Search

When amino acids are plentiful in the diet, the liver upregulates most enzymes responsible for amino acid degradation. In particular, the activity of urea cycle enzymes increases in response to high-protein diets to facilitate the excretion of excess nitrogen. KLF15 has been established as a critical regulator of amino acid catabolism including ureagenesis and we have recently identified FoxO transcription factors as an important upstream regulator of KLF15 in the liver. Therefore, we explored the role of FoxOs in amino acid metabolism under high-protein diet. Our findings revealed that the concentrations of two urea cycle-related amino acids, arginine and ornithine, were significantly altered by FoxOs knockdown. Additionally, using KLF15 knockout mice and an in vivo Ad-luc analytical system, we confirmed that FoxOs directly regulate hepatic Ass1 expression under high-protein intake independently from KLF15. Moreover, ChIP analysis showed that the high-protein diet increased FoxOs DNA binding without altering the nuclear protein amount. Therefore, FoxOs play a direct role in regulating ureagenesis via a KLF15-independent pathway in response to high-protein intake., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Competing Interests: Conflicts of Interest This work was supported by Ibaraki CIC Laboratory Co., Ltd. The funders were not involved in the analysis and interpretation of the data, the writing of the manuscript, or the decision to submit the manuscript for publication.

Introduction: We aimed to determine the effects of exercise on cell-free DNA (cfDNA) levels and concentration changes during the menstrual cycle in participants with regular menstrual cycles and no exercise habits., Methods: Eleven sedentary female students with regular menstrual cycles and ovulation performed bicycle exercises at 60% VO 2max for 30 min during the menstrual, ovulatory, and luteal phases. Blood samples were collected before (Pre), immediately after (Post 0), 30 min after (Post 30), and 60 min after (Post 60) exercise. Blood concentrations of ovarian hormones, cfDNA, prostaglandin F2a (PGF2α), interleukin-6 (IL-6), and aromatase were evaluated., Results: Based on the concentration of ovarian hormones, seven individuals were finally analyzed. No significant phase difference was observed in cfDNA across all time points. cfDNA (menstrual phase: p  = 0.028, ovulatory phase: p  = 0.018, and luteal phase: p  = 0.048) and aromatase concentrations (menstrual phase: p  = 0.040, ovulatory phase: p  = 0.039, and luteal phase: p  = 0.045) significantly increased from Pre to Post 0 in all phases. Serum estradiol (E2) levels were significantly higher in the luteal phase at all time points than in the menstrual phase (Pre: p  < 0.001, Post 0: p  < 0.001, Post 30: p  = 0.005, and Post 60: p  = 0.011); however, serum progesterone (P4) levels were significantly higher in the luteal phase at all time points than in the menstrual (Pre: p  < 0.001, Post 0: p  < 0.001, Post 30: p  < 0.001, and Post 60: p  < 0.001) and ovulatory phases (Pre: p  = 0.005, Post 0: p  = 0.005, Post 30: p  = 0.003, and Post 60: p  = 0.003). E2 levels significantly increased from Pre to Post 0 in the ovulatory and luteal phases, whereas P4 levels increased in the luteal phase. Progesterone to estradiol level ratio (P4/E2) changes from Pre to Post 0 (%baseline) during the luteal phase were significantly negatively correlated ( r  = -0.82, p  = 0.046) with the changes in cfDNA from Pre to Post 0. Furthermore, the repeated measures correlation between P4/E2 and cfDNA level showed a significant negative correlation in ovulatory and luteal phases., Discussion: The results indicate that while resting cfDNA levels are unlikely to be affected by a woman's menstrual cycle, the increase in cfDNA after exercise is higher in the ovulatory phase (when only E2 increases) and lower in the luteal phase (when E2 and P4 increase with exercise) compared to that in the menstrual phase (when E2 and P4 are in low levels), suggesting the contribution of increased ovarian hormone levels after exercise., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Sawai, Shida, Hoshikawa, Hatanaka, Ueda, Kato, Tokinoya, Natsui, Kawakami and Takekoshi.)

During periods of fasting, the body undergoes a metabolic shift from carbohydrate utilization to the use of fats and ketones as an energy source, as well as the inhibition of de novo lipogenesis and the initiation of gluconeogenesis in the liver. The transcription factor sterol regulatory element-binding protein-1 (SREBP-1), which plays a critical role in the regulation of lipogenesis, is suppressed during fasting, resulting in the suppression of hepatic lipogenesis. We previously demonstrated that the interaction of fasting-induced Kruppel-like factor 15 (KLF15) with liver X receptor serves as the essential mechanism for the nutritional regulation of SREBP-1 expression. However, the underlying mechanisms of KLF15 induction during fasting remain unclear. In this study, we show that the glucocorticoid receptor (GR) regulates the hepatic expression of KLF15 and, subsequently, lipogenesis through the KLF15-SREBP-1 pathway during fasting. KLF15 is necessary for the suppression of SREBP-1 by GR, as demonstrated through experiments using KLF15 knockout mice. Additionally, we show that GR is involved in the fasting response, with heightened binding to the KLF15 enhancer. It has been widely known that the hypothalamic-pituitary-adrenal (HPA) axis regulates the secretion of glucocorticoids and plays a significant role in the metabolic response to undernutrition. These findings demonstrate the importance of the HPA-axis-regulated GR-KLF15 pathway in the regulation of lipid metabolism in the liver during fasting., (© 2023 Federation of European Biochemical Societies.)

Background: In patients with atrial fibrillation (AF) and severe blood stasis in the left atrial appendage (LAA), dense spontaneous echo contrast (SEC) disturbs the distinct visualization of the LAA interior, thus making thrombus diagnosis inconclusive. We aimed to prospectively assess the efficacy and safety of a protocol for a low-dose isoproterenol (ISP) infusion to reduce SEC to exclude an LAA thrombus., Methods and results: We enrolled 17 patients with AF and dense SEC (Grade 4 or sludge). ISP was infused with gradually increasing doses of 0.01, 0.02, and 0.03 μg/kg/min at 3-min intervals. After increasing the dose to 0.03 μg/kg/min for 3 min, or when the LAA interior was visible, the infusion was terminated. We reassessed the SEC grade, presence of an LAA thrombus, LAA function, and left ventricular ejection fraction (LVEF) within 1 min of ISP termination. Compared with baseline, ISP significantly increased LAA flow velocity, the LAA emptying fraction, LAA wall velocities, and LVEF (all P<0.01). ISP administration significantly reduced the SEC grade (median) from 4 to 1 (P<0.001). The SEC grade decreased to ≤2 in 15 (88%) patients, and the LAA thrombus was excluded. There were no adverse events., Conclusions: Low-dose ISP infusion may be effective and safe to reduce SEC and exclude an LAA thrombus by improving LAA function and LVEF.

Background: We hypothesized that if computed tomography (CT) images were used as learning data, we could overcome volume underestimation by echocardiography, improving the accuracy of left ventricular (LV) volume measurements., Methods: We utilized a fusion imaging modality consisting of echocardiography with superimposed CT images for 37 consecutive patients to identify the endocardial boundary. We compared LV volumes obtained with and without CT learning trace-lines (TLs). Furthermore, 3D echocardiography was used to compare LV volumes obtained with and without CT learning for endocardial identification. The mean difference between the echocardiography and CT-derived LV volumes and the coefficient of variation were compared pre- and post-learning. Bland-Altman analysis was used to assess the differences in LV volume (mL) obtained from the 2D pre-learning TL and 3D post-learning TL., Results: The post-learning TL was located closer to the epicardium than the pre-learning TL. This trend was particularly pronounced in the lateral and the anterior wall. The post-learning TL was along the inner side of the high echoic layer in the basal-lateral wall in the four-chamber view. CT fusion imaging determined that the difference in LV volume between 2D echocardiography and CT was small (-25.6 ± 14.4 mL before learning, -6.9 ± 11.5 mL after learning) and that CT learning improved the coefficient of variation (10.9 % before learning, 7.8 % after learning). Significant improvements were observed during 3D echocardiography; the difference in LV volume between 3D echocardiography and CT was slight (-20.5 ± 15.1 mL before learning, 3.8 ± 15.7 mL after learning), and the coefficient of variation improved (11.5 % before learning, 9.3 % after learning)., Conclusions: Differences between the LV volumes obtained using CT and echocardiography either disappeared or were reduced after CT fusion imaging. Fusion imaging is useful in training regimens for accurate LV volume quantification using echocardiography and may contribute to quality control., Competing Interests: Declaration of competing interest This study was funded by Canon Medical Systems, Otawara, Japan. The funders had no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication., (Copyright © 2023. Published by Elsevier Ltd.)

We designed and developed two new types of hydrogen fuel cell (HFC) buses (motorcoach and minibus) with a mobile laboratory system. Feasibility studies have been performed for mobile laboratory testing, particularly for the laboratory performance of COVID-19 RT-PCR (PCR). We evaluated the driving range capability, PCR sample size capacity, turnaround time (TAT), and analytical performance for the detection of SARS-CoV-2. Saliva samples were used for the current study, and the analytical performance was compared with that of the reference PCR. The estimated driving range and sample size capacity of the HFC and HFC minibus were 432 km and 2847 samples, respectively, for the HFC motorcoach and 313 km and 1949 samples for the HFC minibus. For the TAT, the median time between sample submission and completion of PCR was 86 min for the motorcoach and 76 min for the minibus, and the median time between sample submission and electronic reporting of the result to each visitor was 182 min for the motorcoach and 194 min for the minibus. A secondary analysis of 1574 HFC mobile laboratory testing samples was conducted, and all negative samples were found to be negative by reference PCR. Furthermore, all samples were confirmed to be positive by reference PCR or other molecular examinations., (© 2023. Springer Nature Limited.)

Mast cells are a significant source of cytokines and chemokines that play a role in pathological processes. Gangliosides, which are complex lipids with a sugar chain, are present in all eukaryotic cell membranes and comprise lipid rafts. Ganglioside GM3, the first ganglioside in the synthetic pathway, is a common precursor of the specifying derivatives and is well known for its various functions in biosystems. Mast cells contain high levels of gangliosides; however, the involvement of GM3 in mast cell sensitivity is unclear. Therefore, in this study, we elucidated the role of ganglioside GM3 in mast cells and skin inflammation. GM3 synthase (GM3S)-deficient mast cells showed cytosolic granule topological changes and hyperactivation upon IgE-DNP stimulation without affecting proliferation and differentiation. Additionally, inflammatory cytokine levels increased in GM3S-deficient bone marrow-derived mast cells (BMMC). Furthermore, GM3S-KO mice and GM3S-KO BMMC transplantation showed increased skin allergic reactions. Besides mast cell hypersensitivity caused by GM3S deficiency, membrane integrity decreased and GM3 supplementation rescued this loss of membrane integrity. Additionally, GM3S deficiency increased the phosphorylation of p38 mitogen-activated protein kinase. These results suggest that GM3 increases membrane integrity, leading to the suppression of the p38 signalling pathway in BMMC and contributing to skin allergic reaction., (© 2023 Federation of European Biochemical Societies.)

Female athletes follow a strict diet and perform rigorous exercise to boost their performance, which induces health issues called the female athlete triad (FAT), defined as the combination of disordered eating, amenorrhoea and low bone mineral density. It is known to have a significant effect on bones. However, its effects on the small intestine, which is responsible for nutrient uptake into the body, remain unclear. In this study, we created an animal model of FAT to examine its effects on digestive and absorptive molecules in the small intestine. Thirty 5-week-old female Sprague-Dawley (sd) rats with an initial body weight of about 147 g were divided into control (Con, n = 7), exercise (Ex, n = 7), food restriction (FR, n = 8) and exercise plus food restriction (FAT, n = 8) groups. The rats were subjected to 4 weeks of wheel running (Ex, FAT) and 50-40 % food restriction (FR, FAT) to examine the effects on bone and typical digestive enzymes and transporters in the jejunum. Two-way ANOVA and the Kruskal-Wallis test were used for statistical analysis of normal and non-normal data, respectively. Four weeks of exercise and food restriction decreased bone weight ( vs . other group P < 0·01) and bone breaking power ( vs . other group P < 0·01). Villus height decreased in the jejunum ( vs . other group P < 0·01), but the expression of typical macronutrients digestive enzyme and absorptive molecules remained unchanged. In contrast, sucrase-isomaltase gene ( v . Ex P = 0·02) and protein expression were increased ( vs . other group P < 0·05). The study findings show that FAT affects sucrase-isomaltase without histone methylation changes.

Background: Mild cognitive impairment (MCI) is not just a prodrome to dementia, but a very important intervention point to prevent dementia caused by Alzheimer's disease (AD). It has long been known that people with AD have a higher frequency of falls with some gait instability. Recent evidence suggests that vestibular impairment is disproportionately prevalent among individuals with MCI and dementia due to AD. Therefore, we hypothesized that the measurement of balance capability is helpful to identify individuals with MCI., Methods: First, we developed a useful method to evaluate balance capability as well as vestibular function using Nintendo Wii balance board as a stabilometer and foam rubber on it. Then, 49 healthy volunteers aged from 56 to 75 with no clinically apparent cognitive impairment were recruited and the association between their balance capability and cognitive function was examined. Cognitive functions were assessed by MoCA, MMSE, CDR, and TMT-A and -B tests., Results: The new balance capability indicator, termed visual dependency index of postural stability (VPS), was highly associated with cognitive impairment assessed by MoCA, and the area under the receiver operating characteristic (ROC) curve was more than 0.8, demonstrating high sensitivity and specificity (app. 80% and 60%, respectively)., Conclusions: Early evidence suggests that VPS measured using Nintendo Wii balance board as a stabilometer helps identify individuals with MCI at an early and preclinical stage with high sensitivity, establishing a useful method to screen MCI., (© 2023. The Author(s).)

COVID-19 has a wide range of clinical presentations, and the susceptibility to SARS-CoV-2 infection and the mortality rate also vary by region and ethnicity. Here, we found that rs12329760 in the TMPRSS2 gene, a missense variant common in East Asian populations, contributes to protection against SARS-CoV-2 infection. TMPRSS2 is a protease responsible for SARS-CoV-2 entry and syncytium formation. rs12329760 (c.478G>A, p. V160M) was associated with a reduced risk of moderate symptoms. The enzymatic activity of Met 160 -TMPRSS2 was lower than that of Val 160 -TMPRSS2, and thus the viral entry and the syncytium formation of SARS-CoV-2 were impaired. Collectively, these results indicate that the genetic variation in TMPRSS2 , which is common in East Asians, is one of the molecular determinants of COVID-19 susceptibility., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sekiya, Ogura, Kai, Kawaguchi, Okawa, Hirohama, Kuroki, Morii, Hara, Hiramatsu, Hitomi, Kawakami, Arakawa, Maruo, Chiba, Suzuki, Kojima, Tachikawa and Yamagata.)

Although branched-chain amino acids (BCAA) are known to stimulate myofibrillar protein synthesis and affect insulin signaling and kynurenine metabolism (the latter being a metabolite of tryptophan associated with depression and dementia), the effects of BCAA supplementation on type 2 diabetes (T2D) are not clear. Therefore, a 24-week, prospective randomized open blinded-endpoint trial was conducted to evaluate the effects of supplementation of 8 g of BCAA or 7.5 g of soy protein on skeletal muscle and glycemic control as well as adverse events in elderly individuals with T2D. Thirty-six participants were randomly assigned to the BCAA group ( n = 21) and the soy protein group ( n = 15). Skeletal muscle mass and HbA1c, which were primary endpoints, did not change over time or differ between groups. However, knee extension muscle strength was significantly increased in the soy protein group and showed a tendency to increase in the BCAA group. Homeostasis model assessment for insulin resistance did not significantly change during the trial. Depressive symptoms were significantly improved in the BCAA group but the difference between groups was not significant. Results suggested that BCAA supplementation may not affect skeletal muscle mass and glycemic control and may improve depressive symptoms in elderly individuals with T2D.

Background: Left-ventricular (LV) global longitudinal strain (GLS) has been reported to be a robust and sensitive marker of chemotherapy-induced cardiac damage. Image quality is paramount for accurate GLS measurements. In real-world cardio-oncology settings, the incidence of suboptimal echocardiography quality and its significance in clinical decision-making have not been well investigated. This prospective study examined the incidence and impact of suboptimal echocardiographic image quality on detecting subtle myocardial damage by chemotherapy., Methods: Seventy-seven consecutive patients with breast cancer (age, 52 ± 12 years, 76 women, 33 with left-sided breast cancer) were included in this study. Echocardiography was performed at 3-month intervals 1 year before and after chemotherapy initiation. We classified the image quality of each echocardiographic acquisition into three groups: optimal, suboptimal, or inadequate for speckle tracking., Results: Among the 376 examinations obtained during the cardiac monitoring, the image quality in 194 (52%) was optimal, suboptimal in 159 (42%), and inadequate in 23 (6%). The interobserver reproducibility was 0.91 in the optimal and 0.21 in the suboptimal group. In contrast, the optimal group showed progressive impairment in both GLS (p = 0.001) and LV ejection fraction (LVEF) (p < 0.001) during follow-up, and the suboptimal group showed a progressive decrease in LVEF (p = 0.006), but not in GLS (p = 0.13). Left-sided mammotomy and/or reconstruction surgery and high body mass index were significant determinants of suboptimal image quality., Conclusions: Even in cases of minor image quality impairment, the physician should assess GLS carefully to avoid errors in crucial clinical decision-making., (© 2022. Japanese Society of Echocardiography.)

We investigated the appropriate D-dimer cutoff value for each brain tumor type for acute or subacute deep vein thrombosis (DVT) following transcranial brain tumor surgery.In this single-center retrospective study, a cumulative total of 128 patients who underwent transcranial brain tumor surgery were enrolled and classified into the glioma group, the other intracranial malignant tumor group, and the intracranial benign tumor group. Venous ultrasonography was performed if the D-dimer plasma levels were positive (≥1 μg/mL) before surgery and on postoperative day (POD) 3 or 7.Of the 128 cases, DVT developed in 32 (25.0%). Among those, acute or subacute DVT was diagnosed in 22 cases on POD 3 and in 8 cases on POD 7. Compared with DVT-negative cases on POD 3, acute or subacute DVT-positive cases on POD 3 revealed a significant increase in the D-dimer level in all groups combined and in the benign tumor group but not in the glioma group. With regard to DVT on POD 3 in all groups, the receiver operating characteristic curve for the D-dimer level on POD 3 demonstrated a cutoff value of 3.3 μg/mL (sensitivity [0.636] and specificity [0.750]). However, if this cutoff value was used in practice, eight cases would be false-negative with a minimum D-dimer level of 1.5 μg/mL.The D-dimer cutoff value for acute or subacute DVT on POD 3 could be set to 3.3 μg/mL; however, the setting resulted in several false-negative cases. Practically, 1.5 μg/mL of the D-dimer cutoff value on POD 3 might be appropriate to avoid false-negative results.

Historically, a right bundle branch block has been considered a benign finding in asymptomatic individuals. However, this conclusion is based on a few old studies with small sample sizes. We examined the association between a complete right bundle branch block (CRBBB) and subsequent cardiovascular mortality in the general population in Japan. In this large community-based cohort study, data of 90,022 individuals (mean age, 58.5 ± 10.2 years; 66.2% women) who participated in annual community-based health check-ups were assessed. Subjects were followed up from 1993 to the end of 2016. Cox proportional hazards' models and log-rank tests were used for the data analysis. CRBBB was documented in 1,344 participants (1.5%). Among all included participants, CRBBB was associated with an increased risk of cardiovascular mortality after adjustment for all potential confounders (hazard ratio [HR] 1.21; 95% confidence interval [CI] 1.06-1.38). The increased risk of cardiovascular mortality was particularly evident in women aged < 65 years (HR 2.00; 95% CI 1.34-2.98) and men aged ≥ 65 years (HR 1.28; 95% CI 1.06-1.55). CRBBB is associated with an increased risk of cardiovascular mortality in women aged < 65 years and men aged ≥ 65 years. Clinicians should be aware of the presence of CRBBB in young women and elderly men, even if they exhibit no symptoms., (© 2021. Springer Japan KK, part of Springer Nature.)

High protein diet (HPD) is an affordable and positive approach in prevention and treatment of many diseases. It is believed that transcriptional regulation is responsible for adaptation after HPD feeding and Kruppel-like factor 15 (KLF15), a zinc finger transcription factor that has been proved to perform transcriptional regulation over amino acid, lipid and glucose metabolism, is known to be involved at least in part in this HPD response. To gain more insight into molecular mechanisms by which HPD controls expressions of genes involved in amino acid metabolism in the liver, we performed RNA-seq analysis of mice fed HPD for a short period (3 days). Compared to a low protein diet, HPD feeding significantly increased hepatic expressions of enzymes involved in the breakdown of all the 20 amino acids. Moreover, using KLF15 knockout mice and in vivo Ad-luc analytical system, we were able to identify Cth (cystathionine gamma-lyase) as a new target gene of KLF15 transcription as well as Ast (aspartate aminotransferase) as an example of KLF15-independent gene despite its remarkable responsiveness to HPD. These findings provide us with a clue to elucidate the entire transcriptional regulatory mechanisms of amino acid metabolic pathways., Competing Interests: Declaration of competing interest The authors declare no competing financial and non-financial interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

KLF15 is a transcription factor that plays an important role in the activation of gluconeogenesis from amino acids as well as the suppression of lipogenesis from glucose. Here we identified the transcription start site of liver-specific KLF15 transcript and showed that FoxO1/3 transcriptionally regulates Klf15 gene expression by directly binding to the liver-specific Klf15 promoter. To achieve this, we performed a precise in vivo promoter analysis combined with the genome-wide transcription-factor-screening method "TFEL scan", using our original Transcription Factor Expression Library (TFEL), which covers nearly all the transcription factors in the mouse genome. Hepatic Klf15 expression is significantly increased via FoxOs by attenuating insulin signaling. Furthermore, FoxOs elevate the expression levels of amino acid catabolic enzymes and suppress SREBP-1c via KLF15, resulting in accelerated amino acid breakdown and suppressed lipogenesis during fasting. Thus, the FoxO-KLF15 pathway contributes to switching the macronutrient flow in the liver under the control of insulin., Competing Interests: The authors declare no competing financial and non-financial interests., (© 2021 The Author(s).)

Background: Brain or B-type natriuretic peptide (BNP) is an objective marker to diagnose the presence of heart failure (HF) and assess its severity. However, the determinants of serum BNP level in elderly patients with severe aortic valve stenosis (AS) referred for transcatheter aortic valve implantation (TAVI) have not been well investigated., Methods: We prospectively studied 106 AS patients who underwent TAVI. Cardiac catheterization, transesophageal echocardiography, and blood collection for plasma BNP level measurements were performed simultaneously just before the TAVI procedures., Results: Ninety-nine patients (83.9±5.0 years, 33% male) were studied. The natural logarithm of BNP (lnBNP) level was 5.4±0.9 pg/mL. Significant correlations with lnBNP level were observed in: 1) the history of syncope, prior HF medication, and New York Heart Association class III or IV (R=0.255, p=0.011) (R=0.210, p=0.037) (R=0.402, p<0.001), 2) albumin and hemoglobin level (R=-0.289, p=0.004) (R=0.263, p=0.009), 3) Left ventricular (LV) ejection fraction and global longitudinal strain (LVGLS) (R=-0.338, p<0.001) (R=0.447, p<0.001), 4) LV end-diastolic volume index (EDVI), LV mass index, and left atrial volume index (R=0.280, p=0.005) (R=0.366, p<0.001) (R=0.337, p<0.001), 5) the catheter-measured pressure gradient across the aortic valve (AVPG) (R=0.365, p<0.001). Note that LV wall stress was not significantly correlated with lnBNP level. LVGLS, AVPG, hemoglobin level, and LVEDVI were independently correlated with ln BNP level (R=0.652, LVGLS; β=0.395, p<0.006, AVPG; β=0.291, p=0.001, hemoglobin level; β=-0.216, p=0.011, and LVEDVI; β=0.203, p=0.016, respectively)., Conclusions: In severe AS patients candidate for TAVI, multiple factors, including the severities of AS and HF conditions and subclinical LV dysfunction determined by LVGLS affects plasma BNP level., Competing Interests: Disclosure The authors declare that there is no conflict of interest., (Copyright © 2021. Published by Elsevier Ltd.)

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< hFST >) has not yet been developed. Therefore, the aim of this study was to develop a method to detect gene doping using rAdV< hFST >., Methods: First, we generated rAdV< hFST > and evaluated the overexpression of the hFST gene, FST protein, and muscle protein synthesis signaling using cell lines. Next, rAdV< hFST > 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< hFST > using TaqMan-qPCR in blood samples. This study may contribute to the development of detection methods for gene doping using rAdV< hFST >., Competing Interests: The authors declare that they have no competing interests., (© 2021 Yanazawa et al.)

Exosomal microRNA (miRNA) in plasma and urine has attracted attention as a novel diagnostic tool for pathological conditions. However, the mechanisms of miRNA dynamics in the exercise physiology field are not well understood in terms of monitoring sports performance. This pilot study aimed to reveal the miRNA dynamics in urine and plasma of full-marathon participants. Plasma and urine samples were collected from 26 marathon participants before, immediately after, 2 h after, and one day after a full marathon. The samples were pooled, and exosomal miRNAs were extracted and analyzed using next-generation sequencing. We determined that the exosomal miRNA expression profile changed under time dependency in full marathon. New uncharacterized exosomal miRNAs such as hsa-miR-582-3p and hsa-miR-199a-3p could be potential biomarkers reflecting physical stress of full marathon in plasma and urine. In addition, some muscle miRNAs in plasma and urine have supported the utility for monitoring physical stress. Furthermore, some inflammation-related exosomal miRNAs were useful only in plasma. These results suggest that these exosomal miRNAs in plasma and/or urine are highly sensitive biomarkers for physical stress in full marathons. Thus, our findings may yield valuable insights into exercise physiology.

Background: The redox modulation of Cys-thiol participates in various pathophysiological processes. We explored the proper index for estimating the redox status of Cys-thiol of serum apolipoprotein E (apoE), named "redox-IDX-apoE," which is necessary to understand the redox biology of age-related diseases., Methods: The fractions of the reduced form (red-), reversible oxidized form (roxi-), and irreversibly oxidized form (oxi-) apoE in serum, obtained from the patients with no apparent disease (controls, n=192) and with atherosclerosis and type 2 diabetes (patients, n=16), were measured by a band-shift assay using a maleimide compound. Redox-IDX-apoE candidates were determined by calculating the values of these fractions and the total apoE concentration., Results: Cys number of apoE significantly increased for the ratio of roxi-apoE to total-apoE (roxi/total) (E2/E3>E3/E3>E3/E4) but decreased for the ratios of red-apoE to roxi-apoE (red/roxi) and [red-apoE + oxi-apoE] to roxi-apoE ([red + oxi]/roxi) (E2/E3

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 (h EPO ) 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 h EPO 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 h EPO gene doping.

The high incidence of germline variants in pheochromocytoma and paraganglioma (PPGL) has been reported mainly in Europe, but not among Japanese populations in Asia. We aimed to study the prevalence of germline variants in Japanese PPGL patients and the genotype-phenotype correlation. We examined 370 PPGL probands, including 43 patients with family history and/or syndromic presentation and 327 patients with apparently sporadic (AS) presentation. Clinical data and blood samples were collected, and the seven major susceptibility genes ( MAX , SDHB , SDHC , SDHD , TMEM127 , VHL , and RET ) were tested using Sanger sequencing. Overall, 120/370 (32.4%) patients had pathogenic or likely pathogenic variants, with 81/327 (24.8%) in AS presentation. SDHB was the most frequently mutated gene (57, 15.4%), followed by SDHD (27, 7.3%), and VHL (18, 4.9%). The incidence of metastatic PPGL was high in SDHB carriers (21/57, 36.8%). A few unique recurrent variants ( SDHB c.137G>A and SDHB c.470delT) were detected in this Japanese cohort, highlighting ethnic differences. In summary, almost a quarter of patients with apparently sporadic PPGL in Japan harboured germline variants of the targeted genes. This study reinforces the recommendation in Western guidelines to perform genetic testing for PPGL and genotype-based clinical decision-making in the Japanese population.

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.

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.

Plasma cell-free DNA (cfDNA) is frequently analyzed using liquid biopsy to investigate cancer markers. We hypothesized that this concept might be applicable in exercise physiology. Here, we aimed to identify specific cfDNA (spcfDNA) sequences in the plasma of healthy humans using next-generation sequencing (NGS) and clearly define the dynamics regarding spcfDNA-fragment levels upon extreme exercises, such as running a full marathon. NGS analysis was performed using cfDNA of pooled plasma collected from healthy participants. We confirmed that the TaqMan-qPCR assay had high sensitivity and found that the spcfDNA sequence abundance was 16,600-fold higher than that in a normal genomic region. We then used the TaqMan-qPCR assay to investigate the dynamics of spcfDNA-fragment levels upon running a full marathon. The spcfDNA fragment levels were significantly increased post-marathon. Furthermore, spcfDNA fragment levels were strongly correlated with white blood cell and plasma myoglobin concentrations. These results suggest the spcfDNA fragments identified in this study were highly sensitive as markers of extreme physical stress. The findings of this study may provide new insights into exercise physiology and genome biology in humans.

Paragonimiasis is a particular foodborne parasitic disease that is endemic to southern Kyushu, including Kagoshima Prefecture, Japan. We previously detected Paragonimus westermani triploid larvae in meat samples obtained from wild boars and sika deer hunted in Akune City, Kagoshima Prefecture. These mammals act as paratenic hosts and their meat is a source of human paragonimiasis. Paratenic host mammals and humans become infected with the lung fluke, P. westermani, following consumption of second intermediate hosts, freshwater crab species, namely, Geothelphusa dehaani or Sawagani in Japanese, and Eriocheir japonica or Mokuzugani in Japanese. Therefore, this study aimed to investigate the current infection status of P. westermani in freshwater crabs in Akune City. We collected freshwater crabs from 15 locations and found that the prevalence of P. westermani metacercariae was 1.6% for Sawagani (15 of 941 examined) and 22.1% for Mokuzugani (21 of 95 examined). Based on the morphological characterization of metacercariae and molecular analyses of the internal transcribed spacer 2 region and mitochondrial 16S rRNA gene region using PCR-restriction fragment length polymorphism and sequencing, all metacercariae were identified as the triploid form of P. westermani. These results indicate that Sawagani and Mokuzugani serve as second intermediate hosts to maintain the life cycle of triploid P. westermani. Further, infection in crabs potentially leads to subsequent P. westermani infections in wild mammals, including wild boars and sika deer, both of which are considered important types of game meat in Japan.

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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 induced oxidative stress. Small intestinal renalase expression was regulated by NF-κB p65 and was considered to be a defense mechanism against oxidative stress., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Tumor hypoxia drastically changes cancer phenotypes, including angiogenesis, invasion, and cell death. Gangliosides are sialic acid-containing glycosphingolipids that are ubiquitously distributed on plasma membranes and are involved in many biological processes, such as the endoplasmic reticulum stress response and apoptosis. In this study, we investigated the regulation and function of glycosphingolipids, which associate with lipid raft on mammalian plasma membranes under hypoxic condition., Methods: B16F10 melanoma cells were subjected to chemical hypoxia and low pO 2 condition, and the effect of hypoxia on expression of GM3 synthase were analyzed. Cellular resistance to oxidative stress was analyzed in GM3S-KO B16F10 cells., Results: Hypoxia treatment decreased the expression of ganglioside GM3 synthase (GM3S; ST3GAL5), which synthesizes the common substrate of ganglioside biosynthesis. RNA interference of hypoxia inducible factor 1 subunit alpha (HIF-1α) inhibited hypoxia-induced GM3S suppression. Additionally, GM3S deficiency increased cellular resistance to oxidative stress and radiation therapy via upregulation of ERK., Conclusions: Altered synthesis of glycosphingolipids downstream of HIF-1α signaling increased the resistance of melanoma cells to oxidative stress. Furthermore, GM3 has important role on cellular adaptive response to hypoxia., General Significance: This study indicates that tumor hypoxia regulates therapy-resistance via modulation of ganglioside synthesis., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Background: In this study, we aimed to investigate the effect of DNA methyltransferase 1 gene (DNMT1) expression on leukemia cell proliferation., Methods: Following stimulation with interferon-α (IFN-α) or methylation inhibitor for three days, we evaluated changes in the DNMT1 expression levels, cell proliferation activity, and Bcl-2-Associated X Protein (BAX) expression levels in the chronic myelogenous leukemia cell line K562 and the acute monocytic leukemia cell line THP-1., Results: DNMT1 expression levels and cell proliferation activity decreased in K562 and THP-1 cells, whereas BAX expression levels increased., Conclusions: These results suggest that the enzymatic activity of DNMT1 promotes the proliferation of tumor cells and that tumor cell proliferation can be suppressed by inhibiting DNMT1 enzymatic activity. Furthermore, because DNA methylation is associated with apoptosis, a process critical to cell growth and injury in leukemia, assessing DNMT1expression levels might help in treatment decisions for leukemia patients.

Redox-mediated modulation of cysteine (Cys) thiols has roles in various pathophysiological functions. We recently found that formation of disulfide-linked complexes of apolipoprotein (apo) E3 prevented apoE3 from irreversible oxidation. In this report, the influence of modification of Cys thiols in apoE2 and apoE3 on interactions with lipids was investigated. The apoE redox status was examined by a band-shift assay using a maleimide compound, and interactions with lipids were evaluated by a kinetic assay using dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and non-denaturing polyacrylamide gel electrophoresis. A reduction in DMPC clearance activity of apoE2 and apoE3 but not apoE4 was observed. Although hydrogen peroxide-induced oxidation decreased the clearance activity of the isoforms, apoE2 showed the greatest residual activity. Both Cys thiol masking and dimerization decreased the activity of apoE2 and apoE3 but not apoE4. In contrast, apoAII preincubation markedly increased the activity (apoE2 > apoE3 > apoE4), in accordance with the formation of apoE-AII and apoAII-E2-AII complexes. ApoAII preincubation also reduced the particle size of apoE-DMPC liposome complexes, especially for apoE2. Redox-mediated modification of Cys thiols of apoE2 or apoE3, especially disulfide bond formation with apoAII, affects lipid metabolism and consequently may be responsible for the diverse isoform specificity of apoE.

To determine the influence of right bundle branch block (RBBB) on right ventricular (RV) size and function, we investigated the association between complete RBBB (CRBBB) and RV volume, function, and dyssynchrony by three-dimensional echocardiography. In this retrospective, cross-sectional study, 103 consecutive patients with adequate three-dimensional echocardiographic images were divided into the CRBBB, middle-range QRS, and narrow QRS group. RV volumetric and functional data were compared between the three groups. Among the 103 patients (44.8 ± 18.7 years, 50 men), the CRBBB group comprised 26 (25%) patients and the middle-range QRS group comprised 48 (47%). The CRBBB group showed a significant contraction delay in the RV inlet free wall and outflow tract; larger RV end-diastolic and systolic volume index (RV-EDVI, RV-ESVI); and lower RV systolic function. On dividing the CRBBB patients into two (with or without mechanical dyssynchrony), those with RV dyssynchrony showed larger RV-EDVI (121 ± 45 vs. 85 ± 25 mL/m 2 , P = 0.019) and RV-ESVI (93 ± 42 vs. 56 ± 20 mL/m 2 , P = 0.009) and smaller RV ejection fraction (24 ± 11 and 34 ± 11%, P = 0.026) than those without RV dyssynchrony. RV dyssynchrony in CRBBB patients might have an adverse effect on RV volume and function. Three-dimensional speckle-tracking echocardiography could provide additional and beneficial data during assessment of RV dyssynchrony.

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 pulse wave transit time (PWTT) is easily measured as the time from the R wave of an electrocardiogram to the arrival of the pulse wave measured by an oxygen saturation monitor at the earlobe. We investigated whether the change of PWTT during exercise testing reflects cardiopulmonary function. Eighty-nine cardiac patients who underwent cardiopulmonary exercise testing (CPX) were enrolled. We analyzed the change of PWTT during exercise and the relationship between the shortening of the PWTT and CPX parameters. PWTT was significantly shortened from rest to peak exercise (204.6 ± 33.6 vs. 145.6 ± 26.4 msec, p < 0.001) in all of the subjects. The patients with heart failure had significantly higher PWTT at peak exercise than the patients without heart failure (152.7 ± 27.1 vs. 140.4 ± 24.8 msec, p = 0.031). The shortening of PWTT from rest to peak exercise showed significant positive correlations with the peak O 2 uptake (VO 2 ) (r = 0.56, p < 0.001), anaerobic threshold (r = 0.40, p = 0.016), and % increase of systolic blood pressure during exercise (r = 0.75, p < 0.001), and a negative correlation with the slope of the increase in ventilation versus the increase in CO 2 output (VE-VCO 2 slope) (r = - 0.42, p = 0.010) in the patients with heart failure. PWTT was shortened during exercise as the exercise intensity increased. In the patients with heart failure, the shortening of PWTT from rest to peak exercise was smaller in those with lower exercise capacity and those with higher VE-VCO 2 slope, an established index known to reflect the severity of heart failure.

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., Competing Interests: The authors declare there are no competing interests., (©2020 Aoki et al.)

Background: There are currently no therapies that can improve prognosis in cases of heart failure (HF) with preserved ejection fraction (EF). We hypothesized that there is a point of no return after which no response to treatment is noted and that for the prevention of hypertensive HF this point may be determined by left ventricle (LV) strain, in the prevention of hypertensive HF. Here an angiotensin-converting enzyme inhibitor (ACE-I) was initiated based on myocardial strain imaging and its effects were determined in an animal model., Methods: Thirty-two male Dahl salt-sensitive rats, age 6 weeks, were divided into six experimental groups and compared with low-salt (n = 8) and high-salt control groups (n = 8). In the early treatment group, ACE-I was administered from the age of 6 weeks (n = 4); in the longitudinal strain (LS) group, at 10-12 weeks when LS impairment was >-21% (n = 4); in the circumferential strain (CS) group, at 16-18 weeks when CS impairment was >-18% (n = 4); and in the EF group, at 20 weeks when EF was <55% (n = 4). Subsequently, all rats were sacrificed at 23 weeks age, the LV and lung weight were measured, and pathologic analyses were performed., Results: At 23 weeks of age, the lung and LV weights increased in the high-salt control, EF, and CS groups, whereas the lung and LV weights in the LS and early groups were similar to those in the low-salt control group. The percentage of area of subendocardial fibrosis was >6% in the high-salt control, EF, and CS groups and <3% in the LS, early, and low-salt groups. Serial echocardiography demonstrated LS improvement in the LS group; however, the CS and EF groups showed no differences., Conclusions: Heart failure-related lung congestion was prevented when ACE-I was administered soon after LS impairment, accompanied by suppression of cardiac hypertrophy and fibrosis, thereby suggesting that the point of no return of myocardial remodeling due to hypertension was present after LS but before CS impairment., (Copyright © 2019 American Society of Echocardiography. Published by Elsevier Inc. All rights reserved.)

Ascaris lumbricoides or roundworm is one of the key soil-transmitted helminths affecting humans. A small number of infections continue to occur in Japan, suggesting plant foodstuff contamination as the source of infection. To understand the current status of ascariasis incidence and to identify potential sources of infection, we extensively surveyed the available literature and collected data from testing facilities that examined clinical samples or foodstuffs. We observed that from 2002 onwards, there was a decrease in the number of ascariasis cases reported in scientific journals. Data from a clinical testing facility indicated that the number of detected cases declined remarkably from 2009. Foodstuff testing facilities reported that 11 of 10,223 plant foodstuff specimens were contaminated with anisakid nematodes but not with Ascaris. Imported kimchi was suspected as the most probable source of ascarid nematode infection, as one Ascaris egg-positive sample was detected among 60 kimchi samples in a testing facility. Therefore, the sources of Ascaris infection are still not fully known and need to be clarified to establish preventive countermeasures to safeguard Ascaris infections that continue to occur in Japan.

With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.

Reversible redox modification of cysteine thiols is crucial for protecting proteins from irreversible detrimental change. However, the physiological significance of the redox modification of apolipoprotein (apo) E is unclear. Here, we hypothesized that the disulfide-linked complexes of apoE3 corresponding to the representative reversible-modified apoE3 play a protective role against oxidative stress. The effects of disulfide bond formation on oxidative stress on apoE3 were evaluated with a band-shift assay. Maleimide-labeled apoE3 and unlabeled apoE3 were defined as the reduced (r)-apoE3 and non-reduced (nr)-apoE3 forms, respectively. Hydrogen peroxide-induced oxidation decreased for reduced-form apoE (r-apoE3) but increased for nr-apoE3. Induction of apoE3-AII complex formation with excess of apoAII markedly suppressed the oxidative stress-induced increase in nr-apoE3 ( P <0.001) and enhanced homodimer formation. The apoE3-AII complex was more dominant in high-density lipoprotein (HDL) than in very low-density lipoprotein. Under oxidative stress, HDL showed a significant decrease, rather than an increase, in nr-apoE3 levels with a concomitant significant increase in apoE3-AII levels ( P <0.005). This finding suggests that the majority of nr-apoE3 in HDL exists in a reversible oxidized form. The apoE3-AII complex, formed from the reversible oxidized apoE3, is beneficial for maintaining the redox equilibrium of apoE3 by preventing the modification of apoE3 to its irreversible oxidized form. The apoE3-AII complex may be possibly implicated in the pathophysiology of various apoE-related diseases., (© 2019 The Author(s).)

Game meat potentially harbors a number of parasitic and bacterial pathogens that cause foodborne disease. It is thus important to monitor the prevalence of such pathogens in game meats before retail and consumption to ensure consumer safety. In particular, Sarcocystis spp. and Shiga toxin-producing Escherichia coli (STEC) have been reported to be causative agents of food poisoning associated with deer meat consumption. To examine the prevalence of these microbiological agents on-site at a slaughterhouse, the rapid, simple and sensitive detection method known as the "DNA strip" has been developed, a novel tool combining loop-mediated isothermal amplification and a lateral flow strip. This assay has achieved higher sensitivity and faster than conventional PCR and is suitable for on-site inspection.

Glucocorticoids have various medical uses but are accompanied by side effects. The glucocorticoid receptor (GR) has been reported to regulate the clock genes, but the underlying mechanisms are incompletely understood. In this study, we focused on the suppressive effect of the GR on the expression of Rev-erbα (Nr1d1), an important component of the clock regulatory circuits. Here we show that the GR suppresses Rev-erbα expression via the formation of a complex with CLOCK and BMAL1, which binds to the E-boxes in the Nr1d1 promoter. In this GR-CLOCK-BMAL1 complex, the GR does not directly bind to DNA, which is referred to as tethering. These findings provide new insights into the role of the GR in the control of circadian rhythm., (© 2019 Federation of European Biochemical Societies.)

It is unclear whether γδ T cells are involved in humoral immunity against Plasmodium infection. Here, we show that B-cell-immunodeficient mice and γδ T-cell-deficient mice were incapable of protecting against Plasmodium berghei XAT parasites. γδ T-cell-deficient mice developed reduced levels of antigen-specific antibodies during the late phase of infection. The numbers of follicular helper T cells and germinal centre B cells in γδ T-cell-deficient mice were lower than in wild-type mice during the late phase of infection. Expression profiling of humoral immunity-related cytokines in γδ T cells showed that interleukin-21 (IL-21) and interferon-γ (IFN-γ) are increased during the early stage of infection. Furthermore, blockade of IL-21 and IFN-γ signalling during the early stage of infection led to reduction in follicular helper T cells and germinal centre B cells. γδ T-cell production of IL-21 and IFN-γ is crucial for the development and maintenance of follicular helper T cells and germinal centre B cells during the late phase of infection. Our data suggest that γδ T cells modulate humoral immunity against Plasmodium infection., (© 2018 John Wiley & Sons Ltd.)

In the management of heart failure (HF), decongestion is critical for improving clinical outcomes in addition to patients' symptoms. Although physicians accept this theory, there are no established markers for the achievement of optimal decongestion status. Organ congestion, in particular liver and kidney congestion, has recently attracted substantial attention. Ultrasound methods have been introduced for assessing organ congestion, although this is merely a preliminary step. Here, we review the clinical implications of the assessment of organ congestion by ultrasound., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

Background: We investigated the effects of the dipeptidyl peptidase 4 inhibitor teneligliptin on cardiac function and hemodynamics during heart failure in hypertensive model rats., Methods and Results: Fifty-five male Dahl salt-sensitive rats were divided into 4 groups: control group (0.3% NaCl chow; n = 13), hypertension (HT) group (8% NaCl chow; n = 20), HT-early TNL group (8% NaCl chow and teneligliptin from 6 weeks; n = 10), and HT-late TNL group (8% NaCl chow and teneligliptin from 10 weeks; n = 12). Hemodynamic measurement and tissue analyses were performed at 18 weeks. In all of the HT groups, systolic blood pressures were similarly elevated (P = .66) and heart weights similarly increased (P = .36) with and without TNL administration. LV end-diastolic dimension was significantly enlarged only in the HT-early TNL group compared with the control group (P = .025). Histologic analysis showed less fibrosis (P = .008) and cardiomyocyte widths (P = .009) in the HT-early TNL group compared with the HT group. On hemodynamic analysis, only the HT group showed significant LV end-diastolic pressure elevation (P = .049) and lung congestion (P < .001) compared with the control group., Conclusions: These results suggest that teneligliptin prevents concentric LV hypertrophy, fibrosis, and development of congestive heart failure in Dahl salt-sensitive rats. Teneligliptin may inhibit pressure-overload hypertrophic adaption and result in LV eccentric hypertrophy with reduced LV ejection fraction., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Accurate assessment of global and regional right ventricular (RV) systolic function is challenging. The aims of this study were to confirm the reliability and feasibility of a three-dimensional (3D) speckle-tracking echocardiography (STE) system, using comparison with cardiac magnetic resonance imaging (CMR), and to assess the contribution of regional RV function to global function., Methods: In a retrospective, cross-sectional study setting, RV volumetric data were studied in 106 patients who were referred for both CMR and 3D echocardiography within 1 month. Three-dimensional STE-derived area strain, longitudinal strain, and circumferential strain were assessed as global, inlet, outflow, apical, and septal segments., Results: Seventy-five patients (70%) had adequate 3D echocardiographic data. RV measurements derived from 3D STE and CMR were closely related (RV end-diastolic volume, R 2  = 0.84; RV end-systolic volume, R 2  = 0.83; RV ejection fraction [RVEF], R 2  = 0.70; P < .001 for all). RVEF and RV end-diastolic volume from 3D STE were slightly but significantly smaller than CMR values (mean differences, -2% and -10 mL for RVEF and RV end-diastolic volume, respectively). Among conventional echocardiographic parameters for RV function (tricuspid annular plane systolic excursion, fractional area change, S' of the tricuspid annulus, RV free wall two-dimensional longitudinal strain), only fractional area change was significantly related to RVEF (r = 0.34, P = .003). Among segmental 3D strain variables, inlet area strain (r = -0.56, P < .001) and outflow circumferential strain (r = -0.42, P < .001) were independent factors associated with CMR-derived RVEF., Conclusions: RV volume and RVEF determined by 3D STE were comparable with CMR measurements. Regional RV wall motion showed that heterogeneous segmental deformations affect global RV function differently; specifically, inlet area strain and outflow circumferential strain were significant factors associated with RVEF in patients with underlying heart diseases., (Copyright © 2017 American Society of Echocardiography. Published by Elsevier Inc. All rights reserved.)

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have both anti-diabetic and anti-obesity effects. However, the precise mechanism of the anti-obesity effect remains unclear. We previously demonstrated that the glycogen depletion signal triggers lipolysis in adipose tissue via liver-brain-adipose neurocircuitry. In this study, therefore, we investigated whether the anti-obesity mechanism of SGLT2 inhibitor is mediated by this mechanism. Diet-induced obese mice were subjected to hepatic vagotomy (HVx) or sham operation and loaded with high fat diet containing 0.015% tofogliflozin (TOFO), a highly selective SGLT2 inhibitor, for 3 weeks. TOFO-treated mice showed a decrease in fat mass and the effect of TOFO was attenuated in HVx group. Although both HVx and sham mice showed a similar level of reduction in hepatic glycogen by TOFO treatment, HVx mice exhibited an attenuated response in protein phosphorylation by protein kinase A (PKA) in white adipose tissue compared with the sham group. As PKA pathway is known to act as an effector of the liver-brain-adipose axis and activate triglyceride lipases in adipocytes, these results indicated that SGLT2 inhibition triggered glycogen depletion signal and actuated liver-brain-adipose axis, resulting in PKA activation in adipocytes. Taken together, it was concluded that the effect of SGLT2 inhibition on weight loss is in part mediated via the liver-brain-adipose neurocircuitry., (Copyright © 2017 Elsevier Inc. All rights reserved.)