Biology subjects

Walter, J.

Publications and source records attributed to Walter, J..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Prediction of Stroke Outcome in Mice Based on Non-Invasive MRI and Behavioral Testing

BackgroundPrediction of post-stroke outcome using the degree of subacute deficit or magnetic resonance imaging is well studied in humans. While mice are frequently used animals in preclinical stroke research, systematic analysis of outcome predictors is lacking. MethodsWe introduced heterogeneity into our study to broaden the applicability of our prediction tools. We analyzed the effect of 30, 45 and 60 minutes of arterial occlusion on the variance of stroke volumes. Next, we built a heterogeneous cohort of 215 mice using data from 15 studies that included 45 minutes of middle cerebral artery occlusion and various genotypes. Motor function was measured using the staircase test of skilled reaching. Phases of subacute and residual deficit were defined. Magnetic resonance images of stroke lesions were co-registered on the Allen Mouse Brain Atlas to characterize stroke topology. Different random forest prediction models that either used motor-functional deficit or imaging parameters were generated for the subacute and residual deficits. ResultsVariance of stroke volumes was increased by 45 minutes of arterial occlusion compared to 60 minutes and including various genotypes. We detected both a subacute and residual motor-functional deficit after stroke and different recovery trajectories. In mice with small cortical lesions, lesion volume was the best predictor of the subacute deficit. The residual deficit was most accurately predicted by the degree of the subacute deficit. When using imaging parameters for the prediction of the residual deficit, including information about the lesion topology increased prediction accuracy. A subset of anatomical regions within the ischemic lesion had particular impact on the prediction of long-term outcome. ConclusionsWe developed and validated a robust tool for the prediction of functional outcome after stroke in mice using a large heterogeneous cohort. Study design and imaging limitations are discussed. In the future, using outcome prediction can improve the design of preclinical studies and guide intervention decisions.

neuroscience↗

bioRxiv2021

Flv3A facilitates O2 photoreduction and affects H2 photoproduction independently of Flv1A in diazotrophic Anabaena filaments

The model heterocyst-forming filamentous cyanobacterium, Anabaena sp. PCC 7120 (Anabaena) represents multicellular organisms capable of simultaneously performing oxygenic photosynthesis in vegetative cells and the O2-sensitive N2-fixation inside the heterocysts. The flavodiiron proteins (FDPs) have been shown to participate in photoprotection of photosynthesis by driving excess electrons to O2 (Mehler-like reaction). Here, we addressed the physiological relevance of the vegetative cell-specific Flv1A and Flv3A on the bioenergetic processes occurring in diazotrophic Anabaena under variable CO2. We demonstrate that both Flv1A and Flv3A are required for proper induction of the Mehler-like reaction upon a sudden increase in light intensity, which is likely important for the activation of carbon-concentrating mechanisms (CCM) and CO2 fixation. Under ambient CO2 diazotrophic conditions, Flv3A is capable of mediating moderate O2 photoreduction, independently of Flv1A, but in coordination with Flv2 and Flv4. Strikingly, the lack of Flv3A resulted in strong downregulation of the heterocyst-specific uptake hydrogenase, which led to enhanced H2 photoproduction under both oxic and micro-oxic conditions. These results reveal a novel regulatory network between the Mehler-like reaction and the diazotrophic metabolism, which is of great interest for future biotechnological applications.

plant biology↗

bioRxiv2021

Beyond traditional visual sleep scoring: massive feature extraction and unsupervised clustering of sleep time series

Sleep is classically measured with electrophysiological recordings, which are then scored based on guidelines tailored for the visual inspection of these recordings. As such, these rules reflect a limited range of features easily captured by the human eye and do not always reflect the physiological changes associated with sleep. Here we present a novel analysis framework that characterizes sleep using over 7700 time-series features from the hctsa software. We used clustering to categorize sleep epochs based on the similarity of their features, without relying on established scoring conventions. The resulting structure overlapped substantially with that defined by visual scoring and we report novel features that are highly discriminative of sleep stages. However, we also observed discrepancies as hctsa features unraveled distinctive properties within traditional sleep stages. Our framework lays the groundwork for a data-driven exploration of sleep and the identification of new signatures of sleep disorders and conscious sleep states.

neuroscience↗

bioRxiv2021

The HMCES DNA-protein cross-link functions as a constitutive DNA repair intermediate

The HMCES protein forms a covalent DNA-protein cross-link (DPC) with abasic (AP) sites in ssDNA, and the resulting HMCES-DPC is thought to suppress double-strand break formation in S phase. However, the dynamics of HMCES cross-linking and whether any DNA repair pathways normally include an HMCES-DPC intermediate remain unknown. Here, we show that an HMCES-DPC forms efficiently on the AP site generated during replication-coupled DNA interstrand cross-link (ICL) repair. We use this system to show that HMCES cross-links form on DNA after the replicative CMG helicase has passed over the AP site, and that HMCES is subsequently removed by the SPRTN protease. The HMCES-DPC suppresses DSB formation, slows translesion synthesis (TLS) past the AP site, and introduces a bias for insertion of deoxyguanosine opposite the AP site. These data show that HMCES-DPCs can form as constitutive intermediates in replication-coupled repair, and they suggest a general model of how HMCES protects AP sites during DNA replication.

molecular biology↗

bioRxiv2021

A secondary metabolite drives intraspecies antagonism in a gut symbiont that is inhibited by peptidoglycan acetylation

The mammalian microbiome encodes numerous secondary metabolite biosynthetic gene clusters, yet their role in microbe-microbe interactions is unclear. Here, we characterized two polyketide synthase gene clusters (fun and pks) in the gut symbiont Limosilactobacillus reuteri. The pks, but not the fun cluster, encodes antimicrobial activity. Forty-one out of 51 L. reuteri strains tested are sensitive to Pks products, which was independent of strains host origin. The sensitivity to Pks was also established in intraspecies competition experiments in gnotobiotic mice. Comparative genome analyses between Pks-resistant and sensitive strains identified an acyltransferase gene (act) that is unique to Pks-resistant strains. Subsequent cell wall analysis of the wild-type and the act mutant strains showed that Act acetylates cell wall components. The pks mutants lost their competitive advantage and act mutants lost their Pks resistance in vivo. Thus, our findings provide insight into how closely related gut symbionts can compete and co- exist in the gastrointestinal tract.

microbiology↗

bioRxiv2021

Identification of tissue-specific and common methylation quantitative trait loci in healthy individuals using MAGAR

BackgroundUnderstanding the influence of genetic variants on DNA methylation is fundamental for the interpretation of epigenomic data in the context of disease. There is a need for systematic approaches not only for determining methylation quantitative trait loci (methQTL) but also for discriminating general from cell-type-specific effects. ResultsHere, we present a two-step computational framework MAGAR, which fully supports identification of methQTLs from matched genotyping and DNA methylation data, and additionally the identification of quantitative cell-type-specific methQTL effects. In a pilot analysis, we apply MAGAR on data in four tissues (ileum, rectum, T-cells, B-cells) from healthy individuals and demonstrate the discrimination of common from cell-type-specific methQTLs. We experimentally validate both types of methQTLs in an independent dataset comprising additional cell types and tissues. Finally, we validate selected methQTLs (PON1, ZNF155, NRG2) by ultra-deep local sequencing. In line with previous reports, we find cell-type-specific methQTLs to be preferentially located in enhancer elements. ConclusionsOur analysis demonstrates that a systematic analysis of methQTLs provides important new insights on the influences of genetic variants to cell-type-specific epigenomic variation.

genetics↗

bioRxiv2021

Nucleocapsid vaccine elicits spike-independent SARS-CoV-2 protective immunity

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing antibodies target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with HAd5 expressing the nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and k18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral antigens, even if they are not a target of neutralizing antibodies, to broaden epitope coverage and immune effector mechanisms.

immunology↗

bioRxiv2021

Human nasal and lung tissues infected ex vivo with SARS-CoV-2 provide insights into differential tissue-specific and virus-specific innate immune responses in the upper and lower respiratory tract

The nasal-mucosa constitutes the primary entry site for respiratory viruses including SARS-CoV-2. While the imbalanced innate immune response of end-stage COVID-19 has been extensively studied, the earliest stages of SARS-CoV-2 infection at the mucosal entry site have remained unexplored. Here we employed SARS-CoV-2 and influenza virus infection in native multi-cell-type human nasal turbinate and lung tissues ex vivo, coupled with genome-wide transcriptional analysis, to investigate viral susceptibility and early patterns of local-mucosal innate immune response in the authentic milieu of the human respiratory tract. SARS-CoV-2 productively infected the nasal turbinate tissues, predominantly targeting respiratory epithelial cells, with rapid increase in tissue-associated viral sub-genomic mRNA, and secretion of infectious viral progeny. Importantly, SARS-CoV-2 infection triggered robust antiviral and inflammatory innate immune responses in the nasal mucosa. The upregulation of interferon stimulated genes, cytokines and chemokines, related to interferon signaling and immune-cell activation pathways, was broader than that triggered by influenza virus infection. Conversely, lung tissues exhibited a restricted innate immune response to SARS-CoV-2, with a conspicuous lack of type I and III interferon upregulation, contrasting with their vigorous innate immune response to influenza virus. Our findings reveal differential tissue-specific innate immune responses in the upper and lower respiratory tract, that are distinct to SARS-CoV-2. The studies shed light on the role of the nasal-mucosa in active viral transmission and immune defense, implying a window of opportunity for early interventions, whereas the restricted innate immune response in early-SARS-CoV-2-infected lung tissues could underlie the unique uncontrolled late-phase lung damage of advanced COVID-19. IMPORTANCEIn order to reduce the late-phase morbidity and mortality of COVID-19, there is a need to better understand and target the earliest stages of SARS-CoV-2 infection in the human respiratory tract. Here we have studied the initial steps of SARS-CoV-2 infection and the consequent innate immune responses within the natural multicellular complexity of human nasal-mucosal and lung tissues. Comparing the global innate response patterns of nasal and lung tissues, infected in parallel with SARS-CoV-2 and influenza virus, we have revealed distinct virus-host interactions in the upper and lower respiratory tract, which could determine the outcome and unique pathogenesis of SARS-CoV-2 infection. Studies in the nasal-mucosal infection model can be employed to assess the impact of viral evolutionary changes, and evaluate new therapeutic and preventive measures against SARS-CoV-2 and other human respiratory pathogens.

microbiology↗

bioRxiv2021

Chromatin accessibility regulates intron retention in a cell type-specific manner

Dynamic intron retention (IR) in vertebrate cells is of widespread biological importance. Aberrant IR is associated with numerous human diseases including cancer. Despite consistent reports demonstrating intrinsic sequence features that predispose introns to become retained, conflicting findings about cell type-specific IR regulation demand a systematic analysis in a controlled experimental setting. We integrated matched transcriptomics and epigenetics data (including DNA methylation, nucleosome occupancy, histone modifications) from primary human myeloid and lymphoid cells. Using machine learning we trained two complementary models to determine the role of epigenetic factors in the regulation of IR. We show that increased chromatin accessibility contributes substantially to the retention of introns in a cell-specific manner. We also confirm that intrinsic characteristics of introns are key for them to evade splicing. With mounting reports linking pathogenic alterations to RNA processing, our findings may have profound implications for the design of therapeutic approaches targeting aberrant splicing.

bioinformatics↗

bioRxiv2020

Wild-type sTREM2 blocks Aβ aggregation and neurotoxicity, while the Alzheimer's R47H mutant does the opposite

Missense mutations (e.g. R47H) of the microglial receptor TREM2 increase risk of Alzheimers disease (AD), and the soluble ectodomain of wild-type TREM2 (sTREM2) appears to protect in vivo, but the underlying mechanisms are unclear. We show that A{beta} oligomers bind to TREM2, inducing shedding of sTREM2. Wild-type sTREM2 inhibits A{beta} oligomerization, fibrillization and neurotoxicity, and disaggregates preformed A{beta} oligomers and protofibrils. In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric A{beta}, but rather promotes A{beta} protofibril formation and neurotoxicity. Thus, in addition to mediating phagocytosis, wild-type TREM2 may protect against amyloid pathology by A{beta}-induced release of sTREM2 that blocks A{beta} aggregation and neurotoxicity; while R47H sTREM2 promotes A{beta} aggregation into neurotoxic forms, which may explain why the R47H variant gene increases AD risk several fold.

biochemistry↗

bioRxiv2020

Single-Strand DNA Breaks Cause Replisome Disassembly

DNA damage impedes replication fork progression and threatens genome stability. Upon encounter with most DNA adducts, the replicative CMG helicase (CDC45-MCM2-7-GINS) stalls or uncouples from the point of synthesis, yet CMG eventually resumes replication. However, little is known about the effect on replication of single-strand breaks or "nicks", which are abundant in mammalian cells. Using Xenopus egg extracts, we reveal that CMG collision with a nick in the leading strand template generates a blunt-ended double-strand break (DSB). Moreover, CMG, which encircles the leading strand template, "runs off" the end of the DSB. In contrast, CMG collision with a lagging strand nick generates a broken end with a single-stranded overhang. In this setting, CMG translocates beyond the nick on double-stranded DNA and is then actively removed from chromatin by the p97 ATPase. Our results show that nicks are uniquely dangerous DNA lesions that invariably cause replisome disassembly, and they argue that CMG cannot be deposited on dsDNA while cells resolve replication stress. HighlightsO_LIThe structures of leading and lagging strand collapsed forks are different C_LIO_LICMG passively "runs off" the broken DNA end during leading strand fork collapse C_LIO_LICMG is unloaded from duplex DNA after lag collapse in a p97-dependent manner C_LIO_LINicks are uniquely toxic lesions that cause fork collapse and replisome disassembly C_LI

molecular biology↗

bioRxiv2020

Genome wide efficiency profiling reveals modulation of maintenance and de novo methylation by Tets

A precise understanding of DNA methylation dynamics on a genome wide scale is of great importance for the comprehensive investigation of a variety of biological processes such as reprogramming of somatic cells to iPSCs, cell differentiation and also cancer development. To date, a complex integration of multiple and distinct genome wide data sets is required to derive the global activity of DNA modifying enzymes. We present GwEEP - Genome-wide Epigenetic Efficiency Profiling as a versatile approach to infer dynamic efficiency changes of DNA modifying enzymes at base pair resolution on a genome wide scale. GwEEP relies on genome wide oxidative Hairpin Bisulfite sequencing (HPoxBS) data sets, which are translated by a sophisticated hidden Markov model into quantitative enzyme efficiencies with reported confidence around the estimates. GwEEP in its present form predicts de novo and maintenance methylation efficiencies of Dnmts, as well as the hydroxylation efficiency of Tets but its purposefully flexible design allows to capture further oxidation processes such as formylation and carboxylation given available data in the future. Applied to a well characterized ES cell model, GwEEP precisely predicts the complex epigenetic changes following a Serum-to-2i shift i.e., (i) instant reduction in maintenance efficiency (ii) gradually decreasing de novo methylation efficiency and (iii) increasing Tet efficiencies. In addition, a complementary analysis of Tet triple knock-out ES cells confirms the previous hypothesized mutual interference of Dnmts and Tets. GwEEP is applicable to a wide range of biological samples including cell lines, but also tissues and primary cell types. MOTIVATIONDynamic changes of DNA methylation patterns are a common phenomenon in epigenetics. Although a stable DNA methylation profile is essential for cell identity, developmental processes require the rearrangement of 5-methylcytosine in the genome. Stable methylation patterns are the result of balanced Dnmts and Tets activities, while methylome transformation results from a coordinated change in Dnmt and Tet efficiencies. Such transformations occur on a global scale, for example during the reprogramming of maternal and paternal methylation patterns and the establishment of novel cell type specific methylomes during embryonic development in vivo, but also in vitro during (re)programming of induced pluripotent stem cells, as well as somatic cells. In addition, local (de)methylation events are key for gene regulation during cell differentiation. A detailed understanding of Dnmt and Tet cooperation is essential for understanding natural epigenetic adaptation as well as optimization of in vitro (re)programming protocols. For this purpose, we developed a pipeline for quantitative and precise estimation of Dnmt and Tet activity. Using only double strand methylation information, GwEEP infers accurate maintenance and de novo methylation efficiency of Dnmts, as well as hydroxylation efficiency of Tets at single base resolution. Thus, we believe GwEEP provides a powerful tool for the investigation of methylome rearrangements in various systems.

genomics↗

bioRxiv2020

Meta-analysis of epigenome-wide association studies in Alzheime's disease highlights 220 differentially methylated loci across cortex

Epigenome-wide association studies of Alzheimers disease have highlighted neuropathology-associated DNA methylation differences, although existing studies have been limited in sample size and utilized different brain regions. Here, we combine data from six DNA methylomic studies of Alzheimers disease (N=1,453 unique individuals) to identify differential methylation associated with Braak stage in different brain regions and across cortex. We identify 236 CpGs in the prefrontal cortex, 95 CpGs in the temporal gyrus and ten CpGs in the entorhinal cortex at Bonferroni significance, with none in the cerebellum. Our cross-cortex meta-analysis (N=1,408 donors) identifies 220 CpGs associated with neuropathology, annotated to 121 genes, of which 84 genes have not been previously reported at this significance threshold. We have replicated our findings using two further DNA methylomic datasets consisting of a further > 600 unique donors. The meta-analysis summary statistics are available in our online data resource (www.epigenomicslab.com/ad-meta-analysis/).

genomics↗

bioRxiv2019

Reference-free deconvolution of complex DNA methylation data - a detailed protocol

Epigenomic profiling enables unique insights into human development and diseases. Often the analysis of bulk samples remains the only feasible option for studying complex tissues and organs in large patient cohorts, masking the signatures of important cell populations in convoluted signals. DNA methylomes are highly cell type-specific, and enable recovery of hidden components using advanced computational methods without the need for reference profiles. We propose a three-stage protocol for reference-free deconvolution of DNA methylomes comprising: (i) data preprocessing, confounder adjustment and feature selection, (ii) deconvolution with multiple parameters, and (iii) guided biological inference and validation of deconvolution results. Our protocol simplifies the analysis and integration of DNA methylomes derived from complex samples, including tumors. Applying this protocol to lung cancer methylomes from TCGA revealed components linked to stromal cells, tumor-infiltrating immune cells, and associations with clinical parameters. The protocol takes less than four days to complete and requires basic R skills.

bioinformatics↗

bioRxiv2019

Parkinson’s disease phenotypes in patient specific brain organoids are improved by HP-β-CD treatment

The etiology of Parkinsons disease (PD) is only partially understood despite the fact that environmental causes, risk factors, and specific gene mutations are contributors to the disease. Biallelic mutations in the PTEN-induced putative kinase 1 (PINK1) gene involved in mitochondrial homeostasis, vesicle trafficking, and autophagy, are sufficient to cause PD. By comparing PD patient-derived cells, we show differences in their energetic profile, imbalanced proliferation, apoptosis, mitophagy, and a reduced differentiation efficiency to dopaminergic neurons compared to control cells. Using CRISPR/Cas9 gene editing, correction of a patients point mutation ameliorated the metabolic properties and neuronal firing rates but without reversing the differentiation phenotype. However, treatment with 2-Hydroxypropyl-{beta}-Cyclodextrin (HP-{beta}-CD) increased the mitophagy capacity of neurons leading to an improved dopaminergic differentiation of patient specific neurons in midbrain organoids. In conclusion, we show that treatment with a repurposed compound is sufficient for restoring dopaminergic differentiation of PD patient-derived cells.

neuroscience↗

bioRxiv2019

Establishing the phenotypic basis of adherent-invasive Escherichia coli (AIEC) pathogenicity in intestinal inflammation

Background & AimsAdherent-invasive Escherichia coli (AIEC) are enriched in ileal Crohns disease patients and implicated in disease etiology. However, AIEC pathogenesis is poorly understood, and it is unclear if the expansion of these organisms contributes to inflammatory bowel disease (IBD). Questions also remain as to what extent the various in vitro phenotypes used to classify AIEC are pathologically relevant.\n\nMethodsWe utilized a combination of in vitro phenotyping and a murine model of intestinal inflammation to systematically relate AIEC phenotypes to pathogenicity for 30 mucosa-associated human-derived E. coli strains. In vitro assays used included survival/replication in and TNF- production by J774 macrophages as well as invasion/replication in Caco2 intestinal epithelial cells.\n\nResultsAIEC do not form a phenotypic group that is clearly separated from non-AIEC. However, E. coli strains displaying in vitro AIEC phenotypes caused, on average, more severe intestinal inflammation. Survival/replication of strains in J774 and Caco2 cells were positively correlated with disease in vivo, while adherence to Caco2 cells and TNF- production by J774 cells were not. Importantly, co-colonization with adherent non-AIEC strains ameliorated AIEC-mediated disease.\n\nConclusionOur findings do not support the existence of an AIEC pathovar that can be clearly separated from commensal E. coli. However, intracellular survival/replication phenotypes do contribute to murine intestinal inflammation, suggesting that the AIEC overgrowth observed in human IBD makes a causal contribution to disease. The ability to differentiate pathologically-relevant AIEC phenotypes from those that are not provides an important foundation for developing strategies to predict, diagnose and treat human IBD through characterizing and modulating patient E. coli populations.

microbiology↗

bioRxiv2019

A novel Ca2+-binding protein influences photosynthetic electron transport in Anabaena sp. PCC 7120

Ca2+ is a potent signalling molecule that regulates many cellular processes. In cyanobacteria, Ca2+ has been linked to cell growth, stress response and photosynthesis, and to the development of specialist heterocyst cells in certain nitrogen-fixing species. Despite this, the pathways of calcium signal transduction in cyanobacteria are poorly understood, and only a few protein components are known. The current study describes a previously unreported calcium-binding protein which was called the Calcium Sensor EF-hand (CSE), which is conserved in filamentous, nitrogen-fixing cyanobacteria. CSE is shown to bind calcium, which induces a conformational change in the protein structure. Poor growth of a strain of Anabaena sp. PCC 7120 overexpressing CSE was attributed to diminished photosynthetic performance. Transcriptomics, biophysics and proteomics analyses revealed modifications in the light-harvesting phycobilisome and photosynthetic reaction center-protein complexes, and downregulated respiration.

plant biology↗

bioRxiv2019

From prevalence to incidence - a new approach in the hospital setting

Point-prevalence surveys (PPSs) are often used to estimate the prevalence of healthcare-associated infections (HAIs). Methods for estimating incidence of HAIs from prevalence have been developed, but application of these methods is often difficult because key quantities, like the average length of infection, cannot be derived directly from the data available in a PPS. We propose a new theory-based method to estimate incidence from prevalence data dealing with these limitations and compare it to other estimation methods in a simulation study. In contrast to previous methods, our method does not depend on any assumptions on the underlying distributions of length of infection and length of stay. As a basis for the simulation study we use data from the second study of nosocomial infections in Germany (Nosokomiale Infektionen in Deutschland, Erfassung und Pravention - NIDEP2) and the European surveillance of HAIs in intensive care units (HAI-Net ICU). The new method compares favourably with the other estimation methods and has the advantage of being consistent in its behaviour across the different setups. It is implemented in an R-package prevtoinc which will be freely available on CRAN (http://cran.r-project.org/).

epidemiology↗