bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural insights into ligand recognition and activation of the melanocortin-4 receptor
Authors: Zhang Huibing , Chen Li-Nan , Yang Dehua , Mao Chunyou , Shen Qingya , Feng Wenbo , Shen Dan-Dan , Dai Antao , Xie Shanshan , Zhou Yan , Qin Jiao , Sun Jinpeng , Scharf Daniel H. , Hou Tingjun , Zhou Tianhua , Wang Ming-Wei , Zhang Yan 
Published: June 2021

SUMMARYMelanocortin-4 receptor (MC4R) plays a central role in the regulation of energy homeostasis. Its high sequence similarity to other MC receptor family members, low agonist selectivity and the lack of structural information concerning receptor activation have hampered the development of MC4R-seletive therapeutics to treat obesity. Here, we report four high-resolution structures of full-length MC4R in complex with the heterotrimeric Gs protein stimulated by the endogenous peptide α-MSH, FDA-approved drugs afamelanotide (Scenesse™) and bremelanotide (Vyleesi™), and selective small-molecule ligand THIQ, respectively. Together with pharmacological studies, our results reveal the conserved binding mode of peptidic agonists, the distinctive molecular details of small-molecule agonist recognition underlying receptor subtype selectivity, and distinct activation mechanism for MC4R, thereby offering new insights into G protein coupling. Our work may facilitate the discovery of selective therapeutic agents targeting MC4R.
High-Resolution Genomic Profiling of Carbapenem-Resistant Klebsiella pneumoniae Isolates: A Multicentric Retrospective Indian Study
Authors: Nagaraj Geetha , Shamanna Varun , Govindan Vandana , Rose Steffimole , Sravani D. , Akshata K. P. , Shincy M.R. , Venkatesha V.T. , Ravikumar K. L. , Abrudan Monica , Argimón Silvia , Kekre Mihir , Underwood Anthony , Aanensen David M 
Published: June 2021

ABSTRACTsummaryWe report insights into genome sequences of Indian K. pneumoniae isolates, highlighting the presence of high-risk international clones and genetic pools different from those predominating in other regions. Identification of multidrug-resistant and hypervirulent K. pneumoniae elicits public health concerns.BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is a threat to public health in India due to its high dissemination, mortality, and limited treatment options. Its genomic variability is reflected in the diversity of sequence types, virulence factors, and antimicrobial resistance (AMR) mechanisms. This study aims to characterize the clonal relationships and genetic mechanisms of resistance and virulence in CRKP isolates in India.Materials and MethodsWe characterized 344 retrospective K. pneumoniae clinical isolates collected from 8 centers across India collected in 2013-2019. Susceptibility to antibiotics was tested with VITEK 2. Capsular types, MLST, virulence genes, AMR determinants, plasmid replicon types, and a single-nucleotide polymorphism (SNP) phylogeny were inferred from their whole genome sequences.ResultsPhylogenetic analysis of the 325 Klebsiella isolates that passed QC revealed 3 groups: K. pneumoniae sensu stricto (n=307), K. quasipneumoniae (n=17), and K. varicolla (n=1). Sequencing and capsular diversity analysis of the 307 K. pneumoniae sensu stricto isolates revealed 28 sequence types, 26 K-locus types, and 11 O-locus types, with ST231, KL51, and O1V2 being predominant. blaOXA-48-like and blaNDM-1/5 were present in 73.2% and 24.4% of isolates respectively. The major plasmid replicon types associated with carbapenase genes were IncF (51.0%), and Col group (35.0%).ConclusionOur study documents for the first time the genetic diversity of K- and O-antigens circulating in India. The results demonstrate the practical applicability of genomic surveillance and its utility in tracking the population dynamics of CRKP. It alerts us to the urgency for longitudinal surveillance of these virulent and transmissible lineages.FundingThis work was supported by Official Development Assistance (ODA) funding from the National Institute of Health Research [grant number 16_136_111].This research was commissioned by the National Institute of Health Research using Official Development Assistance (ODA) funding. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.Conflict of InterestThe authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.
An Empirical Energy Landscape Reveals Mechanism of Proteasome in Polypeptide Translocation
Authors: Fang Rui , Hon Jason , Zhou Mengying , Lu Ying 
Published: June 2021

AbstractThe ring-like ATPase complexes in the AAA+ family perform diverse cellular functions that require coordination between the conformational transitions of their individual ATPase subunits1,2. How the energy from ATP hydrolysis is captured to perform mechanical work by these coordinated movements is not known. In this study, we developed a novel approach for delineating the nucleotide-dependent free-energy landscape (FEL) of the proteasome’s heterohexameric ATPase complex based on complementary structural and kinetic measurements. We used the FEL to simulate the dynamics of the proteasome and quantitatively evaluated the predicted structural and kinetic properties. The FEL model predictions were widely consistent with experimental observations in this and previous studies and suggested novel features of the mechanism of proteasomal ATPase. We find that the cooperative movements of the ATPase subunits result from the design of the ATPase hexamer entailing a unique free-energy minimum for each nucleotide-binding state. ATP hydrolysis dictates the direction of substrate translocation by triggering an energy-dissipating conformational transition of the ATPase complex.
The functional properties of synapses made by regenerated axons across spinal cord lesion sites
Authors: Parker David 
Published: June 2021

AbstractWhile the anatomical properties of regenerated axons across spinal cord lesion sites have been studied extensively, little is known of how the functional properties of regenerated synapses compare to those in unlesioned animals. This comparison has been performed here in the lamprey, a model system for spinal injury research, in which functional locomotor recovery after spinal cord lesions is associated with axonal regeneration across the lesion site.Regenerated synapses below the lesion site did not differ to synapses from unlesioned axons with respect to the amplitude and duration of single excitatory postsynaptic potentials (EPSPs). They also showed the same activity-dependent depression over spike trains. However, regenerated synapses did differ to unlesioned synapses as the estimated number of synaptic vesicles was greater and there was evidence for an increased postsynaptic quantal amplitude. For axons above the lesion site, the amplitude and duration of single synaptic inputs also did not differ significantly to unlesioned animals. However, in this case there was evidence of a reduction in release probability and inputs facilitated rather that depressed over spike trains.Synaptic inputs from single regenerated axons below the lesion site thus do not increase in amplitude to compensate for the reduced number of descending axons after functional recovery. However, the postsynaptic input is maintained at the unlesioned level using different synaptic properties. Conversely, the facilitation from the same initial amplitude above the lesion site will make the synaptic input over spike trains functionally stronger. This may help to increase propriospinal activity across the lesion site to compensate for the lesion-induced reduction in supraspinal inputs.
Complexity of Genomic Epidemiology of Carbapenem-Resistant Klebsiella pneumoniae Isolates in Colombia Urges the Reinforcement of Whole Genome Sequencing-Based Surveillance Programs
Authors: Saavedra Sandra Yamile , Bernal Johan Fabian , Montilla-Escudero Efraín , Arévalo Stefany Alejandra , Prada Diego Andrés , Valencia María Fernanda , Moreno Jaime Enrique , Hidalgo Andrea Melissa , Abrudan Monica , Argimón Silvia , Kekre Mihir , Underwood Anthony , Aanensen David M , Duarte Carolina , Donado-Godoy Pilar ,   the NIHR Global Health Research Unit on Genomic Surveillance of Antimicrobial Resistance
Published: June 2021

ABSTRACTBackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is an emerging public health problem. This study explores the specifics of CRKP epidemiology in Colombia based on whole genome sequencing (WGS) of the National Reference Laboratory at Instituto Nacional de Salud (INS)’s 2013-2017 sample collection.MethodsA total of 425 CRKP isolates from 21 departments were analyzed by HiSeq-X10®Illumina high-throughput sequencing. Bioinformatic analysis was performed, primarily using the pipelines developed collaboratively by the National Institute for Health Research Global Health Research Unit (GHRU) on Genomic Surveillance of AMR, and AGROSAVIA.ResultsOf the 425 CRKP isolates, 91.5% were carbapenemase-producing strains. The data support a recent expansion and the endemicity of CRKP in Colombia with the circulation of 7 high-risk clones, the most frequent being CG258 (48.39% of isolates). We identified genes encoding carbapenemases blaKPC-3, blaKPC-2, blaNDM-1, blaNDM-9, blaVIM-2, blaVIM-4, and blaVIM-24, and various mobile genetic elements (MGE). The virulence of CRKP isolates was low, but colibactin (clb3) was present in 25.2% of isolates, and a hypervirulent CRKP clone (CG380) was reported for the first time in Colombia. ST258, ST512, and ST4851 were characterized by low levels of diversity in the core genome (ANI> 99.9%).ConclusionsThe study outlines complex CRKP epidemiology in Colombia. CG258 expanded clonally and carries specific carbapenemases in specific MGEs, while the other high-risk clones (CG147, CG307, and CG152) present a more diverse complement of carbapenemases. The specifics of the Colombian situation stress the importance of WGS-based surveillance to monitor evolutionary trends of STs, MGE, and resistance and virulence genes.summaryIn Colombia, the dissemination of carbapenemases in carbapenem-resistant Klebsiella pneumoniae is attributed to horizontal gene transfer and successful circulation of CG258, and, to a lesser extent, other clones such as ST307, ST147, and ST152.
EXC-4/CLIC, Gα, and Rho/Rac signaling regulate tubulogenesis in C. elegans
Authors: Arena Anthony F. , Shaye Daniel D. 
Published: June 2021

ABSTRACTThe Rho-family of small GTPases, which play crucial roles in development and disease, are regulated by many signal-transduction cascades, including G-protein-coupled receptor (GPCR)-heterotrimeric G-protein (Gα/β/γ) pathways. Using genetic approaches in C. elegans we identified a new role for Gα and Rho/Rac signaling in cell outgrowth during tubulogenesis and show that the Chloride Intracellular Channel (CLIC) protein EXC-4 is an evolutionarily-conserved player in this pathway. The gene exc-4 was identified by its role in tubulogenesis of the excretory canal (ExCa) cell—a unicellular tube required for osmoregulation and fluid clearance. We identified an exc-4 loss-of-function allele that affects an evolutionarily conserved residue in the C-terminus. Using this mutant we identified genetic interactions between exc-4, Gα, and Rho-family GTPases, defining novel roles for Gα-encoding genes (gpa-12/Gα12/13, gpa-7/Gαi, egl-30/Gαq, gsa-1/Gαs) and the Rho-family members ced-10/Rac and mig-2/RhoG in ExCa outgrowth. EXC-4 and human CLICs have conserved functions in tubulogenesis, and CLICs and Gα-Rho/Rac signaling regulate tubulogenesis during blood vessel development. Therefore, our work defines a primordial role for EXC-4/CLICs in Gα-Rho/Rac-signaling during tubulogenesis.One Sentence SummaryGα and Rho/Rac signaling regulates EXC-4/CLIC-mediated cell outgrowth during tubulogenesis in C. elegans, linking elements of G-protein signaling to the enigmatic CLIC family of proteins.
Transcriptomic Analysis of the Innate Immune Response toin vitroTransfection of Plasmid DNA
Authors: Warga Eric , Tucker Matthew , Harris Emily , Elmer Jacob 
Published: June 2021

ABSTRACTThe innate immune response to cytosolic DNA is intended to protect the host from viral infections, but it can also inhibit the delivery and expression of therapeutic transgenes in gene and cell therapies. The goal of this work was to use mRNA-sequencing to reveal correlations between the transfection efficiencies of four cell types (PC-3, Jurkat, HEK-293T, and primary CD3+T cells) and their innate immune responses to nonviral gene delivery. Overall, the highest transfection efficiency was observed in HEK-293T cells (87%), which upregulated only 142 genes with no known anti-viral functions. Lipofection upregulated a much larger number (n = 1,057) of cytokine-stimulated genes (CSGs) in PC-3 cells, which also exhibited a significantly lower transfection efficiency. However, the addition of serum during Lipofection and electroporation significantly increased transfection efficiencies and decreased the number of upregulated genes in PC-3 cells. Finally, while Lipofection of Jurkat and Primary T cells only upregulated a few genes, several anti-viral CSGs that were absent in HEK and upregulated in PC-3 cells were observed to be constitutively expressed in T cells, which may explain their relatively low Lipofection efficiencies (8-21%). Indeed, overexpression of one such CSG (IFI16) significantly decreased transfection efficiency in HEK cells to 33%.
Nucleotide Dynamics During Flossing of Polycation-DNA-Polycation through a Nanopore using Molecular Dynamics
Authors: Mahalik Jyoti P. , Muthukumar Murugappan 
Published: June 2021

AbstractThe full potential of solid state nanopores is yet to be realized for genome sequencing. Due to its robustness it can handle strong voltage amplitude and frequency. The effect of strong alternating voltage on the dynamics of nucleotides during translocation has been explored. We proposed a setup consisting of single stranded DNA covalently linked with symmetric polycations at both ends fashioned after the proposal of Kasianowicz. 1 Such a setup allows for repeated back and forth motion of the DNA along the nanopore (1.45 nm diameter and 1.53 nm thick) by simply switching the voltage polarity if the polycation tail is sufficiently long (≥ 10) and the applied voltage is below 0.72 volts, but the average residence time of the nucleotides are too small to be of any practical use (6-30 ns). When alternating voltage of higher frequency is applied, it enhances the average residence time of the nucleotides by an order of magnitude to ∼ 0.1 µs relative to direct voltage but the individual trajectories are too stochastic. Since, we are able to collect repeated read on the dynamics of individual nucleotides, we obtained the most probable time of appearance of a nucleotide within the nanopore. With such construct we were able to get almost linear dependence of most probable time versus nucleotide index, after gaussian fitting.
Allosteric activation of CwlD amidase activity by the GerS lipoprotein during Clostridioides difficile spore formation
Authors: Feliciano Carolina Alves , Eckenroth Brian E. , Diaz Oscar R. , Doublié Sylvie , Shen Aimee 
Published: June 2021

AbstractSpore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. Spore germination depends on the degradation of the protective spore peptidoglycan layer known as the spore cortex. Cortex degradation is mediated by enzymes that recognize the spore-specific peptidoglycan modification, muramic-∂-lactam (MAL). In C. difficile, MAL synthesis depends on the activity of the CwlD amidase and the GerS lipoprotein, which directly binds CwlD. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind zinc stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to zinc, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of zinc co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.
TedSim: temporal dynamics simulation of single cell RNA-sequencing data and cell division history
Authors: Pan Xinhai , Li Hechen , Zhang Xiuwei 
Published: June 2021

AbstractRecently, the combined scRNA-seq and CRISPR/Cas9 genome editing technologies have enabled simultaneous readouts of gene expressions and lineage barcodes, which allows for the reconstruction of the cell division tree, and makes it possible to trace the origin of each cell type. Computational methods are emerging to take advantage of the jointly profiled scRNA-seq and lineage barcode data to better reconstruct the cell division history or to infer the cell state trajectories.Here, we present TedSim (single cell Temporal dynamics Simulator), a simulator that simulates the cell division events from the root cell to present-day cells, simultaneously generating the lineage barcodes and scRNA-seq data. In particular, TedSim generates cells from multiple cell types through cell division events. TedSim can be used to benchmark and investigate computational methods which use either or both of the two types of data, scRNA-seq and lineage barcodes, to study cell lineages, ancestral cells or cell trajectories. TedSim is available at: https://github.com/Galaxeee/TedSim.
Deamidation of the human eye lens protein γS-crystallin accelerates oxidative aging
Authors: Norton-Baker Brenna , Mehrabi Pedram , Kwok Ashley O. , Roskamp Kyle W. , Sprague-Piercy Marc A. , Stetten David von , Miller R.J. Dwayne , Martin Rachel W. 
Published: June 2021

ABSTRACTCataract disease, a clouding of the eye lens due to precipitation of lens proteins, affects millions of people every year worldwide. The proteins that comprise the lens, the crystallins, show extensive post-translational modifications (PTMs) in aged and cataractous lenses, most commonly deamidation and oxidation. Although surface-exposed glutamines and asparagines show the highest rates of deamidation, multiple modifications can accumulate over time in these long-lived proteins, even for buried residues. Both deamidation and oxidation have been shown to promote crystallin aggregation in vitro; however, it is not clear precisely how these modified crystallins contribute to insolubilization. Here, we report six novel crystal structures of a major human lens protein, γS-crystallin (γS): one of the wild-type in a monomeric state, and five of deamidated γS variants, ranging from three to nine deamidation sites, after varying degrees of sample aging. Consistent with previous work that focused on single-to triple-site deamidation, the deamidation mutations do not appear to drastically change the fold of γS; however, increasing deamidation leads to accelerated oxidation and disulfide bond formation. Successive addition of deamidated sites progressively destabilized protein structure as evaluated by differential scanning fluorimetry. Light scattering showed the deamidated variants display an increased propensity for aggregation compared to the wild-type protein. The results suggest the deamidated variants are useful as models for accelerated aging; the structural changes observed over time provide support for redox activity of γS-crystallin in the human lens.HighlightsNovel structures of cataract-associated variants of human eye lens protein γS-crystallin reportedIncreasing deamidation of γS-crystallin decreases stability and affects aggregation propensityOverall fold of γS-crystallin maintained among deamidated and disulfide-bonded variantsDeamidated γS variants form disulfide bonds more rapidly than wild-type γSPotential functional advantage of disulfide bonding in the CXCXC motif proposed
Role of collagen-like protein BclA3 in the assembly of the exosporium layer of Clostridioides difficile spores
Authors: Pizarro-Guajardo Marjorie , Ortega-Lizárraga César , Cid-Rojas Francisca , Inostroza Ana , Paredes-Sabja Daniel 
Published: June 2021

AbstractNewly formed spores are essential for persistence of C. difficile in the host, transmission to a new susceptible host (Deakin et al., 2012b) and recurrence of CDI. BclA3 and BclA2 Spore surface proteins are expressed during sporulation under the control of mother-cell specific sigma factors of the RNA polymerase, SigE and SigK. Deletion of bclA3 leads to spores with an electron-dense exosporium layer that lacks bump-like structures in the electron-dense layer and hair-like projections, both structures typically found in the wild type spore. Therefore, in this work, we have addressed the role of the exosporium collagen-like BclA3 glycoprotein in the assembly of the exosporium layer. Immunogold labelling of BclA2CTD and BclA3CTD indicates that both proteins are located in the hairs, with BclA2 located outermost of BclA3. Absence of BclA3 leads to spores with no hair-like projections, and absence of bumps in thick exosporium spores, a phenotype also expressed in by the deletion of the collagen-like region of BclA3. Overall, these results provide insights into the role of BclA3 in the assembly of the exosporium layer of C. difficile spores.
Unfolding of RNA via Translocation Through a Nanopore
Authors: Chalise Sadhana , Muthukumar Murugappan 
Published: June 2021

AbstractRNA unfolding and refolding are important biological phenomena, which occur during the transfer of genetic information from DNA to RNA to proteins. During these processes, RNA is found in single stranded, secondary and tertiary structures, including secondary conformations like hairpins and pseudoknots. Understanding the diverse conformations of RNA and how these influence the dynamics of unfolding and refolding is crucial to gain insight to fundamental biological processes. In this work, we employ coarse-grained Langevin dynamics simulations of a simple model of different RNA hairpins passing through a geometric nanopore to find the influence of structural changes on the translocation dynamics. The threshold voltage of unfolding depends on the length of the hairpin attached to the tail. The lag time to unfold is longer for smaller applied voltages and for the architectures containing a longer hairpin attached to the tail. Chain translocation dynamics for different architectures are largely collapsed by the threshold. A distinct signature for the base unfolding time was observed for the bases around the unpaired bases in all the RNA hairpin models. These results can motivate future technologies or experiments that use translocation to predict secondary structures of polynucleotides.