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The reproductive performance of bulls has a high impact on the beef cattle industry. Scrotal circumference (SC) is the most recorded reproductive trait in beef herds, and is used as a major selection criterion to improve precocity and fertility. The characterization of genomic regions affecting SC can contribute to the identification of diagnostic markers for reproductive performance and uncover molecular mechanisms underlying complex aspects of bovine reproductive biology. In this paper, we report a genome-wide scan for chromosome segments explaining differences in SC, using data of 861 Nellore bulls (Bos indicus) genotyped for over 777,000 single nucleotide polymorphisms. Loci that excel from the genome background were identified on chromosomes 4, 6, 7, 10, 14, 18 and 21. The majority of these regions were previously found to be associated with reproductive and body size traits in cattle. The signal on chromosome 14 replicates the pleiotropic quantitative trait locus encompassing PLAG1 that affects male fertility in cattle and stature in several species. Based on intensive literature mining, SP4, MAGEL2, SH3RF2, PDE5A and SNAI2 are proposed as novel candidate genes for SC, as they affect growth and testicular size in other animal models. These findings contribute to linking reproductive phenotypes to gene functions, and may offer new insights on the molecular biology of male fertility.

Risk factors for atherosclerosis accelerate the senescence of vascular endothelial cells and promote atherogenesis by inducing vascular inflammation. A hallmark of endothelial senescence is the persistent up-regulation of pro-inflammatory genes. We identified CDC42 signaling as a mediator of chronic inflammation associated with endothelial senescence. Inhibition of CDC42 or NF-κB signaling attenuated the sustained up-regulation of pro-inflammatory genes in senescent human endothelial cells. Endothelium-specific activation of the p53/p21 pathway, a key mediator of senescence, also resulted in up-regulation of pro-inflammatory molecules in mice, which was reversed by Cdc42 deletion in endothelial cells. Likewise, endothelial-specific deletion of Cdc42 significantly attenuated chronic inflammation and plaque formation in atherosclerotic mice. While inhibition of NF-κB suppressed the pro-inflammatory responses in acute inflammation, the influence of Cdc42 deletion was less marked. Knockdown of cdc-42 significantly down-regulated pro-inflammatory gene expression and restored the shortened lifespan to normal in mutant worms with enhanced inflammation. These findings indicate that the CDC42 pathway is critically involved in senescence-associated inflammation and could be a therapeutic target for chronic inflammation in patients with age-related diseases without compromising host defenses.

Humans have approximately 400 different olfactory receptors (hORs) and recognize odorants through the repertoire of hOR responses. Although the cell surface expression of hORs is critical to evaluate their response, hORs are poorly expressed on the surface of heterologous cells. To address this problem, previous studies have focused on hOR transportation to the membrane. Nevertheless, the response pattern of hORs to odorants has yet to be successfully linked, and the response sensitivity still remains to be improved. In this study, we demonstrate that increasing the transcriptional level can result in a significant increase in cell surface and functional expression of hORs. We used the TAR-Tat system, which increases the transcription efficiency through positive feedback, and found that OR1A1, OR6N2, and OR51M1 exhibited robust expression. Moreover, this system induces enhanced hOR responses to odorants, thus defining four hORs as novel n-hexanal receptors and n-hexanal is an inverse agonist to one of them. Our results suggested that using the TAR-Tat system and increasing the transcriptional level of hORs can help understanding the relationship between hORs and odorants that were previously undetectable. This finding could facilitate the understanding of the sense of smell by decoding the repertoire of hOR responses., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ojiro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Biomechanical cue within the tissue microenvironment is known to play a critical role in regulating cell behaviors and maintaining tissue homeostasis. As hydrostatic pressure often increases in biliary system under pathological states, we investigated the effect of the moderate elevation of the hydrostatic pressure on biliary epithelial cells, especially on the epithelial-mesenchymal transition (EMT). Human intrahepatic biliary epithelial cells were loaded to hydrostatic pressure using a commercial device. We found that loading the cells to 50 mmHg hydrostatic pressure induced obvious morphological changes and significantly upregulated vimentin, ZEB1, and pSmad2/3, fibronectin, and collagen 1α. All changes induced by hydrostatic pressure loading were effectively mitigated by either ROCK inhibitor (Y-27632) or ALK5 inhibitor (SB-431542). Our in vitro experimental data suggests that hydrostatic pressure loading induces EMT of cholangiocytes through RhoA/ROCK and TGF-β/Smad pathways. Elevated hydrostatic pressure in biliary duct system under pathological states may promote the biliary epithelial cells shifting to profibrotic and mesenchymal characteristics., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Khalifa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

We propose a new method for bipartite link prediction using matrix factorization with negative sample selection. Bipartite link prediction is a problem that aims to predict the missing links or relations in a bipartite network. One of the most popular solutions to the problem is via matrix factorization (MF), which performs well but requires reliable information on both absent and present network links as training samples. This, however, is sometimes unavailable since there is no ground truth for absent links. To solve the problem, we propose a technique called negative sample selection, which selects reliable negative training samples using formal concept analysis (FCA) of a given bipartite network in advance of the preceding MF process. We conduct experiments on two hypothetical application scenarios to prove that our joint method outperforms the raw MF-based link prediction method as well as all other previously-proposed unsupervised link prediction methods., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Peng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Despite the advances in the treatment of cardiovascular diseases, effective treatment remains to be established to improve the quality of life and prognosis of patients with chronic coronary syndromes. This study was aimed to evaluate the effectiveness and safety of the low-intensity pulsed ultrasound (LIPUS) therapy, which we have developed as a novel non-invasive angiogenic therapy through upregulation of endothelial nitric oxide synthase (eNOS)., Methods and Findings: We conducted a randomized, double-blind, placebo-controlled (RCT) pilot trial of the LIPUS therapy for patients with refractory angina pectoris. The patients who received optimal medical therapy without indication of PCI or CABG due to the lack of graftability or complexity of coronary lesions were enrolled. They were randomly divided into the LIPUS treatment group (N = 31) and the placebo group (N = 25) in a 1:1 fashion. The LIPUS therapy was performed in a transthoracic manner for 20 min for 3 sections each (mitral, papillary muscle, and apex levels) under the conditions that we identified; frequency 1.875 MHz, intensity 0.25 MPa, and 32 cycles. The primary endpoint was weekly use of nitroglycerin. Secondary endpoints included stress myocardial perfusion imaging and others. The average weekly nitroglycerin use (times/week) was decreased from 5.50 to 2.44 in the LIPUS group and from 5.94 to 2.83 in the placebo group. The changes in the average weekly nitroglycerin use were comparable; -3.06 (95% CI: -4.481 to -1.648) in the LIPUS group (P<0.01) and -3.10 (95% CI: -4.848 to -1.356) in the placebo group (P<0.01). No adverse effects were noted., Conclusions: In the present study, the LIPUS therapy did not further ameliorate chest pain as compared with optimal medications alone in patients with refractory angina pectoris. The present findings need to be confirmed in another trial with a large number of patients. (Registration ID: UMIN000012369)., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Shindo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background/aims: Although undifferentiated gastric cancer (UGC) diagnosed after Helicobacter pylori eradication (HPE) carries a poor prognosis, characteristics of post-HPE UGC have not been evaluated in detail because of its low incidence. Therefore, we compared the clinicopathologic characteristics of UGC and differentiated gastric cancers (DGC) diagnosed after successful HPE., Methods: GC lesions from patients who had successfully completed HPE and who had undergone upper gastrointestinal endoscopy between January 2004 and March 2016 were analyzed. Tumors were divided into DGC and UGC groups. Clinicopathologic factors of background and tumor characteristics were compared using univariate and multiple logistic analyses., Results: A total of 129 tumors from 115 patients were evaluated; 113 tumors were in the DGC group and 16 in the UGC group. Depressed-type tumors (P = 0.024) and sub-submucosal invasion (P<0.001) were significantly higher in the UGC group. The UGC group had larger tumor diameters (25.9±7.3 mm) than the DGC group (13.2±10.2 mm) (P<0.001). Multivariate analysis showed that female sex (odds ratio [OR] 3.24, 95%CI:1.02-10.37; P = 0.047) and absent follow-up (OR 4.99, 95%CI:1.60-15.57; P = 0.006) were significant independent risk factors for UGC. The DGC group showed a gradually decreasing temporal trend by trend test (P = 0.015), while the UGC group showed a relatively constant incidence over time, although the number of cases was small., Conclusion: UGC was diagnosed even after long time spans following HPE, although the number of cases was small. Female sex, and especially absent follow-up, were risks for post-HPE UGC, suggesting that diligent long-term follow-up after HPE is essential., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kodama et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

To address Tanzania's high maternal mortality ratio, it is crucial to increase women's access to healthcare. To improve access, the quality of antenatal care needs to be improved. Therefore, we conducted a pilot study of a smartphone app for midwives and examined its potential effects on the learning outcomes of midwives and birth preparedness of pregnant women in Tanzania. This mixed-methods, pilot study provided an educational app for midwives in the intervention group, obtained data about the continuous use of the app, measured midwives' learning outcomes, directed focus group discussions on the usability of the app, and conducted surveys among pregnant women about birth preparedness in the intervention and control groups to evaluate if midwives provided proper information to them. The control group received regular antenatal care and answered the same survey. Participants were 23 midwives who participated in the testing and provided learning outcome data. Twenty-one participated in focus group discussions. Results showed that 87.5% of midwives continued to study with the app two months post-intervention. A mini-quiz conducted after using the app showed a significant increase in mean scores (6.9 and 8.4 points, respectively) and a non-significant increase on the questionnaire on women-centered care (98.6 and 102.2 points, respectively). In the focus group discussions, all midwives expressed satisfaction with the app for several reasons, including comprehensive content, feelings of confidence, and reciprocal communication. There were 207 pregnant women included in the analysis. The intervention group had significantly higher knowledge scores and home-based value scores than did controls. The total scores and other subscales did not show statistical significance for group differences. The results indicate the potential impact of the midwifery education app when it is implemented on a larger scale, especially considering that the results show a potential effect on midwives' learning outcomes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Shimpuku et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The number of sperm that reaches the oocytes in mammalian species is limited. In mice, 8-10 oocytes are ovulated, a similar number of sperm reaches the oocytes, and nearly all oocytes are fertilized via natural mating. Meanwhile, our improved superovulation technique (ultrasuperovulation: administration of inhibin antiserum and equine chorionic gonadotropin [IASe]) produced 100 oocytes from a single female C57BL/6 mouse but resulted in only approximately 20 fertilized oocytes via mating. We hypothesized that sperm shortage in the ampulla might cause this low fertilization rate. Mice were mated in the proestrus stage or after hormone injection, but ovulation timing was not considered. In clinical application, the rhythm method supports fertilization by testing the ovulation period and synchronizing the ovulation and copulation timings. Therefore, this study examined the effects of ovulation and copulation timings on in vivo fertilization in female mice with IASe. Synchronization of the ovulation and copulation timings increased fertilization efficiency in female mice with ultrasuperovulation. The number of embryos obtained post ovulation was three times higher than that obtained pre ovulation. This study suggests that synchronized ovulation and copulation timings improve the efficiency of in vivo fertilization in IASe-treated female mice. This technique can be used to produce genetically modified mice and develop technologies for infertility treatment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Nakao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Both Achilles and masticatory muscle tendons are large load-bearing structures, and excessive mechanical loading leads to hypertrophic changes in these tendons. In the maxillofacial region, hyperplasia of the masticatory muscle tendons and aponeurosis affect muscle extensibility resulting in limited mouth opening. Although gene expression profiles of Achilles and patellar tendons under mechanical strain are well investigated in rodents, the gene expression profile of the masticatory muscle tendons remains unexplored. Herein, we examined the gene expression pattern of masticatory muscle tendons and compared it with that of Achilles tendons under tensile strain conditions in the Japanese macaque Macaca fuscata. Primary tenocytes isolated from the masticatory muscle tendons (temporal tendon and masseter aponeurosis) and Achilles tendons were mechanically loaded using the tensile force and gene expression was analyzed using the next-generation sequencing. In tendons exposed to tensile strain, we identified 1076 differentially expressed genes with a false discovery rate (FDR) < 10-10. To identify genes that are differentially expressed in temporal tendon and masseter aponeurosis, an FDR of < 10-10 was used, whereas the FDR for Achilles tendons was set at > 0.05. Results showed that 147 genes are differentially expressed between temporal tendons and masseter aponeurosis, out of which, 125 human orthologs were identified using the Ensemble database. Eight of these orthologs were related to tendons and among them the expression of the glycoprotein nmb and sphingosine kinase 1 was increased in temporal tendons and masseter aponeurosis following exposure to tensile strain. Moreover, the expression of tubulin beta 3 class III, which promotes cell cycle progression, and septin 9, which promotes cytoskeletal rearrangements, were decreased in stretched Achilles tendon cells and their expression was increased in stretched masseter aponeurosis and temporal tendon cells. In conclusion, cyclic strain differentially affects gene expression in Achilles tendons and tendons of the masticatory muscles., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ito et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: Side-effects of medications cause xerostomia. There have been cases where a medication has been discontinued owing to its severe side-effects. Therefore, the xerostomia must be treated to ensure that the primary disease is managed effectively. This study analyzed the actual status of patients with medication-induced xerostomia and investigates factors associated with its improvement., Methods: This study assessed 490 patients diagnosed with medication-induced xerostomia who had an unstimulated salivary flow of ≤0.1 mL/min and received treatment for xerostomia at a xerostomia clinic. Patient age, sex, medical history, medications used, disease duration of xerostomia, and psychological disorders were recorded. The anticholinergic burden was assessed using the Anticholinergic Cognitive Burden scale. The unstimulated salivary flow was measured by the spitting method. According to their symptoms and diagnoses, the patients were introduced to oral lubricants, instructed on how to perform massage, and prescribed Japanese herbal medicines, and sialogogues. Factors associated with the subjective improvement of xerostomia and objective changes in the salivary flow rate were recorded at six months., Results: Xerostomia improved in 338 patients (75.3%). The improvement rate was significantly lower in patients with psychiatric disorders (63.6%) (P = 0.009). The improvement rate decreased as more anticholinergics were used (P = 0.018). However, xerostomia improved in approximately 60% of patients receiving three or more anticholinergics. The unstimulated salivary flow increased significantly more in patients who reported an improvement of xerostomia (0.033±0.053 mL/min) than in those who reported no improvement (0.013±0.02 mL/min) (P = 0.025)., Conclusion: Xerostomia treatment improved oral dryness in 75.3% of patients receiving xerogenic medications in this study. If xerostomia due to side-effects of medications can be improved by treatment, it will greatly contribute to the quality of life of patients with xerogenic medications and may reduce the number of patients who discontinue medications., Competing Interests: This study was funded by Pfizer Japan, Inc. Naoko Izumi, one of the co-authors, is an employee of Pfizer Japan Inc. and has contributed to preparation of the study design, data analysis, decision to publish and preparation of the manuscript. However, Pfizer Japan Inc. did not provided support in the form of salaries for authors other than NI. Pfizer is a marketing authorization holder of fesoterodine, an anti-cholinergic medicine for overactive bladder and pediatric neurogenic bladder. These do not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Ito et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

During submaximal isometric contraction, there are two different load types: maintenance of a constant limb angle while supporting an inertial load (position task) and maintenance of a constant force by pushing against a rigid restraint (force task). Previous studies demonstrated that performing the position task requires more proprioceptive information. The purpose of this study was to investigate whether there would be a difference in cutaneous information processing between the position and force tasks by assessing the gating effect, which is reduction of amplitude of somatosensory evoked potentials (SEPs), and cutaneomuscular reflex (CMR). Eighteen healthy adults participated in this study. They contracted their right first dorsal interosseous muscle by abducting their index finger to produce a constant force against a rigid restraint that was 20% maximum voluntary contraction (force task), or to maintain a target position corresponding to 10° abduction of the metacarpophalangeal joint while supporting a load equivalent to 20% maximum voluntary contraction (position task). During each task, electrical stimulation was applied to the digital nerves of the right index finger, and SEPs and CMR were recorded from C3' of the International 10-20 system and the right first dorsal interosseous muscle, respectively. Reduction of the amplitude of N33 component of SEPs was significantly larger during the force than position task. In addition, the E2 amplitude of CMR was significantly greater for the force than position task. These findings suggest that cutaneous information processing differs with load type during static muscle contraction., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Yunoki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Zinc plays a critical role in many physiological processes, and disruption of zinc homeostasis induces various disorders, such as growth retardation, osteopenia, immune deficiency, and inflammation. However, how the imbalance in zinc homeostasis leads to heart disease is not yet fully understood. Cardiovascular diseases are a major cause of death worldwide, and the development of novel therapeutic targets to treat it is urgently needed. We report that a zinc transporter, ZIP13, regulates cardiovascular homeostasis. We found that the expression level of Zip13 mRNA was diminished in both primary neonatal cardiomyocytes and mouse heart tissues treated with the cardiotoxic agent doxycycline. Primary neonatal cardiomyocytes from Zip13 gene-knockout (KO) mice exhibited abnormal irregular arrhythmic beating. RNA-seq analysis identified 606 differentially expressed genes in Zip13-KO mouse-derived primary neonatal cardiomyocytes and Gene ontology (GO) analysis revealed that both inflammation- and cell adhesion-related genes were significantly enriched. In addition, telemetry echocardiography analysis suggested that arrhythmias were likely to occur in Zip13-KO mice, in which elevated levels of the cardiac fibrosis marker Col1a1, vascular inflammation-related gene eNOS, and Golgi-related molecule GM130 were observed. These results indicate the physiological importance of ZIP13-it maintains cardiovascular homeostasis by resolving inflammation and stress response. Our findings suggest that optimizing ZIP13 expression and/or function may improve cardiovascular disease management., Competing Interests: The authors have declared that no competing interests exist.

Antibiotics disrupt normal gut microbiota and cause dysbiosis, leading to a reduction in intestinal epithelial barrier function. Disruption of the intestinal epithelial barrier, which is known as "leaky gut", results in increased intestinal permeability and contributes to the development or exacerbation of gastrointestinal diseases such as inflammatory bowel disease and irritable bowel syndrome. We have previously reported on a murine model of intestinal epithelial barrier dysfunction associated with dysbiosis induced by the administration of ampicillin and vancomycin. Saireito, a traditional Japanese herbal medicine, is often used to treat autoimmune disorders including ulcerative colitis; the possible mechanism of action and its efficacy, however, remains unclear. In this study, we examined the efficacy of Saireito in our animal model for leaky gut associated with dysbiosis. C57BL/6 mice were fed a Saireito diet for the entirety of the protocol (day1-28). To induce colitis, ampicillin and vancomycin were administered in drinking water for the last seven consecutive days (day22-28). As previously demonstrated, treatment with antibiotics caused fecal occult bleeding, cecum enlargement with black discoloration, colon inflammation with epithelial cell apoptosis, and upregulation of pro-inflammatory cytokines. Oral administration of Saireito significantly improved antibiotics-induced fecal occult bleeding and cecum enlargement by suppressing inflammation in the colon. Furthermore, Saireito treatment ensured the integrity of the intestinal epithelial barrier by suppressing apoptosis and inducing cell adhesion proteins including ZO-1, occludin, and E-cadherin in intestinal epithelial cells, which in turn decreased intestinal epithelial permeability. Moreover, the reduced microbial diversity seen in the gut of mice treated with antibiotics was remarkably improved with the administration of Saireito. In addition, Saireito altered the composition of gut microbiota in these mice. These results suggest that Saireito alleviates leaky gut caused by antibiotic-induced dysbiosis. Our findings provide a potentially new therapeutic strategy for antibiotic-related gastrointestinal disorders., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: [This study received funding from Tsumura & Co. (to K.M.)] This study received funding from Tsumura & Co. This does not alter adherence to PLOS ONE policies on sharing data and materials.

Background: The number of patients aged 80 years or older with diffuse large B-cell lymphoma (DLBCL) is increasing, and the incidence rate of the disease in this population group reaches up to 20%. The risk of infection is higher in older patients than in other patients. Although hypnotic drugs are frequently detected as potentially inappropriate medications, it is unclear whether hypnotic drugs affect the occurrence of infection during chemotherapy. Here, we investigated whether the use of hypnotic drugs is associated with infection during first-line chemotherapy in patients with diffuse large B-cell lymphoma (DLBCL) aged 80 years or older., Methods: Japanese patients aged 80 years or older with diffuse large B-cell lymphoma who had received first-line chemotherapy at Fujita Health University Hospital from January 2005 to March 2020 were enrolled in this retrospective cohort study. The primary study outcome was the identification of the risk factor for infection during first-line chemotherapy., Results: This study included 65 patients received first-line chemotherapy. The proportion of patients with National Comprehensive Cancer Network-international prognostic index ≥ 6 was higher in the infection group than in the non-infection group. The relative dose intensity of each anticancer drug (cyclophosphamide, adriamycin, and vincristine) and dose of prednisolone did not significantly differ between the two groups. Multivariate analysis showed that the use of benzodiazepines was a risk factor for infection (odds ratio, 4.131 [95% confidence interval: 1.225-13.94], P = 0.022)., Conclusion: DLBCL patients using benzodiazepines should be monitored for infection symptoms during chemotherapy., Competing Interests: The authors have declared that no competing interests exist.

Infantile wheezing and eczema are associated with the subsequent onset of asthma and other atopic diseases. However, there are no large population-based surveys on infantile allergic symptoms in Japan. The objective of the study was to determine the prevalence of wheezing and asthma in infants in Nagoya, Japan. This population-based cross-sectional study was performed in the metropolitan city of Nagoya, Japan. We surveyed parents to ascertain the prevalence of wheezing and eczema in infants who attended group health checkups at 3, 18, and 36 months of age. Their parents completed modified questionnaires from the International Study of Asthma and Allergies in Childhood. More than 90% of the approximately 40,000 children in each study group living in the target area were included in the survey. The prevalence of wheezing was 8%, 17%, and 13% at 3, 18, and 36 months, respectively, and was characterized by birth season. The prevalence of eczema was 24%, 30%, and 31%, at 3, 18, and 36 months, respectively. Participants born in autumn and winter had a higher incidence of eczema in each age group. Three-quarters of the children had a parental history of allergic conditions. Parental allergic diseases and male gender are risk factors for wheezing and eczema in children. This survey had a high response rate and covered almost the entire population of the target age groups in a large city. We believe that the results of this study, therefore, provide a much higher level of confidence regarding the prevalence of allergies in infants in Japan than that in previous studies with limited cohorts., Competing Interests: The authors have declared that no competing interests exist.

Patients with type 2 diabetes who have cardiovascular disease and are receiving empagliflozin have a lower rate of primary composite cardiovascular outcomes. In contrast, glimepiride increases cardiovascular hospitalization when combined with metformin. Here, we assessed the effects of empagliflozin and glimepiride on endothelial function using flow-mediated dilation (FMD). In this prospective, open-label, randomized, parallel-group study, 63 patients with type 2 diabetes received metformin and insulin glargine U100 for 12 weeks. This was followed by additional treatment with empagliflozin or glimepiride for 12 weeks. The primary outcome was the change in the FMD measurement (ΔFMDs) at 24 weeks of additional treatment. Secondary outcomes comprised changes in metabolic markers and body composition. The empagliflozin group (n = 33) and glimepiride group (n = 30) showed no significant differences in ΔFMDs (empagliflozin, -0.11 [95%CI: -1.02, 0.80]%; glimepiride, -0.34 [95%CI: -1.28, 0.60]%; P = 0.73). Additionally, changes in glycated hemoglobin were similar between the two groups. However, a significant difference in body weight change was observed (empagliflozin, -0.58 [95%CI: -1.60, 0.43] kg; glimepiride, 1.20 [95%CI: 0.15, 2.26] kg; P = 0.02). Moreover, a body composition analysis revealed that body fluid volume significantly decreased after empagliflozin treatment (baseline, 35.8 ± 6.8 L; after 12 weeks, -0.33 ± 0.72 L; P = 0.03). Hence, although empagliflozin did not improve endothelial function compared with glimepiride for patients with type 2 diabetes, it did decrease body fluid volumes. Thus, the coronary-protective effect of empagliflozin is not derived from endothelial function protection, but rather from heart failure risk reduction. Trial registration: This trial was registered on September 13, 2016; UMIN000024001., Competing Interests: The authors have declared that no competing interests exist.

Drug research with animal models is expensive, time-consuming and translation to clinical trials is often poor, resulting in a desire to replace, reduce, and refine the use of animal models. One approach to replace and reduce the use of animal models is to use in vitro cell-culture models. To study bone physiology, bone diseases and drugs, many studies have been published using osteoblast-osteoclast co-cultures. The use of osteoblast-osteoclast co-cultures is usually not clearly mentioned in the title and abstract, making it difficult to identify these studies without a systematic search and thorough review. As a result, researchers are all developing their own methods, leading to conceptually similar studies with many methodological differences and, as a consequence, incomparable results. The aim of this study was to systematically review existing osteoblast-osteoclast co-culture studies published up to 6 January 2020, and to give an overview of their methods, predetermined outcome measures (formation and resorption, and ALP and TRAP quantification as surrogate markers for formation and resorption, respectively), and other useful parameters for analysis. Information regarding these outcome measures was extracted and collected in a database, and each study was further evaluated on whether both the osteoblasts and osteoclasts were analyzed using relevant outcome measures. From these studies, additional details on methods, cells and culture conditions were extracted into a second database to allow searching on more characteristics. The two databases presented in this publication provide an unprecedented amount of information on cells, culture conditions and analytical techniques for using and studying osteoblast-osteoclast co-cultures. They allow researchers to identify publications relevant to their specific needs and allow easy validation and comparison with existing literature. Finally, we provide the information and tools necessary for others to use, manipulate and expand the databases for their needs., Competing Interests: The authors have declared that no competing interests exist.

The hepato-splanchnic circulation directly influences oxygenation of the abdominal organs and plays an important role in compensating for the blood volume reduction that occurs in the central circulation during hemodialysis (HD) with ultrafiltration. However, the hepato-splanchnic circulation and oxygenation cannot be easily evaluated in the clinical setting of HD therapy. We included 185 HD patients and 15 healthy volunteers as the control group in this study. Before HD, hepatic regional oxygen saturation (rSO2), a marker of hepatic oxygenation reflecting the hepato-splanchnic circulation and oxygenation, was monitored using an INVOS 5100c oxygen saturation monitor. Hepatic rSO2 was significantly lower in patients undergoing HD than in healthy controls (56.4 ± 14.9% vs. 76.2 ± 9.6%, p < 0.001). Multivariable regression analysis showed that hepatic rSO2 was independently associated with body mass index (BMI; standardized coefficient: 0.294), hemoglobin (Hb) level (standardized coefficient: 0.294), a history of cardiovascular disease (standardized coefficient: -0.157), mean blood pressure (BP; standardized coefficient: 0.154), and serum albumin concentration (standardized coefficient: 0.150) in Model 1 via a simple linear regression analysis. In Model 2 using the colloid osmotic pressure (COP) in place of serum albumin concentration, the COP (standardized coefficient: 0.134) was also identified as affecting hepatic rSO2. Basal hepatic oxygenation before HD might be affected by BMI, Hb levels, a history of cardiovascular disease, mean BP, serum albumin concentration, and the COP. Further prospective studies are needed to clarify whether changes in these parameters, including during HD, affect the hepato-splanchnic circulation and oxygenation in HD patients., Competing Interests: The authors have no conflicts of interest to declare.

Advances in intravital microscopy (IVM) have enabled the studies of cellular organization and dynamics in the native microenvironment of intact organisms with minimal perturbation. The abilities to track specific cell populations and monitor their interactions have opened up new horizons for visualizing cell biology in vivo, yet the success of standard fluorescence cell labeling approaches for IVM comes with a "dark side" in that unlabeled cells are invisible, leaving labeled cells or structures to appear isolated in space, devoid of their surroundings and lacking proper biological context. Here we describe a novel method for "filling in the void" by harnessing the ubiquity of extracellular (interstitial) fluid and its ease of fluorescence labelling by commonly used vascular and lymphatic tracers. We show that during routine labeling of the vasculature and lymphatics for IVM, commonly used fluorescent tracers readily perfuse the interstitial spaces of the bone marrow (BM) and the lymph node (LN), outlining the unlabeled cells and forming negative contrast images that complement standard (positive) cell labeling approaches. The method is simple yet powerful, offering a comprehensive view of the cellular landscape such as cell density and spatial distribution, as well as dynamic processes such as cell motility and transmigration across the vascular endothelium. The extracellular localization of the dye and the interstitial flow provide favorable conditions for prolonged Intravital time lapse imaging with minimal toxicity and photobleaching., Competing Interests: The authors have declared that no competing interests exist.

Epicardial adipose tissue may affect hemodynamics and cardiorespiratory fitness as it is a metabolically active visceral adipose tissue and a source of inflammatory bioactive substances that can substantially modulate cardiovascular morphology and function. However, the associations between epicardial adipose tissue and hemodynamics and cardiorespiratory fitness remain unclear. This cross-sectional study aimed to examine the association between epicardial adipose tissue volume and hemodynamics, and cardiorespiratory fitness among Japanese individuals of various ages and of both sexes. Epicardial adipose tissue volume was measured in 120 participants (age, 21-85 years) by cardiac magnetic resonance imaging. To evaluate cardiorespiratory fitness, peak oxygen uptake was measured by cardiopulmonary exercise testing. Peak cardiac output and arteriovenous oxygen difference were calculated by impedance cardiography. The epicardial adipose tissue volume was significantly increased in middle-aged and older women. The epicardial adipose tissue volume was significantly and negatively correlated to peak cardiac output and peak oxygen uptake, regardless of age and sex; furthermore, epicardial adipose tissue showed a strong negative correlation with peak heart rate. Epicardial adipose tissue and peak cardiac output were significantly associated (β = -0.359, 95% confidence interval, -0.119 to -0.049, p < 0.001), even after multivariate adjustment (R2 = 0.778). However, in the multiple regression analysis with peak oxygen uptake as a dependent variable, the epicardial adipose tissue volume was not an independent predictor. These data suggest that increased epicardial adipose tissue volume may be correlated with decreased peak oxygen uptake, which might have mediated the abnormal hemodynamics among Japanese people of various ages and of both sexes. Interventions targeting epicardial adipose tissue could potentially improve hemodynamics and cardiorespiratory fitness., Competing Interests: The authors have declared that no competing interests exist.

Glycolytic metabolism is closely involved in physiological homeostasis and pathophysiological states. Among glycolytic enzymes, phosphoglycerate mutase (PGAM) has been reported to exert certain physiological role in vitro, whereas its impact on glucose metabolism in vivo remains unclear. Here, we report the characterization of Pgam1 knockout mice. We observed that homozygous knockout mice of Pgam1 were embryonic lethal. Although we previously reported that both PGAM-1 and -2 affect global glycolytic profile of cancers in vitro, in vivo glucose parameters were less affected both in the heterozygous knockout of Pgam1 and in Pgam2 transgenic mice. Thus, the impact of PGAM on in vivo glucose metabolism is rather complex than expected before., Competing Interests: The authors have declared that no competing interests exist.

Rationale: Immunoglobulin (Ig) G4-related disease (IgG4-RD) is a novel clinical disease entity characterized by an elevated serum IgG4 concentration and tumefaction or tissue infiltration by IgG4-positive plasma cells. Pathological changes are most frequently seen in the pancreas, lacrimal glands, and salivary glands, but pathological changes in the lung also exist. Linker for activation of T cell (LAT)Y136F knock-in mice show Th2-dominant immunoreactions with elevated serum IgG1 levels, corresponding to human IgG4. We have reported that LATY136F knock-in mice display several characteristic features of IgG4-RD and concluded that they constitute an appropriate model of human IgG4-RD in salivary glands, pancreas, and kidney lesions., Objectives: The aim of this study is to evaluate whether lung lesions in LATY136F knock-in mice can be a model of IgG4-related lung disease., Methods: Lung tissue samples from LATY136F knock-in mice (LAT) and wild-type mice (WT) were immunostained for IgG1 and obtained for pathological evaluation, and cell fractions and cytokine levels in broncho-alveolar lavage fluid (BALF) were analyzed., Results: In the LAT group, IgG1-positive inflammatory cells increased starting at 4 weeks of age and peaked at 10 weeks of age. The total cell count and percentage of lymphocytes increased significantly in BALF in the LAT group compared to the WT group. In BALF, Th2-dominant cytokines and transforming growth factor-β were also increased. In the LAT group, marked inflammation around broncho-vascular bundles peaked at 10 weeks of age. After 10 weeks, fibrosis around broncho-vascular bundles and bronchiectasis were observed in LATY136F knock-in mice but not WT mice., Conclusions: LATY136F knock-in mice constitute an appropriate model of lung lesions in IgG4-RD., Competing Interests: The authors have declared that no competing interests exist.

Bile acid has attracted attention as a signal transmission molecule in energy metabolism. Although a high-fat diet (HFD) or obesity is known to increase hepatic fat content and alter bile acid composition, the changes in bile acid composition due to HFD or obesity remain to be elucidated. We sought to examine the bile acid composition in high fat diet-induced non-alcoholic fatty liver disease (NAFLD) in obese diabetic rats. Eight-week-old male spontaneously diabetic Torii fatty (SDTF) rats or control rats were fed an HFD. Twelve weeks post the commencement of HFD, serum and hepatic bile acid compositions and serum GLP-1 levels, which is stimulated by the secondary bile acid deoxycholic acid (DCA), were measured. The correlation between the bile acid composition and serum GLP-1 levels was also examined. While serum and hepatic levels of cholic acid (CA), a primary bile acid, tended to decrease in HFD-fed control rats, they were significantly decreased in HFD-fed SDTF rats. Hepatic CYP8B1, which plays a role in CA synthesis, the mRNA levels were significantly decreased in HFD-fed control and SDTF rats. In contrast, while serum and hepatic DCA levels were not changed in HFD-fed control rats, they were decreased in HFD-fed SDTF rats. Hepatic DCA/CA did not change in HFD-fed SDTF rats, but significantly increased in HFD-fed control rats. While serum GLP-1 levels were not changed in SDTF rats, they were significantly increased in HFD-fed control rats. Hepatic DCA/CA tended to correlate with serum GLP-1 levels, which tended to negatively correlate with the hepatic triglyceride content in SDTF rats. These results indicate that relatively increased DCA might contribute to an increase in serum GLP-1 levels, which inhibits hepatic steatosis in NAFLD., Competing Interests: The authors have declared that no competing interests exist.

Objectives: This study aimed to clarify the relationship between the white blood cell (WBC) count and hypertension in the general Japanese population., Methods: We conducted a population-based retrospective cohort study using annual health check-up data of residents of Iki City, Nagasaki Prefecture, Japan. A total of 2935 participants without hypertension at baseline were included in the present analysis. WBC counts were classified as tertile 1 (<4700/μL), tertile 2 (4700-5999/μL), and tertile 3 (≥6000/μL). The outcome was incident hypertension (blood pressure ≥140 mmHg). Multivariable-adjusted hazard ratios and 95% confidence intervals (95% CIs) were estimated using the Cox proportional hazards model., Result: During an average follow-up of 4.5 years, 908 participants developed hypertension. The incidence (per 100 person-years) of hypertension increased with an elevation in the WBC count (6.3 in tertile 1, 7.0 in tertile 2, and 7.4 in tertile 3). This association was significant, even after adjustment for other risk factors, including age, sex, current smoking habits, current alcohol intake, exercise habits, obesity, elevated blood pressure, diabetes mellitus, and dyslipidemia. The hazard ratios were 1.07 for tertile 2 (95% CI 0.90-1.26) and 1.27 for tertile 3 (95% CI 1.06-1.51) compared with the reference group of tertile 1 (p = 0.009)., Conclusion: The WBC count was associated with future development of hypertension in the general Japanese population., Competing Interests: The authors have read the journal's policy, and the authors of this study have the following competing interests to declare: HA received funding from Daiichi Sankyo and Takeda, lecture fees from Bayer, Daiichi Sankyo, Fukuda Denshi, MSD, Takeda and Teijin, and fees for consultancy from Kyowa Kirin outside of this work. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare. There are no other conflicts of interest to disclose.

Pseudomonas aeruginosa (Pa) infection is a major cause of airway inflammation in immunocompromised and cystic fibrosis (CF) patients. Mitogen-activated protein (MAP) and tyrosine kinases are integral to inflammatory responses and are therefore potential targets for novel anti-inflammatory therapies. We have determined the involvement of specific kinases in Pa-induced inflammation. The effects of kinase inhibitors against p38MAPK, MEK 1/2, JNK 1/2, Syk or c-Src, a combination of a p38MAPK with Syk inhibitor, or a novel narrow spectrum kinase inhibitor (NSKI), were evaluated against the release of the proinflammatory cytokine/chemokine, IL-6 and CXCL8 from BEAS-2B and CFBE41o- epithelial cells by Pa. Effects of a Syk inhibitor against phosphorylation of the MAPKs were also evaluated. IL-6 and CXCL8 release by Pa were significantly inhibited by p38MAPK and Syk inhibitors (p<0.05). Phosphorylation of HSP27, but not ERK or JNK, was significantly inhibited by Syk kinase inhibition. A combination of p38MAPK and Syk inhibitors showed synergy against IL-6 and CXCL8 induction and an NSKI completely inhibited IL-6 and CXCL8 at low concentrations. Pa-induced inflammation is dependent on p38MAPK primarily, and Syk partially, which is upstream of p38MAPK. The NSKI suggests that inhibiting specific combinations of kinases is a potent potential therapy for Pa-induced inflammation., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Previously Matthew S. Coates was employed by Respivert Ltd. Garth W. Rapeport and Kazuhiro Ito were co-founders and employees of Respivert Ltd. During the period of the research G. W. Rapeport and K. Ito were co-founders and employees of Pulmocide Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The fruit fly Drosophila melanogaster is an important model organism for neuroscience with a wide array of genetic tools that enable the mapping of individual neurons and neural subtypes. Brain templates are essential for comparative biological studies because they enable analyzing many individuals in a common reference space. Several central brain templates exist for Drosophila, but every one is either biased, uses sub-optimal tissue preparation, is imaged at low resolution, or does not account for artifacts. No publicly available Drosophila ventral nerve cord template currently exists. In this work, we created high-resolution templates of the Drosophila brain and ventral nerve cord using the best-available technologies for imaging, artifact correction, stitching, and template construction using groupwise registration. We evaluated our central brain template against the four most competitive, publicly available brain templates and demonstrate that ours enables more accurate registration with fewer local deformations in shorter time., Competing Interests: The authors have declared that no competing interests exist.

Anthropogenic disturbances in forest management practices can affect wild edible plants. Soil scarification is a large-scale disturbance that may cause long-term reduction in productivity of edible dwarf bamboo, Sasa kurilensis, in northern Japan. For their effective and sustainable use, we need to understand the recovery process after such disturbances. At 14 study sites in the Teshio Experimental Forest of Hokkaido University where soil scarification had been conducted between 2 and 44 years prior, the number and stem diameter of old and young (newly emerged, edible) culms was recorded. At sites that were within 11 years of soil scarification, the proportion of old culms (<11%) was lower than in the control area where soil scarification had never been conducted. At sites where more than 15 years had passed since soil scarification, the relative number of old culms was nearly equal to that in control area. Additionally, the number of young culms increased with an increasing number of old culms. These results suggest that recovery of productivity (in term of number) of edible culms may take a few decades. In contrast, the culm diameter of young culms increased linearly with time since soil scarification, but the 95% confidence interval in this relationship suggests that dwarf bamboo can produce thick edible culms soon after soil scarification. These findings will provide useful insights into how to obtain high quality bamboo culms following anthropogenic disturbances in future., Competing Interests: The authors have declared that no competing interests exist.

Hemoglobin (Hb) is associated with cerebral oxygenation status owing to its important role of carrying oxygen to systemic tissues. However, data concerning the associations between Hb levels and cerebral regional oxygen saturation (rSO2) of hemodialysis (HD) patients is limited. We aimed to identify these associations to consider a target Hb level for renal anemia management. This study included 375 HD patients. Cerebral rSO2 before HD was monitored using the INVOS 5100c oxygen saturation monitor. Multivariable linear regression analysis showed that cerebral rSO2 was independently associated with natural logarithm (Ln)-HD duration (standardized coefficient: -0.36), mean blood pressure (standardized coefficient: 0.13), pH (standardized coefficient: -0.10), serum albumin (standardized coefficient: 0.14), presence of diabetes mellitus (standardized coefficient: -0.20), and Hb level (standardized coefficient: 0.29). Furthermore, a generalized linear model with restricted cubic spline function was used to investigate the non-linear association between cerebral rSO2 and Hb levels. In the multivariable analysis for the adjustment with Ln-HD duration, mean blood pressure, pH, serum albumin, and presence of diabetes mellitus, a linear relationship was demonstrated between the two variables (p for linearity = 0.79). Hb levels revealed the positive and significant association with cerebral rSO2 in this study. Moreover, the relationship between cerebral rSO2 and Hb level was proven to be linear. Therefore, the target Hb level in renal anemia management would be considered to be the upper limits for the appropriate management of renal anemia by previous guidelines and position statement from the viewpoint of maintaining cerebral oxygenation in HD patients., Competing Interests: Hakuyukai Medical Corporation provided support in the form of salaries for authors KH, TA, S. Imada, HS, and AW. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

After disasters, issues pertaining to women's health such as irregular periods and bleeding are well surveyed. However, the management of women's health, especially changes in the rate of health checkups, has not been investigated. In the present study, we focused on the change in the cervical cancer screening rates (CCS-Rs) before and after the Great East Japan Earthquake in Miyagi Prefecture, Japan. The earthquake had a magnitude of 9.0, a profound disaster. We examined the CCS-R from 2009 to 2016 in 45 areas of the Miyagi Prefecture. Screening was completed using mobile vans. In the 4 areas impacted by the tsunami after the earthquake, a marked decrease in the CCS-R was observed in 2011 when the earthquake took place (more than a 3% decrease compared with that in the previous year). The CCS-Rs in these 4 regions remained lower in 2016 than in the previous year. In 2009-2016 except for 2014, CCS-Rs in coastal areas (9 areas) were significantly lower than those in the non-coastal areas (36 areas). A delay in seeking healthcare, also known as "patient's delay," is considered as one of the problems of cancer treatment in affected areas. It is possible that a decrease in the CCS-R may lead to low detection of advanced stages of cancer. Therefore, the establishment of a comprehensive medical system including medical screening after a disaster is important for the management of women's health., Competing Interests: The authors have declared that no competing interests exist.

Background: Dietary management is highly important for the maintenance of renal function in patients with chronic kidney disease (CKD). Cerebral oxygen saturation (rSO2) was reportedly associated with the estimated glomerular filtration rate (eGFR) and cognitive function. However, data concerning the association between cerebral rSO2 and dietary intake of CKD patients is limited., Methods: This was a single-center observational study. We recruited 67 CKD patients not undergoing dialysis. Cerebral rSO2 was monitored using the INVOS 5100c oxygen saturation monitor. Energy intake was evaluated by dietitians based on 3-day meal records. Daily protein and salt intakes were calculated from 24-h urine collection., Results: Multivariable regression analysis showed that cerebral rSO2 was independently associated with energy intake (standardized coefficient: 0.370) and serum albumin concentration (standardized coefficient: 0.236) in Model 1 using parameters with p < 0.10 in simple linear regression analysis (body mass index, Hb level, serum albumin concentration, salt and energy intake) and confounding factors (eGFR, serum sodium concentration, protein intake), and the energy/salt index (standardized coefficient: 0.343) and Hb level (standardized coefficient: 0.284) in Model 2 using energy/protein index as indicated by energy intake/protein intake and energy/salt index by energy intake/salt intake in place of salt, protein and energy intake., Conclusions: Cerebral rSO2 is affected by energy intake, energy/salt index, serum albumin concentration and Hb level. Sufficient energy intake and adequate salt restriction is important to prevent deterioration of cerebral oxygenation, which might contribute to the maintenance of cognitive function in addition to the prevention of renal dysfunction in CKD patients., Competing Interests: The authors have declared that no competing interests exist.

The older adult population in developed countries is rapidly increasing, as is the number of older adults with cervical spondylosis. Previous studies on the surgical outcomes of older adults with cervical spondylosis have reported contradictory results. This study aimed to compare the surgical outcomes in adults with cervical spondylosis who were <80 and ≥80 years old. We retrospectively investigated data from adults who underwent surgical treatment for cervical spondylosis between 2006 and 2016. The clinical outcomes and postoperative complications of patients who were <80 years old were compared to those of patients who were ≥80 years old. Of the 108 patients included in the study, 14 (13.0%) were ≥80 years old. The preoperative neurosurgical cervical spine score was significantly different between patients who were <80 (9.1 ± 2.4) and ≥80 (6.1 ± 2.1) years old (p < .001). The recovery rate was 58.2 ± 30.0% and 41.3 ± 24.7% in patients who were <80 and ≥80 years old, respectively (p = .05). However, the number of recovery points scored was 2.8 ± 2.0 and 3.4 ± 2.3 in patients who were <80 and ≥80 years old, respectively, which was not significantly different. Although 12 patients had medical comorbidities, they had no surgical complications. This study clarifies the benefits of surgical treatment for older adults with cervical spondylosis. Generally, older adults have lower recovery rates and are unlikely to experience full recovery; however, surgery for cervical spondylosis appears to improve patients' quality of life., Competing Interests: The authors have declared that no competing interests exist.

Background: The number of patients with Crohn's disease (CD) in Japan has recently been increasing. We examined the association between environmental factors and the development of CD in Japanese focusing on passive smoking., Methods: We conducted a multicenter case-control study and compared the environmental factors of 93 cases who were newly diagnosed with CD to the environmental factors of 132 controls (hospital-, age-, and sex-matched patients with other diseases). The odds ratio (OR) of each factor for the development of CD and the 95% confidence interval (CI) were calculated using a logistic regression model. The association between the details of passive smoking history and the development of CD was examined for those who had an active smoking history "no". Odds ratios of number of passively smoked cigarettes (per day), time of passive smoking (per day) and period of passive smoking (year) were calculated using "passive smoking 'No'" as a reference., Results: History of appendicitis, family history of inflammatory bowel disease, and active smoking history were not significantly associated with the development of CD. Drinking history showed a decreased OR for the development of CD (0.39, 0.19-0.77). "Passive smoking Yes" showed significantly increased OR (2.49, 1.09-5.73). Regarding the association between passive smoking and the development of CD, the OR increased as the number of cigarettes per day, smoking time per day, and smoking duration increased, and there was a dose-response relationship (trend P = 0.024, 0.032, 0.038)., Conclusions: The association between environmental factors and the development of CD among Japanese was examined by case-control study. It was suggested that the passive smoking history may be associated to the development of CD., Competing Interests: The authors have declared that no competing interests exist.

Conophylline (CnP), a vinca alkaloid extracted from the leaves of the tropical plant Tabernaemontana divaricate, attenuates hepatic fibrosis in mice. We have previously shown that CnP inhibits non-alcoholic steatohepatitis (NASH) using a methionine-choline-deficient (MCD) diet-fed mouse model. However, little is known about the CnP mediated inhibition of hepatic steatosis in high-fat diet-induced non-alcoholic fatty liver disease (NAFLD) mouse models. CnP (0.5 and 1 μg/g/body weight) was co-administered along with a high-fat diet to male BALB/c mice. After nine weeks of administering the high-fat diet, hepatic steatosis, triglyceride, and hepatic fat metabolism-related markers were examined. Administration of a high-fat diet for 9 weeks was found to induce hepatic steatosis. CnP dose-dependently attenuated the high-fat diet-induced hepatic steatosis. The diet also attenuated hepatic peroxisome proliferator-activated receptor alpha (PPARA) mRNA levels. PPARA is known to be involved in β-oxidation. CnP upregulated the mRNA levels of hepatic PPARA and its target genes, such as carnitine palmitoyl transferase 1 (CPT1) and CPT2, in a dose-dependent manner in the liver. Furthermore, levels of hepatic β-hydroxybutyrate, which is a type of ketone body, were increased by CnP in a dose-dependent manner. Finally, CnP increased the expression of the autophagosomal marker LC3-II and decreased the expression of p62, which are known to be selectively degraded during autophagy. These results indicate that CnP inhibits hepatic steatosis through the stimulation of β-oxidation and autophagy in the liver. Therefore, CnP might prove to be a suitable therapeutic target for NAFLD., Competing Interests: The authors have declared that no competing interests exist.

Background: Idea density (ID), a natural language processing-based index, was developed to aid in the detection of dementia through the analysis of English narratives. However, it has not been applied to non-English languages due to the difficulties in translating grammatical concepts. In this study, we defined rules to count ideas in Japanese narratives based on a previous study and proposed a novel method to estimate ID in Japanese text using machine translation., Materials: The study participants comprised 42 Japanese patients with dementia aged 69-98 years (mean: 84.95 years). We collected free narratives from the participants to build a speech corpus. The narratives of the patients were translated into English using three machine translation systems: Google Translate, Bing Translator, and Excite Translator. The ID in the translated text was then calculated using the Dependency-based Propositional ID (DEPID), an English ID scoring tool., Results: The maximum correlation coefficient between ID calculated using DEPID-R-ADD (a modified DEPID method to calculate ID after removing vague sentences) and the Mini-Mental State Examination score was 0.473, indicating a moderate correlation., Discussion: The results demonstrate the feasibility of machine translation-based ID measurement. We believe that the basic concept of this translation approach can be applied to other non-English languages., Competing Interests: The authors have declared that no competing interests exist.

Spinal fusion is a standard surgical treatment for patients suffering from low back pain attributed to disc degeneration. However, results are somewhat variable and unpredictable. With fusion the kinematic behaviour of the spine is altered. Fusion and/or stabilizing implants carrying considerable load and prevent rotation of the fused segments. Associated with these changes, a risk for accelerated disc degeneration at the adjacent levels to fusion has been demonstrated. However, there is yet no method to predict the effect of fusion surgery on the adjacent tissue levels, i.e. bone and disc. The aim of this study was to develop a coupled and patient-specific mechanoregulated model to predict disc generation and changes in bone density after spinal fusion and to validate the results relative to patient follow-up data. To do so, a multiscale disc mechanoregulation adaptation framework was developed and coupled with a previously developed bone remodelling algorithm. This made it possible to determine extra cellular matrix changes in the intervertebral disc and bone density changes simultaneously based on changes in loading due to fusion surgery. It was shown that for 10 cases the predicted change in bone density and degeneration grade conforms reasonable well to clinical follow-up data. This approach helps us to understand the effect of surgical intervention on the adjacent tissue remodelling. Thereby, providing the first insight for a spine surgeon as to which patient could potentially be treated successfully by spinal fusion and in which patient has a high risk for adjacent tissue changes., Competing Interests: The authors have declared that no competing interests exist.

Background: A decline in estimated glomerular filtration rate (eGFR) is reportedly associated with increased prevalence rates of cognitive impairment. However, data concerning the association between the cerebral saturation of oxygen (rSO2) and cognitive function of patients with chronic kidney disease (CKD) is limited. This study aimed to (i) elucidate the clinical factors associating with cerebral rSO2 and (ii) investigate the association between cerebral rSO2 and cognitive assessment in CKD patients., Methods: A total of 40 CKD patients not requiring dialysis (26 men and 14 women; mean age, 61.0 ± 2.7 years) were recruited. The numbers of patients at each CKD stage were as follows: G1, 5; G2, 8; G3a, 6; G3b, 5; G4, 11; and G5, 5. Cerebral rSO2 was monitored at the forehead using the oxygen saturation monitor INVOS 5100C. The cognitive function of each patient was confirmed using the Mini-Mental State Examination (MMSE)., Results: Cerebral rSO2 levels were significantly higher in CKD patients than in hemodialysis patients (63.8 ± 1.5% vs. 44.9 ± 2.2%, p < 0.001). Multiple regression analysis showed that cerebral rSO2 was independently associated with eGFR (standardized coefficient: 0.530), serum albumin concentration (standardized coefficient: 0.365), and serum sodium concentration (standardized coefficient: 0.224). Furthermore, MMSE showed a significantly positive correlation with cerebral rSO2 (r = 0.624, p < 0.001)., Conclusions: Cerebral rSO2 was affected by eGFR and serum albumin and sodium concentrations in CKD patients. Furthermore, cerebral rSO2 monitoring, which reflected MMSE scores, might be a useful method for assessing cognitive function in CKD patients., Competing Interests: The authors have declared that no competing interests exist.

Background: The adaptor protein Linker for activation of T cell (LAT) is a key signaling hub used by the T cell antigen receptor. Mutant mice expressing loss-of-function mutations affecting LAT and including a mutation in which tyrosine 136 is replaced by a phenylalanine (LatY136F) develop lymphoproliferative disorder involving T helper type 2 effector cells capable of triggering a massive polyclonal B cell activation that leads to hypergammaglobulinemia G1 and E and to non-resolving inflammation and autoimmunity. The purpose of this study was to evaluate whether the phenotypes of LatY136F knock-in mice resemble the immunohistopathological features of immunoglobulin G4-related disease (IgG4-RD)., Methods: LatY136F knock-in mice were sacrificed at 4-20 weeks of age, and pancreas, kidney, salivary gland and lung were obtained. All organs were stained with hematoxylin-eosin and with Azan for estimation of collagen in fibrosis, and the severity scores of inflammation and fibrosis were evaluated. Immunostainings were performed to analyze the types of infiltrating cells. In addition, the effects of corticosteroid treatment on the development of tissue lesions and serum levels of IgG1 were assessed., Results: Tissue lesions characterized by inflammatory mononuclear cell infiltration and fibrosis were detected in pancreas, kidney, and salivary gland starting from 6 weeks of age. Immunostainings showed pronounced infiltration of plasma cells, CD4-positive T cells, and macrophages. Infiltrating plasma cells predominantly expressed IgG1. The extent of inflammation in pancreas and salivary glands was markedly reduced by corticosteroid treatment., Conclusions: LatY136F knock-in mice displayed increased production of Th2-type IgG1 (a homologue of human IgG4) and developed multiple organ tissue lesions reminiscent of those seen in patients with IgG4-RD. Moreover, the development of these tissue lesions was highly sensitive to corticosteroid treatment like in IgG4-RD. For these reasons we consider the LatY136F knock-in mouse strain to represent a promising model for human IgG4-RD., Competing Interests: The authors have declared that no competing interests exist.

Background: Striatal dopamine is functionally important for the acquisition of motor skills. However, it remains controversial as to whether intrinsic processing of motor learning is impaired in patients with Parkinson's disease (PD), and if yes, whether the impairment is associated with altered striatal dopamine release. Additionally, most neuro-imaging studies of patients with PD have focused on motor sequence learning. In contrast, skill acquisition, specifically, the reconstruction of muscle control of isolated movements, has barely been studied., Method: In this study, we used a repetitive skill training task to measure the peak acceleration of left thumb movement during a process to achieve fine tuning of motor skill. Using 11C-raclopride (RAC) positron emission tomography, we investigated changes in striatal dopamine levels in two conditions of a skill acquisition task: initial skill training (Day 1) and acquired condition (Day 2) with eight patients with PD and age-matched healthy subjects (HS)., Result: In HS, the mean acceleration of each session improved through repeated training sessions on Day 1. However, in patients with PD, the training-associated increase was less than that for HS, and this suggests that repetitive skill training does not result in the effective improvement of motor performance. The regions of interest (ROI) analysis revealed that the RAC-binding potential (BP) was significantly reduced in the right putamen on Day 1 compared with Day 2 in HS. In patients with PD, BP within the right putamen was unchanged. Further, we found that patients with PD had increased dopamine levels within the right ventral striatum (VST) and right caudate (CAU) on Day 2, which was greater than that in HS. These results suggest the impaired activation of the putamen during skill acquisition in patients with PD and compensated hyperactivation of the VST and CAU for the reduced dopamine release within the dorsal putamen (DPU)., Conclusion: Our findings suggest that patients with PD had insufficiency in the process to improve motor skills. Different patterns of striatal dopamine release are relevant to the impairment of these motor functions in patients with PD, at the early stage of the disease., Competing Interests: The authors have declared that no competing interests exist.

Background: Our institution recently started using the femoral neck (FN), as well as the non-shunted distal radius (Rd), to measure bone mineral density (BMD) in patients with chronic kidney disease. We examined the utility and characteristics of this measurement in patients on maintenance dialysis., Methods: We selected 293 patients on chronic dialysis. We measured Rd and FN BMD using dual-energy X-ray absorptiometry, and we reviewed blood test findings, which included hemoglobin, albumin, blood urea nitrogen, creatinine, adjusted calcium, phosphorus, alkaline phosphatase, and intact parathyroid hormone. We conducted a multiple linear regression analysis that was stratified according to sex, age, body weight, height, and dialysis vintage. The Rd and FN BMD values were the dependent variables, and the blood test findings were the independent variables. We compared the areas under the curve (AUCs) of Rd and FN BMD using receiver operating characteristic curve analysis to differentiate between patients with and without fractures., Results: FN BMD was significantly lower than Rd BMD. The general risk factors for osteoporosis, such as low body weight, older age, muscle mass loss, and malnutrition, influenced FN BMD. FN and Rd BMD were not correlated with calcium, phosphorous, or intact parathyroid hormone, whereas a significant, negative correlation with alkaline phosphatase was detected. Both men and women with a history of fragility fractures had significantly lower Rd and FN BMDs than patients without such a history. However, there was no significant difference between the AUCs of FN and Rd BMD for fractures in both men and women., Conclusions: FN BMD was significantly lower than Rd BMD. Additionally, FN BMD was not inferior to Rd BMD for assessing the risk of fracture in patients on maintenance dialysis., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To determine the effect of applied power settings, coil wetness conditions, and e-liquid compositions on the coil heating temperature for e-cigarettes with a "top-coil" clearomizer, and to make associations of coil conditions with emission of toxic carbonyl compounds by combining results herein with the literature., Methods: The coil temperature of a second generation e-cigarette was measured at various applied power levels, coil conditions, and e-liquid compositions, including (1) measurements by thermocouple at three e-liquid fill levels (dry, wet-through-wick, and full-wet), three coil resistances (low, standard, and high), and four voltage settings (3-6 V) for multiple coils using propylene glycol (PG) as a test liquid; (2) measurements by thermocouple at additional degrees of coil wetness for a high resistance coil using PG; and (3) measurements by both thermocouple and infrared (IR) camera for high resistance coils using PG alone and a 1:1 (wt/wt) mixture of PG and glycerol (PG/GL)., Results: For single point thermocouple measurements with PG, coil temperatures ranged from 322 ‒ 1008°C, 145 ‒ 334°C, and 110 ‒ 185°C under dry, wet-through-wick, and full-wet conditions, respectively, for the total of 13 replaceable coil heads. For conditions measured with both a thermocouple and an IR camera, all thermocouple measurements were between the minimum and maximum across-coil IR camera measurements and equal to 74% ‒ 115% of the across-coil mean, depending on test conditions. The IR camera showed details of the non-uniform temperature distribution across heating coils. The large temperature variations under wet-through-wick conditions may explain the large variations in formaldehyde formation rate reported in the literature for such "top-coil" clearomizers., Conclusions: This study established a simple and straight-forward protocol to systematically measure e-cigarette coil heating temperature under dry, wet-through-wick, and full-wet conditions. In addition to applied power, the composition of e-liquid, and the devices' ability to efficiently deliver e-liquid to the heating coil are important product design factors effecting coil operating temperature. Precautionary temperature checks on e-cigarettes under manufacturer-recommended normal use conditions may help to reduce the health risks from exposure to toxic carbonyl emissions associated with coil overheating.

SIRT1 (silent information regulator 2 homolog 1) is a crucial cellular survival protein especially in oxidative stress environments, and has been thought to locate within the nuclei, but also known to shuttle between cytoplasm and nuclei in some cell types. Here, we show for the first time the dynamics of SIRT1 in the presence of single or concurrent cigarette smoke extract (CSE) exposure in human bronchial epithelial cells (HBEC). In BEAS-2B HBEC or primary HBEC, SIRT1 was localized predominantly in cytoplasm, and the CSE (3%) induced nuclear translocation of SIRT1 from cytoplasm in the presence of L-buthionine sulfoximine (an irreversible inhibitor of γ-glutamylcystein synthetase), mainly through the activation of phosphatidylinositol 3-kinase (PI3K) α subunit. This SIRT1 nuclear shuttling was associated with FOXO3a nuclear translocation and the strong induction of several anti-oxidant genes including superoxide dismutase (SOD) 2 and 3; therefore seemed to be an adaptive response. When BEAS-2B cells were pretreated with repeated exposure to a lower concentration of CSE (0.3%), the CSE-induced SIRT1 shuttling and resultant SOD2/3 mRNA induction were significantly impaired. Thus, this result offers a useful cell model to mimic the impaired anti-oxidant capacity in cigarette smoking-associated lung disease such as chronic obstructive pulmonary disease.

The patients' inhalation flow pattern is one of the significant determinants for clinical performance of inhalation therapy. However, the development of inhalation flow meters for various inhalation devices has been unable to keep up with the increasing number of newly launched inhalation devices. In the present study, we developed simple attachment orifices for the inhalation flow pattern monitoring system, which are suitable for all commercial inhalers, and investigated the efficacy of the system on the clinical inhalation instruction for patients co-prescribed dry powder inhaler (DPI) and soft mist inhaler (SMI). First, we constructed simple attachment orifices that were adjusted for 13 commercial inhalers, and examined the correlation between orifice and inhalation device. Second, the inhalation flow patterns (peak inspiratory flow rate, PIFR; inhalation duration time, DT) of patients prescribed a combination of DPI and SMI were monitored before and after inhalation instruction. The inhalation resistance of commercial inhalers are listed in the following order; Twincaps® > Handihaler® > Swinghaler® = Clickhaler® > Twisthaler® > Turbuhaler® > Jenuair® > Diskus® = Ellipta® > Diskhaler® > Breezhaler® > Respimat® = pMDI. The pressure drop via orifice was significantly correlated with that via the commercial inhaler. For the confirmation, all participants achieved the DPI criterion of PIFR. On the other hand, 4 participants (6 clinical visits) of 10 experimented participants could not achieve the essential criterion of DT (> 1.5 sec) for SMI, but all participants improved their duration time after inhalation instruction by pharmacists (P<0.05). In the present study, we successfully developed simple attachment orifice suitable for 13 commercial inhalation devices. These data suggested that our simple attachment orifices for the inhalation flow pattern monitoring system can detect patients with inadequate inhalation patterns via SMI.

Metagenomics uses nucleic acid sequencing to characterize species diversity in different niches such as environmental biomes or the human microbiome. Most studies have used 16S rRNA amplicon sequencing to identify bacteria. However, the decreasing cost of sequencing has resulted in a gradual shift away from amplicon analyses and towards shotgun metagenomic sequencing. Shotgun metagenomic data can be used to identify a wide range of species, but have rarely been applied to fungal identification. Here, we develop a sequence classification pipeline, FindFungi, and use it to identify fungal sequences in public metagenome datasets. We focus primarily on animal metagenomes, especially those from pig and mouse microbiomes. We identified fungi in 39 of 70 datasets comprising 71 fungal species. At least 11 pathogenic species with zoonotic potential were identified, including Candida tropicalis. We identified Pseudogymnoascus species from 13 Antarctic soil samples initially analyzed for the presence of bacteria capable of degrading diesel oil. We also show that Candida tropicalis and Candida loboi are likely the same species. In addition, we identify several examples where contaminating DNA was erroneously included in fungal genome assemblies.

Spinal fusion and bone defect after injuries, removal of bone tumors, and infections need to be repaired by implantation. In an aging society, recovery from these procedures is often difficult. In this study, we found that injection of SPG-178 leads to expression of several bone marker genes and mineralization in vitro, and revealed a significantly higher degree of newly formed bone matrix with use of SPG-178 in vivo. MC3T3-E1 cells were used to evaluate osteoblast differentiation promoted by SPG-178. To analyze gene expression, total RNA was isolated from MC3T3-E1 cells cultured for 7 and 14 days with control medium or SPG-178 medium. Among the several bone marker genes examined, SPG-178 significantly increased the mRNA levels for ALP, BMP-2 and Osteocalcin, OPN, BSP and for the Osterix. Ten-week-old female Wistar rats were used for all transplantation procedures. A PEEK cage was implanted into a bony defect (5 mm) within the left femoral mid-shaft, and stability was maintained by an external fixator. The PEEK cages were filled with either a SPG-178 hydrogel plus allogeneic bone chips (n = 4) or only allogeneic bone chips (n = 4). The rats were then kept for 56 days. Newly formed bone matrix was revealed inside the PEEK cage and there was an increased bone volume per total volume with the cage filled with SPG-178, compared to the control group. SPG-178 has potential in clinical applications because it has several benefits. These include its favorable bone conduction properties its ability to act as a support for various different cells and growth factors, its lack of infection risk compared with materials of animal origin such as ECM, and the ease with which it can be used to fill defects with complex shapes and combined with a wide range of other materials.

Periodontal disease is assessed and its progression is determined via observations on a site-by-site basis. Periodontal data are complex and structured in multiple levels; thus, applying a summary statistical approach (i.e., the mean) for site-level evaluations results in loss of information. Previous studies have shown the availability of mixed effects modeling. However, clinically beneficial information on the progression of periodontal disease during the follow-up period is not available. We conducted a multicenter prospective cohort study. Using mixed effects modeling, we analyzed 18,834 sites distributed on 3,139 teeth in 124 patients, and data were collected 5 times over a 24-month follow-up period. The change in the clinical attachment level (CAL) was used as the outcome variable. The CAL at baseline was an important determinant of the CAL changes, which varied widely according to the tooth surface. The salivary levels of periodontal pathogens, such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, were affected by CAL progression. "Linear"- and "burst"-type patterns of CAL progression occurred simultaneously within the same patient. More than half of the teeth that presented burst-type progression sites also presented linear-type progression sites, and most of the progressions were of the linear type. Maxillary premolars and anterior teeth tended to show burst-type progression. The parameters identified in this study may guide practitioners in determining the type and extent of treatment needed at the site and patient levels. In addition, these results show that prior hypotheses concerning "burst" and "linear" theories are not valid.

Introduction: Degeneration of the intervertebral disc (IVD) is a frequent cause for back pain in humans and dogs. Link-N stabilizes proteoglycan aggregates in cartilaginous tissues and exerts growth factor-like effects. The human variant of Link-N facilitates IVD regeneration in several species in vitro by inducing Smad1 signaling, but it is not clear whether this is species specific. Dogs with IVD disease could possibly benefit from Link-N treatment, but Link-N has not been tested on canine IVD cells. If Link-N appears to be effective in canines, this would facilitate translation of Link-N into the clinic using the dog as an in vivo large animal model for human IVD degeneration., Materials and Methods: This study's objective was to determine the effect of the human and canine variant of Link-N and short (s) Link-N on canine chondrocyte-like cells (CLCs) and compare this to those on already studied species, i.e. human and bovine CLCs. Extracellular matrix (ECM) production was determined by measuring glycosaminoglycan (GAG) content and histological evaluation. Additionally, the micro-aggregates' DNA content was measured. Phosphorylated (p) Smad1 and -2 levels were determined using ELISA., Results: Human (s)Link-N induced GAG deposition in human and bovine CLCs, as expected. In contrast, canine (s)Link-N did not affect ECM production in human CLCs, while it mainly induced collagen type I and II deposition in bovine CLCs. In canine CLCs, both canine and human (s)Link-N induced negligible GAG deposition. Surprisingly, human and canine (s)Link-N did not induce Smad signaling in human and bovine CLCs. Human and canine (s)Link-N only mildly increased pSmad1 and Smad2 levels in canine CLCs., Conclusions: Human and canine (s)Link-N exerted species-specific effects on CLCs from early degenerated IVDs. Both variants, however, lacked the potency as canine IVD regeneration agent. While these studies demonstrate the challenges of translational studies in large animal models, (s)Link-N still holds a regenerative potential for humans.

Introduction: Chronic left ventricular (LV) pressure overload causes relative ischemia with resultant LV dysfunction. We have recently demonstrated that low-intensity pulsed ultrasound (LIPUS) improves myocardial ischemia in a pig model of chronic myocardial ischemia through enhanced myocardial angiogenesis. In the present study, we thus examined whether LIPUS also ameliorates contractile dysfunction in LV pressure-overloaded hearts., Methods and Results: Chronic LV pressure overload was induced with transverse aortic constriction (TAC) in mice. LIPUS was applied to the whole heart three times in the first week after TAC and was repeated once a week for 7 weeks thereafter (n = 22). Animals in the control groups received the sham treatment without LIPUS (n = 23). At 8 weeks after TAC, LV fractional shortening was depressed in the TAC-Control group, which was significantly ameliorated in the TAC-LIPUS group (30.4±0.5 vs. 36.2±3.8%, P<0.05). Capillary density was higher and perivascular fibrosis was less in the LV in the TAC-LIPUS group than in the TAC-Control group. Myocardial relative ischemia evaluated with hypoxyprobe was noted in the TAC-Control group, which was significantly attenuated in the TAC-LIPUS group. In the TAC-LIPUS group, as compared with the control group, mRNA expressions of BNP and collagen III were significantly lower (both P<0.05) and protein expressions of VEGF and eNOS were significantly up-regulated associated with Akt activation (all P<0.05). No adverse effect related to the LIPUS therapy was noted., Conclusions: These results indicate that the LIPUS therapy ameliorates contractile dysfunction in chronically pressure-overloaded hearts through enhanced myocardial angiogenesis and attenuated perivascular fibrosis. Thus, the LIPUS therapy may be a promising, non-invasive treatment for cardiac dysfunction due to chronic pressure overload.

Kawasaki disease (KD) is an acute systemic vasculitis that can potentially cause coronary artery aneurysms in some children. KD occurs approximately 1.5 times more frequently in males than in females. To identify sex-specific genetic variants that are involved in KD pathogenesis in children, we performed a sex-stratified genome-wide association study (GWAS), using the Illumina HumanOmni1-Quad BeadChip data (249 cases and 1,000 controls) and a replication study for the 34 sex-specific candidate SNPs in an independent sample set (671 cases and 3,553 controls). Male-specific associations were detected in three common variants: rs1801274 in FCGR2A [odds ratio (OR) = 1.40, P = 9.31 × 10-5], rs12516652 in SEMA6A (OR = 1.87, P = 3.12 × 10-4), and rs5771303 near IL17REL (OR = 1.57, P = 2.53 × 10-5). The male-specific association of FCGR2A, but not SEMA6A and IL17REL, was also replicated in a Japanese population (OR = 1.74, P = 1.04 × 10-4 in males vs. OR = 1.22, P = 0.191 in females). In a meta-analysis with 1,461 cases and 5,302 controls, a very strong association of KD with the nonsynonymous SNP rs1801274 (p.His167Arg, previously assigned as p.His131Arg) in FCGR2A was confirmed in males (OR = 1.48, P = 1.43 × 10-7), but not in the females (OR = 1.17, P = 0.055). The present study demonstrates that p.His167Arg, a KD-associated FCGR2A variant, acts as a susceptibility gene in males only. Overall, the gender differences associated with FCGR2A in KD provide a new insight into KD susceptibility.