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Substantial numbers of somatic mutations have been found to accumulate with age in different human tissues. Clonal cellular amplification of some of these mutations can cause cancer and other diseases. However, it is as yet unclear if and to what extent an increased burden of random mutations can affect cellular function without clonal amplification. We tested this in cell culture, which avoids the limitation that an increased mutation burden in vivo typically leads to cancer. We performed single-cell whole-genome sequencing of primary fibroblasts from DNA mismatch repair (MMR) deficient Msh2 -/- mice and littermate control animals after long-term passaging. Apart from analyzing somatic mutation burden we analyzed clonality, mutational signatures, and hotspots in the genome, characterizing the complete landscape of somatic mutagenesis in normal and MMR-deficient mouse primary fibroblasts during passaging. While growth rate of Msh2 - / - fibroblasts was not significantly different from the controls, the number of de novo single-nucleotide variants (SNVs) increased linearly up until at least 30,000 SNVs per cell, with the frequency of small insertions and deletions (INDELs) plateauing in the Msh2 - / - fibroblasts to about 10,000 INDELS per cell. We provide evidence for negative selection and large-scale mutation-driven population changes, including significant clonal expansion of preexisting mutations and widespread cell-strain-specific hotspots. Overall, our results provide evidence that increased somatic mutation burden drives significant cell evolutionary changes in a dynamic cell culture system without significant effects on growth. Since similar selection processes against mutations preventing organ and tissue dysfunction during aging are difficult to envision, these results suggest that increased somatic mutation burden can play a causal role in aging and diseases other than cancer., Competing Interests: Competing Interests L.Z., M.L., A.Y.M., J.V., and X.D. are co-founders and shareholders of SingulOmics Corp. J.V. and A.Y.M. are co-founders and shareholders of MutaGenTech Inc. Others declare no conflict of interest.

Purpose: The adequate performance of radiobiological experiments using clinical proton beams typically requires substantial preparations to provide the appropriate setup for specific experiments. Providing radiobiologically interesting low-energy protons is a particular challenge, due to various physical effects that become more pronounced with larger absorber thickness and smaller proton energy. This work demonstrates the generation of decelerated low-energy protons from a clinical proton beam., Methods: Monte Carlo simulations of proton energy spectra were performed for energy absorbers with varying thicknesses to reduce the energy of the clinical proton beam down to the few-MeV level corresponding to μ m-ranges. In this way, a setup with an optimum thickness of the absorber with a maximum efficiency of the proton fluence for the provisioning of low-energy protons is supposed to be found. For the specific applications of 2.5-3.3 MeV protons and α -particle range equivalent protons, the relative depth dose was measured and simulated together with the dose-averaged linear energy transfer (LETd) distribution., Results: The resulting energy spectra from Monte Carlo simulations indicate an optimal absorber thickness for providing low-energy protons with maximum efficiency of proton fluence at an user-requested energy range for experiments. For instance, providing energies lower than 5 MeV, an energy spectrum with a relative total efficiency of 38.6 % to the initial spectrum was obtained with the optimal setup. The measurements of the depth dose, compared to the Monte Carlo simulations, showed that the dosimetry of low-energy protons works and protons with high LETd down to the range of α -particles can be produced., Conclusions: This work provides a method for generating all clinically and radiobiologically relevant energies - especially down to the few-MeV level - at one clinical facility with pencil beam scanning. Thereby, it enables radiobiological experiments under environmentally uniform conditions., (© 2021 The Authors. Medical Physics published by Wiley Periodicals LLC on behalf of American Association of Physicists in Medicine.)

Lipases are among the most frequently used biocatalysts in organic synthesis, allowing numerous environmentally friendly and inexpensive chemical transformations. Here, we present a biomimetic strategy based on iron(III)-catalyzed oxidative coupling and selective ester monohydrolysis using lipases for the synthesis of unsymmetric biphenyl-based esters under mild conditions. The diverse class of biphenyl esters is of pharmaceutical and technical relevance. We explored the potency of a series of nine different lipases of bacterial, fungal, and mammalian origin on their catalytic activities to cleave biphenyl esters, and optimized the reaction conditions, in terms of reaction time, temperature, pH, organic solvent, and water-organic solvent ratios, to improve the chemoselectivity, and hence control the ratio of unsymmetric versus symmetric products. Elevated temperature and increased DMSO content led to an almost exclusive monohydrolysis by the four lipases Candida rugosa lipase (CRL), Mucor miehei lipase (MML), Rhizopus niveus lipase (RNL), and Pseudomonas fluorescens lipase (PFL). The study was complemented by in silico binding predictions to rationalize the observed differences in efficacies of the lipases to convert biphenyl esters. The optimized reaction conditions were transferred to the preparative scale with high yields, underlining the potential of the presented biomimetic approach as an alternative strategy to the commonly used transition metal-based strategies for the synthesis of diverse biphenyl esters.

By means of computational and experimental mechanistic studies the fundamental role of boroxines in the reaction between diazo compounds and boronic acids was elucidated. Consequently, a selective metal-free carbon-carbon homologation of aryl and vinyl boroxines using TMSCHN 2 , giving access to TMS-pinacol boronic ester products, was developed.