Identification of a novel biomarker for sarcopenia diagnosis using serum metabolomic analysis: a pilot study.

PURPOSE: This pilot study compared serum metabolites in participants with and without sarcopenia. METHODS: Metabolomic techniques were applied to identify serum metabolites and novel biomarkers specific to patients with sarcopenia. In accordance with AWGS2019 criteria, sarcopenia was defined as low muscle mass plus either low muscle strength/low physical function, and severe sarcopenia was defined as low muscle mass, low muscle strength, and low physical function all together. RESULTS: The sarcopenia group had higher hypoxanthine, galactose, and mannose levels but lower triethanolamine and homogentisic acid levels than the non-sarcopenia group. The severe sarcopenia group had lower levels of alpha-tocopherol than the mild and moderate sarcopenia groups. CONCLUSION: This study is the first to identify hypoxanthine as a potential biomarker for sarcopenia in humans and provides new insights into the pathophysiology of sarcopenia. Furthermore, the identified metabolites may be useful for the early detection of sarcopenia.

A multi-satellite framework to rapidly evaluate extreme biosphere cascades: The Western US 2021 drought and heatwave.

The increasing frequency and intensity of climate extremes and complex ecosystem responses motivate the need for integrated observational studies at low latency to determine biosphere responses and carbon-climate feedbacks. Here, we develop a satellite-based rapid attribution workflow and demonstrate its use at a 1-2-month latency to attribute drivers of the carbon cycle feedbacks during the 2020-2021 Western US drought and heatwave. In the first half of 2021, concurrent negative photosynthesis anomalies and large positive column CO2 anomalies were detected with satellites. Using a simple atmospheric mass balance approach, we estimate a surface carbon efflux anomaly of 132 TgC in June 2021, a magnitude corroborated independently with a dynamic global vegetation model. Integrated satellite observations of hydrologic processes, representing the soil-plant-atmosphere continuum (SPAC), show that these surface carbon flux anomalies are largely due to substantial reductions in photosynthesis because of a spatially widespread moisture-deficit propagation through the SPAC between 2020 and 2021. A causal model indicates deep soil moisture stores partially drove photosynthesis, maintaining its values in 2020 and driving its declines throughout 2021. The causal model also suggests legacy effects may have amplified photosynthesis deficits in 2021 beyond the direct effects of environmental forcing. The integrated, observation framework presented here provides a valuable first assessment of a biosphere extreme response and an independent testbed for improving drought propagation and mechanisms in models. The rapid identification of extreme carbon anomalies and hotspots can also aid mitigation and adaptation decisions.

Identifying high-risk population of depression: association between metabolic syndrome and depression using a health checkup and claims database.

Depression and metabolic syndrome (MetS) are correlated, leading to an increased healthcare burden and decreased productivity. We aimed to investigate the association between MetS-related factors and depression using a health checkup and claims database. Individuals aged 18-75 years who underwent health examinations between 2014 and 2019 were enrolled in the study. Among 76,277 participants, "ever" and "incident" antidepressant users exhibited worse metabolic profiles and were more likely to be prescribed hypnotics and anxiolytics than "never" users. In a nested case-control study with a 1:10 ratio of incident users to controls, MetS was associated with incident antidepressant use (odds ratio, 1.53 [95% confidence interval 1.24-1.88]) adjusted for lifestyle information obtained from a self-administered questionnaire, medical history, and medications. Other metabolic traits also showed significant associations: body mass index (1.04 [1.02-1.06]), abdominal circumference per 10 cm (1.17 [1.08-1.27]), high blood pressure (1.17 [1.00-1.37]), glucose intolerance (1.29 [1.05-1.58]), and dyslipidemia (1.27 [1.08-1.51]). A bodyweight increase > 10 kg from age 20 years (1.46 [1.25-1.70]) was also significantly associated with incident antidepressant use. In conclusion, metabolic abnormalities were associated with incident antidepressant use and can be useful in identifying populations at high risk of depression.

Associations of circulating xanthine oxidoreductase activity with cardiometabolic risk markers in overweight and obese Japanese men: a cross-sectional pilot study.

Circulating xanthine oxidoreductase (XOR) activity may contribute to the pathogenesis of obesity-related adverse cardiometabolic profiles. This pilot study aimed to examine the cross-sectional associations between plasma XOR activity and cardiometabolic risk (CMR) markers in overweight and obese men. In 64 overweight and obese Japanese men (aged 31-63 years), plasma XOR activity and several CMR markers, such as homeostasis model assessment of insulin resistance (HOMA-IR), and clustered CMR score were measured in each participant. Clustered CMR score was constructed based on waist circumference, triglyceride, blood pressure, fasting plasma glucose, and high-density lipoprotein cholesterol. Plasma XOR activity in overweight and obese men was positively associated with the body mass index, waist circumference, visceral fat area, body fat mass, hemoglobin A1c, serum 8-hydroxy-2'-deoxyguanosine, HOMA-IR, and clustered CMR score and was inversely associated with handgrip strength and high-density lipoprotein cholesterol. Multiple linear regression analysis further demonstrated that the associations of plasma XOR activity with HOMA-IR and the clustered CMR score remained significant after adjustment for covariates including uric acid. Our data demonstrate that circulating XOR activity was independently associated, albeit modestly, with HOMA-IR and the clustered CMR score. These preliminary findings suggest that circulating XOR activity can potentially be one of the preventive targets and biomarkers of cardiometabolic disorders in over-weight and obese men.

Xanthine oxidoreductase activity in marathon runners: potential implications for marathon-induced acute kidney injury.

Excess activation of circulating xanthine oxidoreductase (XOR) may contribute to the pathogenesis of widespread remote organ injury, including kidney injury. The purpose of this study was to determine the acute impact of marathon running on plasma XOR activity and to examine whether plasma XOR activity is associated with marathon-induced elevations in biomarkers of acute kidney injury (AKI). Twenty-three young men (aged 20-25 yr) who participated in the 38th Tsukuba Marathon were included. Blood and urine samples were collected before, immediately, 2 h (only blood sample), and 24 h after a full marathon run. Plasma XOR activity was evaluated using a highly sensitive assay utilizing a combination of [13C2,15N2] xanthine and liquid chromatography-triple quadrupole mass spectrometry. The levels of several AKI biomarkers, such as serum creatinine and urinary liver-type fatty acid-binding protein (L-FABP) were measured in each participant. Marathon running caused a transient elevation in plasma XOR activity and levels of purine degradation products (hypoxanthine, xanthine, and uric acid) as well as serum creatinine, urinary albumin, and urinary L-FABP levels. Immediately after the marathon, individual relative changes in plasma XOR activity were independently correlated with corresponding changes in serum creatinine and urinary L-FABP levels. In addition, the magnitude of marathon-induced elevation in plasma XOR activity and levels of purine degradation products were higher in individuals who developed AKI. These findings collectively suggest that marathon running substantially influences the purine metabolism pathway including XOR activity. Moreover, activated circulating XOR can be partly associated with elevated biomarkers of AKI after marathon running.NEW & NOTEWORTHY This study is the first to show marathon running transiently increases plasma XOR activity and levels of purine degradation products (hypoxanthine, xanthine, and uric acid), and further to demonstrate that activated plasma XOR may contribute to marathon-induced elevations in biomarkers of AKI. These findings significantly extend our prior knowledge of the purine metabolic pathway and several AKI biomarkers under strenuous exercise conditions.

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.

Effects of Aerobic, Resistance, or Combined Exercise Training Among Older Adults with Subjective Memory Complaints: A Randomized Controlled Trial.

BACKGROUND: Physical exercise is suggested to be effective for preventing cognitive decline in older adults, but the relative efficacy of different types of exercise have yet to be clarified. OBJECTIVE: This single-blinded randomized controlled trial was designed to investigate the differential effects of aerobic exercise training (AT), resistance exercise training (RT), and combined exercise training (CT) on cognition in older adults with subjective memory complaints (SMC). METHODS: Community-dwelling older adults with SMC (n = 415; mean age = 72.3 years old) were randomly assigned to one of the four groups: AT, RT, CT, or control group. The study consisted of two phases: a 26-week intervention and a 26-week follow-up. The participants were evaluated at baseline, 26 weeks (postintervention), and 52 weeks (follow-up). The primary outcome of this study was memory function, which was assessed using the Logical Memory II subtest of the Wechsler Memory Scale-Revised (WMS-R) score. The secondary outcomes included global cognitive function, verbal fluency, working memory, processing speed, and executive functions. RESULTS: Intention-to-treat analysis by a mixed-effect model repeated measure showed that the AT group had significantly improved performance on the WMS-R Logical Memory II test (2.74 [1.82-3.66] points) than the control group (1.36 [0.44-2.28] points) at the postintervention assessment (p = 0.037). The effect was more pronounced in those without amnesia than those with amnesia. No significant improvement was observed in the RT and CT groups. CONCLUSION: This study suggests that AT intervention can improve delayed memory in community-dwelling older adults, particularly in individuals without objective memory decline.

Plasma free metanephrine and normethanephrine levels correlated to plasma catecholamine after acute running in amateur runner.

BACKGROUND: Catecholamine is a typical index of exercise intensity, but it is difficult to detect. Plasma metanephrine (MN) and normethanephrine (NMN) levels are more stable than those of catecholamines. This study aimed to investigate plasma MN and NMN levels during acute exercise running in amateur runners. METHODS: Samples were collected from eight healthy male participants. They were either sedentary or running at low or high intensity for 30 min. Blood samples were collected under these conditions. Measurements taken included plasma adrenaline, noradrenaline, MN, and NMN. RESULTS: Plasma adrenaline levels increased after high-intensity exercise compared with sedentary subjects. Plasma noradrenaline, MN, and NMN levels increased after both low- and high-intensity exercise compared with sedentary subjects. In addition, these levels were also significantly higher at high intensity than at low intensity. Plasma adrenaline and noradrenaline levels were positively correlated with plasma free MN and NMN levels after acute running, respectively. CONCLUSION: This study revealed that plasma MN and NMN levels transiently increased depending on exercise intensity in amateur runners. In addition, plasma NMN levels are better markers than plasma MN levels because of their stronger correlation with plasma catecholamine levels.

Validity of "One-size-fits-all" Approaches for the National Health Screening and Education Program: A Large-scale Cohort Study of Corporate Insurance Beneficiaries.

Objective Metabolic syndrome represents a unified condition of atherosclerotic diseases caused by abdominal obesity. The aims of this study were to examine the applicability of the prevalent fixed cut-off values of the abdominal circumference (AC) and body mass index (BMI) to age and gender groups and to identify suitable lifestyle modification factors. Methods We defined an outcome as having ≥ 2 risk components that are necessary to diagnose metabolic syndrome and examined the cross-sectional association of the AC and BMI with the outcome. We also assessed the effects of time-updated lifestyle information on metabolic traits using longitudinal data. Patients We enrolled 22,953 beneficiaries of a corporate health insurance scheme who underwent annual health examinations between January 2004 and December 2014. Results The AC [per 5-cm increase, odds ratio (OR) 1.17, 95% confidence interval (CI) 1.12-1.24] and BMI (OR 1.10, 95% CI 1.07-1.13) were significantly associated with the outcome, adjusted for age, gender, current smoking status, drinking habits, and other lifestyle information. The association between the outcome and AC was modified by gender (p for interaction = 0.033), and the association between the outcome and BMI was modified by age group (p for interaction = 0.049). In the longitudinal analysis, current smoking, drinking habits, and unhealthy eating habits were associated with an increased AC and BMI, whereas regular physical activity was associated with a decreased AC and BMI. Conclusion We showed that the association between the AC or BMI and metabolic syndrome was modified by gender or age group. Further studies will be needed to customize the national health screening and education programs.

Effects of renalase deficiency on liver fibrosis markers in a nonalcoholic steatohepatitis mouse model.

Progression of nonalcoholic steatohepatitis (NASH) is attributed to several factors, including inflammation and oxidative stress. In recent years, renalase has been reported to suppress oxidative stress, apoptosis and inflammation. A number of studies have suggested that renalase may be associated with protecting the liver from injury. The present study aimed to clarify the effects of renalase knockout (KO) in mice with NASH that were induced with a choline­deficient high­fat diet (CDAHFD) supplemented with 0.1% methionine. Wild type (WT) and KO mice (6­week­old) were fed a normal diet (ND) or CDAHFD for 6 weeks, followed by analysis of the blood liver function markers and liver tissues. CDAHFD intake was revealed to increase blood hepatic function markers, lipid accumulation and oxidative stress compared with ND, but no significant differences were observed between the WT and KO mice. However, in the KO­CDAHFD group, the Adgre1 and Tgfb1 mRNA levels were significantly higher, and α­SMA expression was significantly lower compared with the WT­CDAHFD group. Furthermore, the Gclc mRNA and phosphorylated protein kinase B (Akt) levels were significantly lower in the KO­ND group compared with the WT­ND group. The results of the current study indicated that as NASH progressed in the absence of renalase, oxidative stress, macrophage infiltration and TGF­ß expression were enhanced, while α­SMA expression in NASH may be partly suppressed due to the decreased phosphorylation of Akt level.

Non-target environmental analysis by liquid chromatography/high-resolution mass spectrometry with a product ion and neutral loss database.

Despite the increasing detection of emerging substances in the environment, the identity of most are left unknown due to the lack of efficient identification methods. We developed a non-target analysis method for identifying unknown substances in the environment by liquid chromatography/high-resolution mass spectrometry (LC/HRMS) with a product ion and neutral loss database (PNDB). The present analysis describes an elucidation method with elemental compositions of the molecules, product ions, and corresponding neutral losses of the unknown substance: (1) with the molecular formula, possible molecular structures are retrieved from two chemical structure databases (PubChem and ChemSpider); then (2) with the elemental compositions of product ions and neutral losses, possible partial structures are retrieved from the PNDB; and finally, (3) molecular structures that match the possible partial structures are listed in order of number of hits. A molecular structure with a higher number of hits is more similar to the structure of the analyzed substance. The performance of the non-target method was evaluated by simulated analysis of 150 LC/HRMS spectra registered in MassBank. First, all substances of the same mass data (41/41) and 68% (39/57) of the mass data of the same substances not registered in the PNDB were elucidated. It was demonstrated that 14% (7/52) and 31% (16/52) of the substances with no mass spectral data registered in the PNDB were obtained at the first and within the fifth place, respectively. Owing to the fact that 10 of the total hits occurred in product ions and neutral losses, almost 50% of the substances evaluated with this method were placed at the top 4 positions in the similarity ranking. Importantly, the proposed method is effective for analyzing mass spectral data that has not been registered in the PNDB and thus is expected to be used for a variety of non-target analyses.

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.

High Salt Diet Impacts the Risk of Sarcopenia Associated with Reduction of Skeletal Muscle Performance in the Japanese Population.

The World Health Organization has recommended 5 g/day as dietary reference intakes for salt. In Japan, the averages for men and women were 11.0 g/day and 9.3 g/day, respectively. Recently, it was reported that amounts of sodium accumulation in skeletal muscles of older people were significantly higher than those in younger people. The purpose of this study was to investigate whether the risk of sarcopenia with decreased muscle mass and strength was related to the amount of salt intake. In addition, we investigated its involvement with renalase. Four groups based on age and salt intake ("younger low-salt," "younger high-salt," "older low-salt," and "older high-salt") were compared. Stratifying by age category, body fat percentage significantly increased in high-salt groups in both younger and older people. Handgrip strength/body weight and chair rise tests of the older high-salt group showed significant reduction compared to the older low-salt group. However, there was no significant difference in renalase concentrations in plasma. The results suggest that high-salt intake may lead to fat accumulation and muscle weakness associated with sarcopenia. Therefore, efforts to reduce salt intake may prevent sarcopenia.

Abrupt decline in tropospheric nitrogen dioxide over China after the outbreak of COVID-19.

China's policy interventions to reduce the spread of the coronavirus disease 2019 have environmental and economic impacts. Tropospheric nitrogen dioxide indicates economic activities, as nitrogen dioxide is primarily emitted from fossil fuel consumption. Satellite measurements show a 48% drop in tropospheric nitrogen dioxide vertical column densities from the 20 days averaged before the 2020 Lunar New Year to the 20 days averaged after. This decline is 21 ± 5% larger than that from 2015 to 2019. We relate this reduction to two of the government's actions: the announcement of the first report in each province and the date of a province's lockdown. Both actions are associated with nearly the same magnitude of reductions. Our analysis offers insights into the unintended environmental and economic consequences through reduced economic activities.

Influence of Intermittent Cold Stimulations on CREB and Its Targeting Genes in Muscle: Investigations into Molecular Mechanisms of Local Cryotherapy.

Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injuries. The molecular mechanisms are unknown. To clarify these mechanisms, we applied one to three 15-min cold stimulations at 4 °C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cyclic AMP (cAMP) response element binding protein 1 (CREB1) was markedly phosphorylated (p-CREB1), and the CREB-binding protein (CBP) was recruited to p-CREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 °C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes involved in mitochondrial biogenesis. The results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes, in a manner dependent on cold stimulation frequency and duration. This information will inform further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.

LDH isoenzyme 5 is an index of early onset muscle soreness during prolonged running.

BACKGROUND: Muscle soreness is also induced during prolonged running such as a full marathon, and muscle soreness and increased damage markers are detected immediately after such a running. We named this muscle soreness, early onset muscle soreness (EOMS). Additionally, lactate dehydrogenase (LDH) level which has some isoenzyme is increased immediately after prolonged exercise. However, it is unclear that EOMS is related to muscle damage markers on prolonged running. This study aimed to determine at which point EOMS, and muscle damage markers are related to EOMS during prolonged running. METHODS: We studied 11 male subjects who habitually perform aerobic exercise. They ran 30 km at 90% of ventilatory threshold intensity. Every 10 km, we estimated perceived muscle soreness, and sampled blood to measure muscle and liver damage, inflammation, and oxidative stress (d-ROM and BAP) markers. RESULTS: Muscle soreness score lower limbs were significantly appeared at 20 km compared to that at 0 km. Serum lactate dehydrogenase (LDH) level increased at 30 km compared to that at 0 km. LDH isoenzymes 3, 4, and 5, and neutrophils significantly increased at 30 km compared to those at 0 km. Serum LDH isoenzyme 5 and change in aspartate aminotransferase significantly increased at 20 km. In addition, there was a significant correlation between the thigh NRS and amount of serum LDH isoenzyme 5 from 0 km to 20 km. d-ROM and BAP increased at 10 km compared to those at 0 km. CONCLUSIONS: EOMS started to occur at 20 km during a 30 km running task. Our data suggest that LDH isoenzyme 5 is a marker of occurrence in EOMS during prolonged running.

Characterization of Osteoarthritis in a Medial Meniscectomy-Induced Animal Model Using Contrast-Enhanced X-ray Microtomography.

The aim of this study was to clarify degradation characteristics in each tissue of the knee complex of a medial meniscectomy (MMx)-induced knee osteoarthritis (KOA) animal model using classical methods and an alternative comprehensive evaluation method called contrast-enhanced X-ray micro-computed tomography (CEX-µCT), which was developed in the study. Surgical MMx was performed in the right knee joints of five male Wistar rats to induce KOA. At four weeks post-surgery, the synovitis was evaluated using quantitative polymerase chain reaction (qPCR). Degradations of the articular cartilage of the tibial plateau were evaluated using classical methods and CEX-µCT. Evaluation of the synovitis demonstrated significantly increased expression levels of inflammation-associated marker genes in MMx-treated knees compared with those in sham-treated knees. Evaluation of the articular cartilage using classical methods showed that MMx fully induced degradation of the cartilage. Evaluation using CEX-µCT showed that local areas of the medial cartilage of the tibial plateau were significantly reduced in MMx-treated knees compared with those in sham-treated knees. On the other hand, total cartilage volumes were significantly increased in MMx-treated knees. On the basis of the findings of this study, the method could be relevant to study new treatments in KOA research.

The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay.

BACKGROUND: With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. METHODS: Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 µL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. RESULTS: In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. CONCLUSIONS: These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.