Mitochondrial DNA variation along an altitudinal gradient in the greater white-toothed shrew, Crocidura russula.

The distribution of mitochondrial control region-sequence polymorphism was investigated in 15 populations of Crocidura russula along an altitudinal gradient in western Switzerland. High-altitude populations are smaller, sparser and appear to undergo frequent bottlenecks. Accordingly, they showed a loss of rare haplotypes, but unexpectedly, were less differentiated than lowland populations. Furthermore, the major haplotypes segregated significantly with altitude. The results were inconsistent with a simple model of drift and dispersal. They suggested instead a role for historical patterns of colonization, or, alternatively, present-day selective forces acting on one of the mitochondrial genes involved in metabolic pathways.

Hepatitis C virus-mediated mitochondrial dysfunctions are prevented and rescued by cyclophilin inhibition

Alisporivir (Debio-025) is an analogue of cyclosporine A andrepresents the prototype of a new class of non-immunosuppressivecyclophilin inhibitors. In vitro and in vivo studies have shownthat alisporivir inhibits hepatitis C virus (HCV) replication andongoing clinical trials are exploring its therapeutic potential inpatients with chronic hepatitis C. Recent data suggest that theantiviral effect is mediated by inhibition of cyclophilin A whichis an essential host factor in the HCV life cycle. However, alisporiviralso inhibits mitochondrial permeability transition by bindingto cyclophilin D. As HCV is known to affect mitochondrialfunction, we explored the effect of alisporivir on HCV proteinmediatedmitochondrial dysfunction. By the use of inducible celllines, which allow to investigate the effects of HCV polyproteinexpression independent from viral RNA replication and whichrecapitulate the major alterations of mitochondrial bioenergeticsobserved in infectious cell systems, we show that alisporivir preventsHCV protein-mediated cytochrome c redistribution,decrease of cell respiration, collapse of mitochondrial membranepotential, overproduction of reactive oxygen species and mitochondrialcalcium overload. Strikingly, some of the HCV-mediatedmitochondrial dysfunctions could even be rescued byalisporivir. These observations provide new insights into thepathogenesis of HCV-related liver disease and reveal an additionalmechanism of action of alisporivir that is likely beneficialin the treatment of chronic hepatitis C.

[(18)F]FDG-PET Standard Uptake Value as a Metabolic Predictor of Bone Marrow Response to Radiation: Impact on Acute and Late Hematological Toxicity in Cervical Cancer Patients Treated With Chemoradiation Therapy.

PURPOSE: To quantify the relationship between bone marrow (BM) response to radiation and radiation dose by using (18)F-labeled fluorodeoxyglucose positron emission tomography [(18)F]FDG-PET standard uptake values (SUV) and to correlate these findings with hematological toxicity (HT) in cervical cancer (CC) patients treated with chemoradiation therapy (CRT). METHODS AND MATERIALS: Seventeen women with a diagnosis of CC were treated with standard doses of CRT. All patients underwent pre- and post-therapy [(18)F]FDG-PET/computed tomography (CT). Hemograms were obtained before and during treatment and 3 months after treatment and at last follow-up. Pelvic bone was autosegmented as total bone marrow (BMTOT). Active bone marrow (BMACT) was contoured based on SUV greater than the mean SUV of BMTOT. The volumes (V) of each region receiving 10, 20, 30, and 40 Gy (V10, V20, V30, and V40, respectively) were calculated. Metabolic volume histograms and voxel SUV map response graphs were created. Relative changes in SUV before and after therapy were calculated by separating SUV voxels into radiation therapy dose ranges of 5 Gy. The relationships among SUV decrease, radiation dose, and HT were investigated using multiple regression models. RESULTS: Mean relative pre-post-therapy SUV reductions in BMTOT and BMACT were 27% and 38%, respectively. BMACT volume was significantly reduced after treatment (from 651.5 to 231.6 cm(3), respectively; P<.0001). BMACT V30 was significantly correlated with a reduction in BMACT SUV (R(2), 0.14; P<.001). The reduction in BMACT SUV significantly correlated with reduction in white blood cells (WBCs) at 3 months post-treatment (R(2), 0.27; P=.04) and at last follow-up (R(2), 0.25; P=.04). Different dosimetric parameters of BMTOT and BMACT correlated with long-term hematological outcome. CONCLUSIONS: The volumes of BMTOT and BMACT that are exposed to even relatively low doses of radiation are associated with a decrease in WBC counts following CRT. The loss in proliferative BM SUV uptake translates into low WBC nadirs after treatment. These results suggest the potential of intensity modulated radiation therapy to spare BMTOT to reduce long-term hematological toxicity.

Viral inhibitor of apoptosis vFLIP/K13 protects endothelial cells against superoxide-induced cell death.

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes an antiapoptotic viral Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (vFLIP/K13). The antiapoptotic activity of vFLIP/K13 has been attributed to an inhibition of caspase 8 activation and more recently to its capability to induce the expression of antiapoptotic proteins via activation of NF-kappaB. Our study provides the first proteome-wide analysis of the effect of vFLIP/K13 on cellular-protein expression. Using comparative proteome analysis, we identified manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant and an important antiapoptotic enzyme, as the protein most strongly upregulated by vFLIP/K13 in endothelial cells. MnSOD expression was also upregulated in endothelial cells upon infection with HHV-8. Microarray analysis confirmed that MnSOD is also upregulated at the RNA level, though the differential expression at the RNA level was much lower (5.6-fold) than at the protein level (25.1-fold). The induction of MnSOD expression was dependent on vFLIP/K13-mediated activation of NF-kappaB, occurred in a cell-intrinsic manner, and was correlated with decreased intracellular superoxide accumulation and increased resistance of endothelial cells to superoxide-induced death. The upregulation of MnSOD expression by vFLIP/K13 may support the survival of HHV-8-infected cells in the inflammatory microenvironment in KS.

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro.

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.

Mutations of mitochondrial DNA as potential biomarkers in breast cancer.

BACKGROUND: Alterations of mitochondrial DNA (mtDNA) have been found in cancer patients, therefore informative mtDNA mutations could serve as biomarkers for the disease. MATERIALS AND METHODS: The two hypervariable regions HVR1 and HVR2 in the D-Loop region were sequenced in ten paired tissue and plasma samples from breast cancer patients. RESULTS: MtDNA mutations were found in all patients' samples, suggesting a 100% detection rate. Examining germline mtDNA mutations, a total of 85 mutations in the D-loop region were found; 31 of these mutations were detected in both tissues and matched plasma samples, the other 54 germline mtDNA mutations were found only in the plasma samples. Regarding somatic mtDNA mutations, a total of 42 mutations in the D-loop region were found in breast cancer tissues. CONCLUSION: Somatic mtDNA mutations in the D-loop region were detected in breast cancer tissues but not in the matched plasma samples, suggesting that more sensitive methods will be needed for such detection to be of clinical utility.

The cyclophilin inhibitor alisporivir prevents hepatitis C virus-mediated mitochondrial dysfunction.

Alisporivir (Debio-025) is an analogue of cyclosporine A and represents the prototype of a new class of non-immunosuppressive cyclophilin inhibitors. In vitro and in vivo studies have shown that alisporivir inhibits hepatitis C virus (HCV) replication, and ongoing clinical trials are exploring its therapeutic potential in patients with chronic hepatitis C. Recent data suggest that the antiviral effect is mediated by inhibition of cyclophilin A, which is an essential host factor in the HCV life cycle. However, alisporivir also inhibits mitochondrial permeability transition by binding to cyclophilin D. Because HCV is known to affect mitochondrial function, we explored the effect of alisporivir on HCV protein-mediated mitochondrial dysfunction. Through the use of inducible cell lines, which allow to investigate the effects of HCV polyprotein expression independent from viral RNA replication and which recapitulate the major alterations of mitochondrial bioenergetics observed in infectious cell systems, we show that alisporivir prevents HCV protein-mediated decrease of cell respiration, collapse of mitochondrial membrane potential, overproduction of reactive oxygen species and mitochondrial calcium overload. Strikingly, some of the HCV-mediated mitochondrial dysfunctions could even be rescued by alisporivir. Conclusion: These observations provide new insights into the pathogenesis of HCV-related liver disease and reveal an additional mechanism of action of alisporivir that is likely beneficial in the treatment of chronic hepatitis C. (HEPATOLOGY 2012).

Loss of mitochondrial functions associated with azole resistance in Candida glabrata results in enhanced virulence in mice.

Mitochondrial dysfunction is one of the possible mechanisms by which azole resistance can occur in Candida glabrata. Cells with mitochondrial DNA deficiency (so-called "petite mutants") upregulate ATP binding cassette (ABC) transporter genes and thus display increased resistance to azoles. Isolation of such C. glabrata mutants from patients receiving antifungal therapy or prophylaxis has been rarely reported. In this study, we characterized two sequential and related C. glabrata isolates recovered from the same patient undergoing azole therapy. The first isolate (BPY40) was azole susceptible (fluconazole MIC, 4 μg/ml), and the second (BPY41) was azole resistant (fluconazole MIC, >256 μg/ml). BPY41 exhibited mitochondrial dysfunction and upregulation of the ABC transporter genes C. glabrata CDR1 (CgCDR1), CgCDR2, and CgSNQ2. We next assessed whether mitochondrial dysfunction conferred a selective advantage during host infection by testing the virulence of BPY40 and BPY41 in mice. Surprisingly, even with in vitro growth deficiency compared to BPY40, BPY41 was more virulent (as judged by mortality and fungal tissue burden) than BPY40 in both systemic and vaginal murine infection models. The increased virulence of the petite mutant correlated with a drastic gain of fitness in mice compared to that of its parental isolate. To understand this unexpected feature, genome-wide changes in gene expression driven by the petite mutation were analyzed by use of microarrays during in vitro growth. Enrichment of specific biological processes (oxido-reductive metabolism and the stress response) was observed in BPY41, all of which was consistent with mitochondrial dysfunction. Finally, some genes involved in cell wall remodelling were upregulated in BPY41 compared to BPY40, which may partially explain the enhanced virulence of BPY41. In conclusion, this study shows for the first time that mitochondrial dysfunction selected in vivo under azole therapy, even if strongly affecting in vitro growth characteristics, can confer a selective advantage under host conditions, allowing the C. glabrata mutant to be more virulent than wild-type isolates.

Skeletal muscle mitochondrial and lipid droplet content assessed with standardized grid sizes for stereology.

Skeletal muscle mitochondrial (Mito) and lipid droplet (Lipid) content are often measured in human translational studies. Stereological point counting allows computing Mito and Lipid volume density (Vd) from micrographs taken with transmission electron microscopes. Former studies are not specific as to the size of individual squares that make up the grids, making reproducibility difficult, particularly when different magnifications are used. Our objective was to determine which size grid would be best at predicting fractional volume efficiently without sacrificing reliability and to test a novel method to reduce sampling bias. Methods: ten subjects underwent vastus lateralis biopsies. Samples were fixed, embedded, and cut longitudinally in ultrathin sections of 60 nm. Twenty micrographs from the intramyofibrillar region were taken per subject at Ã-33,000 magnification. Different grid sizes were superimposed on each micrograph: 1,000 Ã- 1,000 nm, 500 Ã- 500 nm, and 250 Ã- 250 nm. Results: mean Mito and Lipid Vd were not statistically different across grids. Variability was greater when going from 1,000 Ã- 1,000 to 500 Ã- 500 nm grid than from 500 Ã- 500 to 250 Ã- 250 nm grid. Discussion: this study is the first to attempt to standardize grid size while keeping with the conventional stereology principles. This is all in hopes of producing replicable assessments that can be obtained universally across different studies looking at human skeletal muscle mitochondrial and lipid droplet content.

Mitochondrial apoptosis is dispensable for NLRP3 inflammasome activation but non-apoptotic caspase-8 is required for inflammasome priming.

A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co-deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase-8, a caspase essential for death-receptor-mediated apoptosis, is required for efficient Toll-like-receptor-induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non-apoptotic role for caspase-8 in regulating inflammasome activation and pro-inflammatory cytokine levels.

Molecular phylogenetics of soricid shrews (Mammalia) based on mitochondrial cytochrome b gene sequences: with special reference to the Soricinae

Molecular phylogeny of soricid shrews (Soricidae, Eulipotyphla, Mammalia) based on 1140 bp mitochondrial cytochrome b gene (cytb) sequences was inferred by the maximum likelihood (ML) method. All 13 genera of extant Soricinae and two genera of Crocidurinae were included in the analyses. Anourosorex was phylogenetically distant from the main groupings within Soricinae and Crocidurinae in the ML tree. Thus, it could not be determined to which subfamily Anourosorex should be assigned: Soricinae, Crocidurinae or a new subfamily. Soricinae (excluding Anourosorex) should be divided into four tribes: Neomyini, Notiosoricini, Soricini and Blarinini. However, monophyly of Blarinini was not robust in the present data set. Also, branching orders among tribes of Soricinae and those among genera of Neomyini could not be determined because of insufficient phylogenetic information of the cytb sequences. For water shrews of Neomyini (Chimarrogale, Nectogale and Neomys), monophyly of Neomys and the Chimarrogale-Nectogale group could not be verified, which implies the possibility of multiple origins for the semi-aquatic mode of living among taxa within Neomyini. Episoriculus may contain several separate genera. Blarinella was included in Blarinini not Soricini, based on the cytb sequences, but the confidence level was rather low; hence more phylogenetic information is needed to determine its phylogenetic position. Furthermore, some specific problems of taxonomy of soricid shrews were clarified, for example phylogeny of local populations of Notiosorex crawfordi, Chimarrogale himalayica and Crocidura attenuata.

Identification and potential origin of invasive clawed frogs Xenopus (Anura: Pipidae) in Sicily based on mitochondrial and nuclear DNA

African clawed frogs of the widespread polytypic species Xenopus laevis Daudin, 1802 (ranging large parts of sub-Saharan Africa) have been spreading since the 1940s, and have established reproductive populations in Europe, Asia and the Americas, where they can have negative impact as competitors of native amphibians and as disease vectors for chytridomycosis or ranaviruses. Here we use two mitochondrial (cytochrome b, 16S rDNA) and one nuclear (RAG 1: Recombination Associated Gene 1) DNA markers to infer the potential origin of invasive clawed frogs from Sicily that represent the largest invasive population in Europe. Identical mtDNA haplotypes match with those of Xenopus laevis, and Sicilian clawed frogs very probably belong to a lineage from the Cape Region of South Africa, most likely originating from a laboratory stock. Nuclear data support this conclusion. Identical mtDNA sequences (cyt b, 16S) of frogs sampled across their range in Sicily suggest the occurrence of a single source population and a potential bottleneck at their release, but faster evolving multilocus nuclear data (microsatellites, SNPs) on the population genetics would be important in the future to better support this hypothesis