Biology subjects

Heinrich, S.

Publications and source records attributed to Heinrich, S..

14 recordsLinked to original sources

bioRxiv2025

Laser-driven VHEE pulsed fast fractionation (PFF): second-scale inter-pulse timing differentially modulates normal tissue and tumour toxicity

Radiotherapy (RT) is constrained by the narrow therapeutic window between tumour control and normal-tissue toxicity. While FLASH RT has been reported to reduce normal-tissue toxicity at ultra-high dose rates (UHDR) while preserving tumour control in several preclinical models, the radiobiological potential of intermediate temporal regimes between conventional fractionation and FLASH remains largely unexplored. Here, we show that temporal separation of ultrashort (fs-ps) dose-delivery pulses delivered at ultra-high instantaneous dose rate, with second-scale inter-pulse intervals, can differentially modulate biological response in vitro and in vivo. Samples were irradiated with very high-energy electrons (VHEEs, 50 MeV to 300 MeV) generated by a laser-plasma accelerator (LPA), using a temporal irradiation modality we term pulsed fast fractionation (PFF), with instantaneous dose rates exceeding 10^12 Gy/s. Varying the inter-pulse interval from 1 s to 10 s while maintaining dose per pulse and total dose constant modulated biological response in both in vitro and in vivo models. At the shortest investigated inter-pulse interval ({theta} = 1s), MRC5-hTERT human fibroblasts showed higher viability, whereas HCT116 colorectal carcinoma cells showed lower viability under matched dose conditions. In vivo, the same interval was associated with reduced radiation-induced growth impairment in zebrafish embryos, consistent with the response pattern observed in the non-tumour cell model and extending this timing-dependent response to a whole-organism context. These findings identify second-scale pulse timing as a biologically active degree of freedom for VHEE delivery, positioning PFF as a distinct temporal irradiation regime with the potential to complement spatial dose modulation through temporal optimisation and extend the current fractionation-FLASH framework.

cell biology↗

bioRxiv2025

Enabling antibiotic research: towards selective peptide deformylase inhibitors

Peptide deformylase plays a crucial role in prokaryotic translation and constitutes an antibiotic target previously addressed in clinical trials. In eukaryotes, mitochondrial translation also relies on peptide deformylase, necessitating antibiotic development to aim for selective inhibition of the bacterial enzymes. In the present study, we investigated two compound series: derivatives of actinonin and compounds containing a 5-bromoindole scaffold. Antibacterial activity was evaluated by microdilution-based minimal inhibitory concentration assay and selectivity investigated using human peripheral blood mononuclear cells. In vitro peptide deformylase inhibition was compared for the Escherichia coli and human enzyme. To validate peptide deformylase inhibition in vivo, a mass spectrometric analysis directly coupled to the minimal inhibitory concentration assay was developed for the model organism Bacillus subtilis. Two compounds originating from this work (ZHO-119, ZHO-197) showed antibacterial activity comparable to actinonin, and for the comparator compound BB-3497 superior anti-gram-negative and anti-tubercular activity was confirmed. The three compounds displayed no cytotoxicity and were equally selective in vitro for the bacterial enzyme. The mass spectrometry-based analysis indicates that in addition to peptide deformylase, ZHO-197 very effectively inhibits bacterial methionine aminopeptidase, the metallo-enzyme that removes the deformylated N-terminal methionine.

microbiology↗

bioRxiv2025

Comparative analysis of the radiobiological effects of laser-driven VHEE vs. conventional electrons using in vitro, ex vivo, and in vivo models

PurposeThis study systematically investigates the radiobiological effects of Very High Energy Electrons (VHEE) generated by a laser-plasma accelerator (LPA), in comparison with Conventional Intermediate Energy Electrons (CIEE) from a conventional linear accelerator (LINAC). Using in vitro, ex vivo, and in vivo models, we evaluate and compare their potential toxicity on healthy tissues. Methods and MaterialsCell survival, tissue response, and developmental toxicity were assessed across three biological models. In vitro, human fibroblasts (MRC5-hTERT) were used to generate post-irradiation survival curves. Ex vivo, precision-cut lung slices (PCLS) from mice were analyzed for radiation-induced inhibition of cell proliferation. In vivo, zebrafish embryos were used to evaluate developmental toxicity through body length and spinal curvature measurements. VHEE irradiations were performed using a broadband electron beam spanning 50-300 MeV, using the Salle Jaune LPA (Laboratoire dOptique Appliquee, France), while CIEE exposures were performed with a 7 MeV conventional LINAC (Institut Curie, France). ResultsIn vitro, MRC5-hTERT cells showed no significant difference in radiosensitivity between VHEE and CIEE, with comparable D10 values (p-value = 0.7). In the ex vivo model, both beams induced a dose-dependent decrease in cell division with no significant inter-beam differences at any dose level (p-value > 0.99). In vivo, zebrafish embryos exhibited dose-dependent body shortening and increased spinal curvature following both VHEE and CIEE exposure. No significant differences were observed between the two modalities at matched doses for any measured metric (p-value[≥] 0.5). ConclusionThis study presents the first comprehensive radiobiological evaluation of a laser-driven VHEE beam across multiple biological models. Under the investigated conditions, VHEE and CIEE irradiations exhibit similar biological toxicity. These findings support the feasibility and potential of VHEE generated with LPA for future clinical applications.

cell biology↗

bioRxiv2024

Trans-Translation inhibitors and copper ions synergize for enhanced antibiotic activity

Trans-Translation is the most effective ribosome rescue mechanism and a compelling target for new antimicrobial agents. A recent proteomic study revealed similarities between the responses of Bacillus subtilis to the inhibitors small-molecule inhibitors oxadiazole KKL-40 and tetrazole KKL-55 and divalent cation ionophores, indicating the disturbance of metal homeostasis as potential secondary mechanism of action. Here, we report increased copper levels in KKL-40 and KKL-55-treated B. subtilis. Both inhibitors form copper complexes that enter large unilamellar vesicles. Copper supplementation enhanced the antibacterial activity against B. subtilis by simultaneously increasing inhibitor and copper uptake. The co-treatment of B. subtilis with trans-translation inhibitors and copper concentrations normally benign for trans-translation-competent cells, caused an immediate stalling of growth and translation, as observed at higher KKL-40 and KKL-55 concentrations without copper supplementation. Proteome analysis showed that during translation stalling cells were unable to mount an effective copper toxicity response. Taken together, the synergetic uptake of KKL-40 and KKL-55 with copper leads to a quick-onset translation stalling, preventing B. subtilis from counteracting the toxic effects of rapid copper influx. Significance statementThe challenge of antimicrobial resistance is growing, necessitating an exploration of novel antibiotic targets. Among these, trans-translation has attracted considerable attention due to its ubiquitous presence in bacteria as well as its role in virulence and pathogenesis. Several inhibitors of trans-translation have been identified in a target-based screening using a whole-cell assay. However, recent proteomic profiling studies suggested that the tested trans-translation inhibitors might have an additional mode of action. In this work, we shed light on their previously undiscovered copper ionophore activity and explore the consequences of co-treating B. subtilis with KKL-40 or KKL-55 and CuCl2. This co-treatment results in a rapid antibiotic influx, and, consequently to the stalling of ribosomes, translation, and bacterial growth. Simultaneously, massive amounts of copper accumulate in the cells, the toxic effects of which require a copper stress response to mitigate. However, such a response is averted by the stalled translation. Dual mechanism antibacterial agents are attractive because they are typically associated with slow emergence of resistance. A deep understanding of the complex interplay of KKL-40 and KKL-55 with metal ions will help to fully exploit trans-translation as an antibacterial target and to develop KKL-40 and KKL-55-based antibiotics into novel treatments for bacterial infections.

microbiology↗

bioRxiv2024

Polysomes and mRNA control the biophysical properties of the eukaryotic cytoplasm

The organization and biophysical properties of the cytoplasm influence all cellular reactions, including molecular interactions and the mobility of biomolecules. It has become clear that the cytoplasm does not behave like a simple fluid but instead is a densely crowded and highly organized environment. Yet, the detailed properties of the cytoplasm, the molecular mechanisms that control them and how they influence the biochemistry of cells remain poorly understood. Here, we investigate the diffusive properties of the cytoplasm in silico and in vivo, employing mRNPs (messenger ribonucleoprotein) and GEM (genetically encoded multimeric) particles as rheological probes in proliferating cells. We demonstrate that cytoplasmic diffusivity increases upon polysome disassembly due to translation inhibition or upon a reduction in mRNA levels. Reducing ribosome concentration by up to 20-25% without a change in polysome levels has no effect in vivo. In addition, we show that upon polysome disassembly, mRNA condensation into P-bodies does not affect cytosolic diffusion in budding yeast. Altogether, our results show that mRNAs and their organization into polysomes control the biophysical properties of the eukaryotic cytoplasm. HighlightsO_LIPolysomes control the biophysical properties of cytoplasm. C_LIO_LImRNP and GEM mobility is enhanced upon translation inhibition that leads to polysome disassembly C_LIO_LIPerturbation of mRNA levels leads to an increase in cytosolic diffusion. C_LI

cell biology↗

bioRxiv2024

Tag with Caution - How protein tagging influences the formation of condensates

Fluorescent proteins and peptide tags are essential tools in cellular biology, but can alter the biochemical properties of target proteins. Biomolecular condensates, which have emerged as key principles of cellular organization, are suggested to provide robustness to cells, yet they can also respond sensitively to small changes in environmental conditions--or tagging of their components, as our findings suggest. Here, we investigated the effects of sixteen widely used tags on condensate formation in various model organisms, in vitro, in cells and by computational modelling. We find that tagging strongly influenced condensation for some proteins, while others remained unaffected. Effects varied, with some tags enhancing and others decreasing condensation, and depended on the protein being tagged. Coarse-grained simulations suggest that the charge of the fluorescent protein tags is a critical factor modulating condensation behavior. Together, our results underscore the importance of rigorous experimental design and interpretation in condensate experiments.

molecular biology↗

bioRxiv2024

HOCl Forms Lipid N-Chloramines in Cell Membranes of Bacteria and Immune Cells

Neutrophils orchestrate a coordinated attack on bacteria, combining phagocytosis with a potent cocktail of oxidants, including the highly toxic hypochlorous acid (HOCl), renowned for its deleterious effects on proteins. Here, we examined the occurrence of lipid N-chloramines in vivo, their biological activity and neutralization. Using a chemical probe for N-chloramines, we demonstrate their formation in the membranes of bacteria and monocytic cells exposed to physiologically relevant concentrations of HOCl. N-chlorinated model membranes composed of phosphatidylethanolamine, the major membrane lipid in Escherichia coli and an important component of eukaryotic membranes, exhibited oxidative activity towards the redox-sensitive protein roGFP2, suggesting a role for lipid N-chloramines in protein oxidation. Conversely, the cellular antioxidant glutathione neutralized lipid N-chloramines by removing the chlorine moiety. We propose that lipid N-chloramines, like protein N-chloramines, are involved in inflammation and accelerate the host immune response.

microbiology↗

bioRxiv2024

Newly synthesized mRNA selectively escapes translational repression following acute stress.

When cells encounter environmental stress, they rapidly mount an adaptive response by switching from pro-growth to stress-responsive gene expression programs. It is poorly understood how cells selectively silence pre-existing, pro-growth transcripts, yet efficiently translate transcriptionally-induced stress mRNA, and whether these transcriptional and post-transcriptional responses are coordinated. Here, we show that following acute glucose withdrawal in S. cerevisiae, pre-existing mRNAs are not first degraded to halt protein synthesis, nor are they sequestered away in P-bodies. Rather, their translation is rapidly repressed through a sequence-independent mechanism that differentiates between mRNAs produced before and after stress followed by their decay. Transcriptional induction of endogenous transcripts and reporter mRNAs during stress is sufficient to escape translational repression, while induction prior to stress leads to repression. Our results reveal a timing-controlled coordination of the transcriptional and translational responses in the nucleus and cytoplasm ensuring a rapid and widescale reprogramming of gene expression following environmental stress.

molecular biology↗

bioRxiv2023

Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation

Tardigrades, microscopic animals found in virtually all ecosystems, are renowned for their remarkable ability to withstand extreme conditions. Recent studies have identified novel tardigrade specific protein families that aid in resistance to desiccation and ionizing radiation (IR). Notably, a tardigrade specific DNA binding protein called Dsup (for DNA damage suppressor) has been found to protect from X-ray damage in human cells and from hydroxyl radicals in vitro. However, Dsup has only been found in two species within the Hypsibioidea superfamily. To better understand mechanisms underlying radio-resistance in the Tardigrada phylum, we first characterized DNA damage and repair in response to IR in the model species Hypsibius exemplaris. By analysis of phosphorylated H2AX, we demonstrated the induction and repair of DNA double-strand breaks after IR exposure. Importantly, the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in the remarkable radio-resistance of tardigrades. In order to identify novel tardigrade specific genes involved, we next conducted a comparative transcriptomics across three species, H. exemplaris, Acutuncus antarcticus and Paramacrobiotus fairbanksi, the latter belonging to the Macrobiotoidea superfamily known to lack Dsup homologs. In all three species, many genes of DNA repair were among the most strongly overexpressed genes alongside a novel tardigrade specific gene, named Tardigrade DNA damage Response protein 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and act by preserving chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade specific gene responsible for conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping to cope with high levels of DNA damage. Furthermore, it suggests that at least two tardigrade specific genes, respectively for Dsup and TDR1, have independently evolved DNA-binding functions that contribute to radio-resistance in the Tardigrada phylum.

molecular biology↗

bioRxiv2022

Condensation of a nuclear mRNA export factor regulates mRNA transport during stress

Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that upon glucose withdrawal the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This coincides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro, where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and to control gene expression during stress. HighlightsO_LIThe nuclear poly(A)-binding protein Nab2 forms RNA-containing condensate-like structures upon glucose starvation and upon acute cellular depletion of the DEAD-box ATPase Dbp5 C_LIO_LIThe Nab2 multimerization interface but not the intrinsically disordered regions (IDRs) are essential for condensation in vitro and in vivo C_LIO_LIGlucose stress leads to poly(A) RNA retention in the nucleus, which is affected by the state of the Nab2 condensate C_LI

cell biology↗

bioRxiv2020

Single-cell atlas of tumor clonal evolution in liver cancer

Tumor evolution is a key feature of tumorigenesis and plays a pivotal role in driving intratumor heterogeneity, treatment failure and patients prognosis. Here we performed single-cell transcriptome profiling of 46 primary liver cancers from 37 patients enrolled for interventional studies. We surveyed the landscape of ~57,000 malignant and non-malignant cells and determined tumor cell clonality by developing a machine learning-based consensus clustering method. We found evidence of tumor cell branching evolution using hierarchical clustering, RNA velocity as well as reverse graph embedding methods. Interestingly, an increasing tumor cell clonality was tightly linked to patients prognosis, accompanied by a polarized immune cell landscape. We identified osteopontin as a key player for tumor cell evolution and microenvironmental reprogramming. Our study offers insight into the collective behavior of tumor cell communities in liver cancer as well as potential drivers for tumor evolution in response to therapy.

cancer biology↗

bioRxiv2019

Temperature dependent sex-biased gene expression in the gametophytes of the kelp Saccharina latissima

Saccharina latissima is an economically and ecologically relevant kelp species in Europe and North America. In kelps, the sexuality is expressed during the haploid life stage and the microscopic gametophytes exhibit significant sexual dimorphism. To understand the sex-dependent impact of temperature on the gametophyte stage, we analyzed for the first time, gene expression profiles of male and female gametophytes at three different temperatures (4{degrees}C, 12{degrees}C and 20{degrees}C) characteristic for the species distribution range by using RNA-sequencing. We identified several differentially expressed genes between sexes; while female biased genes were enriched in general metabolism and energy production, male biased genes function within cell cycle and signaling. In our study, temperature modulated sex-biased gene expression, with only a small percentage of differentially expressed genes consistently male (7%) or female-biased (12%) at the three temperatures. Female gametophytes responded stronger to higher temperatures than males, suggesting that males are more heat tolerant. Differences between S. latissima and other brown algal gender-dependent gene expression might mirror the different evolutionary and ecological contexts. Genomic information on kelp gametophyte is still scarce and thus this study adds to our knowledge on sex differences in abiotic stress responses in macroalgae at the transcriptomic level.\n\nHighlightThe transcriptomic basis for sexual dimorphism and associated metabolic needs are described for the kelp Saccharina latissima. Temperature modulates sex-biased gene expression resulting in a stronger stress response in females.

plant biology↗

bioRxiv2018

Transient Hsp90 suppression promotes a heritable change in protein translation

The heat shock protein 90 (Hsp90) chaperone functions as a protein-folding buffer and plays a unique role promoting the evolution of new heritable traits. To investigate the role of Hsp90 in modulating protein synthesis, we screened more than 1200 proteins involved in mRNA regulation for physical interactions with Hsp90 in human cells. Among the top hits was CPEB2, which strongly binds Hsp90 via its prion domain, reminiscent of the prion-like regulation of translation of Aplysia CPEB. In a yeast model of CPEB prion-dependent translation regulation, transient inhibition of Hsp90 amplified CPEB2 prion activity and resulted in persistent translation of the CPEB reporter. Remarkably, inhibition of Hsp90 was sufficient to induce a heritable change in protein translation that persisted for 30 generations, even in the absence of exogenous CPEB. Although we identified a variety of perturbations that enhanced translation of the reporter, only Hsp90 inhibition led to persistent activation. Thus, transient loss of Hsp90 function leads to the non-genetic inheritance of a novel translational state. We propose that, in addition to sculpting the conformational landscape of the proteome, Hsp90 promotes phenotypic variation by modulating protein synthesis.

cell biology↗

bioRxiv2017

Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability

The cytoplasmic abundance of mRNAs is strictly controlled through a balance of production and degradation. Whereas the control of mRNA synthesis through transcription has been well characterized, less is known about the regulation of mRNA turnover, and a consensus model explaining the wide variations in mRNA decay rates remains elusive. Here, we combine non-invasive transcriptome-wide mRNA production and stability measurements with selective and acute perturbations to demonstrate that mRNA degradation is tightly coupled to the regulation of translation, and that a competition between translation initiation and mRNA decay -but not codon optimality or elongation- is the major determinant of mRNA stability in yeast. Our refined measurements also reveal a remarkably dynamic transcriptome with an average mRNA half-life of only 4.8 minutes - much shorter than previously thought. Furthermore, global mRNA destabilization by inhibition of translation initiation induces a dose-dependent formation of processing bodies in which mRNAs can decay over time.

genetics↗