Biology subjects

Thiel, C.

Publications and source records attributed to Thiel, C..

16 recordsLinked to original sources

bioRxiv2026

Cell-envelope conduits enable transfer of megabase-sized double-stranded DNA between cells of the nosocomial pathogen Acinetobacter baumannii

Horizontal gene transfer in Acinetobacter baumannii has been attributed to conjugative pili, transduction, and natural transformation. Here, we describe a previously unrecognized type of chromosomal DNA exchange mediated by cell-envelope conduits that establish direct cytoplasmic continuity between neighboring A. baumannii cells. Cryo-electron tomography reveals that these conduits, which were found in multiple clinical isolates, comprise an outer membrane, a peptidoglycan layer, and an inner membrane. The ~65 nm-thick conduits contain two ~2 nm-thick filaments, which super-resolution microscopy identifies as DNA. Interstrain horizontal gene transfer assays coupled with whole-genome sequencing demonstrate the transfer and homologous recombination of chromosomal segments up to 1.1 Mbp, corresponding to as much as 27% of the A. baumannii genome. Together, these findings provide direct structural and functional evidence for a conduit-mediated large-scale chromosomal DNA exchange, thus expanding the known repertoire of horizontal gene transfer strategies that may contribute to the remarkable genomic plasticity of A. baumannii.

microbiology↗

bioRxiv2025

Domain-Specific Functional Network Adaptations Supporting Dual-Task Performance in Older Adults

Aging is associated with declines in both motor and cognitive functions, which are well captured by dual-task gait paradigms. However, the functional brain network mechanisms supporting motor and cognitive aspects of dual-task performance in aging remain unclear. We examined 40 older adults (50-80 years) and 20 younger adults (20-40 years) who performed a motor single-task (pedaling), a cognitive single-task (Go/NoGo), and a combined cognitive-motor dual-task during functional magnetic resonance Imaging (fMRI) using a custom-built MRI-compatible pedaling device. Behaviorally, older adults showed significant dual-task costs in motor performance, while cognitive performance was preserved. Neurally, older adults showed selective increases in connectivity within executive and motor-planning regions of cognitive networks, consistent with compensatory recruitment, whereas motor networks underwent broader reorganization, with strengthened frontoparietal control circuits but weakened cerebello-parietal and sensorimotor pathways. Multivariate analyses further revealed age-related differences in latent connectivity- behavior relationships: motor-network patterns in older adults were more dispersed, reflecting heterogeneous reorganization, whereas cognitive-network patterns were more overlapping across groups, suggesting relative preservation. These findings suggest that aging involves a domain-specific balance of resilience and vulnerability across brain networks and highlight motor-network adaption as a promising target for understanding why some older adults maintain function while others decline.

neuroscience↗

bioRxiv2025

POMT-dependent O-mannosylation regulates integrin β1 maturation via N-glycan processing

Withdrawal StatementThe authors have withdrawn this manuscript because following a careful internal review conducted in the course of manuscript revision, the authors identified that parts of the presented data could not be reliably traced back to the corresponding original datasets in their current form. While the general biological interpretation is not necessarily invalidated by this finding, the integrity of the present data representation no longer meets the standard required for publication. For this reason, the authors have decided to withdraw this preprint and will refrain from pursuing publication until the underlying data situation has been fully clarified and, if possible, reconstructed. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The authors regret any inconvenience caused to the scientific community.

cell biology↗

bioRxiv2025

Establishing an auxin-inducible GFP nanobody-based acute protein knockdown system to mimic hypomorphic mutations during early medaka embryogenesis

Creating hypomorphic mutations are crucial to study gene function in vivo, especially when null mutations result in (embryonic) lethality. This is especially the case for enzymes involved in glycosylation that, when mutated in human patients, are causing the disease congenital disorders of glycosylation (CDG). To resemble the patient conditions, it would be ideal to acutely modulate the proteins in question to directly interfere with protein levels of such essential enzymes. These methods offer to establish pathogenic enzyme levels resembling net enzyme activity reported from patients suffering from CDG, with Phosphomannomutase 2 - CDG (PMM2-CDG) as the most common form. We established an auxin-inducible acute protein knockdown system for the use in the teleost fish medaka (Oryzias latipes) by combining an improved degron (AID2) technology with a mAID-nanobody targeting endogenously GFP-tagged Pmm2 protein. We generated a fishline expressing a functional Pmm2-GFP fusion protein, by single copy integration of GFP into the pmm2 locus. Upon induction, the degron system efficiently reduced Pmm2-GFP levels and enzyme activity, recapitulating the activity level of the hypomorphic mutations associated with PMM2-CDG in patients. This broadly applicable approach enables the investigation of CDG disease mechanisms during early embryonic development through reduction of protein abundance mimicking hypomorphic mutations and thus substantially expands the range of the genetic toolbox. Summary StatementThe combination of TIR1F74G and mAID-GFP-nanobody enables efficient acute knockdown of endogenously GFP-tagged proteins in medaka. This approach successfully reduced Pmm2 enzyme activity to pathological levels as seen in PMM2-CDG patients.

developmental biology↗

bioRxiv2025

A selective inhibitor of oncogenic JNK signalling perturbs metastatic outgrowth of triple-negative breast cancer through metabolic blockade

Although c-Jun N-terminal Kinase (JNK) represents an attractive anti-cancer target, its pleiotropic functionality limits the use of direct JNK inhibitors. Here, we identify a distinct subcellular pattern of JNK activity as a therapeutic vulnerability in breast cancer, where cytoplasmic JNK activity predicts poor survival outcomes, is elevated in triple-negative breast cancers (TNBC) and is essential for metastatic outgrowth. Mechanistic analyses reveal cytoplasmic JNK acts through multiple mechanisms, with downstream targets involved in cellular metabolism and cytoskeletal regulation. On this basis, we leveraged actin-based phenotypic drug-screening and identified K12, an indirect but selective inhibitor of cytoplasmic JNK that blocks TNBC metastatic outgrowth in vivo. We reveal that K12 inhibits glutaminase-1 and the pyruvate dehydrogenase complex, and that this poly-pharmacology overcomes pyruvate anaplerosis, a known resistance mechanism of existing glutaminase inhibitors. These findings demonstrate the potential of selectively targeting the oncogenic function of JNK, offering new treatment options for early-stage metastatic TNBC.

cancer biology↗

bioRxiv2024

Integrated data from R405W desmin knock-in mice highlight alterations of mitochondrial function, protein quality control, and myofibrillar structure in the initial stages of myofibrillar myopathy

BackgroundMutations in the desmin gene cause skeletal myopathies and cardiomyopathies. The objective of this study was to elucidate the molecular pathology induced by the expression of R405W mutant desmin in murine skeletal muscle tissue. MethodsA comprehensive characterization of the skeletal muscle pathology in hetero- and homozygous R405W desmin knock-in mice was performed, employing grip strength, blood acylcarnitine and amino acid, histological, ultrastructural, immunofluorescence, immunoblot, ribosomal stalling, RNA sequencing and proteomic analyses. ResultsBoth hetero- and homozygous R405W desmin knock-in mice showed classical myopathological features of a myofibrillar myopathy with desmin-positive protein aggregation, degenerative changes of the myofibrillar apparatus, increased autophagic build-up, and mitochondrial alterations. Muscle weakness and increased blood concentrations of acylcarnitines and amino acids were only present in homozygous animals. During its translation, mutant desmin does not induce terminal ribosomal stalling. Analyses of RNA sequencing and proteomic data from soleus muscle of 3-month-old mice depicted 59 up- and 2 down-regulated mRNAs and 101 up- and 18 down-regulated proteins that were shared between the heterozygous and homozygous genotypes in the respective omics datasets compared to the wild-type genotype. Combined analysis of the omics data demonstrated 187 significantly dysregulated candidates distributed across four groups of regulation. A down-regulation on the mRNA and protein levels was observed for a multitude of mitochondrial proteins including essential proton gradient-dependent carriers. Up-regulation on both omics levels was present for the transcription factor Mlf1, which is a binding partner of protein quality control related Dnajb6. Down-regulated on mRNA but up-regulated on the protein level was the sarcomeric lesion marker Xirp2 (xin actin-binding repeat-containing protein 2), whereas Ces2c (acylcarnitine hydrolase) was regulated in the opposite way. ConclusionsThe present study demonstrates that the expression of mutant desmin results in a myofibrillar myopathy in hetero- and homozygous R405W desmin knock-in mice. Combined morphological, transcriptomic and proteomic analyses helped to decipher the complex pattern of early pathological changes induced by the expression of mutant desmin. Our findings highlight the importance of major mitochondrial alterations, including essential proton gradient-dependent carriers as well as Dnajb6-related protein quality control and Xin-related myofibrillar damage, in the molecular pathogenesis of desminopathies.

neuroscience↗

bioRxiv2024

The multiverse of data preprocessing and analysis in graph-based fMRI: A systematic literature review of analytical choices fed into a decision support tool for informed analysis

The large number of different analytical choices researchers use may be partly responsible for the replication challenge in neuroimaging studies. For robustness analysis, knowledge of the full space of options is essential. We conducted a systematic literature review to identify the analytical decisions in functional neuroimaging data preprocessing and analysis in the emerging field of cognitive network neuroscience. We found 61 different steps, with 17 of them having debatable options. Scrubbing, global signal regression, and spatial smoothing are among the controversial steps. There is no standardized order in which different steps are applied, and the options within several steps vary widely across studies. By aggregating the pipelines across studies, we propose three taxonomic levels to categorize analytical choices: 1) inclusion or exclusion of specific steps, 2) distinct sequencing of steps, and 3) parameter tuning within steps. To facilitate access to the data, we developed a decision support app with high educational value called METEOR, which allows researchers to explore the space of choices as reference for well-informed robustness (multiverse) analysis. HighlightsO_LIData analysis variability in neuroimaging hinders replicability. C_LIO_LIAnalysis across multiple defensible options examines the robustness of results. C_LIO_LIWe conducted a systematic literature review to identify analytical options. C_LIO_LIWe identified 61 steps and 102 options in performing graph-fMRI analysis. C_LIO_LIInteractive visualization of these steps and options is available as a Shiny app. C_LI

neuroscience↗

bioRxiv2023

Digitalized organoids: integrated pipeline for 3D high-speed analysis of organoid structures using multilevel segmentation and cellular topology

Analysing the tissue morphogenesis and function is crucial for unravelling the underlying mechanisms of tissue development and disease. Organoids, 3D in vitro models that mimic the architecture and function of human tissues, offer a unique opportunity to study effects of external perturbators that are difficult to replicate in vivo. However, large-scale screening procedures for studying the effects of different stress on cellular morphology and topology of these 3D tissue-like system face significant challenges, including limitations in high-resolution 3D imaging, and accessible 3D analysis platforms. These limitations impede the scale and throughput necessary to accurately quantify the effects of mechanical and chemical cues. Here, we present a novel, fine-tuned pipeline for screening morphology and topology modifications in 3D cell culture using multilevel segmentations and cellular topology, based on confocal microscopy and validated across different image qualities. Our pipeline incorporates advanced image analysis algorithms and artificial intelligence (AI) for multiscale 3D segmentation, enabling quantification of morphology changes at both the nuclear and cytoplasmic levels, as well as at the organoid scale. Additionally, we investigate cell relative position and employ neighbouring topology analysis to identify tissue patterning and their correlations with organoid microniches. Eventually, we have organized all the extracted features, 3D segmented masks and raw images into a single database to allow statistical and data mining approaches to facilitate data analysis, in a biologist-friendly way. We validate our approach through proof-of-concept experiments, including well-characterized conditions and poorly explored mechanical stressors such as microgravity, showcasing the versatility of our pipeline. By providing a powerful tool for discovery-like assays in screening 3D organoid models, our pipeline has wide-ranging interests from biomedical applications in development and aging-related pathologies to tissue engineering and regenerative medicine.

cell biology↗

bioRxiv2023

The non-muscle actinopathy-associated mutation E334Q in cytoskeletal gamma-actin perturbs interaction of actin filaments with myosin and ADF/cofilin family proteins

Various heterozygous cytoskeletal {gamma}-actin mutations have been shown to cause Baraitser-Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal {gamma}-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin {beta}4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q-filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q-filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin-affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover contribute to the observed disease phenotype.

biochemistry↗

bioRxiv2022

Simple, rapid, and sensitive quantification of dolichyl phosphates using phosphate methylation and reverse-phase liquid chromatography-high resolution mass spectrometry

Dolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is crucial for all glycosylation processes, as these lipids serve as membrane-anchored building blocks that various types of glycosyltransferases use to generate complex post-translational modifications of proteins and lipids. Analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) has remained challenging due to their very low abundance and wide range of lipophilicities. Until now, a method for the simultaneous qualitative and quantitative assessment of DolP species from biological membranes has been lacking. Here we describe a novel approach based on simple sample preparation, rapid and efficient trimethylsilyl diazomethane (TMSD)-dependent phosphate methylation and RPLC-MS analysis for quantification of DolP species with different isoprene chain lengths. We used this workflow to selectively quantify DolP species from lipid extracts derived of Saccharomyces cerevisiae, HeLa and human skin fibroblasts from steroid 5--reductase 3-congenital disorders of glycosylation (SRD5A3-CDG) patients and healthy controls. Integration of this workflow with global lipidomics analyses will be a powerful tool to further our understanding of the role of DolPs in pathophysiological alterations of metabolic pathways downstream of HMG-CoA reductase, associated with CDGs, hypercholesterolemia, neurodegeneration, and cancer.

biochemistry↗

bioRxiv2021

Distributed networks for auditory memory contribute differentially to recall precision

Re-directing attention to objects in working memory can enhance their representational fidelity. However, how this attentional enhancement of memory representations is implemented across distinct, sensory and cognitive-control brain network is unspecified. The present fMRI experiment leverages psychophysical modelling and multivariate auditory- pattern decoding as behavioral and neural proxies of mnemonic fidelity. Listeners performed an auditory syllable pitch-discrimination task and received retro-active cues to selectively attend to a to-be-probed syllable in memory. Accompanied by increased neural activation in fronto-parietal and cingulo-opercular networks, valid retro-cues yielded faster and more perceptually sensitive responses in recalling acoustic detail of memorized syllables. Information about the cued auditory object was decodable from hemodynamic response patterns in superior temporal sulcus (STS), fronto-parietal, and sensorimotor regions. However, among these regions retaining auditory memory objects, neural fidelity in the left STS and its enhancement through attention-to-memory best predicted individuals gain in auditory memory recall precision. Our results demonstrate how functionally discrete brain regions differentially contribute to the attentional enhancement of memory representations.

neuroscience↗

bioRxiv2020

Targeted Proteomics Reveals Quantitative Differences in Low Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation

Protein glycosylation is essential in all domains of life and its mutational impairment in humans can result in severe diseases named Congenital Disorders of Glycosylation (CDGs). Studies on molecular level are however challenging, because many glycosyltransferases in the endoplasmic reticulum (ER) are low abundance membrane proteins. We established a comprehensive multiple reaction monitoring (MRM) assay to quantify most human glycosyltransferases involved in the processes of N-glycosylation,O- and C-mannosylation in the ER. To increase reproducibility, a membrane protein fraction of isotopically labeled HEK 293T cells was used as an internal standard. With this internal standard the MRM assay is easily transferable between laboratories. 22 glycosyltransferases could be reliably quantified from whole cell lysates of HEK 293T cells, HeLa cells and skin fibroblast cell lines. We then analyzed fibroblasts derived from CDG type I patients with mutations in the ALG1,ALG2 or ALG11 gene. Mutations in ALG1 or ALG2 gene strongly reduced the levels of the ALG1 and ALG2 protein, respectively. In contrast, the levels of all other glycosyltransferases remained unchanged, which was unexpected given evidence that the ALG1, ALG2 and ALG11 proteins form a stable complex. This study describes an efficient workflow for the development of MRM assays for low abundance proteins, establishes a ready-to-use tool for the comprehensive quantification of ER-localized glycosyltransferases and provides new insight into the organization of disease-relevant glycosylation processes.

biochemistry↗

bioRxiv2020

Glycosyltransferase POMGNT1 deficiency affects N-cadherin-mediated cell-cell adhesion

Defects in protein O-mannosylation lead to severe congenital muscular dystrophies known as -dystroglycanopathy. A hallmark of these diseases is the loss of the O-mannose-bound matriglycan on -dystroglycan, which leads to a reduction in cell adhesion to the extracellular matrix. Mutations in protein O-mannose {beta}1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), which is crucial for the elongation of O-mannosyl glycans, are mainly associated with muscle-eye-brain (MEB) disease. In addition to defects in cell-extracellular matrix adhesion, aberrant cell-cell adhesion has occasionally been observed in response to defects in POMGNT1. However, direct molecular mechanisms are largely unknown. We used POMGNT1 knock-out HEK293T cells and fibroblasts from a MEB patient to gain a deeper insight into the molecular changes in POMGNT1 deficiency. A combination of biochemical and molecular biological techniques with proteomics, glycoproteomics and glycomics revealed that a lack of POMGNT1 activity strengthens cell-cell adhesion. We demonstrate that the altered intrinsic adhesion properties are due to an increased abundance of N-cadherin (N-Cdh). In addition, site-specific changes in the N-glycan structures in the extracellular domain of N-Cdh were detected, which positively impact on homotypic interactions. We found that in POMGNT1 deficient cells ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable to the epithelial-mesenchymal transition (EMT) are triggered, defining a possible molecular mechanism underlying the observed phenotype. Our study indicates that changes in cadherin-mediated cell-cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or -dystroglycanopathy in general, and suggests a previously underestimated impact of changes in O-mannosylation on N-glycosylation.

biochemistry↗

bioRxiv2020

The medaka alg2 mutant is a model for hypo-N-glycosylation-associated retinitis pigmentosa

Patients suffering from Congenital Disorders of Glycosylation (CDG) carry mutations in components of the evolutionarily highly conserved protein-glycosylation-machinery. Employing targeted genome editing, we modeled alleles in medaka fish based on a mutation described in an ALG2-index patient. The multisystemic phenotypes in our alg2 model closely resembled the patients syndromes. Molecularly, the mutation results in a reduction of the abundance of N-glycans without altering the profile of glycan structures in fish as well as in patient fibroblasts. This hypo-N-glycosylation impacted on protein abundance in two directions. We discovered a putative compensatory upregulation of the basic glycosylation and glycoprotein processing machinery highlighting the regulatory topology of the network. Conversely, proteins of the retinal phototransduction machinery were massively downregulated in the alg2 model. Those relate to the specific loss of rod photoreceptors that fail to be maintained in the alg2 mutants, a condition known as retinitis pigmentosa. Transient supply of human or medaka alg2 mRNA efficiently rescued the phenotypic spectrum and restored viability demonstrating that our alg2 model delivers key traits for the potential treatment of the disorder.

developmental biology↗

bioRxiv2018

A physiology-based model of bile acid metabolism shows altered tissue concentrations after drug administration and in specific genotype subgroups

Drug-induced liver injuries (DILI) are an important issue in drug development and patient safety and often lead to termination of drug-development programs or late withdrawals of drugs. Since DILI events are hard to diagnose in preclinical settings, a need for alternative prediction methods such as computational modeling emerges. Impairment of bile acid (BA) metabolism, known as cholestasis, is a frequent form of DILI. Being rather a systemic then a single organ related disease, whole-body physiology-based modeling is a predestined approach for cholestasis modeling. The objectives of the presented study were 1) the development of a physiology-based model for human bile acid metabolism, 2) model validation and characterization for a virtual population, and 3) prediction and quantification of the effects of genetic predispositions and drug interaction on bile acid metabolism. The developed physiology-based bile acid (PBBA) model is based on the standard PBPK model of PKSim(R) and describes the bile acid circulation in a healthy reference individual. Active processes such as the hepatic synthesis, gallbladder emptying upon meal intake, transition through the gastrointestinal tract, reabsorption into the liver, distribution within the body, and excretion are included. The kinetics of active processes for the surrogate BA glycochenodeoxycholic acid were fitted to time-concentration profiles of blood BA levels reported in literature. The robustness of our PBBA model is underlined by the comparison of simulated plasma BA concentrations in a virtual population of 1,000 healthy individuals with reported data. In addition to plasma concentrations, the PBBA model allows simulations of BA exposure in relevant tissues like the liver and can therefore enhance the mechanistic understanding of cholestasis. This feature was used to analyse the reported increased risk of cholestatic DILI in Benign Recurrent Intrahepatic Cholestasis type 2 (BRIC2) patients. Simulations of the PBBA model suggest a higher susceptibility of BRIC2 patients towards cholestatic DILI due to BA accumulation in hepatocytes. Apart from these intrinsic effects, drug-interactions and their effect on the systemic bile acid metabolism were simulated by combining the PBBA model with a drug PBPK model of cyclosporine A (CsA). The results of which confirmed the reported higher risk of developing DILI as a consequence of CsA intake. Altogether, the presented model enhances our mechanistic understanding of cholestasis, allows the identification of drug-interactions leading to altered BA levels in blood and organs, and could be used to prevent clinical cases of cholestasis and enhance patient safety.

systems biology↗

bioRxiv2017

Dopaminergic Modulation Of Brain Signal Variability And The Functional Connectome During Cognitive Performance

Dopamine underlies important aspects of cognition, and has been suggested to boost cognitive performance. However, how dopamine modulates the large-scale cortical dynamics during cognitive performance has remained elusive. Using functional MRI during a working memory task in healthy young human listeners (N=22), we investigated the effect of levodopa (L-dopa) on two aspects of cortical dynamics, blood oxygen-level-dependent (BOLD) signal variability and the functional connectome of large-scale cortical networks. We here show that enhanced dopaminergic signaling modulates the two potentially interrelated aspects of large-scale cortical dynamics during cognitive performance, and the degree of these modulations is able to explain inter-individual differences in L-dopa-induced behavioral benefits. Relative to placebo, L-dopa increased BOLD signal variability in task-relevant temporal, inferior frontal, parietal and cingulate regions. On the connectome level, however, L-dopa diminished functional integration across temporal and cingulo-opercular regions. This hypo-integration was expressed as a reduction in network efficiency and modularity in more than two thirds of the participants and to different degrees. Hypo-integration co-occurred with relative hyper-connectivity in paracentral lobule and precuneus, as well as posterior putamen. Both, L-dopa-induced BOLD signal variability modulation and functional connectome modulations proved predictive of an individuals L-dopa-induced gain in behavioral performance, namely response speed and perceptual sensitivity. Lastly, L-dopa-induced modulations of BOLD signal variability were correlated with L-dopa-induced modulation of nodal connectivity and network efficiency. Our findings underline the role of dopamine in maintaining the dynamic range of, and communication between, cortical systems, and their explanatory power for inter-individual differences in benefits from dopamine during cognitive performance.

neuroscience↗