Propagating and banking genetically diverse human sapovirus strains using a human duodenal cell line: investigating antigenic differences between strains.

There are four genogroups and 18 genotypes of human sapoviruses (HuSaVs) responsible for acute gastroenteritis. To comprehend their antigenic and virological differences, it is crucial to obtain viral stocks of the different strains. Previously, we utilized the human duodenum-derived cell line HuTu80, and glycocholate, a conjugated bile acid, to replicate and propagate GI.1, GI.2, and GII.3 HuSaVs (H. Takagi et al., Proc Natl Acad Sci U S A 117:32078-32085, 2020, https://10.1073/pnas.2007310117). First, we investigated the impact of HuTu80 passage number on HuSaV propagation. Second, we demonstrated that taurocholate improved the initial replication success rate and viral RNA levels in fecal specimens relative to glycocholate. By propagating 15 HuSaV genotypes (GI.1-7, GII.1-5, -8, and GV.1-2) and accomplishing preparation of viral stocks containing 1.0 × 109 to 3.4 × 1011 viral genomic copies/mL, we found that all strains required bile acids for replication, with GII.4 showing strict requirements for taurocholate. The deduced VP1 sequences of the viruses during the scale-up of serial passaged virus cultures were either identical or differed by only two amino acids from the original sequences in feces. In addition, we purified virions from nine strains of different genotypes and used them as immunogens for antiserum production. Enzyme-linked immunosorbent assays (ELISAs) using rabbit and guinea pig antisera for each of the 15 strains of different genotypes revealed distinct antigenicity among the propagating viruses across genogroups and differences between genotypes. Acquisition of biobanked viral resources and determination of key culture conditions will be valuable to gain insights into the common mechanisms of HuSaV infection. IMPORTANCE: The control of human sapovirus, which causes acute gastroenteritis in individuals of all ages, is challenging because of its association with outbreaks similar to those caused by human norovirus. The establishment of conditions for efficient viral propagation of various viral strains is essential for understanding the infection mechanism and identifying potential control methods. In this study, two critical factors for human sapovirus propagation in a conventional human duodenal cell line were identified, and 15 strains of different genotypes that differed at the genetic and antigenic levels were isolated and used to prepare virus stocks. The preparation of virus stocks has not been successful for noroviruses, which belong to the same family as sapoviruses. Securing virus stocks of multiple human sapovirus strains represents a significant advance toward establishing a reliable experimental system that does not depend on limited virus-positive fecal material.

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.

Heritable Tissue-Culture-Free Gene Editing in Nicotiana benthamiana through Viral Delivery of SpCas9 and sgRNA.

Conventional plant gene editing requires laborious tissue-culture-mediated transformation, which restricts the range of applicable plant species. In this study, we developed a heritable and tissue-culture-free gene editing method in Nicotiana benthamiana using tobacco ringspot virus (TRSV) as a vector for in planta delivery of Cas9 and single-guide RNA (sgRNA) to shoot apical meristems. Agrobacterium-mediated inoculation of the TRSV vector induced systemic and heritable gene editing in NbPDS. Transient downregulation of RNA silencing enhanced gene editing efficiency, resulting in an order of magnitude increase (0.8% to 13.2%) in the frequency of transgenerational gene editing. While the TRSV system had a preference for certain sgRNA sequences, co-inoculation of a TRSV vector carrying only Cas9 and a tobacco rattle virus vector carrying sgRNA successfully introduced systemic mutations with all five tested sgRNAs. Extensively gene-edited lateral shoots occasionally grew from plants inoculated with the virus vectors, of which the transgenerational gene editing frequency ranged up to 100%. This virus-mediated heritable gene editing method makes plant gene editing easy, requiring only the inoculation of non-transgenic plants with a virus vector(s) to obtain gene-edited individuals.

Efficacy of robot-assisted partial nephrectomy compared to conventional laparoscopic partial nephrectomy for completely endophytic renal tumor: a multicenter, prospective study.

BACKGROUND: This study aimed to compare the efficacy of robot-assisted partial nephrectomy for completely endophytic renal tumors with the reported outcomes of conventional laparoscopic partial nephrectomy and investigate the transition of renal function after robot-assisted partial nephrectomy. METHODS: We conducted a prospective, multicenter, single-arm, open-label trial across 17 academic centers in Japan. Patients with endophytic renal tumors classified as cT1, cN0, cM0 were included and underwent robot-assisted partial nephrectomy. We defined two primary outcomes to assess functional and oncological aspects of the procedure, which were represented by the warm ischemic time and positive surgical margin, respectively. Comparisons were made using control values previously reported in laparoscopic partial nephrectomy studies. In the historical control group, the warm ischemia time was 25.2, and the positive surgical margin was 13%. RESULTS: Our per-protocol analysis included 98 participants. The mean warm ischemic time was 20.3 min (99% confidence interval 18.3-22.3; p < 0.0001 vs. 25.2). None of the 98 participants had a positive surgical margin (99% confidence interval 0-5.3%; p < 0.0001 vs. 13.0%). The renal function ratio of eGFR before and after protocol treatment multiplied by splits was 0.70 (95% confidence interval: 0.66-0.75). Factors such as preoperative eGFR, resected weight, and warm ischemic time influenced the functional loss of the partially nephrectomized kidney after robot-assisted partial nephrectomy. CONCLUSIONS: Robot-assisted partial nephrectomy for completely endophytic renal tumors offers a shorter warm ischemia time and comparable positive surgical margin rate compared with conventional laparoscopic partial nephrectomy.

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.

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.

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.

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.

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.

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.

Clinical importance of the expression of CD4+CD8+ T cells in renal cell carcinoma.

OBJECTIVE: CD4+CD8+ T cells are expressed in some cancer patients including those with renal cell carcinoma (RCC). However, no reports have mentioned the clinical importance of this expression. We evaluated the expression of CD4+CD8+ T cells in patients with various cancer types to clarify clinical characteristics and prognostic importance significantly correlating with these T cells. METHODS: Expression of CD4+CD8+ T cells was evaluated using flowcytometry in tissue-infiltrating lymphocytes extracted from 260 cancer tissues including 104 RCC samples. RNA sequencing and characterization and regression (Citrus) was used to determine characteristics. The prognostic importance of CD4+CD8+ T cells was evaluated by Cox regression analysis. RESULTS: Among eight cancer types, expression of CD4+CD8+ T cells was significantly highest in RCC patients. According to the expression of CD4+CD8+ T cells in adjacent normal tissue-infiltrating lymphocytes, 24 patients (23.1%) were defined as being positive for CD4+CD8+ with an expression higher than 9.29% in RCC patients. Citrus showed CD8+PD-1+TIM-3+CD103- T cells to be a specific subpopulation of CD4+CD8+ T cells. RNA sequencing revealed that CD4+CD8+ T cells had significantly lower diversity than the other T cells and shared most T-cell receptor clones with CD8+ not CD4+ T cells. Expression of CD4+CD8+ T cells was identified as an independent predictor of overall survival (hazard ratio: 0.11, 95% confidence interval: 0.01-0.86, P = 0.035) in multivariate analysis. CONCLUSIONS: The expression of CD4+CD8+ T cells was significantly up-regulated in RCC patients and correlated significantly with prognostic importance in surgically treated RCC patients.

Complete genome sequence of pleioblastus mosaic virus, a distinct member of the genus Potyvirus.

Pleioblastus mosaic virus (PleMV) is a tentative member of the genus Potyvirus in the family Potyviridae and was discovered in bamboo with mosaic symptoms in Tokyo, Japan. Since no information on the genome sequence of PleMV has been reported, its taxonomic position has long been uncertain. Here, we report the first complete genome sequences of two distinct PleMV isolates. Excluding the 3'-terminal poly(A) tail, their genomic RNA sequences consist of 9,634 and 9,643 nucleotides (nt); both contain a large open reading frame (ORF) encoding a polyprotein and a small ORF termed PIPO. The large ORFs of the two isolates share 79.2% and 87.6% sequence identity at the nucleotide (nt) and amino acid (aa) level, respectively, and were found to have the highest nt and aa sequence identity (69.0% and 69.9%) to the potyvirus johnsongrass mosaic virus (JGMV). Phylogenetic analysis showed that PleMV is most closely related to JGMV but forms its own clade. These results suggest that PleMV is a distinct member of the genus Potyvirus.

Complete genome sequence of viola mottle virus, revealing its synonymous relationship to tulip virus X.

Viola mottle virus (VMoV) was discovered in Viola odorata showing symptoms of reduced growth, leaf mottling, and whitish stripes on flowers in northern Italy in 1977. This virus has been provisionally classified as a member of the genus Potexvirus based on its morphological, serological, and biological characteristics. However, since genetic information of VMoV has never been reported, the taxonomic status of this virus is unclear. Here, we report the first complete genome sequence of VMoV to clarify its taxonomic position. Its genomic RNA is 6,052 nucleotides long, excluding the 3'-terminal poly(A) tail, and has five open reading frames (ORFs) typical of potexviruses. Among potexviruses, VMoV showed the most similarity to tulip virus X (TVX) with 81.1-81.2% nucleotide and 90.4-90.7% amino acid sequence identity in ORF1 and 82.9-83.5% nucleotide and 93.2-95.2% amino acid sequence identity in ORF5. These values are much higher than the species demarcation threshold for the genus. Phylogenetic analysis also indicated that VMoV is nested within the clade of TVX isolates. These data demonstrate that VMoV and TVX are members of the same species.

Nutritional intervention after an early assessment by a flexible endoscopic evaluation of swallowing is associated with a shorter hospital stay for patients with acute cerebral infarction: A retrospective study.

BACKGROUND AND OBJECTIVES: It is important to evaluate the swallowing function of patients with acute cerebral infarction. The effects of nutritional intervention after an early assessment by a flexible endoscopic evaluation of swallowing (FEES) were evaluated. METHODS AND STUDY DESIGN: This retrospective study included 274 patients who were hospitalized for acute cerebral infarction and underwent a FEES between 2016 and 2018. The effects of early nutritional intervention after an assessment by a FEES within 48 h from admission were evaluated. The patients were divided into a shorter hospital stay group (<30 days) and a longer group (≥30 days). A multivariate analysis was performed to identify the predictive factors for a shorter hospital stay. RESULTS: The overall patient characteristics were as follows: 166 men; median age, 81 years old; and median body mass index (BMI), 21.1 kg/m2. No significant differences in the age, sex, or BMI were found between the shorter and longer hospital stay groups. A FEES within 48 h of admission (odds ratio [OR], 2.040; 95% confidence interval [CI], 1.120-3.700; p=0.019), FILS level ≥6 at admission (OR, 2.300; 95% CI, 1.190-4.440; p=0.013), and an administered energy dose of ≥18.5 kcal/kg on hospital day 3 (OR, 2.360; 95% CI, 1.180-4.690; p=0.015) were independently associated with a hospital stay <30 days. CONCLUSIONS: Patients with acute cerebral infarction are more likely to have a shorter hospital stay (<30 days) if they undergo a FEES early after admission and receive optimal nutritional intervention.

Differences in the Repeated Sprint Performance Between the First and Latter Halves of Trials Under Conditions of Several Thermal States in Exercising Muscles.

ABSTRACT: Inoue, K, Yamashita, N, Kume, M, and Yoshida, T. Differences in the repeated sprint performance between the first and latter halves of trials under conditions of several thermal states in exercising muscles. J Strength Cond Res 35(3): 782-790, 2021-The purpose of this study was to determine whether the effects of thermal states in exercising muscle on repeated sprint cycling (RSC) performance differ between the first and latter half of trials. Nine male subjects performed 8 × 8 seconds of RSC with a 40-second rest period. The subjects wore water-perfused trousers with water at 6° C (COLD), 17° C (COOL), 30° C (WARM), or 44° C (HOT). During the first half of trials, the peak power output (PPO), mean power output (MPO), and sum of work output (SWO) were significantly (p < 0.05) greater under the WARM and HOT conditions than under the COLD and COOL conditions, and a difference in the PPO and MPO between WARM and HOT was noted in the second sprint bout during the first half of the exercise. However, during the latter half of trials, there was no significant difference in the PPO, MPO, and SWO among the 4 conditions. The tympanic temperature (Tty) was significantly elevated under the HOT condition but fell under the COLD and COOL conditions, whereas the Tty under the WARM condition did not change significantly (p < 0.05) during the experiment. The total sweat loss was significantly (p < 0.05) greater in the HOT condition than in the other conditions. These results suggest that the effect of thermal states in exercising muscle on the RSC performance is greater in the first half of exercise than in the latter half, possibly because of the elevation of the core temperature and sweat loss under HOT conditions.

Long non-coding RNA MANCR is a target of BET bromodomain protein BRD4 and plays a critical role in cellular migration and invasion abilities of prostate cancer.

Androgen receptor (AR)-negative castration-resistant prostate cancer (CRPC) is highly aggressive and is resistant to most of the current therapies. Bromodomain and extra terminal domain (BET) protein BRD4 binds to super-enhancers (SEs) that drive high expression of oncogenes in many cancers. A BET inhibitor, JQ1, has been found to suppress the malignant phenotypes of prostate cancer cells, however, the target genes of JQ1 remain largely unknown. Here we show that SE-associated genes specific for AR-negative CRPC PC3 cells include genes involved in migration and invasion, and that JQ1 impairs migration and invasion of PC3 cells. We identified a long non-coding RNA, MANCR, which was markedly down-regulated by JQ1, and found that BRD4 binds to the MANCR locus. MANCR knockdown led to a significant decrease in migration and invasion of PC3 cells. Furthermore, RNA sequencing analysis revealed that expression of the genes involved in migration and invasion was altered by MANCR knockdown. In summary, our data demonstrate that MANCR plays a critical role in migration and invasion of PC3 cells.

The Plant Noncanonical Antiviral Resistance Protein JAX1 Inhibits Potexviral Replication by Targeting the Viral RNA-Dependent RNA Polymerase.

Understanding the innate immune mechanisms of plants is necessary for the breeding of disease-resistant lines. Previously, we identified the antiviral resistance gene JAX1 from Arabidopsis thaliana, which inhibits infection by potexviruses. JAX1 encodes a unique jacalin-type lectin protein. In this study, we analyzed the molecular mechanisms of JAX1-mediated resistance. JAX1 restricted the multiplication of a potexviral replicon lacking movement-associated proteins, suggesting inhibition of viral replication. Therefore, we developed an in vitro potato virus X (PVX) translation/replication system using vacuole- and nucleus-free lysates from tobacco protoplasts, and we revealed that JAX1 inhibits viral RNA synthesis but not the translation of the viral RNA-dependent RNA polymerase (RdRp). JAX1 did not affect the replication of a resistance-breaking mutant of PVX. Blue native polyacrylamide gel electrophoresis of fractions separated by sucrose gradient sedimentation showed that PVX RdRp constituted the high-molecular-weight complex that seems to be crucial for viral replication. JAX1 was detected in this complex of the wild-type PVX replicon but not in that of the resistance-breaking mutant. In addition, JAX1 interacted with the RdRp of the wild-type virus but not with that of a virus with a point mutation at the resistance-breaking residue. These results suggest that JAX1 targets RdRp to inhibit potexviral replication.IMPORTANCE Resistance genes play a crucial role in plant antiviral innate immunity. The roles of conventional nucleotide-binding leucine-rich repeat (NLR) proteins and the associated defense pathways have long been studied. In contrast, recently discovered resistance genes that do not encode NLR proteins (non-NLR resistance genes) have not been investigated extensively. Here we report that the non-NLR resistance factor JAX1, a unique jacalin-type lectin protein, inhibits de novo potexviral RNA synthesis by targeting the huge complex of viral replicase. This is unlike other known antiviral resistance mechanisms. Molecular elucidation of the target in lectin-type protein-mediated antiviral immunity will enhance our understanding of the non-NLR-mediated plant resistance system.

A risk stratification model based on four novel biomarkers predicts prognosis for patients with renal cell carcinoma.

BACKGROUND: Accurate prediction of the prognosis of RCC using a single biomarker is challenging due to the genetic heterogeneity of the disease. However, it is essential to develop an accurate system to allow better patient selection for optimal treatment strategies. ARL4C, ECT2, SOD2, and STEAP3 are novel molecular biomarkers identified in earlier studies as survival-related genes by comprehensive analyses of 43 primary RCC tissues and RCC cell lines. METHODS: To develop a prognostic model based on these multiple biomarkers, the expression of four biomarkers ARL4C, ECT2, SOD2, and STEAP3 in primary RCC tissue were semi-quantitatively investigated by immunohistochemical analysis in an independent cohort of 97 patients who underwent nephrectomy, and the clinical significance of these biomarkers were analyzed by survival analysis using Kaplan-Meier curves. The prognostic model was constructed by calculation of the contribution score to prognosis of each biomarker on Cox regression analysis, and its prognostic performance was validated. RESULTS: Patients whose tumors had high expression of the individual biomarkers had shorter cancer-specific survival (CSS) from the time of primary nephrectomy. The prognostic model based on four biomarkers segregated the patients into a high- and low-risk scored group according to defined cut-off value. This approach was more robust in predicting CSS compared to each single biomarker alone in the total of 97 patients with RCC. Especially in the 36 metastatic RCC patients, our prognostic model could more accurately predict early events within 2 years of diagnosis of metastasis. In addition, high risk-scored patients with particular strong SOD2 expression had a much worse prognosis in 25 patients with metastatic RCC who were treated with molecular targeting agents. CONCLUSIONS: Our findings indicate that a prognostic model based on four novel biomarkers provides valuable data for prediction of clinical prognosis and useful information for considering the follow-up conditions and therapeutic strategies for patients with primary and metastatic RCC.

Pediatric voiding cystourethrography: An essential examination for urologists but a terrible experience for children.

Voiding cystourethrography is the most important fluoroscopic examination in pediatric urology for the investigation of lower urogenital tract diseases, such as vesicoureteral reflux or urethral stricture. However, this invasive procedure imposes a significant burden on children and their parents, and recently there has been a paradigm shift in the diagnosis and treatment of vesicoureteral reflux. In the 2011 revision, the American Academy of Pediatrics guidelines on urinary tract infection recommended abandoning routine voiding cystourethrography after the first febrile urinary tract infection. In 2014, the randomized intervention for children with vesicoureteral reflux study recommended discontinuation of routine continuous antibiotic prophylaxis for vesicoureteral reflux. The time is now ripe to radically reconsider indications for voiding cystourethrography and the procedure itself.

The location of the bladder neck in postoperative cystography predicts continence convalescence after radical prostatectomy.

BACKGROUND: This study was conducted to determine whether the location of the bladder neck in postoperative cystography predicts recovery of continence after radical prostatectomy. METHODS: Between 2008 and 2015, 203 patients who underwent laparoscopic radical prostatectomy (LRP, n = 99) and robot assisted radical prostatectomy (RARP, n = 104) were analyzed. The location of the bladder neck was visualized by postoperative routine cystography, and quantitative evaluation of the bladder neck position was performed according to the bladder neck to pubic symphysis (BNPS) ratio proposed by Olgin et al. (J Endourol, 2014). Recovery of continence was defined as no pad use or one security pad per day. To determine the predictive factors for recovery of continence at 1, 3, 6 and 12 months, several parameters were analyzed using logistic regression analysis, including age (≤68 vs. > 68, BMI (≤23.4 vs. > 23.4 kg/m2), surgical procedure (LRP vs. RARP), prostate volume (≤38 vs. > 38 mL), nerve-sparing technique, vesico-urethral anastomosis leakage, and BNPS ratio (≤0.59 vs. > 0.59). RESULTS: The mean postoperative follow-up was 1131 days (79-2880). At 1, 3, 6 and 12 months after surgery, continence recovery rates were 25, 53, 68 and 81%, respectively. Although older age (> 68) and RARP were significant risk factors for incontinence within 3 months, neither was significant after 6 months. A high BNPS ratio (> 0.59) was the only significant risk factor for the persistence of incontinence at all observation points, up to 12 months. CONCLUSIONS: A lower bladder neck position after prostatectomy predicts prolonged incontinence.