ALKBH4 is a novel enzyme that promotes translation through modified uridine regulation.

Epitranscriptomics studies the mechanisms of acquired RNA modifications. The epitranscriptome is dynamically regulated by specific enzymatic reactions, and the proper execution of these enzymatic RNA modifications regulates a variety of physiological RNA functions. However, the lack of experimental tools, such as antibodies for RNA modification, limits the development of epitranscriptomic research. Furthermore, the regulatory enzymes of many RNA modifications have not yet been identified. Herein, we aimed to identify new molecular mechanisms involved in RNA modification by focusing on the AlkB homolog (ALKBH) family molecules, a family of RNA demethylases. We demonstrated that ALKBH4 interacts with small RNA, regulating the formation and metabolism of the (R)-5-carboxyhydroxymethyl uridine methyl ester. We also found that the reaction of ALKBH4 with small RNA enhances protein translation efficiency in an in vitro assay system. These findings indicate that ALKBH4 is involved in the regulation of uridine modification and expand on the role of tRNA-mediated translation control through ALKBH4.

Developmental and functional roles of androgen and interactive signals for external genitalia and erectile tissues.

Background: Recent progress in molecular and signal analyses revealed essential functions of cellular signals including androgen and related growth factors such as Wnt regulators for external genitalia (ExG) development and its pathogenesis. Accumulated data showed their fundamental functions also for erectile tissue (corporal body) development and its abnormalities. The current review focuses on such signals from developmental and functional viewpoints. Methods: Experimental strategies including histological and molecular signal analyses with conditional mutant mice for androgen and Wnt signals have been extensively utilized. Main findings: Essential roles of androgen for the development of male-type ExG and urethral formation are shown. Wnt signals are associated with androgen for male-type ExG organogenesis. Androgen plays essential roles in the development of erectile tissue, the corporal body and it also regulates the duration time of erection. Wnt and other signals are essential for the regulation of mesenchymal cells of erectile tissue as shown by its conditional mutant mouse analyses. Stress signals, continuous erection, and the potential of lymphatic characteristics of the erectile vessels with sinusoids are also shown. Conclusion: Reiterated involvement of androgen, Wnt, and other regulatory factors is stated for the development and pathogenesis of ExG and erectile tissues.

Factors, Including Clinical Trial Eligibility, Associated with Induction of Third-Line Treatment for Advanced Gastric Cancer.

INTRODUCTION: Third-line chemotherapy has been suggested to improve survival in patients with gastric cancer. This study aimed to identify factors associated with the induction of third-line chemotherapy for advanced gastric cancer, focusing on patient eligibility for clinical trial. METHODS: We retrospectively analyzed 335 patients treated for unresectable or recurrent gastric cancer between April 2009 and May 2020. The patients were grouped into those that met the key eligibility criteria for clinical trial (136 patients, 40.6%) and those that did not (199 patients, 59.4%) before receiving first-line chemotherapy. RESULTS: The overall survival (OS) was 16.8 months (95% CI: 14.0-19.6) and 9.3 months (95% CI: 7.8-11.0) in the eligible and ineligible group, respectively. Multivariate analyses to identify the risk factors associated with the induction of third-line chemotherapy revealed ineligibility of clinical trial (OR 1.95; 95% CI: 1.15-3.31), number of metastatic sites (OR 1.99; 95% CI: 1.23-3.22), low albumin concentration (OR 2.24; 95% CI: 1.14-4.38), and a lack of complete or partial response to first-line treatment (OR 1.85; 95% CI: 1.05-3.26). Indeed, in responders to first-line treatment for ineligible patients, the median OS was 17.7 months (95% CI: 10.6-27.9), respectively. CONCLUSIONS: Treatment outcomes were different for those eligible for clinical trials and those who were not. However, this study suggested that patients who responded to first-line treatment have more favorable prognosis when treated with salvage chemotherapy, even if they were deemed ineligible for clinical trials.

Myosteatosis as a novel predictor of urinary incontinence after robot-assisted radical prostatectomy.

OBJECTIVES: To evaluate the impact of sarcopenia and myosteatosis on urinary incontinence after prostatectomy. METHODS: We retrospectively reviewed consecutive patients who underwent robot-assisted radical prostatectomy without nerve sparing between December 2012 and March 2019. Psoas muscle index and average total psoas density, which were measured on preoperative computed tomography images at level L3, were used to evaluate sarcopenia and myosteatosis, respectively. In addition, several magnetic resonance imaging variables associated with pelvic muscles, the urethra and the prostate were measured. Urinary continence was defined as non-use or use of just one incontinence pad per day. Logistic regression analyses aimed to identify the predictors of urinary incontinence 3 and 12 months after surgery. RESULTS: Overall, 121 patients were included in the analysis. The incidence rates of urinary incontinence 3 and 12 months after surgery were 42% (51/121 cases) and 16% (19/121 cases), respectively. Logistic multivariable analysis showed that low average total psoas density was the only significant independent predictor of urinary incontinence 3 months after surgery (P < 0.01), and low obturator internus muscle thickness (P = 0.01), short membranous urethral length (P = 0.01) and low average total psoas density (P < 0.01) were significant independent predictors of urinary incontinence 12 months after surgery. By contrast, psoas muscle index was not statistically associated with urinary incontinence after surgery. CONCLUSIONS: Myosteatosis (low average total psoas density) could be a novel predictor of urinary incontinence after robot-assisted radical prostatectomy.

Establishment of mouse line showing inducible priapism-like phenotypes.

Purpose: Penile research is expected to reveal new targets for treatment and prevention of the complex mechanisms of its disorder including erectile dysfunction (ED). Thus, analyses of the molecular processes of penile ED and continuous erection as priapism are essential issues of reproductive medicine. Methods: By performing mouse N-ethyl-N-nitrosourea mutagenesis and exome sequencing, we established a novel mouse line displaying protruded genitalia phenotype (PGP; priapism-like phenotype) and identified a novel Pitpna gene mutation for PGP. Extensive histological analyses on the Pitpna mutant and intracavernous pressure measurement (ICP) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS)/MS analyses were performed. Results: We evaluated the role of phospholipids during erection for the first time and showed the mutants of inducible phenotypes of priapism. Moreover, quantitative analysis using LC-ESI/MS/MS revealed that the level of phosphatidylinositol (PI) was significantly lower in the mutant penile samples. These results imply that PI may contribute to penile erection by PITPα. Conclusions: Our findings suggest that the current mutant is a mouse model for priapism and abnormalities in PI signaling pathways through PITPα may lead to priapism providing an attractive novel therapeutic target in its treatment.

Pharmacological Inhibition of miR-130 Family Suppresses Bladder Tumor Growth by Targeting Various Oncogenic Pathways via PTPN1.

Previously, we have revealed that the miR-130 family (miR-130b, miR-301a, and miR-301b) functions as an oncomiR in bladder cancer. The pharmacological inhibition of the miR-130 family molecules by the seed-targeting strategy with an 8-mer tiny locked nucleic acid (LNA) inhibits the growth, migration, and invasion of bladder cancer cells by repressing stress fiber formation. Here, we searched for a functionally advanced target sequence with LNA for the miR-130 family with low cytotoxicity and found LNA #9 (A(L)^i^i^A(L)^T(L)^T(L)^G(L)^5(L)^A(L)^5(L)^T(L)^G) as a candidate LNA. LNA #9 inhibited cell growth in vitro and in an in vivo orthotopic bladder cancer model. Proteome-wide tyrosine phosphorylation analysis suggested that the miR-130 family upregulates a wide range of receptor tyrosine kinases (RTKs) signaling via the expression of phosphorylated Src (pSrcTyr416). SILAC-based proteome analysis and a luciferase assay identified protein tyrosine phosphatase non-receptor type 1 (PTPN1), which is implicated as a negative regulator of multiple signaling pathways downstream of RTKs as a target gene of the miR-130 family. The miR-130-targeted LNA increased and decreased PTPN1 and pSrcTyr416 expressions, respectively. PTPN1 knockdown led to increased tumor properties (cell growth, invasion, and migration) and increased pSrcTyr416 expression in bladder cancer cells, suggesting that the miR-130 family upregulates multiple RTK signaling by targeting PTPN1 and subsequent Src activation in bladder cancer. Thus, our newly designed miR-130 family targeting LNA could be a promising nucleic acid therapeutic agent for bladder cancer.

3D reconstruction and histopathological analyses on murine corporal body.

PURPOSE: Erectile dysfunction (ED) is one of the increasing diseases with aging society. The basis of ED derived from local penile abnormality is poorly understood because of the complex three-dimensional (3D) distribution of sinusoids in corpus cavernosum (CC). Understanding the 3D histological structure of penis is thus necessary. Analyses on the status of regulatory signals for such abnormality are also performed. METHODS: To analyze the 3D structure of sinusoid, 3D reconstruction from serial sections of murine CC were performed. Histological analyses between young (2 months old) and aged (14 months old) CC were performed. As for chondrogenic signaling status of aged CC, SOX9 and RBPJK staining was examined. RESULTS: Sinusoids prominently developed in the outer regions of CC adjacent to tunica albuginea. Aged CC samples contained ectopic chondrocytes in such regions. Associating with the appearance of chondrocytes, the expression of SOX9, chondrogenic regulator, was upregulated. The expression of RBPJK, one of the Notch signal regulators, was downregulated in the aged CC. CONCLUSIONS: Prominent sinusoids distribute in the outer region of CC which may possess important roles for erection. A possibility of ectopic chondrogenesis induced by alteration of SOX9/Notch signaling with aging is indicated.

A sensitive and efficient procedure for the high-throughput determination of nine urinary metabolites of pyrethroids by GC-MS/MS and its application in a sample of Japanese children.

Four pyrethroids (PYRs), metofluthrin, profluthrin, tefluthrin, and transfluthrin, which were newly developed and have relatively high vapor activity at ambient temperature, are now playing a key role in safely controlling insects in our daily lives. We developed a sensitive and high-throughput determination method for urinary metabolites derived from the newly developed PYR, e.g., 2,3,5,6-tetrafluoro-1,4-benzenedimethanol (HOCH2-FB-Al), 2,3,5,6-tetrafluorobenzyl alcohol (FB-Al), and other PYR metabolites such as trans-chrysanthemumdicarboxylic acid (trans-CDCA) and 3-phenoxybenzoic acid (3PBA). After high temperature acid hydrolysis of 2 mL urine sample in 24-deep well plate, the PYR metabolites were extracted by semi-automated liquid-liquid extraction with tert-butyl methyl ether. N,O-Bis (trimethylsilyl) trifluoroacetamide containing 1% trimethylchlorosilane or 1,1,1,3,3,3-hexafluoroisopropanol were used for the derivatization of PYR metabolites, and the derivatized metabolites were analyzed separately by GC-MS/MS equipped with dual injector system (DB-5MS and mid- to high-polarity phase Rtx-65 columns). The derivatization and evaporation conditions were mainly optimized for improving sensitivity and reproducibility. The mean within-run day precisions were less than 18.4% (relative standard deviation, %RSD) with low detection limits ranging from 0.01 µg/L for HOCH2-FB-Al to 0.06 µg/L for trans-CDCA. This method was successfully applied to urine samples obtained from 50 3-year-old children with high detection frequencies (e.g., 82% for HOCH2-FB-Al and 84% for FB-Al). This method may be a pivotal tool for developing risk assessment from PYR exposure in the general population.

Expression level of CXCL7 in peripheral blood cells is a potential biomarker for the diagnosis of renal cell carcinoma.

There are no blood biomarkers for the diagnosis of renal cell carcinoma (RCC) in routine clinical use. We focused on the gene expression profile of peripheral blood cells obtained from RCC patients to discover novel biomarkers for RCC diagnosis. Using microarray analysis and quantitative verification, CXCL7 was shown to be significantly upregulated in the peripheral blood cells of RCC patients. Importantly, aberrant CXCL7 expression was confirmed even in peripheral blood cells obtained from early stage (pT1a) RCC patients, and the expression level of CXCL7 in peripheral blood cells was a potential independent biomarker for the diagnosis of RCC by receiver operating characteristic curve analysis (sensitivity, 70.0%; specificity, 64.0%; area under the curve = 0.722; multiple logistic regression analysis: odds ratio, 1.07; 95% confidence interval, 1.03-1.11; P = 0.0004). Moreover, CXCL7 expression in peripheral blood cells significantly decreased after resection of the primary tumor. CXCL7 is more highly expressed in PBMCs than in neutrophils from both healthy controls and RCC patients. Interestingly, CXCL7 expression in PBMCs from healthy volunteers was significantly elevated following coculture with RCC cells compared to those cocultured with normal cells as a control. These results suggest that aberrant CXCL7 expression in peripheral blood cells is induced by RCC cells and may serve as a novel biomarker in the diagnosis of RCC.

TP53 gene status is a critical determinant of phenotypes induced by ALKBH3 knockdown in non-small cell lung cancers.

Human AlkB homolog 3 (ALKBH3) is overexpressed in non-small cell lung cancers (NSCLC) and its high expression is significantly correlated with poor prognosis. While ALKBH3 knockdown induces apoptosis in NSCLC cells, the underlying anti-apoptotic mechanisms of ALKBH3 in NSCLC cells remain unclear. Here we show that ALKBH3 knockdown induces cell cycle arrest or apoptosis depending on the TP53 gene status in NSCLC cells. In comparison to parental cells, TP53-knockout A549 cells showed DNA damage-responsive signal induced by ALKBH3 knockdown. TP53 knockout shifted the phenotypes of A549 cells induced by ALKBH3 knockdown from cell cycle arrest to apoptosis induction, suggesting that the TP53 gene status is a critical determinant of the phenotypes induced by ALKBH3 knockdown in NSCLC cells.

ALKBH8 promotes bladder cancer growth and progression through regulating the expression of survivin.

Human AlkB homolog 8 (ALKBH8) is highly expressed in high-grade, superficially and deeply invasive bladder cancer. Moreover, ALKBH8 knockdown induces apoptosis in bladder cancer cells. However, the underlying anti-apoptotic mechanism of ALKBH8 in bladder cancer cells has thus far remained unclear. Moreover, there is no direct evidence that highly expressed ALKBH8 is involved in tumor progression in vivo. We here show that ALKBH8 knockdown induced apoptosis via downregulating the protein expression of survivin, an anti-apoptotic factor also exhibiting increased levels in bladder cancer. We also clarify that ALKBH8 transgenic mice showed an accelerated rate of bladder tumor mass and invasiveness in an N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced bladder cancer model. These findings suggest that the high expression of ALKBH8 is critical for the growth and progression of bladder cancer.

Design and synthesis of prostate cancer antigen-1 (PCA-1/ALKBH3) inhibitors as anti-prostate cancer drugs.

A series of 1-aryl-3,4-substituted-1H-pyrazol-5-ol derivatives was synthesized and evaluated as prostate cancer antigen-1 (PCA-1/ALKBH3) inhibitors to obtain a novel anti-prostate cancer drug. After modifying 1-(1H-benzimidazol-2-yl)-3,4-dimethyl-1H-pyrazol-5-ol (1), a hit compound found during random screening using a recombinant PCA-1/ALKBH3, 1-(1H-5-methylbenzimidazol-2-yl)-4-benzyl-3-methyl-1H-pyrazol-5-ol (35, HUHS015), was obtained as a potent PCA-1/ALKBH3 inhibitor both in vitro and in vivo. The bioavailability (BA) of 35 was 7.2% in rats after oral administration. As expected, continuously administering 35 significantly suppressed the growth of DU145 cells, which are human hormone-independent prostate cancer cells, in a mouse xenograft model without untoward effects.

DNA methylation of microRNA-124a is a potential risk marker of colitis-associated cancer in patients with ulcerative colitis.

BACKGROUND: Colitis-associated cancer (CAC) is the serious complication of ulcerative colitis (UC), and molecular markers to evaluate the individual risk are required. MicroRNA-124a (miR - 124a) is known to have tumor-suppressive function and be methylation-silenced during exposure to chronic inflammation. AIM: We analyzed whether higher methylation levels of miR-124a genes correlated with the higher epidemiologic risk of CAC development in UC patients. METHODS: Forty UC patients without CAC, four patients with CAC or dysplasia, eight sporadic colorectal cancer (S-CRC) patients, and 12 healthy volunteers (HV) were studied. Methylation status of miR-124a genes (miR-124a-1, -2, and -3) was analyzed by methylation-specific polymerase chain reaction (MSP), and methylation levels were quantified by real-time MSP. Expression of cyclin-dependent kinase 6 (CDK6), a target of miR-124a, was analyzed by immunohistochemistry. RESULTS: Three miR-124a genes were methylated in all neoplastic tissues (CAC, dysplasia, and S-CRC), and CDK6 was highly expressed in those tissues. Regarding disease extent, mean methylation levels of miR-124a-3 in HV, non-pancolitis, and pancolitis were 2.0, 5.3, and 12.3%, respectively, and were significantly higher in pancolitis than in HV (p < 0.01). Regarding disease duration, mean methylation levels in short-term and long-standing UC patients were 2.5 and 13.2%, respectively. Long-standing UC patients had significantly higher methylation levels than HV (p < 0.01). Moreover, UC patients with both pancolitis and long-standing had 7.4-fold higher methylation levels than those without these risk factors. CONCLUSIONS: MiR-124a genes are methylated during carcinogenesis in UC patients. The methylation level of miR-124a-3 is a promising marker for estimating individual risk for CAC.

Difference in protein adsorption onto polymer films with or without thermal annealing.

Protein adsorptions onto non-annealed (NA) and thermally annealed (TA) polyetherimide films were examined by surface plasmon resonance measurements. Proteins adsorbed onto the NA films with smaller adsorption constants in comparison with the TA films. Neutron reflectivity measurements of the two films suggested that the outermost region of the NA films swelled with larger amounts of water molecules than the TA films. It is plausible that the aforementioned difference in the protein adsorption properties is derived from the difference in the interfacial aggregation structures of the two films.

Oral administration of fermented probiotics improves the condition of feces in adult horses.

The effects of probiotics on horses are still controversial. The present study was a randomized, double-blinded, placebo-controlled crossover study designed to evaluate the ability of probiotics to improve intestinal conditions in adult horses. Fermented probiotics were administered to 10 healthy adult geldings for 28 days. The clinical condition of the horses was monitored daily, and the blood and feces were biochemically analyzed every 14 days. In the probiotic-treated group, the concentration of carboxylic acids in the feces was increased at days 14 and 28. In contrast to the fecal pH in the control group, which increased at days 14 and 28, the fecal pH in the probiotic-treated group did not increase. Additionally, the relative amounts of enteropathogenic bacterial DNA were diminished in the probiotic-treated group. These results suggest that probiotic bacteria proliferated in the equine intestine. No instances of abnormal clinical conditions or abnormal values in blood tests were observed throughout the study. Oral administration of fermented probiotics may have the ability to improve the intestinal environment biochemically and microbiologically without the risk of adverse effects.

The Efficacy and Safety of Nivolumab Plus mFOLFOX6 in Gastric Cancer with Severe Peritoneal Metastasis.

(1) Background: Nivolumab plus chemotherapy is established as a first-line treatment for advanced gastric cancer (AGC). While mFOLFOX6 is commonly used for AGC with severe peritoneal metastasis, the efficacy of nivolumab combined with it remains uncertain. We evaluated the outcomes of nivolumab plus mFOLFOX6 for AGC with severe peritoneal metastasis in clinical practice. (2) Methods: This multicenter retrospective study was conducted between December 2021 and June 2023. We investigated AGC patients with massive ascites or inadequate oral intake due to severe peritoneal metastasis and who received nivolumab plus mFOLFOX6. (3) Results: Among 106 patients treated with nivolumab plus chemotherapy, 21 (19.8%) had severe peritoneal metastasis, with 14 receiving nivolumab plus mFOLFOX6. The median progression-free survival was 7.4 months (95%CI 1.9-10.1), and the median overall survival was 10.7 months (95%CI 5.3-NA), with four patients (28.5%) surviving more than 12 months. Improved ascites and oral intake were observed in 6/14 patients (42.8%) and 10/11 patients (90.9%), respectively. The major grade 3 or more adverse events included leukopenia (28.5%) and neutropenia (21.4%), with no severe immune-related adverse events reported. (4) Conclusions: The safety and moderate efficacy of nivolumab plus mFOLFOX6 were suggested even in AGC patients with severe peritoneal metastasis.

CA125 Kinetics as a Potential Biomarker for Peritoneal Metastasis Progression following Taxane-Plus-Ramucirumab Administration in Patients with Advanced Gastric Cancer.

Currently, no established marker exists for predicting peritoneal metastasis progression during chemotherapy, although they are major interruptive factors in sequential chemotherapy in patients with advanced gastric cancer (AGC). This multicenter retrospective study was conducted from June 2015 to July 2019, analyzing 73 patients with AGC who underwent taxane-plus-ramucirumab (TAX/RAM) therapy and had their serum carbohydrate antigen 125 (CA125) concentrations measured. Of 31 patients with elevated CA125 levels above a cutoff of 35 U/mL, 25 (80.6%) had peritoneal metastasis. The CA125 concentrations before TAX/RAM treatment were associated with ascites burden. The overall survival was significantly shorter in the CA125-elevated group. CA125 kinetics, measured at a median of 28 days after chemotherapy, were associated with the ascites response (complete or partial response: -1.86%/day; stable disease: 0.28%/day; progressive disease: 2.33%/day). Progression-free survival in the CA125-increased group, defined by an increase of 0.0067%/day using receiver operating characteristic curve analysis, was significantly poorer among patients with peritoneal metastases. In conclusion, this study highlights that CA125 kinetics can serve as an early predictor for the progression of peritoneal metastasis during TAX/RAM treatment.

Efficacy and Safety of Immune Checkpoint Inhibitor Combination Therapy for Dysphagia in Patients with Advanced Esophageal Cancer.

Background/Objectives: Recently, pembrolizumab plus 5-fluorouracil and cisplatin (FP), nivolumab plus FP, and nivolumab plus ipilimumab have become the first-line treatments for patients with advanced esophageal cancer. However, the treatment efficacy in primary tumors has not been reported. We assessed the outcomes of these treatments in advanced esophageal cancer, specifically focusing on esophageal dysphagia improvements and the primary tumor response. Methods: This retrospective study was conducted between October 2021 and November 2023. We investigated 23 patients with esophageal cancer and dysphagia who received an immune checkpoint inhibitor (ICI) plus FP or nivolumab plus ipilimumab. Results: The median progression-free survival (PFS) was 10.6 months (95% confidence interval [CI]: 9.0-12.5), and the median overall survival was not reached (95%CI: 13.0-NA). Improvement in dysphagia was observed in 19/23 (82.6%) patients, with a median time to improvement of 26 days (range: 15-77 days) and a median dysphagia PFS of 12.6 months (range: 8.1-NA months). Ten patients experienced immune-related adverse events (irAEs): seven had interstitial pneumonia, and three had thyroid dysfunction, pituitary dysfunction, and rash, respectively. Conclusions: Although there was a high frequency of irAEs, ICI for esophageal cancer achieved high response rates and prolonged survival. The observed improvement in dysphagia suggests the potential efficacy of the treatment against primary tumors.

Characteristics of physical activity during beginner-level group tennis lessons and the effect daily activity.

Tennis is a popular leisure sport, and studies have indicated that playing tennis regularly provides many health benefits. We aimed to clarify the characteristics of physical activity during beginner-level group tennis lessons and daily physical activity of the participants. Physical activity was measured using an accelerometer sensor device for four weeks, including the 80-min duration tennis lessons held twice a week. Valid data were categorized for tennis and non-tennis days. The mean physical activity intensity during the tennis lesson was 3.37 METs. The mean ratio of short-bout rest periods to the tennis lesson time in 90 and 120 s was 7% and 4%, respectively. The mean physical activity intensity was significantly higher (p < 0.0001) and the duration of vigorous-intensity physical activity (VPA) was increased in 76% of participants on days with tennis lessons compared to without tennis lessons. Beginner-level tennis lesson has characteristics of less short-bout rest physical activity than previously reported competitive tennis match and increased the duration of VPA in daily activity compared to without tennis lessons, suggesting that beginner-level tennis lessons contribute physical activity of health benefits.