Biology subjects

Roth, F.

Publications and source records attributed to Roth, F..

14 recordsLinked to original sources

bioRxiv2026

The evolution of a Na+-sensitive Vibrio cholerae mutant unmasks the moonlighting aminopeptidase PepA as a regulator of nhaB Na+/H+ antiporter gene expression

The pathogenic bacterium Vibrio cholerae is native to seawater and can therefore be cultivated in nutrient media with increased salt concentration. To maintain osmotic balance, V. cholerae uses Na+/H+ antiporters and an Na+-translocating NADH:quinone oxidoreductase (Na+-NQR). The exact contribution of the various Na+/H+ antiporters to maintain Na+ and H+ homeostasis in V. cholerae is unclear. However, genetic studies indicate the major Na+/H+ antiporter NhaA and the Na+-NQR are required by the bacteria for Na+ resistance at alkaline conditions. Here we show that the growth defect of a V. cholerae nhaA nqr mutant at increased Na+ concentrations and alkaline pH is relieved by the rapid acquisition of suppressor mutations. The suppressor mutants could be assigned to two classes. We identified (i) mutations in the promoter of the nhaB gene encoding a Na+/H+ antiporter and (ii) mutations that either affect the expression level of the pepA gene or DNA binding activity of the encoded multifunctional aminopeptidase PepA. The characterization of the PnhaB and PpepA promoters of the suppressor mutants, membrane potential measurements and comparative proteome analyses identified PepA as a novel factor controlling Na+ homeostasis in V. cholerae. IMPORTANCEThe emergence and spread of multi-resistant bacteria are a major problem for humans and animals. Therefore, the identification of novel targets for the development of novel antibiotics to combat pathogenic bacteria is extremely important. Since sodium ion homeostasis is an essential process in many pathogenic bacteria, especially those living in marine environments, it represents an interesting target for antibiotics. The perturbation of sodium ion homeostasis does indeed impair the fitness of the human pathogenic bacterium Vibrio cholerae but can be restored by the rapid evolution of the bacteria. This suggests that inhibiting multiple targets with different antibiotics might be more effective in preventing the development of resistant bacteria.

microbiology↗

bioRxiv2025

Tissue Engineered Elastic Cartilage-Mimetic Auricular Grafts for Ear Reconstruction

Patients born with microtia, the congenital malformation of the external ear, face substantial psychosocial strain. Current reconstruction relies on harvesting rib cartilage, an invasive procedure associated with donor site morbidity and unnaturally stiff ears due to the use of hyaline cartilage. Tissue engineered auricles could overcome these drawbacks by providing patient-specific elastic cartilage without the need for rib harvest. Yet, key challenges such as fibrocartilage formation, inhomogeneous extracellular matrix formation and mechanical inferiority during ex vivo maturation remain, often leading to graft deformation and degradation in vivo. To address this gap, we integrated approaches maintaining the chondrogenic potential with growth factors, promoting elastic cartilage formation through stress-relaxing materials, and achieving homogeneous maturation by culturing grafts on an elevated bioreactor platform that enables uniform nutrient diffusion, using primary human auricular chondrocytes. Together these approaches resulted in the maturation of bioprinted auricular grafts that closely resemble native human auricular cartilage, demonstrated by the uniform distribution of elastin, glycosaminoglycans, and collagen II, and lack of collagen I. RNA sequencing revealed gene expression patterns consistent with the transition from fibrocartilage towards elastic cartilage. On a functional level, grafts achieved a compressive modulus of 1.1{+/-}0.03 MPa, matching that of native human auricular cartilage (1.0{+/-}0.1 MPa) and maintained their structural integrity for 6 weeks in a subcutaneous rat model, where they transitioned towards mature elastic fibers. These grafts represent the closest approximation of native elastic cartilage achieved ex vivo to date, bringing the field closer to a clinically viable, long-term therapy for children affected by microtia. One-sentence summaryBioprinted auricular grafts develop native-like elastic cartilage and advance towards a durable therapy for children with microtia.

bioengineering↗

bioRxiv2025

Comprehensively Testing the Function of Missense Variation in the STK11 Tumour Suppressor

The tumor suppressor gene STK11 encoding Serine/Threonine Kinase 11 (STK11) is associated with Peutz-Jeghers Syndrome (PJS), a heritable gastrointestinal disease that increases lifetime cancer risk, and with somatic variation that contributes to [~]30% of lung and 20% of cervical cancers. Although identifying pathogenic variants is clinically actionable, over 94% of STK11 missense variants that have been observed clinically lack a definitive classification. We therefore measured the impact of STK11 variants at scale in a mammalian cell-based assay, scoring 6,026 (73% of all possible) amino acid substitutions across the full-length gene. Functional scores--which were consistent with biochemical properties, smaller-scale assays, and pathogenicity annotations--identified a subset of PJS patients with germline STK11 variants diagnosed later in life, as well as somatic STK11 variants found in cancer patients that had comparable overall survival estimates to wild-type STK11. Our scores provided new evidence for 350 annotated VUS STK11 missense variants and [~]80% of missense variants that have not yet been reported clinically, but we might expect to observe in the future. Thus, our effect map provides a proactive resource for gaining sequence-structure-function insights and evidence for actionable interpretation of clinical missense variants.

cancer biology↗

bioRxiv2025

Long-term euxinia hinders microbial ammonium removal in brackish coastal waters

Anthropogenic activities are key drivers of eutrophication and deoxygenation in coastal marine ecosystems. This stimulates the anaerobic degradation of organic matter and the release of reduced products, such as ammonium, methane, and hydrogen sulfide, which may, in turn, exacerbate eutrophication and deoxygenation. In this study, using a combination of chemical and microbial analyses, we assess the nitrogen dynamics in the water column of a eutrophic coastal system (Stockholm Archipelago) at three sites with contrasting redox conditions (oxic to long-term euxinic). At the oxic site, counter gradients of ammonium and oxygen in the water column, low nitrate {delta}15N values in bottom waters, and the 16S rRNA gene-based presence of nitrifiers indicate nitrification near the sediment-water interface. At the seasonally and long-term euxinic sites, nitrification, as inferred from the water column oxygen and nutrient profiles and the relative abundance of nitrifiers, primarily occurred near the oxycline. At these two sites, nitrate was removed below the oxycline through denitrification linked to sulfide oxidation by Sulfurimonas. Nitrous oxide emissions from surface waters in the archipelago reached up to 40 {micro}mol m-2 d-1 and were not directly related to water column redox conditions, indicating that multiple factors control coastal emissions of this greenhouse gas to the atmosphere. The relative abundance of 16S rRNA genes and of N-cycle genes in metagenomes was highest at the seasonally euxinic site. Importantly, nitrifiers were significantly less abundant at the long-term euxinic site. Our results highlight that prolonged euxinia promotes recycling of ammonium over its removal, likely due to sulfide inhibition of nitrification, which sustains eutrophication and deoxygenation of coastal systems.

microbiology↗

bioRxiv2025

Scaled multidimensional assays of variant effect identify sequence-function relationships in hypertrophic cardiomyopathy

BackgroundAn estimated 1 in 500 people live with hypertrophic cardiomyopathy (HCM), a disease for which genetic diagnosis can identify family members at risk, and increasingly guide therapy. Mutations in the myosin binding protein C3 (MYBPC3) gene account for a significant proportion of HCM cases. However, many of these variants are classified as variants of uncertain significance (VUS), complicating clinical decision-making. Scalable methods for variant interpretation in disease-specific cell types are crucial for understanding variant impact and uncovering disease mechanisms. MethodsWe developed a scaled multidimensional mapping strategy to evaluate the functional impact of variants across a critical domain of MYBPC3. We incorporate saturation base editing at the native MYBPC3 locus, a long-read RNA sequencing-enabled assay of variant splice effects, and measurements of HCM-relevant phenotypes, including MYBPC3 abundance, hypertrophic signaling, and ubiquitin-proteasome function in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). ResultsOur multidimensional mapping strategy enabled high-resolution functional analysis of MYBPC3 variants in iPSC-CMs. Targeted transient base editing generated a comprehensive variant library at the native locus, capturing diverse variant effects on cellular HCM-relevant phenotypes. Our massively parallel splicing assay identified novel splice-disrupting variants. Integration of functional assays revealed that decreased MYBPC3 abundance is a key driver of HCM-related phenotypes. In parallel, downregulation of protein degradation was observed as a compensatory response to MYBPC3 loss of function, and novel disease mechanisms were identified for missense variants near a critical binding domain, underscoring their contribution to pathogenesis. Bayesian estimates of variant effects enable the reclassification of clinical variants. ConclusionsThis work provides a platform for extending genome engineering in iPSCs to multiplexed assays of variant effects across diverse disease-relevant cellular phenotypes, enhancing the understanding of variant pathogenicity and uncovering novel biological mechanisms that could inform therapeutic strategies.

genetics↗

bioRxiv2024

Immunometabolic analysis of primary murine Group 2 Innate Lymphoid Cells: a robust step-by-step approach

Group 2 Innate Lymphoid Cells (ILC2s) have recently been shown to exert key regulatory functions in innate and adaptive immune response networks that drive the establishment and progression of type 2 immunity and its associated pathologies. Although mainly tissue resident, ILC2s and their crosstalk within tissue microenvironments influences both local and systemic metabolism. In turn, the metabolic status shapes the diverse ILC2 phenotypes and effector functions. Hence, deciphering the metabolic networks of ILC2s is essential in understanding ILC2s roles in health as well as pathophysiologies. Here we detail a framework of experimental approaches to study key immunometabolic states of primary murine ILC2s and link them to phenotypes and functionality. Utilizing flow cytometry, Single Cell ENergetIc metabolism by ProfilIng Translation inhibition (SCENITH) as well as the Seahorse platform we provide a framework that allows in-depth analysis of cellular bioenergetic states to determine the immunometabolic wiring of ILC2. Linking immunometabolic states and networks to ILC2 phenotypes and effector functions will allow in-depth studies that assess the potential of novel pharmaceutics to alter ILC2 functionality in experimental and clinical settings.

immunology↗

bioRxiv2023

Pervasive mislocalization of pathogenic coding variants underlying human disorders

Widespread sequencing has yielded thousands of missense variants predicted or confirmed as disease-causing. This creates a new bottleneck: determining the functional impact of each variant - largely a painstaking, customized process undertaken one or a few genes or variants at a time. Here, we established a high-throughput imaging platform to assay the impact of coding variation on protein localization, evaluating 3,547 missense variants of over 1,000 genes and phenotypes. We discovered that mislocalization is a common consequence of coding variation, affecting about one-sixth of all pathogenic missense variants, all cellular compartments, and recessive and dominant disorders alike. Mislocalization is primarily driven by effects on protein stability and membrane insertion rather than disruptions of trafficking signals or specific interactions. Furthermore, mislocalization patterns help explain pleiotropy and disease severity and provide insights on variants of unknown significance. Our publicly available resource will likely accelerate the understanding of coding variation in human diseases.

cell biology↗

bioRxiv2022

ZNF524 directly interacts with telomeric DNA and supports telomere integrity

Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing non-homologous end joining (NHEJ) through its subunit TRF2. We here describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity and reveal the base-specific sequence recognition by co-crystallization with telomeric DNA. ZNF524 localizes to telomeres and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity.

cell biology↗

bioRxiv2022

A multiplexed assay of human glucokinase reveals thousands of potential disease variants with both decreased and increased activity

Glucokinase (GCK) regulates insulin secretion to maintain appropriate blood glucose levels. Sequence variants can alter GCK activity to cause hyperinsulinemic hypoglycemia (HH) or hyperglycemia associated with GCK-maturity-onset diabetes of the young (GCK-MODY), collectively affecting up to 10 million people worldwide. Patients with GCK-MODY are frequently misdiagnosed and treated unnecessarily. Genetic testing can prevent this but is hampered by the challenge of interpreting novel missense variants. Here we exploited a multiplexed yeast complementation assay to measure both hyper- and hypoactive GCK variation, capturing 97% of all possible missense and nonsense variants. Activity scores correlated with in vitro catalytic efficiency, fasting glucose levels in carriers of GCK variants and with evolutionary conservation. Hypoactive variants were concentrated at buried positions, near the active site, and at a region of known importance for GCK conformational dynamics. Some hyperactive variants shifted the conformational equilibrium towards the active state through a relative destabilization of the inactive conformation. Our comprehensive assessment of GCK variant activity promises to facilitate variant interpretation and diagnosis, expand our mechanistic understanding of hyperactive variants, and inform development of therapeutics targeting GCK.

genetics↗

bioRxiv2020

No impact of functional connectivity of the motor system on the resting motor threshold: A replication study.

The physiological mechanisms of corticospinal excitability and factors influencing its measurement with transcranial magnetic stimulation are still poorly understood. A recent study reported an impact of functional connectivity between the primary motor cortex and dorsal premotor cortex on the resting motor threshold of the dominant hemisphere. We aimed to replicate these findings in a larger sample of 38 healthy right-handed subjects with data from both hemispheres. Resting-state functional connectivity was assessed between the primary motor cortex and five a-priori defined motor-relevant regions on each hemisphere as well as interhemispherically between both primary motor cortices. Following the procedure by the original authors, we included age, the cortical grey matter volume and coil to cortex distance as further predictors in the analysis. We report replication models for the dominant hemisphere as well as an extension to data from both hemispheres and support the results with Bayes factors. Functional connectivity between the primary motor cortex and dorsal premotor cortex did not explain variability in the resting motor threshold and we obtained moderate evidence for the absence of this effect. In contrast, coil to cortex distance could be confirmed as an important predictor with strong evidence. These findings contradict the previously proposed effect, thus questioning the notion of the dorsal premotor cortex playing a major role in modifying corticospinal excitability.

neuroscience↗

bioRxiv2020

Multiplexed measurement of variant abundance and activity reveals VKOR topology, active site and human variant impact

Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin Despite VKORs pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency 2 or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease.

genomics↗

bioRxiv2018

Guanabenz treatment improves Oculopharyngeal muscular dystrophy phenotype

Oculopharyngeal muscular dystrophy (OPMD) is a rare late onset genetic disease affecting most profoundly eyelid and pharyngeal muscles, leading respectively to ptosis and dysphagia, and proximal limb muscles at later stages. A short abnormal (GCG) triplet expansion in the polyA- binding protein nuclear 1 (PABPN1) gene leads to PABPN1-containing aggregates in the muscles of OPMD patients. It is commonly accepted that aggregates themselves, the aggregation process and/or the early oligomeric species of PABPN1 are toxic in OPMD. Decreasing PABPN1 aggregate load in animal models of OPMD ameliorates the muscle phenotype. In order to identify a potential therapeutic molecule that would prevent and reduce aggregates, we tested guanabenz acetate (GA), an FDA-approved antihypertensive drug, in OPMD cells as well as in the A17 OPMD mouse model. We demonstrate that treating mice with GA reduces the size and number of nuclear aggregates, improves muscle force, protects myofibres from the pathology-derived turnover and decreases fibrosis. GA is known to target various cell processes, including the unfolded protein response (UPR), which acts to attenuate endoplasmic reticulum (ER) stress. Here we used a cellular model of OPMD to demonstrate that GA increases both the phosphorylation of the eukaryotic translation initiator factor 2 subunit (eIF2) and the splicing of Xbp1, key components of the UPR. Altogether these data suggest that modulation of protein folding regulation can be beneficial for OPMD and support the further development of guanabenz or its derivatives for treatment of OPMD in humans.\n\nSignificance StatementOculopharyngeal muscular dystrophy (OPMD) is a rare late onset incurable genetic disease characterized by the formation of insoluble aggregates in skeletal muscles. It has been shown that the reduction of aggregates correlates with an improvement of the disease. Here we used a mouse model of OPMD to show that Guanabenz acetate, the active constituent of a marketed but recently discontinued drug for hypertension, decreases the number and the size of aggregates after systemic delivery and improves many aspects of the disease. We also describe experimental evidences explaining the mechanism behind the efficacy of such compound for OPMD.

pathology↗

bioRxiv2017

Quantifying Immune-Based Counterselection of Somatic Mutations

It is now well established that somatic mutations in protein-coding regions can generate neoantigens, and that these can be recognized by the immune system and contribute to clearance of developing cancers. However, there is currently no model that can quantitatively predict the neoantigenic effect of any given somatic mutation. Here, we examined signatures of immune selection pressure on the distribution of somatic mutations. We quantified the extent to which somatic mutations are significantly depleted in peptides that are predicted to be displayed by major histocompatibility complex (MHC) class I proteins. We characterized the dependence of this depletion on expression level. We then examined whether immune selection pressure on somatic mutations changes depending on whether the patient had either one or two MHC-encoding alleles that can display the peptide. Our results indicate that MHC-encoding alleles are, in general, incompletely dominant, i.e., that having two copies of the display-enabling allele is more effective in displaying that peptide than having just one copy. More generally, a quantitative understanding of counter-selection of identifiable subclasses of neoantigenic somatic variation could guide immunotherapy or aid in developing personalized cancer vaccines.

genetics↗

bioRxiv2017

mTORC1-mediated inhibition of 4EBP1 is essential for Hedgehog (HH) signaling and can be targeted to suppress HH-driven medulloblastoma

Mechanistic target of rapamycin (MTOR) cooperates with Hedgehog (HH) signaling, but the underlying mechanisms are incompletely understood. Here, we provide genetic, biochemical, and pharmacologic evidence that MTOR complex 1 (mTORC1)-dependent translation is a prerequisite for HH signaling. The genetic loss of mTORC1 function inhibited HH signaling- driven growth of the cerebellum and medulloblastoma. Inhibiting translation or mTORC1 blocked HH signaling. Depleting 4EBP1, an mTORC1 target that inhibits translation, alleviated the dependence of HH signaling on mTORC1. Consistent with this, phosphorylated 4EBP1 levels were elevated in HH signaling-driven medulloblastomas in mice and humans. In mice, an mTORC1 inhibitor suppressed medulloblastoma driven by a mutant SMO that is resistant to an SMO inhibitor in the clinic, prolonging the survival of the mice. Our study reveals mTORC1-mediated translation to be a key component of HH signaling and an important target for treating medulloblastoma and other cancers driven by HH signaling.

developmental biology↗