Biology subjects

Steiner, C.

Publications and source records attributed to Steiner, C..

6 recordsLinked to original sources

bioRxiv2026

A Tandem Fluorescent Rh1::eGFP mCherry Reporter Reveals Rhodopsin 1 Trafficking and Turnover In Vivo in Drosophila Photoreceptors

Rhodopsin 1 (Rh1), the major visual pigment of Drosophila photoreceptors, requires a vitamin A-derived chromophore for proper folding, maturation and transport to the rhabdomere. While the endocytic trafficking of mature, light-activated Rh1 to endolysosomal compartments is well characterized, the fate of immature Rh1 during defective ER maturation remains unclear. Here, we developed a Rh1::eGFP-mCherry tandem reporter to monitor Rh1 localization and compartment acidification in vivo. Exploiting the acid stability of mCherry, this reporter enables visualization of Rh1 after delivery to acidic environments and complements conventional antibody-based approaches. Under physiological conditions, pH-dependent quenching of eGFP fluorescence distinguished rhabdomeric Rh1 from Rh1 in acidic endolysosomal compartments. In vitamin A-deficient flies, immature apo-Rh1 accumulated within Calnexin-positive ER membranes, where we identified discrete acidified subdomains characterized by loss of eGFP fluorescence while mCherry fluorescence persisted. These structures remained continuous with the ER but were distinct from conventional endolysosomal compartments, revealing previously unrecognized acidified ER domains associated with immature Rh1. Together, our findings identify acidified ER subdomains as a previously unrecognized stage in Rh1 quality control and establish Rh1::eGFP-mCherry as a robust in vivo reporter for investigating rhodopsin trafficking, maturation and turnover under physiological and disease-relevant conditions.

biochemistry↗

bioRxiv2026

Selection of potent biologic antagonists of the cannabinoid GPCR CB2R from a constrained peptide library

Dysregulated gut homing of leukocytes drives chronic inflammation in Crohns disease (CD). We employed phage display selection campaigns with libraries of stabilized, constrained peptides against endogenous conformation states of the cannabinoid receptor CB2R on human T cells, to discover novel receptor antagonists with potential to inhibit gut homing. Cluster and frequency analysis of 50,000 enriched sequences resulted in expression and functional characterisation of 10 protein candidates using assays of glucose uptake, ERK phosphorylation (pERK) and {beta}-arrestin recruitment. Each candidate antagonised CB2R activity with recorded IC50 values of between 5-10 nM. Cannabinoid receptor nanodisc binding experiments and FSEC confirmed CB2R selectivity. SLKC_09 with an IC50 of 5.4 nM, was studied in a mouse model of chronic ileitis where it significantly inhibited gut homing of CD4+ & CD8+ naive, effector and memory cell types. Our findings highlight an alternative route to therapeutic inhibition of leukocyte trafficking in CD with a biologic inhibitor of CB2R.

immunology↗

bioRxiv2026

Collective protection drives human gut microbiota response to amoxicillin treatment.

The gut microbiota is central to human health, contributing to nutrient processing, metabolite production and protection against pathogens. Yet it is also an unintended target of antibiotic treatments, particularly after oral administration. Antibiotic exposure can therefore disrupt community structure, leading to dysbiosis and promoting the selection of resistant bacteria. Although studies in patients and animal models have shown that these effects vary markedly between individuals, host-related factors have made it difficult to isolate the specific contribution of the microbiota itself. Here, we used a controlled in vitro gut model (MBRA) to examine how 16 human gut microbiotas from the NutriNet-Sante cohort respond to the widely prescribed {beta}-lactam amoxicillin (AMX). By combining dense temporal sampling, 16S amplicon sequencing and mass spectrometry, we observed highly heterogeneous response trajectories, ranging from near-stable communities to strong but reversible disruptions. These differences were not only reflected in the final magnitude of perturbation, but also in the timing, pace and recovery of microbiota change during treatment. Initial community composition partly structured these responses, as Lachnospiraceae/Bacteroidaceae ratio strongly correlated with perturbation. Dynamic quantification of AMX further showed that microbiotas differed in their capacity to deplete the drug over time, thereby altering the duration of exposure above critical concentration thresholds. Supplementation with clavulanic acid that inhibits {beta}-lactamases confirmed that this process was largely mediated by {beta}-lactamase activity. Finally, microbiotas displaying rapid AMX depletion showed reduced selection of resistant Enterobacteriaceae. Together, our results indicate that both initial community composition and the temporal dynamics of antibiotic inactivation jointly determine microbiota resilience and resistance selection.

microbiology↗

bioRxiv2024

EXTRA-seq: a genome-integrated extended massively parallel reporter assay to quantify enhancer-promoter communication

Precise control of gene expression is essential for cellular function, but the mechanisms by which enhancers communicate with promoters to coordinate this process are not fully understood. While sequence-based deep learning models show promise in predicting enhancer-driven gene expression, experimental validation and human-interpretable mechanistic insights lag behind. Here, we present EXTRA-seq, a novel EXTended Reporter Assay followed by sequencing designed to quantify enhancer activity in endogenous contexts over kilobase-scale distances. We demonstrate that EXTRA-seq can be targeted to disease-relevant loci and captures expression changes at the resolution of individual transcription factor binding sites, enabling mechanistic discovery. Using engineered synthetic enhancer-promoter combinations, we reveal that the TATA-box acts as a dynamic range amplifier, modulating expression levels in function of enhancer strength. Importantly, we find that integrating state-of-the-art deep learning models with plasmid-based enhancer assays improves the prediction of gene expression as measured by EXTRA-seq. These findings open new avenues for predictive modeling and therapeutic applications. Overall, our work provides a powerful experimental platform to interrogate the complex interplay between enhancers and promoters, bridging the gap between in silico predictions and human-interpretable biological mechanisms.

genomics↗

bioRxiv2024

Identification of methylation-sensitive human transcription factors using meSMiLE-seq

Transcription factors (TFs) are key players in eukaryotic gene regulation, but the DNA binding specificity of many TFs remains unknown. Here, we assayed 284 mostly poorly characterized, putative human TFs using selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq), revealing 72 new DNA binding motifs. To investigate whether some of the 158 TFs for which we did not find motifs preferably bind epigenetically modified DNA (i.e. methylated CG dinucleotides), we developed methylation-sensitive SMiLE-seq (meSMiLE-seq). This microfluidic assay simultaneously probes the affinity of a protein to methylated and unmethylated DNA, augmenting the capabilities of the original method to infer methylation-aware binding sites. We assayed 114 TFs with meSMiLE-seq and identified DNA-binding models for 48 proteins, including the known methylation-sensitive binding modes for POU5F1 and RFX5. For 11 TFs, binding to methylated DNA was preferred or resulted in the discovery of alternative, methylation-dependent motifs (e.g. PRDM13), while aversion towards methylated sequences was found for 13 TFs (e.g. USF3). Finally, we uncovered a potential role for ZHX2 as a putative binder of Z-DNA, a left-handed helical DNA structure which is adopted more frequently upon CpG methylation. Altogether, our study significantly expands the human TF codebook by identifying DNA binding motifs for 98 TFs, while providing a versatile platform to quantitatively assay the impact of DNA modifications on TF binding.

genomics↗

bioRxiv2018

A high-quality, long-read de novo genome assembly to aid conservation of Hawaii’s last remaining crow species

Genome-level data can provide researchers with unprecedented precision to examine the causes and genetic consequences of population declines, and to apply these results to conservation management. Here we present a high-quality, long-read, de novo genome assembly for one of the worlds most endangered bird species, the Alala. As the only remaining native crow species in Hawaii, the Alala survived solely in a captive breeding program from 2002 until 2016, at which point a long-term reintroduction program was initiated. The high-quality genome assembly was generated to lay the foundation for both comparative genomics studies, and the development of population-level genomic tools that will aid conservation and recovery efforts. We illustrate how the quality of this assembly places it amongst the very best avian genomes assembled to date, comparable to intensively studied model systems. We describe the genome architecture in terms of repetitive elements and runs of homozygosity, and we show that compared with more outbred species, the Alala genome is substantially more homozygous. We also provide annotations for a subset of immunity genes that are likely to be important for conservation applications, and we discuss how this genome is currently being used as a roadmap for downstream conservation applications.

genomics↗