bluuebunny/crossref_metadata_2025_split huggingface dataset:
Allosteric inhibition of the SARS-CoV-2 main protease – insights from mass spectrometry-based assays
Authors: El-Baba Tarick J. , Lutomski Corinne A. , Kantsadi Anastassia L. , Malla Tika R. , John Tobias , Mikhailov Victor , Bolla Jani R. , Schofield Christopher J. , Zitzmann Nicole , Vakonakis Ioannis , Robinson Carol V. 
Published: July 2020

AbstractFollowing translation of the SARS-CoV-2 RNA genome into two viral polypeptides, the main protease Mprocleaves at eleven sites to release non-structural proteins required for viral replication. MProis an attractive target for antiviral therapies to combat the coronavirus-2019 disease (COVID-19). Here, we have used native mass spectrometry (MS) to characterize the functional unit of Mpro. Analysis of the monomer-dimer equilibria reveals a dissociation constant ofKd= 0.14 ± 0.03 μM, revealing MProhas a strong preference to dimerize in solution. Developing an MS-based kinetic assay we then characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, which are effectively “flash-frozen” as they transition from solution to the gas phase. We screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the catalytically active dimer, slow the rate of substrate processing by ~35%. This information was readily obtained and, together with analysis of the x-ray crystal structures of these enzyme-small molecule complexes, provides a starting point for the development of more potent molecules that allosterically regulate MProactivity.
Targeted systematic evolution of an RNA platform neutralizing DNMT1 function and controlling DNA methylation
Authors: Esposito CL , Autiero I , Basal MA , Sandomenico A , Ummarino S , Borchiellini M , Ruvo M , Catuogno S , Ebralidze AK , de Franciscis V , Di Ruscio A 
Published: July 2020

AbstractDNA methylation is a fundamental epigenetic modification regulating gene expression. Aberrant DNA methylation is the most common molecular lesion in cancer cells. However, medical intervention has been limited to the use of toxic, unspecific demethylating drugs. Aptamers are novel high affinity targeting ligand molecules. By conjugating the inherent DNMT1 inhibiting capabilities of RNA to an aptamer platform, we generated a first-of-its kind aptamer approach that can target and neutralize DNMT1 function – the aptaDiR. Molecular modelling of RNA-DNMT1 complexes coupled with biochemical and cellular assays enabled the identification and characterization of aptaDiR. This novel RNA bio-drug blocks DNA methylation and impairs cancer cell viability.Collectively, we present an innovative RNA-based approach to modulate DNMT1 activity in cancer or diseases characterized by aberrant DNA methylation and suggest the first alternative strategy to overcome the limitations of currently approved hypomethylating protocols, which will greatly improve clinical intervention on DNA methylation.
Structures of the germline-specific Deadhead and Thioredoxin T proteins fromDrosophila melanogasterreveal unique features among Thioredoxins
Authors: Freier Regina , Aragón Eric , Bagiński Błażej , Pluta Radoslaw , Martin-Malpartida Pau , Ruiz Lidia , Condeminas Miriam , Gonzalez Cayetano , Macias Maria J. 
Published: July 2020

ABSTRACTThioredoxins (Trxs) are ubiquitous enzymes that regulate the redox state in cells. InDrosophila, there are two germline-specific Trxs, Deadhead (Dhd) and TrxT. Both proteins belong to the L(3)mbt malignant brain tumor signature and to the MMS survival network of genes that mediate the cellular response to DNA damage. Dhd is a maternal protein required for early embryogenesis that promotes protamine-histone exchange in fertilized eggs and midblastula transition. TrxT is testis-specific and associates with the lampbrush loops of the Y chromosome.Here we present the first structures of Dhd and TrxT that unveil new features of these Thioredoxins. Dhd is highly positively charged, unusual in canonical Trxs. This positively charged surface can facilitate its approximation to DNA and to protamine oligomers, to promote chromatin remodeling. On the other hand, TrxT contains a C-terminal extension, mostly unstructured and highly flexible, which wraps the conserved core through a closed conformation. This extension partially covers the catalytic site and modulates the redox activity of the protein.The information provided by these structures can guide future work aimed at understanding how redox inputs modulate the initial steps of embryo development inDrosophilaand may help in the design of molecular inhibitors through a structure-based approach.HighlightsWe have determined the first structures of the germline-specific Trxs Dhd and TrxT.Dhd has a highly positively charged surface that facilitates its approximation to DNA and protamine oligomers, to promote chromatin remodeling.TrxT contains a C-terminal extension, highly unusual in canonical Trxs, mostly unstructured and highly flexible.The TrxT C-terminal extension partially covers the catalytic site and modulates the redox activity of the protein.The differences observed in Thioredoxins can help in fine-tuning specific molecules to be active against selected insect species.
An endometrial organoid model ofChlamydia-epithelial and immune cell interactions
Authors: Dolat Lee , Valdivia Raphael H. 
Published: July 2020

ABSTRACTOur understanding of how the obligate intracellular bacteriumChlamydia trachomatisreprograms the cell biology of host cells in the upper genital tract is largely based on observations made in cell culture with transformed epithelial cell lines. Here we describe a primary spherical organoid system derived from endometrial tissue to recapitulate epithelial cell diversity, polarity, and ensuing responses toChlamydiainfection. Using high-resolution and time-lapse microscopy, we catalogue the infection process in organoids from invasion to egress, including the reorganization of the cytoskeleton and positioning of intracellular organelles. We show this model is amenable to screeningC. trachomatismutants for defects in the fusion of pathogenic vacuoles, the recruitment of intracellular organelles, and inhibition of cell death. Moreover, we reconstructed a primary immune cell response by co-culturing infected organoids with neutrophils, and determined that the effector TepP limits the recruitment of neutrophils to infected organoids. Collectively, our model details a system to study the cell biology ofChlamydiainfections in three dimensional structures that better reflect the diversity of cell types and polarity encountered byChlamydiaupon infection of their animal hosts.Summary statement3D endometrial organoids to modelChlamydiainfection and the role of secreted virulence factors in reprogramming host epithelial cells and immune cell recruitment
XLF acts as a flexible connector during non-homologous end joining
Authors: Carney Sean M. , Moreno Andrew T. , Piatt Sadie C. , Cisneros-Aguirre Metztli , Lopezcolorado Felicia Wednesday , Stark Jeremy M. , Loparo Joseph J. 
Published: July 2020

AbstractNon-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.
Identification and characterization of CD4+ T cell epitopes after Shingrix vaccination
Authors: Voic Hannah , de Vries Rory D. , Sidney John , Rubiro Paul , Moore Erin , Phillips Elizabeth , Mallal Simon , Schwan Brittany , Weiskopf Daniela , Sette Alessandro , Grifoni Alba 
Published: July 2020

AbstractInfections with varicella zoster virus (VZV), a member of the Herpesviridae family, are associated with a range of clinical manifestations. Primary infection with VZV causes chicken pox, and due to the virus’s capacity to remain latent in neurons, it can reactivate later in life causing herpes zoster (HZ), also known as shingles. Two different vaccines have been developed to prevent HZ, one based on a live attenuated VZV strain (Zostavax) and the other on adjuvanted gE recombinant protein (Shingrix). While Zostavax efficacy wanes with age, Shingrix protection retains its efficacy in elderly subjects (80 years of age and beyond). In this context, it is of much interest to understand if there is a role for T cell immunity in differential clinical outcome, and if there is a correlate of protection between T cell immunity and Shingrix efficacy. In this study, we characterized Shingrix specific ex vivo CD4 T cell responses in the context of natural exposure and HZ vaccination using pools of predicted epitopes. We show that T cell reactivity following natural infection and Zostavax vaccination dominantly targets non-structural proteins (NS), while Shingrix vaccination redirects dominant reactivity to target gE. We mapped the gE-specific responses following Shingrix vaccination to 89 different gE epitopes, 34 of which accounted for 80% of the response. Using antigen presentation assays and single HLA molecule transfected lines, we experimentally determined HLA restrictions for 94 different donor/peptide combinations. Finally, we used our results as a training set to assess strategies to predict restrictions based on measured or predicted HLA binding and the corresponding HLA types of responding subjects.ImportanceUnderstanding the T cell profile associated with the protection observed in elderly vaccinees following Shingrix vaccination is relevant to the general definition of correlates of vaccine efficacy. Our study enables these future studies by clarifying patterns of immunodominance associated with Shingrix vaccination, as opposed to natural infection or Zostavax vaccination. Identification of epitopes recognized by Shingrix-induced CD4 T cells and their associated HLA restrictions enables the generation of tetrameric staining reagents and, more broadly, the capability to characterize specificity, magnitude and phenotype of VZV specific T cells.
A spatially-tracked single cell transcriptomics map of neuronal networks in the intrinsic cardiac nervous system
Authors: Moss Alison , Robbins Shaina , Achanta Sirisha , Kuttippurathu Lakshmi , Turick Scott , Nieves Sean , Hanna Peter , Smith Elizabeth H. , Hoover Donald B. , Chen Jin , Cheng Zixi (Jack) , Ardell Jeffrey L. , Shivkumar Kalyanam , Schwaber James S. , Vadigepalli Rajanikanth 
Published: July 2020

AbstractWe developed a spatially-tracked single neuron transcriptomics map of an intrinsic cardiac ganglion - the right atrial ganglionic plexus (RAGP) that is a critical mediator of vagal control of the sinoatrial node (SAN) activity. We developed a 3D representation of RAGP with extensive mapping of neurons and used neuronal tracing to identify the spatial distribution of the subset of neurons that project to the SAN. RNAseq of laser capture microdissected neurons revealed a distinct composition of RAGP neurons compared to CNS neuronal subtypes. High-throughput qPCR of hundreds of laser capture microdissected single neurons led to a surprising finding that cholinergic and catecholaminergic neuronal markersThandChatwere correlated, suggesting multipotential phenotypes that can drive neuroplasticity within RAGP. Interestingly, no single gene or module was an exclusive marker of RAGP neuronal connectivity to SAN. Neuropeptide-receptor coexpression analysis revealed a combinatorial paracrine neuromodulatory network within RAGP, informing follow-on studies on the vagal control of RAGP to regulate cardiac function in health and disease.
Non-parametric modelling of temporal and spatial counts data from RNA-seq experiments
Authors: BinTayyash Nuha , Georgaka Sokratia , John ST , Ahmed Sumon , Boukouvalas Alexis , Hensman James , Rattray Magnus 
Published: July 2020

AbstractMotivationThe negative binomial distribution has been shown to be a good model for counts data from both bulk and single-cell RNA-sequencing (RNA-seq). Gaussian process (GP) regression provides a useful non-parametric approach for modeling temporal or spatial changes in gene expression. However, currently available GP regression methods that implement negative binomial likelihood models do not scale to the increasingly large datasets being produced by single-cell and spatial transcriptomics.ResultsThe GPcounts package implements GP regression methods for modelling counts data using a negative binomial likelihood function. Computational efficiency is achieved through the use of variational Bayesian inference. The GP function models changes in the mean of the negative binomial likelihood through a logarithmic link function and the dispersion parameter is fitted by maximum likelihood. We validate the method on simulated time course data, showing that it is better able to identify changes in over-dispersed counts data than methods based on Gaussian or Poisson likelihoods. To demonstrate temporal inference, we apply GPcounts to single-cell RNA-seq datasets after pseudotime and branching inference. To demonstrate spatial inference, we apply GPcounts to data from the mouse olfactory bulb to identify spatially variable genes and compare to two published GP methods. We also provide the option of modelling additional dropout using a zero-inflated negative binomial. Our results show that GPcounts can be used to model temporal and spatial counts data in cases where simpler Gaussian and Poisson likelihoods are unrealistic.AvailabilityGPcounts is implemented using the GPflow library in Python and is available athttps://github.com/ManchesterBioinference/GPcountsalong with the data, code and notebooks required to reproduce the results presented here.Contactnuha.bintayyash@manchester.ac.ukormagnus.rattray@manchester.ac.uk
Polygenic Risk Score for Smoking is associated with Externalizing Psychopathology and Disinhibited Personality Traits but not Internalizing Psychopathology in Adolescence
Authors: Hicks Brian M. , Clark D. Angus , Deak Joseph D. , Liu Mengzhen , Durbin C. Emily , Schaefer Jonathan D. , Wilson Sylia , Iacono William G. , McGue Matt , Vrieze Scott I. 
Published: July 2020

AbstractImportanceLarge consortia of genome wide association studies have yielded more accurate polygenic risk scores (PRS) that aggregate the small effects of many genetic variants to characterize the genetic architecture of disorders and provide a personalized measure of genetic risk.ObjectiveWe examined whether a PRS for smoking measured genetic risk for general behavioral disinhibition by estimating its associations with externalizing and internalizing psychopathology and related personality traits. We examined these associations at multiple time points in adolescence using more refined phenotypes defined by stable characteristics across time and at young ages, which reduced potential confounds associated with cumulative exposure to substances and reverse causality.MethodsRandom intercept panel models were fit to symptoms of conduct disorder, oppositional defiant disorder, major depressive disorder (MDD), and teacher ratings of externalizing and internalizing problems and personality traits at ages 11, 14, and 17 years-old in the Minnesota Twin Family Study (N= 3225).ResultsThe smoking PRS had strong associations with the random intercept factors for all the externalizing measures (mean standardizedβ= .27), agreeableness (β=−.22, 95% CI: −.28, −.16), and conscientiousness (β=−.19, 95% CI: −.24, −.13), but was not significantly associated with the internalizing measures (meanβ= .06) or extraversion (β=.01, 95% CI: −.05, .07). After controlling for smoking at age 17, the associations with the externalizing measures (meanβ= .13) and personality traits related to behavioral control (meanβ= −.10) remained statistically significant.Conclusions and RelevanceThe smoking PRS measures genetic influences that contribute to a spectrum of phenotypes related to behavioral disinhibition including externalizing psychopathology and normal-range personality traits related to behavioral control, but not internalizing psychopathology. Continuing to identify the correlates and delineate the mechanisms of the genetic influences associated with disinhibition could have substantial impact in mitigating a variety of public health problems (e.g., mental health, academic achievement, criminality).Key PointsQuestionDoes a polygenic risk scores (PRS) for smoking measure genetic risk for behavioral disinhibition in general?FindingsThe smoking PRS was associated with externalizing psychopathology and personality traits related to behavioral control, but not internalizing psychopathology and extraversion during adolescence, even after controlling for smoking status.MeaningThe smoking PRS measures genetic influences on behavioral disinhibition in general which is associated with a variety of important outcomes including mental health, academic success, and criminality.
Genetically Determined Strength of Natural Killer Cells is Enhanced by Adaptive HLA class I Admixture in East Asians
Authors: Deng Zhihui , Zhen Jianxin , Harrison Genelle F. , Zhang Guobin , Chen Rui , Sun Ge , Yu Qiong , Nemat-Gorgani Neda , Guethlein Lisbeth A. , He Liumei , Tang Mingzhong , Gao Xiaojiang , Cai Siqi , Shortt Jonathan A. , Gignoux Christopher R. , Carrington Mary , Zou Hongyan , Parham Peter , Hong Wenxu , Norman Paul J. 
Published: July 2020

AbstractHuman natural killer (NK) cells are essential for controlling infection, cancer and fetal development. NK cell functions are modulated by interactions between polymorphic inhibitory killer cell immunoglobulin-like receptors (KIR) and polymorphic HLA-A, -B and -C ligands expressed on tissue cells. AllHLA-Calleles encode a KIR ligand and contribute to reproduction and immunity. In contrast, only someHLA-Aand-Balleles encode KIR ligands and they focus on immunity. By high-resolution analysis ofKIRandHLA-A,-Band-Cgenes, we show that the Chinese Southern Han are significantly enriched for interactions between inhibitory KIR and HLA-A and -B. This enrichment has had substantial input through population admixture with neighboring populations, who contributedHLA class Ihaplotypes expressing the KIR ligands B*46:01 and B*58:01, which subsequently rose to high frequency by natural selection. Consequently, over 80% of Southern HanHLAhaplotypes encode more than one KIR ligand. Complementing the high number of KIR ligands, the Chinese Southern HanKIRlocus combines a high frequency of genes expressing potent inhibitory KIR, with a low frequency of those expressing activating KIR. The Southern Han centromericKIRregion encodes strong, conserved, inhibitory HLA-C specific receptors, and the telomeric region provides a high number and diversity of inhibitory HLA-A and -B specific receptors. In all these characteristics, the Southern Han represent other East Asians, whose NK cell repertoires are thus enhanced in quantity, diversity and effector strength, likely through natural selection for resistance to endemic viral infections.
Structure and assembly of the diiron cofactor in the heme-oxygenase-like domain of theN-nitrosourea-producing enzyme SznF
Authors: McBride Molly J. , Pope Sarah R. , Hu Kai , Slater Jeffrey W. , Okafor C. Denise , Balskus Emily P. , Bollinger J. Martin , Boal Amie K. 
Published: July 2020

AbstractIn biosynthesis of the pancreatic cancer drug streptozotocin, the tri-domain nonheme-iron oxygenase, SznF, hydroxylatesNδandNω’ ofNω-methyl-L-arginine before oxidatively rearranging the triply modified guanidine to theN-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the mono-iron cofactor in the enzyme’s C-terminal cupin domain, which effects the final rearrangement, but exhibited disorder and minimal metal occupancy in the site of the proposed diiron cofactor in theN-hydroxylating heme-oxygenase-like (HO-like) central domain. Here we leverage our recent report of an intensely absorbingµ-peroxodiiron(III/III) intermediate formed from the Fe2(II/II) complex and O2to understand assembly of the diiron cofactor in the HO-like domain and to obtain structures with both SznF iron cofactors bound. Tight binding at one diiron subsite is associated with a conformational change, which is followed by weak binding at the second subsite and rapid capture of O2by the Fe2(II/II) complex. Differences between iron-deficient and iron-replete structures reveal both the conformational change required to form the O2-reactive Fe2(II/II) complex and the structural basis for cofactor instability, showing that a ligand-harboring core helix dynamically refolds during metal acquisition and release. The cofactor also coordinates an unanticipated Glu ligand contributed by an auxiliary helix implicated in substrate binding by docking and molecular dynamics simulation. The additional ligand is conserved in another experimentally validated HO-likeN-oxygenase but not in two known HO-like diiron desaturases. Among ∼9600 sequences identified bioinformatically as belonging to the emerging HO-like diiron protein (HDO) superfamily, ∼25% have this carboxylate residue and are thus tentatively assigned asN-oxygenases.Significance statementThe enzyme SznF assembles theN-nitrosourea pharmacophore of the drug streptozotocin. Its centralN-oxygenase domain resembles heme-oxygenase (HO) and belongs to an emerging superfamily of HO-like diiron enzymes (HDOs) with unstable metallocofactors that have resisted structural characterization. We investigated assembly of the O2-reactive diiron complex from metal-free SznF and Fe(II) and leveraged this insight to obtain the first structure of a functionally assigned HDO with intact cofactor. Conformational changes accompanying cofactor acquisition explain its instability, and the observation of an unanticipated glutamate ligand that is conserved in only a subset of the HDO sequences provides a potential basis for top-level assignment of enzymatic function. Our results thus provide a roadmap for structural and functional characterization of novel HDOs.
DRAXIN regulates interhemispheric fissure remodelling to influence the extent of corpus callosum formation
Authors: Morcom Laura , Edwards Timothy J , Rider Eric , Jones-Davis Dorothy , Lim Jonathan WC , Chen Kok-Siong , Dean Ryan , Bunt Jens , Ye Yunan , Gobius llan , Suárez Rodrigo , Mandelstam Simone , Sherr Elliott H , Richards Linda J 
Published: July 2020

AbstractCorpus callosum dysgenesis (CCD) is a congenital disorder that incorporates either partial or complete absence of the largest cerebral commissure. Remodelling of the interhemispheric fissure (IHF) provides a substrate for callosal axons to cross between hemispheres, and its failure is the main cause of complete CCD. However, it is unclear whether defects in this process could give rise to the heterogeneity of expressivity and phenotypes seen in human cases of CCD. We identify incomplete IHF remodelling as the key structural correlate for the range of callosal abnormalities in inbred and outcrossed BTBR mouse strains, as well as in humans with partial CCD. We identify an eight base pair deletion inDraxinand misregulated astroglial and leptomeningeal proliferation as genetic and cellular factors for variable IHF remodelling and CCD in BTBR acallosal strains. These findings support a model where genetic events determine corpus callosum structure by influencing leptomeningeal-astroglial interactions at the IHF.