Biology subjects

Müller, P.

Publications and source records attributed to Müller, P..

26 records · Page 2Linked to original sources

bioRxiv2022

In vivo assessment of mechanical properties during axolotl development and regeneration using confocal Brillouin microscopy

In processes such as development and regeneration, where large cellular and tissue rearrangements occur, cell fate and behavior are strongly influenced by tissue mechanics. While most well-established tools probing mechanical properties require an invasive sample preparation, confocal Brillouin microscopy captures mechanical parameters optically with high resolution in a contact-free and label-free fashion. In this work, we took advantage of this tool and the transparency of the highly regenerative axolotl to probe its mechanical properties in vivo for the first time. We mapped the Brillouin frequency shift with high resolution in developing limbs and regenerating digits, the most studied structures in the axolotl. We detected a gradual increase in the cartilage Brillouin frequency shift, suggesting decreasing tissue compressibility during both development and regeneration. Moreover, we were able to correlate such increase with the regeneration stage, which was undetected with fluorescence microscopy imaging. The present work evidences the potential of Brillouin microscopy to unravel the mechanical changes occurring in vivo in axolotls, setting the basis to apply this technique in the growing field of epimorphic regeneration.

developmental biology↗

bioRxiv2021

Predicting the functional effects of voltage-gated potassium channel missense variants with multi-task learning

PurposeVariants in genes encoding voltage-gated potassium channels are associated with a broad spectrum of neurological diseases including epilepsy, ataxia, and intellectual disability. Knowledge of the resulting functional changes, characterized as overall ion channel gain- or loss-of-function, is essential to guide clinical management including precision medicine therapies. However, for an increasing number of variants, little to no experimental data is available. New tools are needed to evaluate variant functional effects. MethodsWe catalogued a comprehensive dataset of 959 functional experiments across 19 voltage-gated potassium channels, leveraging data from 782 unique disease-associated and synthetic variants. We used these data to train a taxonomy-based multi-task learning support vector machine (MTL-SVM), and compared performance to a baseline of standard SVMs. ResultsMTL-SVM maintains channel family structure during model training, improving overall predictive performance (mean balanced accuracy 0.729 {+/-} 0.029, AU-ROC 0.757 {+/-} 0.039) over baseline (mean balanced accuracy 0.645 {+/-} 0.041, AU-ROC 0.710 {+/-} 0.074). We can obtain meaningful predictions even for channels with few known variants (KCNC1, KCNQ5). ConclusionOur model enables functional variant prediction for voltage-gated potassium channels. It may assist in tailoring current and future precision therapies for the increasing number of patients with ion channel disorders.

bioinformatics↗

bioRxiv2021

Ionizing radiation improves RIG-I mediated immunotherapy through enhanced p53 activation in malignant melanoma

Radiation therapy induces cytotoxic DNA damage, which results in cell-cycle arrest and activation of cell-intrinsic death pathways, but its application has been limited by the radioresistance of tumors, such as in malignant melanoma. RIG-I is a cytosolic immune receptor expressed in all somatic cells, including tumor cells, with a key role in sensing viral RNA. RIG-I specific oligonucleotide ligands elicit a robust cell-intrinsic antiviral response and immunogenic cell death in tumor cells and are being tested in clinical trials. Nonetheless, their potential to overcome radioresistance has not yet been explored. Here, we demonstrate that activation of RIG-I enhances the extent and immunogenicity of irradiation-induced tumor cell death in human and murine melanoma cell lines in vitro and improved survival in the murine B16 melanoma model. Pathway analysis of transcriptomic data revealed a central role for p53 downstream of the combination treatment, which was corroborated using p53-/- B16 cells. In vivo, the effect of irradiation on immune-cell activation and inhibition of tumor growth was absent in mice carrying p53-/- B16 tumors, while the response to RIG-I stimulation in those mice was maintained. Our results identify p53 as pivotal for the synergistic antitumoral effect of RIG-I and irradiation, resulting in potent induction of immunogenic tumor-cell death. Thus, the administration of RIG-I ligands in combination with radiotherapy is a promising therapeutic approach to treating radioresistant tumors with a functional p53 pathway, such as malignant melanoma.

immunology↗

bioRxiv2021

Twisting DNA by Salt

The structure and properties of DNA depend on the environment, in particular the ion atmosphere. Here, we investigate how DNA twist -one of the central properties of DNA- changes with concentration and identity of the surrounding ions. To resolve how cations influence the helical twist, we combine single-molecule magnetic tweezer experiments and extensive all- atom molecular dynamics simulations. Two interconnected trends are observed for monovalent alkali and divalent alkaline earth cations. First, DNA twist increases monotonously with increasing concentration for all ions investigated. Second, for a given salt concentration, DNA twist strongly depends on cation identity. At 100 mM concentration, DNA twist increases as Ba2+ < Na+ < K+ < Rb+ < Li+ {approx} Cs+ < Sr2+ < Mg2+ < Ca2+. Our molecular dynamics simulations reveal that preferential binding of the cations to the DNA backbone or the nucleobases has opposing effects on DNA twist and provides the microscopic explanation of the observed ion specificity. However, the simulations also reveal shortcomings of existing force field parameters for Cs+ and Sr2+. The comprehensive view gained from our combined approach provides a foundation for understanding and predicting cation-induced structural changes both in nature and in DNA nanotechnology.

biophysics↗

bioRxiv2020

Combined fluorescence, optical diffraction tomography and Brillouin microscopy

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples -- so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fuorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample -- a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

biophysics↗

bioRxiv2020

SR7 - a dual function antisense RNA from Bacillus subtilis

Here, we describe SR7, a dual-function antisense RNA from the Bacillus subtilis chromosome. This RNA was earlier published as the SigB-dependent regulatory RNA S1136 and reported to reduce the amount of the small ribosomal subunit under ethanol stress. We found that the 5 portion of SR7 encodes a small protein composed of 39 amino acids which we designated SR7P. It is translated from a 185 nt SigB-dependent mRNA under five different stress conditions and a longer SigB-independent RNA constitutively. Two- to three-fold higher amounts of SR7P were detected in B. subtilis cells exposed to salt, ethanol or heat stress. Co-elution experiments with SR7PC-FLAG and Far-Western blotting demonstrated that SR7P interacts with the glycolytic enzyme enolase. Enolase is a scaffolding component of the B. subtilis degradosome where it interacts with RNase Y and phosphofructokinase PfkA. We found that SR7P increases the amount of RNase Y bound to enolase without affecting PfkA. RNA does not bridge the SR7P-enolase-RNase Y interaction. In vitro-degradation assays with the known RNase Y substrates yitJ and rpsO mRNA revealed enhanced enzymatic activity of enolase-bound RNase Y in the presence of SR7P. Northern blots showed a major effect of enolase and a minor effect of SR7P on the half-life of rpsO mRNA indicating a fine-tuning role of SR7P in RNA degradation. Moreover, SR7P impacts survival of B. subtilis under stress conditions. We suggest that the SR7P-dependent modification of the degradosome affects targets in different physiological pathways.

microbiology↗

bioRxiv2019

Using real-time fluorescence and deformability cytometry and deep learning to transfer molecular specificity to label-free sorting

The identification and separation of specific cells from heterogeneous populations is an essential prerequisite for further analysis or use. Conventional passive and active separation approaches rely on fluorescent or magnetic tags introduced to the cells of interest through molecular markers. Such labeling is time- and cost-intensive, can alter cellular properties, and might be incompatible with subsequent use, for example, in transplantation. Alternative label-free approaches utilizing morphological or mechanical features are attractive, but lack molecular specificity. Here we combine image-based real-time fluorescence and deformability cytometry (RT-FDC) with downstream cell sorting using standing surface acoustic waves (SSAW). We demonstrate basic sorting capabilities of the device by separating cell mimics and blood cell types based on fluorescence as well as deformability and other image parameters. The identification of blood sub-populations is enhanced by flow alignment and deformation of cells in the microfluidic channel constriction. In addition, the classification of blood cells using established fluorescence-based markers provides hundreds of thousands of labeled cell images used to train a deep neural network. The trained algorithm, with latency optimized to below 1 ms, is then used to identify and sort unlabeled blood cells at rates of 100 cells/sec. This approach transfers molecular specificity into label-free sorting and opens up new possibilities for basic biological research and clinical therapeutic applications.

biophysics↗

bioRxiv2019

The human α-defensin-derived peptide HD5(1-9) inhibits cellular attachment and entry of human cytomegalovirus.

Human cytomegalovirus (HCMV) infection causes severe illness in newborns and immunocompromised patients. Since treatment options are limited there is an unmet need for new therapeutic approaches. Defensins are cationic peptides, produced by various human tissues, which serve as antimicrobial effectors of the immune system. Furthermore, some defensins are proteolytically cleaved, resulting in the generation of smaller fragments with increased activity. Together, this led us to hypothesize that defensin-derived peptides are natural human inhibitors of virus infection with low toxicity. We screened several human defensin HNP4- and HD5-derived peptides and found HD5(1-9) to be antiviral without toxicity at high concentrations. HD5(1-9) inhibited HCMV cellular attachment and thereby entry and was active against primary as well as a multiresistant HCMV isolate. Moreover, cysteine and arginine residues were identified to mediate the antiviral activity of HD5(1-9). Altogether, defensin-derived peptides, in particular HD5(1-9), qualify as promising candidates for further development as a novel class of HCMV entry inhibitors.\n\nAUTHOR SUMMARYDefensins are peptides produced by various human organs which take part in the natural defense against pathogens. Recently, it has been shown that defensins are further cleaved to smaller peptides that have high intrinsic anti-microbial activity. We here challenged the hypothesis that these peptides might have antiviral activity, and due to their presumably natural occurrence, low toxicity. Indeed, we found one peptide fragment that turned out to block the attachment of the human cytomegalovirus (HCMV) to cells. Furthermore, this peptide did not show toxicity in various cellular assays or impede the embryonic development of zebrafish at the concentrations used to block HCMV. This is important, since HCMV is one of the most important viral congenital infections. Altogether, our results hold promise for the development of a new class of antivirals against HCMV.

microbiology↗