Biology subjects

Wagner, S.

Publications and source records attributed to Wagner, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2020

COVID-19-associated olfactory dysfunction reveals SARS-CoV-2 neuroinvasion and persistence in the olfactory system

While recent investigations have revealed viral, inflammatory and vascular factors involved in SARS-CoV-2 lung pathogenesis, the pathophysiology of neurological disorders in COVID-19 remains poorly understood. Yet, olfactory and taste dysfunction are rather common in COVID-19, especially in pauci-symptomatic patients which constitutes the most frequent clinical manifestation of the infection. We conducted a virologic, molecular, and cellular study of the olfactory system from COVID-19 patients presenting acute loss of smell, and report evidence that the olfactory epithelium represents a highly significant infection site where multiple cell types, including olfactory sensory neurons, support cells and immune cells, are infected. Viral replication in the olfactory epithelium is associated with local inflammation. Furthermore, we show that SARS-CoV-2 induces acute anosmia and ageusia in golden Syrian hamsters, both lasting as long as the virus remains in the olfactory epithelium and the olfactory bulb. Finally, olfactory mucosa sampling in COVID-19 patients presenting with persistent loss of smell reveals the presence of virus transcripts and of SARS-CoV-2-infected cells, together with protracted inflammation. Viral persistence in the olfactory epithelium therefore provides a potential mechanism for prolonged or relapsing symptoms of COVID-19, such as loss of smell, which should be considered for optimal medical management and future therapeutic strategies.

neuroscience↗

bioRxiv2020

Decoding and blocking the bacterial lipid flippase MprF yields a novel anti-virulence strategy

The pandemic of antibiotic resistance represents a major human health threat demanding new antimicrobial strategies. MprF is the synthase and flippase of the phospholipid lysyl-phosphatidylglycerol that increases virulence and resistance of methicillin-resistant Staphylococcus aureus (MRSA) and other pathogens to cationic host defense peptides and antibiotics. With the aim to design MprF inhibitors that could sensitize MRSA to both, human antimicrobials and antibiotics and support the clearance of staphylococcal infections with minimal selection pressure, we developed MprF-targeting monoclonal antibodies, which bound and blocked the MprF flippase subunit. Antibody M-C7.1 targeted a specific loop in the flippase domain that proved to be exposed at both sides of the bacterial membrane, thereby enhancing the mechanistic understanding into bacterial lipid translocation. M-C7.1 rendered MRSA susceptible to host antimicrobial peptides and antibiotics such as daptomycin. Moreover, it impaired MRSA survival in human phagocytes, which recommends MprF inhibitors for new anti-MRSA approaches. MprF-directed monoclonal antibodies provide a proof of concept for development of precisely targeted anti-virulence approaches, which block bacterial antimicrobial resistance mechanisms.

microbiology↗

bioRxiv2020

Recovering genomes and phenotypes using allele-specific gene expression

With the recent increase in RNA sequencing efforts using large cohorts of individuals, studying allele-specific gene expression is becoming increasingly important. Here, we report that, despite not containing explicit variant information, a list of allele-specific gene names of an individual is enough to recover key variants and link the individual back to their genome or phenotype. This creates a privacy conundrum.

bioinformatics↗

bioRxiv2020

Precision medicine for the rescue of specific impairments in social behavior associated with the A350V Iqsec2 mutation

In this study we tested the hypothesis that precision medicine guided therapy targeting glutamatergic neurotransmission could rescue behavioral deficits exhibited by mice carrying a specific mutation in the Iqsec2 gene. The IQSEC2 protein plays a key role in glutamatergic synapses and mutations in the IQSEC2 gene are a frequent cause of neurodevelopmental disorders. We have recently reported on the molecular pathophysiology of one such mutation A350V and demonstrated that this mutation downregulates AMPA type glutamatergic receptors (AMPAR) in A350V mice. Here we sought to identify behavioral deficits in A350V mice and hypothesized that we could rescue these deficits by PF-4778574, a positive AMPAR modulator. We found that A350V Iqsec2 mice exhibit specific deficits in sex preference and emotional state preference behaviors as well as in vocalizations when encountering a female mouse. The social discrimination deficits, but not the impaired vocalization, were rescued with PF-4778574. We conclude that social behavior deficits associated with the A350V Iqsec2 mutation may be rescued by enhancing AMPAR mediated synaptic transmission.

neuroscience↗

bioRxiv2020

Photosynthetic activity triggers pH and NAD redox signatures across different plant cell compartments

A characteristic feature of most plants is their ability to perform photosynthesis, which ultimately provides energy and organic substrates to most life. Photosynthesis dominates chloroplast physiology but represents only a fraction of the tightly interconnected metabolic network that spans the entire cell. Here, we explore how photosynthetic activity affects the energy physiological status in cell compartments beyond the chloroplast. We develop precision live monitoring of subcellular energy physiology under illumination to investigate pH, MgATP2- and NADH/NAD+ dynamics at dark-light transitions by confocal imaging of genetically encoded fluorescent protein biosensors in Arabidopsis leaf mesophyll. We resolve the in vivo signature of stromal alkalinisation resulting from photosynthetic proton pumping and observe a similar pH signature also in the cytosol and the mitochondria suggesting that photosynthesis triggers an alkalinisation wave that affects the pH landscape of large parts of the cell. MgATP2- increases in the stroma at illumination, but no major effects on MgATP2- concentrations in the cytosol were resolved. Photosynthetic activity triggers a signature of substantial NAD reduction in the cytosol that is driven by photosynthesis-derived electron export. Strikingly, cytosolic NAD redox status was deregulated in mutants of chloroplastic NADP- and mitochondrial NAD-dependent malate dehydrogenases even at darkness, pinpointing the participation of the chloroplasts and mitochondria in shaping cytosolic redox metabolism in vivo with a dominant function of malate metabolism. Our data illustrate how profoundly and rapidly changes in photosynthetic activity affect the physiological and metabolic landscape throughout green plant cells. One-sentence summaryDark-light transitions trigger profound re-orchestration of subcellular pH and NAD redox physiology not only in the chloroplast but also beyond, in the cytosol and the mitochondria, as revealed by precision live-monitoring using fluorescent protein biosensors.

plant biology↗

bioRxiv2020

Synapsin-caveolin-1 mitigates cognitive deficits and neurodegeneration in Alzheimer's disease mice

AD presents with severe neurodegeneration which leads to cognitive deficits and dementia. Identifying the molecular signals that attenuate neurodegeneration in AD may be exploited as therapeutic targets. This study revealed that transgenic AD mice (PSAPP) exhibit decreased caveolin-1 (Cav-1), a membrane/lipid raft (MLR) scaffolding protein that organizes synaptic signaling components. Subcellularly, Cav-1 and full length (fl)-TrkB were significantly decreased in MLRs. We thus developed an in vivo gene therapy that re-expresses neuronal-targeted Cav-1 using the synapsin promoter (SynCav1). While AD mice showed significant learning and memory deficits at 9 and 11 months, AD mice that received hippocampal SynCav1 (AD-SynCav1) maintained normal learning and memory at 9 and 11 months respectively. Furthermore, AD-SynCav1 mice showed preserved hippocampal MLR-localized fl-TrkB, synaptic ultrastructure, dendritic arborization and axonal myelin content, all of which occurred independent of reducing amyloid deposit and astrogliosis. Thus, SynCav1 demonstrates translational potential to treat AD by delaying neurodegeneration. SummaryTransgenic PSAPP mice exhibit decreased hippocampal expression of the membrane lipid raft (MLR) scaffolding protein caveolin-1. Synapsin-promoted re-expression of Cav-1 (termed SynCav1) mitigated neuropathology and cognitive deficits. SynCav1 gene therapy has the potential to treat AD and other forms of neurodegeneration.

neuroscience↗

bioRxiv2020

Social recognition in rats and mice requires integration of olfactory, somatosensory and auditory cues

In humans, discrimination between individuals, also termed social recognition, can rely on a single sensory modality, such as vision. By analogy, social recognition in rodents is thought to be based upon olfaction. Here, we hypothesized that social recognition in rodents relies upon integration of olfactory, auditory and somatosensory cues, hence requiring active behavior of social stimuli. Using distinct social recognition tests, we demonstrated that adult male rats and mice do not recognize familiar stimuli or learn the identity of novel stimuli that are inactive due to anesthesia. We further revealed that impairing the olfactory, somatosensory or auditory systems prevents recognition of familiar stimuli. Finally, we found that familiar and novel stimuli generate distinct movement patterns during social discrimination and that subjects react differentially to the movement of these stimuli. Thus, unlike what occurs in humans, social recognition in rats and mice relies on integration of information from several sensory modalities.

animal behavior and cognition↗

bioRxiv2020

Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation reveals multisite regulation of surface expression and gating

Phosphorylation of NaV1.5 channels regulates cardiac excitability, yet the phosphorylation sites regulating channel function and the underlying mechanisms remain largely unknown. Using a systematic quantitative phosphoproteomic approach, we analyzed NaV1.5 channel complexes purified from non-failing and failing mouse left ventricles, and we identified 42 phosphorylation sites on NaV1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic NaV1.5 mutants revealed the roles of three phosphosites in regulating NaV1.5 channel expression and gating. The phosphorylated serines-664 and -667 regulate the voltage-dependence of channel activation in a cumulative manner, whereas phosphorylation of the nearby serine-671, which is increased in failing hearts, decreases cell surface NaV1.5 expression and peak Na+ current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, the results demonstrate that ventricular NaV1.5 is highly phosphorylated, and that the phosphorylation-dependent regulation of NaV1.5-encoded channels is highly complex, site-specific and dynamic. AbbreviationsA, alanine; E, glutamate; HEK-293, Human Embryonic Kidney 293 cells; INa, peak Na+ current; INaL, late Na+ current; IP, immunoprecipitation; mNaVPAN, anti-NaV channel subunit mouse monoclonal antibody; MS, Mass Spectrometry; MS1, mass spectrum of peptide precursors; MS2 or MS/MS, fragmentation mass spectrum of peptides selected in narrow mass range (2 Da) from MS1 scan; NaV, voltage-gated Na+ channel; pS, phosphoserine; pT, phosphothreonine; S, serine; T, threonine; TAC, Transverse Aortic Constriction; TMT, Tandem Mass Tag.

physiology↗

bioRxiv2020

Storing and analyzing a genome on a blockchain

The genomic characterization of individuals promises to be immensely useful for biomedical research and healthcare. However, a critical barrier to expanding personal genome sequencing is achieving secure, high-integrity storage of raw data. While cloud storage offers solutions to access such data from any place and device, the vulnerabilities of centralized storage in relation to security, data integrity, and robustness, such as single points of failure, have not yet been addressed. Blockchain is a potential alternative to these storage modes. However, storing large-scale data on blockchain can be challenging due to slow transaction speeds, the potential for chains to reach large sizes, and limitations on querying data stored on-chain. Currently, several genomic storage applications incorporate blockchain, but likely because of these challenges, many use blockchain only to facilitate and log data-access transactions, rather than to store raw genomic data on-chain. While this secures the process of data access, it does not secure the data itself, which is often stored off-chain (i.e. in a cloud or file-hosting services). Here, we developed a novel method of storing reference-aligned reads on-chain in a private blockchain network. We also developed tools for accessing and analyzing the on-chain data. We addressed the challenges of on-chain data storage by minimizing the data inserted to the chain using reference-based data compression techniques and by binning the on-chain data by genomic location to reduce retrieval times. Our tools provide open-source blockchain-based storage and access for advanced genomic analyses such as variant calling.

bioinformatics↗

bioRxiv2020

Branched-chain amino acid catabolism depends on GRXS15 through mitochondrial lipoyl cofactor homeostasis

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in all life and are used in a wide array of diverse biological processes, including electron transfer chains and several metabolic pathways. Biosynthesis machineries for Fe-S clusters exist in plastids, the cytosol and mitochondria. A single monothiol glutaredoxin (GRX) has been shown to be involved in Fe-S cluster assembly in mitochondria of yeast and mammals. In plants, the role of the mitochondrial homologue GRXS15 has only partially been characterized. Arabidopsis grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype. In an in-depth metabolic analysis, we show that most Fe-S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis and the electron transport chain. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, 2-oxoglutarate, glycine and branched-chain amino acids (BCAAs). The most pronounced accumulation occurred in branched-chain -keto acids (BCKAs), the first degradation products resulting from deamination of BCAAs. In wild-type plants, pyruvate, 2-oxoglutarate, glycine and BCKAs are all metabolized through decarboxylation by four mitochondrial lipoyl cofactor-dependent dehydrogenase complexes. Because these enzyme complexes are very abundant and the biosynthesis of the lipoyl cofactor depends on continuous Fe-S cluster supply to lipoyl synthase, this could explain why lipoyl cofactor-dependent processes are most sensitive to restricted Fe-S supply in GRXS15 K83A mutants. One-sentence summaryDeficiency in GRXS15 restricts protein lipoylation and causes metabolic defects in lipoyl cofactor-dependent dehydrogenase complexes, with branched-chain amino acid catabolism as dominant bottleneck.

plant biology↗

bioRxiv2020

TraCurate: efficiently curating cell tracks

TraCurate is an open-source software tool to curate and manually annotate cell tracking data from time-lapse microscopy. Although many studies of cellular behavior require high-quality, long-term observations across generations of cells, automated cell tracking is often imperfect and typically yields fragmented results that still contain many errors. TraCurate provides the functionality for the curation and correction of cell tracking data with minimal user interaction and expenditure of time and supports the extraction of complete cell tracks and cellular genealogies from experimental data. Source code and binary packages for Linux, macOS and Windows are available at https://tracurate.gitlab.io/, as well as all other complementary tools described herein.

bioinformatics↗

bioRxiv2019

APOBEC3A Catalyzes Mutation and Drives Carcinogenesis In Vivo

The APOBEC3 family of antiviral DNA cytosine deaminases is implicated as the second largest source of mutation in cancer. This mutational process may be a causal driver or inconsequential passenger to the overall tumor phenotype. We show that human APOBEC3A expression in murine colon and liver tissues increases tumorigenesis. All other APOBEC3 family members including APOBEC3B did not as strongly promote liver tumor formation. DNA sequences from APOBEC3A-expressing animals display hallmark APOBEC signature mutations in TCA/T motifs. Bioinformatic comparisons of the observed APOBEC3A mutation signature in murine tumors, previously reported APOBEC3A and APOBEC3B mutation signatures in yeast, and reanalyzed APOBEC mutation signatures in human tumor data sets support cause-and-effect relationships for APOBEC3A-catalyzed deamination and mutagenesis in driving multiple human cancers.

cancer biology↗

bioRxiv2019

BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity

Activity of the NLRP3 inflammasome, a critical mediator of inflammation (1), is controlled by accessory proteins (2, 3), post-translational modifications (4, 5), cellular localization (6, 7) and oligomerization (8). How these factors relate, is unclear. We show that the established drug target, Brutons Tyrosine Kinase (BTK) (2, 9), integrates several levels of NLRP3 regulation: BTK phosphorylation of four conserved tyrosine residues, by neutralizing the charge of a polybasic linker region, weakens the interaction of NLRP3 with Golgi phospholipids and may thus guide NLRP3 cytosolic localization. BTK activity also promotes NLRP3 oligomerization and subsequent formation of inflammasomes. As NLRP3 tyrosine modification ultimately also impacts on IL-1{beta} release, we propose BTK-mediated, charge-switch-based NLRP3 regulation as a novel and therapeutically tractable step in the control of inflammation. One Sentence SummaryMulti-phosphorylation of NLRP3 by Brutons tyrosine kinase modulates NLRP3 cellular localization, inflammasome assembly, and IL-1{beta} release.

immunology↗

bioRxiv2019

Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes

Translational control targeting mainly the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast Translational Complex Profile sequencing (TCP-seq), related to ribosome profiling, and adopted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ses) in addition to 80S ribosomes (80Ses), revealed that mammalian and yeast 40Ses distribute similarly across 5UTRs indicating considerable evolutionary conservation. We further developed a variation called Selective TCP-seq (Sel-TCP-seq) enabling selection for 40Ses and 80Ses associated with an immuno-targeted factor in yeast and human. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5UTRs with scanning 40Ses to successively dissociate upon start codon recognition. Manifesting the Sel-TCP-seq versatility for gene expression studies, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.

molecular biology↗

bioRxiv2019

Human gut mobileome during antibiotic therapy. A trajectory-based approach to analysis of the metagenomic time-series datasets

MotivationAntibiotic resistance is widely recognized as a severe threat to current medical practice. Each antibiotic therapy drives the emergence and subsequent retention of antibiotics resistance genes within the human gut microbiome. However, the details on how the resistance spreads between bacteria within the human gut remain unknown, as does the role of horizontal gene transfer in this process, too.\n\nResultsWe present a novel approach to the analysis of time-series whole-genome metagenomic sequencing data. This involves partitioning the scaffolds from the metagenomic assembly into groups corresponding to bacterial chromosomes, plasmids and those with prophages and transposons. Using specialized sequencing of the bacteriophages we were able to track the flow of ciprofloxacin resistance genes from bacterial chromosomes, through the plasmids, to prophages and phages.\n\nContactanna.gorska@univr.it.

bioinformatics↗

bioRxiv2019

A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a lack of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess secretion and injection through the type III secretion system encoded by Salmonella pathogenicity island 1. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanoliter scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed NanoLuc and split-NanoLuc-based assays that enable the monitoring of type III secretion-dependent injection of effector proteins into host cells.\n\nImportanceThe ability to secrete proteins to the bacterial cell surface, to the extracellular environment, or even into target cells is one of the foundations of interbacterial as well as pathogen-host interaction. While great progress has been made in elucidating assembly and structure of secretion systems, our understanding of their secretion mechanism often lags behind, not last because of the challenge to quantitatively assess secretion function. Here, we developed a luciferase-based assay to enable the simple, quick, quantitative, and high throughput-compatible assessment of secretion and injection through virulence-associated type III secretion systems. The assay allows detection of minute amounts of secreted substrate proteins either in the supernatant of the bacterial culture or within eukaryotic host cells. It thus provides an enabling technology to elucidate the mechanisms of secretion and injection of type III secretion systems and is likely adaptable to assay secretion through other bacterial secretion systems.

microbiology↗

bioRxiv2019

Structural control for the coordinated assembly into functional pathogenic type-3 secretion systems

Functional injectisomes of the type-3 secretion system assemble into highly defined and stoichiometric bacterial molecular machines essential for infecting human and other eukaryotic cells. However, the mechanism that governs the regulated step-wise assembly process from the nucleation-phase, to ring-assembly, and the filamentous phase into a membrane embedded needle complex is unclear. We here report that the formation of a megadalton-sized needle complexes from Salmonella enterica serovar Typhimurium (SPI-1, Salmonella pathogenicity island-1) with proper stoichiometries is highly structurally controlled competing against the self-assembly propensity of injectisome components, leading to a highly unusual structurally-pleiotropic phenotype. The structure of the entire needle complex from pathogenic injectisomes was solved by cryo electron microscopy, focused refinements (2.5-4 [A]) and co-variation analysis revealing an overall asymmetric arrangement containing cyclic, helical, and asymmetric sub-structures. The centrally located export apparatus assembles into a conical, pseudo-helical structure and provides a structural template that guides the formation of a 24-mer cyclic, surrounding ring, which then serves as a docking interface comprising three different conformations for sixteen N-terminal InvG subunits of the outer secretin ring. Unexpectedly, the secretin ring excludes the 16th protein chain at the C-terminal outer ring, resulting in a pleiotropic 16/15-mer ring and consequently to an overall 24:16/15 basal body structure. Finally, we report how the transition from the pseudo-helical export apparatus into the helical filament is structurally resolved to generate the protein secretion channel, which provides the structural basis to restrict access of unfolded effector substrates. These results highlight the diverse molecular signatures required for a highly coordinated assembly process and provide the molecular basis for understanding triggering and transport of unfolded proteins through injectisomes.

molecular biology↗

bioRxiv2019

The flagellar substrate specificity switch protein FlhB assembles onto the extra-membrane export gate to regulate type three secretion

Export of proteins through type three secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are transported through an export gate complex consisting of three proteins (FliPQR in flagellar systems, SctRST in virulence systems) that were initially annotated as membrane proteins, but which we have recently shown assemble into an extra-membranous helical assembly. A fourth putative membrane protein (FlhB/SctU) is essential to the export process, and also functions to \"switch\" secretion substrate specificity once the growing hook/needle structures reach their determined length. Here we present the structure of an export gate containing the switch protein from a Vibrio polar flagellar system at 3.2 [A] resolution by cryo-electron microscopy. The structure reveals that the FlhB/SctU further extends the helical export gate assembly with its four putative transmembrane helices adopting an out-of-membrane location, wrapped around the other export gate components at the base of the structure. The unusual topology of the switch protein helices creates a loop that wraps around the bottom of the closed export gate complex. Structure-informed mutagenesis suggests that this loop is critical in gating secretion and we propose that a series of conformational changes in the type 3 secretion system trigger opening of the export gate through the interactions between FlhB/SctU and FliPQR/SctRST.

microbiology↗