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Abstract Background Diesel exhaust particles (DEP), which contain hazardous compounds, are emitted during the combustion of diesel. As approximately one-third of the vehicles worldwide use diesel, there are growing concerns about the risks posed by DEP to human health. Long-term exposure to DEP is associated with airway hyperresponsiveness, pulmonary fibrosis, and inflammation; however, the molecular mechanisms behind the effects of DEP on the respiratory tract are poorly understood. Such mechanisms can be addressed by examining transcriptional and DNA methylation changes. Although several studies have focused on the effects of short-term DEP exposure on gene expression, research on the transcriptional effects and genome-wide DNA methylation changes caused by long-term DEP exposure is lacking. Hence, in this study, we investigated transcriptional and DNA methylation changes in human adenocarcinoma alveolar basal epithelial A549 cells caused by prolonged exposure to DEP and determined whether these changes are concordant. Results DNA methylation analysis using the Illumina Infinium MethylationEPIC BeadChips showed that the methylation levels of DEP-affected CpG sites in A549 cells changed in a dose-dependent manner; the extent of change increased with increasing dose reaching the statistical significance only in samples exposed to 30 µg/ml DEP. Four-week exposure to 30 µg/ml of DEP significantly induced DNA hypomethylation at 24,464 CpG sites, which were significantly enriched for DNase hypersensitive sites, genomic regions marked by H3K4me1 and H3K27ac, and several transcription factor binding sites. In contrast, 9,436 CpG sites with increased DNA methylation levels were significantly overrepresented in genomic regions marked by H3K27me3 as well as H3K4me1 and H3K27ac. In parallel, gene expression profiling by RNA sequencing demonstrated that long-term exposure to DEP altered the expression levels of 2,410 genes, enriching 16 gene sets including Xenobiotic metabolism, Inflammatory response, and Senescence. In silico analysis revealed that the expression levels of 854 genes correlated with the methylation levels of the DEP-affected cis-CpG sites. Conclusions To our knowledge, this is the first report of genome-wide transcriptional and DNA methylation changes and their associations in A549 cells following long-term exposure to DEP.

Leukocyte immunoglobulin (Ig)-like receptors (LILRs) on human chromosome 19q13.4 encode 11 immunoglobulin superfamily receptors, exhibiting genetic diversity within and between human populations. Among the LILR genes, the genomic region surrounding LILRB3 and LILRA6 has yet to be fully characterized due to their significant sequence homology, which makes it difficult to differentiate between them. To examine the LILRB3 and LILRA6 genomic region, a tool named JoGo-LILR CN Caller, which can call copy number from short-read whole genome sequencing (srWGS) data, was applied to an extensive international srWGS dataset comprising 2,504 samples. During this process, a previously unreported loss of both LILRB3 and LILRA6 was detected in three samples. Using long-read sequencing of these samples, we have discovered a novel large deletion (33,692 bp) in the LILRB3 and LILRA6 genomic regions in the Japanese population. This deletion spanned three genes, LILRB3, LILRA6, and LILRB5, resulting in LILRB3 exons 12-13 being located immediately downstream of LILRB5 exons 1-12 with the loss of LILRA6, suggesting the potential expression of a hybrid gene between LILRB5 and LILRB3 (LILRB5-3). Transcription and subsequent translation of the LILRB5-3 hybrid gene were also verified. The hybrid junction was located within the intracellular domain, resulting in an LILRB5 extracellular domain fused to a partial LILRB3 intracellular domain with three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), suggesting that LILRB5-3 acquired a novel signaling function. Further application of the JoGo-LILR tool to srWGS samples suggested the presence of the LILRB5-3 hybrid gene in the CEU population. Our findings provide insight into the genetic and functional diversity of the LILR family.

Objective To investigate the relationship between oral frailty (OF), nutrient intake and calf circumference (CC) in middle-aged and older adults.Design Cross-sectional study.Setting Residents of four model districts of Shika town, Ishikawa Prefecture, Japan, using data from November 2017 to February 2018.Participants One hundred and ninety-four residents aged ≥50 years in four model districts of Shika town. The OF total score ≥3 was defined as OF. Participants were divided into OF and non-OF groups and divided into the low-CC/kg and the high-CC/kg groups.Outcome measures The primary outcome is to use a two-way analysis of covariance to analyse the interaction between the two CC/kg groups and the two OF groups on nutrition intake. The secondary outcome is to use multiple regression analysis to investigate the nutrients significantly related to CC/kg when stratified by OF, with age, sex, body mass index, drinking status, smoking status and regular exercise as input covariates.Results A two-way analysis of covariance revealed a significant interaction between the two CC/kg groups and the two OF groups on animal protein intake (p=0.039). Multiple comparisons using the Bonferroni analysis revealed a significantly lower animal protein intake in the OF group than in the non-OF group with a low CC/kg (p=0.033) but not in the group with a high CC/kg. The multiple regression analysis stratified by OF revealed a positive correlation between animal protein intake and CC/kg (p=0.002).Conclusions The present results revealed a significantly lower animal protein intake in the OF group than in the non-OF group in the low-CC/kg group, but no such difference was observed in the high-CC/kg group. Further longitudinal studies are needed to elucidate this relationship.

Abstract Differentiation therapy has been proposed as a promising therapeutic strategy for acute myeloid leukemia (AML); thus, the development of more versatile methodologies that are applicable to a wide range of AML subtypes is desired. Although the FOXOs transcription factor represents a promising drug target for differentiation therapy, the efficacy of FOXO inhibitors is limited in vivo. Here, we show that pharmacological inhibition of a common cis-regulatory element of forkhead box O (FOXO) family members successfully induced cell differentiation in various AML cell lines. Through gene expression profiling and differentiation marker-based CRISPR/Cas9 screening, we identified TRIB1, a complement of the COP1 ubiquitin ligase complex, as a functional FOXO downstream gene maintaining an undifferentiated status. TRIB1 is direct target of FOXO3 and the FOXO-binding cis-regulatory element in the TRIB1 promoter, referred to as the FOXO-responsive element in the TRIB1 promoter (FRE-T), played a critical role in differentiation blockade. Thus, we designed a DNA-binding pharmacological inhibitor of the FOXO-FRE-T interface using pyrrole-imidazole polyamides (PIPs) that specifically bind to FRE-T (FRE-PIPs). The FRE-PIPs conjugated to chlorambucil (FRE-chb) inhibited transcription of TRIB1, causing differentiation in various AML cell lines. FRE-chb suppressed the formation of colonies derived from AML cell lines but not from normal counterparts. Administration of FRE-chb inhibited tumor progression in vivo without remarkable adverse effects. In conclusion, targeting cis-regulatory elements of the FOXO family is a promising therapeutic strategy that induces AML cell differentiation.

Abstract Background The prevalence of salvage surgeries after drug therapy for non-small cell lung cancer (NSCLC) has risen, mainly due to recent progress in molecular-targeted drugs and immune checkpoint inhibitors for NSCLC. While the safety and effectiveness of salvage surgery after drug therapy for NSCLC have been studied, its indications remain unclear. We aimed to identify the prognostic factors affecting survival in patients with advanced-stage (stages III–IV) NSCLC treated with salvage surgery after drug therapy. Methods A retrospective investigation was conducted on patients who received salvage surgery after drug therapy at four hospitals between 2007 and 2020. Salvage surgery was defined as surgery after drug therapy for local progression, tumor conversion to resectable status, and discontinuation of prior drug therapy owing to serious complications. Results Thirty-two patients received cytotoxic agents alone (n = 12 [38%]), tyrosine kinase inhibitors (TKIs; n = 16 [50%]), or immune checkpoint inhibitors (n = 4 [13%]) as prior drug therapy. In 11 (34%) and 21 (66%) patients, the clinical stage before treatment was III or IV, respectively. The median initial and preoperative serum carcinoembryonic antigen (CEA) levels were 10.2 (range, 0.5–1024) ng/mL and 4.2 (range, 0.6–92.5) ng/mL, respectively. Among the patients, 28 (88%) underwent lobectomy, 2 (6%) underwent segmentectomy, and 2 (6%) underwent wedge resection. Complete resection of the primary lesion was accomplished in 28 (88%) patients. Postoperative complications were documented in six (19%) patients. Mortality rates were 0% at 30 days and 3% at 90 days post-operation. The 5-year overall survival rate stood at 66%, while the 5-year progression-free survival rate was 21%. Multivariate analyses showed that prior TKI therapy and preoperative serum CEA level < 5 ng/mL were prognostic factors influencing overall survival (hazard ratio [95% confidence interval]: 0.06 [0.006–0.68] and 0.03 [0.002–0.41], respectively). The 5-year overall survival in the 11 patients with both favorable prognosticators was 100%. Conclusions In this study, prior TKI therapy and preoperative serum CEA level < 5 ng/mL were favorable prognostic factors for overall survival in patients with NSCLC treated with salvage surgery. Patients with these prognostic factors are considered good candidates for salvage surgery after drug therapy.

Abstract We studied the pathophysiology of aplastic anaemia (AA) in six different pairs of relatives without a family history of hematologic disorders or congenital AA. Five and four of the six pairs shared the HLA‐DRB1*15:01 and B*40:02 alleles, respectively. Glycosylphosphatidylinositol‐anchored protein‐deficient blood cells were detected in eight of the 10 patients evaluated. In a mother‐daughter pair from one family, flow cytometry detected leukocytes lacking HLA‐A2 due to loss of heterogeneity in chromosome 6p. Whole‐exome sequencing of the family pair revealed a missense mutation in MYSM1. These results suggest that genetic inheritance of immune traits might underlie familial AA in some patients.

Abstract Association rule is a transparent machine learning method expected to share information about risks for chronic kidney disease (CKD) among diabetic patients, but its findings in clinical data are limited. We used the association rule to evaluate the risk for kidney disease in General and Worker diabetic cohorts. The absence of risk factors was examined for association with stable kidney function and worsening kidney function. A confidence value was used as an index of association, and a lift of > 1 was considered significant. Analyses were applied for individuals stratified by KDIGO’s (Kidney Disease: Improving Global Outcomes) CKD risk categories. A General cohort of 4935 with a mean age of 66.7 years and a Worker cohort of 2153 with a mean age of 47.8 years were included in the analysis. Good glycemic control was significantly related to stable kidney function in low-risk categories among the General cohort, and in very-high risk categories among the Worker cohort; confidences were 0.82 and 0.77, respectively. Similar results were found with poor glycemic control and worsening kidney function; confidences of HbA1c were 0.41 and 0.27, respectively. Similarly, anemia, obesity, and hypertension showed significant relationships in the low-risk General and very-high risk Worker cohorts. Stratified risk assessment using association rules revealed the importance of the presence or absence of risk factors.

A germline chimera is a useful model for developing and differentiating germ cells in vivo. Gonadal germ cells (GGCs) collected from chicken embryonic gonads may be used to produce germline chimeras as donor cells. However, the migratory and proliferative abilities of GGCs after transfer into recipient embryos are unclear. Here, the migratory and proliferative abilities of GGCs collected from 7-day-old White Leghorn embryos and fluorescently labeled were analyzed following transfer into the dorsal aorta of 2.5-day-old Rhode Island Red (RIR) embryos. Five days after transfer, the numbers of male and female GGCs were significantly higher in the RIR gonads than those in non-gonadal RIR organs when 50 GGCs were transferred per embryo. To analyze the temporal migration of GGCs in intermediate mesoderm, 50 GGCs were again transferred. The numbers of male and female GGCs in RIR gonads increased significantly from days 3 to 6 after transfer. To analyze GGC migration and proliferation in the gonads, a single GGC was transferred into 100 male and 100 female embryos. Five days after transfer, the frequencies of settled and proliferated GGCs were 37% (37/100) and 24% (24/100) in males, and 23% (23/100) and 8% (8/100) in females, respectively. Thus, GGCs are a heterogeneous cell population that may or may not have migratory and proliferative abilities. The heterogeneity of GGCs may be greater in females than that in males. When 50 GGCs were transplanted, almost all those present in embryos had settled and proliferated in the gonads and mesonephros. The migratory and proliferative abilities of GGCs in recipient gonads were considerably diverse in individual GGCs or between donor sexes.

The effects of oxygen gas injection starting on day 17 of incubation (D17) in a chick shell-less culture system (cSLC) on the subsequent embryo development were examined on day 19 of incubation (D19). On D19 of cSLC, the plasma phosphorus and total cholesterol concentrations of the embryos were significantly higher (P0.05). The percutaneous oxygen saturation was lower in D17-cSLC embryos before oxygen gas supplementation than in the IC (P

iPSCs from three endangered avian species (including Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl) are developed and characterized as a potential resource for their conservation.

To meet the strong quantitative demands for poultry products with high sanitary standards soon after World War II, the Japanese poultry industry experienced a rapid expansion period. However, it should be remembered that the post-war flourishment of the Japanese poultry industry was built on a solid academic and educational platform that took several pre-war decades to construct. In addition, poultry play a special cultural role in Japanese society. In this review, poultry in Japan is illustrated from the following three historical viewpoints: 1) development of the Japanese poultry industry; 2) academic and educational contributions to the poultry industry in Japan; and 3) ritualistic, mythic, and artistic attributes of poultry that are deeply embedded in Japanese society.

This study examined the effects of calcium lactate on the development of chicken embryos in a shell-less culture system (cSLCS) up to the seventeenth day of incubation. In the presence of calcium lactate, a significant reduction in embryo viability was observed during the first week of incubation in cSLCS. On day 17 of embryo development, no significant difference was observed in the blood plasma calcium concentration or tibia bone density between cSLCS and intact control embryos, whereas the tibia length was significantly shorter in cSLCS embryos than in the intact control. These results suggest that calcium lactate supplementation in cSLCS supports bone formation in developing chicken embryos, but has adverse effects on the viability of embryos, particularly during the first week of embryo development.

Abstract Objectives The β3‐adrenergic receptor (ADRB3) gene polymorphism has been implicated in obesity. Therefore, the contribution of ADRB3 Trp64Arg polymorphism to obesity‐related indicators was investigated, taking into account the lifestyle‐related factors in a Japanese rural population. Methods A total of 600 Japanese adults aged ≥40 years in a population‐based cohort study were analyzed. The ADRB3 polymorphism was determined using peripheral blood samples. Associations between genotype and body mass index (BMI), waist circumference (WC), and body fat (BF) percentage were examined, adjusting for lifestyle‐related factors, including daily nutrient intake. Results The frequency of Arg64 allele carriers was 36%. There was no significant difference in BMI, WC, or BF between the groups with or without the Trp64Arg polymorphism. Multivariable logistic regression analysis showed that the Trp64Arg polymorphism was not associated with these three indicators, but lifestyle factors including physical inactivity, higher energy and sodium consumption, and less animal protein intake were significantly related to increased WC and BF percentages. Conclusions The Trp64Arg polymorphism of ADRB3 gene did not contribute to increased BMI, WC, or BF. However, lifestyle‐related factors impacted these indicators in middle‐aged and older Japanese individuals living in rural areas.

Abstract Background Few epidemiological studies have been performed to clarify the association between glucose metabolism disorders in early adults (20 years old) and physiological and environmental factors, including body mass index (BMI) in junior high school days. Therefore, we examined the association between hemoglobin A1c (HbA1c) level and body size (BMI) in early adulthood and lifestyles, including sleep habits and BMI in junior high school days in Shika town, a small town in Japan, by conducting a retrospective cohort study. Methods We examined the HbA1c levels and body size (BMI) of 99 early adults who turned 20 years old between 2016 and 2020 and were residing in Shika town, Ishikawa Prefecture. We obtained the information on lifestyles and living environment factors, including BMI, from a questionnaire survey conducted among the subjects during their junior high school days (13–15 years old) from 2009 to 2013. Results No correlations were observed between the HbA1c levels and the BMI values of the early adults. A two-way analysis of covariance (with the HbA1c levels and BMI values of the early adults as main factors) of the body size and lifestyle habits of the junior high school students revealed that “sleep quality in junior high school” was significantly poorer in the high HbA1c group than in the low HbA1c group in the early adults with high BMI values only. This result was also supported by the logistic regression analysis result. Conclusions The present results indicate that poor sleep quality in junior high school was associated with the high HbA1c levels of the early adults with higher BMI values, which suggests that good sleep quality in junior high school prevents the development of hyperglycemia. However, the present study did not find any relationship between early-adult BMI and HbA1c level.

Multi-bracket appliances have long been established as tools for treating malocclusions. However, the complex construction and difficulty in cleaning due to their long-term intraoral retention have hindered the prevention of caries and periodontitis. In this study, professional mechanical tooth cleaning (PMTC), including tooth-brushing instructions, was continuously performed for 3 months in 24 patients who had worn multi-bracket appliances for more than 6 months, and changes in the oral and gut microbiota were examined using one-way repeated-measures analysis of variance. Additionally, changes in bacterial flora associated with different treatment durations were verified using the Pearson correlation coefficient. The results showed that continuous PMTC significantly reduced the amount of plaque in the oral cavity. No significant changes were observed in the oral or gut microbiota and no significant increase in pathogenic bacteria was observed. Therefore, our results suggest that continuous PMTC during orthodontic treatment with multi-brackets may inhibit the growth of pathogenic bacteria by maintaining a clean oral environment and avoiding dysbiosis in both the oral and gut microbiota. Significant changes in the gut microbiota with different treatment durations suggested that differences in food intake and food choices at each treatment stage of orthodontic treatment may affect the gut microbiota.

ABSTRACT Heterozygous RFX6 mutation has emerged as a potential cause of maturity‐onset diabetes mellitus of the young (MODY). A 16‐year‐old female was diagnosed with diabetes by her family doctor and was referred to our institution for genetic examination. Genetic testing revealed a novel RFX6 heterozygous mutation (NM_173560: exon17: c.1954C>T: p.R652X) in the patient and in her mother and brother. She had no islet‐specific autoantibodies and showed a reduced meal‐induced response of insulin, glucose‐dependent insulinotropic polypeptide, and glucagon‐like peptide‐1, which is consistent with the phenotype of MODY due to heterozygous RFX6 mutation. In conclusion, we report a case of MODY due to a novel heterozygous mutation, p.R652X.

Introduction To present longitudinal data from the Real-world Observational Study on Patient Outcomes in Diabetes (RESPOND) in Japan.Research design and methods In this multicenter, prospective, observational cohort study, patients with type 2 diabetes mellitus (T2DM) newly initiated on monotherapy were followed up for 2 years. Primary outcomes included changes in treatment pattern over time, target hemoglobin A1c (HbA1c) attainment and treatment satisfaction per Oral Hypoglycaemic Agent Questionnaire (OHA-Q).Results Among 1474 enrolled patients (male, 62.1%; mean age, 59.7 years; HbA1c, 8.08%), the oral antidiabetic drug (OAD) monotherapy prescription rate decreased to 47.2% and that of 2 and ≥3 OADs increased to 14.8% and 5.4% at 24 months, respectively. Switch/add-on OAD was associated with higher HbA1c and body mass index (BMI), baseline OAD being non-dipeptidyl peptidase-4 inhibitor (DPP-4i)/non-sodium glucose cotransporter-2 inhibitor (SGLT2i), diabetes complications, no comorbidities and consulting a diabetes specialist. The mean (SD) HbA1c (%) was 6.73 (0.85) at 24 months. Higher HbA1c, diabetes complications, cardiovascular disease, being employed, no hypertension and younger treating physician were associated with ≥2 OAD classes prescription or target HbA1c non-attainment at 24 months. OHA-Q subscale scores were significantly higher in patients achieving (vs not achieving) target HbA1c and in those continuing monotherapy (vs combination therapy). Baseline age (

Background: Although the relationship between social capital (SC) and depressive symptoms has been reported in several studies, resilience and gender differences alter those relationships have not yet been elucidated. Therefore, this study cross-sectionally investigated the relationship between SC and depressive symptoms, including suicidal thoughts, based on resilience and gender differences in community-dwelling middle-aged and elderly individuals in Shika town, Ishikawa Prefecture, Japan. Methods: A total of 312 participants older than forty years (149 males and 163 females) were included in the analysis. We assessed depressive symptoms, social capital, and resilience using the Japanese version of the Beck Depression Inventory (BDI), SC scale, and the resilience scale (RS), respectively. Results: Two-way analysis of covariance (ANCOVA) showed that in the low RS group, the BDI of the low SC group was significantly higher than that of the high SC group (p = 0.001). Furthermore, multiple logistic regression analysis showed that SC was significantly associated with high BDI in the low RS group, but not in the high RS group (odds ratio = 0.498, 95% confidence interval = 0.328, 0.755, p = 0.001). Conclusions: The present study concluded that low RS and low SC were associated with high depressive symptoms, especially among females. Our findings suggest that increasing SC may be useful in preventing and improving depressive symptoms in less resilient females. Further longitudinal studies are needed to emotional that causal relationship among SC, RS, and BDI.

Astrocytes play key roles in supporting the central nervous system structure, regulating synaptic functions, and maintaining brain homeostasis. The number of astrocytes in the cerebrum has markedly increased through evolution. However, the manner by which astrocytes change their features during evolution remains unknown. Compared with the rodent brain, the brain of the ferret, a carnivorous animal, has a folded cerebral cortex and higher white to gray matter ratio, which are common features of the human brain. To further clarify the features of ferret astrocytes, we isolated astrocytes from ferret neonatal brains, cultured these cells, and compared their morphology, gene expression, calcium response, and proliferating ability with those of mouse astrocytes. The morphology of cultured ferret astrocytes differed from that of mouse astrocytes. Ferret astrocytes had longer and more branched processes, smaller cell bodies, and different calcium responses to glutamate, as well as had a greater ability to proliferate, compared to mouse astrocytes. RNA sequencing analysis revealed novel ferret astrocyte-specific genes, including several genes that were the same as those in humans. Astrocytes in the ferret brains had larger cell size, longer primary processes in larger numbers, and a higher proliferation rate compared to mouse astrocytes. Our study shows that cultured ferret astrocytes have different features from rodent astrocytes and similar features to human astrocytes, suggesting that they are useful in studying the roles of astrocytes in brain evolution and cognitive functions in higher animals.

Hepatic small vessel neoplasm (HSVN) is a very rare and recently described entity. Moreover, it is a vascular neoplasm of the liver composed of small vessels with infiltrative borders that mimic hepatic angiosarcoma. Based on its lack of atypical morphological features and high proliferative activity, HSVN is thought to be a benign or low-grade neoplasm; however, there is a lack of follow-up information. Here, we present a 51 year-old man with an incidental segment VIII lesion of the liver that displayed plethoric features, which was suspected to be a neoplasm. The patient underwent a segment anterior sectionectomy. The resected liver tumor demonstrated a 2.0 cm by 1.2 cm tan red and partially ill-defined mass, and microscopical examination confirmed HSVN, although the previously identified mutations in HSVN, GNAQ, GNA11, GNA14, and PIK3CA were not detected by genetic testing. The patient’s postoperative recovery was uncomplicated, and abdominal computed tomography at 6 months and 12 months post-surgery revealed no evidence of recurrence. HSVN is a recently described vascular tumor liver with uncertain malignant potential. More research is warranted to establish guidelines for an accurate diagnosis and to elucidate the clinical course of these tumors.

Abstract While ATF6α plays a central role in the endoplasmic reticulum (ER) stress response, the function of its paralogue ATF6β remains elusive, especially in the central nervous system (CNS). Here, we demonstrate that ATF6β is highly expressed in the hippocampus of the brain, and specifically regulates the expression of calreticulin (CRT), a molecular chaperone in the ER with a high Ca2+-binding capacity. CRT expression was reduced to ~ 50% in the CNS of Atf6b −/− mice under both normal and ER stress conditions. Analysis using cultured hippocampal neurons revealed that ATF6β deficiency reduced Ca2+ stores in the ER and enhanced ER stress-induced death. The higher levels of death in Atf6b −/− neurons were recovered by ATF6β and CRT overexpressions, or by treatment with Ca2+-modulating reagents such as BAPTA-AM and 2-APB, and with an ER stress inhibitor salubrinal. In vivo, kainate-induced neuronal death was enhanced in the hippocampi of Atf6b −/− and Calr +/− mice, and restored by administration of 2-APB and salubrinal. These results suggest that the ATF6β-CRT axis promotes neuronal survival under ER stress and excitotoxity by improving intracellular Ca2+ homeostasis.

Abstract Aims/Introduction A low insulin secretion capacity has been implicated in the high prevalence of non‐obese diabetes in East Asians. As alcohol consumption alters insulin and glucose metabolism, we tested the hypothesis that alcohol consumption contributes to impaired insulin secretion and glucose intolerance in lean/normal‐weight non‐diabetic Japanese men. Materials and Methods This cross‐sectional study was undertaken among the residents of Shika town, Japan, between 2011 and 2017. A total of 402 non‐diabetic men, including participants with normal fasting plasma glucose (FPG) and impaired FPG (FPG 5.6–6.9 mmol/L), and aged ≥40 years, were examined. FPG, the homeostasis model assessment of insulin secretion capacity (HOMA‐B) and alcohol consumption were evaluated and compared between the body mass index (BMI)

Abstract In cancer patients, circulating cell-free DNA (cfDNA) includes tumor-derived DNA (tDNA). cfDNA has been used clinically for non-invasive gene mutation testing. The aim of this study was to characterize the features of the genetic alterations detected in cfDNA. This study included 6 patients with primary lung cancer who died due to cancer progression. Tumors were biopsied at autopsy. Genetic alteration profiles were obtained using next generation sequencing. The features of the tDNA genetic alterations detected in cfDNA included a higher frequency of being present in multiple tumors (67% truncal mutations, 36% shared mutations, and 4% individual mutations) and a higher variant allele frequency (VAF; 47.6% versus 4.1% for tDNA alterations detected in cfDNA versus not detected in cfDNA, respectively). The data revealed that the tumor-derived genetic alterations most easily detected in cfDNA were truncal mutations with a high VAF. These results showed that essential genetic alterations enriched in cfDNA could help to characterize cancer cells and that genetic testing using cfDNA has advantages in the detection of fundamental regulatory aberrations occurring during tumorigenesis.

Abstract PAX2 is a transcription factor essential for kidney development and the main causative gene for renal coloboma syndrome (RCS). The mechanisms of PAX2 action during kidney development have been evaluated in mice but not in humans. This is a critical gap in knowledge since important differences have been reported in kidney development in the two species. In the present study, we hypothesized that key human PAX2-dependent kidney development genes are differentially expressed in nephron progenitor cells from induced pluripotent stem cells (iPSCs) in patients with RCS relative to healthy individuals. Cap analysis of gene expression revealed 189 candidate promoters and 71 candidate enhancers that were differentially activated by PAX2 in this system in three patients with RCS with PAX2 mutations. By comparing this list with the list of candidate Pax2-regulated mouse kidney development genes obtained from the Functional Annotation of the Mouse/Mammalian (FANTOM) database, we prioritized 17 genes. Furthermore, we ranked three genes—PBX1, POSTN, and ITGA9—as the top candidates based on closely aligned expression kinetics with PAX2 in the iPSC culture system and susceptibility to suppression by a Pax2 inhibitor in cultured mouse embryonic kidney explants. Identification of these genes may provide important information to clarify the pathogenesis of RCS, human kidney development, and kidney regeneration.

Although nutrient intake and alcohol consumption are both closely associated with the incidence of diabetes, their interrelationships remain unclear. Therefore, we herein have investigated the interrelationships among nutrient intake, alcohol consumption, and the incidence of diabetes using longitudinal data. This study included 969 residents ≥40 years living in Japan. In 2011 and 2012, a baseline study was conducted using questionnaires on basic demographics, diabetes, nutrient intake, and lifestyle habits. In 2018 and 2019, a follow-up study was performed using questionnaires and medical records on diabetes. Two-way analysis of covariance (two-way ANCOVA) was used to test the interactions of drinking habits and diabetes incidence on nutrients intake. The prospective relationship between nutrient intake at baseline and the incidence of diabetes in the follow-up stratified by drinkers and non-drinkers was evaluated using multiple logistic regression analysis. Interactions were observed for vegetable protein intake (p = 0.023) and animal fat intake (p = 0.016) in males. Vegetable protein intake negatively correlated with the incidence of diabetes in non-drinkers (odds ratio (OR): 0.208; 95% confidence interval (95% CI): 0.046–0.935; p = 0.041). Furthermore, animal fat intake positively correlated with the incidence of diabetes in non-drinkers (OR: 1.625; 95% CI: 1.020–2.589; p = 0.041). Therefore, vegetable protein and animal fat intakes in combination with drinking habits need to be considered for the prevention of diabetes.

Although depression and body weight have individually been associated with chronic pain (CP), it currently remains unclear whether the combination of depressive symptoms (DS) and being underweight/overweight is related to CP. Therefore, we herein investigated the relationships among depression, body mass index (BMI), and CP in community-dwelling middle-aged and elderly individuals. Participants comprised 2216 inhabitants of Shika town in Ishikawa prefecture, Japan, including 1003 males (mean age of 68.72 years, standard deviation (SD) of 8.36) and 1213 females (mean age of 69.65 years, SD of 9.36). CP and DS were assessed using a CP questionnaire and Geriatric Depression Scale-15, respectively. The Breslow–Day test indicated that DS positively correlated with lumbar/knee pain in the BMI < 25 group, but not in the BMI ≥ 25 group. Furthermore, lumber/knee pain was related to a higher BMI. These results were confirmed by a logistic analysis with age, sex, BMI, solitary living, the duration of education, no exercise/hobbies, smoking history, alcohol intake, and medical treatment for diabetes, hyperlipidemia, or hypertension as confounding factors. The present study indicates the importance of considering DS and BMI in the prevention of CP. Further studies are needed to clarify the causal relationships among depression, BMI, and CP.

The association between oral frailty (OFr) and body action has been investigated, but its association with systemic function remains unclear. Therefore, this cross-sectional study examined the association between OFr with decreased bone mineral density (BMD) and renal function in residents of Shika town, Ishikawa Prefecture, Japan aged ≥40 years. This study included 400 inhabitants. The OFr total score was assessed using three oral domains in the Kihon Checklist (a self-reported comprehensive health checklist), the number of teeth, and brushing frequency per day. Measurements were the estimated glomerular filtration rate (eGFR) and the osteo-sono assessment index (OSI). Using a two-way analysis of covariance (p = 0.002), significantly lower OSI was indicated in the eGFR < 60 and OFr group than in the eGFR of < 60 and non-OFr group after adjusting for age, body mass index, and drinking and smoking status as confounding factors. Multiple logistic regression analysis confirmed this relationship (p = 0.006). Therefore, lower BMD seems to be associated with lower renal function only when accompanied by OFr. Further longitudinal studies are needed to confirm these results.

Summary: The killer cell immunoglobulin-like receptor (KIR) recognizes human leukocyte antigen (HLA) class I molecules and modulates the function of natural killer cells. Despite its role in immunity, the complex genomic structure has limited a deep understanding of the KIR genomic landscape. Here we conduct deep sequencing of 16 KIR genes in 1,173 individuals. We devise a bioinformatics pipeline incorporating copy number estimation and insertion or deletion (indel) calling for high-resolution KIR genotyping. We define 118 alleles in 13 genes and demonstrate a linkage disequilibrium structure within and across KIR centromeric and telomeric regions. We construct a KIR imputation reference panel (nreference = 689, imputation accuracy = 99.7%), apply it to biobank genotype (ntotal = 169,907), and perform phenome-wide association studies of 85 traits. We observe a dearth of genome-wide significant associations, even in immune traits implicated previously to be associated with KIR (the smallest p = 1.5 × 10−4). Our pipeline presents a broadly applicable framework to evaluate innate immunity in large-scale datasets.

Although chronic pain (CP) is classified as inflammatory or non-inflammatory, the involvement of fatty acid intake in this process has not yet been examined in detail. Therefore, the present study investigated whether the relationship between CP and fatty acid intake differs between high and low C-reactive protein (CRP) levels in middle-aged and elderly individuals in the Shika study. One-thousand and seven males and 1216 females with mean ages of 68⋅78 and 69⋅65 years, respectively, participated in the present study. CRP was quantified by blood sampling from participants who responded to a CP questionnaire. The brief-type self-administered diet history questionnaire (BDHQ) was used to assess fatty acid intake. Interactions were observed between CP and CRP on monounsaturated fatty acids (MUFA) and eicosadienoic acid in a two-way analysis of covariance adjusted for sex, age, lack of exercise, lack of sleep, current smoking and drinking status, and BMI. MUFA (OR 1⋅359) and eicosadienoic acid (OR 1⋅072) were identified as significant independent variables for CP in a multiple logistic regression analysis, but only in the low CRP group. Only a high intake of MUFA and eicosadienoic acid was associated with chronic neck/shoulder/upper limb pain without elevated CRP. In psychogenic and neuropathic pain without elevated CRP, an increased intake of MUFA and eicosadienoic acid, a family member of n-6 fatty acids, appears to affect CP. Further longitudinal studies are needed to elucidate this relationship.

Abstract In this study, we assessed the relative sensory perception of Wagyu beef using temporal dominance of sensations (TDS), which is a dynamic sensory method that captures the “dominance of sensation” throughout food consumption. In addition, we checked the integrity of the TDS by comparing the TDS results with a physicochemical analysis. Strip loins were obtained from 24‐ and 28‐month‐old Japanese Black cattle (“Wagyu”) and were cooked by grilling (yakiniku) or boiling (shabu‐shabu). Temporal dominance of sensations was then used to evaluate the four types of samples. “Tender and/or soft,” “juicy,” “dry,” “fat melting,” “fat taste,” “umami,” “sweet taste,” and “butter odor” were dominant in at least one of the sample types, with the yakiniku cooking method highlighting texture‐ and fat‐related sensory characteristics, and the shabu‐shabu cooking method highlighting flavor‐related sensory characteristics. In addition, beef obtained from the 24‐month‐old Wagyu was significantly more “dry” than that of the 28‐month‐old cattle, reflecting their different cooking loss. Temporal dominance of sensations successfully demonstrated the dominant sensory perceptions of Wagyu beef prepared with different cooking methods and fattening periods.

The relationship between calcium intake and bone strength in older Asian individuals, including Japanese, is controversial; therefore, we herein investigated this relationship in older Japanese populations. We performed a cross-sectional analysis of 314 participants older than 65 years who voluntarily participated in a medical examination and responded to questionnaires. The osteo-sono assessment index (OSI) measured at the right calcaneus using a quantitative ultrasonic device was used as an indicator of bone strength. The daily dietary intake of calcium was assessed using a brief-type self-administered diet history questionnaire. A two-way analysis of covariance revealed a significant interaction between sex and calcium intake on the OSI (p < 0.01). A multiple regression analysis showed a positive correlation between calcium intake and the OSI in males (p < 0.01), but not females (p = 0.27). In females, grip strength divided by body weight positively correlated with the OSI (p = 0.04). The present results suggest that a higher calcium intake contributes to bone strength in older Japanese males. Although a higher grip strength may contribute to bone strength in females, the potential of estrogen as a confounding factor needs to be considered.

Epidemiological studies reported that resilience, generally regarded as the ability to manage stress in the face of adversity, correlates with mental health in middle-aged and older adults. Currently, there is limited information on eating habits that affect resilience. Therefore, this cross-sectional study investigated the relationship between vitamin intake and resilience based on sex in community-dwelling middle-aged and older individuals in Shika town, Ishikawa Prefecture, Japan. A total of 221 participants (106 men and 115 women) aged 40 years or older were included in the analysis. We assessed vitamin intake and resilience using a brief-type self-administered diet history questionnaire (BDHQ) and the resilience scale (RS), respectively. A two-way analysis of covariance (ANCOVA) revealed that higher intakes of β-carotene and vitamin K were associated with higher RS in women, but not in men. Furthermore, a multiple logistic regression analysis stratified by sex showed that β-carotene and vitamin K were significant independent variables for RS only in women. The present study suggests that higher intakes of β-carotene and vitamin K were associated with higher resilience among middle-aged and older women. The results obtained demonstrate that β-carotene and vitamin K intakes may enhance resilience by strengthening stress tolerance.

The dental anomaly fused teeth is defined as the union of two or more teeth. Its aetiology is unclear; to date no studies have investigated its genetic background. Therefore, this study, on the basis of a hypothesised genetic component, investigated the genetic background of patients with fused teeth using whole exome sequencing. Fifteen individuals from six families, including members with and without fused teeth, provided saliva samples that were analysed using whole exome sequencing. Patients with other congenital diseases were excluded from this study. Rare variants were extracted from the sequencing data and filtered by family grouping to identify candidate variants. As a result, ERCC6, OBSCN, SLC27A3, and KIF25 were identified as candidate variants. Our sequencing analysis identified four candidate gene variants associated with fused teeth, which now require further investigation. A genetic basis for the anomaly appears likely. This may assist in understanding the aetiology of fused teeth, which in turn supports better oral care and treatment, as well as future regenerative medicine and gene therapy.

Although the relationship between hypertension and depression is influenced by several physiological factors, including body weight and other lifestyle factors, such as eating behavior, the specific involvement of depression in hypertension remains unclear. Therefore, this epidemiological study examined the role of body weight in the relationship between hypertension and depressive symptoms among the middle-aged and elderly living in the community of Shika town. In total, 1141 males and 1142 females with mean ages of 69.09 and 70.61 years, respectively, participated this study. Physiological factors, including blood pressure, body mass index (BMI), and lifestyle, were investigated in a medical check-up in Shika town. Depressive symptoms were evaluated using the Geriatric Depression Scale 15 (GDS-15). A two-way analysis of covariance exhibited a significant interaction between the two hypertensive groups and body size groups on GDS in females. The post hoc Bonferroni method showed that in the hypertensive groups, GDS was significantly higher in the underweight group (BMI < 18.5) than in the standard/overweight group; however, this relationship was not observed in the no-hypertensive groups. Multiple regression analysis also verified this relationship. Therefore, it is suggested that the combination of hypertension and being underweight is associated with depressive symptoms only in females.

Dens evaginatus (DE) is a dental abnormality characterized by tubercles on the occlusal surfaces of teeth and is associated with the risk of pulpal inflammation due to fractures. The cause of DE remains unclear, as limited data are available to determine its etiology. The aim of this study was to investigate the genetic background of DE using whole-exome sequencing (WES). Saliva samples were collected from two patients of Family A and three patients of Family B, including an incident case of DE, and analyzed using WES. Rare variants were extracted from the WES data and filtered by family to extract candidate variants. Gene variants of TLR3 and MDC1 were identified as etiologic factors for DE. The variant MDC1 (c.3908C>T) was identified to be damaging, according to the scores from Polymorphism Phenotyping v2. Our findings contribute towards an understanding of the etiology of DE, which would facilitate improved treatment to prevent the risk of DE fractures and pulpal inflammation. Understanding the mechanism of DE development may also be helpful for developing regenerative medicine and gene therapy strategies.

Abstract Aims/Introduction Risk factors of type 2 diabetes mellitus in Japanese women with recent gestational diabetes mellitus are unknown. The objective of the present study was to investigate the clinical and genetic characteristics associated with postpartum abnormal glucose tolerance in Japanese women with gestational diabetes mellitus. Materials and Methods A total of 213 Japanese women with recent gestational diabetes mellitus who underwent a postpartum 2‐h oral glucose tolerance test were investigated. The association between antepartum clinical characteristics and postpartum abnormal glucose tolerance (diabetes or prediabetes based on the Japan Diabetes Society criteria) was examined. Frequencies of 45 known type 2 diabetes mellitus‐associated genetic variants were also compared between women with and without postpartum abnormal glucose tolerance. Results A total of 59 women showed postpartum abnormal glucose tolerance (prediabetes, n = 51; diabetes, n = 8). Plasma glucose levels at 1 or 2 h, the insulinogenic index and the insulin secretion‐sensitivity index‐2 of the antepartum oral glucose tolerance test were independent of postpartum abnormal glucose tolerance risk factors (P = 0.006, P = 0.00002, P = 0.01 and P = 0.006, respectively). Four genetic variants (rs266729 [ADIPOQ], rs6017317 [HNF4A], rs5215 [KCNJ11] and rs7177055 [HMG20A]) showed a nominally significant association with postpartum abnormal glucose tolerance (P

Despite a close relationship between chronic kidney disease (CKD) and uric acid level, few studies have examined the relationship between uric acid level and fat intake by kidney function status. Therefore, we investigated the association between dietary fat intake and hyperuricemia with and without decreased kidney function in males living in Shika Town, Ishikawa Prefecture, Japan. This study included 361 males with a mean age of 60.7 years. Dietary fat and fatty acid intakes were evaluated using the brief-type self-administered diet history questionnaire. Reduced kidney function was defined as an estimated glomerular filtration rate (eGFR) 2, while hyperuricemia was defined as a serum uric acid level >7.0 mg/dL. A two-way analysis of covariance showed that saturated fatty acid (p = 0.026), monounsaturated fatty acid (p = 0.014), and polyunsaturated fatty acid (p = 0.022) were significantly lower in the high uric acid group than in the normal uric acid group. In multiple logistic analysis stratified by renal function, lipid intake was negatively associated with hyperuricemia in the low eGFR group. These findings suggest that higher dietary lipid/fatty acid intake may be effective in the prevention and treatment of hyperuricemia in men with CKD.

Chronic kidney disease (CKD) patients have been advised to take vitamins; however, the effects have been controversial. The individual differences in developing CKD might involve genetic variants of inflammation, including variant rs883484 located upstream of the prostaglandin-endoperoxide synthase 1 (PTGS1) gene. We aimed to identify whether the 12 dietary vitamin intake interacts with genotypes of the rs883484 on developing CKD. The population-based, cross-sectional study had 684 Japanese participants (≥40 years old). The study used a validated, brief, self-administered diet history questionnaire to estimate the intake of the dietary vitamins. CKD was defined as estimated glomerular filtration < 60 mL/min/1.73 m2. The study participants had an average age of 62.1 ± 10.8 years with 15.4% minor homozygotes of rs883484, and 114 subjects had CKD. In the fully adjusted model, the higher intake of vitamins, namely niacin (odds ratio (OR) = 0.74, 95% confidence interval (CI): 0.57–0.96, p = 0.024), α-tocopherol (OR = 0.49, 95% CI: 0.26–0.95, p = 0.034), and vitamin C (OR = 0.97, 95% CI: 0.95–1.00, p = 0.037), was independently associated with lower CKD tendency in the minor homozygotes of rs883484. The results suggested the importance of dietary vitamin intake in the prevention of CKD in middle-aged to older-aged Japanese with minor homozygous of rs883484 gene variant.

Selenoprotein P is a hepatokine with antioxidative properties that eliminate a physiologic burst of reactive oxygen species required for intracellular signal transduction. Serum levels of selenoprotein P are elevated during aging and in people with type 2 diabetes, non-alcoholic fatty liver disease, and hepatitis C. However, how serum levels of full-length selenoprotein P are regulated largely remains unknown, especially in the general population. To understand the significance of serum selenoprotein P levels in the general population, we evaluated intrinsic and environmental factors associated with serum levels of full-length selenoprotein P in 1,183 subjects participating in the Shika-health checkup cohort. Serum levels of selenium were positively correlated with liver enzymes and alcohol intake and negatively correlated with body mass index. Serum levels of selenoprotein P were positively correlated with age, liver enzymes, and alcohol intake. In multiple regression analyses, alcohol intake was positively correlated with serum levels of both selenium and selenoprotein P independently of age, gender, liver enzymes, and fatty liver on ultrasonography. In conclusion, alcohol intake is associated with elevated serum levels of selenium and selenoprotein P independently of liver enzyme levels and liver fat in the general population. Moderate alcohol intake may exert beneficial or harmful effects on health, at least partly by upregulating selenoprotein P. These findings increase our understanding of alcohol-mediated redox regulation and form the basis for the adoption of appropriate drinking guidelines.

Dietary intake modification is important for the treatment of chronic kidney disease (CKD); however, little is known about the association between dietary intake of antioxidant vitamins and kidney function based on gender difference. We examined the relationship of dietary intake of antioxidant vitamins with decreased kidney function according to gender in Japanese subjects. This population-based, cross-sectional study included 936 Japanese participants with the age of 40 years or older. A validated brief self-administered diet history questionnaire was used to measure dietary intakes of vitamin E and its four isoforms, vitamin A and vitamin C. Decreased kidney function was defined as estimated glomerular filtration rate

A polymorphism in the ADRB3 gene (Trp64Arg) has been associated with obesity, insulin resistance, and hypertension. This cross-sectional study investigated the relationships among this polymorphism, hypertension, and insulin resistance values (HOMA-IR) in 719 Japanese subjects aged 40 years and older. The genotype frequencies of Trp64Trp (homozygous, wild), Trp64Arg (heterozygous, variant), and Arg64Arg (homozygous, variant) were 466 (65%), 233 (32%), and 20 (3%), respectively. Insulin resistance was associated with an increased risk of hypertension in a Japanese population. This relationship was dependent on the presence or absence of the Trp64Arg polymorphism (odds ratio, 2.054; confidence interval, 1.191 to 3.541; P value, 0.010). Therefore, the Trp64Arg polymorphism of ADRB3 was associated with hypertension and insulin resistance in a healthy Japanese population. This relationship, which was dependent on the polymorphism, may predict the development of hypertension and diabetes.

Study objectivesAlthough associations between sleep quality and environmental factors and nutrient intake have been reported, interactions between these factors have not been elucidated in detail. Therefore, this cross-sectional study examined the effects of regular exercise and nutrient intake on sleep quality using the Pittsburgh Sleep Quality Index (PSQI), which is the most frequently used index for sleep evaluation.MethodsThe participants included 378 individuals aged 40 years or older living in Shika Town, Ishikawa Prefecture. Of these individuals, 185 met the inclusion criteria. The participants completed a self-administered questionnaire assessing lifestyle habits and frequency and duration of exercise, the PSQI, and the brief-type self-administered diet history questionnaire (BDHQ) on nutrient intake.ResultsA two-way analysis of covariance on regular exercise and PSQI scores indicated that protein intake (17.13% of energy) was significantly higher in the regular exercise and PSQI ≤10 groups than in the non-regular exercise or PSQI ≥11 groups (p = 0.002). In a multiple logistic regression analysis with PSQI scores (≤10 and ≥11), protein intake was a significant independent variable in any of the models adjusted for confounding factors such as age, sex, body mass index, current smoker, and current drinker (OR: 1.357, 95% CI: 1.081, 1.704, p = 0.009) in the regular exercise group but not in the non-regular exercise group.Conclusions We identified a positive relationship between sleep quality and protein intake in the regular exercise group. These findings suggest that regular exercise at least twice a week for 30 minutes or longer combined with high protein intake contributes to good sleep quality.

Apolipoprotein E E4 (APOE4) is a risk factor for cognitive decline. A high blood vitamin C (VC) level reduces APOE4-associated risk of developing cognitive decline in women. In the present study, we aimed to examine the effects of functional variants of VC transporter genes expressed in the brain (SLC2A1, SLC2A3, and SLC23A2) on APOE4-associated risk of developing cognitive decline. This case–control study involved 393 Japanese subjects: 252 cognitively normal and 141 cognitively impaired individuals (87 mild cognitive impairment and 54 dementia). Database searches revealed that rs1279683 of SLC23A2, and rs710218 and rs841851 of SLC2A1 are functional variants that are significantly associated with the altered expression of the respective genes and genotyped as three single nucleotide variants (SNVs). When stratified by SNV genotype, we found a significant association between APOE4 and cognitive decline in minor allele carriers of rs1279683 (odds ratio [OR] 2.02, 95% CI, 1.05–3.87, p = 0.035) but not in the homozygote carriers of the major allele. Significant associations between APOE4 and cognitive decline were also observed in participants with major allele homozygotes of rs710218 (OR 2.35, 95% CI, 1.05–5.23, p = 0.037) and rs841851 (OR 3.2, 95% CI, 1.58–6.46, p = 0.0012), but not in minor allele carriers of the respective SNVs. In contrast, the three functional SNVs showed no significant effect on cognitive decline. Our results imply that functional SNVs of VC transporter genes can affect APOE4-associated risk of developing cognitive decline via altered VC levels in the brain.

Introduction: The somatic mutational profile of oral squamous cell carcinoma (OSCC) among Japanese patients has been less investigated, partly because of the rarity of the tumor. Moreover, previous studies have either used formalin-fixed paraffin-embedded samples or lacked paired normal tissues. We aimed to determine somatic mutations in the exomes of 76 genes, including 50 driver genes of solid cancers and NOTCH-related genes, some of which are previously reported as frequently mutated in head and neck squamous cell carcinoma or OSCC. Materials and Methods: We used fresh-frozen tumor/normal-paired samples from 98 treatment-naïve Japanese patients with OSCC and analyzed their correlations with clinicopathological characteristics and survival. Results: We identified 136 exonic mutations, including 78 non-synonymous mutations, 13 synonymous mutations, 22 nonsense mutations, 2 non-frameshift deletions, 11 frameshift deletion, and 5 each of splice-site and frameshift insertions. The most frequently mutated genes were TP53 (36.7%), FAT1 (9.2%), NOTCH1 (8.2%), CDKN2A (7.1%), ZFHX4 (5.1%), CASP8 (4.1%), EP300 (4.1%), and KMT2D (4.1%). We followed up 90 of the 98 patients for 3 years. Among them, TP53 mutation was associated with significantly shorter 3-year disease-free survival. Most of the identified TP53 mutations occurred in the DNA-binding domain and were functionally deleterious. Discussion: Our findings and the mutation spectra can contribute to the development of a therapeutic strategy for Japanese patients with OSCC.

Leukocytes that lack HLA allelic expression are frequently detected in patients with acquired aplastic anemia (AA) who respond to immunosuppressive therapy (IST), although the exact mechanisms underlying the HLA loss and HLA allele repertoire likely to acquire loss-of-function mutations are unknown. We identified a common nonsense mutation at position 19 (c.19C>T, p.R7X) in exon 1 (Exon1mut) of different HLA-A and -B alleles in HLA-lacking granulocytes from AA patients. A droplet digital PCR (ddPCR) assay capable of detecting as few as 0.07% Exon1mut HLA alleles in total DNA revealed the mutation was present in 29% (101/353) of AA patients, with a median allele frequency of 0.42% (range, 0.071% to 21.3%). Exon1mut occurred in only 12 different HLA-A (n=4) and HLA-B (n=8) alleles, including B*40:02 (n=31) and A*02:06 (n=15), which correspond to 4 HLA supertypes (A02, A03, B07, and B44). The percentages of patients who possessed at least one of these 12 HLA alleles were significantly higher in the 353 AA patients (92%, P

The acrosome reaction (AR) is a strictly-regulated, synchronous exocytosis that is required for sperm to penetrate ova. This all-or-nothing process occurs only once in the sperm lifecycle through a sequence of signaling pathways. Spontaneous, premature AR therefore compromises fertilization potential. Although protein kinase A (PKA) pathways play a central role in AR across species, the signaling network used for AR induction is poorly understood in birds. Mechanistic studies of mammalian sperm AR demonstrate that PKA activity is downstreamly regulated by Src family kinases (SFKs). Using SFK inhibitors, our study shows that in chicken sperm, SFKs play a role in the regulation of PKA activity and spontaneous AR without affecting motility. Furthermore, we examined the nature of SFK phosphorylation using PKA and protein tyrosine phosphatase inhibitors, which demonstrated that unlike in mammals, SFK phosphorylation in birds does not occur downstream of PKA and is primarily regulated by calcium-dependent tyrosine phosphatase activity. Functional characterization of SFKs in chicken sperm showed that SFK activation modulates the membrane potential and plays a role in inhibiting spontaneous AR. Employing biochemical isolation, we also found that membrane rafts are involved in the regulation of SFK phosphorylation. This study demonstrates a unique mechanism for regulating AR induction inherent to avian sperm that ensure fertilization potential despite prolonged storage.

Abstract: Clonal hematopoiesis by hematopoietic stem progenitor cells (HSPCs) that lack an HLA class I allele (HLA− HSPCs) is common in patients with acquired aplastic anemia (AA); however, it remains unknown whether the cytotoxic T lymphocyte (CTL) attack that allows for survival of HLA− HSPCs is directed at nonmutated HSPCs or HSPCs with somatic mutations or how escaped HLA− HSPC clones support sustained hematopoiesis. We investigated the presence of somatic mutations in HLA− granulocytes obtained from 15 AA patients in long-term remission (median, 13 years; range, 2-30 years). Targeted sequencing of HLA− granulocytes revealed somatic mutations (DNMT3A, n = 2; TET2, ZRSR2, and CBL, n = 1) in 3 elderly patients between 79 and 92 years of age, but not in 12 other patients aged 27 to 74 years (median, 51.5 years). The chronological and clonogenic analyses of the 3 cases revealed that ZRSR2 mutation in 1 case, which occurred in an HLA− HSPC with a DNMT3A mutation, was the only mutation associated with expansion of the HSPC clone. Whole-exome sequencing of the sorted HLA− granulocytes confirmed the absence of any driver mutations in 5 patients who had a particularly large loss of heterozygosity in chromosome 6p (6pLOH) clone size. Flow–fluorescence in situ hybridization analyses of sorted HLA+ and HLA− granulocytes showed no telomere attrition in HLA− granulocytes. The findings suggest that HLA− HSPC clones that escape CTL attack are essentially free from somatic mutations related to myeloid malignancies and are able to support long-term clonal hematopoiesis without developing driver mutations in AA patients unless HLA loss occurs in HSPCs with somatic mutations.