Biology subjects

Schmidt, C.

Publications and source records attributed to Schmidt, C..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Sleep-dependent structural neuroplasticity after a spatial navigation task: A diffusion imaging study

Evidence for sleep-dependent changes in micro-structural neuroplasticity remains scarce, despite the fact that it is a mandatory correlate of the reorganization of learning-related functional networks. We investigated the effects of post-training sleep on structural neuroplasticity markers measuring standard diffusion tensor imaging (DTI) mean diffusivity (MD) and the revised biophysical neurite orientation dispersion and density imaging (NODDI) free water fraction (FWF) and neurite density (NDI) parameters that enable disentangling whether MD changes result from modifications in neurites or in other cellular components (e.g., glial cells). Thirty-four healthy young adults were scanned using diffusion weighted imaging [DWI] on Day1 before and after 40-minutes route learning (navigation) in a virtual environment, then were sleep deprived (SD) or slept normally (RS) for the night. After recovery sleep for 2 nights, they were scanned again (Day4) before and after 40-minutes route learning (navigation) in an extended environment. Sleep-related microstructural changes were computed on DTI (MD) and NODDI (NDI and FWF) parameters in the cortical ribbon and subcortical hippocampal and striatal regions of interest (ROIs). Results disclosed navigation learning-related decreased DWI parameters in the cortical ribbon (MD, FWF) and subcortical (MD, FWF, NDI) areas. Post-learning sleep-related changes were found at Day4 in the extended learning session (pre- to post-relearning percentage changes), suggesting a rapid sleep-related remodelling of neurites and glial cells subtending learning and memory processes in basal ganglia and hippocampal structures.

neuroscience↗

bioRxiv2022

SiRCle (Signature Regulatory Clustering) model integration reveals mechanisms of phenotype regulation in renal cancer

Clear cell renal cell carcinoma (ccRCC) tumours develop and progress via complex remodelling of the kidney epigenome, transcriptome, proteome, and metabolome. Given the subsequent tumour and inter-patient heterogeneity, drug-based treatments report limited success, calling for multi-omics studies to extract regulatory relationships, and ultimately, to develop targeted therapies. However, current methods are unable to extract nonlinear multi-omics perturbations. Here, we present SiRCle (Signature Regulatory Clustering), a novel method to integrate DNA methylation, RNA-seq and proteomics data. Applying SiRCle to a case study of ccRCC, we disentangle the layer (DNA methylation, transcription and/or translation) where dys-regulation first occurs and find the primary biological processes altered. Next, we detect regulatory differences between patient subsets by using a variational autoencoder to integrate omics data followed by statistical comparisons on the integrated space. In ccRCC patients, SiRCle allows to identify metabolic enzymes and cell-type-specific markers associated with survival along with the likely molecular driver behind the genes perturbations.

cancer biology↗

bioRxiv2022

FOXA2 controls the antioxidant response in FH-deficient cells independent of NRF2

Hereditary Leiomyomatosis and renal cell cancer (HLRCC) is a cancer syndrome caused by inactivating germline mutations in fumarate hydratase (FH) and subsequent accumulation of fumarate. Fumarate accumulation leads to the activation of an anti-oxidant response via nuclear translocation of the transcription factor NRF2. The activation of the anti-oxidant response is key for cellular survival in FH-deficient cells, yet the extent to which chromatin remodelling shapes the anti-oxidant response is currently unknown. Here, we explored the global effects of FH loss on the chromatin landscape to identify transcription factor networks involved in the highly remodelled chromatin landscape of FH-deficient cells. We identify FOXA2 as a key transcription factor which directly regulates anti-oxidant response genes and subsequent metabolic rewiring. Moreover, we also find that FOXA2 regulates anti-oxidant genes independent of the canonical anti-oxidant regulator NRF2. The identification of FOXA2 as an anti-oxidant regulator provides new insights into the molecular mechanisms behind cell responses to fumarate accumulation, and potentially provides new avenues for therapeutic intervention for HLRCC.

cancer biology↗

bioRxiv2022

HIRA loss transforms FH-deficient cells

Fumarate Hydratase (FH) is a mitochondrial enzyme that catalyses the reversible hydration of fumarate to malate in the TCA cycle. Germline mutations of FH lead to HLRCC, a cancer syndrome characterised by a highly aggressive form of renal cancer(1). Although HLRCC tumours metastasise rapidly, FH-deficient mice develop premalignant cysts in the kidneys, rather than carcinomas (2). How Fh1-deficient cells overcome these tumour suppressive events during transformation is unknown. Here, we perform a genome-wide CRISPR/Cas9 screen to identify genes that, when ablated, enhance the proliferation of Fh1-deficient cells. We found that the depletion of HIRA enhances proliferation and invasion of Fh1-deficient cells in vitro and in vivo. Mechanistically, Hira loss enables the activation of MYC and its target genes, increasing nucleotide metabolism specifically in Fh1-deficient cells, independent of its histone chaperone activity. These results are instrumental for understanding mechanisms of tumorigenesis in HLRCC and the development of targeted treatments for patients.

cancer biology↗

bioRxiv2022

Biogeography and taxonomy of the Hemileuca maia-nevadensis Stretch species complex (Lepidoptera: Saturniidae): insights to the midcontinental populations

An integrated assessment of geographic distribution, genetic variation and interpopulation reproductive barriers of the Great Plains and Great Lakes populations of the Hemileuca maia-nevadensis species complex is presented. The taxonomic circumscription of Hemileuca nevadensis Stretch, key to understanding the complex evolutionary history of the group, has previously been misinterpreted. Detailed distribution maps based on historic, literature, and contemporary occurrence records, combined with analysis of genetic variation using mtDNA-COI barcode sequences and pheromone compatibility trials, indicate that Great Plains H. "nevadensis" are disjunct and genetically divergent from nominate H. nevadensis, instead being more closely related to Great Lakes populations. Despite the genomic differentiation of the bog buckmoth established in recent studies, similar or identical mtDNA-COI haplotypes indicate past genetic linkages among now-isolated populations across the northern range periphery of the maia-complex. Based on the current evidence, northern Great Plains Hemileuca latifascia Barnes & McDunnough stat. nov. and eastern Great Lakes (bog buckmoth) Hemileuca menyanthevora Pavulaan stat. nov. (= Hemileuca iroquois Dirig & Cryan syn. nov.) should be considered as species distinct from others in the maia-group. Post-glacial dispersal hypotheses are evaluated in light of molecular variation and ecology, and key knowledge gaps are identified to guide future research.

zoology↗

bioRxiv2022

Population demography maintains biogeographic boundaries

Global biodiversity is organized into biogeographic regions that comprise distinct biotas. The contemporary factors maintaining differences in species composition between biogeographic regions are poorly understood. Given the evidence that populations with sufficient genetic variation can adapt to fill new habitats, it is surprising that we do not see more homogenization of species assemblages among regions. Theory suggests that the expansion of populations across biogeographic transition zones could be limited by environmental gradients that affect population demography in ways that could limit adaptive capacity, but this has not been empirically explored. Using three independently curated data sets describing continental patterns of mammalian demography and population genetics, we show that populations closer to biogeographic transition zones have lower effective population sizes and genetic diversity, and are more genetically differentiated. These patterns are consistent with reduced adaptive capacity near biogeographic transition zones. The consistency of these patterns across mammalian species suggests they are stable, predictable, and generalizable in their contribution to long-term limits on expansion and homogenization of biodiversity across biogeographic transition zones. Understanding the contemporary processes acting on populations that maintain differences in the composition of regional biotas is crucial for our basic understanding of the current and future organization of global biodiversity. The importance of contemporary, population-level processes on the maintenance of global biogeographic regions suggests that biogeographic boundaries are susceptible to environmental perturbation associated with human-caused global change.

ecology↗

bioRxiv2021

SARS-CoV-2 papain-like protease PLpro in complex with natural compounds reveal allosteric sites for antiviral drug design

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the hosts innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 [A] and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 [A], show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, in the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

biochemistry↗

bioRxiv2021

TRIM67 drives tumorigenesis in oligodendrogliomas through Rho GTPase-dependent membrane blebbing

Oligodendrogliomas are a subtype of isocitrate dehydrogenase (IDH) mutant gliomas defined by the co-deletion of chromosome arms 1p and 19q. Although the somatic genomic alterations of oligodendrogliomas have been well described, transcriptional changes unique to these tumors are not well studied. Here, we identify Tripartite Motif Containing 67 (TRIM67), an E3 ubiquitin ligase with essential roles during neuronal development, as an oncogene distinctly upregulated in oligodendrogliomas. We characterize the function of TRIM67 using high throughput assays, including RNA sequencing, total lysate-mass spectrometry (MS) and co-immunoprecipitation (IP)-MS using human neural progenitor cells and patient-derived glioma tumorspheres constitutively overexpressing TRIM67. Our high throughput data suggest that TRIM67 overexpression alters the abundance of cytoskeletal proteins, which were validated by functional assays, including immunofluorescence (IF) staining, co-IP and western blotting (WB). Additionally, IF staining results indicate that TRIM67 ectopic expression induces formation of membrane blebs in glioma cells, which could be reverted with the nonmuscle class II myosin inhibitor blebbistatin and selective ROCK inhibitor fasudil. GTP pulldown and WB assays further indicate that Rho GTPase/ROCK2 signaling is altered upon TRIM67 ectopic expression. Phenotypically, TRIM67 expression resulted in higher cell motility in wound healing experiments, reduced cell adherence in adhesion assays, accelerated tumor growth and reduced survival in mouse orthotopic implantation models of an oligodendroglioma-derived patient tumorsphere line. Taken together, our results demonstrate that upregulated TRIM67 induces blebbing-based rounded cell morphology through Rho GTPase/ROCK-mediated signaling thereby contributing to glioma pathogenesis. SignificanceWe identify TRIM67 as a novel oncogene in oligodendroglioma that leads to increased cell motility, tumor growth, reduced adhesion, and survival in mice. Our results also show that constitutive TRIM67 expression transforms cell morphology from an adherent to a rounded appearance with membrane blebs. Mechanistic alteration of actin cytoskeleton and Rho GTPase signaling upon TRIM67 upregulation underlies the rounded cell structure and the membrane blebbing phenotype. TRIM67-induced blebbing is specifically regulated by RHOA-RAC1-ROCK2 signaling axis. TRIM67 overexpression also alters pathways associated with cell migration and wound healing in various glioma cell lines and human neural progenitor cells, suggesting a general oncogenic mechanism in gliomas. Overall, our study highlights TRIM67 as a novel player orchestrating cytoskeleton, Rho GTPase signaling and bleb-based cell movement, ultimately causing tumorigenic outcomes in oligodendrogliomas.

cancer biology↗

bioRxiv2021

Fbxo7 promotes Cdk6 activity to inhibit PFKP and glycolysis in T cells.

Deregulated Fbxo7 expression is associated with many pathologies, including anaemia, male sterility, cancer, and Parkinsons disease, demonstrating its critical role in a variety of cell types. Although Fbxo7 is an F-box protein that recruits substrates for SCF-type E3 ubiquitin ligases, it also promotes the formation of cyclin D/Cdk6/p27 complexes in an E3-ligase independent fashion. We discovered PFKP, the major gatekeeper of glycolysis, in a screen for Fbxo7 substrates. PFKP has been previously shown to be a critical substrate of Cdk6 for the viability of T-ALL cells. We investigated the molecular relationships between Fbxo7, Cdk6 and PFKP, and the functional effect Fbxo7 has on T cell metabolism, viability, and activation. Fbxo7 promotes Cdk6-independent ubiquitination and Cdk6-dependent phosphorylation of PFKP. Importantly Fbxo7-deficient cells have reduced Cdk6 activity, and haematopoietic and lymphocytic cell lines show a significant dependency on Fbxo7. Compared to WT cells, CD4+ T cells with reduced Fbxo7 expression show increased glycolysis, despite lower cell viability and activation levels. Metabolomic studies of activated CD4+ T cells confirm increased glycolytic flux in Fbxo7-deficient cells, as well as altered nucleotide biosynthesis and arginine metabolism. We show Fbxo7 expression is glucose-responsive at the mRNA and protein level, and we propose Fbxo7 inhibits PFKP and glycolysis via its activation of Cdk6.

cell biology↗

bioRxiv2021

Neuronal glucose metabolism sets cholinergic tone and controls thermo-regulated signaling at the neuromuscular junction

Cholinergic and sympathetic counter-regulatory networks control numerous physiologic functions including learning/memory/cognition, stress responsiveness, blood pressure, heart rate and energy balance. As neurons primarily utilize glucose as their primary metabolic energy source, we generated mice with increased glycolysis in cholinergic neurons by specific deletion of the fructose-2,6-phosphatase protein TIGAR. Steady-state and stable isotope flux analyses demonstrated increased rates of glycolysis, acetyl-CoA production, acetylcholine levels and density of neuromuscular synaptic junction clusters with enhanced acetylcholine release. The increase in cholinergic signaling reduced blood pressure and heart rate with a remarkable resistance to cold-induced hypothermia. These data directly demonstrate that increased cholinergic signaling through the modulation of glycolysis has several metabolic benefits particularly to increase energy expenditure and heat production upon cold exposure. HighlightsO_LIDeficiency of a negative regulator of glycolysis (TIGAR) in cholinergic neurons increases the biosynthesis and content of the neurotransmitter acetylcholine. C_LIO_LIIncreased cholinergic tone reduces blood pressure and heart rate while enhancing signaling at neuromuscular junction. C_LIO_LIUpregulation of neuromuscular junction activation provides protection against the paralytic curare and cold-induced hypothermia. C_LIO_LIModulation of cholinergic neuron glycolysis may provide a novel therapeutic approach for treatment of diseases stemming from reduced acetylcholine signaling such as myasthenia gravis and sarcopenic pre-synaptic dysfunction. C_LI

cell biology↗

bioRxiv2021

Nitrogen partitioning between branched-chain amino acids and urea cycle enzymes sustains renal cancer progression

Metabolic reprogramming is critical for tumor initiation and progression. However, the exact impact of specific metabolic changes on cancer progression is poorly understood. Here, we combined multi-omics datasets of primary and metastatic clonally related clear cell renal cancer cells (ccRCC) and generated a computational tool to explore the metabolic landscape during cancer progression. We show that a VHL loss-dependent reprogramming of branched-chain amino acid catabolism is required to maintain the aspartate pool in cancer cells across all tumor stages. We also provide evidence that metastatic renal cancer cells reactivate argininosuccinate synthase (ASS1), a urea cycle enzyme suppressed in primary ccRCC, to enable invasion in vitro and metastasis in vivo. Overall, our study provides the first comprehensive elucidation of the molecular mechanisms responsible for metabolic flexibility in ccRCC, paving the way to the development of therapeutic strategies based on the specific metabolism that characterizes each tumor stage. HighlightsO_LIBranched-chain amino acids catabolism is reprogrammed in ccRCC tumors C_LIO_LIBCAT-dependent transamination supplies nitrogen for de novo biosynthesis of amino acids including aspartate and asparagine in ccRCC C_LIO_LIAspartate produced downstream of BCAT is used specifically by metastatic cells through argininosuccinate synthase (ASS1) and argininosuccinate lyase (ASL) to generate arginine, providing a survival advantage in the presence of microenvironments with rate limiting levels of arginine C_LIO_LIASS1 is re-expressed in metastatic 786-M1A through epigenetic remodeling and it is sensitive to arginine levels C_LIO_LISilencing of ASS1 impairs the metastatic potential in vitro and in vivo of ccRCC cells C_LI

cancer biology↗

bioRxiv2021

MEOX2 homeobox gene promotes growth of malignant gliomas

Glioblastoma (GBM) is an aggressive tumor that frequently exhibits gain of chromosome 7, loss of chromosome 10 and aberrantly activated receptor tyrosine kinase signaling pathways. Here, we identify mesenchyme homeobox 2 (MEOX2) on chromosome 7 with increased expression in GBM as a salient oncogenic transcription factor. Specifically, we show that MEOX2 overexpression leads to increased ERK phosphorylation, and we identify a phosphorylation site on MEOX2 that regulates its transcriptional activity by altering its subnuclear localization. We show that MEOX2 overexpression can lead to increased growth in GBM implantation models and cooperates with loss of p53 and PTEN in cerebral organoid models of human malignant gliomas to induce cell proliferation. Furthermore, using high-throughput genomics, we identify transcriptional target genes of MEOX2 in patient-derived GBM tumorsphere models and a fresh frozen GBM tumor. These analyses show that MEOX2 activates several oncogenic pathways involved in MAPK signaling and extracellular matrix organization. Furthermore, MEOX2 binds to oncogenic ETS factors and known glioma oncogenes such as FABP7. In total, we reveal a novel role for MEOX2 in GBM initiation and progression and demonstrate that MEOX2 can enhance ERK signaling through a feed-forward mechanism. Significance StatementGlioblastoma (GBM) harbors gain of chromosome 7 as an early driver event. In this study, we show that mesenchyme homeobox 2 (MEOX2), an aberrantly upregulated transcription factor on chromosome 7, is an oncogene in human glioblastoma. In contrast to GBM, MEOX2 expression is very low in normal brain. We show that MEOX2 cooperates with p53 and PTEN loss to promote tumor initiation in cerebral organoid models. In addition, we identify direct and indirect molecular targets of MEOX2 and demonstrate its role in activating the ERK signaling cascade. These findings identify a novel oncogene in GBM and highlight the transcriptional networks hijacked by these tumors to activate signaling pathways central to GBM biology.

cancer biology↗

bioRxiv2021

Pre-therapeutic Microglia Activation and Sex Determine Therapy Effects of Chronic Immunomodulation

Modulation of the innate immune system is emerging as a promising therapeutic strategy against Alzheimers disease (AD). However, determinants of a beneficial therapeutic effect are ill-understood. Thus, we investigated the potential of 18 kDa translocator protein positron-emission-tomography (TSPO-PET) for assessment of microglial activation in mouse brain before and during chronic immunomodulation. Serial TSPO-PET was performed during five months of chronic microglia modulation by stimulation of peroxisome proliferator-activated receptor (PPAR)-{gamma} with pioglitazone in two different mouse models of AD (PS2APP, AppNL-G-F). Using mixed statistical models on longitudinal TSPO-PET data, we tested for effects of therapy and sex on treatment response. We tested correlations of baseline with longitudinal measures of TSPO-PET, and correlations between PET results with spatial learning performance and {beta}-amyloid accumulation of individual mice. Immunohistochemistry was used to determine the molecular source of the TSPO-PET signal. Pioglitazone-treated female PS2APP and AppNL-G-F mice showed attenuation of the longitudinal increases in TSPO-PET signal when compared to vehicle controls, whereas treated male AppNL-G-F mice showed the opposite effect. Baseline TSPO-PET strongly predicted changes in microglial activation in treated mice (R=-0.874, p<0.0001) but not in vehicle controls (R=-0.356, p=0.081). Reduced TSPO-PET signal upon treatment was associated with better spatial learning and higher fibrillar {beta}-amyloid accumulation. Immunohistochemistry confirmed activated microglia to be the source of the TSPO-PET signal (R=0.952, p<0.0001). TSPO-PET represents a sensitive biomarker for monitoring of immunomodulation and closely reflects activated microglia. Pre-therapeutic assessment of baseline microglial activation and sex are strong predictors of individual immunomodulation effects and could serve for responder stratification.

neuroscience↗

bioRxiv2021

Chronic PPARγ Stimulation Shifts Amyloidosis to Higher Fibrillarity but Improves Cognition

BackgroundWe undertook longitudinal {beta}-amyloid positron emission tomography (A{beta}-PET) imaging as a translational tool for monitoring of chronic treatment with the peroxisome proliferator-activated receptor gamma (PPAR{gamma}) agonist pioglitazone in A{beta} model mice. We thus tested the hypothesis this treatment would rescue from increases of the A{beta}-PET signal while promoting spatial learning and preservation of synaptic density. MethodsPS2APP mice (N=23; baseline age: 8 months) and AppNL-G-F mice (N=37; baseline age: 5 months) were investigated longitudinally for five months using A{beta}-PET. Groups of mice were treated with pioglitazone or vehicle during the follow-up interval. We tested spatial memory performance and confirmed terminal PET findings by immunohistochemical and biochemistry analyses. ResultsSurprisingly, A{beta}-PET and immunohistochemistry revealed a shift towards higher fibrillary composition of A{beta}-plaques during upon chronic pioglitazone treatment. Nonetheless, synaptic density and spatial learning were improved in transgenic mice with pioglitazone treatment, in association with the increased plaque fibrillarity. ConclusionThese translational data suggest that a shift towards higher plaque fibrillarity protects cognitive function and brain integrity. Increases in the A{beta}-PET signal upon immunomodulatory treatments targeting A{beta} aggregation can thus be protective.

neuroscience↗

bioRxiv2021

A chemotactic sensor controls Salmonella-host cell interaction

Intimate cell contact and subsequent type three secretion system-dependent cell invasion are key steps in host colonization of Salmonella. Adhesion to complex glycostructures at the apical membrane of polarized cells is mediated by the giant adhesin SiiE. This protein is secreted by a type 1 secretion system (T1SS) and needs to be retained at the bacterial surface to exert its adhesive function. Here, we show that SiiE surface expression was linked to the presence of L-aspartate sensed by the Salmonella-specific methyl-accepting chemotaxis protein CheM. Bacteria lacking CheM were attenuated for invasion of polarized cells, whereas increased invasion was seen with Salmonella exposed to the non-metabolizable aspartate analog -methyl-D, L-aspartate (MeAsp). While components of the chemotaxis phosphorelay or functional flagella were dispensable for the increased invasion, CheM directly interacted with proteins associated with the SiiE T1SS arguing for a novel non-canonical signaling mechanism. As a result, CheM attractant signaling caused a shift from secreted to surface-retained and adhesion-competent SiiE. Thus, CheM controls the virulence function of SiiE in a precise spatio-temporal fashion depending on the host micro-milieu.

microbiology↗

bioRxiv2021

A multiplex microchamber diffusion assay for the antibody-based detection of microRNAs on randomly ordered microbeads

BackgroundMicroRNAs (miRNAs) are small, conserved, noncoding RNAs regulating gene expression that functions in RNA silencing and post-transcriptional regulation of gene expression. Altered miRNA profiles have been implicated in many human diseases, and due to their circulating abilities, they have excited great interest in their use as clinical biomarkers. The development of innovative methods for miRNA detection has become of high scientific and clinical interest. MethodsWe developed a diffusion-driven microbead assay and combined it with an antibody-based miRNA detection. The diffusion process was carried out in two different approaches a) co-diffusion of miRNA and antibodies (termed diffusion approach I, DAI) and b) diffusion of miRNA in an antibody-saturated environment (DAII). In both approaches, neutravidin-coated microbeads were loaded with specific biotinylated DNA capture probes, which targets either miR-21-5p, miR-30a-3p or miR-93-5p. The miRNAs were time- and dose-dependently detected in a diffusion microchamber by primary anti-DNA:RNA hybrid and fluorescence-labeled secondary antibodies using our in-house developed inverse fluorescence microscope imaging platform VideoScan. ResultsOur assay offers the advantage that several target molecules can be detected simultaneously and in real-time in one reaction environment (multiplex), without any amplification steps. We recorded the diffusion process over a period of 24 h and found that the reaction was almost completed after 2 h. The specificity of the assay was 96.7 % for DAI and 92.3 % for DAII. The detection limits were in a concentration range of 0.03-0.43 nM for DAI and 0.14-1.09 nM for DAII, depending on the miRNA. ConclusionThe miRNAs are successively exposed to the capture probe-loaded randomly ordered microbeads (p value of CSR 0.23-0.96), which leads to microbeads that become saturated with the target molecules first in front rows. Non-bonded miRNAs continue to diffuse further and can therefore subsequently bind to the microbeads with free binding sites. Our detection principle differs from other microbead assays, in which all microbeads are simultaneously mixed with the sample solution, so that all target molecules bind equally distributed to the microbeads, resulting in an averaged signal intensity.

biochemistry↗

bioRxiv2021

Social behaviour and vocalizations of the tent-roosting Honduran White Bat

Bats are highly gregarious animals, displaying a large spectrum of social systems with different organizational structures. One important factor shaping sociality is group stability. To maintain group cohesion and stability, bats often rely on social vocal communication. The Honduran white bat, Ectophylla alba exhibits an unusual social structure compared to other tent-roosting species. This small white-furred bat lives in perennial stable mixed-sex groups. Tent construction requires several individuals and, as the only tent roosting species so far, involves both sexes. The bats social system and ecology render this species an interesting candidate to study social behaviour and social vocal communication. In our study, we investigated the social behaviour and vocalizations of E. alba in the tent by observing two stable groups, including pups, in the wild. We documented 16 different behaviours, among others, play and fur chewing, a behaviour presumably used for scent-marking. Moreover, we found 10 distinct social call types in addition to echolocation calls, and, for seven call types, we were able to identify the corresponding behavioural context. Most of the social call types were affiliative, including two types of contact calls, maternal directives, pup isolation calls and a call type related to the fur-chewing behaviour. In sum, this study entails an ethogram and describes the first vocal repertoire of a tent-roosting phyllostomid bat, providing the basis for further in-depth studies about the sociality and vocal communication in E. alba.

animal behavior and cognition↗

bioRxiv2021

Piezo1 and BKCa channels in human atrial fibroblasts: interplay and remodelling in atrial fibrillation

AimsAtrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging population of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC-presence and -activity associated with AF. Methods and ResultsUsing primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC-signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline and iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent big potassium channels, BKCa. In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of BKCa current during stretch. No co-immunoprecipitation of Piezo1 and BKCa was detected. ConclusionsHuman atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BKCa. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data support the presence and interplay of Piezo1 and BKCa in human atrial fibroblasts in the absence of physical interactions between the two channel proteins.

molecular biology↗