Biology subjects

Vogt, C.

Publications and source records attributed to Vogt, C..

10 recordsLinked to original sources

bioRxiv2026

Substrate-dependent epistasis probes active site intramolecular wiring

Enzyme adaptation toward novel substrates involves the rewiring of intramolecular residue networks, yet how this rewiring differs across multiple substrates, and how it underpins functional trade-offs and promiscuity, remains poorly understood. Here, we profile all 64 combinations of six key mutations in a phosphotriesterase across nine structurally diverse substrates spanning three chemical classes (organophosphates, esters, and lactones), thus generating a multi-dimensional map of epistasis and promiscuity within the phosphotriesterases active site. We developed a statistically robust reference-based analysis pipeline incorporating error propagation and significance testing to move beyond global epistatic trends and resolve idiosyncratic, substrate-dependent intramolecular wiring in specific genetic backgrounds. Simulations confirm that this pipeline reliably identifies genuine higher-order epistatic interactions while minimizing false positives. We reveal that intramolecular network wiring varies substantially between substrates - even within the same chemical class - with notable divergences between the adaptive target substrate 2-naphthyl hexanoate and its shorter-chain ester analogs. Key higher-order networks, including d233E/h254R/l271F and l271F/f306I/i313F, exhibit substrate-specific epistatic signatures that discriminate between subtle structural features such as acyl chain length, leaving group identity, and heteroatom substitution. These substrate-dependent rewiring events account for observed functional trade-offs, particularly the strong anti-correlation between the adaptive and native substrates. Collectively, these findings demonstrate that comprehensive cross-substrate epistatic profiling, paired with rigorous statistical analysis, provides a powerful framework for dissecting the molecular basis of enzyme promiscuity and the trade-offs that define adaptive evolution. Significance of the studyEnzymes are biological catalysts whose function depends on precisely coordinated networks of interacting amino acids. Understanding how these networks change as enzymes evolve to recognize new molecules is fundamental to understanding and predicting evolution. Here, we developed a statistical framework to map these networks across multiple molecules simultaneously, revealing how subtle differences in molecular shape drive functional trade-offs during evolution. These insights could inform the rational design of enzymes with tailored specificities for industrial and therapeutic applications.

biochemistry↗

bioRxiv2026

Profound CD4+ T-Cell Reprogramming by Melphalan-Driven Oxidative Stress in High-Risk Multiple Myeloma

T cell-based immunotherapies have become central to the treatment of multiple myeloma (MM), yet their efficacy depends on the functionality of endogenous T cells. How cumulative treatment exposure, particularly high-dose melphalan, together with disease-intrinsic high-risk features shapes T-cell composition and immune competence remains incompletely understood. Here, we analyzed T cell composition and function in bone marrow (BM) and peripheral blood (PB) samples from MM patients across different stages of their treatment journey using flow cytometry (BM, n=162; PB, n=1,733), single-cell RNA sequencing (n=19), and cytotoxicity assays (n=20). We reveal reduced overall T cell frequencies and CD4+/CD8+ T cell ratio, associated with lines of therapy and driven in part by depletion of naive CD4+ T cells in gene-expression defined high risk (HR) disease. Among therapeutic agents, melphalan exerted the strongest effects on T cell populations and induced pronounced redox stress in both T cells and myeloma cell lines. This oxidative stress signature was enriched in HR patients and was reversible with N-acetyl-L-cysteine treatment. Together, these findings identify immune dysregulation as a defining feature of HR MM that extends beyond tumor-intrinsic genomic alterations and is further shaped by treatment-induced remodeling of the BM microenvironment. Given the association between higher CD4+ T cell numbers and improved CAR-T cell outcomes, our data highlight the translational importance of treatment sequencing, particularly in HR MM. One Sentence SummaryMelphalan-induced redox stress depletes CD4+ naive T cells, particularly in patients with high-risk multiple myeloma.

Cancer Biology↗

bioRxiv2025

Extended poly(A) tails are a shared feature of herpesvirus mRNAs

Poly(A) tails are present on most cellular and viral mRNAs, providing a platform for poly(A)-binding proteins that stimulate translation and regulate the deadenylation and stability of transcripts in the cytoplasm. Here we leverage nanopore direct RNA sequencing to analyse the distribution of poly(A) tail lengths on cellular and viral mRNAs across Herpesviridae and other DNA and RNA virus infections. We find that herpesvirus mRNA poly(A) tails are consistently longer than those on cellular and other viral transcripts, presenting a previously unrecognized yet widespread mechanism to advantage herpesviral gene expression. This contrasts with the templated poly(A) tails on coronavirus RNAs and those on cytoplasmically transcribed poxviral mRNAs, which are more similar in length to those on host mRNAs. Herpesviral noncoding RNAs display differential poly(A) tailing patterns which do not correlate with nuclear localisation while individual herpesviral mRNAs also show variation in the extent to which their poly(A) tail lengths change during the virus lifecycle, suggestive of additional uncharacterised layers of poly(A) tail length regulation. Importantly, while we detect non-adenosine nucleotides within herpesviral poly(A) tails, which are known to oppose deadenylase activity, this "mixed tailing" is not at sufficient frequency to explain the widespread extended tails of herpesvirus mRNAs.

microbiology↗

bioRxiv2025

Diffusive and advective fluid flow shapes chemoautotrophic bacterial communities and sulfur mineralogy in hydrothermal sediments off Milos

Hydrothermal fluid flow not only shapes mineral deposition on the ocean floor but also creates ecological niches by altering temperature and energy availability. In these niches, microbial life thrives and has an additional, often unrecognized impact on mineral formation. In a newly discovered vent field in medium depths off Milos, Greece, we show how contrasting hydrothermal regimes host fundamentally different bacterial metabolisms. Diffusive flow fosters acidic, sulfate-rich conditions that promote kaolinization and pyrite formation. Fatty acid {delta}13C values down to -39{per thousand} indicate acetyl-CoA-based sulfate reduction as the main metabolism, likely contributing to pyrite formation. In contrast, vigorous venting delivers hot, acidic, carbon-rich, and sulfate-depleted fluids that sustain the activity of sulfide-oxidizing chemoautotrophs. Fatty acid {delta}13C values of up to -4{per thousand} and elemental sulfur accumulation provide evidence of their activity. Without such hydrothermal influence, little microbial activity and only quartz-rich sediments can be observed. Combined multivariate lipid analyses highlight Eh as the strongest environmental control, with increasing average chain length of fatty acids as an adaptation to (hydro-)thermal stress. These results illustrate how different types of fluid flow influence the activity of chemoautotrophic bacterial communities that are involved in the formation of characteristic sulfur minerals in hydrothermal environments.

biochemistry↗

bioRxiv2025

Single-molecule localization microscopy reveals the molecular organization of endogenous membrane receptors

Super-resolution microscopy in combination with genetic labeling methods allows imaging of single proteins in cells. However, visualizing endogenous proteins on primary cells remains challenging due to the use of sterically demanding antibodies for labeling. Here, we demonstrate how immunolabeling conditions and antibody crosslinking influence the quantification and identification of membrane receptor stoichiometry on cells using single-molecule localization microscopy. We developed an optimized immunolabeling and analysis protocol and demonstrate the performance of the approach by resolving the molecular organization of endogenous CD45, CD69, and CD38 on Jurkat T cells. To demonstrate the usefulness of the method for immunotherapy applications we investigated the interaction of primary multiple myeloma cells with the therapeutic monoclonal antibodies (mAbs) daratumumab and isatuximab, and a polyclonal anti-CD38 antibody. Our approach might lay the foundation for improved personalized diagnostics and treatment with therapeutic antibodies. One-Sentence SummarySingle-molecule localization microcopy quantifies the expression and resolves the stoichiometry of endogenous membrane receptors

biophysics↗

bioRxiv2024

Repression of varicella zoster virus gene expression during quiescent infection in the absence of detectable histone deposition

Varicella zoster virus (VZV) is a human-specific herpesvirus that establishes latency in peripheral neurons. The only transcripts detected in infected human trigeminal ganglia (TG) obtained shortly after death correspond to the VZV latency-associated transcript (VLT) and associated VLT-ORF63 splice variants. In vitro studies showed that VLT-ORF63 is translated into a protein (pVLT-ORF63) that induces VZV transcription. The mechanisms that lead to this restricted gene expression and the transition to lytic replication remain unknown, partly due to the difficulty of working with human neurons. In this study, we addressed whether the neuroblastoma-derived cell line SH-SY5Y could serve as a model to investigate the mechanisms that lead to repression of VZV gene expression followed by reactivation. VZV productively infected differentiated SH-SY5Y (dSH-SY5Y) whereas incubation with acyclovir (ACV) inhibited virus replication and induced a progressive repression of the virus. Upon removal of ACV there was production of viral particles in a subset of cells, while others contained non-replicating VZV genomes and VLT-containing transcripts for at least 20 days post-infection (dpi). Exogenous expression of VLT-ORF63 induced productive infection, suggesting that the non-replicating and repressed genomes remained functional. Interestingly, histone deposition was undetectable at VZV genomes in quiescently infected dSH-SY5Y cells, pointing to a potential novel mechanism leading to VZV repression in this neuronal setting.

microbiology↗

bioRxiv2023

Anaerobe isolation from denitrifying benzene-degrading enrichment culture and their capacity to mineralize benzene

Only a few benzene-mineralizing anaerobes have been isolated to date. In an attempt using classical isolation techniques to isolate benzene-mineralizing pure cultures from a benzene-mineralizing nitrate-reducing microbial community, two putative isolates were gained under nitrate-reducing conditions spiked separately with acetate and benzene as sole sources of carbon and energy with media containing ammonium or without ammonium. Both putative isolates; Bz4 (with ammonium) and Bz7 (without ammonium) - mineralized 13C-labelled acetate under anoxic conditions at 3.3 and 2.7 M day-1, respectively, revealed by analysis of evolved 13CO2. However, only Bz4 mineralized [13C6]-labelled benzene (0.298 M benzene mineralized day-1) generated up to 960.2 {+/-} 0.3 {per thousand} {delta}13C-CO2 during 184 days while producing only slight amounts of nitrite (4.60 {+/-} 0.004 M); no benzene was mineralized by Bz7 during 184 d, and no nitrite was detected. The 16S rRNA gene amplicon sequencing of the acetate-grown bacteria revealed consortia enriched in Nocardioides (8.9%), Pseudomonas (18.2%), Rhizobiaceae (21.0%), Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium (51.4%) for Bz4 and Simplicispira (96.7%) for Bz7. The gained Bz4 consortium that mineralized benzene under anoxic condition can be further purified and explored for their metabolic potentials.

microbiology↗

bioRxiv2022

Floral humidity as a signal--not a cue--in a nocturnal pollination system

Although visual and olfactory floral signals attract pollinators from a distance, at the flowers threshold, pollinators can use floral humidity as an index cue for nectar presence. We evaluate the role of floral humidity in the Datura wrightii-Manduca sexta nocturnal pollination system. In addition to our finding that M. sexta shows strong innate attraction toward humid flowers, we identify the hygrosensing sensillum on their antennae, demonstrate its extreme sensitivity to minute changes in RH, and observe the elimination of moths behavioral preference towards humid flowers following experimental occlusion of the sensilla. Despite Manducas attraction toward humid flowers, we find that floral humidity is not a reliable cue for nectar presence in this system. While Datura floral headspace sustains an enormous humidity gradient, it is not a consequence of nectar evaporation, but an outcome of gas exchange through floral stomata and is decoupled from nectar presence. Using interdisciplinary tools, we demonstrate the function of floral humidity as an attractive signal, not a cue, in this pollination system, thus showcasing an underappreciated modality by which flowers may manipulate their visitors.

ecology↗

bioRxiv2021

OrtSuite - from genomes to prediction of microbial interactions within targeted ecosystem processes

The high complexity found in microbial communities makes the identification of microbial interactions challenging. To address this challenge, we present OrtSuite, a flexible workflow to predict putative microbial interactions based on genomic content of microbial communities and targeted to specific ecosystem processes. The pipeline is composed of three user-friendly bash commands. OrtSuite combines ortholog clustering with genome annotation strategies limited to user-defined sets of functions allowing for hypothesis-driven data analysis such as assessing microbial interactions in specific ecosystems. OrtSuite matched, on average, 96 % of experimentally verified KEGG orthologs involved in benzoate degradation in a known group of benzoate degraders. Identification of putative synergistic species interactions was evaluated using the sequenced genomes of an independent study which had previously proposed potential species interactions in benzoate degradation. OrtSuite is an easy to use workflow that allows for rapid functional annotation based on a user curated database and can easily be extended to ecosystem processes where connections between genes and reactions are known. OrtSuite is an open-source software available at https://github.com/mdsufz/OrtSuite.

bioinformatics↗

bioRxiv2018

Site specific target binding controls RNA cleavage efficiency by the Kaposi’s sarcoma-associated herpesvirus endonuclease SOX

During lytic replication of Kaposis sarcoma-associated herpesvirus (KSHV), the gene expression landscape of a cell is remodeled to evade the immune response and create an environment favorable to viral replication. A major driver of these gene expression changes is a virally encoded, messenger RNA (mRNA)-specific endonuclease termed SOX. SOX cleaves the majority of cytoplasmic mRNAs, but does so at specific internal sites loosely defined by a degenerate sequence motif. If and how RNA sequence directs SOX targeting remained unknown. To address these questions, we used recombinant, highly purified SOX endonuclease in a series of biochemical assays to reconstitute the cleavage reaction in vitro and gain significant insight into the biochemical mechanism of both SOX target recognition and endonucleolytic cleavage. Using this system, we determined that cut site specificity is preserved with purified SOX and a validated target RNA and thus does not require additional cellular cofactors. Furthermore, we showed that SOX displays robust, sequence-specific RNA binding to residues proximal to the cleavage site, which must be presented in a particular structural context. The strength of SOX binding dictates cleavage efficiency, providing an explanation for the breadth of target RNA susceptibility observed in cells.\n\nSignificance StatementKaposis sarcoma-associated herpesvirus (KSHV) is an oncogenic human virus that causes Kaposis sarcoma, primary effusion lymphoma, and multicentric Castleman disease. During viral replication, KSHV expresses an enzyme called SOX that cuts and inactivates the majority of cellular messenger RNAs, preventing their translation into proteins. Some mRNAs are efficiently cleaved by SOX, while others are poorly cleaved, but the mechanistic basis underlying this selectivity has remained largely unknown. Here, we reveal that the efficiency of RNA cleavage is heavily impacted by RNA sequences proximal to the cleavage site, which serve as a SOX binding platform. This helps explain both the range of RNA cleavage efficiency observed in SOX-expressing cells as well as the sequence specificity underlying SOX targeting.

microbiology↗