Biology subjects

Schmidt, G.

Publications and source records attributed to Schmidt, G..

15 recordsLinked to original sources

bioRxiv2026

Ferritinophagy Contributes to Iron Accumulation and Ferroptosis in FuchsEndothelial Corneal Dystrophy

PurposeFuchs endothelial corneal dystrophy (FECD) is a progressive disease, causing premature death of corneal endothelial cells (CECs). Iron-dependent lipid peroxidation and ferroptosis mediate cell death in FECD. We aimed to determine whether FECD progression is mediated by derangements in ferritinophagy - a form of autophagy that degrades ferritin to release labile ferrous iron - and whether ultraviolet A (UVA) exposure drives FECD progression by activating ferritinophagy. MethodsEndothelium-Descemet membrane (EDM) tissues were collected from patients with end-stage FECD undergoing endothelial keratoplasty and from healthy age-matched donor corneas. Separately, immortalized FECD and healthy control CEC lines were cultured. Cellular levels of NCOA4 production and LC3 activation, both markers of ferritinophagy, were quantified using western blotting and PCR. UVA-exposed immortalized cells were plated on coverslips, stained for immunohistochemistry (IHC), and analyzed using confocal microscopy. Corneal endothelial peels were stained and analyzed using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). ResultsSurgically explanted FECD CECs showed significantly increased levels of NCOA4 compared to healthy controls. LC3 activation was increased in FECD immortalized CECs; UV exposure further increased LC3 activation. Additionally, UVA exposure showed trends of increased expression of NCOA4 in immortalized FECD and healthy CECs. On IHC of FECD surgical explant tissue, ferritin was decreased markedly, NCOA4 localized in a dramatic punctate pattern, and both ferritin and LC3 localized within cell nuclei. Spectrometry images showed higher iron levels correlating with areas of higher FECD disease burden. ConclusionsOur results demonstrate ferritinophagy in FECD indicated by the increase of NCOA4 and LC3 ferritinophagy markers in FECD patient and cell culture models. Our finding that UVA activates ferritinophagy implicates this mechanism in UVA-mediated FECD progression. Altogether, aberrant iron dysregulation associated with FECD and ferroptosis may be mediated by ferritinophagy, providing a biomarker to assess disease severity as well as a potential target for future medical therapeutics.

cell biology↗

bioRxiv2026

The multistep quality control of mitochondrial β-barrel protein import

Mitochondrial {beta}-barrel proteins fulfill essential functions for cell viability including transport of proteins, ions and metabolites across the outer membrane. The translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex) transport these proteins into the outer membrane. Although {beta}-barrel proteins are essential for cell viability, the quality control mechanisms that monitor their import into the outer membrane remain unknown. We uncovered that Msp1 and Ubx2-recruited Cdc48 cooperate to remove a SAM-arrested Tom40 variant. Prior to degradation, the {beta}-barrel protein is deposited in cytosolic protein storages, termed MitoStores, to minimize accumulation of aberrant proteins in the cell. The cytosolic AAA ATPase Hsp104 disaggregates these proteins to facilitate their proteasomal degradation. Altogether, mitochondrial and cytosolic factors cooperate to degrade translocation-arrested mitochondrial {beta}-barrel proteins.

molecular biology↗

bioRxiv2026

Protease suppression by native I9 inhibitor improves recombinant protein production in Trichoderma reesei

Microbes are powerful cell factories for making molecules that are difficult or impossible to produce by other means. Filamentous fungi such as Trichoderma reesei are superior hosts for recombinant protein production, yet secreted proteases often degrade target proteins, reducing yields and limiting process robustness. Preventing proteolysis without relying on expensive commercial inhibitors or compromising strain fitness has been a longstanding challenge in fungal biotechnology, particularly for scaling up production to industrially relevant levels. Here, we report the identification of a native inhibitory protein from T. reesei, TrI9, and show that directing its secretion into the culture medium markedly reduces extracellular protease activity and enables production of the highly protease-sensitive spider silk-like protein CBM-AQ12-CBM, which could be used in high-performance biomaterials. Computational and in vitro analyses provide mechanistic insight, showing that TrI9 functions as a multi-target inhibitor of subtilisin-like proteases (SLPs), including SLP2, a protease that cannot be eliminated by gene deletion due to its crucial role in normal growth and development. This TrI9-based strategy for protease mitigation presents a novel approach for strain improvement, protecting a wide range of protease-labile products beyond silk proteins during large-scale fermentation. Protecting sensitive proteins without added inhibitors offers a cost-effective alternative for scaling up protein production across multiple applications, from protein-based materials manufacturing to pharmaceuticals, as well as enzyme and food applications. SignificanceThe discovery and characterization of an independently encoded I9 inhibitor (TrI9) in the filamentous fungus Trichoderma reesei reveals a new layer of biological regulation of subtilisin-like protease activity in fungi. We show that overexpressing and secreting TrI9 into the culture supernatant can suppress extracellular proteolysis, including activity from the essential SLP2 protease thereby overcoming a major barrier to production of protease-sensitive recombinant proteins without compromising strain fitness. For industrial biomanufacturing, this translates into higher effective titers, more consistent product integrity and reduced reliance on costly commercial protease inhibitors, hence, improving process robustness and economics. As a broadly applicable strain-and-process strategy, TrI9-enabled protease control strengthens T. reesei as a scalable platform for precision fermentation of diverse, otherwise hard-to-produce proteins.

bioengineering↗

bioRxiv2023

Gain efficiency with streamlined and automated data processing: Examples from high-throughput monoclonal antibody production

Data management and sample tracking in complex biological workflows are essential steps to ensure necessary documentation and guarantee the reusability of data and metadata. Currently, these steps pose challenges related to correct annotation and labeling, error detection, and safeguarding the quality of documentation. With growing acquisition of biological data and the expanding automatization of laboratory workflows, manual processing of samples is no longer favorable, as it is time- and resource-consuming, is prone to biases and errors, and lacks scalability and standardization. Thus, managing heterogeneous biological data calls for efficient and tailored systems, especially in laboratories run by biologists with limited computational expertise. Here, we showcase how to meet these challenges with a modular pipeline for data processing, facilitating the complex production of monoclonal antibodies from single B-cells. We present best practices for development of data processing pipelines concerned with extensive acquisition of biological data that undergoes continuous manipulation and analysis. Moreover, we assess the versatility of proposed design principles through a proof-of-concept data processing pipeline for automated induced pluripotent stem cell culture and differentiation. We show that our approach streamlines data management operations, speeds up experimental cycles and leads to enhanced reproducibility. Finally, adhering to the presented guidelines will promote compliance with FAIR principles upon publishing.

cell biology↗

bioRxiv2023

Sea squirt-inspired bio-derived tissue sealants

Sea squirts or tunicates bodies are composed of cellulose nanofibers and gallol- functionalized proteins. These sea creatures are known to heal their injuries under seawater by forming crosslinks between gallols and functional groups from other proteins in their bodies. Inspired by their wound healing mechanism, herein, we have developed a tissue sealant using zein (a plant-based protein) and tannic acid (gallol-containing polyphenol). Except for fibrin- based sealants, most commercial surgical adhesives, and sealants available today are derived from petroleum products that compromise their biodegradability. They often have complicated and multi-step synthesis processes that ultimately affect their affordability. To overcome this challenge, we ensured that these sea squirt-inspired tissue sealants are bio-based, easily synthesized, and low-cost. The sealants were studied on their own and with a food-grade enzyme transglutaminase. The adhesion performances of the sealants were found to be higher than physiological pressures in seven out of nine different tissue substrates studied here. Their performance was also better than or on par with the FDA-approved fibrin sealant Tisseel. Ex vivo models demonstrate instant sealing of leaking wounds in less than a minute. The sealants were not only cytocompatible but also showed complete wound healing on par with sutures and Tisseel when applied in vivo on skin incisions in rats. Overall, these sea squirt-inspired bio-based sealants show great potential to replace currently available wound closure methods.

bioengineering↗

bioRxiv2022

Genome wide CRISPR screen for Pasteurella multocida toxin (PMT) binding proteins reveals LDL Receptor Related Protein1 (LRP1) as crucial cellular receptor

PMT is a protein toxin produced by Pasteurella multocida serotypes A and D. As causative agent of atrophic rhinitis in swine, it leads to rapid degradation of the nasal turbinate bone. The toxin acts as a deamidase to modify a crucial glutamine in heterotrimeric G proteins, which results in constitutive activation of the G proteins and permanent stimulation of numerous downstream signaling pathways. Using a lentiviral based genome wide CRISPR knockout screen in combination with a lethal toxin chimera, consisting of full length inactive PMT and the catalytic domain of diphtheria toxin, we identified the LRP1 gene encoding the Low-Density Lipoprotein Receptor-related protein 1 as a critical host factor for PMT function. Loss of LRP1 reduced PMT binding and abolished the cellular response and deamidation of heterotrimeric G proteins, confirming LRP1 to be crucial for PMT uptake. Expression of LRP1 or cluster 4 of LRP1, respectively, restored intoxication of the knockout cells. In summary our data demonstrate secretory cells as entry site of PMT into airway epithelia and present LRP1 as crucial host entry factor for PMT intoxication by acting as its primary cell surface receptor.

pharmacology and toxicology↗

bioRxiv2022

Multimodal spatiotemporal phenotyping of human organoid development

Organoids generated from human pluripotent stem cells (PSCs) provide experimental systems to study development and disease. However, we lack quantitative spatiotemporal descriptions of organoid development that incorporate measurements across different molecular modalities. Here we focus on the retina and use a single-cell multimodal approach to reconstruct human retinal organoid development. We establish an experimental and computational pipeline to generate multiplexed spatial protein maps over a retinal organoid time course and primary adult human retina, registering protein expression features at the population, cellular, and subcellular levels. We develop an analytical toolkit to segment nuclei, identify local and global tissue units, infer morphology trajectories, and analyze cell neighborhoods from multiplexed imaging data. We use this toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components, and global patterning in each organoid and primary tissue. In addition, we generate a single-cell transcriptome and chromatin accessibility time course dataset and infer a gene regulatory network underlying organoid development. We then integrate genomic data with spatially segmented nuclei into a multi-modal atlas enabling virtual exploration of retinal organoid development. We visualize molecular, cellular, and regulatory dynamics during organoid lamination, and identify regulons associated with neuronal differentiation and maintenance. We use the integrated atlas to explore retinal ganglion cell (RGC) spatial neighborhoods, highlighting pathways involved in RGC cell death. Finally, we show that mosaic CRISPR/Cas genetic perturbations in retinal organoids provide insight into cell fate regulation. Altogether, our work is a major advance toward a virtual human retinal organoid, and provides new directions for how to approach disorders of the visual system. More broadly, our approaches can be adapted to many organoid systems.

systems biology↗

bioRxiv2021

GenOtoScope: Towards automating ACMG classification of variants associated with congenital hearing loss

Since next-generation sequencing (NGS) has become widely available, large gene panels containing up to several hundred genes can be sequenced cost-efficiently. However, the interpretation of the often large numbers of sequence variants detected when using NGS is laborious, prone to errors and often not comparable across laboratories. To overcome this challenge, the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) introduced standards and guidelines for the interpretation of sequencing variants. Further gene- and disease-specific refinements regarding hereditary hearing loss have been developed since then. With more than 200 genes associated with hearing disorders, the manual inspection of possible causative variants is especially difficult and time consuming. We developed an open-source bioinformatics tool GenOtoScope, which automates all ACMG/AMP criteria that can be assessed without further individual patient information or human curator investigation, including the refined loss of function criterion ("PVS1"). Two types of interfaces are provided: (i) a command line application to classify sequence variants in batches for a set of patients and (ii) a user-friendly website to classify single variants. We compared the performance of our tool with two other variant classification tools using two hearing loss data sets, which were manually annotated either by the ClinGen Hearing Loss Gene Curation Expert Panel or the diagnostics unit of our human genetics department. GenOtoScope achieved the best average accuracy and precision for both data sets. Compared to the second-best tool, GenOtoScope improved accuracy metric by 25.75% and 4.57% and precision metric by 52.11% and 12.13% on the two data sets respectively. The web interface is freely accessible. The command line application along with all source code, documentation and example outputs can be found via the project GitHub page. Author summaryNew high-throughput sequencing technologies can produce massive amounts of information and are utilized by laboratories to explain the often complex genetic aetiology of hereditary diseases. The most common sensory disease, hearing loss, is often hereditary and has a high impact on a patients every-day life. To use these sequencing technologies effectively, software tools were developed that can aid researchers interpreting genetic data by semi-automatically classifying the biologic (and thus potentially medical) impact of detected variants (the alterations of the patients genome compared to the human reference genome). The available genetic variant classification tools are either not designed specifically for the interpretation of variants detected in subjects with hearing loss or they do not allow researchers to use them for batch classification of all variants detected, e. g. in a study group. To address this drawback, we developed GenOtoScope, an open-source tool that automates the pathogenicity classification of variants potentially associated with congenital hearing loss. GenOtoScope can be applied for the automatic classification of all variants detected in a set of probands.

bioinformatics↗

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

MinION nanopore sequencing provides similar methylation estimates to Sanger bisulfite sequencing in the TRPA1 promoter region

Bisulfite sequencing has long been considered the gold standard for measurement of DNA methylation at single CpG resolution. In the meantime, several new approaches have been developed, which are regarded as less error-prone. Since these errors were shown to be sequence-specific, we aimed to verify the methylation data of a particular region of the TRPA1 promoter obtained from our previous studies. For this purpose, we compared methylation rates obtained via direct bisulfite sequencing and nanopore sequencing. Thus, we were able to confirm our previous findings to a large extent.

molecular biology↗

bioRxiv2020

An inducible AraC that responds to blue light instead of arabinose

In Escherichia coli, the operon responsible for the catabolism of L-arabinose is regulated by the dimeric DNA-binding protein AraC. In the absence of L-arabinose, AraC binds to the distal I1 and O2 half-sites, leading to repression of the downstream PBAD promoter. In the presence of the sugar, the dimer changes conformation and binds to the adjacent I1 and I2 half-sites, resulting in the activation of PBAD. Here we engineer blue light-inducible AraC dimers in Escherichia coli (BLADE) by swapping the dimerization domain of AraC with blue light-inducible dimerization domains. Using BLADE to overexpress proteins important for cell shape and division site selection, we reversibly control cell morphology with light. We demonstrate the exquisite light responsiveness of BLADE by employing it to create bacteriographs with an unprecedented quality. We then employ it to perform a medium-throughput characterization of 39 E. coli genes with poorly defined or completely unknown function. Finally, we expand the initial library and create a whole family of BLADE transcription factors (TFs), which we characterize using a novel 96-well light induction setup. Since the PBAD promoter is commonly used by microbiologists, we envisage that the BLADE TFs will bring the many advantages of optogenetic gene expression to the field of microbiology.

microbiology↗

bioRxiv2020

Glycosaminoglycans are specific endosomal receptors for Yersinia pseudotuberculosis Cytotoxic Necrotizing Factor

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron–electron double resonance and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. To analyze toxin-glucosaminoglycan interaction we utilized a truncated CNFY (CNFY709-1014). Especially this C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to its protein receptor seems to induce a first conformational change supporting the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface. Lastly, cleavage of heparin sulfate chains by heparanase is likely required for efficient uptake of the toxic enzyme into the cytosol of mammalian cells.Author Summary The RhoA deamidating Cytotoxic Necrotizing Factor Y (CNFY) from Yersinia pseudotuberculosis is a crucial virulence factor that is important for successful infection of mammalian cells by the pathogen. The mode of action by which CNFY is able to intoxicate cells can be divided into the following steps: Binding to the cell surface, internalization, translocation from the endosome to the cytosol and deamidation of RhoA. We show, that CNFY uses heparan sulfates to maximize the amount of molecules entering the cytosol. While not being necessary for toxin binding and uptake, the sugars hold a key role in the intoxication process. We show that CNFY undergoes a conformational change at a low endosomal pH, allowing the C-terminal domain to be released from the endosomal membrane by the action of heparanase. This study reveals new insights into the CNFY-host interaction and promotes understanding of the complex intoxication process of bacterial toxins.View Full Text

pharmacology and toxicology↗

bioRxiv2020

Identification of immunological features enables survival prediction of muscle-invasive bladder cancer patients using machine learning

AO_SCPLOWBSTRACTC_SCPLOWClinical staging and prognosis of muscle-invasive bladder cancer (MIBC) routinely includes assessment of patient tissue samples by a pathologist. Recent studies corroborate the importance of image analysis in identifying and quantifying immunological markers from tissue samples that can provide further insights into patient prognosis. In this paper, we apply multiplex immunofluorescence on MIBC tissue sections to capture whole slide images and quantify potential prognostic markers related to lymphocytes, macrophages, tumour buds, and PD-L1. We propose a machine learning based approach for the prediction of 5 year prognosis with different combinations of image, clinical, and spatial features. An ensemble model comprising several functionally different models successfully stratifies MIBC patients into two risk groups with high statistical significance (p value < 1e - 05). Critical to improving MIBC survival rates, our method classifies correctly 71.4% of the patients who succumb to MIBC within 5 years, significantly higher than the 28.6% of the current clinical gold standard, the TNM staging system.

cancer biology↗

bioRxiv2019

Photorhabdus Luminescens Toxin complex (TCC) a recombinant injection nano-machine - delivery of recombinant Yersinia enterocolitica YopT

Engineering delivery systems for proteins and peptides into mammalian cells is an ongoing challenge for cell biological studies as well as for therapeutic approaches. Photorhabdus luminescens toxin complex (PTC) is a heterotrimeric protein complex able to deliver diverse protein toxins into mammalian cells. We engineered the syringe like nano-machine for delivery of protein toxins from different species. Additionally, we loaded the highly active copepod luciferase Metridia longa M-Luc7 for accurate quantification of injected molecules. We suggest that besides the size also the charge of the cargo defines the efficiency of packing and transport into mammalian cells. Our data show that the Photorhabdus luminescens toxin complex constitutes a powerful system to inject recombinant proteins, peptides and potentially other molecules like aptamers into mammalian cells. In contrast to other protein transporters based on pore formation, the cargo is protected from degradation. The system opens new perspectives for cell research and pharmacology.

bioengineering↗

bioRxiv2019

KCND3 is a novel susceptibility locus for early repolarization

The presence of an early repolarization pattern (ERP) on the surface electrocardiogram (ECG) is associated with risk of ventricular fibrillation and sudden cardiac death. Family studies have shown that ERP is a highly heritable trait but molecular genetic determinants are unknown. We assessed the ERP in 12-lead ECGs of 39,456 individuals and conducted a two-stage meta-analysis of genome-wide association studies (GWAS). In the discovery phase, we included 2,181 cases and 23,641 controls from eight European ancestry studies and identified 19 genome-wide significant (p<5E-8) variants in the KCND3 (potassium voltage gated channel subfamily D member 3) gene with a p-value of 4.6E-10. Replication of two loci in four additional studies including 1,124 cases and 12,510 controls confirmed the association at the KCND3 gene locus with a pooled odds ratio of 0.82, p=7.7E-12 (rs1545300 minor allele T). A subsequent GWAS meta-analysis combining all samples did not reveal additional loci. The lead SNP of the discovery stage (rs12090194) was in strong linkage disequilibrium with rs1545300 (r2=0.96, D=1). Summary statistics based conditional analysis did not reveal any secondary signals. Co-localization analyses indicate causal effects of KCND3 gene expression levels on ERP in both the left ventricle of the heart and in tibial artery.\n\nIn this study we identified for the first time a genome-wide significant association of a genetic variant with ERP. Our findings of a locus in the KCND3 gene not only provide insights into the genetic determinants but also into the pathophysiological mechanism of ERP, revealing a promising candidate for functional studies.

genetics↗