Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice.

BACKGROUND: Gene doping is the misuse of genome editing and gene therapy technologies for the purpose of manipulating specific genes or gene functions in order to improve athletic performance. However, a non-invasive detection method for gene doping using recombinant adenoviral (rAdV) vectors containing human follistatin (hFST) genes (rAdV) has not yet been developed. Therefore, the aim of this study was to develop a method to detect gene doping using rAdV. METHODS: First, we generated rAdV and evaluated the overexpression of the hFST gene, FST protein, and muscle protein synthesis signaling using cell lines. Next, rAdV was injected intravenously or intramuscularly into mice, and whole blood was collected, and hFST and cytomegalovirus promoter (CMVp) gene fragments were detected using TaqMan-quantitative polymerase chain reaction (qPCR). Finally, to confirm the specificity of the primers and the TaqMan probes, samples from each experiment were pooled, amplified using TaqMan-qPCR, and sequenced using the Sanger sequencing. RESULTS: The expression of hFST and FST proteins and muscle protein synthesis signaling significantly increased in C2C12 cells. In long-term, transgene fragments could be detected until 4 days after intravenous injection and 3 days after intramuscular injection. Finally, the Sanger sequencing confirmed that the primers and TaqMan probe specifically amplified the gene sequence of interest. CONCLUSIONS: These results indicate the possibility of detecting gene doping using rAdV using TaqMan-qPCR in blood samples. This study may contribute to the development of detection methods for gene doping using rAdV.

Proof of Gene Doping in a Mouse Model with a Human Erythropoietin Gene Transferred Using an Adenoviral Vector.

Despite the World Anti-Doping Agency (WADA) ban on gene doping in the context of advancements in gene therapy, the risk of EPO gene-based doping among athletes is still present. To address this and similar risks, gene-doping tests are being developed in doping control laboratories worldwide. In this regard, the present study was performed with two objectives: to develop a robust gene-doping mouse model with the human EPO gene (hEPO) transferred using recombinant adenovirus (rAdV) as a vector and to develop a detection method to identify gene doping by using this model. The rAdV including the hEPO gene was injected intravenously to transfer the gene to the liver. After injection, the mice showed significantly increased whole-blood red blood cell counts and increased expression of hematopoietic marker genes in the spleen, indicating successful development of the gene-doping model. Next, direct and potentially indirect proof of gene doping were evaluated in whole-blood DNA and RNA by using a quantitative PCR assay and RNA sequencing. Proof of doping could be detected in DNA and RNA samples from one drop of whole blood for approximately a month; furthermore, the overall RNA expression profiles showed significant changes, allowing advanced detection of hEPO gene doping.

Acute Low-Intensity Treadmill Running Upregulates the Expression of Intestinal Glucose Transporters via GLP-2 in Mice.

The effects of exercise on nutrient digestion and absorption in the intestinal tract are not well understood. A few studies have reported that exercise training increases the expression of molecules involved in carbohydrate digestion and absorption. Exercise was also shown to increase the blood concentration of glucagon-like peptide-2 (GLP-2), which regulates carbohydrate digestion and absorption in the small intestine. Therefore, we investigated the effects of exercise on the expression of molecules involved in intestinal digestion and absorption, including GLP-2. Six-week-old male mice were divided into a sedentary (SED) and low-intensity exercise (LEx) group. LEx mice were required to run on a treadmill (12.5 m/min, 1 h), whereas SED mice rested. All mice were euthanized 1 h after exercise or rest, and plasma, jejunum, ileum, and colon samples were collected, followed by analysis via IHC, EIA, and immunoblotting. The levels of plasma GLP-2 and the jejunum expression of the GLP-2 receptor, sucrase-isomaltase (SI), and glucose transporter 2 (GLUT2) were higher in LEx mice. Thus, we showed that acute low-intensity exercise affects the expression of molecules involved in intestinal carbohydrate digestion and absorption via GLP-2. Our results suggest that exercise might be beneficial for small intestine function in individuals with intestinal frailty.

One Week of CDAHFD Induces Steatohepatitis and Mitochondrial Dysfunction with Oxidative Stress in Liver.

The prevalence of nonalcoholic fatty liver disease (NAFLD) has been rapidly increasing worldwide. A choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) has been used to create a mouse model of nonalcoholic steatohepatitis (NASH). There are some reports on the effects on mice of being fed a CDAHFD for long periods of 1 to 3 months. However, the effect of this diet over a short period is unknown. Therefore, we examined the effect of 1-week CDAHFD feeding on the mouse liver. Feeding a CDAHFD diet for only 1-week induced lipid droplet deposition in the liver with increasing activity of liver-derived enzymes in the plasma. On the other hand, it did not induce fibrosis or cirrhosis. Additionally, it was demonstrated that CDAHFD significantly impaired mitochondrial respiration with severe oxidative stress to the liver, which is associated with a decreasing mitochondrial DNA copy number and complex proteins. In the gene expression analysis of the liver, inflammatory and oxidative stress markers were significantly increased by CDAHFD. These results demonstrated that 1 week of feeding CDAHFD to mice induces steatohepatitis with mitochondrial dysfunction and severe oxidative stress, without fibrosis, which can partially mimic the early stage of NASH in humans.

Renalase is localized to the small intestine crypt and expressed upon the activation of NF-κB p65 in mice model of fasting-induced oxidative stress.

AIMS: Renalase expression is regulated by Nuclear Factor (NF)-κB and hypoxia inducible factor (HIF)-1α, and antioxidative stress function in renal cells were reported. However, dynamics of renalase and localizes in intestine were remain unknown. We evaluated the effects of oxidative stress on renalase expression and localization using model of fasting induced oxidative stress and Caco-2 cell, and examined the its physiological effects. MAIN METHODS: 24 male mice were divided into three groups: Control (Con), 72 h fasting (Fast), and 24 h refeeding after fasting (Refeed). Jejunum and ileum were collected respectively. The structure of jejunum and ileum were observed by hematoxylin and eosin (HE) stain. The expression levels of carbonylated protein, renalase, NF-κB p65 and HIF-1α were measured by immunoblotting. Localization of renalase was observed by immunofluorescent. in vitro assay was performed using Caco-2 cell. Renalase was overexpressed using adenovirus. After that, Caco-2 cell was treated with 2 mM H2O2 for 30 min or 24 h. KEY FINDINGS: Renalase was increased in Fast and it was localized in crypt. HIF-1α did not increase, but NF-κB p65 increased in Fast. Renalase overexpression protects the Caco-2 cells against H2O2 induced oxidative stress. SIGNIFICANCE: Renalase was localized in crypt and increased in Fast. This increase suggested protect response to oxidative stress because undifferenced cells were localized in crypt and need to be protected. Actually, renalase protected Caco-2 cells against H2O2 induced oxidative stress. Small intestinal renalase expression was regulated by NF-κB p65 and was considered to be a defense mechanism against oxidative stress.

Prediction of Japanese children at risk for complications of childhood obesity: gender differences for intervention approaches.

Childhood obesity is one of the most serious public health problems in Japan, especially in Tokushima compared with other prefectures. This study was designed to clarify the life habits which predispose to development of obesity and can be modified through an appropriate intervention program to combat childhood obesity and its lifestyle-related diseases. A total of 216 school children from Itano Town, a municipality of Tokushima Prefecture, Japan, who are attending the fourth grade (9-10 years) of elementary schools, participated in the study from 2004 to 2007. The study included child's life habits questionnaire, investigating physical activity by recording the daily steps using a pedometer, anthropometric measurements, hematological examination and hemodynamometry in a cross-sectional survey during a two-month period from June to July every year. We conclude that there are considerable gender-related differences for developing obesity and other lifestyle-related diseases; and all intervention strategies against obesity must consider such gender differences. For example, restriction of television watching hours must be intervened for controlling obesity in boys, however for girls, promotion of exercise practice or making more steps per day with adequate sleeping periods should be intervened as the proper approaches for preventing and controlling obesity and other lifestyle-related diseases.

Determinants of life satisfaction among Japanese elderly women attending health care and welfare service facilities.

Prolonged life expectancy must be recognized as an excellent achievement of modern medicine, but not all the elderly people are satisfied with their lives. Life satisfaction is a multi-dimensional issue that depends on many objective and subjective characteristics. In this study, we aimed at investigating the factors affecting life satisfaction of 314 elderly Japanese women attending in 28 elderly-care and welfare facilities at Tokushima Prefecture, Japan. Our results indicated that elderly subjects with depression tendencies always show significantly lower degrees of life satisfaction than others who are not depressed (p<0.001) regardless of their cognitive status. Furthermore, elderly women who shared decision for their living place and whose opinions were considered for daily life decisions reported significantly more life satisfaction levels than others. We conclude that elderly life satisfaction is affected by various determinants however, with different influencing weight. Life satisfaction of elderly people, with or without dementia, is greatly affected by their mood status and share in decision making. Avoiding elderly people depressive mood, sharing them in various daily decisions, considering their opinions, and allowing them to decide their elderly-care facility placement are crucial determinants for their life satisfaction and essential for their coping, adaptation, well-being and successful aging.

Weight and height growth velocities of Japanese boys and girls between age 7 and 14 years: a critical window for early adolescent overweight risk.

Childhood overweight is an important worldwide problem of public health concern, with metabolic, physical and psychosocial complications. More and more evidence is accumulating that children who gain weight rapidly earlier in life are at higher risk of becoming overweight later in adulthood life. Therefore, in a seven-year longitudinal study, we studied mid-childhood and early adolescence weight and height growth velocities among 5,024 Japanese 2(nd) grade primary school boys and girls along with its effect on the likelihood of being overweight adolescents by the age of 14 y. Our findings showed that weight growth velocity of both sexes was associated with being overweight at the end of longitudinal study. Boys' risk of accelerated weight growth velocity and becoming overweight adolescents was almost doubled during ages 7 approximately 11 y and become reduced afterward. Otherwise, odds ratios of girls' weight growth velocity peaks were found only at ages from 9 approximately 10 and 10 approximately 11 y. Thus, we suggest a critical window of mid-childhood period associated with adolescence risk of overweight, and we recommend that studying weight growth determinants during 7 approximately 11 y of boys and 9 approximately 11 y of girls may help in developing and applying proper programs for prevention and intervention of overweight problem.

Tracking overweight and obesity in Japanese children; a six years longitudinal study.

Childhood overweight/obesity is growing steeply, globally. It is usually regarded as a risk factor for severe obesity over life-time course. Here, we investigated temporal course of overweight/obesity development in Japanese school children. A six-year longitudinal study was performed on 16,245 Japanese primary school children (8,427 boys and 7,818 girls) comprising three cohorts of 1(st) approximately 3(rd) grade. A baseline survey was conducted at 2001, followed by annual baseline studies from 2002 approximately 2007 to determine the prevalence and track overweight/obesity. Our results showed that the prevalence of overweight was 15 approximately 23% in boys and 15 approximately 18% in girls, however, for obesity it ranged between 4 approximately 7% in boys; and 2 approximately 4% in girls. As regards for tracking status, 60 approximately 80% of overweight and 35 approximately 70% of obese Japanese primary school boys track into overweight or obese junior high school adolescents. However, these percentages are lower among primary school girls, where only 50 approximately 70% overweight and 30 approximately 60% obese primary school girls track into overweight and obese adolescents, respectively. We conclude that Japanese boys are fatter than girls; and approximately 80% of overweight/obese Japanese primary school children track into junior high school overweight/obese adolescents.

A/G heterozygote of the A-3826G polymorphism in the UCP-1 gene has higher BMI than A/A and G/G homozygote in young Japanese males.

UCP-1 is suggested to have important roles for thermogenesis and energy expenditure. To elucidate whether the A-3826G polymorphism that is located in the 5' flanking region of the UCP-1 gene has roles in healthy young people, the polymorphism was genotyped among 251 young Japanese men whose mean age is 22.7 years old. We analyzed relationship between the A-3826G polymorphism and body mass index (BMI) or six biochemical parameters, serum concentration of total cholesterol (TC), high density lipoprotein (HDL) cholesterol, triglyceride (TG), asparatate aminotransferase (AST), alanine aminotransferase (ALT), fasting plasma glucose. The genotype frequencies were observed at the frequencies of 24.3% for AA, 48.2% for AG and 27.5% for GG, respectively. When BMI and the biochemical parameters were compared by ANOVA among individuals with each genotype, the statistical difference was observed only for BMI (P=0.016). Bonferroni's test demonstrated that the men with the AG genotype have higher BMI than those with the AA genotype (22.4+/-2.8 vs. 21.4+/-2.2) (P=0.04). The individuals with the AG genotype also showed trend to have higher BMI than those with the GG, although the difference was not statistically apparent (22.4+/-2.8 vs. 21.5+/-2.3) (P=0.07). Our results indicated that the young healthy Japanese men with the AG heterozygote showed higher BMI than those with other genotypes.

Association of estrogen receptor alpha gene polymorphisms and lifestyle factors with calcaneal quantitative ultrasound and osteoporosis in postmenopausal Vietnamese women.

Genetic and lifestyle factors are important in the pathogenesis of osteoporosis. We investigated the relationships of PvuII and XbaI polymorphisms of the estrogen receptor alpha (ER-alpha) gene, lifestyle factors with speed of sound at the calcaneus (calcaneal SOS) and osteoporosis in a population-based study of 140 healthy postmenopausal women. By an analysis of covariates, women with higher copies of P or X alleles had higher calcaneal SOS compared with others (P=0.012, PP vs. pp; P=0.045, Xx vs. xx). Women with lower copies of px or higher copies of PX haplotypes had higher calcaneal SOS compared with others (P=0.021, 0 px vs. 2 px; P=0.011, 1 PX vs. 0 PX). The px and PX haplotypes, age and years since menopause were found to be independent predictors of calcaneal SOS in multiple linear regression models. Using logistic regression, we found an increased osteoporosis risk with evidence for a px haplotype dose effect (OR=2.82, 95% CI=1.50-5.31, P=0.001) and for a PX haplotype dose effect (OR=0.42, 95% CI=0.19-0.93, P=0.033). An increased educational level was associated with a reduced risk of osteoporosis (P=0.035 in the model with px, P=0.044 in the model with PX). In conclusion, the present study suggests that PvuII and XbaI polymorphims of the ER-alpha gene, age, years since menopause and educational level are associated with bone density, as assessed by calcaneal SOS, and osteoporosis in postmenopausal Vietnamese women.

Expression analysis of a mouse orthologue of HSFY, a candidate for the azoospermic factor on the human Y chromosome.

Heat shock transcription factor on Y (HSFY) is located in one of three candidate regions for azoospermic factor (AZF), AZFb on the Y chromosome. We and others have already revealed that some azoospermic males are missing the regions of the Y chromosome including HSFY. Previously, we showed that murine HSFY-like sequence [mHSFYL (Riken cDNA 4933413G11Rik)], which is the mouse orthologue of HSFY, is exclusively expressed in testis. The sequences encoding the presumed DNA-binding domain in HSFY and mHSFYL were found in other mammals such as dogs, cows and chickens. To elucidate mHSFYL expression in the testes in detail, we carried out in situ hybridization. mHSFYL was predominantly expressed in round spermatids. Furthermore, we clarified the intracellular distribution of mHSFYL in COS1 cells with HA- or GFP-tagged proteins. Both HA-mHSFYL and GFP-mHSFYL were located in the nucleus. Our results suggest that HSFY/mHSFYL may have evolutionarily conserved functions for spermatogenesis.