Transient and lineage-restricted requirement of Ebf3 for sternum ossification.

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues.

Syntheses of Base-Labile Pseudo-Complementary SNA and l-aTNA Phosphoramidite Monomers.

We previously synthesized phosphoramidite monomers bearing Boc-protected 2,6-diaminopurine (D) and 2-methyl-4-methoxybenzyl-protected 2-thiouracil (sU) as building blocks for the preparation of pseudo-complementary serinol nucleic acids (SNAs). Since SNA is stable under acidic conditions, an acid-deprotection step could be inserted into the work-up. However, as the 4,4'-dimethoxytrityl group was concurrently removed at this step, purification of SNA by reversed-phase HPLC was difficult. Here, we report the syntheses of SNA and acyclic l-threoninol nucleic acid (l-aTNA) phosphoramidite monomers with bis(phenoxyacetyl)-protected D and 4-acetoxybenzyl-protected sU, both of which can be deprotected under mild basic conditions. Using these monomers, we prepared pseudo-complementary SNA and l-aTNA in high yield using conventional oligonucleotide synthesis protocols. These monomers can be used for large-scale syntheses of SNAs and l-aTNAs.

PDH-mediated metabolic flow is critical for skeletal muscle stem cell differentiation and myotube formation during regeneration in mice.

Skeletal muscle satellite cells (SMSCs), the major stem cells responsible for the regeneration of skeletal muscle, are normally cell cycle arrested but differentiate to generate myocytes upon muscle damage, forming new myofibers along with self-renewing stem cells in preparation for subsequent injury. In this study, we investigated which factors stimulate the proliferation and differentiation of SMSCs and found that pyruvate, the end product of glycolysis, stimulates their differentiation. Pyruvate antagonizes the effects of hypoxia on preferential self-renewal of SMSCs through dephosphorylation or activation of pyruvate dehydrogenase (PDH), which mediates opening of the gateway from glycolysis to the tricarboxylic acid (TCA) cycle by producing acetyl coenzyme A from pyruvate. PDH kinase 1, highly expressed under hypoxia, is down-regulated under normoxic conditions, leading to an increase in dephosphorylated PDH. Conditional deletion of PDH in SMSCs affects cell divisions generating myocytes and subsequent myotube formation, inefficient skeletal muscle regeneration upon injury, and aggravated pathogenesis of a dystrophin-deficient mouse model of Duchenne muscular dystrophy. Thus, the flow from glycolysis to the TCA cycle mediated by PDH plays a pivotal role in the differentiation of SMSCs, which is critical for the progression of skeletal muscle regeneration.-Hori, S., Hiramuki, Y., Nishimura, D., Sato, F., Sehara-Fujisawa, A. PDH-mediated metabolic flow is critical for skeletal muscle stem cell differentiation and myotube formation during regeneration in mice.

A comparison of laparoendoscopic single-site surgery versus conventional procedures for laparoscopic donor nephrectomy: a Japanese multi-institutional retrospective study.

PURPOSE: Laparoendoscopic single-site donor nephrectomy (LESSDN) is a feasible and effective procedure because of its non-invasiveness and better cosmetic outcomes. However, there have been few multi-institutional studies conducted by multiple surgeons on LESSDN. We retrospectively compared the clinical data and outcomes between LESSDN and conventional laparoscopic donor nephrectomy (LDN) at multiple institutes in Japan. MATERIALS AND METHODS: From 2009 to 2015, the clinical data of 223 donors who underwent LESSDN and 151 donors who underwent LDN were collected from 10 institutes. All LESSDNs were performed transperitoneally, whereas LDNs were performed transperitoneally (P-LDN) in 75 patients and retroperitoneally (R-LDN) in 76 patients. RESULTS: In the LESSDN group, the single-incision site was pararectal in 155 (69.5%) patients and umbilical in 65 (29.1%) patients. Multiple surgeons (one to eight per institute) performed the LESSDN. No significant differences were observed between the three groups regarding estimated blood loss and warm ischemic time. The operative time was significantly shorter in the LESSDN group than in the R-LDN group (p = 0.018). No significant differences were observed regarding the rates of blood transfusion, open conversion, visceral injuries, and postoperative complications. Furthermore, no significant differences were observed regarding the dose of analgesic and the rate of delayed graft function. One patient required open conversion due to injury to the renal artery. Selection of LESS procedure was not an independent risk factor for the median serum creatinine level of above 1.27 mg/dL in recipients at 1 year after kidney transplantation. CONCLUSION: The results showed the technical feasibility of LESSDN compared with the standard LDNs in a multi-institutional and multi-surgeon setting. A few observed non-negligible complications and the significantly higher levels of serum creatinine in patients who underwent LESSDN indicate that this procedure should be employed cautiously when performed by surgeons without ample experience in performing LESS procedures.

Recovery of OATP1B Activity after Living Kidney Transplantation in Patients with End-Stage Renal Disease.

PURPOSE: Recently, several studies have shown that renal failure decreases the metabolic clearance of drugs and the transportation capability of some drug transporters. However, whether organic anion transporting polypeptide (OATP)1B activities decrease in renal failure remains unknown. In this study, we measured plasma concentrations of coproporphyrin-I (CP-I), a specific endogenous OATP1B probe, in patients with end stage renal disease before and after living kidney transplantation and evaluated the effect of renal function on OATP1B activity. METHODS: This prospective study recruited 13 patients with end-stage renal disease. Plasma CP-I concentrations were measured before and 7, 14, 30 and 90 days after living kidney transplantation. RESULTS: Plasma CP-I concentrations decreased over time after living kidney transplantation and showed significant difference on day 90 compared with before living kidney transplantation [1.12 ± 0.59 vs 0.65 ± 0.27 ng/mL, p < 0.05 (95% CI of difference - 0.927, -0.013)]. A significant negative correlation was observed between estimated glomerular filtration rate and plasma CP-I concentration (r = -0.30, p < 0.05), suggesting recovery of OATP1B activity with improvement in renal function. CONCLUSIONS: OATP1B activity may decrease in renal failure and dose adjustment of OATP1B substrates may be needed in patients with renal failure.

CLP1 links tRNA metabolism to progressive motor-neuron loss.

CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1(K/K)) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1(K/K) mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53.

High-sensitivity simultaneous quantification of tacrolimus and 13-O-demethyl tacrolimus in human whole blood using ultra-performance liquid chromatography coupled to tandem mass spectrometry.

Blood concentrations of tacrolimus show large variability among patients and the narrow therapeutic range is related to adverse effects. Therefore, therapeutic drug monitoring is needed for strict management. 13-O-Demethyl tacrolimus (13-O-DMT) was reported as the major metabolite formed by cytochrome P450 (CYP)3A such as CYP3A5. In previous studies, the best lower limit of quantification (LLOQ) was 0.1 ng/mL for both substances. However, this LLOQ may not be low enough now because the dosage of tacrolimus has decreased in recent years. The purpose of this study was to develop and validate a high-sensitivity and high-throughput assay for simultaneous quantification of tacrolimus and 13-O-DMT in human whole blood using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). Thirty-five stable kidney transplant recipients receiving tacrolimus were recruited in this study. The calibration curve range was 0.04-40 ng/mL. All calibration samples and quality control samples fulfilled the requirements of the US Food and Drug Administration and the European Medicines Agency guidelines for assay validation. Trough concentrations of tacrolimus and 13-O-DMT in 35 stable kidney transplant recipients receiving tacrolimus were within the range of the respective calibration curve. Our novel UPLC-MS/MS method is more sensitive than previous methods for quantification of tacrolimus and 13-O-DMT.

Anteroposterior molecular registries in ectoderm of the echinus rudiment.

BACKGROUND: Echinoderms and hemichordates are sister taxa that both have larvae with tripartite coeloms. Hemichordates inherit the coelom plan and ectoderm from larvae, whereas echinoderms form the adult rudiment comprising rearranged coeloms and a vestibule that then develops into adult oral ectoderm. Molecular networks that control patterns of the ectoderm and the central nervous system along the anteroposterior (AP) axis are highly conserved between hemichordates and chordates, respectively. In echinoderms, however, little is known about the AP registry in the ectoderm. RESULTS: We isolated ectodermal AP map genes from the sand dollar Peronella japonica and examined their expression. Comparative expression analyses showed that (1) P. japonica orthologs of hemichordate anterior markers are expressed in the larval apical plate, which degenerates during metamorphosis; (2) P. japonica orthologs of the medial markers are expressed in the ambulacral ectoderm of the rudiment; and (3) few P. japonica orthologs of the posterior markers are expressed in ectoderm. CONCLUSIONS: We suggest that echinoids only inherit the ambulacral ectoderm from a common ambulacrarian ancestor, which largely corresponds to the collar ectoderm in hemichordates. The ectodermal AP registry provides insights into the AP axis and evolutionary processes of echinoderms from a common ambulacrarian ancestor. Developmental Dynamics 247:1297-1307, 2018. © 2018 Wiley Periodicals, Inc.

Immunotherapy with cancer peptides in combination with intravesical bacillus Calmette-Guerin for patients with non-muscle invasive bladder cancer.

PURPOSE: A phase I study using two peptide vaccines derived from M phase phosphoprotein 1 (MPHOSPH1) and DEP domain containing 1 (DEPDC1) demonstrated promising results for the treatment of advanced bladder cancer. Therefore, we further tested the ability of these peptides to prevent recurrence after transurethral resection of the bladder tumor in patients with non-muscle invasive bladder cancer (NMIBC). MATERIALS AND METHODS: 127 patients were enrolled in a multicenter, non-randomized phase II clinical trial. The primary endpoint was recurrence-free survival (RFS) rate, and secondary endpoints were safety and immunological response. HLA-A24-restricted peptides were subcutaneously administered in addition to intravesical BCG therapy. The exploratory endpoint evaluated differences of RFS rate between HLA-A*2402-positive (A24(+)) and -negative (A24(-)) groups. RESULTS: A 2-year RFS rate in all patients was 74.0%. The RFS rate in the A24(+) group (n = 75) and in the A24(-) group (n = 52) were 76.0 and 71.2%, respectively. This vaccine therapy was well-tolerated and feasible. MPHOSPH1 and DEPDC1 peptide-specific cytotoxic T lymphocyte responses were observed in 75.8 and 77.5% of the A24(+) group, respectively. Patients having both peptide-specific CTL responses showed significantly better RFS than patients without CTL response (P = 0.014). In the A24(+) group, patients who had positive reaction at the injection sites (RAI) had significantly lower rates of recurrence than RAI-negative patients (P = 0.0019). CONCLUSIONS: Cancer peptide vaccines in combination with intravesical BCG therapy demonstrated good immunogenicity and safety, and may provide benefit for preventing recurrence of NMIBC.

Secretome analysis to elucidate metalloprotease-dependent ectodomain shedding of glycoproteins during neuronal differentiation.

Many membrane proteins are subjected to limited proteolyses at their juxtamembrane regions, processes referred to as ectodomain shedding. Shedding ectodomains of membrane-bound ligands results in activation of downstream signaling pathways, whereas shedding those of cell adhesion molecules causes loss of cell-cell contacts. Secreted proteomics (secretomics) using high-resolution mass spectrometry would be strong tools for both comprehensive identification and quantitative measurement of membrane proteins that undergo ectodomain shedding. In this study, to elucidate the ectodomain shedding events that occur during neuronal differentiation, we establish a strategy for quantitative secretomics of glycoproteins released from differentiating neuroblastoma cells into culture medium with or without GM6001, a broad-spectrum metalloprotease inhibitor. Considering that most of transmembrane and secreted proteins are N-glycosylated, we include a process of N-glycosylated peptides enrichment as well as isotope tagging in our secretomics workflow. Our results show that differentiating N1E-115 neurons secrete numerous glycosylated polypeptides in metalloprotease-dependent manners. They are derived from cell adhesion molecules such as NCAM1, CADM1, L1CAM, various transporters and receptor proteins. These results show the landscape of ectodomain shedding and other secretory events in differentiating neurons and/or during axon elongation, which should help elucidate the mechanism of neurogenesis and the pathogenesis of neurological disorders.

Urological laparoendoscopic single-site and reduced port surgery: A nationwide survey in Japan.

OBJECTIVES: To evaluate the current status of urological laparoendoscopic single-site and reduced port surgery in Japan. METHODS: Of the 152 institutions to which councilors of the Japanese Society of Endourology belong, 42 (28%) have carried out laparoendoscopic single-site and reduced port surgery. A total of 32 of these institutions agreed to participate in this survey. Patients who had undergone surgery between January 2008 and March 2014 were included in the present study. RESULTS: Overall, 1145 cases of laparoendoscopic single-site and reduced port surgery were recorded during the study period. The most frequent procedures were adrenalectomy and radical nephrectomy. Laparoendoscopic single-site and reduced port surgery represented 12% (872/7311) of all laparoscopic procedures carried out at participating institutions. The number of patients who underwent pyeloplasty, donor nephrectomy and simple nephrectomy tended to increase, whereas those who underwent adrenalectomy, radical nephrectomy and nephroureterectomy peaked in 2012, and then tended to decrease in 2013. The rates of conversion, perioperative and postoperative complications, were 2.7%, 2.2% and 4.5%, respectively. CONCLUSIONS: The number of laparoendoscopic single-site and reduced port urological surgeries in Japan has increased for benign indications, such as pyeloplasty, donor nephrectomy and simple nephrectomy. In contrast, procedures such as adrenalectomy and radical nephrectomy are trending down after reaching a peak in 2012. Overall, laparoendoscopic single-site and reduced port urological surgery in Japan is being safely carried out when compared with other reported series of laparoendoscopic single-site surgery and conventional laparoscopic surgery.

Laparoendoscopic single-site nephrectomy for hemodialysis patients with dialysis-related renal tumors.

PURPOSE: The purpose of this study is to assess the efficacy of laparoendoscopic single-site (LESS) nephrectomy in hemodialysis patients, we compared outcomes between LESS nephrectomy and conventional laparoendoscopic nephrectomy in hemodialysis patients with dialysis-related renal tumors. MATERIAL AND METHODS: A total of 16 hemodialysis patients who underwent LESS nephrectomy (LESS-N; n = 8) or conventional laparoendoscopic nephrectomy (C-N; n = 8) between November 2003 and July 2012 were retrospectively evaluated. Outcomes were compared between the two groups. RESULTS: Patient and tumor characteristics were similar between the LESS-N and C-N groups. The mean operative duration was longer in the LESS-N than in the C-N group (231.0 ± 26.7 min versus 188.6 ± 36.4 min; p = .025). The mean estimated blood loss was lower in the LESS-N compared with the C-N group (26.4 ± 14.4 ml versus 65.6 ± 45.2 ml; p = .047). Postoperative complications were observed in three cases, comprising one case of retroperitoneal hematoma in the LESS-N group and one case each of peritoneal hematoma and retroperitoneal abscess in the C-N group. Surgical scarring was minimal in the LESS-N group. CONCLUSIONS: Although there is a little extension of the operating time, LESS nephrectomy in hemodialysis patients is a feasible procedure compared with the conventional method.

Phase I clinical trial of cell division associated 1 (CDCA1) peptide vaccination for castration resistant prostate cancer.

Cell division associated 1 (CDCA1) was screened as an oncogene that is overexpressed on several cancers, including prostate cancer. A highly immunogenic HLA-A*2402-restricted epitope peptide corresponding to part of the CDCA1 protein was also identified. A phase I clinical trial was conducted for patients with castration resistant prostate cancer (CRPC) using a CDCA1 peptide vaccination. Twelve patients having HLA-A*2402 with CRPC after failure of docetaxel chemotherapy were enrolled. They received subcutaneous administration of the CDCA1 peptide as an emulsion with Montanide ISA51VG once a week in a dose-escalation manner (doses of 1.0 or 3.0 mg/body, six patients received each dose). The primary endpoint was safety, and the secondary endpoints were the immunological and clinical responses. Vaccination with CDCA1 peptide was well tolerated without any serious adverse events. Peptide-specific cytotoxic T lymphocyte (CTL) responses using ELISPOT assay and dextramer assay were observed in three patients receiving the 1.0 mg dose and five patients receiving the 3.0 mg dose. The median overall survival time was 11.0 months and specific CTL reacting to CDCA1 peptide were recognized in long-surviving patients. CDCA1-derived peptide vaccine treatment was tolerable and might effectively induce peptide-specific CTLs for CRPC patients. This novel peptide vaccine therapy for CRPC appears promising. (ClinicalTrials.gov number, NCT01225471).

Effects of Estrogen Receptor ß Stimulation in a Rat Model of Non-Bacterial Prostatic Inflammation.

BACKGROUND: There is increasing evidence showing that chronic non-bacterial prostatic inflammation is involved in the pathogenesis of benign prostatic hyperplasia (BPH) and male lower urinary tract symptoms (LUTS). It has also been reported that estrogen receptor ß (ERß) could have an immunoprotective role in prostatic tissue. Therefore, we investigated the effect of ERß-activation on not only prostatic inflammation, but also bladder overactive conditions in a rat model with nonbacterial prostatic inflammation. METHODS: Male Sprague-Dawley rats (8 weeks, n = 15) were divided into three groups: sham-saline group (n = 5), formalin-vehicle group (n = 5), and formalin-treatment group (n = 5). The sham-saline group had sham operation and 50 µl normal saline injected into each ventral lobe of the prostate. The formalin-vehicle group had 50 µl 5% formalin injection into bilateral ventral lobes of the prostate. The formalin-treatment group was treated with 3α-Adiol (a selective ERß agonist precursor) at a dose of 3 mg/kg daily from 2 days before induction of prostatic inflammation, whereas formalin-vehicle rats received vehicle (olive oil). In each group, conscious cystometry was performed on day 28 after intraprostatic formalin injection or sham treatment. After cystometry, the bladder and prostate were harvested for evaluation of mRNA expression and histological analysis. RESULTS: In cystometric investigation, the mean number of non-voiding contractions was significantly greater and voiding intervals were significantly shorter in formalin-vehicle rats than those in sham-saline rats (P < 0.05). In RT-qPCR analysis, mRNA expression of NGF, P2X2, and TRPA1 receptors was significantly increased in the bladder mucosa, and mRNA expression of TNF-α, iNOS and COX2 in the ventral lobes of prostate was significantly increased in formalin-vehicle rats compared with sham-saline rats (P < 0.05). In addition, relative mRNA expression ratio of ERß to ERα (ERß/ERα) in the ventral lobes of prostate was significantly decreased in formalin-vehicle rats compared with sham-saline rats (P < 0.05). These changes were ameliorated by 3α-Adiol administration in formalin-treatment rats. CONCLUSIONS: These results indicate that ERß activation by 3α-Adiol administration, which normalized the ERß/ERα expression ratio in the prostate, can improve not only prostatic inflammation, but also bladder overactivity. Therefore, ERß agonists might be useful for treating irritative bladder symptoms in patients with symptomatic BPH associated with prostatic inflammation. Prostate 77:803-811, 2017. © 2017 Wiley Periodicals, Inc.

Kidney-specific knockout of Sav1 in the mouse promotes hyperproliferation of renal tubular epithelium through suppression of the Hippo pathway.

We have previously reported that Salvador homologue 1 (SAV1), a component of the Hippo pathway, is significantly down-regulated in high-grade clear cell renal cell carcinoma (ccRCC) due to 14q copy number loss, and that this down-regulation contributes to the proliferation and survival of renal tubular epithelial cells through activation of Yes-associated protein 1 (YAP1), a downstream target of the Hippo pathway. However, the impact of SAV1 loss on the proliferation and survival of kidney cells in vivo remained to be determined. To address this issue, we generated kidney-specific Sav1-knockout (Cdh16-Cre;Sav1(fl/fl) ) mice. Sav1 deficiency enhanced the proliferation of renal tubular epithelial cells in Cdh16-Cre;Sav1(fl/fl) mice, accompanied by nuclear localization of Yap1, suggesting suppression of the Hippo pathway. Sav1 deficiency in renal tubules also caused structural and cellular abnormalities of the epithelial cells, including significant enlargement of their nuclei. Furthermore, Cdh16-Cre;Sav1(fl/fl) mice developed both glomerular and tubular cysts. Although lining cells of the glomerular cysts showed no atypia, those of the tubular cysts showed variations in cell size and nuclear shape, which became more severe as the mice aged. In aged Cdh16-Cre;Sav1(fl/fl) mice, we observed focal disruption of proximal tubules and perivascular lymphocytic infiltration. In conclusion, Sav1 is required for the maintenance of growth, nuclear size and structure of renal tubules under physiological conditions, and its deficiency leads to the acquisition of enhanced proliferation of renal epithelial cells through suppression of Hippo signalling.

A case of anaplastic lymphoma kinase-positive renal cell carcinoma coincident with Hodgkin lymphoma.

We report a case of ALK-positive renal cell carcinoma coincident with Hodgkin lymphoma. The patient was a 19 year-old-girl without sickle cell trait. The right renal tumor was discovered concomitantly with Hodgkin lymphoma (HL). After chemotherapy for HL, right nephrectomy was performed. Microscopically, the tumor showed a solid and focally pseudo-papillary growth pattern studded with tubular structures. Most tumor cells were small bland eosinophilic cells, but rhabdoid cells, vacuolated cells, pleomorphic multinucleated giant cells were also admixed. The variety of growth patterns and cell features led us to speculate a possibility of ALK-positive renal cell carcinoma (ALK + RCC). ALK was immunohistochemically positive, and fluorescence in situ hybridization analysis detected a split signal of the ALK gene. We examined previously reported partner genes (STRN, TPM3, VCL and EML4) by RT-PCR, but fusion gene was not detected. RCC showing solid or cribriform growth patterns with vacuolated cells with intracytoplamic lumina, rhabdoid cells, and mucus production indicates the possibility of ALK + RCC.

A phase III multicenter, randomized, controlled study of combined androgen blockade with versus without zoledronic acid in prostate cancer patients with metastatic bone disease: results of the ZAPCA trial.

OBJECTIVE: To examine the antitumor activity of zoledronic acid (ZA) combined with androgen deprivation therapy (ADT) for men with treatment-naive prostate cancer and bone metastasis. METHODS: We enrolled 227 men with treatment-naive prostate cancer and bone metastasis. Participants were randomly assigned (1:1 ratio) to receive combined androgen blockade alone (CAB group) or ZA with combined androgen blockade (CZ group). Time to treatment failure (TTTF), time to the first skeletal-related event (TTfSRE), and overall survival (OS) rates were estimated using the Kaplan-Meier method. Hazard ratios (HRs) were calculated using the Cox proportional hazards model. Median follow-up duration was 41.5 months. RESULTS: Median TTTFs were 12.4 and 9.7 months for the CZ and CAB groups, respectively (HR 0.75; 95 % CI 0.57-1.00; p = 0.051). For men with baseline prostate-specific antigen levels <200 ng/mL, median TTTFs were 23.7 and 9.8 months for the CZ and CAB groups, respectively (HR 0.58; 95 % CI 0.35-0.93; p = 0.023). Median TTfSREs were 64.7 and 45.9 months for the CZ and CAB groups, respectively (HR 0.58; 95 % CI 0.38-0.88; p = 0.009). OS was similar between the groups. CONCLUSIONS: This study failed to demonstrate that combined use of ZA and ADT significantly prolonged TTTF in men with treatment-naive prostate cancer and bone metastasis. However, it generates a new hypothesis that the combined therapy could delay the development of castration resistance in a subgroup of patients with low baseline prostate-specific antigen values <200 ng/mL. The treatment also significantly prolonged TTfSRE but did not affect OS.

Tumor necrosis factor-alpha inhibits differentiation of myogenic cells in human urethral rhabdosphincter.

OBJECTIVES: To examine the inhibitory effects of tumor necrosis factor-α on myogenic differentiation of human urethral rhabdosphincter cells. METHODS: A rhabdosphincter sample was obtained from a patient who underwent total cystectomy. To expand the lifespan of the primary cultured cells, rhabdosphincter myogenic cells were immortalized with mutated cyclin-dependent kinase 4, cyclin D1 and telomerase. The differential potential of the cells was investigated. The transfected human rhabdosphincter cells were induced for myogenic differentiation with recombinant human tumor necrosis factor-α and/or the tumor necrosis factor-α antagonist etanercept at different concentrations, and activation of signaling pathways was monitored. RESULTS: Human rhabdosphincter cells were selectively cultured for at least 40 passages. Molecular analysis confirmed the expression of myosin heavy chain, which is a specific marker of differentiated muscle cells, significantly increased after differentiation induction. Although tumor necrosis factor-α treatment reduced the myosin heavy chain expression in a concentration-dependent manner, etanercept inhibited this suppression. Tumor necrosis factor-α suppressed phosphorylation of protein kinase B and p38, whereas etanercept pretreatment promoted phosphorylation and myosin heavy chain expression in a concentration-dependent manner. CONCLUSIONS: Tumor necrosis factor-α inhibits differentiation of urethral rhabdosphincter cells in part through the p38 mitogen-activated protein kinase and phosphoinositide 3-kinase pathways. Inhibition of tumor necrosis factor-α might be a useful strategy to treat stress urinary incontinence.

Laparoendoscopic single-site surgeries: A multicenter experience of 469 cases in Japan.

OBJECTIVE: To report on a multi-institutional series of non-robotic urological laparoendoscopic single-site surgery in Japan. METHODS: Consecutive cases of laparoendoscopic single-site surgery carried out between February 2009 and December 2012 at nine academic institutions were included. We examined the surgical outcomes, including conversion and complications rates. RESULTS: Four hundred and sixty-nine cases were included in the analysis. The most common procedure was adrenalectomy (n = 177) and the second most common procedure was radical nephrectomy (n = 143). The procedures also included nephroureterectomy (n = 40), living donor nephrectomy (n = 40), pyeloplasty (n = 30), urachal remnant excision (n = 9), simple nephrectomy (n = 7), radical prostatectomy (n = 6) and others (n = 17). The access sites included umbilicus (n = 248, 53%) and other sites (n = 221, 47%). A transperitoneal approach was used in 385 cases (82%), and retroperitoneal approach in 84 cases (18%). The median operation time of all procedures was 198 min. Conversion to reduced port surgery, conventional laparoscopy, or open surgery was noted in 27 cases (5.8%), 12 cases (2.6%), and two cases (0.4%), respectively, with an overall conversion rate of 8.7%. Intraoperative complications occurred in 10 cases (2.1%). Post-operative complications were noted in 29 cases (6.2%), including five major complications (1.1%). No mortality was recorded in this series. CONCLUSIONS: Non-robotic laparoendoscopic single-site surgery is technically feasible and safe for various urologic diseases in Japan. Furthermore, urological laparoendoscopic single-site surgery is a promising minimally invasive surgical option that is feasible for experienced urological surgeons in intermediate-volume centers as well as high-volume centers.

Downregulation of WDR20 due to loss of 14q is involved in the malignant transformation of clear cell renal cell carcinoma.

Previously, we reported that genomic loss of 14q occurs more frequently in high-grade than in low-grade clear cell renal cell carcinomas (ccRCCs), and has a significant impact on the levels of expression of genes located in this region, suggesting that such genes may be involved in the malignant transformation of ccRCCs. Here, we found that six of the genes located in the minimal common region of 14q loss were significantly downregulated in high-grade ccRCCs due to copy number loss. Using a dataset from The Cancer Genome Atlas Research Network, we found that downregulation of one of these six genes, WDR20, was significantly associated with poorer outcome in patients with ccRCC, suggesting that WDR20 downregulation may be involved in the malignant transformation of ccRCCs. In functional assays, exogenous WDR20 significantly inhibited the growth of RCC cell lines and induced apoptosis. Interestingly, the phosphorylation levels of ERK and protein kinase B/AKT, which reportedly contribute to the malignant phenotype of RCC cells, were clearly reduced by exogenous expression of WDR20. Thus, our data suggest that downregulation of WDR20 due to 14q loss may be involved in the malignant transformation of ccRCCs, in part through activation of the ERK and protein kinase B/AKT pathways.