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Abstract G9a is a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9), which is involved in the regulation of gene expression. We had previously reported that G9a is expressed in developing tendons in vivo and in vitro and that G9a-deficient tenocytes show impaired proliferation and differentiation in vitro. In this study, we investigated the functions of G9a in tendon development in vivo by using G9a conditional knockout (G9a cKO) mice. We crossed Sox9 Cre/+ mice with G9a fl/fl mice to generate G9a fl/fl ; Sox9 Cre/+ mice. The G9a cKO mice showed hypoplastic tendon formation at 3 weeks of age. Bromodeoxyuridine labeling on embryonic day 16.5 (E16.5) revealed decreased cell proliferation in the tenocytes of G9a cKO mice. Immunohistochemical analysis revealed decreased expression levels of G9a and its substrate, H3K9me2, in the vertebral tendons of G9a cKO mice. The tendon tissue of the vertebrae and limbs of G9a cKO mice showed reduced expression of a tendon marker, tenomodulin (Tnmd), and col1a1 genes, suggesting that tenocyte differentiation was suppressed. Overexpression of G9a resulted in enhancement of Tnmd and col1a1 expression in tenocytes in vitro. These results suggest that G9a regulates the proliferation and differentiation of tendon progenitor cells during tendon development. Thus, our results suggest that G9a plays an essential role in tendon development.

Abstract HMGA1 is an abundant non-histone chromatin protein that has been implicated in embryonic development, cancer, and cellular senescence, but its specific role remains elusive. Here, we combine functional genomics approaches with graph theory to investigate how HMGA1 genomic deposition controls high-order chromatin networks in an oncogene-induced senescence model. While the direct role of HMGA1 in gene activation has been described previously, we find little evidence to support this. Instead, we show that the heterogeneous linear distribution of HMGA1 drives a specific 3D chromatin organization. HMGA1-dense loci form highly interactive networks, similar to, but independent of, constitutive heterochromatic loci. This, coupled with the exclusion of HMGA1-poor chromatin regions, leads to coordinated gene regulation through the repositioning of genes. In the absence of HMGA1, the whole process is largely reversed, but many regulatory interactions also emerge, amplifying the inflammatory senescence-associated secretory phenotype. Such HMGA1-mediated fine-tuning of gene expression contributes to the heterogeneous nature of senescence at the single-cell level. A similar ‘buffer’ effect of HMGA1 on inflammatory signalling is also detected in lung cancer cells. Our study reveals a mechanism through which HMGA1 modulates chromatin compartmentalization and gene regulation in senescence and beyond.

Abstract A higher heart rate is recognized as an independent risk factor for all-cause mortality and cardiovascular events in the general population. However, the association between elevated heart rate and clinical adverse outcomes in patients with non-dialysis-dependent chronic kidney disease (CKD) has not been sufficiently investigated. A total of 1353 participants enrolled in the Fukushima CKD Cohort Study were examined to investigate associations between resting heart rate and clinical adverse outcomes using Cox proportional hazards analysis. The primary outcome of the present study was all-cause mortality, with cardiovascular events as the secondary outcome. Participants were stratified into four groups based on resting heart rate levels at baseline (heart rate

Abstract Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

Abstract Background Yokukansan is a traditional Japanese herbal medicine that is widely administered to individuals of various age groups as an effective drug for anxiety, with few side‐effects. While animal studies have yielded promising results concerning yokukansan's potential in treating anxiety disorders, comprehensive validation has remained incomplete. Moreover, most of the clinical investigations regarding yokukansan have primarily focused on Japanese subjects, and its impact on non‐Asian ethnicities remains unverified. Case Presentation We present the case of a 17‐year‐old Caucasian female with panic disorder (PD). Following her relocation to Japan, she experienced panic attacks due to environmental changes, which subsequently prompted her to visit to our clinic. Various medical examinations revealed no abnormalities, which ruled out the possibility of any physical illness other than PD. Significantly, the administration of yokukansan resulted in a notable reduction in panic attacks, as well as in anticipatory anxiety, accompanied by discernible enhancements in psychosocial functioning and overall quality of life. Furthermore, it is imperative to underscore the fact that no noteworthy adverse events took place. Conclusion Panic attacks and profound anxiety in a Caucasian patient with PD were successfully treated with the use of yokukansan. This case study suggests that yokukansan may be effective in treating PD in Caucasians as well as in Asians. However, to substantiate this preliminary observation, further investigations are required.

Abstract Background The data on bosentan were lacking for the treatment of exercise-induced elevation of pulmonary artery pressure (eePAP) or less severe PH in COPD. This study was conducted to investigate long-term efficacy and safety of bosentan for the treatment of eePAP or less severe PH in COPD. Methods COPD patients diagnosed at this hospital as having COPD (WHO functional class II, III or IV) with eePAP or less severe PH whose respiratory symptoms were stable but remained and gradually progressed even after COPD therapy were randomly assigned in a 1:1 ratio to receive either bosentan or no PH treatment for two years and assessed at baseline and every 6 months for respiratory failure, activities of daily living (ADL), lung and heart functions by right heart catheterization (RHC), and other parameters. Results A total of 29 patients who underwent RHC for detail examination were enrolled in the current study between August 2010 and October 2018.No death occurred in drug-treated group (n = 14) for 2 years; 5 patients died in untreated group (n = 15). Significant differences were noted between the 2 group in hospital-free survival (686.00 ± 55.87 days vs. 499.94 ± 53.27 days; hazard ratio [HR], 0.18; P = 0.026) and overall survival (727 days vs. 516.36 ± 55.38 days; HR, 0.095; P = 0.030) in all causes of death analysis, but not in overall survival in analysis of respiratory-related death. Bosentan was not associated with increased adverse events including requiring O2 inhalation. Conclusions This study suggested that the prognosis for COPD patients with eePAP or less severe PH presenting with respiratory symptoms was very poor and that bosentan tended to improve their prognosis and suppress ADL deterioration without worsening respiratory failure. Trial registration This study was registered with UMIN-CTR Clinical Trial as UMIN000004749 . First trial registration at 18/12/2010.

Abstract Predicting the transition of kidney function in chronic kidney disease is difficult as specific symptoms are lacking and often overlooked, and progress occurs due to complicating factors. In this study, we applied time-series cluster analysis and a light gradient boosting machine to predict the trajectories of kidney function in non-dialysis dependent chronic kidney disease patients with baseline estimated glomerular filtration rate (GFR) ≥ 45 mL/min/1.73 m2. Based on 5-year changes in estimated GFR, participants were stratified into groups with similar trajectories by cluster analysis. Next, we applied the light gradient boosting machine algorithm and Shapley addictive explanation to develop a prediction model for clusters and identify important parameters for prediction. Data from 780 participants were available for analysis. Participants were classified into five classes (Class 1: n = 78, mean [± standard deviation] estimated GFR 100 ± 19.3 mL/min/1.73 m2; Class 2: n = 176, 76.0 ± 9.3 mL/min/1.73 m2; Class 3: n = 191, 59.8 ± 5.9 mL/min/1.73 m2; Class 4: n = 261, 52.7 ± 4.6 mL/min/1.73 m2; and Class 5: n = 74, 53.5 ± 12.0 mL/min/1.73 m2). Declines in estimated GFR were 8.9% in Class 1, 12.2% in Class 2, 4.9% in Class 3, 12.0% in Class 4, and 45.1% in Class 5 during the 5-year period. The accuracy of prediction was 0.675, and the top three most important Shapley addictive explanation values were 1.61 for baseline estimated GFR, 0.12 for hemoglobin, and 0.11 for body mass index. The estimated GFR transition of patients with preserved chronic kidney disease mostly depended on baseline estimated GFR, and the borderline for estimated GFR trajectory was nearly 50 mL/min/1.73 m2.

Abstract Aim The mental healthcare system in Japan is transitioning from institution‐based to community‐based treatment. To prevent prolonged hospitalization and community integration of psychiatric patients, mental health social workers (MHSWs) are pivotal in coordinating post‐discharge arrangements for psychiatric inpatients. This study aimed to propose a care model to improve clinical outcomes in psychiatric emergency wards in Japan. Methods We conducted a mail‐in questionnaire survey targeting medical facilities with psychiatric emergency wards. We collected data of the psychiatric care system, including facility profiles, staffing conditions and caseloads, and the provided psychiatric services and treatment options. Using multiple regression analyses, we explored associations between these data and clinical outcomes, focusing on the average number of days for hospitalization and the integration of patients into a community. Results Data were collected from 82 facilities (response rate, 45.8%). The average number of days for hospitalization and community integration were 64.7 and 327.9 days, respectively. The caseloads for MHSWs were significantly associated with longer hospitalization (β = 0.31, p = 0.009) and shorter duration of community living (β = −0.28, p = 0.027). Conclusion The clinical performance in psychiatric emergency wards surpassed the Japanese government's targets regarding these outcomes. We found that heavy caseloads on MHSWs were associated with worse clinical outcomes for patients in psychiatric emergency wards. These findings suggest that reducing MHSW caseloads (≤20 cases) may be a potential interventional strategy to prevent prolonged hospitalization and promote successful community integration of patients.

Abstract Aim The large number of new long‐stay (NLS) patients and high readmission rates in psychiatric hospitals are longstanding concerns in Japan despite reforms to encourage multidisciplinary support of such patients. Staffing shortages of specialists, especially mental health social workers (MHSWs), may be one of the reasons for these problems to remain unsolved. Methods The authors examined the effectiveness of the MHSW‐centered multidisciplinary care model in preventing NLSs and rehospitalization in terms of both patient dynamics and cost by retrospective comparison of before and after program implementation. Results After our program was introduced, NLS was almost completely prevented. In addition, a significant decrease in readmissions of involuntarily admitted patients was also observed. On the other hand, the resulting decrease in treatment costs and hospital revenues was mismatched by an increase in personnel costs. Conclusion While MHSW‐centered multidisciplinary care is effective for the community integration of patients, there are cost challenges. State policy changes are needed to resolve staffing problems, along with the introduction of appropriate indicators of community integration.

Summary: Mitochondria play a vital role in non-shivering thermogenesis in both brown and subcutaneous white adipose tissues (BAT and scWAT, respectively). However, specific regulatory mechanisms driving mitochondrial function in these tissues have been unclear. Here we demonstrate that prolonged activation of β-adrenergic signaling induces epigenetic modifications in scWAT, specifically targeting the enhancers for the mitochondria master regulator genes Pgc1a/b. This is mediated at least partially through JMJD1A, a histone demethylase that in response to β-adrenergic signals, facilitates H3K9 demethylation of the Pgc1a/b enhancers, promoting mitochondrial biogenesis and the formation of beige adipocytes. Disruption of demethylation activity of JMJD1A in mice impairs activation of Pgc1a/b driven mitochondrial biogenesis and limits scWAT beiging, contributing to reduced energy expenditure, obesity, insulin resistance, and metabolic disorders. Notably, JMJD1A demethylase activity is not required for Pgc1a/b dependent thermogenic capacity of BAT especially during acute cold stress, emphasizing the importance of scWAT thermogenesis in overall energy metabolism.

A novel method has been proposed to induce rapid upward movement of colloidal particles with a density lower than water by applying an electric field of several V/mm in water. This phenomenon, known as the Electrically Induced Rapid Sedimentation (ERS) effect, marks the first occurrence of ‘rapid upward movement of colloidal particles’ within the scope of this phenomenon. Focusing on hollow particles, an investigation of the ERS effect was conducted through transmittance measurement. The hollow particles in water showed a drastic increase in ascending velocity through the application of an electric field. The ascending velocity raised when increasing the electric field strength. Utilizing a quasi-DC electric field (an extremely low-frequency AC electric field), aggregate structures were captured for the first time.

Incorporation of bovine serum-derived albumin formulation (AlbuMAX) into a basic culture medium, MEMα, enables the completion of in vitro spermatogenesis through testicular tissue culture in mice. However, this medium was not effective in other animals. Therefore, we sought an alternative approach for in vitro spermatogenesis using a synthetic medium without AlbuMAX and aimed to identify its essential components. In addition to factors known to be important for spermatogenesis, such as retinoic acid and reproductive hormones, we found that antioxidants (vitamin E, vitamin C, and glutathione) and lysophospholipids are vital for in vitro spermatogenesis. Moreover, based on our experience with microfluidic devices (MFD), we developed an alternative approach, the PDMS-ceiling method (PC method), which involves simply covering the tissue with a flat chip made of PDMS, a silicone resin material used in MFD. The PC method, while straightforward, integrates the advantages of MFD, enabling improved and uniform oxygen and nutrient supply via tissue flattening. Furthermore, our studies underscored the significance of lowering the oxygen concentration to 10–15%. Using an integrated cultivation method based on these findings, we successfully achieved in vitro spermatogenesis in rats, which has been a long-standing challenge. Further improvements in culture conditions would pave the way for spermatogenesis completion in diverse animal species.

Abstract Background The purpose of this study was to develop an internet-based Guided Self-Help CBT (iGSH-CBT) for Bulimia Nervosa (BN) / Binge Eating Disorder (BED) for Japanese patients and to test its feasibility. Methods A single-arm feasibility study. After baseline assessment, patients underwent a 16-week iGSH-CBT program, our Japanese adaption of the European-based Salut BN program. During the treatment period, weekly email support from trained counselors was provided. Evaluations were performed at baseline, after 8 weeks, at the end of the 16-week intervention, and at 2 months after treatment had ended. The primary outcome measure was the change in the weekly frequency of objective binging. Secondary outcomes were the change in the weekly frequency of objective purge episodes, responses on self-report questionnaires of the frequencies of binging and purging, psychopathological characteristics of eating disorders found on BITE, EDE-Q, EDI-2, HADS and EQ-5D, measurements of motivation, and completion of intervention (vs. dropout). Results Participants were 9 female outpatients with BN (n = 5) or BED (n = 4), of whom 8 (88.9%) attended the assessment at the end of the 16-week intervention. Mean age was 28 years (SD = 7.9). Percent change of the weekly frequency of objective binging was -4.40%, and at the end of the 16-week intervention 25% of the participants had achieved symptom abstinence. Conclusions No adverse events were observed during the treatment period and follow-up, and the implementation and operation of the program could be performed without any major problems, confirming the feasibility of iGSH-CBT for BN and BED for Japanese patients. Although no significant change was observed in the weekly frequency of objective binging, the abstinence rate from bulimic behaviors of those who completed the assessments was 25.0% at the end of treatment, and the drop-out rate was 11.1%. iGSH-CBT may be an acceptable and possibly even a preferred method of CBT delivery for Japanese patients with BN or BED, and our Japanese adaptation of Salut BN seems feasible. Trial registration UMIN, UMIN000031962. Registered 1 April 2018 - Retrospectively registered, https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000036334

BackgroundMost genetic analyses that have attempted to identify a locus or loci that can distinguish patients with treatment-resistant schizophrenia (TRS) from those who respond to treatment (non-TRS) have failed. However, evidence from multiple studies suggests that patients with schizophrenia who respond well to antipsychotic medication have a higher dopamine (DA) state in brain synaptic clefts whereas patients with TRS do not show enhanced DA synthesis/release pathways.Patients and methodsTo examine the contribution (if any) of genetics to TRS, we conducted a genetic association analysis of DA-related genes in schizophrenia patients (TRS, n = 435; non-TRS, n = 539) and healthy controls (HC: n = 489).ResultsThe distributions of the genotypes of rs3756450 and the 40-bp variable number tandem repeat on SLC6A3 differed between the TRS and non-TRS groups. Regarding rs3756450, the TRS group showed a significantly higher ratio of the A allele, whereas the non-TRS group predominantly had the G allele. The analysis of the combination of COMT and SLC6A3 yielded a significantly higher ratio of the putative low-DA type (i.e., high COMT activity + high SLC6A3 activity) in the TRS group compared to the two other groups. Patients with the low-DA type accounted for the minority of the non-TRS group and exhibited milder psychopathology.ConclusionThe overall results suggest that (i) SLC6A3 could be involved in responsiveness to antipsychotic medication and (ii) genetic variants modulating brain DA levels may be related to the classification of TRS and non-TRS.

Epstein-Barr virus (EBV) is a ubiquitous human lymphotropic herpesvirus that is causally associated with several malignancies. In addition to latent factors, lytic replication contributes to cancer development. In this study, we examined whether the lytic gene BNRF1, which is conserved among gamma-herpesviruses, has an important role in lymphomagenesis. We found that lymphoblastoid cell lines (LCLs) established by BNRF1-knockout EBV exhibited remarkably lower pathogenicity in a mice xenograft model than LCLs produced by wild-type EBV (LCLs-WT). RNA-seq analyses revealed that BNRF1 elicited the expression of interferon-inducible protein 27 (IFI27), which promotes cell proliferation. IFI27 knockdown in LCLs-WT resulted in excessive production of reactive oxygen species, leading to cell death and significantly decreased their pathogenicity in vivo. We also confirmed that IFI27 was upregulated during primary infection in B-cells. Our findings revealed that BNRF1 promoted robust proliferation of the B-cells that were transformed by EBV latent infection via IFI27 upregulation both in vitro and in vivo.

Abstract The red blood cell distribution width–albumin ratio (RAR) is a prognostic factor for adverse outcomes in various populations. However, whether RAR is associated with renal outcomes remains unclear. Therefore, we aimed to investigate the impact of RAR on the prognosis in patients with chronic kidney disease (CKD). We conducted a retrospective cohort study using 997 CKD patients who were enrolled in the Fukushima Cohort Study. Patients were categorized into tertiles (T1-3) according to the baseline RAR. The associations of RAR with end-stage kidney disease (ESKD) were assessed using Kaplan–Meier curves and multivariable cox regression analyses. Receiver operating characteristic (ROC) curves were performed to test whether significant differences were present between red cell distribution width (RDW) and RAR. The median age was 66, 57% were men, the median eGFR was 47.8 ml/min/1.73 m2, and the median value of RAR was 3.5. The higher RAR group showed an increased risk for ESKD in the Kaplan–Meier curve analysis. Compared to the lowest RAR group, higher RAR groups had a higher risk of ESKD (hazard ratio [HR] 1.37, 95% CI 0.68–2.78 and 2.92, 95% CI 1.44–5.94) for T2 and T3 groups, respectively. ROC curve analysis proved that the discriminating ability of RAR for ESKD was superior to RDW. A higher RAR value was associated with worse renal outcomes in patients with CKD. RAR could be a convenient and useful prognostic marker for renal prognosis.

Abstract The classical organ culture method, in which tissue is placed at the gas‒liquid interphase, is effective at inducing mouse spermatogenesis. However, due to reginal variations in the supply of oxygen and nutrients within a tissue, the progress of spermatogenesis was observed only in limited areas of a tissue. In addition, haploid cell formation and its differentiation to spermatozoon, i.e. spermiogenesis, were infrequent and inefficient. Here, we show that the polydimethylsiloxane (PDMS)-chip ceiling (PC) method, which ensures a uniform supply of nutrients and oxygen throughout the tissue by pressing it into a thin, flat shape, can provide control over the culture space. We used this method to culture testis tissue from neonatal mice, aged 1 to 4 days, and found that modulating the culture space during the experiment by replacing one chip with another that had a higher ceiling effectively increased tissue growth. This adjustment also induced more efficient spermatogenesis, with the process of spermiogenesis being particularly promoted. Meiotic cells were observed from culture day 14 onward, and haploid cells were confirmed at the end of each experiment. This technique was also shown to be a sensitive assay for testicular toxicity. Culture-space control will be a critical regulation parameter for sophisticated tissue culture experiments.

Abstract An in vitro spermatogenesis method using mouse testicular tissue to produce fertile sperm was established more than a decade ago. Although this culture method has generally not been effective in other animal species, we recently succeeded in improving the culture condition to induce spermatogenesis of rats up to the round spermatid stage. In the present study, we introduced acrosin-EGFP transgenic rats in order to clearly monitor the production of haploid cells during spermatogenesis in vitro. In addition, a metabolomic analysis of the culture media during cultivation revealed the metabolic dynamics of the testis tissue. By modifying the culture media based on these results, we were able to induce rat spermatogenesis repeatedly up to haploid cell production, including the formation of elongating spermatids, which was confirmed histologically and immunohistochemically. Finally, we performed a microinsemination experiment with in vitro produced spermatids, which resulted in the production of healthy and fertile offspring. This is the first demonstration of the in vitro production of functional haploid cells that yielded offspring in animals other than mice. These results are expected to provide a basis for the development of an in vitro spermatogenesis system applicable to many other mammals.

The sol–gel state of smectite clay dispersions varies with the volume fraction of clay and electrolyte concentration. In this study, it was elucidated that the drying patterns of droplets from four types of smectite clay dispersions vary according to their sol–gel states. Droplets in the sol state exhibited a ring-shaped pattern, while those in the gel state showed a bump-shaped pattern. Near the boundary between the sol and gel states, patterns featuring both ring and bump structures were observed regardless of whether the droplets were on the sol or gel side. When guest particles or molecules were introduced into the clay dispersion, they dispersed uniformly within the system, and the drying pattern depended on the sol–gel state of the droplets. These findings suggest that the presence or absence of convection within the droplets during drying governs the drying pattern.

Abstract Height loss is caused by osteoporosis, vertebral fractures, disc reduction, postural changes, and kyphosis. Marked long-term height loss is reportedly associated with cardiovascular disease and mortality in the elderly. The present study investigated the relationship between short-term height loss and the risk of mortality using the longitudinal cohort data of the Japan Specific Health Checkup Study (J-SHC). Included individuals were aged 40 years or older and received periodic health checkups in 2008 and 2010. The exposure of interest was height loss over the 2 years, and the outcome was all-cause mortality over subsequent follow up. Cox proportional hazard models were used to examine the association between height loss and all-cause mortality. Of the 222,392 individuals (88,285 men, 134,107 women) included in this study, 1436 died during the observation period (mean 4.8 ± 1.1 years). The subjects were divided into two groups based on a cut-off value of height loss of 0.5 cm over 2 years. The adjusted hazard ratio (95% confidence interval) was 1.26 (1.13–1.41) for exposure to height loss ≥ 0.5 cm compared to height loss

ABSTRACT The in vitro growth transformation of primary B cells by Epstein-Barr virus (EBV) is the initial step in the development of posttransplant lymphoproliferative disorder (PTLD). We performed electron microscopic analysis and immunostaining of primary B cells infected with wild-type EBV. Interestingly, the nucleolar size was increased by two days after infection. A recent study found that nucleolar hypertrophy, which is caused by the induction of the IMPDH2 gene, is required for the efficient promotion of growth in cancers. In the present study, RNA-seq revealed that the IMPDH2 gene was significantly induced by EBV and that its level peaked at day 2. Even without EBV infection, the activation of primary B cells by the CD40 ligand and interleukin-4 increased IMPDH2 expression and nucleolar hypertrophy. Using EBNA2 or LMP1 knockout viruses, we found that EBNA2 and MYC, but not LMP1, induced the IMPDH2 gene during primary infections. IMPDH2 inhibition by mycophenolic acid (MPA) blocked the growth transformation of primary B cells by EBV, leading to smaller nucleoli, nuclei, and cells. Mycophenolate mofetil (MMF), which is a prodrug of MPA that is approved for use as an immunosuppressant, was tested in a mouse xenograft model. Oral MMF significantly improved the survival of mice and reduced splenomegaly. Taken together, these results indicate that EBV induces IMPDH2 expression through EBNA2-dependent and MYC-dependent mechanisms, leading to the hypertrophy of the nucleoli, nuclei, and cells as well as efficient cell proliferation. Our results provide basic evidence that IMPDH2 induction and nucleolar enlargement are crucial for B cell transformation by EBV. In addition, the use of MMF suppresses PTLD. IMPORTANCE EBV infections cause nucleolar enlargement via the induction of IMPDH2, which are essential for B cell growth transformation by EBV. Although the significance of IMPDH2 induction and nuclear hypertrophy in the tumorigenesis of glioblastoma has been reported, EBV infection brings about the change quickly by using its transcriptional cofactor, EBNA2, and MYC. Moreover, we present here, for the novel, basic evidence that an IMPDH2 inhibitor, namely, MPA or MMF, can be used for EBV-positive posttransplant lymphoproliferative disorder (PTLD).

Using the newly developed STREAMING-tag system, the authors find that clusters of RNA polymerase II and BRD4 are formed specifically in the transcriptionally active state near the Nanog gene in mouse embryonic stem cells.

Abstract The transtheoretical model (TTM) is a commonly used model of health-related behavioral change. However, the practical effect of using this model for chronic kidney disease (CKD) self-management remains unclear. This study aimed to investigate the association between stages of change for lifestyle behavior and the incidence of CKD in the general Japanese population. A retrospective cohort study was conducted among 178,780 non-CKD participants aged 40–74 years who underwent annual health check-ups for two consecutive years between 2008 and 2009. Health behavior change was determined using questionnaires based on the TTM, which consists of five stages of change (precontemplation, contemplation, preparation, action, and maintenance). The exposure of interest was the change in stages between two years. Participants were categorized into 3 groups ‘improved’, ‘unchanged’, or ‘deteriorated’. The association between the change in stages and the incidence of CKD was examined using logistic regression analysis. After one year of follow-up, 20.0% of participants developed CKD. Participants in the deteriorated group showed a significantly higher risk of CKD incidence than in the improved group. Promoting the stage of change for healthy lifestyle behaviors evaluated by the TTM was associated with a risk reduction for the incidence of CKD.

How β-AR signaling coordinates epigenetic and transcriptional pathways is unknown. Here the authors show that cold-induced β-AR signaling negatively regulates MYPT1-PP1β phosphatase activity to orchestrate both pathways for beige adipogenesis.

A genome-wide CRISPR screen for HSV-1 host factors using near-haploid HAP1 cells revealed PAPSS1 as an essential factor for heparan sulfate biosynthesis and HSV-1 infection, and identified several other host factors also involved in both processes.

Abstract Fractal scaling in animal behavioral activity, where similar temporal patterns appear repeatedly over a series of magnifications among time scales, governs the complex behavior of various animal species and, in humans, can be altered by neurodegenerative diseases and aging. However, the mechanism underlying fractal scaling remains unknown. Here, we cultured C. elegans in a microfluidic device for 3 days and analyzed temporal patterns of C. elegans activity by fractal analyses. The residence-time distribution of C. elegans behaviors shared a common feature with those of human and mice. Specifically, the residence-time power-law distribution of the active state changed to an exponential-like decline at a longer time scale, whereas the inactive state followed a power-law distribution. An exponential-like decline appeared with nutrient supply in wild-type animals, whereas this decline disappeared in insulin-signaling-defective daf-2 and daf-16 mutants. The absolute value of the power-law exponent of the inactive state distribution increased with nutrient supply in wild-type animals, whereas the value decreased in daf-2 and daf-16 mutants. We conclude that insulin signaling differentially affects mechanisms that determine the residence time in active and inactive states in C. elegans behavior. In humans, diabetes mellitus, which is caused by defects in insulin signaling, is associated with mood disorders that affect daily behavioral activities. We hypothesize that comorbid behavioral defects in patients with diabetes may be attributed to altered fractal scaling of human behavior.

Abstract Background Viruses must adapt to the environment of their host cells to establish infection and persist. Diverse mammalian cells, including virus-infected cells, release extracellular vesicles such as exosomes containing proteins and miRNAs, and use these vesicles to mediate intercellular communication. However, the roles of exosomes in viral infection remain unclear. Results We screened viral proteins to identify those responsible for the exosome-mediated enhancement of Epstein–Barr virus (EBV) infection. We identified BGLF2 protein encapsulated in exosomes, which were released by EBV-infected cells. BGLF2 protein is a tegument protein that exists in the space between the envelope and nucleocapsid, and it is released into the cytoplasm shortly after infection. BGLF2 protein-containing exosomes enhanced viral gene expression and repressed innate immunity, thereby supporting the EBV infection. Conclusions The EBV tegument protein BGLF2 is encapsulated in exosomes and released by infected cells to facilitate the establishment of EBV infection. These findings suggest that tegument proteins support viral infection not only between the envelope and nucleocapsid, as well as in extraviral particles such as exosomes. Graphical abstract Video abstract

Mouse spermatogenesis, from spermatogonial stem cell proliferation to sperm formation, can be reproduced in vitro by culturing testis tissue masses of neonatal mice. However, it remains to be determined whether this method is also applicable when testis tissues are further divided into tiny fragments, such as segments of the seminiferous tubule (ST), a minimal anatomical unit for spermatogenesis. In this study, we investigated this issue using the testis of an Acrosin-GFP/Histone H3.3-mCherry (Acr/H3) double-transgenic mouse and monitored the expression of GFP and mCherry as indicators of spermatogenic progression. Initially, we noticed that the cut and isolated stretches of ST shrunk rapidly and conglomerated. We therefore maintained the isolation of STs in two ways: segmental isolation without truncation or embedding in soft agarose. In both cases, GFP expression was observed by fluorescence microscopy. By whole-mount immunochemical staining, meiotic spermatocytes and round and elongating spermatids were identified as Sycp3-, crescent-form GFP-, and mCherry-positive cells, respectively. Although the efficiency was significantly lower than that with tissue mass culture, we clearly showed that spermatogenesis can be induced up to the elongating spermatid stage even when the STs were cut into short segments and cultured in isolation. In addition, we demonstrated that lowered oxygen tension was favorable for spermatogenesis both for meiotic progression and for producing elongating spermatids in isolated STs. Culturing isolated STs rather than tissue masses is advantageous for explicitly assessing the various environmental parameters that influence the progression of spermatogenesis.

Mouse spermatogenesis, from spermatogonial stem cell proliferation to sperm formation, can be reproduced in vitro by culturing testis tissue masses of neonatal mice. However, it remains to be determined whether this method is also applicable when testis tissues are further divided into tiny fragments, such as segments of the seminiferous tubule (ST), a minimal anatomical unit for spermatogenesis. In this study, we investigated this issue using the testis of an Acrosin-GFP/Histone H3.3-mCherry (Acr/H3) double-transgenic mouse and monitored the expression of GFP and mCherry as indicators of spermatogenic progression. Initially, we noticed that the cut and isolated stretches of ST shrunk rapidly and conglomerated. We therefore maintained the isolation of STs in two ways: segmental isolation without truncation or embedding in soft agarose. In both cases, GFP expression was observed by fluorescence microscopy. By whole-mount immunochemical staining, meiotic spermatocytes and round and elongating spermatids were identified as Sycp3-, crescent-form GFP-, and mCherry-positive cells, respectively. Although the efficiency was significantly lower than that with tissue mass culture, we clearly showed that spermatogenesis can be induced up to the elongating spermatid stage even when the STs were cut into short segments and cultured in isolation. In addition, we demonstrated that lowered oxygen tension was favorable for spermatogenesis both for meiotic progression and for producing elongating spermatids in isolated STs. Culturing isolated STs rather than tissue masses is advantageous for explicitly assessing the various environmental parameters that influence the progression of spermatogenesis.

Abstract Background Coronavirus disease 2019 (COVID-19) has heavily affected the economy, industries, and medicine. Local governments and medical institutions have struggled to respond. The purpose of this questionnaire survey was to evaluate strategies for pharmacy services, availability of ethanol for disinfection, and measures adopted for in-house infection control aiming to enhance future infection control efforts. Methods Since pharmacies have been also affected by the COVID-19 pandemic, we surveyed COVID-19 measures taken at 174 pharmacies in Ehime prefecture, Japan. Results The survey showed that pharmacies made changes to facilities and equipment, such as installing partitions at dispensing counters, procuring personal protective equipment for employees, and using ethanol for disinfection, even when these items were in short supply. Pharmacies also adopted new strategies, such as holding meetings with suppliers and internal staff via online platforms. Many pharmacies also undertook COVID-19 preventive measures, such as preparing documentation of infection control measures and disinfectants. Moreover, they held lectures and workshops on disinfection and infection control measures. Conclusions From public health perspectives, pharmacies should adopt measures to prevent infections spread and, if necessary, utilise online tools and other new strategies to achieve this goal. It is also essential to educate the public about infection control, stockpile supplies, and work with hospitals to prevent COVID-19 spreads.

How enhancers transition from a hypoacetylated primed state to a hyperacetylated-active state in response to differentiation stimuli is incompletely understood. Here the authors show that SETD5 forms a complex with NCoR-HDAC3 co-repressor to prevent histone acetylation of master adipogenic gene enhancers, while SETD5 degradation triggers enhancer hyperacetylation and transition to active state.

Shibuta et al develop a live imaging system that allows them to track the spatiotemporal dynamics of RNA polymerase II modification in single cells in Arabidopsis thaliana. This approach potentially enables a more detailed understanding of transcriptional dynamics in plants.

During transcription, RNA polymerase II (RNAP2) is recruited to promoters and phosphorylated stepwise; so far, these steps have not been visualized in a single-copy gene in live cells. Here the authors use single-molecule microscopy to visualize endogenous phosphorylated RNAP2 and nascent mRNA synthesis at a single locus in living cells.

Abstract Objective This study aimed to validate the Sick, Control, One stone, Fat, and Food (SCOFF) questionnaire in relation to the Eating Disorders Examination Questionnaire (EDE-Q) and to examine the appropriateness of a question concerning weight loss among Japanese university students. The psychometric properties of the two Japanese versions were determined among 649 Japanese college students. The original version (SCOFF-O) employed the original item 3, whereas the revised version (SCOFF-2.5) modified the item to “Have you recently lost more than 2.5 kg within three months?” Validity was tested relative to EDE-Q. Results The test–retest reliabilities of SCOFF-O and SCOFF-2.5 were 0.52 and 0.57, while the correlations of SCOFF-O and SCOFF-2.5 with EDE-Q were r = 0.53 and r = 0.56. The sensitivity and specificity of SCOFF-O were 65.2 and 89.7, and those of SCOFF-2.5 were 69.5 and 86.5, respectively. There were significant correlations between the question concerning losing 2.5 kg and the EDE-Q subscales. The Japanese version of SCOFF-2.5 is an appropriate tool for the early screening of eating disorders among Japanese university students.

Abstract The Martian Moons Exploration (MMX) spacecraft is a JAXA mission to Mars and its moons Phobos and Deimos. MMX will be equipped with the Circum-Martian Dust Monitor (CMDM) which is a newly developed light-weight ( $$\mathrm {650\,g}$$ 650 g ) large area ( $$1\,\mathrm {m}^{2}$$ 1 m 2 ) dust impact detector. Cometary meteoroid streams (also referred to as trails) exist along the orbits of comets, forming fine structures of the interplanetary dust cloud. The streams consist predominantly of the largest cometary particles (with sizes of approximately $$100\,\mu \mathrm { m}$$ 100 μ m to 1 cm) which are ejected at low speeds and remain very close to the comet orbit for several revolutions around the Sun. The Interplanetary Meteoroid Environment for eXploration (IMEX) dust streams in space model is a new and recently published universal model for cometary meteoroid streams in the inner Solar System. We use IMEX to study the detection conditions of cometary dust stream particles with CMDM during the MMX mission in the time period 2024 to 2028. The model predicts traverses of 12 cometary meteoroid streams with fluxes of $$100\,\mu \mathrm { m}$$ 100 μ m and bigger particles of at least $$10^{-3}\,\mathrm {m}^{-2}\,\mathrm {day}^{-1}$$ 10 - 3 m - 2 day - 1 during a total time period of approximately 90 days. The highest flux of $$0.15\,\mathrm {m}^{-2}\,\mathrm {day}^{-1}$$ 0.15 m - 2 day - 1 is predicted for comet 114P/Wiseman-Skiff in October 2026. With its large detection area and high sensitivity CMDM will be able to detect cometary meteoroid streams en route to Phobos. Our simulation results for the Mars orbital phase of MMX also predict the occurrence of meteor showers in the Martian atmosphere which may be observable from the Martian surface with cameras on board landers or rovers. Finally, the IMEX model can be used to study the impact hazards imposed by meteoroid impacts onto large-area spacecraft structures that will be particularly necessary for crewed deep space missions.

N6-methyladenosine (m6A) mediates various biological processes by affecting RNA stability, splicing, and translational efficiency. The roles of m6A modification in Epstein-Barr virus (EBV) infection in the lytic phase are unclear. Here, knockout of the m6A methyltransferase, N6-methyladenosine methyltransferase-like 3 (METTL3), or inhibition of methylation by DAA or UZH1a decreased the expression of viral lytic proteins and reduced progeny virion production. Interestingly, cell growth and viability were decreased by induction of the lytic cycle in METTL3-knockout or inhibitor-treated cells. Apoptosis was induced in those conditions possibly because of a decreased level of the anti-apoptotic viral protein, BHRF1. Therefore, m6A shows potential as a target of lytic induction therapy for EBV-associated cancers, including Burkitt lymphoma.

Prognosis for advanced oral carcinoma remains poor. Oncolytic virotherapy uses replication-competent viruses to infect and kill only the tumor cells. However, it has been difficult to investigate the oncolytic activity of viruses against oral carcinomas in mouse models. This study established a mouse model of oral cancer and investigated the in vitro and in vivo anti-tumor effects of HF10, a highly attenuated, replication-competent herpes simplex virus (HSV)-1. Mouse tongue cancer was induced by injecting 4-nitroquinoline 1-oxide into the mouse tongue. The murine oral cancer cell line isolated from this tumor, named NMOC1, formed invasive carcinoma within a week when injected into mouse tongue. HF10 successfully infected, replicated, and spread in the cancer cells in vitro. HF10 was able to kill cancer cells isolated from human or mouse tongue tumor. HF10 injection into tongue carcinomas prolonged mouse survival without any side effects or weight loss. Intertumoral injection of GFP-expressing HF10 confirmed that viral spread was confined within the tumors. Immunohistochemical staining showed that HF10 induced infiltration of CD8-positive T cells around HSV-infected cells in the tumor mass, implying increased anti-tumor immunity. We successfully established an oral cancer cell line and showed that HF10 is a promising therapeutic agent for oral cancer.

Abstract Background Responsiveness to erythropoiesis-stimulating agents (ESAs) is thought to be related to prognosis in patients on hemodialysis. A multi-center, prospective cohort study was conducted to investigate the effects of hyporesponsiveness to long-acting ESAs on cardiovascular events and mortality in Japanese patients on chronic hemodialysis. Methods A total of 127 chronic hemodialysis patients treated with long-acting ESAs were followed-up prospectively. Responsiveness to ESA was evaluated using an erythropoietin resistance index (ERI) calculated by dividing the weekly body-weight-adjusted ESA dose by the hemoglobin concentration. The primary endpoint of this survey was defined as a combination of cardiovascular events and all-cause deaths. The association between hyporesponsiveness to ESAs evaluated by the highest quartile of the ERI and the primary endpoint was investigated. Results During the follow-up period (median 4.6 years), 32 patients reached the primary end point. Kaplan-Meier curve analysis showed that patients with ESA hyporesponsiveness belonging to the highest quartile of the ERI reached the primary end point more frequently than those without (P = 0.031). Cox regression analysis showed that an ERI in the highest quartile was an independent predictor of the primary end point, even after adjustment using a propensity score (hazard ratio 2.76, 95% confidence interval 1.19–6.40). Conclusions ESA hyporesponsiveness in hemodialysis patients treated with long-acting ESAs is related to cardiovascular events and death.

Abstract The morphology of the Golgi complex is influenced by the cellular context, which strictly correlates with nuclear functions; however, the mechanism underlying this association remains elusive. The inner nuclear membrane SUN proteins, SUN1 and SUN2, have diverse functions together with the outer nuclear membrane nesprin proteins, which comprise the LINC complex. We found that depletion of SUN1 leads to Golgi complex dispersion with maintenance of ministacks and retained function for vesicle transport through the Golgi complex. In addition, SUN2 associates with microtubule plus-end-directed motor KIF20A, possibly via nesprin-2. KIF20A plays a role in the Golgi dispersion in conjunction with the SUN2-nesprin-2 LINC complex in SUN1-depleted cells, suggesting that SUN1 suppresses the function of the SUN2-nesprin-2 LINC complex under a steady-state condition. Further, SUN1-knockout mice, which show impaired cerebellar development and cerebellar ataxia, presented altered Golgi morphology in Purkinje cells. These findings revealed a regulation of the Golgi organization by the LINC complex.

Abstract Examining intestine–liver interactions is important for achieving the desired physiological drug absorption and metabolism response in in vitro drug tests. Multi-organ microphysiological systems (MPSs) constitute promising tools for evaluating inter-organ interactions in vitro. For coculture on MPSs, normal cells are challenging to use because they require complex maintenance and careful handling. Herein, we demonstrated the potential of coculturing normal cells on MPSs in the evaluation of intestine–liver interactions. To this end, we cocultured human-induced pluripotent stem cell-derived intestinal cells and fresh human hepatocytes which were isolated from PXB mice with medium circulation in a pneumatic-pressure-driven MPS with pipette-friendly liquid-handling options. The cytochrome activity, albumin production, and liver-specific gene expressions in human hepatocytes freshly isolated from a PXB mouse were significantly upregulated via coculture with hiPS-intestinal cells. Our normal cell coculture shows the effects of the interactions between the intestine and liver that may occur in vivo. This study is the first to demonstrate the coculturing of hiPS-intestinal cells and fresh human hepatocytes on an MPS for examining pure inter-organ interactions. Normal-cell coculture using the multi-organ MPS could be pursued to explore unknown physiological mechanisms of inter-organ interactions in vitro and investigate the physiological response of new drugs.

How euchromatin organisation and transcription are related is unclear. Here, the authors observe the dynamics of euchromatin organization showing that accumulating RNA recruits RNA-binding proteins that together with transcribed euchromatin separate from non-transcribed euchromatin, forming microphases.

Abstract In vitro spermatogenesis (IVS) using air–liquid interphase organ culture method is possible with mouse testis tissues. The same method, however, has been hardly applicable to animals other than mice, only producing no or limited progression of spermatogenesis. In the present study, we challenged IVS of rats with modifications of culture medium, by supplementing chemical substances, including hormones, antioxidants, and lysophospholipids. In addition, reducing oxygen tension by placing tissues in an incubator of lower oxygen concentration and/or applying silicone cover ceiling on top of the tissue were effective for improving the spermatogenic efficiency. Through these modifications of the culture condition, rat spermatogenesis up to round spermatids was maintained over 70 days in the cultured tissue. Present results demonstrated a significant progress in rat IVS, revealing conditions commonly favorable for mice and rats as well as finding rat-specific optimizations. This is an important step towards successful IVS in many animal species, including humans.

Abstract Background Since there was no previous report, we analyzed the relationship between French Risk Stratification parameters in pulmonary arterial hypertension (PAH) and mean pulmonary arterial pressures (mPAP) using Japan PH Registry (JAPHR) national-wide cohort. Methods We enrolled 108 patients with PAH from JAPHR from previous reported cohort and analyzed the relations between French Risk Stratification scores and hemodynamic improvements. Results The ratio meeting 0 to 4 French Risk Stratification score was 21.3%, 31.5%, 32.4%, 13.0%, and 1.9% at baseline, and 6.5%, 23.2%, 33.3%, 23.2%, 13.9% at follow-up, respectively. The improvements in the number of criteria met were associated both with mPAP at follow-up (p = 0.03) and with the improvements in mPAP (p

Abstract As previous studies have reported finding an association between hyperuricemia and the development of cardiovascular and chronic kidney disease, hyperuricemia is thought to be an independent risk factor for hypertension and diabetic mellitus. However, we have not been able to determine whether the use of xanthine oxidase inhibitors can reduce cardiovascular disease. The present study used the longitudinal data of the Fukushima Cohort Study to investigate the relationship between the use of xanthine oxidase inhibitors and cardiovascular events in patients with cardiovascular risks. During the 3-year period between 2012 and 2014, a total of 2724 subjects were enrolled in the study and followed. A total of 2501 subjects had hypertension, diabetic mellitus, dyslipidemia, or chronic kidney disease, and were identified as having cardiovascular risks. The effects of xanthine oxidase inhibitor use on the development of cardiovascular events was evaluated in these patients using a time to event analysis. During the observational periods (median 2.7 years), the incidence of cardiovascular events was 20.7 in subjects with xanthine oxidase inhibitor and 11.2 (/1000 person-years, respectively) in those without. Although a univariate Cox regression analysis showed that the risk of cardiovascular events was significantly higher in subjects administered xanthine oxidase inhibitors (HR = 1.87, 95% CI 1.19–2.94, p = 0.007), the risk was significantly lower in subjects administered a xanthine oxidase inhibitor after adjustment for covariates (HR = 0.48, 95% CI 0.26–0.91; p = 0.024) compared to those without. Xanthine oxidase inhibitor use was associated with reduced risk of cardiovascular disease in patients with cardiovascular risk factors.

Two previously unreported onnamide analogs, 2Z- and 6Z-onnamides A (1 and 2), were isolated from the marine sponge Theonella conica collected at Amami-Oshima Is., Kagoshima Prefecture, Japan. Structures of compounds 1 and 2 were elucidated by spectral analysis. Structure–activity relationships (SARs) for effects on histone modifications and cytotoxicity against HeLa and P388 cells were characterized. The geometry in the polyene systems of onnamides affected the histone modification levels and cytotoxicity.

N6-methyladenosine (m6A) is a post-transcriptional modification of RNA involved in transcript transport, degradation, translation, and splicing. We found that HBV RNA is modified by m6A predominantly in the coding region of HBx. The mutagenesis of methylation sites reduced the HBV mRNA and HBs protein levels. The suppression of m6A by an inhibitor or knockdown in primary hepatocytes decreased the viral RNA and HBs protein levels in the medium. These results suggest that the m6A modification of HBV RNA is needed for the efficient replication of HBV in hepatocytes.

The nuclear lamina regulates chromatin organization and gene positioning. Here the authors show that CROWDED NUCLEI proteins contribute to the meshwork lamina structure in Arabidopsis nuclei and regulate copper tolerance by promoting lamina association and expression of copper response genes.