i-tRAP (individual tRNA acylation PCR): A convenient method for selective quantification of tRNA charging.

Each transfer RNA (tRNA) is aminoacylated (charged) with a genetic codon-specific amino acid at its 3' end. Charged tRNAs are primarily used for translation, whereas fluctuations in charged tRNA fractions are known to reflect cellular response to stress. Here we report the development of individual tRNA-acylation using PCR (i-tRAP), a convenient PCR-based method that can specifically quantify individual tRNA charging ratio. In this i-tRAP method, demethylases remove base methylations which are problematic for reverse transcription reaction, and ß-elimination reaction specifically removes the 3' end of adenine residue in uncharged tRNA. Subsequent TaqMan MGB qRT-PCR can distinguish between cDNA of charged tRNA and uncharged tRNA. By using this method, we revealed that the charging ratio of tRNAGln(CUG) was changed in response to amino acid starvation and also the charging ratio of tRNAGln(CUG) in senescent cells was lower than in young cells under starvation conditions. i-tRAP can be applicable to the quantification of charging ratio of various tRNAs, and provides a simple and convenient method for analyzing tRNA charging.

Impact of the interpregnancy interval after cesarean delivery on subsequent perinatal risks: a retrospective study.

PURPOSE: To assess the impact of the interpregnancy interval (IPI) after cesarean delivery on the risks of adverse perinatal events during subsequent pregnancies. METHODS: We retrospectively examined perinatal outcomes of subsequent pregnancies of women whose most recent birth experience involved cesarean delivery at our hospital between January 2014 and December 2019. IPI was defined as the time between live birth and subsequent conception. Three IPI groups: < 18 months, 18-60 months, and > 60 months, were assessed. The risks of preterm birth, preeclampsia, placenta previa, placental abruption, fetal growth restriction, and successful vaginal birth were compared among the three IPI groups using uni- and multivariate analyses. RESULTS: We registered 592 births after cesarean delivery: 178, 288, and 126 in the IPI < 18 months, 18-60 months, and > 60 months groups, respectively. The groups did not differ significantly regarding perinatal outcomes. The multivariate analysis revealed no significant differences in the risks of adverse perinatal outcomes among all groups. The odds ratios (ORs) for preterm birth at < 37 weeks of gestation were 1.24 and 1.64 for those in the < 18 months and > 60 months groups, respectively (P = 0.362 and P = 0.055, respectively). The groups did not differ significantly regarding vaginal birth success rate (ORs 1.72 for the < 18 months group, 0.49 for the > 60 months group; P = 0.486 and P = 0.446, respectively). CONCLUSION: After cesarean delivery, IPIs shorter than 18 months and longer than 60 months do not significantly impact the risks of adverse perinatal outcomes or successful vaginal birth compared with IPIs of 18-60 months.

Prevalence, Risk Factors, and Perinatal Outcomes of Velamentous Umbilical Cord Insertion in Twin Pregnancies: A Single-Center Retrospective Study.

BACKGROUND: The effect of velamentous cord insertion (VCI) on perinatal outcomes in twin pregnancies is unclear due to conflicting findings. This retrospective study aimed to examine VCI prevalence and related risk factors in twin pregnancies and its association with adverse perinatal outcomes. METHODS: Women with twin pregnancies who delivered between January 2012 and December 2021 in a single tertiary hospital were included. The participants were divided into dichorionic (DC) and monochorionic diamniotic (MCDA) groups, and their maternal and fetal characteristics and VCI rates were compared. Logistic regression models were used to identify risk factors for VCI and VCI-related perinatal outcomes. RESULTS: Among the 694 twin pregnancies included in this study, the VCI rate was significantly higher in MCDA than in DC twins. Body mass index and MCDA twins were significant risk factors for VCI, whereas assisted reproductive technology pregnancy was a significant protective factor against VCI. In DC twins, VCI did not affect perinatal outcomes. In MCDA twins, VCI was a significant risk factor for fetal growth restriction, twin-to-twin transfusion syndrome, and preterm birth at <36 weeks. CONCLUSIONS: VCI was a prominent risk factor for adverse perinatal outcomes only in MCDA twins. Antenatal sonographic assessment of the umbilical cord insertion site would be beneficial.

Inulin-type fructans with different degrees of polymerization improve lipid metabolism but not glucose metabolism in rats fed a high-fat diet under energy restriction.

BACKGROUND: Inulin-type fructan ameliorates metabolic diseases associated with obesity in animals. However, relatively little information is available on the comparative effects of inulins with different degree of polymerization (DP) on the lipid or glucose metabolism. AIM: The objective of this study was to investigate the effect of inulins with various DP on metabolic disorders associated with obesity in rats fed a high-fat diet under food restriction. METHODS: Rats were fed a high-fat diet supplemented with 5 % inulin-GR (Raftiline GR), inulin-Tokachi (Tokachi), or inulin-HP (Raftiline HP) without cellulose for 28 days at normal energy intakes or 14.5 % energy restriction. RESULTS: Under food restriction, the dietary inulin-Tokachi (mean DP 15) and -HP (mean DP 24), but not -GR (mean DP 10), reduced (p < 0.05) the serum cholesterol and triglyceride levels, and liver triglyceride concentration in rats, compared to the control diet. The cecal neutral steroid, bile acid, and propionate concentrations in the Tokachi and HP groups were higher (p < 0.05) than in the CONT group, and the cecal Bifidobacterium count in the Tokachi group was higher (p < 0.05) than in the other groups. CONCLUSIONS: Findings suggest that, depending on DP, dietary supplementation with inulin (DP 15 or DP 24) in rats fed a high-fat diet, regardless of food intake, positively modulates lipid metabolism and fecal microbiota but not glucose metabolism.

Dietary L-cysteine improves the antioxidative potential and lipid metabolism in rats fed a normal diet.

L-cysteine works as a precursor of the antioxidant, glutathione. We investigated the effects of L-cysteine (1% and 2%) on lipid metabolism and the antioxidative system in rats fed a normal diet. Administering L-cysteine dependently decreased the food intake, fat mass weight and body weight dose. Dietary L-cysteine also decreased the triglyceride levels in the serum and liver. However, there were no significant differences in the hepatic TBARS and glutathione (GSH) levels among the groups. The activities of catalase and glutathione reductase in the rats receiving 2% L-cysteine were significantly higher (p<0.05) than in the control rats. These results suggest that dietary L-cysteine dose-dependently affected the antioxidative enzyme activities, and the lipid levels in the serum and liver which might be related to the reduced food intake.

GCN2 kinase-mediated upregulation of ubiquitin C maintains intracellular glutamine level and tRNAGln (CUG) charging under amino acid starvation.

Each tRNA is aminoacylated (charged) with a genetic codon-specific amino acid. It remains unclear what factors are associated with tRNA charging and how tRNA charging is maintained. By using the individual tRNA acylation PCR method, we found that the charging ratio of tRNAGln (CUG) reflects cellular glutamine level. When uncharged tRNAGln (CUG) increased under amino acid starvation, the kinase GCN2, which is a key stimulator of the integrated stress response, was activated. Activation of GCN2 led to the upregulation of ubiquitin C (UBC) expression. Upregulated UBC, in turn, suppressed the further reduction in tRNAGln (CUG) charging levels. Thus, tRNA charging is sensitive to intracellular nutrient status and is an important initiator of intracellular signaling.

Anti-obesity role of adzuki bean extract containing polyphenols: in vivo and in vitro effects.

BACKGROUND: The aim of this work was to evaluate the effects of polyphenol-rich adzuki bean extract on lipid metabolism, triglyceride accumulation and proinflammatory cytokine secretion in vivo and in vitro. RESULTS: For the in vivo study, rats were divided into four groups: group C was fed a control diet, group A was fed the control diet with 1% adzuki bean extract, group CF was fed a high fat diet, and group AF was fed a high fat diet with 1% adzuki bean extract. For the in vitro study, the ability of adzuki bean extract to suppress triglyceride incorporation, glycerol phosphate dehydrogenase activity and inflammatory response was investigated in cultured human adipocytes. Data from the animal study showed that adzuki bean extract improved lipid metabolism in both the normal and high-fat diet groups. Adzuki bean extract treatment in the high-fat group resulted in significant reductions in total hepatic lipid accumulation and lipid secretion into the feces. Incubation of adipocytes with adzuki bean extract significantly decreased triglyceride accumulation, glycerol phosphate dehydrogenase activity and inflammatory responses without affecting cell viability. CONCLUSION: The results of this study demonstrate that adzuki bean extract has high potential to serve as a natural anti-obesity agent.

Generation and functional analysis of monoclonal antibodies against the second extracellular loop of human M3 muscarinic acetylcholine receptor.

The M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the activation of salivary and lachrymal glands. The M3R contains four extracellular domains (the N-terminal, and the first, second, and third extracellular loops), and we recently detected antibodies against each of these four domains in a subgroup of patients with Sjögren's syndrome (SS). Functional analysis indicated that the influence of such anti-M3R antibodies on salivary secretion might differ based on the epitopes to which they bind. To clarify the relationship between B-cell epitopes on the M3R and its function, we generated two hybridomas producing anti-M3R monoclonal antibodies against the second extracellular loop of M3R (anti-M3R(2nd) mAbs) and analyzed their function by Ca(2+)-influx assays, using a human salivary gland (HSG) cell line. These two anti-M3R(2nd) mAbs suppressed Ca(2+)-influx in the HSG cells induced by cevimeline stimulation, suggesting that autoantibodies against the second extracellular loop of M3R could be involved in salivary dysfunction in patients with SS.

Hierarchical surface wrinkles and bumps generated on chitosan films having double-skin layers comprising topmost carrageenan layers and polyion complex layers.

Surface wrinkling to fabricate hierarchical surface topographies has attracted much attention because of the potential and multifunctional applications of hierarchical surface wrinkles beyond uniform wrinkles. Although many reports have described the preparation of hierarchical wrinkles induced by mechanical stress and heat, fabrication through drying-induced shrinkage has hardly been reported. Here we introduce hierarchical surface wrinkles and bumps generated on a chitosan film via the preparation of double-skin layers with κ- and ι-carrageenans, respectively, and subsequent drying. Double-skin layers are fabricated on a swollen chitosan film, called a chitosan hydrogel film, that is soaked first in κ- or ι-carrageenan solution and then in water to remove excess adsorbed κ- or ι-carrageenan. After the film is dried, hierarchical microscopic surface architectures are observed. In the case of the κ-carrageenan system, the wrinkles are hierarchical, consisting of wrinkles (6.2 ± 2.8 µm) that have smaller buckles (0.23 ± 0.09 µm). We reveal that the wrinkles or the smaller buckles are caused by plane inhomogeneous shrinkage between the κ-carrageenan layer and the chitosan film or by the aggregation of the κ-carrageenan layer upon drying, respectively. Interestingly, the ι-carrageenan system showed hierarchical bumps consisting of semispherical bumps (5.6 ± 2.1 µm) that have smaller bumps (0.78 ± 0.27 µm). We reveal that the larger bumps are generated during the immersion of the chitosan hydrogel film into ι-carrageenan solution. The smaller bumps are generated by the aggregation of the ι-carrageenan layer that occurs during drying; this process requires the plane compression strain caused by the shrinkage of the chitosan hydrogel film.

Seasonal variation in the prevalence of Gram-negative bacilli in sputum and urine specimens from outpatients and inpatients.

Objectives: To determine whether the prevalence of gram-negative bacilli (GNB; Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli) in sputum and urine specimens from outpatients and inpatients differed by season and according to temperature and humidity changes. Methods: In this retrospective study, microbiologic data for adult patients from 2008 to 2019 were retrieved from the electronic database of a hospital in Japan. Data were categorized by specimen type (sputum and urine) and specimen collection (outpatient and inpatient). Associations between variables were assessed using Spearman's rank correlation coefficients. Differences between groups were assessed using Pearson's chi-square test and analysis of discrete variance. Results: Among inpatients, the frequencies of P. aeruginosa and K. pneumoniae isolation from sputum specimens were higher in summer and autumn. The frequency of P. aeruginosa isolation from urine specimens was higher in autumn. These seasonal trends were observed in specimens from both outpatients and inpatients. No seasonal trend was observed in the frequency of E. coli isolation. Mean monthly temperature was positively correlated with the frequency of isolating P. aeruginosa (r=0.2198, p=0.0081) and K. pneumoniae (r=0.3443, p=0.00002) from sputum as well as with the frequency of isolating K. pneumoniae (r=0.1905, p=0.0222) from urine. Mean monthly humidity was positively correlated with the frequency of isolating K. pneumoniae (r=0.2602, p=0.0016) from sputum. Conclusions: GNB were isolated more frequently in summer and autumn than in other seasons. These seasonal trends were observed for both outpatient and inpatient specimens. Seasonality should be considered for optimal infection control of GNB in hospitals.

Amelioration of D-galactosamine-induced acute liver injury in rats by dietary supplementation with betaine derived from sugar beet molasses.

The effects of betaine supplementation on D-galactosamine-induced liver injury were examined in terms of hepatic and serum enzyme activities and of the levels of glutathione and betaine-derived intermediates. The rats induced with liver injury showed marked increases in serum enzyme activity, but those receiving dietary supplementation of 1% betaine showed enzyme activity levels similar to a control group without liver injury. Administration of betaine also increased both hepatic and serum glutathione levels, even following D-galactosamine injection. The activity of glutathione-related enzymes was markedly decreased following injection of D-galactosamine, but remained comparable to that of the control group in rats receiving 1% betaine. The concentrations of hepatic S-adenosyl methionine and cysteine showed similar trends to that observed for hepatic glutathione levels. These results indicate that 1% betaine has a hepatoprotective effect by increasing hepatic and serum glutathione levels along with glutathione-related enzyme activities in rats.

Effects of feeding potato pulp on cholesterol metabolism and its association with cecal conditions in rats.

To clarify the functional properties of potato pulp (PP), a waste product resulting from extraction of starch from potatoes, we examined the effects of PP on cholesterol metabolism and cecal conditions in rats. Plasma total cholesterol (T-Chol) levels were lower in rats fed a PP-supplemented diet for four weeks than in those fed a control diet. Cecal pH was lowered due to an increase in the levels of cecal total short-chain fatty acids, especially butyrate, in the PP group compared to the control group. Furthermore, animals fed with the PP-supplemented diet showed increased cecal ratios of Lactobacillus and Clostridia and decreased cecal ratios of Bacteroides and Gammaproteobacteria with slightly negative and positive correlations with plasma T-Chol levels, respectively. In conclusion, ingestion of PP for four weeks is likely to improve both cecal conditions and cholesterol metabolism, suggesting that PP has prebiotic effects.

Estimation of water intake from food moisture in the Japanese diet using a cooking-based conversion factor for water content.

This study aimed to estimate the water intake of Japanese people from food moisture. For this purpose, we described a water conversion factor for cooking (WCFC) that focuses on how cooking changes the water content of complete dishes and not food ingredients, and investigated methods for calculating the water content of cooked dishes. In this study, we utilized 129 dishes commonly consumed by Japanese people. We measured the water content of the dishes before and after cooking and calculated the percentage of residual water after cooking. The dishes were divided into staple foods or side dishes, and categorized based on residual percentage in water content and cooking method. The percentage of residual water after cooking change in water content in each category was expressed in terms of the WCFC, which is the water content of the cooked dish relative to the uncooked dish. In total, there were 43 categories with a WCFC range of 0.40 to 1.33. Our results revealed that the water content of food is not simply changed by heating-based cooking methods, but by a complex combination of cooking operations. Furthermore, by fitting WCFC to a culinary structuralism theory, it can potentially be utilized for all cuisines and not just dishes in Japan. Additionally, we found that the water content of a day's worth of food calculated using WCFC and actual measurements barely differed, indicating that WCFC could potentially be used to estimate water intake from food moisture. PRACTICAL APPLICATION: This research can be used in nutrition surveys to estimate water intake from food moisture.

The effects of trunk endurance training on running kinematics and its variability in novice female runners.

The functional importance of trunk muscle strength for running movement is widely recognised, but the kinematic effects of undertaking specific training are unclear. This study investigated the change in joint angle and its variability during running following trunk muscle training. Eighteen young female and novice runners participated. Using Plug-in-gait model with infrared markers attached to the body surface, the lower limb and lumber angles during running were measured, and the variability was examined by calculating the coefficient variation and Lyapunov exponent. Measurements of trunk endurance were also performed. Over four weeks of training, the subjects performed trunk muscle endurance trainings three times a week. Following this intervention, trunk endurance was found to have significantly increased. The Lyapunov exponent of lumbar flexion-extension angle also significantly increased. Moreover, a decreased range of the ankle angle and increased range of the hip angle were observed following the training. These results demonstrate that the trunk training promoted adjustments to lumbar movement and altered the movement patterns of the participants' lower limbs during running.

Characterization of Plasmodium falciparum Pantothenate Kinase and Identification of Its Inhibitors From Natural Products.

Coenzyme A (CoA) is a well-known cofactor that plays an essential role in many metabolic reactions in all organisms. In Plasmodium falciparum, the most deadly among Plasmodium species that cause malaria, CoA and its biosynthetic pathway have been proven to be indispensable. The first and rate-limiting reaction in the CoA biosynthetic pathway is catalyzed by two putative pantothenate kinases (PfPanK1 and 2) in this parasite. Here we produced, purified, and biochemically characterized recombinant PfPanK1 for the first time. PfPanK1 showed activity using pantetheine besides pantothenate, as the primary substrate, indicating that CoA biosynthesis in the blood stage of P. falciparum can bypass pantothenate. We further developed a robust and reliable screening system to identify inhibitors using recombinant PfPanK1 and identified four PfPanK inhibitors from natural compounds.

Pathogenic role of immune response to M3 muscarinic acetylcholine receptor in Sjögren's syndrome-like sialoadenitis.

The aim of this study was to clarify the role of the immune response to muscarinic type 3 receptor (M3R) in the pathogenesis of Sjögren's syndrome (SS). M3R(-/-) mice were immunized with murine M3R peptides and their splenocytes were inoculated into Rag1(-/-) (M3R(-/-)→Rag1(-/-)) mice. M3R(-/-)→Rag1(-/-) mice had high serum levels of anti-M3R antibodies and low saliva volume. Histological examination showed marked infiltration of mononuclear cells in the salivary glands and immunohistochemistry demonstrated that the majority of these cells were CD4(+) T cells with a few B cells and several IFN-γ- and IL-17-producing cells. Apoptotic cells were present in the salivary glands of M3R(-/-)→Rag1(-/-) mice. Moreover, transfer of only CD3(+) T cells from M3R(-/-) immunized with M3R peptides into Rag1(-/-) mice resulted in cell infiltration and destruction of epithelial cells in the salivary glands, suggesting that M3R reactive CD3(+) T cells play a pathogenic role in the development of autoimmune sialoadenitis. Our findings support the notion that the immune response to M3R plays a crucial role in the pathogenesis of SS-like autoimmune sialoadenitis.

Identification of compounds in red wine that effectively upregulate aquaporin-3 as a potential mechanism of enhancement of skin moisturizing.

In a previous clinical study, the moisture content in the stratum corneum of healthy Japanese women who consumed a beverage rich in oligomeric proanthocyanidins (OPCs) made from red wine extract was found to be higher than that in the control group. This finding suggested that OPCs can increase skin moisture content. In this study, we determined the expression level of aquaporin-3 (AQP3) in keratinocytes to elucidate the mechanism by which compounds in red wine grape increase moisture content in stratum corneum. Through in vitro studies, we confirmed that normal human epidermal keratinocytes (NHEK) incubated with red wine induced AQP3 expression. Furthermore, the supplementation of red wine fractions enriched in OPC was shown to increase AQP3 expression. Besides, the component of OPC-rich fractions that upregulated AQP3 expression was found to be a gallic acid (GA)-binding flavan-3-ol, particularly oligomeric compounds. We found that GA-binding OPC were able to upregulate AQP3 expression and that these compounds were enriched in red wine. Our findings might suggest that the mechanism of enhancement of moisture content in stratum corneum by red wine might be via the upregulation of AQP3 expression in the epidermal keratinocytes.

Effect of Increased Daily Water Intake and Hydration on Health in Japanese Adults.

Increased hydration is recommended as healthy habit with several merits. However, supportive data are sparse. To assess the efficacy of increased daily water intake, we tested the effect of water supplementation on biomarkers in blood, urine, and saliva. Twenty-four healthy Japanese men and 31 healthy Japanese women with fasting blood glucose levels ranging from 90-125 mg/dL were included. An open-label, two-arm, randomized controlled trial was conducted for 12 weeks. Two additional 550 mL bottles of water on top of habitual fluid intake were consumed in the intervention group. The subjects drank one bottle of water (550 mL) within 2 h of waking, and one bottle (550 mL) 2 h before bedtime. Subjects increased mean fluid intake from 1.3 L/day to 2.0 L/day, without changes in total energy intake. Total body water rate increased with associated water supplementation. There were no significant changes in fasting blood glucose and arginine vasopressin levels, but systolic blood pressure was significantly decreased in the intervention group. Furthermore, water supplementation increased body temperature, reduced blood urea nitrogen concentration, and suppressed estimated glomerular filtration rate reduction. Additionally, existence of an intestinal microbiome correlated with decreased systolic blood pressure and increased body temperature. Habitual water supplementation after waking up and before bedtime in healthy subjects with slightly elevated fasting blood glucose levels is not effective in lowering these levels. However, it represents a safe and promising intervention with the potential for lowering blood pressure, increasing body temperature, diluting blood waste materials, and protecting kidney function. Thus, increasing daily water intake could provide several health benefits.

Induced Fetal Human Muscle Stem Cells with High Therapeutic Potential in a Mouse Muscular Dystrophy Model.

Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle-wasting disease caused by DYSTROPHIN deficiency. Cell therapy using muscle stem cells (MuSCs) is a potential cure. Here, we report a differentiation method to generate fetal MuSCs from human induced pluripotent stem cells (iPSCs) by monitoring MYF5 expression. Gene expression profiling indicated that MYF5-positive cells in the late stage of differentiation have fetal MuSC characteristics, while MYF5-positive cells in the early stage of differentiation have early myogenic progenitor characteristics. Moreover, late-stage MYF5-positive cells demonstrated good muscle regeneration potential and produced DYSTROPHIN in vivo after transplantation into DMD model mice, resulting in muscle function recovery. The engrafted cells also generated PAX7-positive MuSC-like cells under the basal lamina of DYSTROPHIN-positive fibers. These findings suggest that MYF5-positive fetal MuSCs induced in the late stage of iPSC differentiation have cell therapy potential for DMD.