Biology subjects

Koch, M.

Publications and source records attributed to Koch, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Age-related structural and functional changes of the intracardiac nervous system

BackgroundAlthough aging is known to be associated with an increased incidence of both atrial and ventricular arrhythmias, there is limited knowledge about how Schwann cells (SC) and the intracardiac nervous system (iCNS) remodel with age. Here we investigate the differences in cardiac SC, parasympathetic nerve fibers, and muscarinic acetylcholine receptor M2 (M2R) expression in young and old mice. Additionally, we examine age-related changes in cardiac responses to sympathomimetic and parasympathomimetic drugs. Methods and ResultsLower SC density, lower SC proliferation and fewer parasympathetic nerve fibers were observed in cardiac and, as a control sciatic nerves from old (20-24 months) compared to young mice (2-3 months). In old mice, CSPG4 was increased in sciatic but not cardiac nerves. Expression of M2R was lower in ventricular myocardium and ventricular conduction system from old mice compared to young mice, while no significant difference was seen in M2R expression in sino-atrial or atrio-ventricular node pacemaker tissue. Heart rate was slower and PQ intervals were longer in Langendorff-perfused hearts from old mice. Ventricular tachycardia and fibrillation were more frequently observed in response to carbachol administration in hearts from old mice versus those from young mice. ConclusionsOn the background of reduced presence of SC and parasympathetic nerve fibers, and of lower M2R expression in ventricular cardiomyocytes and conduction system of aged hearts, the propensity of ventricular arrhythmogenesis upon parasympathomimetic drug application is increased. Whether this is caused by an increase in heterogeneity of iCNS structure and function remains to be elucidated. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/568538v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@11dbccdorg.highwire.dtl.DTLVardef@1563d75org.highwire.dtl.DTLVardef@dcda58org.highwire.dtl.DTLVardef@182e598_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2023

APP substrate ectodomain defines A beta length by restraining gamma-secretase processivity and facilitating product release

Sequential proteolysis of the amyloid precursor protein (APP) by {gamma}-secretases (GSECs) generates amyloid-{beta} (A{beta}) and defines the proportion of short-to-long A{beta} peptides, which is tightly connected to Alzheimers disease (AD) pathogenesis. Here, we study the mechanism controlling substrate processing by GSECs and defining product length. We found that polar interactions established by the APPC99 ectodomain (ECD), involving but not limited to its juxtamembrane region, restrain both the extent and degree of GSEC processive cleavage by destabilizing enzyme-substrate (E-S) interactions. We show that increasing hydrophobicity at APPC99-ECD - due to mutation or ligand binding - attenuates this substrate-driven product release mechanism, and rescues the effects that AD pathogenic variants exert on A{beta} profiles. In addition, our study reveals that APPC99-ECD facilitates the paradoxical production of longer A{beta}s caused by some GSEC inhibitors that act as high-affinity competitors to the substrate. These findings assign a pivotal role to the substrate ECD in the sequential proteolysis by GSEC and suggest it as a sweet spot for the potential design of APP targeting compounds selectively promoting its processing by GSEC.

biochemistry↗

bioRxiv2023

Dual-topology of collagen XV and tenascin C acts in concert to guide and shape developing motor axons

During development, motor axons are guided towards their muscle target by various extrinsic cues including extracellular matrix (ECM) proteins those identities remain poorly documented. Using single-cell RNA-sequencing of differentiating slow muscle progenitors (SMP) in zebrafish, we charaterized the SMP as a major source of ECM proteins that were computationally predicted to form a basement membrane-like structure tailored for motor axon guidance. Multiple in vivo and in vitro approaches further revealed that motor axon shape and growth relies on the timely expression of the attractive cue Collagen XV-B (ColXV-B) that locally provides motor axons with a permissive soft microenvironment and separately organizes the repulsive cue Tenascin C into a unique functional dual topology. Bioprinted micropatterns mimicking their unique topology provide compelling evidence that it represents a sufficient condition to elicit directional motor axon growth. Our study provides the first evidence that ECM topology and stiffness critically influence motor axon navigation in vertebrates with potential applications in regenerative medicine for peripheral nerve injury.

developmental biology↗

bioRxiv2023

Dissecting the impact of somatic hypermutation on SARS-CoV-2 neutralization and viral escape

Somatic hypermutation (SHM) drives affinity maturation and continues over months in SARS-CoV-2 neutralizing antibodies. Yet, several potent SARS-CoV-2 antibodies carry no or only few mutations, leaving the question of how ongoing SHM affects neutralization. Here, we reverted variable region mutations of 92 antibodies and tested their impact on SARS-CoV-2 binding and neutralization. Reverting higher numbers of mutations correlated with decreasing antibody functionality. However, some antibodies, including the public clonotype VH1-58, remained unaffected for Wu01 activity. Moreover, while mutations were dispensable for Wu01-induced VH1-58 antibodies to neutralize Alpha, Beta, and Delta variants, they were critical to neutralize Omicron BA.1/BA.2. Notably, we exploited this knowledge to convert the clinical antibody tixagevimab into a BA.1/BA.2-neutralizer. These findings substantially broaden our understanding of SHM as a mechanism that not only improves antibody responses during affinity maturation, but also counteracts antigenic imprinting through antibody diversification and thus increases the chances of neutralizing viral escape variants.

immunology↗

bioRxiv2023

Reliable Particle Sizing in Vaccine Formulations using Advanced Dynamic LightScattering

Understanding the impact of lipid nanoparticles size on immunogenicity represents an important step for enabling the rapid development of novel vaccines against known or emergent diseases. Dynamic light scattering, also known as quasi-elastic light scattering or photon correlation spectroscopy, has established itself as an optimal analytical method to determine particle size due to its in-situ approach and fast measurements. However, its application to many systems of industrial relevance has been limited due to artifacts arising from multiple scattering. Results interpretation becomes severely compromised depending on the concentration of the system and the size of the particles. In this context, strong sample dilution is often required, bringing additional uncertainties to the formulation development process. Here, we show how advanced dynamic light scattering technology can filter out multiple scattering from the signal and yield fully accurate sizing measurements, regardless of the sample concentration. We illustrate this in a comparative study with standard dynamic light scattering using polystyrene beads as model suspension as well as a concentrated commercial lipid nanoparticle adjuvant (AddaVax).

biophysics↗

bioRxiv2022

Distinct myofibre domains of the human myotendinous junction revealed by single nucleus RNA-seq

The myotendinous junction (MTJ) is a specialized domain of the multinucleated myofibre, faced with the challenge of maintaining robust cell-matrix contact with the tendon under high mechanical stress and strain. Here, we profiled 24,161 nuclei in semitendinosus muscle-tendon samples from 3 healthy males by single nucleus RNA-sequencing (snRNA-seq), alongside spatial transcriptomics, to gain insight into the genes characterizing this specialization in humans. We identified a cluster of MTJ myonuclei, represented by 47 enriched transcripts, of which the presence of ABI3BP, ABLIM1, ADAMTSL1, BICD1, CPM, FHOD3, FRAS1 and FREM2 was confirmed at the MTJ at the protein level by immunofluorescence. Four distinct subclusters of MTJ myonuclei were apparent and segregated into two COL22A1-expressing subclusters and two lacking COL22A1 but with a clear fibre type profile expressing MYH7 or MYH1/2. Our findings reveal distinct myonuclei profiles of the human MTJ, a weak link in the musculoskeletal system, which is selectively affected in pathological conditions, from muscle strains to muscular dystrophies.

molecular biology↗

bioRxiv2022

Single-cell analysis reveals inter- and intratumour heterogeneity in metastatic breast cancer

Metastasis is the leading cause of cancer-related deaths of breast cancer patients. Some cancer cells in a tumour go through successive steps, referred to as the metastatic cascade, and give rise to metastases at a distant site. We know that the plasticity and heterogeneity of cancer cells play critical roles in metastasis but the precise underlying molecular mechanisms remain elusive. Here we aimed to identify molecular mechanisms of metastasis during colonization, one of the most important yet poorly understood steps of the cascade. We performed single-cell RNA-Seq (scRNA-Seq) on tumours and matched lung macrometastases of patient-derived xenografts of breast cancer. After correcting for confounding factors such as the cell cycle and the percentage of detected genes (PDG), we identified cells in three states in both tumours and metastases. Gene-set Enrichment Analysis revealed biological processes specific to proliferation and invasion in two states. Our findings suggest that these states are a balance between epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial transitions (MET) traits that results in so-called partial EMT phenotypes. Analysis of the top Differentially Expressed Genes (DEGs) between these cell states revealed a common set of partial EMT Transcriptions Factors (TFs) controlling gene expression, including ZNF750, OVOL2, TP63, TFAP2C and HEY2. Our data suggest that the TFs related to EMT delineate different cell states in tumours and metastases. The results highlight the marked interpatient heterogeneity of breast cancer but identify common features of single cells from five models of metastatic breast cancer.

cancer biology↗

bioRxiv2022

Development of SARS-CoV-2 mRNA vaccines encoding spike N-terminal and receptor binding domains

With the success of mRNA vaccines against coronavirus disease 2019 (COVID-19), strategies can now focus on improving vaccine potency, breadth, and stability. We present the design and preclinical evaluation of domain-based mRNA vaccines encoding the wild-type spike-protein receptor-binding (RBD) and/or N-terminal domains (NTD). An NTD-RBD linked candidate vaccine, mRNA-1283, showed improved antigen expression, antibody responses, and stability at refrigerated temperatures (2-8{degrees}C) compared with the clinically available mRNA-1273, which encodes the full-length spike protein. In mice administered mRNA-1283 as a primary series, booster, or variant-specific booster, similar or greater immune responses and protection from viral challenge were observed against wild-type, beta, delta, or omicron (BA. 1) compared with mRNA-1273 immunized mice, especially at lower vaccine dosages. These results support clinical assessment of mRNA-1283 (NCT05137236). One Sentence SummaryA domain-based mRNA vaccine, mRNA-1283, is immunogenic and protective against SARS-CoV-2 and emerging variants in mice.

immunology↗

bioRxiv2022

The unique ORF8 protein from SARS-CoV-2 binds to human dendritic cells and induces a hyper-inflammatory cytokine storm

The novel coronavirus pandemic, whose first outbreak was reported in December 2019 in Wuhan, China (COVID-19), is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Tissue damage caused by the virus leads to a strong immune response and activation of antigen-presenting cells, which can elicit acute respiratory distress syndrome (ARDS) characterized by the rapid onset of widespread inflammation, the so-called cytokine storm. In many viral infections the recruitment of monocytes into the lung and their differentiation to dendritic cells (DCs) are seen as a response to the viral infection. DCs are critical players in the development of the acute lung inflammation that causes ARDS. Here we focus on the interaction of the ORF8 protein, a specific SARS-CoV-2 open reading frame protein, with dendritic cells (DCs). We show that ORF8 binds to dendritic cells, causes a pre-maturation of differentiating DCs, and induces the secretion of multiple pro-inflammatory cytokines by these cells. In addition, we identified dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) as a possible interaction partner of ORF8 on dendritic cells. Blockade of ORF8 signaling leads to reduced production of IL-1{beta}, IL-6, IL-12p70, TNF-, MCP-1 (CCL2), and IL-10 by dendritic cells. Analysis of patient sera with high anti-ORF8 antibody titers showed that there was nearly no neutralization of the ORF8 protein and its function. Therefore, a neutralizing antibody that has the capacity of blocking the cytokine and chemokine response mediated by ORF8 protein might be an essential and novel additional step in the therapy of severe SARS-CoV-2 cases.

immunology↗

bioRxiv2022

Melt Electrowriting of Scaffolds with a Porosity Gradient to Mimic the Matrix Structure of the Human Trabecular Meshwork

The permeability of the Human Trabecular Meshwork (HTM) regulates eye pressure via a porosity gradient across its thickness modulated by stacked layers of matrix fibrils and cells. Changes in HTM porosity are associated with increases in intraocular pressure and the progress of diseases like glaucoma. Engineered HTMs could help to understand the structure-function relation in natural tissues, and lead to new regenerative solutions. Here, melt electrowriting (MEW) is explored as a biofabrication technique to produce fibrillar, porous scaffolds that mimic the multilayer, gradient structure of native HTM. Poly(caprolactone) constructs with a height of 125-500 m and fiber diameters of 10-12 m are printed. Scaffolds with a tensile modulus between 5.6 and 13 MPa, and a static compression modulus in the range of 6-360 kPa are obtained by varying the scaffolds design, i.e., density and orientation of the fibers and number of stacked layers. Primary HTM cells attach to the scaffolds, proliferate, and form a confluent layer within 8-14 days, depending on the scaffold design. High cell viability and cell morphology close to that in the native tissue are observed. The present work demonstrates the utility of MEW to reconstruct complex morphological features of natural tissues.

bioengineering↗

bioRxiv2022

Molecular cloning and recombinant protein expression of 9-Lipoxygenase gene from Solanum tuberosum in Pichia pastoris yeast cells

Synthetic fungicides have effectively controlled plant pathogenic fungi but their continued use has been detrimental to natural biological systems, and sometimes resulted into development of fungal resistance. They also have undesirable effects on non-target organisms and foster environmental and human health concerns thus new biodegradable alternatives have to be investigated. Lipoxygenases (LOX) are ubiquitous non-heme Iron containing dioxygenases that catalyze the addition of molecular oxygen to Polyunsaturated Fatty Acids (PUFAs) such as Linoleic acid to form Oxylipins that possess anti-microbial activity.The aim of this study was to generate a recombinant 9-Lipoxygenase protein for chemo enzymatic synthesis of Oxylipin based biodegradable fungicides. Golden gate assembly, a molecular cloning method that allows assembly of many DNA fragments into a complete piece using Type II s restriction enzymes and T4 DNA ligase was used to clone the complete 9-LOX gene into the expression plasmid vector pPICZB. Protein expression in Pichia pastoris yeast cells was induced by addition of absolute methanol every after 24h for up to four days. Analysis of protein expression from cell lysates was achieved by SDS-PAGE and Western blotting probed with anti-histidine antibody which showed putative protein bands of 97kDa representing recombinant 9-LOX protein. It is recommended that optimization studies on the yeast kex2 convertase and the - secretion factor can be done to enable secretion of recombinant Solanum tuberosum 9-LOX protein since the protein in this study was recovered from cell lysates.

molecular biology↗

bioRxiv2021

Ablation of the FACIT collagen XII disturbs musculoskeletal ECM organization and causes patella dislocation and myopathy

Collagen XII, belonging to the fibril-associated collagens with interrupted triple helix (FACIT) family, assembles from three identical -chains encoded by the COL12A1 gene. The trimeric molecule consists of three N-terminal noncollagenous NC3 domains joined by disulfide bonds followed by a short interrupted collagen triple helix at the C-terminus. Collagen XII is expressed widely in the musculoskeletal system and mutations in the COL12A1 gene cause an Ehlers-Danlos/myopathy overlap syndrome, which is associated with skeletal abnormalities and muscle weakness. Our study defines the role of collagen XII in patella development using the Col12a1-/- mouse model. Deficiency in Col12a1 expression causes malformed facies patellaris femoris grooves at an early stage, which leads to patella subluxation and growth retardation. Due to the patella subluxation, more muscle fibers with centralized nuclei occur in the quadriceps than in the gastrocnemius muscles indicating a local effect. To further understand the role of collagen XII in the skeletal tissues single cell RNAseq (scRNA-seq) was performed. Comparison of the gene expression in the tenocyte cell sub-population of wild type and Col12a1-/- mice showed that several matrix genes are altered. Finally, we reinvestigated collagen XII deficient patients and observed a patella instability.

genetics↗

bioRxiv2021

LTBP1 promotes fibrillin incorporation into the extracellular matrix

LTBP1 is a large extracellular matrix protein and an associated ligand of fibrillinmicrofibrils. Knowledge of LTBP1 functions is largely limited to its role in targeting and sequestering TGF{beta} growth factors within the extracellular matrix, thereby regulating their bioavailability. However, the recent description of a wide spectrum of phenotypes in multiple tissues in patients harboring LTBP1 pathogenic variants suggests a multifaceted role of the protein in the homeostasis of connective tissues. To better understand the human pathology caused by LTBP1 deficiency it is important to investigate its functional role in extracellular matrix formation. In this study, we show that LTBP1 coordinates the incorporation of fibrillin-1 and -2 into the extracellular matrix in vitro. We also demonstrate that this function is differentially exerted by the two isoforms, the short and long forms of LTBP1. Thereby our findings uncover a novel TGF{beta}-independent LTBP1 function potentially contributing to the development of connective tissue disorders.

cell biology↗

bioRxiv2021

Consistent predictors of microbial community composition across scales in grasslands reveal low context-dependency

Environmental circumstances shaping soil microbial communities have been studied extensively, but due to disparate study designs it has been difficult to resolve whether a globally consistent set of predictors exists, or context-dependency prevails. Here, we used a network of 18 grassland sites (11 sampled across regional plant productivity gradients) to examine i) if the same abiotic or biotic factors predict both large- and regional-scale patterns in bacterial and fungal community composition, and ii) if microbial community composition differs consistently with regional plant productivity (low vs high) across different sites. We found that there is high congruence between predictors of microbial community composition across spatial scales; bacteria were predominantly associated with soil properties and fungi with plant community composition. Moreover, there was a microbial community signal that clearly distinguished high and low productivity soils that was shared across worldwide distributed grasslands suggesting that microbial assemblages vary predictably depending on grassland productivity.

ecology↗

bioRxiv2021

Lack of evidence for a role of anthrax toxin receptors as surface receptors for collagen VI and for its cleaved off C5 domain (endotrophin)

The widely expressed microfibril-forming collagen VI is subject to proteolytic cleavage and it has been proposed that the cleaved off C-terminal Kunitz domain (C5) of the 3 chain is an adipokine important for tumor progression and fibrosis. Under the name "endotrophin" the C5 fragment has also been shown to be a potent biomarker for fibro-inflammatory diseases. However, the biochemical mechanisms behind endotrophin activity have not been investigated. In earlier studies, the anthrax toxin receptor 1 was found to bind to C5, but this potential interaction has not been further studied. Given the proposed physiological role of endotrophin we aimed to determine how the endotrophin signal is transmitted to the recipient cells. Surprisingly, we could not detect any interaction between endotrophin and anthrax toxin receptor 1 or its close relative, anthrax toxin receptor 2. Moreover, we could not detect binding of fully assembled collagen VI to either anthrax toxin receptor. We also performed similar experiments with the collagen VI surface receptor NG2 (CSPG4). We could confirm that NG2 is a collagen VI receptor that binds to assembled collagen VI, but not to the cleaved C5/endotrophin. A cellular receptor for C5/endotrophin therefore still remains elusive.

cell biology↗

bioRxiv2021

No substantial pre-existing B cell immunity against SARS-CoV-2 in healthy adults

Pre-existing immunity against SARS-CoV-2 may have critical implications for our understanding of COVID-19 susceptibility and severity. Various studies recently provided evidence of pre-existing T cell immunity against SARS-CoV-2 in unexposed individuals. In contrast, the presence and clinical relevance of a pre-existing B cell immunity remains to be fully elucidated. Here, we provide a detailed analysis of the B cell response to SARS-CoV-2 in unexposed individuals. To this end, we extensively investigated the memory B cell response to SARS-CoV-2 in 150 adults sampled pre-pandemically. Comprehensive screening of donor plasma and purified IgG samples for binding and neutralization in various functional assays revealed no substantial activity against SARS-CoV-2 but broad reactivity to endemic betacoronaviruses. Moreover, we analyzed antibody sequences of 8,174 putatively SARS-CoV-2-reactive B cells on a single cell level and generated and tested 158 monoclonal antibodies. None of the isolated antibodies displayed relevant binding or neutralizing activity against SARS-CoV-2. Taken together, our results show no evidence of relevant pre-existing antibody and B cell immunity against SARS-CoV-2 in unexposed adults.

immunology↗

bioRxiv2021

Protection against SARS-CoV-2 Beta Variant in mRNA-1273 Boosted Nonhuman Primates

Neutralizing antibody responses gradually wane after vaccination with mRNA-1273 against several variants of concern (VOC), and additional boost vaccinations may be required to sustain immunity and protection. Here, we evaluated the immune responses in nonhuman primates that received 100 {micro}g of mRNA-1273 vaccine at 0 and 4 weeks and were boosted at week 29 with mRNA-1273 (homologous) or mRNA-1273.{beta} (heterologous), which encompasses the spike sequence of the B.1.351 (beta or {beta}) variant. Reciprocal ID50 pseudovirus neutralizing antibody geometric mean titers (GMT) against live SARS-CoV-2 D614G and the {beta} variant, were 4700 and 765, respectively, at week 6, the peak of primary response, and 644 and 553, respectively, at a 5-month post-vaccination memory time point. Two weeks following homologous or heterologous boost {beta}-specific reciprocal ID50 GMT were 5000 and 3000, respectively. At week 38, animals were challenged in the upper and lower airway with the {beta} variant. Two days post-challenge, viral replication was low to undetectable in both BAL and nasal swabs in most of the boosted animals. These data show that boosting with the homologous mRNA-1273 vaccine six months after primary immunization provides up to a 20-fold increase in neutralizing antibody responses across all VOC, which may be required to sustain high-level protection against severe disease, especially for at-risk populations. One-sentence summarymRNA-1273 boosted nonhuman primates have increased immune responses and are protected against SARS-CoV-2 beta infection.

immunology↗

bioRxiv2021

Serum Neutralizing Activity of mRNA-1273 against SARS-CoV-2 Variants

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has led to growing concerns over increased transmissibility and the ability of some variants to partially escape immunity. Sera from participants immunized on a prime-boost schedule with the mRNA-1273 COVID-19 vaccine were tested for neutralizing activity against several SARS-CoV-2 variants, including variants of concern (VOCs) and variants of interest (VOIs), compared to neutralization of the wild-type SARS-CoV-2 virus (designated as D614G). Results showed minimal effects on neutralization titers against the B.1.1.7 (Alpha) variant (1.2-fold reduction compared with D614G); other VOCs such as B.1.351 (Beta, including B.1.351-v1, B.1.351-v2, and B.1.351-v3), B.1.617.2 (Delta), and P.1 (Gamma) showed decreased neutralization titers ranging from 2.1-fold to 8.4-fold reductions compared with D614G, although all remained susceptible to mRNA-1273-elicited serum neutralization.

microbiology↗