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Mitochondria are dynamic organelles undergoing continuous fusion and fission with Drp1, encoded by the DNM1L gene, required for mitochondrial fragmentation. DNM1L dominant pathogenic variants lead to progressive neurological disorders with early exitus. Herein we report on the case of a boy affected by epileptic encephalopathy carrying two heterozygous variants (in cis) of the DNM1L gene: a pathogenic variant (PV) c.1085G>A (p.Gly362Asp) accompanied with a variant of unknown significance (VUS) c.1535T>C (p.Ile512Thr). Amplicon sequencing of the mother’s DNA revealed the presence of the PV and VUS in 5% of cells, with the remaining cells presenting only VUS. Functional investigations performed on the patient and his mother’s cells unveiled altered mitochondrial respiratory chain activities, network architecture and Ca2+ homeostasis as compared with healthy unrelated subjects’ samples. Modelling Drp1 harbouring the two variants, separately or in combination, resulted in structural changes as compared with Wt protein. Considering the clinical history of the mother, PV transmission by a maternal germline mosaicism mechanism is proposed. Altered Drp1 function leads to changes in the mitochondrial structure and bioenergetics as well as in Ca2+ homeostasis. The novel VUS might be a modifier that synergistically worsens the phenotype when associated with the PV.

Epidemiological studies have shown an increased occurrence of metabolic disorders such as insulin resistance (IR) and steatosis in patients with hepatitis C virus (HCV) infection. IR is believed to represent one of the central clinical features of the “metabolic syndrome” and the major pathogenetic factor for type 2 diabetes mellitus. In patients with chronic HCV hepatitis, IR may have several dangerous consequences such as accelerated progression of liver fibrosis, resistance to antiviral therapy and development of hepatocellular carcinoma. According to recent evidence, the global epidemic of metabolic disorders related to incorrect diets will lead physicians to deal with 1.2 billion patients with diabetes in the world in 2025. Given the high prevalence of HCV infection in several countries, metabolic manifestations will contribute to increasing morbidity and mortality in patients with HCV chronic infection in the near future. HCV treatment, shown able to decrease both the occurrence of HCV-related IR and diabetes, may reduce the risk of the associated morbidities.

Liposomes are nanocarriers able to solubilize and deliver a wide range of hydrophobic pharmaceuticals and to increase drug bioavailability. They show a natural tendency to hepatic accumulation, and thus represent an optimal drug delivery system for the treatment of liver diseases, including chronic virus hepatitis C. Silibinin, the main and more active component of the seed extract from Silybum Marianum, is a hydrophobic flavolignan emerging as an alternative medication for the treatment of hepatitis C virus infection, as it has been shown to inhibit hepatitis C virus entry and replication. In this study we compared cellular delivery and antiviral activity of silibinin encapsulated into phytoliposomes or not, used at the aim to overcome its poor water-solubility and bioavailability. First, it was confirmed the inhibitory activity manifested by lipid-free silibinin in preventing hepatitis C virus entry into the cells. Our data clearly demonstrated that phytoliposome-encapsulated silibinin was absorbed by the cells 2.4 fold more efficiently than the free molecule and showed a three hundreds fold more potent pharmacological activity. Moreover, we surprisingly observed that phytoliposomes themselves inhibited virus entry by reducing the infectivity of viral particles. Based on these observations, phytoliposomes used in this study might be proposed as a delivery system actively contributing to the antiviral efficacy of the encapsulated drug.

Trisomy of chromosome 21 is associated to congenital heart defects in ∼50% of affected newborns. Transcriptome analysis of hearts from trisomic human foeti demonstrated that genes involved in mitochondrial function are globally downregulated with respect to controls, suggesting an impairment of mitochondrial function. We investigated here the properties of mitochondria in fibroblasts from trisomic foeti with and without cardiac defects. Together with the upregulation of Hsa21 genes and the downregulation of nuclear encoded mitochondrial genes, an abnormal mitochondrial cristae morphology was observed in trisomic samples. Furthermore, impairment of mitochondrial respiratory activity, specific inhibition of complex I, enhanced reactive oxygen species production and increased levels of intra-mitochondrial calcium were demonstrated. Seemingly, mitochondrial dysfunction was more severe in fibroblasts from cardiopathic trisomic foeti that presented a more pronounced pro-oxidative state. The data suggest that an altered bioenergetic background in trisomy 21 foeti might be among the factors responsible for a more severe phenotype. Since the mitochondrial functional alterations might be rescued following pharmacological treatments, these results are of interest in the light of potential therapeutic interventions.

MtDNA mutation associated with mitochondrial dysfunction in megakaryoblastic leukaemic cells

The mechanisms of liver injury in chronic hepatitis C virus (HCV) infection are poorly understood though HCV induces a state of hepatic oxidative stress that is more pronounced than that present in many other inflammatory diseases. This mini-review will focus on recent findings revealing an unexpected role of mitochondria in providing a central role in the innate immunity and in addition will illustrate the application of stably transfected human-derived cell lines, inducibly expressing the entire HCV open reading frame for in vitro studies on mitochondria. Results obtained by a comparative analysis of the respiratory chain complexes activities along with mitochondrial morpho-functional confocal microscopy imaging show a detrimental effect of HCV proteins on the cell oxidative metabolism with specific inhibition of complex I activity, decrease of mtΔΨ, increased production of reactive oxygen species. A possible de-regulation of calcium recycling between the endoplasmic reticulum and the mitochondrial network is discussed to provide new insights in the pathogenesis of hepatitis C.

Background Triple therapy including Telaprevir or Boceprevir still represents in many European countries the standard of care for patients with Hepatitis C Virus genotype 1 infection. The number of patients who received this treatment resulted generally lower than expected. We investigated, among naive patients, number and characteristics of treatment candidates who were started on triple or dual therapy in comparison to those who were deferred. Patients and Methods 621 naive treatment candidates were prospectively evaluated at each center. Factors associated with decision to defer or treat with dual or triple therapy were investigated by univariate and multivariate analyses. Rates of Sustained Virological Response and safety profile were analysed. Results Of candidates to treatment, 33% did not received it. It was mostly due to high risk of Interferon-induced decompensation. Of 397 patients who were started on treatment, 266 (67%) received triple, 131 dual. Among patient receiving treatment, unfavorable IL28B, severe liver damage and higher albumin were independently associated with the physician decision to administer triple therapy. Sustained Virological Response after dual therapy was 66.4%, after triple 73.7% (p = 0.14). 142 patients received Telaprevir. The choice of Telaprevir-based therapy was associated with higher Body Mass Index and advanced liver disease. Sustained Virological Response rates were 71.1% after Telaprevir and 76.6% after Boceprevir. Conclusions Individualizing treatment with available regimens allows to maximize Sustained Virological Response and to reduce the number of patients who remain untreated. High proportion of patients with severe liver damage urgently need Interferon free treatment.

Objective: Although the pathogenesis of psoriasis is largely unknown accumulating evidences configure it as an immune-mediated disease determined through cytokines-mediated positive loops between activated lymphocytes subsets and keratinocytes. Mitochondria in addition to their role in the cell bioenergetics are now recognized as a decisional hub in controlling the immunological response. In the present study we compared mitochondria-related functions of PBMC between psoriatic patients and healthy controls. Methods: Freshly isolated PBMC from eleven psoriatic patients and nine healthy controls were subjected to mitochondria-dependent respiratory activity measurements by high-resolution oxymetry and the specific activity of respiratory chain complexes assessed by spectrophotometric assays. Quantitative RT-PCR and immunoblotting were applied to detect the level of selected transcripts and proteins respectively. Results: Respirometric analysis unveiled in patients’ cells a significant three-fold increase of oligomycinsensitive endogenous mitochondria-driven oxygen consumption, which was traceable back to a specific increased activity of the respiratory chain complex I. Analysis by quantitative RT-PCR of transcription factors regulating the mitochondrial biogenesis did not result in significant changes between patients and control cells and was confirmed by the unaffected expression of the complex I subunits. Treatment of either patients’ or control cells with isoproterenol and IBMX ruled out the involvement of a cAMP-PKA-mediated post-transcriptional modification of the respiratory complex. GRIM19 a pleiotropic protein, involved in the structural and functional stabilization of complex I and in the mitochondrial translocation of STAT3 was significantly up-regulated in patients’ cells. Phosphorylation at S727 of STAT3 was increased in patients’cells, which, in addition, unveiled a shift in the relative expression of the STAT3α/β splisoforms. Conclusion: Altogether the results obtained suggest the occurrence in circulating mononucleate cells from psoriatic patients of an altered activity of complex I likely mediated by up-regulation of GRIM19/STAT3β, which might lead to a chronic activation of T-lymphocytes thereby contributing to the development of psoriasis.

Colorectal carcinogenesis relies on loss of homeostasic mechanisms regulating cell proliferation, differentiation and survival. These cell processes have been reported to be influenced independently by transcription factors activated downstream of the Wnt pathway, such as SOX9 and β-catenin, and by the nuclear receptor PPARγ. The purpose of this study was to explore the expression levels and functional link between SOX9, β-catenin and PPARγ in the pathogenesis of colorectal cancer (CRC). We evaluated SOX9, β-catenin and PPARγ expression levels on human CRC specimens by qPCR and immunoblot detection. We tested the hypothesis that PPARγ activation might affect SOX9 and β-catenin expression using four colon cancer cell lines (CaCo2, SW480, HCT116, and HT29 cells). In CRC tissues SOX9 resulted up-regulated at both mRNA and protein levels when compared to matched normal mucosa, β-catenin resulted up-regulated at protein levels, while PPARG mRNA and PPARγ protein levels were down-regulated. A significant relationship was observed between high PPARG and SOX9 expression levels in the tumor tissue and female gender (p=0.005 and p=0.04, respectively), and between high SOX9 expression in the tumor tissue and age (p=0.04) and microsatellite instability (MSI), in particular with MSI-H (p=0.0002). Moreover, treatment with the synthetic PPARγ ligand rosiglitazone induced different changes of SOX9 and β-catenin expression and subcellular localization in the colon cancer cell lines examined. In conclusion, SOX9, β-catenin and PPARγ expression levels are deregulated in the CRC tissue, and in colon cancer cell lines ligand-dependent PPARγ activation unevenly influences SOX9 and β-catenin expression and subcellular localization, suggesting a variable mechanistic role in colon carcinogenesis.

Multiple HCV genotypes have been isolated worldwide. Genotype seems to be involved in the main pathological aspects of HCV infection. Insulin resistance, steatosis and progression toward cirrhosis, fibrosis and hepatocellular carcinoma establish and develop following genotype-specific mechanisms. Moreover genotype influences pharmacological treatment in term of dose and duration. Pathways involved in cell proliferation, apoptosis, lipid metabolism, insulin and interferon signaling are impaired to a different extent among genotypes, leading to distinct pathological settings. Genotype 1 is associated with a more aggressive disease with increased insulin resistance, worst response to therapy, higher risk of cirrhosis and hepatocellular carcinoma development, while genotype 3 is associated with increased steatosis and fibrosis. The identification and characterization of HCV types and subtypes provides insight into the different outcome of HCV infection and responsiveness to therapy. In the present article, we focused on the pathogenicity of HCV genotypes and their effect on disease progression and treatment.

Mutations in DNA double-strand breaks (DSB) repair genes are involved in the pathogenesis of hereditary mammary tumors, it is, however, still unclear whether defects in this pathway may play a role in sporadic breast cancer. In this study, we initially determined mRNA expression of 15 DSB related genes by reverse transcription quantitative polymerase chain reaction in paired normal tissue and cancer specimen from 20 breast cancer cases to classify them into homogeneous clusters. G22P1/ku70, ATR and RAD51 genes were differentially expressed in the three branches recognized by clustering analysis. In particular, a breast cancer subgroup characterized by high RAD51 mRNA levels and estrogen receptor (ER)-positive/progesteron receptor (PR)-negative phenotype was identified. This result was confirmed by the analysis of G22P1/ku70, ATR and RAD51 mRNA levels on paired normal and tumor specimens from an extended breast cancer cohort (n = 75). RAD51 mRNA levels were inversely associated with PR status (p = 0.02) and the highest levels were, indeed, detected in ER-positive/PR-negative tumors (p = 0.03). RAD51 immunostaining of a tissue microarray confirmed the inverse relationship between high RAD51 expression and negative PR status (p = 0.002), as well as, the association with ER-positive/PR-negative phenotype (p = 0.003). Interestingly, the analysis of microarray expression data from 295 breast cancers indicate that RAD51 increased mRNA expression is associated with higher risk of tumor relapse, distant metastases and worst overall survival (p = 0.015, p = 0.009 and p = 0.013 respectively). Our results suggest that RAD51 expression determination could contribute to a better molecular classification of mammary tumors and may represent a novel tool for evaluating postoperative adjuvant therapy for breast cancer patients.

Aims and background: Various genes have been identified for monogenic disorders resembling Parkinson’s disease. The products of some of these genes are associated with mitochondria and have been implicated in cellular protection against oxidative damage. In the present study we analysed fibroblasts from a patient carrying the homozygous mutation p.W437X in the PTEN-induced kinase 1 (PINK1), which manifested a very early onset parkinsonism. Results: Patient’s fibroblasts did not show variation in the mtDNA copy number or in the expression of the oxidative phosphorylation complexes. Sequence analysis of the patient’s mtDNA presented two new missense mutations in the ND5 (m.12397A>G, p.T21A) and ND6 (m. 14319T>C, p.N119D) genes coding for two subunits of complex I. The two mutations were homoplasmic in both the patient and the patient’s mother. Patient’s fibroblasts resulted in enhanced constitutive production of the superoxide anion radical that was abrogated by inhibitor of the complex I. Moreover enzyme kinetic analysis of the NADH:ubiquinone oxidoreductase showed changes in the substrates affinity. Conclusion: To our knowledge, this is the first report showing co-segregation of a Parkinson’s disease related nuclear gene mutation with mtDNA mutation(s). Our observation might shed light on the clinical heterogeneity of the hereditary cases of Parkinson’s disease, highlighting the hitherto unappreciated impact of coexisting mtDNA mutations in determining the development and the clinical course of the disease.