bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro
Authors: Yadav Ruchi , Courouble Valentine V. , Dey Sanjay K. , Harrison Jerry Joe E.K. , Timm Jennifer , Hopkins Jesse B. , Slack Ryan L. , Sarafianos Stefan G. , Ruiz Francesc X. , Griffin Patrick R. , Arnold Eddy 
Published: May 2022

AbstractSARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic. Its ∼30 kb RNA genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro/nsp5). Polyprotein processing is essential yet incompletely understood. We studied Mpro-mediated processing of the nsp7-10/11 polyprotein, whose mature products are cofactors of the viral replicase, identifying the order of cleavages as: 1) nsp9-10, 2) nsp8-9/nsp10-11, and 3) nsp7-8. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and cross-linking) and X-ray scattering yielded three-dimensional models of the nsp7-10/11 polyprotein. Our data suggest that the nsp7- 10/11 structure in complex with Mpro strongly resembles the unbound polyprotein, and that both polyprotein conformation and junction accessibility determine the preference and order of cleavages. Finally, we used limited proteolysis assays to characterize the effect of a series of inhibitors/binders on Mpro processing of nsp7-11 and Mpro inhibition using a polyprotein substrate.TeaserWe elucidated the structural basis of order of cleavage of SARS-CoV-2 nsp7-11 polyprotein, with implications for Mpro inhibition.
Monitoring for SARS-CoV-2 drug resistance mutations in broad viral populations
Authors: Sedova Mayya , Jaroszewski Lukasz , Iyer Mallika , Godzik Adam 
Published: May 2022

AbstractThe search for drugs against COVID-19 and other diseases caused by coronaviruses focuses on the most conserved and essential proteins, mainly the main (Mpro) and the papain-like (PLpro) proteases and the RNA-dependent RNA polymerase (RdRp). Nirmatrelvir, an inhibitor for Mpro, was recently approved by FDA as a part of a two-drug combination, Paxlovid, and many more drugs are in various stages of development. Multiple candidates for the PLpro inhibitors are being studied, but none have yet progressed to clinical trials. Several repurposed inhibitors of RdRp are already in use. We can expect that once anti-COVID-19 drugs become widely used, resistant variants of SARS-CoV-2 will emerge, and we already see that for the drugs targeting SARS-CoV-2 RdRp. We hypothesize that emergence of such variants can be anticipated by identifying possible escape mutations already present in the existing populations of viruses. Our group previously developed the coronavirus3D server (https://coronavirus3d.org), tracking the evolution of SARS-CoV-2 in the context of the three-dimensional structures of its proteins. Here we introduce dedicated pages tracking the emergence of potential drug resistant mutations to Mpro and PLpro, showing that such mutations are already circulating in the SARS-CoV-2 viral population. With regular updates, the drug resistance tracker provides an easy way to monitor and potentially predict the emergence of drug resistance-conferring mutations in the SARS-CoV-2 virus.
M-Risk: A framework for assessing global fisheries management efficacy of sharks, rays, and chimaeras
Authors: Sherman C. Samantha , Sant Glenn , Simpfendorfer Colin A. , Digel Eric D. , Zubick Patrick , Johnson Grant , Usher Michael , Dulvy Nicholas K. 
Published: May 2022

ABSTRACTFisheries management is essential to guarantee sustainable capture of target species and avoid undesirable declines of incidentally captured species. A key challenge is halting and reversing declines of shark and ray species, and specifically assessing the degree to which management is sufficient to avoid declines in relatively data-poor fisheries. While ecological risk analyses focus on intrinsic ‘productivity’ and extrinsic ‘susceptibility’, one would ideally consider the influence of ‘fisheries management’. Currently, there is no single management evaluation that can be applied to a combination of fishery types at the scale of individual country or Regional Fisheries Management Organisations (RFMOs). Here, we outline a management risk (M-Risk) framework for sharks, rays, and chimaeras used to evaluate species’ risk to overfishing resulting from ineffective management. We illustrate our approach with application to one country (Ecuador) and RFMO (Inter-American Tropical Tuna Commission) and illustrate the variation in scores among species. We found that while both management units assessed had similar overall scores, the scores for individual attributes varied. Ecuador scored higher in reporting-related attributes, while the IATTC scored higher in attributes related to data collection and use. We evaluated whether management of individual species was sufficient for their relative sensitivity by combining the management risk score for each species with their intrinsic sensitivity to determine a final M-Risk score. This framework can be applied to determine which species face the greatest risk of overfishing and be used by fisheries managers to identify effective management policies by replicating regulations from countries with lower risk scores.
Iron regulates contrasting toxicity of uropathogenic Eschericia coli in macrophages and epithelial cells
Authors: Dabral Deepti , Ghosh Hiren , Niwa Masato , Hirayama Tasuku , Boer Rinse de , Vos Marjon de , van den Bogaart Geert 
Published: May 2022

AbstractBy far most urinary tract infections are caused by genetically diverse uropathogenic Escherichia coli (UPEC). Knowledge of the virulence mechanisms of UPEC is critical for drug development, but most studies focus on only a single strain of UPEC. In this study, we compared the virulence mechanisms of four antibiotic-resistant and highly pathogenic UPEC isolates in human blood monocyte-derived macrophages and a bladder epithelial cell (BEC) line: ST999, ST131, ST1981 and ST95. We found that while non-pathogenic E. coli strains are efficiently killed by macrophages in bactericidal single membrane vacuoles, the UPEC strains survive within double-membrane vacuoles. On side-by-side comparison, we found that whereas ST999 only carries Fe3+ importers, ST95 carries both Fe2+ and Fe3+ importers and the toxins haemolysin and colibactin. Moreover, we found that ST999 grows in the Fe3+ rich vacuoles of BECs and macrophages with concomitant increased expression of haem receptor chuA and the hydrogen peroxide sensor oxyR. In contrast, ST95 produces toxins in iron-depleted conditions similar to that of the urinary tract. Whereas ST95 also persist in the iron rich vacuoles of BECs, it produces colibactin in response to low Fe3+ contributing to macrophage death. Thus, iron regulates the contrasting toxicities of UPEC strains in macrophages and bladder epithelial cells due to low and high labile iron concentrations, respectively.Key findingsAntibiotics resistant uropathogenic E. coli strains ST999, ST131, ST1981, and ST95 survive within spacious double membrane vacuoles. Non-pathogenic E. coli strains XL1 blue and MG1655 are cleared in single membrane vacuoles in macrophages.ST999 lacks Fe2+ importer and toxins, and grows in iron rich vacuoles of macrophages and bladder epithelial cells.ST95 carries both Fe2+ and Fe3+ importers and grows in iron low conditions.ST95 expresses toxins and induces cell death of infected macrophages, but not of bladder epithelial cells.Bladder epithelial cells have a higher pool of labile iron than macrophages. Differential expression of virulence factors by ST999 and ST95 in bladder epithelial cells and macrophages is dependent on iron concentration.
Inferring light responses of primate retinal ganglion cells using intrinsic electrical signatures
Authors: Zaidi Moosa , Aggarwal Gorish , Shah Nishal P. , Karniol-Tambour Orren , Goetz Georges , Madugula Sasi , Gogliettino Alex R. , Wu Eric G. , Kling Alexandra , Brackbill Nora , Sher Alexander , Litke Alan M. , Chichilnisky E.J. 
Published: May 2022

SummaryReproducing high-acuity vision with an epiretinal implant will likely require inferring the natural light responses of diverse RGC types in the implanted retina, without measuring them directly. Here we demonstrate an approach that exploits intrinsic electrical features of primate RGCs. First, ON-parasol and OFF-parasol RGCs were identified with 95% accuracy using electrical features. Then, the somatic electrical footprint, predicted cell type, and average linear-nonlinear-Poisson model parameters of each cell type were used to infer a light response model for each cell. Across five retinas, these models achieved an average correlation with measured firing rates of 0.49 for white noise visual stimuli and 0.50 for natural scenes stimuli, compared to 0.65 and 0.58 respectively for models fitted to recorded light responses, an upper bound. This finding, and linear decoding of images from predicted RGC activity, suggested that the inference approach may be useful for high-fidelity sight restoration.
A novel approach to single cell analysis to reveal intrinsic differences in immune marker expression in unstimulated macrophages from BALB/c and C57BL/6 mouse strains
Authors: Breda Jeremie , Banerjee Arka , Jayachandran Rajesh , Pieters Jean , Zavolan Mihaela 
Published: May 2022

AbstractMacrophages are cells of the innate immune system that provide the first line of defense against pathogens. Their functional and morphological heterogeneity is well known, though the origin of this heterogeneity is still debated. Furthermore, while mouse strains differ in the type of immune responses that they mount to individual pathogens, the range of gene expression variation among their macrophages in the absence of a specific stimulus is not known. By applying single cell RNA sequencing we here reveal the gene expression variation in pre-stimulation macrophage populations from specific pathogen-free BALB/c and C57BL/6 mice, two mouse strains that give prototypical Th2 and Th1-biased immune responses, directed towards extracellular or intracellular pathogens, respectively. We show that intrinsic differences between the macrophages of these two strains are detectable before any specific stimulation and we place the gene expression profile of these cells within the range of variation that is measured uponin vitrostimulation with pro-inflammatory lipopolysaccharide (LPS) and interferon γ (IFN), or anti-inflammatory IL-4. We find that C57BL/6 mice show stronger evidence of macrophage polarization than BALB/c mice, which could explain their resistance to pathogens such asLeishmania. Our computational methods for analyzing single cell RNA sequencing data, controlling for common sources of stochastic variation, can be more generally adopted to uncover biological variation between cell populations.
Determination of the structure and dynamics of the fuzzy coat of an amyloid fibril of IAPP using cryo-electron microscopy
Authors: Brotzakis Z. Faidon , Löhr Thomas , Truong Steven , Hoff Samuel E. , Bonomi Massimiliano , Vendruscolo Michele 
Published: May 2022

ABSTRACTIn recent years, major advances in cryo-electron microscopy (cryo-EM) have enabled the routine determination of complex biomolecular structures at atomic resolution. An open challenge for this approach, however, concerns large systems that exhibit continuous dynamics. To address this problem, we developed the metadynamic electron-microscopy metainference (MEMMI) method, which incorporates metadynamics, an enhanced conformational sampling approach, into the metainference method of integrative structural biology. MEMMI enables the simultaneous determination of the structure and dynamics of large heterogeneous systems by combining cryo-EM density maps with prior information through molecular dynamics, while at the same time modelling the different sources of error. To illustrate the method, we apply it to elucidate the dynamics of an amyloid fibril of the islet amyloid polypeptide (IAPP). The resulting conformational ensemble provides an accurate description of the structural variability of the disordered region of the amyloid fibril, known as fuzzy coat. The conformational ensemble also reveals that in nearly half of the structural core of this amyloid fibril the side-chains exhibit liquid-like dynamics despite the presence of the highly ordered network backbone of hydrogen bonds characteristic of the cross-β structure of amyloid fibrils.
HMMerge: an Ensemble Method for Improving Multiple Sequence Alignment
Authors: Park Minhyuk , Warnow Tandy 
Published: May 2022

AbstractDespite advances in method development for multiple sequence alignment over the last several decades, the alignment of datasets exhibiting substantial sequence length heterogeneity, especially when the input sequences include very short sequences (either as a result of sequencing technologies or of large deletions during evolution) remains an inadequately solved problem. We present HMMerge, a method to compute an alignment of datasets exhibiting high sequence length heterogeneity, or to add short sequences into a given “backbone” alignment. HMMerge builds on the technique from its predecessor alignment methods, UPP and WITCH, which build an ensemble of HMMs for the backbone alignment and add the remaining sequences into the backbone alignment using the ensemble. HMMerge differs from UPP and WITCH by building a new HMM for each query sequence: it uses a novel ensemble approach to combine the HMMs, each weighted by the probability of generating the query sequence, into a single HMM. Then it applies the Viterbi algorithm to add the query sequence into the backbone alignment. We show that using this “merged” HMM provides better accuracy than the current approach in UPP and matches or improves on WITCH for adding short sequences into backbone alignments. HMMerge is freely available at https://github.com/MinhyukPark/HMMerge.
Huddling nest use are the optimal strategies for heat conservation in a social marsupial: lessons from biophysical models
Authors: Nespolo Roberto F. , Peña Isabella , Mejías Carlos , Ñunque Abel , Altamirano Tomás , Bozinovic Francisco 
Published: May 2022

AbstractEndothermy, understood as the maintenance of continuous and high body temperatures (TB) due to the combination of metabolic heat production and an insulative cover, is severely challenged in small endotherms of cold environments. As a response, social clustering, and nest use (collectively, “communal nesting”) are common strategies for heat conservation in small mammals and birds. To quantify the actual amount of energy that is saved by communal nesting, we studied the social marsupialDromiciops gliroides(monito del monte), a relict marsupial species from the cold forests of southern South America. It is hypothesized that sociability in this marsupial was driven by the cold, for which we calculated the energetic benefits that communal nesting confers. Using biophysical models and experimental coolings, we simulated heat exchanges experienced by grouped or solitary individuals, and also individuals within nests, collected from the field. Assuming a model of passive cooling, we calculated the net energy cost of euthermic maintenance (Ecost: the total energy needed to maintain euthermia). We adjusted 50 cooling curves, to exponential decay models, and found in all cases that the strategy minimizing heat loss is to be clustered within a nest, for which the Ecostwas the minimum. This was significantly lower than the clustered condition, outside the nest, a reduction that represents almost half of energy consumption per day in a resting, thermoneutral condition for this marsupial. Overall, our results suggest that the strategy that significantly maximized heat conservation, compared with alternative strategies, was communal nesting. These findings support the idea that, in this social mammal, sociality is driven by bioenergetic benefits.
Ablation of SYK kinase from primary human Natural Killer cells via CRISPR/Cas9 enhances cytotoxicity and cytokine production
Authors: Dahlvang James D. , Dick Jenna K. , Sangala Jules A. , Pomeroy Emily J. , Snyder Kristin M. , Moushon Juliette M. , Thefaine Claire E. , Wu Jianming , Hamilton Sara E. , Felices Martin , Miller Jeffrey S. , Walcheck Bruce , Webber Beau R. , Moriarity Branden S. , Hart Geoffrey T. 
Published: May 2022

AbstractCytomegalovirus (CMV) infection alters natural killer (NK) cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the Fc receptor γ chain (Gene:FCER1G, FcRγ), PLZF, and SYK. Functionally, adaptive NK cells display enhanced antibody-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives cytotoxicity and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. ADCC by human NK cells is exclusively mediated by the CD16A (FcγRIIIA) signaling apparatus, which includes FcRγ, CD3ζ, SYK, SHP-1, ZAP-70, and the transcription factor PLZF. We ablated the genes encoding these molecules and tested subsequent ADCC and cytokine production. We found that ablating the FcRγ chain caused a modest increase in TNFα production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced both cytotoxicity and cytokine production, while ZAP-70 kinase ablation diminished function. Ablation of the phosphatase SHP-1 resulted in mixed effects on function, with NK cells demonstrating enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcRγ or PLZF. The lack of SYK expression may limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production. In addition to providing mechanistic answers about CMV-induced adaptive NK cell functionality, our results indicate that NK chimeric antigen receptor (CAR) therapeutics that invoke ADCC signaling molecules (e.g., CD3ζ chain) may benefit from ablating SYK, while maintaining ZAP-70, to increase functionality.
Correlations across timing cues in natural vocalizations predict biases in judging synthetic sound burst durations
Authors: Jané Matthew , Pisupati Sashank , Smith Kasey E. , Castro-Tonelli Luan , Melo-Thomas Liana , Schwarting Rainer K.W. , Wohr Markus , Chrobak James J. , Read Heather L. 
Published: May 2022

AbstractIt is well known that animals rely on multiple sources of information in order to successfully identify sounds in natural environments, to make decisions that are optimal for their survival. For example, rats use duration and pitch cues to respond appropriately to prosocial and distress vocalizations (Saito et al., 2019). Vocalization duration cues are known to co-vary with other temporal cues (Khatami et al., 2018), yet little is known about whether animals rely upon such co-variations to successfully discriminate sounds. In the current study, we find natural alarm vocalizations in rats have onset and offset slopes that are correlated with their duration. Accordingly, vocalizations with faster onset slopes are more likely to have shorter durations. Given that vocalization slopes begin and end within milliseconds, they could provide rapid perceptual cues for predicting and discriminating vocalization duration. To examine this possibility, we train rodents to discriminate duration differences in sequences of synthetic vocalizations and examine how artificially changing the slope impacts duration judgments. We find animals are biased to misjudge a range of synthetic vocalizations as being shorter in duration when the onset and offset slopes are artificially fast. Moreover, this bias is reduced when rats are exposed to multiple synthetic vocalization bursts. The observed perceptual bias is accurately captured by a Bayesian decision-theoretic model that utilizes the empirical joint distribution of duration and onset slopes in natural vocalizations as a prior during duration judgements of synthetic vocalizations. This model also explains why the bias is reduced when more evidence is accumulated across multiple bursts, reducing the prior’s influence. These results support the theory that animals are sensitive to fine-grained statistical co-variations in auditory timing cues and integrate this information optimally with incoming sensory evidence to guide their decisions.
Developmental transitions coordinate assembly of the Coxiella burnetii Dot/Icm type IV secretion system
Authors: Park Donghyun , Steiner Samuel , Shao Meng , Roy Craig R. , Liu Jun 
Published: May 2022

ABSTRACTCoxiella burnetii is an obligate intracellular bacterial pathogen that has evolved a unique bi-phasic developmental cycle. The infectious form of C. burnetii is the dormant small cell variant (SCV) that transitions to a metabolically active large cell variant (LCV) that replicates inside the lysosome-derived host vacuole. A Dot/Icm type IV secretion system (T4SS), which can deliver over 100 effector proteins to host cells, is essential for the biogenesis of the vacuole and for intracellular replication. How the distinct C. burnetii life cycle impacts the assembly and function of the Dot/Icm T4SS has remained unknown. Here, we combine advanced cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) imaging to visualize all developmental transitions and assembly of the Dot/Icm T4SS. Importantly, assembled Dot/Icm T4SSs were not present in the infectious SCV. The appearance of an assembled Dot/Icm machine correlated with the transition of the SCV to the LCV intracellularly. Furthermore, temporal characterization of C. burnetii morphological changes revealed regions of the inner membrane that invaginated to form tightly packed stacks during the LCV to SCV transition at late stages of infection, which could enable the SCV to LCV transition that occurs upon infection of a new host cell. Overall, these data establish how C. burnetii developmental transitions control critical bacterial processes to promote intracellular replication and transmission.
Plasticity-induced actin polymerization in the dendritic shaft regulates intracellular AMPA receptor trafficking
Authors: Wong V. C. , Houlihan P.R. , Liu H. , Walpita D. , DeSantis M.C. , Liu Z. , O’Shea E. K. 
Published: May 2022

AbstractAMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing long-term potentiation (LTP) to increase synaptic transmission, but how AMPAR-containing vesicles are selectively trafficked to these synapses during LTP is not known. Here we developed a strategy to label AMPAR GluA1 subunits expressed from the endogenous loci of rat hippocampal neurons such that the motion of GluA1-containing vesicles in time-lapse sequences can be characterized using single-particle tracking and mathematical modeling. We find that GluA1- containing vesicles are confined and concentrated near sites of stimulation-induced plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F- actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of neuronal activity.