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The chirality of amino acids in extraterrestrial materials may provide an insight into the origin of the essential l-enantiopure amino acids in the terrestrial biosphere. In 2020, the Hayabusa2 mission succeeded in bringing back surface materials from the C-type asteroid (162173) Ryugu to the Earth. Amino acids were one of the targeted organic molecules to be studied in the Ryugu samples. To analyze the various structural isomers of amino acids, which were expected to be present, from the limited amount of the returned samples, the development of a highly-sensitive and selective analytical method was necessary. In the present study, a three-dimensional high-performance liquid chromatography (3D-HPLC) system has been developed for the enantioselective determination of five proteinogenic and three non-proteinogenic amino acids in the Ryugu samples, in which amino acids in the sample were separated by reversed-phase, anion-exchange and enantioselective columns after the fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiozole. The applicability of the analytical system to the extraterrestrial samples was evaluated by analyzing several types of carbonaceous meteorites before applying the system to the Ryugu samples. In the analysis of the Ryugu samples, all of the target amino acids were successfully determined quantitatively. Non-proteinogenic amino acids including 2-amino-n-butyric acid, isovaline and norvaline, rarely present in the terrestrial environment, were found as almost racemic mixtures with 47.1 to 55.2%l.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) has potential applications in the qualitative analysis of phospholipids (PLs). However, its capability for quantitative analysis is limited by the unavailability and/or high cost of isotope-labeled internal standards (interSTDs, e.g., 1-oleoyl (d7)-2-hydroxy-sn-glycero-3-phosphocholine, 1-pentadecanoyl-2-oleoyl (d7)-sn-glycero-3-phosphocholine). This study investigated and validated whether only two PL interSTDs could be used to normalize the entire PL species in a complex bio-lipid background (i.e., urinary lipid extracts). The normalized intensities of PL ionization standards (ionSTDs) were found to have better linear regressions (R 2  > 0.984 for all PL subcategories) than those of traditional methods, such as total ion current and matrix-peak normalization methods. Furthermore, the intra-day precision of all the analyte concentrations after normalizing using our ionSTD method was superior to those of traditional methods. The inter-day precision of all the negatively charged analytes also differed statistically between our ionSTD and the two traditional methods. Meanwhile, a comparison of the three normalization methods revealed that the precision of all the positive analytes using the ionSTD method was comparable. Consequently, a cost-effective, fast, simple, convenient, and reliable quantitative method, defined as "qShot MALDI analysis," was developed to analyze PLs that could potentially be applied in clinical biomarker screening, especially in a negative mode., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Press-through packs (PTPs) are widely used for storing drugs. The number of cases involving improper swallowing and esophageal foreign bodies (EFBs) has increased with the increasing use of PTPs. Studies have reported the utility of point-of-care ultrasound (POCUS) for evaluating EFBs. The application of POCUS for esophageal PTPs has not been reported., Case Report: An 83-year-old woman complaining of neck pain and odynophagia that occurred after improperly swallowing 2 PTPs was admitted to the emergency department. EFBs were suspected, and POCUS revealed a hyperechoic material, suggestive of a PTP, in the cervical esophagus. Endoscopy was immediately performed, and the PTPs were successfully removed without complications. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: This study was significant because it documented the application of POCUS to detect esophageal PTPs. POCUS is a simple and noninvasive technique for identifying EFBs without radiation exposure. © 2022 Elsevier Inc., (Copyright © 2022. Published by Elsevier Inc.)

Birt-Hogg-Dubé syndrome is an autosomal dominant disease caused by germline mutations in the folliculin gene (FLCN), characterized by skin fibrofolliculomas, pulmonary cysts, and multiple renal tumors. We report the case of a 51-year-old woman with multiple bilateral renal tumors resected by bilateral open partial nephrectomy. Following pathological diagnosis of hybrid oncocytic/chromophobe tumors, targeted next-generation sequencing of FLCN of the patient's blood revealed a novel missense mutation (c.602A>C, p.Gln201Pro) in exon 6. Sanger sequencing revealed that this mutation was heterozygous. In silico prediction programs consistently indicated the mutation as pathogenic. Western blot analysis and immunohistochemistry revealed suppressed FLCN expression and the upregulation of glycoprotein nonmetastatic B, a downstream target negatively regulated by FLCN, in the tumor tissue, suggesting that the mutation resulted in reduction of functional FLCN expression. Whole-genome sequencing of one of the tumors identified another frameshift mutation in exon 4, suggesting a "second hit" leading to tumorigenesis. We recommend that gene sequencing should be considered in patients with multiple renal tumors to identify their genetic predisposition to renal tumors., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to determine whether a history of treatment for non-muscle invasive bladder cancer (NMIBC), including intravesical bacillus Calmette-Guerin (BCG) therapy, affects the treatment outcomes of pembrolizumab in patients with metastatic, chemo-resistant urothelial carcinoma (UC)., Materials and Methods: The clinicopathological data of 755 patients with metastatic, chemo-resistant UC who received pembrolizumab were retrospectively reviewed. Best overall response and overall survival (OS) from the initiation of pembrolizumab were analyzed with regard to the history of NMIBC treatment and BCG usage using propensity score matching (PSM)., Results: A total of 155 (20.5%) patients had a history of NMIBC treatment, of which 97 (12.8%) had received intravesical BCG therapy. When compared to patients without a NMIBC history (median 10.0 months), the OS from the initiation of pembrolizumab for patients with a NMIBC history (13.3 months, HR [95% CI] 0.79 [0.62-1.02], P = 0.073), those with a NMIBC history and BCG (12.1 months, HR 0.87 [0.64-1.17], P = 0.356), or those with a NMIBC history but not BCG (14.5 months, HR 0.68 [0.45-1.12], P = 0.061) were not significantly different. This tendency was robust after 1:1 or 1:2 PSMs. The objective response rate (ORR, 24.5% vs. 31.0%, P = 0.222) and disease control rate (DCR, 56.1% vs. 52.1%, P = 0.501) of the 155 patients with an NMIBC history did not differ from those of 155 matched patients without an NMIBC history. Among those with an NMIBC history, the prior use of BCG did not affect OS (with vs. without BCG, 12.1 vs. 14.5 months, HR 1.29 [0.80-2.09], P = 0.295), ORR (24.5% vs. 34.0%, P = 0.298) or DCR (57.1% vs. 56.0%, P = 0.908). The ORR in BCG-treated patients was significantly lower than that in those without a NMIBC history (19.8% vs. 33.3%, P = 0.042), whereas DCR between the 2 groups did not differ significantly (55.8% vs. 54.4%, P = 0.855)., Conclusions: Our risk-adjusted analyses revealed that a history of prior NMIBC treatment, including intravesical BCG therapy, did not affect the treatment outcomes of pembrolizumab in metastatic UC patients., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Physiologically based pharmacokinetic (PBPK) modeling is useful for evaluating differences in drug exposure among special populations, but it has not yet been employed to evaluate the absorption process of tacrolimus. In this study, we developed a minimal PBPK model with a compartmental absorption and transit model for renal transplant patients using available data in the literature and clinical data from our hospital. The effective permeability value of tacrolimus absorption and parameters for the single adjusting compartment were optimized via sensitivity analyses, generating a PBPK model of tacrolimus for renal transplant patients with good predictability. Next, we extrapolated the pharmacokinetics of tacrolimus for liver transplant patients by changing the population demographic parameters of the model. When the physiological parameters of a population with normal liver function were changed to those of a population with impaired hepatic function (Child-Pugh class A) in the constructed renal transplant PBPK model, the predicted tacrolimus concentrations were consistent with the observed concentrations in liver transplant patients. In conclusion, the constructed tacrolimus PBPK model for renal transplant patients could predict the pharmacokinetics in liver transplant patients by slightly reducing the hepatic function, even at three weeks post-transplantation., Competing Interests: Declaration of competing interest None of the authors have any competing interests to declare., (Copyright © 2021 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Background: We used real-world and large-scale data to assess the clinical efficacy and safety of pembrolizumab in older patients with advanced urothelial carcinoma (UC)., Methods: A total of 608 patients who received pembrolizumab for the treatment of chemoresistant UC were retrospectively analyzed. All patients were histologically diagnosed with pure UC. Using propensity score matching (PSM) (ECOG performance status, site of metastasis, hemoglobin level and neutrophil-to-lymphocyte ratio, 1:1 matching), the overall survival (OS) and adverse events (AEs) of patients <75 and ≥75 years old were compared., Results: The median follow-up (IQR) period was 16.1 (9.9-20.5) months. After PSM, there were 215 patients each in the aged <75 years and aged ≥75-year-old groups. The median OS of all patients was estimated to be 10.4 months (95% confidence interval [CI] = 8.8-12.1). After PSM, the median OS was 7.8 months (95% CI = 5.2-10.4) in the <75-year-old group and 10.4 months (95% CI = 7.3-13.5) in the ≥75-year-old group (P = 0.186). Any-grade AEs were more frequently reported in the ≥75-year-old group in comparison to the age <75-year-old group (55.3% vs. 41.9%, P = 0.007), whereas there was no significant difference between the two groups in the incidence of grade ≥3 AEs (10.2% vs. 12.6%, P = 0.544). The objective response rate, defined as complete remission or a partial response, was 22.8% in the <75-year-old group and 25.1% in the ≥75-year-old group (P = 0.651)., Conclusions: The present study demonstrates that age does not affect the efficacy and safety of pembrolizumab treatment for advanced chemoresistant UC. Pembrolizumab treatment should not be avoided based on chronological age; however, close monitoring for the development of treatment-related AE should be considered for older patients., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: The aim of this study was to investigate the clinical significance of the effect of age on disease control in men who received high-dose intensity-modulated radiation therapy (IMRT) for nonmetastatic prostate cancer (NMPCa)., Methods: NMPCa patients with favorable intermediate to very high-risk features (National Comprehensive Cancer Network risk classification) treated with IMRT at our institution between September 2000 and May 2011 were analyzed retrospectively. Treatment consisted of high-dose IMRT (74-78 Gy/37-39 fractions) combined with 6 months of neoadjuvant hormonal therapy. Multivariable analysis using Fine and Gray's regression model was performed to evaluate whether age at initiation of IMRT was associated with biochemical failure (BF) and castration-resistant prostate cancer (CRPC) progression., Results: A total of 367 patients were analyzed. The median follow-up period was 8.8 years after IMRT. The 5- and 10-year BF rates were 22.1 and 31.7%, and those of CRPC rates were 4.5 and 12.6%, respectively. Multivariable analysis revealed that a younger age (cut-off: 70 years old) at the initiation of IMRT was significantly correlated with both a higher BF rate (hazard ratio: 1.691, P= 0.0064) and higher CRPC rate (hazard ratio: 2.579, P = 0.0079)., Conclusions: Younger men with NMPCa had increased risks of BF and CRPC after high-dose IMRT, and may benefit from more intensive treatments. Our findings should be further tested in prospective studies., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The biological processes of urothelial carcinogenesis are not fully understood, particularly regarding the relationship between specific genetic events, cell of origin, and molecular subtypes of subsequent tumors. N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced mouse bladder cancer is widely accepted as a useful model that recapitulates the pathway of human bladder tumorigenesis from dysplasia to invasive cancer via carcinoma in situ. However, the long and variable time of tumorigenesis often hinders efficient preclinical or translational research. We hypothesized that Trp53 mutation in specific types of urothelial cells facilitates efficient development of clinically relevant bladder cancer. Using lineage tracing, we showed that Trp53 mutation in Krt5-expressing cells resulted in more efficient tumorigenesis of mouse muscle-invasive bladder cancer (MIBC) with squamous differentiation compared with Trp53 mutation in Upk2-expressing cells, or wild-type or hemizygous Trp53 in the entire urothelium. Mouse MIBC that developed at 24 weeks of BBN treatment showed morphologic and genetic similarities to the basal squamous subtypes of human MIBC, irrespective of pre-induction of Trp53 mutation or whether the cell of origin was Krt5- or Upk2-expressing cells. Our findings suggest that intermediate cells as well as basal cells also can give rise to basal-like MIBC, with pre-induction of Trp53 mutation accelerating MIBC. Thus, in BBN chemical carcinogenesis, pre-induction of Trp53 mutation in basal cells facilitates efficient modeling of the basal squamous subtype of human MIBC., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tertiary lymphoid tissues (TLTs) are inducible ectopic lymphoid tissues in chronic inflammatory states and function as sites of priming local immune responses. We previously demonstrated that aged but not young mice exhibited multiple TLTs after acute kidney injury and that TLTs were also detected in human aged and diseased kidneys. However, the forms of progression and the implication for kidney injury remain unclear. To clarify this we analyzed surgically resected kidneys from aged patients with or without chronic kidney disease as well as kidneys resected for pyelonephritis, and classified TLTs into three distinct developmental stages based on the presence of follicular dendritic cells and germinal centers. In injury-induced murine TLT models, the stages advanced with the extent of kidney injury, and decreased with dexamethasone accompanied with improvement of renal function, fibrosis and inflammation. Kidneys from aged patients with chronic kidney disease consistently exhibited more frequent and advanced stages of TLTs than those without chronic kidney disease. Kidneys of patients with pyelonephritis exhibited more frequent TLTs with more advanced stages than aged kidneys. Additionally, TLTs in both cohorts shared similar locations and components, suggesting that TLT formation may not be a disease-specific phenomenon but rather a common pathological process. Thus, our findings provide the insights into biological features of TLT in the kidney and implicate TLT stage as a potential marker reflecting local injury and inflammation., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: There has been no established clinical evidence for using sequential treatment in castration-resistant prostate cancer (CRPC). Despite evident cross-resistance, androgen receptor axis-targeted agents (ARTAs), namely abiraterone (ABI) and enzalutamide (ENZ), are often used sequentially owing to less toxicity compared with chemotherapy., Patients and Methods: A multicenter retrospective review of chemotherapy-naive patients with CRPC who had received ABI followed by ENZ (ABI-to-ENZ) or ENZ followed by ABI (ENZ-to-ABI) was conducted. Combined progression-free survival (PFS), overall survival (OS), and prostate-specific antigen (PSA) response (≥ 50% PSA decline) to each drug were compared between the 2 groups at the median follow-up of 36.0 months., Results: There were no significant differences in combined PFS (12.4 vs. 10.9 months; hazard ratio [HR], 0.94; 95% confidence interval [CI], 0.72-1.23; P = .6594) or OS (28.3 vs 29.3 months; HR, 0.96; 95% CI, 0.66-1.38; P = .8314) between the ABI-to-ENZ and ENZ-to-ABI groups. PSA response rate was not significantly different in first-line ARTAs (48.9% vs. 58.4%; P = .153) but significantly higher in ENZ as a second-line ARTA (40.4% vs. 13.7%; P < .0001). Although multivariate analysis revealed that the ABI-to-ENZ sequence was associated with favorable PFS on second-line ARTA (HR, 0.65; 95% CI, 0.49-0.85; P = .0019), it was not associated with an increased combined PFS or OS., Conclusion: With relatively longer follow-up, ARTA sequence did not affect clinical outcomes of CRPC treatment except for PSA response and PFS on a second-line ARTA. These findings will be useful information in clinical decision-making, particularly in chemotherapy-unfit patients with CRPC., (Copyright © 2019. Published by Elsevier Inc.)

Background: Serum LacdiNAc-glycosylated prostate-specific antigen (LDN-PSA) and LDN-PSA density together with PSA and PSA density (PSAD) were measured as a diagnostic tool for prostate cancer (PCa)., Patients and Methods: We included 150 patients with PCa without hormonal therapy and 41 patients without PCa obtained from the Kyoto University Hospital between 2012 and 2017. LDN-PSA levels were measured through a WFA-anti-PSA antibody sandwich immunoassay using a highly sensitive surface plasmon field-enhanced fluorescence spectroscopy (SPFS) system. Diagnostic performance of serum LDN-PSA and LDN-PSAD was evaluated by measuring the area under the receiver-operating characteristic curve (AUC)., Results: The AUCs of LDN-PSA, LDN-PSAD, and PSAD levels (0.780, 0.848, and 0.835, respectively) detected in patients with PCa were significantly higher (P = .0001, P < .0001, and P < .0001, respectively) than that of PSA (0.590). Moreover, among 143 patients with PCa who received radical prostatectomy (RP), the AUCs of LDN-PSA, LDN-PSAD, and PSAD levels (0.750, 0.812, and 0.769, respectively) detected in patients with a pathologic Gleason grade group ≥ 2 were significantly higher (P = .0170, P = .0028, and P = .0003, respectively) than that of PSA (0.578). In the group comprising 35 patients who received RP with a Gleason grade group 1-graded biopsy, the LDN-PSA, LDN-PSAD, and PSAD levels were significantly different (P = .0097, P = .0024, and P = .0312, respectively). However, PSA alone could not discriminate cases with adverse features (P = .454)., Conclusions: LDN-PSAD is a potential marker for detecting PCa and selecting candidates for RP., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: Carboplatin and paclitaxel (CP) had shown moderate efficacy in treating castration-resistant prostate cancer (CRPC) before standard first-line docetaxel chemotherapy became available. Currently, for patients with homology-directed repair gene defects as well as for unselected patients, platinum chemotherapy is administered after all standard treatments have been ineffective. Here, we retrospectively studied the efficacy and safety of CP administered as the first-, second-, and third-line chemotherapy in patients with CRPC., Patients and Methods: A retrospective chart review was performed for 58 patients with CRPC who received CP between 2001 and 2018 in a single institution. Twenty-seven patients received CP as the first-line chemotherapy, 21 as the second-line after docetaxel, and 10 as the third-line after docetaxel and cabazitaxel. Prostate-specific antigen (PSA) responses (> 50% decline of PSA from baseline), progression-free survival, overall survival, and adverse events were examined., Results: PSA responses at any time were 55.6%, 19.0%, and 10.0%; PSA responses at 12 weeks were 48.1%, 14.3%, and 10.0%; the median progression-free survival was 3, 1, and 1 month; and the median overall survival was 19, 11, and 6 months, respectively, for the first-, second-, and third-line settings. The only patient who achieved exceptional and durable PSA response in the third-line setting had a deleterious germline BRCA2 mutation (5645C>A). The adverse event profile was favorable., Conclusion: CP shows moderate efficacy against CRPC in the first-line setting, but shows little effect in the third-line setting. CP after docetaxel and cabazitaxel may be recommended in selected patients with CRPC with homology-directed repair gene defects., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Non-muscle-invasive bladder cancer (NMIBC) can be treated using transurethral resection (TUR), but high incidence of intravesical recurrence remains a clinical challenge. Single immediate postoperative instillation of chemotherapy (IPIOC) is controversial for NMIBC patients with intermediate recurrence risk. The aim of the present study was to report the efficacy and toxicity of IPIOC, particularly in intermediate-risk NMIBC patients, in the real-world setting., Patients and Methods: We retrospectively analyzed 363 consecutive patients with primary NMIBC who underwent radical TUR at Kyoto University Hospital between 2007 and 2016., Results: In low-risk patients, recurrence-free survival (RFS) was significantly better for IPIOC than non-IPIOC (2-year RFS: 89.3% vs. 59.4%; P = .001). In intermediate-risk patients, IPIOC was associated with significantly longer RFS compared with non-IPIOC (2-year RFS: 85.5% vs. 58.2%; P = .011). IPIOC and bacillus Calmette-Guérin (BCG) were independent predictors for post-TUR recurrence (non-IPIOC vs. IPIOC: hazard ratio [HR], 2.33; 95% confidence interval [CI], 1.14-4.88; P = .02; non-BCG vs. BCG: HR, 2.22; P = .045, 95% CI, 1.02-5.30). In the high-risk group, only BCG was an independent prognostic factor of recurrence in a multivariate Cox proportional hazards model (HR, 2.55; P = .006, 95% CI, 1.32-4.87). There were no significant differences between the BCG-only group and the IPIOC with BCG group in Grade 3 or more local (16 patients [21%] vs. 21 patients [24%]; P = .61) or systemic (3 patients [4%] vs. 6 patients [7%]; P = .40) toxicity rates., Conclusion: Our study showed the efficacy of IPIOC for the prevention of intravesical recurrence in primary intermediate-risk NMIBC patients regardless of BCG therapy., (Copyright © 2019. Published by Elsevier Inc.)

Collective cell migration during wound healing has been extensively studied in the epidermis. However, it remains unknown whether the urothelium repairs wounds in a manner similar to the epidermis. By in vivo two-photon excitation microscopy of transgenic mice that express fluorescent biosensors, we studied the collective cell migration of the urothelium in comparison with that of the epidermis. In vivo time-lapse imaging revealed that, even in the absence of a wound, urothelial cells continuously moved and sometimes glided as a sheet over the underlying lamina propria. On abrasion of the epithelium, the migration speed of each epidermal cell was inversely correlated with the distance to the wound edge. Repetitive activation waves of extracellular signal-regulated kinase (ERK) were generated at and propagated away from the wound edge. In contrast, urothelial cells glided as a sheet over the lamina propria without any ERK activation waves. Accordingly, the mitogen-activated protein kinase/ERK kinase inhibitor PD0325901 decreased the migration velocity of the epidermis but not the urothelium. Interestingly, the tyrosine kinase inhibitor dasatinib inhibited migration of the urothelium as well as the epidermis, suggesting that the gliding migration of the urothelium is an active, not a passive, migration. In conclusion, the urothelium glides over the lamina propria to fill wounds in an ERK-independent manner, whereas the epidermis crawls to cover wounds in an ERK-dependent manner., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: External beam radiation therapy (EBRT) with short-term androgen deprivation therapy is the standard of care for intermediate-risk prostate cancer patients. However, no study to date has evaluated the hormonal kinetics or sexual and hormonal function recovery after cessation of short-term luteinizing hormone (LH)-releasing hormone (LHRH) antagonist treatment., Patients and Methods: Ten intermediate-risk prostate cancer patients (mean age, 69.9 years) were included. All patients received 4 months of LHRH antagonist (degarelix) treatment followed by EBRT. The testosterone, luteinizing hormone (LH), follicle-stimulating hormone, and prostate-specific antigen levels were measured and Expanded Prostate Cancer Index Composite questionnaires were completed before LHRH antagonist therapy at baseline; 1, 2, 3, and 4 months after the first injection of LHRH antagonist treatment; and every 2 months thereafter until 18 months., Results: The testosterone levels were at the castration level at 1 month after the first LHRH antagonist injection. The median interval to recover a normal testosterone level (> 7.2 nmol/L) was 7 months after the last LHRH antagonist administration. The LH and follicle-stimulating hormone levels decreased but had increased more than twice above baseline at 15 months after the last LHRH antagonist injection. The sexual function and hormonal bother subdomain scores and sexual and hormonal domain scores decreased once after LHRH antagonist treatment but had significantly recovered thereafter (P < .05)., Conclusion: In most patients, the testosterone level had normalized within 9 months after the last LHRH administration. Sexual and hormonal function recovered after short-term LHRH antagonist administration for neoadjuvant therapy before EBRT., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Unlabelled: Despite the favorable toxicity profile at the standard dose of 560 mg daily, the tolerability and toxicology of estramustine phosphate (EMP) have been a cause for concern at administration. Moreover, we do not know whether a lower dose of 280 mg of EMP daily can be administered with some efficacy and fewer side effects. The results of our phase II study suggest that low-dose EMP is a safe treatment option with the same efficacy in patients with castration-resistant prostate cancer., Background: We evaluated the efficacy and safety of low-dose estramustine phosphate (EMP) in Japanese patients with castration-resistant prostate cancer (CRPC)., Patients and Methods: The present study was a single-arm, nonrandomized prospective study in which all patients received EMP orally twice daily for a total dose of 280 mg/day. A total of 31 patients with CRPC were enrolled from December 2009 to December 2012 at 5 institutions in Japan. The primary endpoint was the prostate-specific antigen (PSA) response, defined as a 50% decline in the serum PSA level, confirmed ≥ 3 weeks later. The secondary endpoints included the objective response rate, interval to PSA progression, PSA response duration, progression-free survival, disease-specific survival, overall survival, safety, and quality-of-life assessment using the Functional Assessment of Cancer Therapy-Prostate scores., Results: Ten patients (32%) had a PSA response, and no patient had an objective response. The treatment was well tolerated, and the most frequent toxicities were grade 1 to 2 nausea/vomiting, anorexia, and gynecomastia. The median interval to PSA progression was 140 days (95% confidence interval [CI], 117-260 days). The PSA response duration was 119 days (95% CI, 49-219 days). The median progression-free survival was 213 days (95% CI, 167-422 days). The 3-year disease-specific survival and overall survival rates were 68.6% (median not reached; 95% CI, 33 months to not available) and 59.9% (median 42 months, 95% CI, 28 months to not available), respectively., Conclusion: Low-dose EMP seems to be a safe treatment option with some efficacy in patients with CRPC., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objectives: To improve conventional chemotherapeutic efficacy, it is important to detect new molecular markers for chemosensitivity and possible accelerating cell-killing mechanisms. In this study, we investigated how MK2206, an allosteric Akt inhibitor, enhances the cisplatin (CDDP)-induced cytotoxicity and apoptosis in urothelial cancer cells., Materials and Methods: We examined bladder cancer cell lines for the expression of phospho(p)-Akt and its downstream targets by Western blot. The potential antitumor effects were analyzed by MTT assay in vitro and by subcutaneous xenograft models in vivo. The cell invasion was examined by transwell invasion assay, and the activities of the Akt signaling pathway and expression of apoptosis-related proteins were measured by Western blot., Results: The expression of p-Akt and its downstream targets was increased in invasive bladder cancer cell lines vs. in noninvasive bladder cancer cell lines. MK2206 (500 nM) inhibited cell invasion in UMUC3 cell line and significantly increased the susceptibility of bladder cancer cell lines to CDDP. When used in combination with CDDP, MK2206 (500 nM) enhanced CDDP-induced cytotoxicity and apoptosis, with suppressed expression of p-Akt and its downstream targets. In vivo MK2206 combined with CDDP effectively suppressed tumor growth in subcutaneous xenograft models., Conclusions: These results suggest that concomitant use of MK2206 could promote the CDDP-induced cytotoxicity and apoptosis in urothelial cancer cell lines through the inhibited expression of the Akt pathway. This combined treatment may provide a new therapeutic option to enhance chemosensitivity in bladder cancer., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We prospectively investigated the rates of incidence of surgical site infection (SSI), urinary tract infection (UTI), and remote infection (RI) in 4,677 patients who underwent urological surgery from January to December 2010, including 2,507 endourological cases, 1,276 clean cases, 807 clean-contaminated cases, and 87 contaminated cases involving bowel segments. A single dose of antimicrobial prophylaxis (AMP) was administered in the endourological, clean, and clean-contaminated surgery cases, except for patients who underwent transurethral resection of the prostate (TURP) or percutaneous nephrolithotripsy (PNL). AMP was administered within 72 h in TURP and PNL, and AMP was administered within 48 h in contaminated surgery cases. In cases of endourological surgery, UTI was observed in 4% and RI in 0%, and SSI, UTI, and RI were seen in 1%, 1%, and 1%, respectively, of clean surgery cases, in 3%, 3%, and 2%, respectively, of clean-contaminated surgery cases, and in 17%, 30%, and 10%, respectively, of contaminated surgery cases. In multivariate analysis of the risk factors for infection, operative time was a significant risk factor for UTI in endourological surgery, and American Society of Anesthesiologists score and operative time were significant risk factors for RI in clean surgery. No significant risk factor was found in analyses of clean-contaminated and contaminated surgery cases. A single-dose AMP regimen was shown to be effective and feasible for prevention of perioperative infection in urological surgery.

Organic anion transporters (OATs) and organic cation transporters (OCT) play pivotal roles in the uptake of drugs into epithelial cells at the basolateral membranes, and multidrug and toxin extrusion (MATE) mediates drug secretion into urine at the brush-border membranes. In this study, the expression and distribution of apical MATE1 and MATE2-K, and basolateral OAT1, OAT3, and OCT2 were compared using serial sections of human kidney cortex. First, mRNA expression in the proximal tubules was evaluated using laser microdissection. Levels of OAT, OCT2, and MATE mRNA in the proximal tubules were greatly higher compared with glomerulus. The results quantitatively indicated that these transporters were localized to proximal tubules in the renal cortex. Second, MATE1 and MATE2-K protein were detected in proximal epithelial cells in which OCT2 protein was expressed at the basolateral membranes. In addition, MATE1 was expressed at the brush-border membranes of tubular epithelial cells in which OAT1 and OAT3 were expressed. The results confirmed that OAT1, OAT3, OCT2, MATE1, and MATE2-K were coexpressed in tubular epithelial cells. The cooperation among OAT, OCT, and MATE in renal drug secretion was consistent with their distribution., (Copyright © 2013 Wiley Periodicals, Inc.)

Objective: We created software for patterning uroflowmetry (UFM) curves, and validated its utility., Patients and Methods: The software patterns a given UFM curve upon four parameters: sex, voided volume, maximal flow rate, and amplitude of fluctuation. Using the software, 6 urologists from 4 institutes assessed 30 test curves. Further, 329 UFM curves obtained from children presenting to 3 institutes for daytime and/or nighttime wetting were assessed. Clinical presentation was divided into 3 groups: group A, daytime incontinence; group B, non-monosymptomatic nocturnal enuresis without daytime wetting; and group C, monosymptomatic nocturnal enuresis., Results: Using the software, inter-rater agreement ranged from 0.85 to 1.00 (mean, 0.93 ± 0.04). It could pattern 310 out of 329 clinical curves. In each institute, the tower pattern was prevalent according to severity of daytime symptoms, although not significantly. The merged data showed that the percent tower pattern significantly correlated with presence of daytime symptoms (groups A, B, and C, 29.7%, 27.0%, and 16.3%, respectively; p < 0.05). No correlation with daytime symptoms was noted for fluctuated (staccato and interrupted) and plateau patterns., Conclusion: The software creates a common platform for evaluating pediatric UFM, enabling extraction of common and biased features of different cohorts, and their integration into one single cohort., (Copyright © 2011 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

To elucidate the impact of genetic variations in breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (MDR1/ABCB1) on the pharmacokinetics of sunitinib, we carried out a pharmacogenetic study in a clinical setting and pharmacokinetic analysis using Abcg2(-/-), Abcb1a/1b(-/-) and Abcb1a/1b;Abcg2(-/-) mice. Nineteen renal cell carcinoma patients were enrolled in this study. The plasma concentrations of sunitinib and its active metabolite were determined and the area under the concentration-time curve (AUC) was calculated. Genetic polymorphisms in ABCG2 (421C>A) and ABCB1 (1236C>T, 2677G>T/A and 3435C>T) were examined. The dose-adjusted AUC(0-24) of sunitinib was significantly higher in patients with a heterozygous variant for ABCG2 421C>A than in wild-type patients (p = 0.02), and one homozygous patient showed the highest dose-adjusted AUC(0-24). The ABCB1 polymorphisms were not associated with the dose-adjusted AUC(0-24). The maximum concentration and AUC(0-4) of sunitinib were significantly higher in Abcg2(-/-), Abcb1a/1b(-/-) and Abcb1a/1b;Abcg2(-/-) mice than wild-type mice when sunitinib was given orally but not intraperitoneally. Incidence of thrombocytopenia and hypertension and poor compliance were associated with the systemic exposure to sunitinib and its active metabolite. These results suggest that the loss of protein expression of ABCG2 by genetic polymorphism is associated with an increase in the systemic exposure to sunitinib and sunitinib-induced toxicity.

We describe the longitudinal follow-up of calcineurin activity and its clinical relevance in 4 de novo living-donor kidney transplant recipients treated with cyclosporine (n=1) or tacrolimus (n=3). The calcineurin activity in peripheral blood mononuclear cells was measured in combination with therapeutic drug monitoring during hospitalization. Serial blood samplings were performed after the oral administration of each drug to evaluate the temporal pharmacokinetic and pharmacodynamic profiles. Significant changes in enzyme activity were evaluated in relation to clinical outcomes. A nadir of calcineurin activity occurred at the maximum blood drug concentration within 4 h post-dose in most cases. Unlike cyclosporine, tacrolimus partially suppressed calcineurin activity throughout the dosing interval compared to the pre-dose level (cyclosporine, 62-67% inhibition; tacrolimus, 13-35% inhibition). Notably, calcineurin activity rapidly increased a few days before the onset of acute rejection in 2 patients, 1 receiving cyclosporine and 1 receiving tacrolimus, despite the achievement of therapeutic trough blood concentrations. These preliminary findings indicate that therapeutic monitoring of calcineurin activity in addition to the measurement of blood drug concentrations may be helpful to evaluate the pharmacodynamic effects of cyclosporine and tacrolimus early after renal transplantation.

Despite well known oncogenic function of G1-S cell-cycle progression, cyclin D2 (CCND2) is often silenced epigenetically in prostate cancers. Here we show that CCND2 has an inhibitory potential on the proliferation of androgen receptor (AR)-dependent prostate cancer LNCaP cells. Forced expression of CCND2 suppressed the proliferative ability and induced cell death in LNCaP cells in a cdk-independent manner. Knocking down CCND2 restored the proliferation of LNCaP subclones with relatively high CCND2 expression and low proliferative profiles. Immunoprecipitation using deletion mutants of CCND2 indicated that a central domain of CCND2 is required for binding to AR. A deletion mutant lacking the central domain failed to hinder LNCaP cells. Collectively, our results indicated that CCND2 inhibits cell proliferation of AR-dependent prostate cancer through the interaction with AR. Our study suggests that restoration of CCND2 expression potentially prevents the carcinogenesis of prostate cancer, which is mostly AR-dependent in the initial settings.

We report a patient with a neonatal seizure in whom diffusion-weighted imaging (DWI) at 8 days of age revealed high-intensity areas in the genu and splenium of the corpus callosum. The patient was a 2-day-old girl born at 39 weeks of gestational age. No apparent signs of asphyxia were found at birth. Clinically, she had a clonic seizure of the left hemisphere, with open eyes deviating to the left, and automatism around the mouth. The antiepileptic drug phenobarbital was administered once, her seizure was simultaneously stopped. Because she was a newborn, her corpus callosum was not completely myelinated. Intramyelinic edema was not responsible for these DWI findings; the mechanism of the abnormal DWI findings was clearly unknown. Here, we present abnormal DWI findings in the corpus callosum in a neonatal seizure case that did not meet all the criteria for neonatal hypoxic-ischemic encephalopathy (HIE).

Proton-pump inhibitors (PPIs, e.g. omeprazole and rabeprazole) are often administered to transplant patients as a treatment or prophylaxis for ulcers after surgery. Since tacrolimus and PPIs share the CYP3A4 system for metabolism, pharmacokinetic interactions are anticipated when they are administered simultaneously. We present a Japanese male patient who underwent a living-donor kidney transplantation having received tacrolimus, mycophenolate mofetil, and prednisolone for immunosuppression. The concentration/dose (C/D) ratio for tacrolimus was markedly higher during the period of treatment with omeprazole than ranitidine or rabeprazole. The results of liver functional tests were within the normal range during the use of these three antacid drugs. Since the higher C/D ratio for tacrolimus when omeprazole was being administered did not result from a decrease in the elimination of tacrolimus due to hepatic dysfunction, drug interaction between omeprazole and tacrolimus was strongly suspected. The present case indicates that rabeprazole can be used safely in place of omeprazole in kidney transplant recipients receiving tacrolimus.

To investigate the effect of highly purified eicosapentaenoic acid (EPA) on the progression of diabetic macroangiopathy, we performed an open-label randomized prospective trial. A total of 81 Japanese type 2 diabetes were randomly assigned to the EPA (1800 mg/day) treated group or the control group. Carotid intima-media thickness (IMT) and brachial-ankle pulse wave velocity (baPWV) were evaluated before and after treatment in both groups. Sixty patients (EPA group, n=30; control group, n=30) completed this study. During the study period of 2.1+/-0.2 years, the mean IMT and max IMT of the EPA treated group showed a significant annual decrease compared with that of the control group (mean IMT, -0.029+/-0.112 mm versus 0.016+/-0.109 mm, respectively, P=0.029; max IMT, -0.084+/-0.113 mm versus -0.005+/-0.108 mm, respectively, P=0.0008). The baPWV was also improved significantly in the EPA treated group compared with the control group (-22.1+/-127.9 cm/s versus 62.3+/-223 cm/s, respectively, P=0.021). Multiple regression analysis showed that the administration of EPA was a significant and independent factor associated with an annual improvement of mean IMT (R2=0.067). In summary, this is the first demonstration that administration of purified EPA improves the carotid IMT and the baPWV in patients with type 2 diabetes.

Germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene predispose people to renal cancer, hemangioblastomas, and pheochromocytomas in an allele-specific manner. The best documented function of the VHL gene product (pVHL) relates to its ability to polyubiquitinate, and hence target for destruction, the alpha subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF). pVHL mutants linked to familial pheochromocyctoma (type 2C VHL disease), in contrast to classical VHL disease, appear to be normal with respect to HIF regulation. Using a simple method for identifying proteins that are differentially secreted by isogenic cell line pairs, we confirmed that the HIF targets IGBP3 and PAI-1 are overproduced by pVHL-defective renal carcinoma cells. In addition, cells lacking wild-type pVHL, including cells producing type 2C pVHL mutants, were defective with respect to expression and secretion of clusterin, which does not behave like a HIF target. Decreased clusterin secretion by pVHL-defective tumors was confirmed in vivo by immunohistochemistry. Therefore, clusterin is a secreted marker for a HIF-independent pVHL function that might be especially important in pheochromocytoma development.

Tissue regeneration on acellular matrix grafts has great potential for therapeutic organ reconstruction. However, hollow organs such as the bladder require smooth muscle cell regeneration, the mechanisms of which are not well defined. We investigated the mechanisms by which bone marrow cells participate in smooth muscle formation during urinary bladder regeneration, using in vivo and in vitro model systems. In vivo bone marrow cells expressing green fluorescent protein were transplanted into lethally irradiated rats. Eight weeks following transplantation, bladder domes of the rats were replaced with bladder acellular matrix grafts. Two weeks after operation transplanted marrow cells repopulated the graft, as evidenced by detection of fluorescent staining. By 12 weeks they reconstituted the smooth muscle layer, with native smooth muscle cells (SMC) infiltrating the graft. In vitro, the differential effects of distinct growth factor environments created by either bladder urothelial cells or bladder SMC on phenotypic changes of marrow cells were examined. First, supernatants of cultured bladder cells were used as conditioned media for marrow cells. Second, these conditions were reconstituted with exogenous growth factors. In each case, a growth factor milieu characteristic of SMC induced an SMC-like phenotype in marrow cells, whereas that of urothelial cells failed. These findings suggest that marrow cells differentiate into smooth muscle on acellular matrix grafts in response to the environment created by SMC.

Testicular germ-cell tumours (TGCTs) are the most common malignant diseases among men aged 20-40 years. We developed a DNA tumour marker for TGCTs based on the unmethylated DNA profile of a neoplasm. The 5' end of the XIST gene is mainly hypomethylated in TGCTs irrespective of XIST expression. Male somatic cells, however, show complete methylation through the CpG sites, including the minimum promoter and XIST-conserved repeats. Identification of a XIST unmethylated fragment in male plasma might be diagnostic for TGCTs.

FUBI (failure of ureteric bud invasion) is a highly inbred strain of mouse with a high spontaneous incidence of uni- or bilateral renal agenesis (60%). Bilateral renal agenesis is lethal within 2 days after birth. The primary defect of FUBI is failure of the ureteric bud to penetrate into the metanephric mesenchyme at around embryonic day 11, resulting in apoptosis of metanephric cells and leading to renal agenesis on the affected side. The metanephros seemed to be normal because co-culturing of the FUBI metanephros with homologous spinal cord induced differentiation of the rudiment, but co-culturing with the homologous ureteric bud frequently did not. Genetic analysis revealed that more than two genes were involved in this malformation and we mapped one of the modifier loci, fubi1, on chromosome 2, at approximately 65 cM from the centromere. In this region, there are two possible candidate genes, Wilms' tumor 1 and formin, that play important roles in kidney development. Some of formin mutants shared a similar phenotype with FUBI; however, there was no difference in the expression of formin in embryonic kidneys between FUBI and control NFS/N mice. Studies of fubi1 congenic mice indicated that interaction of two or more loci is essential for the FUBI phenotype.

We previously reported that detection of telomerase activity in urinary exfoliated cells obtained from urothelial cancer patients by telomeric repeat amplification protocol (TRAP) assay is a more sensitive method of diagnosis than conventional urine cytologic examination, particularly in patients with grade 1 tumors. To establish this method as a noninvasive screening test for the diagnosis of urothelial cancers, we performed the semi-quantitative analysis of telomerase activity using Telomerase PCR ELISA™. Spontaneous voided urine samples were obtained from 65 urothelial and 58 non-urothelial cancer patients. When the mean + 2 standard deviation of telomerase activity in urine sediments of non-urothelial cancer patients was arbitrarily determined as a cut-off, the sensitivity of TRAP enzyme-linked immunosorbent assay (ELISA) and the conventional cytology were 57% and 35%, respectively. Detection rate was significantly higher in semi-quantitative TRAP assay than in conventional cytologic examination in grade 1 cancer patients (52% vs. 5%, p = 0.00195). False positives were detected in 5% of non-urothelial cancer patients without pyuria and in 11% of non-urothelial cancer patients with pyuria (p = 0.395). Telomerase activity was enhanced in some cases after phenol extraction or extracting epithelial cells by using Dynabeads of macroscopic hematuria and pyuria, indicating that hematuria and pyuria might contribute to false negatives. In conclusion, the TRAP-ELISA method is superior to the standard TRAP assay in quantitativeness and simplicity of the experimental procedure. Detection of telomerase activity in urine sediments is particularly useful for the diagnosis of low-grade tumors. However, telomerase activity in patients with high grade tumors often might be underestimated due to the excessive amount of exfoliated epithelia with necrotic tissues, hematuria, and pyuria.

We measured serum transforming growth factor β1 (TGF-b1) levels in patients with untreated prostate cancer and compared them with other prognostic indicators, specifically serum prostate-specific antigen (PSA) and the Gleason histopathologic grading score. Prior to treatment, the Sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure TGF-b1 concentrations directly in sera from 55 patients with prostate cancer (21 localized and 34 metastatic), 13 age-matched healthy male control subjects, and 8 patients with benign prostatic hyperplasia (BPH). Serum TGF-b1 levels in patients with lymph node and/or distant metastases were significantly higher than in patients with localized disease (p = 0.0003), but did not differ significantly among localized cancers as to tumor extension. Ten of 11 prostate cancer patients whose serum TGF-b1 was higher than an arbitrary cut-off value of 60 ng/ml had lymph node or distant metastasis (specificity 95.20, although in 24 of 34 metastatic patients serum TGF-b1 levels were less (sensitivity 29.4%). The correlation between serum TGF-b1 and tumor grade as assessed by the Gleason scoring system was weak (r = 0.340). Moreover, serum TGF-b1 was not correlated with the serum PSA level. Interestingly, there was an inverse correlation between serum TGF-b1 in the prostate cancer patients and patient age at diagnosis (r = -0.406). These findings suggest that elevation of the serum TGF-b1 levels reflects certain malignant potentials associated with metastasis that are unpredictable by PSA and the Gleason scoring system.