Effects of exercise on mitochondrial DNA content in skeletal muscle of patients with COPD

Background Exhausting exercise reduces the mitochondrial DNA (mtDNA) content in the skeletal muscle of healthy subjects due to oxidative damage. Since patients with chronic obstructive pulmonary disease (COPD) suffer enhanced oxidative stress during exercise, it was hypothesised that the mtDNA content will be further reduced. Objective To investigate the effects of exercise above and below the lactate threshold (LT) on the mtDNA content of skeletal muscle of patients with COPD. Methods Eleven patients with COPD (676 8 years; forced expiratory volume in 1s (FEV1)456 8%ref) and 10 healthy controls (666 4 years; FEV1 906 7% ref) cycled 45 min above LT (65% peak oxygen uptake (V9O2 peak)and another 7 patients (656 6 years; FEV1 506 4%ref)and 7 controls (566 9 years;FEV1 926 6%ref) cycled 45 min below their LT (50% V9O2 peak). Biopsies from the vastus lateralis muscle were obtained before exercise, immediately after and 1 h, 1 day and 1 week later to determine by PCR the mtDNA/nuclear DNA (nDNA) ratio (a marker of mtDNA content) and the expression of the peroxisome proliferator-activated receptor- g coactivator-1 a (PGC-1a)mRNA and the amount of reactive oxygen species produced during exercise was estimated from total V9O2. Results Skeletal muscle mtDNA/nDNA fell significantly after exercise above the LT both in controls and in patients with COPD, but the changes were greater in those with COPD. These changes correlated with production of reactive oxygen species, increases in manganese superoxide dismutase and PGC-1 a mRNA and returned to baseline values 1 week later. This pattern of response wa was also observed, albeit minimised, in patients exercising below the LT. Conclusions In patients with COPD, exercise enhances the decrease in mtDNA content of skeletal muscle and the expression of PGC-1 a mRNA seen in healthy subjects probably due to oxidative stress.

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options.

TCF3-HLF-positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

Mutation analysis in cystic fibrosis

The article by Lemna et al. (Feb. 1 issue)1 furthers the evaluation of the ΔF508 mutation, which is associated with some cases of cystic fibrosis. Although its real effect may be to help in documenting the substantial clinical variation that can occur among persons who possess the same small genetic deletion, the finding has encouraged calls for general screening...

Bevacizumab, Pemetrexed, and Cisplatin, or Bevacizumab and Erlotinib for Patients With Advanced Non-Small-Cell Lung Cancer Stratified by Epidermal Growth Factor Receptor Mutation: Phase II Trial SAKK19/09.

OBJECTIVE: The goal was to demonstrate that tailored therapy, according to tumor histology and epidermal growth factor receptor (EGFR) mutation status, and the introduction of novel drug combinations in the treatment of advanced non-small-cell lung cancer are promising for further investigation. METHODS: We conducted a multicenter phase II trial with mandatory EGFR testing and 2 strata. Patients with EGFR wild type received 4 cycles of bevacizumab, pemetrexed, and cisplatin, followed by maintenance with bevacizumab and pemetrexed until progression. Patients with EGFR mutations received bevacizumab and erlotinib until progression. Patients had computed tomography scans every 6 weeks and repeat biopsy at progression. The primary end point was progression-free survival (PFS) ≥ 35% at 6 months in stratum EGFR wild type; 77 patients were required to reach a power of 90% with an alpha of 5%. Secondary end points were median PFS, overall survival, best overall response rate (ORR), and tolerability. Further biomarkers and biopsy at progression were also evaluated. RESULTS: A total of 77 evaluable patients with EGFR wild type received an average of 9 cycles (range, 1-25). PFS at 6 months was 45.5%, median PFS was 6.9 months, overall survival was 12.1 months, and ORR was 62%. Kirsten rat sarcoma oncogene mutations and circulating vascular endothelial growth factor negatively correlated with survival, but thymidylate synthase expression did not. A total of 20 patients with EGFR mutations received an average of 16 cycles. PFS at 6 months was 70%, median PFS was 14 months, and ORR was 70%. Biopsy at progression was safe and successful in 71% of the cases. CONCLUSIONS: Both combination therapies were promising for further studies. Biopsy at progression was feasible and will be part of future SAKK studies to investigate molecular mechanisms of resistance.

Effect of aerobic exercise on skeletal muscle mitochondrial content and function in older adults

Regular aerobic exercise training, which is touted as a way to ameliorate metabolic diseases, increases aerobic capacity. Aerobic capacity usually declines with advanced age. The decline in aerobic capacity is typically associated by a decrease in the quality of skeletal muscle. At the molecular level, this decreased quality comes in part from perturbations in skeletal muscle mitochondria. Of particular is a decrease in the total amount of mitochondria that occupy the skeletal muscle volume. What is not well established is if this decrease in mitochondrial content is due to inactive lifestyle or the process of aging. Herein, the work of the thesis shows a clear connection between mitochondrial content and aerobic capacity. This indicates that active individuals with higher VChmax levels also contain higher volumes of mitochondria inside their muscle as opposed to sedentary counterparts who have lower levels of mitochondrial content. Upon taking these previously sedentary individuals and entering them into an aerobic exercise intervention, they are able to recover their mitochondrial content as well as function to similar levels of lifelong athletes of the same age. Furthermore, the results of this thesis show that mitochondrial content and function also correlate with exercise efficiency. If one is more efficient, he/she is able to expend less energy for a similar power output. Furthermore, individuals who increase in efficiency also increase in the ability to oxidize and utilize fat during pro-longed exercise. This increased reliance on fat after the intervention is associated with an increased amount of mitochondria, particularly in the intermyofibrillar region of skeletal muscle. Therefore, elderly adults who were once sedentary were able to recover mitochondrial content and function and are able to reap other health benefits from regular aerobic exercise training. Aging per se does not seem to be the culprit that will lead to metabolic diseases but rather it seems to be a lack of physical activity. -- Un entraînement sportif d'endurance, connu pour réduire le risque de développer des maladies métaboliques, augmente notre capacité aérobie. La capacité aérobie diminue généralement avec l'âge. Ce déclin est typiquement associé d'une diminution de la qualité du muscle squelettique. Au niveau moléculaire, cette diminution est due à des perturbations dans les mitochondries du muscle squelettique,, ce qui conduit à une diminution de la quantité totale des mitochondries présentes dans le muscle squelettique. Il n'a pas encore été établi si cette diminution de la teneur mitochondriale est due à un mode de vie sédentaire ou au processus du vieillissement. Ce travail de thèse montre un lien clair entre le contenu mitochondrial et la capacité aérobie. Il indique que des personnes âgées actives, avec des niveaux de V02max plus élevés, possèdent également un volume plus élevé de mitochondries dans leurs muscles en comparaison à leurs homologues sédentaires. En prenant des individus sédentaires et leur faisant pratiquer une activité physique aérobie, il est possible d'accroître leur contenu de même que leur fonction mitochondriale à des niveaux similaires à ceux d'athlètes du même âge ayant pratiqué une activité physique tout au long de leur vie. De plus, les résultats de ce travail démontrent que le contenu et la fonction mitochondriale sont en corrélation avec l'efficiscience lors d'exercice physique. En agumentant l'effiscience, les personnes sont alors capables de dépenser moins d'énergie pour une puissance d'exercice similaire. Donc, un volume mitochondrial accru dans le muscle squelettique, associé à une fonction mitochondriale améliorée, est associté à une augmentation de l'effiscience. En outre, les personnes qui augmentent leur effiscience, augmentent aussi leur capacité à oxyder les graisses durant l'exercice prolongé. Une augmentation du recours au graisses après l'intervention est associée à une quantité accrue de mitochondries, en particulier dans la région inter-myofibrillaire du muscle squelettique. Par conséquent, les personnes âgées autrefois sédentaires sont en mesure de récupérer leur contenu et leur fonction mitochondriale ainsi que d'autres avantages pour la santé grâce à un entraînement aérobie régulier. Le vieillissement en soi ne semble donc pas être le coupable conduisant aux maladies métaboliques qui semblent plutôt être lié à un manque d'activité physique.

Phylogenetic relationships of Apodemus Kaup, 1829 (Rodentia: Muridae) species in the Eastern Mediterranean inferred from mitochondrial DNA, with emphasis on iranian species

Wood mice of the genus Apodemus are widely distributed in Eurasia, with the Eastern Mediterranean being considered as a hotspot. Indeed, numerous species have been documented in Iran, including A. witherbyi, A. hyrcanicus, A. uralensis, A. avicennicus, A. hermonensis, and A. arianus. In this study, 129 specimens were collected from different Iranian localities and two specimens from Afghanistan. The animals were identified taxonomically and their phylogenetic relationships were investigated using cytochrome b mitochondrial DNA sequences. Five species of the genus Apodemus were identified in Iran, including A. hyrcanicus, A. witherbyi, A. cf. ponticus, A. uralensis, and A. mystacinus, beside, A. pallipes from Afghanistan. This study found no evidence of A. flavicollis or A. sylvaticus in Iran, despite their occurrence in Turkey, shedding doubt on the status of A. flavicollis in Iran, Asia Minor, and the Levant. Phylogenetic analyses imply that A. witherbyi has priority over A. avicennicus, A. hermonensis, and A. iconicus. Estimation of the divergence time for these taxa suggests a separation at around 7.2 Ma for the subgenera Karstomys (including A. mystacinus and A. epimelas) and Sylvaemus (including A. flavicollis, A. sylvaticus, A. uralensis, A. pallipes, A. hyrcanicus, A. witherbyi, and A. cf. ponticus). Within the subgenus Karstomys, the divergence times for A. mystacinus and A. epimelas were between 3.0 and 6.1 Ma, and divergence times within the subgenus Sylvaemus were between 5.2 and 6.9 Ma for A. witherbyi and other species in this subgenus. It is postulated that vicariance events including the uplifting of the Zagros Mountains and Anatolian Plateau in the middle Miocene and climate oscillations during the Messinian Salinity Crisis besides formation of the Hyrcanian tertiary forests during the Neogene probably played substantial roles in the radiation and distribution of the genus Apodemus in the Eastern Mediterranean.

Analysis of BRAF and NRAS Mutation Status in Advanced Melanoma Patients Treated with Anti-CTLA-4 Antibodies: Association with Overall Survival?

Ipilimumab and tremelimumab are human monoclonal antibodies (Abs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4). Ipilimumab was the first agent to show a statistically significant benefit in overall survival in advanced melanoma patients. Currently, there is no proven association between the BRAFV600 mutation and the disease control rate in response to ipilimumab. This analysis was carried out to assess if BRAFV600 and NRAS mutation status affects the clinical outcome of anti-CTLA-4-treated melanoma patients. This is a retrospective multi-center analysis of 101 patients, with confirmed BRAF and NRAS mutation status, treated with anti-CTLA-4 antibodies from December 2006 until August 2012. The median overall survival, defined from the treatment start date with the anti-CTLA-4. Abs-treatment to death or till last follow up, of BRAFV600 or NRAS mutant patients (n = 62) was 10.12 months (95% CI 6.78-13.2) compared to 8.26 months (95% CI 6.02-19.9) in BRAFV600/NRASwt subpopulation (n = 39) (p = 0.67). The median OS of NRAS mutated patients (n = 24) was 12.1 months and although was prolonged compared to the median OS of BRAF mutated patients (n = 38, mOS = 8.03 months) or BRAFV600/NRASwt patients (n = 39, mOS = 8.26 months) the difference didn't reach statistical significance (p = 0.56). 69 patients were able to complete 4 cycles of anti-CTLA-4 treatment. Of the 24 patients treated with selected BRAF- or MEK-inhibitors, 16 patients received anti-CTLA 4 Abs following either a BRAF or MEK inhibitor with only 8 of them being able to finish 4 cycles of treatment. Based on our results, there is no difference in the median OS in patients treated with anti-CTLA-4 Abs implying that the BRAF/NRAS mutation status alone is not sufficient to predict the outcome of patients treated with anti-CTLA-4 Abs.

Mutations in the heat-shock protein A9 (HSPA9) gene cause the EVEN-PLUS syndrome of congenital malformations and skeletal dysplasia.

We and others have reported mutations in LONP1, a gene coding for a mitochondrial chaperone and protease, as the cause of the human CODAS (cerebral, ocular, dental, auricular and skeletal) syndrome (MIM 600373). Here, we delineate a similar but distinct condition that shares the epiphyseal, vertebral and ocular changes of CODAS but also included severe microtia, nasal hypoplasia, and other malformations, and for which we propose the name of EVEN-PLUS syndrome for epiphyseal, vertebral, ear, nose, plus associated findings. In three individuals from two families, no mutation in LONP1 was found; instead, we found biallelic mutations in HSPA9, the gene that codes for mHSP70/mortalin, another highly conserved mitochondrial chaperone protein essential in mitochondrial protein import, folding, and degradation. The functional relationship between LONP1 and HSPA9 in mitochondrial protein chaperoning and the overlapping phenotypes of CODAS and EVEN-PLUS delineate a family of "mitochondrial chaperonopathies" and point to an unexplored role of mitochondrial chaperones in human embryonic morphogenesis.

Skeletal Muscle Mitochondrial Function and Fatigability in Older Adults.

BACKGROUND: Fatigability increases while the capacity for mitochondrial energy production tends to decrease significantly with age. Thus, diminished mitochondrial function may contribute to higher levels of fatigability in older adults. METHODS: The relationship between fatigability and skeletal muscle mitochondrial function was examined in 30 participants aged 78.5 ± 5.0 years (47% female, 93% white), with a body mass index of 25.9 ± 2.7 kg/m(2) and usual gait-speed of 1.2 ± 0.2 m/s. Fatigability was defined using rating of perceived exertion (6-20 point Borg scale) after a 5-minute treadmill walk at 0.72 m/s. Phosphocreatine recovery in the quadriceps was measured using (31)P magnetic resonance spectroscopy and images of the quadriceps were captured to calculate quadriceps volume. ATPmax (mM ATP/s) and oxidative capacity of the quadriceps (ATPmax·Quadriceps volume) were calculated. Peak aerobic capacity (VO2peak) was measured using a modified Balke protocol. RESULTS: ATPmax·Quadriceps volume was associated with VO2peak and was 162.61mM ATP·mL/s lower (p = .03) in those with high (rating of perceived exertion ≥10) versus low (rating of perceived exertion ≤9) fatigability. Participants with high fatigability required a significantly higher proportion of VO2peak to walk at 0.72 m/s compared with those with low fatigability (58.7 ± 19.4% vs 44.9 ± 13.2%, p < .05). After adjustment for age and sex, higher ATPmax was associated with lower odds of having high fatigability (odds ratio: 0.34, 95% CI: 0.11-1.01, p = .05). CONCLUSIONS: Lower capacity for oxidative phosphorylation in the quadriceps, perhaps by contributing to lower VO2peak, is associated with higher fatigability in older adults.

A genotypic mutation system measuring mutations in restriction recognition sequences.

The RFLP/PCR approach (restriction fragment length polymorphism/polymerase chain reaction) to genotypic mutation analysis described here measures mutations in restriction recognition sequences. Wild-type DNA is restricted before the resistant, mutated sequences are amplified by PCR and cloned. We tested the capacity of this experimental design to isolate a few copies of a mutated sequence of the human c-Ha-ras1 gene from a large excess of wild-type DNA. For this purpose we constructed a 272 bp fragment with 2 mutations in the PvuII recognition sequence 1727-1732 and studied the rescue by RFLP/PCR of a few copies of this 'PvuII mutant standard'. Following amplification with Taq-polymerase and cloning into lambda gt10, plaques containing wild-type sequence, PvuII mutant standard or Taq-polymerase induced bp changes were quantitated by hybridization with specific oligonucleotide probes. Our results indicate that 10 PvuII mutant standard copies can be rescued from 10(8) to 10(9) wild-type sequences. Taq polymerase errors originating from unrestricted, residual wild-type DNA were sequence dependent and consisted mostly of transversions originating at G.C bp. In contrast to a doubly mutated 'standard' the capacity to rescue single bp mutations by RFLP/PCR is limited by Taq-polymerase errors. Therefore, we assessed the capacity of our protocol to isolate a G to T transversion mutation at base pair 1698 of the MspI-site 1695-1698 of the c-Ha-ras1 gene from excess wild-type ras1 DNA. We found that 100 copies of the mutated ras1 fragment could be readily rescued from 10(8) copies of wild-type DNA.

Transition mutation in codon 248 of the p53 tumor suppressor gene induced by reactive oxygen species and a nitric oxide-releasing compound.

Exposing the human bronchial epithelial cell line BEAS-2B to the nitric oxide (NO) donor sodium 1-(N,N-diethylamino)diazen-1-ium-1, 2-diolate (DEA/NO) at an initial concentration of 0.6 mM while generating superoxide ion at the rate of 1 microM/min with the hypoxanthine/xanthine oxidase (HX/XO) system induced C:G-->T:A transition mutations in codon 248 of the p53 gene. This pattern of mutagenicity was not seen by 'fish-restriction fragment length polymorphism/polymerase chain reaction' (fish-RFLP/PCR) on exposure to DEA/NO alone, however, exposure to HX/XO led to various mutations, suggesting that co-generation of NO and superoxide was responsible for inducing the observed point mutation. DEA/NO potentiated the ability of HX/XO to induce lipid peroxidation as well as DNA single- and double-strand breaks under these conditions, while 0.6 mM DEA/NO in the absence of HX/XO had no significant effect on these parameters. The results show that a point mutation seen at high frequency in certain common human tumors can be induced by simultaneous exposure to reactive oxygen species and a NO source.