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Muscle loss is a major problem for many in lifetime. Muscle and bone degeneration has also been observed in individuals exposed to microgravity and in unloading conditions. C2C12 myoblst cells are able to form myotubes, and myofibers and these cells have been employed for muscle regeneration purposes and in myogenic regeneration and transplantation studies. We exposed C2C12 cells in an random position machine to simulate microgravity and study the energy and the biochemical challenges associated with this treatment. Simulated microgravity exposed C2C12 cells maintain positive proliferation indices and delay the differentiation process for several days. On the other hand this treatment significantly alters many of the biochemical and the metabolic characteristics of the cell cultures including calcium homeostasis. Recent data have shown that these perturbations are due to the inhibition of the ryanodine receptors on the membranes of intracellular calcium stores. We were able to reverse this perturbations treating cells with thapsigargin which prevents the segregation of intracellular calcium ions in the mitochondria and in the sarco/endoplasmic reticula. Calcium homeostasis appear a key target of microgravity exposure. In conclusion, in this study we reported some of the effects induced by the exposure of C2C12 cell cultures to simulated microgravity. The promising information obtained is of fundamental importance in the hope to employ this protocol in the field of regenerative medicine.

Fanconi anemia (FA) is a complex tumor-prone disease defined by an entangled genotype and phenotype. Despite enormous efforts in the last 20 years, a comprehensive and integrated view of the disease is still missing. The aim of this pilot study was to establish whether a global microRNA (miRNA) analysis approach could be helpful in defining aspects in FA phenotype, which might deserve future attention with the perspective to develop miRNA-based therapies.miRNA array were employed to characterize the global miRNA (miRNoma) profile of FA RNA samples with respect to normal samples.We report and compare miRNA profile from two FA established cell lines and three FA patients. This analysis reveals that 36 and 64 miRNAs, respectively, are found differentially expressed (2-fold variation and P 0.05) in the samples from FA cell lines and FA patients. Overlap of these data results in 24 miRNAs as shared in the two sample populations. Available bioinformatics methods were used to predict target genes for the differentially expressed miRNAs and to perform pathway enrichment analysis.Seven pathway results associated with the FA phenotype. It is interesting to note that some of these pathways were previously unrelated to FA phenotype. It might be important to focus on these pathways not previously emerged as dysfunctional in FA to better define the pathophysiological context of this disease. This is the first report of a global miRNA analysis in FA.

Atypical hemolytic uremic syndrome (aHUS) is an ultra-rare disease characterized by microangiopathic hemolysis, thrombocytopenia, and renal impairment and is associated with dysregulation of the alternative complement pathway on the microvascular endothelium. Outcomes have improved greatly with pharmacologic complement C5 blockade. Abnormalities in complement genes (CFH, CD46, CFI, CFB, C3, and THBD), CFH–CFHR genomic rearrangements, and anti-FH antibodies have been reported in 40–60% of cases. The penetrance of aHUS is incomplete in carriers of complement gene abnormalities; and multiple hits, including the CFH–H3 and CD46GGAAC risk haplotypes and the CFHR1*B risk allele, as well as environmental factors, contribute to disease development. Here, we investigated the determinants of penetrance of aHUS associated with CD46 genetic abnormalities. We studied 485 aHUS patients and found CD46 rare variants (RVs) in about 10%. The c.286+2T>G RV was the most prevalent (13/485) and was associated with G RV who experienced a severe form of aHUS and developed end-stage renal failure. The father and paternal uncle with the same variant in homozygosity and six heterozygous relatives are unaffected. Flow cytometry analysis showed about 50% reduction of CD46 expression on blood mononuclear cells from the heterozygous proband and over 90% reduction in cells from the proband's unaffected homozygous father and aunt. Further genetic studies did not reveal RVs in known aHUS-associated genes or common genetic modifiers that segregated with the disease. Importantly, a specific ex vivo test showed excessive complement deposition on endothelial cells exposed to sera from the proband, and also from his mother and maternal uncle, who do not carry the c.286+2T>G RV, indicating that they share a circulating defect that results in complement dysregulation on the endothelium. These results highlight the complexity of the genetics of aHUS and indicate that CD46 deficiency may not be enough to induce aHUS. We hypothesize that the proband inherited from his mother a genetic abnormality in a complement circulating factor that has not been identified yet, which synergized with the CD46 RV in predisposing him to the aHUS phenotype.

A 6-month-old boy presented with acute renal failure, thrombocytopenia, and severe non-immune hemolytic anemia. Infection by Shiga-like toxin-producing Escherichia coli and other causes of microangiopathic hemolysis were ruled out, leading to a diagnosis of atypical hemolytic uremic syndrome (aHUS). Neither pathogenic variants in HUS-associated genes nor anti-factor H antibodies were identified. Copy number variation analysis uncovered 4 copies of complement factor H related genes, CFHR1-CFHR4, conceivably leading to higher than normal levels of the corresponding proteins. However, this abnormality was also found in the healthy relatives, neither explaining the disease nor the excessive complement deposition on endothelial cells detected by an ex-vivo test. Whole-exome sequencing revealed a pathogenic homozygous variant in GRHPR encoding the glyoxylate and hydroxypyruvate reductase. Recessive GRHPR mutations cause primary hyperoxaluria type 2 (PH2). The presence of renal calculi in the patient and elevated oxalate levels in the urine were consistent with the genetic diagnosis of PH2. We hypothesize that, in this patient, hyperoxaluria caused by the GRHPR genetic defect triggered endothelial perturbation and complement activation, which was amplified by impaired factor H regulatory activity due to the increased ­CFHR1-CFHR4 copy numbers, resulting in aHUS.

Rationale & Objective Although primary atypical hemolytic uremic syndrome (aHUS) is associated with abnormalities in complement genes and antibodies to complement factor H, the role of complement in secondary aHUS remains debatable. We evaluated the usefulness of an ex vivo test to: (1) detect complement activation within the endothelium in primary and secondary aHUS, (2) differentiate active disease from remission, (3) monitor the effectiveness of eculizumab therapy, and (4) identify relapses during eculizumab dosage tapering and after discontinuation of treatment. Study Design Case series. Setting & Participants 121 patients with primary aHUS and 28 with secondary aHUS. Serum samples were collected during acute episodes, following remission, and during eculizumab treatment and were assessed using a serum-induced ex vivo C5b-9 endothelial deposition test. Results Serum-induced C5b-9 deposition on cultured microvascular endothelium was quantified by calculating the endothelial area covered by C5b-9 staining; values were expressed as percentage of C5b-9 deposits induced by a serum pool from healthy controls. Testing with adenosine diphosphate–activated endothelium demonstrated elevated C5b-9 deposits for all untreated patients with aHUS independent of disease activity, while testing with unstimulated endothelium demonstrated deposits only in active disease. Similar findings were observed in secondary aHUS. Serum-induced C5b-9 deposits on activated and unstimulated endothelium normalized during eculizumab treatment. 96% (22/23) of patients receiving eculizumab at extended 3- or 4-week dosing intervals demonstrated normal C5b-9 deposits on activated endothelium, despite most patients having CH50Eq (serum complement activity) > 20 UEq/mL, indicating that adequate complement control was achieved even with incomplete blockade of circulating C5. During eculizumab dosage tapering or after treatment discontinuation, all patients experiencing relapses versus only 6% (1/17) of those in stable remission had elevated C5b-9 deposits on unstimulated endothelium. Limitations The C5b-9 endothelial deposition test can be performed in only specialized laboratories. Findings on eculizumab dosage tapering need to be confirmed with longitudinal monitoring of C5b-9 deposition. Conclusions The C5b-9 endothelial deposition assay may represent an advance in our ability to monitor aHUS activity and individualize therapy.

Oxidative phosphorylation (OXPHOS) is not only the main source of ATP for the cell, but also a major source of reactive oxygen species (ROS), which lead to oxidative stress. At present, mitochondria are considered the organelles responsible for the OXPHOS, but in the last years we have demonstrated that it can also occur outside the mitochondrion. Myelin sheath is able to conduct an aerobic metabolism, producing ATP that we have hypothesized is transferred to the axon, to support its energetic demand. In this work, spectrophotometric, cytofluorimetric, and luminometric analyses were employed to investigate the oxidative stress production in isolated myelin, as far as its respiratory activity is concerned. We have evaluated the levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), markers of lipid peroxidation, as well as of hydrogen peroxide (H2O2), marker of ROS production. To assess the presence of endogenous antioxidant systems, superoxide dismutase, catalase, and glutathione peroxidase activities were assayed. The effect of certain uncoupling or antioxidant molecules on oxidative stress in myelin was also investigated. We report that isolated myelin produces high levels of MDA, 4-HNE, and H2O2, likely through the pathway composed by Complex I-III-IV, but it also contains active superoxide dismutase, catalase, and glutathione peroxidase, as antioxidant defense. Uncoupling compounds or Complex I inhibitors increase oxidative stress, while antioxidant compounds limit ROS generation. Data may shed new light on the role of myelin sheath in physiology and pathology. In particular, it can be presumed that the axonal degeneration associated with myelin loss in demyelinating diseases is related to oxidative stress caused by impaired OXPHOS.

Fanconi's anemia (FA) patients face severe pathological consequences. Bone marrow failure, the major cause of death in FA, accounting for as much as 80-90% of FA mortality, appears to be significantly linked to excessive apoptosis of hematopoietic cells induced by oxidative stress. However, 20-25% of FA patients develop malignancies of myeloid origin. A survival strategy for bone marrow and hematopoietic cells under selective pressure evidently exists. This study reports that lymphoblastoid cell lines derived from two FA patients displayed significant resistance to oxidative stress induced by treatments with H(2) O(2) and various glutathione (GSH) inhibitors that induce production of reactive oxygen species, GSH depletion and mitochondrial membrane depolarization. Among the various GSH inhibitors employed, FA cells appear particularly resistant to menadione (5 μm) and ethacrynic acid (ETA, 50 μm), two drugs that specifically target mitochondria. Even after pre-treatment with buthionine sulfoximine, a GSH synthesis inhibitor that induces enhanced induction of reactive oxygen species, FA cells maintain significant resistance to these drugs. These data suggest that the resistance to oxidative stress and the altered mitochondrial and metabolic functionality found in the FA mutant cells used in this study may indicate the survival strategy that is adopted in FA cells undergoing transformation. The study of redox and mitochondria regulation in FA may be of assistance in diagnosis of the disease and in the care of patients.

Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have ribosome biogenesis and their protein synthesis altered, which are two high-energy consuming cellular processes. The reported changes in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest an energy production defect in SDS cells. In our work, we have demonstrated that SDS cells display a Complex IV activity impairment, which causes an oxidative phosphorylation metabolism defect, with a consequent decrease in ATP production. These data were confirmed by an increased glycolytic rate, which compensated for the energetic stress. Moreover, the signalling pathways involved in glycolysis activation also appeared more activated; i.e. we reported AMP-activated protein kinase hyper-phosphorylation. Notably, we also observed an increase in a mammalian target of rapamycin phosphorylation and high intracellular calcium concentration levels ([Ca2+]i), which probably represent new biochemical equilibrium modulation in SDS cells. Finally, the SDS cell response to leucine (Leu) was investigated, suggesting its possible use as a therapeutic adjuvant to be tested in clinical trials.

Fanconi Anemia (FA) is a rare and complex inherited blood disorder associated with bone marrow failure and malignancies. Many alterations in FA physiology appear linked to red-ox unbalance including alterations in the morphology and structure of nuclei, intermediate filaments and mitochondria, defective respiration, reduced ATP production and altered ATP/AMP ratio. These defects are consistently associated with impaired oxygen metabolism indeed treatment with antioxidants N-acetylcysteine (NAC) and resveratrol (RV) does rescue FA physiology. Due to the importance of the intracellular calcium signaling and its key function in the control of intracellular functions we were interested to study calcium homeostasis in FA. We found that FANCA cells display a dramatically low intracellular calcium concentration ([Ca(2+)]i) in resting conditions. This condition affects cellular responses to stress. The flux of Ca(2+) mobilized by H2O2 from internal stores is significantly lower in FANCA cells in comparison to controls. The low basal [Ca(2+)]i in FANCA appears to be an actively maintained process controlled by a finely tuned interplay between different intracellular Ca(2+) stores. The defects associated with the altered Ca(2+) homeostasis appear consistently overlapping those related to the unbalanced oxidative metabolism in FA cells underlining a contiguity between oxidative stress and calcium homeostasis.

Growing number of publication has proved an increasing of cellular function of the Fanconi anemia proteins. To chromosome stability and DNA repair new roles have been attributed to FA proteins in oxidative stress response and homeostasis, immune response and cytokines sensibility, gene expression. Our work shows a new role for FA-A protein: the organization of the cellular structure. By 2D-PAGE of FA-A and correct fibroblasts treated and untreated with H2O2 we identify different expression of protein involved in the structural organization of nucleus, intermediate filaments and mitochondria. Immunofluorescence and electronic microscopy analysis clearly show an already altered cellular structure in normal culture condition and this worsted after oxidative stress. FA-A cell appears structurally prone to physiologic stress and this could explain part of the phenotype of FA cells.

Fanconi anemia (FA) is a rare and complex inherited blood disorder of the child. At least 15 genes are associated with the disease. The highest frequency of mutations belongs to groups A, C and G. Genetic instability and cytokine hypersensitivity support the selection of leukemic over non-leukemic stem cells. FA cellular phenotype is characterized by alterations in red-ox state, mitochondrial functionality and energy metabolism as reported in the past however a clear picture of the altered biochemical phenotype in FA is still elusive and the final biochemical defect(s) still unknown. Here we report an analysis of the respiratory fluxes in FANCA primary fibroblasts, lymphocytes and lymphoblasts. FANCA mutants show defective respiration through Complex I, diminished ATP production and metabolic sufferance with an increased AMP/ATP ratio. Respiration in FANCC mutants is normal. Treatment with N-acetyl-cysteine (NAC) restores oxygen consumption to normal level. Defective respiration in FANCA mutants appear correlated with the FA pro-oxidative phenotype which is consistent with the altered morphology of FANCA mitochondria. Electron microscopy measures indeed show profound alterations in mitochondrial ultrastructure and shape.

Background Whether microgravity might influence tumour growth and carcinogenesis is still an open issue. It is not clear also if and how normal and transformed cells are differently solicited by microgravity. The present study was designed to verify this issue. Methods Two normal, LB and HSC93, and two transformed, Jurkat and 1310, lymphoblast cell lines were used as representative for the two conditions. Two lymphoblast lines from Fanconi's anemia patients group A and C (FA-A and FA-C, respectively), along with their isogenic corrected counterparts (FA-A-cor and FA-C-cor) were also used. Cell lines were evaluated for their proliferative ability, vitality and apoptotic susceptibility upon microgravity exposure in comparison with unexposed cells. Different parameters correlated to energy metabolism, glucose consumption, mitochondrial membrane potential (MMP), intracellular ATP content, red-ox balance and ability of the cells to repair the DNA damage product 8-OHdG induced by the treatment of the cells with 20 mM KBrO3 were also evaluated. Results Transformed Jurkat and 1310 cells appear resistant to the microgravitational challenge. On the contrary normal LB and HSC93 cells display increased apoptotic susceptibility, shortage of energy storages and reduced ability to cope with oxidative stress. FA-A and FA-C cells appear resistant to microgravity exposure, analogously to transformed cells. FA corrected cells did shown intermediate sensitivity to microgravity exposure suggesting that genetic correction does not completely reverts cellular phenotype. Conclusions In the light of the reported results microgravity should be regarded as an harmful condition either when considering normal as well as transformed cells. Modeled microgravity and space-based technology are interesting tools in the biomedicine laboratory and offer an original, useful and unique approach in the study of cellular biochemistry and in the regulation of metabolic pathways.

Fanconi anemia (FA) is a rare, complex disorder that manifests in childhood. Children with FA suffer bone marrow failure, leukemias, or solid tumors. FA-associated mutations are found in 15 proteins that are involved in DNA repair. Some of these proteins have extranuclear activities involving redox balance, apoptosis, and energy metabolism; and recent data demonstrate respiratory impairment in FA cells, suggesting that altered mitochondrial function is a factor in this disease.

Isomorphic mutation of SBDS gene is the cause of Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have altered ribosome biogenesis and protein synthesis, two high-energy consuming cellular processes. The reported increment in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest a defect in the energy production in SDS cells. In this study, we analyzed the energetic metabolism in SDS cells and find that the oxygen consumption is impaired when it is induced by pyruvate/malate or succinate. This induces poor ATP production and AMP accumulation with a consequent alteration in the ATP/AMP ratio. Also respiratory chain activity was impaired because of faulty function of the complex IV; this defect is not dependent from impaired protein synthesis despite ribosome biogenesis and transduction defects in SDS. In fact, COX5A and Cox2, two subunits of Complex IV encoded respectively by a nuclear and mitochondrial gene, were expressed at normal levels. Impaired function of complex IV could be due to an increment of cytoplasmic calcium concentration that inhibits complex IV activity. Energetic stress induces changes in cellular metabolism, stimulating or inhibiting a network of molecules involved in regulation of energetic balance, such as AMPK and mTOR. In SDS cells as consequence of energetic stress, AMPK is hyper activated and the glycolytic pathway stimulated. Surprisingly, we found that also the AKT/mTOR pathway is aberrantly hyper activated since both these proteins are hyper-phosphorylated. We can speculate that hyper activation of mTOR is a way through which SDS cells support the energy defect and protein synthesis. All these defects were recovered when the SDS cells were complemented with SDS gene. Finally, leucine is an essential amino acid that induces cell proliferation and protein synthesis, restored OXPHOS and ATP synthesis, reduced the cytoplasmic calcium concentration and the AMPK and AKT/mTOR activity, and improved in vitro erythropoiesis from SDS individuals pointing to leucine as potential tool helpful to sustain deranged energetic metabolism and erythropoiesis in SDS patients. In conclusion, we report for the first time that SDS cells suffer of energetic stress and severe respiratory defect that is related to faulty SBSD protein. These defects are compensated by an enhanced activation of AMPK, glycolysis and mTOR/Akt pathways, which appear to adequately support protein synthesis. A pivotal role in the maintenance of this altered metabolism could be played by altered calcium homeostasis. Noteworthy biochemical defects might be largely corrected by leucine which also favourably affects in vitro erythropoiesis thus pointing to biochemical defects as important determinant for impaired hematopoiesis od SDS. Disclosures Dufour: Pfizer: Consultancy.