Subjective perceived risk factors of exertional heat exhaustion-related symptoms in female collegiate athletes in Japan.

Associations of exertional heat exhaustion (EHE)-related symptoms with lifestyle habits and health factors specific to female athletes, ranked by relative risks from high to low, remain elusive. Addressing this issue would benefit athletes and coaches, ensuring safer practices during summer sports activities. To address this issue, we distributed paper-based questionnaires to seven universities in Japan, and 983 respondents completed our survey. The questionnaires covered various personal characteristics, lifestyle habits, perceived health factors, perceived athletic activity, and EHE-related symptoms. In this retrospective case-control study, we analyzed the relationships of EHE-related symptoms (objective variables) with lifestyle habits, health factors, and athletic activity conditions (explanatory variables) using the partial proportional odds model. The questionnaire responses revealed that perceived dehydration, sickness, loss of appetite, perceived accumulated fatigue, perceived mental stress, lack of ambient wind, and insufficient rest breaks were positively associated with EHE-related symptoms, with relative risks ranging from high to low. Using an air conditioner during sleep and having a sleep duration of ≥ 6 h were associated with a reduced risk of EHE-related symptoms. The study results suggest that female athletes should be allowed to postpone exercise or reduce its intensity and volume based on their perceptions of dehydration, sickness, loss of appetite, accumulated fatigue, and mental stress in summer to prevent heat-related illnesses.

Successful treatment of onychomatricoma with minimally invasive surgical procedures based on its pathogenesis: A case report.

Onychomatricoma is a rare, fibroepithelial tumor of the nail. Although it is benign, unnecessary and excessive treatment, such as extensive or total removal of the nail matrix, has been reported in the past. Recently, it was speculated that onychomatricoma is derived from onychomatricodermis, the dermal stroma of the nail matrix. Excision of the stromal rather than the epithelial component of the tumor is important. However, since the boundary between the normal and diseased stroma is usually unclear, minimal excision at the base of the tumor projection should be sufficient. We report a case of onychomatricoma and suggest a method of surgical treatment that would minimize postoperative deformity of the nail plate.

Effects of Exercise on Functional Recovery in Patients with Bipolar Depression: A Study Protocol for a Randomized Controlled Trial.

Treatment of bipolar disorder is prone to prolongation despite various treatments, including medication. The efficacy of exercise treatment (i.e., interventions involving physical exercise and sports intervention) for major depressive disorders has been reported for depressive symptoms, cognitive function, and sleep disturbances. However, its efficacy for bipolar disorder has yet to be established. We designed a randomized, controlled, double-blind clinical trial that includes 100 patients with bipolar disorder aged 20-65 years. This will be a cluster-randomized, two-group trial that will be conducted in ten psychiatric hospitals. The hospitals will be randomly assigned to an exercise intervention + treatment as usual (exercise) group or a placebo exercise intervention (stretching) + treatment as usual (control) group. Patients will be assessed using an extensive battery of clinical tests, physical parameters, sleep status, biological parameters (cytokines, neurotrophic factors), and genetic parameters (DNA and RNA) at baseline after a 6-week intervention period, at 10-week follow-up, and at 6-month follow-up. This innovative study may provide important evidence for the effectiveness of exercise in the treatment of bipolar depression based on clinical, biological, genetic, and physiological markers.

S-531011, a Novel Anti-Human CCR8 Antibody, Induces Potent Antitumor Responses through Depletion of Tumor-Infiltrating CCR8-Expressing Regulatory T Cells.

Although regulatory T cells (Treg) are inhibitory immune cells that are essential for maintaining immune homeostasis, Tregs that infiltrate tumor tissue promote tumor growth by suppressing antitumor immunity. Selective reduction of tumor-infiltrating Tregs is, therefore, expected to activate antitumor immunity without affecting immune homeostasis. We previously reported that selective Treg depletion targeted by a C-C motif chemokine receptor 8 (CCR8) resulted in induction of strong antitumor immunity without any obvious autoimmunity in mouse models. Thus, herein, we developed a novel humanized anti-CCR8 monoclonal antibody, S-531011, aimed as a cancer immunotherapy strategy for patients with cancer. S-531011 exclusively recognized human CCR8 among all chemokine receptors and showed potent antibody-dependent cell-mediated cytotoxicity activity toward CCR8+ cells and neutralization activity against CCR8-mediated signaling. We observed that S-531011 reduced tumor-infiltrating CCR8+ Tregs and induced potent antitumor activity in a tumor-bearing human-CCR8 knock-in mouse model. Moreover, combination therapy with S-531011 and anti-mouse programmed cell death 1 (PD-1) antibody strongly suppressed tumor growth compared with anti-PD-1 antibody alone with no observable adverse effects. S-531011 also depleted human tumor-infiltrating Tregs, but not Tregs derived from human peripheral blood mononuclear cells. These results suggest that S-531011 is a promising drug for inducing antitumor immunity without severe side effects in the clinical setting.

Pembrolizumab plus lenvatinib for radically unresectable or metastatic renal cell carcinoma in the Japanese population.

INTRODUCTION: At present, the combination of immune checkpoint inhibitors (ICIs) or an ICI and a tyrosine kinase inhibitor (TKI) are the main treatment options as first-line therapy for metastatic renal cell cancer (mRCC). Among them, pembrolizumab plus lenvatinib was recently launched in Japanese clinical practice. AREA COVERED: In this review, the efficacies and safety profiles of pembrolizumab plus lenvatinib for mRCC between Japanese and global populations are compared. In addition, lenvatinib is currently available for the treatment of not only mRCC but also of endometrial, thyroid, thymic, and hepatocellular cancers. We briefly summarized the characteristics of pembrolizumab plus lenvatinib or lenvatinib monotherapy for these malignancies. Finally, the characteristics of pembrolizumab plus lenvatinib for mRCC in the Japanese population are briefly elucidated. EXPERT OPINION: In order to develop optimal personalized treatment for mRCC patients, it is necessary for physicians who treat mRCC patients to possess in-depth knowledge of not only the efficacy and safety profile of the respective therapies but also of the interpatient heterogeneities between Japanese and global populations.

Subjective perceived risk factors of exertional heat exhaustion-related symptoms in male collegiate athletes in Japan: a case-control study.

The purpose of this study was to investigate associations between lifestyle habits, health factors, athletic activity conditions, and exertional heat exhaustion (EHE)-related symptoms among male college athletes in Japan based on a self-completed questionnaire. The paper-based questionnaires were distributed to 11 universities in Japan, and 2006 respondents completed the survey. Data of personal characteristics (age, body mass), lifestyle habits (sleep duration, use of air-conditioner while sleeping, and practice duration), perceived health factors (loss of appetite, sleep deprivation, sickness, dehydration, accumulated fatigue, and mental stress), perceived athletic activity (insufficient rest breaks, high ambient temperature, excessive humidity, strong solar radiation, lack of ambient wind, and clothing discomfort), and EHE-related symptoms (dizziness, headaches, nausea, and malaise) were collected. The association between lifestyle habits, health factors, athletic activity conditions (explanatory variables), and EHE-related symptoms (objective variables) was analyzed using the partial-proportional odds model. "Perceived dehydration" (odds ratios [ORs] 1.70-2.31, p < 0.002)," "sickness" (ORs 1.35-1.76), p < 0.001), "perceived accumulated fatigue" (ORs 1.13-1.31, p ≤ 0.001), "perceived mental stress" (ORs 1.17-1.31, p < 0.019), "lack of ambient wind" (ORs 1.12-1.19, p < 0.022), "loss of appetite" (ORs 1.16-1.23, p < 0.037), and "sleep deprivation" (ORs 1.15-1.17, p < 0.025) were positively associated with EHE-related symptoms, whereas "using an air conditioner during sleeping" (ORs 0.91, p = 0.047) during summer seasons was negatively associated. These findings suggest that athletes should be allowed to postpone or downregulate exercise intensity and/or volume based on their perceptions of dehydration, sickness, accumulated fatigue, mental stress, loss of appetite, and/or sleep deprivation in the summer to prevent heat illness.

Prognostic nutritional index of early post-pembrolizumab therapy predicts long-term survival in patients with advanced urothelial carcinoma.

Pembrolizumab has been widely used to treat advanced urothelial carcinoma that has progressed after first-line platinum-based chemotherapy. Because its clinical benefits are limited, biomarkers that can predict a good response to pembrolizumab are required. The prognostic nutritional index (PNI), calculated using the serum albumin level and peripheral lymphocyte count, has been evaluated as a predictive biomarker in cancer immunotherapy. The present study investigated the application of PNI as a predictive biomarker for pembrolizumab response in patients with advanced urothelial cancer. A retrospective study was conducted on 34 patients treated with pembrolizumab at Shiga University of Medical Science Hospital between January 2018 and July 2022. The posttreatment PNI (post-PNI) was calculated within 2 months of starting pembrolizumab. The present study investigated the association between post-PNI and objective response, overall survival (OS) and progression-free survival (PFS). The patient cohort was stratified into two categories, high and low post-PNI groups, with a cutoff value of post-PNI at 40. The higher post-PNI group demonstrated a better disease control rate than the lower post-PNI group (complete response + partial response + stable disease, 75 vs. 21%, P=0.004). Regarding median OS, the higher post-PNI group exhibited a significantly longer survival time than the lower post-PNI group (23.1 vs. 2.9 months, P<0.001). Similarly, the higher post-PNI group exhibited a significantly longer PFS than the lower post-PNI group (10.2 vs.1.9 months, P<0.001). Multivariate analysis showed that a higher post-PNI value was an independent predictor for OS (hazard ratio, 0.04; 95% confidence interval, 0.01-0.14; P<0.001) and PFS (hazard ratio, 0.12; 95% confidence interval, 0.04-0.35; P<0.001). The present study indicated that the post-PNI was a predictor of favorable clinical outcomes in patients treated with pembrolizumab for advanced urothelial carcinoma.

Population­based prostate­specific antigen screening for prostate cancer may have an indirect effect on early detection through opportunistic testing in Kusatsu City, Shiga, Japan.

Prostate cancer is the most common genitourinary cancer in men. Population-based serum prostate-specific antigen (PSA) testing is used to screen men for the early detection of asymptomatic prostate cancer. The present study compared the features of patients with prostate cancer in Kusatsu City, the only municipality in Shiga Prefecture of Japan to implement organized PSA screening, with those in other municipalities. The target population for organized PSA screening by mail invitation was men ≥50 years. Patients were pathologically diagnosed via prostate biopsy because of elevated serum PSA. This multicenter observational study was subsequently conducted in 14 hospitals. The following information was extracted from patient records: age, reason for PSA testing, initial PSA level, Gleason score, clinical stage, and place of residence. Risk classification was defined as low, intermediate, high, and advanced. Each patient was stratified according to their city/town. A total of 984 patients diagnosed with prostate cancer in Shiga in 2012 and 2017 were analyzed, of which 955 (97%) were opportunistically tested, with the remaining 29 (3%) assessed by organized screening. In Kusatsu, 93 patients were diagnosed, of whom 26 (28%) were detected by organized screening. By contrast, only three of 891 patients (0.3%) were detected by organized screening in other municipalities. Of patients in Kusatsu, cases identified by opportunistic testing had a higher initial PSA value (P=0.010) than those identified by organized screening. However, patients detected through opportunistic testing in Kusatsu City were younger (P=0.034), had a lower PSA value (P=0.001), and improved risk classification (P<0.001) than those in other municipalities. It was concluded that more patients were diagnosed with early-stage cancer by organized PSA screening. Furthermore, population-based PSA screening in Kusatsu City may have indirectly affected early detection, even by opportunistic testing.

Spinal Transection Switches the Effect of Metabotropic Glutamate Receptor Subtype 7 from the Facilitation to Inhibition of Ejaculation.

Metabotropic glutamate receptor subtype 7 (mGluR7) is a member of the group III mGluRs, which localize to presynaptic active zones of the central nervous system. We previously reported that mGluR7 knockout (KO) mice exhibit ejaculatory disorders, although they have normal sexual motivation. We hypothesized that mGluR7 regulates ejaculation by potentiating the excitability of the neural circuit in the lumbosacral spinal cord, because administration of the mGluR7-selective antagonist into that region inhibits drug-induced ejaculation. In the present study, to elucidate the mechanism of impaired ejaculation in mGluR7 KO mice, we eliminated the influence of the brain by spinal transection (spinalization). Unexpectedly, sexual responses of male mGluR7 KO mice were stronger than those of wild-type mice after spinalization. Histological examination indicated that mGluR7 controls sympathetic neurons as well as parasympathetic neurons. In view of the complexity of its synaptic regulation, mGluR7 might control ejaculation by multi-level and multi-modal mechanisms. Our study provides insight into the mechanism of ejaculation as well as a strategy for future therapies to treat ejaculatory disorders in humans.

Regeneration of Functional Bladder Using Cell-seeded Amnion and P(LA/CL) Scaffolds.

Long-term bladder regeneration has not been successful instead of augmentation with gastrointestinal segments, as is commonly performed for bladder reconstruction. To evaluate whether or not cell-seeded bioabsorbable materials regenerate half-resected bladder in a rabbit model. Female Japanese white rabbits were divided two groups: cell-seeded material (CSM) group and Control (n = 6 each). Control rabbits underwent resection of half the bladder. CSM rabbits were sutured with cell-seeded amniotic membrane and P(LA/CL) material after bladder resection. After 6, 12, and 18 months, rabbits underwent X-ray and cystometry, and bladder tissues after 18 months were subjected to functional and histological analyses. X-ray confirmed the peristaltic movements of the reconstructed bladders in the CSM group. On cystometry, the mean maximum bladder volume, maximum bladder pressure, and 25 mL bladder volume compliance in the CSM group were significantly greater than in the Control group at 6, 12, and 18 months. In addition, organ bath studies showed good contraction under electrical stimulation with increasing stimulation frequency in the CSM group, while, the Control group showed weak contraction on both tests in the central marginal zone. Furthermore, the rates of neovascularization, urothelial and smooth muscle formation, and neurofilamentation in the CSM group were significantly greater than in the Control group. Oral mucosal cell-seeded amniotic membrane and stomach smooth muscle cell-seeded P(LA/CL) scaffold with omentum after abdominal implantation regenerated functional bladder with satisfactory epithelium and smooth muscle without scarring more than 1 year. Impact Statement Regeneration of functional bladder without using gastrointestinal segments has been a huge challenge to urological reconstruction. Various materials, such as nonbioabsorbable materials and biomaterials have been attempted to reconstruct bladder in animal models. However, the long-term results more than a year failed due to the low biocompatibility, high risks, and difficulty creating the materials. In this study, we revealed long-term bladder regeneration using cell-seeded amniotic membrane and P(LA/CL) material in a rabbit model. The new method of bladder reconstruction seems able to regenerate functional bladder with satisfactory bladder epithelium and bladder smooth muscle function without scarring for more than 1 year successfully.

Inhibition of Cell-Free Translation and Replication of Tobacco Mosaic Virus RNA by Exogenously Added 5'-Proximal Fragments of the Genomic RNA.

Replication proteins of tobacco mosaic virus (TMV), a positive-sense RNA virus, co-translationally bind to a 5'-proximal ~70-nucleotide (nt) region of the genomic RNA, referred to as the nuclease-resistant (NR) region for replication template selection. Therefore, disruption of the interaction between the viral replication proteins and viral genomic RNA is expected to inhibit the replication of TMV. In this study, we demonstrate that the addition of small RNA fragments (18-33 nts in length) derived from different regions within the NR region inhibit the binding of TMV replication proteins to viral RNA and TMV RNA replication in a cell-free system. Intriguingly, some of the small RNA fragments also inhibited the translation of mRNA in a sequence-nonspecific manner. These results highlight the pleiotropic roles of the 5'-proximal region of the TMV genome.

Androgen receptor axis-targeted agents are not superior to conventional hormonal therapy for treatment of metastatic prostate cancer.

The present study aimed to use real-world Japanese data to compare the treatment outcome of conventional hormonal therapy to that of using androgen receptor axis-targeted (ARAT) agents for patients with metastatic castration-resistant prostate cancer. The overall survival between the conventional hormonal therapy group and the ARAT agent therapy group was compared using a group of 75 Japanese patients who were treated for metastatic castration-resistant prostate cancer. A subgroup analysis was carried out and the risk factors that affected overall survival (OS) were determined. The median OS from the time of prostate-specific antigen recurrence was 73.1 months in the ARAT group and 45.2 months in the conventional treatment group (P=0.414). Although OS tended to be slightly longer in the ARAT group, the difference between the groups was not significant. Subgroup analysis suggested that the therapeutic outcome of using ARAT agents tended to be less beneficial in patients who were older, and in those with a higher tumor volume or low Gleason grade. In conclusion, use of ARAT agents did not impart a significant survival benefit to patients with metastatic castration-resistant prostate cancer when compared with survival rates in response to conventional therapy. However, there was some clinical benefit when ARAT agents were used after patients developed castration-resistant prostate cancer. These findings suggest that up-front therapy using ARAT agents at the time of the initial hormone therapy can impart clinical benefit in Japanese patients with metastatic prostate cancer.

Complete Genome Sequence of Tea Plant Necrotic Ring Blotch Virus Detected from a Tea Plant in Japan.

We report the complete genome sequence of a Japanese isolate of Tea plant necrotic ring blotch virus (TPNRBV-J). The predicted TPNRBV-J genes have the same organization as those of a Chinese isolate, and the 5' termini of the segments have conserved nucleotide sequences.

Quantification and Visualization of Reliable Hemodynamics Evaluation Based on Non-Contact Arteriovenous Fistula Measurement.

The condition of arteriovenous fistula (AVF) blood flow is typically checked by using auscultation; however, auscultation should require a qualitative judgment dependent on the skills of doctors, and further attention to contact infection is required. For these reasons, this study developed a non-contact and non-invasive medical device to measure the pulse wave of AVFs by applying optical imaging technology. As a first step toward realization of the quantification judgment based on non-contact AVF measurement, we experimentally validated the developed system, whereby the hemodynamics of 168 subjects were visually and quantitatively evaluated based on clinical tests. Based on the evaluation results, the fundamental statistical characteristics of the non-contact measurement, including the average and median values, and distribution of measured signal-to-noise power ratio, were demonstrated. The clinical test results contributed to the future construction of quantified criteria for the AVF condition with the non-contact measurement.

Tomato brown rugose fruit virus resistance generated by quadruple knockout of homologs of TOBAMOVIRUS MULTIPLICATION1 in tomato.

Tomato brown rugose fruit virus (ToBRFV) is an emerging virus of the genus Tobamovirus. ToBRFV overcomes the tobamovirus resistance gene Tm-22 and is rapidly spreading worldwide. Genetic resources for ToBRFV resistance are urgently needed. Here, we show that clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9)-mediated targeted mutagenesis of four tomato (Solanum lycopersicum) homologs of TOBAMOVIRUS MULTIPLICATION1 (TOM1), an Arabidopsis (Arabidopsis thaliana) gene essential for tobamovirus multiplication, confers resistance to ToBRFV in tomato plants. Quadruple-mutant plants did not show detectable ToBRFV coat protein (CP) accumulation or obvious defects in growth or fruit production. When any three of the four TOM1 homologs were disrupted, ToBRFV CP accumulation was detectable but greatly reduced. In the triple mutant, in which ToBRFV CP accumulation was most strongly suppressed, mutant viruses capable of more efficient multiplication in the mutant plants emerged. However, these mutant viruses did not infect the quadruple-mutant plants, suggesting that the resistance of the quadruple-mutant plants is highly durable. The quadruple-mutant plants also showed resistance to three other tobamovirus species. Therefore, tomato plants with strong resistance to tobamoviruses, including ToBRFV, can be generated by CRISPR/Cas9-mediated multiplexed genome editing. The genome-edited plants could facilitate ToBRFV-resistant tomato breeding.

The impact of CCR8+ regulatory T cells on cytotoxic T cell function in human lung cancer.

Regulatory T cells (Tregs) suppress the host immune response and maintain immune homeostasis. Tregs also promote cancer progression and are involved in resistance to immune checkpoint inhibitor treatments. Recent studies identified selective CCR8 expression on tumor-infiltrating Tregs; CCR8+ Tregs have been indicated as a possible new target of cancer immunotherapy. Here, we investigated the features of CCR8+ Tregs in lung cancer patients. CCR8+ Tregs were highly activated and infiltration of CCR8+ Tregs in tumors was associated with poor prognosis in lung cancer patients. We also investigated their immune suppressive function, especially the influence on cytotoxic T lymphocyte cell function. The Cancer Genome Atlas analysis revealed that CD8 T cell activities were suppressed in high CCR8-expressing tumors. Additionally, depletion of CCR8+ cells enhanced CD8 T cell function in an ex vivo culture of lung tumor-infiltrating cells. Moreover, CCR8+ Tregs, but not CCR8- Tregs, induced from human PBMCs markedly suppressed CD8 T cell cytotoxicity. Finally, we demonstrated the therapeutic effect of targeting CCR8 in a murine model of lung cancer. These findings reveal the significance of CCR8+ Tregs for immunosuppression in lung cancer, especially via cytotoxic T lymphocyte cell suppression, and suggest the potential value of CCR8-targeted therapy for cancer treatment.

CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory.

Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8+ Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8- CCR8+ tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8+ effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.

Transmission Risk of Severe Acute Respiratory Syndrome Coronavirus 2 before and after Symptom Onset of the Infector.

An outbreak of coronavirus 2019 (COVID-19) occurred in Ueda City, Nagano prefecture, Japan, which has a population of 150,000. The residents were a population naïve to COVID-19, and many of them had only one chance of exposure, in which careful epidemiological investigation could reveal attack rates among close contact on the specific date of exposure. We identified 89 cases and 328 close contacts. Among the close contacts, 114 had only one chance of exposure to the 20 index cases. During the follow-up period, 17 close contacts tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), overall attack rate of 15%, after the exposure to 6 infectors. The median number of close contacts for the 6 infectors was 5.5 (range 2-14). Attack rates among these close contacts were 13% (1/8), 20% (2/5), 33% (2/6), 50% (1/2), 64% (4/9 and 5/5), and 100% (2/2), respectively. The transmission risk of SARS-CoV-2 appears to peak one day before symptom onset, and is at similar levels two days before (16%) and on the day (20%) of symptom onset. A multidisciplinary approach is needed to control the COVID-19 outbreak, in addition to investigation, which began after case identification.

Identification of a Proline-Kinked Amphipathic α-Helix Downstream from the Methyltransferase Domain of a Potexvirus Replicase and Its Role in Virus Replication and Perinuclear Complex Formation.

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus, the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales. Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.

Clinical, pathological, and therapeutic features of newly diagnosed prostate cancer predominantly detected by opportunistic PSA screening: A survey of Shiga Prefecture, Japan.

BACKGROUND: In all the prefectures of Japan, with the exception of Shiga Prefecture, more than half of local governments have an organized prostate-specific antigen (PSA) screening system in place. However, in the Shiga Prefecture, only a single city performed PSA screening over the time period of this survey. The purpose of the present study was to determine the clinical, pathological, and therapeutic features of newly diagnosed prostate cancer in localities where a formally organized screening system was almost entirely absent. METHODS: A multicenter observational study was conducted in the Shiga Prefecture, which has the lowest rate of population-based PSA-screening in Japan. Patients' age, initial PSA, reasons for PSA testing, Gleason score, clinical stage, and primary treatments were surveyed. We stratified patients according to the reasons for PSA measurement, and compared the differences between groups subject to organized versus opportunistic screening. RESULTS: In the 2 years 2012 and 2017, 984 newly diagnosed prostate cancer patients were analyzed. Of these, 954 (97%) were opportunistically tested (i.e., not as part of an organized screening system), with the remaining 29 (3%) measured as part of an organized screening program. Patients in the former group exhibited a higher initial PSA value than in the organized screening group (median: 11.49 vs. 5.67 ng/ml). They also had worse clinical features, including higher Gleason score and TNM stage. More patients in the organized screening group were treated curatively than in the nonorganized screening group in terms of the primary treatment. The results were similar in a subanalysis of the patients of age 50-69 years. CONCLUSIONS: Organized PSA screening contributes to increasing the number of patients diagnosed with early-stage cancer who can be treated curatively.