Biology subjects

Rudorf, S.

Publications and source records attributed to Rudorf, S..

5 recordsLinked to original sources

bioRxiv2026

Direct 3D isolation in gelatin methacryloyl microbeads versus conventional 2D isolation and culture: a trade-off between mesenchymal stromal cell purity and niche-associated transcriptional profiles

Bone-marrow mesenchymal stromal cell (MSC) isolation typically couples plastic-adherence selection to planar expansion. Therefore, the recovered cells may reflect culture adaptation as much as native stromal biology. Direct isolation within a three-dimensional matrix could reduce planar selection, but its effects on cellular recovery, composition and MSC transcriptional profiles remain insufficiently resolved. We therefore developed a gelatin methacryloyl (GelMA) workflow progressing from bulk hydrogels to medium-surrounded GelMA microbeads (GMBs) and compared 3D-derived cells with conventional 2D cultures. Qualitative live/dead staining with calcein acetoxymethyl ester (CAM) and propidium iodide (PI) and 5-ethynyl-2'-deoxyuridine (EdU) imaging established cellular activity in both GelMA formats. In bulk GelMA cultures from four human donors, CD73-, CD90- and CD105-associated flow-cytometric readouts did not increase from day 7 to day 21. At day 21, the combined marker readout was numerically higher in donor-matched 2D cultures than in bulk 3D cultures, which retained heterogeneous marker-defined populations. GMBs enabled cell recovery and flow-cytometric characterization and motivated an exploratory one-donor single-cell RNA sequencing (scRNA-seq) analysis. The final dataset contained 37,784 cells across four samples and 23 annotated populations. MSC-focused re-embedding resolved 13,175 cells into two 3D and four 2D clusters. A pooled comparison identified 4,840 differentially expressed genes (DEGs, false discovery rate [FDR]<0.01, |log2FC|[≥]1), indicating a broad method-associated expression shift. The 3D C1 cluster combined progenitor-, extracellular-matrix-, adhesion-, cytokine-, hypoxia/glycolysis- and predicted-interaction-associated programmes, whereas 2D clusters showed mitochondrial, cytoskeletal/mechanoadaptive and proliferative programmes. Thus, 2D culture yielded a more strongly represented and compositionally purer MSC compartment, while 3D isolation contained rare MSC clusters with niche-associated transcriptional profiles within a heterogeneous marrow environment. These findings support a testable native-associated tendency for 3D, but donor replication, matched recovery and direct native-reference mapping are required before inferring greater in-vivo similarity or functional superiority.

cell biology↗

bioRxiv2024

Less is more: Slow-codon windows enhance eGFP mRNA resilience against RNA interference

Extensive efforts have been devoted to enhance the translation efficiency of mRNA delivered to mammalian cells via codon optimization. However, the impact of codon choice on mRNA stability remains underexplored. In this study, we investigated the influence of codon usage on mRNA degradation kinetics in cultured human cell lines using live-cell imaging on single-cell arrays (LISCA). By measuring mRNA lifetimes at the single-cell level for synthetic mRNA constructs, we confirmed that mRNAs containing slowly translated codon windows have shorter lifetimes. Unexpectedly, these mRNAs did not exhibit decreased stability in the presence of siRNA compared to the unmutated sequence, suggesting an interference of different concurrent degradation mechanisms. We employed stochastic simulations to predict ribosome density along the open reading frame, revealing that the ribosome densities correlated with mRNA stability in a cell-type- and codon-position-specific manner. In summary, our results suggest that the effect of codon choice and its influence on mRNA lifetime is context dependent with respect to cell type, codon position, and RNA interference.

biophysics↗

bioRxiv2023

Decomposing bulk signals to reveal hidden information in processive enzyme reactions: A case study in mRNA translation

Processive enzymes, such as polymerases or ribosomes, are often studied in en-semble or bulk experiments through the monitoring of time-dependent signals, such as fluorescence time traces. However, ensemble signals are a superposition of the time traces of all molecules in the reaction and can exhibit less distinct features than individual single-molecule signals due to the stochasticity of biomolecular processes. Here, we demonstrate that under certain conditions, bulk signals from processive reactions can still be decomposed to reveal hidden information about individual reaction steps. Using mRNA translation as a case study for processive biochemical reactions, we explore the limits of least-squares approaches for ensemble fluorescence signal decomposition. Specifically, we show that decomposing a noisy ensemble signal generated by the translation of mRNAs containing more than a few codons represents an ill-posed problem, which can be addressed through Tikhonov regularization. Our findings can help to increase the information content extracted from bulk experiments, thereby expanding the range of these time- and cost-efficient methods.

biochemistry↗

bioRxiv2023

Utilizing high resolution ribosome profiling for the global investigation of gene expression in Chlamydomonas reinhardtii

Ribosome profiling (Ribo-seq) is a powerful method for the deep analysis of translation mechanisms and regulatory circuits during gene expression. Here, we established an optimized and high resolution Ribo-seq protocol for the unicellular model alga Chlamydomonas reinhardtii (Chlamydomonas). Comparing different nuclease treatments for the extraction and sequencing of ribosome-protected fragments (RPFs) and parallel RNA-seq, provided deep insight into translational dynamics and post-transcriptional control of gene expression, thoroughly covering more than 10,000 different transcripts. Our high quality Ribo-seq protocol captures the 3-nucleotide movement of elongating ribosomes along nuclear and chloroplast transcripts. Detailed analysis of the ribosomal offsets on transcripts uncovers presumable transition states during translocation of elongating ribosomes within the 5- and 3-sections of transcripts and features of eukaryotic translation termination. These offsets reveal drastic differences between the nature of cytosolic and chloroplast translation mechanisms. Chloroplast translation is further characterized by heterogenous RPF size distribution. We found that local accumulation of small RPFs correlates with local slowdown of psbA translation, possibly revealing an uncharacterized regulator step during PsbA/D1 synthesis. Further analyses of RPF distribution along specific cytosolic transcripts revealed characteristic patterns of translation elongation exemplified for the major light harvesting complex proteins, LHCs. Moreover, our Ribo-seq data can be utilized to survey coding sequence annotations and the expression preference of alternatively spliced transcripts in Chlamydomonas. We made these features easily accessible for the research community by attaching our Ribo-seq data to the most recent Chlamydomonas reference genome.

systems biology↗

bioRxiv2019

Efficiency of protein synthesis inhibition depends on tRNA and codon compositions

Regulation and maintenance of protein synthesis are vital to all organisms and are thus key targets of attack and defense at the cellular level. Here, we mathematically analyze protein synthesis for its sensitivity to the inhibition of elongation factor EF-Tu and/or ribosomes in dependence of the systems tRNA and codon compositions. We find that protein synthesis reacts ultrasensitively to a decrease in the elongation factors concentration for systems with an imbalance between codon usages and tRNA concentrations. For well-balanced tRNA/codon compositions, protein synthesis is impeded more effectively by the inhibition of ribosomes instead of EF-Tu. Our predictions are supported by re-evaluated experimental data as well as by independent computer simulations. Not only does the described ultrasensitivity render EF-Tu a distinguished target of protein synthesis inhibiting antibiotics. It may also enable persister cell formation mediated by toxin-antitoxin systems. The strong impact of the tRNA/codon composition provides a basis for tissue-specificities of disorders caused by mutations of human mitochondrial EF-Tu as well as for the potential use of EF-Tu targeting drugs for tissue-specific treatments.\n\nAuthor SummaryWe predict and analyze the response of differently composed protein synthesis systems to the inhibition of elongation factor EF-Tu and/or ribosomes. The study reveals a strong interdependency of a protein synthesis systems composition and its susceptibility to inhibition. This interdependency defines a generic mechanism that provides a common basis for a variety of seemingly unrelated phenomena including, for example, persister cell formation and tissue-specificity of certain mitochondrial diseases. The described mechanism applies to simple artificial translation systems as well as to complex protein synthesis in vivo.

cell biology↗