Adaptation of topoisomerase I paralogs to nuclear and mitochondrial DNA

Topoisomerase I is essential for DNA metabolism in nuclei and mitochondria. In yeast, a single topoisomerase I gene provides for both organelles. In vertebrates, topoisomerase I is divided into nuclear and mitochondrial paralogs (Top1 and Top1mt). To assess the meaning of this gene duplication, we targeted Top1 to mitochondria or Top1mt to nuclei. Overexpression in the fitting organelle served as control. Targeting of Top1 to mitochondria blocked transcription and depleted mitochondrial DNA. This was also seen with catalytically inactive Top1 mutants, but not with Top1mt overexpressed in mitochondria. Targeting of Top1mt to the nucleus revealed that it was much less able to interact with mitotic chromosomes than Top1 overexpressed in the nucleus. Similar experiments with Top1/Top1mt hybrids assigned these functional differences to structural divergences in the DNA-binding core domains. We propose that adaptation of this domain to different chromatin environments in nuclei and mitochondria has driven evolutional development and conservation of organelle-restricted topoisomerase I paralogs in vertebrates

Coordinated regulation of mitochondrial topoisomerase IB with mitochondrial nuclear encoded genes and MYC

Mitochondrial DNA (mtDNA) is entirely dependent on nuclear genes for its transcription and replication. One of these genes is TOP1MT, which encodes the mitochondrial DNA topoisomerase IB, involved in mtDNA relaxation. To elucidate TOP1MT regulation, we performed genome-wide profiling across the 60-cell line panel (the NCI-60) of the National Cancer Institute Developmental Therapeutics Program. We show that TOP1MT mRNA expression varies widely across these cell lines with the highest levels in leukemia (HL-60, K-562) and melanoma (SK-MEL-28), intermediate levels in breast (MDA-MB-231), ovarian (OVCAR) and colon (HCT-116, HCT-15, KM-12), and lowest levels in renal (ACHN, A498), prostate (PC-3, DU-145) and central nervous system cell lines (SF-539, SF-268, SF-295). Genome-wide analyses show that TOP1MT expression is significantly correlated with the other mitochondrial nuclear-encoded genes including the mitochondrial nucleoid genes, and demonstrate an overall co-regulation of the mitochondrial nuclear-encoded genes. We also find very high correlation between the expression of TOP1MT and the proto-oncogene MYC (c-myc). TOP1MT contains E-boxes (c-myc binding sites) and TOP1MT transcription follows MYC up- and down-regulation by MYC promoter activation and siRNA against MYC. Our finding implicates MYC as a novel regulator of TOP1MT and confirms its role as a master regulator of MNEGs and mitochondrial nucleoids

Extracellular matrix composition modulates the responsiveness of differentiated and stem pancreatic cancer cells to lipophilic derivate of gemcitabine

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. Gemcitabine (GEM) is used as the gold standard drug in PDAC treatment. However, due to its poor efficacy, it remains urgent to identify novel strategies to overcome resistance issues. In this context, an intense stroma reaction and the presence of cancer stem cells (CSCs) have been shown to influence PDAC aggressiveness, metastatic potential, and chemoresistance. Methods: We used three-dimensional (3D) organotypic cultures grown on an extracellular matrix composed of Matrigel or collagen I to test the effect of the new potential therapeutic prodrug 4-(N)-stearoyl-GEM, called C18GEM. We analyzed C18GEM cytotoxic activity, intracellular uptake, apoptosis, necrosis, and autophagy induction in both Panc1 cell line (P) and their derived CSCs. Results: PDAC CSCs show higher sensitivity to C18GEM treatment when cultured in both two-dimensional (2D) and 3D conditions, especially on collagen I, in comparison to GEM. The intracellular uptake mechanisms of C18GEM are mainly due to membrane nucleoside transporters' expression and fatty acid translocase CD36 in Panc1 P cells and to clathrin-mediated endocytosis and CD36 in Panc1 CSCs. Furthermore, C18GEM induces an increase in cell death compared to GEM in both cell lines grown on 2D and 3D cultures. Finally, C18GEM stimulated protective autophagy in Panc1 P and CSCs cultured on 3D conditions. Conclusion: We propose C18GEM together with autophagy inhibitors as a valid alternative therapeutic approach in PDAC treatment

LETM1 couples mitochondrial DNA metabolism and nutrient preference

The diverse clinical phenotypes of Wolf\u2013Hirschhorn syndrome (WHS) are the result of haploinsufficiency of several genes, one of which, LETM1, encodes a protein of the mitochondrial inner membrane of uncertain function. Here, we show that LETM1 is associated with mitochondrial ribosomes, is required for mitochondrial DNA distribution and expression, and regulates the activity of an ancillary metabolic enzyme, pyruvate dehydrogenase. LETM1 deficiency in WHS alters mitochondrial morphology and DNA organization, as does substituting ketone bodies for glucose in control cells. While this change in nutrient availability leads to the death of fibroblasts with normal amounts of LETM1, WHS-derived fibroblasts survive on ketone bodies, which can be attributed to their reduced dependence on glucose oxidation. Thus, remodeling of mitochondrial nucleoprotein complexes results from the inability of mitochondria to use specific substrates for energy production and is indicative of mitochondrial dysfunction. However, the dysfunction could be mitigated by a modified diet\u2014for WHS, one high in lipids and low in carbohydrates

MPV17 Loss Causes Deoxynucleotide Insufficiency and Slow DNA Replication in Mitochondria

MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients’ quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway

MPV17L2 is required for ribosome assembly in mitochondria.

MPV17 is a mitochondrial protein of unknown function, and mutations in MPV17 are associated with mitochondrial deoxyribonucleic acid (DNA) maintenance disorders. Here we investigated its most similar relative, MPV17L2, which is also annotated as a mitochondrial protein. Mitochondrial fractionation analyses demonstrate MPV17L2 is an integral inner membrane protein, like MPV17. However, unlike MPV17, MPV17L2 is dependent on mitochondrial DNA, as it is absent from ρ(0) cells, and co-sediments on sucrose gradients with the large subunit of the mitochondrial ribosome and the monosome. Gene silencing of MPV17L2 results in marked decreases in the monosome and both subunits of the mitochondrial ribosome, leading to impaired protein synthesis in the mitochondria. Depletion of MPV17L2 also induces mitochondrial DNA aggregation. The DNA and ribosome phenotypes are linked, as in the absence of MPV17L2 proteins of the small subunit of the mitochondrial ribosome are trapped in the enlarged nucleoids, in contrast to a component of the large subunit. These findings suggest MPV17L2 contributes to the biogenesis of the mitochondrial ribosome, uniting the two subunits to create the translationally competent monosome, and provide evidence that assembly of the small subunit of the mitochondrial ribosome occurs at the nucleoid

Predicting needlestick and sharps injuries in nursing students: Development of the SNNIP scale

© 2020 The Authors. Nursing Open published by John Wiley & Sons Ltd. Aim: To develop an instrument to investigate knowledge and predictive factors of needlestick and sharps injuries (NSIs) in nursing students during clinical placements. Design: Instrument development and cross-sectional study for psychometric testing. Methods: A self-administered instrument including demographic data, injury epidemiology and predictive factors of NSIs was developed between October 2018–January 2019. Content validity was assessed by a panel of experts. The instrument's factor structure and discriminant validity were explored using principal components analysis. The STROBE guidelines were followed. Results: Evidence of content validity was found (S-CVI 0.75; I-CVI 0.50–1.00). A three-factor structure was shown by exploratory factor analysis. Of the 238 participants, 39% had been injured at least once, of which 67.3% in the second year. Higher perceptions of “personal exposure” (4.06, SD 3.78) were reported by third-year students. Higher scores for “perceived benefits” of preventive behaviours (13.6, SD 1.46) were reported by second-year students