bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics
Authors: Acevedo-Rocha Carlos G. , Li Aitao , D’Amore Lorenzo , Hoebenreich Sabrina , Sanchis Joaquin , Lubrano Paul , Ferla Matteo P. , Garcia-Borràs Marc , Osuna Sílvia , Reetz Manfred T. 
Published: April 2020

AbstractMultidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. Yet such efforts are rare and focus only on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report the first multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We developed a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative sign and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and beta-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers lessons for engineering P450s.
PD-1 Specific ‘Blocking’ Antibodies That Deplete PD-1+ T Cells Present An Inconvenient Variable In Pre-clinical Immunotherapy Experiments
Authors: Polesso Fanny , Munks Michael W. , Rott Katherine H. , Smart Savannah , Hill Ann B. , Moran Amy E. 
Published: April 2020

AbstractTherapeutic antibodies blocking PD-1-/PD-L1 interaction have achieved remarkable clinical success in cancer. In addition to blocking a target molecule, some isotypes of antibodies can activate complement, NK cells or phagocytes, resulting in death of the cell expressing the antibody’s target. Human anti-PD-1 therapeutics use antibody isotypes designed to minimize such antibody-dependent lysis. In contrast, anti-PD-1 reagents used in mice are derived from multiple species, with different isotypes, and are not engineered to reduce target cell death: few studies analyze or discuss how antibody species and isotype may impact data interpretation. We demonstrate here that anti-PD-1 therapy to promote activation and proliferation of PD-1-expressing CD8 T cells sometimes led instead to a loss of antigen specific cells. This phenomenon was seen in two tumor models and a model of virus infection, and was varied with the clone of anti-PD-1 antibody. Additionally, we compared competition among anti-PD-1 clones to find a combination that allows detection of PD-1-expressing cells despite the presence of blocking anti-PD1 antibodiesin vivo.These data bring attention to the possibility of unintended target cell depletion with some commonly used anti-mouse PD-1 clones, and should provide a valuable resource for the design and interpretation of anti-PD-1 studies in mice. (200)
The evolution of sex-biased gene expression in theDrosophilabrain
Authors: Khodursky Samuel , Svetec Nicolas , Durkin Sylvia , Zhao Li 
Published: April 2020

AbstractGenes with sex-biased expression inDrosophilaare thought to underlie sexually dimorphic phenotypes and have been shown to possess important evolutionary properties. However, the forces and constraints governing the evolution of sex-biased genes in the somatic tissues ofDrosophilaare largely unknown. Using population-scale RNA sequencing data we show that sex-biased genes in theDrosophilabrain are highly enriched on the X Chromosome and that most are biased in a species-specific manner. We show that X-linked male-biased genes, and to a lesser extent female-biased genes, are enriched for signatures of directional selection at the gene expression level. By examining the evolutionary properties of gene flanking regions on the X Chromosome, we find evidence that adaptive cis-regulatory changes are more likely to drive the expression evolution of X-linked male-biased genes than other X-linked genes. Finally, we examine whether constraint due to broad expression across multiple tissues and genetic constraint due to the largely shared male and female genomes could be responsible for the observed patterns of gene expression evolution. We find that expression breadth does not constrain the directional evolution of gene expression in the brain. Additionally, we find that the shared genome between males and females imposes a substantial constraint on the expression evolution of sex-biased genes. Overall, these results significantly advance our understanding of the patterns and forces shaping the evolution of sexual dimorphism in theDrosophilabrain.
De novo 3D models of SARS-CoV-2 RNA elements and small-molecule-binding RNAs to aid drug discovery
Authors: Rangan Ramya , Watkins Andrew M. , Chacon Jose , Kladwang Wipapat , Zheludev Ivan N. , Townley Jill , Rynge Mats , Thain Gregory , Das Rhiju 
Published: April 2020

AbstractThe rapid spread of COVID-19 is motivating development of antivirals targeting conserved SARS-CoV-2 molecular machinery. The SARS-CoV-2 genome includes conserved RNA elements that offer potential small-molecule drug targets, but most of their 3D structures have not been experimentally characterized. Here, we provide a compilation of chemical mapping data from our and other labs, secondary structure models, and 3D model ensembles based on Rosetta’s FARFAR2 algorithm for SARS-CoV-2 RNA regions including the individual stems SL1-8 in the extended 5’ UTR; the reverse complement of the 5’ UTR SL1-4; the frameshift stimulating element (FSE); and the extended pseudoknot, hypervariable region, and s2m of the 3’ UTR. For eleven of these elements (the stems in SL1-8, reverse complement of SL1-4, FSE, s2m, and 3’ UTR pseudoknot), modeling convergence supports the accuracy of predicted low energy states; subsequent cryo-EM characterization of the FSE confirms modeling accuracy. To aid efforts to discover small molecule RNA binders guided by computational models, we provide a second set of similarly prepared models for RNA riboswitches that bind small molecules. Both datasets (‘FARFAR2-SARS-CoV-2’, https://github.com/DasLab/FARFAR2-SARS-CoV-2; and ‘FARFAR2-Apo-Riboswitch’, at https://github.com/DasLab/FARFAR2-Apo-Riboswitch’) include up to 400 models for each RNA element, which may facilitate drug discovery approaches targeting dynamic ensembles of RNA molecules.
A transcription regulatory network within the ACE2 locus may promote a pro-viral environment for SARS-CoV-2 by modulating expression of host factors
Authors: Fadason Tayaza , Gokuladhas Sreemol , Golovina Evgeniia , Ho Daniel , Farrow Sophie , Nyaga Denis , Pan Hong , Karnani Neerja , Wong Conroy , Cooper Antony , Schierding William , O’Sullivan Justin M. 
Published: April 2020

AbstractIntroductionA novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was recently identified as the pathogen responsible for the COVID-19 outbreak. SARS-CoV-2 triggers severe pneumonia, which leads to acute respiratory distress syndrome and death in severe cases. As reported, SARS-CoV-2 is 80% genetically identical to the 2003 SARS-CoV virus. Angiotensin-converting enzyme 2 (ACE2) has been identified as the main receptor for entry of both SARS-CoV and SARS-CoV-2 into human cells. ACE2 is normally expressed in cardiovascular and lung type II alveolar epithelial cells, where it positively modulates the RAS system that regulates blood flow, pressure, and fluid homeostasis. Thus, virus-induced reduction of ACE2 gene expression is considered to make a significant contribution to severe acute respiratory failure. Chromatin remodeling plays a significant role in the regulation of ACE2 gene expression and the activity of regulatory elements within the genome.MethodsHere, we integrated data on physical chromatin interactions within the genome organization (captured by Hi-C) with tissue-specific gene expression data to identify spatial expression quantitative trait loci (eQTLs) and thus regulatory elements located within the ACE2 gene.ResultsWe identified regulatory elements withinACE2that control the expression of PIR, CA5B, and VPS13C in the lung. The gene products of these genes are involved in inflammatory responses,de novopyrimidine and polyamine synthesis, and the endoplasmic reticulum, respectively.ConclusionOur study, although limited by the fact that the identification of the regulatory interactions is putative until proven by targeted experiments, supports the hypothesis that viral silencing ofACE2alters the activity of gene regulatory regions and promotes an intra-cellular environment suitable for viral replication.
Structures of metabotropic GABABreceptor
Authors: Papasergi-Scott Makaía M. , Robertson Michael J. , Seven Alpay B. , Panova Ouliana , Mathiesen Jesper M. , Skiniotis Georgios 
Published: April 2020

AbstractGABA (γ-aminobutyric acid) stimulation of the metabotropic GABABreceptor results in prolonged inhibition of neurotransmission that is central to brain physiology. GABABbelongs to the Family C of G protein-coupled receptors (GPCRs), which operate as dimers to relay synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins. GABAB, however, is unique in its function as an obligate heterodimer in which agonist binding and G protein activation take place on distinct subunits. Here we show structures of heterodimeric and homodimeric full-length GABABreceptors. Complemented by cellular signaling assays and atomistic simulations, the structures reveal an essential role for the GABABextracellular loop 2 (ECL2) in relaying structural transitions by ordering the linker connecting the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of both GABABsubunits caps and interacts with the hydrophilic head of a phospholipid occupying the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G protein. These results provide a starting framework to decipher mechanistic modes of signal transduction mediated by GABABdimers and have important implications for rational drug design targeting these receptors.
Designing a multi-epitope peptide-based vaccine against SARS-CoV-2
Authors: Singh Abhishek , Thakur Mukesh , Sharma Lalit Kumar , Chandra Kailash 
Published: April 2020

AbstractCOVID-19 pandemic has resulted so far 14,395,16 confirmed cases with 85,711 deaths from the 212 countries, or territories. Due to multifacet issues and challenges in implementation of the safety & preventive measures, inconsistent coordination between societies-governments and most importanly lack of specific vaccine to SARS-CoV-2, the spread of Wuhan originated virus is still uprising after taking a heavy toll on human life. In the present study, we mapped several immunogenic epitopes (B-cell, T-cell, and IFN-gamma) over the entire structural proteins of SARS-CoV-2 and by applying various computational and immunoinformatics approaches, we designed a multi-epitope peptide based vaccine that predicted high immunogenic response in the largest proportion of world’s human population. To ensure high expression of the recombinant vaccine inE. coli, codon optimization and in-silico cloning were also carried out. The designed vaccine with high molecular affinity to TLR3 and TLR4, was found capable to initiate effective innate and adaptive immune response. The immune simulation also suggested uprising high levels of both B-cell and T-cell mediated immunity which on subsequent exposure cleared antigen from the system. The proposed vaccine found promising by yielding desired results and hence, should be tested by practical experimentations for its functioning and efficacy to neutralize SARS-CoV-2.
Comparison of motility of H. pylori in broth and mucin reveals the interplay of effect of acid on bacterium and the rheology of the medium it swims in
Authors: Su C. , Bieniek K. , Liao W. , Constantino M. A. , Decker S. M. , Turner B. S. , Bansil R. 
Published: April 2020

ABSTRACTTo colonize on the gastric epitheliumHelicobacter pyloribacteria have to swim across a gradient of pH from 2-7 in the mucus layer. Previous studies ofH. pylorimotility have shown that at pH below 4 do not swim in porcine gastric mucin (PGM) gels. To separately assess the influence of gelation of PGM and that of pH on motors and pH sensitive receptors ofH. pylori, we used phase contrast microscopy to compare the translational and rotational motion ofH. pyloriin PGMversusBrucella broth (BB10) at different pHs. We observed that decreasing pH leads to decreased fraction of motile swimmers with a decrease in the contribution of fast swimmers to the distributions of swimming speeds and length of trajectories. At all pH’s the bacteria swam faster with longer net displacement over the trajectory in BB10 as compared to PGM. While bacteria are stuck in PGM gels at low pH, they swim at low pH in broth,albeitwith reduced speed. The body rotation rate and estimated cell body torque are weakly dependent on pH in BB10, whereas in PGM the torque increases with increasing viscosity and bacteria stuck in the low pH gel rotate faster than the motile bacteria. Our results show thatH. pylorihas optimal swimming under slightly acidic conditions, and exhibits mechanosensing when stuck in low pH mucin gels.
An Autophagy-Dependent Tubular Lysosomal Network Synchronizes Degradative Activity Required for Muscle Remodeling
Authors: Murakawa Tadayoshi , Kiger Amy A. , Sakamaki Yuriko , Fukuda Mitsunori , Fujita Naonobu 
Published: April 2020

AbstractPreviously, we reported that autophagy is critical for Drosophila muscle remodeling during metamorphosis (Fujita et al., 2017). However, little is known about how lysosomes meet increased degradative demand upon cellular remodeling. Here, we found an extensive tubular autolysosomal network in remodeling muscle. The tubular network transiently appeared and exhibited the capacity to degrade autophagic cargoes. The tubular autolysosomal network was uniquely marked by the autophagic SNARE protein, Syntaxin 17, and its formation depended on both autophagic flux and degradative function, with the exception of the Atg12 and Atg8 ubiquitin-like conjugation systems. AmongATG-deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, we revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling.Impact StatementAnalysis of developmentally-regulated Drosophila muscle remodeling revealed autophagy-dependent formation of an extensive, Syntaxin 17-marked, tubular network that synchronizes the abundant degradative activity across a broad region of the remodeling muscle
Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2
Authors: Williamson Brandi N. , Feldmann Friederike , Schwarz Benjamin , Meade-White Kimberly , Porter Danielle P. , Schulz Jonathan , Doremalen Neeltje van , Leighton Ian , Yinda Claude Kwe , Pérez-Pérez Lizzette , Okumura Atsushi , Lovaglio Jamie , Hanley Patrick W. , Saturday Greg , Bosio Catharine M. , Anzick Sarah , Barbian Kent , Cihlar Tomas , Martens Craig , Scott Dana P. , Munster Vincent J. , Wit Emmie de 
Published: April 2020

AbstractBackgroundEffective therapeutics to treat COVID-19 are urgently needed. Remdesivir is a nucleotide prodrug with in vitro and in vivo efficacy against coronaviruses. Here, we tested the efficacy of remdesivir treatment in a rhesus macaque model of SARS-CoV-2 infection.MethodsTo evaluate the effect of remdesivir treatment on SARS-CoV-2 disease outcome, we used the recently established rhesus macaque model of SARS-CoV-2 infection that results in transient lower respiratory tract disease. Two groups of six rhesus macaques were infected with SARS-CoV-2 and treated with intravenous remdesivir or an equal volume of vehicle solution once daily. Clinical, virological and histological parameters were assessed regularly during the study and at necropsy to determine treatment efficacy.ResultsIn contrast to vehicle-treated animals, animals treated with remdesivir did not show signs of respiratory disease and had reduced pulmonary infiltrates on radiographs. Virus titers in bronchoalveolar lavages were significantly reduced as early as 12hrs after the first treatment was administered. At necropsy on day 7 after inoculation, lung viral loads of remdesivir-treated animals were significantly lower and there was a clear reduction in damage to the lung tissue.ConclusionsTherapeutic remdesivir treatment initiated early during infection has a clear clinical benefit in SARS-CoV-2-infected rhesus macaques. These data support early remdesivir treatment initiation in COVID-19 patients to prevent progression to severe pneumonia.
Human SLC46A2 is the dominant cGAMP importer in extracellular cGAMP-sensing macrophages and monocytes
Authors: Cordova Anthony F. , Ritchie Christopher , Böhnert Volker , Li Lingyin 
Published: April 2020

AbstractAdministration of exogenous CDNs to activate the cGAMP-STING pathway is a promising therapeutic strategy to unleash the full potential of cancer immunotherapy. This strategy mirrors the role of endogenous extracellular cGAMP, an immunotransmitter that is transferred from cancer cells to cGAMP-sensing cells in the host, promoting immunity. However, the CDN import mechanisms used by host cells within tumors remain unknown. Here we identified the protein SLC46A2 as the dominant cGAMP importer in primary human monocytes. Furthermore, we discovered that monocytes and M1-polarized macrophages directly sense tumor-derived extracellular cGAMP in murine tumors. Finally, we demonstrated that SLC46A2 is the dominant cGAMP importer in monocyte-derived macrophages. Together, we provide the first cellular and molecular mechanisms of cGAMP as an immunotransmitter, paving the way for effective STING pathway therapeutics.
A multivalent fuzzy interface drives reversible COPII coat assembly
Authors: Stancheva Viktoriya G. , Hutchings Joshua , Li Xiao-Han , Santhanam Balaji , Babu M. Madan , Zanetti Giulia , Miller Elizabeth A. 
Published: April 2020

AbstractProtein secretion is initiated at the endoplasmic reticulum by the COPII coat, which self-assembles to form vesicles. Here, we examine the mechanisms by which the outer scaffolding layer of the coat drives local assembly of a structure rigid enough to enforce membrane curvature, yet able to readily disassemble at the Golgi. An intrinsically disordered region in the outer coat protein, Sec31, drives binding with an inner coat layer via multiple distinct interfaces. Interactions are individually dispensable but combinatorially reinforce each other, suggesting coat oligomerization is driven by the cumulative effects of multivalent interactions. Such a multimodal assembly platform could be readily reversed at the Golgi via perturbation of each individual interface. These design principles provide an explanation for how cells build a powerful yet transient scaffold to direct vesicle traffic.
Complexity of the eukaryotic dolichol-linked oligosaccharide scramblase suggested by activity correlation profiling mass spectrometry
Authors: Verchère Alice , Cowton Andrew , Jenni Aurelio , Rauch Monika , Häner Robert , Graumann Johannes , Bütikofer Peter , Menon Anant K. 
Published: April 2020

AbstractThe canonical pathway ofN-linked protein glycosylation in yeast and humans involves transfer of the oligosaccharide moiety from the glycolipid Glc3Man9GlcNAc2-PP-dolichol to select asparagine residues in proteins that have been translocated into the lumen of the endoplasmic reticulum (ER). Synthesis of Glc3Man9GlcNAc2-PP-dolichol occurs in two stages, producing first the key intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) on the cytoplasmic face of the ER, followed by translocation of M5-DLO across the ER membrane to the luminal leaflet where the remaining glycosyltransfer reactions occur to complete the structure. Despite its critical importance forN-glycosylation, the scramblase protein that mediates the translocation of M5-DLO across the ER membrane has not been identified. Building on our ability to recapitulate scramblase activity in large unilamellar proteoliposomes reconstituted with a crude mixture of ER membrane proteins, we developed a mass spectrometry-based ‘activity correlation profiling’ approach to identify scramblase candidates in the yeastSaccharomyces cerevisiae. Curation of the activity correlation profiling data prioritized six polytopic ER membrane proteins as scramblase candidates, but reconstitution-based assays and gene disruption in the protistTrypanosoma bruceirevealed, unexpectedly, that none of these proteins were necessary for M5-DLO scramblase activity. Our results instead suggest the possibility that the M5-DLO scramblase may be a protein, or protein complex, whose activity is regulated at the level of quaternary structure. This key insight will aid future attempts to identify the scramblase.
Analysis ofTreponema pallidumstrains from China using improved methods for whole-genome sequencing from primary syphilis chancres
Authors: Chen Wentao , Smajs David , Hu Yongfei , Ke Wujian , Pospíšilová Petra , Hawley Kelly L. , Caimano Melissa J. , Radolf Justin D. , Sena Arlene , Tucker Joseph D. , Yang Bin , Juliano Jonathan J. , Zheng Heping , Parr Jonathan B. 
Published: April 2020

ABSTRACTWhole-genome sequencing (WGS) ofTreponema pallidumsubsp.pallidum(TPA) has been constrained by the lack ofin vitrocultivation methods for isolating spirochetes from patient samples. We built upon recently developed enrichment methods to sequence TPA directly from primary syphilis chancre swabs collected in Guangzhou, China. By combining parallel, pooled whole-genome amplification (ppWGA) with hybrid selection, we generated high quality genomes from four of eight chancre-swab samples and two of two rabbit-passaged isolates, all subjected to challenging storage conditions. This approach enabled the first WGS of Chinese samples without rabbit passage and provided insights into TPA genetic diversity in China.
Single-nucleus RNA-seq and FISH reveal coordinated transcriptional activity in mammalian myofibers
Authors: Santos Matthieu Dos , Backer Stéphanie , Saintpierre Benjamin , Relaix Frederic , Sotiropoulos Athanassia , Maire Pascal 
Published: April 2020

AbstractSkeletal muscle fibers are large syncytia but it is currently unknown whether gene expression is coordinately regulated in their numerous nuclei. By snRNA-seq and snATAC-seq, we showed that slow, fast, myotendinous and neuromuscular junction myonuclei each have different transcriptional programs, associated with distinct chromatin states and combinations of transcription factors. In adult mice, identified myofiber types predominantly express either a slow or one of the three fast isoforms of Myosin heavy chain (MYH) proteins, while a small number of hybrid fibers can express more than one MYH. By snRNA-seq and FISH, we showed that the majority of myonuclei within a myofiber are synchronized, coordinately expressing only one fastMyhisoform with a preferential panel of muscle-specific genes. Importantly, this coordination of expression occurs early during post-natal development and depends on innervation. These findings highlight a unique mechanism of coordination of gene expression in a syncytium.