Daniel D. Shapiro

Genetic analysis of the role of yfiR in the ability of Escherichia coli CFT073 to control cellular cyclic dimeric GMP levels and to persist in the urinary tract.

ABSTRACT During urinary tract infections (UTIs), uropathogenic Escherichia coli must maintain a delicate balance between sessility and motility to achieve successful infection of both the bladder and kidneys. Previous studies showed that cyclic dimeric GMP (c-di-GMP) levels aid in the control of the transition between motile and nonmotile states in E. coli. The yfiRNB locus in E. coli CFT073 contains genes for YfiN, a diguanylate cyclase, and its activity regulators, YfiR and YfiB. Deletion of yfiR yielded a mutant that was attenuated in both the bladder and the kidneys when tested in competition with the wild-type strain in the murine model of UTI. A double yfiRN mutant was not attenuated in the mouse model, suggesting that unregulated YfiN activity and likely increased cytoplasmic c-di-GMP levels cause a survival defect. Curli fimbriae and cellulose production were increased in the yfiR mutant. Expression of yhjH, a gene encoding a proven phosphodiesterase, in CFT073 ΔyfiR suppressed the overproduction of curli fimbriae and cellulose and further verified that deletion of yfiR results in c-di-GMP accumulation. Additional deletion of csgD and bcsA, genes necessary for curli fimbriae and cellulose production, respectively, returned colonization levels of the yfiR deletion mutant to wild-type levels. Peroxide sensitivity assays and iron acquisition assays displayed no significant differences between the yfiR mutant and the wild-type strain. These results indicate that dysregulation of c-di-GMP production results in pleiotropic effects that disable E. coli in the urinary tract and implicate the c-di-GMP regulatory system as an important factor in the persistence of uropathogenic E. coli in vivo.

Urosymphyseal Fistulas Resulting From Endoscopic Treatment of Radiation-induced Posterior Urethral Strictures

OBJECTIVE To describe the inciting events leading to urosymphyseal fistulas (UFs) and pubic osteomyelitis (PO) in patients who had radiation-induced urethral strictures. METHODS We retrospectively reviewed patients who underwent simultaneous pubic debridement, simple cystectomy, and urinary diversion for refractory UF and PO from 2014 to 2016. We investigated inciting events leading to UF, as well as patient presenting symptoms, diagnosis, management, and outcomes. RESULTS Five patients were identified over a 2-year period. All patients had a previous history of radiation for prostate cancer. The median age was 67 years. All patients developed UF and PO after endoscopic intervention for urethral stricture. The number of endoscopic interventions per patient for stricture ranged from 1 to 7, including serial dilation, balloon dilation, and urethrotomy. Sterile urine cultures were obtained before all endoscopic interventions. All patients had pelvic pain with ambulation and recurrent urinary tract infections at presentation. Patients were diagnosed using a combination of retrograde urethrography and magnetic resonance imaging. Simultaneous pubic debridement with simple cystectomy and diversion was used for management in all cases. One patient died postoperatively with the remainder recovering well without PO or fistula recurrence, with a median follow-up of 16 months. CONCLUSION UF can occur as a complication of endoscopic treatment of posterior urethral stricture in patients with a history of radiation therapy for prostate cancer. This study demonstrates that UF and PO may develop even with minimally traumatic procedures and sterile urine. All patients treated for posterior stricture must be considered at risk of development of fistulas and osteomyelitis.

Vasectomy Practice Patterns Among Family Medicine Physicians and Compliance With the American Urological Association 2012 Vasectomy Guidelines

OBJECTIVE To survey urologists and family medicine physicians (FMPs) within a single institution to determine current vasectomy practice patterns and determine compliance with 2012 American Urological Association (AUA) vasectomy guidelines. METHODS In 2016, a single-institution survey was conducted to understand the vasectomy practice patterns among urologists and nonurologists. The survey questions and 3 clinical scenarios were designed based on the 2012 AUA vasectomy guidelines. Results of the survey were compiled between urologists and nonurologists and then compared with the guideline recommendations. RESULTS A total of 23 FMPs and 6 urologists responded. Fewer prevasectomy counseling topics were discussed by FMPs compared with urologists. A variety of vasectomy techniques were used among FMPs. Vas deferens segments were more likely to be sent for histology by FMPs than urologists (65% vs 17%, P = .02). FMPs were more likely to send postvasectomy semen analyses earlier than urologists (P = .02) and more likely to send multiple postvasectomy semen analyses (P = .006) before forgoing alternative contraceptive methods. Regarding the clinical scenario questions, FMPs were more likely to answer discordantly from guideline recommendations compared with urologists. CONCLUSION Significant vasectomy practice pattern heterogeneity still exists among nonurologists surveyed within our institution. The 2012 AUA vasectomy guidelines have yet to be broadly implemented within nonurology practices. Further studies are warranted to investigate national trends in nonurologist vasectomy practice patterns and determine how the guidelines can be better implemented in nonurologic practices.

MP39-02 CORE MUSCLE SIZE PREDICTS 6-MONTH MORTALITY IN PATIENTS UNDERGOING RADICAL CYSTECTOMY

INTRODUCTION AND OBJECTIVES: Anticancer agents have been shown to induce endoplasmic reticulum (ER) stress and autophagy in various cancer cells. Autophagy acts as an adaptive mechanism for cancer cell survival. However, the mechanism and the effect of ER stress induced autophagy in urothelial cancer cell survival is not well understood. METHODS: Materials consisted of high and low grade urothelial cancer (UC) tissue and normal urothelium. ER stress was induced by tunicamycin and thapsigargin and measured by expression of various ER stress marker i.e. Grp-78, Ero-1La, PDI, IRE1a and PERK. ER stress induction was also validated by the measurement of Ca+2, IP3 and DAG assay. Autophagy was studied by lysotracker and LC3 immuno-staining. ExpressionofAMP-activatedproteinkinase (AMPK)andmammalian target of rapamycin (mTOR) was also studied for determination of autophagy. ER stress induced autophagy on cell survival was studied in UC derived primary culture cells and T24 bladder cancer cell line, by the measurement of cell viability,mitochondrialmembrane potential, and caspases activation. RESULTS: Treatment of tunicamycin and thapsigargin induced the expression of ER stress marker at six hours and increased up-to 12 hours in UC cells as well as in normal urothelium (Fig.1A). There was increased Ca+2 flux, IP3 and DAG activity. Autophagosomes also increased in number,more so in highgradeUC (Fig.1B). Theexpressionof phophorylated-AMPK increased and the expression of phosphorylatedmTOR decreased. Addition of autophagy inhibitor, wortmannin (Wm) along with tunicamycin or thapsigargin induced cell death in both grade of UC (Fig.2A). Treatment of calcium chelater (EGTA) or pan caspase inhibitor, z-VAD-fmkblocked the cell death induced byWm(Fig.1C). There was increased caspase-12, -9 and -3 expression (Fig.1D). CONCLUSIONS: Ca+2 serves as a potent inducer of autophagy during ER stress in UC as well as in normal urothelium. Ca+2 mediated autophagy is regulated by IP3-AMPK-mTOR pathways and inhibition of Ca+2 mediated autophagy leads to caspase mediated intrinsic apoptotic cell death, suggesting that autophagy plays important roles in cell survival. Further elucidation of the molecular mechanisms linking ER stress to autophagy is needed for therapeutic intervention in urothelial carcinoma.

MP2-05 IDENTIFYING PATIENTS AT HIGH-RISK FOR READMISSION AFTER RADICAL CYSTECTOMY USING THE NATIONAL SURGICAL QUALITY IMPROVEMENT PROGRAM DATABASE

Calculator, we calculated the difference between mean predicted risk and the observed rate of surgical outcomes in our cohort. To assess statistical significance of differences between observed and expected outcomes, we used a bootstrap with 1,000 replicates, calculated confidence intervals via the percentile method, and determined p-values using two-sided pivot tests. RESULTS: 314 patients undergoing RC were included for analysis. All patients had complete data available for pre-and postoperative risk assessment. Comparing observed to expected events, the NSQIP calculator significantly underestimated the risks of death (3.3% [CI 1.3-5.4]; p1⁄40.002), serious complications (19.4% [CI 14.124.5]; p<0.001), overall complications (23.4% [CI 17.6-28.5]; p<0.001), surgical site infections (5.8% [CI 1.8-9.6]; p1⁄40.002), urinary tract infections (15.2% [CI 10.8-19.7]; p<0.001), acute renal failure (3.2% [CI 0.9-6.1]; p1⁄40.008), and hospital length of stay (4.8 days [CI 3.8-6.0]; p<0.001). There were no significant differences between predicted and observed rates of pneumonia, cardiac morbidity, venous thromboembolism, or need for return to the operating room. CONCLUSIONS: Patients undergoing RC had significant differences in several observed versus expected peri-operative outcomes estimated with the ACS NSQIP Surgical Risk Calculator. Growing participation by hospitals in the ACS NSQIP’s Procedure Targeted Option, and creation/incorporation of procedure (e.g. RC)-specific preoperative risk factors and outcomes will likely enhance the accuracy of surgical outcomes estimation by the Surgical Risk Calculator.