We hypothesized that muscle fiber bundles produce reactive oxygen intermediates and that reactive oxidant species contribute to muscular fatigue in vitro. Fiber bundles from rat diaphragm were mounted in chambers containing Krebs-Ringer solution. In studies of intracellular oxidant kinetics, bundles were loaded with 2',7'-dichlorofluorescin, a fluorochrome that emits at 520 nm when oxidized; emissions were quantified using a fluorescence microscope. Emissions from unstimulated muscles increased over time (P < 0.001). Accumulation of fluorescence was slowed by addition of catalase (P < 0.001) or superoxide dismutase (P < 0.001) and was accelerated by repetitive muscular contraction (P < 0.05). To determine effects of reactive oxygen intermediates on fatigue, curarized bundles were stimulated to contract isometrically; force was measured. Catalase, superoxide dismutase, and dimethyl sulfoxide were screened for effects on low- and high-frequency fatigue. Antioxidants inhibited low-frequency fatigue [after 5 min of repetitive contractions, force at 30 Hz was 20% greater than control (P < 0.015)] and increased the variability of fatigue at 30 Hz (P < 0.03). Antioxidants did not alter high-frequency (200-Hz) fatigue. We conclude that 1) diaphragm fiber bundles produce reactive oxygen intermediates, including O2-. and H2O2; 2) muscular contraction increases intracellular oxidant levels; and 3) reactive oxygen intermediates promote low-frequency fatigue in this preparation.

Genetic variants responsible for susceptibility to obesity and its comorbidities among Hispanic children have not been identified. The VIVA LA FAMILIA Study was designed to genetically map childhood obesity and associated biological processes in the Hispanic population. A genome-wide association study (GWAS) entailed genotyping 1.1 million single nucleotide polymorphisms (SNPs) using the Illumina Infinium technology in 815 children. Measured genotype analysis was performed between genetic markers and obesity-related traits i.e., anthropometry, body composition, growth, metabolites, hormones, inflammation, diet, energy expenditure, substrate utilization and physical activity. Identified genome-wide significant loci: 1) corroborated genes implicated in other studies (MTNR1B, ZNF259/APOA5, XPA/FOXE1 (TTF-2), DARC, CCR3, ABO); 2) localized novel genes in plausible biological pathways (PCSK2, ARHGAP11A, CHRNA3); and 3) revealed novel genes with unknown function in obesity pathogenesis (MATK, COL4A1). Salient findings include a nonsynonymous SNP (rs1056513) in INADL (p = 1.2E-07) for weight; an intronic variant in MTNR1B associated with fasting glucose (p = 3.7E-08); variants in the APOA5-ZNF259 region associated with triglycerides (p = 2.5-4.8E-08); an intronic variant in PCSK2 associated with total antioxidants (p = 7.6E-08); a block of 23 SNPs in XPA/FOXE1 (TTF-2) associated with serum TSH (p = 5.5E-08 to 1.0E-09); a nonsynonymous SNP (p = 1.3E-21), an intronic SNP (p = 3.6E-13) in DARC identified for MCP-1; an intronic variant in ARHGAP11A associated with sleep duration (p = 5.0E-08); and, after adjusting for body weight, variants in MATK for total energy expenditure (p = 2.7E-08) and in CHRNA3 for sleeping energy expenditure (p = 6.0E-08). Unprecedented phenotyping and high-density SNP genotyping enabled localization of novel genetic loci associated with the pathophysiology of childhood obesity.

Objective: Deficits in neurocognitive function have been demonstrated in individuals with schizophrenia and in their unaffected family members. Genetic studies of these complementary traits, along with traditional analyses of diagnosis, may help to elucidate the biological pathways underlying liability to schizophrenia. We report a genome-wide screen for schizophrenia and related neurocognitive phenotypes in a multiplex, multigenerational family study. Method:A total of 676 European American individuals in 43 families ascertained through an individual with schizophrenia were examined along with 236 healthy controls. Participants were evaluated clinically and tested with a computerized neurocognitive battery that provides measures of accuracy and speed on domains of abstraction and mental flexibility, attention, verbal, face, and spatial memory, language and reasoning, spatial processing, emotion processing, and sensorimotor dexterity. A genome-wide linkage screen was performed. The controls were used to obtain normative phenotype data, but were not genotyped.Results: Significant evidence of linkage was observed for schizophrenia on chromosome 19q (LOD=3.44). Analysis of cognitive traits revealed significant linkage for abstraction and mental flexibility on chromosome 5q (LOD=3.43). A variety of other neurocognitive traits also showed nominal evidence of linkage (LOD=1.05 − 2.9) in the 5q region. Joint analyses with diagnosis suggested that this QTL may also influence schizophrenia. Conclusions:The chromosome 19 QTL is a novel finding whereas chromosome 5 has been implicated in previous linkage studies of schizophrenia. The identification of this chromosome 5 QTL through linkage to neurocognitive phenotypes in the present study may inform functional hypotheses for how genotypes connect to disease.Cognitive deficits have been observed in individuals with schizophrenia and in their clinically unaffected relatives, particularly in the domains of executive function, learning, and memory (1-7). The appearance of deficits in patients and in unaffected relatives suggests that they are part of the innate underlying individual differences that make some people vulnerable to schizophrenia, rather than an outcome of the disease process. Such complementary biological and behavioral phenotypes may aid in genetic studies of a disorder and may provide valuable (8). Genetic studies of correlated quantitative phenotypes can complement traditional studies of disease outcome because a subset of genes influencing the disorder may have larger genetic effects on a complementary trait than on the disease endpoint. Such genes would be easier to detect at genome-wide significance levels in studies of the quantitative trait. Additionally, genetic analyses of complementary phenotypes can be a way of beginning to form functional hypotheses for how genotype connects to disease for genes that are identified through both diagnosis-and quantitative phenotype-based analyses.In this paper, we report a genome-wide linkage screen for schiz...

Suicide genes such as cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) encode products that convert nontoxic substances (prodrugs) into toxic metabolites. Suicide gene transfer is currently being used in cancer therapy or can be used as a safety modality. To analyze the reliability of suicide genes as a safety modality for a vaccination study with viable cytokine/B7 gene-modified tumor cells, the individual and combined efficacy of the two suicide genes was compared for in vitro and in vivo cell killing of a murine mammary adenocarcinoma cell line (TS/A). To adapt the system to an in vivo gene delivery situation, bulk cultures cotransfected with the CD and TK gene were used instead of selected clones. In vitro, both CD and TK conferred sensitivity to the respective prodrug but the combined cytotoxic effects of both gene products were always superior. For in vivo analysis BALB/c mice were injected subcutaneously with CD- and TK-modified TS/A cells, treated with prodrugs, and tumor size was evaluated for a period of 100 days. In the in vivo situation the combination of both enzyme/prodrug systems was again most effective. The highest single concentration of 5-FC (500 mg/kg) or GCV (100 mg/kg) was not able to fully protect the animals from developing tumors, whereas a combination of 5-FC (250 mg/kg) and GCV (50 mg/kg) resulted in complete tumor eradication. In nude mice treated in the same way, most CD/TK tumors could not be eliminated. Furthermore, BALB/c mice cured of TS/A-CD/TK tumors developed a systemic tumor immunity against challenge with parental TS/A cells. These findings indicate that reliable tumor elimination by the suicide genes depends on T cells. The cooperative effect of both suicide genes was confirmed in vitro with the human renal cell carcinoma line RCC26. We conclude that TK and CD together, but neither gene alone, act as a safety mechanism for the elimination of tumor cells in a reliable fashion and suggest that a rapid and quantitative antigen release by effective TK- and CD-mediated tumor destruction is necessary for T cell immunity to develop.

BackgroundBaboons (Papio hamadryas Sp.) develop features of the cardiometabolic syndrome and represent a clinically-relevant animal model in which to study the aetiology of the disorder. To further evaluate the baboon as a model for the study of the cardiometabolic syndrome, we developed a high sugar high fat diet and hypothesized that it could be used to induce adiposity gain and affect associated circulating biomarkers.MethodsWe developed a diet enriched with monosaccharides and saturated fatty acids that was composed of solid and liquid energy sources. We provided a group of baboons (n = 9) ad libitum access to this diet for 8 weeks. Concurrently, a control group (n = 6) was maintained with ad libitum access to a low sugar low fat baseline diet and normal water for 8 weeks. Body composition was determined by dual-energy X-ray absorptiometry and circulating metabolic biomarkers were measured using standard methodology before and after the 8 week study period.ResultsNeither body composition nor circulating biomarkers changed in the control group. Following the 8 weeks, the intervention group had a significant increase in fat mass (1.71 ± 0.98 vs. 3.23 ± 1.70 kg, p = 0.004), triglyceride (55 ± 13 vs. 109 ± 67 mg/dL, p = 0.006,), and leptin (1.19 ± 1.40 vs. 3.29 ± 2.32 ng/mL, p = 0.001) and a decline in adiponectin concentrations (33530 ± 9744 vs. 23330 ± 7863 ng/mL, p = 0.002). Percentage haemoglobin A1C (4.0 ± 0.3 vs. 6.0 ± 1.4, p = 0.002) also increased in the intervention group.ConclusionsOur findings indicate that when exposed to a high sugar high fat diet, young adult male baboons develop increased body fat and triglyceride concentrations, altered adipokine concentrations, and evidence of altered glucose metabolism. Our findings are in keeping with observations in humans and further demonstrate the potential utility of this highly clinically-relevant animal model for studying diet-induced metabolic dysregulation.

This comprehensive investigation provides strong evidence that MC4R genetic variants are likely to play a functional role in the regulation of weight, not only through energy intake but through energy expenditure.

Background-Consumption of omega-3 fatty acids (FAs) is associated with a reduction in deaths from coronary heart disease, arrhythmia, and sudden death. Although these FAs were originally thought to be anti-atherosclerotic, recent evidence suggests that their benefits are related to reducing risk for ventricular arrhythmia, and that this may be mediated by a slowed heart rate (HR).

Background Smoking remains one of the leading preventable causes of death. Smoking leaves a strong signature on the blood methylome as shown in multiple studies using the Infinium HumanMethylation450 BeadChip. Here, we explore novel blood methylation smoking signals on the Illumina MethylationEPIC BeadChip (EPIC) array, which also targets novel CpG-sites in enhancers. Method A smoking-methylation meta-analysis was carried out using EPIC DNA methylation profiles in 1407 blood samples from four UK population-based cohorts, including the MRC National Survey for Health and Development (NSHD) or 1946 British birth cohort, the National Child Development Study (NCDS) or 1958 birth cohort, the 1970 British Cohort Study (BCS70), and the TwinsUK cohort (TwinsUK). The overall discovery sample included 269 current, 497 former, and 643 never smokers. Replication was pursued in 3425 trans-ethnic samples, including 2325 American Indian individuals participating in the Strong Heart Study (SHS) in 1989–1991 and 1100 African-American participants in the Genetic Epidemiology Network of Arteriopathy Study (GENOA). Results Altogether 952 CpG-sites in 500 genes were differentially methylated between smokers and never smokers after Bonferroni correction. There were 526 novel smoking-associated CpG-sites only profiled by the EPIC array, of which 486 (92%) replicated in a meta-analysis of the American Indian and African-American samples. Novel CpG sites mapped both to genes containing previously identified smoking-methylation signals and to 80 novel genes not previously linked to smoking, with the strongest novel signal in SLAMF7. Comparison of former versus never smokers identified that 37 of these sites were persistently differentially methylated after cessation, where 16 represented novel signals only profiled by the EPIC array. We observed a depletion of smoking-associated signals in CpG islands and an enrichment in enhancer regions, consistent with previous results. Conclusion This study identified novel smoking-associated signals as possible biomarkers of exposure to smoking and may help improve our understanding of smoking-related disease risk.