Biology subjects

Fischer, J.

Publications and source records attributed to Fischer, J..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

No Miracle, Just a Mineral: The Not-So-Magical Antimicrobial World of Chlorine Dioxide

This study evaluates the in vitro antimicrobial efficacy and cytotoxicity of acidified sodium chlorite (ASC), a source of chlorine dioxide. Despite its controversial promotion in alternative medicine as a cure-all solution, known as "Miracle Mineral Solution" (MMS), the data on its factual medicinal activity is very limited. Therefore, we aimed to elucidate the activity of ASC against biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Streptococcus mutans, Pseudomonas aeruginosa, Escherichia coli, and Lactobacillus sp. The study also investigated the in vitro cytotoxic effects of ASC towards eukaryotic fibroblasts and in in vivo model of Galleria mellonella larvae. Our findings demonstrate that these concentrations of ASC which can effectively eradicate biofilms, also pose potential health risks due to their in vitro and in vivo cytotoxicity. It implies that ASC applied in human can lead to damage to the mucous membrane of the gastrointestinal tract. This research contributes to the ongoing debate on the safety and efficacy of chlorine dioxide in clinical applications, highlighting the need for precise dosing to avoid mucosal damage in therapeutic contexts.

microbiology↗

bioRxiv2024

A Novel Imaging Mass Spectrometry Computational Tool for Biomedical Discovery: Untargeted pixel-by-pixel imaging of Metabolite Ratio Pairs

Mass spectrometry imaging (MSI) is a powerful technology used to define the spatial distribution and relative abundance of metabolites across tissue cryosections. While software packages exist for pixel-by-pixel individual metabolite and limited target pairs of ratio imaging, the research community lacks an easy computing and application tool that images any metabolite abundance ratio pairs. Importantly, recognition of correlated metabolite pairs may contribute to the discovery of unanticipated molecules in shared metabolic pathways. Here, we describe the development and implementation of an untargeted R package workflow for pixel-by-pixel ratio imaging of all metabolites detected in an MSI experiment. Considering untargeted MSI studies of murine brain and embryogenesis, we demonstrate that ratio imaging minimizes systematic data variation introduced by sample handling, markedly enhances spatial image contrast, and reveals previously unrecognized metabotype-distinct tissue regions. Furthermore, ratio imaging facilitates identification of novel regional biomarkers and provides anatomical information regarding spatial distribution of metabolite-linked biochemical pathways. The algorithm described herein is applicable to any MSI dataset containing spatial information for metabolites, peptides or proteins, offering a potent hypothesis generation tool to enhance knowledge obtained from current spatial metabolite profiling technologies.

bioinformatics↗

bioRxiv2024

Endoplasmic reticulum stress signaling actively contributes to therapy resistance in colorectal cancer

PurposeWe investigated the involvement of endoplasmic reticulum (ER) stress signaling in cancer cell responses to chemo- and radiotherapy, focusing on three main ER stress mediators, the transcription factors ATF4, XBP1 and ATF6. MethodsPublic cancer genome datasets were assessed for alterations in ER stress mediators. Surgically resected colorectal cancer tissues were tested by flow cytometry and used to generate patient-derived organoids. Human cell lines and organoids were characterized under oxaliplatin treatment, alone or combined with pharmacological inhibitors of the three ER stress branches, or X-ray irradiation, for cytotoxicity, activation of ER stress and proteome changes. To monitor ER stress in real time, stable HEK293 kidney epithelial cell lines were established expressing ATF4, XBP1, or ATF6, fused with a fluorophore. ResultsGenomic amplification of ATF6, but not ATF4 or XBP1, was frequent in solid tumor entities like breast, lung and colorectal cancer and significantly associated with reduced disease-free survival. In colorectal cancer, increased ATF6 was associated with genetic instability. Basal ER stress mediator expression was correlated to chemoresistance in colorectal cancer cell lines, and generally high in cancer cells compared to HEK293 cells. With proteomics and live HEK293-based reporter lines, we noted that oxaliplatin treatment induced ER stress in a remarkably different way from the canonical ER stress inducer thapsigargin. Moreover, modulation of ER stress signaling by exogenous expression of the stress mediators positively affects chemoresistance, and pharmacological inhibition of ATF6 sensitizes ER-stressed HCT116 colorectal cancer cells to chemotherapy. Of note, cellular stress responses was strongly dependent on the individual transcription factor: XBP1-driven response appeared multi-functional, involved in ribosome biogenesis stress and associated with oxaliplatin resistance. ATF6-dependent stress signaling was involved in DNA damage repair, and was essential for radioresistance. Moreover, chemoresistance in HCT116 cancer cells was impaired by pharmacological ATF6 inhibition. ConclusionActivation of the ER stress signaling may be critically involved in acquired chemo- and radioresistance. Due to their apparent cytoprotective roles, ATF6 and XBP1 could be attractive predictive biomarkers and putative therapeutic targets. SUMMARYTo address their roles in the clinical context, genomic alterations of ATF4, XBP1 and/or ATF6 in human solid tumors were assessed with respect to prognosis and genomic instability. Moreover, surgically resected CRC patient tissues were tested for expression of ER stress markers by flow cytometry and associated with clinical characteristics. In addition, a panel of human cell lines and patient-derived colon organoids were characterized under therapeutic conditions for expression and activation of ER stress proteins, and resulting cytotoxicity was determined. To monitor and modulate ER stress activation in live cells with subcellular resolution, stable reporter cell lines expressing ATF4, sXBP1 or ATF6 proteins fused with a fluorophore were established. These lines were tested for gene or protein expression and cytotoxicity assays to analyze how activation or inhibition of ER stress proteins affects the cellular responses to oxaliplatin treatment or X-ray irradiation. Finally, mass spectrometric proteome analysis was performed to obtain an unbiased readout on the cellular responses to chemotherapy driven by the activation of the ER stress proteins.

cancer biology↗

bioRxiv2023

Bayesian Optimized sample-specific Networks Obtained ByOmics data (BONOBO)

Gene regulatory networks (GRNs) are effective tools for inferring complex interactions between molecules that regulate biological processes and hence can provide insights into drivers of biological systems. Inferring co-expression networks is a critical element of GRN inference as the correlation between expression patterns may indicate that genes are coregulated by common factors. However, methods that estimate co-expression networks generally derive an aggregate network representing the mean regulatory properties of the population and so fail to fully capture population heterogeneity. To address these concerns, we introduce BONOBO (Bayesian Optimized Networks Obtained By assimilating Omics data), a scalable Bayesian model for deriving individual sample-specific co-expression networks by recognizing variations in molecular interactions across individuals. For every sample, BONOBO assumes a Gaussian distribution on the log-transformed centered gene expression and a conjugate prior distribution on the sample-specific co-expression matrix constructed from all other samples in the data. Combining the sample-specific gene expression with the prior distribution, BONOBO yields a closed-form solution for the posterior distribution of the sample-specific co-expression matrices, thus making the method extremely scalable. We demonstrate the utility of BONOBO in several contexts, including analyzing gene regulation in yeast transcription factor knockout studies, prognostic significance of miRNA-mRNA interaction in human breast cancer subtypes, and sex differences in gene regulation within human thyroid tissue. We find that BONOBO outperforms other sample-specific co-expression network inference methods and provides insight into individual differences in the drivers of biological processes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/567119v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@f97de5org.highwire.dtl.DTLVardef@1b864a9org.highwire.dtl.DTLVardef@d8a384org.highwire.dtl.DTLVardef@d73091_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2023

ADEVO: Proof-of-concept of Adenovirus Directed EVOlution by random peptide display on the fiber knob

Directed evolution of viral vectors involves the generation of randomized libraries followed by artificial selection of improved variants. Directed evolution only yielded limited results in adenovirus vector (AdV) development until now, mainly due to insufficient complexities of randomized libraries. Clinical applications of AdVs as gene therapy or oncolytic vectors are still hampered by the predetermined tropism of natural types. To overcome this challenge, we hypothesized that the technology of randomized peptide insertions on the capsid surface can be incorporated into the AdV bioengineering toolbox for vector retargeting. Here we developed Adenovirus Directed EVOlution (ADEVO) protocols based on fiber knob peptide display. As a proof-of-concept, HAdV-C5-derived libraries were constructed following three distinct protocols and selected on A549-DCAR cells that lack the HAdV-C5 primary receptor, with the goal of identifying variants able to infect and lyse these tumor cells more efficiently. All protocols enabled the construction of high complexity libraries with up to 9.6x10^5 unique variants, an approximate 100-fold improvement compared to previously published AdV libraries. After selection, the most enriched variants did not display enhanced infectivity but rather more efficient replication and cell lysis. This warrants investigations into potential unsuspected involvement of the fiber protein in adenovirus replication. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/567388v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@187f7baorg.highwire.dtl.DTLVardef@1990555org.highwire.dtl.DTLVardef@12d3c60org.highwire.dtl.DTLVardef@d703da_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Combining CRISPR/Cas mediated terminal resolution with a novel genetic workflow to achieve high diversity adenoviral libraries

While recombinant Adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR/Cas9 mediated in vivo terminal resolution (CTR) aiding high efficiency rescue of rAds from recombinant DNA. Here we report on a genetic workflow that allows construction of BAC-based rAd-libraries employing the efficiency of CTR. We utilized frequent, pre-existing genomic sequences to allow insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In a second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half-site directed fragment replacement (HFR) allows introduction of >106 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of [~]2.5x104 modified rAd per cm2 of transfected cell culture. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/566979v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1db61a7org.highwire.dtl.DTLVardef@1e85764org.highwire.dtl.DTLVardef@c17178org.highwire.dtl.DTLVardef@a299_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Overcoming Pitfalls when Estimating Variation in Parental Investment: A Simulation Study

1. BackgroundIn many animal species, parents allocate resources to enhance offspring survival and thereby reproductive success ( parental investment). Parental care behaviors are a crucial part of parental investment, and one critical question in behavioral ecology is to which degree variation in parental care affects offspring development and survival. Crucially, parental care behaviors vary with offspring age, which creates non-trivial problems for statistical analyses, specifically when sampling is incomplete or biased. 2. MethodsWe conducted a simulation study to illustrate the problem when analyzing average parental care behaviors while not correcting for offspring age ( conventional approach), and propose a modeling approach to correct for offspring age and sampling biases. We simulated a set of different scenarios, varying with regard to sample size, sampling scheme, and variation in parental behavior. To model parent behavior as a function of offspring age, we fitted a Generalized Linear Mixed Model to each simulated data set. We included offspring age as a fixed effect and extracted Best Linear Unbiased Predictors (BLUPs) for the random intercept of parent identity. These BLUPs represent the parental behavior corrected for offspring age. Finally, we assessed how our proposed modeling approach compares to the conventional approach. 3. ResultsThe proposed modeling approach was superior to the conventional approach. More specifically, the conventional approach clearly overestimated the variation between parents, and even diagnosed appreciable variation when there was none in the simulated data. This bias was exacerbated in incomplete or unbalanced sampling schemes. 4. Conclusion.Our simulation study clearly demonstrates the necessity of correcting for offspring age in analyses of parental behavior. We strongly suggest including random slopes of offspring age within parent-offspring dyads, because doing so can reveal parent-specific trajectories of the modeled behavior against offspring age. Our approach allows for the inclusion of further categorical and continuous predictor variables that may affect parental behavior, such as parity or ecological conditions. In summary, the proposed modeling approach has several advantages over conventional methods.

animal behavior and cognition↗

bioRxiv2023

Gene regulatory Networks Reveal Sex Difference in Lung Adenocarcinoma

Lung adenocarcinoma (LUAD) has been observed to have significant sex differences in incidence, prognosis, and response to therapy. However, the molecular mechanisms responsible for these disparities have not been investigated extensively. Sample-specific gene regulatory network methods were used to analyze RNA sequencing data from non-cancerous human lung samples from The Genotype Tissue Expression Project (GTEx) and lung adenocarcinoma primary tumor samples from The Cancer Genome Atlas (TCGA); results were validated on independent data. We observe that genes associated with key biological pathways including cell proliferation, immune response and drug metabolism are differentially regulated between males and females in both healthy lung tissue, as well as in tumor, and that these regulatory differences are further perturbed by tobacco smoking. We also uncovered significant sex bias in transcription factor targeting patterns of clinically actionable oncogenes and tumor suppressor genes, including AKT2 and KRAS. Using differentially regulated genes between healthy and tumor samples in conjunction with a drug repurposing tool, we identified several small-molecule drugs that might have sex-biased efficacy as cancer therapeutics and further validated this observation using an independent cell line database. These findings underscore the importance of including sex as a biological variable and considering gene regulatory processes in developing strategies for disease prevention and management.

cancer biology↗

bioRxiv2023

(De)composing sociality: disentangling individual-specific from dyad-specific propensities to interact

In group-living animals, relationships between group members are often highly differentiated. Some dyads can maintain strong and long-lasting relationships, while others are only connected by weak or fleeting ties. Evidence accumulates that aspects of social relationships are related to reproductive success and survival. Yet, few of these analyses have considered that frequent or prolonged affiliative interactions between two individuals can be principally driven by two distinct processes: namely, the overall gregariousness of individuals, and dyadic affinity, i.e., the preference the members of the dyad have to interact specifically with one another. Crucially, these two axes of sociality cannot be observed directly, although distinguishing them is essential for many research questions, for example, when estimating kin bias or when studying the link between sociality and fitness. We present a principled statistical framework to estimate the two underlying sociality axes using dyadic interaction data. We also provide a basic R package bamoso, which implements models based on the proposed framework and allows visual and numerical evaluation of the estimated sociality axes. We demonstrate critical features of the proposed modeling framework with simulated and empirical data: (1) the possibility of checking model fit against observed data, (2) the assessment of uncertainty in the estimated sociality parameters, and (3) the possibility to extend it to more complex models that use interaction data to estimate the relationship between individual-level social features and individual-level outcomes in a unified model. Our model provides a principled foundation to explain variation in dyadic interactions. This approach allows us to address questions about the relationship between variation in sociality characteristics and other features of interest, both within and across species.

animal behavior and cognition↗

bioRxiv2023

Multimodally trackable and clinically translatable platform for modelling human demyelinating brain diseases by temporally dispersed chemically induced lesions in thepig brain

BackgroundDespite advances in therapy, inflammatory demyelinating diseases of the central nervous system, such as multiple sclerosis, remain important causes of morbidity among young adults. Translation of remyelinating paradigms from current murine models is encumbered by the small size and low white matter content of the brains, limiting the spatial resolution of diagnostic imaging. Large animal models might be more suited for this purpose but pose significant technological, ethical and logistical challenges. MethodWe induced reversible and targeted cerebral demyelinating lesions by controlled injection of lysophosphatidylcholine in the minipig brain. One strength of the approach is the serial induction, allowing parallel imaging of successive stages of de-/remyelination. FindingsWe demonstrate controlled, clinically unapparent, reversible and multimodally trackable brain white matter demyelination in a large animal model. Lesions were amenable to follow-up using the same clinical imaging modalities (3T magnetic resonance imaging, 11C-PIB positron emission tomography) and standard histopathology protocols as for human diagnostics, as well as electron microscopy to compare against biopsy data from two patients with cerebral demyelination. InterpretationBy employing human diagnostic tools and validating the model against data from related human diseases, our platform overcomes one important translational barrier of current animal brain demyelination models while having the potential for developing diagnostic procedures and imaging biomarkers. Remyelination and axon preservation dynamics diverge from classical rodent models. FundingThis work was supported by the DFG under Germanys Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy, ID 390857198) and TRR 274/1 2020, 408885537 (projects B03 and Z01). Research in contextO_ST_ABSEvidence before this studyC_ST_ABSInflammatory demyelinating diseases of the central nervous system (CNS), targeting primarily the white matter (WM) of the brain and spinal cord, such as multiple sclerosis (MS), still represent some of the most important non-traumatic causes of disability in young adults. Current animal models based on murine species, for example, experimental autoimmune encephalomyelitis, have been demonstrated to reliably depict pathophysiological facets of human disease. However, they are nevertheless encumbered by the low WM content and the small size of murine brains, which still pose a translational barrier to diagnostic imaging tools used in a clinical context in human patients. Minipigs are increasingly being used to model human neurological diseases, as yet primarily in the context of neurodegenerative disorders. Added value of this studyHere, we establish a platform for Minipig Stereotactic White-matter Injection using Navigation by Electromagnetism (MiniSWINE) and validate such a tool in a clinical multimodal imaging and microscopy setting against biopsy and imaging data from human demyelinating disorders across different disease stages, as well as against existing and potentially emerging human diagnostic imaging. Moreover, in order to overcome the neuroanatomical challenges of stereotactic injection in the pig brain, we designed a new electromagnetic-guided tracking system whose key advantage is the direct measurement of the injection cannula tip position in situ. Another strength of our study lies in its setup, characterized by the serial induction of successive stages of de- and remyelination, allowing for multimodal assessment via imaging and histopathology or electron microscopy of multiple stages in parallel. The remyelination dynamics inferred in this context diverge from the classical rodent studies, by exhibiting incomplete remyelination at the subacute stage, persistent astroglial and microglial activation as well as a minor degree of secondary axonal degeneration. Thus, they more closely resemble human inflammatory demyelinating brain plaques. Implications of all the available evidenceWe believe that MiniSWINE links evidence from well-established demyelination-induction methods from rodent models of CNS demyelinating disorders, as well as from human imaging and biopsy data, while at the same time providing a novel platform for the potential development of diagnostic procedures, discovery of imaging biomarkers and testing of remyelinating agents in diseases such as MS. Thus, it can have particular relevance to human health in the context of future translational animal model-based research in inflammatory demyelinating disorders of the CNS. Additionally, our electromagnetic-guided injection technique may enhance stereotactic substance delivery in human neurosurgery.

neuroscience↗

bioRxiv2023

Fully Automated Sequential Immunofluorescence (seqIF) for Hyperplex Spatial Proteomics

Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF). The fully automated method uses successive applications of antibody incubation and elution, and in-situ imaging enabled by an integrated microscope and a microfluidic chip that provides optimized optical access to the sample. We show seqIF data on different sample types such as tumor and healthy tissue, including 40-plex on a single tissue section that is obtained in less than 24 hours, using off-the-shelf antibodies. We also present extensive characterization of the developed method, including elution efficiency, epitope stability, repeatability and reproducibility, signal uniformity, and dynamic range, in addition to marker and panel optimization strategies. The streamlined workflow using off-the-shelf antibodies, data quality enabling quantitative downstream analysis, and ease of reaching hyperplex levels make seqIF suitable for immune-oncology research and other disciplines requiring spatial analysis, paving the way for its adoption in clinical settings.

cancer biology↗

bioRxiv2023

Alarm barks of male vervet monkeys as displays of male quality

Research on the vocal behaviour of non-human primates is often motivated by a desire to understand the origins of semantic communication, which led to a partial separation of this research from ecological-evolutionary approaches. To bridge this gap, we returned to the textbook example of semantic communication in animals, the vervet monkey, Chlorocebus pygerythrus, alarm call system, and investigated whether male alarm barks fulfil a dual function of alarming and indicating male quality. Barks are loud calls, produced by adult males, in response to large carnivores. However, since barks occasionally occur in agonistic interactions, we investigated whether barks may also indicate male quality. We recorded natural barking events over 23 months, sampling individual male participation from 45 individuals in six free-ranging groups at the Mawana Game Reserve, KwaZulu-Natal, South Africa. We hypothesized that barking frequency is under intra-sexual selection and predicted that barking frequency would increase with male rank and the degree of male-male competition. We found that the highest-ranking males were more likely to produce barks than lower-ranking males and that the number of daily barking events increased during the mating season. We advocate studying primate communication in its evolutionary context to achieve a comprehensive understanding of call "meaning".

animal behavior and cognition↗

bioRxiv2023

Genome-wide coancestry reveals details of ancient and recent male-driven reticulation in baboons

Baboons (genus Papio) are a morphologically and behaviorally diverse clade of catarrhine monkeys that have experienced hybridization between phenotypically and genetically distinct phylogenetic species. We used high coverage whole genome sequences from 225 wild baboons representing 19 geographic localities to investigate population genomics and inter-species gene flow. Our analyses provide an expanded picture of evolutionary reticulation among species and reveal novel patterns of population structure within and among species, including differential admixture among conspecific populations. We describe the first example of a baboon population with a genetic composition that is derived from three distinct lineages. The results reveal processes, both ancient and recent, that produced the observed mismatch between phylogenetic relationships based on matrilineal, patrilineal, and biparental inheritance. We also identified several candidate genes that may contribute to species-specific phenotypes. One-Sentence SummaryGenomic data for 225 baboons reveal novel sites of inter-species gene flow and local effects due to differences in admixture.

evolutionary biology↗

bioRxiv2023

Inhibition of hyaluronan synthesis prevents β-cell loss in obesity-associated type 2 diabetes

Pancreatic {beta}-cell dysfunction and death are central to the pathogenesis of type 2 diabetes (T2D). We have identified a novel role for the inflammatory extracellular matrix polymer hyaluronan (HA) in this pathophysiology. Low levels of HA are present in healthy pancreatic islets. However, HA substantially accumulates in cadaveric islets of human T2D and islets of the db/db mouse model of T2D in response to hyperglycemia. Treatment with 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, or the deletion of the major HA receptor CD44, preserve glycemic control and insulin levels in db/db mice despite ongoing weight gain, indicating a critical role for this pathway in T2D pathogenesis. 4-MU treatment and the deletion of CD44 likewise preserve glycemic control in other settings of {beta}-cell injury including streptozotocin treatment and islet transplantation. Mechanistically, we find that 4-MU increases the expression of the apoptosis inhibitor survivin, a downstream transcriptional target of CD44 dependent on HA/CD44 signaling, on {beta}-cells such that caspase 3 activation does not result in {beta}-cell apoptosis. These data indicate a role for HA accumulation in diabetes pathogenesis and suggest that it may be a viable target to ameliorate {beta}-cell loss in T2D. These data are particularly exciting, because 4-MU is already an approved drug (also known as hymecromone), which could accelerate translation of these findings to clinical studies.

immunology↗

bioRxiv2023

Mitochondrial translocation of TFEB regulates complex I and inflammation

TFEB is a master regulator of autophagy, lysosome biogenesis and mitochondrial metabolism that works, and immunity, primarily through transcription controlled by cytosol-to-nuclear translocation. Emerging data indicate additional regulatory interactions at the surface of organelles such as lysosomes. Here we show that TFEB has a non-transcriptional role in mitochondria, regulating the electron transport chain complex I to down-modulate inflammation. Proteomic analysis revealed extensive TFEB co-precipitation with several mitochondrial proteins, whose interactions are disrupted upon infection with S. Typhimurium. Localization of TFEB in the mitochondrial matrix was confirmed by high resolution confocal microscopy and biochemistry with translocation dependent on a conserved N-terminal TOMM20-binding motif enhanced by mTOR inhibition. Within the mitochondria, TFEB and protease LONP1 antagonistically co-regulate complex I, reactive oxygen species and the inflammatory response. Consequently, during infection, lack of TFEB specifically in the mitochondria exacerbates the expression of pro-inflammatory cytokines, contributing to innate immune pathogenesis.

cell biology↗

bioRxiv2023

Efficiently Quantifying DNA Methylation for Bulk- and Single-cell Bisulfite Data

MotivationDNA CpG methylation (CpGm) has proven to be a crucial epigenetic factor in the gene regulatory system. Assessment of DNA CpG methylation values via whole-genome bisulfite sequencing (WGBS) is, however, computationally extremely demanding. ResultsWe present FAst MEthylation calling (FAME), the first approach to quantify CpGm values directly from bulk or single-cell WGBS reads without intermediate output files. FAME is very fast but as accurate as standard methods, which first produce BS alignment files before computing CpGm values. We present experiments on bulk and single-cell bisulfite datasets in which we show that data analysis can be significantly sped-up and help addressing the current WGBS analysis bottleneck for large-scale datasets without compromising accuracy. AvailabilityAn implementation of FAME is open source and licensed under GPL-3.0 at https://github.com/FischerJo/FAME.

bioinformatics↗

bioRxiv2022

Deficiency in hyaluronan synthase 3 attenuates ruptures in a murine model of abdominal aortic aneurysms by reduced aortic monocyte infiltration

Abdominal aortic aneurysms (AAA) are a common vascular disorder with a high mortality due to the prevalence of aortic ruptures. The underlying pathomechanisms are complex and involve immune cell infiltration and degradation of the vascular extracellular matrix (ECM). Hyaluronan (HA), synthesized at the plasma membrane by three HA synthase isoenzymes (HAS1-3), is not only a major constituent of the ECM but also known to directly affect the phenotype of vascular smooth muscle cells as well as immunological responses. Specifically, the HAS3 isoenzyme has been reported to play a major role in various inflammatory conditions. Therefore, the aim of the present study was to elucidate the role of HAS3-derived HA in the pathogenesis of abdominal aortic aneurysm. To this end, we used a murine model of Angiotensin II (AngII)-induced abdominal aortic aneurysms and dissections (AAAs/AADs) and could demonstrate that genetic depletion of Has3 improves survival in Apoe/Has3 double deficient (Apoe/Has3-DKO) mice via the reduced occurrence of aortic ruptures. Mechanistically, fewer elastica breaks were observed in Apoe/Has3-DKO mice compared to Apoe-KO littermates. This was associated with a decreased infiltration of myeloid immune cells into the vessel wall of Has3-deficient mice while in parallel elevated numbers of circulating leukocytes were detected. RNA seq analysis from aortic tissue pointed towards a disturbed endothelial-myeloid cell communication as a cause for the diminished recruitment of immune cells to the aortic wall. While endothelial cells were unaffected, upregulation of adhesion receptors as well as the HA receptor CD44, known to mediate leukocyte adhesion to the endothelium, was blunted in monocytes from Apoe/Has3-DKO mice in response to AngII treatment. These findings underline the pivotal detrimental role of monocytes HAS3-dependent pericellular HA matrix for an exaggerated immune cell recruitment to inflammatory foci giving here rise for an increased incidence of ruptured aortic aneurysms.

immunology↗

bioRxiv2022

Anti-integrin αvβ6 autoantibodies are a novel predictive biomarker in ulcerative colitis

Background and AimsBetter biomarkers for prediction of ulcerative colitis (UC) development and prognostication are needed. Anti-integrin v{beta}6 autoantibodies (anti-v{beta}6) have been described in UC patients. Here, we tested for the presence of anti-v{beta}6 antibodies in the pre-clinical phase of UC and studied their association with disease-related outcomes after diagnosis. MethodsAnti-v{beta}6 were measured in 4 longitudinal serum samples collected from 82 subjects who later developed UC and 82 matched controls from a Department of Defense pre-clinical cohort (PREDICTS). In a distinct, external validation cohort (GEM), we tested 12 pre-UC subjects and 49 matched controls. Further, anti-v{beta}6 were measured in 2 incident UC cohorts (COMPASS n=55 and OSCCAR n=104) and associations between anti-v{beta}6 and UC-related outcomes were defined using Cox proportional-hazards model. ResultsAnti-v{beta}6 were significantly higher among individuals who developed UC compared to controls up to 10 years before diagnosis in PREDICTS. The anti-v{beta}6 seropositivity was 12.2% 10 years before diagnosis and increased to 52.4% at the time of diagnosis in subjects who developed UC compared with 2.7% in controls across the 4 timepoints. Anti-v{beta}6 predicted UC development with an AUC of at least 0.8 up to 10 years before diagnosis. The presence of anti-v{beta}6 in pre-clinical UC samples was validated in the GEM cohort. Finally, high anti-v{beta}6 was associated with a composite of adverse UC-outcomes including hospitalization, disease extension, colectomy, systemic steroid use and/or escalation to biologic therapy in recently diagnosed UC. ConclusionAnti-integrin v{beta}6 auto-antibodies precede the clinical diagnosis of UC by up to 10 years and are associated with adverse UC-related outcomes.

immunology↗