Thiol peroxidase deficiency leads to increased mutational load and decreased fitness in saccharomyces cerevisiae

Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (Δ8) is viable. In this study, we employed two independent Δ8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and Δ8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. Δ8 lines showed a significant increase in nonrecurrent point mutations and indels. The original Δ8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all Δ8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of Δ8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness.National Institutes of Health (GM065204

Stability of Metabolomic Content during Sample Preparation: Blood and Brain Tissues

Thermal and enzymatic reactions can significantly change the tissue metabolomic content during the sample preparation. In this work, we evaluated the stability of metabolites in human whole blood, serum, and rat brain, as well as in metabolomic extracts from these tissues. We measured the concentrations of 63 metabolites in brain and 52 metabolites in blood. We have shown that metabolites in the extracts from biological tissues are stable within 24 h at 4 °C. Serum and whole blood metabolomes are also rather stable, changes in metabolomic content of the whole blood homogenate become apparent only after 1–2 h of incubation at 4 °C, and become strong after 24 h. The most significant changes correspond to energy metabolites: the concentrations of ATP and ADP decrease fivefold, and the concentrations of NAD, NADH, and NADPH decrease below the detectable level. A statistically significant increase was observed for AMP, IMP, hypoxanthine, and nicotinamide. The brain tissue is much more metabolically active than human blood, and significant metabolomic changes occur already within the first several minutes during the brain harvest and sample homogenization. At a longer timescale (hours), noticeable changes were observed for all classes of compounds, including amino acids, organic acids, alcohols, amines, sugars, nitrogenous bases, nucleotides, and nucleosides

Use of Environmental Scanning Electron Microscopy for in situ Observation of Interaction of Cells with Micro- and Nanoprobes

Precision intracellular sensing, probing and manipulation offer unprecedented opportunities for advances in biological sciences. Next-generation ultra-fine probes will be capable of targeting individual cell organelles. Development of such probes as well as probes capable of penetrating through tough cell walls requires detailed knowledge of cell-probe interaction. This Letter evaluated the applicability of environmental scanning electron microscopy (ESEM) for cell and cell-probe interaction imaging. Several types of cells (plant and yeast cells as well as mouse spermatozoa) were successfully imaged in their natural state, with mouse spermatozoa observed by ESEM for the first time. Computerized stage applied to image was tough plant cell walls interactions with several probes. Substantial damage to the cell walls was observed as a result of microprobe penetration. The damage persisted after the probe withdrawal and there was residue of cellular content on the withdrawn probes. Several mechanisms of probe failure were observed in situ global buckling, localized bending followed by the tip break-off, and plastic deformation with permanent bending in the case of ultra-fine metal nanoprobe. The results demonstrate applicability of ESEM for high-resolution in situ imaging of cells. Observed mechanisms of cell damage and probe failure provide guidance for future development of probes for minimally-invasive intercellular probing

Thiol peroxidase deficiency leads to increased mutational load and decreased fitness in saccharomyces cerevisiae

Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (Δ8) is viable. In this study, we employed two independent Δ8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and Δ8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. Δ8 lines showed a significant increase in nonrecurrent point mutations and indels. The original Δ8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all Δ8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of Δ8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness.National Institutes of Health (GM065204

Novel Oxidovanadium Complexes with Redox-Active R-Mian and R-Bian Ligands: Synthesis, Structure, Redox and Catalytic Properties

A new monoiminoacenaphthenone 3,5-(CF3)2C6H3-mian (complex 2) was synthesized and further exploited, along with the already known monoiminoacenaphthenone dpp-mian, to obtain oxidovanadium(IV) complexes [VOCl2(dpp-mian)(CH3CN)] (3) and [VOCl(3,5-(CF3)2C6H3-bian)(H2O)][VOCl3(3,5-(CF3)2C6H3-bian)]·2.85DME (4) from [VOCl2(CH3CN)2(H2O)] (1) or [VCl3(THF)3]. The structure of all compounds was determined using X-ray structural analysis. The vanadium atom in these structures has an octahedral coordination environment. Complex 4 has an unexpected structure. Firstly, it contains 3,5-(CF3)2C6H3-bian instead of 3,5-(CF3)2C6H3-mian. Secondly, it has a binuclear structure, in contrast to 3, in which two oxovanadium parts are linked to each other through V=O···V interaction. This interaction is non-covalent in origin, according to DFT calculations. In structures 2 and 3, non-covalent π-π staking interactions between acenaphthene moieties of the neighboring molecules (distances are 3.36–3.40 Å) with an estimated energy of 3 kcal/mol were also found. The redox properties of the obtained compounds were studied using cyclic voltammetry in solution. In all cases, the reduction processes initiated by the redox-active nature of the mian or bian ligand were identified. The paramagnetic nature of complexes 3 and 4 has been proven by EPR spectroscopy. Complexes 3 and 4 exhibited high catalytic activity in the oxidation of alkanes and alcohols with peroxides. The yields of products of cyclohexane oxidation were 43% (complex 3) and 27% (complex 4). Based on the data regarding the study of regio- and bond-selectivity, it was concluded that hydroxyl radicals play the most crucial role in the reaction. The initial products in the reactions with alkanes are alkyl hydroperoxides, which are easily reduced to their corresponding alcohols by the action of triphenylphosphine (PPh3). According to the DFT calculations, the difference in the catalytic activity of 3 and 4 is most likely associated with a different mechanism for the generation of ●OH radicals. For complex 4 with electron-withdrawing CF3 substituents at the diimine ligand, an alternative mechanism, different from Fenton’s and involving a redox-active ligand, is assumed