Biology subjects

Roth, B.

Publications and source records attributed to Roth, B..

4 recordsLinked to original sources

bioRxiv2024

Acid excretion is impaired in calcium oxalate stone formers

BackgroundUrine pH is a key factor in kidney stone formation. We aimed to identify whether acid excretion capacity is disturbed in calcium oxalate (CaOx) or calcium phosphate (CaP) stone formers. MethodUrinary, serum, clinical, and anthropomorphic baseline data were obtained from the Swiss Kidney Stone Cohort, a prospective, longitudinal, and multi-centric observational study. We included in this study 193 non-stone formers (NSF, confirmed by negative CT scan), and 309 CaOx and 28 CaP stone formers. Titratable acids, net acid excretion (NAE), NAE capacity (NAEC) and acid-base (AB) score were calculated. Logistic regression analyses were used to estimate the potential associations of various acid-base variables with the occurrence of CaOx kidney stones. ResultsCaOx stone formers showed a disturbed capacity to excrete acids in comparison to NSF (NAEC NSF = 3.49{+/-}12.6 mmol/24h; CaOx = -1.06{+/-}13.10; CaP = 0.97{+/-}14.70 and AB score NSF = 20.5{+/-}6.36 mmol/24h; CaOx = 17.9{+/-} 6.53; CaP = 18.8{+/-}6.10). The correlation between urine calcium and urine pH was altered in CaOx stone formers and between urine calcium and NAE was stronger in CaP stone formers. Logistic models showed that urinary ammonium was negatively associated with CaOx stone formation (unadjusted model, odds ratio 0.43[0.32-0.58], p< 0.001 for CaOx). Urine calcium was positively associated with CaOx kidney stones (2.85 [2.11-3.92], p<0.001). Similar results were obtained after adjusting for age, sex, and BMI. Replacing urine ammonium, pH, and phosphate with NAEC or ammonium and pH with AB score in our logistic regression models showed that NAEC and AB score are strongly associated with CaOx kidney stone formation. ConclusionAmmonium excretion, NAEC and AB score are associated with the occurrence of CaOx kidney stones suggesting a potential role of proximal tubule dysfunction in their formation. CaP stone formers exhibit a disproportionately higher calcium excretion when acid excretion increases. Key learning pointsO_ST_ABSWhat was knownC_ST_ABSUrine pH is a strong determinant in the formation of various urologically relevant crystals. Impaired urine acidification capacity has been observed in individuals who form calcium phosphate and uric acid stones. This study addsWhen compared to non-stone formers, calcium oxalate stone formers are marked by a reduced capacity of excreting acids when urine pH becomes more acidic. Potential impactThe calculation of net acid excretion capacity and acid-base score are novel tools to identify those under potential higher risk of developing calcium oxalate stones.

physiology↗

bioRxiv2023

AlphaFold2 structures template ligand discovery

AlphaFold2 (AF2) and RosettaFold have greatly expanded the number of structures available for structure-based ligand discovery, even though retrospective studies have cast doubt on their direct usefulness for that goal. Here, we tested unrefined AF2 models prospectively, comparing experimental hit-rates and affinities from large library docking against AF2 models vs the same screens targeting experimental structures of the same receptors. In retrospective docking screens against the {sigma}2 and the 5-HT2A receptors, the AF2 structures struggled to recapitulate ligands that we had previously found docking against the receptors experimental structures, consistent with published results. Prospective large library docking against the AF2 models, however, yielded similar hit rates for both receptors versus docking against experimentally-derived structures; hundreds of molecules were prioritized and tested against each model and each structure of each receptor. The success of the AF2 models was achieved despite differences in orthosteric pocket residue conformations for both targets versus the experimental structures. Intriguingly, against the 5-HT2A receptor the most potent, subtype-selective agonists were discovered via docking against the AF2 model, not the experimental structure. To understand this from a molecular perspective, a cryoEM structure was determined for one of the more potent and selective ligands to emerge from docking against the AF2 model of the 5-HT2A receptor. Our findings suggest that AF2 models may sample conformations that are relevant for ligand discovery, much extending the domain of applicability of structure-based ligand discovery.

pharmacology and toxicology↗

bioRxiv2023

An automated proximity proteomics pipeline for subcellular proteome and protein interaction mapping

Proximity labeling (PL) through biotinylation coupled with mass spectrometry (MS) has emerged as a powerful technique for capturing spatial proteomes within living cells. Large-scale sample processing for proximity proteomics requires a workflow that minimizes hands-on time while enhancing quantitative reproducibility. Here, we present a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. By combining this pipeline with an optimized quantitative MS acquisition method based on data-independent acquisition (DIA), we not only significantly increased sample throughput but also improved the reproducibility of protein identification and quantification. We applied this pipeline to delineate subcellular proteomes across various cellular compartments, including endosomes, late endosomes/lysosomes, the Golgi apparatus, and the plasma membrane. Moreover, employing 5HT2A serotonin receptor as a model, we investigated temporal changes of proximal interaction networks induced by the receptors activation with serotonin. Finally, to demonstrate the applicability of our PL pipeline across multiple experimental conditions, we further modified the PL pipeline for reduced sample input amounts to accommodate CRISPR-based gene knockout, and assessed the dynamics of the 5HT2A network in response to the perturbation of selected proximal interactors. Importantly, the presented PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, increasing both throughput and reproducibility of standard protocols.

systems biology↗

bioRxiv2020

X-ray Irradiation activates immune response in human T-lymphocytes by eliciting a Ca2+ signaling cascade

Radiation therapy is efficiently employed for eliminating cancer cells and reducing tumor growth. To further improving its therapeutic application it is mandatory to unravel the molecular effects of ionizing irradiation and to understand whether they support or counteract tumor therapy. Here we examine the impact of X-ray irradiation on immune activation of human T cells with single doses typically employed in tumor therapy. We discover that exposing cells to radiation triggers in a population of leukemic Jurkat T cells and in peripheral blood mononuclear cells (PBMCs) a canonical Ca2+ signaling cascade, which elicits immune activation of these cells. An early step in the signaling cascade is the initiation of sustained oscillations of the cytosolic Ca2+ concentration, an event mediated by store operated Ca2+ entry (SOCE) via an X-ray induced clustering of the Calcium Release-Activated Calcium Modulator 1 with the stromal interaction molecule 1 (Oari1/STIM1). A functional consequence of the Ca2+ signaling cascade is the translocation of the transcription factor nuclear factor of activated T cells (NFAT) from the cytosol into the nucleus where it elicits the expression of genes required for immune activation. These data imply that a direct activation of blood immune cells by ionizing irradiation has an impact on toxicity and therapeutic effects of radiation therapy.

biophysics↗