Biology subjects

Müller, M.

Publications and source records attributed to Müller, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Differentiation and admixture of Fagus sylvatica L. and Fagus orientalis Lipsky in a northern German forest -- learning from pioneer forest work

Tree species are suffering from changing and stressful environmental conditions worldwide. Fagus sylvatica L., one of the most common Central European deciduous tree species showed symptoms of crown damage, a reduction in growth and increased mortality following the severe recent drought years. For Germany Fagus orientalis Lipsky, a closely related species with higher drought tolerance, originating from south-eastern Europe, Turkey, the Greater Caucasus region and the Hyrcanian forest, has been proposed as an alternative with high future potential. The translocation of pre-adapted planting material has been proposed as a tool to mitigate negative effects of climate change. This approach can be beneficial but might also harbor risks. Taking advantage of F. orientalis trees planted over 100 years ago in the forest district of Memsen, Germany, we set out to study admixture between the two beech species and the direction of gene flow. Furthermore, we used a range-wide dataset of F. sylvatica and F. orientalis provenances to determine the origin of the introduced trees. Using a combination of nuclear EST-SSRs and one chloroplast SSR marker with species-specific variants, we could show that interspecific gene flow was going in both directions. In most cases, F. sylvatica was the pollen donor which is likely explained by the higher abundance of this species producing vast amounts of pollen. The planted trees originated from the Greater Caucasus region and showed strong genetic divergence from German F. sylvatica populations. In the future, gene flow patterns as well as hybrid performance from different provenances should be tested in additional stands and in comparison to F. sylvatica provenances from southern Europe to assess the suitability of Oriental beech for the mitigation of climate change impacts.

plant biology↗

bioRxiv2022

Cerebrovascular damage caused by the gut microbe-derived uraemic toxin p-cresol sulfate isprevented by blockade of the epidermal growth factor receptor

Circulating levels of the gut microbe/host co-metabolite p-cresol sulfate (pCS) correlate with cerebrovascular event risk in individuals with chronic kidney disease, but whether this relationship is mechanistic is unclear. We hypothesised that pCS would impair function of the blood-brain barrier (BBB), the primary brain-vasculature interface. We report that pCS exposure impairs BBB integrity in human cells in vitro and both acutely and chronically in mice, enhancing tracer extravasation, disrupting barrier-regulating tight junction components and ultimately affecting whole-brain transcriptomic activity. In vitro and in vivo mechanistic studies showed that pCS activated epidermal growth factor receptor (EGFR) signalling, resulting in matrix metalloproteinase mobilisation and BBB damage. Furthermore, blockade of EGFR prevented the permeabilising effects of serum from haemodialysis patients upon cerebromicrovascular endothelia in vitro. Our results define a pathway linking the co- metabolite pCS with BBB damage and suggest targeting the EGFR may mitigate against cerebrovascular damage in CKD.

systems biology↗

bioRxiv2022

Heterogeneity in viral replication dynamics shapes the antiviral response

In response to virus infection, host cells can activate antiviral signaling to restrict virus replication and communicate viral infection to neighboring cells. For poorly understood reasons, antiviral response activation is highly heterogeneous among infected cells; both quantitatively (level of pathway activation) and qualitatively (transcribed antiviral gene set). Here, we used live-cell single-molecule imaging to simultaneously visualize viral infection and antiviral signaling, providing quantitative insights into antiviral response activation in single cells; first, the probability of activating an antiviral response varies throughout infection, with most efficient activation occurring several hours after the first viral replication. Second, cell-to-cell heterogeneity in viral replication rates early in infection determine the efficiency of antiviral response activation. Finally, variation in signaling strength of the viral sensing pathway result in qualitatively distinct antiviral responses. Together, this works identifies key parameters that shape the antiviral response and provides quantitative insights into the origin of heterogeneity in the antiviral response.

immunology↗

bioRxiv2022

Influenza A Virus Compromises anti-Streptococcal Innate Immunity

Seasonal influenza epidemics pose a considerable hazard for global health. In the past decades, accumulating evidence revealed that influenza A virus (IAV) renders the host vulnerable to bacterial superinfections which in turn are a major cause for morbidity and mortality. However, whether the impact of influenza on anti-bacterial innate immunity is restricted to the vicinity of the lung or systemically extends to remote sites is underexplored. We therefore sought to investigate intranasal infection of adult C57BL/6J mice with IAV H1N1 in combination with bacteremia elicited by intravenous application of Group A Streptococcus (GAS). Co-infection in vivo was supplemented in vitro by challenging murine bone marrow derived macrophages and exploring gene expression and cytokine secretion. Our results show that viral infection of mice caused mild disease and induced the depletion of CCL2 in the periphery. Influenza preceding GAS infection promoted the occurrence of paw edemas and was accompanied by exacerbated disease scores. In vitro co-infection of macrophages led to significantly elevated expression of TLR2 and CD80 compared to bacterial mono-infection, whereas CD163 and CD206 were downregulated. The GAS-inducible upregulation of inflammatory genes, such as Nos2, as well as the secretion of TNF and IL-1{beta} were notably reduced or even abrogated following co-infection. Our results indicate that IAV primes an innate immune layout that is inadequately equipped for bacterial clearance.

immunology↗

bioRxiv2022

Epigenetic anti-cancer treatment with a stabilized carbocyclic Decitabine analogue

5-Aza-2-deoxycytidine (Decitabine, AzadC) is a nucleoside analogue, which is in clinical use to treat patients with myelodysplastic syndrome or acute myeloid leukemia. Its mode of action is unusual because the compound is one of the few drugs that act at the epigenetic level of the genetic code. AzadC is incorporated as an antimetabolite into the genome and creates covalent, inhibitory links to DNA methyltransferases (DNMTs) that methylate 2-deoxycytidine (dC) to 5-methyl-dC (mdC). Consequently, AzadC treatment leads to a global loss of mdC, which presumably results in a reactivation of silenced genes, among them tumor suppressor and DNA damage response genes. Because AzadC suffers from severe instability, which limits its use in the clinic, a more sophisticated AzadC derivative would be highly valuable. Here, we report that a recently developed carbocyclic AzadC analogue (cAzadC) blocks DNMT1 in the AML cell line MOLM-13 as efficient as AzadC. Moreover, cAzadC has a surprisingly strong anti-proliferative effect and leads to a significantly higher number of double strand breaks compared to AzadC, while showing less off-target toxicity. These results show that cAzadC triggers more deleterious repair and apoptotic pathways in cancer cells than AzadC, which makes cAzadC a promising next generation epigenetic drug.

cancer biology↗

bioRxiv2021

Topologically controlled circuits of human iPSC-derived neurons for electrophysiology recordings

Bottom-up neuroscience, which consists of building and studying controlled networks of neurons in vitro, is a promising method to investigate information processing at the neuronal level. However, in vitro studies tend to use cells of animal origin rather than human neurons, leading to conclusions that might not be generalizable to humans and limiting the possibilities for relevant studies on neurological disorders. Here we present a method to build arrays of topologically controlled circuits of human induced pluripotent stem cell (iPSC)-derived neurons. The circuits consist of 4 to 50 neurons with mostly unidirectional connections, confined by microfabricated polydimethylsiloxane (PDMS) membranes. Such circuits were characterized using optical imaging and microelectrode arrays (MEAs). Electrophysiology recordings were performed on circuits of human iPSC-derived neurons for at least 4.5 months. We believe that the capacity to build small and controlled circuits of human iPSC-derived neurons holds great promise to better understand the fundamental principles of information processing and storing in the brain.

neuroscience↗

bioRxiv2021

The E3 ligase Thin controls homeostatic plasticity through neurotransmitter release repression

Synaptic proteins and synaptic transmission are under homeostatic control, but the relationship between these two processes remains enigmatic. Here, we systematically investigated the role of E3 ligases, key regulators of protein degradation-mediated proteostasis, in presynaptic homeostatic plasticity (PHP). An electrophysiology-based genetic screen of 157 E3 ligase-encoding genes at the Drosophila neuromuscular junction identified thin, an ortholog of human tripartite motif-containing 32 (TRIM32), a gene implicated in several neural disorders, including Autism Spectrum Disorder and schizophrenia. We demonstrate that thin functions presynaptically during rapid and sustained PHP. Presynaptic thin negatively regulates neurotransmitter release under baseline conditions by limiting the number of release-ready vesicles, independent of gross morphological defects. We provide genetic evidence that thin controls release through dysbindin, a schizophrenia-susceptibility gene required for PHP. Thin and Dysbindin localize in close proximity within presynaptic boutons, and Thin degrades Dysbindin in vitro. Thus, the E3 ligase Thin links protein degradation-dependent proteostasis of Dysbindin to homeostatic regulation of neurotransmitter release.

neuroscience↗

bioRxiv2021

Nuclear RNA concentration coordinates RNA production with cell size in human cells

Unlike its DNA template, RNA abundance and synthesis rates increase with cell size, as part of a mechanism of cellular RNA concentration homeostasis. Here, we study this scaling phenomenon in human cells by combining genome-wide perturbations with quantitative single-cell measurements. Despite relative ease in perturbing RNA synthesis, we find that RNA concentrations remain highly constant. Systems-level analysis indicates that perturbations that would lead to increased nuclear mRNA abundance result in downregulation of mRNA synthesis. This is associated with reduced levels of several transcription-associated proteins and protein states that are normally coordinated with RNA production in single cells, including RNA polymerase II (Pol II) itself. Acute shut-down of nuclear RNA degradation, elevation of nuclear mRNA levels, and mathematical modelling indicate that mammalian cells achieve RNA concentration homeostasis by an mRNA-based negative feedback on transcriptional activity in the nucleus. Ultimately, this acts to robustly scale Pol II abundance with cell volume and coordinate mRNA synthesis and degradation.

molecular biology↗

bioRxiv2021

Interferons Reshape the 3D Conformation and Accessibility of Macrophage Chromatin

Engagement of macrophages in innate immune responses is directed and enhanced by type I and type II interferons. An essential component of IFN activity is the use of JAK-STAT signal transduction for the transcriptional control of interferon-stimulated genes (ISG). Here, we study the immediate early nuclear response to type I IFN and IFN-{gamma} in murine macrophages. Despite their distinct immunological activities, both IFN types triggered highly overlapping epigenomic and transcriptional changes. These changes included a rapid rearrangement of the 3D chromatin organization and an increase of DNA accessibility at ISG loci. ISGF3, the major transcriptional regulator of ISG, controlled homeostatic as well as induced-state DNA accessibility at a subset of ISG. Increases in DNA accessibility correlated with the appearance of activating histone marks at surrounding nucleosomes. Collectively our data emphasize changes in the three-dimensional nuclear space and epigenome as an important facet of transcriptional control by the IFN-induced JAK-STAT pathway.

immunology↗

bioRxiv2021

Self-contained and modular structured illumination microscope

We present a modular implementation of structured illumination microscopy (SIM) that is fast, largely self-contained and that can be added onto existing fluorescence microscopes. Our instrument, which we call HIT-SIM, can theoretically deliver well over 50 super-resolved images per second and is readily compatible with existing acquisition software packages. We provide a full technical package consisting of schematics, a list of components and an alignment scheme that provides detailed specifications and assembly instructions. We illustrate the performance of the instrument by imaging optically large samples containing sequence-specifically stained DNA fragments.

biophysics↗

bioRxiv2021

Regulation of blood-brain barrier integrity and cognition by the microbiome-associated methylamines trimethylamine N-oxide and trimethylamine

BackgroundCommunication between the gut microbiota and the brain is primarily mediated via soluble microbe-derived metabolites, but the details of this pathway remain poorly defined. Methylamines produced by microbial metabolism of dietary choline and L-carnitine have received attention due to their proposed association with vascular disease, but their effects upon the cerebrovascular circulation have hitherto not been studied. ResultsHere we use an integrated in vitro/in vivo approach to show that physiologically relevant concentrations of the dietary methylamine trimethylamine N-oxide (TMAO) enhanced blood-brain barrier (BBB) integrity and protected it from inflammatory insult, acting through the tight junction regulator annexin A1. In contrast, the TMAO precursor trimethylamine (TMA) impaired BBB function and disrupted tight junction integrity. Moreover, we show that long-term exposure to TMAO protects murine cognitive function from inflammatory challenge, acting to limit astrocyte and microglial reactivity in a brain region-specific manner. ConclusionOur findings demonstrate the mechanisms through which microbiome-associated methylamines directly interact with the mammalian BBB, with consequences for cerebrovascular and cognitive function.

systems biology↗

bioRxiv2021

Exo-β-N-acetylmuramidase NamZ of Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidase

Endo-{beta}-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that potentially lyse bacterial cells. They catalyze an endo-lytic cleavage of the peptidoglycan, the structural component of the bacterial cell wall; i.e. they hydrolyze glycosidic N-acetylmuramic acid (MurNAc)-{beta}-1,4-N-acetylglucosamine (GlcNAc)-bonds within the heteroglycan backbone of peptidoglycan. In contrast, little is known about exo-{beta}-N-acetylmuramidases, catalyzing an exo-lytic cleavage of {beta}-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We identified ybbC of B. subtilis, renamed namZ, as encoding the reported exo-{beta}-N-acetylmuramidase. A {Delta}namZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl {beta}-MurNAc and the peptidoglycan-derived disaccharide MurNAc-{beta}-1,4-GlcNAc. Together with the exo-{beta}-N-acetylglucosaminidase NagZ and the exo-muramoyl-L-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. NamZ is a member of the DUF1343 protein family of unknown function and shows no significant sequence identity with known glycosidases. A structural model of NamZ revealed a putative active site located in a cleft within the interface of two subdomains, one of which constituting a Rossmann-fold-like domain, unusual for glycosidases. On this basis, we propose that NamZ represents the founding member of a novel family of peptidoglycan hexosaminidases, which is mainly present in the phylum Bacteroidetes and, less frequently, within Firmicutes (Bacilli, Clostridia), Actinobacteria and Gammaproteobacteria.

microbiology↗

bioRxiv2020

Distinct molecular pathways govern presynaptic homeostatic plasticity

Presynaptic homeostatic plasticity (PHP) stabilizes synaptic transmission by counteracting impaired neurotransmitter receptor function through increased presynaptic release. PHP is thought to be triggered by impaired receptor function, and to involve a stereotypic signaling pathway. However, here we demonstrate that different receptor perturbations that similarly reduce synaptic activity result in vastly different responses at the Drosophila neuromuscular junction. While receptor inhibition by the glutamate receptor (GluR) antagonist {gamma}-DGG is not compensated by PHP, the GluR inhibitors PhTx-433 and Gyki induce compensatory PHP. Intriguingly, PHP triggered by PhTx and Gyki involve separable signaling pathways, including inhibition of distinct GluR subtypes, differential modulation of the active-zone scaffold Bruchpilot, and short-term plasticity. Moreover, while PHP upon Gyki treatment does not require genes promoting PhTx-induced PHP, it involves presynaptic Protein Kinase D. Thus, synapses not only respond differentially to similar activity impairments, but achieve homeostatic compensation via distinct mechanisms, highlighting the diversity of homeostatic signaling.

neuroscience↗

bioRxiv2020

Deep-learning based denoising and reconstruction of super-resolution structured illumination microscopy images

Super-resolution structured illumination microscopy (SR-SIM) provides an up to two-fold enhanced spatial resolution of fluorescently labeled samples. The reconstruction of high quality SR-SIM images critically depends on patterned illumination with high modulation contrast. Noisy raw image data, e.g. as a result of low excitation power or low exposure times, result in reconstruction artifacts. Here, we demonstrate deep-learning based SR-SIM image denoising that results in high quality reconstructed images. A residual encoding-decoding convolution neural network (RED-Net) was used to successfully denoise computationally reconstructed noisy SR-SIM images. We also demonstrate the entirely deep-learning based denoising and reconstruction of raw SIM images into high-resolution SR-SIM images. Both image reconstruction methods prove to be very robust against image reconstruction artifacts and generalize very well over various noise levels. The combination of computational reconstruction and subsequent denoising via RED-Net shows very robust performance during inference after training even if the microscope settings change.

biophysics↗

bioRxiv2020

3D reconstruction of SARS-CoV-2 infection in ferrets emphasizes focal infection pattern in the upper respiratory tract

The visualization of viral pathogens in infected tissues is an invaluable tool to understand spatial virus distribution, localization, and cell tropism in vivo. Commonly, virus-infected tissues are analyzed using conventional immunohistochemistry in paraffin-embedded thin sections. Here, we demonstrate the utility of volumetric three-dimensional (3D) immunofluorescence imaging using tissue optical clearing and light sheet microscopy to investigate host-pathogen interactions of pandemic SARS-CoV-2 in ferrets at a mesoscopic scale. The superior spatial context of large, intact samples (> 150 mm3) allowed detailed quantification of interrelated parameters like focus-to-focus distance or SARS-CoV-2-infected area, facilitating an in-depth description of SARS-CoV-2 infection foci. Accordingly, we could confirm a preferential infection of the ferret upper respiratory tract by SARS-CoV-2 and emphasize a distinct focal infection pattern in nasal turbinates. Conclusively, we present a proof-of-concept study for investigating critically important respiratory pathogens in their spatial tissue morphology and demonstrate the first specific 3D visualization of SARS-CoV-2 infection.

microbiology↗

bioRxiv2020

Simulating digital micromirror devices for patterning coherent excitation light in structured illumination microscopy

Digital micromirror devices (DMDs) are spatial light modulators that employ the electro-mechanical movement of miniaturized mirrors to steer and thus modulate the light reflected of a mirror array. Their wide availability, low cost and high speed make them a popular choice both in consumer electronics such as video projectors, and scientific applications such as microscopy. High-end fluorescence microscopy systems typically employ laser light sources, which by their nature provide coherent excitation light. In super-resolution microscopy applications that use light modulation, most notably structured illumination microscopy (SIM), the coherent nature of the excitation light becomes a requirement to achieve optimal interference pattern contrast. The universal combination of DMDs and coherent light sources, especially when working with multiple different wavelengths, is unfortunately not straight forward. The substructure of the tilted micromirror array gives rise to a blazed grating, which has to be understood and which must be taken into account when designing a DMD-based illumination system. Here, we present a set of simulation frameworks that explore the use of DMDs in conjunction with coherent light sources, motivated by their application in SIM, but which are generalizable to other light patterning applications. This framework provides all the tools to explore and compute DMD-based diffraction effects and to simulate possible system alignment configurations computationally, which simplifies the system design process and provides guidance for setting up DMD-based microscopes.

biophysics↗

bioRxiv2020

Light-mediated discovery of surfaceome nanoscale organization and intercellular receptor interaction networks

Delineating the molecular nanoscale organization of the surfaceome is pre-requisite for understanding cellular signaling. Technologies for mapping the spatial relationships of cell surface receptors and their extracellular signaling synapses would open up theranostic opportunities and the possibility to engineer extracellular signaling. Here, we developed an optoproteomic technology termed LUX-MS that exploits singlet oxygen generators (SOG) for the light-triggered identification of acute protein interactions on living cells. Using SOG-coupled antibodies, small molecule-drugs, biologics and intact viral particles, we show that not only ligand-receptor interactions can be decoded across organisms, but also the surfaceome receptor nanoscale organization ligands engage in with direct implications for drug action. Furthermore, investigation of functional immunosynapses revealed that intercellular signaling inbetween APCs and CD8+ T cells can be mapped now providing insights into T cell activation with spatiotemporal resolution. LUX-MS based decoding of surfaceome signaling architectures provides unprecedented molecular insights for the rational development of theranostic strategies.

systems biology↗

bioRxiv2020

Altered central pain processing in fibromyalgia - a multimodal neuroimaging case-control study using arterial spin labelling

Fibromyalgia is characterized by chronic pain and a striking discrepancy between objective signs of tissue damage and severity of pain. Function and structural alterations in brain areas involved in pain processing may explain this feature. Previous case-control studies in fibromyalgia focused on acute pain processing using experimentally-evoked pain paradigms. Yet, these studies do not allow conclusions about chronic, stimulus-independent pain. Resting-state cerebral blood flow (rsCBF) acquired by arterial spin labelling (ASL) may be a more accurate marker for chronic pain. The objective was to integrate four different functional and structural neuroimaging markers to evaluate the neural correlate of chronic, stimulus-independent pain using a resting-state paradigm. In line with the pathophysiological concept of enhanced central pain processing we hypothesized that rsCBF is increased in fibromyalgia in areas involved in processing of acute pain. We performed an age matched case-control study of 32 female fibromyalgia patients and 32 pain-free controls and calculated group-differences in rsCBF, resting state functional connectivity, grey matter density and cortical thickness using whole-brain and region of interest analyses. We adjusted all analyses for depression and anxiety. As centrally acting drugs are likely to interfere with neuroimaging markers, we performed a subgroup analysis limited to patients not taking such drugs. We found no differences between cases and control in rsCBF of the thalamus, the basal ganglia, the insula, the somatosensory cortex, the prefrontal cortex, the anterior cingulum and supplementary motor area as brain previously identified to be involved in acute processing in fibromyalgia. The results remained robust across all four neuroimaging markers and when limiting the study population to patients not taking centrally acting drugs and matched controls. In conclusion, we found no evidence for functional or structural alterations in brain areas involved in pain processing in fibromyalgia that could reflect neural correlates of chronic stimulus-independent pain.

neuroscience↗