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Summary: Single cell sequencing technologies have revolutionized our understanding of biology by mapping cell diversity and gene expression in healthy and diseased tissues. While single-cell RNA sequencing (scRNA-seq) has been widely used, interest in single-nucleus RNA sequencing (snRNA-seq) is growing due to its benefits, including the ability to analyze archival tissues and capture rare cell types that are challenging to dissociate. However, comparative studies across tissues have yielded mixed results, with some reporting enhanced cell type retention using snRNA-seq while others finding cell type identification to be challenging in snRNA-seq data. The GUDMAP consortium aims to construct a molecular atlas of the lower urinary tract (LUT); thus, we set out to determine the strengths and limitations of each approach in characterizing LUT cell types. Using the human bladder, we determined that scRNA-seq offered more discriminative gene sets for identification while snRNA-seq could facilitate capture of previously underrepresented cell types.

Purpose:. According to the National Comprehensive Cancer Network guidelines, segmental ureterectomy (SU) of upper tract urothelial carcinoma (UTUC) is a considerable option for selected mid- and distal ureteral urothelial carcinoma (UC). As a UTUC endemic area, Taiwan lacks treatment outcome analysis of SU. Materials and methods:. This study retrospectively reviewed the treatment outcomes of SU for clinically localized UTUCs. Patients with biopsy or washing cytology-confirmed UTUCs who underwent open, laparoscopic, or robot-assisted management with curative intent were retrospectively reviewed for the eligibility of analysis. Cox regression was applied for univariable and multivariable analyses. Results:. A total of 161 patients who underwent SU were reviewed and analyzed. The median follow-up period was 44.5 (interquartile range, 21.6–84.9) months. After SU, 56/161 (34.8%) patients were free of UTUCs after the follow-up, 25/161 (15.5%) patients had local recurrence, and 35/161 (21.7%) had lymph node or distant metastasis. Surgical margin involvement was a risk factor associated with worse cancer-specific survival. Higher bladder recurrence and local recurrence rates were observed with concurrent bladder UC. Lymphovascular invasion and previous radical nephroureterectomy (RNU) for UC were related to higher local recurrence rates. Patients with pathological T3/T4 stage and end-stage renal disease tended to have higher metastasis rates. For the management of local recurrence, 19 patients received salvage RNU and 25 patients had adjuvant chemotherapy. However, 26/161 (16.1%) patients died of UTUCs and 2/161 (1.2%) patients died of surgery-related complications. Conclusion:. SU provides acceptable oncological outcomes if the surgeons select candidates carefully. SU is not recommended if the patient has T3 or higher stage or comorbidity of end-stage renal disease. Concurrent bladder UC is a risk factor for worse bladder recurrence-free survival and local recurrence-free survival. Lymphovascular invasion and previous RNU for UC were related to higher local recurrence rates. After SU, periodic follow-up is mandatory because the local recurrence rate is higher than radical surgery.

IntroductionFor patients undergoing THR, measuring the postoperative acetabular anteversion precisely plays a pivotal role in the prognosis. However, using elliptical methods mandates computerized equipment that is frequently in shortage in remote areas and developing countries. We invented a laser projector utilizing the ellipse method to measure the acetabular anteversion directly. The aim is to examine the consistency and validity of the laser projector as compared to our original software, Elliversion.Materials and MethodsWe retrospectively collected 50 postoperative pelvis radiographs including acetabulum from our institution. One investigator first measured the anteversion of included radiographs through Elliversion software as the control group. Subsequently, two operators independently used the laser projector for measurements in two separate periods with 1-day intervals as the experimental group. Our analysis was comprised of intra- and inter-observer comparisons and reliability, which investigated both the consistency and validity, by using two-sample student's t-test and intraclass correlation coefficient.ResultsThere was no significant difference in measuring the anteversion through laser projectors between two operators (p = 0.54), with excellent inter-observer reliability (ICC, 0.967). The estimated effect in the anteversion measurement between the Elliversion and laser projector was also comparable, with the ICC level of 0.984, indicating excellent reliability.ConclusionOur study reported the consistency and validity of this laser projector as there is no significant difference between Elliversion and Laser projector, notably with excellent intra- and inter-observer reliability. We look forward to helping elevate clinical acumen when doctors provide care to patients after THR, especially in remote areas.

Objective: To describe in a video format the surgical steps and techniques of single-port robot-assisted extraperitoneal kidney transplantation using the SP da Vinci platform. Methods: We recorded the surgical steps and techniques of this procedure. After induction of anesthesia, and positioning the patient, a 5-cm peri or infra-umbilical incision is made and the extraperitoneal space is developed. GelPOINT access platform is placed and the robot is docked. The external iliac artery and vein are carefully dissected. The bench is performed using a LigaSure™ device to dissect the vessels. A ureteral stent is placed and the posterior surfaces of the artery and the vein are marked. The kidney is then wrapped with a blue surgical glove. After introducing some ice and the kidney into the extraperitoneal space, the robot is redocked, and venous and then arterial anastomoses are performed using 6–0 GORE-TEXⓇ sutures. After repositioning the kidney, ureteroneocystostomy is done. Nephropexy is then performed. The robot is undocked and the wound is closed. Results: Single-port robot-assisted kidney transplantation has been performed for 12 patients with end-stage renal disease. Allograft kidneys were from living and deceased donors in 9 and 3 patients respectively. Surgery was completed successfully in all patients without any complications. Median (IQR) vein and artery anastomosis times were 26 (24–28) and 37 (33–40) minutes, respectively. All grafts were perfused well on the color Doppler ultrasonography. Mean (SD) follow- up and creatinine were 11.25 (6.3) months, and 1.26 (1.16) mg/dL respectively. Conclusion: Extraperitoneal single-port robot-assisted kidney transplantation better duplicates open kidney transplantation and is associated with less number of overall incisions compared to the alternative robotic approaches.

Objective: Quercetin, a natural dietary compound, has been demonstrated with antitumor activities against several types of cancers by disrupting cell cycle and inducing apoptotic cell death. However, human bladder cancer cells such as 5637 and T24 cells expressing mutant p53 are resistant to a 24 hrs quercetin treatment. In this study, the anticancer effect of quercetin was evaluated in these bladder cancer cells. Materials and Methods: The bladder cancer cells treated with quercetin were subjected to evaluated cell apoptosis by caspase activity, TUNEL assay and cell viability assay. The cell autophagy was assessed by detecting procession of LC3-II autophagic marker protein. Results: After 48 and 72 hrs of incubation, quercetin was found to be significantly effective in inhibiting proliferation of 5637 and T24 cells in a dose-dependent manner. Quercetin treatment increased the caspase 3/7 activities, percentage of subG0/G1 cells, and DNA fragmentation, indicating an induced apoptotic cell death. Pretreatment of a pan-caspase inhibitor, Z-VAD-FMK, attenuated the quercetin-decreased cell viability, suggesting that the cytotoxicity caused by quercetin mainly via apoptotic cell death. We also found that quercetin induced autophagy, as evidenced by the increased processing of LC3-II, a specific marker of autophagy. The disruption of autophagic flux by using bafilomycin A1, an autophagy inhibitor, caused significant accumulation of cellular p62 and LC3-II. In addition, the pretreatment of autophagy inhibitors, Baf A1 and chloroquine, strongly augmented apoptosis in 5637 and T24 cells, indicating the suppression of quercetin-induced autophagy enhanced apoptosis. Furthermore, the decreased cell viability and increased LC3-II processing were attenuated in quercetin-treated cells which pretreated with a reactive oxygen species (ROS) scavenger, N-acetyl cystine (NAC) suggested that quercetin-induced cytotoxicity and autophagy were initiated by the generation of ROS. Conclusion: This study proposes that combined treatment of autophagy inhibitor which sensitizes cells to quercetin treatment may be a better therapeutic approach to reduce bladder cancer cells proliferation.

Background: Chloroquine (CQ) is recognized as a potent adjuvant when combined with other chemotherapies to treat cancers. However, the effects of a single treatment of CQ on bladder cancer (BC) cells have not been investigated. Methods: The growth and viability of CQ-treated BC cells were examined. The lysosomal morphology was detected using LysoTracker. The induction of lysosomal membrane permeability (LMP) was detected by acridine orange (AO) translocation, and cathepsin B and D release. The expression of the bid, caspase-3, and cytosolic cytochrome C (Cyto. C) in CQ-treated cells was detected by the Western blot. The pepstatin A and E64d were used to attenuate CQ-induced LMP. Results: A single dose of CQ treatment induced BC cell death, and attenuated by pepstatin A and E64d. The diminishing of fluorescent in CQ-treated cells stained with LysoTracker, suggesting that CQ targets lysosomal functions. This was further supported by increased AO translocation and the releasing of CatB and CatD into the cytosol. The increased level of cleavage bid and cytosolic Cyto. C indicated mitochondrial outer membrane permeabilization and subsequently leading to apoptosis induction judged by the increased level of activated caspase 3. Conclusion: CQ-induced LMP that enhances apoptosis and ultimately leading to BC cell death. The study results demonstrated for the first time that single CQ treatment against BC cells by inducing LMP and subsequent mitochondria membrane permeability that trigger apoptosis, making it a potential treatment for BC therapy in the future.

Background: Prostate cancer (PCa) is a leading cause of cancer-related death in men, which emphasizes the need for novel therapeutic approaches. Targeting microRNA (miRNA) has been considered as a therapeutic strategy against cancers. Human miR-204-5p potentially targeting BCL2 has been reported to be downregulated in various cancers. We hypothesized that miR-204-5p overexpression induces cancer cell apoptosis by repressing BCL2 expression. Methods: A vector harboring mature miR-204-5p was constructed and delivered into human PCa cells. The expression level of miR-204-5p was determined by miRNA quantitative polymerase chain reaction (QPCR). Luciferase reporter assays were performed to verify the function of mature miR-204-5p and its direct binding to BCL2 transcripts. The expression levels of BCL-2 messenger RNA (mRNA) and protein samples were measured by QPCR and Western blot, respectively. Cell viability was detected by WST-1 assays. Induction of apoptosis was determined by increased levels of cleavage caspase 3 and caspase 3/7 activity. Results: The expression levels of miR-204-5p were downregulated in PCa cells compared with normal prostate epithelial cells. Transfection of pSM-204 resulted in up to 6.2-fold higher expression of miR-204-5p when compared with pSM control. The mRNA levels of several potential target genes of miR-204-5p were decreased in pSM-204-transfected PC3 and Rv1 cells. BCL2 mRNA and protein expression decreased in miR-204-5p-transfected cells, which led to cytochrome C release from mitochondria. It subsequently increased cleaved caspase 3 and caspase 3/7 activities and reduced cell viability. Cotransfection of a reporter vector harboring the BCL2 3′-untranslated region to compete with endogenous transcripts partially rescued miR-204-5p-induced apoptosis. Conclusion: Human miR-204-5p targets BCL2 in PCa cells. Restoration of miR-204-5p in PCa could therefore be considered as a novel strategy by targeting antiapoptotic BCL2.

Chloroquine (CQ) and hydroxychloroquine (HCQ), two antimalarial drugs, are suggested to have potential anticancer properties. in the present study, we investigated the effects of CQ and HCQ on cell growth of bladder cancer with emphasis on autophagy inhibition and apoptosis induction in vitro. The results showed that CQ and HCQ inhibited the proliferation of multiple human bladder cell lines (including RT4, 5637, and T24) in a time- and dose-dependent fashion, especially in advanced bladder cancer cell lines (5637 and T24) compared to immortalized uroepithelial cells (SV-Huc-1) or other reference cancer cell lines (PC3 and MCF-7). We found that 24-hour treatment of CQ or HCQ significantly decreased the clonogenic formation in 5637 and T24 cells compared to SV-Huc-1. As human bladder cancer tumor exhibits high basal level of autophagic activities, we detected the autophagic flux in cells treated with CQ and HCQ, showing an alternation in LC3 flux in CQ- or HCQ-treated cells. Moreover, bladder cancer cells treated with CQ and HCQ underwent apoptosis, resulting in increased caspase 3/7 activities, increased level of cleaved poly(ADP-ribose) polymerase (PARP), caspase 3, and DNA fragmentation. Given these results, targeting autophagy with CQ and HCQ represents an effective cancer therapeutic strategy against human bladder cancer.

Background: Transrectal ultrasound-guided prostate (TRUS-P) biopsy is the standard diagnostic procedure for patients with suspected prostate cancer. However, infectious complications can occur. Purpose: To analyze the patients from a single institution who were hospitalized for infectious complications after TRUS-P biopsy. Materials and Methods: From 2003 to 2012, 985 TRUS-P biopsy procedures were performed in a medical center in Northern Taiwan; among these, 28 patients were admitted for infectious complications following the procedure. A retrospective review of the medical records was performed, and data regarding the demographics, details of infectious complications, and hospital course of these patients were collected. Results: The median age of the patients was 62 (27–82) years; the comorbidity rate was 57.1% (16/28); the admission rate was 2.8% (28/985). The most common causative pathogen was Escherichia coli. More than half of the cultured E. coli were not resistant to fluoroquinolones. The median hospital stay was 6 (3–16) days, and one patient was admitted to the intensive care unit. No mortality was observed in this study. Although the number of biopsies increased, no obvious increasing trend in the number of hospitalizations was noted in recent years. Conclusion: TRUS-P biopsy can be safely performed, and no obvious increasing trend in infectious complications and resistant strains has been observed in recent years.

Bladder diverticulum is an outpouching of bladder mucosa through the musculature of the bladder wall. The incidence of bladder diverticulum in Taiwan is about 1.7% in children and 23.4% in adults. Intradiverticular carcinoma of urinary bladder is uncommon. It ranges from 0.8% to 14.3%. Here we report a case of urothelial carcinoma within a bladder diverticulum. A 60-year-old male patient had history of BPH under medical treatment and right ureteral stone treated with extracorporeal shock wave lithotripsy (ESWL). He presented with painless gross hematuria about 3 months after ESWL. Intravenous pyelography showed a filling defect within the bladder diverticulum. Histopathological diagnosis of low grade urothelial carcinoma arising from the bladder diverticulum was made following cystoscopic biopsy. Laparoscopic partial cystectomy was performed with subsequent intravesical chemotherapy. Tumor recurrence was found not from the previous diverticulum but from another area during regular cystoscopy at the 6-month postoperative follow up. He underwent transurethral resection of bladder tumor. Pathology revealed a noninvasive, high grade urothelial carcinoma. There was no further bladder tumor recurrence during the 1-year follow-up period. Bladder-sparing surgery with close cystoscopy follow up for intradiverticular urothelial carcinoma can be applied as an alternative treatment modality.

Objective: Bladder cancer (BC) is one of the most common noncutaneous malignant cancers with high mortality and recurrence rates in the general population, particularly in men. We previously reported detecting dysregulated micro-RNAs (miRNAs) in human BC tissues. Using an miRNA targeting database, we found that miR-204, which is downregulated in BC, targets the B-cell lymphoma 2 gene (BCL2). We hypothesized that the overexpression of miR-204 induces apoptosis by repressing BCL2 expression and causing cancer cell death. Materials and methods: To overexpress miR-204 in cancer cells, a vector that expresses miR-204 was transfected into human BC, embryonic kidney, and cervical cancer cells. The expression of miR-204 in the transfected and mock cells was monitored through miRNA quantitative polymerase chain reactions (Q-PCRs). Luciferase reporter assays were performed to verify the direct binding of miR-204 to targeted sites on the BCL2 transcripts. The BCL2 mRNA and protein expression levels in the miR-204-transfected cells were measured using Q-PCRs and Western blotting, respectively. The cell viability of the miR-204 transfected cells was detected through WST-1 assays. The induction of apoptosis was determined by the increased release of cytochrome c, cleavage caspase 3, caspase 3/7 activity, and DNA fragmentation. Results: The expression of miR-204 in the transfected cells was elevated to 6.2-fold, compared with the controls, and the function of matured miR-204 was confirmed through the luciferase reporter assays. The expressions of BCL2 mRNA and protein were reduced in the miR-204-transfected cells, which generated increased DNA fragmentation, caspase 3 cleavage, caspase 3/7 activity, and reduced cell viability. Cotransfection of the reporter vector that harbored the BCL2 3′-untranslated region to compete with endogenous transcripts partially rescued the apoptosis that was induced by the overexpressed miR-204. This finding further supported that miR-204 targeting BCL2 induces apoptosis in BC cells. Conclusion: miR-204 targets BCL2 in human BC cells. The overexpression of miR-204 in tumors could thus be a novel strategy for inducing cell death by targeting antiapoptotic BCL2.

Objective: Urothelial carcinoma is one of the most common pathological types of bladder cancer. Several studies have shown that dysregulated microRNAs (miRNAs) play an important role in bladder cancer progression. We performed the present miRNA microarray analysis in samples of urothelial carcinoma of the bladder and adjacent normal bladder tissue from Taiwanese patients to investigate dysregulated miRNAs. Materials and methods: To detect dysregulated miRNAs in urothelial carcinoma of the bladder, samples of tumor and adjacent normal tissues were collected from 10 patients. Tissue samples from three patients were subjected to miRNA microarray analysis, and the remaining tissue samples from the other seven patients were used to validate the results obtained from the microarray data. Potential targets of these dysregulated miRNAs were identified using online databases, including MicroCosm and TargetScan. Results: A panel of 30 differentially expressed miRNAs with at least fourfold differences in expression compared with normal controls, including 19 upregulated and 11 downregulated miRNAs, was generated. The expression levels of miR-30a-5p, miR-30a-3p, miR-99a, miR-130b, miR-133b, miR-135b, miR-145, miR-195, miR-204, and miR-214 were experimentally verified using real-time RT-PCR analysis. Using an online miRNA target database, we discovered that these dysregulated miRNAs potentially control components of the insulin-like growth factor 1 receptor (IGF1R) signaling pathway. Conclusion: Our results indicate that dysregulated miRNAs may be involved in bladder cancer pathogenesis and are potential biomarkers.

Background/Objective: Standard laparoscopic adrenalectomy requires early control of the main adrenal vein; however, the small retroperitoneal working space is challenging for beginners to perform this maneuver. We report a technical modification of retroperitoneal laparoscopic adrenalectomy (RLA) for primary hyperaldosteronism (PHA) and the clinical outcomes. Methods: A total of 38 RLAs were performed for the patients with PHA. The patients were placed in true lateral position with mild bending to expand the surgical field. Instead of attempting to control the main adrenal vein initially, we adopted a technical modification that manipulating and freeing the gland first before controlling the main adrenal vein. Results: The RLAs were successfully performed in all but one case, which was converted to open surgery due to pancreatic injury. Mean operative time was 124 minutes and estimated blood loss was 74 ml. Mean maximal fluctuation of systolic blood pressure was 29 mmHg. For the right-side RLA, less operative time (113.5 vs. 137.9 minutes) and estimated blood loss (59.5 vs. 91.2 ml) were noted compared with the left-side procedure. Postoperative complications included cerebrovascular accident in one patient, one surgical site hematoma, and two patients had postoperative fever. Potassium level returned to normal in all patients and 70% of the patients reduced their antihypertensives. Conclusion: Technical modification RLA for PHA without initial control of the main adrenal vein is a safe and feasible procedure. No vigorous blood pressure fluctuation was intraoperatively noted. No vascular injury occurred. Moreover, the right-side procedure became easier.

Stomata play an important role in plant innate immunity by limiting pathogen entry into leaves but molecular mechanisms regulating stomatal closure upon pathogen perception are not well understood. Here we show that the Arabidopsis thaliana L-type lectin receptor kinase-V.5 (LecRK-V.5) negatively regulates stomatal immunity. Loss of LecRK-V.5 function increased resistance to surface inoculation with virulent bacteria Pseudomonas syringae pv tomato DC3000. Levels of resistance were not affected after infiltration-inoculation, suggesting that LecRK-V.5 functions at an early defense stage. By contrast, lines overexpressing LecRK-V.5 were more susceptible to Pst DC3000. Enhanced resistance in lecrk-V.5 mutants was correlated with constitutive stomatal closure, while increased susceptibility phenotypes in overexpression lines were associated with early stomatal reopening. Lines overexpressing LecRK-V.5 also demonstrated a defective stomatal closure after pathogen-associated molecular pattern (PAMP) treatments. LecRK-V.5 is rapidly expressed in stomatal guard cells after bacterial inoculation or treatment with the bacterial PAMP flagellin. In addition, lecrk-V.5 mutants guard cells exhibited constitutive accumulation of reactive oxygen species (ROS) and inhibition of ROS production opened stomata of lecrk-V.5. LecRK-V.5 is also shown to interfere with abscisic acid-mediated stomatal closure signaling upstream of ROS production. These results provide genetic evidences that LecRK-V.5 negatively regulates stomatal immunity upstream of ROS biosynthesis. Our data reveal that plants have evolved mechanisms to reverse bacteria-mediated stomatal closure to prevent long-term effect on CO(2) uptake and photosynthesis.

This report primarily responds to the content of the Articles 10 and 12 regarding gender equity education in the governmental report made by the Taiwanese government in 2014 for CEDAW. In order to observe the obstacles and challenges facing gender equity education in our nation today, this report focuses on three aspects: gender diversity education, sex education, and female participation in exercise and sports. In each topic, the current situation and difficulties are discussed first, followed by concrete recommendations.