Dihydrotestosterone stimulates amino acid uptake and the expression of LAT2 in mouse skeletal muscle fibres through an ERK1/2-dependent mechanism

Dihydrotestosterone (DHT) has acute/non-genomic actions in adult mammalian skeletal muscles whose physiological functions are still poorly understood. Therefore, the primary aim of this study was to investigate the acute/non-genomic effects of DHT on amino acid uptake as well as the cellular signal transduction events underlying these actions in mouse fast- and slow-twitch skeletal muscle fibre bundles. 14C-Labelled amino acids were used to investigate the effects of DHT and testosterone (T) on amino acid uptake and pharmacological interventions were used to determine the cellular signal transduction events mediating these actions. While T had no effect on the uptake of isoleucine (Ile) and a-methylaminoisobutyric acid (MeAIB) in both fibre types, DHT increased their uptake in the fast-twitch fibre bundles. This effect was reversed by inhibitors of protein translation, the epidermal growth factor receptor (EGFR), system A, system L, mTOR and MEK. However, it was relatively insensitive to inhibitors of transcription, androgen receptors and PI3K/Akt. Additionally, DHT treatment increased the expression of LAT2 and the phosphorylation of the EGFR in the fast-twitch fibre bundles and that of ERK1/2, RSK1/2 and ATF2 in both fibre types. Also, it decreased the phosphorylation of eEF2 and increased the incorporation of Ile into proteins in both fibre types. Most of these effects were reversed by EGFR and MEK inhibitors. From these findings we suggest that another physiological function of the acute/non-genomic actions of DHT in isolated mammalian skeletal muscle fibres is to stimulate amino acid uptake. This effect is mediated through the EGFR and involves the activation of the MAPK pathway and an increase in LAT2 expression

Multi-omics Reveals the Lifestyle of the Acidophilic, Mineral-Oxidizing Model Species Leptospirillum ferriphilumT.

Leptospirillum ferriphilum plays a major role in acidic, metal-rich environments, where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as biomining operations. Despite its perceived importance, no complete genome sequence of the type strain of this model species is available, limiting the possibilities to investigate the strategies and adaptations that Leptospirillum ferriphilum DSM 14647T (here referred to as Leptospirillum ferriphilum T) applies to survive and compete in its niche. This study presents a complete, circular genome of Leptospirillum ferriphilum T obtained by PacBio single-molecule real-time (SMRT) long-read sequencing for use as a high-quality reference. Analysis of the functionally annotated genome, mRNA transcripts, and protein concentrations revealed a previously undiscovered nitrogenase cluster for atmospheric nitrogen fixation and elucidated metabolic systems taking part in energy conservation, carbon fixation, pH homeostasis, heavy metal tolerance, the oxidative stress response, chemotaxis and motility, quorum sensing, and biofilm formation. Additionally, mRNA transcript counts and protein concentrations were compared between cells grown in continuous culture using ferrous iron as the substrate and those grown in bioleaching cultures containing chalcopyrite (CuFeS2). Adaptations of Leptospirillum ferriphilum T to growth on chalcopyrite included the possibly enhanced production of reducing power, reduced carbon dioxide fixation, as well as elevated levels of RNA transcripts and proteins involved in heavy metal resistance, with special emphasis on copper efflux systems. Finally, the expression and translation of genes responsible for chemotaxis and motility were enhanced.IMPORTANCE Leptospirillum ferriphilum is one of the most important iron oxidizers in the context of acidic and metal-rich environments during moderately thermophilic biomining. A high-quality circular genome of Leptospirillum ferriphilum T coupled with functional omics data provides new insights into its metabolic properties, such as the novel identification of genes for atmospheric nitrogen fixation, and represents an essential step for further accurate proteomic and transcriptomic investigation of this acidophile model species in the future. Additionally, light is shed on adaptation strategies of Leptospirillum ferriphilum T for growth on the copper mineral chalcopyrite. These data can be applied to deepen our understanding and optimization of bioleaching and biooxidation, techniques that present sustainable and environmentally friendly alternatives to many traditional methods for metal extraction

Stem cell function and stress response are controlled by protein synthesis.

Whether protein synthesis and cellular stress response pathways interact to control stem cell function is currently unknown. Here we show that mouse skin stem cells synthesize less protein than their immediate progenitors in vivo, even when forced to proliferate. Our analyses reveal that activation of stress response pathways drives both a global reduction of protein synthesis and altered translational programmes that together promote stem cell functions and tumorigenesis. Mechanistically, we show that inhibition of post-transcriptional cytosine-5 methylation locks tumour-initiating cells in this distinct translational inhibition programme. Paradoxically, this inhibition renders stem cells hypersensitive to cytotoxic stress, as tumour regeneration after treatment with 5-fluorouracil is blocked. Thus, stem cells must revoke translation inhibition pathways to regenerate a tissue or tumour.This work was funded by Cancer Research UK (CR-UK), Worldwide Cancer Research, the Medical Research Council (MRC), the European Research Council (ERC), and EMBO. Research in Michaela Frye's laboratory is supported by a core support grant from the Wellcome Trust and MRC to the Wellcome Trust-Medical Research Cambridge Stem Cell Institute.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nature1828

Inactivation of the ATMIN/ATM pathway protects against glioblastoma formation

Glioblastoma multiforme (GBM) is the most aggressive human primary brain cancer. Using a Trp53-deficient mouse model of GBM, we show that genetic inactivation of the Atm cofactor Atmin, which is dispensable for embryonic and adult neural development, strongly suppresses GBM formation. Mechanistically, expression of several GBM-associated genes, including Pdgfra, was normalized by Atmin deletion in the Trp53-null background. Pharmacological ATM inhibition also reduced Pdgfra expression, and reduced the proliferation of Trp53-deficient primary glioma cells from murine and human tumors, while normal neural stem cells were unaffected. Analysis of GBM datasets showed that PDGFRA expression is also significantly increased in human TP53-mutant compared with TP53-wild-type tumors. Moreover, combined treatment with ATM and PDGFRA inhibitors efficiently killed TP53-mutant primary human GBM cells, but not untransformed neural stem cells. These results reveal a new requirement for ATMIN-dependent ATM signaling in TP53-deficient GBM, indicating a pro-tumorigenic role for ATM in the context of these tumors

Bioleaching of Pyrite by Iron-Oxidizing Acidophiles under the Influence of Reactive Oxygen Species

After 24h of exposure to acidic media, pyrite generates reactive oxygen species (ROS). Freshly-crushed pyrite with grain sizes between 50-100 μm at a 5 % (w/v), pulp density generated 0.17 ± 0.01 mM H2O2, while 10% pyrite generated 0.29 ± 0.01 mM and 30 % pyrite generated approximately 0.83 ± 0.06 mM. These levels of H2O2 inhibit iron oxidation in iron-grown cells of AcidithiobacillusferrooxidansT but not in pyrite-grown cells. ROS originating from pyrite, which was incubated for 24 h in acidic medium, prohibited pyrite dissolution by iron-grown cells, while pyrite-grown cells were adapted to these concentrations of ROS. Periodical addition of 100 μM H2O2 to pyrite cultures inoculated with pyrite-grown cells did not lower iron dissolution as it was observed with iron-grown cells. By high throughput proteomics analysis, an increased expression of proteins related to oxidative stress management, iron-and sulfur oxidation systems, carbon fixation and biofilm formation was observed in biofilm cells grown on pyrite compared to iron-grown cells.</jats:p

Target-Specific Precision of CRISPR-Mediated Genome Editing

The CRISPR-Cas9 system has successfully been adapted to edit the genome of various organisms. However, our ability to predict the editing outcome at specific sites is limited. Here, we examined indel profiles at over 1,000 genomic sites in human cells and uncovered general principles guiding CRISPR-mediated DNA editing. We find that precision of DNA editing (i.e., recurrence of a specific indel) varies considerably among sites, with some targets showing one highly preferred indel and others displaying numerous infrequent indels. Editing precision correlates with editing efficiency and a preference for single-nucleotide homologous insertions. Precise targets and editing outcome can be predicted based on simple rules that mainly depend on the fourth nucleotide upstream of the protospacer adjacent motif (PAM). Indel profiles are robust, but they can be influenced by chromatin features. Our findings have important implications for clinical applications of CRISPR technology and reveal general patterns of broken end joining that can provide insights into DNA repair mechanisms

LonB Protease Is a Novel Regulator of Carotenogenesis Controlling Degradation of Phytoene Synthase in Haloferax volcanii

Cerletti M, Paggi R, Troetschel C, et al. LonB Protease Is a Novel Regulator of Carotenogenesis Controlling Degradation of Phytoene Synthase in Haloferax volcanii. JOURNAL OF PROTEOME RESEARCH. 2018;17(3):1158-1171.The membrane protease LonB is an essential protein in the archaeon Haloferax volcanii and globally impacts its physiology. However, natural substrates of the archaeal Lon protease have not been identified. The whole proteome turnover was examined in a H. volcanii LonB mutant under reduced and physiological protease levels. LC-MS/MS combined with stable isotope labeling was applied for the identification/quantitation of membrane and cytoplasm proteins. Differential synthesis and degradation rates were evidenced for 414 proteins in response to Lon expression. A total of 58 proteins involved in diverse cellular processes showed a degradation pattern (none/very little degradation in the absence of Lon and increased degradation in the presence of Lon) consistent with a LonB substrate, which was further substantiated for several of these candidates by pull-down assays. The most notable was phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthesis. The rapid degradation of PSY upon LonB induction in addition to the remarkable stabilization of this protein and hyperpigmentation phenotype in the Lon mutant strongly suggest that PSY is a LonB substrate. This work identifies for the first time candidate targets of the archaeal Lon protease and establishes proteolysis by Lon as a novel post-translational regulatory mechanism of carotenogenesis