SBBGR technology for reducing waste sludge production during plastic recycling process: Assessment of potential increase in sludge hazardousness.

Sludge production in the wastewater treatment sector is consistently increasing and represents a critical environmental and economic issue. This study evaluated an unconventional approach for treating wastewater generated from the cleaning of non-hazardous plastic solid waste during the plastic recycling process. The proposed scheme was based on sequencing batch biofilter granular reactor (SBBGR) technology, which was compared with the activated sludge-based treatment currently in operation. These treatment technologies were compared regarding sludge quality, specific sludge production, and effluent quality to highlight whether the reduced sludge production shown by SBBGR corresponded to an increase in the concentration of hazardous compounds in the sludge. The SBBGR technology showed remarkable removal efficiencies (TSS, VSS, and NH3 > 99 %; COD >90 %; TN and TP > 80 %) and a sludge production six-fold lower than the conventional plant (in terms of kgTSS/kg CODremoved). Biomass from the SBBGR did not show a significant accumulation of organic micropollutants (i.e., long-chain hydrocarbons, chlorinated pesticides and chlorobenzenes, PCB, PCDD/F, PAH, chlorinated and brominated aliphatic compounds, and aromatic solvents), whereas a certain accumulation of heavy metals was observed. Furthermore, an initial attempt to compare the operating costs of the two treatment approaches revealed that the SBBGR technology would provide 38 % savings.

Time to harmonize mitochondrial syndrome nomenclature and classification: A consensus from the North American Mitochondrial Disease Consortium (NAMDC).

OBJECTIVE: To harmonize terminology in mitochondrial medicine, we propose revised clinical criteria for primary mitochondrial syndromes. METHODS: The North American Mitochondrial Disease Consortium (NAMDC) established a Diagnostic Criteria Committee comprised of members with diverse expertise. It included clinicians, researchers, diagnostic laboratory directors, statisticians, and data managers. The Committee conducted a comprehensive literature review, an evaluation of current clinical practices and diagnostic modalities, surveys, and teleconferences to reach consensus on syndrome definitions for mitochondrial diseases. The criteria were refined after manual application to patients enrolled in the NAMDC Registry. RESULTS: By building upon published diagnostic criteria and integrating recent advances, NAMDC has generated updated consensus criteria for the clinical definition of classical mitochondrial syndromes. CONCLUSIONS: Mitochondrial diseases are clinically, biochemically, and genetically heterogeneous and therefore challenging to classify and diagnose. To harmonize terminology, we propose revised criteria for the clinical definition of mitochondrial disorders. These criteria are expected to standardize the diagnosis and categorization of mitochondrial diseases, which will facilitate future natural history studies and clinical trials.

Effects of Weed Control Practices on Plant Diversity in a Homogenous Olive-Dominated Landscape (South-East of Italy).

Olive groves represent an important economic, agro-ecological, and cultural resource in the Mediterranean Basin. Weed management plays a fundamental role in their sustainable management. The aim of this work was to characterize and assess the plant diversity associated with different weed control practices, in a homogeneous olive-dominated landscape in the South-East of Italy. Sixty-five vegetation plots were sampled in orchards treated with different weed control practices: mowing, tillage, and use of chemical herbicides. The multi-response permutation procedure was used to test the hypothesis of no difference among the treatments. The relationships between plots were visualized by means of non-metric multidimensional scaling ordination. A generalized linear mixed model was used to analyze the relationships between weed control practices and life forms, chorotypes, and diversity indexes. The results showed that the three weed control practices determined slightly different plant communities. Chemically weeded orchards showed an impoverished floristic composition and the lowest diversity, whereas mowing and tillage yielded similar values. These latter two treatments differed for the percentages of hemicryptophytes and therophytes. Moreover, different from other studies, we did not find plant species of particular concern for biodiversity conservation. We hypothesize that this result is due to the monotonous structure of the agro-landscape we investigated, where natural elements are almost lacking. From this point of view, a correct management of agro-districts should consider both the agronomic practices at the level of the individual olive groves and the structure of the agro-landscape.

Treating and reusing wastewater generated by the washing operations in the non-hazardous plastic solid waste recycling process: Advanced method vs. conventional method.

The effectiveness of an advanced treatment of wastewater generated by non-hazardous plastic solid waste (PSW) washing, based on the Sequencing Batch Biofilter Granular Reactor (SBBGR), was assessed in terms of gross parameters, removal efficiencies and sludge production. The proposed treatment was also compared with the conventional treatment, which was based on primary and secondary treatments, using the activated sludge process, performed by Recuperi Pugliesi, a leading company in the plastic recycling industry located in Bari, Italy. The company produces low-density polyethylene (LDPE) regenerated granules from PSW used in agricultural and floricultural greenhouse activities and industrial packaging after a washing stage in the aqueous phase. The latter generates large volumes of wastewater, the conventional treatment of which is characterised by large quantities of sludge and the associated disposal problems. Under steady-state conditions, the SBBGR provided impressive removal efficiencies regarding the main gross parameters (over 90% for COD and TKN, over 99% for BOD5, TSS, VSS and NH3, and over 80% for TN) with a statistically better effluent quality than that of the conventional treatment. The SBBGR effluent quality was modelled in terms of washing water characteristics by using generalized additive models (GAMs). The SBBGR treatment was characterised by a specific sludge production five times lower than that of the conventional treatment (0.21 kg TSS vs. 1.0 kg TSS per m3 of wastewater treated). Compared with the conventional treatment, the proposed process showed a five-fold reduction in the cost of sludge disposal, which saved 50% of the operating cost.

Regulatory environment for novel therapeutic development in mitochondrial diseases.

At present, there is just one approved therapy for patients with mitochondrial diseases in Europe, another in Japan, and none in the United States. These facts reveal an important and significant unmet need for approved therapies for these debilitating and often fatal disorders. To fill this need, it is critical for clinicians and drug developers to work closely with regulatory agencies. In the United States, mitochondrial disease patients and clinicians, the United Mitochondrial Disease Foundation, and pharmaceutical industry members have engaged with the Food and Drug Administration to educate each other about these complex and heterogeneous diseases and about regulatory requirements to obtain approvals for novel therapies. Clinical development of therapies for rare diseases has been facilitated by the 1983 US Orphan Drug Act (ODA) and similar legislation in Japan and the European Union. Further legislation and regulatory guidance have expanded and refined regulatory flexibility. While regulatory and financial incentives of the ODA have augmented involvement of pharmaceutical companies, clinicians, with patient advocacy groups and industry, need to conduct natural history studies, develop clinical outcome measures, and identify potential supportive surrogate endpoints predictive of clinical benefit, which together are critical foundations for clinical trials. Thus, the regulatory environment for novel therapeutic development is conducive and offers flexibility for mitochondrial diseases. Nevertheless, flexibility does not mean lower standards, as well-controlled rigorous clinical trials of high quality are still required to establish the efficacy of potential therapies and to obtain regulatory agency approvals for their commercial use. This process is illustrated through the authors' ongoing efforts to develop therapy for thymidine kinase 2 deficiency.

Mitochondrial diseases in North America: An analysis of the NAMDC Registry.

OBJECTIVE: To describe clinical, biochemical, and genetic features of participants with mitochondrial diseases (MtDs) enrolled in the North American Mitochondrial Disease Consortium (NAMDC) Registry. METHODS: This cross-sectional, multicenter, retrospective database analysis evaluates the phenotypic and molecular characteristics of participants enrolled in the NAMDC Registry from September 2011 to December 2018. The NAMDC is a network of 17 centers with expertise in MtDs and includes both adult and pediatric specialists. RESULTS: One thousand four hundred ten of 1,553 participants had sufficient clinical data for analysis. For this study, we included only participants with molecular genetic diagnoses (n = 666). Age at onset ranged from infancy to adulthood. The most common diagnosis was multisystemic disorder (113 participants), and only a minority of participants were diagnosed with a classical mitochondrial syndrome. The most frequent classical syndromes were Leigh syndrome (97 individuals) and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (71 individuals). Pathogenic variants in the mitochondrial DNA were more frequently observed (414 participants) than pathogenic nuclear gene variants (252 participants). Pathogenic variants in 65 nuclear genes were identified, with POLG1 and PDHA1 being the most commonly affected. Pathogenic variants in 38 genes were reported only in single participants. CONCLUSIONS: The NAMDC Registry data confirm the high variability of clinical, biochemical, and genetic features of participants with MtDs. This study serves as an important resource for future enhancement of MtD research and clinical care by providing the first comprehensive description of participant with MtD in North America.

Dystonia-Ataxia with early handwriting deterioration in COQ8A mutation carriers: A case series and literature review.

Cerebellar ataxia is a hallmark of coenzyme Q10 (CoQ10) deficiency associated with COQ8A mutations. We present four patients, one with novel COQ8A pathogenic variants all with early, prominent handwriting impairment, dystonia and only mild ataxia. To better define the phenotypic spectrum and course of COQ8A disease, we review the clinical presentation and evolution in 47 reported cases. Individuals with COQ8A mutation display great clinical variability and unpredictable responses to CoQ10 supplementation. Onset is typically during infancy or childhood with ataxic features associated with developmental delay or regression. When disease onset is later in life, first symptoms can include: incoordination, epilepsy, tremor, and deterioration of writing. The natural history is characterized by a progression to a multisystem brain disease dominated by ataxia, with disease severity inversely correlated with age at onset. Six previously reported cases share with ours, a clinical phenotype characterized by slowly progressive or static writing difficulties, focal dystonia, and speech disorder, with only minimal ataxia. The combination of writing difficulty, dystonia and ataxia is a distinctive constellation that is reminiscent of a previously described clinical entity called Dystonia Ataxia Syndrome (DYTCA) and is an important clinical indicator of COQ8A mutations, even when ataxia is mild or absent.

Macrophage derived TNFα promotes hepatic reprogramming to Warburg-like metabolism.

During infection, hepatocytes must undergo a reprioritization of metabolism, termed metabolic reprogramming. Hepatic metabolic reprogramming in response to infection begins within hours of infection, suggesting a mechanism closely linked to pathogen recognition. Following injection with polyinosinic:polycytidylic acid, a mimic of viral infection, a robust hepatic innate immune response could be seen involving the TNFα pathway at 2 h. Repeated doses led to the adoption of Warburg-like metabolism in the liver as determined by in vivo metabolic imaging, expression analyses, and metabolomics. Hepatic macrophages, Kupffer cells, were able to induce Warburg-like metabolism in hepatocytes in vitro via TNFα. Eliminating macrophages in vivo or blocking TNFα in vitro or in vivo resulted in abrogation of the metabolic phenotype, establishing an immune-metabolic axis in hepatic metabolic reprogramming. Overall, we suggest that macrophages, as early sensors of pathogens, instruct hepatocytes via TNFα to undergo metabolic reprogramming to cope with challenges to homeostasis initiated by infection. This work not only addresses a key component of end-organ physiology, but also raises questions about the side effects of biologics in the treatment of inflammatory diseases. KEY MESSAGES: • Hepatocytes develop Warburg-like metabolism in vivo during viral infection. • Macrophage TNFα promotes expression of glycolytic enzymes in hepatocytes. • Blocking this immune-metabolic axis abrogates Warburg-like metabolism in the liver. • Implications for patients being treated for inflammatory diseases with biologics.

In-stream microhabitat mosaic depicts the success of mitigation measures and controls the Ecological Potential of benthic communities in heavily modified rivers.

The positive effect of mitigation measures on in-stream habitat conditions and the benthic community is recognised. In heavily modified rivers, though, the response of aquatic invertebrates to mitigation measures and habitat mosaic changes is scarcely documented. We used non-metric multidimensional scaling to explore the benthic community of leveed rivers in the agricultural lowlands of Northern Italy. The relevance of in-stream substrate microhabitat for the benthic community was assessed, together with the impact of mitigation measures. We proposed a straightforward approach to quantify similarity of microhabitat mosaic between sites testing its statistical significance based on Bayesian statistics. We hypothesised that changes of microhabitat mosaic would reflect the level of implementation of mitigation measures and benthic invertebrates would respond accordingly. Alpha, beta diversity and benthic metrics used to classify ecological status/potential were considered and their variation tested against different levels of measure implementation. Lastly, ecological potential classification was paralleled to both the level of measure implementation and habitat attributes. The microhabitats found at sites where measures were fully implemented differed from those observed elsewhere and they clearly mirrored morphological alteration and mitigation measures. Moreover, alongside morphological alteration, microhabitat diversity and mosaic were the main factors for benthic community structure. While benthic beta diversity strictly reflected microhabitat diversity, alpha diversity and ecological status metrics copied the mosaic gradient. Microhabitat attributes and most benthic metrics showed significant changes following measure implementation and they were accompanied by a gradual shift in ecological potential classes. We demonstrated the importance of in-stream substrate microhabitats as a bridge between mitigation measures and the benthic community. Particularly when ecological classification is under focus, microhabitat mosaic should be evaluated for achieving a better understanding of biological responses. The huge amount of data available worldwide could support a straightforward use of river mosaic information for river management.

Lipomatosis Incidence and Characteristics in an Italian Cohort of Mitochondrial Patients.

Lipomas have often been associated with mtDNA mutations and were mainly observed in patients with mutation in mitochondrial tRNAlysine which is also the most frequent mutation associated with MERRF. Up to date, no systematic studies have been developed in order to assess the incidence of lipomas in large cohorts of mitochondrial patients.The aim of this study is to analyze the incidence and characteristics of lipomas among an Italian cohort of patients with mitochondrial diseases. A retrospective, database-based study (Nation-wide Italian Collaborative Network of Mitochondrial Diseases) of patients with lipomas was performed. A total of 22 (1.7%) patients with lipomas have been identified among the 1,300 mitochondrial patients, enrolled in the Italian database. In about 18% multiple systemic lipomatosis (MSL) was the only clinical manifestation; 54% of patients showed a classical MERRF syndrome. Myopathy, alone or in association with other symptoms, was found in 27% of patients. Lactate was elevated in all the 12 patients in which was measured. Muscle biopsy was available in 18/22 patients: in all of them mitochondrial abnormalities were present. Eighty six percent had mutations in mtDNA coding for tRNA lysine. In most of patients, lipomas were localized along the cervical-cranial-thoracic region. In 68% of the patients were distributed symmetrically. Only two patients had lipomas in a single anatomical site (1 in right arm and 1 in gluteus maximum). MSL is often overlooked by clinicians in patients with mitochondrial diseases where the clinical picture could be dominated by a severe multi-systemic involvement. Our data confirmed that MSL is a rare sign of mitochondrial disease with a strong association between multiple lipomas and lysine tRNA mutations. MSL could be considered, even if rare, a red flag for mitochondrial disorders, even in patients with an apparently isolated MSL.

Anti-Oxidant Drugs: Novelties and Clinical Implications in Cerebellar Ataxias.

BACKGROUND: Hereditary cerebellar ataxias are a group of disorders characterized by heterogeneous clinical manifestations, progressive clinical course, and diverse genetic causes. No disease modifying treatments are yet available for many of these disorders. Oxidative stress has been recurrently identified in different progressive cerebellar diseases, and it represents a widely investigated target for treatment. OBJECTIVE: To review the main aspects and new perspectives of antioxidant therapy in cerebellar ataxias ranging from bench to bedside. METHOD: This article is a summary of the state-of-the-art on the use of antioxidant molecules in cerebellar ataxia treatments. It also briefly summarizes aspects of oxidative stress production and general characteristics of antioxidant compounds. RESULTS: Antioxidants represent a vast category of compounds; old drugs have been extensively studied and modified in order to achieve better biological effects. Despite the vast body of literature present on the use of antioxidants in cerebellar ataxias, for the majority of these disorders conclusive results on the efficacy are still missing. CONCLUSION: Antioxidant therapy in cerebellar ataxias is a promising field of investigations. To achieve the success in identifying the correct treatment more work needs to be done. In particular, a combined effort is needed by basic scientists in developing more efficient molecules, and by clinical researchers together with patients communities, to run clinical trials in order to identify conclusive treatments strategies.

CoQ10 supplementation rescues nephrotic syndrome through normalization of H2S oxidation pathway.

Nephrotic syndrome (NS), a frequent chronic kidney disease in children and young adults, is the most common phenotype associated with primary coenzyme Q10 (CoQ10) deficiency and is very responsive to CoQ10 supplementation, although the pathomechanism is not clear. Here, using a mouse model of CoQ deficiency-associated NS, we show that long-term oral CoQ10 supplementation prevents kidney failure by rescuing defects of sulfides oxidation and ameliorating oxidative stress, despite only incomplete normalization of kidney CoQ levels and lack of rescue of CoQ-dependent respiratory enzymes activities. Liver and kidney lipidomics, and urine metabolomics analyses, did not show CoQ metabolites. To further demonstrate that sulfides metabolism defects cause oxidative stress in CoQ deficiency, we show that silencing of sulfide quinone oxido-reductase (SQOR) in wild-type HeLa cells leads to similar increases of reactive oxygen species (ROS) observed in HeLa cells depleted of the CoQ biosynthesis regulatory protein COQ8A. While CoQ10 supplementation of COQ8A depleted cells decreases ROS and increases SQOR protein levels, knock-down of SQOR prevents CoQ10 antioxidant effects. We conclude that kidney failure in CoQ deficiency-associated NS is caused by oxidative stress mediated by impaired sulfides oxidation and propose that CoQ supplementation does not significantly increase the kidney pool of CoQ bound to the respiratory supercomplexes, but rather enhances the free pool of CoQ, which stabilizes SQOR protein levels rescuing oxidative stress.

Three-Dimensional Analysis of Mitochondrial Crista Ultrastructure in a Patient with Leigh Syndrome by In Situ Cryoelectron Tomography.

Mitochondrial diseases produce profound neurological dysfunction via mutations affecting mitochondrial energy production, including the relatively common Leigh syndrome (LS). We recently described an LS case caused by a pathogenic mutation in USMG5, encoding a small supernumerary subunit of mitochondrial ATP synthase. This protein is integral for ATP synthase dimerization, and patient fibroblasts revealed an almost total loss of ATP synthase dimers. Here, we utilize in situ cryoelectron tomography (cryo-ET) in a clinical case-control study of mitochondrial disease to directly study mitochondria within cultured fibroblasts from a patient with LS and a healthy human control subject. Through tomographic analysis of patient and control mitochondria, we find that loss of ATP synthase dimerization due to the pathogenic mutation causes profound disturbances of mitochondrial crista ultrastructure. Overall, this work supports the crucial role of ATP synthase in regulating crista architecture in the context of human disease.

USMG5 Ashkenazi Jewish founder mutation impairs mitochondrial complex V dimerization and ATP synthesis.

Leigh syndrome is a frequent, heterogeneous pediatric presentation of mitochondrial oxidative phosphorylation (OXPHOS) disease, manifesting with psychomotor retardation and necrotizing lesions in brain deep gray matter. OXPHOS occurs at the inner mitochondrial membrane through the integrated activity of five protein complexes, of which complex V (CV) functions in a dimeric form to directly generate adenosine triphosphate (ATP). Mutations in several different structural CV subunits cause Leigh syndrome; however, dimerization defects have not been associated with human disease. We report four Leigh syndrome subjects from three unrelated Ashkenazi Jewish families harboring a homozygous splice-site mutation (c.87 + 1G>C) in a novel CV subunit disease gene, USMG5. The Ashkenazi population allele frequency is 0.57%. This mutation produces two USMG5 transcripts, wild-type and lacking exon 3. Fibroblasts from two Leigh syndrome probands had reduced wild-type USMG5 mRNA expression and undetectable protein. The mutation did not alter monomeric CV expression, but reduced both CV dimer expression and ATP synthesis rate. Rescue with wild-type USMG5 cDNA in proband fibroblasts restored USMG5 protein, increased CV dimerization and enhanced ATP production rate. These data demonstrate that a recurrent USMG5 splice-site founder mutation in the Ashkenazi Jewish population causes autosomal recessive Leigh syndrome by reduction of CV dimerization and ATP synthesis.

Retrospective natural history of thymidine kinase 2 deficiency.

BACKGROUND: Thymine kinase 2 (TK2) is a mitochondrial matrix protein encoded in nuclear DNA and phosphorylates the pyrimidine nucleosides: thymidine and deoxycytidine. Autosomal recessive TK2 mutations cause a spectrum of disease from infantile onset to adult onset manifesting primarily as myopathy. OBJECTIVE: To perform a retrospective natural history study of a large cohort of patients with TK2 deficiency. METHODS: The study was conducted by 42 investigators across 31 academic medical centres. RESULTS: We identified 92 patients with genetically confirmed diagnoses of TK2 deficiency: 67 from literature review and 25 unreported cases. Based on clinical and molecular genetics findings, we recognised three phenotypes with divergent survival: (1) infantile-onset myopathy (42.4%) with severe mitochondrial DNA (mtDNA) depletion, frequent neurological involvement and rapid progression to early mortality (median post-onset survival (POS) 1.00, CI 0.58 to 2.33 years); (2) childhood-onset myopathy (40.2%) with mtDNA depletion, moderate-to-severe progression of generalised weakness and median POS at least 13 years; and (3) late-onset myopathy (17.4%) with mild limb weakness at onset and slow progression to respiratory insufficiency with median POS of 23 years. Ophthalmoparesis and facial weakness are frequent in adults. Muscle biopsies show multiple mtDNA deletions often with mtDNA depletion. CONCLUSIONS: In TK2 deficiency, age at onset, rate of weakness progression and POS are important variables that define three clinical subtypes. Nervous system involvement often complicates the clinical course of the infantile-onset form while extraocular muscle and facial involvement are characteristic of the late-onset form. Our observations provide essential information for planning future clinical trials in this disorder.

Inhibition of NADPH oxidase 2 (NOX2) prevents sepsis-induced cardiomyopathy by improving calcium handling and mitochondrial function.

Cardiomyopathy frequently complicates sepsis and is associated with increased mortality. Increased cardiac oxidative stress and mitochondrial dysfunction have been observed during sepsis, but the mechanisms responsible for these abnormalities have not been determined. We hypothesized that NADPH oxidase 2 (NOX2) activation could be responsible for sepsis-induced oxidative stress and cardiomyopathy. Treatment of isolated adult mouse cardiomyocytes with low concentrations of the endotoxin lipopolysaccharide (LPS) increased total cellular reactive oxygen species (ROS) and mitochondrial superoxide. Elevated mitochondrial superoxide was accompanied by depolarization of the mitochondrial inner membrane potential, an indication of mitochondrial dysfunction, and mitochondrial calcium overload. NOX2 inhibition decreased LPS-induced superoxide and prevented mitochondrial dysfunction. Further, cardiomyocytes from mice with genetic ablation of NOX2 did not have LPS-induced superoxide or mitochondrial dysfunction. LPS decreased contractility and calcium transient amplitude in isolated cardiomyocytes, and these abnormalities were prevented by inhibition of NOX2. LPS decreased systolic function in mice, measured by echocardiography. NOX2 inhibition was cardioprotective in 2 mouse models of sepsis, preserving systolic function after LPS injection or cecal ligation and puncture (CLP). These data show that inhibition of NOX2 decreases oxidative stress, preserves intracellular calcium handling and mitochondrial function, and alleviates sepsis-induced systolic dysfunction in vivo. Thus, NOX2 is a potential target for pharmacotherapy of sepsis-induced cardiomyopathy.

Update on diagnostics of metabolic myopathies.

PURPOSE OF REVIEW: This review aims to highlight the most relevant clinical and laboratory findings, regarding acute and progressive metabolic myopathies, and to develop an algorithm addressing clinicians to clinical practice. RECENT FINDINGS: Although diagnosis of metabolic myopathies remains still challenging, the recent identification of new disorders has increased the number of patients requiring specific investigations. Nowadays, a more detailed characterization of the clinical spectrum of metabolic myopathies improved awareness as well as a deeper knowledge on their natural history or multisystem involvement. Diagnostic procedures, as first-line screening tests are necessary for an earlier and more accurate diagnostic work up, not only in infantile cases, but also in adults with suspected metabolic myopathies. New generation diagnostic techniques such as NGS (Next Generation Sequencing) and whole exome/genome sequencing have emerged as innovative tools to extensively evaluate either known genes variants or new candidate genes as possible causes of metabolic myopathies. SUMMARY: Diagnosis of metabolic myopathies is still challenging for clinicians because of rarity and clinical heterogeneity which is often overlapping with other neuromuscular disorders. Detailed algorithms supported by advanced laboratory investigations may be helpful to timely reach a diagnosis, so allowing an earlier therapeutic decision.

Risk of Myopathy in Patients in Therapy with Statins: Identification of Biological Markers in a Pilot Study.

Statin therapy may induce skeletal muscle damage ranging from myalgia to severe rhabdomyolysis. Our previous preclinical studies showed that statin treatment in rats involves the reduction of skeletal muscle ClC-1 chloride channel expression and related chloride conductance (gCl). An increase of the activity of protein kinase C theta (PKC theta) isoform, able to inactivate ClC-1, may contribute to destabilize sarcolemma excitability. These effects can be detrimental for muscle function leading to drug-induced myopathy. Our goal is to study the causes of statin-induced muscle side effects in patients at the aim to identify biological markers useful to prevent and counteract statin-induced muscle damage. We examined 10 patients, who experienced myalgia and hyper-CK-emia after starting statin therapy compared to 9 non-myopathic subjects not using lipid-lowering drugs. Western Blot (WB) analysis showed a 40% reduction of ClC-1 protein and increased expression of phosphorylated PKC in muscle biopsies of statin-treated patients with respect to untreated subjects, independently from their age and statin type. Real-time PCR analysis showed that despite reduction of the protein, the ClC-1 mRNA was not significantly changed, suggesting post-transcriptional modification. The mRNA expression of a series of genes was also evaluated. MuRF-1 was increased in accord with muscle atrophy, MEF-2, calcineurin (CN) and GLUT-4 transporter were reduced, suggesting altered transcription, alteration of glucose homeostasis and energy deficit. Accordingly, the phosphorylated form of AMPK, measured by WB, was increased, suggesting cytoprotective process activation. In parallel, mRNA expression of Notch-1, involved in muscle cell proliferation, was highly expressed in statin-treated patients, indicating active regeneration. Also, PGC-1-alpha and isocitrate-dehydrogenase increased expression together with increased activity of mitochondrial citrate-synthase, measured by spectrophotometric assay, suggests mitochondrial biogenesis. Thus, the reduction of ClC-1 protein and consequent sarcolemma hyperexcitability together with energy deficiency appear to be among the most important alterations to be associated with statin-related risk of myopathy in humans. Thus, it may be important to avoid statin treatment in pathologies characterized by energy deficit and chloride channel malfunction. This study validates the measure of ClC-1 expression as a reliable clinical test for assessing statin-dependent risk of myopathy.

Cytochrome c Oxidase Activity Is a Metabolic Checkpoint that Regulates Cell Fate Decisions During T Cell Activation and Differentiation.

T cells undergo metabolic reprogramming with major changes in cellular energy metabolism during activation. In patients with mitochondrial disease, clinical data were marked by frequent infections and immunodeficiency, prompting us to explore the consequences of oxidative phosphorylation dysfunction in T cells. Since cytochrome c oxidase (COX) is a critical regulator of OXPHOS, we created a mouse model with isolated dysfunction in T cells by targeting a gene, COX10, that produces mitochondrial disease in humans. COX dysfunction resulted in increased apoptosis following activation in vitro and immunodeficiency in vivo. Select T cell effector subsets were particularly affected; this could be traced to their bioenergetic requirements. In summary, the findings presented herein emphasize the role of COX particularly in T cells as a metabolic checkpoint for cell fate decisions following T cell activation, with heterogeneous effects in T cell subsets. In addition, our studies highlight the utility of translational models that recapitulate human mitochondrial disease for understanding immunometabolism.

Correction: Inhibition of NAPDH Oxidase 2 (NOX2) Prevents Oxidative Stress and Mitochondrial Abnormalities Caused by Saturated Fat in Cardiomyocytes.

[This corrects the article DOI: 10.1371/journal.pone.0145750.].