Biology subjects

Dietz, T.

Publications and source records attributed to Dietz, T..

3 recordsLinked to original sources

bioRxiv2026

Predicted Bacterial uRBSs Reveal Translational Coupling and Ribosome Mediated RBS Occlusion as Gene-Controlling Mechanisms

Upstream open reading frames (uORFs) in the 5' leader of bacterial mRNAs can modulate gene expression, yet genome-wide identification remains limited. We combined bioinformatic prediction of ribosome-binding sites (RBSs) - a Shine-Dalgarno sequence and a start codon - with experimental validation to uncover new uORFs in Sinorhizobium meliloti 2011. From totally 1106 predicted upstream RBSs (uRBSs), we first examined 15 candidates using eGFP reporters and integrating existing RNA-seq and Ribo-seq data. Translation was detected at 13 sites, with fluorescence intensity broadly correlating with predicted initiation rates. Two uRBSs correspond to gene start sites, thereby refining gene annotations. In nine cases, uRBS mutations affected downstream gene expression in reporter fusions. Among others, the data suggests that a Type I secretion system operon, the RNA chaperone gene hfq, and metabolic genes are regulated by uORFs. Four uORFs acted through translational coupling. We also identified uRBSs that were ribosome-occupied yet (nearly) silent in eGFP assays, and closely spaced to the downstream main RBS (mRBS). These uRBSs probably mediate ribosomal occlusion downregulating lacR and SM2011_RS36230. A re-screen of the prediction set revealed 335 close uRBS/mRBS pairs. Three of them were analyzed, supporting the proposed ribosomal occlusion mechanism for SM2011_RS03630 and SM2011_RS22110, while for glnK translational coupling to an uORF was suggested. These results indicate that uORFs are more widespread in bacteria than previously recognized and suggest that direct ribosomal occlusion of the mRBS is a novel mechanism for down-regulating protein synthesis.

molecular biology↗

bioRxiv2025

A live tumor fragment platform to assess immunotherapy response in core needle biopsies while addressing challenges of tumor heterogeneity

BackgroundImmune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, providing durable and even curative responses. However, most patients do not respond and current biomarkers (eg, programmed death ligand (PD-L1), mismatch repair deficiency (dMMR)/high microsatellite instability (MSI) and tumor mutational burden) lack predictive accuracy. Ex vivo profiling of patient-derived tumor fragments shows promise as a predictive biomarker but relies on substantial surgical tissue to mitigate intra-specimen heterogeneity. Innovations are needed that address these challenges, particularly where limited tissue is available in core needle biopsies (CNBs). MethodsLive tumor fragments (LTFs) were generated from 59 human tumor resections and 31 CNBs from patients enrolled in observational clinical trials (ClinicalTrials.gov identifiers: NCT05478538, NCT05520099, NCT06349642) to assess cytokine induction following ICI treatment. LTFs were encapsulated in hydrogel and cultured ex vivo for up to 72 hours. A sequential treatment strategy that applies control and treatment within the same well was used with response to ICI or CD3/CD28 assessed using a multiplex secretome assay. Viability was assessed using established metabolic assays and dynamic optical coherence microscopy. ResultsLTFs maintained viability and retained T cells responsive to stimulation throughout ex vivo culture. Multiplex immunofluorescence and immunohistochemistry showed key components of the tumor microenvironment, including relative proportions of CD4+ and CD8+ immune cell populations, were preserved. Specimens positive for PD-L1 or dMMR/MSI-high were enriched for cytokine upregulation, including T-cell response cytokines IFN{gamma} and CXCL10, after PD-1 treatment. To demonstrate clinical applicability of the sequential treatment strategy, CNBs from patients with lung, gastrointestinal or kidney cancer were profiled and differential cytokine induction in response to ICI treatment was observed. ConclusionsThe novel ex vivo platform presented is capable of detecting T-cell response to ICI treatment by using a sequential treatment strategy. This approach addresses challenges associated with cross-well heterogeneity in tissue composition and requires half as much tissue as a cross-well comparison, mitigating tissue limitations typically associated with non-surgical biopsies. Importantly, the platform is compatible with established functional assays as well as non-destructive spatial imaging, enabling researchers to characterize response to ICI longitudinally. Ongoing trials will enable clinicians to assess platform performance in predicting response to immunotherapy.

cancer biology↗

bioRxiv2025

Early posttranscriptional response to tetracycline exposure in a gram-negative soil bacterium reveals unexpected attenuation mechanism of a DUF1127 gene

The Gram-negative, soil-dwelling plant symbiont Sinorhizobium meliloti shares its free-living habitat with antibiotic producers. To learn about early steps of its adaptation to antibiotics, we analyzed transcriptome changes after 10 min exposure to subinhibitory amount of tetracycline (Tc). RNA-seq revealed 297 differentially expressed genes. Besides ten upregulated ribosomal genes, there was no recognizable functional pattern in the observed changes. Instead, upregulated genes were mostly first in operons and often small, while downregulated genes were downstream in operons. Furthermore, we detected mRNA stabilization upon Tc exposure for several up- and down-regulated genes. Thus, mRNA stabilization contributed to increased mRNA levels, but for downstream genes its effect was counteracted by premature transcriptional termination caused by disrupted coupling between transcription and translation. Using reporter constructs, we found that a DUF1127 gene, showing highest mRNA increase, is controlled by transcription attenuation depending on the translation of an upstream ORF (uORF). Our data suggest the following model: The attenuation strongly depends on the accessibility of C-rich codons at the begin of the uORF. The accessibility is guaranteed by translation of the uORF, and is possible in a time window after a ribosome moves downstream and before a next ribosome occupies the ribosomal binding site (RBS). The accessibility is blocked either by impaired translation initiation or, in the absence of ribosome binding, by base-pairing between the RBS and the C-rich codons. We propose that this is used by bacteria to monitor ribosome availability and translation efficiency, and to ensure reciprocal expression of the DUF1127 gene.

molecular biology↗