Impact of P-Glycoprotein-Mediated Drug-Endogenous Substrate Interactions on Androgen and Blood-Brain Barrier Permeability.

This report focuses on pharmacokinetic drug-endogenous substrate interactions (DEIs). We hypothesized that P-glycoprotein (P-gp)-mediated DEI might affect androgen kinetics, especially its blood-brain barrier (BBB) permeability. The intracellular accumulation of the endogenous substrates of P-gp, testosterone (TES) and androstenedione (ADO) was increased by several tested drugs in uptake studies using P-gp overexpressing cells, indicating that these drugs inhibit P-gp-mediated efflux of TES of ADO from the cells. In a transport study using rat BBB kit, we found that the BBB limited the penetration of TES and ADO into the central nervous system. In addition, tested drugs that cause DEI were found to increase BBB permeability of TES and ADO via P-gp inhibition. In short, this study provides new findings regarding the possibility that DEI may affect the kinetics of endogenous substrates of P-gp.

Effectiveness of weight-loss prevention with continual nutrition counseling in postoperative outpatients with stage IA and IB gastric cancer.

Outpatient nutritional counseling by a registered dietitian is often performed to prevent weight loss, but evidence supporting this practice is insufficient. In this study, we aimed to clarify the effectiveness of four-time outpatient nutritional counseling in weight-loss prevention compared with conventional intervention limited to one-time nutritional counseling. This study was designed as a retrospective cohort study. The target population was postoperative patients with stage IA and IB gastric cancer. Groups that received one-time and four-time nutritional counseling included patients who underwent gastrectomy from May 2014 to April 2017 and May 2017 to December 2019, respectively. The one-time group received counseling at discharge; the four-time group received counseling at discharge, at the first outpatient visit, and at 3 and 6 months postoperatively. There were 58 patients in the one-time group and 27 patients in the four-time group, with a significant difference in length of hospital stay (p = 0.042). Thirty-six patients (62.1%) in the one-time nutritional counseling group and 12 (44.4%) in the four-time group had a weight loss of 5% or more from hospital discharge to 6 months postoperatively. The adjusted risk ratio for the effectiveness of four counseling sessions compared with one session was 0.69 (95% confidence interval 0.35-1.34). In subgroup analysis, the effect of nutritional guidance was greater for patients with body mass index ≥23 kg/m2, but this depended on the outcome and number of cases, and there was no essential difference between the groups. In postoperative patients with stage IA and stage IB gastric cancer, four sessions of outpatient nutrition counseling may be not superior to one counseling session in preventing weight loss.

Clinical usefulness of phase angle as an indicator of muscle wasting and malnutrition in inpatients with cardiovascular diseases.

Background and Objectives: Extracellular water is increased in patients with edema, such as those with chronic heart failure, and it is difficult to assess skeletal muscle mass with the skeletal muscle mass index when extracellular water is high. We investigated the relationship between phase angle and physical function, nutritional indices, and sarcopenia in patients with cardiovascular diseases, including chronic heart failure. Methods and Study Design: In 590 patients with cardiovascular diseases (372 men), handgrip strength, gait speed, and anterior mid-thigh muscle thickness by ultrasound were measured, and the skeletal muscle mass index, phase angle, and the extracellular water: total body water ratio were measured with a bioelectrical impedance analyzer, and presence of sarcopenia was evaluated. Results: Phase angle, but not the skeletal muscle mass index, was correlated with serum albumin (r = 0.377, p < 0.001) and hemoglobin values in women. Multivariate regression analysis showed that at the extracellular water: total body water ratio below 0.4, both phase angle and skeletal muscle mass index were independent determinants of handgrip strength and log mid-thigh muscle thickness in men, after adjustment for age and presence of chronic heart failure. In contrast, for the ratio of 0.4 or greater, after adjustment for age and presence of chronic heart failure, phase angle was a stronger independent determinant of handgrip strength and log mid-thigh muscle thickness than the skeletal muscle mass index in men. Conclusions: Phase angle is a good marker of muscle wasting and malnutrition in patients with cardiovascular disease, including chronic heart failure.

Development of Ethyl Methanesulfonate Mutant Edamame Soybean (Glycine max (L.) Merr.) Populations and Forward and Reverse Genetic Screening for Early-Flowering Mutants.

Induced mutation is a viable breeding strategy that is widely utilized in the development of elite plant varieties. We aimed to improve a variety of edamame by constructing novel mutant populations using the ethyl methanesulfonate in soybeans (Glycine max (L.) Merr.). In the M2 population, the flowering stage showed a considerable standard deviation compared to the wild type, confirming that the mutant populations had the expected DNA mutations. To identify the DNA mutations in the mutant populations, we used the targeting induced local lesions in genomes (TILLING) method, which is a reverse genetic method, to search for soybean flowering-related gene mutants. A total of 30 mutants from E1, E3, E4, and PhyA1 genes, which are known to be highly effective genes, or their homologous gene for flowering and maturation found in soybean quantitative trait locus analyses were isolated from our TILLING screening. Among these mutants, there were eleven nonsynonymous substitution mutants, one nonsense mutant, and two single nucleotide deletion mutants that could be expected to reduce or eliminate gene function. The e1, e3, and e4 mutants obtained in this study flowered considerably earlier than the wild type. In particular, the e1 mutant with a nonsynonymous substitution flowered approximately 1 month after sowing regardless of the sowing date, and its harvest date was approximately 1 month earlier than that of the wild type. Mutations identified using the TILLING method could not only be used as gel-based DNA markers with the same manipulation method, but the mutations could also be detected as DNA markers by the high-resolution melting method. These results indicate that mutations achieved without chromosome modification by crossbreeding are effective for early and practical improvement of superior varieties and that efficient selection of mutants by reverse genetics is an effective method for the identification of genetic modifications. The edamame mutant populations developed in this study are believed to possess various useful alleles which may be applicable in the search for mutations that lead to improved edamame yield and eating quality beyond the flowering stage.

Phase Angle as an Indicator of Sarcopenia, Malnutrition, and Cachexia in Inpatients with Cardiovascular Diseases.

Malnutrition is associated with sarcopenia, cachexia, and prognosis. We investigated the usefulness of phase angle (PhA) as a marker of sarcopenia, cachexia, and malnutrition in 412 hospitalized patients with cardiovascular disease. We analyzed body composition with bioelectrical impedance analysis, and nutritional status such as controlling nutritional status (CONUT) score. Both skeletal muscle mass index (SMI) and PhA correlated with age, grip strength and knee extension strength (p < 0.0001) in both sexes. The SMI value correlated with CONUT score, Hb, and Alb in males. Phase angle also correlated with CONUT score, Hb, and Alb in males, and more strongly associated with these nutritional aspects. In females, PhA was correlated with Hb and Alb (p < 0.001). In both sexes, sarcopenia incidence was 31.6% and 32.4%; PhA cut-off in patients with sarcopenia was 4.55° and 4.25°; and cachexia incidence was 11.5% and 14.1%, respectively. The PhA cut-off in males with cachexia was 4.15°. Multivariate regression analysis showed that grip strength and brain natriuretic peptide (BNP) were independent determinants of SMI, whereas grip strength, BNP, and Hb were independent determinants of PhA. Thus, PhA appears to be a useful marker for sarcopenia, malnutrition, and cachexia in hospitalized patients with cardiovascular disease.

Trends in Prevalence of Non-Valvular Atrial Fibrillation and Anticoagulation Therapy in a Japanese Region　- Analysis Using the National Health Insurance Database.

BACKGROUND: Direct oral anticoagulants (DOACs) are effective in reducing thromboembolism events in patients with non-valvular atrial fibrillation (NVAF). However, little is known about trends in NVAF prevalence and DOAC prescriptions in daily clinical practice. This study investigated the current status and trends in NVAF prevalence and DOAC prescriptions in a region of Japan.Methods and Results:Annual data for the 4 years from May 2014 to May 2017 in the Tsugaru region of Aomori Prefecture, Japan, were obtained for analysis from the Japanese National Health Insurance database ("Kokuho" database [KDB]). The prevalence of NVAF in subjects aged 40-74 years increased gradually over the 4-year study period (1,094/57,452 [1.90%] in 2014, 1,055/56,018 [1.88%] in 2015, 1,072/54,256 [1.98%] in 2016, and 1,154/52,341 [2.20%] in 2017). The proportion of NVAF patients prescribed warfarin decreased (42%, 33%, 24%, and 21% in 2014, 2015, 2016, and 2017, respectively), the proportion of those prescribed DOACs increased (30%, 42%, 50%, and 57%, respectively), and the proportion not prescribed an oral anticoagulant (OAC) decreased (28%, 25%, 26%, and 22%, respectively). However, 17% of patients with a CHADS2score ≥2 were not prescribed an OAC in 2017. CONCLUSIONS: By using the KDB we found that the prevalence of NVAF has increased gradually from 2014 to 2017. In the Tsugaru region in Japan, DOACs prescriptions increased and warfarin prescriptions decreased over the 4-year period.

Blood Flow Restriction Increases the Neural Activation of the Knee Extensors During Very Low-Intensity Leg Extension Exercise in Cardiovascular Patients: A Pilot Study.

Blood flow restriction (BFR) has the potential to augment muscle activation, which underlies strengthening and hypertrophic effects of exercise on skeletal muscle. We quantified the effects of BFR on muscle activation in the rectus femoris (RF), the vastus lateralis (VL), and the vastus medialis (VM) in concentric and eccentric contraction phases of low-intensity (10% and 20% of one repetition maximum) leg extension in seven cardiovascular patients who performed leg extension in four conditions: at 10% and 20% intensities with and without BFR. Each condition consisted of three sets of 30 trials with 30 s of rest between sets and 5 min of rest between conditions. Electromyographic activity (EMG) from RF, VL, and VM for 30 repetitions was divided into blocks of 10 trials and averaged for each block in each muscle. At 10% intensity, BFR increased EMG of all muscles across the three blocks in both concentric and eccentric contraction phases. At 20% intensity, EMG activity in response to BFR tended to not to increase further than what it was at 10% intensity. We concluded that very low 10% intensity exercise with BFR may maximize the benefits of BFR on muscle activation and minimize exercise burden on cardiovascular patients.

Short Physical Performance Battery for cardiovascular disease inpatients: implications for critical factors and sarcopenia.

We examined the relationship between Short Physical Performance Battery (SPPB) and clinical and laboratory factors and the effect of sarcopenia and sarcopenic obesity (SO) on clinical and laboratory factors for cardiovascular disease (CVD) inpatients. CVD male (n = 318) and female (n = 172) inpatients were recruited. A stepwise multiple-regression analysis was performed to predict total SPPB scores and assess clinical and laboratory factors (physical characteristics, functional and morphological assessments, etc.). Each test outcome were compared among sarcopenia, SO and non-sarcopenic groups. To predict total SPPB scores, the predicted handgrip, Controlling Nutritional Status score, % body fat, anterior mid-thigh muscle thickness, standing height and systolic blood pressure were calculated for males and anterior mid-thigh MTH, BMI, knee extension and fat mass were calculated for females. There were no differences in blood pressure, total SPPB scores and functional assessments between sarcopenia and SO groups for CVD male and female inpatients. In conclusion, the physical performance of CVD inpatients can be predicted by nutritional, functional, clinical and anthropometric variables, regardless the gender and the presence of sarcopenia. Furthermore, the presence of sarcopenia has a negative effect on the clinical and laboratory factors, but there is a difference in impact between sarcopenia and SO regardless the gender.

SOCS1 Is a Key Molecule That Prevents Regulatory T Cell Plasticity under Inflammatory Conditions.

We previously showed that regulatory T cells (Tregs) from T cell-specific Socs1-deficient mice (Socs1fl/flLck-Cre+ mice) easily convert into Th1- or Th17-like cells (ex-Tregs), which lose Foxp3 expression and suppressive functions in vivo. Because Tregs in Socs1fl/flLck-Cre+ mice are constantly exposed to a large amount of inflammatory cytokines produced by non-Tregs in vivo, in this study we analyzed Treg-specific Socs1-deficient mice (Socs1fl/flFoxp3YFP-Cre mice). These mice developed dermatitis, splenomegaly, and lymphadenopathy that were much milder than those in Socs1fl/flLck-Cre+ mice. A fate mapping study revealed that Socs1 deficiency accelerated the conversion of Tregs to Foxp3-IFN-γ+ ex-Tregs in the tumor microenvironment and suppressed tumor growth. When transferred into Rag2-/- mice, Tregs from Socs1fl/flLck-Cre+ mice easily lost Foxp3 expression, whereas those from Socs1fl/flFoxp3YFP-Cre mice maintained Foxp3 expression. Although Tregs from Socs1fl/flLck-Cre+ mice produced IFN-γ after a 3-d culture in response to anti-CD3/CD28 Ab stimulation in vitro, Tregs from Socs1fl/flFoxp3YFP-Cre mice did not. This finding suggested that the inflammatory conditions in Socs1fl/flLck-Cre+ mice modified the born nature of Socs1-deficient Tregs. To investigate this mechanism, Tregs from Socs1fl/flFoxp3YFP-Cre mice were cultured with APCs from Socs1fl/flLck-Cre+ mice. These APCs facilitated STAT4 phosphorylation, IFN-γ production, and loss of Foxp3 expression in Tregs from Socs1fl/flFoxp3YFP-Cre mice in an IL-12-dependent manner. The results indicate that Socs1-deficient Tregs tend to convert into ex-Tregs under the inflammatory conditions in which APCs are highly activated, and that SOCS1 could be a useful target for enhancement of anti-tumor immunity.

Establishment of combined analytical method to extract the genes of interest from transcriptome data.

Techniques for analyzing genome-wide expression profiles, such as the microarray technique and next-generation sequencers, have been developed. While these techniques can provide a lot of information about gene expression, selection of genes of interest is complicated because of excessive gene expression data. Thus, many researchers use statistical methods or fold change as screening tools for finding gene sets whose expression is altered between groups, which may result in the loss of important information. In the present study, we aimed to establish a combined method for selecting genes of interest with a small magnitude of alteration in gene expression by coupling with proteome analysis. We used hypercholesterolemic rats to examine the effects of a crude herbal drug on gene expression and proteome profiles. We could not select genes of interest by using standard methods. However, by coupling with proteome analysis, we found several effects of the crude herbal drug on gene expression. Our results suggest that this method would be useful in selecting gene sets with expressions that do not show a large magnitude of alteration.

Refractory Dermatomyositis Complicated with Myelodysplastic Syndrome.

We herein describe a case of refractory dermatomyositis (DM) complicated with myelodysplastic syndrome (MDS). Despite intensive immunosuppressive therapies, the activity of myositis, skin ulcers, and interstitial pneumonia did not improve. The patient ultimately died following the progression of interstitial pneumonia. There are few reports of DM accompanying MDS to date, and any association in the pathogenesis between the two is still unclear. However, underlying MDS may have the potential to influence the therapeutic response of DM.

SOCS1 and regulation of regulatory T cells plasticity.

Several reports have suggested that natural regulatory T cells (Tregs) lose Forkhead box P3 (Foxp3) expression and suppression activity under certain inflammatory conditions. Treg plasticity has been studied because it may be associated with the pathogenesis of autoimmunity. Some studies showed that a minor uncommitted Foxp3(+) T cell population, which lacks hypomethylation at Treg-specific demethylation regions (TSDRs), may convert to effector/helper T cells. Suppressor of cytokine signaling 1 (SOCS1), a negative regulator of cytokine signaling, has been reported to play an important role in Treg cell integrity and function by protecting the cells from excessive inflammatory cytokines. In this review, we discuss Treg plasticity and maintenance of suppression functions in both physiological and pathological settings. In addition, we discuss molecular mechanisms of maintaining Treg plasticity by SOCS1 and other molecules. Such information will be useful for therapy of autoimmune diseases and reinforcement of antitumor immunity.

Development and application of a loop-mediated isothermal amplification assay for rapid detection of Pythium helicoides.

Root rot of poinsettia, caused by Pythium helicoides at high temperatures in hydroponic cultures, has become a serious problem in many parts of the world. We have developed a species-specific, loop-mediated isothermal amplification (LAMP) assay for the rapid diagnosis of this pathogen. The primers were designed using the ribosomal DNA internal transcribed spacer sequence. Primer specificity was established using 40 Pythium species including P. helicoides, 11 Phytophthora species, and eight other soil-borne pathogens. A sensitivity test was carried out using genomic DNA extracted from P. helicoides, and the detection limit was c. 100 fg which is comparable to that of the polymerase chain reaction (PCR). In addition, we tested the ease of pathogen detection in poinsettia roots. The LAMP results were consistent with those from the conventional plating method and showed more sensitivity than the PCR results. Consequently, the LAMP method developed in this study is effective for the rapid and easy detection of P. helicoides.

Clinical utility of an array comparative genomic hybridization analysis for Williams syndrome.

To reveal the relation between intellectual disability and the deleted intervals in Williams syndrome, we performed an array comparative genomic hybridization analysis and standardized developmental testing for 11 patients diagnosed as having Williams syndrome based on fluorescent in situ hybridization testing. One patient had a large 4.2-Mb deletion spanning distally beyond the common 1.5-Mb intervals observed in 10/11 patients. We formulated a linear equation describing the developmental age of the 10 patients with the common deletion; the developmental age of the patient with the 4.2-Mb deletion was significantly below the expectation (developmental age = 0.51 × chronological age). The large deletion may account for the severe intellectual disability; therefore, the use of array comparative genomic hybridization may provide practical information regarding individuals with Williams syndrome.

Visualizing developmentally programmed endoreplication in mammals using ubiquitin oscillators.

The majority of mammalian somatic cells maintain a diploid genome. However, some mammalian cell types undergo multiple rounds of genome replication (endoreplication) as part of normal development and differentiation. For example, trophoblast giant cells (TGCs) in the placenta become polyploid through endoreduplication (bypassed mitosis), and megakaryocytes (MKCs) in the bone marrow become polyploid through endomitosis (abortive mitosis). During the normal mitotic cell cycle, geminin and Cdt1 are involved in 'licensing' of replication origins, which ensures that replication occurs only once in a cell cycle. Their protein accumulation is directly regulated by two E3 ubiquitin ligase activities, APC(Cdh1) and SCF(Skp2), which oscillate reciprocally during the cell cycle. Although proteolysis-mediated, oscillatory accumulation of proteins has been documented in endoreplicating Drosophila cells, it is not known whether the ubiquitin oscillators that control normal cell cycle transitions also function during mammalian endoreplication. In this study, we used transgenic mice expressing Fucci fluorescent cell-cycle probes that report the activity of APC(Cdh1) and SCF(Skp2). By performing long-term, high temporal-resolution Fucci imaging, we were able to visualize reciprocal activation of APC(Cdh1) and SCF(Skp2) in differentiating TGCs and MKCs grown in our custom-designed culture wells. We found that TGCs and MKCs both skip cytokinesis, but in different ways, and that the reciprocal activation of the ubiquitin oscillators in MKCs varies with the polyploidy level. We also obtained three-dimensional reconstructions of highly polyploid TGCs in whole, fixed mouse placentas. Thus, the Fucci technique is able to reveal the spatiotemporal regulation of the endoreplicative cell cycle during differentiation.

Differential detection of Wheat yellow mosaic virus, Japanese soil-borne wheat mosaic virus and Chinese wheat mosaic virus by reverse transcription loop-mediated isothermal amplification reaction.

A differential detection method for three wheat viruses: Wheat yellow mosaic virus (WYMV), Japanese soil-borne mosaic virus (JSBWMV) and Chinese wheat mosaic virus (CWMV) using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction was developed. All three primer sets, which were designed from the genome sequences of WYMV, JSBWMV and CWMV respectively, worked most efficiently at 65 °C and could detect each virus RNA within 10 min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. Furthermore, these primer sets showed unique annealing curves. The peak denaturing temperatures of WYMV, JSBWMV and CWMV primer sets were 87.6 °C, 84.8 °C and 86.4 °C, respectively and were clearly distinguished by the isothermal DNA amplification and fluorescence detection device. The RT-LAMP assay including all three primer sets was found to be 100 times more sensitive than RT-PCR for WYMV and JSBWMV and as sensitive as RT-PCR for CWMV. The RT-LAMP method was validated for the simultaneous detection of these viruses in wheat and barley leaves.

SOCS1 is essential for regulatory T cell functions by preventing loss of Foxp3 expression as well as IFN-{gamma} and IL-17A production.

Regulatory T cells (T(reg) cells) maintain immune homeostasis by limiting inflammatory responses. SOCS1 (suppressor of cytokine signaling 1), a negative regulator of cytokine signaling, is necessary for the suppressor functions of T(reg) cells in vivo, yet detailed mechanisms remain to be clarified. We found that Socs1(-/-) T(reg) cells produced high levels of IFN-γ and rapidly lost Foxp3 when transferred into Rag2(-/-) mice or cultured in vitro, even though the CNS2 (conserved noncoding DNA sequence 2) in the Foxp3 enhancer region was fully demethylated. Socs1(-/-) T(reg) cells showed hyperactivation of STAT1 and STAT3. Because Foxp3 expression was stable and STAT1 activation was at normal levels in Ifnγ(-/-)Socs1(-/-) T(reg) cells, the restriction of IFN-γ-STAT1 signaling by SOCS1 is suggested to be necessary for stable Foxp3 expression. However, Ifnγ(-/-)Socs1(-/-) T(reg) cells had hyperactivated STAT3 and higher IL-17A (IL-17) production compared with Ifnγ(-/-)Socs1(+/+) T(reg) cells and could not suppress colitis induced by naive T cells in Rag2(-/-) mice. In vitro experiments suggested that cytokines produced by Socs1(-/-) T(reg) cells and Ifnγ(-/-)Socs1(-/-) T(reg) cells modulated antigen-presenting cells for preferential Th1 and Th17 induction, respectively. We propose that SOCS1 plays important roles in T(reg) cell integrity and function by maintaining Foxp3 expression and by suppressing IFN-γ and IL-17 production driven by STAT1 and STAT3, respectively.

Transcription factor Smad-independent T helper 17 cell induction by transforming-growth factor-ß is mediated by suppression of eomesodermin.

Transforming growth factor-ß (TGF-ß) has been shown to be required for Th17 cell differentiation via Smad-independent mechanisms. The molecular mechanism underlying this pathway remains to be clarified, however. We searched for genes regulated by TGF-ß through the Smad-independent pathway by using Smad2 and Smad3 double-deficient T cells and identified the transcription factor Eomesodermin (Eomes), whose expression was suppressed by TGF-ß via the c-Jun N-terminal kinase (JNK)-c-Jun signaling pathway. Inhibition of JNK strongly suppressed disease in an in vivo EAE model as well as in vitro Th17 cell induction. Overexpression of Eomes substantially suppressed Th17 cell differentiation, whereas ablation of Eomes expression could substitute for TGF-ß in Th17 cell induction in primary T cells. Eomes suppressed Rorc and Il17a promoters by directly binding to the proximal region of these promoters. In conclusion, the suppression of Eomes by TGF-ß via the JNK pathway is an important mechanism for Smad-independent Th17 cell differentiation.

Suppressors of cytokine signaling (SOCS) proteins and JAK/STAT pathways: regulation of T-cell inflammation by SOCS1 and SOCS3.

Various cytokines are involved in the regulation of the immune system and inflammation. Dysregulation of cytokine signaling can cause a variety of diseases, including allergy, autoimmune diseases, inflammation, and cancer. Most cytokines use the so-called janus kinase/signal transducer and activator of transcription pathway, and this pathway is negatively regulated by suppressors of cytokine signaling (SOCS) proteins. SOCS proteins bind to janus kinase and to certain cytokine receptors and signaling molecules, thereby suppressing further signaling events. Studies have shown that SOCS proteins are key physiological regulators of inflammation. Recent studies have also demonstrated that SOCS1 and SOCS3 are important regulators of adaptive immunity.