bluuebunny/crossref_metadata_2025 huggingface dataset:
SPPUSM: An MS/MS spectra merging strategy for improved low-input and single-cell proteome identification
Authors: Chen Yongle , Du Zhuokun , Zhao Hongxian , Fang Wei , Liu Tong , Zhang Yangjun , Zhang Wanjun , Qin Weijie
Published: July 2023

Single and rare cell analysis provides unique insights into the investigation of biological processes and disease progress by resolving the cellular heterogeneity that is masked by bulk measurements. Although many efforts have been made, the techniques used to measure the proteome in trace amounts of samples or in single cells still lag behind those for DNA and RNA due to the inherent non-amplifiable nature of proteins and the sensitivity limitation of current mass spectrometry. Here, we report an MS/MS spectra merging strategy termed SPPUSM (same precursor-produced unidentified spectra merging) for improved low-input and single-cell proteome data analysis. In this method, all the unidentified MS/MS spectra from multiple test files are first extracted. Then, the corresponding MS/MS spectra produced by the same precursor ion from different files are matched according to their precursor mass and retention time (RT) and are merged into one new spectrum. The newly merged spectra with more fragment ions are next searched against the database to increase the MS/MS spectra identification and proteome coverage. Further improvement can be achieved by increasing the number of test files and spectra to be merged. Up to 18.2% improvement in protein identification was achieved for 1 ng HeLa peptides by SPPUSM. Reliability evaluation by the “entrapment database” strategy using merged spectra from human andE. colirevealed a marginal error rate for the proposed method. For application in single cell proteome (SCP) study, identification enhancement of 28%-61% was achieved for proteins for different SCP data. Furthermore, a lower abundance was found for the SPPUSM-identified peptides, indicating its potential for more sensitive low sample input and SCP studies.
Tuberculosis susceptibility in genetically diverse mice reveals functional diversity of neutrophils
Authors: Ravesloot-Chávez Mariëtta M. , Van Dis Erik , Fox Douglas , Anaya Sanchez Andrea , Espich Scott , Nguyenla Xammy H. , Rawal Sagar L. , Samani Helia , Ballinger Mallory A. , Thomas Henry , Kotov Dmitri , Vance Russell , Nachman Michael W. , Stanley Sarah A.
Published: July 2023

Tuberculosis (TB) is a heterogenous disease in humans with individuals exhibiting a wide range of susceptibility. This heterogeneity is not captured by standard laboratory mouse lines. We used a new collection of 19 wild-derived inbred mouse lines collected from diverse geographic sites to identify novel phenotypes duringMycobacterium tuberculosis(Mtb) infection. Wild derived mice have heterogenous immune responses to infection that result in differential ability to control disease at early timepoints. Correlation analysis with multiple parameters including sex, weight, and cellular immune responses in the lungs revealed that enhanced control of infection is associated with increased numbers of CD4 T cells, CD8 T cells and B cells. Surprisingly, we did not observe strong correlations between IFN-γ production and control of infection. Although in most lines high neutrophils were associated with susceptibility, we identified a mouse line that harbors high neutrophils numbers yet controls infection. Using single-cell RNA sequencing, we identified a novel neutrophil signature associated with failure to control infection.
Projecting COVID-19 intensive care admissions in the Netherlands for policy advice: February 2020 to January 2021
Authors: Klinkenberg Don , Backer Jantien A. , de Keizer Nicolette F. , Wallinga Jacco
Published: July 2023

0.

Introduction

Model projections of COVID-19 incidence into the future help policy makers about decisions to implement or lift control measures. During 2020, policy makers in the Netherlands were informed on a weekly basis with short-term projections of COVID-19 intensive care unit (ICU) admissions. Here we present the model and the procedure by which it was updated.

Methods

the projections were produced using an age-structured transmission model. A consistent, incremental update procedure that integrated all new surveillance and hospital data was conducted weekly. First, up-to-date estimates for most parameter values were obtained through re-analysis of all data sources. Then, estimates were made for changes in the age-specific contact rates in response to policy changes. Finally, a piecewise constant transmission rate was estimated by fitting the model to reported daily ICU admissions, with a change point analysis guided by Akaike’s Information Criterion.

Results

The model and update procedure allowed us to make mostly accurate weekly projections, accounting for recent and future policy changes, and to adapt the estimated effectiveness of the policy changes based only on the natural accumulation of incoming data.

Discussion

The model incorporates basic epidemiological principles and most model parameters were estimated per data source. Therefore, it had potential to be adapted to a more complex epidemiological situation, as it would develop after 2020.
Three-dimensional genome architecture persists in a 52,000-year-old woolly mammoth skin sample
Authors: Sandoval-Velasco Marcela , Dudchenko Olga , Rodríguez Juan Antonio , Estrada Cynthia Pérez , Dehasque Marianne , Fontsere Claudia , Mak Sarah S.T. , Plotnikov Valerii , Khan Ruqayya , Weisz David , Contessoto Vinícius G. , Oliveira Junior Antonio B. , Kalluchi Achyuth , Omer Arina D. , Batra Sanjit S. , Shamim Muhammad S. , Durand Neva C. , O’Connell Brendan , Roca Alfred L. , Gnirke Andreas , Garcia-Treviño Isabel , Coke Rob , Flanagan Joseph P. , Pletch Kelcie , Ruiz-Herrera Aurora , Lander Eric S. , Rowley M. Jordan , Onuchic José N. , Dalén Love , Marti-Renom Marc A. , Gilbert M. Thomas P. , Aiden Erez Lieberman
Published: July 2023

Ancient DNA (aDNA) sequencing analysis typically involves alignment to a modern reference genome assembly from a related species. Since aDNA molecules are fragmentary, these alignments yield information about small-scale differences, but provide no information about larger features such as the chromosome structure of ancient species. We report the genome assembly of a female Late Pleistocene woolly mammoth (Mammuthus primigenius) with twenty-eight chromosome-length scaffolds, generated using mammoth skin preserved in permafrost for roughly 52,000 years. We began by creating a modified Hi-C protocol, dubbed PaleoHi-C, optimized for ancient samples, and using it to map chromatin contacts in a woolly mammoth. Next, we developed “reference-assisted 3D genome assembly,” which begins with a reference genome assembly from a related species, and uses Hi-C and DNA-Seq data from a target species to split, order, orient, and correct sequences on the basis of their 3D proximity, yielding accurate chromosome-length scaffolds for the target species. By means of this reference-assisted 3D genome assembly, PaleoHi-C data reveals the 3D architecture of a woolly mammoth genome, including chromosome territories, compartments, domains, and loops. The active (A) and inactive (B) genome compartments in mammoth skin more closely resemble those observed in Asian elephant skin than the compartmentalization patterns seen in other Asian elephant tissues. Differences in compartmentalization between these skin samples reveal sequences whose transcription was potentially altered in mammoth. We observe a tetradic structure for the inactive X chromosome in mammoth, distinct from the bipartite architecture seen in human and mouse. Generating chromosome-length genome assemblies for two other elephantids (Asian and African elephant), we find that the overall karyotype, and this tetradic Xi structure, are conserved throughout the clade. These results illustrate that cell-type specific epigenetic information can be preserved in ancient samples, in the form of DNA geometry, and that it may be feasible to perform de novo genome assembly of some extinct species.
Selective consistency of recurrent neural networks induced by plasticity as a mechanism of unsupervised perceptual learning
Authors: Goto Yujin , Kitajo Keiichi
Published: July 2023

Understanding the mechanism by which the brain overcomes its inherent inconsistency in activity to achieve consistent information processing is one of the major challenges in neuroscience. Recently, it has been reported that the consistency of neural responses to stimuli that are presented repeatedly is enhanced implicitly in an unsupervised way, and results in improved perceptual consistency. Here, we propose the term "selective consistency" to describe this input-dependent consistency and hypothesize that it will be acquired in a self-organizing manner by plasticity within the neural system. To test this, we investigated whether a reservoir-based plastic model could acquire selective consistency to repeated stimuli. We used white noise sequences randomly generated in each trial and referenced white noise sequences presented multiple times. The results showed that the plastic network was capable of acquiring selective consistency rapidly, with as little as five exposures to stimuli, even for white noise. The acquisition of selective consistency could occur independently of performance optimization, as the network’s time-series prediction accuracy for referenced stimuli did not improve with repeated exposure and optimization. Furthermore, the network could only achieve selective consistency when in the region between order and chaos. These findings suggest that the neural system can acquire selective consistency in a self-organizing manner and that this may serve as a mechanism for certain types of learning.
Cue- versus reward-encoding basolateral amygdala projections to nucleus accumbens
Authors: He Yi , Huang Yanhua H. , Schlüter Oliver M. , Dong Yan
Published: July 2023

In substance use disorders, drug use as unconditioned stimulus (US) reinforces drug taking. Meanwhile, drug-associated cues (conditioned stimulus, CS) also gain incentive salience to promote drug seeking. The basolateral amygdala (BLA) is implicated in both US- and CS-mediated responses. Here, we show that two genetically distinct BLA neuronal types, expressing Rspo2 versus Ppp1r1b, respectively, project to the nucleus accumbens (NAc) and form monosynaptic connections with both dopamine D1 and D2 receptor-expressing neurons. While intra-NAc stimulation of Rspo2 or Ppp1r1b presynaptic terminals establishes intracranial self-stimulation (ICSS), only Ppp1r1b-stimulated mice exhibit cue-induced ICSS seeking. Furthermore, increasing versus decreasing the Ppp1r1b-to-NAc, but not Rspo2-to-NAc, subprojection increases versus decreases cue-induced cocaine seeking after cocaine withdrawal. Thus, while both BLA- to-NAc subprojections contribute to US-mediated responses, the Ppp1r1b subprojection selectively encodes CS-mediated reward and drug reinforcement. Such differential circuit representations may provide insights into precise understanding and manipulation of drug-versus cue-induced drug seeking and relapse.

Teaser

Two subprojections from the amygdala to nucleus accumbens differentially encode cue-vs reward-mediated reinforcement.
Labile assembly of a tardigrade protein induces biostasis
Authors: Sanchez-Martinez S. , Nguyen K. , Biswas S. , Nicholson V. , Romanyuk A.V. , Ramirez J. , KC S. , Akter A. , Childs C. , Usher E.T. , Ginell G.M. , Yu F. , Gollub E. , Malferrari M. , Francia F. , Venturoli G. , Martin E.W. , Caporaletti F. , Giubertoni G. , Woutersen S. , Sukenik S. , Woolfson D.N. , Holehouse A.S. , Boothby T.C.
Published: July 2023

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins that form gels. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of gelation for CAHS D similar to that of intermediate filaments. We show that gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying.In vivo, we observe that CAHS D forms fiber-like condensates during osmotic stress. Condensation of CAHS D improves survival of osmotically shocked cells through at least two mechanisms: reduction of cell volume change and reduction of metabolic activity. Importantly, condensation of CAHS D is reversible and metabolic rates return to control levels after CAHS condensates are resolved. This work provides insights into how tardigrades induce biostasis through the self-assembly of CAHS gels.
Inference for entomological semi-field experiments: Fitting a mathematical model assessing personal and community protection of vector-control interventions
Authors: Fairbanks Emma L , Saeung Manop , Pongsiri Arissara , Vajda Elodie , Wang Yuqian , McIver David J , Tatarsky Allison , Lobo Neil F , Moore Sarah J , Ponlawat Alongkot , Chareonviriyaphap Theeraphap , Ross Amanda , Chitnis Nakul
Published: July 2023

The effectiveness of vector-control tools is often assessed by experiments as a reduction in mosquito landings using human landing catches (HLCs). However, HLCs alone only quantify a single characteristic and therefore do not provide information on the overall impacts of the intervention product. Using data from a recent semi-field study which used time-stratified HLCs, aspiration of non-landing mosquitoes, and blood feeding, we suggest a Bayesian inference approach for fitting such data to a stochastic model. This model considers both personal protection, through a reduction in biting, and community protection, from mosquito mortality and disarming (prolonged inhibition of blood feeding). Parameter estimates are then used to predict the reduction of vectorial capacity induced by etofenpox-treated clothing, picaridin topical repellents, transfluthrin spatial repellents and metofluthrin spatial repellents, as well as combined interventions forPlasmodium falciparummalaria inAnopleles minimus. Overall, all interventions had both personal and community effects, preventing biting and killing or disarming mosquitoes. This led to large estimated reductions in the vectorial capacity, with substantial impact even at low coverage. As the interventions aged, fewer mosquitoes were killed; however the impact of some interventions changed from killing to disarming mosquitoes. Overall, this inference method allows for additional modes of action, rather than just reduction in biting, to be parameterised and highlights the tools assessed as promising malaria interventions.
The auditory P2 evoked by speech sounds consists of two separate subcomponents
Authors: Steinmetzger Kurt , Rupp André
Published: July 2023

The P2 component of the auditory evoked potential is commonly thought to reflect acoustic stimulus properties as well as prior exposure to the materials, rather than change-related cortical activity. Here, we challenge this view by showing that the P2 is strongly increased in response to voice pitch changes with a stepwise pattern compared to changes in dynamic pitch contours typical for natural speech, and also reflects the magnitude of these pitch changes. Furthermore, it is demonstrated that neither the P2 nor any other component are affected by the harmonicity of the materials. Despite no prior exposure, artificially created inharmonic versions of the speech materials elicited similar activity throughout auditory cortex. This suggests that so-called harmonic template neurons observed in animal studies are either absent or do not exist in sufficient number in human auditory cortex to detect their activity extracranially. Crucially, both morphology and source reconstructions of the EEG data showed that the P2 appears to consist of two separate subcomponents. Whereas source activity for the “P2a” was strongest in right auditory cortex, the subsequent “P2b” included generators spread across auditory cortex and association areas, bilaterally. The two subcomponents thus likely reflect processing at different stages of the auditory pathway.
Spinal facilitation of descending motor input
Authors: Taccola Giuliano , Kissane Roger , Culaclii Stanislav , Apicella Rosamaria , Liu Wentai , Gad Parag , Ichiyama Ronaldo M. , Chakrabarty Samit , Edgerton V. Reggie
Published: July 2023

Highly varying patterns of electrostimulation (Dynamic Stimulation, DS) delivered to the dorsal cord through an epidural array with 18 independent electrodes transiently facilitate corticospinal motor responses, even after spinal injury. To partly unravel how corticospinal input are affected by DS, we introduced a corticospinal platform that allows selective cortical stimulation during the multisite acquisition of cord dorsum potentials (CDPs) and the simultaneous supply of DS. Firstly, the epidural interface was validated by the acquisition of the classical multisite distribution of CDPs on the dorsal cord and their input-output profile elicited by pulses delivered to peripheral nerves. Apart from increased EMGs, DS selectively increased excitability of the spinal interneurons that first process corticospinal input, without changing the magnitude of commands descending from the motor cortex, suggesting a novel correlation between muscle recruitment and components of cortically-evoked CDPs. Finally, DS increases excitability of post-synaptic spinal interneurons at the stimulation site and their responsiveness to any residual supraspinal control, thus supporting the use of electrical neuromodulation whenever the motor output is jeopardized by a weak volitional input, due to a partial disconnection from supraspinal structures and/or neuronal brain dysfunctions.
Loop-extruders alter bacterial chromosome topology to direct entropic forces for segregation
Authors: Harju Janni , van Teeseling Muriel C. F. , Broedersz Chase P.
Published: July 2023

Entropic forces have been argued to drive bacterial chromosome segregation during replication. In many bacterial species, how-ever, specifically evolved mechanisms, such as loop-extruding SMC complexes and the ParABSorigin segregation system, contribute to or are even required for chromosome segregation, suggesting that entropic forces alone may be insufficient. The interplay between and the relative contributions of these segregation mechanisms remain unclear. Here, we develop a biophysical model showing that purely entropic forces actually inhibit bacterial chromosome segregation until late replication stages. By contrast, our model reveals that loop-extruders loaded at the origins of replication, as observed in many bacterial species, alter the effective topology of the chromosome, thereby redirecting and enhancing entropic forces to enable accurate chromosome segregation during replication. We confirm our model predictions with polymer simulations: purely entropic forces do not allow for concurrent replication and segregation, whereas entropic forces steered by specifically loaded loop-extruders lead to robust, global chromosome segregation during replication. Finally, we show how loop-extruders can complement locally acting origin separation mechanisms, such as the ParABSsystem. Together, our results illustrate how changes in the geometry and topology of the polymer, induced by DNA-replication and loop-extrusion, impact the organization and segregation of bacterial chromosomes.
Glycine fermentation byC. difficilepromotes virulence, spore formation, and is induced by host cathelicidin
Authors: Rizvi Arshad , Vargas-Cuebas Germán , Edwards Adrianne N. , DiCandia Michael A. , Carter Zavier A. , Lee Cheyenne D. , Monteiro Marcos P. , McBride Shonna M.
Published: July 2023

The amino acid glycine is enriched in the dysbiotic gut and is suspected to contribute toClostridioides difficileinfection. We hypothesized that the use of glycine as an energy source contributes to colonization of the intestine and pathogenesis ofC. difficile. To test this hypothesis, we deleted the glycine reductase genesgrdAB, renderingC. difficileunable to ferment glycine, and investigated the impact on growth and pathogenesis. We found that thegrdpathway promoted growth, toxin production, sporulation, and pathogenesis ofC. difficilein the hamster model of disease. Further, we determined that thegrdlocus is regulated by host cathelicidin (LL-37) and the cathelicidin-responsive regulator, ClnR, indicating that the host peptide signals to control glycine catabolism. The induction of glycine fermentation by LL-37 demonstrates a direct link between the host immune response and the bacterial reactions of toxin production and spore formation.
Plant Cellulose as a Substrate for 3D Neural Stem Cell Culture
Authors: Couvrette Lauren J. , Walker Krystal L. A. , Bui Tuan V. , Pelling Andrew E.
Published: July 2023

Neural stem cell (NSC) based therapies are at the forefront of regenerative medicine strategies for various neural defects and injuries such as stroke, traumatic brain injury and spinal cord injury. For several clinical applications, NSC therapies require biocompatible scaffolds to support cell survival and to direct differentiation. Here, we investigate decellularized plant tissue as a novel scaffold for three-dimensional (3D)in vitroculture of NSCs. Plant cellulose scaffolds were shown to support attachment and proliferation of adult rat hippocampal neural stem cells (NSCs). Further, NSCs differentiated on the cellulose scaffold had significant increases in their expression of neuron-specific beta-III tubulin and glial fibrillary acidic protein compared to 2D culture on a polystyrene plate, indicating that the scaffold may enhance differentiation of NSCs towards astrocytic and neuronal lineages. Our findings suggest that plant-derived cellulose scaffolds have the potential to be used in neural tissue engineering and can be harnessed to direct differentiation of NSCs.
A CRISPR-based strategy for targeted sequencing in biodiversity science
Authors: Littleford-Colquhoun Bethan , Kartzinel Tyler R.
Published: July 2023

Many applications in molecular ecology require the ability to match specific DNA sequences from single- or mixed-species samples to a diagnostic reference library. Widely used methods for DNA barcoding and metabarcoding require PCR and amplicon sequencing to identify taxa based on target sequences, but the target-specific enrichment capabilities of CRISPR-Cas systems may offer advantages in some applications. We identified 54,837 CRISPR-Cas guide RNAs that may be useful for enriching chloroplast DNA across phylogenetically diverse plant species. We then tested a subset of 17 guide RNAsin vitroto enrich and sequence plant DNA strands ranging in size from diagnostic DNA barcodes of 1,428 bp to entire chloroplast genomes of 121,284 bp. We used an Oxford Nanopore sequencer to evaluate sequencing success based on both single- and mixed-species samples, which yielded mean on-target chloroplast sequence lengths of 5,755-11,367 bp, depending on the experiment. Single-species experiments yielded more on-target sequence reads and greater accuracy, but mixed-species experiments yielded superior coverage. Comparing CRISPR-based strategies to a widely used protocol for plant DNA metabarcoding with the chloroplasttrnL-P6 marker, we obtained a 66-fold increase in sequence length and markedly better estimates of relative abundance for a commercially prepared mixture of plant species. Future work would benefit from developing bothin vitroandin silicomethods for analyses of mixed-species samples, especially when the appropriate reference genomes for contig assembly cannot be knowna priori. Prior work developed CRISPR-based enrichment protocols for long-read sequencing and our experiments pioneered its use for plant DNA barcoding and chromosome assemblies that may have advantages over workflows that require PCR and short-read sequencing.
Mitigation of the effect of high light on the photosynthetic apparatus ofRhodobacter alkalitoleranswhen grown in an alkaline environment
Authors: Zamal Mohammad Yusuf , Ramana Ch Venkata , Subramanyam Rajagopal
Published: July 2023

In the phototrophic alphaproteobacteria, photosynthesis is performed by pigment-protein complexes, including the light-harvesting complexes known as LH1 and LH2. The photosystem also encompasses carotenoids to assist in well-functioning of photosynthesis. Most photosynthetic bacteria are exposed to various abiotic stresses, and here, theRhodobacter (R.) alkalitoleranswere extracted from the alkaline pond. We report the comparative study of the photosynthetic apparatus ofR. alkalitoleransin various light intensities in relation to this bacterium’s high pH tolerance ability. We found that as the light intensity increased, the stability of photosystem complexes decreased in normal pH (npH pH 6.8±0.05) conditions, whereas in high pH (hpH pH 8.6±0.05) acclimation was observed to high light. The content of bacteriochlorophylla, absorbance spectra, and circular dichroism data shows that the integrity of photosystem complexes is less affected in hpH compared to npH conditions. LP-BN of photosystem complexes also shows that LH2 is more affected in npH than hpH, whereas RC-LH1 monomer or dimer has shown interplay between monomer and dimer in hpH although the dimer and monomer both increased in npH. The sucrose density gradient of β-DM solubilized intracytoplasmic membranes, further evidences the pattern of monomer-dimer conversion. Additionally, thin layer chromatographic separation of isolated membrane lipids shows that phosphatidylcholine (PC) levels have increased in hpH conditions which further confirms the integrity of photosystem complexes in hpH conditions. Moreover, qPCR data showed that the subunit -c of ATPase levels was overexpressed in hpH. Consequently, the P515 measurement shows that more ATP production is required in hpH, which dissipates the protons from the chromatophore lumen. This could be the reason the photosystem protein complex destabilized due to more lumen acidification. To maintain homeostasis in hpH, the antiporter NhaD expressed more than in the npH condition.

IMPORTANCE

R. alkalitoleransis an alkaline tolerant species discovered from an alkaline pond in Gujrat India. Being a photoautotrophic photosynthetic organism, it serves as a good model organism to study the photosynthetic apparatus among phototrophic alphaproteobacteria. In nature organisms not only tackle a single abiotic stress but many including temperature, light, salinity, and many other abiotic stresses. Here we investigate how two different abiotic factors light and alkaline conditions modulate the growth and photosynthetic apparatus in a phototrophic alphaproteobacterium,R. alkalitolerans. Our results of this study will give leads in developing alkali-tolerant algae and higher plants.
Genome size evolution and phenotypic correlates in the poison frog family Dendrobatidae
Authors: Douglas Tyler E , Marquez Roberto , Holmes V. Renee D , Johnston J. Spencer , Tarvin Rebecca D
Published: July 2023

Adaptive and neutral processes have produced a spectrum of genome sizes across organisms. Amphibians in particular possess a wide range in C-values, from <1 to over 125 pg. However, the genome size of most amphibians is unknown, and no single family has been comprehensively assessed. We provide new estimates for 32 poison frog species representing the major lineages within Dendrobatidae using Feulgen staining of museum specimens and flow cytometry of fresh tissue. We show that genome size in Dendrobatidae has likely evolved gradually, with potential evolutionary rate shifts in the generaPhyllobatesandHyloxalus, which respectively possess species with the largest (13.0 pg) and second smallest (2.6 pg) genomes in the family. Phylogenetically controlled regression analyses indicate that genome size is positively correlated with snout-vent-length, oocyte number, and clutch size, but negatively correlated with active metabolic rate and metabolic scope. While body size and metabolic rate are also correlates of toxicity, we found no relationship between genome size and evolution of chemical defense within Dendrobatidae. Genome size evolution in Dendrobatidae provides insight into the processes shaping genome size evolution over short timescales and establishes a novel system in which to study the mechanistic links between genome size and organismal physiology.
Efficient Formation of Single-copy Human Artificial Chromosomes
Authors: Gambogi Craig W. , Mer Elie , Brown David M. , Yankson George , Gavade Janardan N. , Logsdon Glennis A. , Heun Patrick , Glass John I. , Black Ben E.
Published: July 2023

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ∼125 bp DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. Here, we describe an approach that efficiently forms single-copy HACs. It employs a ∼750 kb construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells.

One-Sentence Summary

A quarter century after the first human artificial chromosomes, a solution to their uncontrolled multimerization is achieved.
Unconventional Activation of IRE1 Enhances TH17 Responses and Promotes Neutrophilic Airway Inflammation
Authors: Wu Dandan , Zhang Xing , Zimmerly Kourtney M. , Wang Ruoning , Livingston Amanda , Iwawaki Takao , Kumar Ashok , Wu Xiang , Mandell Michael A. , Liu Meilian , Yang Xuexian O.
Published: July 2023

Treatment-refractory severe asthma manifests a neutrophilic phenotype associated with TH17 responses. Heightened unfolded protein responses (UPRs) are associated with the risk of asthma, including severe asthma. However, how UPRs participate in the deregulation of TH17 cells leading to this type of asthma remains elusive. In this study, we investigated the role of the UPR sensor IRE1 in TH17 cell function and neutrophilic airway inflammation. We found that IRE1 is induced in fungal asthma and is highly expressed in TH17 cells relative to naïve CD4+T cells. Cytokine (e.g. IL-23) signals induce the IRE1-XBP1s axis in a JAK2-dependent manner. This noncanonical activation of the IRE1-XBP1s pathway promotes UPRs and cytokine secretion by TH17 cells.Ern1(encoding IRE1)-deficiency decreases the expression of ER stress factors and impairs the differentiation and cytokine secretion of TH17 cells. Genetic ablation ofErn1leads to alleviated TH17 responses and airway neutrophilia in aCandida albicansasthma model. Consistently, IL-23 activates the JAK2-IRE1-XBP1s pathwayin vivoand enhances TH17 responses and neutrophilic infiltration into the airway. Taken together, our data indicate that IRE1, noncanonically activated by cytokine signals, promotes neutrophilic airway inflammation through the UPR- mediated secretory function of TH17 cells. The findings provide a novel insight into the fundamental understanding of IRE1 in TH17-biased TH2-low asthma.
Arf GAP containing dAsap regulates NMJ organization and synaptic calcium through Arf6-dependent signaling inDrosophila
Authors: Mallik Bhagaban , Kushwaha Shikha , Bisht Anjali , Harsha MJ , Frank C. Andrew , Kumar Vimlesh
Published: July 2023

Synaptic morphogenesis involves an interplay of multiple signaling pathways and requires membrane remodeling and cytoskeleton dynamics. We identified the BAR-domain protein dAsap (Arf GAP, SH3, Ankyrin repeat, and PH domain) as one of the regulators of synaptic morphogenesis. Loss of dAsap results in decreased bouton numbers, increased inter-bouton diameter, and disrupted microtubule organization at the nerve terminals. Electrophysiological analysis of the mutants revealed a gain in neurotransmission compared to control neuromuscular junctions (NMJs).dAsapmutant NMJs have increased evoked amplitude, increased spontaneous miniature frequency, and significantly fewer synaptic failures in low calcium. Consistent with these observations,dAsapmutants have increased active zone number. Additional pharmacological and genetic manipulations that are known to impair calcium release from stores suppress the dAsap phenotypes. Finally, we show that expressing a GDP-locked form of Arf6 indAsapmutants restored the NMJ morphological defects, disrupted cytoskeleton, and aberrant neurotransmission. Thus, we propose a model in which dAsap regulates NMJ morphogenesis and synaptic calcium homeostasis through Arf6-dependent neuronal signaling.
Hypoxia blunts angiogenic signaling and upregulates the antioxidant system in elephant seal endothelial cells
Authors: Allen Kaitlin N , Torres-Velarde Julia María , Vazquez Juan Manuel , Moreno-Santillan Diana D , Sudmant Peter H , Vázquez-Medina José Pablo
Published: July 2023

Elephant seals experience extreme hypoxemia during diving bouts. Similar depletions in oxygen availability characterize pathologies including myocardial infarction and ischemic stroke in humans, but seals manage these repeated episodes without injury. However, the real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture system to assess the molecular response to prolonged hypoxia. Seal and human cells exposed to 1% O2for up to 6 h demonstrated differential responses to both acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1α from angiogenic signaling at both the transcriptional and cellular level. Rapid upregulation of genes involved in the glutathione (GSH) metabolism pathway supported maintenance of GSH pools and increases in intracellular succinate in seal but not human cells during hypoxia exposure. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurred in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure. In sum, our studies show that in contrast to human cells, seal cells adapt to hypoxia exposure by dampening angiogenic signaling, increasing antioxidant protection, and maintaining mitochondrial morphological integrity and function.
Binding free-energy landscapes of small molecule binder and non-binder to FMN riboswitch: All-atom molecular dynamics
Authors: Higo Junichi , Bekker Gert-Jan , Kamiya Narutoshi , Fukuda Ikuo , Fukunishi Yoshifumi
Published: July 2023

Binding of a small and flexible molecule, ribocil A (non-binder) or B (binder), to the deep pocket of the aptamer domain of the FMN riboswitch was studied by mD-VcMD, which is a generalized-ensemble method based on molecular dynamics (MD) simulation. Ribocil A and B are structurally similar because they are optical isomers mutually. In the initial conformation of simulation, both ligands and the aptamer were completely dissociated in explicit solvent. The resultant free-energy landscape of ribocil B binding to the aptamer was funnel-like, whereas that of ribocil A was rugged, which agrees qualitatively with an experiment. When entering the gate (named “front gate”) of the pocket, the ligand interacted with the aptamer by native and non-native π-π stackings, and the stackings restrained the molecular orientation of the ligands to be advantageous to reach the binding site smoothly. The simulation showed another pathway, which also led the ligands to the binding site. Its gate (maned “rear gate”) located completely opposite to the front gate on the aptamer’s surface. However, approach from the rear gate required overcoming a free-energy barrier before reaching the binding site, and the ligands should rotate largely and sharply at the free-energy barrier. This ligand’s orientation property is discussed referring to a ligand orientation selection mechanism exserted by a membrane protein capturing its ligand.
Spatial profiling of neuro-immune interactions in gastroenteropancreatic NETs
Authors: Duan Suzann , Sawyer Travis W. , Witten Brandon L. , Song Heyu , Else Tobias , Merchant Juanita L.
Published: July 2023

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are heterogeneous malignancies that arise from complex cellular interactions within the tissue microenvironment. Here, we sought to decipher tumor-derived signals from the surrounding microenvironment by applying Nanostring Digital Spatial Profiling (DSP) to hormone-secreting and non-functional GEP-NETs. DSP was used to evaluate the expression of 40 neural and immune-related proteins in surgically resected duodenal and pancreatic NETs (n=20) primarily comprised of gastrinomas (18/20). A total of 279 regions of interest were examined between tumors, adjacent normal and abnormal-appearing epithelium, and the surrounding stroma. The results were stratified by tissue type andMultiple Endocrine Neoplasia I (MEN1)status and protein expression was validated by immunohistochemical (IHC) staining. A tumor immune cell autonomous inflammatory signature was further evaluated by IHC and RNAscope, while functional pro-inflammatory signaling was confirmed using patient-derived duodenal organoids. Gastrin-secreting and non-functional pancreatic NETs showed a higher abundance of immune cell markers and immune infiltrate compared to duodenal gastrinomas. Tumors displayed strong intra-tissue variation in the expression of neural- and immune-related proteins. Compared to non-MEN1tumors,MEN1gastrinomas showed reduced expression of immune cell markers and upregulated expression of neuropathological proteins. Duodenal gastrinomas showed strong expression of the pro- inflammatory and pro-neural factor IL-17B. Treatment of human duodenal organoids with IL- 17B activated NF-kB and STAT3 signaling and induced the expression of neuroendocrine markers. In conclusion, multiplexed spatial protein analysis identified tissue-specific neuro- immune signatures in GEP-NETs. Moreover, duodenal gastrinomas cell autonomously express immune and pro-inflammatory factors, including tumor-derived IL-17B, that stimulate the neuroendocrine phenotype.
Distinct patterns of connectivity with motor cortex reflect component processes of sensorimotor learning
Authors: Areshenkoff Corson N. , de Brouwer Anouk J. , Gale Daniel J. , Nashed Joseph Y. , Randall Flanagan J. , Smallwood Jonathan , Gallivan Jason P.
Published: July 2023

Sensorimotor learning is supported by multiple competing processes that operate concurrently, making it a challenge to elucidate their neural underpinnings. Here, using human functional MRI, we identify three distinct axes of connectivity between the motor cortex and other brain regions during sensorimotor adaptation. These three axes uniquely correspond to subjects’ degree of implicit learning, performance errors and explicit strategy use, and involve different brain networks situated at increasing levels of the cortical hierarchy. We test the generalizability of these neural axes to a separate form of motor learning known to rely mainly on explicit processes, and show that it is only the Explicit neural axis, composed of higher-order areas in transmodal cortex, that predicts learning in this task. Together, our study uncovers multiple distinct patterns of functional connectivity with motor cortex during sensorimotor adaptation, the component processes that these patterns support, and how they generalize to other forms of motor learning.
Raman micro-spectroscopy reveals the spatial distribution of fumarate in cells and tissues
Authors: Kamp Marlous , Surmacki Jakub , Mondejar Marc Segarra , Young Tim , Chrabaszcz Karolina , Joud Fadwa , Zecchini Vincent , Speed Alyson , Frezza Christian , Bohndiek Sarah E
Published: July 2023

Aberrantly accumulated metabolites such as fumarate elicit intra– and inter-cellular pro-oncogenic cascades, yet current methods to measure them require sample perturbation or disruption and lack spatio-temporal resolution, limiting our ability to fully characterize their function and distribution in cells and within a tissue. Raman spectroscopy (RS) is a powerful bio-analytical tool that directly characterizes the chemical composition of a sample based solely on the optical fingerprint of vibrational modes. Here, we show for the first time that RS can directly detect fumarate in living cellsin vivoand animal tissuesex vivo. Using the observed linear relationship between Raman scattered intensity and fumarate concentration, we demonstrate that RS can distinguish between Fumarate hydratase (Fh1)-deficient and Fh1-proficient cells based on their fumarate concentration. Moreover, RS reveals the spatial compartmentalization of fumarate within cellular organelles: consistent with disruptive methods, in Fh1-deficient cells we observe the highest fumarate concentration (37 ± 19 mM) in the mitochondria, where the TCA cycle operates, followed by the cytoplasm (24 ± 13 mM) and then the nucleus (9 ± 6 mM). Finally, we apply RS to tissues from an inducible mouse model of FH loss in the kidney, demonstrating that RS can accurately classify FH status in these tissues. These results suggest that RS could be adopted as a valuable tool for small molecule metabolic imaging, enablingin situdynamic evaluation of fumarate compartmentalization.
Structural and Biological Evaluations of a Non-Nucleoside STING Agonist Specific for Human STINGA230Variants
Authors: Tang Zhichao , Zhao Junxing , Li Ying , Tomer Shallu , Selvaraju Manikandan , Tien Nicholas , Sun Diyun , Johnson David K. , Zhen Anjie , Li Pingwei , Wang Jingxin
Published: July 2023

Previously we identified a non-nucleotide tricyclic agonist BDW568 that activates human STING (stimulator of interferon genes) gene variant containing A230 in a human monocyte cell line (THP-1). STINGA230alleles, including HAQ and AQ, are less common STING variants in human population. To further characterize the mechanism of BDW568, we obtained the crystal structure of the C-terminal domain of STINGA230complexed with BDW-OH (active metabolite of BDW568) at 1.95 Å resolution and found the planar tricyclic structure in BDW-OH dimerizes in the STING binding pocket and mimics the two nucleobases of the endogenous STING ligand 2’,3’-cGAMP. This binding mode also resembles a known synthetic ligand of human STING, MSA-2, but not another tricyclic mouse STING agonist DMXAA. Structure-activity-relationship (SAR) studies revealed that all three heterocycles in BDW568 and the S-acetate side chain are critical for retaining the compound’s activity. BDW568 could robustly activate the STING pathway in human primary peripheral blood mononuclear cells (PBMCs) with STINGA230genotype from healthy individuals. We also observed BDW568 could robustly activate type I interferon signaling in purified human primary macrophages that were transduced with lentivirus expressing STINGA230, suggesting its potential use to selectively activate genetically engineered macrophages in macrophage-based approaches, such as chimeric antigen receptor (CAR)-macrophage immunotherapies.
Entropy-based decoy generation methods for accurate FDR estimation in large-scale metabolomics annotations
Authors: An Shaowei , Lu Miaoshan , Wang Ruimin , Wang Jinyin , Xie Cong , Tong Junjie , Jiang Hengxuan , Yu Changbin
Published: July 2023

Large-scale metabolomics research faces challenges in accurate metabolite annotation and false discovery rate (FDR) estimation. Recent progress in addressing these challenges has leveraged experience from proteomics and inspiration from other sciences. Although the target-decoy strategy has been applied to metabolomics, generating reliable decoy libraries is difficult due to the complexity of metabolites. Additionally, continuous bioinformatic efforts are necessary to increase the utilization of growing spectra resources while reducing false identifications. Here we introduce the concept of ion entropy and present two entropy-based decoy generation methods. The assessment of public spectral databases using ion entropy validated it as a good metric for ion information content in massive metabolomics data. The decoy generation method developed based on this concept outperformed current representative decoy strategies in metabolomics and achieved the best FDR estimation performance. We analyzed 47 public metabolomics datasets using the constructed workflow to provide instructive suggestions. Finally, we present MetaPhoenix, a tool equipped with a well-constructed FDR estimation workflow that facilitates the development of accurate FDR-controlled analysis in the metabolomics field.
Domain-general and language-specific contributions to speech production in a second language: an fMRI study using functional localizers
Authors: Wolna Agata , Szewczyk Jakub , Diaz Michele , Domagalik Aleksandra , Szwed Marcin , Wodniecka Zofia
Published: July 2023

For bilinguals, speaking in a second language (L2) compared to the native language (L1) is usually more difficult. In this study we asked whether the difficulty in L2 production reflects increased demands imposed on domain-general or core language mechanisms. We compared the brain response to speech production in L1 and L2 within two functionally-defined networks in the brain: the Multiple Demand (MD) network and the language network. We found that speech production in L2 was linked to a widespread increase of brain activity in the domain-general MD network. The language network did not show a similarly robust differences in processing speech in the two languages, however, we found increased response to L2 production in the language-specific portion of the left inferior frontal gyrus (IFG). To further explore our results, we have looked at domain-general and language-specific response within the brain structures postulated to form a Bilingual Language Control (BLC) network. Within this network, we found a robust increase in response to L2 in the domain-general, but also in some language-specific voxels including in the left IFG. Our findings show that L2 production strongly engages domain-general mechanisms, but only affects language sensitive portions of the left IFG. These results put constraints on the current model of bilingual language control by precisely disentangling the domain-general and language-specific contributions to the difficulty in speech production in L2.