomic database mining unveiled additional alleles in this species. Ditelosomic lines with 4DL and 4DS chromosomes revealed a higher similarity with bread and durum wheat respectively. Phylogenetic studies of HKT1;5 orthologs from different Poaceae species revealed the occurrence of five different ortholog groups with taxonomic consistency. Phenotyping based salinity stress experiment distinguished the unknown lines for salinity tolerance and sensitiveness in comparison with known reference lines and possible allelic comparison was made. The salinity stress analysis further revealed that some known drought/heat tolerance lines showed slightly better salinity tolerance with mean values and variability of traits than known saline tolerant wheat line at controlled ambient.

sentences: Inactivation of the Hippo Tumor Suppressor Pathway Promotes Melanoma Human melanomas are commonly driven by activating mutations in BRAF, which promote melanocyte proliferation through constitutive stimulation of the MAPK pathway. However, oncogenic BRAF alone is insufficient to promote melanoma; instead, its expression merely induces a transient burst of proliferation that ultimately ceases with the development of benign nevi (i.e. moles) comprised of growth-arrested melanocytes. The tumor suppressive mechanisms that induce this melanocytic growth arrest remain poorly understood. Recent modeling studies have suggested that the growth arrest of nevus melanocytes is not solely due to oncogene activation in individual cells, but rather due to cells sensing and responding to their collective overgrowth, similar to what occurs in normal tissues. This cell growth arrest is reminiscent of the arrest induced by activation of the Hippo tumor suppressor pathway, which is an evolutionarily conserved pathway known to regulate organ size. Herein, we demonstrate that oncogenic BRAF signaling activates the Hippo pathway in vitro, which leads to inhibition of the pro-growth transcriptional co-activators YAP and TAZ, ultimately promoting the growth arrest of melanocytes. We also provide evidence that the Hippo tumor suppressor pathway is activated in growth-arrested nevus melanocytes in vivo, both from single-cell sequencing of mouse models of nevogenesis and human tissue samples. Mechanistically, we observe that oncogenic BRAF promotes both ERK-dependent alterations in the actin cytoskeleton and whole-genome-doubling events, and that these two effects independently promote Hippo pathway activation. Lastly, we demonstrate that abrogation of the Hippo pathway, via melanocyte-specific deletion of the Hippo kinases Lats1/2, enables oncogenic BRAF-expressing melanocytes to bypass nevus formation, thus leading to the rapid onset of melanoma with 100% penetrance. This model is clinically relevant, as co-heterozygous loss of LATS1/2 is observed in [~]15% of human melanomas. Collectively, our data reveal that the Hippo pathway enforces the stable growth arrest of nevus melanocytes and therefore represents a critical and previously unappreciated barrier to melanoma development.

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Hyperpiliation reduces Pseudomonas aeruginosa pathogenicity by impeding type III secretion Type IVa pili (T4aP) are important virulence factors for many bacterial pathogens. Previous studies suggested that the retraction ATPase, PilT, modulates pathogenicity due to its critical role in pilus dynamics and twitching motility. Here we use a Caenorhabditis elegans slow killing model to show that hyperpiliation, not loss of pilus retraction, reduces virulence of Pseudomonas aeruginosa strains PAK and PA14 by interfering with function of the contact-dependent type III secretion system (T3SS). Hyperactivating point mutations in the P. aeruginosa PilSR two-component system that controls transcription of the major pilin gene, pilA, increased levels of surface pili to the same extent as deleting pilT, without impairing twitching motility. These functionally hyperpiliated PilSR mutants had significant defects in pathogenicity that were rescued by deleting pilA or by increasing the length of T3SS needles via deletion of the needle-length regulator, PscP. Hyperpiliated pilT deletion or pilO point mutants showed similar PilA-dependent impairments in virulence, validating the phenotype. Together, our data support a model where a surfeit of pili prevents effective engagement of contact-dependent virulence factors. These findings suggest that the role of T4aP retraction in virulence should be revised.

SIGNIFICANCEPseudomonas aeruginosa is a major contributor to hospital-acquired infections and particularly problematic due to its intrinsic resistance to many front-line antibiotics. Strategies to combat this and other important pathogens include development of anti-virulence therapeutics. We show that the pathogenicity of P. aeruginosa is impaired when the amount of type IVa pili (T4aP) expressed on the cell surface increases, independent of the bacterias ability to twitch. We propose that having excess T4aP on the cell surface can physically interfere with productive engagement of the contact-dependent type III secretion toxin delivery system. A better understanding of how T4aP modulate interaction of bacteria with target cells will improve the design of therapeutics targeting components involved in regulation of T4aP expression and function, to reduce the clinical burden of P. aeruginosa and other T4aP-expressing bacteria.

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Anti-GD2 antibody therapy alters the neuroblastoma tumor microenvironment and extends survival in TH-MYCN mice BackgroundNeuroblastoma is a commonly lethal solid tumor of childhood and intensive chemoradiotherapy treatment cures ~50% of children with high-risk disease. The addition of immunotherapy using dinutuximab, a monoclonal antibody directed against the GD2 disialoganglioside expressed on neuroblasts, improves survival when incorporated into front-line therapy and shows robust activity in regressing relapsed disease when combined with chemotherapy. Still, many children succumb to neuroblastoma despite receiving dinutuximab-based immunotherapy, and efforts to counteract the immune suppressive signals responsible are warranted. Animal models of human cancers provide useful platforms to study immunotherapies. TH-MYCN transgenic mice are immunocompetent and develop neuroblastomas at autochthonous sites due to enforced MYCN expression in developing neural crest tissues. However, GD2-directed immunotherapy in this model has been underutilized due to the prevailing notion that TH-MYCN neuroblasts express insufficient GD2 to be targeted.

MethodsTH-MYCN mice were treated with 14G2a (anti-GD2 antibody), isotype antibody, or phosphate buffered saline from day 14 of life until day 100 or signs of morbidity. Survival was recorded, and tumors were isolated in terminal surgeries for analysis of GD2 expression and immune cell frequencies. Tumors from untreated mice were explanted for generation into cell lines, and GD2 expression was recorded with serial passage in tissue culture. Immunocytology and immunoblotting were performed to evaluate for adrenergic and mesenchymal markers of neuroblasts. Survival curves compared using Kaplan-Meier method with a log-rank test for significance. Unpaired two-tailed Students t-tests used for comparison of groups in flow cytometry analysis.

Results14G2a markedly extends survival in such TH-MYCN mice. Additionally, neuroblasts in 14G2a-treated mice have reduced GD2 expression and fewer macrophage and myeloid-derived suppressor cells in their tumor microenvironments. Neuroblasts in TH-MYCN-driven tumors express GD2 at levels comparable to human neuroblastomas but rapidly lose GD2 expression when explanted ex vivo to establish tumor cell lines. The loss of GD2 expression ex vivo is associated with a transition from an adrenergic to mesenchymal state that is maintained when reimplanted in vivo.

ConclusionsOur findings support the utility of the TH-MYCN model to inform GD2-directed immunotherapy approaches for neuroblastoma as well as opportunities to investigate drivers of adrenergic to mesenchymal fate decisions.

sentences: Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution NAT10 is an essential enzyme that catalyzes the formation of N4-acetylcytidine (ac4C) in eukaryotic transfer RNA (tRNA) and 18S ribosomal RNA (rRNA). Recent studies in human cells suggested that rRNA acetylation is dependent on SNORD13, a non-canonical box C/D small nucleolar RNA (SNORD) predicted to base-pair with 18S rRNA via two antisense elements. However, the selectivity of SNORD13-dependent cytidine acetylation and its relationship to NAT10s essential function in pre-rRNA processing remain to be defined. Here, we used CRISPR-Cas9 genome editing to formally demonstrate that SNORD13 is required for acetylation of a single cytidine residue of human and zebrafish 18S rRNA. In-depth characterization revealed that SNORD13-dependent ac4C is dispensable for yeast or human cell growth, ribosome biogenesis, translation, and the development of multicellular metazoan model organisms. This loss of function analysis inspired a cross-evolutionary survey of the eukaryotic rRNA acetylation  machinery that led to the characterization of many novel SNORD13 genes in phylogenetically-distant metazoans and more deeply rooted photosynthetic organisms. This includes an atypical SNORD13-like RNA in D. melanogaster which appears to guide ac4C to 18S rRNA helix 45 despite lacking one of the two rRNA antisense elements. Finally, we discover that C. elegans 18S rRNA is not acetylated despite the presence of an essential NAT10 homolog. Altogether, our findings shed light on the molecular mechanisms underlying SNORD13-mediated rRNA acetylation across the eukaryotic tree of life and raise new questions regarding the biological function and evolutionary persistence of this highly conserved rRNA base modification.

sentences: The C. elegans ASPP homolog APE-1 is a junctional protein phosphatase 1 modulator How serine/threonine phosphatases are spatially and temporally tuned by regulatory subunits is a fundamental question in cell biology. Ankyrin repeat, SH3 domain, proline-rich-region-containing proteins (ASPPs) are protein phosphatase 1 (PP1) binding partners associated with cardiocutaneous diseases. ASPPs localize PP1 to cell-cell junctions, but how ASPPs localize and whether they regulate PP1 activity in vivo is unclear. Through a C. elegans genetic screen, we find that loss of the ASPP homolog, APE-1, suppresses a pathology called  jowls, providing us with an in vivo assay for APE-1 activity. Using structure-function analysis, we discover that APE-1s N-terminal half directs the APE-1-PP1 complex to intercellular junctions. Additionally, we isolated mutations in highly conserved residues of APE-1s ankyrin repeats that suppress jowls yet do not preclude PP1 binding, implying ASPPs do more than simply localize PP1. Indeed, in vivo reconstitution of APE-1 suggests the ankyrin repeats modulate phosphatase output, a function we find to be conserved among vertebrate homologs.

sentences: Bayesian inference of population prevalence Within neuroscience, psychology and neuroimaging, the most frequently used statistical approach is null-hypothesis significance testing (NHST) of the population mean. An alternative approach is to perform NHST within individual participants and then infer, from the proportion of participants showing an effect, the prevalence of that effect in the population. We propose a novel Bayesian method to estimate such population prevalence that offers several advantages over population mean NHST. This method provides a population-level inference that is currently missing from study designs with small participant numbers, such as in traditional psychophysics and in precision imaging. Bayesian prevalence delivers a quantitative population estimate with associated uncertainty instead of reducing an experiment to a binary inference. Bayesian prevalence is widely applicable to a broad range of studies in neuroscience, psychology, and neuroimaging. Its emphasis on detecting effects within individual participants can also help address replicability issues in these fields.

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Cerebellar neuronal dysfunction accompanies early motor symptoms in Spinocerebellar Ataxia Type 3 and is partially alleviated upon chronic citalopram treatment Spinocerebellar Ataxia Type 3 (SCA3) is an adult-onset, progressive ataxia. SCA3 presents with ataxia before any gross neuropathology. A feature of many cerebellar ataxias is aberrant cerebellar output that contributes to motor dysfunction. We examined whether abnormal cerebellar output was present in the CMVMJD135 SCA3 mouse model and, if so, whether it correlated with the disease onset and progression. In vivo recordings showed that the activity of deep cerebellar nuclei neurons, the main output of the cerebellum, was altered. The aberrant activity correlated with the onset of ataxia. However, although the severity of ataxia increased with age, the severity of the aberrant cerebellar output was not progressive. The abnormal cerebellar output, however, was accompanied with non-progressive abnormal activity of their upstream synaptic inputs, the Purkinje cells. In vitro recordings indicated that alterations in both intrinsic Purkinje cell pacemaking and in their synaptic inputs contributed to abnormal Purkinje cell activity. These findings implicate abnormal cerebellar physiology as an early, consistent contributor to pathophysiology in SCA3, and suggest that the aberrant cerebellar output could be an appropriate therapeutic target in SCA3.

Summary StatementIn a mouse model of Spinocerebellar ataxia type 3 aberrant cerebellar physiology is apparent early in disease, before overt neuronal pathology or neuronal death.

sentences: Nanobodies identify an activated state of the TRIB2 pseudokinase Tribbles proteins (TRIB1-3) are a pseudokinase-only branch of the human kinome, which recruit substrates to the COP1 ubiquitin-ligase for ubiquitination. TRIB2 was the first Tribbles ortholog to be implicated as a myeloid leukaemia oncogene, by way of recruiting the C/EBPa transcription factor for degradation by COP1. Here we report selection and characterisation of nanobodies against the TRIB2 pseudokinase domain from a synthetic yeast surface-display library. We identified nanobodies that bind the TRIB2 pseudokinase domain with low nanomolar affinity. A crystal structure of Nb4.103 in complex with TRIB2 identified a mode of binding to the N-terminal lobe of the pseudokinase, in a manner that enables specific recognition of TRIB2 over TRIB1 and TRIB3. In the nanobody-stabilised state, TRIB2 adopts an activated conformation that is remarkably similar to the C/EBPa-bound state of TRIB1. Characterization in solution revealed that Nb4.103 can stabilise a TRIB2 pseudokinase domain dimer in a face-to-face manner. Conversely, a distinct nanobody (Nb4.101) binds through a similar epitope but does not readily promote dimerization. In combination, this study identifies features of TRIB2 that could be exploited for the development of inhibitors, and nanobody tools for future investigation of TRIB2 function.

sentences: Detecting intragenic trans-splicing events with hybrid transcriptome sequencing in cancer cells Trans-splicing can generate non-co-linear (NCL) transcripts that consist of exons in an order topologically inconsistent with the corresponding DNA template. Detecting trans-spliced RNAs (ts-RNAs) may be interfered by false positives from experimental artifacts, circular RNAs (circRNAs), and genetic rearrangements. Particularly, intragenic ts-RNAs, which are derived from separate precursor mRNA molecules of the same genes, are often mistaken for circRNAs through analyses of high-throughput transcriptome sequencing (RNA-seq) data. In addition, the biogenesis and function of ts-RNAs remain elusive. Here we developed a bioinformatics pipeline, NCLscan-hybrid, with the integration of long and short RNA-seq reads to minimize false positives and identify intragenic ts-RNAs. We utilized two features of long reads, out-of-circle and rolling circle, to distinguish intragenic ts-RNAs from circRNAs. We also designed multiple experimental validation steps to examine each type of false positives and successfully confirmed an intragenic ts-RNA (ts-ARFGEF1) in breast cancer cells. On the basis of ectopic expression and CRISPR-based endogenous genome modification experiments, we confirmed that ts-ARFGEF1 formation was significantly dependent on the reverse complementary sequences in the flanking introns of the NCL junction. Subsequent in vitro and in vivo experiments demonstrated that ts-ARFGEF1 silencing can significantly inhibit tumor cell growth. We further showed the regulatory role of ts-ARFGEF1 in p53-mediated apoptosis through affecting the PERK/eIF2a/ATF4/CHOP signaling pathway in breast cancer cells. This study thus described both bioinformatics procedures and experimental validation steps for rigorous characterization of transcriptionally non-co-linear RNAs, expanding the discovery of this important but understudied class of RNAs.

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DNA methylome combined with chromosome cluster-oriented analysis provides an early signature for cutaneous melanoma aggressiveness Aberrant DNA methylation is a well-known feature of tumours and has been associated with metastatic melanoma. However, since melanoma cells are highly heterogeneous, it has been challenging to use affected genes to predict tumour aggressiveness, metastatic evolution, and patients outcomes. We hypothesized that common aggressive hypermethylation signatures should emerge early in tumorigenesis and should be shared in aggressive cells, independent of the physiological context under which this trait arises.

We compared paired melanoma cell lines with the following properties: (i) each pair comprises one aggressive counterpart and its parental cell line, and (ii) the aggressive cell lines were each obtained from different host and their environment (human, rat, and mouse), though starting from the same parent cell line. Next, we developed a multi-step genomic pipeline that combines the DNA methylome profile with a chromosome cluster-oriented analysis.

A total of 229 differentially hypermethylated genes were commonly found in the aggressive cell lines. Genome localization analysis revealed hypermethylation peaks and clusters, identifying eight hypermethylated gene promoters for validation in tissues from melanoma patients.

Five CpG identified in primary melanoma tissues were transformed into a DNA methylation score that can predict survival (Log-rank test, p=0.0008). This strategy is potentially universally applicable to other diseases involving DNA methylation alterations.

sentences: Never cared for what they do. High structural stability of Guanine-quadruplexes in presence of strand-break damages DNA integrity is an important factor to assure genome stability and, more generally, cells and organisms viability. In presence of DNA damage, the normal cell cycle is perturbed while cells activate their repair processes. Although efficient, the repair system is not always able to ensure the complete restoration of gene integrity. In these cases, not only mutations may occur, but the accumulation of lesions can either lead to carcinogenesis or reach a threshold which induces apoptosis and the programmed cell death. Among the different types of DNA lesions, strand breaks produced by ionizing radiations are the most toxic, due to their inherently difficult repair, which may lead to genomic instability. In this article we show, by using classical molecular simulations techniques, that differently from the canonical double-helical B-DNA, guanine-quadruplex (G4) arrangements show a remarkable structural stability, even in presence of two strand breaks. Since G4-DNA are recognized for their regulatory roles in cell senescence and gene expression, also involving oncogene, their stability can be related to an evolutionary cellular response aimed at minimizing the effects of ionizing radiation.

sentences: Mapping individual differences across brain network structure to function and behavior with connectome embedding The connectome, a comprehensive map of the brains anatomical connections, is often summarized as a matrix comprising all dyadic connections among pairs of brain regions. This representation cannot capture higher-order relations within the brain graph. Connectome embedding (CE) addresses this limitation by creating compact vectorized representations of brain nodes capturing their context in the global network topology. Here, nodes "context" is defined as random walks on the brain graph and as such, represents a generative model of diffusive communication around nodes. Applied to group-averaged structural connectivity, CE was previously shown to capture relations between inter-hemispheric homologous brain regions and uncover putative missing edges from the network reconstruction. Here we extend this framework to explore individual differences with a novel embedding alignment approach. We test this approach in two lifespan datasets (NKI: n=542; Cam-CAN: n=601) that include diffusion-weighted imaging, resting-state fMRI, demographics and behavioral measures. We demonstrate that modeling functional connectivity with CE substantially improves structural to functional connectivity mapping both at the group and subject level. Furthermore, age-related differences in this structure-function mapping are preserved and enhanced. Importantly, CE captures individual differences by out-of-sample prediction of age and intelligence. The resulting predictive accuracy was higher compared to using structural connectivity and functional connectivity. We attribute these findings to the capacity of the CE to incorporate aspects of both anatomy (the structural graph) and function (diffusive communication). Our novel approach allows mapping individual differences in the connectome through structure to function and behavior.

sentences: Additive manufactured scaffolds for bone tissue engineering: physical characterization of thermoplastic composites with functional fillers Thermoplastic polymer - filler composites are excellent materials for bone tissue engineering (TE) scaffolds, combining the functionality of fillers with suitable load bearing ability, biodegradability, and additive manufacturing (AM) compatibility of the polymer. Two key determinants of their utility are their rheological behavior in the molten state, determining AM processability, and their mechanical load-bearing properties. We report here the characterization of both these physical properties for four bone TE relevant composite formulations with poly(ethylene oxide terephthalate) / poly(butylene terephthalate (PEOT/PBT) as a base polymer, which is often used to fabricate TE scaffolds. The fillers used were reduced graphene oxide (rGO), hydroxyapatite (HA), gentamycin intercalated in zirconium phosphate (ZrP-GTM) and ciprofloxacin intercalated in MgAl layered double hydroxide (MgAl-CFX). The rheological assessment showed that generally the viscous behavior dominated the elastic behavior (G > G) for the studied composites, at empirically determined extrusion temperatures. Coupled rheological-thermal characterization of ZrP-GTM and HA composites showed that the fillers increased the solidification temperatures of the polymer melts during cooling. Both these findings have implications for the required extrusion temperatures and bonding between layers. Mechanical tests showed that the fillers generally made the polymer stiffer but more brittle in proportion to the filler fractions. Furthermore, the elastic moduli of scaffolds did not directly correlate with the corresponding bulk material properties, implying composite-specific AM processing effects on the mechanical properties. Lastly, we show computational models to predict multi-material scaffold elastic moduli using measured single material scaffold and bulk moduli. The reported characterizations are essential for assessing the AM processability and ultimately the suitability of the manufactured scaffolds for the envisioned bone regeneration application.

sentences: Differential dominance of an allele of the Drosophila tbetah gene challenges standard genetic techniques The biogenic amine octopamine (OA) and its precursor tyramine (TA) are involved in controlling a plethora of different physiological and behavioral processes. The tyramine-{beta}-hydroxylase (t{beta}h) gene encodes the enzyme catalyzing the last synthesis step from TA to OA. Here, we report differential dominance (from recessive to overdominant) of the putative null t{beta}hnM18 allele in two behavioral measures in Buridans paradigm (walking speed and stripe deviation) and a proboscis extension assay in the fruit fly Drosophila melanogaster. The behavioral analysis of transgenic t{beta}h expression experiments in mutant and wild type flies as well as of OA- and TA-receptor mutants revealed a complex interaction of both aminergic systems. Our analysis suggests that the different neuronal networks responsible for the three phenotypes show differential sensitivity to t{beta}h gene expression levels. The evidence suggests that this sensitivity is brought about by a TA/OA opponent system modulating the involved neuronal circuits. This conclusion entails important implications for standard transgenic techniques, commonly used in functional genetics.

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Detection of live breast cancer cells in brightfield microscopy images containing white blood cells by image analysis and deep learning SignificanceCirculating tumor cells (CTCs) are important biomarkers for cancer management. Isolated CTCs from blood are stained to detect and enumerate CTCs. However, the staining process is laborious and moreover makes CTCs unsuitable for drug testing and molecular characterization.

AimThe goal is to develop and test deep learning (DL) approaches to detect unstained breast cancer cells in bright field microscopy images that contain white blood cells (WBCs).

ApproachWe tested two convolutional neural network (CNN) approaches. The first approach allows investigation of the prominent features extracted by CNN to discriminate cancer cells from WBCs. The second approach is based on Faster Region-based Convolutional Neural Network (Faster R-CNN).

ResultsBoth approaches detected cancer cells with high sensitivity and specificity with the Faster R-CNN being more efficient and suitable for deployment. The distinctive feature used by the CNN used to discriminate is cell size, however in the absence of size difference, the CNN was found to be capable of learning other features. The Faster R-CNN was found to be robust with respect to intensity and contrast image transformations.

ConclusionsCNN-based deep learning approaches could be potentially applied to detect patient-derived CTCs from images of blood samples.

sentences: Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been extensively studied, mechanisms of autophagosome maturation, i.e., delivery and fusion with the vacuole, remain largely unknown in plants. Here, we have identified an autophagy adaptor, CFS1, that directly interacts with the autophagosome marker ATG8 and localizes on both membranes of the autophagosome. Autophagosomes form normally in Arabidopsis thaliana cfs1 mutants, but their delivery to the vacuole is disrupted. CFS1s function is evolutionarily conserved in plants as it also localizes to the autophagosomes and plays a role in autophagic flux in the liverwort Marchantia polymorpha. CFS1 regulates autophagic flux by connecting autophagosomes with the ESCRT-I component VPS23, leading to the formation of amphisomes. Disrupting the VPS23-CFS1 interaction affects autophagic flux and renders plants sensitive to starvation stress. Altogether, our results reveal a deeply conserved mechanism of vacuolar delivery in plants that is mediated by amphisomes.

sentences: Tumor purity adjusted beta values improve biological interpretability of high-dimensional DNA methylation data A common issue affecting DNA methylation analysis in tumor tissue is the presence of a substantial amount of non-tumor methylation signal derived from the surrounding microenvironment. Although approaches for quantifying and correcting for the infiltration component have been proposed previously, we believe these have not fully addressed the issue in a comprehensive and universally applicable way. We present a multi-population framework for adjusting DNA methylation beta values on the Illumina 450/850K platform using generic purity estimates to account for non-tumor signal. Our approach also provides an indirect estimate of the aggregate methylation state of the surrounding normal tissue. Using whole exome sequencing derived purity estimates and Illumina 450K methylation array data generated by The Cancer Genome Atlas project (TCGA), we provide a demonstration of this framework in breast cancer illustrating the effect of beta correction on the aggregate methylation beta value distribution, clustering accuracy, and global methylation profiles.

sentences: Efficacy of recombinant NP-M1 and NP-M1-CRT DNA vaccines against Influenza A viruses in mice C57/BL6 Effective vaccination against the influenza virus remains a challenge because of antigenic shift and drift in influenza viruses. Conservation is an important feature of the Nucleoprotein (NP) and Matrix protein 1(M1) qualifying them as potential candidates for developing a universal vaccine against the influenza A virus. Carliticulin (CRT), a member of heat shock protein (HSP) family, are conserved and widely distributed in many microorganisms and mammalian cells. In this study, a plasmid vector encoding the NP-M1-CRT sequence was constructed and compared with the NP-M1 sequence with respect to immunogenicity and protective efficacy in a murine model. The potency of the created construct for provoking humoral, cellular immune responses, and its protective immunity against the lethal influenza virus infection were then compared with commercial split vaccine and then evaluated in a murine model system. NP-M1-CRT as a DNA vaccine combined with in vivo electroporation could significantly improve the immunogenicity of constructed vectors. Serological evaluations demonstrated the potency of our approach to provoke strong anti-NP specific antibody responses. Furthermore, our strategy of immunization in prime-boost groups were able to provide protection against lethal viral challenge using H1N1 subtype. The ease of production of these types of vectors and the fact that they would not require annual updating and manufacturing may provide an alternative cost-effective approach to limit the spread of potential pandemic influenza viruses.

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A resistome roadmap: from the human body to pristine environments A comprehensive characterization of the human body resistome (sets of antibiotic resistance genes (ARGs)) is yet to be done and paramount for addressing the antibiotic microbial resistance threat. Here, we study the resistome of 771 samples from five major body parts (skin, nares, vagina, gut and oral cavity) of healthy subjects from the Human Microbiome Project and addressed the potential dispersion of ARGs in pristine environments. A total of 28,731 ARGs belonging to 344 different ARG types were found in the HMP proteome dataset (n=9.1x107 proteins analyzed). Our study reveals a distinct resistome profile (ARG type and abundance) between body sites and high inter-individual variability. Nares had the highest ARG load ({approx}5.4 genes/genome) followed by the oral cavity, while the gut showed one of the highest ARG richness (shared with nares) but the lowest abundance ({approx}1.3 genes/genome). Fluroquinolone resistance genes were the most abundant in the human body, followed by macrolide-lincosamide-streptogramin (MLS) or tetracycline. Most of the ARGs belonged to common bacterial commensals and multidrug resistance trait was predominant in the nares and vagina. Our data also provide hope, since the spread of common ARG from the human body to pristine environments (n=271 samples; 77 Gb of sequencing data and 2.1x108 proteins analyzed) thus far remains very unlikely (only one case found in an autochthonous bacterium from a pristine environment). These findings broaden our understanding of ARG in the context of the human microbiome and the One-Health Initiative of WHO uniting human host-microbes and environments as a whole.

ImportanceThe current antibiotic resistance crisis affects our health and wealth at a global scale and by 2050 predictions estimate 10 million deaths attributed to antibiotic resistance worldwide. Remarkably, a comprehensive analysis of ARG diversity and prevalence in different human body sites is yet to be done. Undoubtedly, our body and human built-environment have antibiotic resistant bacteria than can also be transported to other environments. Hence, the analysis of Human Microbiome Project dataset provides us not only the opportunity to explore in detail the ARGs diversity and prevalence in different parts of our body but also to provide some insights into the dispersion of ARGs from human to natural populations inhabiting pristine environments. Thus, our data would help to stablish a baseline in ARG surveillance protocols to asses further changes in antibiotic resistances in our society.

sentences: A novel automated morphological analysis of microglia activation using a deep learning assisted model There is growing evidence for the key role of microglial activation in brain pathophysiology. Consequently, there is a need for efficient automated methods to measure the morphological changes distinctive of microglia functional states in research settings. Currently, many commonly used automated methods can be subject to sample representation bias, time consuming imaging, specific hardware requirements, and difficulty in maintaining an accurate comparison across research environments. To overcome these issues, we use commercially available deep learning tools (Aiforia(R) Cloud (Aifoira Inc., Cambridge, United States) to quantify microglial morphology and cell counts from histopathological slides of Iba1 stained tissue sections. We provide evidence for the effective application of this method across a range of independently collected datasets in mouse models of viral infection and Parkinsons disease. Additionally, we provide a comprehensive workflow with training details and annotation strategies by feature layer that can be used as a guide to generate new models. In addition, all models described in this work are shared within the Aiforia(R) platform and are available for study-specific adaptation and validation.

sentences: Cerebral perfusion imaging: Hypoxia-induced deoxyhemoglobin or gadolinium? Assessment of resting cerebrovascular perfusion measures (mean transit time, cerebral blood flow and cerebral blood volume) with magnetic resonance imaging currently requires the intravascular injection of the dynamic susceptibility contrast agent gadolinium. An initial comparison between hypoxia-induced deoxyhemoglobin and gadolinium was made for these measures in six healthy participants. A bolus of deoxyhemoglobin is generated in the lung via transient hypoxia induced by an available computer-controlled gas blender technology employing sequential gas delivery (RespirAct). We hypothesised and confirmed perfusion measures from both susceptibility contrast agents would yield similar spatial patterns of cerebrovascular perfusion measures. We conclude that hypoxia-induced deoxyhemoglobin, an endogenously, non-invasively generated, non-allergenic, non-toxic, recyclable, environmentally innocuous molecule, may be suitable to become the first new magnetic resonance imaging susceptibility contrast agent introduction since gadolinium.

sentences: High-fidelity 3D live-cell nanoscopy through data-driven enhanced super-resolution radial fluctuation In recent years, the development of new image analysis approaches has highlighted the possibility of recovering super-resolution information from short sequences of wide-field images. Our recently developed method, SRRF (Super-Resolution Radial Fluctuations), enables long-term live-cell imaging beyond the resolution limit without specialized hardware. Here, we present eSRRF (enhanced-SRRF), a significant improvement over our initial method, enhancing image fidelity to the underlying structure and resolution. Especially, eSRRF uses automated data-driven parameter optimization, including an estimation of the number of frames necessary for optimal reconstruction. We demonstrate the improved fidelity of the images reconstructed with eSRRF and highlight its versatility and ease of use over a wide range of microscopy techniques and biological systems. We also extend eSRRF to 3D super-resolution microscopy by combining it with multi-focus microscopy (MFM), obtaining volumetric super-resolution imaging of live cells with acquisition speed of [~] 1 volume/second.

sentences: Learning from unexpected events in the neocortical microcircuit Scientists have long conjectured that the neocortex learns the structure of the environment in a predictive, hierarchical manner. According to this conjecture, expected, predictable features are differentiated from unexpected ones by comparing bottom-up and top-down streams of information. It is theorized that the neocortex then changes the representation of incoming stimuli, guided by differences in the responses to expected and unexpected events. In line with this conjecture, different responses to expected and unexpected sensory features have been observed in spiking and somatic calcium events. However, it remains unknown whether these unexpected event signals occur in the distal apical dendrites where many top-down signals are received, and whether these signals govern subsequent changes in the brains stimulus representations. Here, we show that both somata and distal apical dendrites of cortical pyramidal neurons exhibit distinct unexpected event signals that systematically change over days. These findings were obtained by tracking the responses of individual somata and dendritic branches of layer 2/3 and layer 5 pyramidal neurons over multiple days in primary visual cortex of awake, behaving mice using two-photon calcium imaging. Many neurons in both layers 2/3 and 5 showed large differences between their responses to expected and unexpected events. Interestingly, these responses evolved in opposite directions in the somata and distal apical dendrites. These differences between the somata and distal apical dendrites may be important for hierarchical computation, given that these two compartments tend to receive bottom-up and top-down information, respectively.

sentences: Limited selection towards lighter seeds in 1 Danish grasslands over an eight-year period Time-series analysis of cover data from 236 Danish grassland sites demonstrated community selection on seed mass. Across four grassland habitat types and during an eight-year period there was significant community selection towards lighter seeds. In the analysis, the continuous seed size variable was used to group plant species into functional types. This method was chosen in order to account for the sampling process of the abundance data, and is in contrast to most other analyses of trait selection, where the community weighted mean of the traits is used as the dependent variable. Generally, little is known on the underlying causes of possible plant trait community selection in semi-natural grasslands that are not subjected to successional processes.

sentences: In vitro and in vivo preclinical studies predict REGEN-COV protection against emergence of viral escape in humans Monoclonal antibodies against SARS-CoV-2 are a clinically validated therapeutic option against COVID-19. As rapidly emerging virus mutants are becoming the next major concern in the fight against the global pandemic, it is imperative that these therapeutic treatments provide coverage against circulating variants and do not contribute to development of treatment emergent resistance. To this end, we investigated the sequence diversity of the spike protein and monitored emergence of minor virus variants in SARS-COV-2 isolates found in COVID-19 patients or identified from preclinical in vitro and in vivo studies. This study demonstrates that a combination of non-competing antibodies, REGEN-COV, not only provides full coverage against current variants of concern/interest but also protects against emergence of new such variants and their potential seeding into the population in a clinical setting.

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Conservation and diversity in transcriptional responses among host plants forming distinct arbuscular mycorrhizal morphotypes O_LIThe morphotype of arbuscular mycorrhizal (AM) roots is distinct mostly depending on AM host species: Arum, Paris, and Intermediate types. We previously reported that gibberellin (GA) promotes the establishment of Paris-type AM symbiosis in Eustoma grandiflorum despite its negative effects on Arum-type AM symbiosis in model plants. However, the molecular mechanisms underlying the differential effects of GA on different morphotypes, including Intermediate-type AM symbiosis, remain elusive.
C_LIO_LIComparative transcriptomics revealed that several symbiosis-related genes were transcriptionally promoted upon AM fungal colonization in Lotus japonicus (Arum-type), Daucus carota (Intermediate-type), and E. grandiflorum (Paris-type). Interestingly, upon GA treatment, the fungal colonization levels and expression of symbiosis-related genes were suppressed in L. japonicus and D. carota but were promoted in E. grandiflorum.
C_LIO_LIExogenous GA transcriptionally inhibited the biosynthetic process of a host-derived signal molecule involved in AM symbiosis, strigolactone, in L. japonicus and E. grandiflorum. Additionally, disaccharides mainly metabolized in AM roots would be different between L. japonicus and D. carota/E. grandiflorum.
C_LIO_LIThis study uncovered the conserved transcriptional responses during mycorrhization and diverse responses to GA in AM roots with distinct morphotypes among phylogenetically distant host plants.
C_LI

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Analysing pneumococcal invasiveness using Bayesian models of pathogen progression rates The disease burden attributable to opportunistic pathogens depends on their prevalence in asymptomatic colonisation and the rate at which they progress to cause symptomatic disease. Increases in infections caused by commensals can result from the emergence of "hyperinvasive" strains. Such pathogens can be identified through quantifying progression rates using matched samples of typed microbes from disease cases and healthy carriers. This study describes Bayesian models for analysing such datasets, implemented in an RStan package (https://github.com/nickjcroucher/progressionEstimation). The models converged on stable fits that accurately reproduced observations from meta-analyses of Streptococcus pneumoniae datasets. The estimates of invasiveness, the progression rate from carriage to invasive disease, in cases per carrier per year correlated strongly with the dimensionless values from meta-analysis of odds ratios when sample sizes were large. At smaller sample sizes, the Bayesian models produced more informative estimates. This identified historically rare but high-risk S. pneumoniae serotypes that could be problematic following vaccine-associated disruption of the bacterial population. The package allows for hypothesis testing through model comparisons with Bayes factors. Application to datasets in which strain and serotype information were available for S. pneumoniae found significant evidence for within-strain and within-serotype variation in invasiveness. The heterogeneous geographical distribution of these genotypes is therefore likely to contribute to differences in the impact of vaccination in between locations. Hence genomic surveillance of opportunistic pathogens is crucial for quantifying the effectiveness of public health interventions, and enabling ongoing meta-analyses that can identify new, highly invasive variants.

Author summaryOpportunistic pathogens are microbes that are commonly carried by healthy hosts, but can occasionally cause severe disease. The progression rate quantifies the risk of such a pathogen transitioning from a harmless commensal to causing a symptomatic infection. The incidence of infections caused by opportunistic pathogens can rise with the emergence of "hyperinvasive" strains, which have high progression rates. Therefore methods for calculating progression rates of different pathogen strains using surveillance data are crucial for rapidly identifying emerging infectious disease threats. Existing methods typically measure progression rates relative to the overall mix of microbes in the population, but these populations can vary substantially between locations and times, making the outputs challenging to combine across studies. This work presents a new method for estimating progression rates from surveillance data that generates values useful for modelling pathogen populations, even from relatively small sample sizes.

sentences: Effects of vestibular stimulation on control of gait stability Vestibular information modulates muscle activity during gait, presumably to contribute to stability. If this is the case, stronger effects of perturbing vestibular stimulation on gait stability can be expected for narrow-base walking (which needs more control) than for normal walking and smaller effects for wide-base walking (which needs less control). An important mechanism to stabilize gait in the mediolateral direction is to coordinate foot placement based on a sensory estimate of the center of mass (CoM) state, to which vestibular information appears to contribute. We, therefore, expected that stochastic vestibular stimulation would decrease the correlation between foot placement and CoM state. In fourteen healthy participants, we measured the kinematics of the trunk (as a proxy of CoM), and feet, while they walked on a treadmill in six conditions: control, narrow-base, and wide-base, each with and without stochastic electrical vestibular stimulation (EVS) (peak amplitude of 5 mA; RMS of ~ 1.2 mA; frequency band of 0 to 25 Hz). Stochastic EVS decreased local dynamic stability. Foot placement correlated stronger with trunk motion during walking with EVS in the control and narrow-base conditions. However, residual variance in foot placement was increased with EVS indicating less accurate foot placement, although this effect was not significantly different between step width conditions. Thus, vestibular information contributes to control of gait stability, but this contribution is not modulated by step width.

sentences: The genetics of specific cognitive abilities Most research on individual differences in performance on tests of cognitive ability focuses on general cognitive ability (g), the highest level in the three-level Cattell-Horn-Carroll (CHC) hierarchical model of intelligence. About 50% of the variance of g is due to inherited DNA differences (heritability) which increases across development. Much less is known about the genetics of the middle level of the CHC model, which includes 16 broad factors such as fluid reasoning, processing speed, and quantitative knowledge. We provide a meta-analytic review of 863,041 monozygotic-dizygotic twin comparisons from 80 publications for these middle-level factors, which we refer to as specific cognitive abilities (SCA). Twin comparisons were available for 11 of the 16 CHC domains. The average heritability across all SCA is 55%, similar to the heritability of g. However, there is substantial differential heritability and the SCA do not show the dramatic developmental increase in heritability seen for g. We also investigated SCA independent of g (g-corrected SCA, which we refer to as SCA.g). A surprising finding is that SCA.g remain substantially heritable (53% on average), even though 25% of the variance of SCA that covaries with g has been removed. Our review frames expectations for genomic research that will use polygenic scores to predict SCA and SCA.g. Genome-wide association studies of SCA.g are needed to create polygenic scores that can predict SCA profiles of cognitive abilities and disabilities independent of g. These could be used to foster childrens cognitive strengths and minimise their weaknesses.

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Enhanced negative response to stress following morphine administration increases wanting of social reward Animal research suggests a central role of the -opioid receptor (MOR) system in regulating affiliative behaviors and in mediating the stress-buffering function of social contact. However, the neurochemistry of stress-related social contact seeking in humans is still poorly understood. In a randomized, double-blind, between-subject design, healthy female volunteers (N = 80) received either 10 mg of the -opioid agonist morphine sulfate, or a placebo. Following a standardized psychosocial stress induction, participants engaged in a social reward task, in which the motivation to obtain skin-to-skin social touch and the hedonic reactions elicited by such touch were assessed.

Morphine prevented the increase of salivary cortisol typically observed following acute stress exposure. Notably, this altered HPA axis responsivity was associated with increased negative affect in response to psychosocial stress, and with enhanced subjective wanting of highly rewarding social contact.

These findings provide novel evidence on the effect of exogenous opioids administration on the reactions to psychosocial stress and point to a state-dependent regulation of social motivation.

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Hippocampal GFAP-positive astrocyte responses to amyloid and tau pathologies IntroductionIn Alzheimers disease clinical research, glial fibrillary acidic protein (GFAP) released in the cerebrospinal fluid and blood is widely measured and perceived as a biomarker of reactive astrogliosis. However, it was recently demonstrated that plasma GFAP levels are associated with amyloid-{beta} (A{beta}) but not tau pathology. The molecular underpinnings behind this specificity are unexplored. Here we investigated biomarker and transcriptomic associations of GFAP-positive astrocytes with A{beta} and tau pathologies in humans and mouse models.

MethodsWe studied 90 individuals with plasma GFAP, A{beta}- and Tau-PET to investigate the association between biomarkers. Then, transcriptomic analysis in hippocampal GFAP-positive astrocytes isolated from mouse models presenting A{beta} (PS2APP) or tau (P301S) pathologies was applied to explore differentially expressed genes (DEGs), Gene Ontology processes, and protein-protein interaction networks associated with each phenotype.

FindingsIn humans, we found that plasma GFAP associates with A{beta} but not tau pathology. Supporting these results, mouse transcriptomics showed scarce overlap of DEGs between the A{beta} and tau mouse models, revealing the unique nature of GFAP-positive astrocytic responses to A{beta} or tau pathology. While A{beta} GFAP-positive astrocytes were overrepresented with genes associated with proteostasis and exocytosis-related processes, tau hippocampal GFAP-positive astrocytes presented greater abnormalities in functions related to DNA/RNA processing and cytoskeleton dynamics.

InterpretationOur results offer insights into A{beta}- and tau-driven specific signatures in GFAP-positive astrocytes. Characterizing how different underlying pathologies distinctly influence astrocyte responses is critical for the biological interpretation of astrocyte-related biomarker and suggests the need to develop context-specific astrocyte targets to study AD.

FundingThis study was supported by Instituto Serrapilheira, Alzheimers Association, CAPES, CNPq and FAPERGS.

Evidence before the studyThe glial fibrillary acidic protein (GFAP) is commonly used as a proxy of reactive astrogliosis in Alzheimers disease (AD) clinical studies. Recent evidence demonstrated that increases in GFAP levels are associated with a specific aspect of AD pathology, namely amyloid-{beta} (A{beta}). However, the biological interpretation behind it was not explored in depth molecularly. Uncovering the phenotypes acquired by GFAP-positive astrocytes under A{beta} or tau influence might directly impact clinical research by improving the biological interpretation of clinical glial biomarkers used in AD.

Added value of this studyThis study revealed molecular signatures driven by AD hallmarks in hippocampal GFAP-positive astrocytes, including 1) a scarce overlap of differentially expressed genes (DEG) driven by A{beta} and tau; 2) abnormalities in proteostasis and exocytosis-related processes in A{beta}-driven reactive astrocytes and 3) impaired DNA/RNA processing and cytoskeleton dynamics in tau-driven astrocytes. Additionally, this study revealed several astrocyte-related DEGs and biological pathways specific to each aspect of AD core pathology.

Implications of all the available evidenceThese results shed light on the existence of GFAP-positive astrocyte signatures driven by A{beta}- and tau pathologies in AD. Characterizing how upstream triggers influence GFAP-positive astrocyte molecular signals may contribute to the biological interpretation of GFAP as a biomarker of AD. Additionally, the signature genes and related biological processes identified in this study might guide other studies to identify context-specific astrocyte biomarkers for AD. Finally, it paves the way to unraveling novel mechanisms and therapeutic targets capable of potentiating the effects of emerging anti[Formula] and anti-tau therapies.

sentences: Assessing methods for geometric distortion compensation in 7T gradient echo fMRI data Echo planar imaging (EPI) is widely used in functional and diffusion-weighted MRI, but suffers from significant geometric distortions in the phase encoding direction caused by inhomogeneities in the static magnetic field (B0). This is a particular challenge for EPI at very high field ([≥] 7T), as distortion increases with higher field strength. A number of techniques for distortion correction exist, including those based on B0 field mapping and acquiring EPI scans with opposite phase encoding directions. However, few quantitative comparisons of distortion compensation methods have been performed using human EPI data, especially at very high field. Here, we compared distortion compensation using B0 field maps and opposite phase encoding scans in two different software packages (FSL and AFNI) applied to 7T gradient echo (GE) EPI data from 31 human participants. We assessed distortion compensation quality by quantifying alignment to anatomical reference scans using Dice coefficients and mutual information. Performance between FSL and AFNI was equivalent. In our whole-brain analyses, we found superior distortion compensation using GE scans with opposite phase encoding directions, versus B0 field maps or spin echo (SE) opposite phase encoding scans. However, SE performed better when analyses were limited to ventromedial prefrontal cortex, a region with substantial dropout. Matching the type of opposite phase encoding scans to the EPI data being corrected (e.g., SE-to-SE) also yielded better distortion correction. While the ideal distortion compensation approach likely varies depending on methodological differences across experiments, this study provides a framework for quantitative comparison of different distortion compensation methods.

sentences: Clinical grade ACE2 as a universal agent to block SARS-CoV-2 variants The recent emergence of multiple SARS-CoV-2 variants has caused considerable concern due to reduced vaccine efficacy and escape from neutralizing antibody therapeutics. It is therefore paramount to develop therapeutic strategies that inhibit all known and future SARS-CoV-2 variants. Here we report that all SARS-CoV-2 variants analyzed, including variants of concern (VOC) Alpha, Beta, Gamma, and Delta, exhibit enhanced binding affinity to clinical grade and phase 2 tested recombinant human soluble ACE2 (APN01). Importantly, soluble ACE2 neutralized infection of VeroE6 cells and human lung epithelial cells by multiple VOC strains with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. Effective inhibition of infections with SARS-CoV-2 variants was validated and confirmed in two independent laboratories. These data show that SARS-CoV-2 variants that have emerged around the world, including current VOC and several variants of interest, can be inhibited by soluble ACE2, providing proof of principle of a pan-SARS-CoV-2 therapeutic.

sentences: Multifactorial remodeling of the cancer immunopeptidome by interferon gamma IFN{gamma} alters the immunopeptidome presented on HLA class I (HLA-I), and its activity on cancer cells is known to be important for effective immunotherapy responses. We performed proteomic analyses of untreated and IFN{gamma}-treated colorectal cancer patient-derived organoids (PDOs) and combined this with transcriptomic and HLA-I immunopeptidomics data to dissect mechanisms that lead to remodeling of the immunopeptidome through IFN{gamma}. IFN{gamma}-induced changes in the abundance of source proteins, switching from the constitutive- to the immunoproteasome, and differential upregulation of different HLA alleles explained some, but not all, observed peptide abundance changes. By selecting for peptides which increased or decreased the most in abundance, but originated from proteins with limited abundance changes, we discovered that the amino acid composition of presented peptides also influences whether a peptide is up- or downregulated on HLA-I through IFN{gamma}. The presence of proline within the peptide core was most strongly associated with peptide downregulation. This was validated in an independent dataset. Proline substitution in relevant core positions did not influence the predicted HLA-I binding affinity or stability, indicating that proline effects on peptide processing may be most relevant. Understanding the multiple factors that influence the abundance of peptides presented on HLA-I in the absence or presence of IFN{gamma} is important to identify the best targets for antigen-specific cancer immunotherapies such as vaccines or T-cell receptor engineered therapeutics.

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Formation of synthetic RNP granules using engineered phage-coat-protein -RNA complexes Observations of RNA-protein (RNP) granules within living cells have generated important insights on intracellular processes in recent years. Here, we demonstrate that PP7-coat protein (PCP) and Q{beta}-coat protein (QCP) co-expressed with synthetic long non-coding RNA (slncRNA) encoded with multiple CP binding sites self-assemble into phase-separated synthetic RNP (SRNP) granules within E. coli cells and in vitro. SNRP granules exhibit hallmarks of RNP granule formation including phase separation from a semi-dilute liquid in vitro, and retention of structure after cell lysis. In vivo real-time tracking of SRNP granules reveals that they are found in cell poles of exponentially growing cells, and that their structure exhibits gel-like properties by the slow stochastic insertion and shedding of CP-slncRNA complexes. We propose a transcriptional model that takes into account the cytosolic structure of E. coli (i.e., the cytosol is divided into a biomolecularly dense domain in the nucleoid-dominated region and a dilute liquid domain in the polar regions), and use it to provide a theoretical framework for explaining the increase in intracellular CP-titer as a function slncRNA structure, and the appearance of SRNP granules in non-polar regions of stationary phase cells. Consequently, the SRNP-granule data and resultant model has implications for various bacterial transcriptional and translational processes, and provides an alternative explanation for the super-Poisson kinetics attributed to transcriptional bursts.

Introductory paragraphsynthetic long non-coding RNA molecules encoded with multiple phage coat protein binding sites form phase separated RNP granules when co-expressed with their cognate binding proteins.

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The Metabolomics Workbench File Status Website: A Metadata Repository Promoting FAIR Principles of Metabolomics Data MotivationAn updated version of the mwtab Python package for programmatic access to the Metabolomics Workbench (MetabolomicsWB) data repository was released at the beginning of 2021. Along with updating the package to match the changes to MetabolomicsWBs  mwTab file format and to enhance the packages functionality, the package included format validation facilities which were used to detect and catalog file inconsistencies and errors across all publically available datasets in MetabolomicsWB.

ResultsThe Metabolomics Workbench File Status website was developed to provide continuous validation of MetabolomicsWB data files and a useful interface to find all inconsistencies and errors that are found. This list of detectable issues/errors include format parsing errors, format compliance issues, access problems via MetabolomicsWBs REST interface, and other small inconsistencies that can hinder reusability. The website uses the mwtab Python package to pull down and validate each available analysis file and then generates an html report. The website is updated on a weekly basis. Moreover, the Python website design utilizes GitHub and GitHub.io, providing an easy to replicate template for implementing other metadata, virtual, and meta-repositories.

AvailabilityMetabolomics Workbench file status website can be accessed at: https://moseleybioinformaticslab.github.io/mwFileStatusWebsite/. The mwtab Python library is available on GitHub: https://github.com/MoseleyBioinformaticsLab/mwtab, PyPI: https://pypi.org/project/mwtab/, and documentation is available on ReadTheDocs: https://mwtab.readthedocs.io/. Metabolomics Workbench analysis data files used for analysis presented here along with the generated HTML files of the website are available on FigShare: https://doi.org/10.6084/m9.figshare.19221159.

Contacthunter.moseley@uky.edu

Supplementary informationSupplementary data are available at FigShare online.

sentences: Trait divergence and trade-offs among Brassicaceae species differing in elevational distribution Species have restricted geographic distributions and the causes are still largely unknown. Temperature has long been associated with distribution limits, suggesting that there are ubiquitous constraints to the evolution of the climate niche. Here we investigated the traits involved in such constraints by macroevolutionary comparisons involving around 100 Brassicaceae species differing in elevational distribution. Plants were grown under three temperature treatments (regular frost, mild, regular heat) and phenotyped for phenological, morphological and thermal resistance traits. Trait values were analysed by assessing the effect of temperature and elevational distribution, by comparing models of evolutionary trajectories, and by correlative approaches to identify trade-offs. Analyses pointed to size, leaf morphology and growth under heat as among the most discriminating traits between low- and high-elevation species, with high-elevation species growing faster under the occurrence of regular heat bouts, at the cost of much reduced size. Mixed models and evolutionary models supported adaptive divergence for these traits, and correlation analysis indicated their involvement in moderate trade-offs. Finally, we found asymmetry in trait evolution, with evolvability across traits being 50% less constrained under regular frost. Overall, results suggest that trade-offs between traits under adaptive divergence contribute to the disparate distribution of species along the elevational gradient.

sentences: Pax3 induces neural circuit repair through a developmental program of directed axon outgrowth. Repairing damaged or dysfunctional human brain circuits remains an ongoing challenge for biomedical science. While surviving neuronal networks can be reorganised after lesion, for example by neurotrophins, these new connections are disorganised and rarely produce clinical improvement. Here we investigate how to promote axonal growth while retaining correct cellular targeting. We show that, in response to brain-derived neurotrophic factor (BDNF) in target-tissue, potential reinnervating neurons upregulate Pax3. Pax3 in turn increases polysialic acid-neural cell adhesion molecule (PSA-NCAM) on their axon terminals, facilitating their outgrowth and pathfinding, and resulting in correctly-targeted neural circuit repair in the mature nervous system. This is a novel role for Pax3, which we confirmed by showing its expression in afferent neurons is essential for spontaneous and BDNF-induced reinnervation in the developing and mature brains, respectively. Together these results suggest that Pax3 contributes to a repair program, in which axon growth is promoted and direction signaling maintained. These data advance our understanding for accurately rebuilding neural circuits: restricting growth-promotion to potential afferent neurons, as opposed to stimulating the whole circuit, allows axon growth without impairing its guidance.

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A genetic switch for male UV-iridescence in an incipient species pair of sulphur butterflies Mating cues evolve rapidly and can contribute to species formation and maintenance. However, little is known about how sexual signals diverge and how this variation integrates with other barrier loci to shape the genomic landscape of reproductive isolation. Here, we elucidate the genetic basis of UV iridescence, a courtship signal that differentiates the males of Colias eurytheme butterflies from a sister species, allowing females to avoid costly heterospecific matings. Anthropogenic range expansion of the two incipient species established a large zone of secondary contact across the eastern US with strong signatures of genomic admixtures spanning all autosomes. In contrast, Z chromosomes are highly differentiated between the two species, supporting a disproportionate role of sex chromosomes in speciation known as the large-X (or large-Z) effect. Within this chromosome-wide reproductive barrier, linkage mapping indicates that cis-regulatory variation of bric a brac (bab) underlies the male UV-iridescence polymorphism between the two species. Bab is expressed in all non-UV scales, and butterflies of either species or sex acquire widespread ectopic iridescence following its CRISPR knock-out, demonstrating that Bab functions as a suppressor of UV-scale differentiation that potentiates mating cue divergence. These results highlight how a genetic switch can regulate a premating signal and integrate with other reproductive barriers during intermediate phases of speciation.

Significance statementIncipient species are at an intermediate stage of speciation where reproductive isolation is counteracted by the homogenizing effects of gene flow. Human activity sometimes leads such species to reunite, as seen in the Orange Sulphur butterfly, which forms large hybridizing populations with the Clouded Sulphur in alfalfa fields. Here we show that the sex chromosome maintains these species as distinct, while the rest of their genome is admixed. Sex chromosomes notably determine which males display to females a bright, iridescent ultraviolet signal on their wings. Genetic mapping, antibody stainings, and CRISPR knock-outs collectively indicate that the gene bric a brac controls whether UV-iridescent nanostructures develop in each species, elucidating how a master switch gene modulates a male courtship signal.

sentences: Pangenome evolution in environmentally transmitted symbionts of deep-sea mussels is governed by vertical inheritance Microbial pangenomes vary across species; their size and structure are determined by genetic diversity within the population and by gene loss and horizontal gene transfer (HGT). Many bacteria are associated with eukaryotic hosts where the host colonization dynamics may impact bacterial genome evolution. Host-associated lifestyle has been recognized as a barrier to HGT in parentally transmitted bacteria. However, pangenome evolution of environmentally acquired symbionts remains understudied, often due to limitations in symbiont cultivation. Using high-resolution metagenomics, here we study pangenome evolution of two co-occurring endosymbionts inhabiting Bathymodiolus brooksi mussels from a single cold seep. The symbionts, sulfur-oxidizing (SOX) and methane-oxidizing (MOX) gamma-proteobacteria, are environmentally acquired at an early developmental stage and individual mussels may harbor multiple strains of each species. We found differences in the accessory gene content of both symbionts across individual mussels, which are reflected by differences in symbiont strain composition. Compared to core genes, accessory genes are enriched in functions involved in genome integrity. We found no evidence for recent horizontal gene transfer between both symbionts. A comparison between the symbiont pangenomes revealed that the MOX population is less diverged and contains fewer accessory genes, supporting that the MOX association with B. brooksi is more recent than that of SOX. Our results show that the pangenomes of both symbionts evolved mainly by vertical inheritance. We conclude that environmentally transmitted symbionts that associate with individual hosts over their lifetime show features of narrow symbioses, where the frequency of HGT is constrained.

sentences: DNA sliding and loop formation by E. coli SMC complex: MukBEF SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.

sentences: High lineage survivorship across the end-Devonian Mass Extinction suggested by a remarkable new Late Devonian actinopterygian Actinopterygian (ray-finned) fishes represent the principal vertebrate group in aquatic settings. This dominance is often attributed to their apparent success in the aftermath of the end-Devonian extinction. Increases in taxonomic and morphological diversity in the early Carboniferous, coupled with phylogenetic hypotheses implying the survival of few Devonian lineages, contribute to a model of explosive post-extinction radiation. However, most actinopterygian fossils from within a ca. 20 million year (Myr) window surrounding the end-Devonian extinction remain poorly known, contributing to uncertainty about these patterns. Here we present detailed anatomical data for an exceptionally preserved but diminutive ray-finned fish from within this gap, ~7 Myr prior to the Devonian-Carboniferous boundary. Representing a new genus and species, it bears a series of derived anatomical features otherwise known only from Carboniferous and younger taxa. It nests phylogenetically within a clade of post-Devonian species and, in an expanded phylogenetic analysis incorporating other previously neglected taxa, draws at least ten lineages of Carboniferous actinopterygians into the Late Devonian. This suggests phenotypically cryptic divergence among ray-finned fishes in the latest Devonian, followed by more conspicuous diversification in feeding and locomotor structures in the Carboniferous. Our revised model matches more complicated patterns of divergence, survival, and diversification around the Devonian-Carboniferous boundary in other vertebrate clades such as tetrapods and lungfishes. It also fundamentally recalibrates the onset of diversification early in the history of a group that today contains half of all living vertebrate species.

sentences: Microbiota induces aging-related leaky gut and inflammation by dampening mucin barriers and butyrate-FFAR2/3 signaling Increased chronic inflammation is one of the key risk factors of aging-related disorders although its precise etiology remains elusive. Here, we demonstrate that aged, but not young, microbiota triggers inflammation by promoting gut permeability (leaky gut) via disruption of mucus barriers. Levels of the beneficial short-chain fatty acid, butyrate, are suppressed in the aged gut. Consistent with feedback regulation, the expression of butyrate-sensing receptors, free fatty acid receptor 2/3 (FFAR2/3), are also reduced in aged gut. Butyrate treatment of aged mice revereses the reduced mucin production, increased gut permeability and inflammation associated with low butyrate levels. In agreement, intestine-specific FFAR2/3 knockout mice manifest a compromised gut phenotype typically seen in aged mice,, such as increased gut permeability and inflammation with reduced mucin production. Taken together, our results demonstrate that an aged gut microbiota causally instigates inflammation by increasing gut permeability due to reduced butyrate levels, FFAR2/3 expression, and mucin barriers. Thus, butyrate-FFAR2/3 agonism could ameliorate the deleterious effects seen in aged gut and their implications on metabolic health.

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Occlusion of lip movements impairs reconstruction of acoustic speech features and higher-level segmentational features in the presence of a distractor speaker Multisensory integration enables stimulus representation even when the sensory input in a single modality is weak. In the context of speech, when confronted with a degraded acoustic signal, congruent visual inputs promote comprehension. When this input is occluded speech comprehension consequently becomes more difficult. But it still remains inconclusive which levels of speech processing are affected under which circumstances by occlusion of the mouth area. To answer this question, we conducted an audiovisual (AV) multi-speaker experiment using naturalistic speech. In half of the trials, the target speaker wore a (surgical) face mask, while we measured the brain activity of normal hearing participants via magnetoencephalography (MEG). We additionally added a distractor speaker in half of the trials in order to create an ecologic difficult listening situation. A decoding model on the clear AV speech was trained and used to reconstruct crucial speech features in each condition. We found significant main effects of face masks on the reconstruction of acoustic features, such as the speech envelope and spectral speech features (i.e. pitch and formant frequencies), while reconstruction of higher level features of speech segmentation (phoneme and word onsets) were especially impaired through masks in difficult listening situations. As we used surgical face masks in our study, which only show mild effects on speech acoustics, we interpret our findings as the result of the occluded lip movements. This idea is in line with recent research showing that visual cortical regions track spectral modulations. Our findings extend previous behavioural results, by demonstrating the complex contextual effects of occluding relevant visual information on speech processing.

HighlightsO_LISurgical face masks impair neural tracking of speech features
C_LIO_LITracking of acoustic features is generally impaired, while higher level segmentational features show their effects especially in challenging listening situations
C_LIO_LIAn explanation is the prevention of a visuo-phonological transformation contributing to audiovisual multisensory integration
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Endometriotic Organoids: A Novel In Vitro Model of Endometriotic Lesion Development. The development and progression of endometriotic lesions are poorly understood, but immune cell dysfunction and inflammation are closely associated with the pathophysiology of endometriosis. A lack of suitable 3D in vitro models permitting the study of interactions between cell types and the microenvironment is a contributing factor. To address this limitation, we developed endometriotic organoids (EO) to explore the role of epithelial-stromal interactions and model peritoneal cell invasion associated with lesion development. Using a non-adherent microwell culture system, spherical organoids were generated with endometriotic epithelial cells (12Z) combined with immortalized endometriotic stromal cells (iEc-ESC) or immortalized uterine stromal cells (iHUF). Organoids self-organized with stromal cells occupying the center and epithelial cells on the periphery of the organoid. Endometriotic organoids (EO), containing iEc-ESC, resulted in the development of stratified 12Z epithelial cells compared to those with iHUF where the 12Z cells developed as a single layered epithelium. Transcriptomic analysis found 4,522 differentially expressed genes (DEG) between EO and 12Z/iHUF organoids, and the top DEG included increased expression of interleukins and prostaglandin synthase enzymes. An overlap of the EO DEG with baboon endometriotic lesions was highly significant. Finally, to mimic invasion of endometrial tissue into the peritoneum, a model was developed using EO and extracellular matrix containing human peritoneal mesothelial cells (LP9). Invasion of EO into the extracellular matrix-LP9 layer was increased in presence of estrogen or THP1-derived proinflammatory macrophages. Taken together, our results strongly support the concept that EO are an appropriate model for dissecting mechanisms that contribute to endometriotic lesion development.

One Sentence SummaryEndometriotic organoids are an appropriate model to study epithelial-stromal interactions and model cell invasion associated with lesion development.

sentences: Bipartite functional fractionation within the neural system for social cognition supports the psychological continuity of self vs. other Research of social neuroscience establishes that regions in the brains default network (DN) and semantic network (SN) are engaged by socio-cognitive tasks. Research of the human connectome shows that DN and SN regions are both situated at the high-order end of cortical gradient but differ in their positions on this gradient. In the present study, we integrated these two bodies of research, used the psychological continuity of self vs. other as a  test-case, and used fMRI to investigate whether these networks would encode social concepts differently. We found a robust dissociation between the DN and SN - while both networks contained sufficient information for decoding broad-stroke distinction of social categories, the DN carried more generalisable information for cross-classifying across social distance and emotive valence than did the SN. We also found that the overarching distinction of self vs. other was a principal divider of the representational space while social distance was an auxiliary factor (subdivision, nested within the principal dimension), and this representational landscape was more manifest in the DN than in the SN. Taken together, our findings demonstrate how insights from connectome research can benefit social neuroscience, and have implications for clarifying the two networks differential contributions to social cognition.

sentences: ATR facilitates the degradation of Api5 through the ubiquitin-proteasome pathway via FBXW2 to regulate apoptosis upon DNA damage Apoptosis inhibitor 5 (Api5) is an inhibitor of apoptosis, which is found to be upregulated in several cancers and promotes invasion as well as metastasis. Over-expression of Api5 is positively co-related with poor survival of cancers and inhibition of DNA damage induced apoptosis in cancerous cells. Acetylation at lysine 251 (K251) on Api5 facilitates the stability of the protein and thus functionally provides resistance to cancer cells against chemotherapeutic or anti-cancerous agents. However, the regulation of Api5 upon DNA damage is not yet known. In this study, we demonstrate that Api5 undergoes degradation following DNA damage via the ubiquitin-proteasome system. Upon DNA damage, ATR was observed to phosphorylate Api5 at serine 138 which led to the cytoplasmic localisation of Api5. The E3-ubiquitin ligase, SCF-FBXW2 ubiquitinates Api5 leading to its proteasomal degradation.

sentences: Millisecond Mix-and-Quench Crystallography (MMQX) Enables Time-Resolved Studies of PEPCK With Remote Data Collection Time-resolved crystallography of biomolecules in action has advanced rapidly as methods for serial crystallography have improved, but the large number of crystals and complex experimental infrastructure required remain serious obstacles to widespread application. We have developed Millisecond Mix-and-Quench crystallography (MMQX), which yields millisecond time-resolved data using far fewer crystals and routine remote synchrotron data collection. To demonstrate the capabilities of MMQX, the conversion of oxaloacetic acid to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase is observed with a time resolution of 40 ms. MMQX, by lowering the entry barrier to time-resolved crystallography, should enable broad expansion in structural studies of protein dynamics.

sentences: The Orbitofrontal Cortex in Temporal Cognition One of the most important factors in decision making is estimating the value of available options. Subregions of the prefrontal cortex, including the orbitofrontal cortex (OFC), have been deemed essential for this process. Value computations require a complex integration across numerous dimensions, including, reward magnitude, effort, internal state, and time. The importance of the temporal dimension is well-illustrated by temporal discounting tasks, in which subjects select between smaller-sooner versus larger-later rewards. The specific role of OFC in telling time and integrating temporal information into decision making remains unclear. Based on the current literature, in this review we reevaluate current theories of OFC function, accounting for the influence of time. Incorporating temporal information into value estimation and decision making requires distinct, yet interrelated, forms of temporal information including the ability to tell time, represent time, create temporal expectations, and the ability to use this information for optimal decision making in a wide range of tasks, including temporal discounting and wagering. We use the term  temporal cognition to refer to the integrated use of these different aspects of temporal information. We suggest that the OFC may be a critical site for the integration of reward magnitude and delay, and thus important for temporal cognition.

sentences: Genetic inactivation of SARM1 axon degeneration pathway improves outcome trajectory after experimental traumatic brain injury based on pathological, radiological, and functional measures Traumatic brain injury (TBI) causes chronic symptoms and increased risk of neurodegeneration. Axons in white matter tracts, such as the corpus callosum (CC), are critical components of neural circuits and particularly vulnerable to TBI. Treatments are needed to protect axons from traumatic injury and mitigate post-traumatic neurodegeneration. The Sarm1 gene is a central driver of axon degeneration through a conserved molecular pathway. Sarm1-/- mice with knockout (KO) of the Sarm1 gene enable genetic proof-of-concept testing of Sarm1 inactivation as a therapeutic target. We evaluated Sarm1 deletion effects after TBI using a concussive model that causes traumatic axonal injury and progresses to CC atrophy at 10 weeks, indicating post-traumatic neurodegeneration. Sarm1 wild-type (WT) mice developed significant CC atrophy that was reduced in Sarm1 KO mice. Using electron microscopy to quantify individual axons demonstrated that Sarm1 KO preserved more intact axons and reduced damaged or demyelinated axons. MRI in live mice identified significantly reduced CC volume after TBI in Sarm1 WT mice that was attenuated in Sarm1 KO mice. MR diffusion tensor imaging detected reduced fractional anisotropy in both genotypes while axial diffusivity remained higher in Sarm1 KO mice. Immunohistochemistry revealed significant attenuation of CC atrophy, myelin loss, and neuroinflammation in Sarm1 KO mice after TBI. Functionally, TBI resulted in late-stage motor learning and sleep deficits that were ameliorated in Sarm1 KO mice. Based on these findings, Sarm1 inactivation can protect axons and white matter tracts to improve translational outcomes associated with CC atrophy and post-traumatic neurodegeneration.

sentences: Dynamical modeling of the H3K27 epigenetic landscape in mouse embryonic stem cells The Polycomb system via the methylation of the lysine 27 of histone H3 (H3K27) plays central roles in the silencing of many lineage-specific genes during development. Recent experimental evidence suggested that the recruitment of histone modifying enzymes like the Polycomb repressive complex 2 (PRC2) at specific sites and their spreading capacities from these sites are key to the establishment and maintenance of a proper epigenomic landscape around Polycomb-target genes. Here, to test whether such mechanisms, as a minimal set of qualitative rules, are quantitatively compatible with data, we developed a mathematical model that can predict the locus-specific distributions of H3K27 modifications based on previous biochemical knowledge. Within the biological context of mouse embryonic stem cells, our model showed quantitative agreement with experimental profiles of H3K27 acetylation and methylation around Polycomb-target genes in wild-type and mutants. In particular, we demonstrated the key role of the reader-writer module of PRC2 and of the competition between the binding of activating and repressing enzymes in shaping the H3K27 landscape around transcriptional start sites. The predicted dynamics of establishment and maintenance of the repressive trimethylated H3K27 state suggest a slow accumulation, in perfect agreement with experiments. Our approach represents a first step towards a quantitative description of PcG regulation in various cellular contexts and provides a generic framework to better characterize epigenetic regulation in normal or disease situations.

sentences: Proapoptotic RHG genes and mitochondria play a key non-apoptotic role in remodelling the Drosophila sensory system Caspases are best known for their role in programmed cell death but have also been found to be important in several non-apoptotic phenomena such as cell fate specification, cell migration and terminal differentiation. The dynamics of such sub-lethal caspase events and the molecular mechanisms regulating them are still largely unknown. As more tools for visualizing and manipulating caspase activation in vivo become available, greater insights into this biology are being made. Using a new and sensitive in vivo effector caspase probe, called SR4VH, we demonstrate that effector caspases are activated in pruning sensory neurons earlier than previously thought and that the level of caspase activation in these neurons is consistently lower than in neurons undergoing cell death. We reveal that Grim and Reaper, two of the four pro-apoptotic RHG proteins, are required for sensory neuron pruning and that disrupting the dynamics of the mitochondrial network prevents effector caspase activation in both pruning and dying sensory neurons. Overall, our findings demonstrate that a sublethal deployment of the  apoptotic machinery is critical for remodelling dendrites and also reveal a direct link between mitochondria and sensory neuron cell death in vivo.

sentences: Live-cell imaging and physical modeling reveal control of chromosome folding dynamics by cohesin and CTCF Physical proximity between genomic sequences in mammalian chromosomes controls key biological processes such as transcriptional regulation and DNA repair. Yet it is currently unknown if chromosomal contacts are rare and stable or instead frequent and dynamic, and how they depend on the loop extrusion activity of cohesin or barriers such as CTCF. By imaging chromosomal locations at high spatial and temporal resolution over several hours in living cells, we show that sequences within topological associating domains (TADs) frequently come into physical proximity during the course of a cell cycle and remain close to each other only for a few minutes. Such contacts become nonetheless substantially longer and more frequent in the presence of convergent CTCF sites, resulting in a suppression of variability in chromosome folding in single cells across time. Supported by physical models of chromosome dynamics, our data additionally suggests that individual CTCF-anchored loops last around 10 minutes. The estimates of chromosomal contact dynamics in our study provide a novel quantitative framework to link chromosome structure to function and show that cohesin and CTCF stabilize otherwise highly dynamic chromosome structures to facilitate selected subsets of chromosomal interactions.

sentences: Hierarchical Bayesian Model for Estimating Migratory Bird Harvest in Canada The Canadian Wildlife Service (CWS) requires reliable estimates of the harvest of migratory game birds, including waterfowl, to effectively manage populations of these hunted species. The National Harvest Survey is an annual survey of hunters who purchase Canadas mandatory migratory game bird hunting permit, integrating information from a survey of hunting activity with information from a separate survey of species composition in the harvest. We use these survey data to estimate the number of birds harvested for each species, as well as hunting activity metrics such as the number of active hunters and days spent hunting. The analytical methods used to generate these estimates have not changed since the survey was first designed in the early 1970s. Here we describe a new hierarchical Bayesian integrated model, which replaces the series of ratio estimators that comprised the old model. We are now using this new model to generate estimates for migratory bird harvests as of the 2019-2020 hunting season, and to generate updated estimates for all earlier years. The hierarchical Bayesian model uses over-dispersed Poisson distributions to model mean hunter activity and harvest (zero inflated Poisson and zero truncated Poisson, respectively). It also includes multinomial distributions to model some key components including, variation in total harvest across periods of the hunting season, the species composition of the harvest within each of those periods, and the age and sex composition in the harvests of a given species. We estimated the parameters of the Poisson and the multinomial distributions for each year as random effects using first-difference time-series. This time-series component allows the model to share information across years and reduces the sensitivity of the estimates to annual sampling noise. The new model estimates are generally very similar to those from the old model, particularly for the species that occur most commonly in the harvest, and so the results do not suggest any major changes to harvest management decisions and regulations. However, estimates for all species from the new model are more precise and less susceptible to annual sampling error, particularly for species that occur less commonly in the harvest (e.g., sea ducks and other species of conservation concern). This new model, with its hierarchical Bayesian framework, will also facilitate future improvements and elaborations, allowing the incorporation of prior information from the rich literature and knowledge in game bird management and biology.

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Muscle activation strategies of the vastus lateralis according to sex AimDespite men exhibiting greater muscle strength and fatigibility than women, it remains unclear if there are sex-based differences in muscle recruitment strategies e.g. motor unit (MU) recruitment and modulation of firing rate (FR) at normalised forces and during progressive increases in force.

MethodsTwenty-nine healthy male and thirty-one healthy female participants (18-35 years) were studied. Intramuscular electromyography was used to record individual motor unit potentials (MUPs) and near fibre MUPs from the vastus lateralis (VL) during 10% and 25% maximum isometric voluntary contractions (MVC), and spike-triggered averaging was used to obtain motor unit number estimates (MUNE) of the VL. Multilevel mixed-effects linear regression models were used to investigate the effects of sex at each contraction level.

ResultsMen exhibited greater muscle strength (p<0.001) and size (p<0.001) than women, with no difference in force steadiness at 10% or 25% MVC. Women had smaller MUs and higher FR at 10% MVC (both p<0.02), similar to that at 25% MVC in MU size (p=0.062) and FR (p=0.031). However, both sexes showed similar increases in MU size and FR when moving from low-to mid-level contractions. There were no sex differences in any near fibre MUP parameters or in MUNE.

ConclusionIn the vastus lateralis, women produce muscle force via different neuromuscular recruitment strategies to men which is characterised by smaller MUs discharging at higher rates. However, similar strategies are employed to increase force production from low to moderate contractions. These findings of similar proportional increases between sexes support the use of mixed sex cohorts in studies of this nature.

Key pointsO_LIIncreases in muscle force production are mediated by motor unit (MU) recruitment, and MU firing rate (FR).
C_LIO_LIWomen are underrepresented in studies of human neuromuscular research and markedly differ to men in a number of aspects of neuromuscular function, yet little is known of the recruitment strategies of each.
C_LIO_LIHere we demonstrate men and women have similar vastus lateralis MU number estimates, yet women recruit smaller MUs with higher FR than men at normalised contraction levels. However, increases in force are achieved via similar trajectories of MU recruitment and MU FR in men and women.
C_LIO_LIAlthough men and women exhibit divergent neuromuscular recruitment strategies to achieve normalised forces, increases in force are achived similarly and support the inclusion of mixed sex cohorts in studies of this nature.
C_LI

sentences: Gonadal sex patterns p21-induced cellular senescence in mouse and human glioblastoma Males exhibit higher incidence and worse prognosis for the majority of cancers, including glioblastoma (GBM). Disparate survival may be related to sex-biased responses to treatment, including radiation. Using a mouse model of GBM, we show that female cells are more sensitive to radiation, and that senescence represents a major component of the radiation therapeutic response in both sexes. Correlation analyses revealed that the CDK inhibitor p21 and irradiation induced senescence were differentially regulated between male and female cells. Indeed, female cellular senescence was more sensitive to changes in p21 levels, a finding that was observed in both wildtype and transformed murine astrocytes and patient-derived GBM cell lines. Using a novel Four Core Genotypes model of GBM, we further show that sex differences in p21-induced senescence are patterned by gonadal sex. These data suggest that sex differences in p21 induced senescence contribute to the female survival advantage in GBM.

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phenopype: a phenotyping pipeline for Python O_LIDigital images are an intuitive way to capture, store and analyze organismal phenotypes. Many biologists are taking images to collect high-dimensional phenotypic information from specimens, which are key to investigating complex ecological, evolutionary and developmental phenomena, such as relationships between trait diversity and ecosystem function, multivariate natural selection, or developmental plasticity. As a consequence, images are being collected at ever increasing rates, but extraction of the contained phenotypic information still poses a veritable analytical bottleneck.
C_LIO_LIphenopype is a high throughput phenotyping pipeline for the programming language Python that aims at alleviating this bottleneck. The package facilitates immediate extraction of high dimensional phenotypic data from laboratory grade digital images with low levels of background noise and complexity. At the core, phenopype provides functionality for rapid signal processing based image preprocessing and segmentation, followed by trait extraction, visualization and numerous options for data export. The functionality is provided by wrapping low-level Python computer vision libraries (e.g. OpenCV) into accessible functions, facilitating their incorporation into scientific image analysis workflows. In addition, phenopype provides a project management ecosystem to further simplify rapid data collection and to increase reproducibility.
C_LIO_LIphenopype offers two different workflows that support users during different stages of scientific image analysis. The low-throughput workflow uses regular Python syntax and has greater flexibility at the cost of reproducibility, which is suitable for prototyping during the initial stage of a research project. The high-throughput workflow allows users to specify and store image-specific settings for analysis in human-readable YAML format, and then execute all functions in one step by means of an interactive parser. This approach facilitates rapid program-user interactions during batch processing, and greatly increases scientific reproducibility.
C_LIO_LIOverall, phenopype intends to make the features of powerful but technically involved low-level CV libraries available to biologists with little or no Python coding experience. Therefore, phenopype is aiming to augment, rather than replace the utility of existing Python CV libraries, allowing biologists to focus on rapid and reproducible data collection. Furthermore, image annotations produced by phenopype can be used as training data, thus presenting a stepping stone towards the application of deep learning architectures.
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Remodeling of dermal adipose tissue alleviates cutaneous toxicity induced by anti-EGFR therapy Anti-epidermal growth factor receptor (EGFR) therapy-associated cutaneous toxicity is a syndrome characterized by papulopustular rash, local inflammation, folliculitis and microbial infection, resulting in a decrease in quality of life and dose interruption. However, no effective clinical intervention is available for this adverse effect. Here, we report the atrophy of dermal white adipose tissue (dWAT), a highly plastic adipose tissue with various skin-specific functions, correlates with rash occurrence and exacerbation in a murine model of EGFR inhibitor-induced rash. The reduction in dWAT is due to the inhibition of adipogenic differentiation by defects in peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) signaling, and increased lipolysis by the induced expression of the lipolytic cytokine IL6. The activation of PPAR{gamma} by rosiglitazone maintains adipogenic differentiation and represses the transcription of IL6, eventually improving skin functions and ameliorating the severity of rash without altering the antitumor effects. Thus, activation of PPAR{gamma} represents a promising approach to ameliorate cutaneous toxicity in patients with cancer who receive anti-EGFR therapy.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

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Base editing derived models of human WDR34 and WDR60 disease alleles replicate retrograde IFT and hedgehog signaling defects and suggest disturbed Golgi protein transport Cytoplasmic Dynein-2 or IFT-dynein is the only known retrograde motor for intraflagellar transport, enabling protein trafficking from the ciliary tip to the base. Dysfunction of WDR34 and WDR60, the two intermediate chains of this complex, causes Short Rib Thoracic Dystrophy (SRTD), human skeletal chondrodysplasias with high lethality. Complete loss of function of WDR34 or WDR60 is lethal in vertebrates and individuals with SRTD carry at least one putative hypomorphic missense allele. Gene knockout is therefore not suitable to study the effect of these human missense disease alleles.

Using CRISPR single base editors, we recreated three different patient missense alleles in cilia-APEX-IMCD3 cells. Consistent with previous findings in dynein-2 full loss of function models and patient fibroblasts, mutant cell lines showed hedgehog signaling defects as well as disturbed retrograde IFT. Transcriptomics analysis revealed differentially regulated expression of genes associated with various biological processes, including G-protein-coupled receptor signaling as well extracellular matrix composition, endochondral bone growth and chondrocyte development. Further, we also observed differential regulation of genes associated with Golgi intracellular transport, including downregulation of Rab6b, a GTPase involved in Golgi-ER retrograde protein trafficking and interacting with components of cytoplasmic dynein-1, in mutant ciliated and non-ciliated clones compared to controls. In addition to providing cellular model systems enabling investigations of the effect of human SRTD disease alleles, our findings indicate non-ciliary functions for WDR34 and WDR60 in addition to the established roles as components of the retrograde IFT motor complex in cilia.

sentences: Microbiota depletion promotes human rotavirus replication in an adult mouse model The study of human rotavirus (RV) infectivity in vivo has been limited by the lack of small animal models able to efficiently replicate the principal human RV genotypes. In recent years, intestinal microbiota-virus-host interaction has emerged as a key factor in mediating enteric virus pathogenicity. With the aim of developing an adult mouse infection model for RV we performed faecal microbiota transplant (FMT) with healthy infants as donors in antibiotic-treated mice. Contrarily to control mice, in the FMT group, but also in antibiotic-treated mice without FMT, challenge with the human RV G1P[8] genotype, Wa strain (RVwa), resulted in viral shedding in the faeces for 6 days. RV titres in faeces were also significantly higher in antibiotic-treated animals with or without FMT. This excluded the hypothesis that donors microbiota promoted infection. Antibiotic treatment followed by self-FMT resulted in incomplete re-establishment of mouse microbiota which partially restored suppression of RVwa infection. Microbial composition analysis revealed profound changes in the intestinal microbiota of antibiotic-treated animals, whereas some bacterial groups, including members of Lactobacillus, Bilophila, Mucispirillum and Oscillospira, reappeared after self-FMT. In antibiotic-treated and FMT animals, differences were observed in gene expression of immune mediators such as IL10, TNF- and IFN{gamma} and the fucosyltransferase FUT2, responsible for H-type antigen synthesis in the small intestine. Collectively, our results suggest that antibiotic-induced microbiota depletion eradicates the microbial taxa that restrict human RV infectivity in mice. Viral permissiveness could involve changes in the innate immune system at the small intestine and alterations in the bacteria population that potentially interact with RV, creating a favourable environment for human RV replication in mice.

sentences: Evolutionary strategies applied to artificial gene regulatory networks Evolution optimizes cellular behavior throughout sequential generations by selecting the successful individual cells in a given context. As gene regulatory networks (GRNs) determine the behavior of single cells by ruling the activation of different processes - such as cell differentiation and death - how GRNs change from one generation to the other might have a relevant impact on the course of evolution. It is not clear, however, which mechanisms that affect GRNs effectively favor evolution and how. Here, we use a population of computational robotic models controlled by artificial gene regulatory networks (AGRNs) to evaluate the impact of different genetic modification strategies in the course of evolution. The virtual agent senses the ambient and acts on it as a bacteria in different phototaxis-like tasks - orientation to light, phototaxis, and phototaxis with obstacles. We studied how the strategies of gradual and abrupt changes on the AGRNs impact evolution considering multiple levels of task complexity. The results indicated that a gradual increase in the complexity of the performed tasks is beneficial for the evolution of the model. Furthermore, we have seen that larger gene regulatory networks are needed for more complex tasks, with single-gene duplication being an excellent evolutionary strategy for growing these networks, as opposed to full-genome duplication. Studying how GRNs evolved in a biological environment allows us to improve the computational models produced and provide insights into aspects and events that influenced the development of life on earth.

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Abnormal molecular signatures of inflammation, energy metabolism and vesicle biology in human Huntington disease peripheral tissues BackgroundA major challenge in neurodegenerative diseases concerns identifying biological disease signatures that track with disease progression or respond to an intervention. Several clinical trials in Huntington disease (HD), an inherited, progressive neurodegenerative disease, are currently ongoing. Therefore, we examined whether peripheral tissues can serve as a source of readily accessible biological signatures at the RNA and protein level in HD patients.

ResultsWe generated large, high-quality human datasets from skeletal muscle, skin and adipose tissue to probe molecular changes in human premanifest and early manifest HD patients - those most likely involved in clinical trials. In-depth single nucleotide polymorphism data across the HTT gene will facilitate the use of the generated primary- and iPSC cell lines in allele-specific targeting approaches. The analysis of the transcriptomics and proteomics data shows robust, stage-dependent dysregulation. Gene ontology analysis confirmed the involvement of inflammation and energy metabolism in peripheral HD pathogenesis. Furthermore, we observed changes in the homeostasis of extracellular vesicles, where we found consistent changes of genes and proteins involved in this process.

ConclusionsOur  omics data document the involvement of inflammation, energy metabolism and extracellular vesicle homeostasis. This demonstrates the potential to identify biological signatures from peripheral tissues in HD suitable as biomarkers in clinical trials. Together with the primary cell lines established from peripheral tissues and a large panel of iPSC lines that can serve as human models of HD, the generated data are a valuable and unique resource to advance the current understanding of molecular mechanisms driving HD pathogenesis.

sentences: A re-emerging arbovirus in livestock in China: Early genomic surveillance and phylogeographic analysis Viruses in livestock represent a great risk to public health due to the close contact of their hosts with humans and the potential long-range transportation though animal trade. Here, we show how to predict outbreaks of potential zoonotic pathogens and use spatially-explicit phylogeographic and phylodynamic approaches to provide estimates of key epidemiological parameters. First, we use metagenomic next generation sequencing (mNGS) to identify a Getah virus (GETV) as the pathogen responsible for a re-emerging swine disease in China. The GETV isolate is able to replicate in a variety of cell lines including human cells and shows high pathogenicity in a mouse model suggesting a potential public health risk. We obtained 16 complete genomes and 78 E2 gene sequences from viral strains collected within China from 2017 to 2021 through large-scale surveillance among livestock, pets and mosquitoes. Moreover, phylogenetic analysis reveals that three major GETV lineages are responsible for the current epidemic in livestock in China. We identify three potential positively selected sites and mutations of interest in E2, which may impact the transmissibility and pathogenicity of the virus. We then reconstruct the evolutionary and dispersal history of the virus and test the impact of several environmental factors on the viral genetic diversity through time and on the dispersal dynamic of viral lineages. Of note, we identify temporal variation in livestock meat consumption as a main predictor of viral genetic diversity through time. Finally, phylogeographic analyses indicate that GETV preferentially circulates within areas associated with relatively higher mean annual temperature and pig population density. Our results highlight the importance of continuous surveillance of GETV among livestock in southern Chinese regions associated with relatively high temperatures, and the need to control mosquitoes in the livestock farms. Our analyses of GETV also provide a baseline for future studies of the molecular epidemiology and early warning of emerging arboviruses in China.

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Resilience in a Natural Model of Metabolic Dysfunction Through Changes in Longevity and Ageing-Related Metabolites Insights from extreme-adapted organisms, which have evolved natural strategies for promoting survivability under severe environmental pressures, may help guide future research into novel approaches for enhancing human longevity. The cave-adapted Mexican tetra,Astyanax mexicanus, has attracted interest as a model system for metabolic resilience, a term we use to denote the property of maintaining robust health and longevity under conditions that would have highly deleterious effects in other organisms (Fig 1). Cave-dwelling populations of Mexi-can tetra exhibit elevated blood glucose and possess a mutation in the insulin receptor that in humans has been linked to Rabson-Mendenhall syndrome, a condition characterized by severe insulin resistance that causes numerous developmental abnormalities, is highly associated with debilitating progression, and drastically reduces lifespan. In addition, cavefish develop large numbers of hypertrophic visceral adipocytes and possess vastly enriched stores of body fat compared to surface-dwelling counterparts. However, cavefish appear to avoid the progression of the respective pathologies typically associated with these conditions, such as accumulation of advanced glycation end products (AGEs), chronic tissue inflammation, impaired growth due to insulin dysregulation, and low survivability due to arterial disease. The metabolic strategies underlying the resilience properties of A. mexicanus cavefish, and how they relate to environmental challenges of the cave environment, are poorly understood. Here, we provide an untargeted metabolomics study of long- and short-term fasting in two A. mexicanus cave populations and one surface population. We find that, although cave-fish share many similarities with metabolic syndrome normally associated with the human state of obesity, important differences emerge, including a reduction in cholesteryl esters and intermediates of protein glycation, and an increase in antioxidants and metabolites associated with hypoxia and longevity. We find important overlaps between metabolic alterations in cave-dwelling Mexican tetra and other models of resilience and extreme longevity, such as naked mole-rats, including enhanced reliance on sugars as an energy source and a trend toward more potent antioxidant activity. This work suggests that certain metabolic features associated with human pathologies are not intrinsically harmful, but are rather consequences of suboptimal adaptation of humans to survival under adverse metabolic conditions, and suggests promising avenues for future investigation into the role of metabolic strategies in evolutionary adaptation and health. We provide a transparent pipeline for reproducing our analysis and a Shiny app for other researchers to explore and visualize our dataset.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/358077v3_fig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@bcaff5org.highwire.dtl.DTLVardef@1c40b89org.highwire.dtl.DTLVardef@1d17d69org.highwire.dtl.DTLVardef@80a483_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFig. 1.C_FLOATNO Metabolic resilience  survivability under a variety of extreme conditions. Certain populations of cavefish have adaptations that cause increased appetite (1) and increased fat accumulation (2) (in cases where nutrients are plentiful, such as in lab-raised populations). These same populations also exhibit robust health and longevity (2, 3) and do not suffer ill-effects due to high levels of visceral fat and hyper-glycemia, both of which are features of most cave populations. However, visceral fat accumulation in cave populations is highly dependent on nutrient availability and is not displayed in wild-caught specimens (4). Thus, cavefish paradoxically appear to tolerate both extremely low and extremely high levels of triglycerides, glucose, and other energy storage metabolites. We argue that these differences can be reconciled under a hypothesis whereby the cave environment selects not for resistance to nutrient deprivation per se, but rather resilience to a variety of nutrient availability states (such as seasonal floods). Survival under such challenging conditions ostensibly favors the ability to tolerate extreme metabolic states, including not only starvation but also high levels of potentially deleterious metabolites such as triglycerides and reactive oxygen species (ROS). We find evidence for elevated antioxidant levels and altered cholesterol / cholesteryl ester homeostasis in cavefish, suggesting that cavefish may use these mechanisms to offset potentially harmful metabolites and tolerate a broad range of metabolic conditions.

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sentences: Short-interval reburns in the boreal forest alter soil bacterial communities, reflecting increased pH and poor conifer seedling establishment Increasing burn rates (percentage area burned annually) in some biomes are leading to fires burning in close succession, triggering rapid vegetation change as well as altering soil properties. Despite the importance of soil microbes for nutrient cycling and as plant symbionts, the effects of increased fire frequency on belowground microbial communities remain largely unknown. We present a study of the effects of short interval reburns (defined here as <20 years between fires) on soil bacterial communities in the boreal forest of northwestern Canada, using a paired site design that spans wetlands and uplands, with 50 sites total. We asked whether short interval reburns significantly alter soil bacterial community composition and richness, and which bacterial taxa are associated with greater or lower fire frequency. We found that, while short interval reburns had no significant effect on bacterial richness, there were significant changes in overall community composition. We did not find correlations between understory vegetation community dissimilarities and bacterial community dissimilarities, suggesting the primary drivers of changes induced by short interval reburns may differ between plants and microbes. We identified an abundant Blastococcus sp. that was consistently enriched in short interval reburns, in both wetlands and uplands, indicating its role as a strongly "pyrophilous" bacterium. We also identified an abundant Callaberonia sordidicola taxon as being consistently depleted in short interval reburns. This endophytic diazotrophic organism is a robust colonizer of pine and spruce seedlings and has the ability to increase seedling growth, due in part to large contributions of fixed nitrogen. Its depletion in short-interval reburn sites raises questions about whether this is contributing to - or merely reflects - poor conifer seedling recolonization post-fire at short-interval reburns.

sentences: Introducing CARATE: Finally speaking chemistry. AO_SCPLOWBSTRACTC_SCPLOWComputer-Aided Drug Design is advancing to a new era. Recent developments in statistical modelling, including Deep Learning, Machine Learning and high throughput simulations, enable workflows and deductions not achievable 20 years ago. The key interaction for many small molecules is via bio-molecules. The interaction between a small molecule and a biological system therefore manifests itself at multiple time and length scales. While the human chemist quite intuitively grasps the concept of multiple scales, most of the computer technologies do not relate multiple scales easily. Therefore, numerous methods in the realm of computational sciences have been developed. However, up to now it was not clear that the problem of multiple scales is not only a mere matter of computational abilities but even more a matter of accurate representation. Because of the amount of already performed simulations at various scales, deep learning (DL) becomes a viable approach to speed up computations by approximating simulations in a data-driven way. However, to accurately approximate the physical (simulated) properties of a given compound, an accurate, uniform representation is mandatory. Therefore, the biochemical and pharmaceutical encoder (CARATE) is introduced. Furthermore, the regression and classification abilities of CARATE are evaluated against benchmarking datasets (ZINC, ALCHEMY, MCF-7, MOLT-4, YEAST, Enzymes, Proteins) and compared to other baseline approaches.

sentences: Internet of Things Architecture for Cellular Biology The Internet of Things (IoT) provides a simple framework to easily control online devices. IoT is now a commonplace tool used by technology companies, but it is rarely used in biology experiments. IoT can benefit research through alarm notifications, automation, and the real-time monitoring of experiments. We developed and implemented an IoT architecture to control biological devices used in experiments. We developed our own electrophysiology, microscopy, and microfluidic devices so that may be controlled through a unified IoT architecture. The system allows each device to be monitored and controlled through an online web tool. We present our IoT architecture so other labs may replicate it for their own experiments.

sentences: Completion of neural crest cell production and emigration is regulated by retinoic acid-dependent inhibition of BMP signaling Production and emigration of neural crest cells is a transient process followed by the emergence of the definitive roof plate. The mechanisms regulating the end of neural crest ontogeny are poorly understood. Whereas early crest development is stimulated by mesoderm-derived retinoic acid, we report that the end of the neural crest period is regulated by retinoic acid synthesized in the dorsal neural tube. Inhibition of retinoic acid signaling in the neural tube prevents the normal upregulation of BMP inhibitors in the nascent roof plate and prolongs the period of BMP responsiveness which otherwise ceases close to roof plate establishment. Consequently, neural crest production and emigration are extended well into the roof plate stage. In turn, extending the activity of neural crest-specific genes inhibits the onset of retinoic acid synthesis in roof plate suggesting a mutual repressive interaction between neural crest and roof plate traits. Although several roof plate-specific genes are normally expressed in the absence of retinoic acid signaling, roof plate and crest markers are co-expressed in single cells and this domain also contains dorsal interneurons. Hence, the cellular and molecular architecture of the roof plate is compromised. Collectively, our results demonstrate that neural tube-derived retinoic acid, via inhibition of BMP signaling, is an essential factor responsible for the end of neural crest generation and the proper segregation of dorsal neural lineages.

sentences: Maturation of distinct neural components of the cognitive control network support early development of inhibitory control Crucial for goal-directed behavior is the capacity to suppress impulses and predominant behavioral responses, called inhibitory control (IC). This ability emerges in early childhood, and a distinction according to neutral ( cold) and emotional ( hot) contexts has been suggested. Here, we ask which maturational changes in the childs brain underlie the emergence of this critical ability. We relate behavioral changes in 3-and 4-year-olds  hot and  cold IC to brain maturation, using a multimodal approach that combines cortical and subcortical grey matter structure with white matter connectivity. Our results show that the maturation of distinct parts of the cognitive control brain network support early development of the different IC domains: Whereas  cold IC is related to frontoparietal regions and the left thalamus,  hot IC is associated with the left supramarginal gyrus and right thalamus. This dissociation of brain networks involved in  cold and  hot IC is confirmed by independent patterns of thalamocortical connectivity, supporting that IC in neutral and emotional settings relies on independent processes.

sentences: Activity in perirhinal and entorhinal cortex predicts perceived visual similarities among category exemplars with highest precision Vision neuroscience has made great strides in understanding the hierarchical organization of object representations along the ventral visual stream (VVS). How VVS representations capture fine-grained visual similarities between objects that observers subjectively perceive has received limited examination so far. In the current study, we addressed this question by focusing on perceived visual similarities among subordinate exemplars of real world-categories. We hypothesized that these perceived similarities are reflected with highest fidelity in neural activity patterns downstream from inferotemporal regions, namely in perirhinal and anterolateral entorhinal cortex in the medial temporal-lobe. To address this issue with fMRI, we administered a modified 1-Back task that required discrimination between category exemplars as well as categorization. Further, we obtained observer-specific ratings of perceived visual similarities, which predicted behavioural performance during scanning. As anticipated, we found that activity patterns in perirhinal and anterolateral entorhinal cortex predicted the structure of perceived visual similarity relationships among category exemplars, including its observer-specific component, with higher precision than any other VVS region. Our findings provide new evidence that subjective aspects of object perception that rely on fine-grained visual differentiation are reflected with highest fidelity in the medial temporal lobe.

sentences: In situ structures of membrane-assisted assembly and selective autophagy of enteroviruses Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. It is unknown how virions assemble around these newly synthesized genomes and how they are then loaded into autophagic membranes for release through secretory autophagy. Here, we use cryo-electron tomography of infected cells to show that poliovirus assembles directly on replication membranes. Pharmacological untethering of capsids from membranes abrogates RNA encapsidation. Our data directly visualize a membrane-bound half-capsid as a prominent virion assembly intermediate. Assembly progression past this intermediate depends on the class III phosphatidylinositol 3-kinase VPS34, a key host-cell autophagy factor. On the other hand, the canonical autophagy initiator ULK1 is shown to restrict virion production since its inhibition leads to increased accumulation of virions in vast intracellular arrays, followed by an increased vesicular release at later time points. Finally, we identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from other types of autophagosome contents. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle.

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Loss of intestinal ChREBP impairs absorption of dietary sugars and prevents glycemic excursion curves Increased sugar consumption is a risk factor for features of the metabolic syndrome including obesity, hypertriglyceridemia, insulin resistance, diabetes, and nonalcoholic fatty liver disease. The gut epithelium, which plays a central role in dietary sugar digestion, absorption and metabolism has emerged a key actor of metabolic disorders. While the transcription factor ChREBP (Carbohydrate response element binding protein) has been established as a key player of the adaptive reprograming of cellular metabolism in various tissues upon glucose or fructose challenge, its specific contribution to the regulation of blood glucose upon dietary sugar intake was not previously addressed.

We demonstrate here that ChREBP is abundantly expressed in the proximal gut epithelium, where carbohydrate digestion and absorption primarily occur and in particular L cells, which produce the glucoincretin GLP-1. The inducible deletion of ChREBP specifically in the mouse gut epithelium (Ch{Delta}GUT mice) resulted in the reduction of early glycemic excursion upon oral glucose load. Surprisingly, despite being associated with reduced GLP-1 production, loss of gut ChREBP activity significantly dampened glucose transepithelial flux, and thereby delayed glucose distribution to peripheral tissues. Among the underlying mechanisms, we unveil that Ch{Delta}GUT mice show an impaired expression of key intestinal hexose (glucose, galactose, fructose) transporters and metabolic enzymes as well as brush border dissacharidases. In agreement, intestinal ChREBP deficiency was accompanied by a precocious intolerance to both high-lactose and high-sucrose diets concomitant with mild galactose and severe fructose malabsorption syndromes.

Altogether, our study demonstrates that, by transcriptionally orchestrating local digestion and absorption of dietary sugars, ChREBP activity in the mouse gut epithelium controls glucose appearance rate into systemic circulation and prevents against intolerance to mono- and disaccharides.

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Fine-scale spatial patterns of wildlife disease are common and understudied O_LIAll parasites are heterogeneous in space, yet little is known about the prevalence and scale of this spatial variation, particularly in wild animal systems. To address this question, we sought to identify and examine spatial dependence of wildlife disease across a wide range of systems.
C_LIO_LIConducting a broad literature search, we collated 31 such datasets featuring 89 replicates and 71 unique host-parasite combinations, only 51% of which had previously been used to test spatial hypotheses. We analysed these datasets for spatial dependence within a standardised modelling framework using Bayesian linear models, and we then meta-analysed the results to identify generalised determinants of the scale and magnitude of spatial autocorrelation.
C_LIO_LIWe detected spatial autocorrelation in 48/89 model replicates (54%) across 21/31 datasets (68%), spread across parasites of all groups. Even some very small study areas (under 0.01km2) exhibited substantial spatial variation.
C_LIO_LIDespite the common manifestation of spatial variation, our meta-analysis was unable to identify host-, parasite-, or sampling-level determinants of this heterogeneity across systems. Parasites of all transmission modes had easily detectable spatial patterns, implying that structured contact networks and susceptibility effects are potentially as important in spatially structuring disease as are environmental drivers of transmission efficiency.
C_LIO_LIOur findings demonstrate that fine-scale spatial patterns of infection manifest frequently and across a range of wild animal systems, and many studies are able to investigate them whether or not the original aim of the study was to examine spatially varying processes. Given the widespread nature of these findings, studies should more frequently record and analyse spatial data, facilitating development and testing of spatial hypotheses in disease ecology. Ultimately, this may pave the way for an a priori predictive framework for spatial variation in novel host-parasite systems.
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sentences: Microtubule-mitochondrial attachment determines cell division symmetry and polarity in fission yeast Association with microtubules inhibits the fission of mitochondria in Schizosachharomyces pombe. Here we show that this attachment of mitochondria to microtubules is an important cell intrinsic factor in determining division symmetry as well as maintaining polarity. By comparing mutant cells that exhibited enhanced attachment and no attachment of mitochondria to microtubules (Dnm1{Delta} and Mmb1{Delta} respectively), we show that microtubules in these mutants displayed aberrant dynamics compared to wild-type cells, which resulted in errors in nuclear positioning. This translated to cell division asymmetry in a significant proportion of both Dnm1{Delta} and Mmb1{Delta} cells. So too, microtubule pivoting was enhanced in both mitochondrial mutants, resulting in a fraction of the cells in these populations displaying polarity defects. The asymmetric division in Dnm1{Delta} and Mmb1{Delta} cells resulted in unequal distribution of mitochondria, with the daughter cell that received more mitochondria growing faster than the other daughter. Taken together, we show the existence of homeostatic feedback controls between mitochondria and microtubules in fission yeast, which directly influence mitochondrial partitioning and thereby, cell growth.

sentences: The tissue differentiation and cancer manifolds in gene and protein expression spaces It is well known that, for a particular tissue, the homeostatic and cancer attractors are well apart both in gene expression and in protein expression spaces. By using data for 15 tissues and the corresponding tumors from The Cancer Genome Atlas, and for 49 normal tissues and 20 tumors from The Human Protein Atlas, we show that the set of normal attractors are also well separated from the set of tumors. Roughly speaking, one may say that there is a cancer progression axis orthogonal to the normal tissue differentiation and cancer manifolds. This separation suggests that therapies targeting common genes, which define the cancer axis, may be effective, irrespective of the tissue of origin.

sentences: Painters in chromatin: a unified quantitative framework to systematically characterize epigenome regulation and memory In eukaryotes, many stable and heritable phenotypes arise from the same DNA sequence, owing to epigenetic regulatory mechanisms relying on the molecular cooperativity of "reader-writer" enzymes. In this work, we focus on the fundamental, generic mechanisms behind the epigenome memory encoded by post-translational modifications of histone tails. Based on experimental knowledge, we introduce a unified modeling framework, the painter model, describing the mechanistic interplay between sequence-specific recruitment of chromatin regulators, chromatin-state-specific reader-writer processes and long-range spreading mechanisms. A systematic analysis of the model building blocks highlights the crucial impact of tridimensional chromatin organization and state-specific recruitment of enzymes on the stability of epigenomic domains and on gene expression. In particular, we show that enhanced 3D compaction of the genome and enzyme limitation facilitate the formation of ultra-stable, confined chromatin domains. The model also captures how chromatin state dynamics impact the intrinsic transcriptional properties of the region, slower kinetics leading to noisier expression. We finally apply our framework to analyze experimental data, from the propagation of{gamma} H2AX around DNA breaks in human cells to the maintenance of heterochromatin in fission yeast, illustrating how the painter model can be used to extract quantitative information on epigenomic molecular processes.

sentences: Regulation of membrane scission in yeast endocytosis During clathrin-mediated endocytosis, a flat plasma membrane is shaped into an invagination that undergoes scission to form a vesicle. In mammalian cells, the force that drives the transition from invagination to vesicle is primarily provided by the GTPase dynamin that acts in concert with crescent-shaped BAR domain proteins. In yeast cells, the mechanism of endocytic scission is unclear. The yeast BAR domain protein complex Rvs161/167 (Rvs) nevertheless plays an important role in this process: deletion of Rvs dramatically reduces scission efficiency. A mechanistic understanding of the influence of Rvs on scission however, remains incomplete. We used quantitative live-cell imaging and genetic manipulation to understand the recruitment and function of Rvs and other late-stage proteins at yeast endocytic sites. We found that arrival of Rvs at endocytic sites is timed by interaction of its BAR domain with specific membrane curvature. A second domain of Rvs167- the SH3 domain-affects localization efficiency of Rvs. We show that Myo3, one of the two type-I myosins in Saccharomyces cerevisiae, has a role in recruiting Rvs167 via the SH3 domain. Removal of the SH3 domain also affects assembly and disassembly of actin and impedes membrane invagination. Our results indicate that both BAR and SH3 domains are important for the role of Rvs as a regulator of scission. We tested other proteins implicated in vesicle formation in Saccharomyces cerevisiae, and found that neither synaptojanins nor dynamin contribute directly to membrane scission. We propose that recruitment of Rvs BAR domains delays scission and allows invaginations to grow by stabilizing them. We also propose that vesicle formation is dependent on the force exerted by the actin network component of the endocytic machinery.

sentences: A comparison of feature selection methodologies and learning algorithms in the development of a DNA methylation-based telomere length estimator The field of epigenomics holds great promise in understanding and treating disease with advances in machine learning (ML) and artificial intelligence being vitally important in this pursuit. Increasingly, research now utilises DNA methylation measures at cytosine-guanine dinucleotides (CpG) to detect disease and estimate biological traits such as aging. Given the high dimensionality of DNA methylation data, feature-selection techniques are commonly employed to reduce dimensionality and identify the most important subset of features. In this study, we test and compare a range of feature-selection methods and ML algorithms in the development of a novel DNA methylation-based telomere length (TL) estimator. We found that principal component analysis in advance of elastic net regression led to the overall best performing estimator when evaluated using a nested cross-validation analysis and two independent test cohorts. In contrast, the baseline model of elastic net regression with no prior feature reduction stage performed worst - suggesting a prior feature-selection stage may have important utility. The variance in performance across tested approaches shows that estimators are sensitive to data set heterogeneity and the development of an optimal DNA methylation-based estimator should benefit from the robust methodological approach used in this study. Additionally, we observed that different DNA methylation-based TL estimators, which have few common CpGs, are associated with many of the same biological entities. Moreover, our methodology which utilises a range of feature-selection approaches and ML algorithms could be applied to other biological markers and disease phenotypes, to examine their relationship with DNA methylation and predictive value.

sentences: Single cell analysis of population-wide nuclear and cytosolic drug responses using high-content FRET imaging: measuring protein kinase activation in rat primary striatal neurons Genetically-encoded biosensors are used to track biochemical activities in living cells by measuring changes in fluorescence emitted by one or more fluorescent proteins. In the present article, we describe the application of genetically-encoded FRET biosensors with high content microscopy to image the signaling responses of thousands of neurons in response to drug treatments. We applied this approach to reveal intercellular variation in signaling responses among cultured striatal neurons stimulated with multiple drugs. The striatum is largely composed of medium-spiny GABAergic neurons which are divided into two broad sub-types based in part on their expression of dopamine D1 vs. D2 receptors. Using high content FRET imaging and immunofluorescence, we identified neuronal sub-populations with unique responses to pharmacological manipulation. Focusing on dopamine- and glutamate-regulated PKA and ERK1/2 signaling in both the cytoplasm and nucleus, we identified pronounced intercellular differences, in both the magnitude and kinetics of signaling responses to drug application. Importantly, we found that a conventional "bulk" analysis that included all cells in culture yielded a different rank order of drug potency than that revealed by our single-cell analysis. The high degree of heterogeneity that we observed at the single cell level would not have been detectable using common population-level analyses, derived for example from western blotting or plate reader-based measurements. In conclusion, our single-cell analytical approach highlights the limitations of population-level analyses, and provides a novel way to study signaling biology.

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Intraspecific diversity based on low temperature induced arrhythmia in embryonic heart of medaka (Oryzias latipes) BackgroundCold tolerance during embryonic development, especially blood circulation is important for growth in poikilothermic animals. Medaka (Oryzias latipes), has cold tolerance and is distributed in the highest latitudes occupied by the genus Oryzias. Regarding cold tolerance in embryogenesis, Hd-rR strain belonging to the southern Japanese (S.JPN) group showed arrhythmia when the embryo was exposed to 15 {degrees}C in the heartbeat initiation period (st. 24), whereas the embryo of Odate medaka, which belongs to the northern Japanese (N.JPN) group, showed stable heartbeats.

ResultsIn this study, to verify the sensitivity to low temperature in the medaka wild populations, heartbeat intervals of st. 24 embryos derived from three N.JPN, eight S.JPN, and two western Korean/Chinese (W.KOR) populations were investigated at an arbitrary temperature range of 12-15 {degrees}C. There was no significant difference in the mean values of the heart rates and the coefficient of variation (CV) of interbeat intervals in the N.JPN, S.JPN, and W.KOR groups. A temperature dependency of the CV within 12-15 {degrees}C was observed only in five S.JPN specimens, whereas three S.JPN, the N.JPN and the W.KOR specimens showed no alteration in CV with temperature. Temperature dependency of the heart rate was also varied in the S.JPN specimens.

ConclusionsThese results suggest that the temperature dependency of the CV under 12-15 {degrees}C during the heartbeat initiation period is a variation within the S.JPN after the divergence from the N.JPN.

sentences: NOMIS: Quantifying morphometric deviation from normality over the lifetime in the adult human brain We present NOMIS (https://github.com/medicslab/NOMIS), a comprehensive open MRI tool to assess morphometric deviation from normality in the adult human brain. Based on MR anatomical images from 6,909 cognitively healthy individuals aged 18-100 years, we modeled 1,344 measures computed using the open access FreeSurfer pipeline, considering account personal characteristics (age, sex, intracranial volume) and image quality (resolution, contrast-to-noise ratio and surface reconstruction defect holes), and providing expected values for any new individual. Then, for each measure, the NOMIS tool was built to generate Z-score effect sizes denoting the extent of deviation from the normative sample. Depending on the user need, NOMIS offers four versions of Z-score adjusted on different sets of variables. While all versions consider head size and image quality, they can also incorporate age and/or sex, thereby facilitating multi-site neuromorphometric research across adulthood.

sentences: Input-Output Slope Curve Estimation in Neural Stimulation Based on Optimal Sampling Principles This paper discusses some of the practical limitations and issues, which exist for the input-output (IO) slope curve estimation (SCE) in neural, brain and spinal, stimulation techniques. The drawbacks of the SCE techniques by using existing uniform sampling and Fisher-information-based optimal IO curve estimation (FO-IOCE) methods are elaborated. A novel IO SCE technique is proposed with a modified sampling strategy and stopping rule which improve the SCE performance compared to these methods. The effectiveness of the proposed IO SCE is tested on 1000 simulation runs in transcranial magnetic stimulation (TMS), with a realistic model of motor evoked potentials (MEPs). The results show that the proposed IO SCE method successfully satisfies the stopping rule, before reaching the maximum number of TMS pulses in 79.5% of runs, while the estimation based on the uniform sampling technique never converges and satisfies the stopping rule. At the time of successful termination, the proposed IO SCE method decreases the 95th percentile (mean value in the parentheses) of the absolute relative estimation errors (AREs) of the slope curve parameters up to 7.45% (2.2%), with only 18 additional pulses on average compared to that of the FO-IOCE technique. It also decreases the 95th percentile (mean value in the parentheses) of the AREs of the IO slope curve parameters up to 59.33% (16.71%), compared to that of the uniform sampling method. The proposed IO SCE also identifies the peak slope with higher accuracy, with the 95th percentile (mean value in the parentheses) of AREs reduced by up to 9.96% (2.01%) compared to that of the FO-IOCE method, and by up to 46.29% (13.13%) compared to that of the uniform sampling method.

sentences: Multiple classes of bactericidal antibiotics cause DNA double strand breaks in Staphylococcus aureus Antibiotics inhibit essential bacterial processes, resulting in arrest of growth and in some cases cell death. Many antibiotics are also reported to trigger endogenous production of reactive oxygen species (ROS), which damage DNA and other macromolecules. However, the type of DNA damage that arises and the mechanisms used by bacteria to repair it are largely unclear. We found that several different classes of antibiotic triggered dose-dependent DNA damage in Staphylococcus aureus, including some bacteriostatic drugs. Damage was heterogenous and varied in magnitude between strains. However, antibiotic-triggered DNA damage led to double strand breaks, the processing of which by the RexAB helicase/nuclease complex triggered the SOS response and reduced staphylococcal susceptibility to most of the antibacterials tested. In most cases, DNA DSBs occurred under aerobic but not anaerobic conditions, suggesting a role for ROS. We conclude that DNA double strand breaks are a common occurrence during bacterial exposure to several different antibiotic classes and that repair of this damage by the RexAB complex promotes bacterial survival.

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Distinct within-host bacterial populations ensure function, colonization and transmission in leaf symbiosis Hereditary symbioses have the potential to drive transgenerational effects, yet the mechanisms responsible for transmission of heritable plant symbionts are still poorly understood. The leaf symbiosis between Dioscorea sansibarensis and the bacterium Orrella dioscoreae offers an appealing model system to study how heritable bacteria are transmitted to the next generation. Here, we demonstrate that inoculation of apical buds with a bacterial suspension is sufficient to colonize newly-formed leaves and propagules, and to ensure transmission to the next plant generation. Flagellar motility is not required for movement inside the plant, but is important for the colonization of new hosts. Further, stringent tissue-specific regulation of putative symbiotic functions highlight the presence of two distinct subpopulations of bacteria in the leaf gland and at the shoot meristem. We propose that bacteria in the leaf gland dedicate resources to symbiotic functions, while dividing bacteria in the shoot tip ensure successful colonization of meristematic tissue, glands and propagules. Compartmentalization of intra-host populations, together with tissue-specific regulation may serve as a robust mechanism for the maintenance of mutualism in leaf symbiosis.

ImportanceSeveral plant species form associations with bacteria in their leaves, called leaf symbiosis. These associations are highly specific, but the mechanisms responsible for symbiont transmission are poorly understood. Using the association between the yam species Dioscorea sansibarensis and Orrella dioscoreae as a model leaf symbiosis, we provide experimental evidence that bacteria are transmitted vertically and distributed to specific leaf structures via association with shoot meristems. Flagellar motility is required for initial infection, but does not contribute to spread within host tissue. We also provide evidence that bacterial subpopulations at the meristem or in the symbiotic leaf gland differentially express key symbiotic genes. We argue that this separation of functional symbiont populations, coupled to tight control over bacterial infection and transmission, explain the evolutionary robustness of leaf symbiosis. These findings may provide insights into how plants may recruit and maintain beneficial symbionts at the leaf surface.

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The visual cortex produces a signal in the gamma band in response to broadband visual flicker The aim of this study is to uncover the network dynamics of the human visual cortex by driving it with a broadband random visual flicker. We here applied a broadband flicker (1-720 Hz) while measuring the MEG and then estimated the temporal response function (TRF) between the visual input and the MEG response. This TRF revealed an early response in the 40-60 Hz gamma range as well as in the 8-12 Hz alpha band. While the gamma band response is novel, the latter has been termed the alpha band perceptual echo. The gamma echo preceded the alpha perceptual echo. The dominant frequency of the gamma echo was subject-specific thereby reflecting the individual dynamical properties of the early visual cortex. To understand the neuronal mechanisms generating the gamma echo, we implemented a pyramidal-interneuron gamma (PING) model that produces gamma oscillations in the presence of constant input currents. Applying a broadband input current mimicking the visual stimulation allowed us to estimate TRF between the input current and the population response (akin to the local field potentials). The TRF revealed a gamma echo that was similar to the one we observed in the MEG data. Our results suggest that the visual gamma echo can be explained by the dynamics of the PING model even in the absence of sustained gamma oscillations.

Author SummaryThe properties of the neuronal dynamics governing the visual system are highly debated. While some emphasize the neuronal firing rate and evoked activity in response to visual stimuli, others emphasize the oscillatory neuronal dynamics. To investigate the dynamical properties of the visual system, we recorded the magnetoencephalography while stimulating the visual system using a broadband (1-720 Hz) visual flicker. By estimating the temporal response function (similar to cross-correlation) between the visual input and neuronal activity, we demonstrated a clear response in the gamma band that we term the gamma echo. We then constructed a physiologically realistic network model that could generate gamma-band oscillations by a pyramidal-interneuron gamma (PING) mechanism. This model allowed us to account for empirically observed response in the gamma band, and to provide novel insight on the neuronal dynamics governing the early visual system. The stage is now set for further investigating how the gamma echo is modulated by tasks such as spatial attention as well as uncovering how the echo might propagate in the visual hierarchy.

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Region-specific effects of early-life status epilepticus on the adult hippocampal CA3 - medial entorhinal cortex circuitry in vitro: focus on interictal spikes and concurrent high-frequency oscillations. Convulsive status epilepticus (SE) in immature life is often associated with lasting neurobiological changes. We provoked SE by pentylenetetrazole in postnatal day 20 rat pups and examined communication modalities between the temporal hippocampus and medial entorhinal cortex (mEC) in vitro. After a minimum of 40 days post-SE, we prepared combined temporal hippocampal - medial entorhinal cortex (mEC) slices from conditioned (SE) and naive (N) adult rats and recorded 4-aminopyridine-induced spontaneous epileptiform interictal-like discharges (IED) simultaneously from CA3 and mEC layer V-VI. We analyzed IED frequency and high frequency oscillations (HFOs) in intact slices and after surgical separation of hippocampus from mEC, by two successive incisions (Schaffer collateral cut, Parasubiculum cut). In all slices, IED frequency was higher in CA3 vs mEC and Raster plots indicated no temporal coincidence between them either in intact or in CA1-cut slices. IED frequency was significantly higher in SE mEC, but similar in SE and N CA3, independently of connectivity state. Ripples (R) and Fast Ripples (FR) coincided with IEDs and their power differed between SE and N intact slices, both in CA3 and mEC. CA3 FR/R ratios were higher in the absence of mEC. Moreover, SE (vs N) slices showed significantly higher FR/R ratios independently of the presence of mEC. Taken together, these findings suggest lasting effects of immature SE in network dynamics governing hippocampal-entorhinal communication which may impact adult cognitive, behavioral and/or seizure threshold sequalae.

HIGHLIGHTSO_LIEarly-life Status Epilepticus (SE) impacts on the adult hippocampal - entorhinal communication in the in vitro 4-AP model
C_LIO_LIPost-SE CA3 output decreases in HFO power with no change in interictal discharge frequency
C_LIO_LIPost-SE mEC output increases both in HFO power and interictal discharge frequency
C_LIO_LIInterictal HFO dynamics in CA3-mEC change upon the connectivity state of the two areas and priorhistory of early-life SE
C_LI

sentences: A novel positive feedback mechanism of ABI5 phosphorylation by mitogen activated protein kinase-3 regulates ABA signaling in Arabidopsis Seed germination is the crucial physiological process regulated by both environmental and endogenous phytohormones. ABA negatively regulates seed germination, post-germination growth and floral transition, however, the cross talk between multiple regulatory pathways are still unclear. Here, we show that ABA activates two MAP kinases, AtMPK3/AtMPK6 and selectively regulates the transcript of AtMPK3 through ABI5, a master regulator of ABA signaling. As a feedback loop, AtMPK3 interacts and phosphorylates ABI5 at the serine-314 position. ABI5 phosphorylation by MAP kinases positively regulates ABI5 nuclear localization and negatively regulates its dimerization. Subcellular localization of ABI5 phospho-null protein further suggests the role of phosphorylation in regulation of its cytoplasmic stability and its nuclear dimerization. Overexpression of phosphor-null ABI5 in abi5-8 mutant restored the ABA sensitivity during seed germination and delayed the floral transition as compared to phospho-mimic ABI5. Additionally, overexpression of constitutive phosphorylated ABI5 in abi5-8 mutants suggest that phosphorylation makes ABI5 partially inactive. Furthermore, phospho-null ABI5 plants showed drought sensitive phenotype whereas, mpk3, mkk4, mkk5, abi5-8 and phosphor-mimic plants showed drought tolerant phenotype. Our findings present a new insight between MAP kinase cascade and ABA signaling which collectively regulates the ABA response through ABI5 phosphorylation.

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Semaphorin 3C exacerbates liver fibrosis Background & AimsChronic liver disease is a growing epidemic leading to fibrosis and cirrhosis. TGF-{beta} is the pivotal pro-fibrogenic cytokine which activates hepatic stellate cells (HSC), yet, other molecules can substantially modulate TGF-{beta} signaling in the course of liver fibrosis. Expression of the axon guidance molecules Semaphorins (SEMAs), which signal through Plexins and Neuropilins (NRPs), have been associated with liver fibrosis in HBV-induced chronic hepatitis. This study aims at determining their function in the regulation of HSCs.

Approach & ResultsWe analyzed publicly available patient databases and liver biopsies. We employed transgenic mice where genes are deleted only in activated HSCs to perform ex vivo analysis and animal models. SEMA3C is the most enriched member of the Semaphorin family in liver samples from cirrhotic patients. Higher expression of SEMA3C in patients with NASH, alcoholic hepatitis or HBV-induced hepatitis discriminates those with a more pro-fibrotic transcriptomic profile. SEMA3C expression is also elevated in different mouse models of liver fibrosis and in isolated HSCs upon activation. In keeping with this, deletion of SEMA3C in activated HSCs reduces myofibroblast marker expression. Conversely, SEMA3C overexpression exacerbates TGF-{beta}-mediated myofibroblast activation, as shown by increased SMAD2 phosphorylation and target gene expression. Among SEMA3C receptors, only NRP2 expression is maintained upon activation of isolated HSCs. Interestingly, lack of NRP2 in those cells reduces myofibroblast marker expression. Finally, deletion of either SEMA3C or NRP2, specifically in activated HSCs, reduces liver fibrosis in mice.

ConclusionSEMA3C is a novel marker for activated HSCs that plays a fundamental role in the acquisition of the myofibroblastic phenotype and liver fibrosis.

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A Large-Scale Brain Network Mechanism for Increased Seizure Propensity in Alzheimer's Disease People with Alzheimers disease (AD) are 6-10 times more likely to develop seizures than the healthy aging population. Leading hypotheses largely consider increased excitability of local cortical tissue as primarily responsible for increased seizure prevalence in AD. However, both local dynamics and large-scale brain network structure are believed to be crucial for determining seizure likelihood and phenotype. In this study, we combine computational modelling with electrophysiological data to demonstrate a potential large-scale brain network mechanism for increased seizure propensity in people with AD. EEG was recorded from 21 people with probable AD and 26 healthy controls. At the time of EEG acquisition, all participants were free from seizures. Whole brain functional connectivity derived from source-reconstructed EEG recordings was used to build subject-specific brain network models of seizure transitions using an approach previously validated on participants with epilepsy vs controls. As cortical tissue excitability was increased in the simulations, network models of AD simulations were more likely to transition into seizures than simulations from healthy controls. Our results suggest an increased group-level probability of developing seizures at a future time for AD participants. We subsequently used the model to assess seizure propensity of different regions across the cortex. We found the most important regions for seizure generation were those typically burdened by amyloid-beta at the early stages of AD, as previously reported by in-vivo and post-mortem staging of amyloid plaques. These included cingulate, medial temporal, and orbital regions. Analysis of these spatial distributions also give potential insight into mechanisms of increased susceptibility to generalized (as opposed to focal) seizures in AD vs controls. This research suggests avenues for future studies testing patients with seizures, e.g. co-morbid AD/epilepsy patients, and comparisons with PET and MRI scans to relate regional seizure propensity with amyloid/tau pathology and cortical atrophy.

Author summaryPeople with Alzheimers disease (AD) are more likely to develop seizures than cognitively healthy people. In this study, we aimed to understand whether whole-brain network structure is related to this increased seizure likelihood. We used electroencephalography (EEG) to estimate brain networks from people with AD and healthy controls. We subsequently inserted these networks into a model brain and simulated disease progression by increasing the excitability of brain tissue. We found the simulated AD brains were more likely to develop seizures than the simulated control brains. No participants had seizures when we collected data, so our results suggest an increased probability of developing seizures at a future time for AD participants. Therefore functional brain network structure may play a role in increased seizure likelihood in AD. We also used the model to examine which brain regions were most important for generating seizures, and found that the seizure-generating regions corresponded to those typically affected in early AD. Our results also provide a potential explanation for why people with AD are more likely to have generalized seizures (i.e. seizures involving the whole brain, as opposed to  focal seizures which only involve certain areas) than the general population with epilepsy.

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The role of competition versus cooperation in microbial community coalescence New microbial communities often arise through the mixing of two or more separately assembled parent communities, a phenomenon that has been termed "community coalescence". Understanding how the interaction structures of complex parent communities determine the outcomes of coalescence events is an important challenge. While recent work has begun to elucidate the role of competition in coalescence, that of cooperation, a key interaction type commonly seen in microbial communities, is still largely unknown. Here, using a general consumer-resource model, we study the combined effects of competitive and cooperative interactions on the outcomes of coalescence events. In order to do so, we simulate coalescence events between pairs of communities with different degrees of competition for shared carbon resources and cooperation through cross-feeding on leaked metabolic by-products (facilitation). We also study how structural and functional properties of post-coalescence communities evolve when they are subjected to repeated coalescence events. We find that in coalescence events, the less competitive and more cooperative parent communities contribute a higher proportion of species to the new community, because this endows superior ability to deplete resources and resist invasions. Consequently, when a community is subjected to repeated coalescence events, it gradually evolves towards being less competitive and more cooperative, as well as more species rich, robust and efficient in resource use. Encounters between microbial communities are becoming increasingly frequent as a result of anthropogenic environmental change, and there is great interest in how the coalescence of microbial communities affects environmental and human health. Our study provides new insights into the mechanisms behind microbial community coalescence, and a framework to predict outcomes based on the interaction structures of parent communities.

Author summaryIn nature, new microbial communities often arise from the fusion of whole, previously separate communities (community coalescence). Despite the crucial role that interactions among microbes play in the dynamics of complex communities, our ability to predict how these affect the outcomes of coalescence events remains limited. Here, using a general mathematical model, we study how the structure of species interactions confers an advantage upon a microbial community when it encounters another, and how communities evolve after undergoing repeated coalescence events. We find that competitive interactions between species preclude their survival upon a coalescence event, while cooperative interactions are advantageous for post-coalescence survival. Furthermore, after a community is exposed to many coalescence events, the remaining species become less competitive and more cooperative. Ultimately, this drives the community evolution, yielding post-coalescence communities that are more species-rich, productive, and resistant to invasions. There are many potential environmental and health implications of microbial community coalescence, which will benefit from the theoretical insights that we offer here about the fundamental mechanisms underlying this phenomenon.

sentences: Signatures of copy number alterations in human cancer The gains and losses of DNA that emerge as a consequence of mitotic errors and chromosomal instability are prevalent in cancer. These copy number alterations contribute to cancer initiaition, progression and therapeutic resistance. Here, we present a conceptual framework for examining the patterns of copy number alterations in human cancer using whole-genome sequencing, whole-exome sequencing, and SNP6 microarray data making it widely applicable to diverse datasets. Deploying this framework to 9,873 cancers representing 33 human cancer types from the TCGA project revealed a set of 19 copy number signatures that explain the copy number patterns of 93% of TCGA samples. 15 copy number signatures were attributed to biological processes of whole-genome doubling, aneuploidy, loss of heterozygosity, homologous recombination deficiency, and chromothripsis. The aetiology of four copy number signatures are unexplained and some cancer types have unique patterns of amplicon signatures associated with extrachromosomal DNA, disease-specific survival, and gains of proto-oncogenes such as MDM2. In contrast to base-scale mutational signatures, no copy number signature associated with known cancer risk factors. The results provide a foundation for exploring patterns of copy number changes in cancer genomes and synthesise the global landscape of copy number alterations in human cancer by revealing a diversity of mutational processes giving rise to copy number changes.

sentences: The cortical bone metabolome of C57BL/6J mice is sexually dimorphic Cortical bone quality, which is sexually dimorphic, depends on bone turnover and therefore the activities of remodeling bone cells. However, sex differences in cortical bone metabolism are not yet defined. Adding to the uncertainty about cortical bone metabolism, the metabolomes of whole bone, isolated cortical bone without marrow, and bone marrow have not been compared. We hypothesized that the metabolome of isolated cortical bone would be distinct from that of bone marrow and would reveal sex differences. Metabolite profiles from LC-MS of whole bone, isolated cortical bone, and bone marrow were generated from humeri from 20-week-old female C57Bl/6J mice. The cortical bone metabolomes were then compared for 20-week-old female and male C57Bl/6J mice. Femurs from male and female mice were evaluated for flexural material properties and were then categorized into bone strength groups. The metabolome of isolated cortical bone was distinct from both whole bone and bone marrow. We also found sex differences in the isolated cortical bone metabolome. Based on metabolite pathway analysis, females had higher lipid metabolism, and males had higher amino acid metabolism. High-strength bones, regardless of sex, had greater tryptophan and purine metabolism. For males, high strength bones had upregulated nucleotide metabolism, whereas lower strength bones had greater pentose phosphate pathway metabolism. Since the higher strength groups (females compared with males, high strength males compared with lower strength males) had higher serum CTX1/P1NP, we estimate that the metabolomic signature of bone strength in our study at least partially reflects differences in bone turnover. These data provide novel insight into bone bioenergetics and the sexual dimorphic nature of bone material properties in C57Bl/6 mice.

sentences: Ancestral chromosomes for the Peronosporaceae inferred from a telomere-to-telomere genome assembly of Peronospora effusa. We report the first telomere-to-telomere genome assembly for an oomycete. This assembly has extensive synteny with less complete genome assemblies of other oomycetes and will therefore serve as a reference genome for this taxon. Downy mildew disease of spinach, caused by the oomycete Peronospora effusa, causes major losses to spinach production. The 17 chromosomes of P. effusa were assembled telomere-to-telomere using Pacific Biosciences High Fidelity reads. Sixteen chromosomes are complete and gapless; Chromosome 15 contains one gap bridging the nucleolus organizer region. Putative centromeres were identified on all chromosomes. This new assembly enables a re-evaluation of the genomic composition of Peronospora spp.; the assembly was almost double the size and contained more repeat sequences than previously reported for any Peronospora spp. Genome fragments consistently under-represented in six previously reported assemblies of P. effusa typically encoded repeats. Some genes annotated as encoding effectors were organized into multigene clusters on several chromosomes. At least two effector-encoding genes were annotated on every chromosome. The intergenic distances between annotated genes were consistent with the two-speed genome hypothesis, with some effectors located in gene-sparse regions. The near-gapless assembly revealed apparent horizontal gene transfer from Ascomycete fungi. Gene order was highly conserved between P. effusa and the genetically oriented assembly of the oomycete Bremia lactucae. High levels of synteny were also detected with Phytophthora sojae. Many oomycete species may have similar chromosome organization; therefore, this genome assembly provides the foundation for genomic analyses of diverse oomycetes.

sentences: A Focused Small-Molecule Screen Identifies PHA-680626 as an Amphosteric Inhibitor Disrupting the Interaction between Aurora-A and N-Myc Neuroblastoma is a severe childhood disease, accounting for ~10% of all infant cancers. The amplification of the MYCN gene, coding for the N-Myc transcriptional factor, is an essential marker correlated with tumor progression and poor prognosis. In neuroblastoma cells, the mitotic kinase Aurora-A (AURKA), also frequently overexpressed in cancer, prevents N-Myc degradation, by directly binding to a highly conserved N-Myc region, i.e. Myc Box I. As a result, elevated levels of N-Myc, which are required for the growth of MYCN amplified cells, are observed. During the last years, it has been demonstrated that the ATP competitive inhibitors of AURKA CD532, MLN8054 and Alisertib also cause essential conformational changes in the structure of the activation loop of the kinase that prevent N-Myc binding, thus impairing the formation of the AURKA/N-Myc complex. In this study, starting from a screening of crystal structures of AURKA in complex with known inhibitors, we identified additional compounds affecting the conformation of the kinase activation loop. We assessed the ability of such compounds to disrupt the interaction between AURKA and N-Myc in vitro, using Surface Plasmon Resonance competition assays, and in tumor cell lines overexpressing MYCN, by performing Proximity Ligation Assays. Finally, their effects on N-Myc cellular levels and cell viability were investigated. Our results, identifying PHA-680626 as an amphosteric inhibitor both in vitro and MYCN overexpressing cell lines, expand the repertoire of known conformational disrupting inhibitors of the AURKA/N-Myc complex, and confirm that altering the conformation of the activation loop of AURKA with a small molecule is an effective strategy to destabilize the AURKA/N-Myc interaction in neuroblastoma cancer cells.

sentences: Patching Science - amending the literature through version control The ideal of self-correction in science is not well served by the current culture and system surrounding amendments to published literature. Here we report on a survey (N = 132) that highlights academics dissatisfaction with the status quo and their support for an alternative approach. We then describe our view of how amendments could and should work by drawing on the idea of an author-led version control system. Here authors would include a link in their published manuscripts to an updatable website (e.g. a GitHub repository or similar) that could be disseminated in the event of any amendment. Such a system is already in place for computer code and, as such, requires nothing but buy-in from the scientific community - a community that is already evolving towards various open science frameworks. This would remove a number of frictions that discourage amendments thus leading to an improved scientific literature and a healthier academic climate.

sentences: Pulvinar Slits: Cellulose-deficient and De-esterified Homogalacturonan-rich Structures in a Legume Motor Cell The cortical motor cells (CMCs) in a legume pulvinus execute the reversible deformation in leaf movement that is driven by changes in turgor pressure. In contrast to the underlying osmotic regulation, the cell wall structure of CMCs that contributes to the movement has yet to be characterized in detail. Here, we report that the cell wall of CMCs has tangential slits with low levels of cellulose deposition. This structure is unique and distinct from any other primary cell wall reported so far; thus, we named them " pulvinar slits." Notably, we predominantly detected de-methyl esterified homogalacturonan inside pulvinar slits, with a low deposition of highly methyl-esterified homogalacturonan inside the slits, as with cellulose. In addition, Fourier-transform infrared spectroscopy analysis indicated that the cell wall composition of pulvini is different from that of other axial organs, i.e. petioles or stems. Moreover, monosaccharide analysis showed that pulvini are pectin-rich organs like developing stems. Computer modeling suggested that pulvinar slits facilitate anisotropic extension in the direction perpendicular to the slits in the presence of turgor pressure. Mechanical tests revealed that the proximo-distal extensibility of pulvini is superior to that of the other axial organs. In this study, we thus established the distinctive cell wall structure of CMCs that contribute to plant movement, adding new knowledge to the structural diversity and function of the plant cell wall.

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Mechanical loading and hyperosmolarity as a daily resetting cue for skeletal circadian clocks Daily rhythms in mammalian behaviour and physiology are generated by a multi-oscillator circadian system entrained through environmental cues (e.g. light). Presence of niche-dependent physiological time cues has been proposed, allowing local tissues flexibility of phase adjustment. However, to date, such stimuli have remained elusive. Here we show that cycles of mechanical loading and osmotic stimuli within physiological range drive rhythmic expression of clock genes and reset clock phase and amplitude in cartilage and intervertebral disc tissues. Hyperosmolarity (not hypo-osmolarity) resets clocks in young and ageing skeletal tissues through mTORC2-AKT-GSK3{beta} pathway, leading to genome-wide induction of rhythmic genes. These results advocate diurnal patterns of mechanical loading and consequent daily surges in osmolarity as a bona fide tissue niche-specific time cue to maintain skeletal circadian rhythms in sync.

One-Sentence SummaryCircadian clocks in aneural skeletal tissues sense the passage of time through rhythmic patterns of loading and osmolarity

sentences: Kernel Local Fisher Discriminant Analysis of Principal Components (KLFDAPC) significantly improves the accuracy of predicting geographic origin of individuals Geographic patterns of human genetic variation provide important insights into human evolution and disease. A commonly used tool to detect geographic patterns from genetic data is principal components analysis (PCA) or the supervised linear discriminant analysis of principal components (DAPC). However, genetic features produced from both approaches could fail to correctly characterize population structure for complex scenarios involving admixture. In this study, we introduce Kernel Local Fisher Discriminant Analysis of Principal Components (KLFDAPC), a supervised nonlinear approach for inferring individual geographic genetic structure that could rectify the limitations of these approaches by preserving the multimodal space of samples. We tested the power of KLFDAPC to infer population structure and to predict individual geographic origin using neural networks. Simulation results showed that KLFDAPC significantly improved the population separability compared with PCA and DAPC. The application to POPRES and CONVERGE datasets indicated that the first two reduced features of KLFDAPC correctly recapitulated the geography of individuals, and significantly improved the accuracy of predicting individual geographic origin when compared to PCA and DAPC. Therefore, KLFDAPC can be useful for geographic ancestry inference, design of genome scans and correction for spatial stratification in GWAS that link genes to adaptation or disease susceptibility.

sentences: Spatial organization of chromosomes leads to heterogeneous chromatin motion and drives the liquid- or gel-like dynamical behavior of chromatin Chromosome organization and dynamics are involved in regulating many fundamental processes such as gene transcription and DNA repair. Experiments unveiled that chromatin motion is highly heterogeneous inside cell nuclei, ranging from a liquid-like, mobile state to a gel-like, rigid regime. Using polymer modeling, we investigate how these different physical states and dynamical heterogeneities may emerge from the same structural mechanisms. We found that the formation of topologically-associating domains (TADs) is a key driver of chromatin motion heterogeneity. In particular, we demonstrated that the local degree of compaction of the TAD regulates the transition from a weakly compact, fluid state of chromatin to a more compact, gel state exhibiting anomalous diffusion and coherent motion. Our work provides a comprehensive study of chromosome dynamics and a unified view of chromatin motion enabling to interpret the wide variety of dynamical behaviors observed experimentally across different biological conditions, suggesting that the  liquid or  solid behaviour of chromatin are in fact two sides of the same coin.

sentences: Effects of Platycodon grandiflorum on Gut Microbiota and Immune System of Immunosuppressed Mouse Platycodon grandiflorum (PG) has been used as a traditional remedy to control immune related diseases. However, there is limited information about its immune stimulating effects on the immunosuppressed model. The main bioactive components such as saponins are known to con-tribute to controlling immune activity. Thus, we developed an aged red PG (PGS) with 2.6 times of platycodin D, one of the saponins. We treated PG and PGS (PG-diets) to immunosuppressed mice via cyclophosphamide (CPA) injection. After 2weeks of the supplement, 16S rRNA sequencing was performed to investigate the effects of PG-diets on the gut microbiota and immune system in the immune suppressed model. PG-diets groups showed an increased abundance of microorganism in immune-deficient mice compared to the control NC group, indicating PG-diets have a distinct effect on microbial communities. Detection of specific genera related to the immune related biomarkers in PG-diets groups can support their effects on the immune system. Especially, the Akkermansia showed a significant decrease of abundance in response to the CPA treatment in the NC group at the genus level, but its abundance increased in response to the PG-diets treatment in the PG-diets groups. We also found that the modulation of gut microbiome by PG-diets was correlated with body weight as one of important immune biomarkers, though not much difference was found between PG and PGS effects. The results demonstrate that PG-diets may improve the health benefits of immune suppressed mice by altering the gut microbiome.

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The Requirement of Ubiquitin C-Terminal Hydrolase L1 (UCHL1) in Mouse Ovarian Development and Fertility Ubiquitin C-Terminal Hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme enriched in neuronal and gonadal tissues known to regulate the cellular stores of mono-ubiquitin and protein turnover. While its function in maintaining proper motor neuron function is well-established, investigation into its role in the health and function of reproductive processes is only just beginning to be studied. Single-cell-sequencing analysis of all ovarian cells from the murine perinatal period revealed that Uchl1 is very highly expressed in the developing oocyte population, an observation which was corroborated by high levels of oocyte-enriched UCHL1 protein expression in oocytes of all stages throughout the mouse reproductive lifespan. To better understand the role UCHL1 may be playing in oocytes, we utilized a UCHL1-deficient mouse line, finding reduced number of litters, reduced litter sizes, altered folliculogenesis, morphologically abnormal oocytes, disrupted estrous cyclicity and apparent endocrine dysfunction in these animals compared to their wild-type and heterozygous littermates. These data reveal a novel role of UCHL1 in female fertility as well as overall ovarian function, and suggest a potentially essential role for the ubiquitin proteasome pathway in mediating reproductive health.

Summary sentenceUbiquitin C-Terminal Hydrolase L1 (UCHL1) is required for proper ovarian folliculogenesis, estrous cyclicity, and fertility in the female mouse.

sentences: Analysis of next- and third-generation RNA-Seq data reveals the structures of alternative transcription units in bacterial genomes Alternative transcription units (ATUs) are dynamically encoded under different conditions or environmental stimuli in bacterial genomes, and genome-scale identification of ATUs is essential for studying the emergence of human diseases caused by bacterial organisms. However, it is unrealistic to identify all ATUs using experimental techniques, due to the complexity and dynamic nature of ATUs. Here we present the first-of-its-kind computational framework, named SeqATU, for genome-scale ATU prediction based on next-generation RNA-Seq data. The framework utilizes a convex quadratic programming model to seek an optimum expression combination of all of the to-be-identified ATUs. The predicted ATUs in E. coli reached a precision of 0.77/0.74 and a recall of 0.75/0.76 in the two RNA-Sequencing datasets compared with the benchmarked ATUs from third-generation RNA-Seq data. We believe that the ATUs identified by SeqATU can provide fundamental knowledge to guide the reconstruction of transcriptional regulatory networks in bacterial genomes.

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Fiber-parenchyma trade-off underlies changes in tropical forest structure and xylem architecture across a soil water gradient O_LIWood anatomical traits can underpin tropical forest structural and functional changes across soil water gradients and therefore could improve our mechanistic understanding of how plants adapt to environmental change.
C_LIO_LIWe assessed how the variation in the forest maximum height (Hmax), stem diameter, and wood density (WD) is associated with variation in xylem traits (area of fibers and parenchyma, conductive area [CondA, sum of all vessels lumens], vessel lumen area [VLA], vessel density [VD], and vessel wall reinforcement [VWR]) across 42 plots of a Brazilian Atlantic Forest habitat that span strong soil water gradients.
C_LIO_LIWe found that in wetter communities, greater height and lower WD were associated with greater parenchyma area (capacitance), and lower fibers, VD, VWR. Contrastingly, in drier communities, lower height was associated with higher fiber area (xylem reinforcement), WD, VD, and VWR, while parenchyma area and vessels are reduced.
C_LIO_LITree communities vary from conservative resource-use and structurally dependent hydraulic safety (Fibers) to acquisitive resource-use and capacitance dependent hydraulic safety (parenchyma). Such a fiber-parenchyma trade-off (FPT) underlies the variation in tree height across a soil water gradient. Wood anatomy is fundamental to understanding and predicting the impacts of environmental change on forest structure.
C_LI

sentences: The AMA1-RON complex drives Plasmodium sporozoite invasion in the mosquito and mammalian hosts Plasmodium sporozoites that are transmitted by blood-feeding female Anopheles mosquitoes invade hepatocytes for an initial round of intracellular replication, leading to the release of merozoites that invade and multiply within red blood cells. Sporozoites and merozoites share a number of proteins that are expressed by both stages, including the Apical Membrane Antigen 1 (AMA1) and the Rhoptry Neck Proteins (RONs). Although AMA1 and RONs are essential for merozoite invasion of erythrocytes during asexual blood stage replication of the parasite, their function in sporozoites is still unclear. Here we show that AMA1 interacts with RONs in mature sporozoites. By using DiCre-mediated conditional gene deletion in P. berghei, we demonstrate that loss of AMA1, RON2 or RON4 in sporozoites impairs colonization of the mosquito salivary glands and invasion of mammalian hepatocytes, without affecting transcellular parasite migration. Our data establish that AMA1 and RONs facilitate host cell invasion across Plasmodium invasive stages, and suggest that sporozoites use the AMA1-RON complex to safely enter the mosquito salivary glands without causing cell damage, to ensure successful parasite transmission. These results open up the possibility of targeting the AMA1-RON complex for transmission-blocking antimalarial strategies.

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Coding Recognition of the Dose-Effect Interdependence of Small Biomolecules Encrypted on Paired Chromatographic-Based Microassay Arrays The discovery of small biomolecules suffers from the lack of a comprehensive framework to express the intrinsic correlation between bioactivity and contributing small molecules in complex samples with molecular and bioactivity diversity. Here, by mapping a samples 2D-HPTLC fingerprint to microplates, the paired chromatographic-based microassay arrays are created, which can be used as quasi-chip to characterize multiple attributes of chromatographic components; and as the array differential expression of the bioactivity and molecular attributes of irregular chromatographic spots for dose-effect interdependent encoding; and also as the automatic-collimated array mosaics of the multi-attributes of each component itself encrypted by its chromatographic fingerprint. Based on this homologous framework, we propose a correlating recognition strategy for small-biomolecules through their self-consistent chromatographic behavior characteristics. In the approach, the small-biomolecule recognition in diverse compounds is transformed into a constraint satisfaction problem, which is addressed through examining the dose-effect interdependence of the homologous 2D code pairs by array matching algorithm, instead of preparing diverse compound monomers of complex test sample for identifying item-by-item. Furtherly, considering the dose-effect interdependent 2D code pairs as links and the digital-specific quasimolecular ions as nodes, an extendable self-consistent framework that correlates mammalian cell phenotypic and target-based bioassays with small biomolecules is established. Therefore, the small molecule contributions and the correlations of bioactivities, as well as their pathway can be comprehensively revealed, so as to improve the reliability and efficiency of screening. This strategy was successfully applied to galangal, and practiced the high-throughput digital preliminary screening of small-biomolecules in a natural product.

HighlightRapid self-recognition of small biomolecules in diverse samples without pre-isolation Recognized by dose-effect interdependency developed from homologous TLC fingerprint Matching of HPTLC-based molecular imprinting and bioautography on microassay arrays Microarray-based differential expression of substance attributes instead of spot scan An array framework for combining phenotype-based and target-based assays with TLC-MS

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sentences: Stochastic model of T Cell repolarization during target elimination (II) Cytotoxic T lymphocytes (T cells) and natural killer cells form a tight contact, the immunological synapse (IS), with target cells, where they release their lytic granules containing perforin/granzyme and cytokine containing vesicles. During this process the cell repolarizes and moves the microtubule organizing center (MTOC) towards the IS. In the first part of our work we developed a computational model for the molecular-motor-driven motion of the MT cytoskeleton during T cell polarization and analyzed effects of cortical sliding and capture-shrinkage mechanisms. Here we use this model to analyze the dynamics of the MTOC repositioning in situations in which a) the IS is in an arbitrary position with respect to the initial position of the MTOC and b) the T cell has two IS at two arbitrary positions. In the case of one IS, we found that the initial position determines which mechanism is dominant and that the time of repositioning does not rise monotonously with the MTOC-IS distance. In the case of two IS, we observe several scenarios that have also been reported experimentally: the MTOC alternates stochastically (but with a well defined average transition time) between the two IS; it wiggles in between the two IS without transiting to one of the two; or it is at some point pulled to one of the two IS and stays there. Our model allows to predict which scenario emerges in dependency of the mechanisms in action and the number of dyneins present. We report that the presence of capture-shrinkage mechanism at least in one IS is necessary to assure the transitions in every cell configuration. Moreover, the frequency of transitions does not decrease with the distance between the two IS and is the highest when both mechanisms are present in both IS.

sentences: A gain and dynamic range independent index to quantify spillover spread to aid panel design in flow cytometry In conventional flowcytometry one detector (primary) is dedicated for one fluorochrome. However, photons usually end up in other detectors too (fluorescence spillover).  Compensation is a process that corrects the spillover signal from all detectors except the primary detector. Post  compensation, the photon counting error of spillover signals become evident as spreading of the data. The spreading induced by spillover impairs the ability to resolve stained cell population from the unstained one, potentially reducing or completely losing cell populations. For successful multi-color panel design, it is important to know the expected spillover to maximize the data resolution. The Spillover Spreading Matrix (SSM) can be used to estimate the spread, but the outcome is dependent on detector sensitivity. Simply, the same single stained sample produces different spillover spread values when detector(s) sensitivity is altered. Many researchers mistakenly use this artifact to "reduce" the spread by decreasing detector sensitivity. This can result in diminished capacity to resolve dimly expressing cell populations. Here, we introduce SQI (Spread Quantification Index), that can quantify the spillover spread independent of detector sensitivity and independent of dynamic range. This allows users to compare spillover spread between instruments having different types of detectors, which is not possible using SSM.

sentences: The SPICA Coarse-Grained Force Field for Proteins and Peptides A coarse-grained (CG) model for peptides and proteins was developed as an extension of the SPICA (Surface Property fItting Coarse grAined) force field (FF). The model was designed to examine membrane proteins that are fully compatible with the lipid membranes of the SPICA FF. A preliminary version of this protein model was created using thermodynamic properties, including the surface tension and density in the SPICA (formerly called SDK) FF. In this study, we improved the CG protein model to facilitate molecular dynamics (MD) simulation with a reproduction of multiple properties from both experiments and all-atom (AA) simulations. The side chain analogs reproduced the transfer free energy profiles across the lipid membrane and demonstrated reasonable dimerization free energies in water compared to those from AA-MD. A series of peptides/proteins adsorbed or penetrated into the membrane simulated by the CG-MD correctly predicted the penetration depths and tilt angles of peripheral and transmembrane peptides/proteins comparable to those in the orientation of protein in membrane (OPM) database. In addition, the dimerization free energies of several transmembrane helices within a lipid bilayer were comparable to those from experimental estimation. Application studies on a series of membrane protein assemblies, scramblases, and poliovirus capsids demonstrated a good performance of the SPICA FF.

sentences: Bridging brain and cognition: A multilayer network analysis of brain structural covariance and general intelligence in a developmental sample of struggling learners Network analytic methods that are ubiquitous in other areas, such as systems neuroscience, have recently been used to test network theories in psychology, including intelligence research. The network or mutualism theory of intelligence proposes that the statistical associations among cognitive abilities (e.g., specific abilities such as vocabulary or memory) stem from causal relations among them throughout development. In this study, we used network models (specifically LASSO) of cognitive abilities and brain structural covariance (grey and white matter) to simultaneously model brain-behavior relationships essential for general intelligence in a large (behavioral, N=805; cortical volume, N=246; fractional anisotropy, N=165), developmental (ages 5-18) cohort of struggling learners (CALM). We found that mostly positive, small partial correlations pervade our cognitive, neural, and multilayer networks. Moreover, using community detection (Walktrap algorithm) and calculating node centrality (absolute strength and bridge strength), we found convergent evidence that subsets of both cognitive and neural nodes play an intermediary role  between brain and behavior. We discuss implications and possible avenues for future studies.

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Permethylation of ribonucleosides provides enhanced mass spectrometry quantification of post-transcriptional modifications Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, degradation, as well as human disease states such as cancer. However, the analysis of ribonucleoside modifications is impeded due to the hydrophilicity of the ribonucleoside molecules. In this research, we used solid-phase permethylation to derivatize the ribonucleosides, and the permethylated ribonucleosides, which were then quantitively analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. The solid-phase permethylation efficiently derivatized the ribonucleosides, and more than 60 RNA modifications were simultaneously monitored using ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. Because of the increased hydrophobicity of permethylated ribonucleosides, this method enhanced retention, separation, and ionization efficiency, resulting in improved detection and quantification when compared to existing analytical strategies of RNA modifications. We applied this new approach to measure the extent of cytosine methylation and hydroxymethylation in RNA obtained from mouse embryonic stem cells with genetic deficiencies in ten-eleven translocation (TET) enzymes. The results matched previously performed analyses and highlighted the sensitivity, efficacy, and robustness of the new method. The advantage of this method enables comprehensive analysis of RNA modifications in biological samples.

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sentences: Single-cell genetic heterogeneity linked to immune infiltration in glioblastoma. Glioblastoma (GBM) is the most aggressive brain tumor with a median survival of ~15 months. Targeted approaches have not been successful in this tumor type due to the large extent of intratumor heterogeneity. Mosaic amplification of oncogenes suggests that multiple genetically distinct clones are present in each tumor. To uncover the relationships between genetically diverse subpopulations of GBM cells and their native tumor microenvironment, we employed highly multiplexed spatial protein profiling, coupled with single-cell spatial mapping of fluorescence in situ hybridization (FISH) for EGFR, CDK4, and PDGFRA. Single-cell FISH analysis of a total of 35,843 single nuclei (~2,100 per tumor) revealed that tumors in which amplifications of EGFR and CDK4 more frequently co-occur in the same cell exhibit higher infiltration of CD163+ immunosuppressive macrophages. Our results suggest that high throughput assessment of genomic alterations at the single cell level could provide a measure for predicting the immune state of GBM.

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Glycine receptor α3K governs mobility and conductance of L/K splice variant heteropentamers Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR-3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearsons and dual-color spatiotemporal correlation analysis to prove that GlyR-3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via single-channel on-cell patch clamp we show heteropentameric conductances resemble those of the 3K splice variant. Our data are compatible with a model where 3 heteropentamerization fine-tunes mobility and activity of GlyR 3 channels, which is important to understand and tackle 3 related diseases.

SummaryThe glycine receptor 3 is key to the central nervous systems physiology and involved in chronic pain and epilepsy. In this paper, Lemmens et al. reveal and functionally characterize 3 splice variant heteropentamerization via advanced single-molecule fluorescence image analysis.

DeclarationsO_ST_ABSFundingC_ST_ABSWe acknowledge the UHasselt Advanced Optical Microscopy Centre (AOMC). Prof. Em. Marcel Ameloot, the Research Foundation Flanders (FWO, project G0H3716N) and the province of Limburg (Belgium) (tUL Impuls II) are acknowledged for funding the microscopy hardware. V. Lemmens is grateful for a doctoral scholarship from the UHasselt (17DOC11BOF) and KU Leuven (C14/16/053) Special Research Funds (BOF).

Conflicts of interest / competing interestsNo conflicts of interest apply.

Ethics approvalNot applicable

Availability of data and materialAll data and material are available upon request.

Code availabilityFluctuation imaging and co-localization analyses were performed in the software package PAM [71]. The software is available as source code, requiring MATLAB to run, or as pre-compiled standalone distributions for Windows or MacOS at http://www.cup.uni-muenchen.de/pc/lamb/software/pam.html or hosted in Git repositories under http://www.gitlab.com/PAM-PIE/PAM and http://www.gitlab.com/PAM-PIE/PAMcompiled. A detailed user manual is available at http://pam.readthedocs.io.

Author contributionsConceptualization Meier J.C., Brone B. and Hendrix J.; Investigation and formal analysis Lemmens V. and Thevelein B.; Software development Hendrix J.; Writing the original draft Lemmens V., Thevelein B and Hendrix, J.; Review and editing by all authors.

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Allometric tissue-scale forces activate mechanoresponsive immune cells to drive pathological foreign body response to biomedical implants For decades, it has been assumed that the foreign body response (FBR) to biomedical implants is primarily a reaction to the chemical and mechanical properties of the implant. Here, we show for the first time that a third independent variable, allometric tissue-scale forces (which increase exponentially with body size), can drive the biology of FBR in humans. We first demonstrate that pathological FBR in humans is mediated by immune cell-specific Rac2 mechanotransduction signaling, independent of implant chemistry or mechanical properties. We then show that mice, which are typically poor models of human FBR, can be made to induce a strikingly human-like pathological FBR by altering these extrinsic tissue forces. Altering these extrinsic tissue forces alone activates Rac2 signaling in a unique subpopulation of immune cells and results in a human-like pathological FBR at the molecular, cellular, and local tissue levels. Finally, we demonstrate that blocking Rac2 signaling negates the effect of increased tissue forces, dramatically reducing FBR. These findings highlight a previously unsuspected mechanism for pathological FBR and may have profound implications for the design and safety of all implantable devices in humans.

One-Sentence SummaryAllometric tissue-scale forces at the implant-tissue interface drive pathological foreign body response.

sentences: BNIP3 upregulation characterizes cancer cell subpopulation with increased fitness and proliferation BNIP3 is a BH3 only protein with both pro-apoptotic and pro-survival roles depending on the cellular context. It remains unclear how BNIP3 RNA level dictates cell fate decisions of cancer cells. Here we undertook a quantitative analysis of BNIP3 expression and functions in single cell datasets of various epithelial malignancies. Our results demonstrated that BNIP3 upregulation characterizes cancer cell subpopulations with increased fitness and proliferation. We further validated the upregulation of BNIP3 in liver cancer organoids compared with 2D culture. Taken together, the combination of in silico perturbations using public single cell datasets and experimental cancer modeling using organoids ushered in a new approach to address cancer heterogeneity.

sentences: Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2 Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and Nucleocapsid (N) proteins from SARS-CoV-2, and obtained cross-link sets with an average density of one cross-link per twenty residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites, and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and inter-domain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryo-EM structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

sentences: Normalizing and denoising protein expression data from droplet-based single cell profiling Multimodal single-cell protein and transcriptomic profiling (e.g. CITE-seq) holds promise for comprehensive dissection of cellular heterogeneity, yet protein counts measured by oligo-conjugated-antibody can have substantial noise that masks biological variations. Here we integrated experiments and computational analysis to reveal two major noise sources: protein-specific noise from unbound antibodies and cell-specific noise captured by the shared variance of isotype controls and background protein counts. We provide an open source R package (dsb) to denoise and normalize CITE-seq data based on these findings. (https://cran.r-project.org/web/packages/dsb/index.html).

sentences: Significance of Oct-4 transcription factor as a pivotal therapeutic target for CD44+/24- mammary tumour initiating cells; aiming at the root of the recurrence Breast cancer (BC) remains one of the deadliest and frequently diagnosed metastatic cancers worldwide. Cancer stem cells (CSCs) are the cell population within the tumour niche, having an epithelial to mesenchymal (EMT) transition phenotype, high self-renewal, vigorous metastatic capacity, drug resistance, and tumour relapse. Identification of targets for induction of apoptosis is essential to provide novel therapeutic approaches in BC. Our earlier studies showed that Vitamin C induces apoptotic cell death by losing redox balance in TNBC CSCs. In this study, we have attempted to identify previously unrecognized CSC survival factors that can be used as druggable targets for bCSCs apoptosis regulators isolated from the TNBC line, MDA MB 468. After a thorough literature review, Oct-4 was identified as the most promising marker for its unique abundance in cancer and absence in normal cells and the contribution of Oct-4 to the sustenance of cancer cells. We then validated a very high expression of Oct-4 in the MDA MB 468 bCSCs population using flow-cytometry. The loss of Oct-4 was carried out using siRNA-mediated knockdown in the bCSCs, followed by assessing for cellular apoptosis. Our results indicated that Oct-4 knockdown induced cell death, changes in cellular morphology, inhibited mammosphere formation, and positive for Annexin-V expression, thereby indicating the role of Oct-4 in bCSC survival. Moreover, our findings also suggest the direct interaction between Oct-4 and Vitamin C using in silico docking. This data, hence, contributes towards novel information about Oct-4 highlighting this molecule as a novel survival factor in bCSCs.

sentences: Dissection of the ATPase active site of McdA reveals the sequential steps essential for carboxysome distribution Carboxysomes, the most prevalent and well-studied anabolic bacterial microcompartment, play a central role in efficient carbon fixation by cyanobacteria and proteobacteria. In previous studies, we identified the two-component system called McdAB that spatially distributes carboxysomes across the bacterial nucleoid. McdA, a ParA-like ATPase, forms a dynamic oscillating gradient on the nucleoid in response to carboxysome-localized McdB. As McdB stimulates McdA ATPase activity, McdA is removed from the nucleoid in the vicinity of carboxysomes, propelling these proteinaceous cargos toward regions of highest McdA concentration via a Brownian-ratchet mechanism. However, how the ATPase cycle of McdA governs its in vivo dynamics and carboxysome positioning remains unresolved. Here, by strategically introducing amino acid substitutions in the ATP-binding region of McdA, we sequentially trap McdA at specific steps in its ATP cycle. We map out critical events in the ATPase cycle of McdA that allows the protein to bind ATP, dimerize, change its conformation into a DNA-binding state, interact with McdB-bound carboxysomes, hydrolyze ATP and release from the nucleoid. We also find that McdA is a member of a previously unstudied subset of ParA family ATPases, harboring unique interactions with ATP and the nucleoid for trafficking their cognate intracellular cargos.

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Modeling Cell Biological Features of Meiotic Chromosome Pairing During meiosis, homologous chromosomes become associated side by side in a process known as homologous chromosome pairing. Pairing requires long range chromosome motion through a nucleus that is full of other chromosomes. It remains unclear how the cell manages to align each pair of chromosomes quickly while mitigating and resolving interlocks. Here, we use a coarse-grained molecular dynamics model to investigate how specific features of meiosis, including motor-driven telomere motion, nuclear envelope interactions, and increased nuclear size, affect the rate of pairing and the mitigation/resolution of interlocks. By creating in silico versions of three yeast strains and comparing the results of our model to experimental data, we find that a more distributed placement of pairing sites along the chromosome is necessary to replicate experimental findings. Active motion of the telomeric ends speeds up pairing only if binding sites are spread along the chromosome length. Adding a meiotic bouquet significantly speeds up pairing but does not significantly change the number of interlocks. An increase in nuclear size slows down pairing while greatly reducing the number of interlocks. Interestingly, active forces increase the number of interlocks, which raises the question: How do these interlocks resolve? Our model gives us detailed movies of interlock resolution events which we then analyze to build a step-by-step recipe for interlock resolution. In our model, interlocks must first translocate to the ends, where they are held in a quasi-stable state by a large number of paired sites on one side. To completely resolve an interlock, the telomeres of the involved chromosomes must come in close proximity so that the cooperativity of pairing coupled with random motion causes the telomeres to unwind. Together our results indicate that computational modeling of homolog pairing provides insight into the specific cell biological changes that occur during meiosis.

Author SummaryEarly in meiosis, homologous chromosomes must find each other within the crowded nuclear space and become aligned along their entire length in a process known as homologous chromosome pairing. It remains unclear how the cell manages to align each pair of chromosomes quickly while mitigating and resolving interlocks. Here, we study this process by using a computational model. Our model attempts to capture the large-scale cell biological picture of meiotic pairing including the random initial 3D search, active motion of the chromosome ends, and meiosis specific constraints such as telomere attachment to the nuclear envelope. We use our model to study how these different features of meiosis affect the rate of pairing and the mitigation/resolution of interlocks. Importantly, our model gives us detailed movies of interlock resolution events, which we then analyze to build a step-by-step recipe for interlock resolution. We believe computational modeling of homolog pairing provides valuable insight into this complex biological process.

sentences: Thermodynamic stabilization of the von Willebrand Factor A1 domain due to loss-of-function disease-related mutations. The von Willebrand disease (vWD) is the most common hereditary bleeding disorder, caused by defects of the von Willebrand Factor (vWF), a large extracellular protein in charge of adhering platelets at sites of vascular lesion. vWF carries out this essential homeostatic task, via the specific protein-protein interaction between the vWF A1 domain and the platelet receptor, the glycoprotein Ib alpha (GPIB). Upon the vWF activation triggered by the shear of the flowing blood. The two naturally occurring mutations G1324A and G1324S at the A1 domain, near the GPIB binding site, result in a dramatic decrease of platelets adhesion, a bleeding disorder classified as type 2M vWD. However, it remained unclear how these two supposedly minor modifications lead to this drastic phenotypic response. We addressed this question using a combination of equilibrium-molecular dynamics (MD) and non-equilibrium MD-based free energy simulations. Our data confirm that both mutations maintain the highly stable Rossmann fold of the vWF A1 domain. These mutations locally diminished the flexibility of the binding site to GPIB and induced a conformational change that affected the nearby secondary structure elements. Furthermore, we observed two significant changes in the vWF A1 domain upon mutation, the global redistribution of the internal mechanical stress and the increased thermodynamic stability of the A1 domain. These observations are consistent with previously-reported mutation-augmented melting temperatures. Overall, our results support the idea of thermodynamic conformational restriction of A1-- before the binding to GPIB--as a crucial factor determining the loss-of-function of the G1324A(S) vWD mutants.

sentences: Effects of variability in manually contoured spinal cord masks on fMRI co-registration and interpretation Functional magnetic resonance imaging (fMRI) of the human spinal cord (SC) is an emerging method for understanding neurovascular responses to stimuli. Group-analysis of SC fMRI data involves co-registration of subject-level data to standard space, which requires manual masking of the cord and may result in bias of group-level SC fMRI results. To test this, we examined variability in SC masks drawn in fMRI data from 21 healthy participants from a completed study mapping responses to sensory stimuli of the C7 dermatome. Masks were drawn on temporal mean functional image by eight raters with varying levels of neuroimaging experience, and the rater from the original study acted as an expert reference. Spatial agreement between rater and expert masks was measured using the Dice Similarity Coefficient, and the influence of rater and dataset was examined using ANOVA. Each raters masks were used to register functional data to the PAM50 template. Gray matter-white matter signal contrast of registered functional data was used to evaluate the spatial normalization accuracy across raters. Subject- and group-level analyses of activation during left- and right-sided sensory stimuli were performed for each raters co-registered data. Agreement with the experts SC mask was associated with both rater experience level (F(7,140) = 32.12, P < 2x10-16,{eta} 2 = 0.29) and dataset (F(20,140) = 20.58, P < 2x10-16,{eta} 2 = 0.53). Dataset variations may reflect image quality metrics: the ratio between the signal intensity of spinal cord voxels and surrounding cerebrospinal fluid was correlated with DSC results (p<0.001). As predicted, variability in the manually-drawn masks influenced spatial normalization, and GM:WM contrast in the registered data showed significant effects of rater and dataset (rater: F(8,160) = 23.57, P < 2x10-16,{eta} 2 = 0.24; dataset: F(20,160) = 22.00, P < 2x10-16,{eta} 2 = 0.56). Registration differences propagated into subject-level activation maps which showed rater-dependent agreement with the expert. Although group-level activation maps differed between raters, no systematic bias was identified. Increasing consistency in manual contouring of spinal cord fMRI data improved co-registration and inter-rater agreement in activation mapping, however our results suggest that improvements in image acquisition and post-processing are also critical to address.

sentences: Dynamic interplay between structural variations and 3D chromosome organization in pancreatic cancer Structural variations (SVs) are the greatest source of variation in the genome and can lead to oncogenesis. However, the identification and interpretation of SVs in human pancreatic cancer remain largely undefined due to technological limitations. Here, we investigate the spectrum of SVs and three-dimensional (3D) chromatin architecture in human pancreatic ductal epithelial cell carcinogenesis by using state-of-the-art long- read single-molecule real-time (SMRT) and high-throughput chromosome conformation capture (Hi-C) sequencing techniques. We find that the 3D genome organization is remodeled and correlated with gene expressional change. The bulk remodeling effect of cross-boundary SVs in the 3D genome partly depends on intercellular genomic heterogeneity. Meanwhile, contact domains tend to minimize these disrupting effects of SVs within local adjacent genomic regions to maintain overall stability of 3D genome organization. Moreover, our data also demonstrates complex genomic rearrangements involving two key driver genes CDKN2A and SMAD4, and elucidates their influence on cancer-related gene expression from both linear view and 3D perspective. Overall, this study provides a valuable resource and highlights the impact, complexity and dynamicity of the interplay between SVs and 3D genome organization, which further expands our understanding of pathogenesis of SVs in human pancreatic cancer.

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A locus conferring tolerance to Theileria infection in African cattle East Coast fever, a tick-borne cattle disease caused by the Theileria parva parasite, is among the biggest natural killers of cattle in East Africa, leading to over 1 million deaths annually. Here we report on the genetic analysis of a cohort of Boran cattle demonstrating heritable tolerance to infection by T. parva (h2 = 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region on Bos taurus chromosome 15 that is significantly associated with survival outcome following T. parva exposure. Testing this locus in an independent cohort of animals replicates this association with survival following T. parva infection. A stop gained polymorphism in this region was found to be highly associated with survival across both related and unrelated animals, with only one of the 20 homozygote carriers (T/T) of this change succumbing to the disease in contrast to 44 out of 97 animals homozygote for the reference allele (C/C). Consequently, we present a genetic locus linked to tolerance of one of Africas most important cattle diseases, raising the promise of marker-assisted selection for cattle that are less susceptible to infection by T. parva.

Author SummaryMore than a million cattle die of East Coast fever in Africa each year, the impact of which disproportionately falls onto low-income, smallholder farmers. The lack of a widely accessible vaccine, heavy reliance on chemicals to control the tick vector and inadequate drug treatments means that new approaches for controlling the disease are urgently required. Through a genetic study of an extended pedigree of Boran cattle that are more than three times less likely to succumb to the disease than matched controls, we identify a region on chromosome 15 of the cattle genome associated with a high level of tolerance to the disease. We show that a variant in this region is also associated with survival in an independent cohort, and is linked to rates of cell expansion during infection. This genetic variant can therefore support marker-assisted selection, allowing farmers to breed tolerant cattle and offers a route to introduce this beneficial DNA to non-native breeds, enabling reduced disease incidence and increased productivity, which would be of benefit to millions of rural smallholder farmers across Africa.

sentences: Single-Cell Analysis Identifies Distinct Immune and Smooth Muscle Cell Populations that Contribute to Chronic Thromboembolic Pulmonary Hypertension Chronic thromboembolic pulmonary hypertension (CTEPH) is a sequelae of acute pulmonary embolism (PE) in which the PE remodels into a chronic scar in the pulmonary arteries. This results in vascular obstruction, small vessel arteriopathy and pulmonary hypertension. Our current understanding of CTEPH pathobiology is primarily derived from cell-based studies limited by the use of specific cell markers or phenotypic modulation in cell culture. Here we used single cell RNA sequencing (scRNAseq) of tissue removed at the time of pulmonary thromboendarterectomy (PTE) surgery to identify the multiple cell types, including macrophages, T cells, and smooth muscle cells, that comprise CTEPH thrombus. Notably, multiple macrophage subclusters were identified but broadly split into two categories, with the larger group characterized by an upregulation of inflammatory signaling predicted to promote pulmonary vascular remodeling. Both CD4+ and CD8+ T cells were identified and likely contribute to chronic inflammation in CTEPH. Smooth muscle cells were a heterogeneous population, with a cluster of myofibroblasts that express markers of fibrosis and are predicted to arise from other smooth muscle cell clusters based on pseudotime analysis. Additionally, cultured endothelial, smooth muscle and myofibroblast cells isolated from CTEPH thrombus have distinct phenotypes from control cells with regards to angiogenic potential and rates of proliferation and apoptosis. Lastly, our analysis identified protease-activated receptor 1 (PAR1) as a potential therapeutic target that links thrombosis to chronic PE in CTEPH, with PAR1 inhibition decreasing smooth muscle cell and myofibroblast proliferation and migration. These findings suggest a model for CTEPH similar to atherosclerosis, with chronic inflammation promoted by macrophages and T cells driving vascular remodeling through smooth muscle cell modulation, and suggest new approaches for pharmacologically targeting this disease.

sentences: Hoslundia opposita and other Nigerian plants inhibit sickle hemoglobin polymerization and prevent erythrocyte sickling: a virtual screening-guided identification of bioactive plants. In sickle cell disease, a hereditary hemoglobinopathy, clinically observed disease presentations are the endpoint of a point mutation involving the substitution of glutamic acid with valine at the position 6 of the beta globin chain. With about 4.4 million people globally being affected, and another 43 million people bearing the sickle cell trait, several research efforts have been made to discover new and affordable treatment and possibly cure for the disease. Africa is endowed with a large flora population and traditional healers and citizens have over time depended on the use of herbs in folkloric medicine for different ailments including sickle cell disease (SCD). Such native knowledge has often formed the basis for different research exploration into the anti-sickling activities of selected African plants. These plants that have been so far investigated for their anti-sickling properties represent about 0.05 % of the 45,000 plant species enriching the flora landscape in Sub-Saharan Africa. Some of these have yielded potent anti-sickling profiles. In the current work we seek to achieve a more extensive search of the African plant diversity with anti-sickling properties: for this we have adopted a hybrid computational-cum-experimental protocol that employed computer-aided drug design (CADD) means for identifying plants with at least one constituent capable of interacting with the sickle hemoglobin, followed by extractive procedures and anti-sickling experiments for validating the predictions. Over two thousand (or 2,000) African natural products, representing over 200 plant species, were first virtually screened against the crystal structure of the dimerized human sickle hemoglobin. The natural products with the best computed sickle hemoglobin interaction energetics were found to belong to five plant species including Catharanthus roseus, Rauvolfia vomitoria, Hoslundia opposita, Lantana camara and Euphorbia hirta. The leaves of these plants were each collected subsequently and subjected to standard processing and extraction procedures. Using both HbSS polymerization inhibition and sickling reversal tests significant reductions in polymerization of erythrocyte hemolysate of the HbSS genotype were observed with the methanolic extracts of the plants, as well as sickling reversal levels of up to 68.50 % (H. opposita) was observed.

sentences: Serine 182 on RORγt regulates T helper 17 and regulatory T cell functions to resolve inflammation Intestine homeostasis is maintained by the delicate balance of Th17 effector cells and Treg cells. Dysregulation of these cell populations contributes to inflammation, tissue damage, and chronic conditions. ROR{gamma}t is essential for the differentiation of Th17 and a subset of Treg (ROR{gamma}t+ Treg) cells involved in intestinal inflammation. ROR{gamma}t belongs to the nuclear receptor family of transcription factors with hinge regions that are highly flexible for co-activator/co-repressor interactions. Serine 182 at the hinge region of ROR{gamma}t is phosphorylated. This study aims to uncover how S182 on ROR{gamma}t contributes to mucosal homeostasis and diseases. We used CRISRP technology to generate a phosphor-null knock-in mutant mouse line (ROR{gamma}tS182A) to assess its role in intestine physiology. scRNA-seq was performed on WT and ROR{gamma}tS182A cohoused littermates to evaluate colonic T cell heterogeneity under steady state and colitis settings. Single-cell transcriptomics revealed that ROR{gamma}tS182 maintains colonic T cell heterogeneity under steady state, without interfering T cell development and differentiation. In inflamed tissues, ROR{gamma}tS182 simultaneously restricts IL-1{beta}-mediated Th17 activities and promotes anti-inflammatory cytokine IL-10 production in LT-like Treg cells. Phospho-null ROR{gamma}tS182A knock-in mice challenged with DSS induced colitis and EAE experienced delayed recovery and exacerbated pathology. The double switch role of ROR{gamma}tS182 is critical in resolving T cell-mediated inflammation and provides a potential therapeutic target to combat autoimmune diseases.

sentences: GRAMD1C promotes autophagy initiation and mitochondrial bioenergetics through regulation of ER-mitochondria cholesterol transport During autophagy, cytosolic cargo is sequestered into double-membrane vesicles called autophagosomes. The origin and identity of the membranes that form the autophagosome remain to be fully characterized. Here, we investigated the role of cholesterol in starvation- induced autophagy and identify a role for the ER-localized cholesterol transport protein GRAMD1C in the regulation of autophagy and mitochondrial function. We demonstrate that cholesterol depletion leads to a rapid induction of autophagy, possibly caused by a corresponding increased abundance of curved autophagy membranes. We further show that GRAMD1C is a negative regulator of starvation-induced autophagy. Similar to its yeast orthologue, GRAMD1C is recruited to mitochondria through its GRAM domain. Additionally, we find that GRAMD1C depletion leads to increased mitochondrial cholesterol accumulation and mitochondrial oxidative phosphorylation. Finally, we demonstrate that expression of GRAM family genes is linked to clear cell renal carcinoma survival, highlighting the pathophysiological relevance of cholesterol transport proteins.

sentences: Sex-specific life extension in tauopathy mice by CSF1R inhibition causing selective microglial depletion and suppressed pathogenesis Microglia are a fundamental component of pathogenesis in many neurological conditions and have specialized functions that vary by disease stage or specific pathology. Drugs targeting colony-stimulating factor-1 receptor (CSF1R) to block microglial proliferation in preclinical disease models have shown mixed outcomes, thus the therapeutic potential of this approach remains unclear. Here, we evaluated CSF1R inhibitors in tauopathy mice using multiple dosing schemes, drug analogs, and longitudinal measurements in the brain and plasma. In both spontaneous disease and in tau fibril inoculation models, we found a region-dependent reduction in insoluble phosphorylated tau and replication-competent tau in mice treated with CSF1R inhibitors. Surprisingly, despite greater drug exposure and microglial depletion in male mice, we observed a rescue of aberrant behavior, reduced plasma neurofilament light chain, and extended survival in female mice only. Gene expression patterns in CSF1R inhibitor-treated tauopathy mice reverted toward a normal wildtype signature, and in vivo imaging revealed suppressed astrogliosis. However, we observed drug dose-dependent upregulation of immediate early genes in male mice only, indicating excitotoxicity, which may have masked functional benefits. Drug-resilient microglia in tauopathy mice exhibited a ramified morphology similar to wildtype microglia but with greater territory occupied per cell, and their transcriptome was neither disease-associated nor homeostatic, suggesting a unique microglial subtype. Our data argue that complete or continuous microglial ablation is neither required nor desired for neuroprotection, and that selective depletion of detrimental, tauopathy-activated microglia may be achieved by precise timing and dosing of CSF1R inhibitors. Importantly, therapeutics targeting microglia must consider sex-dependent effects on functional outcomes when weighing their translational potential for neurological disease.

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Heparin promotes fibrillation of most phenol soluble modulin peptides from S. aureus: a possible strengthening of the bacterial biofilm Phenol soluble modulins (PSMs) are virulence peptides secreted by different Staphylococcus aureus strains. In addition, PSMs are able to form amyloid fibrils which may strengthen the biofilm matrix. The highly sulfated glycosaminoglycan heparin promotes S.aureus infection but the basis for this is unclear. We hypothesized that heparin promotes PSM fibrillation and in this way aids bacterial colonization. Here we address this hypothesis using a combination of different biophysical techniques along with peptide microarrays. We find that heparin accelerates fibrillation of all -PSMs (except PSM2) and {delta}-toxin, but inhibits {beta}-PSMs fibrillation by blocking nucleation. Given that S. aureus secretes higher levels of -PSMs than {beta}-PSMs peptidess, heparin is likely to overall promote fibrillation. Heparin binding is driven by multiple positively charged lysine residues in -PSMs and {delta}-toxins, whose removal strongly reduces affinity. Binding of heparin does not alter the final fibril conformation. Rather, heparin provides a scaffold to catalyze or inhibit fibrillation. Our findings suggest that heparin may strengthen bacterial biofilm through increased PSM fibrillation.

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sentences: Ligand GA: a genetic algorithm for automated protein inhibitor design Ligand GA is introduced in this work and approaches the problem of finding small molecules inhibiting protein functions by using the protein site to find close to optimal or optimal small molecule binders. Genetic algorithms (GA) are an effective means for approximating or solving computationally hard mathematics problems with large search spaces such as this one. The algorithm is designed to include constraints on the generated molecules from ADME restriction, localization in a binding site, specified hydrogen bond requirements, toxicity prevention from multiple proteins, sub-structure restrictions, and database inclusion. This algorithm and work is in the context of computational modeling, ligand design and docking to protein sites.

sentences: A study of thermally-induced sex reversal in casque-headed lizards Non-avian reptiles, unlike mammals and birds, have undergone numerous sex determination turnovers. For example, casque-headed lizards replaced the ancestral XY system shared across pleurodonts with a new pair of XY chromosomes. However, the evolutionary forces that triggered this transition have remained unclear. An interesting evolutionary hypothesis suggests that species with intermediate states, with sex chromosomes but also thermal-induced sex reversal at specific incubation temperatures, could be more susceptible to sex determination turnovers. We contrasted genotypic data (presence/absence of the Y chromosome) against the histology of gonads of embryos from stages 35-37 incubated at various temperatures, including typical male-producing (26{degrees}C) and female-producing (32{degrees}C) temperatures. We observed perfect concordance between genotype and phenotype at all temperatures. However, analysis of transcriptomic data from embryos incubated at 26{degrees}C and 32{degrees}C identified transcript variants of the chromatin modifiers JARID2 and KDM6B that have been linked to temperature-dependent sex determination in other reptiles. Besides, our work reports for the first time to our knowledge the histology of gonads, including morphological changes, from stages 35-37 of development in the Corytophanidae family. We also observed that all embryos developed hemipenes, suggesting sex-linked developmental heterochrony.Our work tested the validity of a mixed sex determination system in the Corytophanidae family. We showed that XY chromosomes are dominant, however, our work supports the hypothesis of a conserved transcriptional response to incubation temperatures across non-avian reptiles that could be the reminiscence of an ancestral sex determination system.

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Transcriptomic and rRNA:rDNA signatures of environmental vs. enteric Enterococcus faecalis isolates under oligotrophic freshwater conditions The use of enterococci as a fecal indicator bacterial group for public health risk assessment has been brought into question by recent studies showing that "naturalized" populations of E. faecalis exist in the extraenteric environment in a viable but not culturable (VBNC) state. The extent to which these naturalized or VBNC E. faecalis can confound water quality monitoring is unclear. To determine if strains isolated from different habitats display different survival strategies and responses, we compared the decay patterns of three E. faecalis isolates from the natural environment (environmental strains) against three human gut isolates (enteric strains) in laboratory mesocosms that simulate an oligotrophic, aerobic freshwater environment. Our results showed similar overall decay rates between enteric and environmental isolates based on viable plate and qPCR counts. However, the enteric isolates exhibited a spike in rRNA:rDNA ratios between days 1 and 3 of the mesocosm incubations that was not observed in environmental isolates, which could indicate a different stress response. Nevertheless, there was no strong evidence of differential expression of genes thought to be related to habitat adaptation in the accompanying mesocosm metatranscriptomes when compared between environmental and enteric isolates. Overall, our results provide novel information on how rRNA levels may vary over different metabolic states (i.e., alive vs. VBNC) for this important indicator bacteria. We also observed some evidence for habitat adaptation in E. faecalis; however, this adaptation may not be substantial or consistent enough for integration in water quality monitoring.

IMPORTANCEEnterococci are commonly used worldwide to monitor environmental fecal contamination and public health risk for waterborne diseases. However, some species within this group can enter an inactive, viable but not culturable (VBNC) state that make it difficult to accurately quantify during routine monitoring. Furthermore, lower-risk, environmental enterococci strains may also confound water quality estimates. We developed an rRNA:rDNA viability assay for E. faecalis (a predominant species within this fecal group) and tested it against both enteric and environmental isolates in freshwater mesocosms to assess whether this approach can serve as a more sensitive water quality monitoring tool. We were unable to reliably distinguish the different isolate types using this assay under the conditions tested here; thus, environmental strains should continue to be counted during routine water monitoring. However, this assay could be useful for distinguishing more recent (i.e., higher risk) fecal pollution because rRNA levels significantly decreased after one week in all isolates.

sentences: Rescuing Low Frequency Variants within Intra-Host Viral Populations directly from Oxford Nanopore sequencing data Infectious disease monitoring on Oxford Nanopore Technologies (ONT) platforms offers rapid turnaround times and low cost, exemplified by well over a half of million ONT SARS-COV-2 datasets. Tracking low frequency intra-host variants has provided important insights with respect to elucidating within host viral population dynamics and transmission. However, given the higher error rate of ONT, accurate identification of intra-host variants with low allele frequencies remains an open challenge with no viable solutions available. In response to this need, we present Variabel, a novel approach and first method designed for rescuing low frequency intra-host variants from ONT data alone. We evaluated Variabel on both within patient and across patient paired Illumina and ONT datasets; our results show that Variabel can accurately identify low frequency variants below 0.5 allele frequency, outperforming existing state-of-the-art ONT variant callers for this task. Variabel is open-source and available for download at: www.gitlab.com/treangenlab/variabel.

sentences: High Resolution Single Cell Maps Reveals Distinct Cell Organization and Function Across Different Regions of the Human Intestine The colon is a complex organ that promotes digestion, extracts nutrients, participates in immune surveillance, maintains critical symbiotic relationships with microbiota, and affects overall health. To better understand its organization, functions, and its regulation at a single cell level, we performed CODEX multiplexed imaging, as well as single nuclear RNA and open chromatin assays across eight different intestinal sites of four donors. Through systematic analyses we find cell compositions differ dramatically across regions of the intestine, demonstrate the complexity of epithelial subtypes, and find that the same cell types are organized into distinct neighborhoods and communities highlighting distinct immunological niches present in the intestine. We also map gene regulatory differences in these cells suggestive of a regulatory differentiation cascade, and associate intestinal disease heritability with specific cell types. These results describe the complexity of the cell composition, regulation, and organization for this organ, and serve as an important reference map for understanding human biology and disease.

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Compartment-specific energy requirements of photosynthetic carbon metabolism in Camelina sativa leaves Detailed knowledge about plant energy metabolism may aid crop improvements. Using published estimates of flux through central carbon metabolism, we phenotype energy metabolism in illuminated Camelina sativa leaves (grown at 22 {degrees}C, 500 mol photons m-2 s-1) and report several findings. First, the oxidative pentose phosphate pathway (OPPP) transfers 3.3% of the NADPH consumed in the Calvin-Benson cycle to the cytosol. NADPH supply proceeds at about 10% of the rate of net carbon assimilation. However, concomitantly respired CO2 accounts for 4.8% of total rubisco activity. Hence, 4.8% of the flux through the Calvin-Benson cycle and photorespiration is spent on supplying cytosolic NADPH, a significant investment. Associated energy requirements exceed the energy output of the OPPP. Thus, autotrophic carbon metabolism is not simply optimised for flux into carbon sinks but sacrifices carbon and energy use efficiency to support cytosolic energy metabolism. To reduce these costs, we suggest bioengineering plants with a repressed cytosolic OPPP, and an inserted cytosolic NADPH-dependent malate dehydrogenase tuned to compensate for the loss in OPPP activity (if required). Second, sucrose cycling is a minor investment in overall leaf energy metabolism but a significant investment in cytosolic energy metabolism. Third, leaf energy balancing strictly requires oxidative phosphorylation, cofactor export from chloroplasts, and peroxisomal NADH import. Fourth, mitochondria are energetically self-sufficient. Fifth, carbon metabolism has an ATP/NADPH demand ratio of 1.52 which is met if [≤]21.7% of whole electron flux is cyclic. Sixth, electron transport has a photon use efficiency of [≥]62%. Lastly, we discuss interactions between the OPPP and the cytosolic oxidation-reduction cycle in supplying leaf cytosolic NADPH.

Main ConclusionThe oxidative pentose phosphate pathway provides cytosolic NADPH yet reduces carbon and energy use efficiency. Repressing this pathway and introducing cytosolic NADPH-dependent malate dehydrogenase may increase crop yields by {approx}5%.

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Reduced Dimension, Biophysical Neuron Models Constructed From Observed Data Using methods from nonlinear dynamics and interpolation techniques from applied mathematics, we show how to use data alone to construct discrete time dynamical rules that forecast observed neuron properties. These data may come from from simulations of a Hodgkin-Huxley (HH) neuron model or from laboratory current clamp experiments. In each case the reduced dimension data driven forecasting (DDF) models are shown to predict accurately for times after the training period.

When the available observations for neuron preparations are, for example, membrane voltage V(t) only, we use the technique of time delay embedding from nonlinear dynamics to generate an appropriate space in which the full dynamics can be realized.

The DDF constructions are reduced dimension models relative to HH models as they are built on and forecast only observables such as V(t). They do not require detailed specification of ion channels, their gating variables, and the many parameters that accompany an HH model for laboratory measurements, yet all of this important information is encoded in the DDF model.

As the DDF models use only voltage data and forecast only voltage data, they can be used in building networks with biophysical connections. Both gap junction connections and ligand gated synaptic connections among neurons involve presynaptic voltages and induce postsynaptic voltage response. Biophysically based DDF neuron models can replace other reduced dimension neuron models, say of the integrate-and-fire type, in developing and analyzing large networks of neurons.

When one does have detailed HH model neurons for network components, a reduced dimension DDF realization of the HH voltage dynamics may be used in network computations to achieve computational efficiency and the exploration of larger biological networks.

sentences: Does extracellular DNA mask microbial responses to a pulse disturbance? A major goal in microbial ecology is to predict how microbial communities will respond to global change. However, DNA-based sequencing that is intended to characterize live microbial communities includes extracellular DNA (exDNA) from non-viable cells. This could obscure relevant microbial responses, particularly to pulse disturbances which kill bacteria and have disproportionate effects on ecosystems. Here, we characterize bacterial communities before and after a drying-rewetting pulse disturbance, using an improved method for exDNA exclusion. We find that exDNA removal is important for detecting subtle yet significant changes in microbial abundance, diversity, and community composition across the disturbance. However, inclusion of exDNA did not obscure results to a large extent, only sometimes altering statistical significance but rarely changing the direction of the response or general conclusions about bacterial disturbance dynamics. Although there may be instances where exDNA removal is essential for accurate representation of microbial communities, our study suggests these scenarios will be difficult to predict a priori. Overall, we found no evidence that certain time points across the distrubance were more affected by exDNA inclusion, nor did the size or composition of exDNA pools accurately predict when exDNA would alter significance levels. However, exDNA dynamics did vary strongly across the two soil types tested.

sentences: Phylogenetic and protein prediction analysis reveals the taxonomically diverse distribution of virulence factors in the Bacillus cereus group Bacillus cereus is a food contaminant with widely varying enterotoxic potential of its virulence proteins. In this article, phylogenetic analysis of the whole-genome amino acid sequences of 41 strains, evolutionary distance calculation of the amino acid sequences of the virulence genes, and functional and structural prediction of the virulence proteins were performed to reveal the taxonomically diverse distribution of virulence factors. The genome evolution of the strains showed a clustering trend based on the coding virulence genes. The strains of B. cereus have evolved into non-toxic risk and toxic risk clusters with medium-high- and medium-low-risk clusters. The distances of evolutionary transfer relative to housekeeping genes of incomplete virulence genes were greater than those of complete virulence genes, and the distance values of HblACD were higher than those of nheABC and CytK among the complete virulence genes. Cytoplasmic localization was impossible for all the virulence proteins, and NheB, NheC, Hbl-B, and Hbl-L1 were extracellular according to predictive analysis. Nhe and Hbl proteins except CytK had similar spatial structures. The predicted structures of Nhe and Hbl mainly showed  head and  tail domains. The  head of NheA and Hbl-B, including two -helices separated by {beta}-tongue strands, might play a special role in Nhe trimers and Hbl trimers, respectively. The  cap of CytK, which includes two  latches with many {beta}-sheets, formed a {beta}-barrel structure with pores, and a  rim balanced the structure. The evolution of B. cereus strains showed a clustering tendency based on the coding virulence genes, and the complete virulence-gene operon combination had higher relative genetic stability. The beta-tongue or latch associated with {beta}-sheet folding might play an important role in the binding of virulence structures and pore-forming toxins in B. cereus.

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DNA damage induces senescent phenotypes in mouse NIT1 beta cells and human islets ObjectiveType 1 Diabetes (T1D) is characterized by progressive loss of insulin-producing pancreatic {beta} cells as a result of autoimmune destruction. In addition to {beta} cell death, recent work has shown that subpopulations of {beta} cells acquire dysfunction during T1D. We previously reported that some {beta} cells adopt a senescent fate involving a DNA damage response (DDR) during the pathogenesis of T1D, however, the question of how senescence develops in {beta} cells has not been investigated.

MethodsHere, we tested the hypothesis that unrepaired DNA damage triggers {beta} cell senescence using culture models including the mouse NIT1 {beta} cell line derived from the T1D-susceptible nonobese diabetic (NOD) strain, human donor islets and EndoC {beta} cells. DNA damage was chemically induced using etoposide or bleomycin and cells or islets were analyzed by a combination of molecular assays for senescence phenotypes including Western blotting, qRT-PCR, Luminex assays, flow cytometry and histochemical staining. RNA-seq was carried out to profile global transcriptomic changes in human islets undergoing DDR and senescence. Insulin ELISAs were used to quantify glucose stimulated insulin secretion from chemically-induced senescent islets and cells in culture.

ResultsSub-lethal DNA damage in NIT1 cells led to several classical hallmarks of senescence including sustained DDR activation, growth arrest, enlarged flattened morphology and a senescence-associated secretory phenotype (SASP) resembling what occurs in primary {beta} cells during T1D in NOD mice. Some of these phenotypes differed between NIT1 cells and the MIN6 {beta} cell line derived from a non-T1D susceptible mouse strain. RNA-seq analysis of human islets undergoing DDR and early senescence revealed a coordinated p53-p21 transcriptional program and upregulation of prosurvival signaling and SASP genes, which were confirmed in independent islet preparations at the protein level. Importantly, chemically induced DNA damage also led to DDR activation and senescence phenotypes in the EndoC-{beta}H5 human {beta} cell line, confirming that this response can occur directly in human {beta} cells. Finally, DNA damage and senescence in both mouse {beta} cell lines and human islets led to decreased insulin content.

ConclusionsTaken together, these findings suggest that some of the phenotypes of {beta} cell senescence during T1D can be modeled by chemically induced DNA damage in mouse {beta} cell lines and human islets and {beta} cells in culture. These culture models will be useful tools to understand some of the mechanisms of {beta} cell senescence in T1D.

HighlightsO_LIDNA damage induces senescent phenotypes in mouse {beta} cell lines
C_LIO_LIDNA damage induces a p53-p21 transcriptional program and senescent phenotypes in human islets and EndoC cells
C_LIO_LIDNA damage and senescence leads to decreased insulin content
C_LIO_LIDNA damage models some aspects of {beta} cell senescence in Type 1 Diabetes
C_LI

sentences: Complete metamorphosis and microbiota turnover in insects The insects constitute the majority of animal diversity. Most insects are holometabolous: during complete metamorphosis their bodies are radically re-organized. This re-organization poses a significant challenge to the gut microbiota, as the gut is replaced during pupation, a process that does not occur in hemimetabolous insects. In holometabolous hosts, it offers the opportunity to decouple the gut microbiota between the larval and adult life stages resulting in high beta diversity whilst limiting alpha diversity. Here we studied 18 different herbivorous insect species from 5 orders of holometabolous and 3 orders of hemimetabolous insects. Comparing larval and adult specimens, we find a much higher beta-diversity and hence microbiota turnover in holometabolous insects compared to hemimetabolous insects. Alpha diversity did not differ between holo-and hemimetabolous insects nor between developmental stages within these groups. Our results support the idea that pupation offers the opportunity to change the gut microbiota and hence facilitates ecological niche shifts. This effect of niche shift facilitation could explain a selective advantage of the evolution of complete metamorphosis, which is a defining trait of the most speciose insect taxon, the holometabola.

sentences: Erasing motion: Scrambling direction selectivity in visual cortex during saccades Distinguishing sensory stimuli caused by changes in the environment from those caused by an animals own actions is a hallmark of sensory processing. Saccades are rapid eye movements that shift the image on the retina. How visual systems differentiate motion of the image induced by saccades from actual motion in the environment is not fully understood. Here, we discovered that in mouse primary visual cortex (V1), the two types of motion evoke distinct activity patterns. This is because during saccades, V1 combines the visual input with a strong non-visual input arriving from the thalamic pulvinar nucleus. Thus, the pulvinar input ensures differential V1 responses to external and self-generated motion and may allow the animal to interpret sensory stimuli within the context of its actions.

sentences: Decitabine Reactivation of FoxM1-Dependent Endothelial Regeneration and Vascular Repair for Potential Treatment of Elderly ARDS and COVID-19 Patients Aging is a major risk factor of high incidence and increased mortality of acute respiratory distress syndrome (ARDS) and COVID-19. We repot that aging impairs the intrinsic FoxM1-dependent endothelial regeneration and vascular repair program and causes persistent lung injury and high mortality following sepsis. Therapeutic gene transduction of FOXM1 in vascular endothelium or treatment with FDA-approved drug Decitabine was sufficient to reactivate FoxM1-dependent lung endothelial regeneration in aged mice, reverse aging-impaired resolution of inflammatory injury, and promote survival. In COVID-19 lung autopsy samples, FOXM1 expression was not induced in vascular endothelial cells of elderly patients in contrast to mid-age patients. Thus, Decitabine reactivation of FoxM1-dependent vascular repair represents a potential effective therapy for elderly COVID-19 and non-COVID-19 ARDS patients.

sentences: Proximal Exploration for Model-guided Protein Sequence Design Designing protein sequences with a particular biological function is a long-lasting challenge for protein engineering. Recent advances in machine-learning-guided approaches focus on building a surrogate sequence-function model to reduce the demand for expensive in-lab experiments. In this paper, we study the exploration mechanism of model-guided sequence design. We leverage a natural property of protein fitness landscape that a concise set of mutations upon the wild-type sequence are usually sufficient to enhance the desired function. By utilizing this property, we propose Proximal Exploration (PEX) algorithm that conducts a proximal optimization framework to prioritize the search for advantageous mutants with low mutation counts. In addition, we construct a specialized model architecture to predict low-order mutational effects, which further improves the sample efficiency of model-guided evolutionary search. We extensively evaluate our method on a suite of in-silico protein sequence design tasks and demonstrate substantial improvement over baseline algorithms.

sentences: Lateral habenula M2 muscarinic receptor control of neuronal activity and cocaine seeking behavior The lateral habenula (LHb) plays a central role in balancing reward and aversion by opposing the contributions of brain reward nuclei. Using a rat cocaine self-administration model, we previously found that LHb inhibition or non-selective blockade of LHb muscarinic acetylcholine receptors (mAChRs) led to persistent cocaine seeking despite its signaled unavailability. As understanding roles for the LHb and cholinergic signaling in behavioral control is important to psychiatric illness and addiction, we examine how mAChRs act on LHb neurons using in vitro electrophysiology. We find that different groups of LHb neurons are depolarized or hyperpolarized by the cholinergic agonist carbachol (CCh), and that CCh could inhibit GABAergic and glutamatergic synaptic inputs to these cells. Presynaptic CCh effects were reversed by the M2 mAChR (M2R) antagonist AFDX-116, but not by pirenzepine, an M1R antagonist. Contemporaneous measurement of CCh effects on synaptic inhibition and excitation in LHb neurons showed a smaller effect on inhibition, suggesting a net shift in synaptic integration toward greater inhibition by mAChRs. Synaptic currents elicited by light-activation of ventral tegmental area (VTA) axons in the LHb, following channelrhodopsin-2 transfection of VTA, were also inhibited by M2Rs, suggesting the VTA as at least one M2R-sensitive LHb afferent. Finally, Go-NoGo cocaine seeking studies showed that blockade of LHb M2Rs, and not M1Rs, triggered continued cocaine seeking. These data identify LHb M2Rs as a potential control point of LHb function that enables withholding responses for cocaine and define cellular mechanisms through which mAChRs modulate LHb activity.

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Host-symbiont population genomics provide insights into partner fidelity, transmission mode and habitat adaptation in deep-sea hydrothermal vent snails Symbiont specificity, both at the phylotype and strain level, can have profound consequences for host ecology and evolution. However, except for insights from a few model symbiosis systems, the degree of partner fidelity and the influence of host versus environmental factors on symbiont composition are still poorly understood. Nutritional symbioses between invertebrate animals and chemosynthetic bacteria at deep-sea hydrothermal vents are examples of relatively selective associations, where hosts affiliate only with particular, environmentally acquired phylotypes of gammaproteobacterial or campylobacterial symbionts. In hydrothermal vent snails of the sister genera Alviniconcha and Ifremeria this phylotype specificity has been shown to play a role in habitat distribution and partitioning among different holobiont species. However, it is currently unknown if fidelity goes beyond species level associations that might influence genetic structuring, connectivity and habitat adaptation of holobiont populations. We used metagenomic analyses to assess sequence variation in hosts and symbionts and identify correlations with geographic and environmental factors. Our analyses indicate that host populations are not differentiated across a ~800 km gradient, while symbiont populations are clearly structured between vent locations due to a combination of neutral and selective processes. Overall, these results suggest that host individuals flexibly associate with locally adapted strains of their specific symbiont phylotypes, which supports a long-standing but untested paradigm of the benefits of horizontal transmission. Strain flexibility in these snails likely enables host populations to exploit a range of habitat conditions, which might favor wide-spread genetic connectivity and ecological resilience unless physical dispersal barriers are present.

Significance StatementSymbiont composition in horizontally transmitted symbioses is influenced by a combination of host genetics, environmental conditions and geographic barriers. Yet the relative importance of these factors and the effects of adaptive versus neutral evolutionary forces on symbiont population structure remain unknown in the majority of marine symbioses. To address these questions, we applied population genomic approaches in four species of deep-sea hydrothermal vent snails that live in obligate association with chemosynthetic bacteria. Our analyses show that host genetics plays a minor role compared to environment for symbiont strain composition despite specificity to symbiont species and corroborate a long-standing hypothesis that vent invertebrates affiliate with locally adapted symbiont strains to cope with the variable habitat conditions characterizing hydrothermal vents.

sentences: Heterogeneous mutation rates and spectra within and between species of yeast and their hybrids Mutation rates and spectra vary between species and among populations. Hybridization can contribute to this variation, but its role remains poorly understood. Estimating mutation rates requires controlled conditions where the effect of natural selection can be minimized. One way to achieve this is through mutation accumulation experiments coupled with genome sequencing. Here we investigate 400 mutation accumulation lines initiated from 11 genotypes spanning intra-lineage, inter-lineage and interspecific crosses of the yeasts Saccharomyces paradoxus and S. cerevisiae and propagated for 770 generations. We find significant differences in mutation rates and spectra among crosses, which are not related to the level of divergence of parental strains but are specific to some genotype combinations. Differences in number of generations and departures from neutrality play a minor role, whereas polyploidy and loss of heterozygosity impact mutation rates in some of the hybrid crosses in an opposite way.

sentences: Conformist social learning leads to self-organised prevention against adverse bias in risky decision making Given the ubiquity of potentially adverse behavioural bias owing to myopic trial-and-error learning, it seems paradoxical that improvements in decision-making performance through conformist social learning, a process widely considered to be bias amplification, still prevail in animal collective behaviour. Here we show, through model analyses and large-scale interactive behavioural experiments with 585 human subjects, that conformist influence can indeed promote favourable risk taking in repeated experience-based decision making, even though many individuals are systematically biased towards adverse risk aversion. Although strong positive feedback conferred by copying the majoritys behaviour could result in unfavourable informational cascades, our differential equation model of collective behavioural dynamics identified a key role for increasing exploration by negative feedback arising when a weak minority influence undermines the inherent behavioural bias. This  collective behavioural rescue, emerging through coordination of positive and negative feedback, highlights a benefit of collective learning in a broader range of environmental conditions than previously assumed and resolves the ostensible paradox of adaptive collective behavioural flexibility under conformist influences.

sentences: Epigenetic therapy to enhance therapeutic effects of PD-1 inhibition in uveal melanoma Targeted therapy and immunotherapy have revolutionized the treatment of metastatic skin melanoma but none of the treatments are approved for patients with metastatic uveal melanoma (UM). Here we hypothesized that the poor responses to immunotherapy of UM can be enhanced by epigenetic modulation using HDAC or BET inhibitors (BETi). Cultured uveal melanoma cells were treated with the HDAC inhibitor (HDACi) entinostat or BETi JQ1. Entinostat induced HLA expression and PD-L1, but JQ1 did not. A syngenic mouse model carrying B16-F10 melanoma cells were treated with PD-1 and CTLA-4 inhibitors, which was curative. Co-treatment with the bioavailable BETi iBET-726 impaired the immunotherapy effect. Monotherapy of a B16-F10 mouse model with anti-PD-1 resulted in a moderate therapeutic effect that could be enhanced by entinostat. Mice carrying PD-L1 knockout B16-F10 cells were also sensitive to entinostat. This suggests HDAC inhibition and immunotherapy could work in concert. Indeed, co-cultures of UM with HLA-matched melanoma-specific tumor-infiltrating lymphocytes (TILs) resulted in higher TIL-mediated melanoma killing when entinostat was added. Further exploration of combined immunotherapy and epigenetic therapy in metastatic UM is warranted.

sentences: Interactions between anthropogenic stressors and recurring perturbations mediate ecosystem resilience or collapse Insights into declines in ecosystem resilience, their causes and effects, can inform pre-emptive action to avoid ecosystem collapse and loss of biodiversity, ecosystem services and human well-being. Empirical studies of ecosystem collapse are rare and hampered by ecosystem complexity, non-linear and lagged responses, and interactions across scales. We investigated how an anthropogenic stressor could diminish ecosystem resilience to a recurring perturbation by altering a critical ecosystem driver. We studied groundwater-dependent, peat-accumulating, fire-prone wetlands in southeastern Australia. We hypothesised that underground mining (stressor) reduced resilience of these wetlands to landscape fires (perturbation) by diminishing groundwater, a key ecosystem driver. We monitored soil moisture as an indicator of ecosystem resilience during and after underground mining and, after a landscape fire, we compared the responses of multiple state variables representing ecosystem structure, composition and function in wetlands within the mining footprint to unmined reference wetlands. Soil moisture showed very strong evidence of decline without recovery in mined swamps, but was maintained in reference swamps through eight years. Relative to burnt reference swamps, burnt and mined swamps showed greater loss of peat via substrate combustion, reduced cover, height and biomass of regenerating vegetation, reduced post-fire plant species richness and abundance, altered plant species composition, increased mortality rates of woody plants, reduced post-fire seedling recruitment, and local extinction of a hydrophilc fauna species. Mined swamps therefore showed strong symptoms of post-fire ecosystem collapse, while reference swamps regenerated vigorously. We conclude that an anthropogenic stressor may diminish the resilience of an ecosystem to recurring perturbations, predisposing it to collapse. Avoidance of ecosystem collapse hinges on early diagnosis of mechanisms and preventative risk reduction. It may be possible to delay or ameliorate symptoms of collapse or to restore resilience, but the latter appears unlikely in our study system due to fundamental alteration of a critical ecosystem driver.

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Cancer models and statistical analysis of age distribution of cancers In this paper, mathematical mutational models of the age distribution of cancers are obtained. These are two models - a simple model and a complex model, which takes into account the growth of the cell population and the transmission of mutations to daughter cells. Using the resulting formulas, we approximated real age-specific cancer incidence datasets in women (colon, lung, mammary, stomach) and men (colon, lung, prostate, stomach). We estimated parameters such as the average number of mutations (per cell per unit of time) and number of mutations required for cancer to occur.

The number of mutations averaged (over four types of cancer) required for cancer to occur is 5.5 (mutations per cell for women) and 6.25 (mutations per cell for men) for the complex mutational model.

As an alternative to mutational models, we also consider the model of delayed carcinogenic event.

sentences: A hymenopteran odorant alerts flies to bury eggs Ants are ubiquitous and consume insects at all life stages, presumably creating a strong selective pressure for ant avoidance behaviors across insects. The insect egg stage can be especially defenseless against predation given that eggs are usually immobile and unguarded, suggesting insect mothers may have evolved oviposition strategies to minimize the ant predation risk to their offspring. Given the lack of parental care in most insects, these oviposition strategies would likely be innate rather than learned, since insect mothers are not usually present to assess predation of their eggs. Here, we use the vinegar fly Drosophila melanogaster as a model system for examining parental defensive responses to ant presence. Flies usually lay eggs partially inserted into the food substrate, although some are laid on top of the food and a few are inserted deeply into the food. We found that exposure to ants significantly alters fly oviposition depth: the proportion of eggs on the food surface decreased while the proportion of buried eggs increased. Buried eggs survive ant foraging bouts better than surface eggs, showing that this oviposition depth behavior is adaptive. This induced behavior is conserved across the genus Drosophila and is dependent on the fly olfactory system: anosmic mutant flies fail to bury their eggs in the presence of ants, and ant odor extracts are sufficient to induce egg burying. To further delineate the ant lineages to which flies respond, we exposed flies to the odors from numerous species of ants and other insects. Surprisingly, flies buried their eggs in response to the odors of nearly all hymenopterans tested, including hymenopteran groups that flies rarely interact with in nature like bees and paper wasps. Our data suggest that hymenopterans possess a conserved and ancient odorant, and that drosophilids evolved a mechanism for sensing this odorant early in their evolution as a means of protecting their offspring from ant predation. This study sheds light on the ecology and mechanisms underlying a sscommon biotic interaction in nature, that between insect parents ands the ants that would consume their offspring.

sentences: An atlas of gene expression variation across the Caenorhabditis elegans species Phenotypic variation in diverse organism-level traits have been studied in Caenorhabditis elegans wild strains, but differences in gene expression and the underlying variation in regulatory mechanisms are largely unknown. Here, we use natural variation in gene expression to connect genetic variants to differences in organismal- level traits, including drug and toxicant responses. We performed transcriptomic analysis on 207 genetically distinct C. elegans wild strains to study natural regulatory variation of gene expression. Using this massive dataset, we performed genome-wide association mappings to investigate the genetic basis underlying gene expression variation and revealed complex genetic architectures. We found a large collection of hotspots enriched for expression quantitative trait loci across the genome. We further used mediation analysis to understand how gene expression variation could underlie organism-level phenotypic variation for a variety of complex traits. These results reveal the natural diversity in gene expression and possible regulatory mechanisms in this keystone model organism, highlighting the promise of gene expression variation in shaping phenotypic diversity.

sentences: PALB2 maintains redox and mitochondrial homeostasis in the brain and cooperates with ATG7 to suppress p53 dependent neurodegeneration The PALB2 tumor suppressor plays key roles in DNA repair and has been implicated in redox homeostasis. Autophagy maintains mitochondrial quality, mitigates oxidative stress and suppresses neurodegeneration. Here we show that Palb2 deletion in the mouse brain leads to motor deficits and that co-deletion of Palb2 with the essential autophagy gene Atg7 accelerates and exacerbates neurodegeneration induced by ATG7 loss. Palb2 deletion leads to elevated DNA damage, oxidative stress and mitochondrial markers, especially in Purkinje cells, and co-deletion of Palb2 and Atg7 results in accelerated Purkinje cell loss. Further analyses suggest that the accelerated Purkinje cell loss and severe neurodegeneration in the double deletion mice are due to oxidative stress and mitochondrial dysfunction, rather than DNA damage, and partially dependent on p53 activity. Our studies uncover a role of PALB2 in mitochondrial regulation and a cooperation between PALB2 and ATG7/autophagy in maintaining redox and mitochondrial homeostasis essential for neuronal survival.

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Colouring coral larvae for tracking dispersal O_LIEcologists have long sought to understand larval dispersal characteristics of benthic marine invertebrates. Direct quantification of coral larvae dispersal has proven challenging, in part due to their complex life-history, minute size, and widespread dispersal at the scale of kilometres. Instead, indirect methods such as particle modelling, chemical signatures, and genetic correlation are often used in dispersal studies.
C_LIO_LIHere, we develop a direct method of quantifying larval dispersal by applying vital stains to coral larvae, allowing differentiation and direct tracking of millions of larvae from the pelagic dispersal stage through to the sedentary stages of attachment and metamorphosis on coral reefs.
C_LIO_LINeutral red and Nile blue stains were extremely effective at staining coral larvae, while alizarin red and calcein blue showed no visible results. Differences in toxicity to vital stains was noted among species, with Acropora spp. exhibiting decreased larval survival and settlement, while Merulinidae spp. were unaffected. By experimenting with different incubation times and concentrations, our results indicate that neutral red can be effectively applied for short periods (<20 minutes) at low concentrations (1-100 mg l-1), whereas Nile blue requires longer stain times (>60 minutes) at higher concentrations (100-1000 mg l-1).
C_LIO_LIThe strong colour of both neutral red and Nile blue stains was retained by newly settled larvae in lab settings upwards of five days following settlement, providing a direct method of differentiating between newly settled larvae on reefs. Field-validation of Nile blue applied to coral larvae from wild-captured coral slicks demonstrates the efficacy of staining across a diverse range of coral taxa.
C_LIO_LIVital staining provides a simple, rapid (<60 mins), and low cost (<AUD$0.00001 per larva) method of colouring coral larvae that allows for direct tracking of dispersal and recruitment in studies of reef connectivity and restoration.
C_LI

sentences: Rapid and strain-specific resistance evolution of Staphylococcus aureus against inhibitory molecules secreted by Pseudomonas aeruginosa Pseudomonas aeruginosa and Staphylococcus aureus frequently occur together in polymicrobial infections, and there is evidence that their interactions negatively affect disease outcome in patients. At the molecular level, interactions between the two bacterial taxa are well-described, with P. aeruginosa usually being the dominant species suppressing S. aureus through a variety of inhibitory molecules. However, in polymicrobial infections, the two species interact over prolonged periods of time, and S. aureus might evolve resistance against inhibitory molecules deployed by P. aeruginosa. Here, we used experimental evolution to test this hypothesis by exposing three different S. aureus strains (Cowan I, 6850, JE2) to the growth-inhibitory supernatant of P. aeruginosa PAO1 over 30 days. We found that all three S. aureus strains rapidly evolved resistance against inhibitory molecules and show that (i) adaptations were strain-specific; (ii) resistance evolution affected the expression of virulence traits; and (iii) mutations in membrane transporters were the most frequent evolutionary targets. Our work indicates that adaptations of S. aureus to co-infecting pathogens could increase virulence and decrease antibiotic susceptibility, because both virulence traits and membrane transporters involved in drug resistance were under selection. Thus, pathogen co-evolution could exacerbate infections and compromise treatment options.

sentences: Antifragile therapy Antifragility is a recently coined word used to describe the opposite of fragility. Systems or organisms can be described as antifragile if they derive a benefit from systemic variability, volatility, randomness, or disorder. Herein, we introduce a mathematical framework to quantify the fragility or antifragility of cancer cell lines in response to treatment variability. This framework enables straightforward prediction of the optimal dose treatment schedule for a range of treatment schedules with identical cumulative dose. We apply this framework to non-small-cell lung cancer cell lines with evolved resistance to ten anti-cancer drugs. We show the utility of this antifragile framework when applied to 1) treatment resistance, and 2) collateral sensitivity of sequential monotherapies.

sentences: High-resolution spatiotemporal transcriptomic maps of developing Drosophila embryos and larvae Drosophila has long been a successful model organism in multiple fields such as genetics and developmental biology. Drosophila genome is relatively smaller and less redundant, yet largely conserved with mammals, making it a productive model in studies of embryogenesis, cell signaling, disease mechanisms, etc. Spatial gene expression pattern is critical for understanding of complex signaling pathways and cell-cell interactions, whereas temporal gene expression changes need to be tracked during highly dynamic activities such as tissue development and disease progression. Systematic studies in Drosophila as a whole are still impeded by lack of these spatiotemporal transcriptomic information. Drosophila embryos and tissues are of relatively small size, limiting the application of current technologies to comprehensively resolve their spatiotemporal gene expression patterns. Here, utilizing SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq), we dissected the spatiotemporal transcriptomic changes of developing Drosophila with high resolution and sensitivity. Our data recapitulated the spatial transcriptomes of embryonic and larval development in Drosophila. With these data, we identified known and previously undetected subregions in several tissues during development, and revealed known and potential gene regulatory networks of transcription factors within their topographic background. We further demonstrated that Stereo-seq data can be used for 3D reconstruction of Drosophila embryo spatial transcriptomes. Our data provides Drosophila research community with useful resources of spatiotemporally resolved transcriptomic information across developmental stages.

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Chained structure of dimeric F1-like ATPase in Mycoplasma mobile gliding machinery Mycoplasma mobile, a fish pathogen, exhibits gliding motility using ATP hydrolysis on solid surfaces, including animal cells. The gliding machinery can be divided into surface and internal structures. The internal structure of the motor is composed of 28 so-called  chains that are each comprised of 17 repeating protein units called  particles. These proteins include homologs of the catalytic - and {beta}-subunits of F1-ATPase. In this study, we isolated the particles and determined their structures using negative-staining electron microscopy and high-speed atomic force microscopy. The isolated particles were composed of five proteins, MMOBs 1660 (-subunit homolog), 1670 ({beta}-subunit homolog), 1630, 1620, and 4530, and showed ATP hydrolyzing activity. The 2D structure, with dimensions of 35 and 26 nm, showed a hexameric ring dimer about 12 nm in diameter, resembling F1-ATPase catalytic ({beta})3. We isolated the F1-like ATPase unit, which is composed of MMOBs 1660, 1670, and 1630. Furthermore, we isolated the chain and analyzed the 3D structure, showing that dimers of mushroom-like structures resembling F1-ATPase were connected and aligned along the dimer axis at 31 nm intervals. An atomic model of F1-ATPase catalytic ({beta})3 from Bacillus PS3 was successfully fitted to each hexameric ring of the mushroom-like structure. These results suggest that the motor for M. mobile gliding shares an evolutionary origin with F1-ATPase. Based on the obtained structure, we propose possible force transmission processes in the gliding mechanism.

IMPORTANCEF1Fo- ATPase, a rotary ATPase, is widespread in the membranes of mitochondria, chloroplasts, and bacteria, and converts ATP energy with a proton motive force across the membrane by its physical rotation. Homologous protein complexes play roles in ion and protein transport. Mycoplasma mobile, a pathogenic bacterium, was recently suggested to have a special motility system evolutionarily derived from F1-ATPase. The present study isolated the protein complex from Mycoplasma cells and supported this conclusion by clarifying the detailed structures containing common and novel features as F1-ATPase relatives.

sentences: Population structure and association mapping for hundred seed weight in mungbean minicore Mungbean is an important legume rich in protein and carbohydrates. It is cultivated mostly in south to south east Asia and east Africa. One of the important yield contributing traits in mungbean is 100 seed weight. Variation for this trait is available in the mungbean germplasm, and to facilitate improvement of this trait through breeding, genome-wide association mapping for 100 seed weight was carried out in the World Vegetable Center mungbean minicore collection. A total of 24,870 single nucleotide polymorphic markers were tested for association with 100 see weight in plants grown in Pakistan, Bangladesh, Myanmar and Taiwan and candidate loci associated with seed weight on chromosomes 1, 4, 6, 8 and 9 were identified. None of the QTLs was stable across all environments, but loci on chromosomes 4 and 8 were significant in Pakistan and Bangladesh and other loci on chromosome 8 were significantly associated in plants grown in Myanmar and Taiwan.

sentences: Transgenic mice for in vivo epigenome editing with CRISPR-based systems The discovery, characterization, and adaptation of the RNA-guided clustered regularly interspersed short palindromic repeat (CRISPR)-Cas9 system has greatly increased the ease with which genome and epigenome editing can be performed. Fusion of chromatin-modifying domains to the nuclease-deactivated form of Cas9 (dCas9) has enabled targeted gene activation or repression in both cultured cells and in vivo in animal models. However, delivery of the large dCas9 fusion proteins to target cell types and tissues is an obstacle to widespread adoption of these tools for in vivo studies. Here we describe the generation and validation of two conditional transgenic mouse lines for targeted gene regulation, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. Using the dCas9p300 and dCas9KRAB transgenic mice we demonstrate activation or repression of genes in both the brain and liver in vivo, and T cells and fibroblasts ex vivo. We show gene regulation and targeted epigenetic modification with gRNAs targeting either transcriptional start sites (TSS) or distal enhancer elements, as well as corresponding changes to downstream phenotypes. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.

sentences: Cortical Silent Period reflects individual differences in action stopping performance. Inhibitory control is the ability to suppress inappropriate movements and unwanted actions, allowing to regulate impulses and responses. This ability can be measured via the Stop Signal Task, which provides a temporal index of response inhibition, the stop signal reaction time (SSRT). At the neural level, Transcranial Magnetic Stimulation (TMS) allows to investigate of motor inhibition within the primary motor cortex (M1), such as the Cortical Silent period (CSP). CSPs length is an index of GABAB-mediated intracortical inhibition within M1. Although there is strong evidence that intracortical inhibition varies during action stopping, it is still not clear whether differences in the neurophysiological markers of intracortical inhibition contribute to behavioral differences in actual inhibitory capacities. Hence, we here explored the relationship between intracortical inhibition within M1 and behavioral response inhibition. GABABergic-mediated inhibition in M1 was determined by the length of CSP, while behavioral inhibition was assessed by the SSRT. We found a significant positive correlation between CSPs length and SSRT, namely that individuals with greater levels of GABABergic-mediated inhibition seem to perform overall worse in inhibiting behavioral responses. These results support the assumption that individual differences in intracortical inhibition are mirrored by individual differences in action stopping abilities.

sentences: Addressing the Embeddability Problem in Transition Rate Estimation Markov State Models (MSM) and related techniques have gained significant traction as a tool for analyzing and guiding molecular dynamics (MD) simulations due to their ability to extract structural, thermodynamic, and kinetic information on proteins using computationally feasible MD simulations. The MSM analysis often relies on spectral decomposition of empirically generated transition matrices. Here, we discuss an alternative approach for extracting the thermodynamic and kinetic information from the so-called rate/generator matrix rather than the transition matrix. Although the rate matrix itself is built from the empirical transition matrix, it provides an alternative approach for estimating both thermodynamic and kinetic quantities, particularly in diffusive processes. We particularly discuss a fundamental issue with this approach, known as the embeddability problem and offer ways to address this issue. We describe eight different methods to overcome the embeddability problem, including a novel approach developed for this work. The algorithms were tested on data from a one-dimensional toy model to show the workings of these methods and discuss the robustness of each method in terms of its dependence in lag time and trajectory length.

sentences: Goal-directed recruitment of Pavlovian biases through selective visual attention Prospective outcomes bias behavior in a "Pavlovian" manner: Reward prospect invigorates action, while punishment prospect suppresses it. Theories have posited Pavlovian biases as global action "priors" in unfamiliar or uncontrollable environments. However, this account fails to explain the strength of these biases--causing frequent action slips--even in well-known environments. We propose that Pavlovian control is additionally useful if flexibly recruited by instrumental control. Specifically, instrumental action plans might shape selective attention to reward/ punishment information and thus the input to Pavlovian control. In two eye-tracking samples (N = 35/ 64), we observed that Go/ NoGo action plans influenced when and for how long participants attended to reward/ punishment information, which in turn biased their responses in a Pavlovian manner. Participants with stronger attentional effects showed higher performance. Thus, humans appear to align Pavlovian control with their instrumental action plans, extending its role beyond action defaults to a powerful tool ensuring robust action execution.

sentences: Deconstructing the role of iron and heme in the structure of healthy human gut microbial communities The response of the human gut microbiome to disruptions is often difficult to discern without model systems that remove the complexity of the host environment. Fluctuations in iron availability provide a case in point: the responses of pathogenic bacteria to iron in vitro are much better understood than those of the indigenous human gut commensal microbiota. In a clinical study of iron supplementation in healthy humans, we identified gradual, participant-specific microbiota shifts correlated with bacterial iron internalization. To identify direct effects due to taxon-specific iron sensitivity, we used stool samples from these participants as inocula to derive in vitro communities. Iron supplementation of these communities caused small shifts in structure, similar to in vivo, whereas iron deprivation dramatically inhibited growth with irreversible and cumulative reduction in diversity and replacement of some dominant species. Sensitivity of individual species to iron deprivation during growth in axenic culture generally predicted iron dependency in a community context. Finally, exogenous heme acted as a source of inorganic iron to prevent depletion of some community members. Our results highlight the complementarity of in vivo and in vitro studies in deconstructing how environmental factors affect gut microbiome structure.

sentences: Generalization in data-driven models of primary visual cortex AO_SCPLOWBSTRACTC_SCPLOWDeep neural networks (DNN) have set new standards at predicting responses of neural populations to visual input. Most such DNNs consist of a convolutional network (core) shared across all neurons which learns a representation of neural computation in visual cortex and a neuron-specific readout that linearly combines the relevant features in this representation. The goal of this paper is to test whether such a representation is indeed generally characteristic for visual cortex, i.e. gener-alizes between animals of a species, and what factors contribute to obtaining such a generalizing core. To push all non-linear computations into the core where the generalizing cortical features should be learned, we devise a novel readout that reduces the number of parameters per neuron in the readout by up to two orders of magnitude compared to the previous state-of-the-art. It does so by taking advantage of retinotopy and learns a Gaussian distribution over the neurons receptive field po-sition. With this new readout we train our network on neural responses from mouse primary visual cortex (V1) and obtain a gain in performance of 7% compared to the previous state-of-the-art network. We then investigate whether the convolutional core indeed captures general cortical features by using the core in transfer learning to a different animal. When transferring a core trained on thousands of neurons from various animals and scans we exceed the performance of training directly on that animal by 12%, and outperform a commonly used VGG16 core pre-trained on imagenet by 33%. In addition, transfer learning with our data-driven core is more data-efficient than direct training, achieving the same performance with only 40% of the data. Our model with its novel readout thus sets a new state-of-the-art for neural response prediction in mouse visual cortex from natural images, generalizes between animals, and captures better characteristic cortical features than current task-driven pre-training approaches such as VGG16.

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Variance misperception under skewed empirical noise statistics explains overconfidence in the visual periphery Perceptual confidence typically corresponds to accuracy. However, observers can be overconfident relative to accuracy, termed  subjective inflation. Inflation is stronger in the visual periphery relative to central vision, especially under conditions of peripheral inattention. Previous literature suggests inflation stems from errors in estimating noise, i.e.  variance misperception. However, despite previous Bayesian hypotheses about metacognitive noise estimation, no work has systematically explored how noise estimation may critically depend on empirical noise statistics which may differ across the visual field, with central noise distributed symmetrically but peripheral noise positively skewed. Here we examined central and peripheral vision predictions from five Bayesian-inspired noise-estimation algorithms under varying usage of noise priors, including effects of attention. Models that failed to optimally estimate noise exhibited peripheral inflation, but only models that explicitly used peripheral noise priors -- but used them incorrectly -- showed increasing peripheral inflation under increasing peripheral inattention. Further, only one model successfully captured previous empirical results which showed a selective increase in confidence in incorrect responses under performance reductions due to inattention accompanied by no change in confidence in correct responses; this was the model that implemented Bayesian estimation of peripheral noise, but using an (incorrect) symmetric rather than the correct positively skewed peripheral noise prior. Our findings explain peripheral inflation, especially under inattention, and suggest future experiments that might reveal the noise expectations used by the visual metacognitive system.

SignificancePerceptual confidence can dissociate from accuracy in peripheral visual perception, a phenomenon known as peripheral inflation. No previous model has considered how this phenomenon may arise from metacognitive noise estimation which depends on empirical noise statistics. Here we simulate Bayesian-inspired noise estimation algorithms to show that the systems erroneous beliefs about distributions of noise in the visual periphery can explain the occurrence of peripheral inflation, including how inflation varies with attentional manipulations in surprising ways. Our results explain why peripheral inflation occurs by positing a new Bayesian metacognitive noise estimation mechanism, paving the way for future psychophysical studies.

sentences: Wavelength-dependent DNA photodamage in a 3-D human skin model over the far-UVC and germicidal-UVC wavelength ranges from 215 to 255 nm The effectiveness of UVC to reduce airborne-mediated disease transmission is well-established. However conventional germicidal UVC (~254 nm) cannot be used directly in occupied spaces because of the potential for damage to the skin and eye. A recently studied alternative with the potential to be used directly in occupied spaces is far-UVC (200 to 235 nm, typically 222 nm), as it cannot penetrate to the key living cells in the epidermis. Optimal far-UVC use is hampered by limited knowledge of the precise wavelength dependence of UVC-induced DNA damage, and thus we have used a monochromatic UVC exposure system to assess wavelength-dependent DNA damage in a realistic 3-D human skin model. We exposed a 3-D human skin model to mono-wavelength UVC exposures of 100 mJ/cm2, at UVC wavelengths from 215 to 255 nm (5-nm steps). At each wavelength we measured yields of DNA-damaged keratinocytes, and their distribution within the layers of the epidermis. No increase in DNA damage was observed in the epidermis at wavelengths from 215 to 235 nm, but at higher wavelengths (240-255 nm) significant levels of DNA damage were observed. These results support use of far-UVC light to safely reduce the risk of airborne disease transmission in occupied locations.

sentences: Impaired astrocytic Ca2+ signalling in awake Alzheimer's disease transgenic mice Increased astrocytic Ca2+ signaling related to amyloid plaques has been shown in Alzheimers disease mouse models, but to date no reports have characterized behaviorally induced astrocytic Ca2+ signalling in such mice without the confounding effects of anesthesia. Here, we employ an event-based algorithm to assess astrocytic Ca2+ signals in the neocortex of awake-behaving tg-ArcSwe mice and non-transgenic wildtype littermates while monitoring pupil responses and behavior. We demonstrate an attenuated astrocytic Ca2+ response to locomotion and an uncoupling of pupil responses and astrocytic Ca2+ signalling in 15-months old plaque-bearing mice. This points to a potential decoupling of neuromodulatory activation and astrocytic Ca2+ activity, which may account for some of the cognitive dysfunctions observed in Alzheimers disease.

sentences: Effects of order on memory of event times Memorizing time of an event may employ two processes (i) encoding of the absolute time of events within an episode, (ii) encoding of its relative order. Here we study interaction between these two processes. We performed experiments in which one or several items were presented, after which participants were asked to report the time of occurrence of items. When a single item was presented, the distribution of reported times was quite wide. When two or three items were presented, the relative order among them strongly affected the reported time of each of them. Bayesian theory that takes into account the memory for the events order is compatible with the experimental data, in particular in terms of the effect of order on absolute time reports. Our results suggest that people do not deduce order from memorized time, instead peoples memory for absolute time of events relies critically on memorized order of the events.

sentences: A comparative recombination analysis of human coronaviruses and implications for the SARS-CoV-2 pandemic The SARS-CoV-2 pandemic prompts evaluation of recombination in human coronavirus (hCoV) evolution. We undertook recombination analyses of 158,118 public seasonal hCoV, SARS-CoV-1, SARS-CoV-2 and MERS-CoV genome sequences using the RDP4 software. We found moderate evidence for 8 SARS-CoV-2 recombination events, two of which involved the spike gene, and low evidence for one SARS-CoV-1 recombination event. Within MERS-CoV, 229E, OC43, NL63 and HKU1 datasets, we noted 7, 1, 9, 14, and 1 high-confidence recombination events, respectively. There was propensity for recombination breakpoints in structural genes, and recombination severely skewed the temporal structure of these data, especially for NL63 and OC43. Bayesian time-scaled analyses on recombinant-free data indicated the sampled diversity of seasonal CoVs emerged in the last 70 years, with 229E displaying continuous lineage replacements. These findings emphasize the importance of genomic based surveillance to detect recombination in SARS-CoV-2, particularly if recombination may lead to immune evasion.

sentences: Deformable Mirror based Optimal PSF Engineering for 3D Super-resolution Imaging Point spread function (PSF) engineering is an important technique to encode the properties (e.g., 3D positions, color, and orientation) of single molecule in the shape of the PSF, often with the help of a programmable phase modulator. Deformable mirror (DM) is currently the most widely used phase modulator for fluorescence detection as it shows negligible photon loss. However, it relies on careful calibration for precise wavefront control. Therefore, design of an optimal PSF not only relies on the theoretical calculation of maximum information content, but also the physical behavior of the phase modulator, which is often ignored during the optimization process. Here, we developed a framework of PSF engineering which could generate a device specific optimal PSF for 3D super-resolution imaging using DM. We used our method to generate two types of PSFs with depths of field comparable to the widely used astigmatism and Tetrapod PSFs, respectively. We demonstrated the superior performance of the DM specific optimal PSF over the conventional astigmatism and Tetrapod PSF both theoretically and experimentally.

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Mechanisms involved in the active secretion of CTX-M-15 β-lactamase by pathogenic E. coli ST131 Infections caused by antimicrobial resistant bacterial pathogens are fast becoming an important global health issue. Strains of Escherichia coli are common causal agents of urinary tract infection and can carry multiple resistance genes. This includes the gene blaCTX-M-15 that encodes for an extended spectrum beta-lactamase (ESBL). While studying antimicrobial resistance (AMR) in the environment we isolated several strains of E. coli ST131 downstream of a WWTP in a local river. These isolates were surviving in the river sediment and characterisation proved that a multi-resistant phenotype was evident. Here, we show that E. coli strain 48 (river isolate ST131), provided a protective effect against a third-generation cephalosporin (cefotaxime) for a susceptible E. coli strain 33 (river isolate ST3576) through secretion of a functional ESBL into the growth medium. Furthermore, extracellular ESBL activity was stable for at least 24 h after secretion. Proteomic and molecular genetic analyses identified CTX-M-15 as the major secreted ESBL responsible for the observed protective effect. In contrast to previous studies, OMVs were not the sole route for CTX-M-15 secretion. Indeed, mutation of the Type I secretion system led to a significant reduction in the growth of the ESBL-producing strain as well as a significantly reduced ability to confer protective effect. We speculate that CTX-M-15 secretion, mediated through active secretion using molecular machinery provides a public goods service by facilitating the survival of otherwise susceptible bacteria in the presence of cefotaxime.

Abstract importanceInfections caused by antimicrobial resistant bacterial pathogens have become an important global health issue. Wastewater treatment plants (WWTPs) have been identified as hotspots for the dissemination of antimicrobial resistant genes/bacteria into the environment. In this study, we investigated resistance enzyme secretion by a multi-drug resistant human pathogenic E. coli, isolated from a UK river, downstream of a WWTP. We present evidence that the resistant strain actively secreted an important resistance enzyme into the surrounding medium which degraded the antibiotic cefotaxime. This research provided evidence for the mechanism for secretion of this enzyme which could indicate a new target to tackle antibiotic resistance pathogens.

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Cytolytic memory CD4+ T cell clonotypes are expanded during Plasmodium falciparum infection Plasmodium falciparum (Pf) malaria causes high rates of morbidity and mortality and lacks a sufficiently effective vaccine. Clinical immunity develops in residents of malaria endemic regions which confers reduced clinical symptoms during infection and protection against severe disease. We hypothesized that understanding the immune mechanisms of clinical immunity could inform vaccine design to improve efficacy. We compared the peripheral blood cellular and humoral immune responses during a mild episode of Pf malaria infection. Participants were classified as either clinically susceptible or clinically protected, based on the number of recurrent clinical infections over an 18-month longitudinal study in a malaria endemic region in Malawi. Susceptible participants had three or more recurrent clinical episodes while clinically immune individuals had one or none. Protected participants exhibited higher plasma immunoglobulin G (IgG) breadth and titers against Pf antigens, and greater antibody (Ab)-dependent Pf opsonization compared to susceptible participants. Using high dimensional mass cytometry (CyTOF), spectral flow cytometry and single-cell transcriptomic analyses, we identified expanded memory CD4+ T cell clones sharing identical T cell receptor clonotypes in the blood of protected participants during malaria infection. These cells express a strong cytolytic T helper 1 effector program with transcripts encoding granzymes (A, B, H, M), granulysin, NKG7 and the Zeb2 master transcriptional regulator of terminally differentiated effector T cells. Memory CD4+ T cells expressing Zeb2+ were CD39hiTIGIThi and expressed multiple chemotactic and checkpoint inhibitory receptors, although the cellular levels of several of these receptors were reduced in protected compared to susceptible individuals. We propose that clonally expanded Zeb2+ cytolytic memory CD4+ Th1 cells could represent essential contributors to clinical immunity against Pf malaria.

One Sentence SummaryA population of cytolytic memory CD4+ T cells is clonally expanded in patients with Plasmodium falciparum malaria and has reduced chemotactic and inhibitory receptor expression in patients with naturally acquired clinical malaria immunity.

sentences: Engineering a super-secreting strain of Escherichia coli by directed co-evolution of the multiprotein Tat translocation machinery Escherichia coli remains one of the preferred hosts for biotechnological protein production due to its robust growth in culture and ease of genetic manipulation. It is often desirable to export recombinant proteins into the periplasmic space for reasons related to proper disulfide bond formation, prevention of aggregation and proteolytic degradation, and ease of purification. One such system for expressing heterologous secreted proteins is the twin-arginine translocation (Tat) pathway, which has the unique advantage of delivering correctly folded proteins into the periplasm. However, transit times for proteins through the Tat translocase, comprised of the TatABC proteins, are much longer than for passage through the SecYEG pore, the translocase associated with the more widely utilized Sec pathway. To date, a high protein flux through the Tat pathway has yet to be demonstrated. To address this shortcoming, we employed a directed co-evolution strategy to isolate mutant Tat translocases for their ability to deliver higher quantities of heterologous proteins into the periplasm. Three super-secreting translocases were selected that each exported a panel of recombinant proteins at levels that were significantly greater than that observed for wildtype TatABC or SecYEG translocases. Interestingly, all three of the evolved Tat translocases exhibited quality control suppression, suggesting that increased translocation flux was gained by relaxation of substrate proofreading. Overall, our discovery of highly efficient translocase variants paves the way for the use of the Tat system as a powerful complement to the Sec pathway for secreted production of both commodity and high value-added proteins.

sentences: Curtobacterium glycinis sp. nov. from Glycine max, Curtobacterium gossypii sp. nov. from Gossypium hirsutum and Curtobacterium oryzae sp. nov. from Oryza sativa, three new Curtobacterium species and endophytes from agricultural crops Three new Curtobacterium species from healthy tissues of agricultural crop plants in the United States are reported. They are Curtobacterium glycinis sp. nov. from soybean in Missouri, Curtobacterium gossypii from cotton in Puerto Rico and corn in Missouri, and Curtobacterium oryzae sp. nov. from rice in Texas.

sentences: Antioxidants green tea extract and nordihydroguaiaretic acid confer species and strain specific lifespan and health effects in Caenorhabditis nematodes The Caenorhabditis Intervention Testing Program (CITP) is an NIH-funded research consortium of investigators who conduct analyses at three independent sites to identify chemical interventions that reproducibly promote health and lifespan in a robust manner. The founding principle of the CITP is that compounds with positive effects across a genetically diverse panel of Caenorhabditis species and strains are likely engaging conserved biochemical pathways to exert their effects. As such, interventions that are broadly efficacious might be considered prominent compounds for translation for pre-clinical research and human clinical applications. Here, we report results generated using a recently streamlined pipeline approach for the evaluation of the effects of chemical compounds on lifespan and health. We studied five compounds previously shown to extend C. elegans lifespan or thought to promote mammalian health: 17-estradiol, acarbose, green tea extract, nordihydroguaiaretic acid, and rapamycin. We found that green tea extract and nordihydroguaiaretic acid extend Caenorhabditis lifespan in a species-specific manner. Additionally, these two antioxidants conferred assay-specific effects in some studies--for example, decreasing survival for certain genetic backgrounds in manual survival assays in contrast with extended lifespan as assayed using automated C. elegans Lifespan Machines. We also observed that GTE and NDGA impact on older adult mobility capacity is dependent on genetic background, and that GTE reduces oxidative stress resistance in some Caenorhabditis strains. Overall, our analysis of the five compounds supports the general idea that genetic background and assay type can influence lifespan and health effects of compounds, and underscores that lifespan and health can be uncoupled by chemical interventions.

sentences: A role for the immune system-released activating agent (ISRAA) in the ontogenetic development of brain astrocytes The Immune System-Released Activating Agent (ISRAA) was discovered as a novel molecule that functions as a mediator between the nervous and immune systems in response to a nervous stimulus following an immune challenge. This research investigated the role of ISRAA) in promoting the ontogeny of the mouse brain astrocytes. Astrocyte cultures were prepared from two-month-old BALB/c mice. Recombinant ISRAA protein was used to stimulate astrocyte cultures. Immunohistochemistry and ELISA were utilized to measure ISRAA and IFN-{gamma} levels, IFN-{gamma}R expression and STAT1 nuclear translocation. MTT-assay was used to evaluate cellular survival and proliferation. To assess astrocyte cell lysates and tyrosine-phosphorylated proteins, SDS-PAGE and western blot were used. ISRAA was highly expressed in mouse embryonic astrocytes, depending on cell age. Astrocytes aged seven days (E7) showed increased proliferation and diminished differentiation, while 21-day-old (E21) astrocytes depicted reversed effects. ISRAA stimulated the tyrosine phosphorylation of numerous cellular proteins and the nuclear translocation of STAT1. IFN-{gamma} was involved in the ISRAA action as ISRAA induced IFN-{gamma} in both age groups, but only E21 astrocytes expressed IFN-{gamma}R. The results suggest that ISRAA is involved in mouse brain development through the cytokine network involving IFN-{gamma}.

sentences: High trait variability in optimal polygenic prediction strategy within multiple-ancestry cohorts Polygenic scores (PGSs) are individual-level measures that aggregate the genome-wide genetic predisposition to a given trait. As PGS have predominantly been developed using European-ancestry samples, trait prediction using such European ancestry-derived PGS is less accurate in non-European ancestry individuals. Although there has been recent progress in combining multiple PGS trained on distinct populations, the problem of how to maximize performance given a multiple-ancestry cohort is largely unexplored. Here, we investigate the effect of sample size and ancestry composition on PGS performance for fifteen traits in UK Biobank. For some traits, PGS estimated using a relatively small African-ancestry training set outperformed, on an African-ancestry test set, PGS estimated using a much larger European-ancestry only training set. We observe similar, but not identical, results when considering other minority-ancestry groups within UK Biobank. Our results emphasise the importance of targeted data collection from underrepresented groups in order to address existing disparities in PGS performance.

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Lactate Signaling Mediated by AMD3100 Ameliorates Astrocyte Pathology and Remyelination Without Additional Extension of SOD1G93A Mice' Life-Span BackgroundAmyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of motor neurons accompanied by the accumulation of the morphologically and functionally altered glial cells. Reactive astrocytes and microglia secrete pro-inflammatory mediators and contribute to disease progression. Notably, oligodendrocyte functions are disrupted in ALS, including trophic support, myelination, and oligodendrocyte differentiation. ALS patients and mutant SOD1 mice models display reduced levels of myelin basic protein (MBP) and monocarboxylate transporter 1 (MCT1) contributing to impairment of oligodendrocyte function. Lactate, an active metabolite capable of moving into or out of cells, acts as a signaling molecule, transported exclusively by monocarboxylate transporters (MCTs). We showed previously, that AMD3100 increases the lactate transporter MCT1 in an animal models of Alzheimers disease (AD) and ALS.

Methods and ResultsAMD3100 is a reversible antagonist of CXCR4, and therefore inhibits the CXCR4/CXCL12 axis. AMD3100 was shown to have beneficial effects on extension of SOD1G93A mice life-span and enabling migration of hematopoietic stem cells (HSPCs) from bone marrow to periphery. The low content of lactate and transporters in SOD1G93A mice model led us to propose a combined treatment with AMD3100 and exogeneous L-lactate.

ConclusionsThe combined treatment attenuated neuroinflammation and remyelination but did not have a significant extension of SOD1G93A mice life-span compared with AMD3100 treatment alone.

sentences: Unleashing a Novel Function of Endonuclease G in Mitochondrial Genome Instability Having its own genome makes mitochondria a unique and semiautonomous organelle within cells. Mammalian mitochondrial DNA (mtDNA) is double-stranded closed circular molecule of about 16 kb coding 37 genes. Mutations, including deletions in the mitochondrial genome can culminate in different human diseases. Mapping of the deletion junctions suggests that the breakpoints are generally seen at hotspots.  9-bp deletion (8271-8281), seen in the intergenic region of cytochrome c oxidase II/tRNALys, is the most common mitochondrial deletion. While it is associated with several diseases like myopathy, dystonia, and hepatocellular carcinoma, it has also been used as an evolutionary marker. However, the mechanism responsible for its fragility is unclear. In the current study, we show that Endonuclease G, a mitochondrial nuclease responsible for nonspecific cleavage of nuclear DNA during apoptosis, can induce breaks at sequences associated with  9-bp deletion, when it is present on a plasmid or in the mitochondrial genome. Through a series of in vitro and intracellular studies, we show that Endonuclease G binds to G-quadruplex structures formed at the hotspot and induces DNA breaks. Besides, we reconstitute the whole process of  9-bp deletion using purified Endonuclease G to induce breaks in mtDNA, followed by mitochondrial extract-mediated DSB repair and establish that microhomology-mediated end joining is responsible for the generation of mtDNA deletion. Finally, we show that the whole process is regulated by different stress conditions, which may modulate release of Endonuclease G to the mitochondrial matrix. Therefore, we uncover a new role for Endonuclease G in generating deletions, which is dependent on the formation of G4 DNA within the mitochondrial genome. Thus, in this study we identify a novel property of Endonuclease G, besides its role in apoptosis, and the recently described  elimination of paternal mitochondria during fertilization.

sentences: Spindle reorientation in response to mechanical stress is an emergent property of the spindle positioning mechanisms Proper orientation of the mitotic spindle plays a crucial role in embryos, during tissue development, and in adults, where it functions to dissipate mechanical stress to maintain tissue integrity and homeostasis. While mitotic spindles have been shown to reorient in response to external mechanical stresses, the subcellular cues that mediate spindle reorientation remain unclear. Here, we have used a combination of optogenetics and computational modelling to better understand how mitotic spindles respond to inhomogeneous tension within the actomyosin cortex. Strikingly, we find that the optogenetic activation of RhoA only influences spindle orientation when it is induced at both poles of the cell. Under these conditions, the sudden local increase in cortical tension induced by RhoA activation reduces pulling forces exerted by cortical regulators on astral microtubules. This leads to a perturbation of the torque balance exerted on the spindle, which causes it to rotate. Thus, spindle rotation in response to mechanical stress is an emergent phenomenon arising from the interaction between the spindle positioning machinery and the cell cortex.

sentences: Verbally mediated cooperation is consistently associated with inter-brain synchrony in frontal and temporoparietal areas: A mini-review and meta-analysis Single-brain neuroimaging studies have shown that human cooperation is associated with neural activity in frontal and temporoparietal regions. However, it remains unclear whether single-brain studies are informative about cooperation in real life, where people interact dynamically. Such dynamic interactions have become the focus of inter-brain studies. An advantageous technique in this regard is functional near-infrared spectroscopy (fNIRS) because it is less susceptible to movement artifacts than more conventional techniques like EEG or fMRI. We conducted a systematic review and the first quantitative meta-analysis of fNIRS hyperscanning of cooperation, based on thirteen studies with 890 participants. Overall, the meta-analysis revealed evidence of statistically significant inter-brain synchrony while people were cooperating, with large overall effect sizes in both frontal and temporoparietal areas. All thirteen studies observed significant inter-brain synchrony in the prefrontal cortex (PFC), suggesting that this region is particularly relevant for cooperative behavior. The consistency in these findings is unlikely to be due to task-related activations, given that the relevant studies used diverse cooperation tasks. Together, the present findings support the importance of inter-brain synchronization of frontal and temporoparietal regions in interpersonal cooperation. Moreover, the present article highlights the usefulness of meta-analyses as a tool for discerning patterns in inter-brain dynamics.

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Protection from lethal Clostridioides difficile infection via intraspecies competition for co-germinant Clostridioides difficile, a Gram-positive, spore-forming bacterium, is the primary cause of infectious nosocomial diarrhea. Antibiotics are a major risk factor for C. difficile infection (CDI) as they disrupt the gut microbial community, enabling increased germination of spores and growth of vegetative C. difficile. To date the only single-species bacterial preparation that has demonstrated efficacy in reducing recurrent CDI in humans is non-toxigenic C. difficile. Using multiple infection models we determined that pre-colonization with a less virulent strain is sufficient to protect from challenge with a lethal strain of C. difficile, surprisingly even in the absence of adaptive immunity. Additionally, we showed that protection is dependent on high levels of colonization by the less virulent strain and that it is mediated by exclusion of the invading strain. Our results suggest that reduction of amino acids, specifically glycine following colonization by the first strain of C. difficile is sufficient to decrease germination of the second strain thereby limiting colonization by the lethal strain.

ImportanceAntibiotic-associated colitis is often caused by infection with the bacterium Clostridioides difficile. In this study we found that reduction of the amino acid glycine by pre-colonization with a less virulent strain of C. difficile is sufficient to decrease germination of a second strain. This finding demonstrates that the axis of competition for nutrients can include multiple life stages. This work is important, as it is the first to identify a possible mechanism through which pre-colonization with C. difficile, a current clinical therapy, provides protection from reinfection. Furthermore, our work suggests that targeting nutrients utilized by all life stages could be an improved strategy for bacterial therapeutics that aim to restore colonization resistance in the gut.

sentences: Caenorhabditis elegans ETR-1/CELF has broad effects on the muscle cell transcriptome, including genes that regulate translation and neuroblast migration Migration of neuroblasts and neurons from their birthplace is central to the formation of neural circuits and networks. ETR-1 is the Caenorhabditis elegans homolog of the CELF1 (CUGBP, ELAV-like family 1) RNA-processing factor involved in neuromuscular disorders. etr-1 regulates body wall muscle differentiation. Our previous work showed that etr-1 in muscle has a non-autonomous role in neuronal migration, suggesting that ETR-1 is involved in the production of a signal emanating from body wall muscle that controls neuroblast migration and that interacts with Wnt signaling. etr-1 is extensively alternatively-spliced, and we identified the viable etr-1(lq61) mutant, caused by a stop codon in alternatively-spliced exon 8 and only affecting etr-1 isoforms containing exon 8. We took advantage of viable etr-1(lq61) to identify potential RNA targets of ETR-1 in body wall muscle using a combination of fluorescence activated cell sorting (FACS) of body wall muscles from wild-type and etr-1(lq61) and subsequent RNA-seq. This analysis revealed genes whose splicing and transcript levels were controlled by ETR-1 exon 8 isoforms, and represented a broad spectrum of genes involved in muscle differentiation, myofilament lattice structure, and physiology. Genes with transcripts underrepresented in etr-1(lq61) included those involved in ribosome function and translation, similar to potential CELF1 targets identified in chick cardiomyocytes. This suggests that at least some targets of ETR-1 might be conserved in vertebrates, and that ETR-1 might generally stimulate translation in muscles. As proof-of-principle, a functional analysis of a subset of ETR-1 targets revealed genes involved in AQR and PQR neuronal migration. One such gene, lev-11/tropomyosin, requires ETR-1 for alternative splicing, and another, unc-52/perlecan, requires ETR-1 for the production of long isoforms containing 3 exons. In sum, these studies identified gene targets of ETR-1/CELF1 in muscles, which included genes involved in muscle development and physiology, and genes with novel roles in neuronal migration.

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A stress-reduced passaging technique improves the viability of human pluripotent cells Xeno-free culture systems have expanded the clinical and industrial application of human pluripotent stem cells (PSCs). However, yet some problems, such as the reproducibility among the experiments, remain. Here we describe an improved method for the subculture of human PSCs. The revised method significantly enhanced the viability of human PSCs by lowering DNA damage and apoptosis, resulting in more efficient and reproducible downstream applications such as gene editing, gene delivery, and directed differentiation. Furthermore, the method did not alter PSC characteristics after long-term culture and attenuated the growth advantage of abnormal subpopulations. This robust passaging method minimizes experimental error and reduces the rate of PSCs failing quality control of human PSC research and application.

HighlightsO_LIThe revised passaging method significantly increases the viability of human PSCs.
C_LIO_LIThe method triggers less DNA damage and apoptosis signals compared to the conventional method.
C_LIO_LIThe stress-reduced method improves the results of downstream applications.
C_LIO_LIThe method does not alter PSC characters and attenuates the overgrowth of abnormal subpopulations.
C_LI

sentences: Recurrent circuit based neural population codes for stimulus representation and inference A large part of the synaptic input received by cortical neurons comes from local cortico-cortical connectivity. Despite their abundance, the role of local recurrent connections in cortical function is unclear, and in simple coding schemes it is often the case that a circuit with no recurrent connections performs optimally. We consider a recurrent excitatory-inhibitory circuit model of a cortical hypercolumn which performs sampling-based Bayesian inference to infer latent hierarchical stimulus features. We show that local recurrent connections can store an internal model of the correlations between stimulus features that are present in the external world. When the resulting recurrent input is combined with feedforward input it produces a population code from which the posterior over the stimulus features can be linearly read out. Internal Poisson spiking variability provides the proper fluctuations for the population to sample stimulus features, yet the resultant population variability is aligned along the stimulus feature direction, producing differential correlations. Importantly, the amplitude of these internally generated differential correlations is determined by the associative prior in the model stored in the recurrent connections. This provides experimentally testable predictions for how population connectivity and response variability are related to the structure of latent external stimuli.

sentences: GNNQQNY: Methodology for biophysical and structural understanding of aggregation GNNQQNY sequence offers crucial information about the formation and structure of an amyloid fibril. In this study, we demonstrate a reproducible solubilisation protocol where the reduction of pH to 2.0 resulted in the generation of GNNQQNY monomers. The subsequent ultracentrifugation step removes the residual insoluble peptide from the homogeneous solution. This procedure ensures and allows the peptides to remain monomers till their aggregation is triggered by adjusting the pH to 7.2. The aggregation kinetics analysis showed a distinct lag-phase that is concentration-dependent, indicating nucleation-dependent aggregation kinetics. Nucleation kinetics analysis suggested a critical nucleus of size [~]7 monomers at physiological conditions. The formed nucleus acts as a template for further self-assembly leading to the formation of highly ordered amyloid fibrils. These findings suggest that the proposed solubilisation protocol provides the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation in GNNQQNY sequences. This procedure can also be used for solubilising such small amyloidogenic sequences for their biophysical studies.

sentences: Sexual dimorphic responses of C57BL/6 mice to Fisetin or Dasatinib and Quercetin cocktail oral treatment Senolytic treatment in aged mice clears senescent cell burden leading to functional improvements. We hypothesized that administering senotherapeutics in young adulthood of mice would slow physiological markers of aging through mid-life. C57BL/6 mice were treated monthly with either Fisetin or a Dasatinib (D) plus Quercetin (Q) cocktail from 4-13 months of age. Fisetin treated male mice had reduced senescence-associated secretory phenotype (SASP), enhanced glucose and energy metabolism, improved cognitive performance, and increased hippocampal expression of adiponectin 1 receptor and glucose transporter 4. D+Q treated females had increased SASP expression along with accumulation of white adipose tissue, reduced energy metabolism, and cognitive performance. Senotherapeutics in young adulthood, has beneficial, negligible, or detrimental effects in mice dependent upon sex and treatment.

sentences: Neuro-molecular characterization of fish cleaning interactions Coral reef fish exhibit a large variety of behaviours crucial for fitness and survival. The cleaner wrasse Labroides dimidiatus displays cognitive abilities during interspecific interactions by providing services of ectoparasite cleaning, thus serving as a good example to understand the processes of complex social behaviour. However, little is known about the molecular underpinnings of cooperative behaviour between L. dimidiatus and a potential client fish (Acanthurus leucosternon). Therefore, we investigated the molecular mechanisms in three regions of the brain (fore-, mid-, and hindbrain) during the interaction of these fishes. Here we show, using transcriptomics, that most of the transcriptional response in both species was regulated in the hindbrain and forebrain regions and that the interacting behaviour responses of L. dimidiatus involved immediate early gene alteration, dopaminergic and glutamatergic pathways, the expression of neurohormones (such as isotocin) and steroids (e.g. progesterone and estrogen). In contrast, in the client, fewer molecular alterations were found, mostly involving pituitary hormone responses. The particular pathways found suggested learning and memory processes in the cleaner wrasse, while the client indicated stress relief.

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Surface-Shaving Proteomics of Mycobacterium marinum Identifies Biofilm Subtype-Specific Changes Affecting Virulence, Tolerance and Persistence The complex cell wall and biofilm matrix (ECM) act as key barriers to antibiotics in mycobacteria. Here, the ECM-proteins of Mycobacterium marinum ATCC927, a non-tuberculous mycobacterial model, was monitored over three months by label-free proteomics and compared with cell-surface proteins on planktonic cells to uncover pathways leading to virulence, tolerance, and persistence. We show that ATCC927 forms pellicle-type (PBFs) and submerged-type (SBFs) biofilms after two weeks and two days of growth, respectively, and that the increased CelA1 synthesis in this strain prevents biofilm formation and leads to reduced rifampicin tolerance. The proteomic data suggests that specific changes in mycolic acid synthesis (cord factor), Esx1-secretion, and cell-wall adhesins explain the appearance of PBFs as ribbon-like cords and SBFs as lichen-like structures. A subpopulation of cells resisting the 64 x MIC rifampicin (persisters) were detected in both biofilm subtypes, and already in one-week-old SBFs. The key forces boosting their development could include subtype-dependent changes in asymmetric cell division, cell wall biogenesis, tricarboxylic acid/glyoxylate cycle activities, and energy/redox/iron metabolisms. The effect of varying ambient oxygen tensions on each cell type and non-classical protein secretion are likely factors explaining majority of the subtype-specific changes. The proteomic findings also imply that Esx1-type protein secretion is more efficient in PL and PBF cells, while SBF may prefer both the Esx5- and non-classical pathways to control virulence and prolonged viability/persistence. In conclusion, this study reports a first proteomic insight into aging mycobacterial biofilm-ECMs and indicates biofilm subtype-dependent mechanisms conferring increased adaptive potential and virulence on non-tuberculous mycobacteria.

IMPORTANCEMycobacteria are naturally resilient and mycobacterial infections are notoriously difficult to treat with antibiotics, with biofilm formation being the main factor complicating the successful treatment of TB. The present study shows that non-tuberculous Mycobacterium marinum ATCC927 forms submerged- and pellicle-type biofilms with lichen- and ribbon-like structures, respectively, as well as persister cells under the same conditions. We show that both biofilm subtypes differ in terms of virulence-, tolerance- and persistence-conferring activities, highlighting the fact that both subtypes should be targeted to maximize the power of antimycobacterial treatment therapies.

sentences: Extreme longevity of highly fecund termite queens achieved by mitochondrial and insulin upregulation without harmful lipid signatures or accumulation Kings and queens of eusocial termites can live for decades, while queens sustain a nearly maximal fertility. To investigate the molecular mechanisms underlying their long lifespan, we carried out transcriptomics, lipidomics and metabolomics in Macrotermes natalensis on sterile short-lived workers, long-lived kings and five stages spanning twenty years of adult queen maturation. Reproductives share gene expression differences from workers in agreement with a reduction of several aging-related processes, involving upregulation of DNA damage repair and mitochondrial functions. Anti-oxidant gene expression is downregulated, while peroxidability of membranes in queens decreases. Against expectations, we observed an upregulated gene expression in fat bodies of reproductives of several components of the IIS pathway, including an insulin-like peptide, Ilp9. This pattern does not lead to deleterious fat storage in physogastric queens, while simple sugars dominate in their hemolymph and large amounts of resources are allocated towards oogenesis. Our findings support the notion that all processes causing aging need to be addressed simultaneously in order to prevent it.

sentences: HSP70-binding motifs function as protein quality control degrons Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for degradation through the ubiquitin-proteasome system (UPS). To uncover how PQC degrons function, we performed a screen in Saccharomyces cerevisiae by fusing a library of flexible tetrapeptides to the C-terminus of the Ura3-HA-GFP reporter. The identified degrons exhibited high sequence variation but with marked hydrophobicity. Notably, the best scoring degrons constitute predicted Hsp70-binding motifs. When directly tested, a canonical Hsp70 binding motif (RLLL) functioned as a dose-dependent PQC degron that was targeted by Hsp70, Hsp110, Fes1, several Hsp40 J-domain co-chaperones and the PQC E3 ligase Ubr1. Our results suggest that multiple PQC degrons overlap with chaperone-binding sites and that PQC-linked degradation achieves specificity via chaperone binding. Thus, the PQC system has evolved to exploit the intrinsic capacity of chaperones to recognize misfolded proteins, thereby placing them at the nexus of protein folding and degradation.

sentences: CLCd and CLCf act redundantly at the TGN/EE and prevent acidification of the Golgi stack The trans-Golgi network/early endosome (TGN/EE) serves as the central hub in which exo- and endocytic trafficking pathways converge and specificity of cargo routing needs to be achieved. Acidification is a hallmark of the TGN/EE and is maintained by the vacuolar H+-ATPase (V-ATPase) with support of proton-coupled antiporters. We show here that CLCd and CLCf, two distantly related members of the Arabidopsis chloride channel (CLC)-family that colocalize in the TGN/EE act redundantly and are essential for male gametophyte development. Combining an inducible knock-down approach and in vivo pH-measurements, we show here that reduced CLC-activity does not affect pH in the TGN/EE but causes accumulation of the V-ATPase in trans-Golgi cisternae leading to their hyper-acidification. Taken together, our results show that CLC-mediated anion transport into the TGN/EE is essential and affects spatio-temporal aspects of TGN/EE-maturation as well as its functional separation from the Golgi stack.

sentences: Exploring the mobilome and resistome of Enterococcus faecium in a One Health context across two continents Enterococcus faecium is a ubiquitous opportunistic pathogen that is exhibiting increasing levels of antimicrobial resistance (AMR). Many of the genes that confer resistance and pathogenic functions are localized on mobile genetic elements (MGEs), which facilitate their transfer between lineages. Here, features including resistance determinants, virulence factors, and MGEs were profiled in a set of 1273 E. faecium genomes from two disparate geographic locations (in the UK and Canada) from a range of agricultural, clinical, and associated habitats. Neither lineages of E. faecium nor MGEs are constrained by geographic proximity, but our results show evidence of a strong association of many profiled genes and MGEs with habitat. Many features were associated with a group of clinical and municipal wastewater genomes that are likely forming a new human-associated ecotype. The evolutionary dynamics of E. faecium make it a highly versatile emerging pathogen, and its ability to acquire, transmit, and lose features presents a high risk for the emergence of new pathogenic variants and novel resistance combinations. This study provides a workflow for MGE-centric surveillance of AMR in Enterococcus that can be adapted to other pathogens.

sentences: Cation-chloride cotransporters, Na/K pump, and channels in cell water and ions regulation: in silico and experimental studies of the U937 cells under stopping the pump and during RVD Cation-coupled chloride cotransporters play a key role in generating the Cl electrochemical gradient on the cell membrane which is important for regulation of many cellular processes. However, the cooperation of transporters and channels of the plasma membrane in holding the ionic homeostasis of the whole cell remains poorly characterized because of the lack of a suitable tool for its computation. Our software successfully predicted in real-time changes in the ion homeostasis of U937 cells after stopping the Na/K pump, but so far considered the model with only NC cotransporter. Here the model with all main types of cotransporters is used in computation of the rearrangements of ionic homeostasis due to stopping the pump and associated with the regulatory volume decrease (RVD) of cells swollen in hypoosmolar medium. The parameters obtained for the real U937 cells are used. Successful prediction of changes in ion homeostasis in real-time after stopping the pump using the model with all major cotransporters indicates that the model is reliable. Using this model for analysis RVD showed that there is a "physical" RVD, associated with the time-dependent changes in electrochemical ion gradients, but not with alteration of channels and transporters of the plasma membrane that should be considered in studies of truly active regulatory processes mediated by the intracellular signaling network. The developed software can be useful for calculation of the balance of the partial unidirectional fluxes of monovalent ions across the cell membrane of various cells under various conditions.

sentences: Neural ADMIXTURE: rapid population clustering with autoencoders Characterizing the genetic substructure of large cohorts has become increasingly important as genetic association and prediction studies are extended to massive, increasingly diverse, biobanks. ADMIXTURE and STRUCTURE are widely used unsupervised clustering algorithms for characterizing such ancestral genetic structure. These methods decompose individual genomes into fractional cluster assignments with each cluster representing a vector of DNA marker frequencies. The assignments, and clusters, provide an interpretable representation for geneticists to describe population substructure at the sample level. However, with the rapidly increasing size of population biobanks and the growing numbers of variants genotyped (or sequenced) per sample, such traditional methods become computationally intractable. Furthermore, multiple runs with different hyperparameters are required to properly depict the population clustering using these traditional methods, increasing the computational burden. This can lead to days of compute. In this work we present Neural ADMIXTURE, a neural network autoencoder that follows the same modeling assumptions as ADMIXTURE, providing similar (or better) clustering, while reducing the compute time by orders of magnitude. Indeed, the equivalent of one month of continuous compute can be reduced to hours. In addition, Neural ADMIXTURE can include multiple outputs, providing the equivalent results as running the original ADMIXTURE algorithm many times with different numbers of clusters. Our models can also be stored, allowing later cluster assignment to be performed with a linear computational time. The software implementation of Neural ADMIXTURE can be found at https://github.com/ai-sandbox/neural-admixture.

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The role of parasympathetic mechanisms in the infarct-limiting effect of SGLT2 inhibitor ertugliflozin IntroductionBased on data that outcome in patients with acute myocardial infarction is predicted by final infarct size (IS), reducing IS is of paramount importance. Recent experimental studies have demonstrated a strong infarct-sparing effect of SGLT2 inhibitors - a class of drugs which have proved to be safe and beneficial in patients with heart failure. Repurposing SGLT2 inhibitors for the benefit of patients presenting with acute myocardial infarction should be preceded by investigation of the underlying mechanisms of this infarct limitation. Experimental and clinical data indicate a potential role for autonomic modulation in these mechanisms, specifically sympatho-inhibition. The aim of this study was to investigate the role of the parasympathetic mechanism in the infarct-limiting effect of SGLT2 inhibition.

MethodsFortyeight Sprague Dawley male rats were fed with a standard diet containing either the SGLT2 inhibitor ertugliflozin or vehicle, for 5-7 days. Myocardial ischaemia/reperfusion injury was initiated by a 40-min occlusion of the left anterior descending coronary artery followed by a 2hr period of reperfusion under isoflurane anaesthesia. Bilateral cervical vagotomy was performed 10min prior to myocardial ischaemia. Alternatively, muscarinic receptors were blocked systemically with the non-selective blocker atropine sulphate (2 mg/kg bolus, then 1 mg/kg/h) or the M3-selective blocker 4-DAMP (2 mg/kg bolus).

ResultsPre-treatment with ertugliflozin reduced IS in comparison with the vehicle-treated controls (p<0.001). Bilateral vagotomy, atropine sulphate and 4-DAMP all abolished this infarct-limiting effect (IS 35{+/-}10%, 44{+/-}8%, and 35{+/-}4% respectively; P<0.01 vs. Ertu for vagotomy, P<0.001 vs. Ertu for both atropine sulphate and 4-DAMP).

ConclusionThese results suggest that the Infarct-limiting effect of the SGLT2 inhibitor ertugliflozin, may be mediated via activation of the vagus nerve and M3-cholinoreceptors.

sentences: Seed banks alter the molecular evolutionary dynamics of Bacillus subtilis Fluctuations in the availability of resources constrains the growth and reproduction of individuals, which subsequently effects the evolution of their respective populations. Many organisms contend with such fluctuations by entering a reversible state of reduced metabolic activity, a phenomenon known as dormancy. This pool of dormant individuals (i.e., a seed bank) does not reproduce and is expected to act as an evolutionary buffer, though it is difficult to observe this effect directly over an extended evolutionary timescale. Through genetic manipulation, we analyze the molecular evolutionary dynamics of Bacillus subtilis populations in the presence and absence of a seed bank over 700 days. The ability of these bacteria to enter a dormant state increased the accumulation of genetic diversity over time and altered the trajectory of mutations, findings that were recapitulated using simulations based on a mathematical model of evolutionary dynamics. While the ability to form a seed bank did not alter the degree of negative selection, we found that it consistently altered the direction of molecular evolution across genes. Together, these results show that the ability to form a seed bank can affect the direction and rate of molecular evolution over an extended evolutionary timescale.

sentences: Global Dynamics as Communication Sensors in Peptide Synthetase Cyclization Domains. Structural biology is the foundation for deriving molecular mechanisms, where snapshots of macromolecules and binding partners inform on mutations that test or modify function. However, frequently, the impact of mutations violates the underpinnings of structural models, and mechanisms become cryptic. This conundrum applies to multidomain enzymatic systems called nonribosomal peptide synthetases (NRPSs), which assemble simple substrates into complex metabolites often with pharmaceutical properties. Engineering NRPSs can generate new pharmaceuticals1-3 but a dynamic domain organization challenges rational design.4-8 Using nuclear magnetic resonance (NMR), we determined the solution structure of a 52 kDa cyclization domain and demonstrate that global intra-domain dynamics enable sensing of substrates tethered to partner domains and draw an allosteric response encompassing the enzymes buried active site and two binding sites 40 [A] apart. We show that a point-site mutation that impedes the domain dynamics globally hampers the allosteric response. We demonstrate this mechanism through NMR experiments that provide atomic-level read-outs of allosteric responses during biochemical transformations in situ. Our results establish global structural dynamics as sensors of molecular events that can remodel domain interactions and illustrate the need for integrating structural dynamics explicitly when deriving molecular mechanisms through mutagenesis and structural biology.

sentences: Observation of a single protein by ultrafast X-ray diffraction The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. According to theory1, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes2. This was first demonstrated on biological samples a decade ago on the giant mimivirus3. Since then a large collaboration4 has been pushing the limit of the smallest sample that can be imaged5,6. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that of Escherichia coli GroEL which at 14 nm in diameter7 is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.

sentences: Chemical interplay and complementary adaptative strategies toggle bacterial antagonism and co-existence Bacterial communities are in a continuous adaptive and evolutionary race for survival. A myriad of molecules that kill, defend, or mediate communication between bacterial cells of different lineages shape the final structure of the microbial community. In this work we expand our knowledge on the chemical interplay and specific mutations that modulate the transition from antagonism to co-existence between two plant-beneficial bacteria, Pseudomonas chlororaphis PCL1606 and Bacillus amyloliquefaciens FZB42. We reveal that the bacteriostatic activity of bacillaene produced by Bacillus relies on an interaction with the protein elongation factor FusA and how mutations in this protein lead to tolerance to bacillaene and other protein translation inhibitors. Additionally, we describe how the unspecific tolerance to antimicrobials associated with mutations in the glycerol kinase GlpK is provoked mainly by a decrease of Bacillus cell membrane permeability among other pleiotropic cellular responses. We conclude that nutrient specialization and mutations in basic biological functions are bacterial evolutive and adaptive strategies that lead to the coexistence of two primary competitive bacterial species rather than their mutual eradication.

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MIG-23 is involved in sperm migration in Ascaris suum In nematodes, spermiogenesis, which is also called sperm activation, is a process in which nonmotile spermatids are transformed into crawling spermatozoa, which is accompanied by a series of morphological, physiological and biochemical changes. Sperm motility acquisition during this process is essential for successful fertilization. However, the mechanisms of sperm motility regulation in nematodes remain to be clarified. Herein, we found that extracellular adenosine-5-triphosphate (ATP) level mediation by MIG-23, which is a homolog of human ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), was required for major sperm protein (MSP) filament dynamics and sperm motility in the nematode Ascaris suum. MIG-23 was localized on the sperm plasma membrane. During sperm activation, mitochondrial activity was increased dramatically, and a large amount of ATP was produced and stored in refringent granules (RGs). In addition, a portion of the produced ATP was released to the extracellular space through ATP channels, which were composed of innexins and localized on the sperm plasma membrane. Spermatozoa, instead of spermatids, hydrolyzed exogenous ATP and processed ecto-ATPase activity. MIG-23 contributed to the ecto-ATPase activity of spermatozoa. Once MIG-23 activity was interrupted, spermatozoa also decreased their ATP hydrolysis activity. Blocking MIG-23 activity resulted in an increase in the depolymerization rate of MSP filaments in pseudopodia, which eventually affected nematode sperm migration. Overall, our data imply that MIG-23, which contributes to the ecto-ATPase activity of spermatozoa, regulates sperm migration by modulating extracellular ATP levels.

HighlightsO_LIATP is released to extracellular space through innexin channels which are identified in worm sperm.
C_LIO_LIWorm spematozoa show ecto-ATPase activity.
C_LIO_LIMIG-23 contributes to the ecto-ATPase activity of spermatozoa and regulates extracellular ATP level.
C_LIO_LIMIG-23 is required for MSP-based filament dynamics and sperm migration.
C_LI

sentences: GRAMMAR-Lambda: An Extreme Simplification for Genome-wide Mixed Model Association Analysis A highly efficient genome-wide association method, GRAMMAR-Lambda is proposed to make simple genomic control for the test statistics deflated by GRAMMAR, producing statistical power as high as exact mixed model association method. Using the simulated and real phenotypes, we show that at a moderate or above genomic heritability, polygenic effects can be estimated using a small number of randomly selected markers, which extremely simplify genome-wide association analysis with an approximate computational complexity to naive method in large-scale complex population. Upon a test at once, joint association analysis offers significant increase in statistical power over existing methods.

sentences: Species and strain cultivation of skin, oral, and gut microbiota Genomics-driven discovery of microbial species have provided extraordinary insights into the biodiversity of human microbiota. High resolution genomics to investigate species- and strain-level diversity and mechanistic studies, however, rely on the availability of individual microbes from a complex microbial consortia. Here, we describe and validate a streamlined workflow for cultivating microbes from the skin, oral, and gut microbiota, informed by metagenomic sequencing, mass spectrometry, and strain profiling.

sentences: New auranofin analogs with antibacterial properties against Burkholderia clinical isolates Bacteria of the genus Burkholderia include pathogenic Burkholderia mallei, Burkholderia pseudomallei and the Burkholderia cepacia complex (Bcc). These Gram-negative pathogens have intrinsic drug resistance, which makes treatment of infections difficult. Bcc affects individuals with cystic fibrosis (CF) and the species B. cenocepacia is associated with one of the worst clinical outcomes. Following the repurposing of auranofin as an antibacterial against Gram-positive bacteria, we previously synthetized auranofin analogs with activity against Gram-negatives. In this work, we show that two auranofin analogs, MS-40S and MS-40, have antibiotic activity against Burkholderia clinical isolates. The compounds are bactericidal against B. cenocepacia and kill stationary-phase cells and persisters without selecting for multistep resistance. Caenorhabditis elegans and Galleria mellonella tolerated high concentrations of MS-40S and MS-40, demonstrating that these compounds have low toxicity in these model organisms. In summary, we show that MS-40 and MS-40S have the potential to be effective therapeutic options to treat Burkholderia infections.

sentences: Peptide-RNA Coacervates as a Cradle for the Evolution of Folded Domains Peptide-RNA coacervates can result in the concentration and compartmentalization of simple biopolymers. Given their primordial relevance, peptide-RNA coacervates may have also been a key site of early protein evolution. However, the extent to which such coacervates might promote or suppress the exploration of novel peptide conformations is fundamentally unknown. To this end, we used electron paramagnetic resonance (EPR) spectroscopy to characterize the structure and dynamics of an ancient and ubiquitous nucleic acid binding element, the helix-hairpin-helix (HhH) motif, alone and in the presence of RNA, with which it forms coacervates. Double electron-electron resonance (DEER) spectroscopy applied to singly labeled peptides containing one HhH motif reveals the presence of dimers, even in the absence of RNA, and transient -helical character. Moreover, dimer formation is promoted upon RNA binding and was detectable within peptide-RNA coacervates. The distance distributions between spin labels are consistent with the symmetric (HhH)2-Fold, which is generated upon duplication and fusion of a single HhH motif and traditionally associated with dsDNA binding. These results support the hypothesis that coacervates are a unique testing ground for peptide oligomerization and that phase-separating peptides could have been a resource for the construction of complex protein structures via common evolutionary processes, such as duplication and fusion.

sentences: TAS-Seq: a robust and sensitive amplification method for bead-based scRNA-seq Single-cell RNA-sequencing (scRNA-seq) is valuable for analyzing cellular heterogeneity. Cell composition accuracy is critical for analyzing cell-cell interaction networks from scRNA-seq data. We developed terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq) for scRNA-seq based on a terminator, terminal transferase, and nanowell/bead-based scRNA-seq platform; TAS-Seq showed high tolerance to variations in the terminal transferase reaction, which complicate the handling of existing terminal transferase-based scRNA-seq methods. In murine and human lung samples, TAS-Seq yielded scRNA-seq data that were highly correlated with flow-cytometric data, showing higher gene-detection sensitivity and more robust detection of important cell-cell interactions and expression of growth factors/interleukins in cell subsets than 10X Chromium v2 and Smart-seq2. Expanding TAS-Seq application will improve understanding and atlas construction of lung biology at the single-cell level.

sentences: Anther cones increase pollen release in buzz-pollinated Solanum flowers The widespread evolution of tube-like anthers releasing pollen from apical pores is associated with buzz pollination, in which bees vibrate flowers to remove pollen. The mechanical connection among anthers in buzz-pollinated species varies from loosely held conformations, to anthers tightly held together with trichomes or bio-adhesives forming a functionally joined conical structure (anther cone). Joined anther cones in buzz-pollinated species have evolved independently across plant families and via different genetic mechanisms, yet their functional significance remains mostly untested. We used experimental manipulations to compare vibrational and functional (pollen release) consequences of joined anther cones in three buzz-pollinated species of Solanum (Solanaceae). We applied bee-like vibrations to focal anthers in flowers with ("joined") and without ("free") experimentally created joined anther cones, and characterised vibrations transmitted to other anthers and the amount of pollen released. We found that joined anther architectures cause non-focal anthers to vibrate at higher amplitudes than free architectures. Moreover, in the two species with naturally loosely held anthers, anther fusion increases pollen release, while in the species with a free but naturally compact architecture it does not. We discuss hypotheses for the adaptive significance of the convergent evolution of joined anther cones.

sentences: Chromatin accessibility determines cell-type specific intron retention Dynamic intron retention (IR) in vertebrate cells is of widespread biological importance. Aberrant IR is associated with numerous human diseases including cancer. Despite consistent reports demonstrating intrinsic sequence features that predispose introns to become retained, conflicting findings about cell type-specific IR regulation demand a systematic analysis in a controlled experimental setting. We integrated matched transcriptomics and epigenetics data (including DNA methylation, nucleosome occupancy, histone modifications) from primary human myeloid and lymphoid cells. Using machine learning we trained two complementary models to determine the role of epigenetic factors in the regulation of IR. We show that increased chromatin accessibility contributes substantially to the retention of introns in a cell-specific manner. We also confirm that intrinsic characteristics of introns are key for them to evade splicing. With mounting reports linking pathogenic alterations to RNA processing, our findings may have profound implications for the design of therapeutic approaches targeting aberrant splicing.

sentences: Minimal functional alignment of ventromedial prefrontal cortex intracranial EEG signals during naturalistic viewing The ventromedial prefrontal cortex (vmPFC) has been thought to play an important role in processing endogenous information such as generating subjective affective meaning. Unlike sensory cortex, which processes exogenous information about the external world similarly across individuals, prior work has posited that vmPFC activity may be idiosyncratic to each individual, even when exposed to the same external stimulus. In this study, we recorded local field potentials (LFPs) from intracranial stereotactic electrodes implanted in patients with intractable epilepsy while they watched an emotionally engaging television show episode and evaluated temporal synchronization of these signals across participants in auditory cortex and vmPFC. Overall, we observed markedly lower intersubject synchronization of signals recorded from electrodes implanted in vmPFC compared to auditory cortex. A subset of patients, however, appeared to share similar vmPFC states during the more emotionally salient scenes. This work suggests that the vmPFC is involved in processing affective responses to ongoing experience in a state-like manner, but the specific states and temporal sequences are idiosyncratic to each individual, even when viewing the same television episode.

sentences: In silico approach on drug repurposing - Antimalarial drugs against HIV-1 protease Acquired Immunodeficiency Syndrome (AIDS), belonging to the retrovirus family is one of the most devastating contagious diseases of this century. Most of the available approved drugs are small molecules which are used in antiretroviral therapy (ART) that trigger the therapeutic response through binding to a targeted protein, HIV-1 protease (PR). This protein represents the most important antiretroviral drug target due to its key role in viral development inhibition. Computational tools using computer-aided technologies have proven useful in accelerating the drug discovery. In this study we evaluated selected FDA (USA) approved antimalarial drugs against HIV-1 protease to find a potential inhibitor candidate for HIV-1 PR (PDB 6DJ1). Binding affinities and Ki inhibition constant of an AutoDock 4.2 study suggest that of all assessed antimalarial agents, Lumefantrine (LUM) would be a most promising HIV-1 PR inhibitor.

sentences: Learning and organization of memory for evolving patterns Storing memory for molecular recognition is an efficient strategy for responding to external stimuli. Biological processes use different strategies to store memory. In the olfactory cortex, synaptic connections form when stimulated by an odor, and establish distributed memory that can be retrieved upon re-exposure. In contrast, the immune system encodes specialized memory by diverse receptors that recognize a multitude of evolving pathogens. Despite the mechanistic differences between the olfactory and the immune memory, these systems can still be viewed as different information encoding strategies. Here, we present a theoretical framework with artificial neural networks to characterize optimal memory strategies for both static and dynamic (evolving) patterns. Our approach is a generalization of the energy-based Hopfield model in which memory is stored as a networks energy minima. We find that while classical Hopfield networks with distributed memory can efficiently encode a memory of static patterns, they are inadequate against evolving patterns. To follow an evolving pattern, we show that a distributed network should use a higher learning rate, which in turn, can distort the energy landscape associated with the stored memory attractors. Specifically, narrow connecting paths emerge between memory attractors, leading to misclassification of evolving patterns. We demonstrate that compartmentalized networks with specialized subnetworks are the optimal solutions to memory storage for evolving patterns. We postulate that evolution of pathogens may be the reason for the immune system to encoded a focused memory, in contrast to the distributed memory used in the olfactory cortex that interacts with mixtures of static odors.

sentences: A wMel Wolbachia variant in Aedes aegypti from field-collected Drosophila melanogaster with increased phenotypic stability under heat stress Mosquito-borne diseases such as dengue, Zika and chikungunya remain a major cause of morbidity and mortality across tropical regions. Population replacement strategies involving the wMel strain of Wolbachia are being used widely to control mosquito-borne diseases transmitted by Aedes aegypti. However, these strategies may be influenced by environmental temperature because wMel is vulnerable to heat stress. wMel infections in their native host Drosophila melanogaster are genetically diverse, but few transinfections of wMel variants have been generated in Ae. aegypti mosquitoes. Here we successfully transferred a wMel variant (termed wMelM) originating from a field-collected D. melanogaster population from Victoria, Australia into Ae. aegypti. The new wMelM variant (clade I) is genetically distinct from the original wMel transinfection (clade III) generated over ten years ago, and there are no genomic differences between wMelM in its original and transinfected host. We compared wMelM with wMel in its effects on host fitness, temperature tolerance, Wolbachia density, vector competence, cytoplasmic incompatibility and maternal transmission under heat stress in a controlled background. wMelM showed a higher heat tolerance than wMel, with stronger cytoplasmic incompatibility and maternal transmission when eggs were exposed to heat stress, likely due to higher overall densities within the mosquito. Both wMel variants had minimal host fitness costs, complete cytoplasmic incompatibility and maternal transmission, and dengue virus blocking under standard laboratory conditions. Our results highlight phenotypic differences between closely related Wolbachia variants. wMelM shows potential as an alternative strain to wMel in dengue control programs in areas with strong seasonal temperature fluctuations.

sentences: HIV corruption of the Arp2/3-Cdc42-IQGAP1 axis to hijack cortical F-Actin to promote cell-cell viral spread. F-Actin remodelling is important for the spread of HIV via cell-cell contacts, yet the mechanisms by which HIV corrupts the actin cytoskeleton are poorly understood. Through live cell imaging and focused ion beam scanning electron microscopy (FIB-SEM), we observed F-Actin structures that exhibit strong positive curvature to be enriched for HIV buds. Virion proteomics, gene silencing, and viral mutagenesis supported a Cdc42-IQGAP1-Arp2/3 pathway as the primary intersection of HIV budding, membrane curvature and F-Actin regulation. Whilst HIV egress activated the Cdc42-Arp2/3 filopodial pathway, this came at the expense of cell-free viral release. Importantly, release could be rescued by cell-cell contact, provided Cdc42 and IQGAP1 were present. From these observations we conclude that a proportion out-going HIV has corrupted a central F-Actin node that enables initial coupling of HIV buds to cortical F-Actin to place HIV at the leading cell edge. Whilst this initially prevents particle release, maturation of cell-cell contacts signals back to this F-Actin node to enable viral release & subsequent infection of the contacting cell.

sentences: Optogenetic stimulation of lateral hypothalamic orexin/dynorphin inputs in the ventral tegmental area potentiates mesolimbic dopamine neurotransmission and promotes reward-seeking behaviours. Reward and reinforcement processes are critical for survival and propagation of genes. While numerous brain systems underlie these processes, a cardinal role is ascribed to mesolimbic dopamine. However, ventral tegmental area (VTA) dopamine neurons receive complex innervation and various neuromodulatory factors, including input from lateral hypothalamic (LH) orexin/hypocretin neurons which also express and co-release the neuropeptide, dynorphin. Dynorphin in the VTA induces aversive conditioning through the Kappa opioid receptor (KOR) and decreases dopamine when administered intra-VTA. Exogenous application of orexin or orexin 1 receptor (oxR1) antagonists in the VTA bidirectionally modulates dopamine-driven motivation and reward-seeking behaviours, including the attribution of motivational value to primary rewards and associated conditioned stimuli. However, the effect of endogenous stimulation of LH orexin/dynorphin-containing projections to the VTA and the potential contribution of co-released dynorphin on mesolimbic dopamine and reward related processes remains uncharacterised. We combined optogenetic, electrochemical, and behavioural approaches to examine this. We found that optical stimulation of LH orexin/dynorphin inputs in the VTA potentiates mesolimbic dopamine neurotransmission in the nucleus accumbens (NAc) core, produces real time and conditioned place preference, and increases the food cue-directed orientation in a Pavlovian conditioning procedure. LH orexin/dynorphin potentiation of NAc dopamine release and real time place preference was blocked by an oxR1, but not KOR antagonist. Thus, rewarding effects associated with optical stimulation of LH orexin/dynorphin inputs in the VTA are predominantly driven by orexin rather than dynorphin.

sentences: A novel hybrid protein promotes Aβ clearance and reduces inflammatory response through MerTK Alzheimers disease (AD) is the worlds leading cause of dementia and the most common neurodegenerative disorder. Its major pathological features are amyloid beta (A{beta}) plaques, tau tangles, and neuroinflammation that eventually leads to massive death of nerve cells. Even with the multifactorial aspect of AD, the most accepted theory is that A{beta} is the driving force of AD pathogenesis. We engineered a novel hybrid protein that facilitates the phagocytosis of A{beta} and redirect its clearance to the noninflammatory Mer tyrosine kinase (MerTK) pathway. The novel hybrid protein facilitates robust uptake and clearance of A{beta} in BV2 microglia through MerTK receptor with reduced production of inflammatory factors and oxidative products. In APP/PS1 transgenic AD mouse model, intraperitoneal administration of the hybrid protein for two months results in significant reduction of A{beta} burden in the brain and protection of nerve cells from dying. Taken together, our results suggest that the novel hybrid may have the potential for AD treatment by targeting both A{beta} clearance and reduction of inflammation.

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Phenotypic heterogeneity is adaptive for microbial populations under starvation To persist in variable environments populations of microorganisms have to survive periods of starvation and be able to restart cell division in nutrient-rich conditions. Typically, starvation signals initiate a transition to a quiescent state in a fraction of individual cells, while the rest of the cells remain non-quiescent. It is widely believed that, while quiescent cells (Q) help the population to survive long starvation, the non-quiescent cells (NQ) are a side effect of imperfect transition. We analysed regrowth of starved monocultures of Q and NQ cells compared to mixed, heterogeneous cultures in simple and complex starvation environments. Our experiments, as well as mathematical modelling, demonstrate that Q monocultures benefit from better survival during long starvation, and from a shorter lag phase after resupply of rich medium. However, when the starvation period is very short, the NQ monocultures outperform Q and mixed cultures, due to their short lag phase. In addition, only NQ monocultures benefit from complex starvation environments, where nutrient recycling is possible. Our study suggests that phenotypic heterogeneity in starved populations could be a form of bet hedging, which is adaptive when environmental determinants, such as the length of the starvation period, the length of the regrowth phase, and the complexity of the starvation environment vary over time.

ImportanceNon-genetic cell heterogeneity is present in glucose starved yeast populations in the form of quiescent (Q) and nonquiescent (NQ) phenotypes. There is evidence that Q cells help the population to survive long starvation. However, the role of the NQ cell type is not known, and it has been speculated that the NQ phenotype is just a side effect of imperfect transition to the Q phenotype. Here we show that, in contrast, there are ecological scenarios in which NQ cells perform better than monocultures of Q cells or naturally occuring mixed populations containing both Q and NQ. NQ cells benefit when the starvation period is very short and environmental conditions allow nutrient recycling during starvation. Our experimental and mathematical modeling results suggest a novel hypothesis: the presence of both Q and NQ phenotypes within starved yeast populations may reflect a form of bet hedging, where different phenotypes provide fitness advantages depending on environmental conditions.

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Intact rDNA arrays of Potentilla-origin detected in Erythronium nucleus suggest recent eudicot-to-monocot horizontal transfer The occurrence of horizontal gene transfer (HGT) in Eukarya is increasingly gaining recognition. Nuclear-to-nuclear jump of DNA between plant species at high phylogenetic distance and devoid of intimate association (e.g., parasitism) is still scarcely reported. Within eukaryotes, components of ribosomal DNA (rDNA) multigene family have been found to be horizontally transferred in protists, fungi and grasses. However, in neither case HGT occurred between phylogenetic families, nor the transferred rDNA remained tandemly arrayed and transcriptionally active in the recipient organism. This study aimed to characterize an alien eudicot-type of 45S nuclear rDNA, assumingly transferred horizontally to the genome of monocot European Erythronium (Liliaceae). Genome skimming coupled by PacBio HiFi sequencing of a BAC clone were applied to determine DNA sequence of the alien rDNA. A clear phylogenetic signal traced the origin of the alien rDNA of Erythronium back to the Argentea clade of Potentilla (Rosaceae) and deemed the transfer to have occurred in the common ancestor of E. dens-canis and E. caucasicum. Though being discontinuous, transferred rDNA preserved its general tandemly arrayed feature in the host organism. Southern blotting, molecular cytogenetics, and sequencing of a BAC clone derived from flow-sorted nuclei indicated integration of the alien rDNA into the recipients nuclear genome. Unprecedently, dicot-type alien rDNA was found to be transcribed in the monocot Erythronium albeit much less efficiently than the native counterpart. This study adds a new example to the growing list of naturally transgenic plants while holding the scientific community continually in suspense about the mode of DNA transfer.

Significance StatementRibosomal DNA is an essential component of all cellular genomes. In plants, accidental movement of rDNA via horizontal gene transfer has only been reported in sexually incompatible grasses (monocots) where it involved non-functional rDNA units. In this study, we propose that evolutionary trajectories of eudicots and monocots were bypassed by the jump of rDNA from a Potentilla species (Rosaceae) to a common ancestor of Erythronium dens-canis and E. caucasicum (Liliaceae). The alien eudicot-type rDNA appeared relatively well conserved in the examined host Erythronium genome, being able to be expressed while preserving its general tandemly repeated feature, evidences that have no match in earlier literature.

sentences: The Impact of Protection Measures and Treatment onPneumonia Infection Model Pneumonia has been a major airborne transmitted disease and continues to pose a major public health burden in both developed and developing countries of the world. In this study, we constructed and analyzed a nonlinear deterministic compartmental mathematical model for assessing the community-level impacts of vaccination, other protection measures like practicing good hygiene, avoiding close contacts with sick people and limiting exposure to cigarette smoke, etc. and treatment on the transmission dynamics of pneumonia disease in a population of varying size. Our model exhibits two kinds of equilibrium points: pneumonia disease-free equilibrium point, and pneumonia endemic equilibrium point(s). Using center manifold criteria, we have verified that the pneumonia model exhibits backward bifurcations whenever its effective reproduction number [R]P < 1 and in the same region, the model shows the existence of more than one endemic equilibrium point where some of which are stable and others are unstable. Thus, for pneumonia infection, the necessity of the pneumonia effective reproduction number [R]P < 1, although essential, it might not be enough to completely eradicate the pneumonia infection from the considered community. Our examination of sensitivity analysis shows that the pneumonia infection transmission rate denoted by {beta} plays a crucial role to change the qualitative dynamics of pneumonia infection. By taking standard data from published literature, our numerical computations show that the numerical value of pneumonia infection model effective reproduction number is [R]P = 8.31 at {beta} = 4.21 it implies that the disease spreads throughout the community. Finally, our numerical simulations show that protection, vaccination, and treatment against pneumonia disease have the effect of decreasing pneumonia expansion.

sentences: Modern microbial mats from the Chihuahuan Desert provide insights into ecological stability throughout Earth's history Microbial mats are complex ecological assemblages that are found in the Precambrian fossil record and in extant extreme environments. Hence, these structures are regarded as highly stable ecosystems. In this work, we assess the ecological stability in a modern, fluctuating, hypersaline pond from the Cuatro Cienegas Basin. From the 2016 to 2019 metagenomic sampling of this site, we found that this microbial site is sensitive to disturbances, which leads to high taxonomic replacement. Additionally, the mats have shown to be functionally stable throughout time, and could be differentiated between dry and rainy seasonal states. We speculate that this microbial system could represent modern analogs of ancient microbial mats where functions were preserved over time, whereas composition was subject to diversification in the face of local and planetary perturbations.

sentences: The Relative Binding Position of Nck and Grb2 Adaptors Dramatically Impacts Actin-Based Motility of Vaccinia Virus Phosphotyrosine (pTyr) motifs in unstructured polypeptides orchestrate important cellular processes by engaging SH2-containing adaptors to nucleate complex signalling networks. The concept of phase separation has recently changed our appreciation of such multivalent networks, however, the role of pTyr motif positioning in their function remains to be explored. We have now explored this parameter in the assembly and operation of the signalling cascade driving actin-based motility and spread of Vaccinia virus. This network involves two pTyr motifs in the viral protein A36 that recruit the adaptors Nck and Grb2 upstream of N-WASP and Arp2/3-mediated actin polymerization. We generated synthetic networks on Vaccinia by manipulating pTyr motifs in A36 and the unrelated p14 from Orthoreovirus. In contrast to predictions, we find that only specific spatial arrangements of Grb2 and Nck binding sites result in robust N-WASP recruitment, Arp2/3 driven actin polymerization and viral spread. Our results suggest that the relative position of pTyr adaptor binding sites is optimised for signal output. This finding may explain why the relative positions of pTyr motifs are usually conserved in proteins from widely different species. It also has important implications for regulation of physiological networks, including those that undergo phase transitions.

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Cross-linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) Bovine serum albumin, (ii) E. coli ribosome, and (iii) HEK293T cell nuclear lysates. We identified a total of 508 unique cross-linking sites for BSA, 868 for the E. coli ribosome, and 1,623 unique cross-links for nuclear lysates, corresponding to 1,088 intra- and 535 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving 3D-structures of single proteins, but also for performing system-wide protein interaction studies.

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sentences: Inducing oncolytic cell death in human cancer cells by the long non-coding RNA let-A Long non-coding (lnc) RNAs contain functional elements that play important regulatory roles in a variety of processes during development, normal physiology, as well as disease. We recently discovered a new lncRNA, we named let-A, expressed from the evolutionary conserved let-7-Complex locus in Drosophila. This RNA induces cell death in Drosophila cancer cells. Here we show that ectopic expression of Drosophila let-A is also exerting an oncolytic toxicity in several human cancer cell lines, but shows almost no effect in more differentiated or cell lines derived from normal tissue. We demonstrate that let-A RNA prepared by in vitro transcription and provided in the growth medium is sufficient to induce cell death both in human and Drosophila cancer cells. The activity of in vitro transcribed let-A is most efficient in its full length, but requires prior modification/processing to become active. let-A induces a reduction of nucleolar size in treated cells. We show exo/endocytosis and Toll signaling pathway to be necessary for let-A-induced toxicity. Our findings indicate let-A exhibits an evolutionary conserved anti-cancer function, making it a promising molecule for tumor treatments.

sentences: Paired single-cell host profiling with multiplex-tagged bacterial mutants reveals intracellular virulence-immune networks Encounters between host cells and intracellular bacterial pathogens lead to complex phenotypes that determine the outcome of infection. Single-cell RNA-sequencing (scRNA-seq) are increasingly used to study the host factors underlying diverse cellular phenotypes. But current approaches do not permit the simultaneous unbiased study of both host and bacterial factors during infection. Here, we developed scPAIR-seq, an approach to analyze both host and pathogen factors during infection by combining multiplex-tagged mutant bacterial library with scRNA-seq to identify mutant-specific changes in host transcriptomes. We applied scPAIR-seq to macrophages infected with a library of Salmonella Typhimurium secretion system effector mutants. We developed a pipeline to independently analyze redundancy between effectors and mutant-specific unique fingerprints, and mapped the global virulence network of each individual effector by its impact on host immune pathways. ScPAIR-seq is a powerful tool to untangle bacterial virulence strategies and their complex interplay with host defense strategies that drive infection outcome.

sentences: The Alzheimer's disease genetic risk factor BIN1 induces isoform-dependent neurotoxicity through early endosome defects The Bridging Integrator 1 (BIN1) gene is a major susceptibility gene for Alzheimers disease (AD). Deciphering its pathophysiological role is challenging due to its numerous isoforms. Here we observed in Drosophila that human BIN1 isoform1 (BIN1iso1) overexpression, contrary to BIN1iso8 and BIN1iso9, induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level is responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs. Altogether, our data demonstrate that the AD susceptibility gene BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation, which is an early pathophysiological hallmark of AD pathology.

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Expansive and diverse phenotypic landscape of field Aedes aegypti larvae with differential susceptibility to temephos: beyond metabolic detoxification Arboviruses including dengue, Zika and chikungunya are amongst the most significant public health concerns worldwide and their control relies heavily on the use of insecticides to control the vector mosquito Aedes aegypti. The success of controlling these vector-pathogen systems is threatened by widespread insecticide resistance. The work presented here profiled the gene expression of the larvae from two field populations of Ae. aegypti with differential susceptibility to temephos. The contrasting phenotypes originated from two Colombian urban locations, Bello and Cucuta, that we have previously reported to have distinctive disease incidence, socioeconomics, and climate. The closeness of the geographical origin of the study populations was suspected to be highly influential in the profiling of the gene expression of resistance since the mosquitos resistance levels themselves are highly dependent upon environmental variables. We demonstrated that an exclusive field-to-lab (Ae. aegypti reference strain New Orleans) comparison generates an over estimation of differential gene expression (DGE) and that the inclusion of a geographically relevant field control, as used here, yields a more discrete, and likely, more specific set of genes. The composition of the obtained DGE profiles is varied, with commonly reported resistance associated genes such as detoxifying enzymes having only a small representation. We identify cuticle biosynthesis, ion exchange homeostasis, an extensive number of long non-coding RNAs, and chromatin modelling among the specifically and differentially expressed genes in field resistant Ae. aegypti larvae. It was also shown that temephos resistant larvae undertake further gene expression responses when temporarily exposed to this insecticide. The results from the sampling triangulation approach undertaken here contributes a discrete DGE profiling with reduced noise that permitted the observation of a greater gene diversity. This deeper gene granularity significantly increases the number of potential targets for the control of insecticide resistant mosquitoes and widens our knowledge base on the complex phenotypic network of the Ae. aegypti mosquito responses to insecticides.

Author SummaryAedes aegypti mosquitoes are vectors for several significant human viruses including dengue, Zika and chikungunya. The lack of widely available vaccines and specific antiviral treatments for these viruses means that the principal method for reducing disease burden is through controlling the vector mosquitoes. Mosquito control relies heavily on the use of insecticides and successful vector control is threatened by widespread insecticide resistance in Ae. aegypti. Here, we examined changes in gene expression that occur in temephos resistant populations of Ae. aegypti from two field populations in Colombia. We compare gene expression in resistant larvae from Cucuta with susceptible larvae from Bello and a susceptible laboratory strain of Ae. aegypti (New Orleans). We also compare mosquitoes from Cucuta with and without temephos exposure. We report several differentially expressed genes beyond those usually reported in resistant mosquitoes. We also demonstrate the over estimation in differential gene expression that can occur when field resistant populations are compared against lab susceptible populations only. The identification of new mechanisms involved in the development of insecticide resistance is crucial to fully understanding how resistance occurs and how best it can be reduced.

sentences: A northern range extension of a Canadian species of special concern, Dielis pilipes (Hymenoptera: Scoliidae), in the Okanagan Valley of British Columbia The only known Canadian records of the yellow scarab hunter wasp, Dielis pilipes (Saussure) (Hymenoptera: Scoliidae), are from the southern Okanagan and Similkameen valleys of British Columbia. We report a 25-km northern range extension of the species into the Ponderosa Pine Biogeoclimatic Ecosystem Classification zone, collected in an unmanaged agricultural field in Summerland, British Columbia. This finding is of conservation importance and has implications for natural biological control of ten-lined June beetles, Polyphylla decemlineata (Say) and P. crinita LeConte (Coleoptera: Scarabaeidae), incidental agricultural pests in the Okanagan.

sentences: Forkhead transcription factor FKH-8 is a master regulator of primary cilia in C. elegans Cilia, either motile or non-motile (a.k.a primary or sensory), are complex evolutionary conserved eukaryotic structures composed of hundreds of proteins required for their assembly, structure and function that are collectively known as the ciliome. Ciliome mutations underlie a group of pleiotropic genetic diseases known as ciliopathies. Proper cilium function requires the tight coregulation of ciliome gene transcription, which is only fragmentarily understood. RFX transcription factors (TF) have an evolutionarily conserved role in the direct activation of ciliome genes both in motile and non-motile cilia cell types. In vertebrates, FoxJ1 and FoxN4 Forkhead (FKH) TFs work with RFX in the direct activation of ciliome genes, exclusively in motile cilia cell-types. No additional TFs have been described to act together with RFX in primary cilia cell-types in any organism. Here we describe FKH-8, a FKH TF, as master regulator of the primary ciliome in Caenorhabditis elegans. fkh-8 is expressed in all ciliated neurons in C. elegans, binds the regulatory regions of ciliome genes, regulates ciliome gene expression, cilium morphology and a wide range of behaviours mediated by sensory cilia. Importantly, we find FKH-8 function can be replaced by mouse FOXJ1 and FOXN4 but not by members of other mouse FKH subfamilies. In conclusion, our results show that RFX and FKH TF families act as master regulators of ciliogenesis also in sensory ciliated cell types and suggest that this regulatory logic could be an ancient trait predating functional cilia sub-specialization.

sentences: Modulation of the renin-angiotensin system inhibits memory advantage for negative emotional material via decreasing hippocampus activation and its coupling with the amygdala Exaggerated arousal and dysregulated emotion-memory interactions are key pathological dysregulations that accompany the development of post-traumatic stress disorder (PTSD). Current treatments for PTSD are of moderate efficacy and preventing the dysregulations already during exposure to threatening events may attenuate the development of PTSD-symptomatology. In a preregistered double-blind, between-subject, placebo-controlled pharmaco-fMRI design, the present proof-of-concept study examined the potential of a single dose of angiotensin II type 1 receptor (AT1R) antagonist losartan (LT) to attenuate the mnemonic advantage of threatening stimuli and the underlying neural mechanism via combining an emotional subsequent memory paradigm with LT (n=29) or placebo treatment (n=30) and a surprise memory test after 24h washout. LT generally improved memory performance and abolished emotional memory enhancement for negative yet not positive material while emotional experience during encoding remained intact. LT further suppressed hippocampus activity during encoding of subsequently remembered negative stimuli. On the network level LT reduced coupling between hippocampus and basolateral amygdala during successful memory formation of negative stimuli. Our findings suggest that LT may have the potential to attenuate memory formation for negative yet not positive information by decreasing hippocampus activity and its functional coupling strength with amygdala. These findings suggest a promising potential of LT to prevent preferential encoding and remembering of negative events, a mechanism that could prevent the emotion-memory dysregulations underlying the development of PTSD-symptomatology.

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Individual Stability of Pain- and Touch-Related Neuronal Gamma Oscillations BackgroundThe processing of brief pain and touch stimuli has been associated with an increase of neuronal oscillations in the gamma range (40-90 Hz). However, some studies report divergent gamma effects across single participants.

MethodsIn two repeated sessions we recorded gamma responses to pain and touch stimuli using EEG. Individual gamma responses were extracted from EEG channels and from ICA components that contain a strong gamma amplitude.

ResultsWe observed gamma responses in the majority of the participants. If present, gamma synchronisation was always bound to a component that contained a laser-evoked response. We found a broad variety of individual cortical processing: some participants showed a clear gamma effect, others did not exhibit any gamma. For both modalities, the effect was reproducible between sessions. In addition, participants with a strong gamma response showed a similar time-frequency pattern across sessions.

ConclusionsOur results indicate that current measures of reproducibility of research results do not reflect the complex reality of the diverse individual processing pattern of applied pain and touch. The present findings raise the question of whether we would find similar quantitatively different processing patterns in other domains in neuroscience: group results would be replicable but the overall effect is driven by a subgroup of the participants.

sentences: Liquid-liquid phase separation of florigen activation complex induces flowering Floral transition, regulated by the systemic action of the mobile florigen protein FLOWERING LOCUS T (FT), is essential for successful plant reproduction1. How FT controls downstream gene expression remains incompletely understood, although it relies on the florigen activation complex (FAC), a core component of FT function2-4. The FAC is a nucleus-localized transcriptional activator of genes encoding MADS-box transcription factors critical to reproductive development and consists of florigen FT; a scaffold 14-3-3 protein that is a key component for complex assembly; and FD, a basic leucine-zipper protein that recruits the FAC to DNA. Here we report that the FAC exhibits phase separation. In rice shoot apical meristem cells, rice florigen Heading date 3a (Hd3a) fused to the green fluorescent protein formed speckles in the nucleus. The FAC speckle is formed in a FAC-dependent manner in tobacco cells. Recombinant Hd3a, but not OsFD1, phase-separated in vitro, and this effect was enhanced in the presence of 14-3-3 protein. Furthermore, mutations affecting functionally important residues in the pocket region or C-terminal disordered region of Hd3a affected FAC phase separation, providing a biochemical framework for the proteins effect on flowering. The ability to form condensates via phase transition represents a previously unknown mechanism for gene activation by the FAC.

sentences: CatSper mediates the chemotactic behavior and motility of the ascidian sperm Sperm motility, including chemotactic behavior, is regulated by changes in the intracellular Ca2+ concentration. The cation channel of sperm (CatSper), plays an important role in the regulation of intracellular Ca2+ concentration. In mammals, CatSper is the only Ca2+ channel that functions in the sperm, and the mice that lack the genes for the subunits of CatSper, which make up the pore region of the Ca2+ channel, are infertile due to the inhibition of hyperactivation of the sperm. CatSper is also thought to be involved in chemotaxis in sea urchins. In contrast, in the ascidian, Ciona intestinalis, the sperm-activating and -attracting factor (SAAF) interacts with Ca2+/ATPase, which is a Ca2+-pump. Although the existence of CatSper genes has been reported, it is not clear whether CatSper is the specific Ca2+ channel that functions in the ascidian sperm. Therefore, in this study, we generated Catsper3 knockout (KO) animals that found that they were significantly less motile, with few motile sperms not exhibiting any chemotactic behavior. These results suggest that CatSper plays important roles in the spermatogenesis and basic motility mechanisms of sperms in both ascidians and mammals.

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Effect sizes and test-retest reliability of the fMRI-based Neurologic Pain Signature Identifying biomarkers that predict mental states with large effect sizes and high test-retest reliability is a growing priority for fMRI research. We examined a well-established multivariate brain measure that tracks pain induced by nociceptive input, the Neurologic Pain Signature (NPS). In N = 295 participants across eight studies, NPS responses showed a very large effect size in predicting within-person single-trial pain reports (d = 1.45) and medium effect size in predicting individual differences in pain reports (d = 0.49). The NPS showed excellent shortterm (within-day) test-retest reliability (ICC = 0.84, with average 69.5 trials/person). Reliability scaled with the number of trials within-person, with [≥]60 trials required for excellent test-retest reliability. Reliability was tested in two additional studies across 5-day (N = 29, ICC = 0.74, 30 trials/person) and 1-month (N = 40, ICC = 0.46, 5 trials/person) test-retest intervals. The combination of strong within-person correlations and only modest between-person correlations between the NPS and pain reports indicate that the two measures have different sources of between-person variance. The NPS is not a surrogate for individual differences in pain reports but can serve as a reliable measure of pain-related physiology and mechanistic target for interventions.

Significance statementCurrent efforts towards translating brain biomarkers require identifying brain measures that can strongly and reliably predict outcomes of interest. We systematically examined the performance of a well-established brain activity pattern, the Neurological Pain Signature (NPS), in a large and diverse sample of participants. The NPS showed excellent reliability, and the reliability scaled with the number of trials within-person. The NPS responses showed strong correlations with pain reports at the within-person level but only modest correlations at the between-person level. The findings suggest that the NPS is not a surrogate for individual differences in pain reports but can serve as a reliable measure of a pain-related physiological target.

Author NoteThis project was supported by grants R01MH076136 (T.D.W.), R01DA046064, R01EB026549, and R01DA035484. Elizabeth A. Reynolds Losin was supported by a Mentored Research Scientist Development award from National Institute On Drug Abuse of the National Institutes of Health (K01DA045735). Lauren Y. Atlas was supported in part by funding from the Intramural Research Program of the National Center for Complementary and Integrative Health. Yoni K. Ashar was supported by NCATS Grant # TL1-TR-002386. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Code for all analyses and figures is available at https://github.com/XiaochunHan/NPS_measurement_properties. Data for all analyses and figures is available at https://osf.io/v9px7/.

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Network potential identifies therapeutic miRNA cocktails in Ewings Sarcoma MicroRNA (miRNA)-based therapies are an emerging class of targeted therapeutics with many potential applications. Ewing Sarcoma patients could benefit dramatically from personalized miRNA therapy due to inter-patient heterogeneity and a lack of druggable (to this point) targets. However, because of the broad effects miRNAs may have on different cells and tissues, trials of miRNA therapies have struggled due to severe toxicity and unanticipated immune response. In order to overcome this hurdle, a network science-based approach is well-equipped to evaluate and identify miRNA candidates and combinations of candidates for the repression of key oncogenic targets while avoiding repression of essential housekeeping genes. We first characterized 6 Ewing sarcoma cell lines using mRNA sequencing. We then estimated a measure of tumor state, which we term network potential, based on both the mRNA gene expression and the underlying protein-protein interaction network in the tumor. Next, we ranked mRNA targets based on their contribution to network potential. We then identified miRNAs and combinations of miRNAs that preferentially act to repress mRNA targets with the greatest influence on network potential. Our analysis identified TRIM25, APP, ELAV1, RNF4, and HNRNPL as ideal mRNA targets for Ewing sarcoma therapy. Using predicted miRNA-mRNA target mappings, we identified miR-3613-3p, let-7a-3p, miR-300, miR-424-5p, and let-7b-3p as candidate optimal miRNAs for preferential repression of these targets. Ultimately, our work, as exemplified in the case of Ewing sarcoma, describes a novel pipeline by which personalized miRNA cocktails can be designed to maximally perturb gene networks contributing to cancer progression.

Author SummaryPrecision medicine in cancer aims to find the right treatment, for the right patient, at the right time. Substantial variation between patient tumors, even of the same disease site, has limited the application of precision medicine in the clinic. In this study, we present novel computational tools for the identification of targets for cancer therapy using widely available sequencing data. We used a network-science based approach that leveraged multiple types of omic data to identify functionally relevant disease targets. Further, we developed algorithms to identify potential miRNA-based therapies that inhibit these predicted disease targets. We applied this pipeline to a novel Ewing Sarcoma transcriptomics data-set as well as publicly available patient data from the St. Jude Cloud. We identified a number of promising therapeutic targets for this rare disease, including EWSR1, the proposed driver of Ewing Sarcoma development. These novel data and methods will provide researchers with new tools for the development of precision medicine treatments in a variety of cancer systems.

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Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step towards a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented.

SUMMARYThis protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided along-side directions to prevent potential pitfalls.

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Flanker: a tool for comparative genomics of gene flanking regions Analysing the flanking sequences surrounding genes of interest is often highly relevant to understanding the role of mobile genetic elements (MGEs) in horizontal gene transfer, particular for antimicrobial resistance genes. Here, we present Flanker, a Python package which performs alignment-free clustering of gene flanking sequences in a consistent format, allowing investigation of MGEs without prior knowledge of their structure. These clusters, known as  flank patterns, are based on Mash distances, allowing for easy comparison of similarity across sequences. Additionally, Flanker can be flexibly parameterised to finetune outputs by characterising upstream and downstream regions separately and investigating variable lengths of flanking sequence. We apply Flanker to two recent datasets describing plasmid-associated carriage of important carbapenemase genes (blaOXA-48 and blaKPC-2/3) and show that it successfully identifies distinct clusters of flank patterns, including both known and previously uncharacterised structural variants. For example, Flanker identified four Tn4401 profiles that could not be sufficiently characterised using TETyper or MobileElementFinder, demonstrating the utility of Flanker for flanking gene characterisation. Similarly, using a large (n=226) European isolate dataset, we confirm findings from a previous smaller study demonstrating association between Tn1999.2 and blaOXA-48 upregulation and demonstrate 17 flank patterns (compared to the 5 previously identified). More generally the demonstration in this study that flank patterns are associated with to geographical regions and antibiotic susceptibility phenotypes suggests that they may be useful as epidemiological markers. Flanker is freely available under an MIT license at https://github.com/wtmatlock/flanker.

Data SummaryNCBI accession numbers for all sequencing data used in this study is provided in Supplementary Table 1. The analysis performed in this manuscript can be reproduced in a binder environment provided on the Flanker Github page (https://github.com/wtmatlock/flanker).

sentences: Extinction of the Thylacine Like the Dodo and Passenger Pigeon before it, the predatory marsupial Thylacine (Thylacinus cynocephalus), or  Tasmanian tiger, has become an iconic symbol of human-caused extinction. The last captive animal died in 1936, but even today reports of the Thylacines possible ongoing survival in remote regions of Tasmania are newsworthy and capture the publics imagination. Extirpated from mainland Australia in the mid-Holocene, the large island of Tasmania became the species final stronghold. Following European settlement in the 1800s, the Thylacine was heavily persecuted and pushed to the margins of its range, although many sightings were reported thereafter--even well beyond the 1930s. To gain a new depth of insight into the extinction of the Thylacine, we assembled an exhaustive database of 1,237 observational records from Tasmania (from 1910 onwards), quantified their uncertainty, and charted the patterns these revealed. We also developed a new method to visualize the species 20th-century spatio-temporal dynamics, to map potential post-bounty refugia and pinpoint the most-likely location of the final persisting subpopulation. A direct reading of the high-quality records (confirmed kills and captures, in combination with sightings by past Thylacine hunters and trappers, wildlife professionals and experienced bushmen) implies a most-likely extinction date within four decades following the last capture (i.e., 1940s to 1970s). However, uncertainty modelling of the entire sighting record, where each observation is assigned a probability and the whole dataset is then subject to a sensitivity analysis, suggests that extinction might have been as recent as the late 1980s to early 2000s, with a small chance of persistence in the remote south-western wilderness areas. Beyond the intrinsically fascinating problem of reconstructing the final fate of the Thylacine, the new spatio-temporal mapping of extirpation developed herein would also be useful for conservation prioritization and search efforts for other rare taxa of uncertain status.

sentences: Adduction Induces Large Optic Nerve Head Deformations in Subjects with Normal Tension Glaucoma Purpose: To assess optic nerve head (ONH) deformations and strains during adduction, abduction, and intraocular pressure (IOP) elevation in subjects with high-tension glaucoma (HTG) and normal-tension glaucoma (NTG). Design: Clinic-based cross-sectional study. Participants: 114 HTG subjects and 114 NTG subjects. Methods. We recruited 228 subjects (114 subjects with HTG [pre-treatment IOP > 21mmHg] and 114 with NTG [pre-treatment IOP < 21mmHg]). For each subject, we imaged the ONH using spectral-domain optical coherence tomography (OCT) under the following conditions: (1) primary gaze, (2) 20 degree adduction, (3) 20 degree abduction, and (4) primary gaze with acute IOP elevation (to approximately 33 mmHg) achieved through ophthalmodynamometry. For each OCT volume, we automatically segmented the prelaminar tissue (PLT), the choroid, the sclera and the lamina cribrosa (LC) using a deep learning algorithm. We also digitally aligned the OCT volumes obtained from (2)-(4) to the primary gaze volume (1) before performing digital volume correlation (DVC) analysis to quantify IOP- and gaze-induced ONH tissues three-dimensional displacements and effective strain (a local measure of tissue deformation) for all scenarios. Main Outcome Measures: Three-dimensional ONH displacements and strains. Results: Across all subjects, adduction generated high effective strain (4.2 {+/-} 1.4%) in the ONH tissues with no significant difference (p>0.05) with those induced by IOP elevation (4.5 {+/-} 1.5%); while abduction generated significantly lower (p = 0.014) effective strain (3.8 {+/-} 1.1%). Interestingly, the LC of HTG subjects exhibited significantly higher effective strain than those of NTG subjects under IOP elevation (HTG:4.6 {+/-} 1.7% vs NTG:4.1 {+/-} 1.5%, p = 0.047). Conversely, the LC tissue of NTG subjects exhibited significantly higher effective strain than those of HTG subjects under adduction (NTG: 4.9 {+/-} 1.9% vs HTG: 4.0 {+/-} 1.4%, p = 0.041). Conclusion: We found that adduction produced comparable strains and displacements as IOP elevation. We also found that NTG subjects experienced higher strains due to adduction than HTG subjects, while HTG subjects experienced higher strain due to IOP elevation than NTG subjects - and that these differences were most pronounced in the LC tissue.

sentences: Sequential action of a tRNA base editor in conversion of cytidine to pseudouridine Post-transcriptional RNA editing modulates gene expression in a condition-dependent fashion. We recently discovered C-to-{Psi} editing in Vibrio cholerae tRNA. Here, we characterize the biogenesis, regulation, and functions of this previously undescribed RNA editing process. We show that an enzyme, TrcP, mediates the editing of C-to-U followed by the conversion of U to {Psi}, consecutively. AlphaFold-2 predicts that TrcP consists of two globular domains, including a novel cytidine deaminase and a pseudouridylase, along with a long helical domain. The latter domain tethers tRNA substrates during both the C-to-U editing and pseudouridylation, likely enabling a substrate channeling mechanism for efficient catalysis all the way to the terminal product. C-to-{Psi} editing both requires and suppresses other modifications, creating an interdependent network of modifications in the tRNA anticodon loop that facilitates coupling of tRNA modification states to iron availability. Our findings provide mechanistic insights into an RNA editing process that likely promotes environmental adaptation.

sentences: SOCS3 limits endotoxin-induced endothelial dysfunction by blocking a required autocrine interleukin 6 signal in human umbilical vein endothelial cells Increased circulating levels of soluble interleukin (IL)-6 receptor  (sIL-6R) are commonly observed during inflammatory responses, allowing for IL-6 signaling to occur in cells that express the ubiquitous receptor subunit gp130 but not IL-6R, such as endothelial cells. Activation of Toll-like receptor (TLR)-4 or the tumor necrosis factor (TNF) receptor leads to NF-{kappa}B-dependent increases in endothelial IL-6 expression. Thus, we hypothesize that danger signals may induce autocrine IL-6 signaling within the endothelium via sIL-6R-mediated trans-signaling. In support of this hypothesis, we recently demonstrated that conditional deletion in the endothelium of the IL-6 signaling inhibitor SOCS3 leads to rapid mortality in mice challenged with the TLR-4 agonist endotoxin through increases in vascular leakage, thrombosis, leukocyte adhesion, and a type I-like interferon response. Here, we sought to directly test a role for sIL-6R in LPS-treated human umbilical vein endothelial cells. We show that cotreatment with sIL-6R dramatically increases the loss of barrier function and the expression of COX2 and tissue factor mRNA levels induced by LPS. This co-treatment led to a strong activation of STAT1 and STAT3 while not affecting LPS-induced activation of p38 and NF-{kappa}B signaling. Similar results were obtained when sIL-6R was added to a TNF challenge. JAK inhibition by pretreatment with ruxolitinib or by SOCS3 overexpression blunted LPS and sIL-6R synergistic effects, while SOCS3 knockdown further increased the response. Together, these findings demonstrate that IL-6 signaling downstream of NF-kB activation leads to a strong endothelial activation and may explain the acute endotheliopathy observed during critical illness.

sentences: BigBrainWarp: Toolbox for integration of BigBrain 3D histology with mutlimodal neuroimaging AO_SCPLOWBSTRACTC_SCPLOWNeuroimaging stands to benefit from emerging ultrahigh-resolution histological atlases of the human brain; the first of which is "BigBrain". Ongoing research aims to characterise regional differentiation of cytoarchitecture with BigBrain and to optimise registration of BigBrain with standard neuroimaging templates. Together, this work paves the way for multi-scale investigations of brain organisation. However, working with BigBrain can present new challenges for neuroimagers, including dealing with cellular resolution neuroanatomy and complex transformation procedures. To simplify workflows and support adoption of best practices, we developed BigBrainWarp, a toolbox for integration of BigBrain with multimodal neuroimaging. The primary BigBrainWarp function wraps multiple state-of-the-art deformation matrices into one line of code, allowing users to easily map data between BigBrain and standard MRI spaces. Additionally, the toolbox contains ready-to-use cytoarchitectural features to improve accessibility of histological information. The present article discusses recent contributions to BigBrain-MRI integration and demonstrates the utility of BigBrainWarp for further investigations.

sentences: Do pesticide residues have enduring negative effect on macroinvertebrates and vertebrates in fallow rice paddies? Rice is one of the most important staple food in the world, with irrigated rice paddies largely converted from natural wetlands. The effectiveness of rice fields in help preserve species depends partially on management practices, including the usage of pesticides. However, related studies have focused predominately on the cultivation period, leaving the effects of soil pesticide residues on aquatic invertebrates during the fallow periods little explored; other animals, such as waterbirds, also rely on aquatic invertebrates in flooded fallow fields for their survival. We therefore investigated vertebrates and macroinvertebrates (terrestrial and aquatic) on rice stands and in flooded water during cultivation and fallow periods in organic and conventional rice fields in Taiwan. Association of environmental factors with terrestrial and aquatic organisms was also analyzed. In total, 32,880 individuals of 144 invertebrate families and 381 individuals of 15 vertebrate families were recorded after nine samplings each for six organic and six conventional rice fields. Family richness and abundance of all invertebrates (terrestrial and aquatic) were higher in organic than in conventional fields during the cultivation period, but were similar between the two agricultural practices during the fallow period. Richness and abundance of terrestrial invertebrates in both organic and conventional fields increased with the progression of rice cultivation, so did the differences between the two practices. Richness of aquatic invertebrates was mostly constant across the sampling period, while abundance increased but differences decreased during the fallow period. Richness and abundance of terrestrial invertebrates were positively associated with ambient temperature and height of rice stand. Abundance of aquatic invertebrates were positively associated with pH value and amount of dissolved oxygen but negatively associated with water temperature. Richness and abundance of all vertebrates and each of the constituting groups (fish, amphibian, reptile, bird, and migratory waterbird) were statistically similar between the two practices although abundance of migratory waterbirds in organic fields were two times those in conventional fields during the fallow period. Our study suggested accumulated effects of pesticides on suppressing terrestrial invertebrates during the cultivation period, but diminishing effects of pesticide residues on repressing aquatic invertebrates during the fallow period. This comprehensive study provided a holistic picture on macroinvertebrate and vertebrate fauna, as well as ramifications of pesticide usage, in a representative Southeast Asia rice paddy ecosystem. Further study should compare rice fields with natural wetlands to better assess how to capitalize on agroecosystems for biodiversity conservation.

sentences: Real-time object locator for cryo-EM data collection--- You only navigate EM once --- In cryo-electron microscopy (cryo-EM) data collection, locating a target object is the most error-prone. Here, we present a machine learning-based approach with a real-time object locator named yoneoLocr using YOLO, a well-known object detection system. Implementation showed its effectiveness in rapidly and precisely locating carbon holes in single particle cryo-EM and for locating crystals and evaluating electron diffraction (ED) patterns in automated cryo-electron crystallography (cryo-EX) data collection.

sentences: Structure-function conservation between the methyltransferases SETD3 and SETD6 Among the protein lysine methyltransferases family members, it appears that SETD6 is highly similar and closely related to SETD3. The two methyltransferases show high similarity in their structure, which raised the hypothesis that they share cellular functions. Using a proteomic screen, we identified 52 shared interacting-proteins. Gene Ontology (GO) analysis of the shared proteins revealed significant enrichment of proteins involved in transcription. Our RNA-seq data of SETD6 KO and SETD3 KO HeLa cells identified [~]100 up-regulated and down-regulated shared genes. We have also identified a substantial number of genes that changed dramatically in the double KO cells but did not significantly change in the single KO cells. GO analysis of these genes revealed enrichment of apoptotic genes. Accordingly, we show that the double KO cells displayed high apoptotic levels, suggesting that SETD6 and SETD3 inhibit apoptosis. Collectively, our data strongly suggest a functional link between SETD6 and SETD3 in the regulation of apoptosis.

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Protein sequence sampling and prediction from structural data The increasing integration between protein engineering and machine learning has led to many interesting results. A problem still to solve is to evaluate the likelihood that a sequence will fold into a target structure. This problem can be also viewed as sequence prediction from a known structure.

In the current work, we propose improvements in the recent architecture of Geometric Vector Perceptrons [1] in order to optimize the sampling of sequences from a known backbone structure. The proposed model differs from the original in that there is: (i) no updating in the vectorial embedding, only in the scalar one, (ii) only one layer of decoding. The first aspect improves the accuracy of the model and reduces the use of memory, the second allows for training of the model with several tasks without incurring data leakage.

We treat the trained classifier as an Energy-Based Model and sample sequences by sampling amino acids in a non-autoreggresive manner in the empty positions of the sequence using energy-guided criteria and followed by random mutation optimization. We improve the median identity of samples from 40.2% to 44.7%.

An additional question worth investigating is whether sampled and original sequences fold into similar structures independent of their identity. We chose proteins in our test set whose sampled sequences show low identity (under 30%) but for which our model predicted favorable energies. We used AlphaFold [2, 3] and observed that the predicted structures for sampled sequences highly resemble the predicted structures for original sequences, with an average TM-score of 0.84.

sentences: A butterfly pan-genome reveals a large amount of structural variation underlies the evolution of chromatin accessibility Despite insertions and deletions being the most common structural variants (SVs) found across genomes, not much is known about how much these SVs vary within populations and between closely related species, nor their significance in evolution. To address these questions, we characterized the evolution of indel SVs using genome assemblies of three closely related Heliconius butterfly species. Over the relatively short evolutionary timescales investigated, up to 18.0% of the genome was composed of indels between two haplotypes of an individual H. charithonia butterfly and up to 62.7% included lineage-specific SVs between the genomes of the most distant species (11 Mya). Lineage-specific sequences were mostly characterized as transposable elements (TEs) inserted at random throughout the genome and their overall distribution was similarly affected by linked selection as single nucleotide substitutions. Using chromatin accessibility profiles (i.e., ATAC-seq) of head tissue in caterpillars to identify sequences with potential cis-regulatory function, we found that out of the 31,066 identified differences in chromatin accessibility between species, 30.4% were within lineage-specific SVs and 9.4% were characterized as TE insertions. These TE insertions were localized closer to gene transcription start sites than expected at random and were enriched for several transcription factor binding site candidates with known function in neuron development in Drosophila. We also identified 24 TE insertions with head-specific chromatin accessibility. Our results show high rates of structural genome evolution that were previously overlooked in comparative genomic studies and suggest a high potential for structural variation to serve as raw material for adaptive evolution.

sentences: Inferring Mycobacterium bovis transmission between cattle and badgers using isolates from the Randomised Badger Culling Trial Mycobacterium bovis (M. bovis) is a causative agent of bovine tuberculosis, a significant source of morbidity and mortality in the global cattle industry. The Randomised Badger Culling Trial was a field experiment carried out between 1998 and 2005 in the South West of England. As part of this trial, M. bovis isolates were collected from contemporaneous and overlapping populations of badgers and cattle within ten defined trial areas. We combined whole genome sequences from 1,442 isolates with location and cattle movement data, identifying transmission clusters and inferred rates and routes of transmission of M. bovis. Most trial areas contained a single transmission cluster that had been established shortly before sampling, often contemporaneous with the expansion of bovine tuberculosis in the 1980s. The estimated rate of transmission from badger to cattle was approximately two times higher than from cattle to badger, and the rate of within-species transmission considerably exceeded these for both species. We identified long distance transmission events linked to cattle movement, recurrence of herd breakdown by infection within the same transmission clusters and superspreader events driven by cattle but not badgers. Overall, our data suggests that the transmission clusters in different parts of South West England that are still evident today were established by long-distance seeding events involving cattle movement, not by recrudescence from a long-established wildlife reservoir. Clusters are maintained primarily by within-species transmission, with less frequent spill-over both from badger to cattle and cattle to badger.

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Human neural progenitors establish a diffusion barrier in the ER membrane during cell division Asymmetric segregation of cellular components regulates the fate and behavior of somatic stem cells. Similar to dividing budding yeast and precursor cells in C. elegans, it has been shown that mouse neural progenitors establish a diffusion barrier in the membrane of the endoplasmic reticulum (ER), which has been associated with asymmetric partitioning of damaged proteins and cellular age. However, the existence of an ER-diffusion barrier in human cells remains unknown. Here we used fluorescence loss in photobleaching (FLIP) imaging to show that human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells establish an ER-diffusion barrier during cell division. The human ER-diffusion barrier is regulated via Lamin-dependent mechanisms and is associated with asymmetric segregation of mono- and polyubiquitinated, damaged proteins. Further, forebrain regionalized organoids derived from hESCs were used to show the establishment of an ER-membrane diffusion barrier in more naturalistic tissues mimicking early steps of human brain development. Thus, the data provided here show that human neural progenitors establish a diffusion barrier during cell division in the membrane of the ER, which may allow for asymmetric segregation of cellular components, contributing to the fate and behavior of human neural progenitor cells.

SummaryHuman neural progenitors (NPCs) establish a diffusion barrier during cell division in the membrane of the endoplasmic reticulum, allowing for asymmetric segregation of cellular components, which may contribute to the fate and behavior of human NPCs.

sentences: Timing and localization of myasthenia gravis-related gene expression Myasthenia gravis (MG) is an acquired autoimmune disorder caused by autoantibodies binding acetylcholine receptors (AChR), muscle-specific kinase (MuSK), agrin or low-density lipoprotein receptor-related protein 4 (Lrp4). These autoantibodies inhibit neuromuscular transmission by blocking the function of these proteins, and thereby cause fluctuating skeletal muscle weakness. Several reports suggest that these autoantibodies might also affect the central nervous system (CNS) in MG patients. A comprehensive overview of the timing and localization of the expression of MG-related antigens in other organs is currently lacking. To investigate the spatio-temporal expression of MG-related genes outside skeletal muscle, we used in silico tools to assess public expression databases. Acetylcholine esterase, nicotinic AChR 1 subunit, agrin, collagen Q, Dok7, Lrp4, MuSK and rapsyn were included as MG-related genes because of their well-known involvement in either congenital or autoimmune MG. We investigated expression of MG-related genes in 1) all human tissues using GTEx data, 2) specific brain regions, 3) neurodevelopmental stages, and 4) cell types using datasets from the Allen Institute for Brain Sciences. MG-related genes show heterogenous spatio-temporal expression patterns in the human body as well as in the CNS. For each of these genes several (new) tissues, brain areas and cortical cell types with (relatively) high expression were identified suggesting a potential role for these genes outside skeletal muscle. The possible presence of MG-related antigens outside skeletal muscle suggests that autoimmune MG, congenital MG or treatments targeting the same proteins may affect MG-related protein function in other organs.

sentences: Environment-driven reprogramming of gamete DNA methylation occurs during maturation and is transmitted intergenerationally in salmon An epigenetic basis for transgenerational plasticity is widely theorized, but convincing empirical support is limited by taxa-specific differences in the presence and role of epigenetic mechanisms. In teleost fishes, DNA methylation generally does not undergo extensive reprogramming and has been linked with environmentally-induced intergenerational effects, but solely in the context of early life environmental differences. Using whole genome bisulfite sequencing, we demonstrate that differential methylation of sperm occurs in response to captivity during the maturation of Atlantic Salmon (Salmo salar), a species of major economic and conservation significance. We show that adult captive exposure further induces differential methylation in an F1 generation that is associated with fitness-related phenotypic differences. Genes targeted with differential methylation were consistent with genes differential methylated in other salmonid fishes experiencing early-life hatchery rearing, as well as genes under selection in domesticated species. Our results support a mechanism of transgenerational plasticity mediated by intergenerational inheritance of DNA methylation acquired late in life for salmon. To our knowledge, this is the first-time environmental variation experienced later in life has been directly demonstrated to influence gamete DNA methylation in fish.

sentences: Impact of sequencing technologies on long non-coding RNA computational identification The correct annotation of non-coding RNAs, especially long non-coding RNAs (lncRNAs), is still an important critial challenge in genome analyses. One crucial issue in lncRNA transcript annotation is the transcriptome resource that supports lncRNA loci. Long-read technologies now bring the potential to improve the quality of transcriptome annotation. Consequently, long non-coding RNAs (lncRNA) are probably the most benefited class of transcripts that would have improved annotation using this novel technology. However, there is a gap regarding benchmarking studies that highlighted if the direct use of lncRNA predictors in long-reads makes more precise identification of these transcripts. Considering that these lncRNA tools were not trained with these reads, we want to address: how is the performance of these tools? Are they also able to efficiently identify lncRNAs? We could provide evidence of where and how to make potential better approaches for the lncRNA annotation by understanding these issues. Keywords: Non-coding RNAs, high-throughput sequencing technologies, coding, methods, benchmarking, tools, NGS, transcripts

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Prediction of Kinase-Substrate Associations Using The Functional Landscape of Kinases and Phosphorylation Sites MotivationProtein phosphorylation is a key post-translational modification that plays a central role in many cellular processes. With recent advances in biotechnology, thousands of phosphorylated sites can be identified and quantified in a given sample, enabling proteome-wide screening of cellular signaling. However, the kinase(s) that phosphorylate most (> 90%) of the identified phosphorylation sites are unknown. Knowledge of kinase-substrate associations is also mostly limited to a small number of well-studied kinases, with 20% of known kinases accounting for the phosphorylation of 87% of currently annotated sites. The scarcity of available annotations calls for the development of computational algorithms for more comprehensive and reliable prediction of kinase-substrate associations.

ResultsTo broadly utilize available structural, functional, evolutionary, and contextual information in predicting kinase-substrate associations, we develop a network-based machine learning framework. Our framework integrates a multitude of data sources to characterize the landscape of functional relationships and associations among phosphosites and kinases. To construct a phosphosite-phosphosite association network, we use sequence similarity, shared biological pathways, co-evolution, co-occurrence, and co-phosphorylation of phosphosites across different biological states. To construct a kinase-kinase association network, we integrate protein-protein interactions, shared biological pathways, and membership in common kinase families. We use node embeddings computed from these heterogeneous networks to train machine learning models for predicting kinase-substrate associations. Our systematic computational experiments using the PhosphositePLUS database shows that the resulting algorithm, NetKSA, outperforms state-of-the-art algorithms and resources, including KinomeXplorer and LinkPhinder, in reliably predicting KSAs. By stratifying the ranking of kinases, NetKSA also enables annotation of phosphosites that are targeted by relatively less-studied kinases. Finally, we observe that the performance of NetKSA is robust to the choice of network embedding algorithms, while each type of network contributes valuable information that is complementary to the information provided by other networks.

ConclusionRepresentation of available functional information on kinases and phosphorylation sites, along with integrative machine learning algorithms, has the potential to significantly enhance our knowledge on kinase-substrate associations.

AvailabilityThe code and data are available at compbio.case.edu/NetKSA.

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Cerebrospinal fluids from healthy pregnant women does not harbor a detectable microbial community Cerebrospinal fluids circulating human central nervous system have long been considered aseptic in healthy individuals, because normally the blood-brain barrier protects against microbial invasions. However, this dogma has been questioned by several reports that microbes were identified in human brains, raising the question whether a microbial community is present in cerebrospinal fluids of healthy individuals without neurological diseases. Here, we collected and analyzed metagenomic and metatranscriptomic sequencing data of cerebrospinal fluid specimens from a cohort of 23 pregnant women aged between 23 and 40 and one-to-one matched contamination controls. From data analysis of 116 specimens of eight different types, we detected 619 nonredundant microbial taxa which were dominated by bacteria (75%) and viruses (24%). In cerebrospinal fluids metagenomic samples, a total of 76 redundant species were detected including four (one nonredundant) eukaryota taxa, eleven (four nonredundant) bacteria, and 61 (21 nonredundant) viruses that were mostly bacteriophages. Metagenomic data analysis found no significant difference between cerebrospinal fluid specimens and negative controls in terms of microbial species diversity. In addition, no active or viable microbiome were present in the cerebrospinal fluid samples after subtracting microbes detected in contamination controls. In conclusion, we found no strong evidence that colonized microbial community exist in the cerebrospinal fluids of healthy individuals.

IMPORTANCEMicrobiome are prevalent throughout human bodies with profound health implications. However, it remains unclear whether a microbiome is present and active in human cerebrospinal fluids that are long considered aseptic given the blood-brain barrier. Here, we applied unbiased metagenomic and metatranscriptomic sequencing to detect microbiome in cerebrospinal fluids collected from a cohort of 23 pregnant women with matched controls. By analyzing 116 specimens of eight types, no strong evidence was found to support a presence of colonized microbiome in the cerebrospinal fluids. Our findings have profound implications to human immunity against neurological infections and disorders, providing a guide for disease diagnostics, prevention and therapeutics in clinical settings.

sentences: Single-Cell Multiomics Defines Tolerogenic Extrathymic Aire-Expressing Populations with Unique Homology to Thymic Epithelium The Autoimmune Regulator (Aire) gene, well defined for its role in medullary thymic epithelial cells (mTECs) and immune self-tolerance, is also expressed in extrathymic Aire-expressing cells (eTACs) in the secondary lymphoid organs. eTACs have been shown to be hematopoietic antigen presenting cells (APCs) and potent inducers of immune tolerance (1-3). However, the precise identity and function of these cells remain unclear. Here, we use high-dimensional single-cell multiomics and functional approaches to define eTACs at the transcriptional, genomic, and proteomic level. We find that eTACs consist of two similar cell types: CCR7+ Aire-expressing migratory dendritic cells (AmDCs) and a unique Aire-hi population co-expressing Aire and RAR-related orphan receptor gamma-t (ROR{gamma}t). The latter, which have significant transcriptional and genomic homology to migratory dendritic cells (migDCs) and mTECs, we term Janus cells (JCs). All eTACs, and JCs in particular, have a highly accessible chromatin structure and high levels of broad gene expression, including tissue-specific antigens, as well as remarkable transcriptional and genomic homology to thymic medullary epithelium. As in the thymus, Aire expression in eTACs is also dependent on RANK-RANK-ligand interactions. Furthermore, lineage-tracing shows that JCs are not precursors to the majority of AmDCs. Finally, self-antigen expression by eTACs is sufficient to mediate negative selection of T cells escaping thymic selection and can prevent autoimmune diabetes in non-obese diabetic mice. This transcriptional, genomic, and functional symmetry between a hematopoietic Aire-expressing population in the periphery and an epithelial Aire-expressing population in the thymus suggests that a core biological program may influence self-tolerance and self-representation across the spectrum of immune development.

sentences: Site-specific O-glycosylation analysis of SARS-CoV-2 spike protein produced in insect and human cells Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S - insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.

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Caspase-1-driven neutrophil pyroptosis promotes an incomplete NETosis upon Pseudomonas aeruginosa infection Multiple neutrophil death programs contribute to host defense against infections. Although expressing all necessary components, neutrophils specifically fail to undergo pyroptosis, a lytic form of cell death triggered by the activation of the pro-inflammatory complex inflammasome. In the light of the arm race, we hypothesized that intrinsic neutrophil pyroptosis resistance might be bypassed in response to specific microbial species. We show that Pseudomonas aeruginosa (P. aeruginosa) stimulates Caspase-1-dependent pyroptosis in human and murine neutrophils. Mechanistically, activated NLRC4 inflammasome supports Caspase-1-driven Gasdermin-D (GSDMD) activation, IL-1{beta} cytokine release and neutrophil pyroptosis. Furthermore, GSDMD activates Peptidyl Arginine Deaminase-4 which drives an "incomplete NETosis" where neutrophil DNA fills the cell cytosol but fails crossing plasma membrane. Finally, we show that neutrophil Caspase-1 account for IL-1{beta} production and contributes to various P. aeruginosa strains spread in mice. Overall, we demonstrate that neutrophils are fully competent for Caspase-1-dependent pyroptosis, which drives an unsuspected "incomplete NETosis".

SummaryNeutrophils play an essential roles against infections. Although multiple neutrophil death programs contribute to host defense against infections, they fail to undergo pyroptosis, a pro-inflammatory form of cell death. Upon Infections, pyroptosis can be induced in macrophages or epithelial cells upon activation of pro-inflammatory complexes, inflammasomes that trigger Caspase-1-driven Gasdermin dependent plasma membrane lysis. In the light of host-microbe interactions, we hypothesized that yet to find microbial species might hold the capacity to overcome neutrophil resistance to inflammasome-driven pyroptosis. Among several bacterial species, we describe that the bacterium Pseudomonas aeruginosa specifically engages the NLRC4 inflammasome, which promotes Caspase-1-dependent Gasdermin-D activation and subsequent neutrophil pyroptosis. Furthermore, inflammasome-driven pyroptosis leads to DNA decondensation and expansion into the host cell cytosol but not to the so called Neutrophil Extracellular Trap (NET) release as DNA fails breaching the plasma membrane. Finally, in vivo P. aeruginosa infections highlight that Caspase-1-driven neutrophil pyroptosis is functional and is detrimental upon P. aeruginosa infection. Altogether, our results unexpectedly underline neutrophil competence for Caspase-1-dependent pyroptosis, a process that contributes to host susceptibility to P. aeruginosa infection.

sentences: Feature-based encoding of face identity by single neurons in the human amygdala and hippocampus Neurons in the human amygdala and hippocampus that are selective for the identity of specific people are classically thought to encode a persons identity invariant to visual features (e.g., skin tone, eye shape). However, it remains largely unknown how visual information from higher visual cortical areas is translated into such a semantic representation of an individual person. Here, we show that some amygdala and hippocampal neurons are selective to multiple different unrelated face identities based on shared visual features. The encoded identities form clusters in the representation of a deep neural network trained to recognize faces. Contrary to prevailing views, these neurons thus represent an individuals face with a visual feature-based code rather than one based on association with known concepts. Feature neurons encoded faces regardless of their identity, race, gender, familiarity, or pixel-level visual features; and the region of feature space to which feature neurons are tuned predicted their response to new face stimuli. Our results reveal a new class of neurons that bridge the perception-driven representation of facial features in the higher visual cortex with mnemonic semantic representations in the MTL, which may form the basis for declarative memory.

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Repair of APOBEC3G-mutated retroviral DNA in vivo is facilitated by the host enzyme uracil DNA glycosylase 2 Apolipoprotein B mRNA Editing Enzyme Catalytic Subunit 3 (APOBEC3) proteins are critical for the control of infection by retroviruses. These proteins deaminate cytidines in negative strand DNA during reverse transcription, leading to G to A changes in coding strands. Uracil DNA glycosylase (UNG) is a host enzyme that excises uracils in genomic DNA, which the base excision repair machinery then repairs. Whether UNG removes uracils found in retroviral DNA after APOBEC3-mediated mutation is not clear, and whether this occurs in vivo has not been demonstrated. To determine if UNG plays a role in the repair of retroviral DNA, we used APOBEC3G (A3G) transgenic mice which we showed previously had extensive deamination of murine leukemia virus (MLV) proviruses. The A3G transgene was crossed onto an UNG and mouse APOBEC3 knockout background (UNG-/-APO-/-) and the mice were infected with MLV. We found that virus infection levels were decreased in A3G UNG-/-APO-/- compared to A3G APO-/- mice. Deep sequencing of the proviruses showed that there were significantly higher levels of G-to-A mutations in proviral DNA from A3G transgenic UNG-/-APO-/- than A3G transgenic APO-/- mice, suggesting that UNG plays a role in the repair of uracil-containing proviruses. In in vitro studies, we found that cytoplasmic viral DNA deaminated by APOBEC3G was uracilated. In the absence of UNG, the uracil-containing proviruses integrated at higher levels into the genome than did those made in the presence of UNG. Thus, UNG also functions in the nucleus prior to integration by nicking uracil-containing viral DNA, thereby blocking integration. These data show that UNG plays a critical role in the repair of the damage inflicted by APOBEC3 deamination of reverse-transcribed DNA.

ImportanceWhile APOBEC3-mediated mutation of retroviruses is well-established, what role the host base excision repair enzymes play in correcting these mutations is not clear. This question is especially difficult to address in vivo. Here, we use a transgenic mouse developed by our lab that expresses human APOBEC3G and also lacks the endogenous uracil DNA glycosylase (Ung) gene, and show that UNG removes uracils introduced by this cytidine deaminase in MLV reverse transcripts, thereby reducing G-to-A mutations in proviruses. Furthermore, our data suggest that UNG removes uracils at two stages in infection - in unintegrated nuclear viral reverse transcribed DNA, resulting in its degradation and second, in integrated proviruses, resulting in their repair. These data suggest that retroviruses damaged by host cytidine deaminases take advantage of the host DNA repair system to overcome this damage.

sentences: Identification of Novel Long non-coding RNAs and Expression Analyses in Rectal Neuroendocrine Tumors Altered expression of long-non-coding RNAs (lncRNAs) is associated with various pathological conditions, including cancers. LncRNAs associated with rectal neuroendocrine tumors (rNETs) remain unexplored. Here, we report the identification of 418 high confidence novel lncRNAs by analyzing publicly available RNA-Seq datasets of primary rNETs and rNETs with lymph node or liver metastases. Novel lncRNAs had basic characteristics similar to the known lncRNAs and showed individual-specific expressions. Expression analysis in the normal rectal mucosa and pancreas NETs (pNETs) and small intestine NETs (siNETs) revealed high expression of two lncRNAs, namely MSTRG30689.1 and MSTRG341161.1. Functional characterization revealed that 9 lncRNAs could work as the ce-RNA and one as miRNAs precursor. Cis- and trans- analysis of the 9 PCG-associated lncRNAs revealed that these lncRNAs could regulate various important cancer-related genes. Further, an integrated regulatory mechanism for selected cancer hallmark genes such as SMAD3, ZEB1, TCF4 and ATM through lncRNAs is predicted. Taken together, our analyses reveal common and unique lncRNAs associated with rNETs and their metastatic variants, their PCG targets, and predict mechanisms of lncRNA mediated regulation of target PCGs.

sentences: ICoRD: Iterative Correlation-Based ROI Detection Method for the Extraction of Neural Signals in Calcium Imaging In vivo calcium imaging is a standard neuroimaging technique that allows selective observation of target neuronal activities. In calcium imaging, neuron activation signals provide key information for the investigation of neural circuits. For efficient extraction of the calcium signals of neurons, selective detection of the region of interest (ROI) pixels corresponding to the active subcellular region of the target neuron is essential. However, current ROI detection methods for calcium imaging data exhibit a relatively low signal extraction performance from neurons with a low signal-to-noise power ratio (SNR). This is problematic because a low SNR is unavoidable in many biological experiments. Therefore, we propose an iterative correlation-based ROI detection (ICoRD) method that robustly extracts the calcium signal of the target neuron from a calcium imaging series with severe noise. ICoRD extracts calcium signals closer to the ground-truth calcium signal than the conventional method from simulated calcium imaging data in all low SNR ranges. Additionally, this study confirmed that ICoRD robustly extracts activation signals against noise, even within in vivo environments. ICoRD showed reliable detection from neurons with a low SNR and sparse activation, which were not detected by conventional methods. ICoRD will facilitate our understanding of neural circuit activity by providing significantly improved ROI detection in noisy images.

sentences: Extensive neutralization against SARS-CoV-2 variants elicited by Omicron-specific subunit vaccine booster The currently dominant variant of SARS-CoV-2 Omicron, carrying a great number of mutations, has been verified its strong capacity of immune escape in COVID-19 convalescents and vaccinated individuals. An increased risk of SARS-CoV-2 reinfection or breakthrough infection should be concerned. Here we reported higher humoral immune response elicited by Delta and Omicron variants after breaking through previous infection and cross-neutralization against VOCs, compared to the ancestral wild-type (WT) virus infection. To overcome the immune escape of Omicron, Omicron-specific vaccine was considered as a novel and potential strategy. Mouse models were used to verify whether Omicron-specific RBD subunit boost immune response by immunizing Omicron-RBD recombinant proteins. Three doses of Omicron-RBD immunization elicit comparable neutralizing antibody (NAb) titers with three doses of WT-RBD immunization, but the neutralizing activity was not cross-active. By contrast, two doses of WT-RBD with an Omicron-RBD booster increased the NAb geometric mean titers against Omicron by 9 folds. Moreover, an additional boost vaccination with Omicron-RBD protein could increase humoral immune response against both WT and current VOCs. These results suggest that the Omicron-specific subunit booster shows its advantages in the immune protection from both WT and current VOCs, and that SARS-CoV-2 vaccines administration using two or more virus lineages as antigens might improve the NAb response.

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Mitochondrial ATP Synthase Subunit d, a Component of the Peripheral Stalk, is Essential for Growth and Heat Stress Tolerance in Arabidopsis thaliana. As rapid changes in climate threaten global crop yields, an understanding of plant heat stress tolerance is increasingly relevant. Heat stress tolerance involves the coordinated action of many cellular processes and is particularly energy demanding. We acquired a knockout mutant and generated knockdown lines in Arabidopsis thaliana of the d subunit of mitochondrial ATP synthase (gene name: ATPQ, AT3G52300, referred to hereafter as ATPd), a subunit of the peripheral stalk, and used these to investigate the phenotypic significance of this subunit in normal growth and heat stress tolerance. Homozygous knockout mutants for ATPd could not be obtained due to gametophytic defects, while heterozygotes possess no visible phenotype. Therefore, we used RNAi to create knockdown plant lines for further studies. Proteomic analysis and blue native gels revealed that ATPd downregulation impairs only subunits of the mitochondrial ATP synthase (complex V of the electron transport chain). Knockdown plants were more sensitive to heat stress, had abnormal leaf morphology, and were severely slow growing compared to wild type. These results indicate that ATPd plays a crucial role in proper function of the mitochondrial ATP synthase holoenzyme, which, when reduced, leads to wide-ranging defects in energy-demanding cellular processes. In knockdown plants, more hydrogen peroxide accumulated and mitochondrial dysfunction stimulon (MDS) genes were activated. These data establish the essential structural role of ATPd and provide new information about complex V assembly and quality control, as well as support the importance of mitochondrial respiration in normal plant growth and heat stress tolerance.

SIGNIFICANCE STATEMENTThe energy converter, mitochondrial ATP synthase, is critical for all organisms, but the functional importance of ATP synthase subunit d remains largely unknown in plants. We demonstrate the contributions of subunit d to plant growth, development, and heat stress tolerance, as well as to ATP synthase stability, ROS signaling and mitochondrial dysfunction regulation.

sentences: Validity of freehand three-dimensional ultrasound system in measurement of three-dimensional surface shape of shoulder muscles Freehand three-dimensional ultrasound (3DUS) system is a promising technique for accurately assessing muscle morphology. However, its accuracy has been validated mainly in terms of volume by examining lower limb muscles. This study aimed to validate 3DUS in the measurements of 3D surface shape and volume by comparing them with MRI measurements while ensuring the reproducibility of participant posture by focusing on the shoulder muscles. The supraspinatus, infraspinatus, and posterior deltoid muscles of 10 healthy males were scanned using 3DUS and MRI while secured by an immobilization support customized for each participant. A 3D surface model of each muscle was created from the 3DUS and MRI methods, and the agreement between them was assessed. For the muscle volume, the mean difference between the two models was within -0.51 cm3. For the 3D surface shape, the distances between the closest points of the two models and Dice similarity coefficient were calculated. The results showed that the median surface distance was less than 1.21 mm and Dice similarity coefficient was larger than 0.85. These results suggest that, given the above error is permitted, 3DUS can be used as an alternative to MRI in measuring volume and surface shape, even for the shoulder muscles.

sentences: Alternative Cdc20 translational isoforms bypass the spindle assembly checkpoint to control mitotic arrest duration Mitotic chromosome segregation defects activate the Spindle Assembly Checkpoint (SAC), which inhibits the APC/C co-activator Cdc20 to induce a prolonged cell cycle arrest. Once errors are corrected, the SAC is silenced thereby allowing anaphase onset and mitotic exit to proceed. However, in the presence of persistent, unresolvable errors, cells can undergo "mitotic slippage", exiting mitosis into a tetraploid G1 state and escaping the cell death that results from a prolonged arrest. The molecular logic that allows cells to balance these dueling mitotic arrest and slippage behaviors remains unclear. Here we demonstrate that human cells modulate their mitotic arrest duration through the presence of conserved, alternative Cdc20 translational isoforms. Translation initiation at downstream start sites results in truncated Cdc20 isoforms that are resistant to SAC-mediated inhibition and promote mitotic exit even in the presence of mitotic perturbations. Targeted molecular changes or naturally-occurring mutations in cancer cells that alter the relative Cdc20 isoform levels or its translational regulatory control modulate both mitotic arrest duration and anti-mitotic drug sensitivity. Our work reveals a critical role for the differential translational regulation of Cdc20 in mitotic arrest timing, with important implications for the diagnosis and treatment of human cancers.

sentences: Mesowestern Blot: Simultaneous Analysis of Hundreds of Sub-Microliter Lysates Western blotting is a widely-used technique for molecular-weight-resolved analysis of proteins and their post-translational modifications, but has been refractory to affordable scale-up. Here, we report the Mesowestern blot, which uses a 3D-printable gel-casting mold to enable affordable, high-throughput Western blotting with standard sample preparation and small (<1 uL) sample sizes. The casted polyacrylamide gel contains 336, 0.5 uL micropipette-loadable sample wells arranged within a standard microplate footprint. Polyacrylamide % can be altered to change molecular weight resolution range. Proof-of-concept experiments using both infrared-fluorescent molecular weight protein ladder as well as cell lysate (RIPA buffer) demonstrate protein loaded in Mesowestern gels is amenable to the standard Western blotting steps. The main difference between Mesowestern and traditional Western is that semi-dry horizontal instead of immersed vertical gel electrophoresis is used. The linear range of detection is approximately 2 orders of magnitude, with a limit of detection (for {beta}-actin) of around 30 ng of total protein from mammalian cell lysates (~30-3000 cells). Because the gel mold is 3D-printable, users have significant design freedom for custom layouts, and there are few barriers to adoption by the typical cell and molecular biology laboratory already performing Western blots.

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Habituation, task solving and memorization may facilitate biological invasions: the Starling example Invasions ecology deals more and more with behavioural characteristics of invasive species. Particularly, research have focused on the personality of invaders and on their way of coping with novelty in new habitats. Traits of neophobia may limit individuals in their exploration of novel objects or the consumption of novel foods, they may stop the access to valuable ressources. Actually, in novel environments like cities, food can be unreachable in throwaway dishes with lids or hidden in the garbage or even close to frightening objects. Animals may either left the place and waste these resources, or they can express low neophobia from the beginning and manipulate the objects to reach food. They may also habituate progressively to the context and use the ressources.

Here we analyzed the behavioural responses of individuals from three populations of European starling Sturnus vulgaris: a population anciently settled in a rural region, a population that has recently colonized a urban area and a population of wintering migrant birds.

We used a series of tests in order to explore if individuals would habituate to a novel object and if they could remember it eight months later. We explored if individuals would be less neophobic when confronted to two novel objects successively and we tested them in a learning task involving a novel object and an attractive food. Our results show that Sturnus vulgaris habituates rapidly to novel objects and that categorization facilitates neophobia lost when confronted to two different novel objects. Initially young birds appeared to be more skilled than adults in the learning task. Individuals from this species seem to be able to remember an object durably. We suggest that habituation, task solving and memorization are three mechanisms enhancing biological invasions.

sentences: Spatiotemporal changes in genetic diversity and structure of a recent fish invasion in eastern North America Introduced and geographically expanding populations experience similar eco-evolutionary challenges, including founder events, genetic bottlenecks, and novel environments. Theory predicts that reduced genetic diversity resulting from such genetic phenomena limits the colonization success of introduced populations. We examined an invasive population of a Eurasian freshwater fish, Tench (Tinca tinca), that has been expanding geographically in eastern North America for three decades. Using genomic data, we evaluated evidence for single versus multiple introductions and the connectivity of the population across the entire range in which it has been spreading. Tench exhibited low levels of genetic diversity, a lack of marked population subdivision across time and space, and evidence of a recent genetic bottleneck. These results suggest that the invasion stemmed from a single introduction, consistent with the reported invasion history. Furthermore, the large genetic neighbourhood size and weak within-population genetic substructure suggest high connectivity across the invaded range, despite the large area occupied, and no evidence of substantial diminution of genetic diversity from the invasion core to the margins. As eradicating the species within a ~112 km radius would be necessary to prevent recolonization, eradicating Tench is likely not feasible at watershed--and possibly local--scales. Management should instead focus on reducing abundance in priority conservation areas to mitigate adverse impacts. Our study supports the argument that introduced populations can thrive despite recent bottlenecks and low levels of genetic diversity, and it suggests that landscape heterogeneity and population demographics can generate variability in spatial patterns of genetic diversity within a single range expansion.

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Cryptic pathogen-sugar interactions revealed by universal saturation transfer analysis Many host pathogen interactions such as human viruses (including non-SARS-coronaviruses) rely on attachment to host cell-surface glycans. There are conflicting reports about whether the Spike protein of SARS-CoV-2 binds to sialic acid commonly found on host cell-surface N-linked glycans. In the absence of a biochemical assay, the ability to analyze the binding of glycans to heavily- modified proteins and resolve this issue is limited. Classical Saturation Transfer Difference (STD) NMR can be confounded by overlapping sugar resonances that compound with known experimental constraints. Here we present  universal saturation transfer analysis (uSTA), an NMR method that builds on existing approaches to provide a general and automated workflow for studying protein-ligand interactions. uSTA reveals that B-origin-lineage-SARS-CoV-2 spike trimer binds sialoside sugars in an  end on manner and modelling guided by uSTA localises binding to the spike N-terminal domain (NTD). The sialylated-polylactosamine motif is found on tetraantennary human N-linked-glycoproteins in deeper lung and may have played a role in zoonosis. Provocatively, sialic acid binding is abolished by mutations in some subsequent SARS- CoV-2 variants-of-concern. A very high resolution cryo-EM structure confirms the NTD location and  end on mode; it rationalises the effect of NTD mutations and the structure-activity relationship of sialic acid analogues. uSTA is demonstrated to be a robust, rapid and quantitative tool for analysis of binding, even in the most demanding systems.

Extended AbstractThe surface proteins found on both pathogens and host cells mediate entry (and exit) and influence disease progression and transmission. Both types can bear host-generated post- translational modifications such as glycosylation that are essential for function but can confound biophysical methods used for dissecting key interactions. Several human viruses (including non- SARS-coronaviruses) attach to host cell-surface N-linked glycans that include forms of sialic acid (sialosides). There remains, however, conflicting evidence as to if or how SARS-associated coronaviruses might use such a mechanism. Here, we demonstrate quantitative extension of  saturation transfer protein NMR methods to a complete mathematical model of the magnetization transfer caused by interactions between protein and ligand. The method couples objective resonance-identification via a deconvolution algorithm with Bloch-McConnell analysis to enable a structural, kinetic and thermodynamic analysis of ligand binding beyond previously-perceived limits of exchange rates, concentration or system. Using an automated and openly available workflow this  universal saturation transfer analysis (uSTA) can be readily-applied in a range of even heavily-modified systems in a general manner to now obtain quantitative binding interaction parameters (KD, kEx). uSTA proved critical in mapping direct interactions between natural sialoside sugar ligands and relevant virus-surface attachment glycoproteins - SARS-CoV-2-spike and influenza-H1N1-haemagglutinin variants - by quantitating ligand signal in spectral regions otherwise occluded by resonances from mobile protein glycans (that also include sialosides). In B- origin-lineage-SARS-CoV-2 spike trimer  end on-binding to sialoside sugars was revealed contrasting with  extended surface-binding for heparin sugar ligands; uSTA-derived constraints used in structural modelling suggested sialoside-glycan binding sites in a beta-sheet-rich region of spike N-terminal domain (NTD). Consistent with this, uSTA-glycan binding was minimally- perturbed by antibodies that neutralize the ACE2-binding domain (RBD) but strongly disrupted in spike from the B1.1.7/alpha and B1.351/beta variants-of-concern, which possess hotspot mutations in the NTD. Sialoside binding in B-origin-lineage-NTD was unequivocally pinpointed by cryo-EM to a site that is created from residues that are notably deleted in variants (e.g. H69,V70,Y145 in alpha). An analysis of beneficial genetic variances in cohorts of patients from early 2020 suggests a model in which this site in the NTD of B-origin-lineage-SARS-CoV-2 (but not in alpha/beta-variants) may have exploited a specific sialylated-polylactosamine motif found on tetraantennary human N-linked-glycoproteins in deeper lung. Together these confirm a novel binding mode mediated by the unusual NTD of SARS-CoV-2 and suggest how it may drive virulence and/or zoonosis via modulation of glycan attachment. Since cell-surface glycans are widely relevant to biology and pathology, uSTA can now provide ready, quantitative, widespread analysis of complex, host-derived and post-translationally modified proteins with putative ligands relevant to disease even in previously confounding complex systems.

sentences: Translational Proteomics for Transfusion Medicine: Resolution of the IVIG Proteomes of Different Geographically Sourced and Prepared IVIG Immunotherapies. Intravenous Immunoglobulins (IVIGs) are prepared from thousands of donor plasmas and as a result comprise an extreme broad mix and depth of Antibody (Ab) specificities. IVIG formulations available in Australia are from both local and overseas donor sources and extracted using a variety of purification methods and immunoglobulin purities, with the Australia-derived and prepared IVIG listed at >98% and the overseas-derived preparations at [≥] 95%. Because of these differences it was predicted that the formulations might individually vary in composition and that together with obvious genetic and geographic antigenic (Ag) environment differences may result in notable variability between formulations. Hence a focussed comparative proteomic profiling of IVIG formulations was undertaken to identify notable similarities and differences across products. Comparisons between formulations did reveal marked qualitative differences in 2D-gel Antibody profiling that included parameters of isoelectric charge (pI), as well as immunoglobulin (Ig) monomer to dimer ratio variability between products, including high molecular weight (MW) immunoglobulin multimers for some. These notable differences were in part quite likely a product of the respective purification methods used, and capacity to select (or de-select) for antibodies of such different properties. Furthermore, for identification of non-Ig proteins carried over from plasma through purifications Mass spectrometry was performed. This identified a few such ancillary proteins, and their identities, in general, differed between formulations. Proteins detected included the most abundant protein of plasma, albumin, as well as other mostly large and abundant proteins; RAG1 - V(D)J recombination activating protein1, gelsolin, complement Factor-B, serotransferrin, tetranectin, NADH ubiquinone oxidoreductase, caspase 3 and VEGFR1. An alternate strategy used commercial Multiplex xMAP assay to detect cytokines, which are small and present in plasma at trace but highly active quantities. This revealed various different cytokine profiles across the formulations studied. The identification of additional proteins, and especially cytokines in IVIGs, is particularly notable, and the positive, negative or null biological relevance for clinical use, needs resolution. Collectively these findings reveal marked differences between Australian and overseas-derived (non-Australian) IVIGs in immunoglobulin composition and biochemical characteristics, and presence of additional carry-over proteins from plasma. These findings prompt the need for further evaluation of the micro-compositions of individual formulations. Perhaps detailed mining and improved comparative understanding of each IVIG formulation, may enable highly tailored and strategic clinical use of certain formulations that are personalised best-fit treatments for particular conditions. Such as in treatment of a neuropathy, as compared to another formulation, more suited for treating a particular infectious disease. The most salient and overarching study conclusion is need for caution in attributing equivalence across IVIGs.

sentences: Food interactions observed in a pharmacokinetic investigation comparing two marketed cold preparations (BNO1016 and ELOM-080) after administration to male beagle dogs The cold remedies Sinupret extract (BNO 1016) and Gelomyrtol forte (ELOM-080) represent the two top-selling cold remedies in Germany nowadays. Whereas BNO 1016 is a typical immediate release coated tablet, ELOM-080 is an enteric-coated soft gelatin capsule. The latter formulation, however, is at risk of pharmacokinetic interactions affecting absorption especially in the case of concomitant food intake. In the present pilot study, we investigated the risk of a possible food effect on BNO 1016 in comparison to ELOM-080 in three male beagle dogs. Single doses of BNO 1016 and ELOM-80 at 80 mg/kg and 160 mg/kg, respectively, were administered under fasting and fed conditions according to a 4-period, 4-treatment within-design. Blood sampling took place until up to 30 h p.a. and plasma concentrations of gentiopicroside, verbenalin and from a further marker analyte (BNO 1016 analytes) as well as 1,8-cineole, limonene and perillic acid (ELOM-080 analytes) were determined. Pharmacokinetic parameters focusing on rate and extent of absorption were derived. BNO 1016 analytes demonstrated a homogenous course in all animals in both, the fasted and fed state. Pharmacokinetic characteristics of a typical immediate release drug formulation were observed for all analytes and a food effect could be ruled out. ELOM-080 analytes also showed a homogeneous picture in the fasted state. However, lag-times (tlag) of up to 2 h p.a. with corresponding tmax values of 3 to 4 h were observed, reflecting a longer gastric residence time of the formulation. In the fed state, ELOM-080 showed significant pharmacokinetic characteristics suggesting a clear food effect. A major observation was a double peak phenomenon that could be observed in two out of three dogs. Furthermore, lag-times of some analytes up to 3-4 h and corresponding tmax values of up to 6-8 h occurred. In contrast to BNO 1016, these findings suggest that, as with other enteric-coated formulations, there may be a significant risk for food effects with ELOM-080 also in humans.

sentences: Phylotranscriptomics reveals discordance in the phylogeny of Hawaiian Drosophila and Scaptomyza (Diptera: Drosophilidae) Island radiations present natural laboratories for studying the evolutionary process. The Hawaiian Drosophil-idae are one such radiation, with nearly 600 described species and substantial morphological and ecological diversification. These species are largely divided into a few major clades, but the relationship between these clades remains uncertain. Here we present 12 new assembled transcriptomes from across these clades, and use these transcriptomes to resolve the base of the evolutionary radiation. We recover a new hypothesis for the relationship between clades, and demonstrate its support over previously published hypotheses. We then use the evolutionary radiation to explore dynamics of concordance in phylogenetic support, by analyzing the gene and site concordance factors for every possible topological combination of major groups. We show that high bootstrap values mask low evolutionary concordance, and we demonstrate that the most likely topology is distinct from the topology with the highest support across gene trees and from the topology with highest support across sites. We then combine all previously published genetic data for the group to estimate a time-calibrated tree for over 300 species of drosophilids. Finally, we digitize dozens of published Hawaiian Drosophilidae descriptions, and use this to pinpoint probable evolutionary shifts in reproductive ecology as well as body, wing, and egg size. We show that by examining the entire landscape of tree and trait space, we can gain a more complete understanding of how evolutionary dynamics play out across an island radiation.

sentences: Driving singing behaviour in songbirds using multi-modal, multi-agent virtual reality Interactive biorobotics provides unique experimental potential to study the mechanisms underlying social communication but is limited by our ability to build expressive robots that exhibit the complex behaviours of birds and small mammals. An alternative to physical robots is to use virtual reality (VR). Here, we designed and built a modular, audio-visual virtual reality environment that allows online, multi-modal, multi-agent interaction for social communication. We tested this system in songbirds, which provide an exceptionally powerful and tractable model system to study social communication. We show that zebra finches (Taeniopygia guttata) communicating through the VR environment exhibit normal call timing behaviour, males sing female directed song and both males and females display high-intensity courtship behaviours to their mates. These results suggest that the VR system provides a sufficiently natural environment to elicit normal social communication behaviour. Furthermore, we developed a fully unsupervised online song motif detector and used it to manipulate the virtual social environment of male zebra finches based on the number of motifs sung. Our VR setup represents a first step in taking automatic behaviour annotation into the online domain and allows for animal-computer interaction using higher level behaviours such as song. Our unsupervised acoustic analysis eliminates the need for annotated training data thus reducing labour investment and experimenter bias.

sentences: Neutralizing antibodies elicited by the Ad26.COV2.S COVID-19 vaccine show reduced activity against 501Y.V2 (B.1.351), despite protection against severe disease by this variant. The emergence of SARS-CoV-2 variants, such as 501Y.V2, with immune evasion mutations in the spike has resulted in reduced efficacy of several COVID-19 vaccines. However, the efficacy of the Ad26.COV2.S vaccine, when tested in South Africa after the emergence of 501Y.V2, was not adversely impacted. We therefore assessed the binding and neutralization capacity of n=120 South African sera (from Day 29, post-vaccination) from the Janssen phase 3 study, Ensemble. Spike binding assays using both the Wuhan-1 D614G and 501Y.V2 Spikes showed high levels of cross-reactivity. In contrast, in a subset of 27 sera, we observed significantly reduced neutralization of 501Y.V2 compared to Wuhan-1 D614G, with 22/27 (82%) of sera showing no detectable neutralization of 501Y.V2 at Day 29. These data suggest that even low levels of neutralizing antibodies may contribute to protection from moderate/severe disease. In addition, Fc effector function and T cells may play an important role in protection by this vaccine against 501Y.V2.

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Longitudinal Assessment of SARS-CoV-2 Specific T Cell Cytokine-Producing Responses for 1 Year Reveals Persistence of Multi-Cytokine Proliferative Responses, with Greater Immunity Associated with Disease Severity Cellular-mediated immunity is critical for long-term protection against most viral infections, including coronaviruses. We studied 23 SARS-CoV-2-infected survivors over a one year post symptom onset (PSO) interval by ex vivo cytokine ELISpot assay. All subjects demonstrated SARS-CoV-2-specific IFN-{gamma}, IL-2, and Granzyme B (GzmB) T cell responses at presentation, with greater frequencies in severe disease. Cytokines, mainly produced by CD4+ T cells, targeted all structural proteins (Nucleocapsid, Membrane, Spike) except Envelope, with GzmB > IL-2 > IFN-{gamma}. Mathematical modeling predicted that: 1) cytokine responses peaked at 6 days for IFN-{gamma}, 36 days for IL-2, and 7 days for GzmB, 2) severe illness was associated with reduced IFN-{gamma} and GzmB, but increased IL-2 production rates, 3) males displayed greater production of IFN-{gamma}, whereas females produced more GzmB. Ex vivo responses declined over time with persistence of IL-2 in 86% and of IFN-{gamma} and GzmB in 70% of subjects at a median of 336 days PSO. The average half-life of SARS-CoV-2-specific cytokine-producing cells was modelled to be 139 days ([~]4.6 months). Potent T cell proliferative responses persisted throughout observation, were CD4 dominant, and were capable of producing all 3 cytokines. Several immunodominant CD4 and CD8 epitopes identified in this study were shared by seasonal coronaviruses or SARS-CoV-1 in the Nucleocapsid and Membrane regions. Both SARS-CoV-2-specific CD4+ and CD8+ T cell clones were able to kill target cells, though CD8 tended to be more potent.

ImportanceOur findings highlight the relative importance of SARS-CoV-2-specific GzmB-producing T cell responses in SARS-CoV-2 control, shared CD4 and CD8 immunodominant epitopes in seasonal coronaviruses or SARS-CoV-1, and indicate robust persistence of T cell memory at least one year after infection. Our findings should inform future strategies to induce T cell vaccines against SARS-CoV-2 and other coronaviruses.

sentences: Drug Contraindications in Comorbid Diseases: a Protein Interactome Perspective Adverse drug reactions (ADRs) are leading causes of death and drug withdrawals and frequently cooccur with comorbidities. However, systematic studies on the effects of drugs in comorbidities are lacking. Drug interactions with the cellular protein-protein interaction (PPI) network give rise to ADRs. We selected 6 comorbid disease pairs, identified the drugs used in the treatment of the individual diseases  A and  B- 44 drugs in anxiety and depression, 128 in asthma and hypertension, 48 in chronic obstructive pulmonary disease and heart failure, 58 in type 2 diabetes and obesity, 58 in Parkinsons disease and schizophrenia, and 84 in rheumatoid arthritis and osteoporosis - and categorized them based on whether they aggravate the comorbid condition. We constructed drug target networks (DTNs) and examined their enrichment among genes in disease A/B PPI networks, expressed across 53 tissues and involved in ~1000 pathways. To pinpoint the biological features characterizing the DTNs, we performed principal component analysis and computed the Euclidean distance between DTN component scores and feature loading values. DTNs of disease A drugs not contraindicated in B were affiliated with proteins common to A/B networks or uniquely found in the B network, similarly regulated common pathways, and disease-B specific pathways and tissues. DTNs of disease A drugs contraindicated in B were affiliated with common proteins or those uniquely found in the A network, differentially regulated common pathways, and disease A-specific pathways and tissues. Hence, DTN enrichment in pathways, tissues, and PPI networks of comorbid diseases will help identify drugs contraindications in comorbidities.

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The Arabidopsis thaliana PeptideAtlas; harnessing world-wide proteomics data for a comprehensive community proteomics resource We developed a new resource, the Arabidopsis PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/), to solve central questions about the Arabidopsis proteome, such as the significance of protein splice forms, post-translational modifications (PTMs), or simply obtain reliable information about specific proteins. PeptideAtlas is based on published mass spectrometry (MS) analyses collected through ProteomeXchange and reanalyzed through a uniform processing and metadata annotation pipeline. All matched MS-derived peptide data are linked to spectral, technical and biological metadata. Nearly 40 million out of [~]143 million MSMS spectra were matched to the reference genome Araport11, identifying [~]0.5 million unique peptides and 17858 uniquely identified proteins (only isoform per gene) at the highest confidence level (FDR 0.0004; 2 non-nested peptides [≥] 9 aa each), assigned canonical proteins, and 3543 lower confidence proteins. Physicochemical protein properties were evaluated for targeted identification of unobserved proteins. Additional proteins and isoforms currently not in Araport11 were identified, generated from pseudogenes, alternative start, stops and/or splice variants and sORFs; these features should be considered for updates to the Arabidopsis genome. Phosphorylation can be inspected through a sophisticated PTM viewer. This new PeptideAtlas is integrated with community resources including TAIR, tracks in JBrowse, PPDB and UniProtKB. Subsequent PeptideAtlas builds will incorporate millions more MS data.

One sentence summaryA new web resource providing the global community with mass spectrometry-based Arabidopsis proteome information and its spectral, technical and biological metadata integrated with TAIR and JBrowse

sentences: Behavioral evidence for nested central pattern generator control of Drosophila grooming Central pattern generators (CPGs) are neurons or neural circuits that produce periodic output without requiring patterned input. More complex behaviors can be assembled from simpler subroutines, and nested CPGs have been proposed to coordinate their repetitive elements, organizing control over different time-scales. Here, we use behavioral experiments to establish that Drosophila grooming may be controlled by nested CPGs. On a short time-scale (5-7 Hz), flies execute periodic leg sweeps and rubs. More surprisingly, transitions between bouts of head cleaning and leg rubbing are also periodic on a longer time-scale (0.3 - 0.6 Hz). We examine grooming at a range of temperatures to show that the frequencies of both oscillations increase - a hallmark of CPG control - and also that the two time-scales increase at the same rate, indicating that the nested CPGs may be linked. This relationship also holds when sensory drive is held constant using optogenetic activation, but the rhythms can decouple in spontaneously grooming flies, showing that alternative control modes are possible. Loss of sensory feedback does not disrupt periodicity but slows the longer time-scale alternation. Nested CPGs simplify generation of complex but repetitive behaviors, and identifying them in Drosophila grooming presents an opportunity to map the neural circuits that constitute them.

sentences: Whole Genome Variation of Transposable Element Insertions in a Maize Diversity Panel Intact transposable elements (TEs) account for 65% of the maize genome and can impact gene function and regulation. Although TEs comprise the majority of the maize genome and affect important phenotypes, genome wide patterns of TE polymorphisms in maize have only been studied in a handful of maize genotypes, due to the challenging nature of assessing highly repetitive sequences. We implemented a method to use short read sequencing data from 509 diverse inbred lines to classify the presence/absence of 445,418 non-redundant TEs that were previously annotated in four genome assemblies including B73, Mo17, PH207, and W22. Different orders of TEs (i.e. LTRs, Helitrons, TIRs) had different frequency distributions within the population. LTRs with lower LTR similarity were generally more frequent in the population than LTRs with higher LTR similarity, though high frequency insertions with very high LTR similarity were observed. LTR similarity and frequency estimates of nested elements and the outer elements in which they insert revealed that most nesting events occurred very near the timing of the outer element insertion. TEs within genes were at higher frequency than those that were outside of genes and this is particularly true for those not inserted into introns. Many TE insertional polymorphisms observed in this population were tagged by SNP markers. However, there were also 19.9% of the TE polymorphisms that were not well tagged by SNPs (R2 < 0.5) that potentially represent information that has not been well captured in previous SNP based marker-trait association studies. This study provides a population scale genome-wide assessment of TE variation in maize, and provides valuable insight on variation in TEs in maize and factors that contribute to this variation.

sentences: Identification of candidate susceptibility genes to Puccinia graminis f. sp. tritici in wheat Wheat stem rust disease caused by Puccinia graminis f. sp. tritici (Pgt) is a global threat to wheat production. Fast evolving populations of Pgt limit the efficacy of plant genetic resistance and constrain disease management strategies. Understanding molecular mechanisms that lead to rust infection and disease susceptibility could deliver novel strategies to deploy crop resistance through genetic loss of disease susceptibility. We used comparative transcriptome-based and orthology-guided approaches to characterize gene expression changes associated with Pgt infection in susceptible and resistant Triticum aestivum genotypes as well as the non-host Brachypodium distachyon. We targeted our analysis to genes with differential expression in T. aestivum and genes suppressed or not affected in B. distachyon and report several processes potentially linked to susceptibility to Pgt, such as cell death suppression and impairment of photosynthesis. We complemented our approach with a gene co-expression network analysis to identify wheat targets to deliver resistance to Pgt through removal or modification of putative susceptibility genes.

sentences: Distribution modeling of Lantana camara and identification of hotspot for future management of the species in the lower Shivalik region of Uttarakhand, India Invasive alien species are one of the most significant threats to biodiversity of the earth ecosystem. Spatial modeling has played a vital role in predicting and mapping the suitable areas of the species habitat for management implementation. Lantana camara is one of the ten worst invasive weed enlisted by IUCN., and has its distribution in more than 154000 sq. km area in India. We predicted the distribution of Lantana camara using Ecological Niche Modeling (ENM) in MaxEnt software for Rajaji- Corbett landscape in the lower Shivalik region of the state of Uttarakhand, India. The predicted area for the invasion was classified into four categories high, good, medium, and low potential areas. The distribution hotspots were identified using Getis Ord G* method in ArcGIS 10.5 by calculating plot-wise species occurrence. Major hotspot areas under threat of high infestation are Dholkhand, Gohri, Ramgarh, Motichur, and Chilla from Rajaji Tiger Reserve, Palain, Mandal and Maidavan from Kalagarh Tiger Reserve and Bijrani, Dheela, Dhikala, Jhirna and Kalagarh from Corbett Tiger Reserve. This study provides a current status of the invasive species in the landscape with identified areas that need immediate actions to control the spread of the species. The study results can help the policymakers and stakeholders build an efficient and strategically weed management plan.

sentences: Set4 coordinates the activity of histone deacetylases and regulates stress-responsive gene expression within subtelomeric regions in yeast The yeast chromatin protein Set4 is a member of the Set3-subfamily of SET domain proteins which play critical roles in the regulation of gene expression in diverse developmental and environmental contexts, although they appear to lack methyltransferase activity. The molecular functions of Set4 are relatively unexplored, likely due to its low abundance in standard growth conditions. We previously reported that Set4 promotes survival during oxidative stress and regulates expression of stress response genes via stress-dependent chromatin localization. In this study, global gene expression analysis and investigation of histone modification status has revealed a role for Set4 in maintaining gene repressive mechanisms within yeast subtelomeres under both normal and stress conditions. We show that Set4 works in a partially overlapping pathway to the SIR complex and the histone deacetylase Rpd3 to maintain proper levels of histone acetylation and expression of stress response genes encoded in subtelomeres. This role for Set4 is particularly critical for cells under hypoxic conditions, and the loss of Set4 decreases cell fitness and cell wall integrity in hypoxia. These findings uncover a new regulator of subtelomeric chromatin that is key to stress defense pathways and demonstrate a function for yeast Set4 in regulating repressive, heterochromatin-like environments.

sentences: Analog of the Hutchinson equation in biophysical neurodynamics: from the Morris-Lecar model to a delay differential equation Starting with the classical biophysical Morris-Lecar model of neuronal excitability, we introduce a functional analog of the Hutchinson equation initially obtained for population dynamics with delayed negative feedback. It is shown that the resulting equation with a fixed time delay qualitatively reproduces the dynamics of the original model upon direct current stimulation, preserving both the initial type of neuronal excitability and biophysically realistic spike shape within a wide range of the delay values. If the delay becomes very small (2 ms or less), the simplified delay-based model exhibits a distinct transition from the 1st to the 2nd excitability type.

sentences: Mechanisms underlying the cooperation between loss of epithelial polarity and Notch signaling during neoplastic growth in Drosophila Aggressive neoplastic growth can be initiated by a limited number of genetic alterations, such as the well-established cooperation between loss of cell architecture and hyperactive signaling pathways. However, our understanding of how these different alterations interact and influence each other remains very incomplete. Using Drosophila paradigms of imaginal wing disc epithelial growth, we have monitored the changes in Notch pathway activity according to the polarity status of cells (scrib mutant). We show that the scrib mutation impacts the direct transcriptional output of the Notch pathway, without altering the global distribution of Su(H), the Notch dedicated transcription factor. The Notch-dependent neoplasms require however, the action of a group of transcription factors, similar to those previously identified for Ras/scrib neoplasm (namely AP-1, Stat92E, Ftz-F1, and bZIP factors), further suggesting the importance of this transcription factor network during neoplastic growth. Finally our work highlights some Notch/scrib specificities, in particular the role of the PAR domain containing bZIP transcription factor and Notch direct target Pdp1 for neoplastic growth.

sentences: Genome Mining and Biochemical Characterization of a Bifunctional Type I Diterpene Synthase from a Marine-Derived Fungus We report the identification of the tnd biosynthetic cluster from the marine-derived fungal strain Aspergillus flavipes CNL-338 and in vivo characterization of a cryptic type I diterpene synthase. Heterologous expression of the bifunctional terpene synthase TndC in Saccharomyces cerevisiae led to the discovery of a new diterpene backbone, talarodiene 8, harboring a benzo[a]cyclopenta[d]cyclooctane tricyclic fused ring system. The cyclization mechanism that converts geranylgeranyl diphosphate to the tricyclic hydrocarbon skeleton was investigated using 13C-labeling studies and stable isotope tracer experiments showed the biotransformation of 8 into the natural product talaronoid C.

sentences: Single-cell transcriptome analysis of embryonic and adult endothelial cells allows to rank the hemogenic potential of post-natal endothelium Hematopoietic stem cells are crucial for the continuous production of blood cells during life. The transplantation of these cells is one of the most common treatments to cure patient suffering of blood diseases. However, the lack of suitable donors is a major limitation. One option to get hematopoietic stem cells matching perfectly a patient is cellular reprogramming. Hematopoietic stem cells emerge from endothelial cells in blood vessels during embryogenesis through the endothelial to hematopoietic transition. Here, we used single-cell transcriptomics analysis to compare embryonic and post-natal endothelial cells to investigate the potential of adult vasculature to be reprogrammed in hematopoietic stem cells. Although transcriptional similarities have been found between embryonic and adult endothelial cells, we found some key differences in term of transcription factors expression. There is a deficit of expression of Runx1, Tal1, Lyl1 and Cbfb in adult endothelial cells compared to their embryonic counterparts. Using a combination of gene expression profiling and gene regulatory network analysis, we found that endothelial cells from the pancreas, brain, kidney and liver appear to be the most suitable targets for cellular reprogramming into hematopoietic stem cells. Overall, our work provides an important resource for the rational design of a reprogramming strategy for the generation of hematopoietic stem cells.

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A novel role for the extraembryonic area opaca in positioning the primitive streak of the early chick embryo Classical studies have established that the marginal zone, a ring of extraembryonic epiblast immediately surrounding the embryonic epiblast (area pellucida) of the chick embryo is important in setting embryonic polarity by positioning the primitive streak, the site of gastrulation. The more external extraembryonic region (area opaca) was only thought to have nutritive and support functions. Using experimental embryology approaches, this study reveals three separable functions for this outer region: first, juxtaposition of the area opaca directly onto the area pellucida induces a new marginal zone from the latter; this induced domain is entirely posterior in character. Second, ablation and grafting experiments using an isolated anterior half of the blastoderm and pieces of area opaca suggest that the area opaca can influence the polarity of the adjacent marginal zone. Finally, we show that the loss of the ability of such isolated anterior half-embryos to regulate (re-establish polarity spontaneously) at the early primitive streak stage can be rescued by replacing the area opaca by one from a younger stage. These results uncover new roles of chick extraembryonic tissues in early development.

Summary statementTwo adjacent extraembryonic tissues, the area opaca and the marginal zone, interact to influence the polarity of the early chick embryo.

sentences: A de novo transcription-dependent TAD boundary underpins critical multiway interactions during antibody class switch recombination Conflicts between transcription and cohesin-mediated loop extrusion can majorly influence 3D chromatin architecture but whether these structural changes affect biological function is unknown. Here, we show that a critical step in antibody class switch recombination (CSR) in activated B cells, namely, the juxtaposition (synapsis) of donor and acceptor switch (S) recombination sequences at the immunoglobulin heavy chain locus (Igh), occurs at the interface of a de novo topologically associating domain (TAD) boundary formed via transcriptional activity at acceptor S regions. Using Tri-C to capture higher-order multiway chromatin conformations, we find that synapsis occurs predominantly in the proximity of distal 3 CTCF-binding sites and that this multiway conformation is abolished upon downregulation of transcription and loss of the TAD boundary at the acceptor S region. Thus, an insulating de novo TAD boundary created by the conflict between transcription and loop extrusion plays a direct role in the mechanism of CSR.

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Glycopeptide antibiotic teicoplanin inhibits cell entry of SARS-CoV-2 by suppressing the proteolytic activity of cathepsin L Since the outbreak of the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the public health worldwide has been greatly threatened. The development of an effective treatment for this infection is crucial and urgent but is hampered by the incomplete understanding of the viral infection mechanism and the lack of specific antiviral agents. We previously reported that teicoplanin, a glycopeptide antibiotic that has been commonly used in the clinic to treat bacterial infection, significantly restrained the cell entry of Ebola virus, SARS-CoV and MERS-CoV by specifically inhibiting the activity of cathepsin L (CTSL). Here, we found that the cleavage sites of CTSL on the Spike of SARS-CoV-2 were highly conserved among all the variants. The treatment with teicoplanin suppressed the proteolytic activity of CTSL on Spike and prevented the cellular infection of different pseudotyped SARS-CoV-2 viruses. Teicoplanin potently prevented the entry of authentic SARS-CoV-2 into the cellular cytoplasm with an IC50 of 2.038 M for the Wuhan-Hu-1 reference strain and an IC50 of 2.116 M for the SARS-CoV-2 (D614G) variant. The pre-treatment of teicoplanin also prevented SARS-CoV-2 infection in hACE2 mice. In summary, our data reveal that CTSL is required for both SARS-CoV-2 and SARS-CoV infection and demonstrate the therapeutic potential of teicoplanin for universal anti-CoVs intervention.

ImportanceDisease prevention and treatment are two important countermeasures to end the coronavirus disease 2019 (COVID-19). However, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, evolves all the time, resulting in the emerging of many epidemic SARS-CoV-2 mutants, which significantly impairs the effectiveness of early strain-based vaccines and antibodies. Developing universal vaccines and broad-spectrum antiviral drugs are essential to confront SARS-CoV-2 mutants including those may emerge in the future. Our study reported here showed that the cleavage sites of cellular cathepsin L (CTSL) are highly conserved among all the SARS-CoV-2 mutants and SARS-CoV. The CTSL inhibitor teicoplanin not only inhibited the cell entry of two live SARS-CoV-2 strains and various pseudotyped viruses but also prevented live virus infection in animal models. Based on our previous finding that teicoplanin also inhibited SARS-CoV and MERS-CoV infection, we believe that teicoplanin possesses the potential to become a universal anti-CoVs drug.

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Ionotropic receptors in the turnip moth Agrotis segetum respond to repellent medium-chain fatty acids BackgroundIn insects, airborne chemical signals are mainly detected by two receptor families, odorant receptors (ORs) and ionotropic receptors (IRs). Functions of ORs have been intensively investigated in Diptera and Lepidoptera, while the functions and evolution of the more ancient IR family remain largely unexplored beyond Diptera.

ResultsHere, we identified a repertoire of 26 IRs from transcriptomes of female and male antennae, and ovipositors in the moth Agrotis segetum. We observed that a large clade formed by IR75p and IR75q expansions is closely related to the acid-sensing IRs identified in Diptera. We functionally assayed each of the five AsegIRs from this clade using Xenopus oocytes and found that two receptors responded to the tested ligands. AsegIR75p.1 responded to several compounds but hexanoic acid was revealed to be the primary ligand, and AsegIR75q.1 responded primarily to octanoic acid, and less so to nonanoic acid. It has been reported that the C6-C10 medium-chain fatty acids repel various insects including many drosophilids and mosquitos. Our GC-EAD recordings showed that C6-C10 medium-chain fatty acids elicited antennal responses of both sexes of A. segetum, while only octanoic acid had repellent effect to the moths in a behavioural assay. In addition, using fluorescence in situ hybridization, we demonstrated that the five IRs and their co-receptor AsegIR8a are not located in coeloconic sensilla as found in Drosophila, but in basiconic or trichoid sensilla.

ConclusionsOur results significantly expand the current knowledge of the insect IR family. Based on the functional data in combination with phylogenetic analysis, we propose that subfunctionalization after gene duplication plays an important role in the evolution of ligand specificities of the acid-sensing IRs in Lepidoptera.

sentences: Deconvolving clinically relevant cellular immune crosstalk from bulk gene expression using CODEFACS and LIRICS The tumor microenvironment (TME) is a complex mixture of cell types whose interactions affect tumor growth and clinical outcome. To discover such interactions, we developed CODEFACS (COnfident DEconvolution For All Cell Subsets), a tool deconvolving cell-type-specific gene expression in each sample from bulk expression, and LIRICS (LIgand Receptor Interactions between Cell Subsets), a statistical framework prioritizing clinically relevant ligand-receptor interactions between cell types from the deconvolved data. We first demonstrate the superiority of CODEFACS versus the state-of-the-art deconvolution method, CIBERSORTx. Second, analyzing the TCGA, we uncover cell-type-specific interactions of mismatch-repair-deficient tumors that are associated with their higher anti-PD1 response rates, including specific T-cell co-stimulating interactions that enhance immunotherapy response independently of the tumors mutation burden levels. Finally, we identify a subset of ligand-receptor interactions in the melanoma TME that predict patient response to anti-PD1 therapy better than recently published transcriptomics-based methods.

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Image segmentation and separation of spectrally similar dyes in fluorescence microscopy by dynamic mode decomposition of photobleaching kinetics BackgroundImage segmentation in fluorescence microscopy is often based on spectral separation of fluorescent probes (color-based segmentation) or on significant intensity differences in individual image regions (intensity-based segmentation). These approaches fail, if dye fluorescence shows large spectral overlap with other employed probes or with strong cellular autofluorescence.

ResultsHere, a novel model-free approach is presented which determines bleaching kinetics based on dynamic mode decomposition (DMD) and uses the inferred photobleaching kinetics to distinguish different probes or dye molecules from autofluorescence. DMD is a data-driven computational method for detecting and quantifying dynamic events in complex spatiotemporal data. Here, DMD is used to determine photobleaching characteristics of a fluorescent sterol probe, dehydroergosterol (DHE), compared to that of cellular autofluorescence in the nematode Caenorhabditis elegans. It is shown that decomposition of those dynamic modes allows for precise image segmentation, thereby separating probe from autofluorescence without invoking a particular model for the bleaching process. In a second application, DMD of dye-specific photobleaching is used to separate two green-fluorescent dyes, an NBD-tagged sphingolipid and Alexa488-transferrin, thereby assigning them to different cellular compartments.

ConclusionsData-based decomposition of dynamic modes can be employed to analyze spatially varying photobleaching of fluorescent probes in cells and tissues for image segmentation, discrimination of probe from autofluorescence and image denoising. The new method should find wide application in analysis of dynamic fluorescence imaging data.

sentences: Cardiotoxic effects of methamphetamine associated with electrophysiological and epigenetic aberrations in Zebrafish Long-term methamphetamine (Meth) abuse damages functional and molecular changes in the brain that causes chronic and relapsing disease. In this study, we sought to investigate a relationship between cardiotoxicity and arrhythmia with associated Meth abuse in zebrafish to identify and to understand the adverse cardiac symptoms associated with Meth as well as to assess the applicability of zebrafish as an appropriate model for cardiac-related drug screening studies. Over a two-week duration, zebrafish were first treated with various concentrations of Meth, ranging from 0 to 50 M. Immediately after treatment, zebrafish underwent electrocardiogram (ECG) measurement for electrophysiological analysis. Results show that a higher incidence of increased heart rate over the duration of the experiment, corroborating with results from previous human case studies involving Meth users. However, abnormalities commonly cited in those same case studies, such as prolongation of QTc, were not significantly presented in obtained ECG recordings. We have also conducted genetic, epigenetic, and histochemical analysis in an attempt to understand the cardiotoxic effects of Meth on zebrafish cardiac function. These results suggested myocardial damage and decrease in gene expression associated with normal physiological function. Finally, this paper provides insights into potential reasons for the apparent discrepancies in our data with prior research as well as an outlook of zebrafish cardiotoxic drug screening studies.

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Effects of microplastics mixed with natural particles on Daphnia magna populations The toxicity of microplastics on Daphnia magna as key model for freshwater zooplankton is well described. While several studies predict population-level effects based on short-term, individual-level responses, only very few have validated these predictions experimentally. Thus, we exposed D. magna populations to irregular polystyrene microplastics and diatomite as natural particle (both [≤]63 {micro}m) over 50 days. We used mixtures of both particle types at fixed particle concentrations (50,000 mL-1) and recorded the overall population density, the size of the individual animals, and resting egg production. Particle exposure adversely affected the population density and structure and induced resting egg production. The terminal population size was 31-42% lower in exposed compared to control populations. Interestingly, mixtures containing diatomite induced stronger effects than microplastics alone highlighting that natural particles are not per se less toxic than microplastics. Our results demonstrate that an exposure to synthetic and natural particles has negative population-level effects on zooplankton. Understanding the mixture toxicity of microplastics and natural particles is important given that aquatic organisms will experience exposure to both. Just as for chemical pollutants, better knowledge of such joint effects is essential to fully understand the environmental risks of complex particle mixtures.

Environmental ImplicationsWhile microplastics are commonly considered hazardous based on individual-level effects, there is a dearth of information on how they affect populations. Since the latter is key for understanding the environmental impacts of microplastics, we investigated how particle exposures affect the population size and structure of Daphnia magna. In addition, we used mixtures of microplastics and natural particles because neither occurs alone in nature and joint effects can expected in an environmentally realistic scenario. We show that such mixtures adversely affect daphnid populations and highlight that population-level and mixture-toxicity designs are one important step towards more environmental realism in microplastics research.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/490562v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@13cf281org.highwire.dtl.DTLVardef@552921org.highwire.dtl.DTLVardef@ff461corg.highwire.dtl.DTLVardef@153f479_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIDaphnia populations exposed to mixtures of microplastics and diatomite
C_LIO_LIEffects on population density, structure, and resting egg production
C_LIO_LIDiatomite as natural particle was more toxic than microplastics
C_LIO_LIParticle mixtures induce negative population-level effects
C_LIO_LIParticle mixtures represent more realistic exposure scenario
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Emergent spatiotemporal population dynamics with cell-length control of synthetic microbial consortia AO_SCPLOWBSTRACTC_SCPLOWIncreased complexity of engineered microbial biocircuits highlights the need for distributed cell functionality due to concomitant increases of metabolic and regulatory burdens imposed on single-strain topologies. Distributed systems, however, introduce additional challenges since consortium composition and spatiotemporal dynamics of constituent strains must be robustly controlled to achieve desired circuit behaviors. Here, we address these challenges with a modeling-based investigation of emergent spatiotemporal population dynamics that result from cell-length control of monolayer, two-strain bacterial consortia. We demonstrate that with dynamic control of a strains division length, nematic cell alignment in close-packed monolayers can be destabilized. We found this destabilization conferred an emergent, competitive advantage on smaller-length strains--but by mechanisms that differed depending on the spatial patterns of the population. We used complementary modeling approaches to elucidate underlying mechanisms: an agent-based model to simulate detailed mechanical and signaling interactions between the competing strains and a reductive, stochastic lattice model to represent cell-cell interactions with a single rotational parameter. Our modeling suggests that spatial strain-fraction oscillations can be generated when cell-length control is coupled to quorum-sensing signaling in negative feedback topologies. Our research employs novel methods of population control and points the way to programming strain fraction dynamics in consortial synthetic biology.

Engineered microbial collectives are more versatile and robust than single strain populations. However, the function of such collectives is sensitive to their spatiotemporal organization. Here, we demonstrate control of the spatiotemporal composition of synthetic microbial consortia by dynamically modulating the average cell length of constituent strains. Such modulation confers an emergent "mechanical fitness" advantage upon the shorter length strain. We used both a biophysically realistic agent-based model to test the impact of cell shape on spatiotemporal dynamics and a conceptually simpler stochastic lattice model to explain the essential mechanisms driving the dynamics.

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Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks Complex gene regulatory networks ensure that important genes are expressed at precise levels. When gene expression is sufficiently perturbed it can lead to disease. To understand how gene expression disruptions percolate through a network, we must first map connections between regulatory genes and their downstream targets. However, we lack comprehensive knowledge of the upstream regulators of most genes. Here we developed an approach for systematic discovery of upstream regulators of critical immune factors - IL2RA, IL-2, and CTLA4 - in primary human T cells. Then, we mapped the network of these regulators target genes and enhancers using CRISPR perturbations, RNA-Seq, and ATAC-Seq. These regulators form densely interconnected networks with extensive feedback loops. Furthermore, this network is highly enriched for immune-associated disease variants and genes. These results provide insight into how immune-associated disease genes are regulated in T cells and broader principles about the structure of human gene regulatory networks.

HighlightsO_LIA systematic approach to identify upstream regulators of key immune genes in primary human cells
C_LIO_LIComprehensive RNA-Seq and ATAC-Seq perturbation maps after KO of individual discovered regulators
C_LIO_LIAnalysis uncovers a highly interconnected regulatory network of enhancers and genes in T cells
C_LIO_LIThis network is highly enriched for immune disease variants and genes shedding light on the trans-regulatory connections among key immune genes in health and disease
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sentences: Characterizing Human Mesenchymal Stromal Cells Immune Modulatory Potency Using Targeted Lipidomic Profiling of Sphingolipids Cell therapies are expected to increase over the next decade due to increasing demand for clinical applications. Mesenchymal stromal cells (MSCs) have been explored to treat a number of diseases, with some successes in early clinical trials. Despite early successes, poor MSC characterization results in lessened therapeutic capacity once in vivo. Here, we characterized bone-marrow (BM), adipose derived and umbilical cord tissue MSCs sphingolipids (SLs), a class of bioactive lipids, using liquid chromatography - tandem mass spectrometry. We found ceramide levels differed based upon donors sex in BM-MSCs. We detected fatty acyl chain variants in MSCs from all 3 sources. Linear discriminant analysis revealed that MSCs separated based on tissue source. Principal component analysis showed IFN-{gamma} primed and unstimulated MSCs separated according to their SL signature. Lastly, we detected higher ceramide levels in low IDO MSCs, indicating sphingomeylinase or ceramidase enzymatic activity may be involved in their immune potency.

sentences: Nanopore quality score resolution can be reduced with little effect on downstream analysis We investigate the effect of quality score information loss on downstream analysis from nanopore sequencing FASTQ files. We polished denovo assemblies for a mock microbial community and a human genome, and we called variants on a human genome. We repeated these experiments using various pipelines, under various coverage level scenarios, and various quality score quantizers. In all cases we found that the quantization of quality scores cause little difference on (or even improves) the results obtained with the original (non-quantized) data. This suggests that the precision that is currently used for nanopore quality scores is unnecessarily high, and motivates the use of lossy compression algorithms for this kind of data. Moreover, we show that even a non-specialized compressor, like gzip, yields large storage space savings after quantization of quality scores.

sentences: PLETHORA-WOX5 interaction and subnuclear localisation control Arabidopsis root stem cell maintenance Maintenance and homeostasis of the stem cell niche (SCN) in the Arabidopsis root is essential for growth and development of all root cell types. The SCN is organized around a quiescent center (QC) maintaining the stemness of cells in direct contact. The key transcription factors (TFs) WUSCHEL-RELATED HOMEOBOX 5 (WOX5) and PLETHORAs (PLTs) are expressed in the SCN where they maintain the QC and regulate distal columella stem cell (CSC) fate. Here, we describe the concerted mutual regulation of the key TFs WOX5 and PLTs on a transcriptional and protein interaction level. Additionally, by applying a novel SCN staining method, we demonstrate that both WOX5 and PLTs regulate root SCN homeostasis as they control QC quiescence and CSC fate interdependently. Moreover, we uncover that some PLTs, especially PLT3, contain intrinsically disordered prion-like domains (PrDs) that are necessary for complex formation with WOX5 and its recruitment to subnuclear microdomains/nuclear bodies (NBs) in the CSCs. We propose that this partitioning of PLT-WOX5 complexes to NBs, possibly by phase separation, is important for CSC fate determination.

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Insights into the microbial strain mediated impact on pest insect development Molecular analyses of host-associated microorganisms have demonstrated the essential role that the microbiome plays in host development. Approaches targeting the sequencing of ribosomal genes have successfully identified key species of the host-associated microbiome. However, it remains unclear to what extent the strain-specific characteristics influence the outcome of the host-microbiome interactions. This is particularly important for insect pests, where microbial species might be used as targets for biocontrol purposes. Understanding strain-level variation represents thus a crucial step in determining the microbial impact on hosts. To investigate the microbial strain-level effects on an invasive insect pest, Drosophila suzukii, we compared the impact of monocultures and cocultures of different bacterial and yeast strains. We investigated whether different strains of Gluconobacter and Pichia differentially influenced the larval development of the pest. Fly trait measurements demonstrated beneficial, although variable, impact of these microbial strains on the fitness of suzukii. Using cocultures of microbial strains, we found that in some combinations, the beneficial effects were intermediate between those of the respective monocultures. In contrast, in other cases, strong inhibitory effects were observed. Hence, our study reports that strain-level effects within species are present in D. suzukii, reinforcing the importance of assessing the impact of associated microbiota on pest insects at the strain level.

HighlightsO_LIMicrobial strains make up an essential part of the diversity of an insect hosts
C_LIO_LICharacterizing and accounting for strain-specific impact on a pests life-history traits and different combinations of strains constitute an important step in our understanding of the pest management strategies.
C_LIO_LIWe investigated whether there was any strain-specific impact of bacteria and yeasts on the larval development of a frugivorous pest.
C_LIO_LIWe observed that strains varied in their impact, both as monocultures and cocultures, indicating their importance in modifying the host ecology.
C_LIO_LIOur study adds to the growing literature on the importance of strains in pest insects.
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sentences: A small-molecule fluorescent probe for live-cell imaging of endocytosis Endocytosis involves plasma membrane-derived vesicles for the recycling of intra- and extracellular components. Increasing evidence suggests that endocytosis is related to maintaining intracellular homeostasis and defense against disease. Consequently, investigation of the endocytic pathway attracts considerable scientific interest. This study reports live-cell imaging of endocytosis using the newly-developed fluorescent probe ECGreen. We demonstrate that ECGreen is not membrane permeable and its fluorescence signal increases in acidic conditions. Because of these characteristics, ECGreen remains on the plasma membrane, and then shows increased fluorescence when it is internalized into the acidic vesicles formed in the endocytic process. ECGreen allows direct observation of the internalized vesicle; it is a valuable new probe for endocytic imaging.

sentences: A new early-branching armoured dinosaur from the Lower Jurassic of southwestern China The early evolutionary history of the armoured dinosaurs (Thyreophora) is obscured by its patchily distributed fossil record and by conflicting views on the relationships of its Early Jurassic representatives. Here, we describe an early-diverging thyreophoran from the Lower Jurassic Fengjiahe Formation of Yunnan Province, China, on the basis of an associated partial skeleton that includes skull, axial, limb and armour elements. It can be diagnosed as a new taxon based on numerous cranial and postcranial autapomorphies and is further distinguished from all other thyreophorans by a unique combination of character states. Although the robust postcranium is similar to that of more deeply nested ankylosaurs and stegosaurs, phylogenetic analysis recovers it as either the sister taxon of Emausaurus or of the clade Scelidosaurus+Eurypoda. This new taxon, XXXX XXXX, represents the first valid thyreophoran dinosaur to be described from the Early Jurassic of Asia and confirms the rapid geographic spread and diversification of the clade after its first appearance in the Hettangian. Its heavy build and distinctive armour also hint at previously unrealised morphological diversity early in the clades history.

sentences: A Meta-Analysis of fMRI Studies of Semantic Cognition in Children Our capacity to derive meaning from things that we see and words that we hear is unparalleled in other animal species and current AI systems. Despite a wealth of functional magnetic resonance imaging (fMRI) studies on where different semantic features are processed in the adult brain, the development of these systems in children is poorly understood. Here we conducted an extensive database search and identified 50 fMRI experiments investigating semantic world knowledge, semantic relatedness judgments, and the differentiation of visual semantic object categories in children (total N = 1,018, mean age = 10.1 years, range 4-15 years). Synthesizing the results of these experiments, we found consistent activation in the bilateral inferior frontal gyri (IFG), fusiform gyri (FG), and supplementary motor areas (SMA), as well as in the left middle and superior temporal gyri (MTG/STG). Within this system, we found little evidence for age-related changes across childhood and high overlap with the adult semantic system. In sum, the identification of these cortical areas provides the starting point for further research on the mechanisms by which the developing brain learns to make sense of its environment.

sentences: Dynamic rewiring of biological activity across genotype and lineage revealed by context-dependent functional interactions Coessentiality networks derived from CRISPR screens in cell lines provide a powerful framework for identifying functional modules in the cell and for inferring the role of uncharacterized genes. However, these networks integrate signal across all underlying data, and can mask strong interactions that occur in only a subset of the cell lines analyzed. Here we decipher dynamic functional interactions by identifying significant cellular contexts, primarily by oncogenic mutation, lineage, and tumor type, and discovering coessentiality relationships that depend on these contexts. We recapitulate well-known gene-context interactions such as oncogene-mutation, paralog buffering, and tissue-specific essential genes, show how mutation rewires known signal transduction pathways, including RAS/RAF and IGF1R-PIK3CA, and illustrate the implications for drug targeting. We further demonstrate how context-dependent functional interactions can elucidate lineage-specific gene function, as illustrated by the maturation of proreceptors IGF1R and MET by proteases FURIN and CPD. This approach advances our understanding of context-dependent interactions and how they can be gleaned from these data. We provide an online resource to explore these context-dependent interactions at diffnet.hart-lab.org.

sentences: Brain connectivity at rest predicts individual differences in normative activity during movie watching When multiple individuals are exposed to the same sensory event, some are bound to have less typical experiences than others. These atypical experiences are underpinned by atypical stimulus-evoked brain activity, the extent of which is often indexed by intersubject correlation (ISC). Previous research has attributed individual differences in ISC to variation in trait-like behavioral phenotypes. Here, we extend this line of work by showing that an individuals degree and spatial distribution of ISC are closely related to their brains intrinsic functional architecture. Using resting state and movie watching fMRI data from 176 Human Connectome Project participants, we reveal that resting state functional connectivity (RSFC) profiles can be used to predict cortex-wide ISC with considerable accuracy. Similar region-level analyses demonstrate that the amount of ISC a brain region exhibits during movie watching is associated with its connectivity to others at rest, and that the nature of these connectivity-activity relationships varies as a function of the regions role in sensory information processing. Finally, we show that an individuals unique spatial distribution of ISC, independent of its magnitude, is also related to their RSFC profile. These findings suggest that the brains ability to process complex sensory information is tightly linked to its baseline functional organization and motivate a more comprehensive understanding of individual responses to naturalistic stimuli.

sentences: Life-stage and sex influence Philornis ectoparasitism in a Neotropical woodpecker (Melanerpes striatus) with essential male parental care The nestlings of many Neotropical bird species suffer from Philornis (Diptera: Muscidae) ectoparasitism. While nestlings are typically considered the intended targets, recent work indicates that Philornis infest adult birds more frequently than previously appreciated, yet few studies have concurrently surveyed nestlings and adults for Philornis in the same population. Over six field seasons (2012-2017), I documented the presence of current or recent subcutaneous Philornis infestations on adult and nestling Hispaniolan Woodpeckers Melanerpes striatus from the same population. I tested the following three non-mutually exclusive hypotheses regarding occurrence of Philornis on adult birds: (1) nestlings are more vulnerable to Philornis parasitism than adults, (2) nesting is associated with Philornis parasitism in adults, and (3) Philornis parasitism is associated with incubation and brooding investment. While nestling and adult woodpeckers exhibited similar prevalence of parasitism, parasitized nestlings hosted on average 3.5 times more Philornis wounds (larvae plus empty wounds) than parasitized adults. Nesting per se was not significantly associated with parasitism among adults, as breeding and non-breeding adults showed similar prevalence and intensity. However, adult males, which perform overnight incubation and brooding, were significantly more likely to be parasitized than adult females. This last result supports the hypothesis that incubation and brooding investment increase the risk of Philornis parasitism for adults, but this conclusion is complicated by the lack of an association between parasitism and nesting status. Together, these results raise questions about the degree of host life-stage specialization and whether adult parasitism is incidental or part of an alternative parasitic strategy for Philornis.

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Mayaro Virus Infection Elicits an Innate Immune Response and Represses Autophagy in Anopheles stephensi Mayaro virus (MAYV) is an arboviral pathogen in the genus Alphavirus that is circulating in South America with potential to spread to naive regions. MAYV is also one of the few viruses with the ability to be transmitted by mosquitoes in the genus Anopheles, as well as the typical arboviral transmitting mosquitoes in the genus Aedes. Few studies have investigated the infection response of Anopheles mosquitoes. In this study we detail the transcriptomic and small RNA responses of An. stephensi to infection with MAYV via infectious bloodmeal at 2, 7, and 14 days post infection (dpi). 487 unique transcripts were significantly regulated, 78 putative novel miRNAs were identified, and an siRNA response is observed targeting the MAYV genome. Gene ontology analysis of transcripts regulated at each timepoint suggested activation of the Toll pathway at 7 dpi and repression of pathways related to autophagy and apoptosis at 14 dpi. These findings provide a basic understanding of the infection response of An. stephensi to MAYV and help to identify host factors which might be useful to target to inhibit viral replication in Anopheles mosquitoes.

AUTHOR SUMMARYMayaro virus (MAYV) is a mosquito-borne Alphavirus responsible for outbreaks in South America and the Caribbean. In this study we infected Anopheles stephensi with MAYV and sequenced mRNA and small RNA to understand how MAYV infection impacts gene transcription and the expression of small RNAs in the mosquito vector. Genes involved with innate immunity and signaling pathways related to cell death are regulated in response to MAYV infection of An. stephensi, we also discover novel miRNAs and describe the expression patterns of miRNAs, siRNAs, and piRNAs following bloodmeal ingestion. These results suggest that MAYV does induce a molecular response to infection in its mosquito vector species.

sentences: Placental uptake and metabolism of 25(OH)Vitamin D determines its activity within the fetoplacental unit Pregnancy 25-hydroxyvitamin D (25(OH)D) concentrations are associated with maternal and fetal health outcomes. Using physiological human placental perfusion and villous explants, we investigate the role of the placenta in regulating the relationships between maternal 25(OH)D and fetal physiology. We demonstrate active placental uptake of 25(OH)D3 by endocytosis, placental metabolism of 25(OH)D3 into 24,25-dihydroxyvitamin D3 and active 1,25-dihydroxyvitamin D [1,25(OH)2D3], with subsequent release of these metabolites into both the maternal and fetal circulations. Active placental transport of 25(OH)D3 and synthesis of 1,25(OH)2D3 demonstrate that fetal supply is dependent on placental function rather than simply the availability of maternal 25(OH)D3. We demonstrate that 25(OH)D3 exposure induces rapid effects on the placental transcriptome and proteome. These map to multiple pathways central to placental function and thereby fetal development, independent of vitamin D transfer. Our data suggest that the underlying epigenetic landscape helps dictate the transcriptional response to vitamin D treatment. This is the first quantitative study demonstrating vitamin D transfer and metabolism by the human placenta, with widespread effects on the placenta itself. These data demonstrate a complex interplay between vitamin D and the placenta and will inform future interventions using vitamin D to support fetal development and maternal adaptations to pregnancy.

sentences: Kinesin-8-specific loop-2 controls the dual activities of the motor domain according to tubulin protofilament shape Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains unclear. We addressed this by obtaining cryo-EM structures (2.6-3.9 [A]) of Candida albicans Kip3 in different catalytic states on the microtubule lattice and on a curved microtubule end mimic, as well as a microtubule-unbound CaKip3-ADP crystal structure (2.0 [A]). Together with biochemical analyses of CaKip3 and kinesin-1 mutants, we define a model that explains the kinesin-8 mechanism. The microtubule depolymerization activity originates in conformational changes of the kinesin-8 motor core that are amplified by its dynamic loop-2. On curved microtubule ends, loop-1 assists depolymerization by inserting into preceding motor domains, forming head-to-tail arrays of kinesin-8s that complement loop-2 contacts with curved tubulin. On straight tubulin protofilaments in the microtubule lattice, extended loop-2-tubulin contacts inhibit conformational changes in the motor core, but in the ADP-Pi state these contacts are relaxed, allowing neck-linker docking for motility. These tubulin shape-induced alternations between pro-microtubule-depolymerization and pro-motility kinesin states, regulated by loop-2, are the key to the dual activity of kinesin-8 motors.

sentences: Joint structural annotation of small molecules using liquid chromatography retention order and tandem mass spectrometry data We present LC-MS2Struct, a machine learning framework for structural annotation of small molecule data arising from liquid chromatography-tandem mass spectrometry (LC-MS2) measurements. LC-MS2Struct jointly predicts the annotations for a set of mass spectrometry features in a sample, using a novel structured prediction model trained to optimally combine the output of state-of-the-art MS2 scorers and observed retention orders. We evaluate our method on a dataset covering all publicly available reversed phase LC-MS2 data in the MassBank reference database, including 4327 molecules measured using 18 different LC conditions from 16 contributors, greatly expanding the chemical analytical space covered in previous multi-MS2 scorer evaluations. LC-MS2Struct obtains significantly higher annotation accuracy than earlier methods and improves the annotation accuracy of state-of-the-art MS2 scorers by up to 106%. The use of stereochemistry-aware molecular fingerprints improves prediction performance, which highlights limitations in existing approaches and has strong implications for future computational LC-MS2 developments.

sentences: DiversityScanner: Robotic discovery of small invertebrates with machine learning methods Invertebrate biodiversity remains poorly explored although it comprises much of the terrestrial animal biomass, more than 90% of the species-level diversity and supplies many ecosystem services. The main obstacle is specimen- and species-rich samples. Traditional sorting techniques require manual handling and are slow while molecular techniques based on metabarcoding struggle with obtaining reliable abundance information. Here we present a fully automated sorting robot, which detects each specimen, images and measures it before moving it from a mixed invertebrate sample to the well of a 96-well microplate in preparation for DNA barcoding. The images are then used by a newly trained convolutional neural network (CNN) to assign the specimens to 14 particularly common, usually family-level "classes" of insects in Malaise trap samples and an "other-class" (N=15). The average assignment precision for the classes is 91.4% (75-100%). In order to obtain biomass information, the specimen images are also used to measure specimen length and estimate body volume. We outline how the DiversityScanner robot can be a key component for tackling and monitoring invertebrate diversity. The robot generates large numbers of images that become training sets for CNNs once the images are labelled with identifications based on DNA barcodes. In addition, the robot allows for taxon-specific subsampling of large invertebrate samples by only removing the specimens that belong to one of the 14 classes. We conclude that a combination of automation, machine learning, and DNA barcoding has the potential to tackle invertebrate diversity at an unprecedented scale.

sentences: Feed-forward inhibition fine-tunes response timing in auditory-vocal interactions The ability to regulate vocal timing is a fundamental aspect of communicative interactions for many species, including conversational speech among humans, yet little is known about the neural circuitry that regulates the input-dependent timing of vocal replies. Exploring this topic in the zebra finch premotor area HVC, we identify feed-forward inhibition as a key regulator of vocal response timing. Based on a spiking network model informed by behavioral and electrophysiological data from communicating zebra finches, we predicted that two different patterns of inhibition regulate vocal-motor responses. In one scenario, the strength of production-related premotor inhibition translates into plasticity in vocal response delays. In the other scenario, fast transient interneuron activity in response to auditory input results in the suppression of call production while a call is heard, thereby reducing acoustic overlap between callers. Extracellular recordings in HVC during the listening phase confirm the presence of auditory-evoked response patterns in putative inhibitory interneurons, along with corresponding signatures of auditory-evoked activity suppression. The proposed model provides a parsimonious framework to explain how auditory-vocal transformations can give rise to vocal turn-taking and highlights multiple roles of local inhibition for behavioral modulation at different time scales.

sentences: Modified E. coli strains enhance baculovirus production by elimination of aberrant transposition events Baculovirus technology has been the most commonly used expression system for insect cells both due to its potential to generate a large amount of recombinant protein as well as the benefit of post-translational modifications. The most commonly used system to generate recombinant baculoviruses is the Tn7 transposition-based technology known as Bac-to-Bac. Although improvements have been made to this system to further improve quality and reproducibility of baculovirus production, recent data suggests that improved strains still have potential issues with contamination of non-recombinant baculovirus caused by improper transposition into a Tn7 site in the E. coli chromosome. Here we describe a new option for alteration of the E. coli genome to completely block the native Tn7 attachment site, leading to far fewer false positive bacmid colonies being selected and eliminating all risk of non-recombinant baculovirus production.

sentences: Evaluating the reliability, validity, and utility of overlapping networks: Implications for cognitive control Brain regions can be consistently clustered together into a small number of large-scale networks based on the correlation of their activity across time. However, the methods typically used to assign regions to these networks typically force non- or minimal overlap between the networks, losing information about how particular regions may connect to multiple networks. However, new methods are emerging that allow networks to overlap. Here, we examined the reliability and validity of one such assignment method, the mixed membership algorithm, and the utility of overlapping assignment for identifying potential gaps in existing network models of behavior. Analyses were conducted on resting state data from a large set of participants. First, we split the data into two halves and generated group-level overlapping networks for each subgroup. We then used a bootstrapped Dice similarity analysis to demonstrate that the binarized network topographies showed good agreement between subgroups. Next, we generated nonoverlapping networks in the combined data using a typical k-means clustering method. Using the same Dice Similarity analysis, we compared these nonoverlapping networks both with previously published nonoverlapping networks (Yeo et al, 2011) and with overlapping networks likewise generated for the combined data. The nonoverlapping networks generally recapitulated known networks in the literature, and the overlapping networks captured large portions of one to three nonoverlapping network topographies. Following this, we used a relative entropy analysis to assess the uniformity of each regions distribution of connections across the overlapping networks. While many regions were indeed assigned to one network, more were assigned to multiple networks. Furthermore, regions assigned to multiple networks did not necessarily show stronger connection to one network over the others. Finally, we assessed the network membership of regions of the fronto-parietal and cingulo-opercular networks of the Dual-Networks model of cognitive control. In combination, the regions were members of all of the overlapping networks, and the majority were assigned to multiple networks. Together, these results indicate that overlapping assignment produces consistent and biologically plausible network topographies, and that a complete network model of cognitive control, or indeed any network model of behavior needs to be updated to account for multiple-network membership and the complex interactions that occur between the relevant networks as a function of their overlapping nature.

sentences: Misoprostol Treatment Prevents Hypoxia-Induced Cardiac Dysfunction Through a 14-3-3 and PKA regulatory motif on Bnip3 Systemic hypoxia is a common element in most perinatal emergencies and is a known driver of Bnip3 expression in the neonatal heart. Bnip3 plays a prominent role in the evolution of necrotic cell death, disrupting ER calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests a cardioprotective role for the prostaglandin E1 analogue misoprostol during periods of hypoxia, but the mechanisms for this protection are not completely understood. Using a combination of mouse and cell models, we tested if misoprostol is cardioprotective during neonatal hypoxic injury by altering Bnip3 function. Here we report that hypoxia elicits mitochondrial-fragmentation, MPT, reduced ejection fraction, and evidence of necroinflammation, which were abrogated with misoprostol treatment or Bnip3 knockout. Through molecular studies we show that misoprostol leads to PKA-dependent Bnip3 phosphorylation at threonine-181, and subsequent redistribution of Bnip3 from mitochondrial Opa1 and the ER through an interaction with 14-3-3 proteins. Taken together, our results demonstrate a role for Bnip3 phosphorylation in the regulation of cardiomyocyte contractile/metabolic dysfunction, and necroinflammation. Furthermore, we identify a potential pharmacological mechanism to prevent neonatal hypoxic injury.

sentences: Catheterized bladder environment induces Candida albicans hyphal formation and promotes colonization and persistence through Als1. When Candida is found in the urine, physicians face the dilemma to treat or not, since its presence could represent commensalism or infection, resulting in inappropriate management. Candida spp., specifically Candida albicans, are the second most common cause of catheter-associated urinary tract infections (CAUTIs). Yet, the cellular and molecular details of C. albicans CAUTI are poorly understood. With the steady increase in catheter usage, combined with fungal infection mismanagement and treatment challenges, this question is of utmost public health importance. Here, for the first time, we characterized fungal mechanisms during CAUTI, finding that the catheterized bladder environment triggers Candida virulent programing, inducing Efg1-dependent hyphal morphogenesis, required for CAUTI establishment. Moreover, Als1, an Efg1-downstream effector, is necessary for in vitro and in vivo fibrinogen-biofilm formation and Als1 expression levels were crucial for fungal persistence during CAUTI. These findings unveil the mechanisms for fungal CAUTIs and identify potential treatment targets.

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FcγRIIB regulates (auto)antibody responses by limiting marginal zone B cell activation Fc{gamma}RIIB is an inhibitory receptor expressed throughout B cell development. Diminished expression or function is associated with lupus in mice and humans, in particular through an effect on autoantibody production and plasma cell differentiation. Here, we analysed the effect of B cell-intrinsic Fc{gamma}RIIB expression on B cell activation and plasma cell differentiation.

Loss of Fc{gamma}RIIB on B cells (Fcgr2b cKO mice) led to a spontaneous increase in autoantibody titers. This increase was most striking for IgG3, suggestive of increased extrafollicular responses. Marginal zone (MZ) and IgG3+ B cells had the highest expression of Fc{gamma}RIIB and the increase in serum IgG3 was linked to increased MZ B cell signaling and activation in the absence of Fc{gamma}RIIB. Likewise, human circulating MZ-like B cells had the highest expression of Fc{gamma}RIIB, and their activation was most strongly inhibited by engaging Fc{gamma}RIIB. Finally, marked increases in IgG3+ plasma cells and B cells were observed during extrafollicular plasma cell responses with both T-dependent and T-independent antigens in Fcgr2b cKO mice. The increased IgG3 response following immunization of Fcgr2b cKO mice was lost in MZ-deficient Notch2/Fcgr2b cKO mice.

Thus, we present a model where high Fc{gamma}RIIB expression in MZ B cells prevents their hyperactivation and ensuing autoimmunity.

Graphical abstract

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sentences: RNA G-quadruplex structure contributes to cold adaptation in plants Nucleotide composition is suggested to infer gene functionality and ecological adaptation of species to distinct environments. However, the underlying biological function of nucleotide composition dictating environmental adaptations is largely unknown. Here, we systematically analyzed the nucleotide compositions of transcriptomes across 1000 plants (1KP) and their corresponding habitats. Intriguingly, we found that plants growing in cold climates have guanine (G)-enriched transcriptomes, which are prone to forming RNA G-quadruplex structures. Both immunofluorescence detection and in vivo structure profiling revealed that RNA G-quadruplex formation in plants was globally enhanced in response to cold. Cold-responsive RNA G-quadruplexes strongly enhanced mRNA stability, rather than affecting translation. Disruption of individual RNA G-quadruplex promoted mRNA decay in the cold, leading to impaired plant cold response. Therefore, we propose that plants adopted RNA G-quadruplex structure as a molecular marker to facilitate their adaptation to the cold during evolution.

sentences: Robust transcriptional indicators of plant immune cell death revealed by spatio-temporal transcriptome analyses Recognition of a pathogen by the plant immune system often triggers a form of regulated cell death traditionally known as the hypersensitive response. This type of immune cell death occurs precisely at the site of pathogen recognition, and it is restricted to a few cells. Extensive research has shed light into how plant immune receptors are mechanistically activated. However, a central key question remains largely unresolved: how does cell death zonation take place and what are the mechanisms that underpin this phenomenon? As a consequence, bona fide transcriptional indicators of immune cell death are lacking, which prevents gaining a deeper insight of its mechanisms before cell death becomes macroscopic and precludes any early or live observation. We addressed this question using the paradigmatic Arabidopsis thaliana-Pseudomonas syringae pathosystem, by performing a spatio-temporally resolved gene expression analysis that compared infected cells that will undergo immune cell death upon pathogen recognition vs by-stander cells that will stay alive and activate immunity. Our data revealed unique and time-dependent differences in the repertoire of differentially expressed genes, expression profiles and biological processes derived from tissue undergoing immune cell death and that of its surroundings. Further, we generated a pipeline based on concatenated pairwise comparisons between time, zone and treatment that enabled us to define 13 robust transcriptional immune cell death markers. Among these genes, the promoter of an uncharacterized AAA-ATPase has been used to obtain a fluorescent reporter transgenic line, which displays a strong spatio-temporally resolved signal specifically in cells that will later undergo pathogen-triggered cell death. In sum, this valuable set of genes can be used to define those cells that are destined to die upon infection with immune cell death-triggering bacteria, opening new avenues for specific and/or high-throughput techniques to study immune cell death processes at a single-cell level.

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A Developmentally Prometastatic Niche to Hepatoblastoma in Neonatal Liver mediated by the Cxcl1/Cxcr2 Axis Background and RationaleHepatoblastoma (HB) is the most common pediatric liver cancer. Its predominant occurrence in very young children led us to investigating whether the neonatal liver provides a protumorigenic niche to HB development.

MethodsHB development was compared between orthotopic transplantation models established in postnatal day 5 and 60 mice (P5Tx and P60Tx models). Single-cell RNA-sequencing was performed using tumor and liver tissues from both models and the top candidate cell types and genes identified are investigated for their roles in HB cell growth, migration, and survival.

ResultsWe found that various HB cell lines including HepG2 cells were consistently and considerably more tumorigenic and metastatic in the P5Tx model than in the P60Tx models. Sc-RNAseq of the P5Tx and P60Tx HepG2 models revealed that the P5Tx tumor was more hypoxic and had a larger number of activated hepatic stellate cells (aHSCs) in the tumor-surrounding liver which express significantly higher levels of Cxcl1 than those from the P60Tx model. We found these differences were developmentally present in normal P5 and P60 liver. We showed that the Cxcl1/Cxcr2 axis mediated HB cell migration and was critical to HB cell survival under hypoxia. Treating HepG2 P60Tx model with recombinant CXCL1 protein induced intrahepatic and pulmonary metastasis and CXCR2 knockout in HepG2 cells abolished their metastatic potential in the P5Tx model. Lastly, we showed that in metastatic HB patient tumors there was a similar larger population of aHSCs in the tumor-surrounding liver than in localized tumors, and tumor hypoxia was uniquely associated with HB patient prognosis among pediatric cancers.

ConclusionWe demonstrated that the neonatal liver provides a prometastatic niche to HB development via the Cxcl1/Cxcr2 axis.

sentences: Fast processing of environmental DNA metabarcoding sequence data using convolutional neural networks 1The intensification of anthropogenic pressures have increased consequences on biodiversity and ultimately on the functioning of ecosystems. To monitor and better understand biodiversity responses to environmental changes using standardized and reproducible methods, novel high-throughput DNA sequencing is becoming a major tool. Indeed, organisms shed DNA traces in their environment and this "environmental DNA" (eDNA) can be collected and sequenced using eDNA metabarcoding. The processing of large volumes of eDNA metabarcoding data remains challenging, especially its transformation to relevant taxonomic lists that can be interpreted by experts. Speed and accuracy are two major bottlenecks in this critical step. Here, we investigate whether convolutional neural networks (CNN) can optimize the processing of short eDNA sequences. We tested whether the speed and accuracy of a CNN are comparable to that of the frequently used OBITools bioinformatic pipeline. We applied the methodology on a massive eDNA dataset collected in Tropical South America (French Guiana), where freshwater fishes were targeted using a small region (60pb) of the 12S ribosomal RNA mitochondrial gene. We found that the taxonomic assignments from the CNN were comparable to those of OBITools, with high correlation levels and a similar match to the regional fish fauna. The CNN allowed the processing of raw fastq files at a rate of approximately 1 million sequences per minute which was 150 times faster than with OBITools. Once trained, the application of CNN to new eDNA metabarcoding data can be automated, which promises fast and easy deployment on the cloud for future eDNA analyses.

sentences: Antithrombin glycoforms from type II antithrombin deficient patients selectively adsorb onto the surface of plasma extracellular vesicles Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clotting proteases. A deficit in AT function leads to AT qualitative deficiency, challenging to diagnose. Here we report that active AT may travel physiosorbed on the surface of plasma extracellular vesicles (EVs), contributing to form the "EV-protein corona". The corona is enriched in specific AT glycoforms, thus suggesting glycosylation to play a key role in AT partitioning between EVs and plasma. Differences in AT glycoform composition of the corona of EVs separated from plasma of healthy and AT qualitative deficiency-affected subjects were also noticed. This suggests deconstructing the plasma into its nanostructured components, as extracellular vesicles, could help to unravel pathophysiological mechanisms otherwise undiscovered.

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Parasite-driven cascades or hydra effects: susceptibility and foraging depression shape parasite-host-resource interactions O_LIWhen epidemics kill hosts and increase their resources, should the density of hosts decrease (with a resource increase, this constitutes a trophic cascade) or increase (a hydra effect)? Seeking answers, we integrate trait measurements, a resource-host-parasite model, and experimental epidemics with plankton. This combination reveals how a spectrum from cascades to hydra effects can arise. It reflects tension between parasite-driven mortality (a density-mediated effect) and foraging depression upon contact with parasite propagules (a trait-mediated one).
C_LIO_LIIn the model, mortality rises when higher susceptibility to infection increases infection prevalence. Epidemics release resources while suppressing hosts (creating a cascade). In contrast, when hosts are less susceptible and parasites depress their foraging, a resource feedback can elevate host density during epidemics (creating a hydra effect), particularly at higher carrying capacity of resources. This combination elevates primary production relative to per-host consumption of resources (two key determinants of host density).
C_LIO_LIWe test these predictions of the qualitative effects of host traits and resource carrying capacity with trait measurements and a mesocosm experiment. Trait measurements show clonal lines of zooplankton hosts differ in their foraging depression and susceptibility. We seeded resource-host-parasite mesocosms with different host genotypes and provided different nutrient supplies to test model predictions. Hydra effects and trophic cascades arose under different conditions, as predicted by the model.
C_LIO_LIHence, tension between trait-mediated and density-mediated effects of parasites governs the fate of host density during epidemics - from cascades to hydra effects - via feedbacks with resources.
C_LI

sentences: Structure of mycobacterial CIII2CIV2 respiratory supercomplex bound to the tuberculosis drug candidate telacebec (Q203) The imidazopyridine telacebec, also known as Q203, is one of only a few new classes of compounds in more than fifty years with demonstrated antituberculosis activity in humans. Telacebec inhibits the mycobacterial respiratory supercomplex composed of complexes III and IV (CIII2CIV2). In mycobacterial electron transport chains, CIII2CIV2 replaces canonical CIII and CIV, transferring electrons from the intermediate carrier menaquinol to the final acceptor, molecular oxygen, while simultaneously transferring protons across the inner membrane to power ATP synthesis. We show that telacebec inhibits the menaquinol:oxygen oxidoreductase activity of purified Mycobacterium smegmatis CIII2CIV2 at concentrations similar to those needed to inhibit electron transfer in mycobacterial membranes and Mycobacterium tuberculosis growth in culture. We then used electron cryomicroscopy (cryoEM) to determine structures of CIII2CIV2 both in the presence and absence of telacebec. The structures suggest that telacebec prevents menaquinol oxidation by blocking two different menaquinol binding modes to prevent CIII2CIV2 activity.

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Fasting reverses drug-resistance in hepatocellular carcinoma through p53-dependent metabolic synergism Cancer cells voraciously consume nutrients to support their growth, exposing a metabolic vulnerability that can be therapeutically exploited. Here we show in hepatocellular carcinoma (HCC) cells, xenografts, and in patient-derived HCC organoids that fasting can synergistically sensitize resistant HCC to sorafenib. Mechanistically, sorafenib acts non-canonically as an inhibitor of mitochondrial respiration, causing resistant cells to depend on glycolysis for survival. Fasting, through reduction in glucose and impeded AKT/mTOR-signaling, prevents this Warburg shift. Regulating glucose transporter and pro-apoptotic protein expression, p53 is necessary and sufficient for the sorafenib-sensitizing effect of fasting. p53 is also crucial for fasting-mediated improvement of sorafenib efficacy in an orthotopic HCC mouse model. Together, our data suggest intermittent fasting and sorafenib as rational combination therapy for HCC with intact p53 signaling. As HCC therapy is currently severely limited by resistance, these results should instigate clinical studies aimed at improving therapy response in advanced-stage, and possibly even early-stage, HCC.

HIGHLIGHTSO_LIFasting sensitizes resistant HCC xenografts and patient-derived organoids to sorafenib
C_LIO_LISorafenib-mediated Warburg shift is prevented by glucose limitation upon fasting
C_LIO_LIFasting synergistically improves sorafenib efficacy in non-resistant models
C_LIO_LIp53 is required for synergism by regulating glucose uptake and apoptosis
C_LI

GRAPHICAL ABSTRACT

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sentences: Introduction of probiotic bacterial consortia promotes plant growth via impacts on the resident rhizosphere microbiome Plant growth depends on a range of functions provided by their associated rhizosphere microbiome, including nutrient mineralization, hormone co-regulation and pathogen suppression. Improving the ability of plant associated microbiome to deliver these functions is thus important for developing robust and sustainable crop production. However, it is yet unclear how beneficial effects of probiotic microbial inoculants can be optimised and how they are mediated. Here, we sought to enhance the tomato plant growth by targeted introduction of probiotic bacterial consortia consisting of up to eight plant-associated Pseudomonas strains. We found that the effect of probiotic consortium inoculation was richness-dependent: consortia that contained more Pseudomonas strains, reached higher densities in the tomato rhizosphere and had clearer beneficial effects on multiple plant growth characteristics. Crucially, these effects were best explained by changes in the resident community diversity, composition and increase in the relative abundance of initially rare taxa, instead of introduction of plant-beneficial traits into the existing community along with probiotic consortia. Together, our results suggest that beneficial effects of microbial introductions can be driven indirectly through effects on the diversity and composition of resident plant rhizosphere microbiome.

sentences: Acetazolamide modulates intracranial pressure directly by its action on the cerebrospinal fluid secretion apparatus Elevated intracranial pressure (ICP) is observed in many neurological pathologies, e.g. hydrocephalus and stroke. This condition is routinely relieved with neurosurgical approaches, since effective and targeted pharmacological tools are still lacking. The carbonic anhydrase inhibitor, acetazolamide (AZE), may be employed to treat elevated ICP. However, its effectiveness is questioned, its location of action unresolved, and its tolerability low. Here, we employed in vivo and ex vivo approaches to reveal the efficacy and mode of action of AZE in the rat brain. The drug effectively reduced the ICP, irrespective of the mode of drug administration and level of anaesthesia. The effect occurred via a direct action on the choroid plexus and an associated decrease in cerebrospinal fluid secretion, and not indirectly via the systemic action of AZE on renal and vascular processes. Upon a single administration, the reduced ICP endured for approximately 10 h post-AZE delivery with no long-term changes of brain water content or choroidal transporter expression. However, a persistent reduction of ICP was secured with repeated AZE administrations throughout the day. Future specific targeting of choroidal carbonic anhydrases may limit the systemic side effects, and therefore enhance the treatment tolerability and effectiveness in select patient groups experiencing elevated ICP.

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EsMeCaTa: Estimating metabolic capabilities from taxonomic affiliations SummaryPredicting the functional potential of microorganisms in environmental samples from cultivation-independent techniques is a major challenge. A persistent difficulty lies in associating taxonomic profiles obtained from metabarcoding experiment with accurate functional profiles, particularly for poorly-resolved taxonomic groups. In this paper, we present EsMeCaTa a python package predicting shared proteins from taxonomic affiliations. EsMeCaTa relies on the UniProt database to retrieve the public proteomes associated with a taxon and then uses MMseqs2 in order to compute the set of proteins shared in the taxon. Finally, Es-MeCaTa extracts the functional annotations of these proteins to provide an accurate estimate of the functional potential associated to taxonomic affiliations.

AvailabilityEsMeCaTa is available at: https://github.com/AuReMe/esmecata under the GPL-3 license.

sentences: The Statistical Trends of Protein Evolution: A Lesson from AlphaFold Database The recent development of artificial intelligence provides us with new and powerful tools for studying the mysterious relationship between organism evolution and protein evolution. In this work, based on the AlphaFold Protein Structure Database (AlphaFold DB), we perform comparative analyses of the protein structures of different species. The statistics of AlphaFold-predicted structures show that, as species evolve towards higher complexity (e.g., from prokaryotes to eukaryotes, from unicellular to multicellular organisms, etc.), the proteins within them evolve toward larger radii of gyration, higher coil fractions, higher modularity, and slower relaxations, indicating that the average flexibility of proteins gradually increases in the species evolution. With the scaling analyses, the size dependence of proteins shape, topology, and dynamics suggest a decreasing fractal dimension of the proteins in species evolution. This evolutionary trend is accompanied by the increasing eigengaps in the vibration spectra of proteins, implying that the proteins are statistically evolving toward higher functional specificity and lower dimensionality in the native dynamics. Furthermore, we also uncover the topology and sequence bases of this evolutionary trend. The residue contact networks of proteins evolve toward higher assortativity, and the hydrophobic and hydrophilic amino acid residues evolve to have increasing segregations in the sequences. This study provides new insights into how the diversity in the functionality of proteins increases and how the dimensionality of the manifold of protein dynamics reduces during evolution. The statistical evolutionary trends also shed light on the study of protein design.

sentences: Accurate spliced alignment of long RNAsequencing reads Long-read RNA sequencing techniques are establishing themselves as the primary sequencing technique to study the transcriptome landscape. Many such analyses are dependent on read alignments. However, the error rate and sequencing length of long-read technologies create new challenges for accurately aligning these reads. We present an alignment method uLTRA based on a novel two-pass collinear chaining algorithm. Furthermore, uLTRA can be used both as a stand-alone aligner and as a wrapper around minimap2 for improved alignments in gene regions. We show that uLTRA produces higher accuracy over state-of-the-art aligners with substantially higher accuracy for small exons on simulated and synthetic data. On biological data where true read location is unknown, we show several examples where uLTRA aligns to known and novel isoforms with exon structures that are not detected with other aligners. uLTRA is available at https://github.com/ksahlin/ultra.

sentences:
Machine Learning to Summarize and Provide Context for Sleep and Eating Schedules. The relative timing of sleep and of eating within the circadian day is important for human health. Despite much data on sleep, and a growing data set for eating, there remains a need for an interpretative framework for the understanding of this data for health decisions. This study provides a new statistical and machine learning analysis of more than 500 participants in the Daily24 project. From their data, and the analysis, we propose a framework for determining the classification of participants into different chronotypes and with that the ability to realize the potential impact of daily circadian habits on health. We propose that our resulting distribution curves could be used, similar to NHANES (National Health and Nutrition Examination Survey) data for pediatric growth, as a measure for circadian misalignment and used to help guide re-entrainment schedules.

Author summaryDaily habits can be positive, negative or neutral for human health. Generally sleep and eating schedules are assumed without thought for their potential to help or interfere with health. In this study we propose a framework, based on data from more than 500 participants, for evaluating the relative timing of meals and sleep schedules. This evaluation, similar to pediatric growth charts, can guide clinical suggestions for those at the extremes, while helping others to realize that they are unusual relative to the population average

sentences: Metacommunity stability and persistence for predation turnoff in selective patches Predation as an important trophic interaction of ecological communities controls the large-scale patterns of species distribution, population abundance and community structure. Numerous studies address that predation can mediate diversity and regulate the ecological community and food web stability through changes in the behaviour, morphology, development, and abundance of prey. Since predation has large effects on persistence and diversity, the local loss or removal of predation in a community can trigger a cascade of extinctions. In ecological theory, the effect of predation removal has been well studied in foodwebs, but it remains unclear in the case of a spatially distributed community connected by dispersal. In this study, the interaction between local and spatial processes is taken into account, we present how a predation turnoff in selective patches affects the stability and persistence of a metacommunity. Using a simple predator-prey metacommunity with a diffusive dispersal, we show the impact of predation on synchronized, asynchronized and source-sink dynamics. Our results reveal that predation turnoff in very few patches alters a perfectly synchronized oscillatory state into multicluster states consisting of various patterns. In a source-sink behaviour, predation turnoff in a source patch reduces the number of sink patches and changes the clusters. In general, predation turnoff in a finite number of patches increases the number of clusters through asynchronized (inhomogeneous) states, whereas predation turnoff in a larger number of patches can lead to the complete extinction of predators. Typically, there exists a critical number of patches below which the predation turnoff results in asynchronized states and above that predation turnoff leads to a synchronized state in prey population with complete extinction of predators. Further, our results identify the network configurations that exhibit a unique number of clusters. Moreover, prey density from the patches where predation is absent goes to a saturating state near the carrying capacity. Thus, this study stresses that predation turnoff in selective patches acts as a stabilizing mechanism that can promote metacommunity persistence.

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Generation, characterization and drug sensitivities of twelve patient-derived IDH1 mutant glioma cell cultures BackgroundMutations of the isocitrate dehydrogenase (IDH) gene occur in over 80% of low-grade gliomas and secondary glioblastomas. Despite considerable efforts, endogenous in vitro IDH-mutated glioma models remain scarce. Availability of these models is key for the development of new therapeutic interventions.

MethodsCell cultures were established from fresh tumor material and expanded in serum-free culture media. D-2-Hydroxyglutarate levels were determined by mass-spectrometry. Genomic and transcriptomic profiling were carried out on the Illumina Novaseq platform, methylation profiling was performed with the Infinium MethylationEpic BeadChip array. Mitochondrial respiration was measured with the Seahorse XF24 Analyzer. Drug screens were performed with an NIH FDA-approved anti-cancer drug set and two IDH-mutant specific inhibitors.

ResultsA set of twelve patient-derived IDHmt cell cultures was established. We confirmed high concordance in driver mutations, copy number and methylation profiles between the tumors and derived cultures. Homozygous deletion of CDKN2A/B was observed in all cultures. IDH-mutant cultures had lower mitochondrial reserve capacity. IDH-mutant specific inhibitors did not affect cell viability or global gene expression. Screening of 107 FDA-approved anti-cancer drugs identified nine compounds with potent activity against IDHmt gliomas, including three compounds with favorable pharmacokinetic characteristics for CNS penetration: teniposide, omacetaxine mepesuccinate, and marizomib.

ConclusionsOur twelve IDH-mutant cell cultures show high similarity to the parental tissues and offer a unique tool to study the biology and drug sensitivities of high-grade IDHmt gliomas in vitro. Our drug screening studies reveal lack of sensitivity to IDHmt inhibitors, but sensitivity to a set of nine available anti-cancer agents.

Key pointsO_LIIDHmt glioma cultures closely resemble their parental tumors
C_LIO_LIMicroscopic monitoring of early passages and colony isolation increases IDH1mt culture success
C_LIO_LIDrug screening identified nine candidate repurposed drugs for IDHmt glioma
C_LI

Importance of the studyIDH-mutations are highly prevalent in low grade and secondary high-grade gliomas. Despite this high frequency however, very few in vitro models have been reported for IDH-mutated gliomas. In this manuscript we describe and characterize in detail twelve primary cultures from IDH-mutant astrocytomas. We show that these cultures retain most of the genetic, epigenetic and metabolic features of their respective parental tumors. Because of these similarities, these independent model systems will not only help understand the molecular defects driven by the mutation, but are also vital to identify means to target these tumors. Screening of 107 FDA-approved anti-cancer agents on these cultures identified a set of highly effective agents that may offer candidates for either systemic or assisted delivery treatment of this tumor subtype.

sentences: Yolk sac-derived bipotent endothelial-macrophage progenitor cells are a self-renewing tissue-resident source of inflammation and neovascularization postnatally Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these yolk sac-derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic, self-renewing endothelial-macrophage (EndoMac) progenitor cells in postnatal mouse aorta, heart and lung, that are independent of definitive hematopoiesis and derive from a CX3CR1+ and CSF1R+ yolk sac source. These bipotent progenitors are highly proliferative and vasculogenic, contributing to adventitial neovascularization in the aortic wall and forming perfused blood vessels after adoptive transfer into ischemic tissue. We establish a regulatory role for angiotensin II, which enhances their clonogenic, self-renewal and differentiation properties. Our findings demonstrate that tissue-resident EndoMac progenitors drive local inflammatory and vasculogenic responses by contributing to the renewal and expansion of yolk sac-derived macrophages and endothelial cells postnatally.

sentences: Benchmarking functional connectivity by the structure and geometry of the human brain The brains structural connectivity supports the propagation of electrical impulses, manifesting as patterns of co-activation, termed functional connectivity. Functional connectivity emerges from the underlying sparse structural connections, particularly through poly-synaptic communication. As a result, functional connections between brain regions without direct structural links are numerous, but their organization is not completely understood. Here we investigate the organization of functional connections without direct structural links. We develop a simple, data-driven method to benchmark functional connections with respect to their underlying structural and geometric embedding. We then use this method to re-weigh and re-express functional connectivity. We find evidence of unexpectedly strong functional connectivity within the canonical intrinsic networks of the brain. We also find unexpectedly strong functional connectivity at the apex of the unimodal-transmodal hierarchy. Our results suggest that both phenomena - functional modules and functional hierarchies - emerge from functional interactions that transcend the underlying structure and geometry. These findings also potentially explain recent reports that structural and functional connectivity gradually diverge in transmodal cortex. Collectively, we show how structural connectivity and geometry can be used as a natural frame of reference with which to study functional connectivity patterns in the brain.

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Multimodal profiling of lung granulomas reveals cellular correlates of tuberculosis control Mycobacterium tuberculosis lung infection results in a complex multicellular structure, the granuloma. In some granulomas, immune activity promotes bacterial clearance; in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal PET-CT imaging, single-cell RNA-sequencing, and measures of bacterial clearance. We find that bacterial persistence occurs in granulomas enriched for mast, endothelial, fibroblast and plasma cells, signaling amongst themselves via Type II immunity and wound healing pathways. In contrast, these interactions are largely absent in granulomas that drive bacterial control, which are often those that form later in the course of infection; these restrictive lesions are characterized by cellular ecosystems enriched for Type1-Type17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks that involve diverse myeloid and non-immune cell populations. There is also a temporal aspect to bacterial control, in that granulomas that arise later in infection (in the context of an established immune response) share the functional characteristics of restrictive granulomas and are more capable of killing Mtb. Taken together, our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.

One-Sentence SummaryBacterial control in TB lung granulomas correlates with distinct cellular immune microenvironments and time of formation after infection.

sentences: The cell adhesion molecule Sdk1 shapes assembly of a retinal circuit that detects localized edges. Nearly 50 different mouse retinal ganglion cell (RGC) types sample the visual scene for distinct features. RGC feature selectivity arises from its synapses with a specific subset of amacrine (AC) and bipolar cell (BC) types, but how RGC dendrites arborize and collect input from these specific subsets remains poorly understood. Here we examine the hypothesis that RGCs employ molecular recognition systems to meet this challenge. By combining calcium imaging and type-specific histological stains we define a family of circuits that express the recognition molecule Sidekick 1 (Sdk1) which include a novel RGC type (S1-RGC) that responds to local edges. Genetic and physiological studies revealed that Sdk1 loss selectively disrupts S1-RGC visual responses which result from a loss of excitatory and inhibitory inputs and selective dendritic deficits on this neuron. We conclude that Sdk1 shapes dendrite growth and wiring to help S1-RGCs become feature selective.

sentences: The gradient model of brain organization in decisions involving 'empathy for pain' Recent meta-analytic studies of social cognition and the functional imaging of empathy have exposed the overlap between their neural substrates and heteromodal association areas. The  gradient model of cortical organization proposes a close relationship between these areas and highly connected hubs in the default mode network, a set of cortical areas deactivated by demanding tasks. Here, we used a decision-making task and representational similarity analysis with classic  empathy for pain visual stimuli to probe the relationship between high-level representations of imminent pain in others and the high end of the gradient of this model. High-level representations were found to co-localize with task deactivations or the transitions from activations to deactivations. These loci belonged to two groups: those that loaded on the high end of the principal cortical gradient and were associated by meta-analytic decoding with the default mode network, and those that appeared to accompany functional repurposing of somatosensory cortex in the presence of visual stimuli. In contrast to the nonspecific meta-analytic decoding of these loci, low-level representations, such as those of body parts involved in pain or of pain itself, were decoded with matching topics terms. These findings suggest that that task deactivations may set out cortical areas that host high-level representations, but whose functional characterization in terms of simple mappings is unlikely. We anticipate that an increased understanding of the cortical correlates of high-level representations may improve neurobiological models of social interactions and psychopathology.

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Comparative biofilm assays using Enterococcus faecalis OG1RF identify new determinants of biofilm formation Enterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by 6 Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA, tig, OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions, and identifies multiple new genetic determinants of biofilm formation.

ImportanceE. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.

sentences: χ-separation: Magnetic susceptibility source separation toward iron and myelin mapping in the brain Obtaining a histological fingerprint from the in-vivo brain has been a long-standing target of magnetic resonance imaging (MRI). In particular, non-invasive imaging of iron and myelin, which are involved in normal brain functions and are histopathological hallmarks in neurodegenerative diseases, has practical utilities in neuroscience and medicine. Here, we propose a biophysical model that describes the individual contribution of paramagnetic (e.g., iron) and diamagnetic (e.g., myelin) susceptibility sources to the frequency shift and transverse relaxation of MRI signals. Using this model, we develop a method,{chi} -separation, that generates the voxel-wise distributions of the two sources. The method is validated using computer simulation and phantom experiments, and applied to ex-vivo and in-vivo brains. The results delineate the well-known histological features of iron and myelin in the specimen, healthy volunteers, and multiple sclerosis patients. This new technology may serve as a practical tool for exploring the microstructural information of the brain.

sentences: Evolution of diversity in metabolic strategies Understanding the origin and maintenance of biodiversity is a fundamental problem. Many theoretical approaches have been investigating ecological interactions, such as competition, as potential drivers of diversification. Classical consumer-resource models predict that the number of coexisting species should not exceed the number of distinct resources, a phenomenon known as the competitive exclusion principle. It has recently been argued that including physiological tradeoffs in consumer-resource models can lead to violations of this principle and to ecological coexistence of very high numbers of species. Here we show that these results crucially depend on the functional form of the tradeoff. We investigate the evolutionary dynamics of resource use constrained by tradeoffs and show that if the tradeoffs are non-linear, the system either does not diversify, or diversifies into a number of coexisting species that does not exceed the number of resources. In particular, very high diversity can only be observed for linear tradeoffs.

sentences: Visceral mesoderm signaling regulates assembly position and function of the Drosophila testis niche Tissue homeostasis often requires a properly placed niche to support stem cells. Morphogenetic processes that position a niche are just being described. For the Drosophila testis we recently showed that pro niche cells, specified at disparate positions during early gonadogenesis, must assemble into one collective at the anterior of the gonad. We now find that Slit and FGF signals emanating from adjacent visceral mesoderm regulate assembly. In response to signaling, niche cells express islet, which we find is also required for niche assembly. Without signaling, niche cells specified furthest from the anterior are unable to migrate, remaining dispersed. Function of such niches is severely disrupted, with niche cells evading cell cycle quiescence, compromised in their ability to signal the incipient stem cell pool, and failing to orient stem cell divisions properly. Our work identifies both extrinsic signaling and intrinsic responses required for proper assembly and placement of the testis niche.

sentences: Maintenance of Bound or Independent Features in Visual Working Memory is Task-dependent Over the last decade, seemingly conflicting results were obtained regarding the question of whether features of an object are stored separately, or bound together, in visual working memory. Many of these studies are based on an implicit assumption about a default, or fixed, mode of working memory storage. In contrast, here we asked whether the anticipated memory probes used in a given experiment might determine the format in which information is maintained in working memory, consistent with its task-oriented function. In order to test this flexible maintenance hypothesis, we recorded EEG while subjects performed a delayed-match-to-sample task with different requirements and load. In Experiment 1, by contrasting conditions with and without the requirement of maintaining bound features, we found significant differences in EEG signals recorded in central-parietal channels while controlling for differences in visual stimulation. Such difference was confirmed by multivariate pattern analysis (MVPA). Moreover, behavior results, topographical distributions and cross-contrast MVPA decoding jointly suggested that this effect is not the same as task load effect. These results were confirmed in Experiment 2 with an independent group of subjects with the same paradigm, providing reliable evidence that the format of object maintenance in visual working memory could be flexibly shaped by the current task.

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The role of the orbitofrontal cortex in creating cognitive maps We use internal models of the external world to guide behavior, but little is known about how these cognitive maps are created. The orbitofrontal cortex (OFC) is typically thought to access these maps to support model-based decision-making, but it has recently been proposed that its critical contribution may be instead to integrate information into existing and new models. We tested between these alternatives using an outcome-specific devaluation task and a high-potency chemogenetic approach. We found that selectively inactivating OFC principal neurons when rats learned distinct cue-outcome associations, but prior to outcome devaluation, disrupted subsequent model-based inference, confirming that the OFC is critical for creating new cognitive maps. However, OFC inactivation surprisingly led to generalized devaluation. Using a novel reinforcement learning framework, we demonstrate that this effect is best explained not by a switch to a model-free system, as would be traditionally assumed, but rather by a circumscribed deficit in defining credit assignment precision during model construction. We conclude that the critical contribution of the OFC to learning is regulating the specificity of associations that comprise cognitive maps.

One Sentence SummaryOFC inactivation impairs learning of new specific cue-outcome associations without disrupting model-based learning in general.

sentences: Individual differences in visual search performance extend from artificial arrays to naturalistic environments. Visual search is a universal human activity in naturalistic environments. Traditionally, visual search is investigated under tightly controlled conditions, where head-restricted participants locate a minimalistic target in a cluttered array presented on a computer screen. Do classic findings of visual search extend to naturalistic settings, where participants actively explore complex, real-world scenes? Here, we leverage advances in virtual reality (VR) technology to relate individual differences in classic visual search paradigms to naturalistic search behavior. In a naturalistic visual search task, participants looked for an object within their environment via a combination of head-turns and eye-movements using a head-mounted display. Then, in a classic visual search task, participants searched for a target within a simple array of colored letters using only eye-movements. We tested how set size, a property known to limit visual search within computer displays, predicts the efficiency of search behavior inside immersive, real-world scenes that vary in levels of visual clutter. We found that participants search performance was impacted by the level of visual clutter within real-world scenes. Critically, we also observed that individual differences in vi1-3sual search efficiency in classic search predicted efficiency in real-world search, but only when the comparison was limited to the forward-facing field of view for real-world search. These results demonstrate that set size is a reliable predictor of individual performance across computer-based and active, real-world visual search behavior.

sentences: Identifying Causal Subsequent Memory Effects Over 40 years of accumulated research has detailed associations between neuroimaging signals measured during a memory encoding task and later memory performance, across a variety of brain regions, measurement tools, statistical approaches and behavioral tasks. But the interpretation of these Subsequent Memory Effects (SMEs) remains unclear: if the identified signals reflect cognitive and neural mechanisms of memory encoding then the underlying neural activity must be causally related to future memory. However, almost all previous SME analyses do not control for potential confounders of this causal interpretation, such as serial position and item effects. We collect a large fMRI dataset and use a novel experimental design and analysis approach that allows us to statistically adjust for all exogenous confounding variables. We find that, using standard approaches without adjustment, we replicate several univariate and multivariate subsequent memory effects and are able to predict memory performance across people. However, we are unable to identify any signal that reliably predicts subsequent memory after adjusting for confounding variables, bringing into doubt the causal status of these effects. We apply the same approach to subjects judgments of learning collected during an encoding period, and show that these behavioral measures of encoding quality do predict memory after adjustments, suggesting that it is possible to measure signals at the time of encoding that reflect causal mechanisms but that existing neuroimaging measures may not have the precision and specificity to do so.

sentences: Fission of megamitochondria into multiple smaller well-defined mitochondria in the ageing zebrafish retina Mitochondria are essential adenosine triphosphate (ATP)-generating cellular organelles. In the retina, they are highly numerous in the photoreceptors and retinal pigment epithelium (RPE) due to their high energetic requirements. Fission and fusion of the mitochondria within these cells allow them to adapt to changing demands over the lifespan of the organism. Using transmission electron microscopy, we examined the mitochondrial ultrastructure of zebrafish photoreceptors and RPE from 5 days post fertilisation (dpf) through to late adulthood (3 years). Notably, mitochondria in the youngest animals were large and irregular shaped with a loose cristae architecture, but by 8 dpf they had reduced in size and expanded in number with more defined cristae. When investigating temporal gene expression of several mitochondrial-related markers, they indicated fission as the dominant mechanism contributing to these changes observed over time. This is likely to be due to continued mitochondrial stress resulting from the oxidative environment of the retina and prolonged light exposure. We have characterised retinal mitochondrial ageing in a key vertebrate model organism, that provides a basis for future studies of retinal diseases that are linked to mitochondrial dysfunction.

sentences: Release of HIV-1 particles from the viral compartment in macrophages requires an associated cytoskeleton and is driven by mechanical constraints A defining feature of HIV-1 replication in macrophages is that viral assembly occurs at the limiting membrane of a compartment often named VCC (virus-containing compartments) that is connected to the extracellular medium. The newly formed viral progeny pinches of the membrane and accumulates in the lumen of the VCC. While HIV budding has been extensively studied, very little is known about how viral particles present in the lumen of VCC are released in the extracellular medium. Here we show that the actin dynamics are critical for this process by combining ultrastructural analyses, time-lapse microscopy and perturbations of the actin cytoskeleton. We found that jasplakinolide, which stabilizes actin fibres, inhibited viral release from HIV-1-infected macrophages. Furthermore, in jasplakinolide-treated macrophages, VCC became scattered and no longer co-localized with the integrin CD18, nor the phosphorylated form of the focal adhesion kinase PYK2. Inhibition of PYK2 activity in infected macrophages promoted intracellular retention of viral particles in VCC that were no longer connected to the plasma membrane. Finally, we stimulated the rapid release of viral particles from the VCC by subjecting infected macrophages to frustrated phagocytosis. As macrophages spread on IgG-coated glass surfaces, VCC rapidly migrated to the basal membrane and released their viral content in the extracellular medium, which required their association with CD18 and the actin cytoskeleton. These results highlight that VCC trafficking and virus release are intimately linked to the reorganization of the macrophage actin cytoskeleton in response to external physical cues, suggesting that it might be regulated in tissues by the mechanical stress to which these cells are exposed.

sentences: An empirical evaluation of functional alignment using inter-subject decoding Inter-individual variability in the functional organization of the brain presents a major obstacle to identifying generalizable neural coding principles. Functional alignment--a class of methods that matches subjects neural signals based on their functional similarity--is a promising strategy for addressing this variability. To date, however, a range of functional alignment methods have been proposed and their relative performance is still unclear. In this work, we benchmark five functional alignment methods for inter-subject decoding on four publicly available datasets. Specifically, we consider three existing methods: piecewise Procrustes, searchlight Procrustes, and piecewise Optimal Transport. We also introduce and benchmark two new extensions of functional alignment methods: piecewise Shared Response Modelling (SRM), and intra-subject alignment. We find that functional alignment generally improves inter-subject decoding accuracy though the best performing method depends on the research context. Specifically, SRM and Optimal Transport perform well at both the region-of-interest level of analysis as well as at the whole-brain scale when aggregated through a piecewise scheme. We also benchmark the computational efficiency of each of the surveyed methods, providing insight into their usability and scalability. Taking inter-subject decoding accuracy as a quantification of inter-subject similarity, our results support the use of functional alignment to improve inter-subject comparisons in the face of variable structure-function organization. We provide open implementations of all methods used.

sentences: An Activator Locks Kv7.1 Channels Open by Electro-Mechanical Uncoupling and Allosterically Modulates its Pore with similarity to phosphatidylinositol-4,5-bisphosphat Loss-of-function mutations in Kv7.1 often lead to long QT syndrome (LQTS), a cardiac repolarization disorder associated with increased risk of arrhythmia and subsequent sudden cardiac death. The discovery of agonistic IKs modulators may offer a new potential strategy in pharmacological treatment of this disorder. The benzodiazepine (R)-L3 potently activates Kv7.1 channels and shortens action potential duration, thus may represent a starting point for drug development. However, the molecular mechanisms underlying modulation by (R)-L3 are still unknown. By combining alanine scanning mutagenesis, non-canonical amino acid incorporation, voltage-clamp electrophysiology and fluorometry, and in silico protein modelling, we showed that (R)-L3 not only stimulates currents by allosteric modulation of the pore domain but also alters the kinetics independently from the pore domain effects. We identified novel (R)-L3-interacting key residues in the lower S4-segment of Kv7.1 and observed an uncoupling of the outer S4 segment with the inner S5, S6 and selectivity filter segments. Summarizing, we provide structural and functional evidence for two independent Kv7.1 activating mechanisms by a single modulator.

sentences: KDM6B sensitizes PARthanatos via suppression of O6-methylguanine DNA methyltransferase repair and sustained checkpoint response Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA damage sensor and contributes to both DNA repair and cell death processes. However, how PARP-1 signaling is regulated to switch its function from DNA repair to cell death remains largely unknown. Here, we found that PARP-1 plays a central role in alkylating agent-induced PARthanatic cancer cell death. Lysine demethylase 6B (KDM6B) was identified as a key cell death effector in PARthanatos. Knockout of KDM6B or loss of KDM6B demethylase activity conferred cancer cells resistance to PARthanatic cell death in response to alkylating agents. Mechanistically, KDM6B knockout suppressed methylation at the promoter of O6-methylguanine-DNA methyltransferase (MGMT) to enhance MGMT expression and its direct DNA repair function, thereby inhibiting DNA damage-evoked PARP-1 hyperactivation and subsequent cell death. Moreover, KDM6B knockout triggered sustained Chk1 phosphorylation and activated a second repair machinery to fix DNA damage evading from MGMT repair. Inhibition of MGMT or checkpoint response re-sensitized KDM6B deficient cells to PARthanatos induced by alkylating agents. These findings provide new molecular insights into epigenetic regulation of PARP-1 signaling mediating DNA repair or cell death and identify KDM6B as a biomarker for prediction of cancer cell vulnerability to alkylating agent treatment.

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Effect of landscape structure on genetic structure of the Lesser horseshoe bat (Rhinolophus hipposideros) in Britanny colonies Some species are difficult to observe and others, need to be not disturbed because of their vulnerability. In response to the difficulty of studying the dispersal behaviors of these species, some areas of biology have been combined in order to access the information despite practical limitations. Here we present the combination of several methodologies from landscape ecology to non- invasive population genetics that allow us to obtain important information on Rinolophus hipposideros, a vulnerable European bat. We genotyped 18 georeferrenced colonies in Brittany (France) from droppings collected in their refuges. We used 6 microsatellite markers in order to obtain the genetic distances between them. On the other hand we calculated Euclidian distances between the refuges occupied by these colonies and some ecological distances with the Pathmatrix module of ArcGis 3.2. We tested hypothesis about the difficulty of dispersal of the species in areas without forest cover or with a low density of hedges. Thanks to the Monmonier algorithm we could infer possible genetic barriers between the colonies and we could compare their location to the presence of landscape barriers (areas with little tree cover). We detected a pattern of isolation by distance that reveals limited dispersal capacities in the species but no pattern linked to ecological distances. We found that some of the neighboring colonies with greater genetic distances between them were located in areas with low density of hedges which could suggest an impact of this landscape element in their movements. Finer studies should allow us to conclude on the need or not of forest cover in the dispersal of this species.

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Deep mining of the Sequence Read Archive reveals bipartite coronavirus genomes and inter-family Spike glycoprotein recombination Genetic variation in RNA viruses is generated by point mutation and recombination as well as reassortment in the case of viruses with segmented genomes. While point mutation concerns only few sites per genome copy, recombination and reassortment can affect large genome regions, possibly facilitating the sudden emergence of novel traits. The contribution of recombination and reassortment to genomic plasticity and their rates remain poorly understood and might be underappreciated because of the lack of a comprehensive description of the virosphere.

Here we employed a computational approach that directly queries primary sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy. By screening more than 213,000 data sets from the Sequence Read Archive repository and using two metrics that quantitatively assess assembly quality we discovered 25 novel nidoviruses from a wide range of vertebrate hosts. These include eight fish coronaviruses with bipartite genomes, a giant 36.1 kilobase coronavirus genome with a duplicated Spike glycoprotein (S) gene, and 16 additional so far undescribed vertebrate nidoviruses. Some of these novel virus genomes encode protein domains that have not been described for nidoviruses. We provide evidence for a possible inter-family homologous recombination event involving S between ancestral bipartite coronaviruses and unsegmented tobaniviruses and report a case example of an individual fish simultaneously infected with members from both virus families. Our results shed light on the evolution and genomic plasticity of coronaviruses and identify recombinants with a possibly improved ability to cross species barriers, which might elevate their pandemic potential.

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A mosaic bulk-solvent model improves density maps and the fit between model and data Bulk solvent is a major component of bio-macromolecular crystals and therefore contributes significantly to diffraction intensities. Accurate modeling of the bulk-solvent region has been recognized as important for many crystallographic calculations, from computing of R-factors and density maps to model building and refinement. Owing to its simplicity and computational and modeling power, the flat (mask-based) bulk-solvent model introduced by Jiang & Brunger (1994) is used by most modern crystallographic software packages to account for disordered solvent. In this manuscript we describe further developments of the mask-based model that improves the fit between the model and the data and aids in map interpretation. The new algorithm, here referred to as mosaic bulk-solvent model, considers solvent variation across the unit cell. The mosaic model is implemented in the computational crystallography toolbox and can be used in Phenix in most contexts where accounting for bulk-solvent is required. It has been optimized and validated using a sufficiently large subset of the Protein Data Bank entries that have crystallographic data available.

SynopsisA mosaic bulk-solvent method models disordered solvent more accurately than current flat bulk solvent model. This improves the fit between the model and the data, improves map quality and allows for the solution of problems previously inaccessible.

sentences: The post-transcriptional regulation of TFs in immature motoneurons shapes the axon-muscle connectome Temporal factors expressed sequentially in neural stem cells, such as RNA binding proteins (RBPs) or transcription factors (TFs), are key elements in the generation of neuronal diversity. The molecular mechanism underlying how the temporal identity of stem cells is decoded into their progeny to generate neuronal diversity is largely unknown. Here, we used genetic and new computational tools to study with precision the unique fates of the progeny of a stem cell producing 29 morphologically distinct leg motoneurons (MNs) in Drosophila. We identified 40 TFs expressed in this MN lineage, 15 of which are expressed in a combinatorial manner in immature MNs just before their morphological differentiation. By following TF expression patterns at an earlier developmental stages, we discovered 19 combinatorial codes of TFs that were progressively established in immature MNs as a function of their birth order. The comparison of the RNA and protein expression profiles of 6 TFs revealed that post-transcriptional regulation plays an essential role in shaping these TF codes. We found that the two known RBPs, Imp and Syp, expressed sequentially in neuronal stem cells, are upstream regulators of the TF codes. Both RBPs are key players in the construction of axon-muscle connectome through the post-transcriptional regulation of 5 of the 6 TFs examined. By deciphering the function of Imp in the immature MNs with respect to the stem cell of the same lineage, we propose a model where RBPs shape the morphological fates of MNs through post-transcriptional regulation of TF codes in immature MNs. Taken together, our study reveals that immature MNs are plastic cells that have the potential to acquire many morphological fates. The molecular basis of MN plasticity originates in the broad expression of different TF mRNA, that are post-transcriptionally shaped into TF codes by Imp and Syp, and potentially by other RBPs that remain to be discovered, to determine their morphological fates.

sentences: Music-Based Intervention Connects Auditory and Reward Systems Listening to pleasurable music is known to engage the brains reward system, but little is known about how this engagement develops over time. Here we show for the first time that brain network connectivity can change longitudinally as a result of a personalized receptive music-based intervention (MBI) in cognitively unimpaired older adults. Using a combination of whole-brain regression, seed-based connectivity analysis, and representational similarity analysis (RSA), we compared fMRI responses during a simple music listening task in older adults before and after an eight-week personalized music listening program. Participants rated self-selected and researcher-selected musical excerpts on liking and familiarity. Parametric effects of liking, familiarity, and selection showed significant activation of auditory, reward, default mode, and sensorimotor areas both pre- and post-intervention. Seed-based connectivity comparing pre- and post-intervention showed a significant increase in functional connectivity between auditory regions and the medial prefrontal cortex (mPFC), and this auditory-mPFC connectivity was modulated by participant liking and familiarity ratings. RSA showed significant representations of selection and novelty in auditory regions at both time-points, and an increase in striatal representation of musical stimuli following intervention. Taken together, results show a sensitivity of auditory, reward, default, and sensorimotor regions to individual differences in music familiarity and liking, as well as a shift in brain network dynamics following the personalized MBI. Results show how regular music listening can provide an auditory channel towards the mPFC, thus offering a potential neural mechanism supporting healthy brain aging.

sentences: Rhythmic coordination of hippocampal-prefrontal ensembles for odor-place associative memory and decision making Memory-guided decision making involves long-range coordination across sensory and cognitive brain networks, with key roles for the hippocampus and prefrontal cortex (PFC). To investigate these coordination mechanisms, we monitored activity in hippocampus (CA1), PFC, and olfactory bulb in rats performing an odor-place associative memory guided decision task on a T-maze. During odor sampling, the beta (20-30 Hz) and respiratory (7-8 Hz) rhythms (RR) were prominent across the three regions, with CA1-PFC beta and RR coherence enhanced during the odor-cued decision making period. Beta phase modulation of CA1 and PFC neuronal activity during this period was linked to accurate decisions, suggesting that this temporal modulation influences sensory-cued decision making. Single neurons and ensembles in both CA1 and PFC encoded and predicted animals upcoming choices. Our findings indicate that rhythmic coordination within the hippocampal-prefrontal network supports utilization of odor cues for memory-guided decision making.

sentences: Opposing subclasses of Drosophila ellipsoid body neurons promote and suppress sleep Recent work in Drosophila has uncovered several neighboring classes of sleep-regulatory neurons within the central complex. However, the logic of connectivity and network motifs remains limited by the incomplete examination of relevant cell types. Using a recent genetic-anatomical classification of ellipsoid body ring neurons, we conducted a thermogenetic screen to assess sleep/wake behavior and discovered two opposing populations: sleep-promoting R3m and wake-promoting R3d neurons. Activation of these neurons influences sleep duration and architecture by prolonging or shortening sleep bouts, suggesting a key role in sleep maintenance. R3m and R3d neurons are GABAergic and require GABA synthesis for their effects on sleep. Finally, we use a fluorescent reporter for putative synaptic partners to embed these neurons within the known sleep-regulatory network; R3m and R3d neurons lay downstream of wake-active Helicon cells, and R3m neurons likely inhibit R3d neurons. Together, the data presented herein suggest a neural mechanism by which previously uncharacterized circuit elements operate within the sleep homeostat to stabilize sleep-wake states.

sentences: Nano-Insecticides Against the Black Cutworm Agrotis ipsilon (Lepidoptera: Noctuidae): Toxicity, Development, Enzyme activity, and DNA Mutagenicity High-frequency doses of chemical pesticides cause environmental pollution with high pesticide residues. In addition, increasing insecticide resistance in many insect pests requires novel pest control methods. Nanotechnology could be a promising field of modern agriculture, and is receiving considerable attention in the development of novel nano-agrochemicals, such as nanoinsectticides and nanofertilizers. This study assessed the effects of the lethal and sublethal concentrations of chlorantraniliprole, thiocyclam, and their nano-forms on the development, reproductive activity, oxidative stress enzyme activity, and DNA changes at the molecular level of the polyphagous species of black cutworm Agrotis ipsilon. The results revealed that A. ipsilon larvae were more susceptible to the nano-formsthan the regular forms of both nano chlorine and sulfur within the chlorantraniliprole and thiocyclam insecticides, respectively, with higher toxicities than the regular forms (ca. 3.86, and ca.2.06-fold, respectively). Significant differences in biological parameters, including developmental time and reproductive activity (fecundity and hatchability percent) were also observed. Correspondingly, increases in oxidative stress enzyme activities were observed, as were mutagenic effects on the genomic DNA of A. ipsilon after application of the LC50 of the nano-forms of both insecticides compared to the control. The positive results obtained here have led us to apply these nano-forms indifferent insect models in additional studies.

sentences: Cloning of Nine Glucocorticoid Receptor Isoforms from the Slender African lungfish (Protopterus dolloi) We wanted to clone the glucocorticoid receptor (GR) from slender African lungfish (Protopterus dolloi) for comparison to P. dolloi MR, which we had cloned and were characterizing, as well as for comparison to the GRs from humans, elephant shark and zebrafish. However, although sequencing of the genome of the Australian lungfish (Neoceratodus forsteri), as well as, that of the West African lungfish (Protopterus annectens) were reported in the first three months of 2021, we could not retrieve a GR sequence with a BLAST search of GenBank, when we submitted our research for publication in July 2021. Moreover, we were unsuccessful in cloning the GR from slender African lungfish using a cDNA from the ovary of P. dolloi and PCR primers that had successfully cloned a GR from elephant shark, Xenopus and gar GRs. On October 21, 2021 the nucleotide sequence of West African lungfish (P. annectens) GR was deposited in GenBank. We used this GR sequence to construct PCR primers that successfully cloned the GR from the slender spotted lungfish. Here, we report the sequences of nine P. dolloi GR isoforms and explain the basis for the previous failure to clone a GR from slender African lungfish using PCR primers that cloned the GR from elephant shark, Xenopus and gar. Studies are underway to determine corticosteroid activation of these slender African lungfish GRs.

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Tau assemblies enter the cytosol of neurons in a cholesterol sensitive manner Accumulating evidence supports a prion-like mechanism in the spread of assembled tau in neurodegenerative diseases. Prion-like spread is proposed to require the transit of tau assemblies to the interior of neurons in order to seed aggregation of native, cytosolic tau. This process is poorly understood and remains largely hypothetical. Here, we develop sensitive techniques to quantify the cytosolic entry of tau in real-time. We find that tau does not promote its own entry but, rather, is wholly dependent on cellular machinery. We find that entry to the widely used reporter cell line HEK293 requires clathrin whereas entry to neurons does not. Cholesterol depletion or knockdown of cholesterol transport protein Niemann-Pick type C1 in neurons renders cells highly vulnerable to cytosolic entry and seeded aggregation. Our findings establish entry as the rate-limiting step in seeded aggregation and demonstrate that dysregulated cholesterol, a feature of several neurodegenerative diseases, potentiates tau aggregation.

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sentences: How voluntary and involuntary attention differentially shape spatial resolution Attention alters perception across the visual field. Typically, endogenous (voluntary) and exogenous (involuntary) attention similarly improve performance in many visual tasks, but they have differential effects in some tasks. Extant models of visual attention assume that the effects of these two types of attention are identical and consequently do not explain differences between them. Here, we develop a model of spatial resolution and attention that distinguishes between endogenous and exogenous attention. We focus on texture-based segmentation as a model system because it has revealed a clear dissociation between both attention types. For a texture for which performance peaks at parafoveal locations, endogenous attention improves performance across eccentricity, whereas exogenous attention improves performance where the resolution is low (peripheral locations) but impairs it where the resolution is high (foveal locations) for the scale of the texture. Our model emulates sensory encoding to segment figures from their background and predict behavioral performance. To explain attentional effects, endogenous and exogenous attention require separate operating regimes across visual detail (spatial frequency). Our model reproduces behavioral performance across several experiments and simultaneously resolves three unexplained phenomena: (1) the parafoveal advantage in segmentation, (2) the uniform improvements across eccentricity by endogenous attention and (3) the peripheral improvements and foveal impairments by exogenous attention. Overall, we unveil a computational dissociation between each attention type and provide a generalizable framework for predicting their effects on perception across the visual field.

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Fine multi-coil electronic control of transcranial magnetic stimulation: effects of stimulus orientation and intensity BackgroundTranscranial magnetic stimulation (TMS) coils allow only a slow, mechanical adjustment of the stimulating electric field (E-field) orientation in the cerebral tissue. Fast E-field control is needed to synchronize the stimulation with the ongoing brain activity. Also, empirical models that fully describe the relationship between evoked responses and the stimulus orientation and intensity are still missing.

ObjectiveWe aimed to (1) develop a TMS transducer for manipulating the E-field orientation electronically with high accuracy at the neuronally meaningful millisecond-level time scale and (2) devise and validate a physiologically based model describing the orientation selectivity of neuronal excitability.

MethodsWe designed and manufactured a two-coil TMS transducer. The coil windings were computed with a minimum-energy optimization procedure, and the transducer was controlled with our custom-made electronics. The electronic E-field control was verified with a TMS characterizer. The motor evoked potential amplitude and latency of a hand muscle were mapped in 3{degrees} steps of the stimulus orientation in 16 healthy subjects for three stimulation intensities. We fitted a logistic model to the motor response amplitude.

ResultsThe two-coil TMS transducer allows one to manipulate the pulse orientation accurately without manual coil movement. The motor response amplitude followed a logistic function of the stimulus orientation; this dependency was strongly affected by the stimulus intensity.

ConclusionThe developed electronic control of the E-field orientation allows exploring new stimulation paradigms and probing neuronal mechanisms. The presented model helps to disentangle the neuronal mechanisms of brain function and guide future non-invasive stimulation protocols.

sentences: A method for simultaneously monitoring phloem and xylem reconnections in grafted watermelon seedlings Grafting is an effective way to increase watermelon tolerance to biotic and abiotic stresses. However, the survival of grafted seedlings largely depends on successful graft formation. Therefore, understanding the graft formation process, particularly the vascular reconnection process is of critical importance. This study found that lignin in watermelon stem shows strong auto-fluorescence under blue-light excitation which makes blue-light excited fluorescent tracers (FTs) such as 5(6)-carboxy fluorescein diacetate (CFDA) become unsuitable for assaying vascular connectivity in watermelon. In contrast, UV-light excited esculin and red-light excited acid fuchsin were proved to be efficient FTs for monitoring the phloem and xylem connectivity, respectively, in self-grafted watermelon. Furthermore, a combined application of esculin to the scion cotyledon and acid fuchsin to the rootstock root enabled simultaneous monitoring of the phloem and xylem connectivity in individual self-grafted watermelon seedlings. In addition, this method is also applicable in investigating the phloem and xylem reconnections in self-grafted melon and cucumber, and heterograft of watermelon, melon and cucumber onto pumpkin rootstock. Based on this established method, we found that phloem and xylem reconnections are not timely separated in self-grafted watermelon. Furthermore, low temperature and removal of the rootstock cotyledons both delayed the vascular reconnection process in watermelon. In conclusion, this new method provides a convenient, accurate and rapid way to analyze the vascular connectivity not only in watermelon, but also in other cucurbit crops.

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Mutation rate of SARS-CoV-2 and emergence of mutators during experimental evolution Background and objectivesTo understand how organisms evolve, it is fundamental to study how mutations emerge and establish. Here, we estimated the rate of mutation accumulation of SARS-CoV-2 in vitro and investigated the repeatability of its evolution when facing a new cell type but no immune or drug pressures.

MethodologyWe performed experimental evolution with two strains of SARS-CoV-2, one carrying the originally described spike protein (CoV-2-D) and another carrying the D614G mutation that has spread worldwide (CoV-2-G). After 15 passages in Vero cells and whole genome sequencing, we characterized the spectrum and rate of the emerging mutations and looked for evidences of selection across the genomes of both strains.

ResultsFrom the mutations accumulated, and excluding the genes with signals of selection, we estimate a spontaneous mutation rate of 1.25x10-6 nt-1 per infection cycle for both lineages of SARS-CoV-2. We further show that mutation accumulation is heterogeneous along the genome, with the spike gene accumulating mutations at rate five-fold higher than the genomic average. We also observe the emergence of mutators in the CoV-2-G background, likely linked to mutations in the RNA-dependent RNA polymerase and/or in the error-correcting exonuclease protein.

Conclusions and implicationsThese results provide valuable information on how spontaneous mutations emerge in SARS-CoV-2 and on how selection can shape its genome towards adaptation to new environments.

Lay summaryMutation is the ultimate source of variation. We estimated how the SARS-COV-2 virus--cause of the COVID-19 pandemic--mutates. Upon infecting cells, its genome can change at a rate of 0.04 per replication. We also find that this rate can change and that its spike protein can adapt, even within few replications.

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Enrichment analysis on regulatory subspaces: a novel direction for the superior description of cellular responses to SARS-CoV-2 StatementThe enrichment analysis of discriminative cell transcriptional responses to SARS-CoV-2 infection using biclustering produces a broader set of superiorly enriched GO terms and KEGG pathways against alternative state-of-the-art machine learning approaches, unraveling novel knowledge.

Motivation and methodsThe comprehensive understanding of the impacts of the SARS-CoV-2 virus on infected cells is still incomplete. This work identifies and analyses the main cell regulatory processes affected and induced by SARS-CoV-2, using transcriptomic data from several infectable cell lines available in public databases and in vivo samples. We propose a new class of statistical models to handle three major challenges, namely the scarcity of observations, the high dimensionality of the data, and the complexity of the interactions between genes. Additionally, we analyse the function of these genes and their interactions within cells to compare them to ones affected by IAV (H1N1), RSV and HPIV3 in the target cell lines.

ResultsGathered results show that, although clustering and predictive algorithms aid classic functional enrichment analysis, recent pattern-based biclustering algorithms significantly improve the number and quality of the detected biological processes. Additionally, a comparative analysis of these processes is performed to identify potential pathophysiological characteristics of COVID-19. These are further compared to those identified by other authors for the same virus as well as related ones such as SARS-CoV-1. This approach is particularly relevant due to a lack of other works utilizing more complex machine learning tools within this context.

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A Unit Pipe Pneumatic model to simulate gas kinetics during measurements of embolism in excised angiosperm xylem O_LIThe Pneumatic method has been introduced to quantify embolism resistance in plant xylem of various organs. Despite striking similarity in vulnerability curves between the Pneumatic and hydraulic methods, a modeling approach is highly needed to demonstrate that xylem embolism resistance can be accurately quantified based on gas diffusion kinetics.
C_LIO_LIA Unit Pipe Pneumatic (UPPn) model was developed to estimate gas diffusion from intact conduits, which were axially interconnected by interconduit pit membranes. The physical laws used included Ficks law for diffusion, Henrys law for gas concentration partitioning between liquid and gas phases at equilibrium, and the ideal gas law.
C_LIO_LIThe UPPn model showed that 91% of the extracted gas came from the first two series of embolized, intact conduits, and only 9% from the aqueous phase after 15 s of simulation. Embolism resistance measured with a Pneumatic apparatus was systematically overestimated by 2 to 17%, corresponding to a typical measuring error of 0.11 MPa for P50 (the water potential equivalent to 50% of the maximum amount of gas extracted).
C_LIO_LIBecause results from the UPPn model are supported by experimental evidence, there is a good theoretical and experimental basis for applying the pneumatic method to research on embolism resistance of angiosperms.
C_LI

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Neuronal mechanism of a BK channelopathy in absence epilepsy and movement disorders A growing number of gain-of-function (GOF) BK channelopathy have been identified in patients with epilepsy and paroxysmal movement disorders. Nevertheless, the underlying pathophysiology and corresponding therapeutics remain obscure. Here we utilized a knock-in mouse model carrying human BK-D434G channelopathy to investigate the neuronal mechanism of BK GOF in the pathogenesis of epilepsy and movement disorders. We found that the BK-D434G mice manifest the clinical features of absence epilepsy and exhibit severe motor deficits. BK-D434G mutation causes hyperexcitability of cortical pyramidal neurons and cerebellar Purkinje cells, which contributes to the pathogenesis of absence seizures and the motor defects, respectively. A BK channel blocker paxilline potently suppresses BK-D434G-induced hyperexcitability and effectively mitigates absence seizures in mice. Our study thus uncovered a neuronal mechanism of BK GOF in absence epilepsy and provided the evidence that BK inhibition is a promising therapeutic strategy to mitigate BK GOF-induced neurological disorders.

SignificanceDysfunction of BK channels or BK channelopathy has been increasingly implicated in diverse neurological disorders including epilepsy, movement, cognitive and neurodevelopmental disorders. However, precision medicine to treat BK channelopathy is lacking. Here we characterized a mouse model carrying a gain-of-function BK channelopathy D434G from a large family of patients with absence epilepsy and involuntary movement disorders. The BK-D434G mice resemble the clinical manifestations of absence seizures and exhibit severe motor defects. The hyperexcitability in BK-D434G cortical neurons and cerebellar Purkinje cells underscores the neuronal mechanism of BK gain-of-function induced absence epilepsy and movement disorders. The effectiveness of a BK channel blocker on preventing absence seizures suggests that BK inhibition is a promising strategy to treat gain-of-function BK channelopathy.

sentences: Establishment of Strigolactone-Producing Bacterium-Yeast Consortium Strigolactones (SLs) are a class of phytohormones playing diverse roles in plant growth and development, yet the limited access to SLs is largely impeding SL-based foundational investigations and applications. Here, we developed Escherichia coli-Saccharomyces cerevisiae consortia to establish a microbial biosynthetic platform for the synthesis of various SLs, including carlactone, carlactonic acid, 5-deoxystrigol (5DS, 6.65{+/-}1.71 {micro}g/L), 4-deoxyorobanchol (4DO, 3.46{+/-}0.28 {micro}g/L), and orobanchol (OB, 19.36{+/-}5.20 {micro}g/L). The SL-producing platform enabled us to conduct functional identification of CYP722Cs from various plants as either OB or 5DS synthase. It also allowed us to quantitatively compare known variants of plant SL biosynthetic enzymes in the microbial system. The titer of 5DS was further enhanced through pathway engineering to 47.3 {micro}g/L. This work provides a unique platform for investigating SL biosynthesis and evolution and lays the foundation for developing SL microbial production process.

sentences: Binding Free energy Decomposition and Multiple Unbinding Paths of Buried Ligands in a PreQ1 Riboswitch Riboswitches are naturally occurring RNA elements that control bacterial gene expression by binding to specific small molecules. They serve as important models for RNA-small molecule recognition and have also become a novel class of targets for developing antibiotics. Here, we carried out conventional and enhanced-sampling molecular dynamics (MD) simulations, totaling 141.5 s, to characterize the determinants of binding free energies and unbinding paths for the cognate and synthetic ligands of a PreQ1 riboswitch. Binding free energy analysis showed that two triplets of nucleotides U6-C15-A29 and G5-G11-C16, contribute the most to the binding of the cognate ligands, by hydrogen bonding and by base stacking, respectively. Mg2+ ions are essential in stabilizing the binding pocket. For the synthetic ligands, the hydrogen-bonding contributions of the U6-C15-A29 triplet are significantly compromised, and the bound state resembles the apo state in several respects, including the disengagement of the C15-A14-A13 and A32-G33 base stacks. The bulkier synthetic ligands lead to significantly loosening of the binding pocket, including extrusion of the C15 nucleobase and a widening of the C15-C30 groove. Enhanced-sampling simulations further revealed that the cognate and synthetic ligands unbind in almost opposite directions. Our work offers new insight for designing riboswitch ligands.

sentences: A pipeline for assessing the quality of images and metadata from crowd-sourced databases. Crowd-sourced biodiversity databases provide easy access to data and images for ecological education and research. One concern with using publicly sourced databases; however, is the quality of their images, taxonomic descriptions, and geographical metadata. To address this concern and allow researchers and educators to make informed decisions about using crowd-sourced data, I developed a suite of pipelines to evaluate taxonomic consistency, how well geo-tagging fits known distributions, and image quality of crowd-sourced biodiversity data of the order Araneae (spiders) from iNaturalist. This pipeline allows users to analyze multiple images from iNaturalist and their associated metadata; to determine the level of taxonomic identification (family, genera, species) for each occurrence; whether the taxonomy label for an image matches accepted nesting of families, genera, and species; and whether geo-tags match the distribution of the taxon described using occurrence data from the Global Biodiversity Infrastructure Facility (GBIF) as a reference. Additionally, I assessed image quality with the MatLab algorithm, BRISQUE. I used entries from the order Araneae (spiders) as a case study. At the time of my analyses (July 2021), I found that iNaturalist contained at least one observation for 124 of the 129 families of Araneae, and 115 families had three or more unique observations, with relatively similar quality of metadata and image quality across families. Taxonomic consistency was similar for observations identified at the genus and species level, but lower in observations with only family level identification. Observations with species level identifications had higher precision for geo-tags compared to those identified to the family or genus level and the highest image quality according to the BRISQUE scores. Overall, the results suggest that iNaturalist can provide large metadata and images sets for research. Given the inevitability of some low-quality observations, this pipeline provides a valuable resource for researchers and educators to evaluate the quality of iNaturalist and other crowd-sourced data.

sentences: Temporary prey storage along swarm columns of army ants: an adaptive strategy for successful raiding? While pillaging the brood of other ant colonies, Eciton army ants accumulate prey in piles, or caches, along their foraging trails. Widely documented, these structures have historically been considered as byproducts of heavy traffic or aborted relocations of the ants temporary nest, or bivouac. However, we recently observed that caches of the hook-jawed army ant, Eciton hamatum, appeared independently from heavy traffic or bivouac relocations. In addition, the flow of prey through caches varied based on the quantity of prey items workers transported. As this suggested a potential adaptive function, we developed agent-based simulations to compare raids of caching and non-caching virtual army ants. We found that caches increased the amount of prey that relatively low numbers of raiders were able to retrieve. However, this advantage became less conspicuous - and generally disappeared - as the number of raiders increased. Based on these results, we hypothesize that caches maximize the amount of prey that limited amounts of raiders can retrieve, especially as prey colonies coordinately evacuate their brood. In principle, caches also allow workers to safely collect multiple prey items and efficiently transport them to the bivouac. Further field observations are needed to test this and other hypotheses emerging from our study.

sentences: Can fMRI reveal the representation of syntactic structure in the brain? While studying semantics in the brain, neuroscientists use two approaches. One is to identify areas that are correlated with semantic processing load. Another is to find areas that are predicted by the semantic representation of the stimulus words. However, in the domain of syntax, most studies have focused only on identifying areas correlated with syntactic processing load. One possible reason for this discrepancy is that representing syntactic structure in an embedding space such that it can be used to model brain activity is a non-trivial computational problem. Another possible reason is that it is unclear if the low signal-to-noise ratio of neuroimaging tools such as functional Magnetic Resonance Imaging (fMRI) can allow us to reveal correlates of complex (and perhaps subtle) syntactic representations. In this study, we propose novel multi-dimensional features that encode information about the syntactic structure of sentences. Using these features and fMRI recordings of participants reading a natural text, we model the brain representation of syntax. First, we find that our syntactic structure-based features explain additional variance in the brain activity of various parts of the language system, even after controlling for complexity metrics that capture processing load. At the same time, we see that regions well-predicted by syntactic features are distributed in the language system and are not distinguishable from those processing semantics.

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Efficient Coding in the Economics of Human Brain Connectomics In systems neuroscience, most models posit that brain regions communicate information under constraints of efficiency. Yet, evidence for efficient communication in structural brain networks characterized by hierarchical organization and highly connected hubs remains sparse. The principle of efficient coding proposes that the brain transmits maximal information in a metabolically economical or compressed form to improve future behavior. To determine how structural connectivity supports efficient coding, we develop a theory specifying minimum rates of message transmission between brain regions to achieve an expected fidelity, and we test five predictions from the theory based on random walk communication dynamics. In doing so, we introduce the metric of compression efficiency, which quantifies the trade-off between lossy compression and transmission fidelity in structural networks. In a large sample of youth (n = 1,042; age 8-23 years), we analyze structural networks derived from diffusion weighted imaging and metabolic expenditure operationalized using cerebral blood flow. We show that structural networks strike compression efficiency trade-offs consistent with theoretical predictions. We find that compression efficiency prioritizes fidelity with development, heightens when metabolic resources and myelination guide communication, explains advantages of hierarchical organization, links higher input fidelity to disproportionate areal expansion, and shows that hubs integrate information by lossy compression. Lastly, compression efficiency is predictive of behavior--beyond the conventional network efficiency metric--for cognitive domains including executive function, memory, complex reasoning, and social cognition. Our findings elucidate how macroscale connectivity supports efficient coding, and serve to foreground communication processes that utilize random walk dynamics constrained by network connectivity.

Author SummaryMacroscale communication between interconnected brain regions underpins most aspects of brain function and incurs substantial metabolic cost. Understanding efficient and behaviorally meaningful information transmission dependent on structural connectivity has remained challenging. We validate a model of communication dynamics atop the macroscale human structural connectome, finding that structural networks support dynamics that strike a balance between information transmission fidelity and lossy compression. Notably, this balance is predictive of behavior and explanatory of biology. In addition to challenging and reformulating the currently held view that communication occurs by routing dynamics along metabolically efficient direct anatomical pathways, our results suggest that connectome architecture and behavioral demands yield communication dynamics that accord to neurobiological and information theoretical principles of efficient coding and lossy compression.

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TOWARDS QUANTITATIVE FORECASTING OF SPECIES YIELDS WITH INCOMPLETE INFORMATION ON MODEL PARAMETERS Predicting both the absolute and the relative abundance of species in a spatial patch is of paramount interest in areas like, agriculture, ecology and environmental science.

The linear Lotka-Volterra generalized equations (LLVGE) serve for describing the dynamics of communities of species connected by negative as well as positive interspecific interactions.

Here we particularize these LLVGE to the case of single trophic ecological communities, like mixtures of plants, with S >2 species. Thus, by estimating the LLVGE parameters from the yields in monoculture and biculture experiments, the LLVGE are able to produce decently accurate predictions for species yields.

However, a common situation we face is that we dont know all the parameters appearing in the LLVGE. Indeed, for large values of S, only a fraction of the experiments necessary for estimating the model parameters is commonly carried out. We then analyze which quantitative predictions are possible with an incomplete knowledge of the parameters.

sentences: Experimental evolution of extremophile levels of radiation resistance in Escherichia coli Recent human development of high-level sources of ionizing radiation (IR) prompts a corresponding need to understand the effects of IR on living systems. One approach has focused on the capacity of some organisms to survive astonishing levels of IR exposure. Using experimental evolution, we have generated populations of Escherichia coli with IR resistance comparable to the extremophile Deinococcus radiodurans. Every aspect of cell physiology is affected. Cellular isolates exhibit approximately 1,000 base pair changes plus major genomic and proteomic alterations. The IR resistance phenotype is stable without selection for at least 100 generations. Defined and probable contributions include alterations in cellular systems involved in DNA repair, amelioration of reactive oxygen species, Fe metabolism and repair of iron-sulfur centers, DNA packaging, and intermediary metabolism. A path to new mechanistic discoveries, exemplified by an exploration of rssB function, is evident. Most important, there is no single molecular mechanism underlying extreme IR resistance.

sentences: New intranasal and injectable gene therapy for healthy life extension As the global elderly population grows, it is socioeconomically and medically critical to have diverse and effective means of mitigating the impact of aging on human health. Previous studies showed that adenovirus-associated virus (AAV) vector induced overexpression of certain proteins can suppress or reverse the effects of aging in animal models. Here, we sought to determine whether the high-capacity cytomegalovirus vector can be an effective and safe gene delivery method for two such-protective factors: telomerase reverse transcriptase (TERT) and follistatin (FST). We found that the mouse cytomegalovirus (MCMV) carrying exogenous TERT or FST (MCMVTERT or MCMVFST) extended median lifespan by 41.4% and 32.5%, respectively. This is the first report of CMV being used successfully as both an intranasal and injectable gene therapy system to extend longevity. Treatment significantly improved glucose tolerance, physical performance, and prevented loss of body mass and alopecia. Telomere shortening seen with aging was ameliorated by TERT, and mitochondrial structure deterioration was halted in both treatments. Intranasal and injectable preparations performed equally well in safely and efficiently delivering gene therapy to multiple organs, with long-lasting benefits and without carcinogenicity or unwanted side effects. Translating this research to humans could have significant benefits associated with increased health span.

sentences: Deconvoluting signals downstream of growth and immune receptor kinases by phosphocodes of the BSU1 family phosphatases Hundreds of leucine-rich repeat receptor kinases (LRR-RKs) have evolved to control diverse processes of growth, development, and immunity in plants; the mechanisms that link LRR-RKs to distinct cellular responses are not understood. Here we show that two LRR-RKs, the brassinosteroid hormone receptor BRI1 (BRASSINOSTEROID INSENSITIVE 1) and the flagellin receptor FLS2 (FLAGELLIN SENSING 2), regulate downstream glycogen synthase kinase 3 (GSK3) and mitogen-activated protein (MAP) kinases, respectively, through phosphocoding of the BRI1-SUPPRESSOR1 (BSU1) phosphatase. BSU1 was previously identified as a component that inactivates GSK3s in the BRI1 pathway. We found surprisingly that loss of the BSU1 family phosphatases activates effector-triggered immunity (ETI) and impairs flagellin-triggered MAP kinase activation and immunity. The flagellin-activated BOTRYTIS-INDUCED KINASE 1 (BIK1) phosphorylates BSU1 at serine-251. Mutation of serine-251 reduces the ability of BSU1 to mediate flagellin-induced MAP kinase activation and immunity, but not its abilities to suppress ETI and interact with GSK3, which is enhanced through the phosphorylation of BSU1 at serine-764 upon brassinosteroid signaling. These results demonstrate that BSU1 plays an essential role in immunity and transduces brassinosteroid-BRI1 and flagellin-FLS2 signals using different phosphorylation sites. Our study illustrates that phosphocoding in shared downstream components provides signaling specificities for diverse plant receptor kinases.

sentences: Coevolution of the Tlx homeobox gene with medusa development (Cnidaria: Medusozoa) The jellyfish, or medusa, is a life cycle stage characteristic of the cnidarian subphylum Medusozoa. By contrast, the other cnidarian subphyla Anthozoa and Endocnidozoa lack a medusa stage. Of the medusozoan classes, Hydrozoa is the most diverse in terms of species number and life cycle variation. A notable pattern in hydrozoan evolution is that the medusa stage has been lost or reduced several times independently. Although this loss of the jellyfish stage is thought to be due to heterochrony, the precise developmental mechanisms underlying this complex pattern of medusa evolution are unknown. We found that the presence of the homeobox gene Tlx in cnidarian genomes is correlated with those medusozoans that have a medusa stage as part of their life cycle. Although Tlx is conserved in Bilateria and Cnidaria, it is missing in the genomes of anthozoans, endocnidozoans, and those hydrozoans that have lost the medusa stage. Selection analyses of Tlx across medusozoans revealed that hydrozoans undergo relatively relaxed selection compared to the other medusozoan classes, which may in part explain the pattern of multiple medusa losses. Differential expression analyses on three distantly related medusozoan representatives indicate an upregulation of Tlx during medusa development. In addition, Tlx expression is spatially restricted to regions of active development in medusae of the hydrozoan Podocoryna carnea. Our results suggest that Tlx plays a key role in medusa development and that the loss of this gene is likely linked to the repeated loss of the medusa life cycle stage.

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A novel innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins Sensing of pathogens by ubiquitination is critical for maintaining cytosolic sanctity. However, universal ubiquitination targets on bacteria, especially of proteinaceous origin, remain unidentified. Here, we unveil a novel strategy, involving recognition of degron-like motifs for identification of first protein-based ubiquitination substrates on phylogenetically distinct bacteria. Such motifs can form a new class of intra-cytosolic pathogen associated molecular patterns (PAMPs) as their incorporation enables identification of non-ubiquitin targets by host Ub-ligases. We find SCFFBW7 E3-ligase, supported by the regulatory kinase, GSK3{beta}, is crucial for effective pathogen detection and clearance. This may explain the enhanced risk of infections in Chronic Lymphocytic Leukaemia patients bearing FBXW7 mutations. We conclude that exploitation of such ubiquitous pathogen sensing strategy allows conservation of cellular resources and boost anti-microbial immunity.

One Sentence SummaryUbiquitination of bacterial surface proteins fosters sensing and clearance of diverse pathogens

sentences: Robust IgM responses following vaccination are associated with prevention of Mycobacterium tuberculosis infection in macaques Development of an effective tuberculosis (TB) vaccine has suffered from an incomplete understanding of the correlates of protection against Mycobacterium tuberculosis (Mtb). However, recent work has shown that compared to standard intradermal Bacille Calmette-Guerin (BCG) vaccination, intravenous (IV) BCG vaccination provides nearly complete protection against TB in rhesus macaques. While studies have focused on cellular immunity in this setting, the antibody response elicited by IV BCG vaccination remains incompletely defined. Using an agnostic antibody profiling approach, here we show that IV BCG drives superior antibody responses in the plasma and the bronchoalveolar lavage fluid (BAL). While IV BCG immunization resulted in the expansion of a robust IgM, IgG, IgA, Fc-receptor binding antibodies, and antibody effector functions in the BAL, IgM titers were among the strongest markers of reduced bacterial burden in the plasma and BAL of BCG immunized animals. Moreover, IgM immunity was also enriched among animals receiving protective vaccination with an attenuated Mtb strain. Finally, a LAM-specific IgM monoclonal antibody reduced Mtb survival in vitro. Collectively, these data highlight the potential importance of IgM responses as a marker and as a functional mediator of protection against TB.

sentences: Endogenous memory reactivation during sleep in humans is clocked by slow oscillation spindle complexes. Sleep is thought to support memory consolidation via reactivation of prior experiences, with particular electrophysiological sleep signatures (slow oscillations (SOs) and sleep spindles) gating the information flow between relevant brain areas. However, empirical evidence for a role of endogenous memory reactivation (i.e., without experimentally delivered memory cues) for consolidation in humans is lacking. Here, we devised a paradigm in which participants acquired associative memories before taking a nap. Multivariate decoding was then used to capture endogenous memory reactivation during non-rapid eye movement (NREM) sleep in surface EEG recordings. Our results reveal reactivation of learning material during SO-spindle complexes, with the precision of SO-spindle coupling predicting reactivation strength. Critically, reactivation strength (i.e. classifier evidence in favor of the previously studied stimulus category) in turn predicts the level of consolidation across participants. These results elucidate the memory function of sleep in humans and emphasize the importance of SOs and spindles in clocking endogenous consolidation processes.

sentences: No evidence for changes in GABA concentration, functional connectivity, or working memory following continuous theta burst stimulation over dorsolateral prefrontal cortex Continuous theta burst stimulation (cTBS) is thought to reduce cortical excitability and modulate functional connectivity, possibly by altering cortical inhibition at the site of stimulation. However, most evidence comes from the motor cortex and it remains unclear whether similar effects occur following stimulation over other brain regions. We assessed whether cTBS over left dorsolateral prefrontal cortex altered gamma aminobutyric acid (GABA) concentration, functional connectivity and brain dynamics at rest, and brain activation and memory performance during a working memory task. Seventeen healthy individuals participated in a randomised, sham-controlled, cross-over experiment. Before and after either real or sham cTBS, magnetic resonance spectroscopy was obtained at rest to measure GABA concentrations. Functional magnetic resonance imaging (fMRI) was also recorded at rest and during an n-back working memory task to measure functional connectivity, regional brain activity (low-frequency fluctuations), and task-related patterns of brain activity. We could not find evidence for changes in GABA concentration (P=0.66, Bayes factor [BF10]=0.07), resting-state functional connectivity (P(FWE)>0.05), resting-state low-frequency fluctuations (P=0.88, BF10=0.04), blood-oxygen level dependent activity during the n-back task (P(FWE) >0.05), or working memory performance (P=0.13, BF10=0.05) following real or sham cTBS. Our findings add to a growing body of literature suggesting the effects of cTBS are highly variable between individuals and question the notion that cTBS is a universal  inhibitory paradigm.

sentences: DNA Self-Assembled Plasmonic Nanodiamonds for Biological Sensing Nitrogen-vacancy (NV) centers in diamonds are promising solid-state quantum emitters for developing superior biological imaging modalities. They possess desired bio-compatibility, photostability and electronic spin-related photophysical properties that are optically accessible at room temperature. Yet, bare nanodiamond-based imaging modalities are limited by the brightness and temporal resolution due to the intrinsically long lifetime of NV centers. Moreover, it remains a technological challenge using top-down fabrication to create freestanding hybrid nanodiamond imaging probes with enhanced performance. In this study, we leverage the bottom-up DNA self-assembly to develop a hybrid plasmonic nanodiamond construct, which we coin as the plasmon-enhanced nanodiamond (PEN), for biological imaging. The PEN nano-assembly features a closed plasmonic nanocavity that completely encapsulates a single nanodiamond, thus enabling the largest possible plasmonic enhancement to accelerate the emission dynamics of NV centers. Creation of the PEN nano-assembly is size-independent, so is its broadband scattering spectrum that is optimally overlapped with the emission spectrum of NV centers. Study of the structure-property correlation reveals that the optimal condition for emission dynamics modification is causally linked to that for a plasmonic nanocavity. The cellular internalization and cytotoxicity studies further confirm the delivery efficiency and biological safety of PEN nano-assemblies. Collectively, the PEN nano-assembly provides a promising approach for manipulating photophysical properties of solid-state quantum emitters and could serve as a versatile platform to uncover non-trivial quantum effects in biological systems.

sentences: Basal Ganglia Responses to Electrical Stimulation of the Posterior Hypothalamic Nucleus Electrical or chemical stimulation of the posterior hypothalamic nucleus (PH) elicits highly adaptive locomotion, demonstrating both evidence of flexibility and variety in exhibited motor behaviours. However, the neural substrates of PH stimulation elicited behavioural changes are poorly understood. The basal ganglia are postulated to be critically involved in the process of action selection in conjunction with thalamo-cortical systems. The present study examines changes in basal ganglia activities in response to the high-frequency stimulation of the PH. Under urethane anaesthesia, ensemble and single-unit recordings were obtained from the striatum (STR), globus pallidus externa (GPe), entopeduncular nucleus (EP), subthalamic nucleus (STN) and the substantia nigra pars reticulata (SNr). Upon PH stimulation, increases in firing rates were observed in the STR, GPe, and STN, a decrease was observed in the SNr and no changes were seen in the EP. The increase in spike rate in the STR and GPe was dependent on the stimulation intensity but not duration. Despite the differences in the direction of firing changes during PH stimulation, all examined areas including those not part of the basal ganglia demonstrated an elevated spiking rate upon stimulus train termination. Taking into account the known anatomical connections between the PH and the basal ganglia, it is hypothesized responses seen during PH stimulus trains are mediated through thalamic and cortical relays whereas the overall post-stimulus excitatory response is related to the impact of the PH on brainstem arousal systems.

sentences: MarcoPolo: a clustering-free approach to the exploration of differentially expressed genes along with group information in single-cell RNA-seq data A common approach to analyzing single-cell RNA-sequencing data is to cluster cells first and then identify differentially expressed genes based on the clustering result. However, clustering has an innate uncertainty and can be imperfect, undermining the reliability of differential expression analysis results. To overcome this challenge, we present MarcoPolo, a clustering-free approach to exploring differentially expressed genes. To find informative genes without clustering, MarcoPolo exploits the bimodality of gene expression to learn the group information of the cells with respect to the expression level directly from given data. Using simulations and real data analyses, we showed that our method puts biologically informative genes at high ranks more robustly than other existing methods. As our method provides information on how cells can be grouped for each gene, it can help identify cell types that are not separated well in the standard clustering process. Our method can also be used as a feature selection method to improve the robustness of the dimension reduction against changes in the parameters involved in the process.

sentences: Vacuole-specific lipid release for tracking intracellular lipid metabolism and transport in Saccharomyces cerevisiae Lipid metabolism is spatiotemporally regulated within cells, yet intervention into lipid functions at subcellular resolution remains difficult. Here we report a method that enables site-specific release of sphingolipids and cholesterol inside the vacuole in Saccharomyces cerevisiae. Using this approach, we monitored real-time sphingolipid metabolic flux out of the vacuole by mass spectrometry and found that the ER-vacuole tethering protein Mdm1 facilitated the metabolism of sphingoid bases into ceramides. In addition, we showed that cholesterol, once delivered into yeast using our method, could restore cell proliferation induced by ergosterol deprivation, overcoming the previously described sterol-uptake barrier under aerobic conditions. Together, these data define a new way to study intracellular lipid metabolism and transport from the vacuole in yeast.

sentences: Re-evaluating the role of nucleosomal bivalency in early development Nucleosomes, composed of DNA and histone proteins, represent the fundamental repeating unit of the eukaryotic genome1; posttranslational modifications of these histone proteins influence the activity of the associated genomic regions to regulate cell identity2-4. Traditionally, trimethylation of histone 3-lysine 4 (H3K4me3) is associated with transcriptional initiation5-10, whereas trimethylation of H3K27 (H3K27me3) is considered transcriptionally repressive11-15. The apparent juxtaposition of these opposing marks, termed "bivalent domains"16-18, was proposed to specifically demarcate of small set transcriptionally-poised lineage-commitment genes that resolve to one constituent modification through differentiation, thereby determining transcriptional status19-22. Since then, many thousands of studies have canonized the bivalency model as a chromatin hallmark of development in many cell types. However, these conclusions are largely based on chromatin immunoprecipitations (ChIP) with significant methodological problems hampering their interpretation. Absent direct quantitative measurements, it has been difficult to evaluate the strength of the bivalency model. Here, we present reICeChIP, a calibrated sequential ChIP method to quantitatively measure H3K4me3/H3K27me3 bivalency genome-wide, addressing the limitations of prior measurements. With reICeChIP, we profile bivalency through the differentiation paradigm that first established this model16,18: from naive mouse embryonic stem cells (mESCs) into neuronal progenitor cells (NPCs). Our results cast doubt on every aspect of the bivalency model; in this context, we find that bivalency is widespread, does not resolve with differentiation, and is neither sensitive nor specific for identifying poised developmental genes or gene expression status more broadly. Our findings caution against interpreting bivalent domains as specific markers of developmentally poised genes.

sentences: Barth syndrome cellular models have dysregulated respiratory chain complex I and mitochondrial quality control due to abnormal cardiolipin Barth syndrome (BTHS) is an X-linked genetic condition caused by defects in TAZ, which encodes a transacylase involved in the remodeling of the inner mitochondrial membrane phospholipid, cardiolipin (CL). As such, CL has been implicated in numerous mitochondrial functions, and the role of defective CL in the clinical pathology of BTHS is under intense investigation. We used untargeted proteomics, shotgun lipidomics, gene expression analysis, and targeted metabolomics to identify novel areas of mitochondrial dysfunction in a new model of TAZ deficiency in HEK293 cells. Functional annotation analysis of proteomics data revealed abnormal regulation of mitochondrial respiratory chain complex I (CI), driven by the reduced abundance of 6 CI associated proteins in TAZ-deficient HEK293 cells: MT-ND3, NDUFA5, NDUFAB1, NDUFB2, NDUFB4, and NDUFAF1. This resulted in reduced assembly and function of CI in TAZ-deficient HEK293 cells as well as BTHS patient derived lymphoblast cells. We also identified increased abundance of PARL, a rhomboid protein involved in the regulation of mitophagy and apoptosis, and abnormal downstream processing of PGAM5, another mediator of mitochondrial quality control, in TAZ-deficient cells. Lastly, we modulated CL via the phospholipase inhibitor bromoenol lactone and the CL targeted SS-peptide, SS-31, and showed that each is able to remediate abnormalities in CI abundance as well as PGAM5 processing. Thus, mitochondrial respiratory chain CI and PARL/PGAM5 regulated mitochondrial quality control, both of whose functions localize to the inner mitochondrial membrane, are dysregulated due to TAZ deficiency and are partially remediated via modulation of CL.

sentences: Role of Lateral Inhibition on Visual Number Sense Lateral inhibition is a basic principle of information processing and widely exists in the human and animal nervous systems. Lateral inhibition is also involved in processing visual information because it travels through the retina, primary visual cortex, and visual nervous system. This finding suggests that lateral inhibition is associated with visual number sense in humans and animals. Here, we show a number-sensing neural network model based on lateral inhibition. The model can reproduce the size and distance effects of the output response of human and animal number-sensing neurons when the network connection weights are set randomly without adjustment. The number sense of the model disappears when lateral inhibition is removed. Our study shows that the first effect of lateral inhibition is to strengthen the linear correlation between the total response intensity of the input layer and the number of objects. The second one is to allow the output cells to prefer different numbers. Results indicate that lateral inhibition plays an indispensable role in untrained spontaneous number sense.

sentences: Effect of reduced graphene oxide (rGO) compaction degree and concentration on rGO-polymer composites printability and cell interactions Graphene derivatives combined with polymers have attracted enormous attention for bone tissue engineering applications. Among others, reduced graphene oxide (rGO) is one of the preferred graphene-based fillers for the preparation of composites via melt compounding, and their further processing into 3D scaffolds, due to its established large-scale production method, thermal stability, and electrical conductivity. In this study, rGO (low bulk density 10g/L) was compacted by densification using a solvent (either acetone or water) prior to melt compounding, to simplify its handling and dosing into a twin-screw extrusion system. The effects of rGO bulk density (medium and high), densification solvent, and rGO concentration (3, 10 and 15% in weight) on rGO dispersion within the composite, electrical conductivity, printability and cell-material interactions were studied. High bulk density rGO (90 g/L) occupied a low volume fraction within polymer composites, offering poor electrical properties but a reproducible printability up to 15 wt% rGO. On the other hand, the volume fraction within the composites of medium bulk density rGO (50 g/L) was higher for a given concentration, enhancing rGO particle interactions and leading to enhanced electrical conductivity, but compromising the printability window. For a given bulk density (50 g/L), rGO densified in water was more compacted and offered poorer dispersability within the polymer than rGO densified in acetone, and resulted in scaffolds with poor layer bonding or even lack of printability at high rGO percentages. A balance in printability and electrical properties was obtained for composites with medium bulk density rGO densified in acetone. Here, increasing rGO concentration led to more hydrophilic composites with a noticeable increase in protein adsorption. Moreover, scaffolds prepared with such composites presented antimicrobial properties even at low rGO contents (3 wt%). In addition, the viability and proliferation of human mesenchymal stromal cells (hMSCs) was maintained on scaffolds with up to 15% rGO and with enhanced osteogenic differentiation on 3% rGO scaffolds.

sentences: Acceleration of Evolutionary Processes by Learning and Extended Fisher's Fundamental Theorem Natural selection is general and powerful concept not only to explain evolutionary processes of biological organisms but also to design engineering systems such as genetic algorithms and particle filters. There is a surge of interest, both from biology and engineering, in considering natural selection of intellectual agents that can learn individually. Learning by individual agents of better behaviors for survival may accelerate the evolutionary processes by natural selection. We have accumulating pieces of evidence that organisms can transmit its information to the next generation via epigenetic states or memes. Also, such idea is important for engineering applications to improve the genetic algorithms and the particle filter. To accelerate the evolutionary process, an agent should change their strategy so that the population fitness increases the most. Equivalently, an agent should update the strategy towards a gradient (derivative) of the population fitness with respect to the strategy. However, it has not yet been clarified whether and how an agent can estimate the gradient and accelerate the evolutionary process. We also lack methodology to quantify the acceleration to understand and predict the impact of learning. In this paper, we address these problems. We show that an learning agent can accelerate the evolutionary process by proposing ancestral learning, which uses the information transmitted from the ancestor (ancestral information) via epigenetic states or memes. Numerical experiments show that ancestral learning actually accelerates the evolutionary process. We next show that the ancestral information is sufficient to estimate the gradient. In particular, learning can accelerate the evolutionary process without communications between agents. Finally, to quantify the acceleration, we extend the Fishers fundamental theorem (FF-thm) for natural selection to ancestral learning. The conventional FF-thm relates the speed of evolution by natural selection to the variety of the individual fitness in the population. Our extended FF-thm relates the acceleration of the evolutionary process to the variety of individual fitness of the agent. By the theorem, we can quantitatively understand when and why learning is beneficial.

sentences: Coevolution of brain size and longevity in parrots Previous studies have demonstrated a correlation between longevity and brain size in a variety of taxa. Little research has been devoted to understanding this link in parrots; yet parrots are well-known for both their exceptionally long lives and cognitive complexity. We employed a large-scale comparative analysis that investigated the influence of brain size and life history variables on longevity in parrots. Specifically, we addressed two hypotheses for evolutionary drivers of longevity: the Cognitive Buffer Hypothesis, which proposes that increased cognitive abilities enable longer life spans, and the Expensive Brain Hypothesis, which holds that increases in life span are caused by prolonged developmental time of, and increased parental investment in, large brained offspring. We estimated life expectancy from detailed zoo records for 133,818 individuals across 244 parrot species. Using a principled Bayesian approach that addresses data uncertainty and imputation of missing values, we found a consistent correlation between relative brain size and life expectancy in parrots. This correlation was best explained by a direct effect of relative brain size. Notably, we found no effects of developmental time, clutch size, or age at first reproduction. Our results suggest that selection for enhanced cognitive abilities in parrots have in turn promoted longer lifespans.

sentences: Novel Hendra virus variant detected by sentinel surveillance of Australian horses. A novel Hendra virus (HeV) variant, not detected by routine testing, was identified and isolated from a Queensland horse that suffered acute, fatal disease consistent with HeV infection. Whole genome sequencing and phylogenetic analysis demonstrated the variant to have ~83% nucleotide identity to the prototype HeV strain. An updated RT-qPCR assay was designed for routine HeV surveillance. In silico and in vitro comparison of the receptor-binding protein with prototypic HeV showed that the human monoclonal antibody m102.4 used for post-exposure prophylaxis, as well as the current equine vaccine, should be effective against this variant. Genetic similarity of this virus to sequences detected from grey-headed flying-foxes suggests the variant circulates at-least in this species. Studies determining infection kinetics, pathogenicity, reservoir-species associations, viral-host co-evolution and spillover dynamics for this virus are urgently needed. Surveillance and biosecurity practices should be updated to appreciate HeV spillover risk across all regions frequented by flying foxes.

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Disruption of the gut microbiota attenuates epithelial ovarian cancer sensitivity to cisplatin therapy Epithelial Ovarian Cancer (EOC) is the leading cause of gynecologic cancer death. Despite many patients achieving remission with first-line therapy, up to 80% of patients will recur and require additional treatment. Retrospective clinical analysis of OC patients indicates antibiotic use during chemotherapy treatment is associated with poor overall survival. We assessed whether antibiotic (ABX) therapy would impact growth of EOC and sensitivity to cisplatin in murine models. Immune competent or compromised mice were given control or ABX containing water (metronidazole, ampicillin, vancomycin, and neomycin) before being intraperitoneally injected with murine EOC cells. Stool was collected to confirm microbiome disruption and tumors were monitored, and cisplatin therapy was administered weekly until endpoint. EOC tumor-bearing mice demonstrate accelerated tumor growth and resistance to cisplatin therapy in ABX treated compared with nonABX treatment. Stool analysis indicated most gut microbial species were disrupted by ABX treatment except for ABX resistant bacteria. To test for role of the gut microbiome, cecal microbiome transplants (CMTs) of microbiota derived from ABX or nonABX treated mice were used to recolonize the microbiome of ABX treated mice. nonABX cecal microbiome was sufficient to ameliorate the chemoresistance and survival of ABX treated mice indicative of a gut derived tumor suppressor. Mechanistically, tumors from ABX treated compared to nonABX treated mice contained a high frequency of cancer stem cells that were augmented by cisplatin. These studies indicate an intact microbiome provides a gut derived tumor suppressor and maintains chemosensitivity that is disrupted by ABX treatment.

SignificancePlatinum resistance is associated with poor prognosis and reduced therapeutic options for ovarian cancer patients. We identifed a tumor suppressive role of the gut microbiome that is disrupted upon antibiotic therapy.

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The ternary complex CEP90, FOPNL and OFD1 specifies the future location of centriolar distal appendages, and promotes their assembly Cilia assembly starts with centriole to basal body maturation, migration to the cell surface and docking to the plasma membrane. The basal body docking process involves the interaction of both the distal end of the basal body and the transition fibers (or mature distal appendages), with the plasma membrane. During this process, the transition zone assembles and forms the structural junction between the basal body and the nascent cilium. Mutations in numerous genes involved in basal body docking and transition zone assembly are associated with the most severe ciliopathies, highlighting the importance of these events in cilium biogenesis.

The conservation of this sequence of events across phyla is paralleled by a high conservation of the proteins involved. We identified CEP90 by BioID using FOPNL as a bait. Ultrastructure expansion microscopy showed that CEP90, FOPNL and OFD1 localized at the distal end of both centrioles/basal bodies in Paramecium and mammalian cells. These proteins are recruited early after duplication on the procentriole. Finally, functional analysis performed both in Paramecium and mammalian cells demonstrate the requirement of this complex for distal appendage assembly and basal body docking. Altogether, we propose that this ternary complex is required to determine the future position of distal appendages.

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A simplified method for CRISPR-Cas9 engineering of Bacillus subtilis The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system from Streptococcus pyogenes has been widely deployed as a tool for bacterial strain construction. Conventional CRISPR-Cas9 editing strategies require design and molecular cloning of an appropriate guide RNA (gRNA) to target genome cleavage and a repair template for introduction of the desired site-specific genome modification. Here, we present a streamlined method that leverages the existing collection of nearly 4000 Bacillus subtilis strains (the BKE collection) with individual genes replaced by an integrated erythromycin (erm) resistance cassette. A single plasmid (pAJS23) with a gRNA targeted to erm allows cleavage of the genome at any non-essential gene, and at sites nearby to many essential genes. This plasmid can be engineered to include a repair template, or the repair template can be co-transformed with the plasmid as either a PCR product or genomic DNA. We demonstrate the utility of this system for generating gene replacements, site-specific mutations, modification of intergenic regions, and introduction of gene-reporter fusions. In sum, this strategy bypasses the need for gRNA design and allows the facile transfer of mutations and genetic constructions with no requirement for intermediate cloning steps.

ImportanceBacillus subtilis is a well-characterized Gram-positive model organism and a popular platform for biotechnology. Although many different CRISPR-based genome editing strategies have been developed for B. subtilis, they generally involve the design and cloning of a specific gRNA and repair template for each application. By targeting the erm resistance cassette with an anti-erm gRNA, genome-editing can be directed to any of nearly 4000 gene disruptants within the existing BKE collection of strains. Repair templates can be engineered as PCR products, or specific alleles and constructions can be transformed as chromosomal DNA, thereby bypassing the need for plasmid construction. The described method is rapid, free of off-target effects, and facilitates a wide-range of genome manipulations.

"Healing is a matter of time, but it is sometimes also a matter of opportunity." - Hippocrates

sentences: Edaphic controls on genome size and GC content of bacteria in soil microbial communities Genomic traits, such as genome size, GC content, codon usage, and amino acid content, shed insight into the evolutionary processes of bacteria and selective forces behind microbial community composition. Nutrient limitation has been shown to reduce bacterial genome size and influence nucleotide composition, yet little research has been conducted in the soil environment, and the factors which shape soil bacterial genomic traits remain largely unknown. Here we determined average genome size, GC content, codon usage, and amino acid content from 398 soil metagenomes across the United States from the National Ecological Observation Network (NEON) and observed the distribution of these traits across numerous environmental gradients. We found that genomic trait averages were most strongly related to pH, which we suggest results in both physiological constraints on growth as well as affects availability of nutrients in soil. Low pH soils had higher carbon to nitrogen ratios (C:N) and tended to have communities with larger genomes and lower GC-content, potentially a result of increased physiological stress and increased metabolic diversity. Conversely, smaller genomes with high GC content were associated with high pH and low soil carbon to nitrogen ratios, indicating potential resource driven selection against carbon-rich AT base pairs. We found that this relationship of nutrient conservation also applied to amino acid stoichiometry, where bacteria in soils with C:N ratios tended to code for amino acids with lower C:N. Together, these relationships point towards fundamental mechanisms which underpin nucleotide and amino acid selection in soil bacterial communities.

sentences: Escherichia coli recombinant expression of SARS-CoV-2 protein fragments. We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach used the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline-threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All fusion proteins were expressed in E. coli at about 0.1 g/L, and could be purified with a single affinity binding step using inexpensive cellulose powder. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibody directed to the appropriate SARS-CoV-2 antigenic region. The largest intact CBM9 fusion protein incorporates spike protein amino acids 540-588, which is a conserved region immediately C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.

sentences: Serial Dependence in Temporal Perception Reveals the Dynamics of Constructing an Internal Reference Frame Temporal perception is crucial to cognitive functions. To better estimate temporal durations, the observers need to construct an internal reference frame based on past experience and apply it to guide future perception. However, how this internal reference frame is constructed remains largely unclear. Here we showed the dynamics of the internal reference construction from the perspective of serial dependence in temporal reproduction tasks. We found the current duration estimation is biased towards both perceived and reproduced durations in previous trials. Moreover, this effect is regulated by the variability of sample durations. The influence of previous trials was stronger when the observers were exposed to context with more variable durations, which is inconsistent with previous theories that the similarity between successive stimuli induces serial dependence. We proposed a Bayesian model with an adaptive reference updated continuously after each observation, which can better explain the serial dependence observed in temporal perception.

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Characterization in mice of the stromal niche maintaining AT2 stem cell self-renewal in homeostasis and disease Resident mesenchymal cells (rMCs defined as Cd31NegCd45NegEpcamNeg) control the self-renewal and differentiation of alveolar epithelial type 2 (AT2) stem cells in vitro. The identity of these rMCs is still elusive. Among them, Axin2Pos mesenchymal alveolar niche cells (MANCs), which are expressing Fgf7, have been previously described. We propose that an additional population of rMCs, expressing Fgf10 (called rMC-Sca1PosFgf10Pos) are equally important to maintain AT2 stem cell self-renewal.

The alveolosphere model, based on the AT2-rMC co-culture in growth factor reduced Matrigel, was used to test the efficiency of different rMC subpopulations isolated by FACS from adult murine lung to sustain the self-renewal and differentiation of AT2 stem cells.

We demonstrate that rMC-Sca1PosFgf10Pos cells are efficient to promote the self-renewal and differentiation of AT2 stem cells. Co-staining of adult lung for Fgf10 mRNA and Sftpc protein respectively, indicate that 28% of Fgf10Pos cells are located close to AT2 cells. Co-ISH for Fgf7 and Fgf10 indicate that these two populations do not significantly overlap. Gene arrays comparing rMC-Sca1PosAxin2Pos and rMC-Sca1PosFgf10Pos support that these two cell subsets express differential markers. In addition, rMC function is decreased in diabetic and obese ob/ob mutant compared to WT mice with a much stronger loss of function in males compared to females.

In conclusion, rMC-Sca1PosFgf10Pos cells play important role in supporting AT2 stem cells self-renewal and differentiation. This result sheds a new light on the subpopulations of rMCs contributing to the AT2 stem cell niche in homeostasis and in the context of COVID-19 pathogenesis.

Key messageO_ST_ABSWhat is already known about the subject?C_ST_ABSResident mesenchymal cells (rMCs defined as Cd31NegCd45NegEpcamNeg) control the self-renewal and differentiation of alveolar epithelial type 2 (AT2) stem cells in vitro. The identity of these rMCs is still elusive. Among them, Axin2Pos mesenchymal alveolar niche cells (MANCs), which are expressing Fgf7, have been previously described.

What does this study add?Our study shows that an additional population of rMCs, expressing Fgf10 (called rMC-Sca1PosFgf10Pos) is equally important to maintain AT2 stem cell self-renewal. rMC-Sca1PosFgf10Pos are LipidToxHigh and are located close to AT2s. In addition, rMC-Sca1PosFgf10Pos cells support AT2 stem cell self-renewal and differentiation thereby identifying these cells as bone fide functional lipofibroblasts (LIFs). We have previously reported that LIF can transdifferentiate into activated MYF in the context of bleomycin-induced fibrosis in mice [1] and that activated MYF isolated from the lungs of end stage idiopathic fibrosis human patients can respond to Metformin to undergo transdifferentiation back to the LIF phenotype [2]. We also show that the function of rMCs-Sca1Pos is negatively impacted by gender and obesity, which represent two major aggravating factors for COVID-19 pathogenesis, leading to either death or major complications after infection recovery such as lung fibrosis.

How might this impact on clinical practice and future development?By establishing that rMC-Sca1PosFgf10Pos are different from the MANCs, our study opens the way for a new key mesenchymal cell population that should be targeted to either prevent or reverse fibrosis. In addition, as this population maintains the AT2 stem cells self-renewal and differentiation, such targeting will also allow to progressively recover the loss in respiratory function.

sentences: Cascading indirect genetic effects in a clonal vertebrate Understanding how individual differences among organisms arise and how their effects propagate through groups of interacting individuals are fundamental questions in biology.Individual differences can arise from genetically-based variation in the conspecifics with which an individual interacts, and these effects might then be propagated to other individuals. Using a clonal species, the Amazon molly (Poecilia formosa), we test the hypothesis that such indirect genetic effects (IGE) propagate beyond individuals that experience them firsthand. We tested this hypothesis by exposing genetically identical Amazon mollies to social partners of different genotypes, and then moving these individuals to new social groups in which they were the only member to have experienced the IGE. We found that genetically different social environments induced different levels of aggression experienced by the focal animals, and that these genetically-based social effects carried over into new social groups to influence the behavior of individuals that did not directly experience the previous social environments. Our data reveal that IGE can cascade beyond the individuals that directly experience them to influence phenotypes even when there is no genetically-based variation present within interacting groups. Theoretical and empirical expansion of the quantitative genetic framework developed for IGE to include cascading and other types of carry-over effects will improve understanding of social behavior and its evolution.

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The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation BackgroundA hallmark of heart failure is cardiac fibrosis, which results from the injury-induced differentiation response of resident fibroblasts to myofibroblasts that deposit extracellular matrix. During myofibroblast differentiation, fibroblasts progress through polarization stages of early pro-inflammation, intermediate proliferation, and late maturation, but the regulators of this progression are poorly understood. Planar cell polarity receptors, receptor tyrosine kinase like orphan receptor 1 and 2 (Ror1/2), can function to promote cell differentiation and transformation. In this study, we investigated the role of the Ror1/2 in a model of heart failure with emphasis on myofibroblast differentiation.

Methods and ResultsThe role of Ror1/2 during cardiac myofibroblast differentiation was studied in cell culture models of primary murine cardiac fibroblast activation and in knockout mouse models that underwent transverse aortic constriction (TAC) surgery to induce cardiac injury by pressure overload. Expression of Ror1 and Ror2 were robustly and exclusively induced in fibroblasts in hearts after TAC surgery, and both were rapidly upregulated after early activation of primary murine cardiac fibroblasts in culture. Cultured fibroblasts isolated from Ror1/2-KO mice displayed a pro-inflammatory phenotype indicative of impaired myofibroblast differentiation. Although the combined ablation of Ror1/2 in mice did not result in a detectable baseline phenotype, TAC surgery led to the death of all mice by day 6 that was associated with myocardial hyper-inflammation and vascular leakage.

ConclusionsTogether, these results show that Ror1/2 are essential for the progression of myofibroblast differentiation and for the adaptive remodeling of the heart in response to pressure overload.

sentences: A conserved role of Parkinson-associated DJ-1 metabolites in sperm motility, mitosis, and embryonic development Fertility rates in the developing world have dramatically dropped in the last decades. This drop is likely due to a decline in sperm quality and women having children at older ages. Loss of function mutations in DJ-1, a Parkinsons associated gene, are linked to alterations in multiple cellular processes such as mitochondrial activity, ROS production or sperm motility and lead to an early onset of Parkinsons disease and male infertility in humans and other species. Glycolate (GA) and D-lactate (DL), products of DJ-1 glyoxalase activity, sustain mitochondrial function and protect against environmental aggressions. We, therefore, tested whether these substances could also have a rescue effect on these phenotypes. Here, we show that DJ-1 loss of function not only affects sperm motility but also leads to defects in mitosis and an age-dependent increase in the abortion rate. Remarkably, whereas DL was only able to rescue embryonic lethality in C. elegans, GA rescued these phenotypes in all model systems tested and even increased sperm motility in wild-type sperm. These positive effects seem to be mediated through an increase in NAD(P)H production and the regulation of intracellular calcium. These findings not only strongly suggest GA as a new therapeutic candidate to improve male and female fertility but also show its potential to treat diseases associated with a decline in mitochondrial function or to improve mitochondrial function in aging.

sentences: The decoy SNARE Tomosyn sets tonic versus phasic release properties and is required for homeostatic synaptic plasticity Synaptic vesicle release probability (Pr) is a key presynaptic determinant of synaptic strength established by cell intrinsic properties and further refined by plasticity. To characterize mechanisms that generate Pr heterogeneity between distinct neuronal populations, we examined glutamatergic tonic (Ib) and phasic (Is) motoneurons in Drosophila with stereotyped differences in Pr and synaptic plasticity. We found the decoy SNARE Tomosyn is differentially expressed between these motoneuron subclasses and contributes to intrinsic differences in their synaptic output. Tomosyn expression enables tonic release in Ib motoneurons by reducing SNARE complex formation and suppressing Pr to generate decreased levels of synaptic vesicle fusion and enhanced resistance to synaptic fatigue. In contrast, phasic release dominates when Tomosyn expression is low, enabling high intrinsic Pr at Is terminals at the expense of sustained release and robust presynaptic potentiation. In addition, loss of Tomosyn disrupts the ability of tonic synapses to undergo presynaptic homeostatic potentiation (PHP).

sentences: Predicting functional consequences of mutations using molecular interaction network features Variant interpretation remains a central challenge for precision medicine. Missense variants are particularly difficult to understand as they change only a single amino acid in protein sequence yet can have large and varied effects on protein activity. Numerous tools have been developed to identify missense variants with putative disease consequences from protein sequence and structure. However, biological function arises through higher order interactions among proteins and molecules within cells. We therefore sought to capture information about the potential of missense mutations to perturb protein interaction networks by integrating protein structure and interaction data. We developed 16 network-based annotations for missense mutations that provide orthogonal information to features classically used to prioritize variants. We then evaluated them in the context of a proven machine-learning framework for variant effect prediction across multiple benchmark datasets to demonstrate their potential to improve variant classification. Interestingly, network features resulted in larger performance gains for classifying somatic mutations than for germline variants, possibly due to different constraints on what mutations are tolerated at the cellular versus organismal level. Our results suggest that modeling variant potential to perturb context-specific interactome networks is a fruitful strategy to advance in silico variant effect prediction.

sentences: The initial response of females towards congeneric males matches the propensity to hybridize in Ophthalmotilapia. AO_SCPLOWBSTRACTC_SCPLOWCichlid radiations often harbour closely related species with overlapping niches and distribution ranges. Such species sometimes hybridise in nature, which raises the question how can they coexist. This also holds for the Tanganyika mouthbrooders Ophthalmotilapia ventralis and O. nasuta. Earlier studies found indications of asymmetrical hybridisation with females of O. ventralis accepting males of O. nasuta, but not the other way around. We hypothesised that this was due to differences in the capacity for species recognition. Given the higher propensity of O. ventralis females towards hybridisation, we expect a reduced ability for species recognition in O. ventralis females, compared to O. nasuta females. We staged two experiments, one focusing on 22 female O. nasuta and one on 21 female O. ventralis. These fish were placed in one half of a tank and briefly exposed to a conspecific or a heterospecific male, a conspecific female, or nothing (control). Female response was evaluated by scoring six tracking parameters and by noting the occurrence of ten discrete behaviours before and during the encounter. Females always responded to the presence of another fish by approaching it. Remarkably, for both O. nasuta and O. ventralis, we did not find a different response between encounters with conspecific males and females. However, in agreement with our hypothesis, O. nasuta females behaved differently towards conspecific or heterospecific males, whereas O. ventralis females did not. When presented with a heterospecific male, O. nasuta females performed a lower number of  ram behaviours. Additionally, they never displayed the  flee behaviour, a component of the species mating repertoire that was seen in all but one of the presentations with a conspecific male. Our findings show that differences in species recognition at first encounter predict to a large degree the outcome of the mating process, even in the absence of mating behaviour.

sentences: The angiotensin antagonist Losartan shifts social reward motivation and punishment feedback sensitivity via modulating midbrain-striato-frontal circuits Social deficits and dysregulations in dopaminergic midbrain-striato-frontal circuits represent transdiagnostic symptoms across psychiatric disorders. Animal models suggest that modulating interactions between the dopamine and renin-angiotensin system with the angiotensin receptor antagonist Losartan (LT) can modulate learning and reward-related processes. We have therefore determined the behavioral and neural effects of LT on social reward and punishment processing in humans. A pre-registered randomized double-blind placebo-controlled between-subject pharmacological design was combined with a social incentive delay fMRI paradigm during which subjects could avoid social punishment or gain social reward. Healthy volunteers received a single-dose of LT (50mg, n=43) or placebo (n=44). Reaction times and emotional ratings served as behavioral outcomes, on the neural level activation, connectivity and social feedback prediction errors were modelled. Relative to placebo, LT switched reaction times and arousal away from prioritizing punishment towards social reward. On the neural level the LT-enhanced motivational salience of social rewards was accompanied by stronger ventral striatum-prefrontal connectivity during reward anticipation and attenuated activity in the ventral tegmental area (VTA) and associated connectivity with the bilateral insula in response to punishment during the outcome phase. Computational modelling further revealed an LT-enhanced social reward prediction error signal in VTA and dorsal striatum. LT shifted motivational and emotional salience away from social punishment towards social reward via modulating distinct core nodes of the midbrain-striato-frontal circuits. The findings document a modulatory role of the renin-angiotensin system in these circuits and associated social processes, suggesting a promising treatment target to alleviate social dysregulations.

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A chromosome-level reference genome of Ensete glaucum gives insight into diversity, chromosomal and repetitive sequence evolution in the Musaceae BackgroundEnsete glaucum (2n = 2x = 18) is a giant herbaceous monocotyledonous plant in the small Musaceae family along with banana (Musa). A high-quality reference genome sequence of E. glaucum offers a vital genomic resource for functional and evolutionary studies of Ensete, the Musaceae, and more widely in the Zingiberales.

FindingsUsing a combination of Illumina and Oxford Nanopore Technologies (ONT) sequencing, genome-wide chromosome conformation capture (Hi-C), and RNA survey sequence, we report a high-quality assembly of the 481.5Mb genome with 9 pseudochromosomes and 36,836 genes (BUSCO 94.7%). A total of 55% of the genome is composed of repetitive sequences with LTR-retroelements (37%) and DNA transposons (7%) predominant. The 5S and 45S rDNA were each present at one locus, and the 5S rDNA had an exceptionally long monomer length of c.1,056 bp, contrasting with the c. 450 bp monomer at multiple loci in Musa. A tandemly repeated c. 134 bp satellite, 1.1% of the genome (with no similar sequence in Musa), was present around all nine centromeres, with a LINE retroelement also found at Musa centromeres. The assembly, including centromeric positions, enabled us to characterize in detail the chromosomal rearrangements occurring between the x = 9 species and x = 11 species of Musa. Only one chromosome has the same gene content as M. acuminata (ma). Three ma chromosomes represent part of only one E. glaucum (eg) chromosome, while the remaining seven ma chromosomes are fusions of parts of two, three, or four eg chromosomes, demonstrating complex and multiple evolutionary rearrangements in the change between x = 9 and x = 11.

ConclusionsThe advance towards a Musaceae pangenome including E. glaucum, tolerant of extreme environments, makes a complete set of gene alleles available for crop breeding and understanding environmental responses. The chromosome-scale genome assembly show how chromosome number evolves, and features of the rapid evolution of repetitive sequences.

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Detection of an unstable minor QTL linked to fire blight resistance on linkage group 16 of Malus fusca ObjectiveErwinia amylovora causes fire blight disease in Malus. A strong resistance QTL (Mfu10) was previously detected on linkage group 10 of Malus fusca accession MAL0045, using several strains of the bacterium. As no strain capable of breaking the resistance of MAL0045 has been found, it was hypothesized that a second resistance factor contributes to the fire blight resistance of MAL0045. However, to date, no minor locus has been detected with previously published strains of the bacterium. We detected a minor QTL only on a subset of a population following inoculation with strain Ea1038, which heterologously expresses an effector in a derivative of isolate Ea3049. Two genetic maps of MAL0045, one scarce, the other dense with markers, were used for QTL analyses.

ResultsMfu10 was detected on LG10 with Ea1038, as was previously with Ea3049. Although no other QTLs of significant LOD was previously detected in other linkage groups with Ea3049, a QTL of significant LOD was detected on LG16 (Mfu16) after inoculation of a subset of 76 individuals with Ea1038, but only using the dense genetic map. Mfu16 improved the effect of Mfu10. However, when the number of individuals inoculated with Ea1038 was increased to 121, Mfu16 was no longer detected in the dense genetic map. We hypothesize some factors, which might be responsible for the instability of this QTL.

sentences: Integrated Regulation of PKA by Fast and Slow Neurotransmission in the Nucleus Accumbens Controls Plasticity and Stress Responses Cortical glutamate and midbrain dopamine neurotransmission converge to mediate striatum-dependent behaviors, while maladaptations in striatal circuitry contribute to mental disorders. Here we uncover a molecular mechanism by which glutamatergic and dopaminergic signaling integrate to regulate cAMP-dependent protein kinase (PKA) via phosphorylation of the PKA regulatory subunit, RII{beta}. We find that glutamate-dependent reduction in Cdk5-dependent RII{beta} phosphorylation alters the PKA holoenzyme auto-inhibitory state to increase PKA signaling in response to dopamine. Disruption of RII{beta} phosphorylation by Cdk5, consequently, enhances cortico-ventral striatal synaptic plasticity. Acute and chronic stress in rats inversely modulate RII{beta} phosphorylation and ventral striatal infusion of a small interfering peptide that selectively targets RII{beta} regulation by Cdk5 improves behavioral response to stress. This new signaling mechanism integrating ventral striatal glutamate and dopamine neurotransmission is likely important to brain function, may contribute to neuropsychiatric conditions, and serves as a possible target for the development of novel therapeutics for stress-related disorders.

sentences: Transmit Field Bias Correction of T1w/T2w Myelin Maps T1-weighted divided by T2-weighted (T1w/T2w) myelin maps were initially developed for neuroanatomical analyses such as identifying cortical areas, but they are increasingly used in statistical comparisons across individuals and groups with other variables of interest. Existing T1w/T2w myelin maps contain radiofrequency transmit field (B1+) biases, which may be correlated with these variables of interest, leading to potentially spurious results. Here we propose two empirical methods for correcting these transmit field biases using either explicit measures of the transmit field or alternatively a 'pseudo-transmit' approach that is highly correlated with the transmit field at 3T. We find that the resulting corrected T1w/T2w myelin maps are both better neuroanatomical measures (e.g., for use in cross-species comparisons), and more appropriate for statistical comparisons of relative T1w/T2w differences across individuals and groups (e.g., sex, age, or body-mass-index) within a consistently acquired study at 3T. We recommend that investigators who use the T1w/T2w approach for mapping cortical myelin use these B1+ transmit field corrected myelin maps going forward.

sentences: Precise measurement of nanoscopic septin ring structures in deep learning-assisted quantitative superresolution microscopy The combination of image analysis and fluorescence superresolution microscopy methods allows for unprecedented insight into the organization of macromolecular assemblies in cells. Advances in deep learning-based object recognition enables the automated processing of large amounts of data, resulting in high accuracy through averaging. However, while the analysis of highly symmetric structures of constant size allows for a resolution approaching the dimensions of structural biology, deep learning methods are prone to different forms of bias. A biased recognition of structures may prohibit the development of readouts for processes that involve significant changes in size or shape of amorphous macromolecular complexes. What is required to overcome this problem is a detailed investigation of potential sources of bias and the rigorous testing of trained models using real or simulated data covering a wide dynamic range of possible results. Here we combine single molecule localization-based superresolution microscopy of septin ring structures with the training of several different deep learning models for a quantitative investigation of bias resulting from different training approaches and finally quantitative changes in septin ring structures. We find that trade-off exists between measurement accuracy and the dynamic range of recognized phenotypes. Using our trained models, we furthermore find that septin ring size can be explained by the number of subunits they are assembled from alone. Our work provides a new experimental system for the investigation of septin polymerization.

sentences: On the hormonal control of posterior regeneration in the annelid Platynereis dumerilii Regeneration is the process by which many animals are able to restore lost or injured body parts. After amputation of the posterior part of its body, the annelid Platynereis dumerilii is able to regenerate the pygidium, the posteriormost part of its body that bears the anus, and a subterminal growth zone containing stem cells that allows the subsequent addition of new segments. The ability to regenerate their posterior part (posterior regeneration) is promoted in juvenile worms by a hormone produced by the brain and is lost when this hormonal activity becomes low at the time the worms undergo their sexual maturation. By characterizing posterior regeneration at the morphological and molecular levels in worms that have been decapitated, we show that the presence of the head is essential for multiple aspects of posterior regeneration, as well as for the subsequent production of new segments. We also show that methylfarnesoate, the molecule proposed to be the brain hormone, can partially rescue the posterior regeneration defects observed in decapitated worms. Our results are therefore consistent with a key role of brain hormonal activity in the control of regeneration and growth in P. dumerilii, and support the hypothesis of the involvement of methylfarnesoate in this control.

sentences: Regeneration of the larval sea star nervous system by wounding induced respecification to the sox2 lineage The ability to restore lost body parts following traumatic injury is a fascinating area of biology that challenges current understanding of the ontogeny of differentiation. The origin of new cells needed to regenerate lost tissue, and whether they are pluripotent stem cells, tissue-specific stem cells or have de- or trans- differentiated, remains one of the most important open questions in regeneration. Additionally, it is not clearly known whether developmental gene regulatory networks (GRNs) are reused to direct specification in these cells or whether regeneration specific networks are deployed. Echinoderms, including sea stars, have extensive ability for regeneration and have therefore been the subject of many thorough studies on the ultrastructural and molecular properties of cells needed for regeneration. However, the technologies for obtaining transgenic echinoderms are limited and tracking cells involved in regeneration, and thus identifying the cellular sources and potencies has proven challenging. In this study we develop new transgenic tools for cell tracking in the regenerating bipinnaria larva of the sea star Patira minaita. We show that the larval serotonergic nervous system can regenerate following decapitation. Using a BAC-transgenesis approach with photoconvertible fluorescent proteins, we show that expression of the pan ectodermal marker, sox2, is induced in previously sox2 minus cells at the wound site, even when cell division is inhibited. sox2+ cells give rise to new sox4+ neural precursors that then proceed along an embryonic neurogenesis pathway to reform the anterior nervous systems. sox2+ cells contribute to only neural and ectoderm lineages, indicating that these progenitors maintain their normal, embryonic lineage restriction. This indicates that sea star larval regeneration uses a combination of existing lineage restricted stem cells, as well as respecification of cells into neural lineages, and at least partial reuse of developmental GRNs to regenerate their nervous system.

sentences: No influence of antibiotic on locomotion in Drosophila nigrosparsa after recovery, but influence on microbiome, possibly mediating wing-morphology change Antibiotics, such as tetracycline, has been frequently used to cure endosymbiont Wolbachia in arthropods. After the symbionts had been removed, the hosts must be waited for some generations to recover from side effects of the antibiotics. Knowledge of potential long-term effects of the antibiotic is important. Here, we treated Drosophila nigrosparsa with and without antibiotic tetracycline for three generations and two generations recovering time to investigate effects of the tetracycline on the flies concerning locomotion of larvae and adults, wing morphology, and gut microbiome of adults. In addition, gut-microbiome restoration was tested as a solution to reducing potential side effects of tetracycline on the flies microbiome more quickly. We found significant differences in larval and adult locomotion within groups but no significant differences among the control, antibiotic-treated, and gut-restoration groups. We found a slight differentiation of wing morphology into the three groups and significant differences in bacterial abundance among groups. The influence of tetracycline on the gut microbiome may have contributed to wing-morphology differences among groups, which would be an indirect effect of the antibiotic. Together with the absence of an effect on locomotion, this suggests that checking for both direct and indirect effects of tetracycline after a particular recovery time before using tetracycline curing is important. The microbiome of the gut-restoration group was not like that of the control group. Therefore, gut restoration cannot be used to remove effects of tetracycline in D. nigrosparsa, at least in the setup used here.

sentences: Molecular profiling of retinal pigment epithelial cell differentiation for therapeutic use Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, detailed single cell mapping of the transient cellular and molecular dynamics from the pluripotent state to mature RPE has not been performed. Here we conduct single-cell transcriptomic analyses of 25,718 cells during differentiation as well as in embryonic and adult retina references, revealing differentiation progression through an un-expected initial cell diversification recapitulating early embryonic development before converging towards an RPE lineage. We also identified NCAM1 to track and capture an intermediate retinal progenitor with the potential to give rise to multiple neuroepithelial lineages. Finally, we profiled hESC-RPE cells after subretinal transplantation into the rabbit eye, uncovering robust in vivo maturation towards an adult state. Our detailed evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.

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ALDH2 DEFICIENCY INCREASES SUSCEPTIBILITY TO BINGE ALCOHOL-INDUCED GUT LEAKINESS, ENDOTOXEMIA, AND ACUTE LIVER INJURY IN MICE THROUGH THE GUT-LIVER AXIS Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is the major enzyme responsible for metabolizing toxic acetaldehyde to acetate and acts as a protective or defensive protein against various disease states associated with alcohol used disorder (AUD), including alcoholic liver disease (ALD), and elevated oxidative stress. We hypothesized that Aldh2-knockout (KO) mice are susceptible to binge alcohol-mediated liver injury than wild-type (WT) mice through increased gut leakiness and endotoxemia. Therefore, this study aimed to investigate the protective role of ALDH2 in binge alcohol-induced gut permeability, endotoxemia, and acute inflammatory liver injury by exposing Aldh2-KO or WT mice to a single oral dose of binge alcohol 3.5, 4.0, or 5.0 g/kg. Our findings showed for the first time that ALDH2 deficiency in Aldh2-KO mice increases their sensitivity to alcohol-induced oxidative and nitrative stress, enterocyte apoptosis, and nitration of gut tight junction (TJ) and adherent junction (AJ) proteins, leading to their degradation. These resulted in gut leakiness and endotoxemia in Aldh2-KO mice after exposure to a single dose of ethanol even at 3.5 g/kg, while no changes were observed in the corresponding WT mice. The elevated serum endotoxin (lipopolysaccharide, LPS) and/or bacterial translocation contributed to systemic inflammation, hepatocyte apoptosis, and subsequently acute liver injury, indicating the disruption in the gut-liver axis. Furthermore, treatment with Daidzin, an ALDH2 inhibitor, exacerbated ethanol-induced cell permeability and reduced TJ/AJ proteins in T84 human colonic cells. These changes were reversed by Alda-1, an ALDH2 activator, indicating a crucial role of ALDH2 in protecting against alcohol-induced epithelial barrier dysfunction. All these findings suggest that ALDH2 deficiency or gene mutation in humans is a risk factor to alcohol-mediated gut and liver injury, and ALDH2 could be an important therapeutic target against alcohol-associated tissue/organ damage.

HighlightsO_LIBinge alcohol increases oxidative and nitrative stress in the intestine and liver.
C_LIO_LIBinge alcohol causes gut leakiness, endotoxemia, and acute liver injury.
C_LIO_LILeaky gut is caused by elevated degradation of nitrated intestinal TJ/AJ proteins.
C_LIO_LIAldh2-KO mice are susceptible to binge-alcohol-induced leaky gut and liver injury.
C_LIO_LIALDH2 inhibition increases alcohol-induced T84 colonic epithelial cell permeability.
C_LI

sentences: Network dynamics underlying OFF responses in the auditory cortex Across sensory systems, complex spatio-temporal patterns of neural activity arise following the onset (ON) and offset (OFF) of stimuli. While ON responses have been widely studied, the mechanisms generating OFF responses in cortical areas have so far not been fully elucidated. We examine here the hypothesis that OFF responses are single-cell signatures of recurrent interactions at the network level. To test this hypothesis, we performed population analyses of two-photon calcium recordings in the auditory cortex of awake mice listening to auditory stimuli, and compared linear single-cell and network models. While the single-cell model explained some prominent features of the data, it could not capture the structure across stimuli and trials. In contrast, the network model accounted for the low-dimensional organisation of population responses and their global structure across stimuli, where distinct stimuli activated mostly orthogonal dimensions in the neural state-space.

sentences: Interferon-regulated genetic programs and JAK/STAT pathway activate the intronic promoter of the short ACE2 isoform in renal proximal tubules Recently, a short, interferon-inducible isoform of Angiotensin-Converting Enzyme 2 (ACE2), dACE2 was identified. ACE2 is a SARS-Cov-2 receptor and changes in its renal expression have been linked to several human nephropathies. These changes were never analyzed in context of dACE2, as its expression was not investigated in the kidney. We used Human Primary Proximal Tubule (HPPT) cells to show genome-wide gene expression patterns after cytokine stimulation, with emphasis on the ACE2/dACE2 locus. Putative regulatory elements controlling dACE2 expression were identified using ChIP-seq and RNA-seq. qRT-PCR differentiating between ACE2 and dACE2 revealed 300- and 600-fold upregulation of dACE2 by IFN and IFN{beta}, respectively, while full length ACE2 expression was almost unchanged. JAK inhibitor ruxolitinib ablated STAT1 and dACE2 expression after interferon treatment. Finally, with RNA-seq, we identified a set of genes, largely immune-related, induced by cytokine treatment. These gene expression profiles provide new insights into cytokine response of proximal tubule cells.

sentences: Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model The etiology of necrotizing enterocolitis (NEC) is not known. Alterations in gut microbiome, mucosal barrier function, immune cell activation, and blood flow are characterized events in its development, with stress as a contributing factor. The hormone corticotropin-releasing factor (CRF) is a key mediator of stress responses and influences these aforementioned processes. CRF signaling is modulated by NECs main risk factors of prematurity and formula feeding. Using an established neonatal rat model of NEC, we tested hypotheses that: (i) increased CRF levels--as seen during stress--promote NEC in formula-fed newborn rats, and (ii) antagonism of CRF action ameliorates NEC. Newborn pups were formula-fed to initiate gut inflammation and randomized to: no stress, no stress with subcutaneous CRF administration, stress (acute hypoxia followed by cold exposure--NEC model), or stress after pretreatment with the CRF peptide antagonist Astressin. Dam-fed unstressed and stressed littermates served as controls. NEC incidence and severity in the terminal ileum were determined using a histologic scoring system. Changes in CRF, CRF receptor (CRFRs), and toll-like receptor 4 (TLR4) expression levels were determined by immunofluorescence and immunoblotting, respectively. Stress exposure in FF neonates resulted in 40.0% NEC incidence, whereas exogenous CRF administration resulted in 51.7% NEC incidence compared to 8.7% in FF non-stressed neonates (p<0.001). Astressin prevented development of NEC in FF-stressed neonates (7.7% vs. 40.0%; p=0.003). CRF and CRFR immunoreactivity increased in the ileum of neonates with NEC compared to dam-fed controls or FF unstressed pups. Immunoblotting confirmed increased TLR4 protein levels in FF stressed (NEC model) animals vs. controls, and Astressin treatment restored TLR4 to control levels. Peripheral CRF may serve as specific pharmacologic target for the prevention and treatment of NEC.

sentences: Robust maintenance of cell surface tension in mitosis by RhoA-driven myosin II mechanoresponse As cells enter mitosis, cell cortex contraction generates surface tension to establish a geometry feasible for division in a physically confined environment. Cell surface tension rises in prophase and continues to stay constant during metaphase to support mitosis. How the cell surface tension is maintained throughout mitosis is not well explored. We show that the cell surface tension is actively maintained by a mechanosensitive RhoA pathway at the cell cortex during mitosis. Mechanical activation of RhoA leads to non-muscle myosin IIB (NMIIB) stabilization and mechanosensitive accumulation at the cell cortex via Rho kinase (ROCK) regulation of the NMIIB head domain. Interestingly, when the NMIIB tail domain regulation is perturbed, the NMIIB has reduced ability to generate tension but could still support mitotic cells to withstand compressive stress by undergoing mechanosensitive accumulation at the cell cortex. Thus, mechanical RhoA activation drives NMIIB mechanoresponse via its head domain regulation to maintain cell surface tension during mitosis.

sentences: Psychological stress disrupts intestinal epithelial cell function and mucosal integrity through microbe and host-directed processes Psychological stress alters the gut microbiota and predisposes individuals to increased risk for enteric infections and chronic bowel conditions. Intestinal epithelial cells (IECs) are responsible for maintaining homeostatic interactions between the gut microbiota and its host. In this study, we hypothesized that disruption to colonic IECs is a key factor underlying stress-induced disturbances to intestinal homeostasis. Conventionally raised (CONV-R) and germ-free (GF) mice were exposed to a social disruption stressor (Str) to ascertain how stress modifies colonic IECs, the mucosal layer, and the gut microbiota. RNA sequencing of IECs isolated from CONV-R mice revealed a robust pro-inflammatory (Saa1, Il18), pro-oxidative (Duox2, Nos2), and antimicrobial (Reg3b/g) transcriptional profile as a result of Str. This response occurred concomitant to mucus layer thinning and signs of microbial translocation. In contrast to their CONV-R counterparts, IECs from GF mice or mice treated with broad spectrum antibiotics exhibited no detectable transcriptional changes in response to Str. Nevertheless, IECs from Str-exposed GF mice exhibited an altered response to ex vivo bacterial challenge (increased dual Oxidase-2 [Duox2] and nitric oxide synthase-2 (Nos2)), indicating that STR primes host IEC pro-oxidative responses. In CONV-R mice stress-induced increases in colonic Duox2 and Nos2 (ROS generating enzymes) strongly paralleled changes to microbiome composition and function, evidencing Str-mediated ROS production as a primary factor mediating gut-microbiota dysbiosis. In conclusion, a mouse model of social stress disrupts colonic epithelial and mucosal integrity, a response dependent on an intact microbiota and host stress signals. Together these preclinical findings may provide new insight into mechanisms of stress-associated bowel pathologies in humans.

sentences: Allosteric interactions prime androgen receptor dimerization and activation The androgen receptor (AR) is a steroid receptor and master transcription factor that governs gene expression programs required for luminal development of prostate epithelium, formation of muscle tissue and maintenance of the male phenotype. AR misregulation is a hallmark of multiple malignancies, including prostate cancer, where AR hyperactivation and expansion of its transcriptome occur in part through AR gene amplification and interaction with oncoprotein cofactors. Despite its biological importance, how ARs individual domains and its protein cofactors cooperate to bind DNA have remained elusive. Using a combination of reconstitution biochemistry and single particle cryo-electron microscopy (EM), we have isolated three conformational states of AR bound to DNA. We observe that AR forms a non-obligate dimer, with the buried dimer interface utilized by related ancestral nuclear receptors repurposed to facilitate cooperative DNA binding. We identify surfaces bridging ARs domains responsible for allosteric communication, that are compromised in partial androgen insensitivity syndrome (PAIS), and are reinforced by ARs oncoprotein cofactor, ERG, and DNA binding site motifs. Finally, we present evidence that this plastic dimer interface for transcriptional activation may have been adopted by AR at the expense of DNA binding. Our work highlights how fine-tuning of ARs cooperative interactions translate to consequences in development and disease.

sentences: Epigenetic scores for the circulating proteome as tools for disease prediction Protein biomarkers have been identified across many age-related morbidities. However, characterising epigenetic influences could further inform disease predictions. Here, we leverage epigenome-wide data to study links between the DNAm signatures of the circulating proteome and incident diseases. Using data from four cohorts, we trained and tested epigenetic scores (EpiScores) for 953 plasma proteins, identifying 109 scores that explained between 1% and 58% of the variance in protein levels after adjusting for known protein quantitative trait loci (pQTL) genetic effects. By projecting these EpiScores into an independent sample, (Generation Scotland; n=9,537) and relating them to incident morbidities over a follow-up of 14 years, we uncovered 137 EpiScore - disease associations. These associations were largely independent of immune cell proportions, common lifestyle and health factors and biological aging. Notably, we found that our diabetes-associated EpiScores highlighted previous top biomarker associations from proteome-wide assessments of diabetes. These EpiScores for protein levels can therefore be a valuable resource for disease prediction and risk stratification.

sentences: Discordant genotype calls across technology platforms elucidate variants with systematic errors in next-generation sequencing Large-scale next-generation sequencing (NGS) datasets have been transformative for informing clinical variant interpretation and as reference panels for statistical and population genetic efforts. While such resources are often treated as ground truth, we find that in widely used reference datasets such as the Genome Aggregation Database (gnomAD), some variants pass gold standard filters yet are systematically different in their genotype calls across sequencing technologies. The inclusion of such discordant sites in study designs involving multiple sequencing platforms (e.g. whole genome and/or different whole-exome captures) could bias results and lead to false-positive hits in association studies due to technological artifacts rather than a true relationship to the phenotype. Here, we describe this phenomenon of discordant genotype calls across sequencing technologies, characterize the error mode of wrong calls, provide a blacklist of discordant sites identified in gnomAD that should be treated with caution in analyses, and present a metric and machine learning classifier trained on gnomAD data to identify likely discordant variants in other datasets. We find that different NGS technologies have different sets of variants at which this problem occurs but that there are characteristic variant features that can be used to predict discordant behavior. Discordant sites are largely shared across ancestry groups, though different populations are powered for discovery of different variants. We find that the most common error mode is that of a variant being heterozygous for one platform and homozygous for the other, with heterozygous in the genomes and homozygous reference in the exomes making up the majority of miscalls.

sentences: EEG evidence that morally relevant autobiographical memories can be suppressed Remembering unpleasant events can trigger negative feelings. Fortunately, research indicates that unwanted memories can be suppressed to prevent them from intruding into awareness, improving our mental state. The current scientific understanding of memory suppression is, however, based mostly on simpler memories such as associations between words or pictures, which may not reflect how people control unpleasant memory intrusions in everyday life. Here, we investigated the neural and behavioural dynamics of suppressing personal and emotional autobiographical memories using a modified version of the Think/No-Think task. We asked participants to suppress memories of their own past immoral actions, which were hypothesised to be both highly intrusive and motivating to suppress. We report novel evidence from behavioural, ERP and EEG oscillation measures that autobiographical memory retrieval can be suppressed and suggest that autobiographical suppression recruits similar neurocognitive mechanisms as suppression of simple laboratory associations. Suppression did fail sometimes, and EEG oscillations indicated that such memory intrusions occurred from lapses in sustained control. Importantly however, participants improved at limiting intrusions with repeated practice. Furthermore, both behavioural and EEG evidence indicated that intentional suppression may be more difficult for memories of our morally wrong actions than memories of our morally right actions. The findings elucidate the neurocognitive correlates of autobiographical retrieval suppression and have implications for theories of morally motivated memory control.

sentences: Population dynamics of free-roaming dogs in Europe and implications for population control Changes in free-roaming dog population size are important indicators of the effectiveness of dog population management. Assessing the effectiveness of different management methods also requires estimating the processes that change population size, such as the rates of recruitment into and removal from a population. This is one of the first studies to quantify the size, rates of recruitment and removal, and health and welfare status of free-roaming dog populations in Europe. We determined the size, dynamics, and health status of free-roaming dog populations in Pescara, Italy, and Lviv, Ukraine, over a 15-month study period. Both study populations had ongoing dog population management through catch-neuter-release and sheltering programmes. Average monthly apparent survival probability was 0.93 (95% CI 0.81-1.00) in Pescara and 0.93 (95% CI 0.84-0.99) in Lviv. An average of 7 dogs km-2 were observed in Pescara and 40 dogs km-2 in Lviv. Per capita entry probabilities varied between 0.09 and 0.20 in Pescara, and 0.12 and 0.42 in Lviv. In Lviv, detection probability was lower on weekdays (odds ratio: 0.74, 95% CI 0.53-0.96) and higher on market days (odds ratio: 2.58, 95% CI 1.28-4.14), and apparent survival probability was lower in males (odds ratio: 0.25, 95% CI 0.03-0.59). Few juveniles were observed in the study populations, indicating that recruitment may be occurring by movement between dog subpopulations (e.g. from local owned or neighbouring free-roaming dog populations), with important consequences for population control. This study provides important data for planning effective dog population management and for informing population and infectious disease modelling. The results emphasise the importance of dog population management efforts that encourage responsible ownership through reduced abandonment and cover the entire city to reduce the risk of dog movement between sections of the city hindering management efforts.

sentences: Yolk platelets impede nuclear expansion in Xenopus embryos During metazoan early embryogenesis, the intracellular properties of proteins and organelles change dynamically through rapid cleavage. In particular, a change in the nucleus size is known to contribute to embryonic development-dependent cell cycle and gene expression regulation. Here, we compared the nuclear sizes of various blastomeres from developing Xenopus embryos and analyzed the mechanisms that control the nuclear expansion dynamics by manipulating the amount of intracellular components in a cell-free system. There was slower nuclear expansion during longer interphase durations in blastomeres from vegetal hemispheres than those from animal hemispheres. Furthermore, upon recapitulating interphase events by manipulating the concentration of yolk platelets, which are originally rich in the vegetal blastomeres, in cell-free cytoplasmic extracts, there was slower nuclear expansion and DNA replication as compared to normal yolk-free conditions. Under these conditions, the supplemented yolk platelets accumulated around the nucleus in a microtubule-dependent manner and impeded organization of the endoplasmic reticulum network. Overall, we propose that yolk platelets around the nucleus reduce membrane supply from the endoplasmic reticulum to the nucleus, resulting in slower nuclear expansion in the yolk-rich vegetal blastomeres.

sentences: PGC-1α in the myofibers regulates the balance between myogenic and adipogenic progenitors affecting muscle regeneration Skeletal muscle repair is accomplished by satellite cells (MuSC) in cooperation with interstitial stromal cells (ISCs). So far, the relationship between the function of these cells and the metabolic state of myofibers remains unclear. The present study reports alterations in the proportion of both MuSCs and adipogenesis regulators (Aregs) induced by overexpression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1) in the myofibers (MCK-PGC-1 mice). Although PGC-1-driven increase of MuSCs does not accelerate muscle regeneration, myogenic progenitors isolated from MCK-PGC-1 mice and transplanted into intact and regenerating muscles are more prone to fuse with recipient myofibers than those derived from WT donors. Moreover, both young and aged MCK-PGC-1 animals show reduced perilipin-positive areas when challenged with an adipogenic stimulus, demonstrating low propensity to accumulate adipocytes within the muscle. These results provide new insights on the role played by PGC-1 in promoting myogenesis and hindering adipogenesis in the skeletal muscle.

sentences: ANANSE: An enhancer network-based computational approach for predicting key transcription factors in cell fate determination Proper cell fate determination is largely orchestrated by complex gene regulatory networks centered around transcription factors. However, experimental elucidation of key transcription factors that drive cellular identity is currently often intractable. Here, we present ANANSE (ANalysis Algorithm for Networks Specified by Enhancers), a network-based method that exploits enhancer-encoded regulatory information to identify the key transcription factors in cell fate determination. As cell type-specific transcription factors predominantly bind to enhancers, we use regulatory networks based on enhancer properties to prioritize transcription factors. First, we predict genome-wide binding profiles of transcription factors in various cell types using enhancer activity and transcription factor binding motifs. Subsequently, applying these inferred binding profiles, we construct cell type-specific gene regulatory networks, and then predict key transcription factors controlling cell fate transitions using differential networks between cell types. This method outperforms existing approaches in correctly predicting major transcription factors previously identified to be sufficient for trans-differentiation. Finally, we apply ANANSE to define an atlas of key transcription factors in 18 normal human tissues. In conclusion, we present a ready-to-implement computational tool for efficient prediction of transcription factors in cell fate determination and to study transcription factor-mediated regulatory mechanisms. ANANSE is freely available at https://github.com/vanheeringen-lab/ANANSE.

sentences: Intact reading ability in spite of a spatially distributed visual word form 'area' in an individual born without the left superior temporal lobe The visual word form area (VWFA) is an experience-dependent region in the left ventral temporal cortex (VTC) of literate adults that responds selectively to visual words. Why does it emerge in this stereotyped location? Past research shows the VWFA is preferentially connected to the left-lateralized frontotemporal language network. However, it remains unclear whether the presence of a typical language network and its connections with VTC are critical for the VWFAs emergence, and whether alternative functional architectures may support reading ability. We explored these questions in an individual (EG) born without the left superior temporal lobe but exhibiting normal reading ability. We recorded fMRI activation to visual words, objects, faces, and scrambled words in EG and neurotypical controls. We did not observe word selectivity either in EGs right homotope of the VWFA (rVWFA)--the most expected location given that EGs language network is right-lateralized--or in her spared left VWFA (lVWFA), despite typical face selectivity in both the right and left fusiform face area (rFFA, lFFA). We replicated these results across scanning sessions (5 years apart). Moreover, in contrast with the idea that the VWFA is simply part of the language network that responds to general linguistic information, no part of EGs VTC showed selectivity to higher-level linguistic processing. Interestingly, multivariate pattern analyses revealed sets of voxels in EGs rVWFA and lVWFA that showed 1) higher within- than between-category correlations for words (e.g., Words-Words>Words-Faces), and 2) higher within-category correlations for words than other categories (e.g., Words-Words>Faces-Faces). These results suggest that a typical left-hemisphere language network may be necessary for the emergence of focal word selectivity within the VTC, and that orthographic processing can be supported by a distributed neural code.

sentences: Physical localization of 45S rDNA in Cymbopogon and the analysis of differential distribution of rDNA in homologous chromosomes of Cymbopogon winterianus. Cymbopogon, commonly known as lemon grass, is one of the most important aromatic grasses having therapeutic and medicinal values. FISH signals on somatic chromosome spreads off Cymbopogon species indicated the localization of 45S rDNA on the terminal region of short arms of a chromosome pair. A considerable interspecific variation in the intensity of 45S rDNA hybridization signals was observed in the cultivars of Cymbopogon winterianus and Cymbopogon flexuosus. Furthermore, in all the varieties of Cymbopogon winterianus namely Bio-13, Manjari and Medini, a differential distribution of 45S rDNA was observed in a heterologous pair of chromosome 1. The development of Cymbopogon winterianus var. Manjari through gamma radiation may be responsible for breakage of fragile rDNA site from one of the chromosomes of this heterologous chromosome pair. While, in other two varieties of Cymbopogon winterianus (Bio-13 and Medini), this variability may be because of evolutionary speciation due to natural cross among two species of Cymbopogon which was fixed through clonal propagation. However, in both the situations these changes were fixed by vegetative method of propagation which is general mode of reproduction in the case of Cymbopogon winterianus.

sentences: Copper selects for siderophore-mediated virulence in Pseudomonas aeruginosa Iron is essential for almost all bacterial pathogens and consequently it is actively withheld by their hosts. However, the production of extracellular siderophores enables iron sequestration by pathogens, increasing their virulence. Another function of siderophores is extracellular detoxification of non-ferrous metals. Here, we experimentally link the detoxification and virulence roles of siderophores by testing whether the opportunistic pathogen Pseudomonas aeruginosa displays greater virulence after exposure to copper. To do this, we incubated P. aeruginosa under different environmentally relevant copper regimes for either two or twelve days. Subsequent growth in a copper-free environment removed phenotypic effects, before we quantified pyoverdine production (the primary siderophore produced by P. aeruginosa), and virulence using the Galleria mellonella infection model. Copper selected for increased pyoverdine production, which was positively correlated with virulence. This effect increased with time, such that populations incubated with high copper for twelve days were the most virulent. Replication of the experiment with a non-pyoverdine producing strain of P. aeruginosa demonstrated that pyoverdine production was largely responsible for the change in virulence. Therefore we here show a direct link between metal stress and bacterial virulence, highlighting another dimension of the detrimental effects of metal pollution on human health.

sentences: Personalized Virus Load Curves of SARS-CoV-2 Infection This manuscript has been withdrawn due to submission errors. Therefore, the authors do not wish this work to be cited as a reference. Please see and cite for reference the manuscript posted on medRxiv https://doi.org/10.1101/2021.01.21.21250268 If you have any questions, please contact the corresponding author.

sentences: Comparative analysis of transcriptomic profiles among ascidians, zebrafish, and mice: insights from tissue-specific gene expression Tissue/organ-specific genes (TSGs) are important not only for understanding organ development and function, but also for investigating the evolutionary lineages of organs in animals. Here, we investigate the TSGs of 9 adult tissues of an ascidian, Ciona intestinalis Type A (Ciona robusta), which lies in the important position of being the sister group of vertebrates. RNA-seq and qRT-PCR identified the Ciona TSGs in each tissue, and BLAST searches identified their homologs in zebrafish and mice. Tissue distributions of the vertebrate homologs were analyzed and clustered using public RNA-seq data for 12 zebrafish and 30 mouse tissues. Among the vertebrate homologs of the Ciona TSGs in the neural complex, 48% and 63% showed high expression in the zebrafish and mouse brain, respectively, suggesting that the central nervous system is evolutionarily conserved in chordates. In contrast, vertebrate homologs of Ciona TSGs in the ovary, pharynx, and intestine were not consistently highly expressed in the corresponding tissues of vertebrates, suggesting that these organs have evolved in Ciona-specific lineages. Intriguingly, more TSG homologs of the Ciona stomach were highly expressed in the vertebrate liver (17-29%) and intestine (22-33%) than in the mouse stomach (5%). Expression profiles for these gene suggest that the biological roles of the Ciona stomach are distinct from those of their vertebrate counterparts. Collectively, Ciona tissues were categorized into 3 groups: i) high similarity to the corresponding vertebrate tissues (neural complex and heart), ii) low similarity to the corresponding vertebrate tissues (ovary, pharynx, and intestine), and iii) low similarity to the corresponding vertebrate tissues, but high similarity to other vertebrate tissues (stomach, endostyle, and siphons). The present study provides transcriptomic catalogs of adult ascidian tissues and significant insights into the evolutionary lineages of the brain, heart, and digestive tract of chordates.

sentences: Intranasal Immunization with a Proteosome-Adjuvanted SARS-CoV2 Spike Protein-Based Vaccine is Immunogenic and Efficacious in Mice & Hamsters With the persistence of the SARS-CoV-2 pandemic and the emergence of novel variants, the development of novel vaccine formulations with enhanced immunogenicity profiles could help reduce disease burden in the future. Intranasally delivered vaccines offer a new modality to prevent SARS-CoV-2 infections through the induction of protective immune responses at the mucosal surface where viral entry occurs. Herein, we evaluated a novel protein subunit vaccine formulation containing a resistin-trimerized prefusion Spike antigen (SmT1v3) and a proteosome-based mucosal adjuvant (BDX301) formulated to enable intranasal immunization. In mice, the formulation induced robust antigen-specific IgG and IgA titers, in the blood and lungs, respectively. In addition, the formulations were highly efficacious in a hamster challenge model, reducing viral load and body weight loss. In both models, the serum antibodies had strong neutralizing activity, preventing the cellular binding of the viral Spike protein based on the ancestral reference strain, the Beta (B.1.351) and Delta (B.1.617.2) variants of concern. As such, this intranasal vaccine formulation warrants further development as a novel SARS-CoV-2 vaccine.

sentences: Stem cell regulators control a G1 duration gradient in the plant root meristem In meristems, where new plant organs initiate, key stem cell regulators have been identified, but their link to cell cycle progression remains unclear. Here, we show that the root meristem has a positional gradient of G1 duration that ranges from [~]2 h near the meristem boundary to more than 20 h in stem cells and early derivatives. Mutants in the PLETHORA (PLT) genes shortened G1 length and flattened its gradient. Computer modeling of an incoherent feed-forward loop (IFFL) predicted the inference of a negative regulatory pathway. We propose that PLT genes play opposing roles, maintaining meristem and stem cell activity and inhibiting G1 progression through the CDK inhibitor KRP5, a PLT target, and RBR1. This establishes a previously undescribed proximal-distal feature of the root meristem in which a G1 duration gradient is shaped by stem cell and meristem maintenance regulators.

sentences: Retinal OFF ganglion cells allow detection of quantal shadows at starlight Perception of light in darkness requires no more than a handful of photons and this remarkable behavioral performance can be directly linked to a particular retinal circuit - the retinal ON pathway. However, the neural limits to shadow detection in very dim light have remained unresolved. Here, we unravel the neural mechanisms that determine the sensitivity of mice to the dimmest light decrements by measuring signals from the most sensitive ON and OFF retinal ganglion cell types and by correlating their signals with visually guided behavior. We show that mice can detect shadows when only a few photon absorptions are missing among thousands of rods. Behavioral detection of "quantal" shadows relies only on the retinal OFF pathway and is limited by noise and losses of single-photon signals in retinal processing. Thus, in the dim-light regime, light increments and decrements are encoded separately via the ON and OFF retinal pathways, respectively.

sentences: Effects of recombination on the evolvability, genetic diversity and mutational robustness of neutrally evolving populations Many effects attributed to recombination have been invoked to explain the advantage of sex. The most prominent arguments focus on either evolvability, genetic diversity, or mutational robustness to justify why the benefit of recombination overcomes its costs, with partially contradicting results. As a consequence, understanding which aspects of recombination are most important in a given situation remains an open problem for theoretical and experimental research. In this study, we focus on finite populations evolving on neutral networks, which already display remarkably complex behavior. We aim to provide a comprehensive overview of the effects of recombination by jointly considering different measures of evolvability, genetic diversity, and mutational robustness over a broad parameter range, such that many evolutionary regimes are covered. We find that several of these measures vary non-monotonically with the rates of mutation and recombination. Moreover, the presence of lethal genotypes that introduce inhomogeneities in the network of viable states qualitatively alters the effects of recombination. We conclude that conflicting trends induced by recombination can be explained by an emerging trade-off between evolvability and genetic diversity on the one hand, and mutational robustness and fitness on the other. Finally, we discuss how different implementations of the recombination scheme in theoretical models can affect the observed dependence on recombination rate through a coupling between recombination and genetic drift.

sentences: Smc3 acetylation, Pds5 and Scc2 control the translocase activity that establishes cohesin dependent chromatin loops Chromosome spatial organization and dynamics influence DNA-related metabolic processes. SMC complexes like cohesin are essential instruments of chromosome folding. Cohesin-dependent chromatin loops bring together distal loci to regulate gene transcription, DNA repair and V(D)J recombination processes. Here we characterize further the roles of members of the cohesin holocomplex in regulating chromatin loop expansion, showing that Scc2, which stimulates cohesin ATPase activity, is essential for the translocation process required to extend DNA loop length. Eco1-dependent acetylation of Smc3 during S phase counteracts this activity through the stabilization of Pds5, to finely tune loop sizes and stability during G2. Inhibiting Pds5 in G2 leads to a strong enlargement of pre-established, stable DNA loops, in a Scc2-dependent manner. Altogether, the study strongly supports a Scc2-mediated translocation process driving expansion of DNA loops in living cells.

sentences: Emergence of Probabilistic Representation in the Neural Network of Primary Visual Cortex During the early development of mammalian visual system, the distribution of neuronal preferred orientations in the primary visual cortex (V1) gradually shifts to match the major orientation features of an environment, achieving optimal representation of the environment. By combining the computational modeling and experimental electrophysiological recording, we provide a circuitry plasticity mechanism that underlies the developmental emergence of such matched representation in the visual cortical network. Specifically, in a canonical circuit of densely interconnected pyramidal cells and inhibitory parvalbumin-expressing (PV+) fast-spiking interneurons in the V1 layer 2/3, our model successfully simulates the experimental observations and further reveals that the non-uniform inhibition, mediated by local interneurons, exerts a key role in shaping the network representation through spike timing-dependent synaptic modifications. The experimental results confirm that PV+ interneurons in the V1 are capable of providing such non-uniform inhibition during a short period after the vision onset. Thus, our study elucidates a circuitry mechanism for acquisition of the prior knowledge of environment for optimal inference in sensory neural system.

sentences: Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein: a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to and stabilises the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we define the conformational repertoire of this element and measure changes in unfolding pathways arising from mutation and 2A binding. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, expand our understanding of the 2A-dependent gene expression switch and reveal how 2A accumulation may shut down translation during virus infection.

sentences: Statistical learning of frequent distractor locations in visual search involves regional signal suppression in early visual cortex Observers can learn the locations where salient distractors appear frequently to reduce potential interference - an effect attributed to better suppression of distractors at frequent locations. But how distractor suppression is implemented in the visual cortex and frontoparietal attention networks remains unclear. We used fMRI and a regional distractor-location learning paradigm (Sauter et al. 2018, 2020) with two types of distractors defined in either the same (orientation) or a different (colour) dimension to the target to investigate this issue. fMRI results showed that BOLD signals in early visual cortex were significantly reduced for distractors (as well as targets) occurring at the frequent versus rare locations, mirroring behavioural patterns. This reduction was more robust with same-dimension distractors. Crucially, behavioural interference was correlated with distractor-evoked visual activity only for same- (but not different-) dimension distractors. Moreover, with different- (but not same-) dimension distractors, a colour-processing area within the fusiform gyrus was activated more when a colour distractor was present versus absent and with a distractor occurring at a rare versus frequent location. These results support statistical learning of frequent distractor locations involving regional suppression in the early visual cortex and point to differential neural mechanisms of distractor handling with different-versus same-dimension distractors.

sentences: A lack of repeatability creates the illusion of a trade-off between basal and plastic cold tolerance. The thermotolerance-plasticity trade-off hypothesis predicts that ectotherms with greater basal thermal tolerance have a lower acclimation capacity. This hypothesis has been tested at both high and low temperatures but the results often conflict. If basal tolerance constrains plasticity (e.g. through shared mechanisms that create physiological constraints), it should be evident at the level of the individual, provided the trait measured is repeatable. Here, we used chill-coma onset temperature and chill-coma recovery time (CCO and CCRT; non-lethal thermal limits) to quantify cold tolerance of Drosophila melanogaster across two trials (pre- and post-acclimation). Cold acclimation improved cold tolerance, as expected, but individual measurements of CCO and CCRT in non-acclimated flies were not (or only slightly) repeatable. Surprisingly, however, there was still a strong correlation between basal tolerance and plasticity in cold-acclimated flies. We argue that this relationship is a statistical artefact (specifically, a manifestation of regression to the mean; RTM) and does not reflect a true trade-off or physiological constraint. Thermal tolerance trade-off patterns in previous studies that used similar methodology are thus likely to be impacted by RTM. Moving forward, controlling and/or correcting for RTM effects is critical to determining whether such a trade-off or physiological constraint truly exists.

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Fast event-related mapping of population fingertip tuning properties in human sensorimotor cortex at 7T fMRI studies that investigate somatotopic tactile representations in the human cortex typically use either block or phase-encoded stimulation designs. Event-related (ER) designs allow for more flexible and unpredictable stimulation sequences than the other methods, but they are less efficient. Here we compared an efficiency-optimized fast ER design (2.8s average intertrial interval, ITI) to a conventional slow ER design (8s average ITI) for mapping voxelwise fingertip tactile tuning properties in the sensorimotor cortex of 6 participants at 7 Tesla. The fast ER design yielded more reliable responses compared to the slow ER design, but with otherwise similar tuning properties. Concatenating the fast and slow ER data, we demonstrate in each individual brain the existence of two separate somatotopically-organized representations of the fingertips, one in the primary somatosensory cortex (S1) on the post-central gyrus, and the other shared across the motor and pre-motor cortices on the pre-central gyrus. In both S1 and motor representations, fingertip selectivity decreased progressively, from narrowly-tuned Brodmann areas 3b and 4a respectively, towards associative parietal and frontal regions that responded equally to all fingertips, suggesting increasing information integration along these two pathways. In addition, fingertip selectivity in S1 decreased from the cortical representation of the thumb to that of the pinky.

Significance StatementSensory and motor cortices in the human brain contain map-like representations of the body in which adjacent brain regions respond to adjacent body parts. The properties of these somatotopic maps provide important insight into how tactile and motor information is processed by the brain. Here, we describe an efficient mapping method using functional magnetic resonance imaging (fMRI) to measure somatotopic maps and their tuning properties. We used a fast event-related sequence to map the five fingers of the left hand in six human participants, and show that this method is superior to a conventional, slower event-related design. Furthermore, we confirm previously-identified tuning properties of fingertip representations in somatosensory cortex, and reveal a hitherto unknown fingertip map in the motor cortex.

sentences: A structural analysis of amyloid polymorphism in disease: clues for selective vulnerability? The increasing amount of amyloid structures offers an opportunity to investigate the general principles determining amyloid stability and polymorphism in disease. We find that amyloid stability is dominated by about 30% of residues localized in few segments interspersed with regions that are often structurally frustrated in the cross-{beta} conformation. These stable segments correspond to known aggregation-nucleating regions and constitute a cross-{beta} structural framework that is shared among polymorphs. Alternative tertiary packing of these segments within the protofibril results in conformationally different but energetically similar polymorphs. This combination of a conserved structural framework along the axis and energetic ambiguity across the axis results in polymorphic plasticity that explains a number of fundamental amyloid properties, including fibril defects and brittleness but also the polymorphic instability of amyloids in simple aqueous buffers. Together these findings suggest a structural model for in vivo polymorphic bias and selective cellular vulnerability whereby (1) polymorphic bias is induced by particular templating interactions in susceptible cells, (2) once formed specific polymorphs are entropically primed to selectively bind similar targets in neighbouring cells, (3) conservation of polymorphic bias during pathological spreading implies the continued presence of similar templating interactions in successive susceptible cells and (4) absence of templating interactions relaxes polymorphic bias possibly allowing for the modification of cellular susceptibilities during disease progression by novel templating interactions.

sentences: Empirical study on software and process quality in bioinformatics tools Software quality in computational tools impacts research output in a variety of scientific disciplines. Biology is one of these fields, especially for High Throughput Sequencing (HTS) data, such tools play an important role. This study therefore characterises the overall quality of a selection of tools which are frequently part of HTS pipelines, as well as analyses the maintainability and process quality of a selection of HTS alignment tools. Our findings highlight the most pressing issues, and point to software engineering best practices developed for the improvement of maintenance and process quality. To help future research, we share the tooling for the static code analysis with SonarCloud which we used to collect data on the maintainability of different alignment tools. The results of the analysis show that the maintainability level is generally high but trends towards increasing technical debt over time. We also observed that the development activities on alignment tools are generally driven by very few developers and are not utilising modern tooling to their advantage. Based on these observations, we recommend actions to improve both maintainability and process quality in open source alignment tools. Those actions include improvements in tooling like the use of linters as well as better documentation of architecture and features. We encourage developers to use these tools in order to ease future maintenance efforts, increase user experience, support reproducibility, and ultimately increase the quality of research through increasing the quality of research software tools.

sentences: Wnt/β-catenin signaling and p68 conjointly regulate CHIP in colorectal cancer The differential expression pattern of Carboxy terminus of Hsc70 Interacting Protein (CHIP, alias STIP1 Homology and U-box Containing Protein 1 or STUB1) in cancers is associated with ubiquitination mediated degradation of its client proteins. Emerging evidences suggest its abundant expression of CHIP in colorectal cancer compared to normal tissues, but the mechanistic detail of this augmented expression pattern is unclear. The signature driver of canonical Wnt pathway, {beta}-catenin, and its co-activator RNA helicase p68, are also overexpressed in colorectal cancer. In this study, we describe a novel mechanism of Wnt/{beta}-catenin and p68 mediated transcriptional activation of CHIP gene leading to enhanced proliferation of colorectal cancer cells. Wnt3A treatment and pharmacological activation of canonical Wnt signaling pathway resulted in increased nuclear translocation of {beta}-catenin and elevated expression of CHIP. Likewise, overexpression and knockdown of {beta}-catenin and p68 upregulated and downregulated CHIP expression, respectively, at both mRNA and protein levels. After cloning CHIP promoter, the increased and decreased promoter activities of CHIP induced by overexpression and knockdown of either {beta}-catenin or p68 further confirmed transcriptional regulation of CHIP gene by Wnt/{beta}-catenin signaling cascade. p68 along with {beta}-catenin were found to occupy Transcription Factor 4 (TCF4) binding sites on endogenous CHIP promoter and regulate its transcription. Finally, enhanced cellular propagation and migration of colorectal cancer cells induced by  Wnt/{beta}-catenin-p68-CHIP axis established the significance of this pathway in oncogenesis. To the best of our knowledge, this is the first report elucidating the mechanistic details of transcriptional regulation of CHIP (STUB1) gene expression.

sentences: A mathematical framework for dissecting normative foundations of objective-driven conservation decisions Perspectives in conservation are based on a variety of value systems. Such differences in how people value nature and its components lead to different evaluations of the morality of conservation goals and approaches, and often underlie disagreements in the formulation and implementation of environmental management policies. Specifically, whether a conservation action (e.g. killing feral cats to reduce predation on bird species threatened with extinction) is viewed as appropriate or not can vary among people with different value systems. Here, we present a conceptual, mathematical framework intended as a tool to systematically explore and clarify core value statements in conservation approaches. Its purpose is to highlight how fundamental differences between these value systems can lead to different prioritizations of available management options and offer a common ground for discourse. The proposed equations decompose the question underlying many controversies around management decisions in conservation: what or who is valued, how, and to what extent? We compare how management decisions would likely be viewed under three different idealised value systems: ecocentric conservation, which aims to preserve biodiversity; new conservation, which considers that biodiversity can only be preserved if it benefits humans; and sentientist conservation, which aims at minimising suffering for sentient beings. We illustrate the utility of the framework by applying it to case studies involving invasive alien species, rewilding, and trophy hunting. By making value systems and their consequences in practice explicit, the framework facilitates debates on contested conservation issues, and complements philosophical discursive approaches about moral reasoning. We believe dissecting the core value statements on which conservation decisions are based will provide an additional tool to understand and address conservation conflicts.

sentences: Temporal responses of bumblebee gustatory neurons encode sugar identity The sense of taste permits the recognition of valuable nutrients and the avoidance of potential toxins. Models of gustatory coding propose that within modalities (e.g. sweet, bitter, etc.), taste ligands are not distinct stimuli. However, these models are based on data from mice or flies that have omnivorous, non-specialist diets. A specialist feeder might, however, be expected to have acuity within modality if stimulus resolution was critical to survival. Previously, we found that bumblebees have a specialized mechanism for sensing sugars whereby two gustatory receptor neurons (GRNs) within the galeal sensilla of the bees mouthparts exhibit burst of spikes. Here, we show that the temporal firing patterns of these GRNs separate sugars into four distinct groups that correlate with sugar nutritional value and palatability. We also identified a third GRN that responded to stimulation with relatively high concentrations of fructose, sucrose, and maltose. Sugars that were non-metabolizable or toxic suppressed the responses of bursting GRNs to sucrose. These abilities to encode information about sugar value are a refinement to the bumblebees sense of sweet taste that could be an adaptation that enables precise calculations of the nature and nutritional value of floral nectar.

sentences: Removal of disulfide from acid stress chaperone HdeA does not wholly eliminate structure or function at low pH HdeA is an acid-stress chaperone that operates in the periplasm of various strains of pathogenic gram-negative bacteria. Its primary function is to prevent irreversible aggregation of other periplasmic proteins when the bacteria enter the acidic environment of the stomach after contaminated food is ingested; its role is therefore to help the bacteria survive long enough to enter and infect the intestines. The mechanism of operation of HdeA is unusual in that this helical homodimer is inactive when folded at neutral pH but becomes activated at low pH after the dimer dissociates and becomes partially unfolded. Studies with chemical reducing agents have previously suggested that the intramolecular disulfide bond of HdeA aids in the maintenance of residual structure at low pH; it is believed that this residual structure is important for clustering exposed hydrophobic residues together for the purpose of binding unfolded client proteins. In order to explore its role in HdeA structure and chaperone function we performed a conservative cysteine to serine mutation of the disulfide. We found that, although residual structure is greatly diminished at pH 2 without the disulfide, it is not completely lost; conversely, the mutant is almost completely random coil at pH 6. Aggregation assays showed that mutated HdeA, although less successful as a chaperone than wild type, still maintains a surprising level of function. These studies highlight that we still have much to learn about the factors that stabilize residual structure at low pH and the role of disulfide bonds.

sentences: Prime Editing Efficiency and Fidelity are Enhanced in the Absence of Mismatch Repair Prime editing (PE) is a powerful genome engineering approach that enables the introduction of base substitutions, insertions and deletions into any given genomic locus. However, the efficiency of PE varies widely and depends not only on the genomic region targeted, but also on the genetic background of the edited cell. To determine which cellular factors affect PE efficiency, we carried out a focused genetic screen targeting 32 DNA repair factors, spanning all reported repair pathways. We show that, depending on cell line and type of edit, ablation of mismatch repair (MMR) affords a 2-17 fold increase in PE efficiency, across several human cell lines, types of edits and genomic loci. The accumulation of the key MMR factors MLH1 and MSH2 at PE sites argues for direct involvement of MMR in PE control. Our results shed new light on the mechanism of PE and suggest how its efficiency might be optimised.

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The race goes on: A fall armyworm-resistant maize inbred line influences insect oral secretion elicitation activity and nullifies herbivore suppression of plant defense O_LIFall armyworm (Spodoptera frugiperda) is an invasive lepidopteran pest with strong feeding preference towards maize (Zea mays). Its success on maize is facilitated by a suite of specialized detoxification and manipulation mechanisms that curtail host plant defense responses.
C_LIO_LIIn this study, we identified a Chinese maize inbred line Xi502 that was able to mount effective defense in response to fall armyworm attack. Comparative transcriptomics analyses, phytohormonal measurements, and targeted benzoxazinoid quantification consistently demonstrate significant inducible defense responses in Xi502, but not in the susceptible reference inbred line B73.
C_LIO_LIIn 24 hours, fall armyworm larvae feeding on B73 showed accelerated maturation-oriented transcriptomic responses and more changes in detoxification gene expression compared to their Xi502-fed sibling. Interestingly, oral secretions collected from larvae fed on B73 and Xi502 leaves demonstrated distinct elicitation activity when applied on either host genotypes, suggesting that variation in both insect oral secretion composition and host plant alleles could influence plant defense response.
C_LIO_LIThese results revealed host plant adaptation towards counter-defense mechanisms in a specialist insect herbivore, adding yet another layer to the evolutionary arms race between maize and fall armyworm. This could facilitate future investigation into the molecular mechanisms in this globally important crop-pest interaction system.
C_LI

sentences: Homotopic contralesional excitation suppresses spontaneous circuit repair and global network reconnections following ischemic stroke Understanding circuit-level changes that affect the brains capacity for plasticity will inform the design of targeted interventions for treating stroke recovery. We combine optogenetic photostimulation with optical neuroimaging to examine how contralesional excitatory activity affects cortical remodeling after stroke in mice. Following photothrombosis of left primary somatosensory forepaw (S1FP) cortex, mice received chronic excitation of right S1FP, a maneuver mimicking the use of the unaffected limb during recovery. Contralesional excitation suppressed perilesional S1FP remapping and was associated with abnormal patterns of evoked activity in the unaffected limb. Contralesional stimulation prevented the restoration of resting-state functional connectivity (RSFC) within the S1FP network, RSFC in several networks functionally-distinct from somatomotor regions, and resulted in persistent limb-use asymmetry. In stimulated mice, perilesional tissue exhibited suppressed transcriptional changes in several genes important for recovery. These results suggest that contralesional excitation impedes local and global circuit reconnection through suppression of several neuroplasticity-related genes after stroke.

sentences: Development of a Cell Surface Display System in Chlamydomonas reinhardtii Cell-surface display systems are biotechnological techniques used to express heterologous proteins on the cell surface. Their application depends directly on the cell system used, as well as on the anchoring point for the surface displayed protein. To meet most application demands an inexpensive, safe, and scalable production platform, that reduces the economic barriers for large scale use is needed. Towards this goal, we screened three possible cell surface anchoring points in the green algae Chlamydomonas by fusing mVenus to prospective anchors moieties. The vectors harboring mVenus:anchor were screened for mVenus fluorescence and tested for cellular localization by confocal laser scanning microscopy. This strategy allowed the identification of two functional anchors, one for the cytoplasmic membrane using the MAW8 GPI-anchor signal, and one for the cell wall using the GP1 protein. We also exploited GP1 chemical and biological traits to release the fused proteins efficiently during cell wall shedding. Our work provides a foundation for surface engineering of C reinhardtii supporting both cell biology studies and biotechnology applications.

sentences: The systematic assessment of completeness of public metadata accompanying omics studies The scientific community has accumulated enormous amounts of genomic data stored in specialized public repositories. Genomic data is easily accessible and available from public genomic repositories allowing the biomedical community to effectively share the omics datasets. However, improperly annotated or incomplete metadata accompanying the raw omics data can negatively impact the utility of shared data for secondary analysis. In this study, we perform a comprehensive analysis under 137 studies over 18,559 samples across six therapeutics fields to assess the completeness of metadata accompanying omics studies in both publication and its related online repositories across and make observations about how the process of data sharing could be made reliable. This analysis involved finding studies based on the six therapeutic fields, that are Alzheimers disease, acute myeloid leukemia, cystic fibrosis, cardiovascular diseases, inflammatory bowel disease, sepsis, and tuberculosis. We carefully examined the availability of metadata over nine clinical variables, that included disease condition, age, organism, sex, tissue type, ethnicity, country, mortality, and clinical severity. By comparing the metadata availability in both original publications and online repositories, we observed discrepancies in sharing the metadata. We determine that the overall availability of metadata is 72.8%, where the most complete reported phenotypes are disease condition and organism, and the least is mortality. Additionally, we examined the completeness of metadata reported separately in original publications and online repositories. The completeness of metadata from the original publication across the nine clinical phenotypes is 71.1%. In contrast, the overall completeness of metadata information from the public repositories is 48.6%. Our study is the first one to systematically assess the completeness of metadata accompanying raw data across a large number of studies and phenotypes and opens a crucial discussion about solutions to improve completeness and accessibility of metadata accompanying omics studies.

sentences: Functional genomics analysis identifies impairment of HNF1B function as a cause of Mayer-Rokitansky-Küster-Hauser syndrome. Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome is a congenital condition characterized by aplasia or hypoplasia of the uterus and vagina in women with a typical 46,XX karyotype. This condition can occur as type I when isolated or as type II when associated with extragenital anomalies including kidney and skeletal abnormalities. The genetic basis of MRKH syndrome remains unexplained and several candidate genes have been proposed to play a role in its etiology, including HNF1B, LHX1, and WNT4. Here, we conducted a genomic analysis of 13 women affected by MRKH syndrome, resulting in the identification of candidate genes, including several novel candidates. We focused on HNF1B for further investigation due to its known association with, but unknown etiological role in, MRKH syndrome. We ablated Hnf1b specifically in the epithelium of the Mullerian ducts in mice, and found that this caused hypoplastic development of both the epithelial and stromal compartments of the uterus, as well as kidney anomalies, closely mirroring the MRKH type II phenotype. Using single-cell RNA sequencing of uterine tissue in the Hnf1b-ablated embryos, we analyzed the molecules and pathways downstream of Hnf1b, revealing a dysregulation of processes associated with cell proliferation, migration, and differentiation. Thus, we establish that loss of Hnf1b function leads to an MRKH phenotype, and generate the first mouse model of MRKH syndrome type II. Our results support the diagnostic value of HNF1B in clinical genetic testing for MRKH syndrome, and shed new light on the genetic causes of this poorly understood condition in womens reproductive health.

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Spatio-temporal, optogenetic control of gene expression in organoids Organoids derived from stem cells become increasingly important to study human development and to model disease. However, methods are needed to control and study spatio-temporal patterns of gene expression in organoids. To this aim, we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes, at single-cell resolution and in programmable spatio-temporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. High-resolution spatial transcriptomic and single-cell analyses showed that this local induction was sufficient to generate stereotypically patterned organoids in three dimensions and revealed new insights into SHHs contribution to gene regulation in neurodevelopment.

With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.

sentences: Crystal structures and functional analysis of the ZnF5-WWE1-WWE2 region of PARP13/ZAP define a new mode of engaging poly(ADP-ribose) PARP13/ZAP acts against multiple viruses through recognizing and promoting degradation of cytoplasmic viral mRNA. PARP13 has four N-terminal Zn-finger motifs that bind CG-rich nucleotide sequences, and a C-terminal ADP ribosyltransferase fold similar to other PARPs. A central region predicted to contain a fifth Zn-finger and two tandem WWE domains is implicated in binding poly(ADP-ribose); however, there are limited insights into the structure and function of this PARP13 region (ZnF5-WWE1-WWE2). Here, we present crystal structures of ZnF5-WWE1-WWE2 from mouse PARP13 in complex with ADP-ribose and with ATP. ZnF5-WWE1-WWE2 crystallized as a dimer with major contacts formed between WWE1 and WWE2 originating from different monomers, indicative of a more compact monomeric arrangement of the tandem WWE domains. Solution scattering experiments and biophysical analysis indicated a monomer in solution, suggesting that the crystal dimer represents domain swapping that could potentially represent a PARP13 conformation assumed when signaling viral RNA detection. The crystal structure and binding studies demonstrate that WWE2 interacts with ADP-ribose and ATP, whereas WWE1 does not have a functional binding site. The shape of the WWE2 binding pocket disfavors interaction with the ribose-ribose linkage of poly(ADP-ribose). Binding studies with poly(ADP-ribose) ligands indicate that WWE2 serves as an anchor for preferential binding to the terminal end of poly(ADP-ribose), and the composite structure of ZnF5-WWE1-WWE2 forms an extended surface to engage polymer chains of ADP-ribose. This model represents a novel mode of poly(ADP-ribose) recognition and provides a structural framework for investigating poly(ADP-ribose) impact on PARP13 function.

sentences: Synapse type-specific molecular nanoconfigurations of the presynaptic active zone in the hippocampus identified by systematic nanoscopy Neurotransmitters are released by exocytosis from synaptic vesicles at the active zone in the presynaptic terminal. The scaffold of the active zone consists of only a few evolutionarily conserved proteins, including RIM, CAST/ELKS, and RIM-BP, and tethers Munc13 and Ca2+ channels. The molecular principles that enable these proteins to mediate synaptic diversity have remained unclear. Here, we identified synapse type-specific molecular nanoconfigurations in the active zone by systematic quantification of active zone proteins using nanoscopy at two types of excitatory synapses and two types of inhibitory synapses in the rat hippocampal CA3 region. Quantitative analysis revealed that Munc13 content was particularly varied among the various synapse types and that the physical proximity of Ca2+ channels to the active zone scaffolds correlated with the efficacy of neurotransmitter release. We propose that the active zone is a flexible supramolecular assembly that can tune its composition and spatial configuration to adjust neurotransmitter release.

sentences: SBP-SITA: A sequence-based prediction tools for S-itaconation As a recently-reported post-translational modification, S-itaconation plays an important role in inflammation suppression. In order to understand its regulatory mechanism in many life activities, the essential step is the recognition of S-itaconation. However, it is difficult to identify S-itaconation in the proteome for the high cost, which limits further investigation. In this study, we constructed an ensemble algorithm based on Soft Voting Classifier. The area under the ROC curve (AUC) value 0.73 for ensemble model. Accordingly, we constructed the on-line prediction tool dubbed SBP-SITA for easily identifying Cystine sites. SBP-SITA is available at http://www.bioinfogo.org/sbp-sita.

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Regulation of PDF Receptor signaling controlling daily locomotor rhythms in Drosophila Each day and in conjunction with ambient daylight conditions, neuropeptide PDF regulates the phase and amplitude of locomotor activity rhythms in Drosophila through its receptor, PDFR, a Family B G protein-coupled receptor (GPCR). We studied the in vivo process by which PDFR signaling turns off, by converting as many as half of the 28 potential sites of phosphorylation in its C terminal tail to a non-phosphorylatable residue (alanine). We report that many such sites are conserved evolutionarily, and their conversion creates a specific behavioral syndrome opposite to loss-of-function phenotypes previously described for pdfr. That syndrome includes increases in the amplitudes of both Morning and Evening behavioral peaks, as well as multi-hour delays of the Evening phase. The precise behavioral effects were dependent on day-length, and most effects mapped to conversion of only a few, specific serine residues near the very end of the protein and specific to its A isoform. Behavioral phase delays under entraining conditions were often, but not always correlated with increases in circadian period. The behavioral phenotypes produced by the most severe PDFR variant were ligand-dependent in vivo, and not a consequence of changes to their pharmacological properties, nor of changes in their surface expression, as measured in vitro. The mechanisms underlying termination of PDFR signaling are complex, subject to regulation that is modified by season, and central to a better understanding of the peptidergic modulation of behavior.

AUTHOR SUMMARYIn multi-cellular organisms, circadian pacemakers create output as a series of phase markers across the 24 hour day to allow other cells to pattern diverse aspects of daily rhythmic physiology and behavior. Within circadian pacemaker circuits, neuropeptide signaling is essential to help promote coherent circadian outputs. In the fruit fly Drosophila 150 neurons are dedicated circadian clocks and they all tell the same time. In spite of such strong synchronization, they provide diverse phasic outputs in the form of their discrete, asynchronous neuronal activity patterns. Neuropeptide signaling breaks the clock-generated symmetry and drives many pacemakers away from their preferred activity period in the morning. Each day, neuropeptide PDF is released by Morning pacemakers and delays the phase of activity of specific other pacemakers to later parts of the day or night. When and how the PDF that is released in the morning stops acting is unknown. Furthermore, timing of signal termination is not fixed because day length changes each day, hence the modulatory delay exerted by PDF must itself be regulated. Here we test a canonical model of G protein-coupled receptor physiology to ask how PDF receptor signaling is normally de-activated. We use behavioral measures to define sequence elements of the receptor whose post-translational modifications (e.g., phosphorylation) may define the duration of receptor signaling.

sentences: Homologous BHLH transcription factors induce distinct deformations of torsionally-stressed DNA: a potential transcription regulation mechanism. Changing torsional restraints on DNA is essential for the regulation of transcription. Torsional stress, introduced by RNA polymerase, can propagate along chromatin facilitating topological transitions and modulating the specific binding of transcription factors (TFs) to DNA. Despite the importance, the mechanistic details on how torsional stress impacts the TFs-DNA complexation remain scarce. Herein we address the impact of torsional stress on DNA complexation with homologous human basic-helix-loop-helix (BHLH) hetero- and homodimers: MycMax, MadMax, and MaxMax. The three TF dimers exhibit specificity towards the same DNA consensus sequences, the E-box response element, while regulating different transcriptional pathways. Using microseconds-long atomistic molecular dynamics simulations together with the torsional restraint that controls DNA total helical twist, we gradually over- and underwind naked and complexed DNA to a maximum of {+/-}5{degrees}/b.p. step. We observe that the binding of the BHLH dimers results in a similar increase in DNA torsional rigidity. However, under torsional stress the BHLH dimers induce distinct DNA deformations, characterised by changes in DNA grooves geometry and a significant asymmetric DNA bending. Supported by bioinformatics analyses, our data suggest that torsional stress may contribute to the execution of differential transcriptional programs of the homologous TFs by modulating their collaborative interactions.

sentences: mmSIM: An open toolbox for accessible structured illumination microscopy Since the first practical super-resolution structured illumination fluorescence microscopes (SIM) were demonstrated more than two decades ago the method has become increasingly popular for a wide range of bioimaging applications. The high cost and relative inflexibility of commercial systems, coupled with the conceptual simplicity of the approach and the desire to exploit and customise existing hardware, have led to the development of a large number of home-built systems. Several detailed hardware designs are available in the scientific literature, complemented by open-source software tools for SIM image validation and reconstruction. However, there remains a lack of simple open-source software to control these systems and manage the synchronization between hardware components, which is critical for effective SIM imaging. This article describes a new suite of software tools based on the popular Micro-Manager package, which enable the keen microscopist to develop and run a SIM system. We use the software to control two custom-built, high-speed, spatial light modulator-based SIM systems, evaluating their performance by imaging a range of fluorescent samples. By simplifying the process of SIM hardware development, we aim to support wider adoption of the technique.

sentences: Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS Reversed-phase ultrahigh-performance liquid chromatography q mass spectrometry (RP-UHPLC/MS) method was developed with the aim to unambiguously identify a large number of lipid species from multiple lipid classes in human plasma. The optimized RP-UHPLC/MS method employed the C18 column with sub-2 m particles with the total run time of 25 min. The chromatographic resolution was investigated with 42 standards from 18 lipid classes. The UHPLC system was coupled to high-resolution quadrupole - time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) measuring full scan and tandem mass spectra (MS/MS) in positive- and negative-ion modes with high mass accuracy. Our identification approach was based on m/z values measured with mass accuracy within 5 ppm tolerance in the full scan mode, characteristic fragment ions in MS/MS, and regularity in chromatographic retention dependences for individual lipid species, which provides the highest level of confidence for reported identifications of lipid species including regioisomeric and other isobaric forms. The graphs of dependences of retention times on the carbon number or on the number of double bond(s) in fatty acyl chains were constructed to support the identification of lipid species in homologous lipid series. Our list of identified lipid species is also compared with previous publications investigating human blood samples by various MS based approaches. In total, we have reported more than 500 lipid species representing 26 polar and nonpolar lipid classes detected in NIST Standard reference material 1950 human plasma.

sentences: E2F4 as a single multifactorial target against Alzheimer's disease Alzheimers disease (AD) has a multifactorial etiology, which requires a single multi-target approach for an efficient treatment. We have focused on E2F4, a transcription factor that regulates cell quiescence and tissue homeostasis, controls gene networks affected in AD, and is upregulated in the brain of Alzheimers patients and of APPswe/PS1dE9 and 5xFAD transgenic mice. E2F4 contains an evolutionarily-conserved Thr-motif that, when phosphorylated, modulates its activity, thus constituting a potential target for intervention. Here we show that neuronal expression in 5xFAD mice of a dominant negative form of E2F4 lacking this Thr-motif (E2F4DN) potentiates a transcriptional program consistent with the attenuation of the immune response and global brain homeostasis. This correlates with reduced microgliosis and astrogliosis, modulation of A{beta} proteostasis, and blockade of neuronal tetraploidization. Moreover, E2F4DN prevents cognitive impairment and body weight loss, a known somatic alteration associated with AD. Our finding is relevant for AD, since E2F4 is expressed in cortical neurons from Alzheimer patients in association with Thr-specific phosphorylation, as evidenced by an anti-E2F4/anti-phosphoThr proximity ligation assay. We propose E2F4DN-based gene therapy as a promising multifactorial approach against AD.

sentences: Bulkhead-like apical membrane structures between hepatocytes are required for anisotropic lumen expansion and liver tissue morphogenesis Lumen morphogenesis is key to the function of organs and results from the integration of molecular pathways and mechanical forces1-3. The mechanisms governing anisotropic lumen expansion remain elusive4-6. In contrast to epithelial cells which have simple apico-basal polarity and form tubes, hepatocytes are multi-polar and form narrow lumina that grow anisotropically between adjacent cells, collectively generating a complex 3D network of bile canaliculi (BC)7,8. Here, we studied lumen elongation and BC morphogenesis in differentiating primary mouse hepatoblasts in vitro. Remarkably, we discovered a pattern of specific extensions of the apical membrane traversing the lumen between adjacent hepatocytes and sealed by tight junctions, reminiscent of the bulkheads of boats. These structures were also present in the developing liver. A targeted screen revealed that silencing of Rab35 caused loss of the bulkheads, conversion of hepatocyte into simple epithelial polarity and formation of spherical lumina in vitro. Strikingly, we could re-engineer hepatocyte polarity and tissue morphogenesis in vivo in the embryonic liver, converting BC into simple epithelial tubes. Our results suggest that the apical bulkheads of hepatocytes are cell-intrinsic anisotropic mechanical elements that ensure stability of the elongating lumen between two cells, thus determining the structure of BC during liver tissue morphogenesis.

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An IPI based immune prognostic model for diffuse large B-cell lymphoma BackgroundInternational Prognostic Index (IPI) was widely used to better discriminate prognosis of patients with diffuse large B-cell lymphoma (DLBCL). However, there is a significant need to identify novel valuable biomarkers in the context of targeted therapies, such as immune checkpoint blockade (ICB) therapy.

MethodsGene expression data and clinical information of DLBCL were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. 371 immune-related hub genes in DLBCL patients with different IPI levels were identified by weighted gene co-expression network analysis (WGCNA), and 8 genes were selected to construct an IPI-based immune prognostic model (IPI-IPM). Afterward, the genetic, somatic mutational and molecular profiles of IPI-IPM subgroups were analyzed, as well as the potential clinical response of ICB in different IPI-IPM subgroups.

ResultsThe IPI-IPM was constructed based on the expression of CMBL, TLCD3B, SYNDIG1, ESM1, EPHA3, HUNK, PTX3 and IL12A, where high-risk patients had shorter overall survival (OS) than low-risk patients, consistent with the results in the GEO cohorts. The comprehensive results showed that high IPI-IPM risk scores were correlated with immune-related signaling pathways, high KMT2D and CD79B mutation rates, as well as up-regulation of inhibitory immune checkpoints including PD-L1, BTLA and SIGLEC7, indicating more potential response to ICB therapy.

ConclusionThe IPI-IPM has independent prognostic significance for DLBCL patients, which provides an immunological perspective to elucidate the mechanisms on tumor progression, also sheds a light on developing immunotherapy for DLBCL.

sentences: Differential impact of TNFRSF co-stimulation on CD8+ T cell cytokine production by feedback control of surface receptor expression T cell responses towards infections and cancers are regulated by a host of co-signalling receptors that are largely grouped into the binary categories of co-stimulation and co-inhibition. The TNF receptor superfamily (TNFRSF) members 4-1BB, CD27, GITR, and OX40 are well-established co-stimulation receptors with largely shared molecular pathways raising the question of whether they also have a similar impact on quantitative T cell responses, such as the efficacy, sensitivity, and duration of T cell responses. Here, we systematically stimulated primary human CD8+ T cell blasts with dose ranges of antigen and ligands for TNFRSF members to screen for their quantitative effects on cytokine production. Although both 4-1BB and CD27 increased efficacy, only 4-1BB was able to prolong the duration of cytokine production, and both had only a modest impact on antigen sensitivity. An operational model could explain these divergent quantitative phenotypes using a shared signalling mechanism based on the surface expression of 4-1BB, but not CD27, being regulated through a signalling feedback. The model predicted that CD27 co-stimulation would increase 4-1BB expression and subsequent 4-1BB co-stimulation, which we confirmed experimentally. Although GITR and OX40 produced only minor changes in cytokine production on their own, we found that like 4-1BB, CD27 could enhance GITR expression and subsequent GITR co-stimulation. Thus, feedback control of induced TNFRSF surface expression explains both synergy and differential impact on cytokine production. The work highlights that different co-stimulation receptors can have different quantitative phenotypes on the same output allowing for highly regulated control of T cell responses.

sentences: Feedback processing in attack and defense conflicts: a neurophysiological study Despite being extensively modelled to explore decision making processes in economic tasks, there are no studies exploring the neurophysiological correlates of the Predator-Prey Game, a game theory paradigm designed to operationalize attack and defence conflicts. In the present study, we explored the relationship between the ERP components associated with feedback, namely feedback-related negativity (FRN) and feedback -elated P3b, and investment in an adapted version of the Predator-Prey Game (PPG), operationalizing attack and defence. Forty-seven (28 female) community-dwelling volunteers without history of neurological disease were recruited. Participants played the PPG game both as attackers and defenders while EEG signal was recorded with a 128 channels sensor net. Behavioural results showed that individuals tend to invest more and decide faster when playing in defence, rather than in attack. Electrophysiological data showed FRN to be sensitive to the valence of feedback, with increased amplitudes of FRN loss feedbacks. On the other hand, the P3b amplitudes were sensitive to the role, with increased amplitude for attack when compared with defence. The integration of the behavioural and ERP findings supports the theoretical model positing that attack elicits more deliberate and less automatic brain processes than defence.

sentences: A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered a novel class of small molecule ketobenzothiazole TMPRSS2 inhibitors with significantly improved activity over existing irreversible inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East Respiratory Syndrome Coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice with a half-life of 8.6 hours in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

sentences: Probing adaptation and spontaneous firing in human auditory-nerve fibers with far-field peri-stimulus time responses Information in sound stimuli is conveyed from sensory hair cells to the cochlear nuclei by the firing of auditory nerve fibers (ANFs). For obvious ethical reasons, single unit recordings from the cochlear nerve have never been performed in human, thus functional hallmarks of ANFs are unknown. By filtering and rectifying the electrical signal recorded at the round window of gerbil cochleae, we reconstructed a peri-stimulus time response (PSTR), with a waveform similar to the peri-stimulus time histograms (PSTHs) recorded from single ANFs. Pair-by-pair analysis of simultaneous PSTR and PSTH recordings in gerbil provided a model to predict the rapid adaptation and spontaneous discharge rates (SR) in a population of ANFs according to their location in the cochlea. We then probed the model in the mouse, in which the SR-based distribution of ANFs differs from the gerbil. We show that the PSTR-based predictions of the rapid adaptation time constant and mean SR across frequency again matched those obtained by recordings from single ANFs. Using PSTR recorded from the human cochlear nerve in 8 normal-hearing patients who underwent cerebellopontine angle surgeries for a functional cranial-nerve disorders (trigeminal neuralgia or hemifacial spasm), we predicted a rapid adaptation of about 3 milliseconds and a mean SR of 23 spikes/s in the 4 kHz frequency range in human ANFs. Together, our results support the use of PSTR as a promising diagnostic tool to map the auditory nerve in humans, thus opening new avenues to better understanding neuropathies, tinnitus, and hyperacusis.

sentences: Translational suppression via IFG-1/eIF4G confers resistance to stress-induced RNA alternative splicing in Caenorhabditis elegans Splicing of pre-mRNA is an essential process for dividing cells and splicing defects have been linked to aging and various chronic diseases. Environmental stress has recently been shown to alter splicing fidelity and molecular mechanisms that protect against splicing disruption remains unclear. Using an in vivo RNA splicing reporter, we performed a genome-wide RNAi screen in Caenorhabditis elegans and found that protein translation suppression via silencing of the conserved initiation factor 4G (IFG-1/eIF4G) protects against cadmium-induced splicing disruption. Transcriptome analysis of an ifg-1 deficient mutant revealed an overall increase in splicing fidelity and resistance towards cadmium-induced alternative splicing compared to the wild-type. We found that the ifg-1 mutant up-regulates >80 RNA splicing regulatory genes that are controlled by the TGF-{beta} transcription factor SMA-2. The extended lifespan of the ifg-1 mutant is partially reduced upon sma-2 depletion and completely nullified when core spliceosome genes including snr-1, snr-2, and uaf-2 are knocked down. Together, these data describe a molecular mechanism that provides resistance towards stress-induced alternative splicing and demonstrate an essential role for RNA homeostasis in promoting longevity in a translation-compromised mutant.

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A short ERK5 isoform modulates nucleocytoplasmic shuttling of active ERK5 and associates with poor survival in breast cancer BackgroundThe nucleocytoplasmic shuttling of ERK5 has gained recent attention as a regulator of its diverse roles in cancer progression but the exact mechanisms for this shuttling are still under investigation.

MethodsUsing in vitro, in vivo and in silico studies, we investigated the roles of shorter ERK5 isoforms in regulating the nucleocytoplasmic shuttling of active phosphorylated-ERK5 (pERK5). Retrospective cohorts of primary and metastatic breast cancer cases were used to evaluate the association of the subcellular localization of pERK5 with clinicopathological features.

ResultsExtranuclear localization of pERK5 was observed during cell migration in vitro and at the invasive fronts of metastatic tumors in vivo. The nuclear and extranuclear cell fractions contained different isoforms of pERK5, which are encoded by splice variants expressed in breast and other cancers in the TCGA data. One isoform, isoform-3, lacks the C-terminal transcriptional domain and the nuclear localization signal. The co-expression of isoform-3 and full-length ERK5 associated with high epithelial-to-mesenchymal transition (EMT) and poor patient survival. Experimentally, expressing isoform-3 with full-length ERK5 in breast cancer cells increased cell migration, drove EMT and led to tamoxifen resistance. In breast cancer patient samples, pERK5 showed variable subcellular localizations where its extranuclear localization associated with aggressive clinicopathological features, metastasis, and poor survival.

ConclusionOur studies support a model of ERK5 nucleocytoplasmic shuttling driven by splice variants in an interplay between mesenchymal and epithelial states during metastasis. Using ERK5 as a biomarker and a therapeutic target should account for its splicing and context-dependent biological functions.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/436061v1_ufig1.gif" ALT="Figure 1">
View larger version (58K):
org.highwire.dtl.DTLVardef@1491ed5org.highwire.dtl.DTLVardef@1875fbeorg.highwire.dtl.DTLVardef@9edbbcorg.highwire.dtl.DTLVardef@16c34c8_HPS_FORMAT_FIGEXP  M_FIG C_FIG ERK5 isoform-3 expression deploys active ERK5 (pERK5) outside the nucleus to facilitate EMT and cell migration. In cells dominantly expressing isoform-1, pERK5 shuttles to the nucleus to drive cell expansion.

sentences: BigBrainWarp: Toolbox for integration of BigBrain 3D histology with multimodal neuroimaging AO_SCPLOWBSTRACTC_SCPLOWNeuroimaging stands to benefit from emerging ultrahigh-resolution histological atlases of the human brain; the first of which is "BigBrain". Ongoing research aims to characterise regional differentiation of cytoarchitecture with BigBrain and to optimise registration of BigBrain with standard neuroimaging templates. Together, this work paves the way for multi-scale investigations of brain organisation. However, working with BigBrain can present new challenges for neuroimagers, including dealing with cellular resolution neuroanatomy and complex transformation procedures. To simplify workflows and support adoption of best practices, we developed BigBrainWarp, a toolbox for integration of BigBrain with multimodal neuroimaging. The primary BigBrainWarp function wraps multiple state-of-the-art deformation matrices into one line of code, allowing users to easily map data between BigBrain and standard MRI spaces. Additionally, the toolbox contains ready-to-use cytoarchitectural features to improve accessibility of histological information. The present article discusses recent contributions to BigBrain-MRI integration and demonstrates the utility of BigBrainWarp for further investigations.

sentences: Experimental demonstration of tethered gene drive systems for confined population modification or suppression Homing gene drives hold great promise for the genetic control of natural populations. However, current homing systems are capable of spreading uncontrollably between populations connected by even marginal levels of migration. This could represent a substantial sociopolitical barrier to the testing or deployment of such drives and may generally be undesirable when the objective is only local population control, such as suppression of an invasive species outside of its native range. Tethered drive systems, in which a locally confined gene drive provides the CRISPR nuclease needed for a homing drive, could provide a solution to this problem, offering the power of a homing drive and confinement of the supporting drive. Here, we demonstrate the engineering of a tethered drive system in Drosophila, using a regionally confined CRISPR Toxin-Antidote Recessive Embryo (TARE) drive to support modification and suppression homing drives. Each drive was able to bias inheritance in its favor, and the TARE drive was shown to spread only when released above a threshold frequency in experimental cage populations. After the TARE drive had established in the population, it facilitated the spread of a subsequently released split homing modification drive (to all individuals in the cage) and of a homing suppression drive (to its equilibrium frequency). Our results show that the tethered drive strategy is a viable and easily engineered option for providing confinement of homing drives to target populations.

sentences: A mitochondrial long-chain fatty acid oxidation defect leads to uncharged tRNA accumulation and activation of the integrated stress response in the mouse heart The heart relies mainly on mitochondrial fatty acid {beta}-oxidation (FAO) for its high energy requirements. Cardiomyopathy and arrhythmias can be severe complications in patients with inherited defects in mitochondrial long-chain FAO, reinforcing the importance of FAO for cardiac health. However, the pathophysiological mechanisms that underlie the cardiac abnormalities in long-chain FAO disorders remain largely unknown. Here, we investigated the cardiac transcriptional adaptations to the FAO defect in the long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mouse. We found a prominent activation of the integrated stress response (ISR) mediated by the eIF2/ATF4 axis in both fed and fasted states, accompanied by a reduction in cardiac protein synthesis during a short period of food withdrawal. Notably, we found an accumulation of uncharged tRNAs in LCAD KO hearts, consistent with a reduced availability of cardiac amino acids, in particular, glutamine. We replicated the activation of the cardiac ISR in hearts of mice with a muscle-specific deletion of carnitine palmitoyltransferase 2 deletion (Cpt2M-/-). Our results show that perturbations in amino acid metabolism caused by long-chain FAO deficiency impact cardiac metabolic signaling, in particular the ISR, and may play a role in the associated cardiac pathology.

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Vicia faba TPC1, a genetically encoded variant of the vacuole Two Pore Channel 1, is hyperexcitable To fire action-potential-like electrical signals, the vacuole membrane requires the depolarization-activated two-pore channel TPC1, also called Slowly activating Vacuolar SV channel. The TPC1/SV channel, encoded by the TPC1 gene, functions as a voltage-dependent and Ca2+-regulated potassium channel. TPC1 currents are activated by a rise in cytoplasmic Ca2+ but inhibited by luminal Ca2+. In search for species-dependent functional TPC1 channel variants, we studied polymorphic amino acids contributing to luminal Ca2+ sensitivity. We found that the acidic residues E457, E605 and D606 of the Ca2+-sensitive Arabidopsis AtTPC1 channel were neutralized by either asparagine or alanine in Vicia faba and many other Fabaceae as well. When expressed in the Arabidopsis loss-of-AtTPC1 function background, the wild type VfTPC1 was hypersensitive to vacuole depolarization and insensitive to blocking luminal Ca2+. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the Arabidopsis At-VfTPC1 channel mutant gained VfTPC1-like voltage and luminal Ca2+ insensitivity that together made vacuoles hyperexcitable. These findings indicate that natural TPC1 channel variants in plant families exist which differ in vacuole excitability and very likely respond to changes in environmental settings of their ecological niche.

Significance statementVacuolar electrical excitability and stress-related Ca2+ signaling depends on the activity of the vacuolar cation channel TPC1. Until now, the regulatory features of AtTPC1 from the model plant Arabidopsis thaliana was believed to apply to the TPC1 channels of other species. However, here we now show that, surprisingly, the VfTPC1 channel of the economic broad bean, in contrast to AtTPC1, proves to be hyperactive and confers hyperexcitability to the vacuole. The different gating behavior is most likely related to an impaired Ca2+ sensor site in the vacuolar pore vestibule, rising the probability to open at more negative membrane voltages. These natural variants of the TPC1 channel could help the plant adapt and respond to environmental challenges.

sentences: Ionizing radiation improves RIG-I mediated immunotherapy through enhanced p53 activation in malignant melanoma Radiation therapy induces cytotoxic DNA damage, which results in cell-cycle arrest and activation of cell-intrinsic death pathways, but its application has been limited by the radioresistance of tumors, such as in malignant melanoma. RIG-I is a cytosolic immune receptor expressed in all somatic cells, including tumor cells, with a key role in sensing viral RNA. RIG-I specific oligonucleotide ligands elicit a robust cell-intrinsic antiviral response and immunogenic cell death in tumor cells and are being tested in clinical trials. Nonetheless, their potential to overcome radioresistance has not yet been explored. Here, we demonstrate that activation of RIG-I enhances the extent and immunogenicity of irradiation-induced tumor cell death in human and murine melanoma cell lines in vitro and improved survival in the murine B16 melanoma model. Pathway analysis of transcriptomic data revealed a central role for p53 downstream of the combination treatment, which was corroborated using p53-/- B16 cells. In vivo, the effect of irradiation on immune-cell activation and inhibition of tumor growth was absent in mice carrying p53-/- B16 tumors, while the response to RIG-I stimulation in those mice was maintained. Our results identify p53 as pivotal for the synergistic antitumoral effect of RIG-I and irradiation, resulting in potent induction of immunogenic tumor-cell death. Thus, the administration of RIG-I ligands in combination with radiotherapy is a promising therapeutic approach to treating radioresistant tumors with a functional p53 pathway, such as malignant melanoma.

sentences: Transcriptomic profiles of Plasmodium falciparum and Plasmodium vivax-infected individuals in Indonesia Malaria is one of the leading causes of illness and death globally. The vast majority of transcriptomic studies of the impact of malaria on human hosts have been conducted on populations of African ancestry suffering from Plasmodium falciparum infection, making it unclear whether biological responses observed in these studies can be generalised to other populations. Here, we perform differential expression analysis between healthy controls and malaria-infected patients within Indonesia, a country of over 260 million people which has substantial morbidity due to endemic malaria. We find that in samples infected with P. falciparum and P. vivax, there is an upregulation of genes involved in inflammation, the immediate early immune response, translation, and apoptosis. When comparing these findings to transcriptomic studies conducted in Africa (on P. falciparum) and South America (on P. vivax), we find that many pathways are shared. This is particularly apparent for receptor recognition and inflammation-related genes in P. falciparum and innate immune and chemokine-related genes in P. vivax infection. However, we also find that many genes are unique to the Indonesian population, particularly those involved in RNA processing, splicing, and cell surface receptor genes. This study provides a more comprehensive view of malaria infection outside of Africa and contributes to a better characterisation of malaria pathogenesis within humans across a range of genetic architectures.

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Hypereosinophilia causes progressive cardiac pathologies in mice Hypereosinophilic syndrome is a progressive disease with extensive eosinophilia that results in organ damage. Cardiac pathologies are the main reason for its high mortality rate. A better understanding of the mechanisms of eosinophil-mediated tissue damage would benefit therapeutic development. Here, we describe the cardiac pathologies that developed in a mouse model of hypereosinophilic syndrome. These IL-5 transgenic mice exhibited decreased left ventricular function at a young age which worsened with age. Mechanistically, we demonstrated infiltration of activated eosinophils into the heart tissue that led to an inflammatory environment. Gene expression signatures showed tissue damage as well as repair and remodeling processes. Cardiomyocytes from IL-5Tg mice exhibited significantly reduced contractility relative to WT controls. This impairment may result from the inflammatory stress experienced by the cardiomyocytes and suggest that dysregulation of contractility and Ca2+ reuptake in cardiomyocytes contributes to cardiac dysfunction at the whole organ level in hypereosinophilic mice.

TeaserToo many eosinophils cause inflammation in the heart and change cardiomyocyte contraction leading to poor heart function.

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Hidden viral sequences in public sequencing data and warning for future emerging diseases RNA viruses cause numerous emerging diseases, mostly due to transmission from mammalian and avian reservoirs. Large-scale surveillance of RNA viral infections in these animals is a fundamental step for controlling viral infectious diseases. Metagenomic analysis is a powerful method for virus identification with low bias and has substantially contributed to the discovery of novel viruses. Deep sequencing data have been collected from diverse animals and accumulated in public databases, which can be valuable resources for identifying unknown viral sequences. Here, we screened for infections of 33 RNA viral families in publicly available mammalian and avian sequencing data and found approximately 900 hidden viral infections. We also discovered six nearly complete viral genomes in livestock, wild, and experimental animals: hepatovirus in a goat, hepeviruses in blind mole-rats and a galago, astrovirus in macaque monkeys, parechovirus in a cow, and pegivirus in tree shrews. Some of these viruses were phylogenetically close to human pathogenic viruses, suggesting the potential risk of causing disease in humans upon infection. Furthermore, the infections of five novel viruses were identified in several different individuals, indicating that their infections may have already spread in the natural host population. Our findings demonstrate the reusability of public sequencing data for surveying viral infections and identifying novel viral sequences, presenting a warning about a new threat of viral infectious disease to public health.

ImportanceMonitoring the spread of viral infections and identifying novel viruses capable of infecting humans through animal reservoirs are necessary to control emerging viral diseases. Massive amounts of sequencing data collected from various animals are publicly available, and these data may contain sequences originating from a wide variety of viruses. Here, we analyzed more than 46,000 public sequencing data and identified approximately 900 hidden RNA viral infections in mammalian and avian samples. Some viruses discovered in this study were genetically similar to pathogens that cause hepatitis, diarrhea, or encephalitis in humans, suggesting the presence of new threats to public health. Our study demonstrates the effectiveness of reusing public sequencing data to identify known and unknown viral infections, indicating that future continuous monitoring of public sequencing data by metagenomic analyses would help prepare and mitigate future viral pandemics.

sentences: A Synthesis of Game Theory and Quantitative Genetic Models of Social Evolution Two popular approaches for modeling social evolution, evolutionary game theory and quantitative genetics, ask complementary questions but are rarely integrated. Game theory focuses on evolutionary outcomes, with models solving for evolutionarily stable equilibria, whereas quantitative genetics provides insight into evolutionary processes, with models predicting short-term responses to selection. Here we draw parallels between evolutionary game theory and interacting phenotypes theory, which is a quantitative genetic framework for understanding social evolution. First, we show how any evolutionary game may be translated into two quantitative genetic selection gradients, nonsocial and social selection, which may be used to predict evolutionary change from a single round of the game. We show that synergistic fitness effects may alter predicted selection gradients, causing changes in magnitude and sign as the population mean evolves. Second, we show how evolutionary games involving plastic behavioral responses to partners can be modeled using indirect genetic effects, which describe how trait expression changes in response to genes in the social environment. We demonstrate that repeated social interactions in models of reciprocity generate indirect effects and conversely, that estimates of parameters from indirect genetic effect models may be used to predict the evolution of reciprocity. We argue that a pluralistic view incorporating both theoretical approaches will benefit empiricists and theorists studying social evolution. We advocate the measurement of social selection and indirect genetic effects in natural populations to test the predictions from game theory, and in turn, the use of game theory models to aid in the interpretation of quantitative genetic estimates.

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An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure BackgroundUrinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical analysis methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine.

Methods3 EV isolation methods were compared: ultracentrifugation, magnetic bead-based, and size-exclusion chromatography from 0.5 mL or 1 mL of rat and human urine. EV yield and mass spectrometry signals (Q-ToF and Triple Quad) were evaluated from each method. Metabolomic profiling was performed on EVs isolated from the urine of rats exposed to ionizing radiation 1-, 14-, 30- or 90-days post-exposure, and human urine from patients receiving thoracic radiotherapy for the treatment of lung cancer pre- and post-treatment.

ResultsSize-exclusion chromatography is the preferred method for EV isolation from 0.5 mL of urine. Mass spectrometry-based metabolomic analyses of EV cargo identified biochemical changes induced by radiation, including altered nucleotide, folate, and lipid metabolism. We have provided standard operating procedures for implementation of these methods in other laboratories.

ConclusionsWe demonstrate that EVs can be isolated from small volumes of urine and analytically investigated for their biochemical contents to detect radiation induced metabolomic changes. These findings lay a groundwork to develop future methods to monitor response to radiotherapy and can be extended to an array of molecular phenotyping studies aimed at characterizing EV cargo.

sentences: Tuftsin: a natural molecule against SARS-CoV-2 infection Coronavirus disease 2019 (COVID-19) continuously proceeds despite the application of a variety of vaccines. It is still urgent to find effective ways to treat COVID-19. Recent studies indicate that NRP1, an important receptor of the natural peptide tuftsin, facilitates SARS-CoV-2 infection. Importantly, tuftsin is a natural human molecule released from IgG. Here, we found 91 overlapping genes between tuftsin targets and COVID-19-associated genes. Bioinformatics analyses indicated that tuftsin could also target ACE2 and exert some immune-related functions to treat COVID-19. Using surface plasmon resonance (SPR) analysis, we confirmed that tuftsin can bind ACE2 and NRP1 directly. Moreover, tuftsin effectively impairs the binding of SARS-CoV-2 S1 to ACE2. Thus, tuftsin is an attractive drug against COVID-19. And tuftsin as natural immunostimulating peptide in human, we speculate that tuftsin may has crucial roles in asymptomatic carriers or mild cases of COVID-19.

sentences: Unexpected growth of a classic yeast auxotroph In budding yeast, the Met15 enzyme has long been assumed to be the sole homocysteine synthase facilitating de novo synthesis of sulfur-containing organic compounds (organosulfurs) from inorganic precursors. Here we show that an alternative homocysteine synthase encoded by the previously uncharacterized gene YLL058W supports growth of mutants lacking MET15 in the absence of exogenous organosulfurs. This growth is observed specifically when cells are deposited in an automated fashion to seed colonies, but not with traditional cell propagation techniques such as thick patches of cells or liquid cultures. We show that the lack of growth in these contexts, which has historically justified the status of MET15 as a classic auxotrophic marker, is largely due to toxic levels of hydrogen sulfide accumulation rather than an inability to perform de novo homocysteine biosynthesis. These data suggest that cell fate decisions in yeast are influenced by hydrogen sulfide levels in the microenvironment. Our results have broad implications for investigations of sulfur metabolism, including studies of methionine restriction and aging.

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Ending animal agriculture would stabilize greenhouse gas levels for 30 years and offset 70 percent of CO2 emissions this century Animal agriculture contributes significantly to global warming through ongoing emissions of the potent greenhouse gases methane and nitrous oxide, and displacement of biomass carbon on the land used to support livestock. However, because estimates of the magnitude of the effect of ending animal agriculture often focus on only one factor, the full potential benefit of a more radical change remains underappreciated. Here we quantify the full "climate opportunity cost" of current global livestock production, by modeling the combined, long-term effects of emission reductions and biomass recovery that would be unlocked by a phaseout of animal agriculture. We show that, even in the absence of any other emission reductions, persistent drops in atmospheric methane and nitrous oxide levels, and slower carbon dioxide accumulation, following a phaseout of livestock production would, through the end of the century, have the same cumulative effect on the warming potential of the atmosphere as a 25 gigaton per year reduction in anthropogenic CO2 emissions, providing half of the net emission reductions necessary to limit warming to 2{degrees}C. The magnitude and rapidity of these potential effects should place the reduction or elimination of animal agriculture at the forefront of strategies for averting disastrous climate change.

Significance StatementThe use of animals to produce food has a negative impact on the climate, but the benefits of a global switch to a plant based diet are underappreciated. We show that the global warming impact, through the rest of this century, of eliminating greenhouse gas emissions from livestock and allowing native ecosystems to regrow on the land currently used to house and feed livestock, would be equivalent to a 68% reduction in carbon dioxide emissions. We hope putting clearer numbers on the "climate opportunity cost" of our continued use of animals as food technology will help policymakers and the public properly prioritize dietary change as a climate defense strategy.

Declaration of Conflict of InterestPatrick Brown is the founder and CEO of Impossible Foods, a company developing alternatives to animals in food-production. Michael Eisen is an advisor to Impossible Foods. Both are shareholders in the company and thus stand to benefit financially from reduction of animal agriculture.

sentences: Diffusion-Informed Spatial Smoothing of fMRI Data in White Matter Using Spectral Graph Filters Brain activation mapping using functional magnetic resonance imaging (fMRI) has been extensively studied in brain gray matter (GM), whereas in large disregarded for probing white matter (WM). This unbalanced treatment has been in part due to controversies in relation to the nature of the blood oxygenation level-dependent (BOLD) contrast in WM and its detachability. However, an accumulating body of studies has provided solid evidence of the functional significance of the BOLD signal in WM and has revealed that it exhibits anisotropic spatio-temporal correlations and structure-specific fluctuations concomitant with those of the cortical BOLD signal. In this work, we present an anisotropic spatial filtering scheme for smoothing fMRI data in WM that accounts for known spatial constraints on the BOLD signal in WM. In particular, the spatial correlation structure of the BOLD signal in WM is highly anisotropic and closely linked to local axonal structure in terms of shape and orientation, suggesting that isotropic Gaussian filters conventionally used for smoothing fMRI data are inadequate for denoising the BOLD signal in WM. The fundamental element in the proposed method is a graph-based description of WM that encodes the underlying anisotropy observed across WM, derived from diffusion-weighted MRI data. Based on this representation, and leveraging graph signal processing principles, we design subject-specific spatial filters that adapt to a subjects unique WM structure at each position in the WM that they are applied at. We use the proposed filters to spatially smooth fMRI data in WM, as an alternative to the conventional practice of using isotropic Gaussian filters. We test the proposed filtering approach on two sets of simulated phantoms, showcasing its greater sensitivity and specificity for the detection of slender anisotropic activations, compared to that achieved with isotropic Gaussian filters. We also present WM activation mapping results on the Human Connectome Projects 100-unrelated subject dataset, across seven functional tasks, showing that the proposed method enables the detection of streamline-like activations within axonal bundles.

sentences: Halophilic bacteria Bacillus altitudinis MIM2 producing melanin isolated from Mundra port, Kutch, Gujarat, India Melanins are ubiquitous black or brown color pigments exhibiting a wide variety of bioactivities. They are stable and insoluble in nature. Melanin are industrially important pigment currently used in cosmetics, medicine and pharmaceuticals, industries. Bacteria mainly produce three types of melanin namely, Eumelanin, pheomelanin and pyomelanin which is usually extracellularly secreted. This makes the downstream processing of bacterial melanin easier. Stress conditions like salt stress, radiation stress etc. triggers the bacteria to produce melanin and several bacterial species were found to produce melanin under different stress induced conditions. In this present study we had isolated melanin producing bacteria from saline sediment sample of Mundra port, Kutch region of Gujarat state of India. The melanin producing bacteria was characterized using staining, biochemical and molecular methods. The melanin produced was extracted and analyzed using physicochemical techniques. To our knowledge this is the first report on Bacillus altitudinis producing melanin.

sentences: Evaluating an internet-delivered fear conditioning and extinction protocol using response times and affective ratings. Pavlovian fear conditioning is widely used to study mechanisms of fear learning, but high-throughput studies are hampered by the labor-intensive nature of examining participants in the lab. To circumvent this bottle-neck, fear conditioning tasks have been developed for remote delivery. Previous studies have examined remotely delivered fear conditioning protocols using expectancy and affective ratings. Here we replicate and extend these findings using an internet-delivered version of the Screaming Lady paradigm, evaluating the effects on negative affective ratings and response time to an auditory probe during stimulus presentations. In a sample of 80 adults, we observed clear evidence of both fear acquisition and extinction using affective ratings. Response times were faster when probed early, but not later, during presentation of stimuli paired with an aversive scream. The response time findings are at odds with previous lab-based studies showing slower responses to threat-predicting cues. The findings underscore the feasibility of employing remotely delivered fear conditioning paradigms with affective ratings as outcome, and highlight the need for further research examining optimal parameters for concurrent response time measures or alternate modes of non-verbal estimation of conditioned responses in Pavlovian conditioning protocols.

sentences: Mixed Effects Machine Learning Models for Colon Cancer Metastasis Prediction using Spatially Localized Immuno-Oncology Markers Spatially resolved characterization of the transcriptome and proteome promises to provide further clarity on cancer pathogenesis and etiology, which may inform future clinical practice through classifier development for clinical outcomes. However, batch effects may potentially obscure the ability of machine learning methods to derive complex associations within spatial omics data. Profiling thirty-five stage three colon cancer patients using the GeoMX Digital Spatial Profiler, we found that mixed-effects machine learning (MEML) methods{dagger} may provide utility for overcoming significant batch effects to communicate key and complex disease associations from spatial information. These results point to further exploration and application of MEML methods within the spatial omics algorithm development life cycle for clinical deployment.

sentences: Automated Analysis of Neuronal Morphology through an Unsupervised Classification Model of Neurites Brain function emerges from a highly complex network of specialized cells that are interlinked by billions of synapses. The synaptic connectivity between neurons is established between the elongated processes of their axons and dendrites or, together, neurites. To establish these billions of often far-reaching connections, cellular neurites have to grow in highly specialized, cell-type dependent patterns covering often mm distances and connecting with thousands of other neurons. The outgrowth and branching of neurites are tightly controlled during development and are a commonly used functional readout of imaging in the neurosciences. Manual analysis of neuronal morphology from microscopy images, however, is very time intensive and error prone. Especially fully automated segmentation and classification of all neurites remain unavailable in open-source software. Here we present a standalone, GUI-based software for batch-quantification of neuronal morphology in fluorescence micrographs with minimal requirements for user interaction. Neurons are segmented using a Hessian-based algorithm to detect thin neurite structures combined with intensity- and shape-based detection of the cell body. To measure the number of branches in a neuron accurately, rather than just determining branch points, neurites are classified into axon, dendrites and their branches of increasing order by their length using a geodesic distance transform of the cell skeleton. The software was benchmarked against a large, published dataset and reproduced the phenotype observed after manual annotation before. Our tool promises greatly accelerated and improved morphometric studies of neuronal morphology by allowing for consistent and automated analysis of large datasets.

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Profiling acetogenic community dynamics in anaerobic digesters - comparative analyses using next-generation sequencing and T-RFLP Acetogens play a key role in anaerobic degradation of organic material and in maintaining biogas process efficiency. Profiling this community and its temporal changes can help evaluate process stability and function, especially under disturbance/stress conditions, and avoid complete process failure. The formyltetrahydrofolate synthetase (FTHFS) gene can be used as a marker for acetogenic community profiling in diverse environments. In this study, we developed a new high-throughput FTHFS gene sequencing method for acetogenic community profiling and compared it with conventional T-RFLP of the FTHFS gene, 16S rRNA gene-based profiling of the whole bacterial community, and indirect analysis via 16S rRNA profiling of the FTHFS gene-harbouring community. Analyses and method comparisons were made using samples from two laboratory-scale biogas processes, one operated under stable control and one exposed to controlled overloading disturbance. Comparative analysis revealed satisfactory detection of the bacterial community and its changes for all methods, but with some differences in resolution and taxonomic identification. FTHFS gene sequencing was found to be the most suitable and reliable method to study acetogenic communities. These results pave the way for community profiling in various biogas processes and in other environments where the dynamics of acetogenic bacteria have not been well studied.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/427894v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@14ac78org.highwire.dtl.DTLVardef@1472ea3org.highwire.dtl.DTLVardef@46fc19org.highwire.dtl.DTLVardef@50ce7a_HPS_FORMAT_FIGEXP  M_FIG C_FIG ONE SENTENCE SUMMARYOur high-throughput FTHFS gene AmpSeq method for barcoded samples and unsupervised analysis with AcetoScan accurately reveals temporal dynamics of acetogenic community structure in anaerobic digesters.

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Development of an in vitro model for animal species susceptibility to SARS-CoV-2 replication based on expression of ACE2 and TMPRSS2 in avian cells The SARS-CoV-2 (SC2) virus has caused a worldwide pandemic because of the viruss ability to transmit efficiently human-to-human. A key determinant of infection is the attachment of the viral spike protein to the host receptor angiotensin-converting enzyme 2 (ACE2). Because of the presumed zoonotic origin of SC2, there is no practical way to assess every species susceptibility to SC2 by direct challenge studies. In an effort to have a better predictive model of animal host susceptibility to SC2, we expressed the ACE2 and/or transmembrane serine protease 2 (TMPRSS2) genes from humans and other animal species in the avian fibroblast cell line, DF1, that is not permissive to infection. We demonstrated that expression of both human ACE2 and TMPRSS2 genes is necessary to support SC2 infection and replication in DF1 and a non-permissive sub-lineage of MDCK cells. Titers of SC2 in these cell lines were comparable to those observed in control Vero cells. To further test the model, we developed seven additional transgenic cell lines expressing the ACE2 and TMPRSS2 derived from Felis (cat), Equus (horse), Sus (pig), Capra (goat), Mesocricetus (Golden hamster), Myotis lucifugus (Little Brown bat) and Hipposideros armiger (Great Roundleaf bat) in DF1 cells. Results demonstrate permissive replication of SC2 in cat, Golden hamster, and goat species, but not pig or horse, which correlated with the results of reported challenge studies. The development of this cell culture model allows for more efficient testing of the potential susceptibility of many different animal species for SC2 and emerging variant viruses.

IMPORTANCESARS-CoV-2 (SC2) is believed to have originated in animal species and jumped into humans where it has produced the greatest viral pandemic of our time. Identification of animal species susceptible to SC2 infection would provide information on potential zoonotic reservoirs, and transmission potential at the human-animal interface. Our work provides a model system to test the ability of the virus to replicate in an otherwise non-permissive cell line by transgenic insertion of the ACE2 and TMPRSS2 genes from human and other animal species. The results from our in vitro model positively correlate with animal infection studies enhancing the predicative capability of the model. Importantly, we demonstrate that both proteins are required for successful virus replication. These findings establish a framework to test other animal species for susceptibility to infection that may be critical zoonotic reservoirs for transmission, as well as to test variant viruses that arise over time.

sentences: Evaluation of Vicinity-based Hidden Markov Models for Genotype Imputation The decreasing cost of DNA sequencing has led to a great increase in our knowledge about genetic variation. While population-scale projects bring important insight into genotype-phenotype relationships, the cost of performing whole-genome sequencing on large samples is still prohibitive. In-silico genotype imputation coupled with genotyping-by-arrays is a cost-effective and accurate alternative for genotyping of common and uncommon variants. Imputation methods compare the genotypes of the typed variants with the large population-specific reference panels and estimate the genotypes of untyped variants by making use of the linkage disequilibrium patterns. Most accurate imputation methods are based on the Li-Stephens hidden Markov model, HMM, that treats the sequence of each chromosome as a mosaic of the haplotypes from the reference panel. Here we assess the accuracy of local-HMMs, where each untyped variant is imputed using the typed variants in a small window around itself (as small as 1 centimorgan). Locality-based imputation is used recently by machine learning-based genotype imputation approaches. We assess how the parameters of the local-HMMs impact the imputation accuracy in a comprehensive set of benchmarks and show that local-HMMs can accurately impute common and uncommon variants and can be relaxed to impute rare variants as well. The source code for the local HMM implementations is publicly available at https://github.com/harmancilab/LoHaMMer.

sentences: Structural origins of Escherichia coli RNA polymerase open promoter complex stability The first step of gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms "open" complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo but their structural origins remain largely unknown. Here we use cryo-electron microscopy to determine structures of RPo formed de novo at three promoters with widely differing lifetimes at 37{degrees}C: {lambda}PR (t1/2 [~] 10 hours), T7A1 (t1/2 [~] 4 minutes), and a point mutant in {lambda}PR ({lambda}PR-5C) (t1/2 [~] 2 hours). Two distinct RPo conformers are populated at {lambda}PR, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA-RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate-strand "scrunches" inside the active site cleft; the template-strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate RPo lifetime and affect the subsequent steps of the transcription cycle.

sentences: Acquisition of alveolar fate and differentiation competence by human fetal lung epithelial progenitor cells Variation in lung alveolar development is strongly linked to disease susceptibility. However, the cellular and molecular mechanisms underlying alveolar development are difficult to study in humans. Using primary human fetal lungs we have characterized a tip progenitor cell population with alveolar fate potential. These data allowed us to benchmark a self-organising organoid system which captures key aspects of lung lineage commitment and can be efficiently differentiated to alveolar type 2 cell fate. Our data show that Wnt and FGF signalling, and the downstream transcription factors NKX2.1 and TFAP2C, promote human alveolar or airway fate respectively. Moreover, we have functionally validated cell-cell interactions in human lung alveolar patterning. We show that Wnt signalling from differentiating fibroblasts promotes alveolar type 2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. Our organoid system recapitulates key aspects of human lung development allowing mechanistic experiments to determine the underpinning molecular regulation.

sentences: Condensin-driven loop extrusion on supercoiled DNA Condensin, a structural maintenance of chromosomes (SMC) complex, has been shown to be a molecular motor protein that organizes chromosomes by extruding loops of DNA. In cells, such loop extrusion is challenged by many potential conflicts, e.g., the torsional stresses that are generated by other DNA-processing enzymes. It has so far remained unclear how DNA supercoiling affects loop extrusion. Here, we use time-lapse single-molecule imaging to study condensin-driven DNA loop extrusion on supercoiled DNA. We find that condensin binding and DNA looping is stimulated by positive supercoiled DNA where it preferentially binds near the tips of supercoiled plectonemes. Upon loop extrusion, condensin collects all nearby plectonemes into a single supercoiled loop that is highly stable. Atomic force microscopy imaging shows that condensin generates supercoils in the presence of ATP. Our findings provide insight into the topology-regulated loading and formation of supercoiled loops by SMC complexes and clarify the interplay of loop extrusion and supercoiling.

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Discoidin domain receptor 2 drives melanoma drug resistance through AXL-dependent phenotype switching Anti-BRAF plus anti-MEK are used as first-line treatment of patients with metastatic melanomas harboring BRAF V600E mutation. The main issue with targeted therapy is acquired cellular resistance. In 70% of acquired resistance, melanoma cells switch their phenotype and become more aggressive and invasive. The molecular signature of this phenotype is MITF low, AXL high associated with actin cytoskeleton reorganization. After this switch, resistant cells present an anarchic cell proliferation due to MAP kinase pathway hyper-activation. We demonstrate that resistant cell lines presenting phenotype switching overexpress DDR1 and DDR2. We show that DDR2 inhibition induces a decrease in AXL and reduces actin stress fiber formation. Once this phenotype switching is acquired, we report that both DDRs promotes tumor cell proliferation, but only DDR2 can over-activate the MAP kinase pathway in resistant invasive cells in vitro and in vivo. Therefore, DDRs inhibition could be a promising strategy for countering this resistance mechanism.

SignificanceOur results show that DDR2 is a relevant target in melanoma resistance. DDR2 is required at the beginning of resistance for melanoma cell phenotype switching to occur. After phenotype switching, DDRs promote tumor cell proliferation of resistant invasive melanoma cells, but only DDR2 is able to over-activate the MAP kinase pathway. We put forward dasatinib (a DDR inhibitor) as a potential second-line treatment after targeted dual therapy for resistant patients overexpressing DDRs.

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SNAI2-mediated direct repression of BIM protects rhabdomyosarcoma from ionizing radiation Ionizing radiation (IR) and chemotherapy are the mainstays of treatment for patients with rhabdomyosarcoma (RMS). Yet, the molecular mechanisms that underlie the success or failure of radiotherapy remain unclear. The transcriptional repressor SNAI2 was previously identified as a key regulator of IR sensitivity in normal and malignant stem cells through its repression of the proapoptotic BH3-only gene PUMA. Here, we demonstrate a clear correlation between SNAI2 expression levels and radiosensitivity across multiple RMS cell lines. Moreover, modulating SNAI2 levels in RMS cells through its overexpression or knockdown can alter radiosensitivity in vitro and in vivo. SNAI2 expression reliably promotes overall cell growth and inhibits mitochondrial apoptosis following exposure to IR, with either variable or minimal effects on differentiation and senescence, respectively. Importantly, SNAI2 knockdown results in a striking increase in expression of the proapoptotic BH3-only gene BIM, and ChIP-seq experiments establish that SNAI2 is a direct repressor of BIM. Since the P53 pathway is nonfunctional in the RMS cells used in this study, we have identified a new, P53-independent SNAI2/BIM axis that could potentially predict clinical responses to IR treatment and be exploited to improve RMS therapy.

HighlightsO_LISNAI2 expression levels are directly correlated with protection from radiation in rhabdomyosarcoma.
C_LIO_LILoss of SNAI2 primes rhabdomyosarcomas for IR-induced apoptosis.
C_LIO_LISNAI2 directly represses the expression of the proapoptotic BH3-only gene BIM.
C_LI

sentences: Transcriptional expression changes during compensatory plasticity in the prothoracic ganglion of the adult cricket Gryllus bimaculatus Most adult organisms are limited in their capacity to recover from neurological damage. The auditory system of the Mediterranean field cricket, Gryllus bimaculatus, presents a compelling model for investigating neuroplasticity due to its unusual capabilities for structural reorganization into adulthood. Specifically, the dendrites of the central auditory neurons of the prothoracic ganglion sprout in response to the loss of auditory afferents. Deafferented auditory dendrites grow across the midline, a boundary they normally respect, and form functional synapses with the contralateral auditory afferents, restoring tuning-curve specificity. The molecular pathways underlying these changes are entirely unknown. Here, we used a multiple k-mer approach to re-assemble a previously reported prothoracic ganglion transcriptome that included ganglia collected one, three, and seven days after unilateral deafferentation in adult, male animals. We used EdgeR and DESeq2 to perform differential expression analysis and we examined Gene Ontologies to further understand the potential molecular basis of this compensatory anatomical plasticity. Enriched GO terms included those related to protein translation and degradation, enzymatic activity, and Toll signaling. Extracellular space GO terms were also enriched and included the upregulation of several protein yellow family members one day after deafferentation. Investigation of these regulated GO terms help to provide a broader understanding of the types of pathways that might be involved in this compensatory growth and can be used to design hypotheses around identified molecular mechanisms that may be involved in this unique example of adult structural plasticity.

sentences: Independent control of mean and noise by convolution of gene expression distributions Gene expression noise can reduce cellular fitness or facilitate processes such as alternative metabolism, antibiotic resistance, and differentiation. Unfortunately, efforts to study the impacts of noise have been hampered by a scaling relationship between noise and expression level from a single promoter. Here, we use theory to demonstrate that mean and noise can be controlled independently by expressing two copies of a gene from separate inducible promoters in the same cell. We engineer low and high noise inducible promoters to validate this result in Escherichia coli, and develop a model that predicts the experimental distributions. Finally, we use our method to reveal that the response of a promoter to a repressor is less sensitive with higher repressor noise and explain this result using a law from probability theory. Our approach can be applied to investigate the effects of noise on diverse biological pathways or program cellular heterogeneity for synthetic biology applications.

sentences: The Actin Bundling Protein Fascin Promotes Lung Cancer Growth and Metastasis by Enhancing Glycolysis and PFKFB3 Expression Fascin is a pro-metastasis actin bundling protein upregulated in essentially all the metastatic carcinoma. It is believed that fascin promotes cancer cell migration and invasion by facilitating membrane protrusions such as filopodia and invadopodia. Aerobic glycolysis is a key feature of cancer metabolism that provides critical intermediate metabolites for tumor growth and cell proliferation. Here we report that fascin increase glycolysis in lung cancer to promote tumor growth and metastasis. Fascin promotes glycolytic flux by increasing the expression and activities of phosphofructose kinase 1 and 2 (PFK1 and 2). The glycolytic function of fascin depends on activation of YAP1 through its canonical actin bundling activity. Fascin promotes the binding of YAP1 to a TEAD1/4 binding motif located 30 bp upstream of the PFKFB3 transcription start site to activate its transcription. Our interrogation of the TCGA database suggest that the fascin-YAP1-PFKFB3 circuit is likely conserved across different types of cancer. We further showed that the glycolytic function of fascin is essential for promotion of lung cancer growth and metastasis. Importantly, pharmacological inhibitors of fascin could be used to suppress the YAP1-PFKFB3 signaling and inhibit glycolysis in cancer cell lines, organoid cultures and xenograft metastasis models. Taken together, our data reveal an important glycolytic role of fascin in lung cancer metabolism, and suggest that pharmacological inhibitors of fascin could be used to reprogram cancer metabolism in lung cancer and potentially other cancer with fascin upregulation.

sentences: Untangling the complexity of priority effects in multi-species communities The history of species immigration can dictate how species interact in local communities, thereby causing historical contingency in community assembly. Since immigration history is rarely known, these historical influences, or priority effects, pose a major challenge in predicting community assembly. Here, we provide a graph-based, non-parametric, theoretical framework for understanding the predictability of community assembly as affected by priority effects. To develop this frame-work, we first show that the diversity of possible priority effects increases super-exponentially with the number of species. We then point out that, despite this diversity, the consequences of priority effects for multispecies communities can be classified into four basic types, each of which reduces community predictability: alternative stable states, alternative transient paths, compositional cycles, and the lack of escapes from compositional cycles to stable states. Using a neural network, we show that this classification of priority effects enables accurate explanation of community predictability, particularly when each species immigrates repeatedly. We also demonstrate the empirical utility of our theoretical framework by applying it to two experimentally derived assembly graphs of algal and ciliate communities. Based on these analyses, we discuss how the framework proposed here can help guide experimental investigation of the predictability of history-dependent community assembly.

sentences: Tropical land use alters functional diversity of soil food webs and leads to monopolization of the detrital energy channel Agricultural expansion is among the main threats to biodiversity and functions of tropical ecosystems. Conversion of rainforest into plantations erodes biodiversity with little-explored consequences for food-web structure and energetics of belowground communities, and associated ecosystem functions and services. We used a unique combination of approaches in stable isotope analysis and food web energetics to analyze in a comprehensive way consequences of the conversion of rainforest into plantations on the structure of and channeling of energy through soil animal food webs in Sumatra, Indonesia. Across the 23 animal groups studied, the channeling of energy shifted towards freshly-fixed plant carbon in plantations, indicating fast energy channeling as opposed to slow energy channeling in rainforests. Earthworms as major detritivores stayed unchanged in their trophic niche and monopolized the detrital pathway in plantations. Functional diversity metrics of soil food webs reflected the reduced amount of litter, tree density and species richness in plantations, providing guidelines how to improve the complexity of the structure of and channeling of energy through soil food webs. Our results highlight the strong restructuring of soil food webs with the conversion of rainforest into plantations threatening soil functioning and ecosystem stability in the long term.

sentences: Structural Insights into the Venus flytrap Mechanosensitive Ion Channel Flycatcher1 Flycatcher1 (FLYC1), a MscS homolog, has recently been identified as a candidate mechanosensitive (MS) ion channel involved in Venus flytrap prey recognition. FLYC1 is larger and its sequence diverges from previously studied MscS homologs, suggesting it has unique structural features that contribute to its function. Here, we characterized FLYC1 by cryo-electron microscopy, molecular dynamics simulations, and electrophysiology. Akin to bacterial MscS and plant MSL1 channels, we find that FLYC1 central core includes side portals in the cytoplasmic cage that regulate ion conduction, by identifying critical residues that modulate channel conductance. Topologically unique cytoplasmic flanking regions can adopt  up or  down conformations, making the channel asymmetric. Disruption of an up conformation-specific interaction severely delays channel deactivation by 40-fold likely due to stabilization of the channel open state. Our results illustrate novel structural features and likely conformational transitions that regulate mechano-gating of FLYC1.

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SurvBenchmark: comprehensive benchmarking study of survival analysis methods using both omics data and clinical data Survival analysis is a branch of statistics that deals with both, the tracking of time and of the survival status simultaneously as the dependent response. Current comparisons of survival model performance mostly center on clinical data with classic statistical survival models, with prediction accuracy often serving as the sole metric of model performance. Moreover, survival analysis approaches for censored omics data have not been thoroughly investigated. The common approach is to binarise the survival time and perform a classification analysis.

Here, we develop a benchmarking framework, SurvBenchmark, that evaluates a diverse collection of survival models for both clinical and omics datasets. SurvBenchmark not only focuses on classical approaches such as the Cox model, but it also evaluates state-of-art machine learning survival models. All approaches were assessed using multiple performance metrics, these include model predictability, stability, flexibility and computational issues. Our systematic comparison framework with over 320 comparisons (20 methods over 16 datasets) shows that the performances of survival models vary in practice over real-world datasets and over the choice of the evaluation metric. In particular, we highlight that using multiple performance metrics is critical in providing a balanced assessment of various models. The results in our study will provide practical guidelines for translational scientists and clinicians, as well as define possible areas of investigation in both survival technique and benchmarking strategies.

Contactjean.yang@sydney.edu.au

sentences: dynDeepDRIM: a dynamic deep learning model to infer direct regulatory interactions using single cell time-course gene expression data Time-course single-cell RNA sequencing (scRNA-seq) data have been widely applied to reconstruct the cell-type-specific gene regulatory networks by exploring the dynamic changes of gene expression between transcription factors (TFs) and their target genes. The existing algorithms were commonly designed to analyze bulk gene expression data and could not deal with the dropouts and cell heterogeneity in scRNA-seq data. In this paper, we developed dynDeepDRIM that represents gene pair joint expression as images and considers the neighborhood context to eliminate the transitive interactions. dynDeepDRIM integrated the primary image, neighbor images with time-course into a four-dimensional tensor and trained a convolutional neural network to predict the direct regulatory interactions between TFs and genes. We evaluated the performance of dynDeepDRIM on five time-course gene expression datasets. dynDeepDRIM outperformed the state-of-the-art methods for predicting TF-gene direct interactions and gene functions. We also observed gene functions could be better performed if more neighbor images were involved.

sentences: PharmaNet: Pharmaceutical discovery with deep recurrent neural networks. The discovery and development of novel pharmaceuticals is an area of active research mainly due to the large investments required and long payback times. As of 2016, the development of a novel drug candidate required up to $ USD 2.6 billion in investment for only 10% rate of approval by the FDA. To help decreasing the costs associated with the process, a number of in silico approaches have been developed with relatively low success due to limited predicting performance. Here, we introduced a machine learning-based algorithm as an alternative for a more accurate search of new pharmacological candidates, which takes advantage of Recurrent Neural Networks (RNN) for active molecule prediction within large databases. Our approach, termed PharmaNet was implemented here to search for ligands against specific cell receptors within 102 targets of the DUD-E database, which contains 22886 active molecules. PharmaNet comprises three main phases. First, a SMILES representation of the molecule is converted into a raw molecular image. Second, a convolutional encoder processes the data to obtain a fingerprint molecular image that is finally analyzed by a Recurrent Neural Network (RNN). This approach enables precise predictions of the molecules target on the basis of the feature extraction, the sequence analysis and the relevant information filtered out throughout the process. Molecule Target prediction is a highly unbalanced detection problem and therefore, we propose that an adequate evaluation metric of performance is the area under the Normalized Average Precision (NAP) curve. PharmaNet largely surpasses the previous state-of-the-art method with 97.7% in the Receiver Operating Characteristic curve (ROC-AUC) and 65.5% in the NAP curve. We obtained a perfect performance for human farnesyl pyrophosphate synthase (FPPS), which is a potential target for antimicrobial and anticancer treatments. We decided to test PharmaNet for activity prediction against FPPS by searching in the CHEMBL data set. We obtained three (3) potential inhibitors that were further validated through both molecular docking and in silico toxicity prediction. Most importantly, one of this candidates, CHEMBL2007613, was predicted as a potential antiviral due to its involvement on the PCDH17 pathway, which has been reported to be related to viral infections.

sentences: Development and Validation of Fluorescently Labeled, Functional Type I Collagen Molecules While de novo collagen fibril formation is well-studied, there are few investigations into the growth and remodeling of extant fibrils, where molecular collagen incorporation into and erosion from the fibril surface must delicately balance during fibril growth and remodeling. Observing molecule/fibril interactions is difficult, requiring the tracking of molecular dynamics while, at the same time, minimizing the effect of the observation on fibril structure and assembly. To address the observation-interference problem, exogenous collagen molecules are tagged with small fluorophores and the fibrillogenesis kinetics of labeled collagen molecules as well as the structure and network morphology of assembled fibrils are quantified for the first time. While excessive labeling significantly disturbs fibrillogenesis kinetics and network morphology of assembled fibrils, adding less than ~1.2 labels preserves them. Applications of the functional, labeled collagen probe are demonstrated in both cellular and acellular systems. The functional, labelled collagen associates strongly with native fibrils and when added to an in vitro model of corneal stromal development, is endocytosed rapidly by cells and is translocated into synthesized matrix networks within 24 hours.

sentences: Evidence for adaptive evolution in the receptor-binding domain of seasonal coronaviruses OC43 and 229E Seasonal coronaviruses (OC43, 229E, NL63 and HKU1) are endemic to the human population, regularly infecting and reinfecting humans while typically causing asymptomatic to mild respiratory infections. It is not known to what extent reinfection by these viruses is due to waning immune memory or antigenic drift of the viruses. Here, we address the influence of antigenic drift on immune evasion of seasonal coronaviruses. We provide evidence that at least two of these viruses, OC43 and 229E, are undergoing adaptive evolution in regions of the viral spike protein that are exposed to human humoral immunity. This suggests that reinfection may be due, in part, to positively-selected genetic changes in these viruses that enable them to escape recognition by the immune system. It is possible that, as with seasonal influenza, these adaptive changes in antigenic regions of the virus would necessitate continual reformulation of a vaccine made against them.

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Single domain antibodies against enteric pathogen virulence factors are active as curli fiber fusions on probiotic E. coli Nissle 1917 Enteric microbial pathogens, including Escherichia coli, Shigella and Cryptosporidium species, take a particularly heavy toll in low-income countries and are highly associated with infant mortality. We describe here a means to display anti-infective agents on the surface of a probiotic bacterium. Because of their stability and versatility, VHHs, the variable domains of camelid heavy-chain-only antibodies, have potential as components of novel agents to treat or prevent enteric infectious disease. We isolated and characterized VHHs targeting several enteropathogenic Escherichia.coli (EPEC) virulence factors: flagellin (Fla), which is required for bacterial motility and promotes colonization; both intimin and the translocated intimin receptor (Tir), which together play key roles in attachment to enterocytes; and E. coli secreted protein A (EspA), an essential component of the type III secretion system (T3SS) that is required for virulence. Several VHHs that recognize Fla, intimin, or Tir blocked function in vitro. The probiotic strain E. coli Nissle 1917 (EcN) produces on the bacterial surface curli fibers, which are the major proteinaceous component of E. coli biofilms. A subset of Fla-, intimin-, or Tir-binding VHHs, as well as VHHs that recognize either a T3SS of another important bacterial pathogen (Shigella flexneri), a soluble bacterial toxin (Shiga toxin or Clostridioides difficile toxin TcdA), or a major surface antigen of an important eucaryotic pathogen (Cryptosporidium parvum) were fused to CsgA, the major curli fiber subunit. Scanning electron micrographs indicated CsgA-VHH fusions were assembled into curli fibers on the EcN surface, and Congo Red binding indicated that these recombinant curli fibers were produced at high levels. Ectopic production of these VHHs conferred on EcN the cognate binding activity and, in the case of anti-Shiga toxin, was neutralizing. Taken together, these results demonstrate the potential of the curli-based pathogen sequestration strategy described herein and contribute to the development of novel VHH-based gut therapeutics.

Author SummaryEnteric pathogens are the causative agents of diarrheal disease - a leading cause of infant morbidity and mortality worldwide. While treatment and prevention options such as drugs or vaccines exist for some pathogens, their efficacy and availability are often limited. New therapeutic strategies are therefore needed, especially inexpensive agents in low-income countries where enteric disease burdens are highest. One promising avenue for novel treatments uses VHHs - highly stable, well-expressed, antibody domains derived from camelid species such as llamas and alpacas. The small size, high stability and simple structure of these antibody fragments enables their streamlined production by bacteria such as E. coli, potentially reducing cost and improving scalability. In this work, we describe the development of VHHs targeting multiple virulence factor proteins of pathogenic E. coli and other leading causes of diarrheal disease. These VHHs provide new tools for the research community and may serve as promising components of agents that prevent or treat pathogen infections. Towards that goal, we engineered a novel system in which the probiotic, mucus-establishing bacterial strain E. coli Nissle 1917 (EcN) is used to express and display VHHs at high density on its surface. By demonstrating the ability of these engineered EcN to bind to pathogens, we provide a first step toward using such probiotics as a cheap, simple, and effective treatment for enteric pathogen infections.

sentences: Cross-seeding Controls Aβ Fibril Populations and Resulting Function Amyloid peptides nucleate from monomers to aggregate into fibrils through primary nucleation; pre-existing fibrils can then act as seeds for additional monomers to fibrillize through secondary nucleation. Both nucleation processes can occur simultaneously, yielding a distribution of fibril polymorphs that can generate a spectrum of neurodegenerative effects. Understanding the mechanisms driving polymorph structural distribution during both nucleation processes is important for uncovering fibril structure-function relationships, as well creating polymorph distributions in vitro that better match distributions found in vivo. Here, we explore how cross-seeding WT A{beta}1-40 with A{beta}1-40 mutants E22G (Arctic) and E22{Delta} (Osaka), as well as with WT A{beta}1-42 affects the distribution of fibril structural polymorphs, and how changes in structural distribution impact toxicity. Transmission electron microscopy analysis reveals that fibril seeds derived from mutants of A{beta}1-40 impart their structure to WT A{beta}1-40 monomer during secondary nucleation, but WT A{beta}1-40 fibril seeds do not affect the structure of fibrils assembled from mutant A{beta}1-40 monomers, despite kinetics data indicating accelerated aggregation when cross-seeding of any combination of mutants. Additionally, WT A{beta}1-40 fibrils seeded with mutant fibrils to produce similar structural distributions to the mutant seeds also produced similar cytotoxicity on neuroblastoma cell lines. This indicates that mutant fibril seeds not only impart their structure to growing WT A{beta}1-40 aggregates, but they also impart cytotoxic properties. Our findings provide clear evidence that there is a relationship between fibril structure and phenotype on a polymorph population level, and that these properties can be passed on through secondary nucleation of succeeding generations of fibrils.

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Omicron-Based Vaccine Candidate Elicits Potent Neutralizing Antibodies in the Animal Model The B.1.1.529 (Omicron) variant of SARS-CoV-2 with multiple novel mutations has reduced the neutralization potential of vaccinated individuals sera. World Health Organization has suggested that a vaccination strategy based on repeated booster doses is unlikely to be sustainable. The objective of this commentary was to investigate the safety and neutralizing antibody induction of the Omicron-based SARS-CoV-2 vaccine candidate, BIV1-CovIran Plus, against the Omicron variant on mice and guinea pig models.

After isolation and characterization, the Omicron variant underwent chemical inactivation, purification, and was then formulated with alum adjuvant. A full human dose of BIV1-CovIran Plus was injected intraperitoneally to five female mice and two Guinea pigs for abnormal toxicity reactions evaluation and pathologic investigations. For potency evaluation, four groups of ten mice received two doses of BIV1-CovIran Plus or phosphate-buffered-saline at 7-day and 14-day intervals. The conventional virus-neutralizing test was conducted on sera acquired from vaccinated mice groups seven days after the second injection.

There was no evidence of abnormal clinical symptoms macroscopic or microscopic tissue alterations among the animal models. In all samples from the study group that received two doses of BIV1-CovIran Plus at a 7-day interval, the sera at [≥]1/32 times dilution would neutralize the Omicron variant SARS-CoV-2. Similarly, the sera of all samples from the study group, which received two doses of BIV1-CovIran Plus at a 14-day interval, at [≥]1/64 times dilution, would neutralize the Omicron variant SARS-CoV-2. Moreover, six out of ten (60%) of the samples in this group would neutralize the Omicron variant of SARS-CoV-2 at 1/128 times dilution. CPE formation was observed in all samples from the control group, and no neutralizing activity was detected at any sera dilutions.

BIV1-CovIran Plus was well-tolerated in the animal models, and no safety concerns were raised. Moreover, the vaccine candidate elicited protective neutralization against the Omicron variant. Future reports will focus on the use of the updated vaccine as a booster dose and the potency of the vaccine candidate on other SARS-CoV-2 variants.

sentences: Alprazolam modulates persistence energy during emotion processing in first-degree relatives of individuals with schizophrenia: a network control study Schizophrenia is marked by deficits in facial affect processing associated with abnormalities in GABAergic circuitry, deficits also found in first-degree relatives. Facial affect processing involves a distributed network of brain regions including limbic regions like amygdala and visual processing areas like fusiform cortex. Pharmacological modulation of GABAergic circuitry using benzodiazepines like alprazolam can be useful for studying this facial affect processing network and associated GABAergic abnormalities in schizophrenia. Here, we use pharmacological modulation and computational modeling to study the contribution of GABAergic abnormalities toward emotion processing deficits in schizophrenia. Specifically, we apply principles from network control theory to model persistence energy - the control energy required to maintain brain activation states - during emotion identification and recall tasks, with and without administration of alprazolam, in a sample of first-degree relatives and healthy controls. Here, persistence energy quantifies the magnitude of theoretical external inputs during the task. We find that alprazolam increases persistence energy in relatives but not in controls during threatening face processing, suggesting a compensatory mechanism given the relative absence of behavioral abnormalities in this sample of unaffected relatives. Further, we demonstrate that regions in the fusiform and occipital cortices are important for facilitating state transitions during facial affect processing. Finally, we uncover spatial relationships (i) between regional variation in differential control energy (alprazolam versus placebo) and (ii) both serotonin and dopamine neurotransmitter systems, indicating that alprazolam may exert its effects by altering neuromodulatory systems. Together, these findings reveal differences in emotion-processing circuitry associated with genetic vulnerability to schizophrenia.

sentences: Megakaryocyte derived immune-stimulating cells regulate host-defense immunity against bacterial pathogens Megakaryocytes (MKs) continuously produce platelets to support hemostasis and form a niche for hematopoietic stem cell maintenance in the bone marrow. MKs are also involved in inflammation responses; however, the mechanism remains poorly understood. Here, using single-cell sequencing we identified an MK-derived immune-stimulating cell (MDIC) population exhibiting both MK-specific and immune characteristics, which highly expresses CXCR4 and immune response genes to participate in host-protective response against bacteria. MDICs interact with myeloid cells to promote their migration and stimulate the bacterial phagocytosis of macrophages and neutrophils by producing TNF and IL-6. CXCR4high MDICs egress circulation and infiltrate into the spleen, liver, and lung upon bacterial infection. Ablation of MKs suppresses the innate immune response and T cell activation to impair the anti-bacterial effects in mice under the Listeria monocytogenes challenge. Using hematopoietic stem/progenitor cell lineage-tracing mouse line, we show that MDICs are generated from infection-induced emergency megakaryopoiesis in response to bacterial infection. Overall, we identify MDICs as an MK subpopulation, which regulates host-defense immune response against bacterial infection.

sentences: Snake Fungal Disease Caused by the Fungal Pathogen Ophidiomyces ophidiicola in Texas The pathogen Ophidiomyces ophidiicola (O.o.), widely known as the primary cause of snake fungal disease (SFD) has been detected in Texass naive snakes. Our team set out to begin to characterize O. ophidiicolas spread in east Texas. From July 2019 until October 2021, we sampled 176 snakes across east Texas and detected 27 positives cases (qPCR confirmed 27/176). From a ribbon snake with clear clinical display, we isolated and cultured what we believe to be the Texas isolate of O. ophidiicola. With over 1/10 snakes that may be infected in East Texas, gives credence to the onset of SFD in Texas.

sentences: Characterization of a miniaturized ultrasound transducer customized for oral health applications Periodontitis affects nearly 50% of Americans. Conventional clinical assessment based on periodontal probing remains the gold standard despite being operator-dependent, time-consuming, and often painful. In contrast, ultrasound imaging is non-invasive, portable, low-cost, and free of ionizing radiation. Unfortunately, most conventional ultrasound transducers can only access anterior teeth due to the size of the imaging equipment. Here, we characterized a novel and customized periodontal ultrasound transducer SS-19-128 that can examine the entire human mouth. The transducer is 1.8 cm width by 1 cm height, including 128 channels with a center frequency of 19 MHz. The axial and lateral spatial resolution at 5.5 mm depth is 102.1 m and 142.9 m, respectively. The penetration depth in tissue mimicking phantom is over 31 mm. Imaging was performed on 3 human subjects, and statistical analysis on swine jaws indicated a high agreement with the gold standard.

sentences: Lysosome Lipid Signaling from the Periphery to Neurons Regulates Longevity Lysosomes are key cellular organelles that metabolize extra- and intracellular substrates. Alterations in lysosomal metabolism are implicated in aging-associated metabolic and neurodegenerative diseases. However, how lysosomal metabolism actively coordinates the metabolic and nervous systems to regulate aging remains unclear. Here, we report a fat-to-neuron lipid signaling pathway induced by lysosomal metabolism and its longevity promoting role in Caenorhabditis elegans. We discovered that lysosomal lipolysis in peripheral fat storage tissue up-regulates the neuropeptide signaling pathway in the nervous system to promote longevity. This cell-non-autonomous regulation requires the secretion from the fat storage tissue of a lipid chaperone protein LBP-3 and polyunsaturated fatty acids (PUFAs). LBP-3 binds to specific PUFAs, and acts through a nuclear hormone receptor NHR-49 and neuropeptide NLP-11 in neurons to extend lifespan. Together, these results reveal lysosomes as a signaling hub to coordinate metabolism and aging, and a lysosomal signaling mechanism that mediates intertissue communication to promote longevity.

sentences: Contextual associations represented both in neural networks and human behavior Contextual associations facilitate object recognition in human vision. However, the role of context in artificial vision remains elusive as does the characteristics that humans use to define context. We investigated whether contextually related objects (bicycle-helmet) are represented more similarly in convolutional neural networks (CNNs) used for image understanding than unrelated objects (bicycle-fork). Stimuli were of objects against a white background and consisted of a diverse set of contexts (N=73). CNN representations of contextually related objects were more similar to one another than to unrelated objects across all CNN layers. Critically, the similarity found in CNNs correlated with human behavior across three experiments assessing contextual relatedness, emerging significant only in the later layers. The results demonstrate that context is inherently represented in CNNs as a result of object recognition training, and that the representation in the later layers of the network tap into the contextual regularities that predict human behavior.

sentences: Stress relaxation amplitude of hydrogels determines migration, proliferation, and morphology of cells in 3-D The viscoelastic behavior of hydrogel matrices sensitively influences the cell behavior in 3D culture and biofabricated tissue model systems. Previous reports have demonstrated that cells tend to adhere, spread, migrate and proliferate better in hydrogels with pronounced stress relaxation. However, it is currently unknown if cells respond more sensitively to the amplitude of stress relaxation, or to the relaxation time constant. To test this, we compare the behavior of fibroblasts cultured for up to 10 days in alginate and oxidized alginate hydrogels with similar Youngs moduli but diverging stress relaxation behavior. We find that fibroblasts elongate, migrate and proliferate better in hydrogels that display a higher stress relaxation amplitude. By contrast, the cells response to the relaxation time constant was less pronounced and less consistent. Together, these data suggest that it is foremost the stress relaxation amplitude of the matrix that determines the ability of cells to locally penetrate and remodel the matrix, which subsequently leads to better spreading, faster migration, and higher cell proliferation. We conclude that the stress relaxation amplitude is a central design parameter for optimizing cell behavior in 3-D hydrogels.

sentences: Structure-based identification of naphthoquinones and derivatives as novel inhibitors of main protease Mpro and papain-like protease PLpro of SARS-CoV-2 The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In the present work, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 {micro}M and 66 {micro}M. In addition, eight compounds inhibited PLpro with IC50 ranging from 1.7 {micro}M to 46 {micro}M. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

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Pathogen-derived 9-methyl sphingoid base is perceived by a lectin receptor kinase in Arabidopsis In plants, many invading microbial pathogens are recognized by cell-surface pattern recognition receptors (PRRs), inducing defense responses; yet how PRRs perceive pathogen sphingolipids remains unclear. Here, we show that the ceramide Pi-Cer D from a plant pathogenic oomycete Phytophthora infestans triggers defense responses in Arabidopsis. Pi-Cer D is cleaved by an Arabidopsis apoplastic ceramidase, NCER2, and the resulting 9-methyl-branched sphingoid base is recognized by a plasma membrane lectin receptor-like kinase, RDA2. Importantly, 9-methyl-branched sphingoid base, which is unique to microbes, induces plant immune responses by interacting with RDA2. Loss of RDA2 or NCER2 function compromised Arabidopsis resistance against an oomycete pathogen, indicating that these are crucial for defense. We provide new insights that help elucidate the recognition mechanisms of pathogen-derived lipid molecules in plants.

One Sentence SummaryOomycete-derived ceramide is cleaved into sphingoid base by ceramidase and recognized by an Arabidopsis receptor kinase.

sentences: Super-enhancer signature reveals key mechanisms associated with resistance to non-alcoholicsteatohepatitis in humans with obesity The molecular underpinnings of non-alcoholic steatohepatitis (NASH) development in patients are poorly understood. Active enhancer landscapes are known to determine cell states and behaviors. Super-enhancers, in particular, have helped reveal key disease drivers in several cancer types; however, they remain unexplored in human NASH. To define the enhancer signature of NASH-prone (NP) and NASH-resistant (NR) phenotypes in humans with obesity, we performed chromatin run-on sequencing (ChRO-seq) analysis on liver biopsies of individuals with obesity who were stratified into either NP or NR. We first demonstrated that NP and NR groups exhibit distinct active enhancer signatures. The subsequent identification of NP- and NR-specific super-enhancers revealed the specific genes that are likely the most critical for each of the phenotypes, including HES1 for NP and GATM for NR. Integrative analysis with results from genome-wide association studies of NAFLD and related traits identified disease/trait-loci specific to NP or NR enhancers. Further analysis of the ChRO-seq data pointed to critical roles for serine/glycine metabolism in NASH resistance, which was corroborate by profiling of circulating amino acids in the same patients. Overall, the distinct enhancer signatures of human NP and NR phenotypes revealed key genes, pathways, and transcription factor networks that promote NASH development.

sentences: The Specious Art of Single-Cell Genomics Dimensionality reduction is standard practice for filtering noise and identifying relevant dimensions in large-scale data analyses. In biology, single-cell expression studies almost always begin with reduction to two or three dimensions to produce  all-in-one visuals of the data that are amenable to the human eye, and these are subsequently used for qualitative and quantitative analysis of cell relationships. However, there is little theoretical support for this practice. We examine the theoretical and practical implications of low-dimensional embedding of single-cell data, and find extensive distortions incurred on the global and local properties of biological patterns relative to the highdimensional, ambient space. In lieu of this, we propose semi-supervised dimensionality reduction to higher dimension, and show that such targeted reduction guided by the metadata associated with single-cell experiments provides useful latent space representations for hypothesis-driven biological discovery.

sentences: Epidermal TRPV4 ion channels regulate UVB induced sunburn by triggering inflammmasome activation and MAPK signaling Skin inflammation is an evolutionary-honed protective mechanism that serves to clear noxious cues and irritants and initiate regeneration. Calcium-permeable transient-receptor-potential (TRP) ion channels have critical functions in sensory transduction which is sensitized in skin inflammation. Skin sensory transduction relies on skin-innervating sensory neurons in the dorsal root ganglion (DRG), but also on innervated keratinocytes (KC). The multimodally-activated TRPV4 is robustly expressed in KC, where it can readily be activated by Ultraviolet-B (UVB). Our goal was to deconstruct keratinocyte TRPV4-mediated signaling, specifically how TRPV4 can facilitate inflammatory injury, thus lowering pain thresholds and rendering KC into pain-generator cells. We wanted to uncover the effect of TRPV4-mediated signaling on UVB-induced inflammasome activation in KC given the powerful impact of the activated inflammasome on pro-inflammatory/pro-algesic secretory signaling, using mouse models and cultured human KC. In mice, our evidence suggests that TRPV4 functions as calcium-permeable channel upstream of the KC inflammasome. Furthermore, we found that UVB induced activation of TRPV4 caused rapid - within minutes - Extracellular Signal Regulated Kinase (ERK) phosphorylation, caspase-1 activation and Interleukin-1{beta} (IL-1{beta}) secretion. In human primary KC we demonstrated that UVB induced secretion of IL-1{beta} was dependent on the NLR family pyrin domain containing 1 (NLRP1) inflammasome. Direct chemical TRPV4 activation could also activate NLRP1 and to lesser extent NLPR3. Building on our previous work, we now define at increased resolution TPRV4-dependent forefront signaling mechanisms in KC in response to UVB, showing TRPV4 upstream of the NLRP1 inflammasome, subsequent rapid ERK activation and pro-inflammatory/pro-algesic secretory function.

sentences: Cytotoxic and micronuclei inducing effects of petroleum ether fraction of leaf aqueous extract of Clerodendrum viscosum Vent. in Allium cepa root tip cells Clerodendrum viscosum is a traditionally used medicinal plant. The present study aimed to analyze a detailed cytotoxic effect of the nonpolar petroleum ether fraction (AQPEF) of leaf aqueous extract of C. viscosum Vent. (LAECV) in Allium cepa root tip cells. The LAECV was fractionated with petroleum ether and tested for A. cepa toxicity at early hours (2 and 4 h treatment) at concentrations 0, 50, 100, 150 and 200 {micro}g mL-1. The highest aberrant cell percentage (10.45{+/-}0.46%) was scored from 150 {micro}g mL-1 followed by100 {micro}g mL-1 (8.75{+/-}0.26%) concentration at 4 h treated samples. The AQPEF treatment induced a significant (p<0.0001) increase in micronuclei frequency; 4.31{+/-}0.33, 5.08{+/-}0.13, 5.05{+/-}0.22 and 3.05{+/-}0.37% respectively at concentrations 50, 100, 150, and 200 {micro}g mL-1. The highest polyploid frequency (20.14{+/-}0.68 %) induced with 100 {micro}g mL-1 of AQPEF at 16 h recovery. 150 {micro}g mL-1 is the most effective concentration of AQPEF to decipher its activity similar to colchicine (150 {micro}g mL-1). In summary, the present study indicates petroleum ether is suitable for extraction of the active phytochemicals of LAECV having cytotoxic effects on A. cepa root tip cells. The AQPEF has colchicine like micronuclei, polyploidy, and mitotic abnormality inducing potentials in A. cepa root apical meristem cells.

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Absence of muricholic acid due to Cyp2c-deficiency protects against high fat diet-induced obesity in male mice but promotes liver damage ObjectiveMurine-specific muricholic acids (MCAs) are reported to protect against obesity and associated metabolic disorders. However, the response of mice with genetic depletion of MCA to an obesogenic diet has not been evaluated. We used Cyp2c-deficient (Cyp2c-/-) mice, which lack MCAs and thus have a human-like bile acid (BA) profile, to directly investigate the potential role of MCAs in diet-induced obesity.

MethodsMale and female Cyp2c-/- mice and wild-type controls were fed a standard chow diet or a high fat diet (HFD) for 18 weeks. We measured BA composition from a pool of liver, gallbladder, and intestine, as well as weekly body weight, food intake, lean and fat mass, systemic glucose homeostasis, energy expenditure, intestinal lipid absorption, fecal lipid, and energy content.

ResultsCyp2c deficiency depleted MCAs and caused other changes in BA composition, namely a decrease in the ratio of 12-hydroxylated (12-OH) BAs to non-12-OH BAs, without altering the total BA levels. While wild-type male mice became obese after HFD-feeding, Cyp2c-/- male mice were protected from obesity and associated metabolic dysfunctions. Cyp2c-/- male mice also showed reduced intestinal lipid absorption and increased lipid excretion, which was reversed by oral gavage with the 12-OH BA, taurocholic acid. Cyp2c-/- mice also showed increased liver damage, which appeared stronger in females.

ConclusionMCA does not protect against diet-induced obesity but may protect against liver injury. Reduced lipid absorption in Cyp2c-deficient male mice is potentially due to a reduced ratio of 12-OH/non-12-OH BAs.

sentences: S-Palmitoylation-Dependent Regulation of Cardiomyocyte Rac1 Signaling Activity and Cardiac Hypertrophy S-palmitoylation is a reversible lipid modification that regulates trafficking, localization, activity, and/or stability of protein substrates by serving as a fatty acid anchor to cell membranes. However, S-palmitoylation-dependent control of signal transduction in cardiomyocytes and its effects on cardiac physiology are not well understood. We performed an in vivo gain-of-function screen of zinc finger Asp-His-His-Cys (zDHHC) family S-acyl transferases that catalyze S-palmitoylation and identified the Golgi-localized enzyme zDHHC3 as a critical regulator of cardiac maladaptation. The closely-related enzyme, zDHHC7, also induced severe cardiomyopathy but this effect was not observed with overexpression of plasma membrane enzyme zDHHC5, endoplasmic reticulum enzyme zDHHC6, or Golgi enzyme zDHHC13. To identify effectors that may underlie zDHHC3-induced cardiomyopathy we performed quantitative site-specific S-acyl proteomics in zDHHC3-overexpressing cells that revealed the small GTPase Rac1 as a novel substrate. We generated cardiomyocyte-specific transgenic mice overexpressing zDHHC3, which develop severe cardiac disease. Cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice are preceded by enhanced S-palmitoylation of Rac1 and induction of additional Rho family small GTPases including RhoA, Cdc42, and the Rho family-specific chaperone RhoGDI. In contrast, transgenic mice overexpressing an enzymatically-dead mutant of zDHHC3 do not exhibit this profound induction of RhoGTPase signaling or develop cardiac disease. Rac1 S-palmitoylation, plasma membrane localization, activity, and downstream hypertrophic signaling were substantially increased in zDHHC3 overexpressing hearts. Taken together, these data suggest inhibition of zDHHC3/7 S-acyl transferase activity at the cardiomyocyte Golgi or disruption of Rac1 S-palmitoylation as novel therapeutic strategies to treat cardiac disease or other diseases associated with enhanced RhoGTPase signaling.

sentences: Plasmodium falciparum vacuolar pyrophosphatase 1 for ring stage development and its transition to trophozoite The malaria parasite relies on anaerobic glycolysis for energy supply when growing inside RBCs as its mitochondrion does not produce ATP. The ring stage lasts [~] 22 hours and is traditionally thought to be metabolically quiescent. However, recent studies show that the ring stage is active for several energy-costly processes including gene transcription/translation, protein export, and movement inside the RBC. It has remained unclear if a low glycolytic flux can meet the energy demand of the ring stage. Here we show that the metabolic by-product, pyrophosphate, is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to pump protons across the parasite plasma membrane to maintain the membrane potential and cytosolic pH. Conditional deletion of PfVP1 leads to delayed ring stage development and a complete blockage of the ring-to-trophozoite transition, which can be partially rescued by Arabidopsis thaliana vacuolar pyrophosphatase 1, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae. Proton-pumping pyrophosphatases are absent in humans, which highlights the possibility of developing highly selective VP1 inhibitors against the malaria parasite.

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The evolutionary origin of host association in an ancient bacterial clade The evolution of obligate host-association of bacterial symbionts and pathogens remains poorly understood. The Rickettsiales represent an order of obligate alphaproteobacterial endosymbionts and parasites that infect a wide variety of eukaryotic hosts, including humans, livestock, insects and protists. Induced by their host-associated lifestyle, Rickettsiales genomes have undergone reductive evolution, leading to small, AT-rich genomes with limited metabolic capacities. We describe several genomes of deep-branching, environmental alphaproteobacteria that branch basal to previously sampled Rickettsiales, and whose genome content are reminiscent of free-living and biofilm-associated lifestyles. Ancestral genome content reconstruction across the Rickettsiales tree revealed that the free-living to host-association transition of this group occurred more recently than previously anticipated, and likely involved the repurposing of a type IV secretion system.

One-Sentence SummaryDeep-branching Rickettsiales provide insights into the evolution of obligate host-associated lifestyle

sentences: Simultaneous Morphology, Motility and Fragmentation Analysis of Live Individual Sperm Cells for Male Fertility Evaluation We present a new technique for sperm analysis on individual unstained live cells, measuring DNA fragmentation, morphology with virtual staining, and motility. The method relies on quantitative stain-free interferometric imaging and deep-learning frameworks, and is utilized for male fertility evaluation. In the common clinical practice, only motility evaluation is carried out on live human cells, while full morphological evaluation and DNA fragmentation assays require different staining protocols, and therefore cannot be performed on the same cell, resulting in inconsistencies in fertility evaluation. We use a clinic-ready interferometric module to acquire dynamic sperm cells without chemical staining, and deep learning to evaluate all three scores per cell. We show that the number of cells that pass each criterion separately does not accurately predict how many would pass all criteria. This stain-free evaluation method is expected to decrease uncertainty in infertility diagnosis, increasing treatment success rates.

sentences: mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits comparable B cell expansion, neutralizing antibodies and protection against Omicron SARS-CoV-2 Omicron is highly transmissible and has substantial resistance to antibody neutralization following immunization with ancestral spike-matched vaccines. It is unclear whether boosting with Omicron-specific vaccines would enhance immunity and protection. Here, nonhuman primates that received mRNA-1273 at weeks 0 and 4 were boosted at week 41 with mRNA-1273 or mRNA-Omicron. Neutralizing antibody titers against D614G were 4760 and 270 reciprocal ID50 at week 6 (peak) and week 41 (pre-boost), respectively, and 320 and 110 for Omicron. Two weeks after boost, titers against D614G and Omicron increased to 5360 and 2980, respectively, for mRNA-1273 and 2670 and 1930 for mRNA-Omicron. Following either boost, 70-80% of spike-specific B cells were cross-reactive against both WA1 and Omicron. Significant and equivalent control of virus replication in lower airways was observed following either boost. Therefore, an Omicron boost may not provide greater immunity or protection compared to a boost with the current mRNA-1273 vaccine.

sentences: Bioinformatics analyses of significant genes, related pathways and candidate prognostic biomarkers in Alzheimer disease Alzheimers disease (AD) is one of the most common causes of dementia and frailty. This study aimed to use bioinformatics analysis to identify differentially expressed genes (DEGs) in AD. The Expression profiling by high throughput sequencing dataset GSE125583 was downloaded from the Gene Expression Omnibus (GEO) database and DEGs were identified. After assessment of Gene Ontology (GO) terms and pathway enrichment for DEGs, a protein-protein interaction (PPI) network, module analysis, miRNA-hub gene regulatory network construction and TF-hub gene regulatory network were conducted via comprehensive target prediction and network analyses. Finally, we validated hub genes by receiver operating characteristic curve (ROC). In total, 956 DEGs were identified in the AD samples, including 479 up regulated genes and 477 down regulated genes. Functional enrichment analysis showed that these DEGs are mainly involved in the neuronal system, GPCR ligand binding, regulation of biological quality and cell communication. The hub genes of PAK1, ELAVL2, NSF, HTR2C, TERT, UBD, MKI67, HSPB1, PYHIN1 and TES might be associated with AD. The diagnostic value and expression levels of these hub genes in AD were further confirmed by ROC analysis. In conclusion, we identified pathways and crucial candidate genes that affect the outcomes of patients with AD, and these genes might serve as potential therapeutic targets.

sentences: Coupled protein synthesis and ribosome-guided piRNA processing on mRNAs PIWI-interacting small RNAs (piRNAs) protect the germline genome and are essential for fertility. Previously, we showed that ribosomes guide the biogenesis of piRNAs from long non-coding RNAs (lncRNAs) after translating the short open reading frames (ORFs) near their 5 cap. It remained unclear, however, how ribosomes proceed downstream of ORFs and how piRNA precursors distinguish from other RNAs. It is thus important to test whether a short ORF length is required for substrate recognition for ribosome guided-piRNA biogenesis. Here, we characterized a poorly understood class of piRNAs that originate from the 3 untranslated regions (3UTRs) of protein coding genes in mice and chickens. We demonstrate that their precursors are full-length mRNAs and that post-termination 80S ribosomes guide piRNA production on 3UTRs after translation of upstream long ORFs. Similar to non-sense mediated decay (NMD), piRNA biogenesis degrades mRNA right after pioneer rounds of translation and fine-tunes protein production from mRNAs. Interestingly, however, we found that NMD, along with other surveillance pathways for ribosome recycling are temporally sequestered during the pachytene stage to allow for robust piRNA production. Although 3UTR piRNA precursor mRNAs code for distinct proteins in mice and chickens, they all harbor embedded transposable elements (TEs) and produce piRNAs that cleave TEs, suggesting that TE suppression, rather than the function of proteins, is the primary evolutionary force maintaining a subset of mRNAs as piRNA precursors. Altogether, we discover a function of the piRNA pathway in fine-tuning protein production and reveal a conserved, general piRNA biogenesis mechanism that recognizes translating RNAs regardless of their ORF length in amniotes.

sentences: Scrutiny of human lung infection by SARS-CoV-2 and associated human immune responses in humanized mice There is an urgent need for animal models of COVID-19 to study immunopathogenesis and test therapeutic intervenes. In this study we showed that NSG mice engrafted with human lung (HL) tissue (NSG-L mice) could be infected efficiently by SARS-CoV-2, and that live virus capable of infecting Vero cells was found in the HL grafts and multiple organs from infected NSG-L mice. RNA-seq examination identified a series of differentially expressed genes, which are enriched in viral defense responses, chemotaxis, interferon stimulation, and pulmonary fibrosis between HL grafts from infected and control NSG-L mice. Furthermore, when infecting humanized mice with human immune system (HIS) and autologous HL grafts (HISL mice), the mice had bodyweight loss and hemorrhage and immune cell infiltration in HL grafts, which were not observed in immunodeficient NSG-L mice, indicating the development of anti-viral immune responses in these mice. In support of this possibility, the infected HISL mice showed bodyweight recovery and lack of detectable live virus at the later time. These results demonstrate that NSG-L and HISL mice are susceptible to SARS-CoV-2 infection, offering a useful in vivo model for studying SARS-CoV-2 infection and the associated immune response and immunopathology, and testing anti-SARS-CoV-2 therapies.

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Adeno-associated virus type 2 (AAV2) uncoating is a stepwise process and is linked to structural reorganization of the nucleolus Nucleoli are membrane-less structures located within the nucleus and are known to be involved in many cellular functions, including stress response and cell cycle regulation. Besides, many viruses can employ the nucleolus or nucleolar proteins to promote different steps of their life cycle such as replication, transcription and assembly. While adeno-associated virus type 2 (AAV2) capsids have previously been reported to enter the host cell nucleus and accumulate in the nucleolus, both the role of the nucleolus in AAV2 infection, and the viral uncoating mechanism remain elusive. In all prior studies on AAV uncoating, viral capsids and viral genomes were not directly correlated on the single cell level, at least not in absence of a helper virus. To elucidate the properties of the nucleolus during AAV2 infection and to assess viral uncoating on a single cell level, we combined immunofluorescence analysis for detection of intact AAV2 capsids and capsid proteins with fluorescence in situ hybridization for detection of AAV2 genomes. The results of our experiments provide evidence that uncoating of AAV2 particles occurs in a stepwise process that is completed in the nucleolus and supported by alteration of the nucleolar structure.

Author SummaryAdeno-associated virus (AAV) capsids have been reported to enter the host cell nucleus and accumulate in the nucleolus. However, both the role of the nucleolus in AAV2 infection as well as the viral uncoating mechanism remain unknown. Here, we provide evidence that uncoating of the AAV2 particle is a stepwise process that is completed in the nucleolus and supported by alteration of the nucleolar morphology.

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Cell Surface Glycan Engineering Reveals that Matriglycan Alone can Recapitulate Dystroglycan Binding and Function -Dystroglycan (-DG) is uniquely modified on O-mannose sites by a repeating disaccharide (-Xyl1,3-GlcA{beta}1,3-)n termed matriglycan, which is a receptor for laminin-G domain-containing proteins and employed by old-world arenaviruses for infection. Using chemoenzymatically synthesized matriglycans printed as a microarray, we demonstrated length-dependent binding to Laminin, Lassa virus GP1, and the clinically-important antibody IIH6. Utilizing an enzymatic engineering approach, an N-linked glycoprotein was converted into a IIH6-positive Laminin-binding glycoprotein. Engineering of the surface of cells deficient for either -DG or O-mannosylation with matriglycans of sufficient length recovered infection with a Lassa-pseudovirus. Finally free matriglycan in a dose and length dependent manner inhibited viral infection of wildtype cells. These results indicate that matriglycan alone is necessary and sufficient for IIH6 staining, Laminin and LASV GP1 binding, and Lassa-pseudovirus infection and support a model in which it is a tunable receptor for which increasing chain length enhances ligand-binding capacity. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Graphical Abstract

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sentences: Meta-analysis of orthogonal OMICs data from COVID-19 patients unveils prognostic markers and antiviral factors. While our battle with the COVID-19 pandemic continues, a multitude of Omics data has been generated from patient samples in various studies, which remains to be translated. We conducted a meta-analysis of published transcriptome and proteome profiles of nasal swab and bronchioalveolar lavage fluid (BALF) samples of COVID-19 patients, to shortlist high confidence upregulated host factors. Subsequently, mRNA overexpression of selected genes was validated in nasal swab/BALF samples from a cohort of COVID-19 positive/negative, symptomatic/asymptomatic individuals. Analysis of these data revealed S100 family genes (S100A6, S100A8, S100A9, and S100P) as prognostic markers of COVID-19 disease. Furthermore, Thioredoxin gene (TXN) was identified as a significant upregulated host factor in our overlap analysis. An FDA-approved drug Auranofin, which inhibits Thioredoxin reduction, was found to mitigate SARS-CoV-2 replication in vitro and in vivo in the hamster challenge model. Overall, this study translates COVID-19 host response Big Data into potential clinical interventions.

sentences: SARS-CoV-2 B.1.351 (beta) variant shows enhanced infectivity in K18-hACE2 transgenic mice and expanded tropism to wildtype mice compared to B.1 variant SARS-CoV-2 variants display enhanced transmissibility and/or immune evasion and can be generated in humans or animals, like minks, thus generating new reservoirs. The continuous surveillance of animal susceptibility to new variants is necessary to predict pandemic evolution. In this study we demonstrate that, compared to the B.1 SARS-CoV-2 variant, K18-hACE2 transgenic mice challenged with the B.1.351 variant displayed a faster progression of infection. Furthermore, we also report that B.1.351 can establish infection in wildtype mice, while B.1 cannot. B.1.351-challenged wildtype mice showed a milder infection than transgenic mice, confirmed by detectable viral loads in oropharyngeal swabs and tissues, lung pathology, immunohistochemistry and serology. In silico models supported these findings by demonstrating that the Spike mutations in B.1.351 resulted in increased affinity for both human and murine ACE2 receptors. Overall, this study highlights the plasticity of SARS-CoV-2 animal susceptibility landscape, which may contribute to viral persistence and expansion.

sentences: From Diversity to Complexity: Microbial Networks in Soils Network analysis has been used for many years in ecological research to analyze organismal associations, for example in food webs, plant-plant or plant-animal interactions. Although network analysis is widely applied in microbial ecology, only recently has it entered the realms of soil microbial ecology, shown by a rapid rise in studies applying co-occurrence analysis to soil microbial communities. While this application offers great potential for deeper insights into the ecological structure of soil microbial ecosystems, it also brings new challenges related to the specific characteristics of soil datasets and the type of ecological questions that can be addressed. In this Perspectives Paper we assess the challenges of applying network analysis to soil microbial ecology due to the small-scale heterogeneity of the soil environment and the nature of soil microbial datasets. We review the different approaches of network construction that are commonly applied to soil microbial datasets and discuss their features and limitations. Using a test dataset of microbial communities from two depths of a forest soil, we demonstrate how different experimental designs and network constructing algorithms affect the structure of the resulting networks, and how this in turn may influence ecological conclusions. We will also reveal how assumptions of the construction method, methods of preparing the dataset, and definitions of thresholds affect the network structure. Finally, we discuss the particular questions in soil microbial ecology that can be approached by analyzing and interpreting specific network properties. Targeting these network properties in a meaningful way will allow applying this technique not in merely descriptive, but in hypothesis-driven research.

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Cofactor specificity of glucose-6-phosphate dehydrogenase isozymes in Pseudomonas putida reveals a general principle underlying glycolytic strategies in bacteria Glucose-6-phosphate dehydrogenase (G6PDH) is widely distributed in nature and catalyzes the first committing step in the oxidative branch of the pentose phosphate (PP) pathway, feeding either the reductive PP or the Entner-Doudoroff pathway. Besides its role in central carbon metabolism, this dehydrogenase also provides reduced cofactors, thereby affecting redox balance. Although G6PDH is typically considered to display specificity towards nicotinamide adenine dinucleotide phosphate (NADP+), some variants accept nicotinamide NAD+ similarly (or even preferentially). Furthermore, the number of G6PDH isozymes encoded in bacterial genomes varies from none to more than four orthologues. On this background, we systematically analyzed the interplay of the three G6PDH isoforms of the soil bacterium Pseudomonas putida KT2440 from a genomic, genetic and biochemical perspective. P. putida represents an ideal model to tackle this endeavor, as its genome encodes numerous gene orthologues for most dehydrogenases in central carbon metabolism. We show that the three G6PDHs of strain KT2440 have different cofactor specificities, and that the isoforms encoded by zwfA and zwfB carry most of the activity, acting as metabolic  gatekeepers for carbon sources that enter at different nodes of the biochemical network. Moreover, we demonstrate how multiplication of G6PDH isoforms is a widespread strategy in bacteria, correlating with the presence of an incomplete Embden-Meyerhof-Parnas pathway. Multiplication of G6PDH isoforms in these species goes hand-in-hand with low NADP+ affinity at least in one G6PDH isozyme. We propose that gene duplication and relaxation in cofactor specificity is an evolutionary strategy towards balancing the relative production of NADPH and NADH.

ImportanceProtein families have likely arisen during evolution by gene duplication and divergence followed by neo-functionalization. While this phenomenon is well documented for catabolic activities (typical of environmental bacteria that colonize highly polluted niches), the co-existence of multiple isozymes in central carbon catabolism remains relatively unexplored. We have adopted the metabolically-versatile soil bacterium Pseudomonas putida KT2440 as a model to interrogate the physiological and evolutionary significance of co-existing glucose-6-phosphate dehydrogenase (G6PDH) isozymes. Our results show that each of the three G6PDHs encoded in this bacterium display distinct biochemical properties, especially at the level of cofactor preference, impacting bacterial physiology in a carbon source-dependent fashion. Furthermore, the presence of multiple G6PDHs differing in NAD+- or NADP+-specificity in bacterial species strongly correlates with their predominant metabolic lifestyle. Our findings support the notion that multiplication of genes encoding cofactor-dependent dehydrogenases is a general evolutionary strategy towards achieving redox balance according to the growth conditions.

sentences: Niche differences, not fitness differences, explain coexistence across ecological groups Understanding the drivers of species coexistence is an important objective in ecology. Yet, the multitude of methods to study coexistence hampers cross-community comparisons. Here, we standardized niche and fitness differences (i.e how species limit themselves compared to others and their competitive ability, respectively) across 1018 species pairs to investigate species coexistence across ecological groups and methodological settings (experimental setup, natural co-occurrence, population model used, and growth method). We find that, first, coexistence is driven by large niche differences, not by small fitness differences. Second, species group into clear clusters of coexisting and non-coexisting species along the niche axis. Finally, these clusters are not driven by ecological or methodological settings. This suggests differences between coexisting and non-coexisting communities transcending those measured in our empirical systems. Overall, our results show that species coexistence is mainly influenced by mechanisms acting on niche differences.

sentences: Cortical microtubule pulling forces contribute to the union of the parental genomes in the C. elegans zygote Previously, we reported that the Polo-like kinase PLK-1 phosphorylates the single C. elegans lamin (LMN-1) to trigger lamina depolymerization during mitosis. We showed that this event is required for the formation of a pronuclear envelopes scission event that removes membranes on the juxtaposed oocyte and sperm pronuclear envelopes in the zygote, allowing the parental chromosomes to merge in a single nucleus after segregation (Velez-Aguilera, 2020). Here we show that cortical microtubule pulling forces contribute to pronuclear envelopes scission by promoting mitotic spindle elongation. We also demonstrate that weakening of the pronuclear envelopes, via PLK-1-mediated lamina depolymerization, is a prerequisite for the astral microtubule pulling forces to trigger pronuclear membranes scission. Finally, we provide evidence that PLK-1 mainly acts via lamina depolymerization in this process. These observations thus indicate that temporal coordination between lamina depolymerization and mitotic spindle elongation facilitates pronuclear envelopes scission and parental genomes unification.

sentences: Haplotype-aware variant calling enables high accuracy in nanopore long-reads using deep neural networks Long-read sequencing has the potential to transform variant detection by reaching currently difficult-to-map regions and routinely linking together adjacent variations to enable read based phasing. Third-generation nanopore sequence data has demonstrated a long read length, but current interpretation methods for its novel pore-based signal have unique error profiles, making accurate analysis challenging. Here, we introduce a haplotype-aware variant calling pipeline PEPPER-Margin-DeepVariant that produces state-of-the-art variant calling results with nanopore data. We show that our nanopore-based method outperforms the short-read-based single nucleotide variant identification method at the whole genome-scale and produces high-quality single nucleotide variants in segmental duplications and low-mappability regions where short-read based genotyping fails. We show that our pipeline can provide highly-contiguous phase blocks across the genome with nanopore reads, contiguously spanning between 85% to 92% of annotated genes across six samples. We also extend PEPPER-Margin-DeepVariant to PacBio HiFi data, providing an efficient solution with superior performance than the current WhatsHap-DeepVariant standard. Finally, we demonstrate de novo assembly polishing methods that use nanopore and PacBio HiFi reads to produce diploid assemblies with high accuracy (Q35+ nanopore-polished and Q40+ PacBio-HiFi-polished).

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High levels of Dorsal transcription factor downregulate, not promote, snail expression by regulating enhancer action In Drosophila embryos, genes expressed along the dorsal-ventral axis are responsive to concentration of the Dorsal (Dl) transcription factor, which varies in space; however, levels of this morphogen also build over time. Since expression of high-threshold Dl target genes such as snail (sna) is supported before Dl levels peak, it is unclear what role increasing levels have if any. Here we investigated action of two enhancers that control sna expression in embryos, demonstrating using genome editing that Dl binding sites within one enhancer located promoter proximally, sna.prox, can limit the ability of the other distally-located enhancer, sna.dis, to increase sna levels. In addition, MS2-MCP live imaging was used to study sna transcription rate in wildtype, dl heterozygote, and a background in which a photo-sensitive degron is fused to Dl (dl-BLID). The results demonstrate that, when Dl levels are high, Dl acts through sna.prox to limit the activity of sna.dis and thereby influence sna transcription rate. In contrast, when Dl levels are kept low using dl-BLID, sna.prox positively influences sna transcription rate. Collectively, our data support the view that Dls effect on gene expression changes over time, switching from promoting sna expression at low concentration to dampening sna expression at high concentration by regulating enhancer interactions. We propose this differential action of the Dl morphogen is likely supported by occupancy of this factor first to high and then low affinity binding sites over time as Dl levels rise to coordinate action of these two co-acting enhancers.

Significance statementA gradient of the maternal transcription factor Dorsal is important for establishing spatial expression of target genes along the dorsal-ventral axis of Drosophila embryos. Dorsal levels are also dynamic as nuclear concentration builds in time. Surprisingly, expression of high-threshold target genes such as snail is supported before levels peak, raising the question why levels continue to build. Our data support the view that peak Dorsal levels act to preferentially support activity of one enhancer over another to effectively decrease snail expression. In addition, while the morphogen Dorsal acts early to support gene expression, later it effectively acts as a damper to limit gene expression. Our results suggest other morphogens also have effects on gene expression that change over time.

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Recombinant adenovirus causes prolonged mobilization of macrophages in the anterior chamber of mice PurposeOcular tissues of mice have been studied in many ways using replication deficient species C type 5 adenoviruses (Ad5) as tools for manipulating gene expression. While refinements to injection protocols and tropism have led to several advances in targeting cells of interest, there remains a relative lack of information concerning how Ad5 may influence other ocular cell types capable of confounding experimental interpretation. Here, a slit-lamp is used to thoroughly photodocument the sequelae of intraocular Ad5 injections over time in mice, with attention to potentially confounding indices of inflammation.

MethodsA cohort of C57BL/6J mice was randomly split into 3 groups (Virus, receiving unilateral intracameral injection with 5x107 pfu of a cargo-less Ad5 construct; Saline, receiving unilateral balanced salt solution injection; and Naive, receiving no injections). From this initial experiment, a total of 52 eyes from 26 mice were photodocumented via slit-lamp at four time points (baseline, 1, 3, and 10 weeks following initiation of the experiment) by an observer masked to treatments and other parameters of the experimental design. Following the last in vivo exam, tissues were collected. Based on the slit-lamp data, tissues were studied via immunostaining with the macrophage marker F4/80. Subsequently, three iterations of the original experiment were performed with otherwise identical experimental parameters testing the effect of age, intravitreal injection, and A195 buffer, adding slit-lamp photodocumentation of an additional 32 eyes from 16 mice.

ResultsThe masked investigator was able to use the sequential images from each mouse in the initial experiment to assign each mouse into its correct treatment group with near perfect fidelity. Virus injected eyes were characterized by corneal damage indicative of intraocular injection and a prolonged mobilization of clump cells on the surface of the iris. Saline injected eyes had only transient corneal opacities indicative of intraocular injections, and Naive eyes remained normal. Immunostaining with F4/80 was consistent with ascribing the clump cells visualized via slit-lamp imaging as a type of macrophage. Experimental iterations using Ad5 indicate that all virus injected eyes had the distinguishing feature of a prolonged presence of clump cells on the surface of the iris regardless of injection site. Mice receiving an intraocular injection of Ad5 at an advanced age displayed a protracted course of corneal cloudiness that prevented detailed visualization of the iris at the last time point.

ConclusionsBecause the eye is often considered an "immune privileged site", we suspect that several studies have neglected to consider that the presence of Ad5 in the eye might evoke strong reactions from the innate immune system. Ad5 injection caused a sustained mobilization of clump cells, i.e. macrophages. This change is likely a consequence of either direct macrophage transduction or a secondary response to cytokines produced locally by other transduced cells. Regardless of how these cells were altered, the important implication is that the adenovirus led to long lasting changes in the environment of the anterior chamber. Thus, these findings describe a caveat of Ad5-mediated studies involving macrophage mobilization, which we encourage groups to use as a bioassay in their experiments and consider in interpretation of their ongoing experiments using adenoviruses.

sentences: ChexMix: A Literature Content Extraction Tool for Bioentities Biomedical databases grow by more than a thousand new publications every day. The large volume of biomedical literature that is being published at an unprecedented rate hinders the discovery of relevant knowledge from keywords of interest to gather new insights and form hypotheses. A text-mining tool, PubTator, helps to automatically annotate bioentities, such as species, chemicals, genes, and diseases, from PubMed abstracts and full-text articles. However, the manual re-organization and analysis of bioentities is a non-trivial and highly time-consuming task. ChexMix was designed to extract the unique identifiers of bioentities from query results. Herein, ChexMix was used to construct a taxonomic tree with allied species among Korean native plants and to extract the medical subject headings unique identifier of the bioentities, which co-occurred with the keywords in the same literature. ChexMix discovered the allied species related to a keyword of interest and experimentally proved its usefulness for multi-species analysis.

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ALS and FTD-associated missense mutations in TBK1 differentially disrupt mitophagy TANK-binding kinase 1 (TBK1) is a multi-functional kinase with an essential role in mitophagy, the selective clearance of damaged mitochondria. More than 90 distinct mutations in TBK1 are linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD), including missense mutations that disrupt the ability of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), auto-activate, or catalyze phosphorylation. We investigated how ALS-associated mutations in TBK1 affect Parkin-dependent mitophagy using imaging to dissect the molecular mechanisms involved in clearing damaged mitochondria. Some mutations cause severe dysregulation of the pathway, while others induce limited disruption. Mutations that abolish either TBK1 dimerization or kinase activity were insufficient to fully inhibit mitophagy, while mutations that reduced both dimerization and kinase activity were more disruptive. Ultimately, both TBK1 recruitment and OPTN phosphorylation at S177 are necessary for engulfment of damaged mitochondra by autophagosomal membranes. Surprisingly, we find that ULK1 activity contributes to the phosphorylation of OPTN in the presense of either WT- or kinase inactive TBK1. In primary neurons, TBK1 mutants induce mitochondrial stress under basal conditions; network stress is exacerbated with further mitochondrial insult. Our study further refines the model for TBK1 function in mitophagy, demonstrating that some ALS-linked mutations likely contribute to disease pathogenesis by inducing mitochondrial stress or inhibiting mitophagic flux. Other TBK1 mutations exhibited much less impact on mitophagy in our assays, suggesting that cell-type specific effects, cumulative damage, or alternative TBK1-dependent pathways such as innate immunity and inflammation also factor into the development of ALS in affected individuals.

SIGNIFICANCE STATEMENTMissense mutations in TANK-binding kinase 1 (TBK1) have various biophysical and biochemical effects on the molecule, and are associated with the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). TBK1 plays an essential role in clearing damaged mitochondria. Here, we investigate the impact of 10 ALS-linked TBK1 mutations on the critical early stage of mitophagy. We find that both TBK1 recruitment and kinase activity contribute to the clearance of the damaged mitochondria. Furthermore, in neurons, expression of TBK1 mutants alone affects mitochondrial network health. Our investigation utilizes disease-linked mutations to further refine the current model of mitophagy, identifying crosstalk between the regulatory kinases TBK1 and ULK1, and providing new insights into the roles of TBK1 in neurodegenerative pathogenesis.

sentences: Multi-tier mechanics control stromal adaptations in swelling lymph nodes Lymph nodes (LNs) comprise two main structural elements: Fibroblastic reticular cells (FRCs) that form dedicated niches for immune cell interaction and capsular fibroblasts that build a shell around the organ. While LNs are fairly stable in size during homeostatic conditions, immunological challenge causes more than 10-fold increase in size within only a few days. How a solid organ can accommodate such extreme volumetric changes is poorly understood. Here, we characterize the biomechanics of LN swelling on the cellular and organ scale. We identify lymphocyte trapping by influx and proliferation as drivers of an outward pressure force, causing FRCs and their associated conduits to stretch. After an initial phase of relaxation, FRCs sense the resulting strain via cell matrix adhesions, which coordinates local growth and remodeling of the stromal network. While the expanded FRC network adopts its typical configuration, a massive fibrotic reaction of the organ capsule sets in and counters further organ expansion. Thus, different fibroblast populations mechanically control LN swelling in a multi-tier fashion.

sentences: The role of hub neurons in modulating cortical dynamics Many neurodegenerative diseases are associated with the death of specific neuron types in particular brain regions. What makes the death of specific neuron types particularly harmful for the integrity and dynamics of the respective network is not well understood. To start addressing this question we used the most up-to-date biologically-realistic dense neocortical microcircuit (NMC) of rodent, which has reconstructed a volume of 0.3 mm3 and containing 31,000 neurons, 36 million synapses, and 55 morphological cell types arranged in 6 cortical layers. Using modern network science tools, we identified "hub-neurons" in the NMC, that are connected synaptically to a large number of their neighbors and systematically examined the impact of abolishing these cells. In general, the structural integrity of the network is robust to cells attack; yet, attacking hub neurons strongly impacted the "small worldness" topology of the network, whereas similar attacks on random neurons have a negligible effect. Such hub-specific attacks are also impactful on the network dynamics, both when the network is at its spontaneous synchronous state and when it was presented with synchronized thalamo-cortical visual-like input. We found that attacking layer 5 hub neurons are most harmful to the structural and functional integrity of the NMC. The significance of our results for understanding the role of specific neuron types and cortical layers for disease manifestation is discussed.

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PD-1 in hippocampal neurons regulates excitability, synaptic plasticity, and cognition Immunotherapy using monoclonal antibodies against programmed cell death protein 1 (PD-1) demonstrated improved survival in cancer patients through immune activation. Here we show that functional PD-1 is expressed in mouse and primate hippocampal neurons and PD-1 inhibition improves cognition in physiological and pathological conditions. Mice lacking the Pdcd1 gene encoding PD-1 exhibit enhanced long-term potentiation (LTP) and learning and memory. These behavioral and cellular changes can be recapitulated by selective deletion of Pdcd1 in hippocampal excitatory neurons but not in microglia. Perfusion of mouse or nonhuman primate brain slices with anti-PD-1 antibody is sufficient to increase excitability in CA1 hippocampal neurons. Conversely, re-expression of Pdcd1 in PD-1 deficient hippocampal neurons suppresses memory and LTP. Traumatic brain injury impairs learning and memory, which is improved by intraventricular administration of anti-PD-1. These findings suggest that anti-PD-1 treatment has therapeutic potential to counteract cognitive decline.

HighlightsO_LIAdult mice lacking Pdcd1 in hippocampal neurons exhibit enhanced memory and LTP
C_LIO_LIAnti-PD-1 antibody treatment increases CA1 neuron excitability in brain slices of mice and primates
C_LIO_LIRe-expression of Pdcd1 in PD-1 deficient hippocampal neurons impairs memory and LTP
C_LIO_LICognitive deficits after traumatic brain injury are improved by anti-PD-1 treatment
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Effect of nandrolone decanoate on healing of experimentally induced radial nonunion in rabbits (Oryctolagus cuniculus) The aim of this study was to evaluate the effect of nandrolone decanoate (ND) on treatment of bone nonunion in the radius of rabbits. Thirty-one, young adult, New Zealand White rabbits (Oryctolagus cuniculus) were allocated to one of four groups: nandrolone males (NMG), nandrolone females (NFG), placebo males (NPM), and placebo females (NPF). After bone nonunion of a 10 mm ostectomy of the radius was confirmed (45 days after surgery), the animals in the NMG and NFG groups received 10 mg/kg ND intramuscular once a week for four weeks, while placebo groups received intramuscular 0.9% NaCl solution. Radiographic, histopathologic, and densitometric parameters (DXA) were used to compared groups.

ResultsNo significant differences were observed radiographically. However, ND groups showed greater area (P=0.0258) and BMC (P=0.0140) in the densitometric evaluation. Histologically, the placebo group showed a predominance of primary bone tissue. Whereas, lamellary organizations of secondary bone and the presence of fibrocartilage were found in the ND group (P =0.006). In conclusion, ND promoted bone regeneration after the creation of a large defect in the radius of rabbits.

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Interaction between theta-phase and spike-timing dependent plasticity simulates theta induced memory effects Rodent studies suggest that spike timing relative to hippocampal theta activity determines whether potentiation or depression of synapses arise. Such changes also depend on spike timing between pre- and post-synaptic neurons, known as spike-timing-dependent plasticity (STDP). STDP, together with theta-phase-dependent learning, has inspired several computational models of learning and memory. However, evidence to elucidate how these mechanisms directly link to human episodic memory is lacking. In a computational model, we modulate long-term potentiation (LTP) and long-term depression (LTD) of STDP, by opposing phases of a simulated theta rhythm. We fit parameters to a hippocampal cell culture study in which LTP and LTD were observed to occur in opposing phases of a theta rhythm. Further, we modulated two inputs by cosine waves with synchronous and asynchronous phase offsets and replicate key findings in human episodic memory. Learning advantage was found for the synchronous condition, as compared to the asynchronous conditions, and was specific to theta modulated inputs. Importantly, simulations with and without each mechanism suggest that both STDP and theta-phase-dependent plasticity are necessary to replicate the findings. Together, the results indicate a role for circuit-level mechanisms, which bridges the gap between slice preparation studies and human memory.

Author SummaryLong-lasting changes in synaptic connectivity between neurons have been suggested to support learning and memory processes at the cellular level in the brain. Such synaptic modifications depend on synchronous activation of neurons, which leads to generate brain oscillations. Human memory studies focus on the relationships between brain oscillations and memory processes. Direct evidence on how the cellular mechanism links to human memory behaviour is lacking. To investigate the direct link between synaptic plasticity mechanisms and human memory formation, we built a computational neural network that implements two synaptic plasticity mechanisms, which are well-established in the rodents hippocampus. One mechanism shows that strengthening or weakening in synaptic connectivity depends on the phases of ongoing brain oscillation at theta frequency (4 - 8 Hz), which is a dominant signal in the hippocampus. The other mechanism suggests that synaptic modification depends on the precise timing of action potentials between two neurons. Our model successfully reproduces results from rodents, as well as several human episodic memory studies which demonstrated that human associative memory performance depends on phase synchronisation in theta frequency. These findings suggest a link between specific learning mechanisms at cellular level and human memory behaviour.

sentences: Genetic and epigenetic characteristics associated with. the rapid radiation of Aquilegia species Elucidating the genetic and epigenetic bases underlying species diversification is crucial to understanding the evolution and persistence of biodiversity. As a well-known horticultural plant grown worldwide, the genus Aquilegia (columbine) is also a model system in adaptive radiation research. In this study, we surveyed the genomes and DNA methylomes of ten representative Aquilegia species from the Asian, European and North American lineages. Our inferences of the phylogenies and population structure revealed clearly high genetic and DNA methylomic divergence across the three lineages. By multi-levelled genome-wide scanning, we identified candidate genes exhibiting lineage-specific genetic or epigenetic variation patterns that are signatures of inter-specific divergence. We demonstrated that these species diversification-associated genetic variations and epigenetic variabilities were partially independent but were both functionally related to various biological processes vital to adaptation, including stress tolerance, cell reproduction and DNA repair. Our study provides an exploratory overview of how the established genetic and epigenetic signatures are associated with the rapid radiation of Aquilegia species.

sentences: Transcriptional and Functional Activity of Hemangiosarcoma Support Bone Marrow Nurse Cell Ontogeny Hemangiosarcoma and angiosarcoma are soft tissue sarcomas of malignant blood vessel-forming cells in dogs and humans, respectively. These vasoformative sarcomas are aggressive and highly metastatic, with disorganized, irregular blood-filled vascular spaces. Our objective was to define molecular programs that support the niche, enabling progression of canine hemangiosarcoma and human angiosarcoma. Here, we show that the transcriptional landscape of canine hemangiosarcoma and human angiosarcoma included comparable angiogenic and inflammatory programs. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized, and they were lined by both donor and host cells, a trait that was not observed in xenografts from canine osteosarcoma and lymphoma. In some cases, the xenografted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. We did not uncover a definitive transmissible etiology, but our data indicate that transcriptional programs of hemangiosarcoma cells resemble those of hematopoietic nurse cells, and these malignant cells support expansion and differentiation of human hematopoietic progenitors. We conclude that canine hemangiosarcoma, and possibly human angiosarcoma, originate from nurse cells that make up the stromal bone marrow niche, and that these cells may also support the growth of hematopoietic tumors.

sentences: The scaffold-dependent function of RIPK1 in dendritic cells promotes injury-induced colitis Receptor interacting protein kinase 1 (RIPK1) is a cytosolic multidomain protein that controls cell life and death. While RIPK1 promotes cell death through its kinase activity, it also functions as a scaffold protein to promote cell survival by inhibiting FADD-caspase 8-dependent apoptosis and RIPK3-MLKL-dependent necroptosis. This pro-survival function is highlighted by excess cell death and a perinatal lethality in Ripk1-/- mice. Recently, loss of function mutation of RIPK1 was found in patients with immunodeficiency and inflammatory bowel diseases. Hematopoietic stem cell transplantation restored not only immunodeficiency but also intestinal inflammatory pathology, indicating that RIPK1 in hematopoietic cells is critical to maintain intestinal immune homeostasis. Here, we generated dendritic cell (DC)-specific Ripk1-/- mice in a genetic background with loss of RIPK1 kinase activity and found that the mice developed spontaneous colonic inflammation characterized by increased neutrophil infiltration. In addition, these mice were highly resistant to injury-induced colitis. The increased neutrophil infiltration in the colon and the resistance to colitis were restored by dual inactivation of RIPK3 and FADD, but not by inhibition of RIPK3, MLKL, or ZBP1 alone. Altogether, these results reveal a scaffold activity-dependent role of RIPK1 in protecting colonic DCs from apoptotic insults and maintenance of colonic immune homeostasis.

sentences: Patch-Clamp Proteomics of Single Neuronal Somas in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry Understanding of the relationship between cellular function and molecular composition holds a key to next-generational therapeutics but requires measurement of all types of molecules in cells. Developments in sequencing enabled semi-routine measurement of single-cell genomes and transcriptomes, but analytical tools are scarce for detecting diverse proteins in tissue-embedded cells. To bridge this gap for neuroscience research, we report the integration of patch-clamp electrophysiology with subcellular shot-gun proteomics by high-resolution mass spectrometry (HRMS). Recording of electrical activity permitted identification of dopaminergic neurons in the substantia nigra pars compacta. Ca. 20-50% of the neuronal soma content, containing an estimated 100 pg of total protein, was aspirated into the patch pipette filled with ammonium bicarbonate. About ~1 pg of somal protein, or ~0.25% of the total cellular proteome, was analyzed on a custom-built capillary electrophoresis (CE) high-resolution mass spectrometer (HRMS). A series of experiments were conducted to systematically enhance detection sensitivity through refinements in sample processing and detection, allowing us to quantify ~275 different proteins from somal aspirate-equivalent protein digests. From single neurons, patch-clamp proteomics of the soma quantified 91, 80, and 95 different proteins from 3 different dopaminergic neurons, or 157 proteins in total. Quantification revealed detectable proteomic differences between the somal protein samples. Analysis of canonical knowledge predicted rich interaction networks between the proteins. The integration of patch-clamp electrophysiology with subcellular CE-HRMS proteomics expands the analytical toolbox of neuroscience.

sentences: Coordination of di-acetylated histone ligands by the ATAD2 bromodomain The ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four -helices. ATAD2 functions as a co-activator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized histones. In addition, ATAD2 is shown to be up regulated in multiple forms of cancer including breast, lung, gastric, endometrial, renal, and prostate. Furthermore, up-regulation of ATAD2 is strongly correlated with poor prognosis in many types of cancer, making the ATAD2 bromodomain an innovative target for cancer therapeutics. In this study, we describe the recognition of histone acetyllysine modifications by the ATAD2 bromodomain. Residue-specific information on the complex formed between the histone tail and the ATAD2 bromodomain, obtained through nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, illustrates key residues lining the binding pocket, which are involved in coordination of di-acetylated histone tails. Analytical ultracentrifugation, NMR relaxation data, and isothermal titration calorimetry further confirm the monomeric state of the functionally active ATAD2 bromodomain in complex with di-acetylated histone ligands. Overall, we describe histone tail recognition by ATAD2 BRD and illustrate that one acetyllysine group is primarily engaged by the conserved asparagine (N1064), the "RVF" shelf residues, and the flexible ZA loop. Coordination of a second acetyllysine group also occurs within the same binding pocket, but is essentially governed by unique hydrophobic and electrostatic interactions making the di-acetyllysine histone coordination more specific than previously presumed.

sentences: APOE4 is associated with elevated blood lipids and lower levels of innate immune biomarkers in a tropical Amerindian subsistence population In post-industrial settings, APOE4 is associated with increased cardiovascular and neurological disease risk. However, the majority of human evolutionary history occurred in environments with higher pathogenic diversity and low cardiovascular risk. We hypothesize that in high-pathogen and energy-limited contexts, the APOE4 allele confers benefits by reducing baseline innate inflammation when uninfected, while maintaining higher lipid levels that buffer costs of immune activation during infection. Among Tsimane forager-farmers of Bolivia (N=1266), APOE4 is associated with 30% lower C-reactive protein, and higher total cholesterol and oxidized-LDL. Blood lipids were either not associated, or negatively associated with inflammatory biomarkers, except for associations of oxidized-LDL and inflammation which were limited to obese adults. Further, APOE4 carriers maintain higher levels of total and LDL cholesterol at low BMIs. These results suggest the relationship between APOE4 and lipids is likely beneficial for pathogen-driven immune responses, and unlikely to increase cardiovascular risk in an active subsistence population.

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Aspirin Protective Effect Against Cyclophosphamide Hematological Toxicity In Experimental Animals Bone marrow toxicity is the most important factor limiting the use of cytotoxic drugs like alkylating agents in cancer treatment. Recently PG synthase enzyme inhibitors have been reported to potentiate the cytotoxic effects of these agents on cancer cells but little is known if they can affect the toxicity of these agents on bone marrow or other tissues. Cyclophosphamide is one of the most commonly used alkylating agent.

In the present work, the effect of these PG synthase enzyme inhibitors, aspirin on cyclophosphamide myelotoxicity was determined employing the peripheral blood count to reflect bone marrow injury. The effect on body weight changes caused by cyclophosphamide was also determined.

O_LICyclophosphamide in doses of 25, 50 and 75 mg/kg i. v. produced as a dose dependent reduction in total WBC count, granulocyte, non granulocyte, and Hb% which was maximum on second day after injection and still present on 5th day post injection. It also produced a dose dependent reduction in body weight on day 5 after injection.
C_LIO_LIAspirin in doges of 75, 150 and 300 mg/kg i. m. protected against the reduction in WBC counts  measured for 5 days after injection of cyclophosphamide (50 mg/kg). This protection was not dose dependent, though it was more optimum with 300 mg/kg and disappeared largely when a dose of 450 mg/kg was used. Aspirin did not prevent the changes in Hb% but retard the reduction in body weight caused by cyclophosphamide.
C_LIO_LIIt is concluded that aspirin can help to reduce injury and enhance recovery from bone marrow toxicity caused by cytotoxic agents such as the alkylating drugs cyclophosphamide for which no specific antidote is available. Aspirin produces this effect possibly by eliminating the harmful inhibitory effect of excess PGs or leukotrienes, released by bone marrow injury on growth factors of haemopoietic progenitor cells.
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The magnitude of this protection on WBC counts does not seem to differ between either PG synthase enzyme inhibitors or steroids when used alone or in combination although a synergistic effect in protecting erythropoiesis is observed.

sentences: Database and deep learning toolbox for noise-optimized, generalized spike inference from calcium imaging data Calcium imaging is a key method to record patterns of neuronal activity across populations of identified neurons. Inference of temporal patterns of action potentials ( spikes) from calcium signals is, however, challenging and often limited by the scarcity of ground truth data containing simultaneous measurements of action potentials and calcium signals. To overcome this problem, we compiled a large and diverse ground truth database from publicly available and newly performed recordings. This database covers various types of calcium indicators, cell types, and signal-to-noise ratios and comprises a total of >35 hours from 298 neurons. We then developed a novel algorithm for spike inference (CASCADE) that is based on supervised deep networks, takes advantage of the ground truth database, infers absolute spike rates, and outperforms existing model-based algorithms. To optimize performance for unseen imaging data, CASCADE retrains itself by resampling ground truth data to match the respective sampling rate and noise level. As a consequence, no parameters need to be adjusted by the user. To facilitate routine application of CASCADE we developed systematic performance assessments for unseen data, we openly release all resources, and we provide a user-friendly cloud-based implementation.

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A multilayer network model of neuron-astrocyte populations in vitro reveals mGluR5 inhibition is protective following traumatic injury Astrocytes communicate bidirectionally with neurons, enhancing synaptic plasticity and promoting the synchronization of neuronal microcircuits. Despite recent advances in understanding neuron-astrocyte signaling, little is known about astrocytic modulation of neuronal activity at the population level, particularly in disease or following injury. We used high-speed calcium imaging of mixed cortical cultures in vitro to determine how population activity changes after disruption of glutamatergic signaling and mechanical injury. We constructed a multilayer network model of neuron-astrocyte connectivity, which captured distinct topology and response behavior from single cell type networks. mGluR5 inhibition decreased neuronal activity, but did not on its own disrupt functional connectivity or network topology. In contrast, injury increased the strength, clustering, and efficiency of neuronal but not astrocytic networks, an effect that was not observed in networks pre-treated with mGluR5 inhibition. Comparison of spatial and functional connectivity revealed that functional connectivity is largely independent of spatial proximity at the microscale, but mechanical injury increased the spatial-functional correlation. Finally, we found that astrocyte segments of the same cell often belong to separate functional communities based on neuronal connectivity, suggesting that astrocyte segments function as independent entities. Our findings demonstrate the utility of multilayer network models for characterizing the multiscale connectivity of two distinct but functionally dependent cell populations.

AUTHOR SUMMARYAstrocytes communicate bidirectionally with neurons, enhancing synaptic plasticity and promoting the synchronization of neuronal microcircuits. We constructed a multilayer network model of neuron-astrocyte connectivity based on calcium activity in mixed cortical cultures, and used this model to evaluate the effect of glutamatergic inhibition and mechanical injury on network topology. We found that injury increased the strength, clustering, and efficiency of neuronal but not astrocytic networks, an effect that was not observed in injured networks pre-treated with a glutamate receptor antagonist. Our findings demonstrate the utility of multilayer network models for characterizing the multiscale connectivity of two distinct but functionally dependent cell populations.

sentences: Reproducible, high-dimensional imaging in archival human tissue by Multiplexed Ion Beam Imaging by Time-of-Flight (MIBI-TOF) Multiplexed ion beam imaging by time-of-flight (MIBI-TOF) is a form of mass spectrometry imaging that uses metal labeled antibodies and secondary ion mass spectrometry to image dozens of proteins simultaneously in the same tissue section. Working with the National Cancer Institutes (NCI) Cancer Immune Monitoring and Analysis Centers (CIMAC), we undertook a validation study, assessing concordance across a dozen serial sections of a tissue microarray of 21 samples that were independently processed and imaged by MIBI-TOF or single-plex immunohistochemistry (IHC) over 12 days. Pixel-level features were highly concordant across all 16 targets assessed in both staining intensity (R2 = 0.94 {+/-} 0.04) and frequency (R2 = 0.95 {+/-} 0.04). Comparison to digitized, single-plex IHC on adjacent serial sections revealed similar concordance (R2 = 0.85 {+/-} 0.08) as well. Lastly, automated segmentation and clustering of eight cell populations found that cell frequencies between replicates yielded an average correlation of R2 = 0.92 {+/-} 0.06. Taken together, we demonstrate that MIBI-TOF, with well-vetted reagents and automated analysis, can generate consistent and quantitative annotations of clinically relevant cell states in archival human tissue, and more broadly, present a scalable framework for benchmarking multiplexed IHC approaches.

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Alevin-fry unlocks rapid, accurate, and memory-frugal quantification of single-cell RNA-seq data The rapid growth of high-throughput single-cell and single-nucleus RNA sequencing technologies has produced a wealth of data over the past few years. The available technologies continue to evolve and experiments continue to increase in both number and scale. The size, volume, and distinctive characteristics of these data necessitate the development of new software and associated computational methods to accurately and efficiently quantify single-cell and single-nucleus RNA-seq data into count matrices that constitute the input to downstream analyses.

We introduce the alevin-fry framework for quantifying single-cell and single-nucleus RNA-seq data. Despite being faster and more memory frugal than other accurate and scalable quantification approaches, alevin-fry does not suffer from the false positive expression or memory scalability issues that are exhibited by other lightweight tools. We demonstrate how alevin-fry can be effectively used to quantify single-cell and single-nucleus RNA-seq data, and also how the spliced and unspliced molecule quantification required as input for RNA velocity analyses can be seamlessly extracted from the same pre-processed data used to generate regular gene expression count matrices.

sentences: Numerical uncertainty can critically affect simulations of mechanistic models in neuroscience Understanding neural computation on the mechanistic level requires models of neurons and neuronal networks. To analyze such models one typically has to solve coupled ordinary differential equations (ODEs), which describe the dynamics of the underlying neural system. These ODEs are solved numerically with deterministic ODE solvers that yield single solutions with either no, or only a global scalar bound on precision. It can therefore be challenging to estimate the effect of numerical uncertainty on quantities of interest, such as spike-times and the number of spikes. To overcome this problem, we propose to use recently developed sampling-based probabilistic solvers, which are able to quantify such numerical uncertainties. They neither require detailed insights into the kinetics of the models, nor are they difficult to implement. We show that numerical uncertainty can affect the outcome of typical neuroscience simulations, e.g. jittering spikes by milliseconds or even adding or removing individual spikes from simulations altogether, and demonstrate that probabilistic solvers reveal these numerical uncertainties with only moderate computational overhead.

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