Biology subjects

Herzog, M.

Publications and source records attributed to Herzog, M..

15 recordsLinked to original sources

bioRxiv2026

Towards forecasting antimicrobial resistance evolution: concordance of in vitro and in vivo studies

Antimicrobial resistance is a major global health concern, and current measures to address the problem are new drugs, prudent use and rapid diagnostics. Recently, the idea of forecasting resistance evolution experimentally to prevent resistance a priori has gained attention. But how well do experimental data on antimicrobial resistance evolution reflect resistance in strains from patients? We conducted a systematic review and analysis to compare in vitro and in vivo data. We collect-ed and analysed literature on experimental resistance evolution published between 1977 and 2024 available on PubMed for ESKAPE pathogens plus E. coli (ESKAPEE). We catalogued the magnitude of resistance evolution via minimum inhibitory concentration (MIC) changes across antimicrobial classes. We then focussed on P. aeruginosa and E. coli under ciprofloxacin (CIP) selection, as these were most frequent bacterial species used in experimental evolution. CIP-selected gene mutations and amino acid substitutions, as well as the associated MICs in experimentally evolved P. aeruginosa and E. coli isolates, were compared with corresponding in vivo data. In vitro and in vivo isolates of E. coli showed high concordance in the selected gene mutations and the specific amino acid changes. Similar results were found for P. aeruginosa Evolved MIC values differed significantly depending on the respective gene mutation and the total num-ber of mutations, but not due to the origin of the strains, in vitro or in vivo. Our findings clearly show that experimental resistance evolution in vitro could predict the emergence of antimicrobial resistance evolution in vivo. This study supports the idea that experimental data can be used to forecast the risks of resistance emergence and therefore contribute to antimicrobial stewardship. It will be important to investigate how generalizable these findings are for other bacteria-antimicrobial combinations.

evolutionary biology↗

bioRxiv2025

Quantification of H3.1-nucleosomes using a chemiluminescent immunoassay: a reliable method for neutrophil extracellular trap detection

Neutrophil extracellular traps (NETs) are chromatin-based web-like structures released by activated neutrophils in response to infectious agents. Overproduction or insufficient clearance of NETs contributes to dysfunction of immune response and disease pathogenesis, underlying the importance of early detection and monitoring of NET levels in clinical samples. While existing methods for NETs detection and quantification face limitations, there is a pressing need for a reliable, sensitive, and clinically applicable assay. Since NETs consist of long strains of decondensed chromatin, with nucleosomes as their basic units, we propose circulating H3.1-nucleosomes as biomarkers for NETs detection in clinical plasma samples. In the initial phase of our study, we confirmed the presence of H3.1-nucleosomes by immunofluorescence and immunoprecipitation experiments in two in vitro NET models: neutrophil-like cells differentiated from the HL-60 cell line and primary neutrophils isolated from whole blood, both treated with phorbol 12-myristate 13-acetate to induce NET formation. Subsequently, we developed and analytically validated a chemiluminescent immunoassay for the quantification of circulating H3.1-nucleosomes in plasma. This fully automated assay demonstrates impressive analytical performance in parameters including sensitivity, precision, linearity and reproducibility. Overall, by measuring the H3.1-nucleosome levels in plasma samples from patients suffering from NETs-related diseases compared to healthy donors, we demonstrated the assays potential as a groundbreaking diagnostic tool for disease management.

molecular biology↗

bioRxiv2024

Changes in Muscle Synergy Structure and Activation Patterns Underlie Force Field Adaptation, Retention, and Generalization

1Humans can adapt their motor commands in response to errors when they perform reaching movements in new dynamic conditions, a process called motor adaptation. They acquire knowledge about the new dynamics, which they can use when they are re-exposed and, limitedly, generalize to untrained reaching directions. While force field adaptation, retention, and generalization have been thoroughly investigated at a kinematic and kinetic task level, the underlying coordination at a muscular level remains unclear. Many studies propose that the central nervous system uses low- dimensional control, i.e., coordinates muscles in functional groups: so-called muscle synergies. Accordingly, we hypothesized that changes in muscle synergy structure and activation patterns represent the acquired knowledge underlying force field adaptation, retention, and generalization. To test this, 36 male humans practiced reaching to a single target in a viscous force field and were tested for retention and generalization to new directions, while we simultaneously measured muscle activity from 13 upper-body muscles. We found that muscle synergies used for unperturbed reaching cannot explain the muscle patterns when adapted. Instead, muscle synergies specific to this adapted state were necessary, alongside a novel four-phasic pattern of muscle synergy activation. Furthermore, these structural changes and patterns were also evident during retention and generalization. Our results suggest that reaching in an environment with altered dynamics requires structural changes to muscle synergies compared to unperturbed reaching, and that these changes facilitate retention and generalization. These findings provide new insights into how the central nervous system coordinates the muscles underlying motor adaptation, retention, and generalization. 2 Significance StatementHumans can adapt their reaching movements to new dynamic conditions. They acquire knowledge about the new dynamics and can use it not only when re-exposed to these conditions but also, in part, to generalize unpracticed reaching directions. While adaptation, retention, and generalization in a force field with new dynamics have been thoroughly investigated at a kinematic and kinetic task level, coordination of the underlying muscles remains elusive. Our results show how muscle synergies - functional groupings of co-activated muscles - underlie adaptation, retention, and generalization. In particular, we observed structural changes in the muscle synergies after adaptation compared to unperturbed reaching. These changes facilitate retention and spatial generalization. Thus, muscle synergies provide new insights into human motor adaptation.

neuroscience↗

bioRxiv2024

High-Throughput Epigenetic Profiling Immunoassays for Accelerated Disease Research and Clinical Development

Epigenetics, which examines the regulation of genes without modification of the DNA sequence, plays a crucial role in various biological processes and disease mechanisms. Among the different forms of epigenetic modifications, histone post-translational modifications (PTMs) are important for modulating chromatin structure and gene expression. Aberrant levels of histone PTMs are implicated in a wide range of diseases, including cancer, making them promising targets for biomarker discovery and therapeutic intervention. In this context, blood, tissues, or cells serve as valuable resources for epigenetic research and analysis. Traditional methods such as mass spectrometry and western blotting are widely used to study histone PTMs, providing qualitative and (semi)quantitative information. However, these techniques often face limitations that could include throughput and scalability, particularly when applied to clinical samples. To overcome these challenges, we developed and validated 13 Nu.Q(R) immunoassays to detect and quantify specific histone PTM-nucleosomes from K2EDTA plasma samples. Then, we tested these assays on other types of samples, including chromatin extracts from frozen tissues, as well as cell lines and white blood cells Our findings demonstrate that the Nu.Q(R) assays offer high specificity, sensitivity, precision and linearity, making them effective tools for epigenetic profiling. A comparative analysis of HeLa cells using mass spectrometry, Western blot, and Nu.Q(R) immunoassays revealed a consistent histone PTMs signature, further validating the effectiveness of these assays. Additionally, we successfully applied Nu.Q(R) assays across various biological samples, including human tissues from different organs and specific white blood cell subtypes, highlighting their versatility and applicability in diverse biological contexts.

molecular biology↗

bioRxiv2024

Cross-linked agarose-gelatine beads as a substrate for investigating biofilms of bacterial pathogens

Treating chronic bacterial infections is challenging due to the formation of biofilms, making bacteria less susceptible to antimicrobials. In vitro models have limitations in replicating biofilm physiology. To address this problem, we have created a hydrogel substrate that combines crosslinked agarose and gelatine presented as beads, providing stability and resistance to autoclaving. Bacterial pathogens rapidly colonise these biogel beads when submerged in liquid culture. The substrate was tested with Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, showing more robust biofilm growth than its glass bead counterpart. Additionally, this led to increased virulence factor production and served as a reservoir for biofilm quorum sensing molecules. These features closely resemble clinical situations, suggesting a more accurate representation of biofilm-associated infections than current approaches. This new substrate offers a practical and convenient model for studying biofilms of bacterial pathogens, providing an efficient solution to the research community and holding promise for future breakthroughs.

microbiology↗

bioRxiv2023

CO2 Reduction by the Iron Nitrogenase Competes with N2 Fixation Under Physiological Conditions

Nitrogenases are the only known enzymes that reduce molecular nitrogen (N2) to ammonia. Recent findings have demonstrated that nitrogenases also reduce the greenhouse gas carbon dioxide (CO2), suggesting CO2 to be a competitor of N2. Intriguingly, nitrogenase isoforms (i.e., molybdenum (Mo), vanadium and iron (Fe) nitrogenase) differ significantly in their ability to reduce CO2, but the mechanisms underlying these differences remain elusive. Here, we study the competing reduction of CO2 and N2 by the two nitrogenases of Rhodobacter capsulatus, the Mo and Fe nitrogenase. Analyzing their full CO2 reduction product spectrum in vitro, we find the Fe nitrogenase almost three-fold more efficient in CO2 reduction than the Mo isoform. Furthermore, the in vitro competition experiments reveal the Fe nitrogenase to be profoundly less selective for the reduction of N2 than the Mo nitrogenase. We observe the same effects in vivo, where adding CO2 drastically increases the doubling times of diazotrophically grown R. capsulatus strains that rely on the Fe nitrogenase. The Fe nitrogenase-dependent R. capsulatus strains reduce CO2 to methane under physiological conditions, highlighting the potential of the Fe nitrogenase for the biotechnological conversion of CO2 into value-added compounds. Furthermore, both products are secreted into the surrounding, potentially influencing the composition of microbial communities in Mo-deficient environments.

biochemistry↗

bioRxiv2023

Monitoring plasma nucleosome concentrations to measure of disease response and progression in dogs with hematopoietic malignancies.

BackgroundHematopoietic malignancies are extremely common in pet dogs and represent nearly 30% of the malignancies diagnosed in this population each year. Clinicians commonly use existing tools such as physical exam findings, radiographs, ultrasound and baseline blood work to monitor these patients for treatment response and remission. Circulating biomarkers, such as prostate specific antigen or carcinoembryonic antigen, can be useful tools for monitoring treatment response and remission status in human cancer patients. To date, there has a been a lack of useful circulating biomarkers available to veterinary oncology patients. MethodsCirculating plasma nucleosome concentrations were evaluated at diagnosis, throughout treatment and during remission monitoring for 40 dogs with lymphoma, acute myelogenous leukemia and multiple myeloma. Additionally, C-reactive protein and thymidine kinase-1 levels were recorded. ResultsPlasma nucleosome concentrations were significantly higher at diagnosis and progressive disease than they were when dogs were in remission. All but two dogs had plasma nucleosome concentrations that returned to the low range during treatment. These two dogs had the shortest progression free and overall survival times. Dogs with the highest plasma nucleosome concentrations had a significantly shorter first progression free survival than dogs with lower plasma nucleosome concentrations at diagnosis. Plasma nucleosome concentrations correlated better with disease response and progression than either thymidine kinase or C reactive protein. ConclusionsPlasma nucleosome concentrations can be a useful tool for treatment monitoring and disease progression in dogs with hematopoietic malignancies.

cancer biology↗

bioRxiv2020

Mutagenic mechanisms of cancer-associated DNA polymerase ε alleles

A single amino acid residue change in the exonuclease domain of human DNA polymerase {varepsilon}, P286R, is associated with the development of colorectal cancers, and has been shown to impart a mutagenic phenotype. Perhaps unexpectedly, the corresponding Pol {varepsilon} allele in the yeast Saccharomyces cerevisiae (pol2-P301R), was found to drive greater mutagenesis than exonuclease-deficient Pol {varepsilon} (pol2-4), a phenotype sometimes termed ultra-mutagenesis. By studying the impact on mutation frequency, type, replication-strand bias, and sequence context, we show that ultra-mutagenesis is commonly observed in cells carrying a range of cancer-associated Pol {varepsilon} exonuclease domain alleles. Similarities between mutations generated by these alleles and those generated in pol2-4 cells indicate a shared mechanism of mutagenesis that yields a mutation pattern similar to cancer Signature 14. Comparison of POL2 ultra-mutator with pol2-M644G, a mutant in the polymerase domain decreasing Pol {varepsilon} fidelity, revealed unexpected analogies in the sequence context and strand bias of mutations. Analysis of mutational patterns unique to exonuclease domain mutant cells suggests that backtracking of the polymerase, when the mismatched primer end cannot be accommodated in the proofreading domain, results in the observed increase in insertions and T>A mutations in specific sequence contexts.

molecular biology↗

bioRxiv2020

Parallel genetics of regulatory sequences in vivo

Understanding how regulatory sequences control gene expression is fundamental to explain how phenotypes arise in health and disease. Traditional reporter assays inform about function of individual regulatory elements, typically in isolation. However, regulatory elements must ultimately be understood by perturbing them within their genomic environment and developmental- or tissue-specific contexts. This is technically challenging; therefore, few regulatory elements have been characterized in vivo. Here, we used inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3' UTRs of 16 genes in C. elegans. To quantify the consequences of mutations on expression, we developed a targeted RNA sequencing strategy across hundreds of mutant animals. We were also able to systematically and quantitatively assign fitness cost to mutations. Finally, we identified and characterized sequence elements that strongly regulate phenotypic traits. Our approach enables highly parallelized, functional analysis of regulatory sequences in vivo.

genetics↗

bioRxiv2019

Synaptic weight decay with selective consolidation enables fast learning without catastrophic forgetting

Learning can interfere with pre-existing memories that in classical neural networks may lead to catastrophic forgetting. Different from these networks, biological synapses show an early decay of long-term potentiation, combined with a tag & capture mechanism for selective consolidation. We present a 2-component synaptic plasticity model that, by an early decay and a tag & capture mechanism, enables context-dependent fast learning without catastrophic forgetting. For reinforcement learning in multiple contexts, the fast learning combined with an early weight decay maximizes the expected reward while minimizing interferences between subsequent contexts. Fast learning, enabled by a highly plastic weight component, improves performance for a given context. Between contexts this plastic component decays to prevent interference, but selective consolidation into a stable component protects old memories. As a downside of these mechanisms, learning is hampered when consolidation is triggered prematurely by interleaving easy and difficult tasks, consistent with human psychophysical experiments.

neuroscience↗

bioRxiv2018

Failure to learn during roving, analysing the unsupervised bias hypothesis.

We examine the unsupervised bias hypothesis [11] as an explanation for failure to learn two bisection tasks, when task sequencing is randomly alternating (roving). This hypothesis is based on the idea that a covariance based synaptic plasticity rule, which is modulated by a reward signal, can be biased when reward is averaged across multiple tasks of differing difficulties. We find that, in our hands, the hypothesis in its original form can never explain roving. This drives us to develop an extended mathematical analysis, which demonstrates not one but two forms of unsupervised bias. One form interacts with overlapping task representations and the other does not. We find that overlapping task representations are much more susceptible to unsupervised biases than non-overlapping representations. Biases from non-overlapping representations are more likely to stabilise learning. But this in turn is incompatible with the experimental understanding of perceptual learning and task representation, in bisection tasks. Finally, we turn to alternative network encodings and find that they also are unlikely to explain failure to learn during task roving as a result of unsupervised biases. As a solution, we present a single critic hypothesis, which is consistent with recent literature and could explain roving by a, much simpler, certainty normalised reward signalling mechanism.

neuroscience↗

bioRxiv2018

OUTRIDER: A statistical method for detecting aberrantly expressed genes in RNA sequencing data

RNA sequencing (RNA-seq) is gaining popularity as a complementary assay to genome sequencing for precisely identifying the molecular causes of rare disorders. A powerful approach is to identify aberrant gene expression levels as potential pathogenic events. However, existing methods for detecting aberrant read counts in RNA-seq data either lack assessments of statistical significance, so that establishing cutoffs is arbitrary, or rely on subjective manual corrections for confounders. Here, we describe OUTRIDER (OUTlier in RNA-seq fInDER), an algorithm developed to address these issues. The algorithm uses an autoencoder to model read count expectations according to the co-variation among genes resulting from technical, environmental, or common genetic variations. Given these expectations, the RNA-seq read counts are assumed to follow a negative binomial distribution with a gene-specific dispersion. Outliers are then identified as read counts that significantly deviate from this distribution. The model is automatically fitted to achieve the best correction of artificially corrupted data. Precision-recall analyses using simulated outlier read counts demonstrated the importance of combining correction for co-variation and significance-based thresholds. OUTRIDER is open source and includes functions for filtering out genes not expressed in a data set, for identifying outlier samples with too many aberrantly expressed genes, and for the P-value-based detection of aberrant gene expression, with false discovery rate adjustment. Overall, OUTRIDER provides a computationally fast and scalable end-to-end solution for identifying aberrantly expressed genes, suitable for use by rare disease diagnostic platforms.

bioinformatics↗

bioRxiv2018

ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks

Mutations in the ATM tumor suppressor confer hypersensitivity to DNA-damaging agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase poison topotecan. Thus, we establish that loss of terminal components of the non-homologous end-joining (NHEJ) machinery or the BRCA1-A complex specifically confers topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase inhibitor olaparib is due to delayed homologous recombination repair at DNA-replication-fork-associated double-strand breaks (DSBs), resulting in toxic NHEJ-mediated chromosome fusions. Accordingly, restoring legitimate repair in ATM-deficient cells, either by preventing NHEJ DNA ligation or by enhancing DSB-resection by BRCA1-A complex inactivation, markedly suppresses this toxicity. Our work suggests opportunities for patient stratification in ATM-deficient cancers and when using ATM inhibitors in the clinic, and identifies additional therapeutic vulnerabilities that might be exploited when such cancers evolve drug resistance.\n\nOne Sentence SummaryATM counteracts toxic NHEJ at broken replication forks

cell biology↗

bioRxiv2018

Loss-of-function in IRF2BPL is associated with neurological phenotypes

The Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) gene encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals affected with neurological symptoms who carry damaging heterozygous variants in IRF2BPL. Five cases carrying nonsense variants in IRF2BPL resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The bioinformatics signature for IRF2BPL based on population genomics is consistent with a gene that is intolerant to variation. We show that the IRF2BPL ortholog in the fruit fly, called pits (protein interacting with Ttk69 and Sin3A), is broadly expressed including the nervous system. Complete loss of pits is lethal early in development, whereas partial knock-down with RNA interference in neurons leads to neurodegeneration, revealing requirement for this gene in proper neuronal function and maintenance. The nonsense variants in IRF2BPL identified in patients behave as severe loss-of-function alleles in this model organism, while ectopic expression of the missense variants leads to a range of phenotypes. Taken together, IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

genetics↗

bioRxiv2017

Detection of functional protein domains by unbiased genome-wide forward genetic screening

Genetic and chemo-genetic interactions have played key roles in elucidating the molecular mechanisms by which certain chemicals perturb cellular functions. Many studies have employed gene knockout collections or gene disruption/depletion strategies to identify routes for evolving resistance to chemical agents. By contrast, searching for point-mutational genetic suppressors that can identify separation- or gain-of-function mutations, has been limited even in simpler, genetically amenable organisms such as yeast, and has not until recently been possible in mammalian cell culture systems. Here, by demonstrating its utility in identifying suppressors of cellular sensitivity to the drugs camptothecin or olaparib, we describe an approach allowing systematic, large-scale detection of spontaneous or chemically-induced suppressor mutations in yeast and in haploid mouse embryonic stem cells in a short timeframe, and with potential applications in essentially any other haploid system. In addition to its utility for molecular biology research, this protocol can be used to identify drug targets and to predict mechanisms leading to drug resistance. Mapping suppressor mutations on the primary sequence or three-dimensional structures of protein suppressor hits provides insights into functionally relevant protein domains, advancing our molecular understanding of protein functions, and potentially helping to improve drug design and applicability.

molecular biology↗