Biology subjects

Mueller, S.

Publications and source records attributed to Mueller, S..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

HP1 deficiency results in De-Repression of Endogenous Retroviruses and Induction of Neurodegeneration via Complement

In aging cells and animal models of premature aging, heterochromatin loss coincides with transcriptional disruption including the activation of normally silenced endogenous retroviruses (ERVs). Here we show that loss of heterochromatin maintenance and de-repression of ERVs results in a chronic inflammatory environment characterized by neurodegeneration and cognitive decline. We discovered differential contributions of HP1 proteins to ERV silencing where HP1{gamma} is necessary and sufficient for H4K20me3 deposition and HP1{beta} deficiency causes aberrant DNA methylation. Combined loss of HP1{beta} and HP1{gamma} resulted in loss of DNA methylation at ERVK elements. Progressive ERV de-repression in HP1{beta}/{gamma} DKO mice was followed by stimulation of the integrated stress response, an increase of Complement 3+ reactive astrocytes and phagocytic microglia. This chronic inflammatory state coincided with age-dependent reductions in dendrite complexity and cognition. Our results demonstrate the importance of preventing loss of epigenetic maintenance, as this will be the only way postmitotic neuronal genomes can be protected and/or renewed. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/505641v4_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@f5feb7org.highwire.dtl.DTLVardef@25d73dorg.highwire.dtl.DTLVardef@5604a4org.highwire.dtl.DTLVardef@14ad84a_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

HSPA8 chaperone complex drives chaperone-mediated autophagy regulation in acute promyelocytic leukemia differentiation

Acute myeloid leukemia (AML) is a cancer of the hematopoietic system characterized by the hyperproliferation of undifferentiated cells of the myeloid lineage. While most of AML therapy are focusing on tumor debulking, all-trans retinoic acid (ATRA) induces differentiation in acute promeylocytic leukemia (APL) a particular subtype. Macroautophagy has been extensively investigated in the context of various cancers and is often dysregulated in AML where it can have context-dependent pro- or anti-leukemogenic effects. On the contrary, the implications of chaperone-mediated autophagy (CMA) on the pathophysiology of diseases are still being explored and its role in AML has remained elusive. To answer our questions we took advantages of human AML primary samples and databases. Furthermore, we used ATRA-sensitive (NB4) and -resistant (NB4-R1) cells to further dissect a potential function for CMA in ATRA-mediated neutrophil differentiation. NB4-R1 cells are unique in that they do respond to retinoic acid transcriptionally, but do not mature in response to retinoid signaling alone unless maturation is triggered by adding cAMP. Here, we report that CMA related mRNA transcripts are higher expressed in immature hematopoietic cells as compared to neutrophils. In line, lysosomal degradation of mCherry-KFERQ CMA reporter decreases during ATRA-induced differentiation of APL cells. On the other hand, using NB4-R1 cells we found that macroautophagy flux primed the ATRA resistant NB4-R1 cells to differentiate upon ATRA treatment, but reduced association of LAMP-2A and HSPA8 is necessary for complete neutrophil maturation. Accordingly, depletion of HSPA8 attenuated CMA activity and facilitated APL differentiation. In contrast, maintaining high CMA activity by ectopic expression of LAMP-2A impeded APL differentiation. Overall, our findings demonstrate that both normal and APL neutrophil differentiation require CMA downregulation and this pathway is predominantly dependent on HSPA8 assisted by other co-chaperones.

cancer biology↗

bioRxiv2022

Deep learning-based automated lesion segmentation on mouse stroke magnetic resonance images

Magnetic resonance imaging (MRI) is widely used for ischemic stroke lesion detection in mice. A challenge is that lesion segmentation often relies on manual tracing by trained experts, which is labor-intensive, time-consuming, and prone to inter- and intra-rater variability. Here, we present a fully automated ischemic stroke lesion segmentation method for mouse T2-weighted MRI data. As an end-to-end deep learning approach, the automated lesion segmentation requires very little preprocessing and works directly on the raw MRI scans. We randomly split a large dataset of 382 MRI scans into a subset (n = 293) to train the automated lesion segmentation and a subset (n = 89) to evaluate its performance. We compared Dice coefficients and accuracy of lesion volume against manual segmentation, as well as its performance on an independent dataset from an open repository with different imaging characteristics. The automated lesion segmentation produced segmentation masks with a smooth, compact, and realistic appearance that are in high agreement with manual segmentation.

neuroscience↗

bioRxiv2022

The slow evolving genome of the xenacoelomorph worm Xenoturbella bocki

The evolutionary origins of Bilateria remain enigmatic. One of the more enduring proposals highlights similarities between a cnidarian-like planula larva and simple acoel-like flatworms. This idea is based in part on the view of the Xenacoelomorpha as an outgroup to all other bilaterians which are themselves designated the Nephrozoa (protostomes and deuterostomes). Genome data can help to elucidate phylogenetic relationships and provide important comparative data. Here we assemble and analyse the genome of the simple, marine xenacoelomorph Xenoturbella bocki, a key species for our understanding of early bilaterian and deuterostome evolution. Our highly contiguous genome assembly of X. bocki has a size of [~]111 Mbp in 18 chromosome like scaffolds, with repeat content and intron, exon and intergenic space comparable to other bilaterian invertebrates. We find X. bocki to have a similar number of genes to other bilaterians and to have retained ancestral metazoan synteny. Key bilaterian signalling pathways are also largely complete and most bilaterian miRNAs are present. We conclude that X. bocki has a complex genome typical of bilaterians, in contrast to the apparent simplicity of its body plan. Overall, our data do not provide evidence supporting the idea that Xenacoelomorpha are a primitively simple outgroup to other bilaterians and gene presence/absence data support a relationship with Ambulacraria.

evolutionary biology↗

bioRxiv2022

The role of auxiliary domains in modulating CHD4 activity suggests mechanistic commonality between enzyme families

CHD4 is an essential, widely conserved ATP-dependent translocase that is also a broad tumour dependency. In common with other SF2-family chromatin remodelling enzymes, it alters chromatin accessibility by repositioning histone octamers. Besides the helicase and adjacent tandem chromodomains and PHD domains, CHD4 features 1000 residues of N- and C-terminal sequence with unknown structure and function. We demonstrate that these regions regulate CHD4 activity through different mechanisms. An N-terminal intrinsically disordered region (IDR) promotes remodelling integrity in a manner that depends on the composition but not sequence of the IDR. The C-terminal region harbours an auto-inhibitory region that contacts the helicase domain. Auto-inhibition is relieved by a previously unrecognized C-terminal SANT-SLIDE domain split by [~]150 residues of disordered sequence, most likely by binding of this domain to substrate DNA. Our data shed light on CHD4 regulation and reveal strong mechanistic commonality between CHD family members, as well as with ISWI-family remodellers.

biochemistry↗

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↗

bioRxiv2022

The DNA methylation landscape of the human oxytocin receptor gene (OXTR): Recommendations for future research

The oxytocin receptor gene (OXTR) is of interest when investigating the effects of early adversity on DNA methylation. However, there is heterogeneity regarding the selection of the most promising CpG sites to target for analyses. The goal of this study was to determine functionally relevant clusters of CpG sites within the OXTR CpG island in 113 mother-infant dyads, with 58 of the mothers having experienced childhood maltreatment (CM). OXTR DNA methylation and gene expression was analyzed in peripheral/umbilical blood mononuclear cells. Different complexity reduction approaches were used to reduce the 188 CpG sites into clusters of co-methylated sites. Furthermore, associations between OXTR DNA methylation (cluster- and site-specific level) and OXTR gene expression and CM were investigated. Results showed that, first, CpG sections differed strongly regarding their statistical utility for research of individual differences in DNA methylation. Second, cluster analyses and Partial Least Squares (PLS) suggested two clusters consisting of intron1/exon2 and the protein-coding region of exon 3, respectively, as most strongly associated with outcome measures. Third, cross-validated PLS regression explained 7% of variance in CM, with low cross-validated variance explained for the prediction of gene expression. Fourth, very high mother-child correspondence was observed in correlation patterns within the identified clusters, but only modest correspondence outside these clusters. This study characterized the DNA methylation landscape of the OXTR CpG island by highlighting clusters of CpG sites that show desirable statistical properties and predictive value. We provide a Companion Web Application to guide future studies in their choice of CpG sites.

molecular biology↗

bioRxiv2022

Impaired functional connectivity of the hippocampus in murine models of NMDA-receptor antibody associated pathology

IntroductionWhile decreased hippocampal connectivity and disruption of functional networks are established MRI features in human anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis, the underlying pathophysiology for brain network alterations remains poorly understood. Application of patient-derived monoclonal antibodies against the NR1 subunit of the NMDAR allows for the investigation of potential functional connectivity alterations in experimental murine NMDAR antibody disease models. ObjectiveTo explore functional connectivity changes in NR1 antibody mouse models using resting-state functional MRI (rs-fMRI). MethodsAdult C57BL/6J mice (n=10) were intrathecally injected with a recombinant human NR1 antibody over 14 days and then studied using rs-fMRI at 7 Tesla. In addition, a newly established mouse model with in utero exposure to a human recombinant NR1 antibody characterized by a neurodevelopmental disorder (NR1-offspring) was investigated with rs-fMRI at the age of 8 weeks (n=15) and 10 months (n=14). Mice exposed to isotype-matched control antibodies served as controls. Independent component analysis (ICA) and dual regression analysis were performed to compare functional connectivity between NMDAR antibody mouse models and control mice. ResultsAdult NR1-antibody injected mice showed significantly impaired functional connectivity within the dentate gyrus of the left hippocampus in comparison to controls, resembling impaired hippocampal functional connectivity patterns observed in human patients with NMDAR encephalitis. Similarly, analyses showed significantly reduced functional connectivity in the dentate gyrus in NR1-offspring compared after 8 weeks, and impaired connectivity in the dentate gyrus and CA3 hippocampal subregion in NR1-offspring at the age of 10 months. ConclusionFunctional connectivity changes within the hippocampus resulting from both direct application and in utero exposure to NMDAR antibodies can be modeled in experimental murine systems. With this translational approach, we successfully reproduced functional MRI alterations previously observed in human NMDAR encephalitis patients. Future experimental studies will identify the detailed mechanisms that cause functional network alterations and may eventually allow for non-invasive monitoring of disease activity and therapeutic effects in autoimmune encephalitis.

neuroscience↗

bioRxiv2022

Viscoelastic properties of suspended cells measured with shear flow deformation cytometry

Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher-level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 {micro}m wide microfluidic channel. The fluid shear stress induces large, near ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe1 that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.

biophysics↗

bioRxiv2021

Fibroblastic reticular cells provide a supportive niche for lymph node-resident macrophages

The lymph node (LN) is home to resident macrophage populations that are essential for immune function and homeostasis. The T cell paracortical zone is a major site of macrophage efferocytosis of apoptotic cells, but key factors controlling this niche are undefined. Here we show that fibroblastic reticular cells (FRCs) are an essential component of the LN macrophage niche. Macrophages co-localised with FRCs in human LNs, and murine single-cell RNA-sequencing revealed that most reticular cells expressed master macrophage regulator CSF1. Functional assays showed that CSF1R signalling was sufficient to support macrophage development. In the presence of LPS, FRCs underwent a mechanistic switch and maintained support through CSF1R-independent mechanisms. These effects were conserved between mouse and human systems. Rapid loss of macrophages and monocytes from LNs was observed upon genetic ablation of FRCs. These data reveal a critically important role for FRCs in the creation of the parenchymal macrophage niche within LNs.

immunology↗

bioRxiv2021

Proteomics of spatially identified tissues in whole organs

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of biological specimens imaged in 3D as a whole are lacking. Here, we present DISCO-MS, a technology combining whole-organ imaging, deep learning-based image analysis, and ultra-high sensitivity mass spectrometry. DISCO-MS yielded qualitative and quantitative proteomics data indistinguishable from uncleared samples in both rodent and human tissues. Using DISCO-MS, we investigated microglia activation locally along axonal tracts after brain injury and revealed known and novel biomarkers. Furthermore, we identified initial individual amyloid-beta plaques in the brains of a young familial Alzheimers disease mouse model, characterized the core proteome of these aggregates, and highlighted their compositional heterogeneity. Thus, DISCO-MS enables quantitative, unbiased proteome analysis of target tissues following unbiased imaging of entire organs, providing new diagnostic and therapeutic opportunities for complex diseases, including neurodegeneration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/466753v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1e37035org.highwire.dtl.DTLVardef@dbaa98org.highwire.dtl.DTLVardef@19cece1org.highwire.dtl.DTLVardef@183c032_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDISCO-MS combines tissue clearing, whole-organ imaging, deep learning-based image analysis, and ultra-high sensitivity mass spectrometry C_LIO_LIDISCO-MS yielded qualitative and quantitative proteomics data indistinguishable from fresh tissues C_LIO_LIDISCO-MS enables identification of rare pathological regions & their subsequent molecular analysis C_LIO_LIDISCO-MS revealed core proteome of plaques in 6 weeks old Alzheimer s disease mouse model Supplementary Video can be seen at: http://discotechnologies.org/DISCO-MS/ C_LI

bioengineering↗

bioRxiv2021

mRNA Vaccines Induce Rapid Antibody Responses in Mice

mRNA vaccines can be developed and produced quickly, making them attractive for immediate outbreak responses. Furthermore, clinical trials have demonstrated rapid protection following mRNA vaccination. We sought to investigate how quickly mRNA vaccines elicit antibody responses compared to other vaccine modalities. We first examined immune kinetics of mRNA and DNA vaccines expressing SARS-CoV-2 spike in mice. We observed rapid induction of antigen-specific binding and neutralizing antibodies by day 5 following mRNA, but not DNA, immunization. The mRNA vaccine also induced increased levels of IL-5, IL-6 and MCP-1. We then evaluated immune kinetics of an HIV-1 mRNA vaccine in comparison to DNA, protein, and rhesus adenovirus 52 (RhAd52) vaccines with the same HIV-1 envelope antigen in mice. Induction of envelope-specific antibodies was observed by day 5 following mRNA vaccination, whereas antibodies were detected by day 7-14 following DNA, protein, and RhAd52 vaccination. Eliciting rapid humoral immunity may be an advantageous property of mRNA vaccines for controlling infectious disease outbreaks. IMPORTANCEmRNA vaccines can be developed and produced in record time. Here we demonstrate induction of rapid antibody responses by mRNA vaccines encoding two different viral antigens by day 5 following immunization in mice. The rapid immune kinetics of mRNA vaccines can be an advantageous property that makes them well suited for rapid control of infectious disease outbreaks.

microbiology↗

bioRxiv2021

Impairment of the cellulose degradation machinery enhances fungal virulence but limits reproductive fitness

Fungal endophytes grow in the apoplastic space, in constant contact with the plant cell wall (CW) that hinders microbe progression, while representing a source of nutrients. Although numerous fungal CW modifying proteins have been identified, their role during host colonization remains underexplored. Here we show that the root-infecting plant pathogen Fusarium oxysporum (Fo) does not require its complete arsenal of cellulases to infect the host plant. Quite the opposite, Fo mutants impaired in cellulose degradation become hypervirulent by enhancing the secretion of virulence factors. On the other hand, the reduction on cellulase activity had a severe negative effect on saprophytic growth and microconidia production during the final stages of the Fo infection cycle. These findings enhance our understanding on the function of plant CW degradation on the outcome of host-microbe interactions and reveal an unexpected role of cellulose degradation in a pathogens evolutionary success. TeaserUnexpectedly, fungi compromised in their capacity to degrade plant cellulose are hypervirulent but impaired in sporulation.

microbiology↗

bioRxiv2021

Biomarker Candidates for Tumors Identified from Deep-Profiled Plasma Stem Predominantly from the Low Abundant Area

The plasma proteome has the potential to enable a holistic analysis of the health state of an individual. However, plasma biomarker discovery is difficult due to its high dynamic range and variability. Here, we present a novel automated analytical approach for deep plasma profiling and applied it to a 180-sample cohort of human plasma from lung, breast, colorectal, pancreatic, and prostate cancer. Using a controlled quantitative experiment, we demonstrate a 257% increase in protein identification and a 263% increase in significantly differentially abundant proteins over neat plasma. In the cohort, we identified 2,732 proteins. Using machine learning, we discovered biomarker candidates such as STAT3 in colorectal cancer and developed models that classify the disease state. For pancreatic cancer, a separation by stage was achieved. Importantly, biomarker candidates came predominantly from the low abundance region, demonstrating the necessity to deeply profile because they would have been missed by shallow profiling.

cancer biology↗

bioRxiv2021

Mechanical and possible auxetic properties of human Achilles tendon during in vitro testing to failure

The Achilles tendon is the strongest tendon in the human body, but the basis of its high tensile strength has not been elucidated in detail. Here we have loaded healthy, human, Achilles tendons to failure in an anatomically authentic fashion while studying the local three-dimensional deformation and strains in real time, with very high precision, using digital image correlation (DIC). These studies identified a remarkable degree of anisotropic, medio-lateral auxetic behavior, with Poissons ratios not exceeding minus 1 in any part of the tendon at any time; under certain loads, discrete areas within the tendon had a Poissons ratio below minus 6. Early in the loading cycle, the proximal region of the tendon accumulated high lateral strains while longitudinal strains remained low. This behavior shielded the mid-substance of the tendon, its weakest part, from high longitudinal strains until immediately before rupture. These new insights are of great relevance to understanding the material basis of tendon injuries, designing improved prosthetic replacements, and developing regenerative strategies.

bioengineering↗

bioRxiv2021

Methylothon: a versatile course-based high school research experience in microbiology and bioinformatics-- with pink bacteria

Methylothon is an inquiry-based high school learning module in microbial ecology, molecular biology, and bioinformatics that centers around pink-pigmented plant-associated methylotrophic bacteria. Here we present an overview of the modules learning goals, describe course resources (available for public use on http://methylothon.com), and relate lessons learned from adapting Methylothon for remote learning during the pandemic in spring of 2021. The original in-person version of the module allows students to isolate their own strains of methylotrophic bacteria from plants they sample from the environment, to identify these using PCR, sequencing, and phylogenetic analysis, and to contribute their strains to original research in a university lab. The adapted version strengthens the focus on bioinformatics and increases its flexibility and accessibility by making the lab portion optional and adopting free web-based tools. Student feedback and graded assignments from Spring 2021 revealed that the lesson was especially effective at introducing the concepts of BLAST and phylogenetic trees, and that students valued and felt inspired by the opportunity to conduct hands-on work and to participate in community science.

scientific communication and education↗

bioRxiv2021

Metabolic control of adult neural stem cell self-renewal by the mitochondrial protease YME1L

The transition between quiescence and activation in neural stem and progenitor cells (NSPCs) is coupled to reversible changes in energy metabolism with key implications for life-long NSPC self-renewal and neurogenesis. How this metabolic plasticity is ensured between NSPC activity states is unclear. We found that a state-dependent rewiring of the mitochondrial proteome by the peptidase YME1L is required to preserve NSPC self-renewal in the adult brain. YME1L-mediated proteome rewiring regulates the rate of fatty acid oxidation (FAO) for replenishing Krebs cycle intermediates and dNTP precursors, which are required to sustain NSPC amplification. Yme1l deletion irreversibly shifts the metabolic profile of NSPCs away from a FAO-dependent state resulting in defective self-renewal, premature differentiation and NSPC pool depletion. Our results disclose an important role for YME1L in coordinating the switch between metabolic states of NSPCs and suggest that NSPC fate is regulated by compartmentalized changes in protein network dynamics.

neuroscience↗

bioRxiv2021

Optimization of Non-Coding Regions Improves Protective Efficacy of an mRNA SARS-CoV-2 Vaccine in Nonhuman Primates

The CVnCoV (CureVac) mRNA vaccine for SARS-CoV-2 has recently been evaluated in a phase 2b/3 efficacy trial in humans. CV2CoV is a second-generation mRNA vaccine with optimized non-coding regions and enhanced antigen expression. Here we report a head-to-head study of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in nonhuman primates. We immunized 18 cynomolgus macaques with two doses of 12 ug of lipid nanoparticle formulated CVnCoV, CV2CoV, or sham (N=6/group). CV2CoV induced substantially higher binding and neutralizing antibodies, memory B cell responses, and T cell responses as compared with CVnCoV. CV2CoV also induced more potent neutralizing antibody responses against SARS-CoV-2 variants, including B.1.351 (beta), B.1.617.2 (delta), and C.37 (lambda). While CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded robust protection with markedly lower viral loads in the upper and lower respiratory tract. Antibody responses correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of an mRNA SARS-CoV-2 vaccine in nonhuman primates.

immunology↗