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10.1101/2022.05.06.490781 | Alone in a crowd: Effect of a nonfunctional lateral line on expression of the social hormone parathyroid hormone 2. | Parathyroid hormone 2 (Pth2) is a vertebrate-specific neuropeptide whose thalamic expression is upregulated by social contact with conspecifics. However, social interactions fail to stimulate pth2 expression in isolated zebrafish whose lateral line hair cells have been chemically ablated. These results suggest that modulation of pth2 by social context is acutely dependent on mechanosensory information from the lateral line. However, it is unclear how a congenital loss of lateral line function influences the ability of zebrafish to interpret their social environment. In this study, we measure pth2 levels in zebrafish mutants lacking hair cell function in either the lateral line only, or in both the inner ear and lateral line. Socially-raised lateral line mutants express lower levels of pth2 relative to wild type siblings, but there is no further reduction when all sensory hair cells are nonfunctional. However, social isolation of hair cell mutants causes a further reduction in pth2 expression, pointing to additional unidentified sensory cues that influence pth2 production. Lastly, we report that social context modulates fluorescent transgenes driven by the pth2 promoter. Altogether, these data suggest that lateral line mutants experience a chronic sense of isolation, even when raised in a social environment.
SUMMARY STATEMENTExpression of the pro-social neuropeptide pth2 is downregulated in larval zebrafish with a congenital loss of lateral line function. Thus, even in social environments, fish with compromised lateral lines may experience chronic loneliness. | neuroscience |
10.1101/2022.05.07.490872 | Phase separation of competing memories along the human hippocampal theta rhythm. | Competition between overlapping memories is considered one of the major causes of forgetting and it is still unknown how the human brain resolves such mnemonic conflict. In the present MEG study, we empirically tested a computational model that leverages an oscillating inhibition algorithm to minimise overlap between memories. We used a proactive interference task, where a reminder word could be associated with either a single image (non-competitive condition) or two competing images, and participants were asked to always recall the most recently learned word-image association. Time-resolved pattern classifiers were trained to detect the reactivated content of target and competitor memories from MEG sensor patterns, and the timing of these neural reactivations was analysed relative to the phase of the dominant hippocampal 3Hz theta oscillation. In line with our preregistered hypotheses, target and competitor reactivations locked to different phases of the hippocampal theta rhythm after several repeated recalls. Participants who behaviourally experienced lower levels of interference also showed larger phase separation between the two overlapping memories. The findings provide evidence that the temporal segregation of memories, orchestrated by slow oscillations, plays a functional role in resolving mnemonic competition by separating and prioritising relevant memories under conditions of high interference. | neuroscience |
10.1101/2022.05.07.491015 | Preconfigured dynamics in the hippocampus are guided by embryonic birthdate and rate of neurogenesis | The incorporation of novel information into the hippocampal network is likely be constrained by its innate architecture and internally generated activity patterns. However, the origin, organization, and consequences of such patterns remain poorly understood. Here, we show that hippocampal network dynamics are affected by sequential neurogenesis. We birthdated CA1 pyramidal neurons with in-utero electroporation over 4 embryonic days encompassing the peak of hippocampal neurogenesis, and compared their functional features in freely moving, adult mice. Neurons of the same birthdate displayed distinct connectivity, coactivity across brain states, and assembly dynamics. Same birthdate hippocampal neurons were topographically organized, in that anatomically clustered (<500{micro}m) neurons exhibited overlapping spatial representations. Overall, the wiring and functional features of CA1 pyramidal neurons reflected a combination of birthdate and the rate of neurogenesis. These observations demonstrate that sequential neurogenesis in embryonic development shapes the preconfigured forms of adult network dynamics. | neuroscience |
10.1101/2022.05.07.491032 | Exploring the utility of recombinantly expressed snake venom serine protease toxins as immunogens for generating experimental snakebite antivenoms | Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies. | immunology |
10.1101/2022.05.07.490979 | Pnpt1 mediates NLRP3 inflammasome activation by MAVS and metabolic reprogramming in macrophages | Polyribonucleotide nucleotidyltransferase 1 (Pnpt1) plays critical roles in mitochondrial homeostasis by controlling mitochondrial RNA (mt-RNA) processing, trafficking and degradation. Pnpt1 deficiency results in mitochondrial dysfunction that triggers a Type I interferon response, suggesting a role in inflammation. However, the role of Pnpt1 in inflammasome activation remains largely unknown. In this study, we generated myeloid-specific Pnpt1-knockout mice, and demonstrated that Pnpt1 depletion enhanced interleukin-1 beta (IL-1{beta}) and interleukin-18 (IL-18) secretion in mouse sepsis models. Using cultured peritoneal and bone marrow-derived macrophages we demonstrated that Pnpt1 regulated NLRP3 inflammasome dependent IL-1{beta} release in response to lipopolysaccharides (LPS), followed by nigericin, ATP or poly (I:C) treatment. Pnpt1 deficiency in macrophages increased glycolysis after LPS, and mt-reactive oxygen species (mt-ROS) after NLRP3 inflammasome activation. Pnpt1 activation of the inflammasome was dependent on both increased glycolysis and expression of the mitochondrial antiviral-signaling protein (MAVS), but not NF-{kappa}B signaling. Collectively, these data strengthen the concept that Pnpt1 is an important mediator of inflammation as shown by activation of the NLRP3 inflammasome in mouse sepsis and cultured macrophages. | immunology |
10.1101/2022.05.06.490988 | Defining the HIV Capsid Binding Site of Nucleoporin 153 | The interaction between the HIV-1 capsid (CA) and human nucleoporin 153 (NUP153) is vital for delivering the HIV-1 preintegration complex into the nucleus via the nuclear pore complex. The interaction with CA requires a phenylalanine/glycine-containing motif in the C-terminus of NUP153. This study used molecular modeling and biochemical assays to determine the amino acids of NUP153 that are essential for its interactions with CA. Molecular dynamics, FoldX, and PyRosetta simulations delineated the minimal CA binding motif of NUP153 based on the known structure of NUP153 bound to the HIV-1 CA hexamer. Computational predictions were experimentally validated by testing the interaction of NUP153 with CA using an in vitro binding assay and a cell-based TRIM-NUP153C restriction assay. This multidisciplinary approach identified eight amino acids from P1411 to G1418 that stably engage with CA, with significant correlations between molecular models and empirical experiments. Specifically, P1411, V1414, F1415, T1416, F1417, and G1418 were confirmed as critical amino acids required to interact NUP153 with CA. | molecular biology |
10.1101/2022.05.07.489984 | DNA metabarcoding of gut contents reveals key habitat and seasonal drivers of trophic networks involving generalist predators in agricultural landscapes | BACKGROUND: Understanding the networks of trophic interactions into which generalist predators are embedded is key to assessing their ecological role of in trophic networks and the biological control services they provide. The advent of affordable DNA metabarcoding approaches greatly facilitates quantitative understanding of trophic networks and their response to environmental drivers. Here, we examine how key environmental gradients interact to shape predation by Lycosidae in highly dynamic vegetable growing systems in China. RESULTS: For the sampled Lycosidae, crop identity, pesticide use, and seasons shape the abundance of preydetected in spider guts. For the taxonomic richness of prey, local- and landscape-scale factors gradients were more influential. Multivariate ordinations confirm that these crop-abundant spiders dynamically adjust their diet to reflect environmental constraints and seasonal availability to prey. CONCLUSION: The plasticity in the diet composition is likely to account for the persistence of spiders in relatively ephemeral brassica crops. Our findings provide further insights into the optimization of habitat management for predator-based biological control practices. | ecology |
10.1101/2022.05.07.491005 | Food resources drive rodent population demography mediated by seasonality and inter-specific competition | O_LIAs fast reproducing species, rodents present proximate numerical responses to resource availability that have been assessed by experimental manipulation of food, with contrasting results. Other intrinsic and extrinsic factors, such as climate severity, species life cycles, and sympatry of potential competitors in the community, may interplay to modulate such responses, but their effects have rarely been evaluated ensemble.
C_LIO_LIWe applied a niche-based approach to experimentally determine the effect of bottom-up (food availability) and top-down (climate severity) extrinsic factors, as well as intrinsic seasonal cycles, on rodent demography, also in presence of sympatry between species in the community.
C_LIO_LITo this end, we live-trapped rodents at two latitudinal extremes of the boreal-temperate gradient (Italian Alps and Norway) deploying control/treatment designs of food manipulation. We applied a multistate open robust design model to estimate population patterns and survival rate.
C_LIO_LIYellow-necked and wood mouse (Apodemus spp.) were sympatric with bank vole in Italy, while the latter was the only species trapped in Norway. At northern latitudes, where harsher climatic conditions occurred, vole survival was principally regulated by intrinsic seasonal cycles, with a positive effect of food also on population abundance. At southern latitudes, mice and voles exhibited asynchronous population patterns across seasons, with survival depending from seasonal cycles. When concentrated ad libitum food was experimentally provided, though, population size and survival of voles strongly decreased, while mice abundance benefited from food supplementation.
C_LIO_LIOur results evidence that rodent demography is regulated by a combination of top-down (climate severity) and bottom-up (food availability) extrinsic factors, together with intrinsic seasonal ones. Moreover, we showed that the seasonal niche partitioning of mice and voles could be disrupted by availability of abundant resources that favour the demography of the more opportunistic Apodemus spp. at the expense of Myodes glareolus, suggesting competitive mechanisms. We conclude putting our results in the context of climate change, where shifts in vegetation productivity may affect the diversity of the rodent community via demographic effects.
C_LI | ecology |
10.1101/2022.05.07.490295 | Potential future climate change effects on global reptile distribution and diversity | AimUntil recently, complete information on global reptile distributions has not been widely available. Here, we provide the first comprehensive climate impact assessment for reptile distributions at a global scale.
LocationGlobal, excluding Antarctica
Time period1995, 2050, 2080
Major taxa studiedReptiles
MethodsWe performed species distribution models for 6296 reptile species and assessed potential global as well as realm-specific changes in species richness, the change in global species richness across climatic space and species-specific changes in distribution and range extent and overlap, under future climate change. To assess the future climatic impact of 3768 non-modeled species, we compared the future change in climatic conditions between both modeled and non-modeled species.
ResultsReptile richness was projected to decline significantly over time, globally but also for most zoogeographic realms, with the strongest decrease in Brazil, Australia and South Africa. Species richness was highest in warm, but moist regions, which were projected to shift further to climate extremes in the future. Extents of occurrence were projected to decline considerably in the future, with a low overlap between projected current and future ranges. Shifts in range centroids differed among realms and taxa, with a dominating global poleward shift. Non-modeled species were significantly stronger affected by climatic changes than modeled species.
Main conclusionsReptile richness was projected to decrease significantly across most parts of the world with ongoing future climate change. This effect is visible across lizards, snakes and turtles alike and has considerable impact on species extent of occurrence (EOO) and range distribution. Together with other anthropogenic impacts, such as habitat loss and harvesting, this is cause for concern. Given the historical lack of information on global reptile distributions, this calls for an re-assessment of global conservation efforts towards reptile species, with specific focus on anticipated future climatic changes. | ecology |
10.1101/2022.05.07.490865 | Significant phylogenetic signal is not enough to trust phylogenetic predictions | In a recent study, Cantwell-Jones et al. (2022) proposed a list of 1044 species as promising key sources of B vitamins based primarily on phylogenetic predictions. To identify candidate plants, they fitted lambda models of evolution to edible species with known values in each of six B vitamins (232 to 280 species) and used the estimated parameters to predict B-vitamin profiles of edible plants lacking nutritional data (6460 to 6508 species). The latter species were defined as potential sources of a B vitamin if the predicted vitamin content was [≥]15% towards recommended dietary allowances for active females between 31-50 years per 100 g of fresh edible plant material consumed. Unfortunately, the reliability of the predictions that informed the list of candidate species is questionable due to insufficient phylogenetic signal in the data (Pagels {lambda} between 0.171 and 0.665) and a high incidence of species with missing values (over 95% of all the species analyzed in the study). We found that of the 1044 species proposed as promising B-vitamin sources, 626 to 993 species showed accuracies that were indistinguishable from those obtained under a white noise model of evolution (i.e. random predictions conducted in absence of any phylogenetic structure) in at least one of the vitamins, which proves the weakness of the inference drawn from imputed information in the original study. We hope this commentary serves as a cautionary note for future phylogenetic imputation exercises to carefully assess whether the data meet the requirements for the predictions to be valuable, or at least more accurate than expected by chance. | evolutionary biology |
10.1101/2022.05.07.490996 | Allele-specific knockouts reveal a role for apontic-like in the evolutionary loss of larval pigmentation in Bombyx mori | The domesticated silkworm, Bombyx mori, and its wild progenitor, B. mandarina, are extensively studied as a model case of the evolutionary process of domestication. A conspicuous difference between these species is the dramatic reduction in pigmentation in both larval and adult B. mori. Here we evaluate the efficiency of CRISPR/Cas9-targeted knockouts of pigment-related genes as a tool to understand their potential contributions to domestication-associated pigmentation loss in B. mori. To demonstrate the efficacy of targeted knockouts in B. mandarina, we generated a homozygous CRISPR/Cas9-targeted knockout of yellow-y. In yellow-y knockout mutants, black body color became lighter throughout the larval, pupal and adult stages, confirming a role for this gene in pigment formation. Further, we performed allele-specific CRISPR/Cas9-targeted knockouts of the pigment-related transcription factor, apontic-like (apt-like) in B. mori x B. mandarina F1 hybrid individuals. Knockout of the B. mandarina allele of apt-like in F1 embryos results in depigmented patches on the dorsal integument of larvae, whereas corresponding knockouts of the B. mori allele consistently exhibit normal F1 larval pigmentation. These results demonstrate a contribution of apt-like to the evolution of reduced pigmentation in B. mori. Together, our results demonstrate the feasibility of CRISPR/Cas9-targeted knockouts as a tool for understanding the genetic basis of traits associated with B. mori domestication.
Brief abstractBombyx mori and its wild progenitor are an important model for the study of phenotypic evolution associated with domestication. As proof-of-principle, we used CRISPR/Cas9 to generate targeted knockouts of two pigmentation-related genes. By generating a homozygous knockout of yellow-y in B. mandarina, we confirmed this gene"s role in pigment formation. Further, by generating allele-specific knockouts of apontic-like (apt-like) in B. mori x B. mandarina F1 hybrids, we establish that evolution of apt-like contributed to reduced pigmentation during B. mori domestication.
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org.highwire.dtl.DTLVardef@acae15org.highwire.dtl.DTLVardef@d9143borg.highwire.dtl.DTLVardef@1bf54e7org.highwire.dtl.DTLVardef@5f2a63_HPS_FORMAT_FIGEXP M_FIG C_FIG | genetics |
10.1101/2022.05.06.490983 | MegaBayesianAlphabet: Mega-scale Bayesian Regression methods for genome-wide prediction and association studies with thousands of traits | Large-scale phenotype data are expected to increase the accuracy of genome-wide prediction and the power of genome-wide association analyses. However, genomic analyses of high-dimensional, highly correlated data are challenging. We developed MegaBayesianAlphabet to simultaneously analyze genetic variants underlying thousands of traits using the flexible priors of the Bayesian Alphabet family. As a demonstration, we implemented the BayesC prior in the R package MegaLMM and applied it to both simulated and real data sets. Our analyses show that the resulting model MegaBayesC can effectively use high-dimensional phenotypic data to improve the accuracy of genetic value prediction, the reliability of marker discovery, and the accuracy of marker effect size estimation in genome-wide analyses. | genetics |
10.1101/2022.05.07.491045 | Limited overlap of eQTLs and GWAS hits due to systematic differences in discovery | Most signals in genome-wide association studies (GWAS) of complex traits point to noncoding genetic variants with putative gene regulatory effects. However, currently identified expression quantitative trait loci (eQTLs) explain only a small fraction of GWAS signals. By analyzing GWAS hits for complex traits in the UK Biobank, and cis-eQTLs from the GTEx consortium, we show that these assays systematically discover different types of genes and variants: eQTLs cluster strongly near transcription start sites, while GWAS hits do not. Genes near GWAS hits are enriched in numerous functional annotations, are under strong selective constraint and have a complex regulatory landscape across different tissue/cell types, while genes near eQTLs are depleted of most functional annotations, show relaxed constraint, and have simpler regulatory landscapes. We describe a model to understand these observations, including how natural selection on complex traits hinders discovery of functionally-relevant eQTLs. Our results imply that GWAS and eQTL studies are systematically biased toward different types of variants, and support the use of complementary functional approaches alongside the next generation of eQTL studies. | genomics |
10.1101/2022.05.07.491026 | A detailed landscape of genomic alterations in malignant peripheral nerve sheath tumor cell lines challenges the current MPNST diagnosis | Background: Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that arise from the peripheral nervous system. Half of the tumors develop in the context of the genetic disease Neurofibromatosis type 1 (NF1) and the rest are sporadic sarcomas. MPNSTs have a dismal prognosis due to their aggressiveness and tendency to metastasize, and new treatment options are needed. The diagnosis of MPNSTs can be challenging, especially outside of the NF1 context since specific histological criteria have not been completely established. Genomic analysis may both facilitate differential diagnoses and suggest precision medicine strategies. Methods: We generated a complete genomic resource of a set of widely used human NF1-related and sporadic MPNST cell lines by applying ploidy analysis, whole genome and whole exome sequencing and SNP-array analysis, complemented by methylome-based classification and immunofluorescence of cell identity markers (SOX9, SOX10, S100B). Results: NF1 MPNST cell lines faithfully recapitulated the genomic copy number profile of primary MPNSTs. Structural variants were key players in the complete inactivation of most recurrently altered tumor suppressor genes (TSGs) (NF1, CDKN2A, SUZ12/EED), while small variants played a minor role in the NF1 context, both concerning TSG inactivation and due to the absence of gain-of-function mutations. In clear contrast, the sporadic cell lines (STS-26T, HS-Sch-2, HS-PSS) did not recapitulate the copy number profile of primary MPNSTs. They carried different TSG inactivation and exhibited gain-of-function mutations by predicted kinase activation or generation of fusion genes. Mutational frequencies and signatures emerged as promising informative tools for aiding in MPNST differential diagnosis. Due to the multiple genomic differences exhibited, we complemented their characterization using a methylome-based classifier. All NF1-related cell lines were assigned within the MPNST group, while sporadic cell lines clustered either with melanomas or with an uncertain MPNST-like sarcoma group. The staining of cell identity markers reinforced the idea of a potential misdiagnose of the MPNSTs used to derive the sporadic cell lines analyzed. Conclusions: Deep genomic analysis, together with methylome-based sarcoma classification and cell identity marker analysis, challenged the MPNST identity of sporadic cell lines. Results presented here open an opportunity to revise MPNST differential diagnosis and classification. | genomics |
10.1101/2022.05.06.490969 | The first glimpse of Homo sapiens hereditary fusion genes | Family-inherited fusion genes have been known to be associated with human disease for decades. However, only a small number of them have been discovered so far. In this report, monozygotic (MZ) twins are used as a genetic model to investigate hereditary fusion genes (HFG). We have analyzed RNA-Seq from 37 MZ twins and discovered 1,180 HFGs, the maximum of which is 608 per haploid genome. Eight HFGs associated with MZ twin inheritance range from 52.7% to 67.6%, some of which are previously-studied cancer fusion genes and indicate hereditary cancer genes. These data suggest that HFGs are major genetic factors for human diseases and complex traits. This study gives us the first glimpse of human HFGs and lays theoretical and technological foundations for future genetic and medical studies. | genomics |
10.1101/2022.05.06.490973 | Amplification is the Primary Mode of Gene-by-Sex Interaction in Complex Human Traits | Sexual dimorphism is observed in many complex traits and diseases and is suspected to be in part due to widespread gene-by-sex interactions (GxSex). To date, empirical evidence for GxSex in GWAS data has been elusive. We hypothesized that GxSex may be pervasive but largely missed by current approaches if it acts primarily through sex differences in the magnitude of many genetic effects ("amplification"), regulated by a shared cue such as a sex hormone, rather than differences in the identity of causal variants or the direction of their effect. To test this hypothesis, we inferred the genetic covariance structure between males and females across 27 physiological traits in the UK Biobank. We found amplification to be a pervasive mode of GxSex across traits. As one example, we estimate that 38% of variants have a greater effect on urate levels in females than males. For some traits, notably those related to body mass, testosterone levels are associated with the magnitude of genetic effects in both males and females, but the association is opposite in sign between the sexes. Finally, we developed a novel test of sexually-antagonistic viability selection linking GxSex signals to allele frequency divergence between adult males and females. Using independent allele frequency data, we find marginally-significant evidence for contemporary sexually-antagonistic selection on genetic variation associated with testosterone. In summary, our results suggest that the systematic amplification of genetic effects is a common mode of GxSex that may contribute to sexual dimorphism and fuel its evolution. | genomics |
10.1101/2022.05.06.490975 | Computational prediction and characterization of cell-type-specific and shared binding sites | Cell-type-specific gene expression is maintained in large part by transcription factors (TFs) selectively binding to distinct sets of sites in different cell types. Recent research works have provided evidence that such cell-type-specific binding is determined by TFs intrinsic sequence preferences, cooperative interactions with cofactors, cell-type-specific chromatin landscapes, and 3D chromatin interactions. However, computational prediction and characterization of cell-type-specific and shared binding sites is rarely studied. In this paper, we propose two computational approaches for predicting and characterizing cell-type-specific and shared binding sites by integrating multiple types of features, in which one is based on XGBoost and another is based on convolutional neural network (CNN). To validate the performance of our proposed approaches, ChIP-seq datasets of 10 binding factors were collected from the GM12878 (lymphoblastoid) and K562 (erythroleukemic) human hematopoietic cell lines, each of which was further categorized into cell-type-specific (GM12878-specific and K562-specific) and shared binding sites. Then, multiple types of features for these binding sites were integrated to train the XGBoost-based and CNN-based models. Experimental results show that our proposed approaches significantly outperform other competing methods on three classification tasks. To explore the contribution of different features, we performed ablation experiments and feature importance analysis. Consistent with previous studies, we find that chromatin features are major contributors in which chromatin accessibility is the best predictor. Moreover, we identified independent feature contribution for cell-type-specific and shared sites through SHAP values, observing that chromatin features play a main role in the cell-type-specific sites while motif features play a main role in the shared sites. Beyond these observations, we explored the ability of the CNN-based model to predict cell-type-specific and shared binding sites by excluding or including DNase signals, showing that chromatin accessibility significantly improves the prediction performance. Besides, we investigated the generalization ability of our proposed approaches to different binding factors in the same cellular environment or to the same binding factors in the different cellular environments. | bioinformatics |
10.1101/2022.05.06.490832 | Hemodynamic transient and functional connectivity follow structural connectivity and cell type over the brain hierarch | The neural circuit of the brain is organized as a hierarchy of functional units with wide-ranging connections that support the information flow and functional connectivity. Studies using magnetic resonance imaging (MRI) indicate a moderate coupling between structural and functional connectivity at the system level. However, how do connections of different directions (feedforward and feedback) and regions with different excitatory and inhibitory (E/I) neurons shape the hemodynamic activity and functional connectivity over the hierarchy are unknown. Here, we used functional MRI to detect optogenetic-evoked and resting-state activities over a somatosensory pathway in the mouse brain and compared with axonal projection and E/I distribution. With a highly sensitive ultrafast imaging, we identified extensive activation in regions up to the third order of axonal projections following optogenetic excitation of the ventral posteriomedial nucleus of the thalamus. The evoked response and functional connectivity correlated with feedforward projections but less with the feedback and weakened with the hierarchy. The hemodynamic signal exhibited regional and hierarchical differences, with slower and more variable responses in high-order areas and bipolar responses predominantly in the contralateral cortex. Importantly, the positive and negative parts of hemodynamics correlated with E/I neuronal densities, respectively. Furthermore, resting-state functional connectivity more associated with E/I distribution whereas stimulus-evoked effective connectivity followed structural wiring. These findings indicate that structure-function relationship is projection-, cell-type- and hierarchy-dependent. Hemodynamic transients could reflect E/I activity and the increased complexity of hierarchical processing.
Significance statementThe neural circuit of the brain is organized as a hierarchy of functional units with complicated feedforward and feedback connections to selectively enhance (excitation) or suppress (inhibit) information from massive sensory inputs. How brain activity is shaped by the structural wiring and excitatory and inhibitory neurons are still unclear. We characterize how brain-wide hemodynamic responses reflect these structural constituents over the hierarchy of a somatosensory pathway. We find that functional activation and connectivity correlate with feedforward connection strengths and neuronal distributions. This association subsides with hierarchy due to slower and more variable hemodynamic responses, reflecting increased complexity of processing and neuronal compositions in high-order areas. Our findings indicate that hemodynamics follow the hierarchy of structural wiring and neuronal distribution. | neuroscience |
10.1101/2022.05.07.491000 | Multi-tasking Deep Network for Tinnitus Classification and Severity Prediction from Multimodal Structural Images | Subjective tinnitus is an auditory phantom perceptual disorder without an objective biomarker. Fast and efficient diagnostic tools will advance clinical practice by detecting or confirming the condition, tracking change in severity, and monitoring treatment response. Motivated by evidence of subtle anatomical or functional morphological information in magnetic resonance images (MRI) of the brain, we examined data-driven machine learning methods for joint tinnitus classification (tinnitus or no tinnitus) and tinnitus severity prediction. We propose a deep multi-task multi-modal framework for joint functionalities using structural MRI (sMRI) data. To leverage cross-information multimodal neuroimaging data, we integrated two modalities of 3-dimensional sMRI - T1 weighted (T1w) and T2 weighted (T2w) images. To explore the key components in the MR images that drove task performance, we segmented both T1w and T2w images into three different components - cerebrospinal fluid (CSF), grey matter (GM) and white matter (WM), and examined performance of each segmented image. Results demonstrate that our multimodal framework capitalizes on the information across both modalities (T1w and T2w) for the joint task of tinnitus classification and severity prediction. Our model outperforms existing learning-based and conventional methods in terms of accuracy, sensitivity, specificity, and negative predictive value. | neuroscience |
10.1101/2022.05.07.491033 | Nitric oxide-induced tyrosine nitration of TrkB impairs BDNF signaling and restrains neuronal plasticity | Nitric oxide has been long recognized as an important modulator of neural plasticity, but characterization of the molecular mechanisms involved - specially the guanylyl cyclase-independent ones - has been challenging. There is evidence that NO could modify BDNF-TRKB signaling, a key mediator of neuronal plasticity. However, the mechanism underlying the interplay of NO and TRKB remains unclear. Here we show that nitric oxide induces nitration of the tyrosine 816 in the TRKB receptor in vivo and in vitro, and that post-translational modification inhibits TRKB phosphorylation and binding of phospholipase C{gamma}1 (PLC{gamma}1) to this same tyrosine residue. Additionally, nitration triggers clathrin-dependent endocytosis of TRKB through the adaptor protein AP2M and ubiquitination, thereby increasing translocation of TRKB away from the neuronal surface and directing it towards lysosomal degradation. Accordingly, inhibition of nitric oxide increases TRKB phosphorylation and TRKB-dependent neurite branching in neuronal cultures. In vivo, chronic inhibition of neuronal nitric oxide synthase (nNOS) dramatically reduced TRKB nitration and facilitated TRKB signaling in the primary visual cortex, and promoted a shift in ocular dominance upon monocular deprivation in the visual cortex - an indicator of increased plasticity. Altogether, our data describe and characterize a new molecular brake on plasticity, namely nitration of TRKB receptors.
Significance statementWe described the nitration of TRKB receptors at the tyrosine residue 816 as a new post-translational modification (PTM) that restrains the signaling of the neurotrophic factor BDNF in neurons. This new PTM leads to endocytosis and degradation of the TRKB receptors. Intriguingly, this mechanism is tonically active under physiological conditions in vivo, and it is important for restricting ocular dominance plasticity in the visual cortex. This mechanism directly links two major systems involved in brain plasticity, BDNF/TRKB and nitric oxide. Our data provides a model for how NO production from nNOS can compromise TRKB function, and for the effects of nNOS inhibitors promoting plasticity. | neuroscience |
10.1101/2022.05.07.491043 | Reconstruction of the cell pseudo-space from single-cell RNA sequencing data with scSpace | Tissues are highly complicated with spatial heterogeneity in gene expression. However, the cutting-edge single-cell RNA-seq technology eliminates the spatial information of individual cells, which contributes to the characterization of cell identities. Herein, we propose single-cell spatial position associated co-embeddings (scSpace), an integrative algorithm to distinguish spatially variable cell subclusters by reconstructing cells onto a pseudo-space with spatial transcriptome references (Visium, STARmap, Slide-seq, etc.). We demonstrated that scSpace can define biologically meaningful cell subpopulations neglected by single-cell RNA-seq or spatially resolved transcriptomics. The use of scSpace to uncover the spatial association within single-cell data, reproduced, the hierarchical distribution of cells in the brain cortex and liver lobules, and the regional variation of cells in heart ventricles and the intestinal villus. scSpace identified cell subclusters in intratelencephalic neurons, which were confirmed by their biomarkers. The application of scSpace in melanoma and Covid-19 exhibited a broad prospect in the discovery of spatial therapeutic markers. | bioinformatics |
10.1101/2022.05.06.490927 | Molecular dynamics of spike variants in the locked conformation: RBD interfaces, fatty acid binding and furin cleavage sites. | Since December 2019 the SARS-CoV-2 virus has infected billions of people around the world and caused millions of deaths. The ability for this RNA virus to mutate has produced variants that have been responsible for waves of infections across the globe. The spike protein on the surface of the SARS-CoV-2 virion is responsible for cell entry in the infection process. Here we have studied the spike proteins from the Original, Alpha (B.1.1.7), Delta (B1.617.2), Delta-plus (B1.617.2-AY1), Omicron BA.1 and Omicron BA.2 variants. Using models built from cryo-EM structures with linoleate bound (6BZ5.pdb) and the N-terminal domain from 7JJI.pdb, each is built from the first residue, with missing loops modelled and 45 disulphides per trimer. Each spike variant was modified from the same Original model framework to maximise comparability. Three replicate, 200 ns atomistic molecular dynamics simulations were performed for each case. (These data also provide the basis for further, non-equilibrium molecular dynamics simulations, published elsewhere.) The analysis of our equilibrium molecular dynamics reveals that sequence variation at the closed receptor binding domain interface particularly for Omicron BA.2 has implications for the avidity of the locked conformation, with potential effects on Omicron BA.1 and Delta-plus. Linoleate binding has a mildly stabilizing effect on furin cleavage site motions in the Original and Alpha variants, but has no effect in Delta, Delta-plus and slightly increases motions at this site for Omicron BA.1, but not BA.2, under these simulation conditions. | biochemistry |
10.1101/2022.05.06.490956 | Fixation Can Change the Appearance of Phase Separation in Living Cells | Fixing cells with paraformaldehyde (PFA) is an essential step in numerous biological techniques as it is thought to preserve a snapshot of biomolecular transactions in living cells. Fixed cell imaging techniques such as immunofluorescence have been widely used to detect liquid-liquid phase separation (LLPS) in vivo. Here, we compared images, before and after fixation, of cells expressing intrinsically disordered proteins that are able to undergo LLPS. Surprisingly, we found that PFA fixation can both enhance and diminish putative LLPS behaviors. For specific proteins, fixation can even cause their droplet-like puncta to artificially appear in cells that do not have any detectable puncta in the live condition. Fixing cells in the presence of glycine, a molecule that modulates fixation rates, can reverse the fixation effect from enhancing to diminishing LLPS appearance. We further established a kinetic model of fixation in the context of dynamic protein-protein interactions. Simulations based on the model suggest that protein localization in fixed cells depends on an intricate balance of protein-protein interaction dynamics, the overall rate of fixation, and notably, the difference between fixation rates of different proteins. Our work reveals that PFA fixation changes the appearance of LLPS from living cells, presents a caveat in studying LLPS using fixation-based methods, and suggests a mechanism underlying the fixation artifact. | cell biology |
10.1101/2022.05.08.491072 | On the limits of fitting complex models of population history to genetic data | Our understanding of human population history in deep time has been assisted by fitting 'admixture graphs' to data: models that specify the ordering of population splits and mixtures which is the only information needed to capture the patterns of allele frequency correlation among populations. Not needing to specify population size changes, split times, or whether admixture events were sudden or drawn out simplifies the space of models that need to be searched. However, the space of possible admixture graphs relating populations is vast and cannot be sampled fully, and thus most published studies have identified fitting admixture graphs through a manual process driven by prior hypotheses, leaving the vast majority of alternative models unexplored. Here, we develop a method for systematically searching the space of all admixture graphs that can incorporate non-genetic information in the form of topology constraints. We implement this findGraphs tool within a software package, ADMIXTOOLS 2, which is a reimplementation of the ADMIXTOOLS software with new features and large performance gains. We apply this methodology to identify alternative models to admixture graphs that played key roles in eight published studies and find that graphs modeling more than six populations and two or three admixture events are often not unique, with many alternative models fitting nominally or significantly better than the published one. Our results suggest that strong claims about population history from admixture graphs should only be made when all well-fitting and temporally plausible models share common topological features. Our re-evaluation of published data also provides insight into the population histories of humans, dogs, and horses, identifying features that are stable across the models we explored, as well as scenarios of populations relationships that differ in important ways from models that have been highlighted in the literature, that fit the allele frequency correlation data, and that are not obviously wrong. | evolutionary biology |
10.1101/2022.05.08.491072 | On the limits of fitting complex models of population history to genetic data | Our understanding of human population history in deep time has been assisted by fitting 'admixture graphs' to data: models that specify the ordering of population splits and mixtures which is the only information needed to capture the patterns of allele frequency correlation among populations. Not needing to specify population size changes, split times, or whether admixture events were sudden or drawn out simplifies the space of models that need to be searched. However, the space of possible admixture graphs relating populations is vast and cannot be sampled fully, and thus most published studies have identified fitting admixture graphs through a manual process driven by prior hypotheses, leaving the vast majority of alternative models unexplored. Here, we develop a method for systematically searching the space of all admixture graphs that can incorporate non-genetic information in the form of topology constraints. We implement this findGraphs tool within a software package, ADMIXTOOLS 2, which is a reimplementation of the ADMIXTOOLS software with new features and large performance gains. We apply this methodology to identify alternative models to admixture graphs that played key roles in eight published studies and find that graphs modeling more than six populations and two or three admixture events are often not unique, with many alternative models fitting nominally or significantly better than the published one. Our results suggest that strong claims about population history from admixture graphs should only be made when all well-fitting and temporally plausible models share common topological features. Our re-evaluation of published data also provides insight into the population histories of humans, dogs, and horses, identifying features that are stable across the models we explored, as well as scenarios of populations relationships that differ in important ways from models that have been highlighted in the literature, that fit the allele frequency correlation data, and that are not obviously wrong. | evolutionary biology |
10.1101/2022.05.08.491080 | Mosaic patterns of selection in genomic regions associated with diverse human traits | Natural selection shapes the genetic architecture of many human traits. However, the prevalence of different modes of selection on genomic regions associated with variation in traits remains poorly understood. To address this, we developed an efficient computational framework to calculate enrichment of different evolutionary measures among regions associated with complex traits. We applied the framework to summary statistics from >900 genome-wide association studies (GWASs) and 11 evolutionary measures of sequence constraint, population differentiation, and allele age while accounting for linkage disequilibrium, allele frequency, and other potential confounders. We demonstrate that this framework yields consistent results across GWASs with variable sample sizes, numbers of trait-associated SNPs, and analytical approaches. The resulting evolutionary atlas maps diverse signatures of selection on genomic regions associated with complex human traits on an unprecedented scale. We detected positive enrichment for sequence conservation among trait-associated regions for the majority of traits (>77% of 290 high power GWASs), which was most dominant in reproductive traits. Many traits also exhibited substantial enrichment for population differentiation and recent positive selection, especially among hair, skin, and pigmentation traits. In contrast, we detected widespread negative enrichment for balancing selection (51% GWASs) and no evidence of enrichment for selection signals in regions associated with late-onset Alzheimer's disease. These results support a pervasive role for negative selection on regions of the human genome that contribute to variation in complex traits, but also demonstrate where diverse modes of selection have shaped trait-associated loci. This atlas of signatures of different modes of natural selection across the diversity of available GWASs will enable exploration of the relationship between the genetic architecture and selection in the human genome. | evolutionary biology |
10.1101/2022.05.07.491022 | Cell cycle independent role of cyclin D3 in host restriction of SARS-CoV-2 infection | The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents a great threat to human health. The interplay between the virus and host plays a crucial role in successful virus replication and transmission. Understanding host-virus interactions is essential for development of new COVID-19 treatment strategies. Here we show that SARS-CoV-2 infection triggers redistribution of cyclin D1 and cyclin D3 from the nucleus to the cytoplasm, followed by its proteasomal degradation. No changes to other cyclins or cyclin dependent kinases were observed. Further, cyclin D depletion was independent from SARS-CoV-2 mediated cell cycle arrest in early S phase or S/G2/M phase. Cyclin D3 knockdown by small interfering RNA specifically enhanced progeny virus titres in supernatants. Finally, cyclin D3 co-immunoprecipitated with SARS-CoV-2 Envelope and Membrane proteins. We propose that cyclin D3 inhibits virion assembly and is depleted during SARS-CoV-2 infection to restore efficient assembly and release of newly produced virions. | microbiology |
10.1101/2022.05.08.491059 | FIB-SEM analysis on three-dimensional structures of growing organelles in wild Chlorella pyrenoidosa cells | To clarify dynamic changes of organelle microstructures in Chlorella pyrenoidosa cells during photosynthetic growth with CO2 fixation, three-dimensional organelle microstructures in three growth periods of meristem, elongation and maturity were quantitatively determined and comprehensively reconstructed with focused ion beam scanning electron microscopy (FIB-SEM). The single round-pancake mitochondria in each cell split into a dumbbell and then into a circular ring, while barycenter distance of mitochondria to chloroplast and nucleus was reduced to 45.5% and 88.3% to strengthen energy transfer, respectively. The single pyrenoid consisting of a large part and another small part in each chloroplast gradually developed to a mature state in which the two parts were nearly equal in size. The nucleolus progressively became larger with euchromatin replication. The number of starch grains gradually increased, but average grain volume remained nearly unchanged. | cell biology |
10.1101/2022.05.06.490962 | The bacterial lysate Lantigen B reduces the expression of ACE2 on primary oropharyngeal cells | Background: Vercelli and coworkers recently observed that a well-established bacterial lysate (OM-85, Vifor Pharma; CH) was able to downregulate the expression of Angiotensin-Converting Enzyme 2 (ACE2) on epithelial cells. This downregulation was also associated with a reduced infectivity of cells, resulting in a reduced viral titre. We evaluated whether another bacterial lysate (Lantigen B, Bruschettini Ltd; Italy) may have similar activities. However, while OM-85 is given per os and has a systemic effect after absorption at the gut level, Lantigen B is given locoregionally. Thus, the concentration that the bacterial lysate can reach at the mucosal level seems to be promising. Methods: Oropharyngeal cells were collected from healthy donors. After 24 hours of treatment in vitro with doses of Lantigen B comparable to those that are reached in vivo, the expression of ACE2 was evaluated by direct fluorescence and flow cytometry. Results: A reduction in the number of ACE2-positive cells was observed in 80% of treated samples. Only a few donors had poor expression of ACE2, and in these donors, the downregulation was less evident or absent. Conclusions: These results suggest that Lantigen B, at pharmacological doses, could be an interesting drug to reduce ACE2 expression on oropharyngeal cells, thus contributing to the prophylaxis of COVID-19 in humans. | cell biology |
10.1101/2022.05.08.490683 | Capacitation promotes a shift in the energy metabolism in murine sperm | In mammals, sperm acquire fertilization ability after a series of physiological and biochemical changes, collectively known as capacitation, that occur inside the female reproductive tract. In addition to other requirements, sperm bioenergetic metabolism has been identified as a fundamental component in the acquisition of the capacitated status. Mammalian sperm produce ATP by means of two main metabolic processes, oxidative phosphorylation (OXPHOS) and aerobic glycolysis, that are localized in two different flagellar compartments, midpiece and principal piece, respectively. In mouse sperm, the occurrence of many events associated with capacitation depends on the activity of these two energy-producing pathways, leading to the hypothesis that some of these events may impose changes in sperm energetic demands. In the present study, we used extracellular flux analysis to evaluate the changes in the glycolytic and respiratory parameters of murine sperm that occur as a consequence of capacitation. Furthermore, we examined whether these variations affect sperm ATP sustainability. Our results show that capacitation promotes a shift in the usage ratio of the two main metabolic pathways, from oxidative to glycolytic. However, this metabolic rewiring does not seem to affect the rate at which the sperm consume ATP. We conclude that the probable function of the metabolic switch is to increase the ATP supply in the distal flagellar regions, thus sustaining the energetic demands that arise from capacitation. | cell biology |
10.1101/2022.05.08.491074 | A Drosophila larvae-inspired vacuum-actuated soft robot | Peristalsis is one of the most common locomotion patterns in limbless animals. This motion is generated by propagating muscular contraction and relaxation along the body axis. While the kinematics of peristalsis has been examined intensively, the kinetics and mechanical control of peristalsis remain unclear, partially due to the lack of suitable physical models to analyse the force and temporal control in soft-bodied animals' locomotion. Here, based on a soft-bodied animal, Drosophila larvae, we proposed a vacuum-actuated soft robot replicating their crawling behaviour. The soft structure, made with hyperelastic silicon rubber, was designed to mimic the larval hydrostatic structure. To estimate the adequate range of pressures and time scales for control of the soft robots, a numerical simulation by the finite element method was conducted. Pulse-Width-Modulation (PWM) was used to generate time-series signals to control the vacuum pressure in each segment. Based on this control system, the soft robots could exhibit the peristaltic pattern resembling fly larval crawling. The soft robots reproduced two previous experimental results on fly larvae: slower crawling speed in backward crawling than in forward crawling, and the involvement of segmental contraction duration and intersegmental delay in crawling speed. Furthermore, the soft robot provided a novel prediction that the larger the contraction force, the faster the crawling speed. These observations indicate that the use of soft robots could serve to examine the kinetics and mechanical regulation of crawling behaviour in soft-bodied animals. | biophysics |
10.1101/2022.05.07.491038 | Simultaneous and sequential multi-species coronavirus vaccination | Although successful COVID-19 vaccines have been developed, multiple pathogenic coronavirus species exist, urging for development of multi-species coronavirus vaccines. Here we developed prototype LNP-mRNA vaccine candidates against SARS-CoV-2 (Delta variant), SARS-CoV and MERS-CoV, and test how multiplexing of these LNP-mRNAs can induce effective immune responses in animal models. A triplex scheme of LNP-mRNA vaccination induced antigen-specific antibody responses against SARS-CoV-2, SARS-CoV and MERS-CoV, with a relatively weaker MERS-CoV response in this setting. Single cell RNA-seq profiled the global systemic immune repertoires and the respective transcriptome signatures of multiplexed vaccinated animals, which revealed a systemic increase in activated B cells, as well as differential gene expression signatures across major adaptive immune cells. Sequential vaccination showed potent antibody responses against all three species, significantly stronger than simultaneous vaccination in mixture. These data demonstrated the feasibility, antibody responses and single cell immune profiles of multi-species coronavirus vaccination. The direct comparison between simultaneous and sequential vaccination offers insights on optimization of vaccination schedules to provide broad and potent antibody immunity against three major pathogenic coronavirus species. | immunology |
10.1101/2022.05.08.491081 | Effects of larval and adult crowding on fitness components in Drosophila populations adapted to larval crowding experienced under different combinations of food amount and egg number | Since the realization in the 1970s that simple discrete-time population growth models can show complex unstable dynamics of population size, many explanations were proposed for the evolution of enhanced population stability. The most plausible of these was density-dependent selection, suggested to favour greater stability due to r-K trade-offs. However, the first experiment aimed at testing this prediction revealed that Drosophila melanogaster populations adapted to larval crowding did not evolve greater constancy stability than their ancestral controls. A subsequent study showed that D. ananassae populations adapted to larval crowding had evolved greater constancy and persistence than ancestral controls. These D. ananassae populations had experienced chronic larval crowding in conditions of very low amounts of food, whereas the earlier studied D. melanogaster populations had experienced chronic larval crowding at fairly high food amounts. Further theoretical work also suggested that populations adapting to crowding could evolve greater stability even in the absence of r-K trade-offs. Most recently, studies in our laboratory showed that two sets of crowding adapted D. melanogaster populations, derived from a common ancestral lineage, which differed in the food amounts at which they experienced larval crowding, evolved different patterns of constancy and persistence stability. These two sets of populations also differed in the traits, e.g. larval feeding rate, that evolved as they became more competitive. Here, we examine the response of key fitness components to larval and adult densities in these two sets of populations, to see whether differences in their stability attributes can be explained by variation in how their life-histories respond to crowding at different life stages. Of all traits examined, only pre-adult survivorship responded differently to larval density across the two sets of populations. The populations that adapted to larval crowding at low food amounts showed reduced sensitivity of pre-adult survivorship to larval density, compared to those that adapted to larval crowding at high food amounts. We discuss our results in the context of different ways in which density-dependent selection may facilitate the evolution of greater constancy or persistence, depending on the ecological details of how crowding was experienced. | evolutionary biology |
10.1101/2022.05.08.491058 | High-throughput discovery of TRAF6-interacting peptides identifies determinants of positive and negative design and shows known and candidate human interaction partner motifs are not optimized for affinity | TRAF6 is an adapter protein and E3 ubiquitin ligase that is involved in signaling downstream of cell receptors important for development and immune system activation and maintenance. TRAF6 participates in hundreds of protein-protein interactions, some of which are mediated by a C-terminal MATH domain that recruits TRAF6 to cell-surface receptors and associated proteins. The TRAF6 MATH domain binds to short peptide segments containing the motif PxExx[FYWHDE], where x is any amino acid. Blocking TRAF6 interactions is associated with favorable effects in a variety of disease models. To better define TRAF6 binding preferences, we generated a bacterial cell-surface peptide display library to sample the TRAF6 motif sequence space. We performed sorting experiments to identify 236 of the best TRAF6-interacting peptides and a set of 1,200 peptides that match the sequence PxE but do not bind TRAF6. Selected binders tested by single-clone bacterial display titrations and by bio-layer interferometry bound TRAF6 tighter than previously measured native peptides. We built all-atom structural models of the TRAF6 MATH domain in complex with high-affinity binders and motif-matching nonbinders identified by screening to elucidate the structural basis for TRAF6 interaction preferences. We identified motif features that favor binding to TRAF6 and also negative design elements, distributed across the motif, that can disfavor or preclude binding. Searching the human proteome for matches to the library screening-defined binding motif revealed that most known, biologically relevant TRAF6 binders occupy a different sequence space from the most enriched hits that we discovered in combinatorial library screening. Our experimentally determined binding preferences and structural models can support the design of peptide-based interaction inhibitors with higher affinities than endogenous TRAF6 ligands. | biochemistry |
10.1101/2022.05.04.490646 | AA-amyloidosis in cats (Felis catus) housed in shelters | Systemic AA-amyloidosis is a protein-misfolding disease that is characterized by fibril deposition of serum amyloid-A protein (SAA) in several organs in humans and many animal species. Fibril deposits originate from abnormally high serum levels of SAA during chronic inflammation. In domestic short-hair cats, AA-amyloidosis has only been anecdotally reported and is considered a rare disease. Here we report that an astonishing 57-73% of early deceased short-hair cats kept in three independent shelters suffer from amyloid deposition in the liver, spleen, or kidney. Histopathology and mass spectrometry of post-mortem extracted deposits identified SAA as the major protein source. The duration of stay in the shelters was positively associated with a histological score of AA-amyloidosis (B=0.026, CI95%=0.007-0.046; p=0.010). Presence of SAA fragments in bile secretions raises the possibility of fecal-oral transmission of the disease. | pathology |
10.1101/2022.05.08.490841 | Cardiac patch treatment alleviates ischemic cardiomyopathy correlated with reverting Piezo1/2 expression by unloading left ventricular myocardium | Pathologically elevated mechanical load promotes the adverse remodeling of left ventricle (LV) post myocardial infarction, which results in the progression from ischemic cardiomyopathy to heart failure. Cardiac patches could attenuate adverse LV remodeling by providing mechanical support to infarcted myocardium and border zone tissue. However, the mechanism of the translation from mechanical effects to favorable therapeutic outcome is still not clear. By transcriptome analysis, we found that the myocardial transcription levels of mechanosensitive ion channel proteins Piezo1 and Piezo2 significantly increased in patients with ischemic cardiomyopathy. In vitro tensile tests with local tissue information revealed a significant decrease in local strain and mechanical load in rat infarct. Cardiac function and geometry were preserved compared to non-treated control. Further, in LV myocardium of the patch-treated group, MI induced expression levels of Piezo1/2 were significantly reverted to the similar levels of the sham group, indicating that cardiac patch beneficial effects were correlated with suppressing mechanosensitive genes, particularly Piezo1/2. These findings demonstrated the potential of cardiac patches in treating ICM patients with remodeling risks, and could provide guidance for improvement in next generation of patch devices. | pathology |
10.1101/2022.05.08.491034 | Adipokine C1q/Tumor necrosis factor-related protein 3 (CTRP3) Attenuates Intestinal Inflammation via Histone Deacetylase Sirtuin 1 (SIRT1)/NF-κB Signaling | BACKGROUND & AIMS: The adipokine C1q/tumor necrosis factor-related protein 3 (CTRP3) has anti-inflammatory effects in several non-intestinal disorders. Although CTRP3 is reduced in the serum of patients with inflammatory bowel disease (IBD), its function in IBD has not been established. We aimed to elucidate the function of CTRP3 and related molecular mechanisms in intestinal inflammation using a colitis model of genetically-modified CTRP3 mice and intestinal epithelial tissue from patients with Crohns disease (CD), one of the two main forms of IBD. METHODS: CTRP3 knockout (KO) and overexpressing transgenic (Tg) mice along with their corresponding wild-type (WT) littermates were subjected to drinking water containing dextran sulfate sodium (DSS) for 6-10 days to induce acute colitis. Mouse colitis symptoms and histological data were analyzed. CTRP3-mediated signaling was examined in the intestinal tissue of mice and patients with CD. RESULTS: CTRP3 mRNA and protein were detected in murine and human intestinal epithelial cells, as well as in murine intestinal smooth muscle cells and mesenteric fat. In DSS-induced acute colitis models, CTRP3 KO mice developed more severe colitis than their WT littermates, while CTRP3 overexpressing Tg mice developed less severe colitis than their WT littermates. In both water- and DSS-treated CTRP3 KO mice, reduced CTRP3 levels correlated with decreased levels of Sirtuin 1 (SIRT1), a histone deacetylase, increased levels of phosphorylated nuclear factor kappa B (NF-kB) subunit p65, resulting in increased expression of pro-inflammatory cytokines tumor necrosis factor-a (TNF-a) and interleukin 6 (IL-6). The results from CTRP3 Tg mice mirrored those from CTRP3 KO mice in most respects. This CTRP3/SIRT1/NF-kB relationship was also observed in the intestinal epithelial tissue of patients with active and inactive CD. CONCLUSIONS: CTRP3 expression levels correlate negatively with intestinal inflammation in mouse colitis models and CD patients. CTRP3 attenuates intestinal inflammation via SIRT1/NF-kB signaling to suppress pro-inflammatory cytokines in mouse colitis models and patients with IBD. The manipulation of CTRP3 signaling, including through the use of SIRT1 agonists, may offer translational potential in the treatment of IBD. | pathology |
10.1101/2022.05.08.491083 | A data-driven approach to quantifying meal characteristics influencing energy intake | We used a data-driven approach to determine the influence of energy density, hyper-palatability, protein content, and eating rate on ad libitum non-beverage energy intake during 2733 meals consumed by 35 inpatient adults who participated in two 28-day feeding studies. All four meal characteristics significantly contributed to energy intake, but their relative importance varied by the prevailing dietary pattern according to macronutrient composition and degree of processing. | physiology |
10.1101/2022.05.07.491047 | Responses of Agricultural plants to Lithium pollution: Trends, Meta-Analysis, and Perspectives | Lithium (Li) is gaining attention due to rapid rise of modern industries but their ultimate fingerprints on plants are not well established. Herein, we executed a meta-analysis of the existing recent literature investigating the impact of Li sources and levels on plant species under different growth conditions to understand the existing state of knowledge. Toxic effects of Li exposure in plants varies as a function of medium and interestingly, more negative responses are reported in hydroponic media as compared to soil and foliar application. Additionally, toxic effects of Li vary with Li source materials and LiCl more negatively affected plant development parameters such as plant germination (n=48) and root biomass (n=57) and recorded highly uptake in plants (n=78), while LiNO3 has more negative effects on shoot biomass. The Li at <50 mg L-1 concentrations significantly influenced the plant physiological indicators including plant germination and root biomass, while 50-500 mg L-1 Li concentration influence the biochemical parameters. The uptake potential of Li is dose dependent and their translocation/bioaccumulation remains unknown. Future work should include complete lifespan studies of the crop to elucidate the bioaccumulation of Li in edible tissues and to investigate possible trophic transfer of Li. | plant biology |
10.1101/2022.05.07.491053 | Dynamic emergence of relational structure network in human brains | Reasoning the hidden relational structure from sequences of events is a crucial ability humans possess, which help them to predict the future and make inferences. Besides simple statistical properties, humans also excel in learning more complex relational networks. Several brain regions are engaged in the process, yet the time-resolved neural implementation of relational structure learning and its behavioral relevance remains unknown. Here human subjects performed a probabilistic sequential prediction task on image sequences generated from a transition graph-like network, with their brain activities recorded using electroencephalography (EEG). We demonstrate the emergence of two key aspects of relational knowledge - lower-order transition probability and higher-order community structure, which arise around 840 msec after image onset and well predict behavioral performance. Furthermore, computational modeling suggests that the formed higher-order community structure, i.e., compressed clusters in the network, could be well characterized by a successor representation operation. Overall, human brains are constantly computing the temporal statistical relationship among discrete inputs, based on which new abstract knowledge could be inferred. | neuroscience |
10.1101/2022.05.06.490941 | Predicting Working Memory performance based on specific individual EEG spatiotemporal features | Working Memory (WM) is a limited capacity system for storing and processing information, which varies from subject to subject. Several works show the ability to predict the performance of WM with machine learning (ML) methods, and although good prediction results are obtained in these works, ignoring the intersubject variability and the temporal and spatial characterization in a WM task to improve the prediction in each subject. In this paper, we take advantage of the spectral properties of WM to characterize the individual differences in visual WM capacity and predict the subject's performance. Feature selection was implemented through the selection of electrodes making use of methods to treat unbalanced classes. The results show a correlation between the accuracy achieved with an Regularized Linear Discriminant Analysis (RLDA) classifier using the power spectrum of the EEG signal and the accuracy achieved by each subject in the behavioral experiment response of a WM task with retro-cue. The proposed methodology allows identifying spatial and temporal characteristics in the WM performance in each subject. Our methodology shows that it is possible to predict the WM performance in each subject. Finally, our results showed that by knowing the spatiotemporal characteristics that predict WM performance, it is possible to customize a WM task and optimize the use of electrodes for agile processing adapted to a specific subject. Thus, we pave the way for implementing neurofeedback through a Brain-Computer Interface. | neuroscience |
10.1101/2022.05.08.491082 | A sequential two-step priming scheme reproduces diversity in synaptic strength and short-term plasticity | Glutamatergic synapses display variable strength and diverse short-term plasticity (STP), even for a given type of connection. Using non-negative tensor factorization (NTF) and conventional state modelling, we demonstrate that a kinetic scheme consisting of two sequential and reversible steps of release-machinery assembly and a final step of synaptic vesicle (SV) fusion reproduces STP and its diversity among synapses. Analyzing transmission at calyx of Held synapses reveals that differences in synaptic strength and STP are not primarily caused by variable fusion probability (pfusion) but determined by the fraction of docked synaptic vesicles equipped with a mature release machinery. Our simulations show, that traditional quantal analysis methods do not necessarily report pfusion of SVs with a mature release machinery but reflect both pfusion and the distribution between mature and immature priming states at rest. Thus, the approach holds promise for a better mechanistic dissection of the roles of presynaptic proteins in the sequence of SV docking, two-step priming and fusion and suggests a mechanism for activity-induced redistribution of synaptic efficacy. | neuroscience |
10.1101/2022.05.07.491044 | Opposing associations of Internet Use Disorder symptom domains with structural and functional organization of the striatum: a dimensional neuroimaging approach | Background: Accumulating evidence suggests brain structural and functional alterations in Internet Use Disorder (IUD). However, conclusions are strongly limited due to the retrospective case-control design of the studies, small samples, and the focus on general rather than symptom-specific approaches. Methods: We here employed a dimensional multi-methodical MRI-neuroimaging design in a final sample of n = 203 subjects to examine associations between levels of IUD and its symptom-dimensions (loss of control/time management, craving/social problems) with brain structure, resting state and task-based (pain empathy, affective go/no-go) brain function. Results: Although the present sample covered the entire range of IUD, including normal, problematic as well as pathological levels, general IUD symptom load was not associated with brain structural or functional alterations. However, the symptom-dimensions exhibited opposing associations with the intrinsic and structural organization of the brain, such that loss of control/time management exhibited negative associations with intrinsic striatal networks and hippocampal volume, while craving/social problems exhibited a positive association with intrinsic striatal networks and caudate volume. Conclusions: Our findings provided the first evidence for IUD symptom-domain specific associations with progressive alterations in the intrinsic structural and functional organization of the brain, particularly of striatal systems involved in reward, habitual and cognitive control processes. | neuroscience |
10.1101/2022.05.08.491092 | m6A epitranscriptomic modification regulates neural progenitor-to-glial cell transition in the retina | N6-methyladenosine (m6A) is the most prevalent mRNA internal modification and has been shown to regulate the development, physiology and pathology of various tissues. However, the functions of the m6A epitranscriptome in the visual system remain unclear. In this study, using a retina-specific conditional knockout mouse model, we show that retinas deficient in Mettl3, the core component of the m6A methyltransferase complex, exhibit structural and functional abnormalities beginning at the end of retinogenesis. Immunohistological and scRNA-seq analyses of retinogenesis processes reveal that retinal progenitor cells (RPCs) and Muller glial cells are the two cell types primarily affected by Mettl3 deficiency. Integrative analyses of scRNA-seq and MeRIP-seq data suggest that m6A fine-tunes the transcriptomic transition from RPCs to Muller cells by promoting the degradation of RPC transcripts, the disruption of which leads to abnormalities in late retinogenesis and compromises the glial functions of Muller cells. Finally, overexpression of m6A-regulated RPC-enriched transcripts in late RPCs partially recapitulates the Mettl3- deficient retinal phenotype. Collectively, our study reveals an epitranscriptomic mechanism governing progenitor-to-glial cell transition during late retinogenesis, which is essential for the homeostasis of the mature retina. The mechanism revealed in this study might also apply to other nervous systems. | developmental biology |
10.1101/2022.05.08.491078 | Human variation impacting MCOLN2 restricts Salmonella Typhi replication by magnesium deprivation | Human genetic diversity can reveal critical factors in host-pathogen interactions. This is especially useful for human-restricted pathogens like Salmonella enterica serovar Typhi (S. Typhi), the cause of Typhoid fever. One key dynamic during infection is competition for nutrients: host cells attempt to restrict intracellular replication by depriving bacteria of key nutrients or delivering toxic metabolites in a process called nutritional immunity. Here, a cellular genome-wide association study of intracellular replication by S. Typhi in nearly a thousand cell lines from around the world--and extensive follow-up using intracellular S. Typhi transcriptomics and manipulation of magnesium concentrations--demonstrates that the divalent cation channel mucolipin-2 (MCOLN2) restricts S. Typhi intracellular replication through magnesium deprivation. Our results reveal natural diversity in Mg2+ limitation as a key component of nutritional immunity against S. Typhi. | genetics |
10.1101/2022.05.08.491077 | MuSiC2: cell type deconvolution for multi-condition bulk RNA-seq data | Cell type composition of intact bulk tissues can vary across samples. Deciphering cell type composition and its changes during disease progression is an important step towards understanding disease pathogenesis. To infer cell type composition, existing cell type deconvolution methods for bulk RNA-seq data often require matched single-cell RNA-seq (scRNA-seq) data, generated from samples with similar clinical conditions, as reference. However, due to the difficulty of obtaining scRNA-seq data in diseased samples, only limited scRNA-seq data in matched disease conditions are available. Using scRNA-seq reference to deconvolve bulk RNA-seq data from samples with different disease conditions may lead to biased estimation of cell type proportions. To overcome this limitation, we propose an iterative estimation procedure, MuSiC2, which is an extension of MuSiC [1], to perform deconvolution analysis of bulk RNA-seq data generated from samples with multiple clinical conditions where at least one condition is different from that of the scRNA-seq reference. Extensive benchmark evaluations indicated that MuSiC2 improved the accuracy of cell type proportion estimates of bulk RNA-seq samples under different conditions as compared to the traditional MuSiC [1] deconvolution. MuSiC2 was applied to two bulk RNA-seq datasets for deconvolution analysis, including one from human pancreatic islets and the other from human retina. We show that MuSiC2 improves current deconvolution methods and provides more accurate cell type proportion estimates when the bulk and single-cell reference differ in clinical conditions. We believe the condition-specific cell type composition estimates from MuSiC2 will facilitate downstream analysis and help identify cellular targets of human diseases. | genomics |
10.1101/2022.05.08.491088 | Specific binding of Hsp27 and phosphorylated Tau mitigates abnormal Tau aggregation-induced pathology | Amyloid aggregation of phosphorylated Tau (pTau) into neurofibrillary tangles is closely associated with Alzheimers disease (AD). Several molecular chaperones have been reported to bind Tau and impede its pathological aggregation. Recent findings of elevated levels of Hsp27 in the brains of patients with AD suggested its important role in pTau pathology. However, the molecular mechanism of Hsp27 in pTau aggregation remains poorly understood. Here, we show that Hsp27 partially co-localizes with pTau tangles in the brains of patients with AD. Notably, phosphorylation of Tau by microtubule affinity regulating kinase 2 (MARK2), dramatically enhances the binding affinity of Hsp27 to Tau. Moreover, Hsp27 efficiently prevents pTau fibrillation in vitro and mitigates neuropathology of pTau aggregation in a Drosophila tauopathy model. Further mechanistic study reveals that Hsp27 employs its N-terminal domain to directly interact with multiple phosphorylation sites of pTau for specific binding. Our work provides the structural basis for the specific recognition of Hsp27 to pathogenic pTau, and highlights the important role of Hsp27 in preventing abnormal aggregation and pathology of pTau in AD. | biochemistry |
10.1101/2022.05.08.491096 | Characterization of a nanobody-epitope tag interaction and its application for receptor engineering | Peptide epitope tags offer a valuable means for detection and manipulation of protein targets for which high quality detection reagents are not available. Most commonly used epitope tags are bound by conventional, full-size antibodies (Abs). The complex architecture of Abs complicates their application in protein engineering and intracellular applications. To address these shortcomings, single domain antibodies (nanobodies, Nbs) that recognize short peptide epitopes have become increasingly prized. Here we characterize the interaction between a Nb (Nb6E) and a 14-mer peptide epitope. We identify residues in the peptide epitope essential for high affinity binding. Using this information in combination with computational modeling we propose a mode of interaction between Nb6E and this epitope. We apply this nanobody-epitope pair to augment the potency of a ligand at an engineered adenosine A2A receptor. This characterization of the nanobody-epitope pair opens the door to diverse applications including mechanistic studies of G protein-coupled receptor function. | biochemistry |
10.1101/2022.05.08.491075 | Attention-wise masked graph contrastive learning for predicting molecular property | Accurate and efficient prediction of the molecular property is one of the fundamental problems in drug research and development. Recent advancements in representation learning have been shown to greatly improve the performance of molecular property prediction. However, due to limited labeled data, supervised learning-based molecular representation algorithms can only search limited chemical space and suffer from poor generalizability. In this work, we proposed a self-supervised learning method, ATMOL, for molecular representation learning and properties prediction. We developed a novel molecular graph augmentation strategy, referred to as attention-wise graph masking, to generate challenging positive samples for contrastive learning. We adopted the graph attention network (GAT) as the molecular graph encoder, and leveraged the learned attention weights as masking guidance to generate molecular augmentation graphs. By minimization of the contrastive loss between original graph and augmented graph, our model can capture important molecular structure and higher-order semantic information. Extensive experiments showed that our attention-wise graph mask contrastive learning exhibited state-of-the-art performance in a couple of downstream molecular property prediction tasks. We also verified that our model pretrained on larger scale of unlabeled data improved the generalization of learned molecular representation. Moreover, visualization of the attention heatmaps showed meaningful patterns indicative of atoms and atomic groups important to specific molecular property. | bioinformatics |
10.1101/2022.05.08.491094 | A framework for summarizing chromatin state annotations within and identifying differential annotations across groups of samples | Motivation: Genome-wide maps of epigenetic modifications are powerful resources for non-coding genome annotation. Maps of multiple epigenetics marks have been integrated into cell or tissue type-specific chromatin state annotations for many cell or tissue types. With the increasing availability of multiple chromatin state maps for biologically similar samples, there is a need for methods that can effectively summarize the information about chromatin state annotations within groups of samples and identify differences across groups of samples at a high resolution. Results: We developed CSREP, which takes as input chromatin state annotations for a group of samples and then probabilistically estimates the state at each genomic position and derives a representative chromatin state map for the group. CSREP uses an ensemble of multi-class logistic regression classifiers to predict the chromatin state assignment of each sample given the state maps from all other samples. The difference of CSREP s probability assignments for two groups can be used to identify genomic locations with differential chromatin state patterns. Using groups of chromatin state maps of a diverse set of cell and tissue types, we demonstrate the advantages of using CSREP to summarize chromatin state maps and identify biologically relevant differences between groups at a high resolution. Availability and implementation: The CSREP source code is openly available under http://github.com/ernstlab/csrep. | bioinformatics |
10.1101/2022.05.08.491060 | Integrated Analysis of Tissue-specific Gene Expression in Diabetes by Tensor Decomposition-based Unsupervised Feature Extraction with Standard Deviation Optimization Can Identify Possible Associated Diseases. | In the field of gene expression analysis, methods of integrating multiple gene expression profiles are still being developed and the existing methods have scope for improvement. The previously proposed tensor decomposition-based unsupervised feature extraction method was improved by introducing standard deviation optimization. The improved method was applied to perform an integrated analysis of three tissue-specific gene expression profiles (namely, adipose, muscle, and liver) for diabetes mellitus, and the results showed that it can detect diseases that are associated with diabetes (e.g., neurodegenerative diseases) but that cannot be predicted by individual tissue expression analyses using state-of-the-art methods. Although the selected genes differed from those identified by the individual tissue analyses, the selected genes are known to be expressed in all three tissues. Thus, compared with individual tissue analyses, an integrated analysis can provide more in-depth data and identify additional factors, namely, the association with other diseases. | bioinformatics |
10.1101/2022.05.07.491050 | Spatial cumulant models enable spatially informed treatment strategies and analysis of local interactions in cancer systems | Theoretical and applied cancer studies that use individual-based models (IBMs) have been limited by the lack of a mathematical formulation that enables rigorous analysis of these models. However, spatial cumulant models (SCMs), which have arisen from recent advances in theoretical ecology, can be used to describe population dynamics generated by a specific family of IBMs, namely spatio-temporal point processes (STPPs). SCMs are spatially resolved population models formulated by a system of ordinary differential equations that approximate the dynamics of two STPP-generated summary statistics: first order spatial cumulants (densities), and second order spatial cumulants (spatial covariances). Here, we exemplify how SCMs can be used in mathematical oncology by modelling a theoretical cancer cell population comprising interacting growth factor producing and non-producing cells. To formulate our models, we use the Unified Framework Software which was recently developed by Cornell et al. (2019) and enables the generation of STPPs, SCMs and mean-field population models (MFPMs) from user-defined model descriptions. Our results demonstrate that SCMs can capture STPP-generated population density dynamics, even when MFPMs fail to do so. From both MFPM and SCM equations, we derive treatment-induced cell death rates required to achieve non-growing cell populations. When testing these treatment strategies in STPP-generated cell populations, our results demonstrate that SCM-informed strategies outperform MFPM-informed strategies in terms of inhibiting population growths. We argue that SCMs provide a new framework in which to study cell-cell interactions, and can be used to deepen the mathematical analysis of IBMs and thereby increase IBMs' applicability in cancer research. | cancer biology |
10.1101/2022.05.06.490923 | Intracortical remodelling increases in highly-loaded bone after exercise cessation | Resorption within cortices of long bones removes excess mass and damaged tissue, and increases during periods of reduced mechanical loading. Returning to high-intensity exercise may place bones at risk of failure due to increased porosity caused by bone resorption. We used microradiographic images of bone slices from highly-loaded (metacarpal, tibia, humerus) and minimally-loaded (rib) bones from 12 racehorses, 6 in active high-intensity exercise and 6 in a period of rest following intense exercise, and measured intracortical canal cross-sectional area (Ca.Ar) and number (N.Ca) to infer remodelling activity across sites and exercise groups. Large canals representing resorption spaces (Ca.Ar > 0.04 mm2 were 5- to 18-fold greater in number and area in the third metacarpal bone from rested than exercised animals (p = 0.005-0.008), but were similar in number and area in ribs from rested and exercised animals (p = 0.575-0.688). A weaker, intermediate relationship was present in tibia and humerus, and when resorption spaces and partially-infilled canals (Ca.Ar > 0.002 mm2) were considered together. The mechanostat may override targeted remodelling during periods of high mechanical load by enhancing bone formation, reducing resorption and suppressing turnover, but both systems may work synergistically in rest periods to remove excess and damaged tissue. | physiology |
10.1101/2022.05.08.491089 | Characteristics and Impact of the rNST GABA Network on Neural and Behavioral Taste Responses | The rostral nucleus of the solitary tract (rNST), the initial CNS site for processing gustatory information, is comprised of two major cell types, glutamatergic excitatory and GABAergic inhibitory neurons. Many investigators have described taste responses of rNST neurons, but the phenotypes of these cells were unknown. The current investigation used mice expressing ChR2 under the control of GAD65, a synthetic enzyme for GABA. In vivo single-unit recording of rNST cells during optogenetic stimulation allowed us to address two important questions: (1) what are the gustatory response characteristics of 'optotagged', putative GABAergic (G+TASTE) neurons? and (2) how does optogenetic activation of the rNST GABA network impact taste responses in non-GABAergic (G-TASTE) neurons? We observed that chemosensitive profiles of G+TASTE neurons were similar to non-GABA taste neurons but had much lower response rates. We further observed that there was a population of GABA cells unresponsive to taste stimulation (G+UNR) and located more ventrally in the nucleus. Activating rNST inhibitory circuitry suppressed gustatory responses of G-TASTE neurons across all qualities and types of chemosensitive neurons. Tuning curves were modestly sharpened but the overall shape of response profiles and the ensemble pattern remained highly stable. These neurophysiological effects were consistent with the behavioral consequences of activating GAD65-expressing inhibitory neurons using DREADDs. In a brief-access licking task, concentration-response curves to both palatable (sucrose, maltrin) and unpalatable (quinine) stimuli were shifted to the right when GABA neurons were activated. Thus, the rNST GABAergic network is poised to modulate taste intensity across the qualitative and hedonic spectrum. | neuroscience |
10.1101/2022.05.08.491087 | Adversarial interspecies relationships facilitate population suppression by gene drive in spatially explicit models | Suppression gene drives are designed to bias their inheritance and increase in frequency in a population, disrupting an essential gene in the process. When the frequency is high enough, the population will be unable to reproduce above the replacement level and could be eliminated. CRISPR suppression drives based on the homing mechanism have already seen success in the laboratory, particularly in malaria mosquitoes. However, several models predict that the use of these drives in realistic populations with spatial structure may not achieve complete success. This is due to the ability of wild-type individuals to escape the drive and reach empty areas with reduced competition, allowing them to achieve high reproductive success and leading to extinction-recolonization cycles across the landscape. Here, we extend our continuous space gene drive framework to include two competing species or predator-prey species pairs. We find in both discrete-generation and mosquito-specific models that the presence of a competing species or predator can greatly facilitate drive-based suppression, even for drives with modest efficiency. However, the presence of a competing species also substantially increases the frequency of outcomes in which the drive is lost before suppression is achieved. These results are robust in models with seasonal population fluctuations. We also found that suppression can be somewhat more difficult if targeting a predator with strong predator-prey interactions. Our results illustrate the difficulty of predicting outcomes of interventions that could substantially affect the populations of interacting species in complex ecosystems. However, our results are also potentially promising for the prospects of less powerful gene drives in achieving successful elimination of target pest populations. | ecology |
10.1101/2022.05.08.490699 | Resting-state network hubs are causally required in memory consolidation | Resting-state networks (RSNs) detected by fMRI have been associated with cognitive function, providing insights to the relationship between brain and behavior. However, whether altered RSNs are epiphenomena or essential constructs of behavior remains unclear. Here we investigated whether post-encoding RSN hubs that are commonly engaged after similar tasks or integrate distributed areas into large networks are causally involved in memory consolidation. RSN changes following two types of spatial memory training in mice, allowing us to distinguish post-encoding hubs related to spatial memory, and verify their behavioral impact by hub inhibition. We found that functional connectivity with sensory areas was commonly strengthened in both tasks whereas frontal and striatal areas were influential in network integration. Chemogenetic suppression of each hub after learning resulted in retrograde amnesia. These results demonstrate causal and functional roles of RSN hubs in system consolidation and validate fMRI as a means to track this process. | animal behavior and cognition |
10.1101/2022.05.06.490971 | Assembly-free discovery of human novel sequences using long reads | DNA sequences that are absent in the human reference genome are classified as novel sequences. The discovery of these missed sequences is crucial for exploring the genomic diversity of populations and understanding the genetic basis of human diseases. However, various DNA lengths of reads generated from different sequencing technologies can significantly affect the results of novel sequences. In this work, we designed an Assembly-Free Novel Sequence (AF-NS) approach to identify novel sequences from Oxford Nanopore Technology long reads. Among the newly detected sequences using AF-NS, more than 95% were omitted from those using long-read assemblers, and 85% were not present in short reads of Illumina. We identified the common novel sequences among all the samples and revealed their association with the binding motifs of transcription factors. Regarding the placements of the novel sequences, we found about 70% enriched in repeat regions and generated 430 for one specific subpopulation that might be related to their evolution. Our study demonstrates the advance of the Assembly-Free approach to capture more novel sequences over other assembler-based methods. Combining the long-read data with powerful analytical methods can be a robust way to improve the completeness of novel sequences. | bioinformatics |
10.1101/2022.05.06.490992 | MAGNETO: an automated workflow for genome-resolved metagenomics | Metagenome-Assembled Genomes (MAGs) represent individual genomes recovered from metagenomic data. MAGs are extremely useful to analyse uncultured microbial genomic diversity, as well as to characterize associated functional and metabolic potential in natural environments. Recent computational developments have considerably improved MAGs reconstruction but also emphasized several limitations, such as the non-binning of sequence regions with repetitions or distinct nucleotidic composition. Different assembly and binning strategies are often used, however, it still remains unclear which assembly strategy in combination with which binning approach, offers the best performance for MAGs recovery. Several workflows have been proposed in order to reconstruct MAGs, but users are usually limited to single-metagenome assembly or need to manually define sets of metagenomes to co-assemble prior to genome binning. Here, we present MAGNETO, an automated workflow dedicated to MAGs reconstruction, which includes a fully-automated co-assembly step informed by optimal clustering of metagenomic distances, and implements complementary genome binning strategies, for improving MAGs recovery. MAGNETO is implemented as a Snakemake workflow and is available at: https://gitlab.univ-nantes.fr/bird_pipeline_registry/magneto. | bioinformatics |
10.1101/2022.05.08.489936 | Cryo-EM structure of gas vesicles for buoyancy-controlled motility | Gas vesicles allow a diverse group of bacteria and archaea to move in the water column by controlling their buoyancy. These gas-filled cellular nanocompartments are formed by up to micrometers long protein shells that are permeable only to gas. The molecular basis of their unique properties and mechanism of assembly remains unknown. Here, we solve the 3.2 [A] cryo-EM structure of the B.megaterium gas vesicle shell made from the structural protein GvpA that self-assembles into hollow helical cylinders closed off by cone-shaped tips. Remarkably, the unique fold adopted by GvpA generates a corrugated cylinder surface typically found in force-bearing thin-walled structures. We identified pores in the vesicle wall that enable gas molecules to freely diffuse in and out of the GV shell, while the exceptionally hydrophobic interior surface effectively repels water. Our results show that gas vesicles consist of two helical half-shells connected through a unique arrangement of GvpA monomers, suggesting a mechanism of gas vesicle biogenesis. Comparative structural analysis confirms the evolutionary conservation of gas vesicle assemblies and reveals molecular details of how the secondary structural protein GvpC reinforces the GvpA shell. Our findings provide a structural framework that will further research into the biology of gas vesicles, and enable rational molecular engineering to harness their unique properties for acoustic imaging. | biophysics |
10.1101/2022.05.08.490752 | Multi-policy models of interregional communication in the human connectome | Network models of communication, e.g. shortest paths, diffusion, navigation, have become useful tools for studying structure-function relationships in the brain. These models generate estimates of communication efficiency between all pairs of brain regions, which can then be linked to the correlation structure of recorded activity, i.e. functional connectivity (FC). At present, however, communication models have a number of limitations, including difficulty adjudicating between models and the absence of a generic framework for modeling multiple interacting communication policies at the regional level. Here, we present a framework that allows us to incorporate multiple region-specific policies and fit them to empirical estimates of FC. Briefly, we show many communication policies, including shortest paths and greedy navigation, can be modeled as biased random walks, enabling these policies to be incorporated into the same multi-policy communication model alongside unbiased processes, e.g. diffusion. We show that these multi-policy models outperform existing communication measures while yielding neurobiologically interpretable regional preferences. Further, we show that these models explain the majority of variance in time-varying patterns of FC. Collectively, our framework represents an advance in network-based communication models and establishes a strong link between these patterns and FC. Our findings open up many new avenues for future inquiries and present a flexible framework for modeling anatomically-constrained communication. | neuroscience |
10.1101/2022.05.08.491098 | Comparative brain structure and the neural network features of cuttlefish and squid | Cuttlefishes, like their octopus cousins, are masters of camouflage by control of body pattern and skin texture to blend in with their surroundings for prey ambush and threat avoidance. Aside from significant progress on the cuttlefish visual perception and communication, a growing number of studies have focused on their behavioural neurobiology and the remarkably rapid and apparently cognitively complex reactions to novel challenges such as spatial learning to solve maze tasks and vertebrate-like cognitive capabilities (e.g. object recognition, number sense and episodic-like memory). Despite intense interest of cuttlefish, much of our knowledge of its neuroanatomy and links to behaviour and ecology comes from one temperate species, the European common cuttlefish, Sepia officinalis. Here we present the first detailed comparison of neuroanatomical features between the tropical cuttlefish and squid and describe differences in basic brain and wiring anatomy using MRI-based techniques and conventional histology. Furthermore, comparisons amongst nocturnal and diurnal cuttlefish species suggest that the characteristic neuroanatomical features infer interspecific variation in visual capabilities, the importance of vision relative to the less utilised chemosensory system and clear links with life modes (e.g. diurnal vs nocturnal), ecological factors (e.g. living depth and ambient light condition) as well as to an extent, phylogeny. These findings link brain heterogeneity to ecological niches and lifestyle, feeding hypotheses around evolutionary history and provide a timely, new technology update to older literature. | neuroscience |
10.1101/2022.05.08.491097 | Closed-loop and automatic tuning of pulse amplitude and width in EMG-guided controllable transcranial magnetic stimulation (cTMS) | This paper proposes a tool for automatic and optimal tuning of pulse amplitude and width for sequential parameter estimation (SPE) of the membrane time constant and input-output curve in closed-loop electromyography-guided (EMG-guided) controllable transcranial magnetic stimulation (cTMS). A normalized depolarization factor is defined which separates the optimization of the pulse amplitude and width. Then, the pulse amplitude is chosen by the maximization of the Fisher information matrix (FIM), while the pulse width is chosen by the maximization of the normalized depolarization factor. The simulation results confirm satisfactory estimation. The results show that the normalized depolarization factor maximization can identify the critical pulse width, which is an important parameter in the identifiability analysis, without any prior neurophysiological or anatomical knowledge of the neural membrane. | neuroscience |
10.1101/2022.05.08.491107 | Two complete genomes of male-killing Wolbachia infecting Ostrinia moth species illuminate their evolutionary dynamics and association with hosts | Wolbachia is an extremely widespread endocellular symbiont which causes reproductive manipulation on various arthropod hosts. Male progenies are killed in Wolbachia-infected lineages of the Japanese Ostrinia moth population. While the mechanism of male killing and the evolutionary interaction between host and symbiont are significant concerns for this system, the absence of Wolbachia genomic information has limited approaches to these issues. We determined the complete genome sequences of wFur and wSca, the male-killing Wolbachia of O. furnacalis and O. scapulalis. The two genomes shared an extremely high degree of homology, with over 95% of the predicted protein sequences being identical. A comparison of these two genomes revealed nearly minimal genome evolution, with a strong emphasis on the frequent genome rearrangements and the rapid evolution of ankyrin repeat-containing proteins. Additionally, we determined the mitochondrial genomes of both species' infected lineages and performed phylogenetic analyses to deduce the evolutionary dynamics of Wolbachia infection in the Ostrinia clade. According to the inferred phylogenetic relationship, Wolbachia infection was established in the Ostrinia clade prior to the speciation of related species such as O. furnacalis and O. scapulalis. Simultaneously, the relatively high homology of mitochondrial genomes suggested recent Wolbachia introgression between infected Ostrinia species. The findings of this study collectively shed light on the host-symbiont interaction from an evolutionary standpoint. | microbiology |
10.1101/2022.05.06.490963 | Spontaneously produced lysogenic phages are an important component of the soybean Bradyrhizobium mobilome | The ability to nodulate and fix atmospheric nitrogen in soybean root nodules makes soybean Bradyrhizobium spp. (SB) critical in supplying humanity's nutritional needs. The intricacies of SB-plant interactions have been studied extensively; however, bradyrhizobial ecology as influenced by phages has received somewhat less attention even though these interactions may significantly impact soybean yield. In batch culture four SB strains, S06B (B. japonicum, S06B-Bj), S10J (B. japonicum, S10J-Bj), USDA 122 (B. diazoefficiens, USDA 122-Bd), and USDA 76T (B. elkanii, USDA 76-Be), spontaneously (without apparent exogenous chemical or physical induction) produced phages throughout the growth cycle; for three strains, phage concentrations exceeded cell numbers by ca. 3-fold after 48 h incubation. Observed spontaneously produced phages (SPP) were tailed. Phage terminase large-subunit protein phylogeny revealed possible differences in phage packaging and replication mechanisms. Bioinformatic analyses predicted multiple prophage regions within each SB genome preventing accurate identification of SPP genomes. A DNA sequencing approach was developed that accurately delineated the boundaries of four SPP genomes within three of the SB chromosomes. Read mapping suggested that the SPP are capable of transduction. In addition to the phages, bacterial strains S06B-Bj and USDA 76-Be were rich in mobile elements consisting of insertion sequences (IS) and large, conjugable, broad host range plasmids. The prevalence of SPP along with IS and plasmids indicate that horizontal gene transfer likely plays an outsized role in SB ecology and may subsequently impact soybean productivity. Importance: Previous studies have shown that IS and plasmids mediate horizontal gene transfer (HGT) of symbiotic nodulation (nod) genes in SB; however, these events require close cell to cell contact which could be limited in soil environments. Bacteriophage assisted gene transduction through spontaneously produced prophages could provide stable means of HGT not limited by the constraints of proximal cell to cell contact. Phage mediated HGT events could be important in SB population ecology with concomitant impacts on soybean agriculture. | microbiology |
10.1101/2022.05.08.489343 | Skeletal muscle cells derived from induced pluripotent stem cells: a platform for limb girdle muscular dystrophies. | Limb-girdle muscular dystrophies (LGMD), caused by mutations in 29 different genes, are the fourth most prevalent group of genetic muscle diseases, leading to progressive weakness and atrophy of the skeletal muscles. Although the link between LGMD and their genetic origins has been determined, LGMD still represent an unmet medical need. In this paper, we describe a platform for modeling LGMD based on the use of human induced pluripotent stem cells (hiPSC). Thanks to the self-renewing and pluripotency properties of hiPSC, this platform provides an alternative and renewable source of skeletal muscle cells (skMC) to primary, immortalized or overexpressing cells. We report that skMC derived from hiPSC express the majority of the genes and proteins causing LGMD. As a proof of concept, we demonstrate the importance of this cellular model for studying LGMDR9 by evaluating disease-specific phenotypes in skMC derived from hiPSC obtained from four patients. | cell biology |
10.1101/2022.05.08.491100 | Evolution and diversity of the TopoVI and TopoVI-like subunits with extensive divergence of the TOPOVIBL subunit | Type II DNA topoisomerases regulate topology by double-stranded DNA cleavage and ligation. The TopoVI family of DNA topoisomerase, first identified and biochemically characterized in Archaea, represents, with TopoVIII and mini-A, the type IIB family. TopoVI has several intriguing features in terms of function and evolution. TopoVI has been identified in some eucaryotes, and a global view is lacking to understand its evolutionary pattern. In addition, in eucaryotes, the two TopoVI subunits (TopoVIA and TopoVIB) have duplicated and evolved to give rise to Spo11 and TopoVIBL, forming TopoVI-like (TopoVIL), a complex essential for generating DNA breaks that initiation homologous recombination during meiosis. TopoVIL is essential for sexual reproduction. How the TopoVI subunits have evolved to ensure this meiotic function is unclear. Here, we investigated the phylogenetic conservation of TopoVI and TopoVIL. We demonstrate that BIN4 and RHL1, potentially interacting with TopoVIB, have co-evolved with TopoVI. Based on model structures, this observation supports the hypothesis for a role of TopoVI in decatenation of replicated chromatids and predicts that in eucaryotes the TopoVI catalytic complex includes BIN4 and RHL1. For TopoVIL, the phylogenetic analysis of Spo11, which is highly conserved among Eukarya, highlighted a eukaryal-specific N-terminal domain that may be important for its regulation. Conversely, TopoVIBL was poorly conserved and rapidly evolving, giving rise to ATP hydrolysis-mutated or -truncated protein variants, or was undetected in some species. This remarkable plasticity of TopoVIBL provides important information for the activity and function of TopoVIL during meiosis. | evolutionary biology |
10.1101/2022.05.07.490914 | Population genomics provide insights into the global genetic structure of Colletotrichum graminicola, the causal agent of maize anthracnose | Background: Colletotrichum graminicola, the causal agent of maize anthracnose, is an important crop disease worldwide. Understanding the genetic diversity and mechanisms underlying genetic variation in pathogen populations is crucial to the development of effective control strategies. The genus Colletotrichum is largely recognized as asexual, but several species have been reported to have a sexual cycle. Here, we employed a population genomics approach to investigate the genetic diversity and reproductive biology of C. graminicola isolates infecting maize. We sequenced 108 isolates of C. graminicola collected in 14 countries using restriction site-associated DNA sequencing (RAD-Seq) and whole-genome sequencing (WGS). Results: Clustering analyses based on single-nucleotide polymorphisms showed populational differentiation at a global scale, with three genetic groups delimited by continental origin, compatible with short-dispersal of the pathogen, and geographic subdivision. Distinct levels of genetic diversity were observed between these clades, suggesting different evolutionary histories. Intra and inter-continental migration was predicted between Europe and South America, likely associated with the movement of contaminated germplasm. Low clonality and evidence of genetic recombination were detected from the analysis of linkage disequilibrium and the pairwise homoplasy index (PHI) test for clonality. We show evidence that even if rare (possibly due to losses of sex and meiosis-associated genes) C. graminicola can undergo sexual recombination based on lab assays and genomic analyses. Conclusions: Our results support hypotheses of intra and intercontinental pathogen migration and genetic recombination with great impact on C. graminicola population structure. | genomics |
10.1101/2022.05.09.491186 | Single-molecule architecture and heterogeneity of human telomeric DNA and chromatin | Telomeres are essential for linear genomes, yet their repetitive DNA content and somatic variability has hindered attempts to delineate their chromatin architectures. We performed single-molecule chromatin fiber sequencing (Fiber-seq) on human cells with a fully resolved genome, enabling nucleotide-precise maps of the genetic and chromatin structure of all telomeres. Telomere fibers are predominantly comprised of three distinct chromatin domains that co-occupy individual DNA molecules - multi-kilobase telomeric caps, highly accessible telomeric-subtelomeric boundary elements, and subtelomeric heterochromatin. Extended G-rich telomere variant repeats (TVRs) punctuate nearly all telomeres, and telomere caps imprecisely bridge these degenerate repeats. Telomeres demonstrate pervasive somatic alterations in length, sequence, and chromatin composition, with TVRs and adjacent CTCF-bound promoters impacting their stability and composition. Our results detail the structure and function of human telomeres. | genomics |
10.1101/2022.05.06.490968 | Single-Cell RNAseq Analysis Reveals Robust Anti-PD-1-Mediated Increase of Immune Infiltrate in Metastatic Castration-Sensitive Prostate Cancer | Compared to other malignancies, the tumor microenvironment (TME) of primary and castration-resistant prostate cancer (CRPC) is relatively devoid of immune infiltrates. While androgen deprivation therapy (ADT) induces a complex immune infiltrate in localized prostate cancer, both in animal models and humans, the TME composition of metastatic, castration-sensitive prostate cancer (mCSPC) is relatively unknown and the effects of ADT and other treatments are poorly characterized in this context. To address this challenge, we analyzed metastatic sites from patients enrolled on a phase 2 clinical trial (NCT03951831), in which men were treated with standard-of-care chemo-hormonal therapy with anti-PD-1 immunotherapy, at the single cell level. Longitudinal protein activity-based analysis of TME subpopulations identified immune subpopulations conserved across multiple metastatic sites, their dynamic, treatment-mediated evolution, and associated clinical response features. Our study revealed a therapy-resistant, transcriptionally distinct tumor subpopulation, which comprises an increasing number of cells in treatment-refractory patients, and identified several druggable targets in both tumor and immune cells as candidates to advance treatment and improve outcomes for patients with mCSPC. | cancer biology |
10.1101/2022.05.09.491042 | Reproducibility of in-vivo electrophysiological measurements in mice | Understanding whole-brain-scale electrophysiological recordings will rely on the collective work of multiple labs. Because two labs recording from the same brain area often reach different conclusions, it is critical to quantify and control for features that decrease reproducibility. To address these issues, we formed a multi-lab collaboration using a shared, open-source behavioral task and experimental apparatus. We repeatedly inserted Neuropixels multi-electrode probes targeting the same brain locations (including posterior parietal cortex, hippocampus, and thalamus) in mice performing the behavioral task. We gathered data across 9 labs and developed a common histological and data processing pipeline to analyze the resulting large datasets. After applying stringent behavioral, histological, and electrophysiological quality-control criteria, we found that neuronal yield, firing rates, spike amplitudes, and task-modulated neuronal activity were reproducible across laboratories. To quantify variance in neural activity explained by task variables (e.g., stimulus onset time), behavioral variables (timing of licks/paw movements), and other variables (e.g., spatial location in the brain or the lab ID), we developed a multi-task neural network encoding model that extends common, simpler regression approaches by allowing nonlinear interactions between variables. We found that within-lab random effects captured by this model were comparable to between-lab random effects. Taken together, these results demonstrate that across-lab standardization of electrophysiological procedures can lead to reproducible results across labs. Moreover, our protocols to achieve reproducibility, along with our analyses to evaluate it are openly accessible to the scientific community, along with our extensive electrophysiological dataset with corresponding behavior and open-source analysis code. | neuroscience |
10.1101/2022.05.09.491042 | Reproducibility of in-vivo electrophysiological measurements in mice | Understanding whole-brain-scale electrophysiological recordings will rely on the collective work of multiple labs. Because two labs recording from the same brain area often reach different conclusions, it is critical to quantify and control for features that decrease reproducibility. To address these issues, we formed a multi-lab collaboration using a shared, open-source behavioral task and experimental apparatus. We repeatedly inserted Neuropixels multi-electrode probes targeting the same brain locations (including posterior parietal cortex, hippocampus, and thalamus) in mice performing the behavioral task. We gathered data across 9 labs and developed a common histological and data processing pipeline to analyze the resulting large datasets. After applying stringent behavioral, histological, and electrophysiological quality-control criteria, we found that neuronal yield, firing rates, spike amplitudes, and task-modulated neuronal activity were reproducible across laboratories. To quantify variance in neural activity explained by task variables (e.g., stimulus onset time), behavioral variables (timing of licks/paw movements), and other variables (e.g., spatial location in the brain or the lab ID), we developed a multi-task neural network encoding model that extends common, simpler regression approaches by allowing nonlinear interactions between variables. We found that within-lab random effects captured by this model were comparable to between-lab random effects. Taken together, these results demonstrate that across-lab standardization of electrophysiological procedures can lead to reproducible results across labs. Moreover, our protocols to achieve reproducibility, along with our analyses to evaluate it are openly accessible to the scientific community, along with our extensive electrophysiological dataset with corresponding behavior and open-source analysis code. | neuroscience |
10.1101/2022.05.09.491197 | Single-cell profiling coupled with lineage analysis reveals distinct sacral neural crest contributions to the developing enteric nervous | During development, the enteric nervous system (ENS) arises from neural crest cells that emerge from the neural tube, migrate to and along the gut, and colonize the entire intestinal tract. While much of the ENS arises from vagal neural crest cells that originate from the caudal hindbrain, there is a second contribution from the sacral neural crest that migrates from the caudal end of the spinal cord to populate the post-umbilical gut. By coupling single cell transcriptomics with axial-level specific lineage tracing in avian embryos, we compared the contributions between embryonic vagal and sacral neural crest cells to the ENS. The results show that the two neural crest populations form partially overlapping but also complementary subsets of neurons and glia in distinct ganglionic units. In particular, the sacral neural crest cells appear to be the major source of adrenergic/dopaminergic and serotonergic neurons, melanocytes and Schwann cells in the post-umbilical gut. In addition to neurons and glia, the results also reveal sacral neural crest contributions to connective tissue and mesenchymal cells of the gut. These findings highlight the specific properties of the sacral neural crest population in the hindgut and have potential implications for understanding development of the complex nervous system in the hindgut environment that may influence congenital neuropathies. | developmental biology |
10.1101/2022.05.09.491207 | Scaled-expansion of the membrane associated cytoskeleton requires conserved kinesin adaptors | A periodic lattice of actin rings and spectrin tetramers scaffolds the axonal membrane. How spectrin is delivered to this structure to scale its size to that of the growing axon is unknown. We found that endogenous spectrin, visualized with singe axon resolution in vivo, is delivered to hotspots in the lattice that support its expansion at rates set by axon stretch-growth. Unlike other cytoskeletal proteins, whose apparent slow movement consists of intermittent bouts of fast movements, spectrin moves slowly and processively. We identified a pair of coiled coil proteins that mediate this slow movement and the expansion of the lattice by linking spectrin to kinesin-1. Thus, processive slow transport and local lattice incorporation support scaled cytoskeletal expansion during axon stretch-growth. | cell biology |
10.1101/2022.05.09.491117 | A new model of Notch signaling: Control of Notch receptor cis-inhibition via Notch ligand dimers | All tissue development and replenishment relies upon the breaking of symmetries leading to the morphological and operational differentiation of progenitor cells into more specialized cells. One of the main engines driving this process is the Notch signal transduction pathway, a ubiquitous signalling system found in the vast majority of metazoan cell types characterized to date. Broadly speaking, Notch receptor activity is governed by a balance between two processes: 1) intercellular Notch transactivation triggered via interactions between receptors and ligands expressed in neighbouring cells; 2) intracellular cis inhibition caused by ligands binding to receptors within the same cell. Additionally, recent reports have also unveiled evidence of cis activation. Whilst context-dependent Notch receptor clustering has been hypothesized, to date, Notch signalling has been assumed to involve an interplay between receptor and ligand monomers. In this study, we demonstrate biochemically, through a mutational analysis of DLL4, both in vitro and in tissue culture cells, that Notch ligands can efficiently self-associate. We found that the membrane proximal EGF-like repeat of DLL4 was necessary and sufficient to promote oligomerization/dimerization. Mechanistically, our experimental evidence supports the view that DLL4 ligand dimerization is specifically required for cis-inhibition of Notch receptor activity. To further substantiate these findings, we have adapted and extended existing ordinary differential equation-based models of Notch signalling to take account of the ligand dimerization-dependent cis-inhibition reported here. Our new model faithfully recapitulates our experimental data and improves predictions based upon published data. Collectively, our work favours a model in which net output following Notch receptor/ligand binding results from ligand monomer-driven Notch receptor transactivation (and cis activation) counterposed by ligand dimer-mediated cis-inhibition. | cell biology |
10.1101/2022.05.09.491019 | Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain | Capturing mitochondria's intricate and dynamic structure poses a daunting challenge for optical nanoscopy. Different labeling strategies have been demonstrated for live-cell stimulated emission depletion (STED) microscopy of mitochondria, but orthogonal strategies are yet to be established, and image acquisition has suffered either from photodamage to the organelles or from rapid photobleaching. Therefore, live-cell nanoscopy of mitochondria has been largely restricted to 2D single-color recordings of cancer cells. Here, by conjugation of cyclooctatetraene to a benzo-fused cyanine dye, we report a mitochondrial inner-membrane (IM) fluorescent marker, PK Mito Orange (PKMO), featuring efficient STED at 775 nm, strong photostability and markedly reduced phototoxicity. PKMO enables super-resolution recordings of inner-membrane dynamics for extended periods in immortalized mammalian cell lines, primary cells, and organoids. Photostability and reduced phototoxicity of PKMO open the door to live-cell 3D STED nanoscopy of mitochondria for three-dimensional analysis of the convoluted IM. PKMO is optically orthogonal with green and far-red markers allowing multiplexed recordings of mitochondria using commercial STED microscopes. Using multi-color STED, we demonstrate that imaging with PKMO can capture the sub-mitochondrial localization of proteins, or interactions of mitochondria with different cellular components, such as the ER or the cytoskeleton at sub-100 nm resolution. Thereby, this work offers a versatile tool for studying mitochondrial inner-membrane architecture and dynamics in a multiplexed manner. | cell biology |
10.1101/2022.05.09.490039 | Visinity: Visual Spatial Neighborhood Analysis for Multiplexed Tissue Imaging Data | New multiplexed tissue imaging technologies have enabled the study of normal and diseased tissues in unprecedented detail. These methods are increasingly being applied to understand how cancer cells and immune response change during tumor development, progression, and metastasis as well as following treatment. Yet, existing analysis approaches focus on investigating small tissue samples on a per-cell basis, not taking into account the spatial proximity of cells, which indicates cell-cell interaction and specific biological processes in the larger cancer microenvironment. We present Visinity, a scalable visual analytics system to analyze cell interaction patterns across cohorts of whole-slide multiplexed tissue images. Our approach is based on a fast regional neighborhood computation, leveraging unsupervised learning to quantify, compare, and group cells by their surrounding cellular neighborhood. These neighborhoods can be visually analyzed in an exploratory and confirmatory workflow. Users can explore spatial patterns present across tissues through a scalable image viewer and coordinated views highlighting the neighborhood composition and spatial arrangements of cells. To verify or refine existing hypotheses, users can query for specific patterns to determine their presence and statistical significance. Findings can be interactively annotated, ranked, and compared in the form of small multiples. In two case studies with biomedical experts, we demonstrate that Visinity can identify common biological processes within a human tonsil and uncover novel white-blood networks and immune-tumor interactions. | cell biology |
10.1101/2022.05.08.491095 | Prenatal inflammation perturbs fetal hematopoietic development and causes persistent changes to postnatal immunity | Adult hematopoietic stem and progenitor cells (HSPCs) respond directly to inflammation and infection, resulting in both acute and persistent changes to quiescence, mobilization, and differentiation. Here we show that fetal HSPCs respond to prenatal inflammation in utero, and that the fetal response shapes postnatal hematopoiesis and immunity. Heterogenous fetal HSPCs showed divergent responses to maternal immune activation (MIA), including changes in quiescence, expansion, and lineage-biased output. Single cell transcriptomic analysis of fetal HSPCs in response to MIA revealed specific upregulation of inflammatory gene profiles in discrete, transient HSC populations, that propagated expansion of lymphoid-biased progenitors. Beyond fetal development, MIA caused the inappropriate expansion and persistence of fetal lymphoid-biased progenitors postnatally, concomitant with increased cellularity and hyperresponsiveness of fetal-derived innate-like lymphocytes. Our investigation demonstrates how inflammation in utero can direct the trajectory of output and function of fetal-derived immune cells by reshaping fetal HSC establishment. | cell biology |
10.1101/2022.05.09.491166 | Rapid covalent labeling of a GPCR on living cells using a nanobody-epitope tag pair to interrogate receptor pharmacology | Peptide epitope tags offer a valuable means for detection and manipulation of protein targets for which high quality detection reagents are not available. Most commonly used epitope tags are bound by conventional, full-size antibodies (Abs). The complex architecture of Abs complicates their application in protein engineering and intracellular applications. To address these shortcomings, single domain antibodies (nanobodies, Nbs) that recognize short peptide epitopes have become increasingly prized. Here we characterize the interaction between a Nb (Nb6E) and a 14-mer peptide epitope. We identify residues in the peptide epitope essential for high affinity binding. Using this information in combination with computational modeling we propose a mode of interaction between Nb6E and this epitope. We apply this nanobody-epitope pair to augment the potency of a ligand at an engineered adenosine A2A receptor. This characterization of the nanobody-epitope pair opens the door to diverse applications including mechanistic studies of G protein-coupled receptor function. | biochemistry |
10.1101/2022.05.09.491144 | Sampling biases obscure the early diversification of the largest living vertebrate group | Extant ray-finned fishes (Actinopterygii) dominate marine and freshwater environments, yet their spatiotemporal diversity dynamics following their origin in the Palaeozoic are poorly understood. Previous studies investigate face-value patterns of richness, with only qualitative assessment of potential biases acting on the Palaeozoic actinopterygian fossil record. Here, we investigate palaeogeographic trends and apply richness estimation techniques to a recently-assembled occurrence database for Palaeozoic ray-finned fishes. We reconstruct patterns of local richness of Palaeozoic actinopterygians, alongside sampling standardised estimates of 'global' diversity. We identify substantial fossil record biases, such as geographic bias in the sampling of actinopterygian occurrences centred around Europe and North America. Similarly, estimates of diversity are skewed by extreme unevenness in the abundance distributions of occurrences, reflecting past taxonomic practices and historical biases in sampling. Increasing sampling of poorly represented regions and expanding sampling beyond the literature to include museum collection data will be critical in obtaining accurate estimates of Palaeozoic actinopterygian diversity. In conjunction, applying diversity estimation techniques to well-sampled regional subsets of the 'global' dataset may identify accurate local diversity trends. | paleontology |
10.1101/2022.05.09.490051 | Centrosome Amplification promotes cell invasion via cell-cell contact disruption and Rap-1 activation | Centrosome amplification (CA), a prominent feature of human cancers linked to genomic instability and tumourigenesis in vivo, is observed as early as pre-malignant metaplasia, increasing with progression from dysplasia to neoplasia. However, the mechanistic contributions of CA to tumourigenesis are not fully understood. Using non-tumourigenic breast cells (MCF10A), we demonstrate that induction of CA (by CDK1 inhibition or PLK4 overexpression) increased both the migration and invasion of non-tumourigenic cells. Mechanistically, we found small GTPase Rap-1 was activated upon CA induction. Rap-1 inhibition (using GGTI-298) blocked CA-induced migration, invasion and ECM attachment, demonstrating the role of Rap-1 in CA induced tumourigenesis. Induction of CA in a long-term cell culture system disrupted epithelial cell-cell junction integrity, via dysregulation of expression and subcellular localisation of cell junction proteins (ZO-1, Occludin, JAM-A & {beta}-catenin). Physically, CA inhibited apical junctional complex formation, visualised by transmission electron microscopy. Furthermore, CA induction in non-tumourigenic cells elevated {beta}-integrin 3 expression explaining increased cell attachment to the extracellular matrix (ECM). Simultaneously, CA induced elevated expression of matrix metalloprotease MMP1 and MMP13 facilitating ECM degradation and cell invasion. In vivo validation in a Chicken Embryo xenograft model, showed CA+ MCF10A cells invaded into the chicken mesodermal layer, characterised by inflammatory cell infiltration and a marked focal reaction between chorioallantoic membrane and cell graft. This reaction was inhibited by pre-treatment of CA+ MCF10A cells with Rap-1 inhibitor GGTI-298. Inhibition of CA in metastatic breast cancer cells with high levels of endogenous CA (triple negative cell line MDA-MB-231), using PLK4 inhibitor Centrinone B, abrogated their metastatic capacity in vitro. Here, we demonstrated CA induction in normal cells confers early tumourigenic changes which promote tumour progression, mediated by ECM disruption, altered cell-cell contacts, and Rap-1-dependent signaling. These insights fundamentally demonstrate the mechanism of how CA induces tumourigenesis in normal cells, alone and without requiring additional pre-tumourigenic alterations. | cell biology |
10.1101/2022.05.09.491210 | A sporulation-independent way of life for Bacillus thuringiensis in the late stages of an infection | The formation of endospores has been considered as the unique mode of survival and transmission of sporulating Firmicutes due to the exceptional resistance and persistence of this bacterial form. However, the persistence of non-sporulated bacteria (Spo-) was reported during infection in Bacillus thuringiensis, an entomopathogenic sporulating Gram-positive bacterium. In this study, we investigated the behavior of a bacterial population in the late stages of an infection as well as the characteristics of the Spo- bacteria in the B. thuringiensis/Galleria mellonella infection model. Using fluorescent reporters coupled to flow cytometry as well as molecular markers, we demonstrated that the Spo- cells constitute about half of the population two weeks post-infection (pi) and that these bacteria present vitality signs. However, a protein synthesis and a growth recovery assay indicated that they are in a metabolically slowed-down state. Interestingly, they were extremely resistant to the cadaver environment which proved deadly for in vitro-grown vegetative cells and, strikingly, did not support spore germination. A transcriptomic analysis of this subpopulation at 7 days pi revealed a signature profile of this state. The expression analysis of individual genes at the cell level suggests that iron homeostasis is important at all stages of the infection, whereas the oxidative stress response seems of particular importance as the survival time increases. Altogether, these data show that non-sporulated bacteria are able to survive for a prolonged period of time and indicate that they engage in a profound adaptation process that leads to their persistence in the host cadaver. | microbiology |
10.1101/2022.05.08.491110 | Thermal endurance by a hot-spring-dwelling phylogenetic relative of the mesophilic Paracoccus | High temperature growth/survival was revealed in a phylogenetic relative (strain SMMA_5) of the mesophilic Paracoccus isolated from the 78-85{degrees}C water of a Trans-Himalayan sulfur-borax spring. After 12 h at 50{degrees}C, or 45 minutes at 70{degrees}C, in mineral salts thiosulfate (MST) medium, SMMA_5 retained ~2% colony-forming units (CFUs), whereas comparator Paracoccus had 1.5% and 0% CFU left at 50{degrees}C and 70{degrees}C respectively. After 12 h at 50{degrees}C, the thermally-conditioned sibling SMMA_5_TC exhibited ~1.5 time increase in CFU-count; after 45 minutes at 70{degrees}C, SMMA_5_TC had 7% of the initial CFU-count intact. 1000-times diluted Reasoner's 2A medium, and MST supplemented with lithium, boron or glycine-betaine (solutes typical of the SMMA_5 habitat), supported higher CFU-retention/CFU-growth than MST. With or without lithium/boron/glycine-betaine in MST, a higher percentage of cells always remained viable (cytometry data), compared with what percentage remained capable of forming single colonies (CFU data). SMMA_5, compared with other Paracoccus, contained 335 unique genes, mostly for DNA replication/recombination/repair, transcription, secondary metabolites biosynthesis/transport/catabolism, and inorganic ion transport/metabolism. It's also exclusively enriched in cell wall/membrane/envelope biogenesis, and amino acid metabolism, genes. SMMA_5 and SMMA_5_TC mutually possessed 43 nucleotide polymorphisms, of which 18 were in protein-coding genes with 13 nonsynonymous and seven radical amino acid replacements. Such biochemical and biophysical mechanisms could be involved in thermal stress mitigation which streamline the cells' energy and resources towards system-maintenance and macromolecule-stabilization, thereby relinquishing cell-division for cell-viability. Thermal conditioning apparently helped memorize those potential metabolic states which are crucial for cell-system maintenance, while environmental solutes augmented the indigenous stability-conferring mechanisms. | microbiology |
10.1101/2022.05.09.491123 | Chemoenzymatic fluorescence labeling of intercellularly contacting cells using lipidated sortase A | Methods to label intercellular contact attract particular attention due to their potential in cell biological and medical applications through analysis of intercellular communications. In this study, a simple and versatile method for chemoenzymatically labeling the intercellularly contacting cell was developed by using a cell-surface anchoring reagent of poly(ethylene glycol)(PEG)-lipid conjugate. The surfaces of each cell in cell pairs of interest were efficiently decorated with sortase A (SrtA) and triglycine peptide that were lipidated with PEG-lipid, respectively. In the mixture of the two cell populations, the triglycine-modified cells were enzymatically labeled with a fluorescent labeling reagent by contacting with the SrtA-modified cells both on the substrate and in cell suspensions. Such selective labeling of the contacting cells was confirmed by confocal microscopy and flow cytometry. The results show a proof of principle that the present method is a promising tool for selective visualization and quantification of the intercellularly contacting cells among cell mixtures in cell-cell communication analysis. | biochemistry |
10.1101/2022.05.09.491177 | Roughening instability of growing 3D bacterial colonies | How do growing bacterial colonies get their shapes? While colony morphogenesis is well-studied in 2D, many bacteria grow as large colonies in 3D environments, such as gels and tissues in the body, or soils, sediments, and subsurface porous media. Here, we describe a morphological instability exhibited by large colonies of bacteria growing in 3D. Using experiments in transparent 3D granular hydrogel matrices, we show that dense colonies of four different species of bacteria--Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, and Komagataeibacter sucrofermentans--generically roughen as they consume nutrients and grow beyond a critical size, eventually adopting a characteristic branched, broccoli-like, self-affine morphology independent of variations in the cell type and environmental conditions. This behavior reflects a key difference between 2D and 3D colonies: while a 2D colony may access the nutrients needed for growth from the third dimension, a 3D colony inevitably becomes nutrient-limited in its interior, driving a transition to rough growth at its surface. We elucidate the onset of roughening using linear stability analysis and numerical simulations of a continuum model that treats the colony as an 'active fluid' whose dynamics are driven by nutrient-dependent cellular growth. We find that when all dimensions of the growing colony substantially exceed the nutrient penetration length, nutrient-limited growth drives a 3D morphological instability that recapitulates essential features of the experimental observations. Our work thus provides a framework to predict and control the organization of growing colonies--as well as other forms of growing active matter, such as tumors and engineered living materials--in 3D environments. | biophysics |
10.1101/2022.05.09.491071 | Deformable Mirror based Optimal PSF Engineering for 3D Super-resolution Imaging | Point spread function (PSF) engineering is an important technique to encode the properties (e.g., 3D positions, color, and orientation) of single molecule in the shape of the PSF, often with the help of a programmable phase modulator. Deformable mirror (DM) is currently the most widely used phase modulator for fluorescence detection as it shows negligible photon loss. However, it relies on careful calibration for precise wavefront control. Therefore, design of an optimal PSF not only relies on the theoretical calculation of maximum information content, but also the physical behavior of the phase modulator, which is often ignored during the optimization process. Here, we developed a framework of PSF engineering which could generate a device specific optimal PSF for 3D super-resolution imaging using DM. We used our method to generate two types of PSFs with depths of field comparable to the widely used astigmatism and Tetrapod PSFs, respectively. We demonstrated the superior performance of the DM specific optimal PSF over the conventional astigmatism and Tetrapod PSF both theoretically and experimentally. | biophysics |
10.1101/2022.05.09.490905 | Computational modeling of TGF-β2:TβRI:TβRII receptor complex assembly as mediated by the TGF-β co-receptor betaglycan | Transforming growth factor-{beta}1, -{beta}2, and -{beta}3 (TGF-{beta}1, -{beta}2, and -{beta}3) are secreted signaling ligands that play essential roles in tissue development, tissue maintenance, immune response, and wound healing. TGF-{beta} homodimers signal by assembling a heterotetrameric complex comprised of two type I receptor (T{beta}RI):type II receptor (T{beta}RII) pairs. TGF-{beta}1 and TGF-{beta}3 signal with high potency due to their high affinity for T{beta}RII, which engenders high affinity binding of T{beta}RI through a composite TGF-{beta}:T{beta}RII binding interface. However, TGF-{beta}2 binds T{beta}RII 200-500 more weakly than T{beta}RII and signals with lower potency compared to TGF-{beta}1 and -{beta}3. Remarkably, potency of TGF-{beta}2 is increased to that of TGF-{beta}1 and -{beta}3 in the presence of an additional membrane-bound co-receptor, known as betaglycan (BG), even though betaglycan does not directly participate in the signaling mechanism and is displaced as the signaling receptors, T{beta}RI and T{beta}RII, bind. To determine the role of betaglycan in the potentiation of TGF-{beta}2 signaling, we developed deterministic computational models with different modes of betaglycan binding and varying cooperativity between receptor subtypes. The models, which were developed using published kinetic rate constants for known quantities and optimization to determine unknown quantities, identified conditions for selective enhancement of TGF-{beta}2 signaling. The models provide support for additional receptor binding cooperativity that has been hypothesized, but not evaluated in the literature. The models further showed that betaglycan binding to TGF-{beta}2 ligand through two domains provides an effective mechanism for transfer to the signaling receptors that has been tuned to efficiently promote assembly of the TGF-{beta}2(T{beta}RII)2(T{beta}RI)2 signaling complex. | biophysics |
10.1101/2022.05.09.491086 | De novo design of peptides that form transmembrane barrel pores killing antibiotic resistant bacteria | De novo design of peptides that self-assemble into transmembrane barrel pores is challenging due to the complexity of several competing interactions involving peptides, lipids, water, and ions. Here, we develop a computational approach for the de novo design of -helical peptides that self-assemble into stable transmembrane barrel pores with a central functional channel. We formulate the previously missing design guidelines and report 52 sequence patterns that can be tuned for specific applications using the identified role of each residue. Atomic force microscopy and fluorescent dye leakage experiments confirm that the designed peptides form leaky membrane pores in vitro. Customized designed peptides act as antimicrobial agents able to kill even antibiotic-resistant ESKAPE bacteria at micromolar concentrations, while exhibiting low toxicity to human cells. The peptides can be similarly fine-tuned for other medical and biotechnological applications. | biophysics |
10.1101/2022.05.09.491161 | Derivation of the Relationship Between the Mass and Lifespan of Living Organisms | The relation p=1-a between exponents, by which the dependencies of lifespan p and growth intensity a on masses of living organisms are described, is obtained and testified. | biophysics |
10.1101/2022.05.09.491135 | Sequence-based pH-dependent prediction of protein solubility using CamSol | Solubility is a property of central importance for the use of proteins in research and in applications in biotechnology and medicine. Since experimental methods for measuring protein solubility are resource-intensive and time-consuming, computational methods have recently emerged to enable the rapid and inexpensive screening of large libraries of proteins, as it is routinely required in development pipelines. Here, we describe the extension of one of such methods, CamSol, to include in the predictions the effect of the pH of the solubility. We illustrate the accuracy of the pH-dependent predictions on a variety of antibodies and other proteins. | biophysics |
10.1101/2022.05.09.490804 | Evolution avoids a pathological stabilizing interaction in the immune protein S100A9 | Protein stability constrains protein evolution. While much is known about evolutionary constraints on destabilizing mutations, much less is known about evolutionary constraints on mutations that increase protein stability. We recently found that the M63F mutation in the innate immune protein S100A9 increases its stability but disrupts its natural ability to activate inflammation. Here we show, through careful biophysical and functional studies, that this mutation stabilizes a nonfunctional form of the protein through a direct interaction with another amino acid, Phe37. Phe can be tolerated at position 37 or 63, but not at both sites simultaneously. We find that this pattern has been conserved over millions of years of evolution. Our work highlights an underappreciated evolutionary constraint on stabilizing mutations: they must avoid inappropriately stabilizing non-functional protein conformations. | biophysics |
10.1101/2022.05.09.491136 | Ubiquitous bacterial polyketides induce cross-kingdom microbial interactions | Although the interaction of prokaryotic and eukaryotic microorganisms is critical for the functioning of ecosystems, knowledge of the processes driving microbial interactions within communities is in its infancy. We previously reported that the soil bacterium Streptomyces iranensis specifically triggers the production of natural products in the fungus Aspergillus nidulans. Here, we discovered that arginine-derived polyketides serve as the bacterial signals for this induction. Arginine-derived polyketide-producing bacteria occur world wide. These producer bacteria and the fungi that decode and respond to this signal can be co-isolated from the same soil sample. Arginine-derived polyketides impact surrounding microorganisms both directly as well as indirectly, by inducing the production of natural products in fungi that further influence the composition of microbial consortia. | microbiology |
10.1101/2022.05.09.491233 | Corynebacterium matruchotii fitness enhancement of adjacent streptococci by multiple mechanisms | Polymicrobial biofilms are present in many environments particularly in the human oral cavity where they can prevent or facilitate the onset of disease. While recent advances have provided a clear picture of both the constituents and their biogeographical arrangement, it is still unclear what mechanisms of interaction occur between individual species in close proximity within these communities. In this study we investigated two mechanisms of interaction between the highly abundant supragingival plaque (SUPP) commensal Corynebacterium matruchotii and Strepto-coccus mitis which are directly adjacent in vivo. We discovered that C. matruchotii enhanced the fitness of streptococci dependent on its ability to detoxify streptococcal-produced hydrogen per-oxide and its ability to oxidize lactate also produced by streptococci. We demonstrate that the fitness of adjacent streptococci was linked to that of C. matruchotii and that these mechanisms support the previously described corncob arrangement between these species but that this is favorable only in aerobic conditions. Further we utilized scanning electrochemical microscopy (SECM) to quantify lactate production and consumption between individual bacterial cells for the 1st time, revealing that lactate oxidation provides a fitness benefit to S. mitis and not pH mitigation. This study describes mechanistic interactions between two highly abundant human com-mensals that can explain their observed in vivo spatial arrangements and suggest a way by which they may help preserve a healthy oral bacterial community. | microbiology |
10.1101/2022.05.09.491063 | In-silico identification of Tyr232 in AMPKα2 as a dephosphorylation site for the protein tyrosine phosphatase PTP-PEST | The AMP-activated protein kinase (AMPK) is known to be activated by the protein tyrosine phosphatase non-receptor type 12 (PTP-PEST) under hypoxic conditions. This activation is mediated by tyrosine dephosphorylation of the AMPK subunit. However, the identity of the phosphotyrosine residues remains unknown. In this study we first predicted the structure of the complex of the AMPK2 subunit and PTP-PEST catalytic domain using bioinformatics tools and further confirm the stability of the complex using molecular dynamics simulations. Evaluation of the protein-protein interfaces indicates that residue Tyr232 is the most likely site of dephosphorylation on AMPK2. In addition, we explored the effect of phosphorylation of PTP-PEST residue Tyr64 on the stability of the complex. The phosphorylation of Tyr64, an interface residue, enhances the stability of the complex via the rearrangement of a network of electrostatic interactions in conjunction with conformational changes in the catalytic WPD loop. Our findings present a plausible structural basis of AMPK regulation mediated by PTP-PEST and shows how phosphorylation of PTP-PEST could be involved in its activation. | biochemistry |
10.1101/2022.05.09.491169 | Dynamic HIV-1 spike motion creates vulnerability for its membrane-bound tripod to antibody attack | Vaccines targeting HIV-1's gp160 spike protein are stymied by high viral mutation rates and structural chicanery. gp160's membrane-proximal external region (MPER) is the target of naturally arising broadly neutralizing antibodies (bnAbs), yet MPER-based vaccines fail to generate bnAbs. Here, nanodisc-embedded spike protein was investigated by cryo-electron microscopy and molecular-dynamics simulations, revealing spontaneous ectodomain tilting that creates vulnerability for HIV-1. While each MPER protomer radiates centrally towards the three-fold axis contributing to a membrane-associated tripod structure that is occluded in the upright spike, tilting provides access to the opposing MPER. Structures of spike proteins with bound 4E10 bnAb Fabs reveal that the antibody binds exposed MPER, thereby altering MPER dynamics, modifying average ectodomain tilt, and imposing strain on the viral membrane and the spike's transmembrane segments, resulting in the abrogation of membrane fusion and informing future vaccine development. | biophysics |
10.1101/2022.05.09.491209 | Decoding The Nuclear Genome of The Human Pathogen Babesia duncani Shed Light on its Virulence, Drug Susceptibility and Evolution among Apicomplexa | Babesia species are tick-transmitted apicomplexan pathogens and the causative agents of babesiosis, a malaria-like disease of major medical and veterinary importance. Of the different species of Babesia reported so far, Babesia duncani causes severe to lethal infection in patients. Despite the highly virulent nature of this parasite and the risk it may pose as an emerging pathogen, little is known about its biology, metabolic requirements, and pathogenesis. B. duncani is unique among apicomplexan parasites that infect red blood cells in that it can be continuously cultured in vitro in human erythrocytes but can also infect mice leading to fulminant babesiosis infection and death. Here we have taken advantage of the recent advances in the propagation of this parasite in vitro and in vivo to conduct detailed molecular, genomic and transcriptomic analyses and to gain insights into its biology. We report the assembly, 3D structure, and annotation of the nuclear genome of this parasite as well as its transcriptomic profile and an atlas of its metabolism during its intraerythrocytic life cycle. Detailed examination of the B. duncani genome and comparative genomic analyses identified new classes of candidate virulence factors, suitable antigens for diagnosis of active infection, and several attractive drug targets. Translational analyses and efficacy studies identified highly potent inhibitors of B. duncani thus enriching the pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis. | microbiology |
10.1101/2022.05.09.491188 | Identification of Banana heat responsive long non-coding RNAs and their gene expression analysis. | Identification and characterization of long non-coding RNAs (lncRNAs) in the last decade has attained great attention because of their importance in gene regulatory functions in response to various plant stresses. Rising temperature is a potential threat to the agriculture world. Banana being an important economic crop, identification and characterization of genes and RNAs that regulate high temperature stress is imperative. As the prediction of lncRNAs in response to heat stress in banana is largely unknown, the present study was focused to identify the heat stress responsive lncRNA in banana (DH Pahang). Perl script was written to identify the novel transcripts, using the work flow. StringTie and CPC softwares were used and a total of 363 novel HS-lncRNA were identified in banana. Further, lncRNA were classified as 288 lincRNAs, 71 antisense LncRNA, 5 sense lncRNAs. The functional classification was done and transcripts were broadly classified into molecular function, cellular components and biological processes. Differential expression of lncRNA showed the varied patterns at different stages of heat stress. Finally, qPCR results confirmed DGE expression pattern of lncRNAs. Further, the Cytoscape analysis was performed which showed protein coding genes involved in membrane integrity and other signal transduction pathways. Taken together, these findings expand our understanding of lncRNAs as ubiquitous regulators under heat stress conditions in banana. | molecular biology |
10.1101/2022.05.09.491187 | Chronic clinical signs of upper respiratory tract disease shape gut and respiratory microbiomes in cohabitating domestic felines. | Feline upper respiratory tract disease (FURTD), often caused by infections etiologies, is a multifactorial syndrome affecting feline populations worldwide. Because of its highly transmissible nature, infectious FURTD is most prevalent anywhere cats are housed in groups such as animal shelters, and is associated with negative consequences such as decreasing adoption rates, intensifying care costs, and increasing euthanasia rates. Understanding the etiology and pathophysiology of FURTD is thus essential to best mitigate the negative consequences of this disease. Clinical signs of FURTD include acute respiratory disease, with a small fraction of cats developing chronic sequelae. It is thought that nasal mucosal microbiome changes play an active role in the development of acute clinical signs, but it remains unknown if the microbiome may play a role in the development and progression of chronic clinical disease. To address the knowledge gap surrounding how microbiomes link to chronic FURTD, we asked if microbial community structure of upper respiratory and gut microbiomes differed between cats with chronic FURTD signs and clinically normal cats. We selected 8 households with at least one cat exhibiting chronic clinical FURTD, and simultaneously collected samples from cohabitating clinically normal cats. Microbial community structure was assessed via 16S rDNA sequencing of both gut and nasal microbiome communities. Using a previously described ecophylogenetic method, we identified 37 and 27 microbial lineages within gut and nasal microbiomes respectively that significantly associated with presence of active FURTD clinical signs in cats with a history of chronic signs. Overall, we find that nasal and gut microbial communities may contribute to the development of chronic clinical course, but more research is needed to confirm our observations. | microbiology |
10.1101/2022.05.09.491178 | The Drosophila ZAD zinc finger protein Kipferl guides Rhino to piRNA clusters | RNA interference systems depend on the synthesis of small RNA precursors whose sequences define the target spectrum of these silencing pathways. The Drosophila Heterochromatin Protein 1 (HP1) variant Rhino permits transcription of PIWI-interacting RNA (piRNA) precursors within transposon-rich heterochromatic loci in germline cells. Current models propose that Rhino's specific chromatin occupancy at piRNA source loci is determined by histone marks and maternally inherited piRNAs, but also imply the existence of other, undiscovered specificity cues. Here, we identify a member of the diverse family of zinc finger associated domain (ZAD)-C2H2 proteins, Kipferl, as critical Rhino cofactor in ovaries. By binding to guanosine-rich DNA motifs and interacting with the Rhino chromodomain, Kipferl recruits Rhino to specific loci and stabilizes it on chromatin. In kipferl mutant flies, Rhino is lost from most of its target chromatin loci and instead accumulates on pericentromeric satellite arrays, resulting in decreased levels of transposon targeting piRNAs and impaired fertility. Our findings reveal that DNA sequence, in addition to the H3K9me3 mark, determines the identity of piRNA source loci and provide insight into how Rhino might be caught in the crossfire of genetic conflicts. | genetics |
10.1101/2022.05.09.491114 | Real-time single-molecule observation of chaperone-assisted protein folding | The ability of Hsp70 molecular chaperones to remodel the conformation of their clients is central to their biological function; however, questions remain regarding the precise molecular mechanisms by which Hsp70 machinery interacts with the client and how this contributes towards efficient protein folding. Here, we used Total Internal Reflection Fluorescence (TIRF) microscopy and single-molecule Fluorescence Resonance Energy Transfer (smFRET) to temporally observe the conformational changes that occur to individual firefly luciferase (Fluc) proteins as they are folded by the bacterial Hsp70 system. For the first time, we observed multiple cycles of chaperone binding-and-release to an individual client during refolding and that high rates of chaperone cycling improves refolding yield. Furthermore, we demonstrate that DnaJ remodels misfolded proteins via a conformational selection mechanism whereas DnaK resolves misfolded states via mechanical unfolding. This study illustrates that the temporal observation of chaperone-assisted folding enables the elucidation of key mechanistic details inaccessible using other approaches. | biochemistry |
10.1101/2022.05.09.491239 | Design of an ultrafast pulsed ponderomotive phase plate for cryo-electron tomography | Ponderomotive phase plates have shown temporally consistent phase contrast is possible within electron microscopes via high fluence static laser modes resonating in Fabry-Perot cavities. Here, we explore using pulsed laser beams as an alternative method of generating high fluences. We find through forward-stepping finite element models that picosecond-or-less interactions are required for meaningful fluences phase shifts, with higher pulse energies and smaller beam waists leading to the predicted higher fluences. An additional model based on quasiclassical assumptions is used to discover the shape of the phase plate by incorporating the oscillatory nature of the electric field. From these results, we find the transient nature of the laser pulses removes the influence of Kapitza-Dirac diffraction patterns that appear in the static resonator cases. The addition of a second laser aligned 90 degrees to the first induces anisotropy to the shape of the phase plate. By incorporating a shifting-electron-beam algorithm, the effects of a finite electron beam crossover are also simulated. A total pulse energy of 8.7 microJoules is enough to induce the required pi/2 phase shift for Zernike-like phase microscopy. As a brief thought experiment, we also explore the usage of high frequency lasers in a standard electron emission scheme to see if a pulsed electron beam is even necessary. Ultimately, frequency requirements limit the laser to nanosecond pulse durations, causing the required pulse energies to reach unreasonable levels before adequate phase shifts are achieved. | biophysics |