bluuebunny/crossref_metadata_2025_split huggingface dataset:
Changes in protonation states of in-pathway residues can alter ligand binding pathways obtained from spontaneous binding molecular dynamics simulations
Authors: Girame Helena , Garcia-Borràs Marc , Feixas Ferran 
Published: May 2022

ABSTRACTProtein-ligand binding processes often involve changes in protonation states that can be key to recognize and orient the ligand in the binding site. The pathways through which (bio)molecules interplay to attain productively bound complexes are intricate and involve a series of interconnected intermediate and transition states. Molecular dynamics (MD) simulations and enhanced sampling techniques are commonly used to characterize the spontaneous binding of a ligand to its receptor. However, the effect of protonation state changes of in-pathway residues in spontaneous binding MD simulations remained mostly unexplored. Here, we used molecular dynamics simulations to reconstruct the trypsin-benzamidine binding pathway considering different protonation states of His57. This residue is part of the trypsin catalytic triad and is located more than 10 Å away from Asp189, which is responsible for benzamidine binding in the trypsin S1 pocket. Our MD simulations showed that the binding pathways that benzamidine follow to target the S1 binding site are critically dependent on the His57 protonation state. Binding of benzamidine frequently occurs when His57 is protonated in the delta nitrogen while the binding process is significantly less frequent when His57 is positively charged. Constant-pH MD simulations retrieved the equilibrium populations of His57 protonation states at trypsin active pH offering a clearer picture of benzamidine recognition and binding. These results indicate that properly accounting for protonation states of distal residues can be important in spontaneous binding MD simulations.
Imaging the structure and dynamic activity of retinal microglia and macrophage-like cells in the living human eye
Authors: Rui Yuhua , Lee Daniel M.W. , Zhang Min , Snyder Valerie C , Raghuraman Rashmi , Gofas-Salas Elena , Mecê Pedro , Yadav Sanya , Tiruveedhula Pavan , Grieve Kate , Sahel José-Alain , Errera Marie-Hélène , Rossi Ethan A. 
Published: May 2022

AbstractPurposeWe recently showed how a refined sequential detection pattern and image processing pipeline for multi-offset adaptive optics scanning light ophthalmoscopy (AOSLO) can increase the contrast of weakly scattering inner retinal structures, including microglia. However, sequential detection was still time-consuming, preventing dynamics from being monitored over short intervals (< 3 mins). Here we show that simultaneous fiber-bundle (FB) detection can overcomes this limitation to reveal the structure and dynamic activity of microglia and macrophage-like cells in healthy and diseased retinae.MethodsWe designed and implemented a custom 7-fiber optical FB with one central confocal fiber and six larger fibers for multi-offset detection in AOSLO at a single focal plane. We imaged the ganglion cell layer at several locations at multiple timepoints (from minutes to weeks) in 8 healthy participants and in 4 patients with ocular infections or inflammation, including ocular syphilis and posterior uveitis. Microglia and immune cells were manually segmented to quantify cell morphometry and motility.ResultsFiber-bundle detection reduced single acquisition time to 20-30 seconds, enabling imaging over larger areas and monitoring of dynamics over shorter intervals. Presumed microglia in healthy retinas had an average diameter of 12.8 μm and with a spectrum of morphologies including circular cells and elongated cells with visible processes. We also detected the somas of putative macroglia, potentially astrocytes, near the optic nerve head. Microglia moved slowly in normal eyes (0.02μm/sec, on average) but speed increased in patients with active infections or inflammation (up to 2.37μm/sec). Microglia activity was absent in a patient with chronic uveitis that was quiescent but apparent over short intervals in an active uveitis retina. In a patient with ocular syphilis imaged at multiple timepoints during treatment, macrophage-like cells containing granular internal structures were seen. Decreases in the quantity and motility of these immune cells were correlated with improvements to vision and other structural and systemic biomarkers.ConclusionsFB-AOSLO enable simplified optical setup with easy alignment and implementation. We imaged the fine-scale structure and dynamics of microglia and macrophage-like cells during active infection and inflammation in the living eye for the first time. In healthy eyes we also detected putative glia cell near the optic nerve head. FB-AOSLO offers promise as a powerful tool for detecting and monitoring retinal inflammation and infection in the living eye over short response to treatment.
Exploratory Meta-Analysis of Hypoxic Transcriptomes Using Precise Transcript Reference Sequence Set
Authors: Ono Yoko , Bono Hidemasa 
Published: May 2022

AbstractGene expression studies are intrinsically biased, with many studies influenced by concomitant information such as gene-disease associations. This limitation can be overcome using a data-driven analysis approach without relying on ancillary information. The FANTOM CAGE Associated Transcriptome (FANTOM-CAT) project provides a comprehensive meta-assembly of the human transcriptome using coding and non-coding genes. Hypoxia strongly influences gene expression; additionally, non-coding RNA (ncRNA) metabolism is downregulated in response to hypoxic stimuli. We evaluated the differential response of various transcripts to hypoxia by determining their hypoxia responsiveness scores. Enrichment analysis revealed that several genes associated with ncRNA metabolism, particularly those involved in ribosomal RNA processing, were downregulated in response to hypoxia. Previously published information from the FANTOM-CAT project was suitable for meta-analysis of the transcriptome sequencing data from both coding and ncRNAs, and evaluate the hypoxia responsiveness of target transcripts and relationship between sense-antisense transcripts from the same locus. Our results may facilitate functional annotation of various transcripts including ncRNAs, allowing for both sense and antisense and coding and non-coding evaluations.Summary blurbExploratory meta-analysis of hypoxic RNA-seq data using FANTOM-CAT as a reference transcriptome facilitates the evaluation of relationship between sense-antisense transcripts from the same locus.
Genomic, transcriptomic and proteomic depiction of iPSC-derived smooth muscle cells as emerging cellular models for arterial diseases
Authors: Liu Lu , Jouve Charlène , Henry Joséphine , Berrandou Takiy-Eddine , Hulot Jean-Sébastien , Georges Adrien , Bouatia-Naji Nabila 
Published: May 2022

AbstractBackgroundVascular smooth muscle cells (VSMCs) plasticity is a central mechanism in cardiovascular health and disease. We aimed at providing deep cellular phenotyping, epigenomic and proteomic depiction of SMCs derived from induced pluripotent stem cells (iPSCs) and evaluating their potential as cellular models in the context of complex genetic arterial diseases.MethodsWe differentiated 3 human iPSC lines using either RepSox (R-SMCs) or PDGF-BB and TGF-β (TP-SMCs), during the second half of a 24-days-long protocol. In addition to cellular assays, we performed RNA-Seq and assay for transposase accessible chromatin (ATAC)-Seq at 6 time-points of differentiation. The extracellular matrix content (matrisome) generated by iPSCs derived SMCs was analyzed using mass spectrometry.ResultsBoth iPSCs differentiation protocols generated SMCs with positive expression of SMC markers. TP-SMCs exhibited greater capacity of proliferation, migration and lower calcium release in response to contractile stimuli compared to R-SMCs. RNA-Seq data showed that genes involved in the contractile function of arteries were highly expressed in R-SMCs compared to TP-SMCs or primary SMCs. Matrisome analyses supported an overexpression of proteins involved in wound repair in TP-SMCs and a higher secretion of basal membrane constituents by R-SMCs. Open chromatin regions of R-SMCs and TP-SMCs were significantly enriched for variants associated with coronary artery disease and blood pressure, while only TP-SMCs were enriched for variants associated with peripheral artery disease.ConclusionsOur study portrayed two iPSCs derived SMCs models presenting complementary cellular phenotypes of high relevance to SMC plasticity. In combination with genome-editing tools, our data supports high relevance of the use of these cellular models to the study of complex regulatory mechanisms at genetic risk loci involved in several arterial diseases.Graphical Abstract
Combined MD and QM/MM Calculations reveal Allostery Driven Promiscuity in Dipeptide Epimerases of Enolase Family
Authors: Tripathi Ankita , Dubey Kshatresh Dutta 
Published: May 2022

ABSTRACTThe adaptability of the active site to amplify the secondary function is supposed to be the fundamental cause of the promiscuity and the evolution of new functions in the enzymes. In most cases, mutations occur close to the active site and/or in the catalytic site to change the active site plasticity to accommodate the non-native substrate. In the present study, using MD simulations and hybrid QM/MM calculations, we have shown a new way to enhance the promiscuity, i.e., the allostery-driven promiscuity. Using a case study of the AEE enzyme where the capping loop recognizes the substrate, herein, we show that a single site mutation (D321G) far from the capping loop can induce a large conformational change in the capping loop to recognize different substrates for different functions. The QM/MM calculations for the WT and mutated enzyme provide a first validation of the mechanism of 1,1-proton transfer and dehydration by the AEE enzyme. Since AEE epimerase possesses a highly conserved TIM-barrel fold, we believe that our study provides a crucial lead to understanding the mechanism of emergence of secondary function which can be useful to repurpose ancient enzymes for modern usage.
Qualification of Identity, Quality-Attribute Monitoring and New Peak Detection in An Improved Multi-Attribute Method with Lys-C Digestion for Characterization and Quality Control of Therapeutic Monoclonal Antibodies
Authors: Li Xuanwen , Pierson Nicholas A. , Hua Xiaoqing , Patel Bhumit A. , Olma Michael H. , Strulson Christopher A. , Letarte Simon , Richardson Douglas D. 
Published: May 2022

AbstractThe use of Multi-attribute method (MAM) for identity and purity testing of biopharmaceuticals offers the ability to complement and replace multiple conventional analytical technologies with a single mass spectrometry (MS) method. Method qualification and phase-appropriate validation is one major consideration for the implementation of MAM in a current Good Manufacturing Practice (cGMP) environment. We developed an improved MAM workflow with optimized sample preparation using Lys-C digestion for therapeutic monoclonal antibodies. In this study, we qualified the enhanced MAM workflow for mAb-1 identity, product quality attributes (PQAs) monitoring and new peak detection (NPD). The qualification results demonstrated the full potential of the MAM for its intended use in mAb-1 characterization and quality control in regulated labs. To the best of our knowledge, this is the first report of MAM qualification for mAb identity, PQA monitoring, and new peak detection (NPD) in a single assay, featuring 1) the first full qualification of MAM using Lys-C digestion without desalting using a high-resolution MS, 2) a new approach for mAb identity testing using MAM, and 3) the first qualification of NPD for MAM. The developed MAM workflow and the approaches for MAM qualification may serve as a reference for other labs in the industry.
Omicron infection induces low-level, narrow-range SARS-CoV-2 neutralizing activity
Authors: Turelli Priscilla , Zaballa María-Eugenia , Raclot Charlène , Fenwick Craig , Kaiser Laurent , Eckerle Isabella , Pantaleo Giuseppe , Guessous Idris , Stringhini Silvia , Trono Didier 
Published: May 2022

AbstractBackgroundThe rapid worldwide spread of the mildly pathogenic SARS-CoV-2 Omicron variant has led to the suggestion that it will induce levels of collective immunity that will help putting an end to the COVID19 pandemics.MethodsConvalescent serums from non-hospitalized individuals previously infected with Alpha, Delta or Omicron BA.1 SARS-CoV-2 or subjected to a full mRNA vaccine regimen were evaluated for their ability to neutralize a broad panel of SARS-CoV-2 variants.FindingsPrior vaccination or infection with the Alpha or to a lesser extent Delta strains conferred robust neutralizing titers against most variants, albeit more weakly against Beta and even more Omicron. In contrast, Omicron convalescent serums only displayed low level of neutralization activity against the cognate virus and were unable to neutralize other SARS-CoV-2 variants.InterpretationModerately symptomatic Omicron infection is only poorly immunogenic and does not represent a substitute for vaccination.FundingEPFL COVID Fund; private foundation advised by CARIGEST SA; Private Foundation of the Geneva University Hospitals; General Directorate of Health of the canton of Geneva, the Swiss Federal Office of Public Health.
An Epitope-Focused Trypanosome-Derived Vaccine Platform Elicits High-Affinity Antibodies and Immunity Against Fentanyl Effects
Authors: Triller Gianna , Vlachou Evi P. , Hashemi Hamidreza , van Straaten Monique , Zeelen Johan , Kelemen Yosip , Baehr Carly , Marker Cheryl L. , Ruf Sandra , Svirina Anna , Kruse Sebastian , Baumann Andreas , Miller Aubry K. , Bartel Marc , Pravetoni Marco , Stebbins C. Erec , Papavasiliou F. Nina , Verdi Joseph P. 
Published: May 2022

SummaryPoorly antigenic small molecules pose challenges for the production of clinically efficacious antibodies. To address this, we have developed an immunization platform derived from the antigenic surface coat of the African trypanosome. Through sortase-based conjugation of antigens to the trypanosome surface coat protein variant surface glycoprotein (VSG), we created the VAST (VSG-immunogen Array by Sortase Tagging). We used VAST to elicit protective immunity to the effects fentanyl. Immunizing mice with fentanyl-VAST generated serological memory and protection from fentanyl challenge. Employing single-cell RNAseq, we then developed a streamlined method that synergizes with the VAST to identify immunization-elicited memory B cells and the antibodies they encode. All antibodies selected by this method displayed picomolar affinities for fentanyl. Passive immunization protected animals from fentanyl, while crystallography revealed that the mAbs bind fentanyl in an unusually deep pocket suited to small molecules, demonstrating the ability of the VAST to elicit high-quality therapeutic antibodies.
Integrated viral elements suggest the dual lifestyle of Tetraselmis spp. polinton-like viruses
Authors: Chase Emily E. , Desnues Christelle , Blanc Guillaume 
Published: May 2022

AbstractIn this study, we aimed at exploring horizontal gene transfer between viruses and Chlorodendraceae green algae (Chlorophyta) using available genomic and transcriptomic sequences for 20 algal strains. We identified a significant number of genes sharing a higher sequence similarity with viral homologues, thus signaling their possible involvement in HGTs with viruses. Further characterization showed that many of these genes were clustered in DNA regions of several tens to hundreds of kilobases in size, originally belonging to viruses related to known Tetraselmis spp. viruses (TetV and TsV). In contrast, the remaining candidate HGT genes were randomly dispersed in the algal genomes, more frequently transcribed and belonged to large multigene families. The presence of homologs in Viridiplantae suggested that these latter were more likely of algal rather than viral origin. We found a remarkable diversity Polinton- like virus (PLV) elements inserted in Tetraselmis genomes, all of which were most similar to the Tetrasemis striata virus TsV. The genes of PLV elements are transcriptionally inactive with the notable exception of the homologue of the TVSG_00024 gene of TsV whose function is unknown. We suggest that this gene may be involved in a sentinel process to trigger virus reactivation and excision in response to an environmental stimulus. Altogether, these results provide evidence that TsV- related viruses have a dual lifestyle, alternating between a free viral phase (i.e., virion) and a phase integrated into host genomes.
Evaluating and mitigating clinical samples matrix effects on TX-TL cell-free performance
Authors: Voyvodic Peter L. , Conejero Ismael , Mesmoudi Khouloud , Renard Eric , Courtet Philippe , Cattoni Diego I. , Bonnet Jerome 
Published: May 2022

AbstractCell-free biosensors are promising tools for medical diagnostics, yet their performance can be affected by matrix effects arising from the sample itself or from external components. Here we systematically evaluate the performance and robustness of cell-free systems in serum, plasma, urine, and saliva using two reporter systems, sGFP and luciferase. In all cases, clinical samples have a strong inhibitory effect. Of different inhibitors, only the RNase inhibitor mitigated matrix effects. However, we found that the recovery potential of RNase inhibitor was partially muted by interference from glycerol contained in the commercial buffer. We solved this issue by designing a strain producing an RNase inhibitor protein requiring no additional step in extract preparation. Furthermore, our new extract yielded higher reporter levels than previous conditions and tempered interpatient variability associated with matrix effects. This systematic evaluation and improvements of cell-free system robustness unified across many types of clinical samples is a significant step towards developing cell-free diagnostics for a wide range of conditions.
Quantitative spatial analysis of neuroligin-3 mRNA expression in the enteric nervous system reveals a potential role in neuronal-glial synapses and reduced expression inNlgn3R451Cmice
Authors: Herath Madushani , Cho Ellie , Franks Ashley E , Bornstein Joel C , Hill-Yardin Elisa L 
Published: May 2022

ABSTRACTMutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction but its cellular expression in the GI tract remains to be characterised. Localisation of NLGN3 protein is challenging in intestinal tissue due to the lack of target-specific antibodies. Here, we combined RNAScopein situhybridization forNlgn3mRNA and immunofluorescence for markers of all enteric neurons, cholinergic submucosal neurons, non-cholinergic submucosal neurons, nitregic and calretinin-containing myenteric neurons as well as glial cells. We also developed a quantitative 3-dimensional image analysis method to measureNlgn3mRNA cellular expression levels in enteric neurons and glia. We show thatNlgn3mRNA is expressed in most submucosal and myenteric neurons as well as in enteric glia. The R451C mutation reducesNlgn3mRNA expression levels in cholinergic, nitrergic and calretinin enteric neuronal subpopulations but does not affectNlgn3mRNA expression in VIPergic submucosal neurons. In summary, we show that the autism-associated R451C mutation inNlgn3reducesNlgn3mRNA expression in the mouse ENS. These findings could shed light on the pathophysiology of GI dysfunction in ASD.
Heterologously secreted MbxA fromMoraxella bovisinduces a membrane blebbing response of the human host cell
Authors: Erenburg Isabelle N. , Hänsch Sebastian , Chacko Feby M. , Hamacher Anna , Wintgens Sebastian , Stuhldreier Fabian , Poschmann Gereon , Spitz Olivia , Stühler Kai , Wesselborg Sebastian , Hegemann Johannes , Smits Sander H. J. , Weidtkamp-Peters Stefanie , Schmitt Lutz 
Published: May 2022

AbstractMany proteins of the Repeats in Toxins (RTX) protein family are toxins of Gram-negative pathogens including hemolysin A (HlyA) of uropathogenicE. coli. RTX proteins are secreted via Type I secretion systems (T1SS) and adopt their native conformation in the Ca2+-rich extracellular environment. Here we employed theE. coliHlyA T1SS as a heterologous surrogate system for the RTX toxin MbxA from the bovine pathogenMoraxella bovis. InE. colithe HlyA system successfully activates the heterologous MbxA substrate by acylation and secretes the precursor proMbxA and active MbxA allowing purification of both species in quantities sufficient for a variety of investigations. The activatingE. coliacyltransferase HlyC recognizes the acylation sites in MbxA, but unexpectedly in a different acylation pattern as for its endogenous substrate HlyA. HlyC-activated MbxA shows host species-independent activity including a so-far unknown toxicity against human lymphocytes and epithelial cells. Using live- cell imaging, we show an immediate MbxA-mediated permeabilization and a rapidly developing blebbing of the plasma membrane in epithelial cells, which is associated with immediate cell death.
Deep trans-omic network fusion reveals altered synaptic network in Alzheimer’s Disease
Authors: Xie Linhui , Raj Yash , Varathan Pradeep , He Bing , Nho Kwangsik , Risacher Shannon L. , Salama Paul , Saykin Andrew J. , Yan Jingwen 
Published: May 2022

AbstractMulti-omic data spanning from genotype, gene expression to protein expression have been increasingly explored to interpret findings from genome wide association studies of Alzheimer’s disease (AD) and to gain more insight of the disease mechanism. However, each -omics data type is usually examined individually and the functional interactions between genetic variations, genes and proteins are only used after discovery to interpret the findings, but not beforehand. In this case, multi-omic findings are likely not functionally related and therefore give rise to challenges in interpretation. To address this problem, we propose a new interpretable deep neural network model MoFNet to jointly model the prior knowledge of functional interactions and multi-omic data set. It aims to identify a subnetwork of functional interactions predictive of AD evidenced by multi-omic measures. Particularly, prior functional interaction network was embedded into the architecture of MoFNet in a way that it resembles the information flow from DNA to gene and protein. The proposed model MoFNet significantly outperformed all other state-of-art classifiers when evaluated using multi-omic data from the ROS/MAP cohort. Instead of individual markers, MoFNet yielded multi-omic sub-networks related to innate immune system, clearance of misfolded proteins, and neurotransmitter release respectively. Around 50% of these findings were replicated in another independent cohort. Our identified gene/proteins are highly related to synaptic vesicle function. Altered regulation or expression of these genes/proteins could cause disruption in neuron-neuron or neuron-glia cross talk and further lead to neuronal and synapse loss in AD. Further investigation of these identified genes/proteins could possibly help decipher the mechanisms underlying synaptic dysfunction in AD, and ultimately inform therapeutic strategies to modify AD progression in the early stage.